£EPA
EPA/635 /R-16/331b
Final Agency/Interagency Draft
www.epa.gov/iris
Toxicological Review of Benzo[a]pyrene
(CASRN 50-32-8]
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
Supplemental Information
November 2016
NOTICE
This document is a Final Agency Review/Interagency Science Discussion draft. This
information is distributed solely for the purpose of pre-dissemination peer review under applicable
information quality guidelines. It has not been formally disseminated by EPA. It does not represent
and should not be construed to represent any Agency determination or policy. It is being circulated
for review of its technical accuracy and science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. Mention of trade names or
commercial products does not constitute endorsement of recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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CONTENTS
APPENDIX A. CHEMICAL PROPERTIES AND EXPOSURE INFORMATION A-l
APPENDIX B. ASSESSMENTS BY OTHER NATIONAL AND INTERNATIONAL HEALTH AGENCIES B-l
APPENDIX C. LITERATURE SEARCH STRATEGY C-l
APPENDIX D. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-RESPONSE
ANALYSIS D-l
D.l. TOXICOKINETICS D-l
D.l.l. Overview D-l
D.l.2. Absorption D-l
D.l.3. Distribution D-3
D.1.4. Metabolism D-4
D.l.5. Elimination D-ll
D.2. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS D-12
D.2.1. Recommendations for the Use of PBPK Models in Toxicity Value
Derivation D-15
D.3. HUMAN STUDIES D-15
D.3.1. Noncancer Endpoints D-15
D.3.2. Cancer-related Endpoints D-26
D.3.3. Epidemiologic Findings in Humans D-29
D.4. ANIMAL STUDIES D-42
D.4.1. Oral Bioassays D-42
D.4.2. Inhalation Studies D-61
D.4.3. Dermal studies D-64
D.4.4. Reproductive and Developmental Toxicity Studies D-74
D.4.5. Inhalation D-90
D.5. OTHER PERTINENT TOXICITY INFORMATION D-94
D.5.1. Genotoxicity Information D-94
D.5.2. Tumor Promotion and Progression D-122
D.5.3. Benzo[a]pyrene Transcriptomic Microarray Analysis D-126
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APPENDIX E. DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES FOR
EFFECTS OTHER THAN CANCER AND THE DERIVATION OF CANCER RISK
ESTIMATES E-l
E.l. NONCANCER ENDPOINTS E-l
E.l.l. Data Sets E-l
E.l.2. Dose Response Modeling for Noncancer Endpoints E-3
E.l.3. Dosimetry Modeling for Estimation of Human Equivalent
Concentrations for Reference Concentration (RfC) E-41
E.2. Cancer Endpoints E-44
E.2.1. Dose-Response Modeling for the Oral Slope Factor E-44
E.2.2. Dose-Response Modeling for the Inhalation Unit Risk E-90
APPENDIX F. SUMMARY OF SAB PEER REVIEW COMMENTS AND EPA'S DISPOSITION F-l
REFERENCES FOR APPENDICES 1
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TABLES
Table A-l. Chemical and physical properties of benzo[a]pyrene A-2
Table A-2. Benzo[a]pyrene concentrations in air A-4
Table A-3. Benzo[a]pyrene levels in food A-5
Table A-4. Levels of benzo[a]pyrene in soil A-7
Table B-l. Health assessments and regulatory limits by other national and international
agencies B-l
Table C-l. Summary of detailed search strategies for benzo[a]pyrene comprehensive literature
searches (Pubmed, Toxline, Toxcenter, TSCATS) C-l
Table C-2. Summary of detailed literature search strategies for benzo(a)pyrene cardiovascular
toxicity C-9
Table D-l. Exposure to benzo[a]pyrene and mortality from cardiovascular diseases in a
European cohort of asphalt paving workers D-16
Table D-2. Exposure to benzo[a]pyrene and mortality from cardiovascular diseases in a
Canadian cohort of male aluminum smelter workers D-18
Table D-3. Exposure-related effects in Chinese coke oven workers or warehouse controls
exposed to benzo[a]pyrene in the workplace D-24
Table D-4. Exposure-related effects in Chinese coke oven workers or warehouse controls
exposed to benzo[a]pyrene in the workplace, stratified by urinary metabolite
levels D-24
Table D-5. Background information on Chinese coke oven workers or warehouse controls
exposed to benzo[a]pyrene in the workplace D-26
Table D-6. Studies examining skin cancer risk in relation to therapeutic coal tar D-34
Table D-7. Exposure-related effects in male Wistar rats exposed to benzo[a]pyrene by gavage 5
days/week for 5 weeks D-43
Table D-8. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene by gavage 5
days/week for 5 weeks D-46
Table D-9. Means ± SDa for liver and thymus weights in Wistar rats exposed to benzo[a]pyrene
by gavage 5 days/week for 90 days D-48
Table D-10. Incidences of exposure-related neoplasms in Wistar rats treated by gavage with
benzo[a]pyrene, 5 days/week, for 104 weeks D-50
Table D-ll. Incidences of alimentary tract tumors in Sprague-Dawley rats chronically exposed to
benzo[a]pyrene in the diet or by gavage in caffeine solution D-53
Table D-12. Incidence of nonneoplastic and neoplastic lesions in female B6C3Fi mice fed
benzo[a]pyrene in the diet for up to 2 years D-56
Table D-13. Other oral exposure cancer bioassays in mice D-57
Table D-14. Tumor incidence in the respiratory tract and upper digestive tract for male Syrian
golden hamsters exposed to benzo[a]pyrene via inhalation for lifetime—
Thyssen et al. (1981)a D-63
Table D-15. Skin tumor incidence and time of appearance in male C57L mice dermally exposed
to benzo[a]pyrene for up to 103 weeks D-66
Table D-16. Skin tumor incidence and time of appearance in male SWR, C3HeB, and A/He mice
dermally exposed to benzo[a]pyrene for life or until a skin tumor was detected D-67
Table D-17. Tumor incidence in female Swiss mice dermally exposed to benzo[a]pyrene for up
to 93 weeks D-68
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Table D-18. Skin tumor incidence in female NMRI and Swiss mice dermally exposed to
benzo[a]pyrene D-69
Table D-19. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene D-70
Table D-20. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene D-70
Table D-21. Skin tumor incidence and time of appearance in female CFLP mice dermally
exposed to benzo[a]pyrene for 104 weeks D-71
Table D-22. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene for
life D-72
Table D-23. Skin tumor incidence in male C3H/HeJ mice dermally exposed to benzo[a]pyrene for
24 months D-73
Table D-24. Mortality and cervical histopathology incidences in female ICR mice exposed to
benzo[a]pyrene via gavage for 14 weeks D-77
Table D-25. Means ± SD for ovary weight in female Sprague-Dawley rats D-80
Table D-26. Reproductive effects in male and female CD-I F1 mice exposed in utero to
benzo[a]pyrene D-82
Table D-27. Effect of prenatal exposure to benzo[a]pyrene on indices of reproductive
performance in F1 female NMRI mice D-83
Table D-28. Exposure-related effects in Long-Evans Hooded rats exposed to benzo[a]pyrene by
gavage daily in utero from GD 14 to 17 D-87
Table D-29. Exposure-related pup body weight effects in Swiss Albino OF1 mice exposed as pups
to benzo[a]pyrene in breast milk from dams treated by gavage daily from PND 1
to 14 D-88
Table D-30. Pregnancy outcomes in female F344 rats treated with benzo[a]pyrene on GDs
11-21 by inhalation D-91
Table D-31. Select PAH-DNA adduct detection methods3 D-94
Table D-32. In vitro genotoxicity studies of benzo[a]pyrene in non-mammalian cells D-95
Table D-33. In vitro genotoxicity studies of benzo[a]pyrene in mammalian cells D-97
Table D-34. Studies of benzo[a]pyrene-induced genotoxicity in humans exposed to PAHs D-102
Table D-35. Non-human in vivo genotoxicity studies of benzo[a]pyrene D-107
Table D-36. Search terms and the number of studies retrieved from the gene expression
omnibus and array express microarray repositories D-126
Table D-37. Mapping of group numbers to time/dose groups D-129
Table E-l. Noncancer endpoints selected for dose-response modeling for benzo[a]pyrene: RfD E-2
Table E-2. Noncancer endpoints selected for dose-response modeling for benzo[a]pyrene: RfC E-3
Table E-3. Summary of BMD modeling results for decreased thymus weight in male Wistar rats
exposed to benzo[a]pyrene by gavage for 90 days (Kroese et al., 2001); BMR = 1
SD change from the control mean E-5
Table E-4. Summary of BMD modeling results for decreased thymus weight in female Wistar
rats exposed to benzo[a]pyrene by gavage for 90 days (Kroese et al., 2001); BMR
= 1 SD change from the control mean E-8
Table E-5. Summary of BMD modeling results for decreased ovary weight in female Sprague-
Dawley rats exposed to benzo[a]pyrene by gavage for 60 days (Xu et al., 2010);
BMR = 1 SD change from the control mean E-ll
Table E-6. Summary of BMD modeling results for decreased primordial follicles in female
Sprague-Dawley rats exposed to benzo[a]pyrene by gavage for 60 days (Xu et
al., 2010); BMR = 1 SD change from the control mean E-14
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Table E-7. Summary of BMD modeling results for mean number of squares crossed on PND 69
by male and female Sprague Dawley rats exposed to benzo[a]pyrene by gavage,
PNDs 5-11 (Chen et al., 2012); BMR = 1 SD change from control mean E-18
Table E-8. Summary of BMD modeling results for elevated plus maze: open arm entries at PND
70 for female Sprague Dawley rats exposed to benzo[a]pyrene by gavage on
PNDs 5-11 (Chen et al., 2012); BMR = 1 SD E-20
Table E-9. Summary of BMD Modeling Results for escape latency of male and female Sprague-
Dawley rats at PND 71 exposed to benzo[a]pyrene by gavage on PNDs 5-11,
(Chen et al., 2012); BMR = 1 SDa change from the control mean E-22
Table E-10. Summary of BMD Modeling Results for escape latency of male and female Sprague-
Dawley rats at PND 72 exposed to benzo[a]pyrene by gavage on PNDs 5-11
(Chen et al., 2012); BMR = 1 SDa from control mean E-24
Table E-ll. Summary of BMD Modeling Results for escape latency of male and female Sprague-
Dawley rats at PND 73 exposed to benzo[a]pyrene by gavage on PNDs 5-11
(Chen et al., 2012); BMR = 1 SDa change from control mean E-26
Table E-12. Summary of BMD modeling results for escape latency at PND 74 for male and
female Sprague-Dawley rats exposed to benzo[a]pyrene by gavage PNDs 5-11
(Chen et al., 2012); BMR = 1 SDa change from control mean E-28
Table E-13. Summary of BMD modeling results for incidence of cervical epithelial hyperplasia in
female ICR mice exposed to benzo[a]pyrene by oral exposure for 98 days (Gao
et al., 2011); BMR = 10% extra risk E-30
Table E-14. Summary of BMD modeling results of embryo/fetal survival for female F344 rats
exposed to benzo[a]pyrene via inhalation on GDs 11-20 (Archibong et al., 2002);
BMR = 10 percentage points absolute deviation from control mean E-33
Table E-15. Derivation of incidence data adjusted for design effect, for embryo/fetal resorption
data in Archibong et al. (2002) E-34
Table E-16. Summary of BMD modeling results for estimated incidence of embryo/fetal
resorptions (Archibong et al., 2002), adjusted for design effect; BMR=1, 5, or
20% extra risk3 E-35
Table E-17. Summary of BMD Modeling Results for ovarian weight in F344 rats exposed to
benzo[a]pyrene via inhalation for 14 days prior to mating (Archibong et al.,
2012); BMR = 10% relative deviation from control mean E-38
Table E-18. Summary of BMD modeling results for ovulation rate (ovulated oocytes/dam) in
female F344 rats following inhalation exposure to benzo[a]pyrene for 14 days
(Archibong et al., 2012); BMR = 1 or 10% relative deviation from control mean E-39
Table E-19. Tumor incidence data, with time to death with tumor for male Wistar rats exposed
by gavage to benzo[a]pyrene for 104 weeks (Kroese et al., 2001) E-47
Table E-20. Tumor incidence data, with time to death with tumor for female Wistar rats
exposed by gavage to benzo[a]pyrene for 104 weeks (Kroese et al., 2001) E-50
Table E-21. Tumor incidence, with time to death with tumor; B6C3Fifemale mice exposed to
benzo[a]pyrene via diet for 2 years (Beland and Culp, 1998) E-53
Table E-22. Derivation of HEDs to use for BMD modeling of Wistar rat tumor incidence data
from Kroese et al. (2001) E-54
Table E-23. Derivation of HEDs for dose-response modeling of B6C3Fi female mouse tumor
incidence data from Beland and Culp (1998) E-54
Table E-24. Summary of BMD modeling results for best-fitting multistage-Weibull models, using
time-to-tumor data for Wistar rats exposed to benzo[a]pyrene via gavage for
104 weeks (Kroese et al., 2001); BMR = 10% extra risk E-56
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Table E-27. Summary of human equivalent overall oral slope factors, based on tumor incidence
in male and female Wistar rats exposed to benzo[a]pyrene by gavage for 104
weeks (Kroese et al., 2001) E-85
Table E-28. Summary of BMD model selection among multistage-Weibull models fit to
alimentary tract tumor data for female B6C3Fi mice exposed to benzo[a]pyrene
for 2 years (Beland and Culp, 1998) E-86
Table E-29. Summary of alternative BMD modeling results for alimentary tract squamous cell
tumors in female B6C3Fi mice exposed to benzo[a]pyrene for 2 years (Beland
and Culp, 1998): poly-3 adjusted incidences3 E-89
Table E-30. Individual pathology and tumor incidence data for male Syrian golden hamsters
exposed to benzo[a]pyrene via inhalation for lifetime—Thyssen et al. (1981)a E-91
Table E-31. Summary of BMD model selection among multistage-Weibull models fit to tumor
data for male Syrian golden hamsters exposed to benzo[a]pyrene via inhalation
for lifetime (Thyssen et al., 1981) E-94
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FIGURES
Figure A-l. Structural formula of benzo[a]pyrene A-l
Figure D-l. Metabolic pathways for benzo[a]pyrene D-5
Figure D-2. The stereospecific activation of benzo[a]pyrene D-7
Figure D-3. Interaction of PAHs with the AhR D-123
Figure D-4. AhR pathway D-130
Figure D-5. DNA damage pathway D-131
Figure D-6. Nrf2 pathway D-132
Figure E-l. Fit of linear model (nonconstant variance) to data on decreased thymus weight in
male Wistar rats—90 days (Kroese et al., 2001) E-5
Figure E-2. Fit of linear model (constant variance) to decreased thymus weight in female Wistar
rats exposed to benzo[a]pyrene by gavage for 90 days (Kroese et al., 2001) E-8
Figure E-3. Fit of linear/polynomial (1°) model to data on decreased ovary weight in female
Sprague-Dawley rats exposed to benzo[a]pyrene by gavage for 60 days (Xu et
al., 2010) E-ll
Figure E-4. Fit of linear/polynomial (1°) model to primordial follicle count data for female
Sprague-Dawley rats exposed to benzo[a]pyrene by gavage for 60 days (Xu et
al., 2010) E-14
Figure E-5. Plot of mean squares crossed on PND 69 by male and female Sprague Dawley rats
exposed to benzo[a]pyrene by gavage on PNDs 5-11, by dose, with fitted curve
for Exponential (M4) model with constant variance (Chen et al., 2012); BMR = 1
SD change from control mean; dose shown in mg/kg E-18
Figure E-6. Fit of exponential 4 model for elevated plus maze, open arm maze entries on PND 70
for female Sprague Dawley rats exposed to BaP by oral gavage PNDs 5 - PND 11
(Chen et al., 2012); BMR = 1 SD E-20
Figure E-7. Plot of escape latency at PND 71 by dose, with fitted curve for Hill model using
constant variance, for male and female Sprague-Dawley rats exposed to
benzo[a]pyrene by gavage on PNDs 5-11 (Chen et al., 2012); BMR = 1 SD from
control mean; dose shown in mg/kg E-22
Figure E-8. Plot of mean escape latency at PND 72 by dose, with fitted curve for Hill model with
constant variance for male and female Sprague-Dawley rats exposed to
benzo[a]pyrene by gavage on PNDs 5-11 (Chen et al., 2012); BMR = 1 SD from
control mean; dose shown in mg/kg E-24
Figure E-9. Plot of mean escape latency at PND 73 by dose, with fitted curve for Hill model with
constant variance, for male and female Sprague-Dawley rats exposed to
benzo[a]pyrene by gavage PNDs 5-11 (Chen et al., 2012); BMR = 1 SD change
from control mean; dose shown in mg/kg E-26
Figure E-10. Plot of mean response by dose with fitted curve for Hill model with modeled
variance for escape latency of male and female Sprague-Dawley rats at PND 74
exposed to benzo[a]pyrene by gavage on PNDs 5-11 (Chen et al., 2012); BMR =
9 absolute deviation from control mean; dose shown in mg/kg E-28
Figure E-ll. Fit of log-logistic model to data on cervical epithelial hyperplasia (Gao et al., 2011) E-30
Figure E-12. Plot of incidence of embryo/fetal resorptions by dose, with fitted curve for
Quantal-Linear model, for F344 female rats exposed to benzo[a]pyrene by
inhalation on GDs 11-20 (Archibong et al., 2012); dose shown in ng/m3 E-36
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Figure E-13. Plot of mean ovarian weight by dose, with fitted curve for Exponential (M4) model
with constant variance for female F344 rats exposed to benzo[a]pyrene for
14 days prior to mating (Archibong et al., 2012); BMR = 10% relative deviation
from control mean; dose shown in ng/m3 E-38
Figure E-14. Plot of mean ovulation rate by dose, with fitted curve for Polynomial 2° model with
constant variance, for female F344 rats following inhalation exposure to
benzo[a]pyrene for 14 days (Archibong et al., 2012); BMR = 10% relative
deviation from control mean; dose shown in ng/m3 E-39
Figure E-15. Human fractional deposition E-42
Figure E-16. Rat fractional deposition E-43
Figure E-17. Fit of multistage Weibull model to squamous cell papillomas or carcinomas in oral
cavity or forestomach of male rats exposed orally to benzo[a]pyrene (Kroese et
al., 2001) E-59
Figure E-18. Fit of multistage Weibull model to hepatocellular adenomas or carcinomas in male
rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-63
Figure E-19. Fit of multistage Weibull model to duodenum or jejunum adenocarcinomas in male
rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-66
Figure E-20. Fit of multistage Weibull model to skin or mammary gland basal cell tumors of male
rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-69
Figure E-21. Fit of multistage Weibull model to skin or mammary gland squamous cell tumors of
male rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-71
Figure E-22. Fit of multistage Weibull model to kidney urothelial tumors of male rats exposed
orally to benzo[a]pyrene (Kroese et al., 2001) E-74
Figure E-23. Fit of multistage Weibull model to squamous cell papillomas or carcinomas in oral
cavity or forestomach of female rats exposed orally to benzo[a]pyrene (Kroese
et al., 2001) E-77
Table E-26. Summary of alternative BMD modeling results for squamous cell papillomas or
carcinomas in oral cavity or forestomach of female rats exposed orally to
benzo[a]pyrene (Kroese et al., 2001): poly-3 adjusted incidences3 E-78
Figure E-24. Fit of multistage Weibull model to hepatocellular adenomas or carcinomas in
female rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-81
Figure E-25. Fit of multistage Weibull model to duodenum or jejunum adenocarcinomas in
female rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-84
Figure E-26. Fit of multistage Weibull model to duodenum or jejunum adenocarcinomas in male
rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-88
Figure E-27. Fit of multistage Weibull model to respiratory tract tumors in male hamsters
exposed via inhalation to benzo[a]pyrene (Thyssen et al., 1981); tumors treated
as incidental to death E-96
Figure E-28. Fit of multistage Weibull model to respiratory tract tumors in male hamsters
exposed via inhalation to benzo[a]pyrene (Thyssen et al., 1981); tumors treated
as cause of death E-99
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ABBREVIATIONS
1-OH-Py
1-hydroxypyrene
Fe203
ferrous oxide
AchE
acetylcholine esterase
FSH
follicle stimulating hormone
ADAF
age-dependent adjustment factor
GABA
gamma-aminobutyric acid
Ah
aryl hydrocarbon
GD
gestational day
AHH
aryl hydrocarbon hydroxylase
GI
gastrointestinal
AhR
aryl hydrocarbon receptor
GJIC
gap junctional intercellular
AIC
Akaike's Information Criterion
communication
AKR
aldo-keto reductase
GSH
reduced glutathione
AMI
acute myocardial infarction
GST
glutathione-S-transferase
ANOVA
analysis of variance
GSTM1
glutathione-S-transferase Ml
ARNT
Ah receptor nuclear translocator
hCG
human chorionic gonadotropin
AST
aspartate transaminase
HEC
human equivalent concentration
ATSDR
Agency for Toxic Substances and
HED
human equivalent dose
Disease Registry
HERO
Health and Environmental Research
BMC
benchmark concentration
Online
BMCL
benchmark concentration lower
HFC
high-frequency cell
confidence limit
HPLC
high-performance liquid
BMD
benchmark dose
chromatography
BMDL
benchmark dose, 95% lower bound
hprt
hypoxanthine guanine phosphoribosyl
BMDS
Benchmark Dose Software
transferase
BMR
benchmark response
HR
hazard ratio
BPDE
benzo[a]pyrene-7,8-diol-9,10-epoxide
Hsp90
heat shock protein 90
BPQ
benzo[a]pyrene semiquinone
i.p.
intraperitoneal
BrdU
bromodeoxyuridine
i.v.
intravenous
BSM
benzene-soluble matter
Ig
immunoglobulin
BUN
blood urea nitrogen
IHD
ischemic heart disease
BW
body weight
IRIS
Integrated Risk Information System
CA
chromosomal aberration
LDH
lactate dehydrogenase
CAL/EPA
California Environmental Protection
LH
luteinizing hormone
Agency
LOAEL
lowest-observed-adverse-effect level
CASRN
Chemical Abstracts Service Registry
MAP
mitogen-activated protein
Number
MCL
Maximum Contaminant Level
CERCLA
Comprehensive Environmental
MCLG
Maximum Contaminant Level Goal
Response, Compensation, and Liability
MIAME
Minimum Information About a
Act
Microarray Experiment
CHO
Chinese hamster ovary
MLE
maximum likelihood estimate
CI
confidence interval
MMAD
mass median aerodynamic diameter
CYP
cytochrome
MN
micronucleus
CYP450
cytochrome P450
MPPD
Multi-Path Particle Deposition
DAF
dosimetric adjustment factor
mRNA
messenger ribonucleic acid
dbcAMP
dibutyl cyclic adenosine
MS
mass spectrometry
monophosphate
NCE
normochromatic erythrocyte
DMSO
dimethyl sulfoxide
NCEA
National Center for Environmental
DNA
deoxyribonucleic acid
Assessment
EC
European Commission
NIOSH
National Institute for Occupational
EH
epoxide hydrolase
Safety and Health
ELISA
enzyme-linked immunosorbent assay
NK
natural-killer
EPA
Environmental Protection Agency
NMDA
N-methyl-D-aspartate
EROD
7-ethoxyresorufin-0-deethylase
NOAEL
no-observed-adverse-effect level
ETS
environmental tobacco smoke
NPL
National Priorities List
EU
European Union
NQO
NADPH:quinone oxidoreductase
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NRC
National Research Council
NTP
National Toxicology Program
OECD
Organisation for Economic
Co-operation and Development
OR
odds ratio
ORD
Office of Research and Development
PAH
polycyclic aromatic hydrocarbon
PBMC
peripheral blood mononuclear cell
PBPK
physiologically based pharmacokinetic
PCA
Principal Components Analysis
PCE
polychromatic erythrocyte
PCNA
proliferating cell nuclear antigen
PND
postnatal day
POD
point of departure
PUVA
psoralen plus ultraviolet-A
RBC
red blood cell
RDDRer
regional deposited dose ratio for
extrarespiratory effects
RfC
inhalation reference concentration
RfD
oral reference dose
RNA
ribonucleic acid
ROS
reactive oxygen species
RR
relative risk
s.c.
subcutaneous
see
squamous cell carcinoma
SCE
sister chromatid exchange
SCSA
sperm chromatin structure assay
SD
standard deviation
SE
standard error
SEM
standard error of the mean
SHE
Syrian hamster embryo
SIR
standardized incidence ratio
SMR
standardized mortality ratio
SOAR
Systematic Omics Analysis Review
SOD
superoxide dismutase
SRBC
sheep red blood cells
SSB
single-strand break
TCDD
2,3,7,8-tetrachlorodibenzo-p-dioxin
TK
thymidine kinase
ToxR
Toxicological Reliability Assessment
TPA
12-0-tetradecanoylphorbol-13-acetate
TUNEL
terminal deoxynucleotidyl transferase
dUTP nick end labeling
TWA
time-weighted average
UCL
upper confidence limit
UDP-UGT
uridine diphosphate-
glucuronosyltransferase
UDS
unscheduled DNA synthesis
UF
uncertainty factor
UFa
interspecies uncertainty factor
UFd
database deficiencies uncertainty factor
UFh
intraspecies uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFs
subchronic-to-chronic uncertainty
factor
UVA
ultraviolet-A
UVB
ultraviolet-B
WBC
white blood cell
WESPOC
water escape pole climbing
WT
wild type
WTC
World Trade Center
XPA
xeroderma pigmentosum group A
This document is a draft for review purposes only and does not constitute Agency policy.
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APPENDIX A. CHEMICAL PROPERTIES AND
EXPOSURE INFORMATION
Benzo[a]pyrene is a five-ring polycyclic aromatic hydrocarbon (PAH) (Figure A-l). It is a
pale yellow crystalline solid with a faint aromatic odor. It is relatively insoluble in water and has
low volatility. Benzo[a]pyrene is released to the air from both natural and anthropogenic sources
and removed from the atmosphere by photochemical oxidation; reaction with nitrogen oxides,
hydroxy and hydroperoxy radicals, ozone, sulfur oxides, and peroxyacetyl nitrate; and wet and dry
deposition to land or water. In air, benzo[a]pyrene is predominantly adsorbed to particulates, but
may also exist as a vapor at high temperatures fHSDB. 20121. The half-lives for degradation of
benzo[a]pyrene in soil, air, water, and sediment are 229-309, 0.02-7, 39-71, and 196-2,293 days,
respectively (HSDB. 2012: GLC. 20071.
The structural formula is presented in Figure A-l. The physical and chemical properties of
benzo[a]pyrene are shown in Table A-l.
Benzo[a]pyrene
Figure A-l. Structural formula of benzo[a]pyrene.
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Table A-l. Chemical and physical properties of benzo[a]pyrene
CASRN 50-32-8
Synonyms
Benzo[d,e,f]chrysene;
3,4-benzopyrene,
3,4-benzpyrene; benz[a]pyrene; BP; BaP
ChemlDplus (2012)
Melting point
179-179.3°C
O'Neil etal. (2001)
Boiling point
310-312°C at 10 mm Hg
O'Neil etal. (2001)
Vapor pressure, at 20°C
5 x 10"7 mm Hg
Verschueren (2001)
Density
1.351 g/cm3
IARC (1973)
Flashpoint (open cup)
No data
Water solubility at 25°C
1.6-2.3 x 10"3 mg/L
Howard and Mevlan (1997); ATSDR
(1995)
Log Kow
6.04
Verschueren (2001)
Odor threshold
No data
Molecular weight
252.32
O'Neil etal. (2001)
Conversion factors3
1 ppm = 10.32 mg/m3
Verschueren (2001)
Empirical formula
C20H12
ChemlDplus (2012)
Calculated based on the ideal gas law, PV = nRT at 25°C: ppm = mg/m3 x 24.45 -f molecular weight.
No reference to any commercial use for purified benzo[a]pyrene, other than for research
purposes, was found. The earliest research reference for benzo[a]pyrene was related to the
identification of coal tar constituents associated with human skin tumors (Phillips. 1983: Cook et
al.. 19331. It is found ubiquitously in the environment, primarily as a result of incomplete
combustion emissions (Bostrom etal.. 20021. It is released to the environment via both natural
sources (such as forest fires) and anthropogenic sources including stoves/furnaces burning fossil
fuels (especially wood and coal), motor vehicle exhaust, cigarette smoke, and various industrial
combustion processes fATSDR. 19951. Benzo[a]pyrene is also found in soot and coal tars. Studies
have reported that urban run-off from asphalt-paved car parks treated with coats of coal-tar
emulsion seal could account for the majority of PAHs in many watersheds (Rowe and O'Connor.
2011: Van Metre and Mahler. 2010: Mahler etal.. 20051. Occupational exposure to PAHs occurs
primarily through inhalation and skin contact during the production and use of coal tar and coal-
tar-derived products, such as roofing tars, creosote, and asphalt (IARC. 20101. Chimney sweeping
can result in exposure to benzo[a]pyrene-contaminated soot (ATSDR. 19951. Workers involved in
the production of aluminum, coke, graphite, and silicon carbide may also be exposed to
benzo[a]pyrene (see Table A-2).
Benzo[a]pyrene concentrations have been well documented in samples of ground, drinking,
and surface water (HSDB. 20121. An assessment of benzo[a]pyrene emissions in the Great Lakes
This document is a draft for review purposes only and does not constitute Agency policy.
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Region in 2002 indicated that the largest source categories are metal production (33%), petroleum
refineries (11%), residential wood burning (28%), open burning (13%), on-road vehicles (6%), and
off-highway gasoline engines (3%) fGLC. 20071.
Inhalation Exposure. The Agency for Toxic Substances and Disease Registry (ATSDR. 1995)
reported average indoor concentrations of benzo[a]pyrene of 0.37-1.7 ng/m3 for smokers and
0.27-0.58 ng/m3 for nonsmokers. Naumova etal. f20021 measured PAHs in 55 nonsmoking
residences in three urban areas during June 1999-May 2000. Mean indoor benzo[a]pyrene levels
ranged from 0.02 to 0.078 ng/m3; outdoor levels were 0.025-0.14 ng/m3. The authors concluded
that indoor levels of the 5-7-ring PAHs (such as benzo[a]pyrene) were dominated by outdoor
sources and observed an average indoor/outdoor ratio of approximately 0.7 f Naumova etal..
2002). Mitra and Wilson (1992) measured benzo[a]pyrene air levels in Columbus, Ohio, and found
elevated indoor levels in homes with smokers. The measured average concentration was
1.38 ng/m3 for outdoor air; indoor concentrations were 0.07 ng/m3 for homes with electrical
utilities, 0.91 ng/m3 for homes with gas utilities, 0.80 ng/m3 for homes with gas utilities and a
fireplace, 2.75 ng/m3 for homes with gas utilities and smokers, and 1.82 ng/m3 for homes with gas
utilities, smokers, and a fireplace (Mitra and Wilson. 1992). Mitra and Ray (1995) evaluated data
on benzo[a]pyrene air levels in Columbus, Ohio, and reported average concentrations of 0.77 ng/m3
inside homes and 0.23 ng/m3 outdoors. Park etal. (2001) measured an average ambient level of
benzo[a]pyrene in Seabrook, Texas during 1995-1996 of 0.05 ng/m3 (vapor plus particulate). Park
etal. (2001) also reported average ambient air levels from earlier studies as 1.0 ng/m3 for Chicago,
0.19 ng/m3 for Lake Michigan, 0.01 ng/m3 for Chesapeake Bay, and 0.02 ng/m3 for Corpus Christie,
Texas. Petrv etal. (1996) conducted personal air sampling during 1992 at five workplaces in
Switzerland: carbon anode production, graphite production, silicon carbide production, bitumen
paving work, and metal recycling. Table A-2 summarizes the benzo[a]pyrene air concentration
data from the previous studies.
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Table A-2. Benzo[a]pyrene concentrations in air
Setting
Years
n
Concentration
(ng/m3)
Reference
Outdoor, urban
Los Angeles, California
1999-2000
19
0.065
Naumova et al. (2002)
Houston, Texas
1999-2000
21
0.025
Naumova et al. (2002)
Elizabeth, New Jersey
1999-2000
15
0.14
Naumova et al. (2002)
Seabrook, Texas
1995-1996
NA
0.05
Park et al. (2001)
Columbus, Ohio
1986-1987
8
0.23
Mitra and Rav (1995)
Indoor, residential
Los Angeles, California
1999-2000
19
0.078
Naumova et al. (2002)
Houston, Texas
1999-2000
21
0.020
Naumova et al. (2002)
Elizabeth, New Jersey
1999-2000
15
0.055
Naumova et al. (2002)
Columbus, Ohio
1986-1987
8
0.77
Mitra and Rav (1995)
Columbus, Ohio
10
0.07-2.75
Mitra and Wilson (1992)
Homes with smokers
0.37-1.7
ATSDR (1995)
Homes without smokers
0.27-0.58
ATSDR (1995)
Occupational
Aluminum production
30-530
ATSDR (1995)
Coke production
150-6,720; 8,000
Petrv et al. (1996); ATSDR (1995)
Carbon anode production, Switzerland
1992
30
1,100
Petrv et al. (1996)
Graphite production, Switzerland
1992
16
83
Petrv et al. (1996)
Silicon carbide production, Switzerland
1992
14
36
Petrv et al. (1996)
Metal recovery, Switzerland
1992
5
14
Petrv et al. (1996)
Bitumen paving, Switzerland
1992
9
10
Petrv et al. (1996)
NA = not available.
Santodonato etal. (19811 estimated the adult daily intake from inhalation as 9-43 ng/day.
The European Commission (EC. 20021 reported benzo[a]pyrene air levels in Europe during the
1990s as 0.1-1 ng/m3 in rural areas and 0.5-3 ng/m3 in urban areas. The amount of
benzo[a]pyrene is reported to be 5-80 ng per cigarette in mainstream cigarette smoke, but
significantly higher, 25-200 ng per cigarette in sidestream smoke. Concentrations of
400-760,000 ng/m3 have been reported in a cigarette smoke-polluted environment fCal/EPA.
20101. The mean intake via inhalation for an adult nonsmoker was estimated as 20 ng/day.
Naumova etal. (20021 focused on nonsmoking residences and suggested that typical air exposures
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are <0.14 ng/m3, which would result in an intake of <3 ng/day assuming an inhalation rate of
20 m3/day.
Oral Exposure. The processing and cooking of foods is viewed as the dominant pathway of
PAH contamination in foods (Bostrom et al.. 20021. Among the cooking methods that lead to PAH
contamination are the grilling, roasting, and frying of meats. Raw meat, milk, poultry, and eggs
normally do not contain high levels of PAHs due to rapid metabolism of these compounds in the
species of origin. However, some marine organisms, such as mussels and lobsters, are known to
adsorb and accumulate PAHs from contaminated water (e.g., oil spills). Vegetables and cereal
grains can become contaminated primarily through aerial deposition of PAHs present in the
atmosphere fLi etal.. 20091.
Kazerouni etal. (20011 measured benzo[a]pyrene in a variety of commonly consumed foods
collected from grocery stores and restaurants in Maryland (analyzed as a composite from
4-6 samples of each food type). The foods were tested after various methods of cooking; the
results are reported in Table A-3. The concentrations were combined with food consumption data
to estimate intake. The intakes of the 228 subjects ranged from approximately 10 to 160 ng/day,
with about 30% in the 40-60 ng/day range. The largest contributions to total intake were reported
as bread, cereal, and grain (29%) and grilled/barbecued meats (21%).
Table A-3. Benzo[a]pyrene levels in food
Food
Concentration (ng/g)
Meat
Fried or broiled beef
0.01-0.02
Grilled beef
0.09-4.9
Fried or broiled chicken
0.08-0.48
Grilled chicken
0.39-4.57
Fish
0.01-0.24
Smoked fish
0.1
Bread
0.1
Breakfast cereals
0.02-0.3
Vegetable oil
0.02
Eggs
0.03
Cheese
<0.005
Butter
<0.005
Milk
0.02
Fruit
0.01-0.17
Source: Kazerouni et al. (2001).
This document is a draft for review purposes only and does not constitute Agency policy.
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Kishikawa et al. (20031 measured benzo[a]pyrene levels in cow milk, infant formula, and
human milk from Japan, with means of 0.03 ng/g (n = 14) in cow milk, 0.05 ng/g (n = 3) in infant
formula, and 0.002 (n = 51) in human milk.
From the surveys conducted in six EU countries, the mean or national-averaged dietary
intake of benzo[a]pyrene for an adultperson was estimated in the range of 0.05-0.29 ng/day fEC.
2002). Children may be subject to higher oral intake of benzo[a]pyrene. In a Spanish study in
which benzo[a]pyrene was detected in foods, children ages 4-9 years old were found to have the
highest estimated daily intake, as compared to adults and adolescents fFalco etal.. 20031. In the
United Kingdom, average intakes on a ng kg1 day1 basis were estimated for the following age
groups: adults, 1.6; 15-18 years, 1.4; 11-14 years, 1.8; 7-10 years, 2.6; 4-6 years, 3.3; and toddlers,
3.1-3.8. The major contributors were the oils and fats group (50%), cereals (30%), and vegetables
(8%) fEC. 20021. The contribution from grilled foods appeared less important in Europe than in the
United States because grilled foods are consumed less often (EC. 2002). In the United States, the
ingested dose of benzo[a]pyrene may be much higher than the amount inhaled. A study in New
Jersey estimated a daily median total ingested dose of 176 ng based on a urinary biomarker study
of 14 adult volunteers over 14 consecutive days, which exceeded the winter inhalation dose
(11 ng/day) by 16-fold and the summer/fall inhalation dose (2.3 ng/day) by 122-fold (Buckley et
al.. 19951.
Dermal Exposure. The general population can be exposed dermally to benzo[a]pyrene when
contacting soils or materials that contain benzo[a]pyrene, such as soot or tar. Exposure can also
occur via the use of dermally applied pharmaceutical products that contain coal tars, including
shampoos and formulations used to treat conditions such as eczema and psoriasis (IARC. 2010).
PAHs are commonly found in all types of soils. ATSDR (1995) reported benzo[a]pyrene
levels in soil of 2-1,300 |J.g/kg in rural areas, 4.6-900 |J.g/kg in agricultural areas, 165-220 |J.g/kg in
urban areas, and 14,000-159,000 |J.g/kg at contaminated sites (before remediation). The soil levels
for all land uses appear highly variable. The levels are affected by proximity to roads/combustion
sources, use of sewage-sludge-derived amendments on agricultural lands, particle size, and organic
carbon content. Weinberg etal. (1989) reported that PAH levels in soils generally increased during
the 1900s and that sediment studies suggest that some declines may have occurred since the 1970s.
An illustration of benzo[a]pyrene levels in soil is presented in Table A-4.
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Table A-4. Levels of benzo[a]pyrene in soil
Reference
Location
Land type
Concentration
mean (ng/kg)
Butler et al. (1984)
United Kingdom
Urban
1,165
Vogt et al. (1987)
Norway
Industrial
321
Norway
Rural
14
Yang et al. (1991)
Australia
Residential
363
Poland
Agricultural
22
Trapido (1999)
Estonia
Urban
106
Estonia
Urban
398
Estonia
Urban
1,113
Estonia
Urban
1,224
Estonia
Rural
6.8
Estonia
Rural
15
Estonia
Rural
27
Estonia
Rural
31
Nam et al. (2008)
United Kingdom
Rural
46
Norway
Rural
5.3
Mielke et al. (2001)
New Orleans
Urban
276
Nadal et al. (2004)
Spain
Industrial-chemical
100
Spain
Industrial-petrochemical
18
Spain
Residential
56
Spain
Rural
22
Maliszewska-Kordvbach et al.
(2009)
Poland
Agricultural
30
Wilcke (2000)
Various temperate
Arable
18
Various temperate
Grassland
19
Various temperate
Forest
39
Various temperate
Urban
350
Bangkok
Urban-tropical
5.5
Brazil
Forest-tropical
0.3
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2 APPENDIX B. ASSESSMENTS BY OTHER NATIONAL
3 AND INTERNATIONAL HEALTH AGENCIES
4 Table B-l. Health assessments and regulatory limits by other national and
5 international agencies
Organization
Toxicity value or determination
Oral value
WHO (2003);
WHO (1996)
The guideline value for benzo[a]pyrene in drinking water of 0.7 ng/L was based on a cancer
slope factor of 0.46 (mg/kg-d) 1 derived from Neal and Rigdon (1967) and a lifetime excess
cancer risk of 10~5.
Health Canada
(2010); Health
Canada (1998)
The Maximum Acceptable Concentration for benzo[a]pyrene in drinking water of 0.01 ng/L
was derived from Neal and Rigdon (1967) using a drinking water consumption rate of
1.5 L/day, a body weight of 70 kg, and a lifetime cancer risk of 5 x 10"7. (The concentrations of
2, 0.2, and 0.02 |ig/L benzo[a]pyrene correspond to lifetime excess cancer risks of 10~A, 10~5,
and 10"6.)
Inhalation value
WHO (1997);
WHO (2000)
Does not recommend specific guideline values for polycyclic aromatic hydrocarbons (PAHs) in
air. A unit risk of 87 (mg/m3) 1 for benzo[a]pyrene, as an indicator a PAH mixtures, was
derived from U.S. EPA's inhalation unit risk from coke oven emissions.
EU (2005)
Target value of 1 ng/m3 benzo[a]pyrene (averaged over 1 calendar year) as a marker of PAH
carcinogenic risk. Does not include information for how target value was derived.
Cancer characterization
IARC (2010)
Carcinogenic to humans (Group 1) (based on mechanistic data).
NTP (2011)
Reasonably anticipated to be a human carcinogen. (First classified in 1981.)
Health Canada
(1998)
Probably carcinogenic to man.
6
7 EU = European Union; IARC = International Agency for Research on Cancer; NTP = National Toxicology Program;
8 WHO = World Health Organization.
This document is a draft for review purposes only and does not constitute Agency policy.
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2 APPENDIX C. LITERATURE SEARCH STRATEGY
3 Table C-l. Summary of detailed search strategies for benzo[a]pyrene
4 comprehensive literature searches (Pubmed, Toxline, Toxcenter, TSCATS)
Database
Search Date
Query String
PubMed
08/08/2016
((("Benzo(a)pyrene"[MeSH Terms]) AND (("Benzo(a)pyrene/adverse effects"[MeSH Terms] OR
"Benzo(a)pyrene/antagonists and inhibitors"[MeSH Terms] OR "Benzo(a)pyrene/blood"[MeSH
Terms] OR "Benzo(a)pyrene/pharmacokinetics"[MeSH Terms] OR
"Benzo(a)pyrene/poisoning"[MeSH Terms] OR "Benzo(a)pyrene/toxicity"[MeSH Terms] OR
"Benzo(a)pyrene/urine"[MeSH Terms]) OR ("chemically induced"[Subheading] OR "environmental
exposure"[MeSH Terms] OR "endocrine system"[MeSH Terms] OR "hormones, hormone
substitutes, and hormone antagonists"[MeSH Terms] OR "endocrine disruptors"[MeSH Terms] OR
"dose-response relationship, drug"[MeSH Terms] OR ((pharmacokinetics[MeSH Terms] OR
metabolism[MeSH Terms]) AND (humans[MeSH Terms] OR animals[MeSH Terms])) OR risk[MeSH
Terms] OR (cancer[sb] AND "Benzo(a)pyrene"[majr]) OR ("benzo a pyrene/metabolism"[MeSH
Terms] AND (humans[MeSH Terms] OR animals[MeSH Terms]))))) AND 2011/12/01: 3000[mhda])
OR ((("Benzo a pyrene"[tw] OR "Benzo d, e, f chrysene"[tw] OR "Benzo def chrysene"[tw] OR "3,4-
Benzopyrene"[tw] OR "1,2-Benzpyrene"[tw] OR "3,4-BP"[tw] OR "Benz(a)pyrene"[tw] OR "3,4-
Benzpyren"[tw] OR "3,4-Benzpyrene"[tw] OR "4,5-Benzpyrene"[tw] OR "6,7-Benzopyrene"[tw] OR
Benzopirene[tw] OR "benzo[alpha]pyrene"[tw] OR (("B(a)P"[tw] OR BaP[tw]) AND (pyrene*[tw] OR
benzopyrene*[tw] OR pah[tw] OR pahs[tw] OR polycyclic aromatic hydrocarbon[tw] OR polycyclic
aromatic hydrocarbons[tw]))) NOT medline[sb]) AND (2011/12/01: 3000[crdat] OR 2011/12/01:
3000[edat]))
02/14/2012
("Benzo(a)pyrene"[MeSH Terms] AND (("Benzo(a)pyrene/adverse effects"[MeSH Terms] OR
"Benzo(a)pyrene/antagonists and inhibitors"[MeSH Terms] OR "Benzo(a)pyrene/blood"[MeSH
Terms] OR "Benzo(a)pyrene/pharmacokinetics"[MeSH Terms] OR
"Benzo(a)pyrene/poisoning"[MeSH Terms] OR "Benzo(a)pyrene/toxicity"[MeSH Terms] OR
"Benzo(a)pyrene/urine"[MeSH Terms]) OR ("chemically induced"[Subheading] OR "environmental
exposure"[MeSH Terms] OR "endocrine system"[MeSH Terms] OR "hormones, hormone
substitutes, and hormone antagonists"[MeSH Terms] OR "endocrine disruptors"[MeSH Terms] OR
"dose-response relationship, drug"[MeSH Terms] OR ((pharmacokinetics[MeSH Terms] OR
metabolism[MeSH Terms]) AND (humans[MeSH Terms] OR animals[MeSH Terms])) OR risk[MeSH
Terms] OR (cancer[sb] AND "Benzo(a)pyrene"[majr]) OR ("benzo a pyrene/metabolism"[MeSH
Terms] AND (humans[MeSH Terms] OR animals[MeSH Terms])))) AND 2008/10/01: 3000[mhda])
OR ((("Benzo a pyrene"[tw] OR "Benzo d, e, f chrysene"[tw] OR "Benzo def chrysene"[tw] OR "3,4-
Benzopyrene"[tw] OR "1,2-Benzpyrene"[tw] OR "3,4-BP"[tw] OR "Benz(a)pyrene"[tw] OR "3,4-
Benzpyren"[tw] OR "3,4-Benzpyrene"[tw] OR "4,5-Benzpyrene"[tw] OR "6,7-Benzopyrene"[tw] OR
Benzopirene[tw] OR "benzo[alpha]pyrene"[tw] OR (("B(a)P"[tw] OR BaP[tw]) AND (pyrene*[tw] OR
benzopyrene*[tw] OR pah[tw] OR pahs[tw] OR polycyclic aromatic hydrocarbon[tw] OR polycyclic
aromatic hydrocarbons[tw]))) NOT medline[sb]) AND 2008/10/01: 3000[edat]) OR
((("Benzo(a)pyrene"[MeSH Terms] AND (("Benzo(a)pyrene/adverse effects"[MeSH Terms] OR
"Benzo(a)pyrene/antagonists and inhibitors"[MeSH Terms] OR "Benzo(a)pyrene/blood"[MeSH
Terms] OR "Benzo(a)pyrene/pharmacokinetics"[MeSH Terms] OR
"Benzo(a)pyrene/poisoning"[MeSH Terms] OR "Benzo(a)pyrene/toxicity"[MeSH Terms] OR
This document is a draft for review purposes only and does not constitute Agency policy.
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Database
Search Date
Query String
"Benzo(a)pyrene/urine"[MeSH Terms]) OR ("chemically induced"[Subheading] OR "environmental
exposure"[MeSH Terms] OR "endocrine system"[MeSH Terms] OR "hormones, hormone
substitutes, and hormone antagonists"[MeSH Terms] OR "endocrine disruptors"[MeSH Terms] OR
"dose-response relationship, drug"[MeSH Terms] OR ((pharmacokinetics[MeSH Terms] OR
metabolism[MeSH Terms]) AND (humans[MeSH Terms] OR animals[MeSH Terms])) OR risk[MeSH
Terms] OR (cancer[sb] AND "Benzo(a)pyrene"[majr]) OR ("benzo a pyrene/metabolism"[MeSH
Terms] AND (humans[MeSH Terms] OR animals[MeSH Terms]))))) OR (("Benzo a pyrene"[tw] OR
"Benzo d, e, f chrysene"[tw] OR "Benzo def chrysene"[tw] OR "3,4-Benzopyrene"[tw] OR "1,2-
Benzpyrene"[tw] OR "3,4-BP"[tw] OR "Benz(a)pyrene"[tw] OR "3,4-Benzpyren"[tw] OR "3,4-
Benzpyrene"[tw] OR "4,5-Benzpyrene"[tw] OR "6,7-Benzopyrene"[tw] OR Benzopirene[tw] OR
"benzo[alpha]pyrene"[tw] OR (("B(a)P"[tw] OR BaP[tw]) AND (pyrene*[tw] OR benzopyrene*[tw]
OR pah[tw] OR pahs[tw] OR polycyclic aromatic hydrocarbon[tw] OR polycyclic aromatic
hydrocarbons[tw])))AND ("Benzopyrenes/adverse effects"[MeSH Terms] OR
"Benzopyrenes/antagonists and inhibitors"[MeSH Terms] OR "Benzopyrenes/blood"[MeSH Terms]
OR "Benzopyrenes/pharmacokinetics"[MeSH Terms] OR "Benzopyrenes/poisoning"[MeSH Terms]
OR "Benzopyrenes/toxicity"[MeSH Terms] OR "Benzopyrenes/urine"[MeSH Terms] OR
("benzopyrenes"[MeSH Terms] AND ("chemically induced"[Subheading] OR "environmental
exposure"[MeSH Terms])) OR "benzopyrenes/metabolism"[Mesh Terms]) AND 1966[PDAT] :
1984[PDAT])) AND (cancer[sb] OR "genes"[MeSH Terms] OR "genetic processes"[MeSH Terms] OR
"mutagenicity tests"[MeSH Terms] OR "mutagenesis"[MeSH Terms] OR "mutagens"[MeSH Terms]
OR "mutation"[MeSH Terms] OR "neurotoxicity syndromes"[MeSH Terms] OR "nervous
system"[MeSH Terms] OR "nervous system diseases"[MeSH Terms] OR "immune system"[MeSH
Terms] OR "immune system diseases"[MeSH Terms] OR "immunologic factors"[MeSH Terms] OR
"reproductive physiological phenomena"[MeSH Terms] OR ("growth and
development"[Subheading] OR "urogenital system"[MeSH Terms] OR "congenital, hereditary, and
neonatal diseases and abnormalities"[MeSH Terms] OR "teratogens"[MeSH Terms]))
Toxline
08/08/2016
"benzo a pyrene" OR "benzo def chrysene" OR "benzo def chrysene" OR "3 4 benzopyrene" OR "1
2 benzpyrene" OR "3 4 bp" OR "benz(a)pyrene" OR "3 4 benzpyren" OR "3 4 benzpyrene" OR "4 5
benzpyrene" OR "6 7 benzopyrene" OR benzopirene OR "benzo(alpha)pyrene" OR 50-32-8 [rn]
AND 2011:2016 [yr] AND (ANEUPL [org] OR BIOSIS [org] OR CIS [org] OR DART [org] OR PUBDART
[org] OR EMIC [org] OR EPIDEM [org] OR FEDRIP [org] OR HEEP [org] OR HMTC [org] OR IPA [org]
OR RISKUNE [org] OR MTGABS [org] OR NIOSH [org] OR NTIS [org] OR PESTAB [org] OR PPBIB [org]
OR PubMed [org]) AND NOT PubMed [org] AND NOT pubdart [org]
02/14/2012
(((50-32-8 [rn] OR "benzo a pyrene" OR "benzo def chrysene" OR "benzo def chrysene" OR "3 4
benzopyrene" OR "12 benzpyrene" OR "3 4 bp" OR "benz ( a ) pyrene" OR "3 4 benzpyren" OR "3 4
benzpyrene" OR "4 5 benzpyrene" OR "6 7 benzopyrene" OR benzopirene OR "benzo ( alpha )
pyrene") AND 2008:2012 [yr] NOT PubMed [org] NOT pubdart [org]) NOT crisp[org]) OR (((50-32-8
[rn] OR "benzo a pyrene" OR "benzo def chrysene" OR "benzo def chrysene" OR "3 4
benzopyrene" OR "12 benzpyrene" OR "3 4 bp" OR "benz ( a ) pyrene" OR "3 4 benzpyren" OR "3 4
benzpyrene" OR "4 5 benzpyrene" OR "6 7 benzopyrene" OR benzopirene OR "benzo ( alpha )
pyrene") NOT PubMed [org] NOT pubdart [org]) AND (brain OR brains OR cephalic OR cerebral OR
cerebrum OR cognition OR cognitive OR corpus OR encephalopathies OR encephalopathy OR nerve
OR nerves OR nervous OR neural OR neurologic OR neurological OR neurology OR neuronal OR
neuropathies OR neuropathy OR neurotoxic OR neurotoxicities OR neurotoxicity OR neurotoxin OR
neurotoxins OR spinal cord) OR (antibodies OR antibody OR antigen OR antigenic OR antigens OR
autoimmune OR autoimmunities OR autoimmunity OR cytokine OR cytokines OR granulocyte OR
granulocytes OR immune OR immunities OR immunity OR immunologic OR immunological OR
immunology OR immunoproliferation OR immunosuppression OR immunosuppressive OR
This document is a draft for review purposes only and does not constitute Agency policy.
C-2 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Database
Search Date
Query String
inflammation OR inflammatory OR interferon OR interferons OR interleukin OR interleukins OR
leukocyte OR leukocytes OR lymph OR lymphatic OR lymphocyte OR lymphocytes OR
lymphocytosis OR lymphokines OR monocyte OR monocytes) OR (abnormal OR abnormalities OR
abnormality OR abort OR aborted OR abortion OR aborts OR cleft OR clefts OR development OR
developmental OR embryo OR embryologic OR embryology OR embryonic OR embryos OR fertile
OR fertilities OR fertility OR fetal OR fetus OR fetuses OR foetal OR foetus OR foetuses OR
gestation OR gestational OR infertile OR infertility OR malform OR malformation OR malformations
OR malformed OR malforms OR neonatal OR neonatally OR neonate OR neonates OR newborn OR
newborns OR ova OR ovaries OR ovary OR ovum OR perinatal OR perinatally OR placenta OR
placental OR placentas OR postnatal OR postnatally OR pregnancies OR pregnancy OR pregnant OR
prenatal OR prenatally OR reproduction OR reproductive OR sperm OR spermatid OR spermatids
OR spermatocidal OR spermatocyte OR spermatocytes OR spermatogenesis OR spermatogonia OR
spermatozoa OR sterile OR sterility OR teratogen OR teratogenesis OR teratogenic OR
teratogenicities OR teratogenicity OR teratogens OR weaned OR weaning OR weanling OR
weanlings OR zygote OR zygotes) OR (ames OR aneuploid OR aneuploidy OR chromosomal OR
chromosome OR chromosomes OR clastogen OR clastogenesis OR clastogenic OR clastogenicities
OR clastogenicity OR clastogens OR cytogenesis OR cytogenetic OR cytogenetics OR dna OR
dominant lethal OR gene OR genes OR genetic OR genotoxic OR genotoxicities OR genotoxicity OR
genotoxin OR genotoxins OR hyperploid OR hyperploidy OR micronuclei OR micronucleus OR
mitotic OR mutagen OR mutagenesis OR mutagenicities OR mutagenicity OR mutagens OR mutate
OR mutated OR mutating OR mutation OR mutations OR recessive lethal OR sister chromatid) OR
(cancer OR cancerous OR cancers OR carcinogen OR carcinogenesis OR carcinogenic OR
carcinogenicities OR carcinogenicity OR carcinogens OR carcinoma OR carcinomas OR
cocarcinogen OR cocarcinogenesis OR cocarcinogenic OR cocarcinogens OR lymphoma OR
lymphomas OR neoplasm OR neoplasms OR neoplastic OR oncogene OR oncogenes OR oncogenic
OR precancerous OR tumor OR tumorigenesis OR tumorigenic OR tumorigenicities OR
tumorigenicity OR tumors OR tumour OR tumourigenesis OR tumourigenic OR tumourigenicity OR
tumours))
Toxcenter
08/08/2016
LI S 50-32-8
L2 S LI NOT (PATENT/DT OR TSCATS/FS)
L3 S L2 AND (PY>2011 OR ED>20111201)
L15 QUE (CHRONIC OR IMMUNOTOX? OR NEUROTOX? ORTOXICOKIN? OR BIOMARKER? OR
NEUROLOG? OR PHARMACOKIN? OR SUBCHRONIC OR PBPK OR EPIDEMIOLOGY/ST,CT,IT OR
ACUTE OR SUBACUTE OR LD50# OR LC50# OR (TOXICITY OR ADVERSE OR POISONING)/ST,CT,IT)
L16 QUE (INHAL? OR PULMON? OR NASAL? OR LUNG? OR RESPIR? OR OCCUPATION? OR
WORKPLACE? OR WORKER? OR ORAL OR ORALLY OR INGEST? OR GAVAGE? OR DIET OR DIETS OR
DIETARY OR DRINKING(W)WATER OR (MAXIMUM AND CONCENTRATION? AND (ALLOWABLE OR
PERMISSIBLE)))
L17 QUE (ABORT? OR ABNORMALIT? OR EMBRYO? OR CLEFT? OR FETUS? OR FOETUS? OR
FETAL? OR FOETAL? OR FERTIL? OR MALFORM? OR OVUM OR OVA OR OVARY OR PLACENTA? OR
PREGNAN? OR PRENATAL OR PERINATAL? OR POSTNATAL? OR REPRODUC? OR STERIL? OR
TERATOGEN?)
L18 QUE (SPERM OR SPERMAC? OR SPERMAG? OR SPERMATI? OR SPERMAS? OR SPERMATOB?
OR SPERMATOC? OR SPERMATOG? OR SPERMATOI? OR SPERMATOL? OR SPERMATOR? OR
SPERMATOX? OR SPERMATOZ? OR SPERMATU? OR SPERMI? OR SPERMO?)
L19 QUE (NEONAT? OR NEWBORN OR DEVELOPMENT OR DEVELOPMENTAL? OR ZYGOTE? OR
CHILD OR CHILDREN OR ADOLESCEN? OR INFANT OR WEAN? OR OFFSPRING OR AGE(W)FACTOR?
OR DERMAL? OR DERMIS OR SKIN OR EPIDERM? OR CUTANEOUS?)
This document is a draft for review purposes only and does not constitute Agency policy.
C-3 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Database
Search Date
Query String
L20 QUE (CARCINOG? OR COCARCINOG? OR CANCER? OR PRECANCER? OR NEOPLAS? OR
TUMOR? OR TUMOUR? OR ONCOGEN? OR LYMPHOMA? OR CARCINOM? OR GENETOX? OR
GENOTOX? OR MUTAGEN? OR GENETIC(W)TOXIC?)
L21 QUE (NEPHROTOX? OR HEPATOTOX? OR ENDOCRIN? OR ESTROGEN? OR ANDROGEN? OR
HORMON?)
L22 QUE (RAT OR RATS OR MOUSE OR MICE OR MURIDAE OR DOG OR DOGS OR RABBIT? OR
HAMSTER? OR PIG OR PIGS OR SWINE OR PORCINE OR GOAT OR GOATS OR SHEEP OR MONKEY?
OR MACAQUE? OR MARMOSET? OR PRIMATE? OR MAMMAL? OR FERRET? OR GERBIL?)
L23 QUE (RODENT? OR LAGOMORPHA OR BABOON? OR BOVINE OR CANINE OR CAT OR CATS
OR FELINE OR PIGEON? OR OCCUPATION? OR WORKER? OR EPIDEM?)
L24 QUE L15 OR L16 OR L17 OR L18 OR L19 OR L20 OR L21 OR L22 OR L23
L25 SL3ANDL24
L26 S L25 AND BIOSIS/FS
L28 S L25 AND CAPLUS/FS
L29 S L28 AND (RAT OR RATS OR MOUSE OR MICE OR GUINEA PIG OR MURIDAE OR DOG OR
DOGS OR RABBIT? OR HAMSTER? OR PIG OR PIGS OR SWINE OR PORCINE OR GOAT OR GOATS OR
SHEEP OR MONKEY? OR MACAQUE? OR MARMOSET? OR PRIMATE?)
L30 S L28 AND (MAMMAL? OR FERRET? OR GERBIL? OR RODENT? OR LAGOMORPHA OR
BABOON? OR BOVINE OR CANINE OR CAT OR CATS OR FELINE OR PIGEON? OR OCCUPATION? OR
WORKER? OR EPIDEM? OR HUMAN?)
L31 S L28 AND (HOMINIDAE OR MAMMAL? OR SUBJECT? OR PATIENT? OR GENOTOX? OR
MUTAT? OR MUTAG?)
L32 S L29 OR L30OR L31
L33 S L26 OR L32
L34 DUP REM L33
02/14/2012
L48 QUE RAT OR RATS OR MOUSE OR MICE OR GUINEA PIG OR MURIDAE OR DOG OR DOGS OR
RABBIT? OR HAMSTER? OR PIG OR PIGS OR SWINE OR PORCINE OR GOAT OR GOATS OR SHEEP OR
MONKEY? OR MACAQUE?
L49 QUE MARMOSET? OR PRIMATE? OR MAMMAL? OR FERRET? OR GERBIL? OR HAMSTER? OR
RODENT? OR LAGOMORPHA OR BABOON? OR BOVINE OR CANINE OR CAT OR CATS OR FELINE OR
PIGEON?
L50 QUE OCCUPATION? OR WORKER? OR EPIDEM?
L51 QUE HUMAN? OR HOMINIDAE OR MAMMAL?
L52 QUE SUBJECT? OR PATIENT?
L53 QUE GENOTOX? OR MUTAT? OR MUTAG?
L54 QUE L48 OR L49 OR L50 OR L51 OR L52 OR L53
L57 S 50-32-8
L58 S L57 NOT PATENT/DT
L59 S L58 AND ED>20080930
L60 S L58 AND PY>2007
L61 S L59 OR L60
L62 QUE (CHRONIC OR IMMUNOTOX? OR NEUROTOX? ORTOXICOKIN? OR BIOMARKER? OR
NEUROLOG?)
L63 QUE (PHARMACOKIN? OR SUBCHRONIC OR PBPK OR EPIDEMIOLOGY/ST,CT,IT)
L64 QUE (ACUTE OR SUBACUTE OR LD50# OR LC50#)
L65 QUE (TOXICITY OR ADVERSE OR POISONING)/STCTIT
L66 QUE (INHAL? OR PULMON? OR NASAL? OR LUNG? OR RESPIR?)
L67 QUE (OCCUPATION? OR WORKPLACE? OR WORKER?) AND EXPOS?
L68 QUE (ORAL OR ORALLY OR INGEST? OR GAVAGE? OR DIET OR DIETS OR DIETARY OR
DRINKING(W)WATER?)
This document is a draft for review purposes only and does not constitute Agency policy.
C-4 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Database
Search Date
Query String
L69 QUE MAXIMUM AND CONCENTRATION? AND (ALLOWABLE OR PERMISSIBLE)
L70 QUE (ABORT? OR ABNORMALIT? OR EMBRYO? OR CLEFT? OR FETUS?)
L71 QUE (FOETUS? OR FETAL? OR FOETAL? OR FERTIL? OR MALFORM? OR OVUM?)
L72 QUE (OVA OR OVARY OR PLACENTA? OR PREGNAN? OR PRENATAL?)
L73 QUE (PERINATAL? OR POSTNATAL? OR REPRODUC? OR STERIL? OR TERATOGEN?)
L74 QUE (SPERM OR SPERMAC? OR SPERMAG? OR SPERMATI? OR SPERMAS? OR SPERMATOB?
OR SPERMATOC? OR SPERMATOG?)
L75 QUE (SPERMATOI? OR SPERMATOL? OR SPERMATOR? OR SPERMATOX? OR SPERMATOZ?
ORSPERMATU?)
L76 QUE (SPERMI? ORSPERMO?)
L77 QUE (NEONAT? OR NEWBORN OR DEVELOPMENT OR DEVELOPMENTAL?)
L78 QUE ENDOCRIN? AND DISRUPT?
L79 QUE (ZYGOTE? OR CHILD OR CHILDREN OR ADOLESCEN? OR INFANT?)
L80 QUE (WEAN? OR OFFSPRING OR AGE(W)FACTOR?)
L81 QUE (DERMAL? OR DERMIS OR SKIN OR EPIDERM? OR CUTANEOUS?)
L82 QUE (CARCINOG? OR COCARCINOG? OR CANCER? OR PRECANCER? OR NEOPLAS?)
L83 QUE (TUMOR? OR TUMOUR? OR ONCOGEN? OR LYMPHOMA? OR CARCINOM?)
L84 QUE (GENETOX? OR GENOTOX? OR MUTAGEN?)
L85 QUE GENETIC(W)TOXIC?
L86 QUE L62 OR L63 OR L64 OR L65 OR L66 OR L67 OR L68 OR L69 OR L70 OR L71 OR L72 OR L73
OR L74
L87 QUE L75 OR L76 OR L77 OR L78 OR L79 OR L80 OR L81 OR L82 OR L83 OR L84 OR L85
L88 QUE L86 OR L87
L89 QUE NEPHROTOX? OR HEPATOTOX? OR ENDOCRIN? OR ESTROGEN? OR ANDROGEN? OR
HORMON?
L90 QUE L88 OR L89
L91 QUE RAT OR RATS OR MOUSE OR MICE OR GUINEA PIG OR MURIDAE OR DOG OR DOGS OR
RABBIT? OR HAMSTER? OR PIG OR PIGS OR SWINE OR PORCINE OR GOAT OR GOATS OR SHEEP OR
MONKEY? OR MACAQUE?
L92 QUE MARMOSET? OR FERRET? OR GERBIL? OR HAMSTER? OR RODENT? OR LAGOMORPHA
OR BABOON? OR BOVINE OR CANINE OR CAT OR CATS OR FELINE OR PIGEON?
L93 QUE OCCUPATION? OR WORKER? OR WORKPLACE? OR EPIDEM?
L94 QUE L90 OR L91 OR L92 OR L93
L99 S L61 AND L94
L100 S L99 AND MEDLINE/FS
L101 S L99 AND BIOSIS/FS
L102 S L99 AND CAPLUS/FS
L103 S L99 AND IPA/FS
L104 DUP REM L100 L101 L102
L108 S (L104) AND BIOSIS/FS
L112 S (L104) AND CAPLUS/FS
L113 S L112 AND L54
LI 14 S L112 NOT L113
L115 S L108 OR L113 OR L114
02/14/2012
LI S 50-32-8
L2 S LI NOT PATENT/DT
L3 S L2 NOTTSCATS/FS
L4 S L3 AND ED>20080930
L5 S L3 AND PY>2007
L6 S L4 OR L5
This document is a draft for review purposes only and does not constitute Agency policy.
C-5 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Database
Search Date
Query String
17
S L3 NOT L6
L8
S L7 AND (CANCER? OR CARCINOG? OR CARCINOM? OR COCARCINOG? OR LYMPHOMA? OR
NEOPLAS? OR ONCOGEN? OR PRECANCER? OR TUMOR? OR TUMOUR?)/TI,CT,ST,IT
L9
S L7 AND (AMES OR ANEUPLOID? OR CHROMOSOM? OR CLASTOGEN? OR CYTOGEN? OR DNA
OR DOMINANT LETHAL OR GENETIC OR GENE? OR GENOTOX? OR HYPERPLOID? OR MICRONUCLE?
OR MITOTIC OR MUTAGEN? OR MUTAT? OR RECESSIVE LETHAL OR SISTER
CHROMATID)/TI,CT,ST,IT
L10
S L7 AND (BRAIN OR CEREBRAL OR COGNITION OR COGNITIVE OR ENCEPHAL? OR NERVE?
OR NERVOUS OR NEURAL OR NEUROLOG? OR NEURON? OR NEUROP? OR NEUROTOX? OR SPINAL
CORD)/TI,CT,ST,IT
Lll
S L7 AND (ANTIBOD? OR ANTIGEN? OR AUTOIMMUN? OR CYTOKINE? OR GRANULOCYTE?
OR IMMUN? OR INFLAMM? OR INTERFERON? OR INTERLEUKIN? OR LEUKOCYTE? OR LYMPH? OR
LYMPHOCYT? OR MONOCYT?)/Tl,CT,ST,IT
L12
S L7 AND (ABNORMAL? OR ABORT? OR CLEFT? OR DEVELOPMENT OR DEVELOPMENTAL OR
EMBRYO? OR ENDOCRINE OR FERTIL? OR FETAL? OR FETUS? OR FOETAL? OR FOETUS? OR
GESTATION? OR INFERTIL? OR MALFORM? OR NEONAT? OR NEWBORN? OR OVA OR OVARIES OR
OVARY OR OVUM)/Tl,CT,ST,IT
L13
S L7 AND (PERINATAL? OR PLACENTA? OR POSTNATAL? OR PREGNAN? OR PRENATAL? OR
REPRODUC? OR SPERM? OR STERIL? OR TERATOGEN? OR WEAN? OR ZYGOTE?)/Tl,CT,ST,IT
L14
S L8 OR L9 OR L10 OR Lll OR L12 OR L13
L15
S L14 AND MEDLINE/FS
L16
S L14 AND BIOSIS/FS
L17
S L14 AND CAPLUS/FS
L18
S L14 AND IPA/FS
L19
DUP REM L15 L16 L18 L17
L29
S L19 NOT MEDLINE/FS
L30
S L29 AND (ED>=20000801 OR PY>2000)
L31
S L29 AND PY>1999
L32
S 50-32-8
L33
S L32 NOT PATENT/DT
L34
S L33 NOT TSCATS/FS
L35
S L34 AND ED>20080930
L36
S L34 AND PY>2007
L37
S L35 OR L36
L38
S L34 NOT L37
L39
QUE (CHRONIC OR IMMUNOTOX? OR NEUROTOX? ORTOXICOKIN? OR BIOMARKER? OR
NEUROLOG?)
L40
QUE (PHARMACOKIN? OR SUBCHRONIC OR PBPK OR EPIDEMIOLOGY/ST,CT,IT)
L41
QUE (ACUTE OR SUBACUTE OR LD50# OR LC50#)
L42
QUE (TOXICITY OR ADVERSE OR POISONING)/ST,CT,IT
L43
QUE (INHAL? OR PULMON? OR NASAL? OR LUNG? OR RESPIR?)
L44
QUE (OCCUPATION? OR WORKPLACE? OR WORKER?) AND EXPOS?
L45
QUE (ORAL OR ORALLY OR INGEST? OR GAVAGE? OR DIET OR DIETS OR DIETARY OR
DRINKING(W)WATER?)
L46
QUE MAXIMUM AND CONCENTRATION? AND (ALLOWABLE OR PERMISSIBLE)
L47
QUE (ABORT? OR ABNORMALIT? OR EMBRYO? OR CLEFT? OR FETUS?)
L48
QUE (FOETUS? OR FETAL? OR FOETAL? OR FERTIL? OR MALFORM? OR OVUM?)
L49
QUE (OVA OR OVARY OR PLACENTA? OR PREGNAN? OR PRENATAL?)
L50
QUE (PERINATAL? OR POSTNATAL? OR REPRODUC? OR STERIL? OR TERATOGEN?)
This document is a draft for review purposes only and does not constitute Agency policy.
C-6 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Database
Search Date
Query String
L51 QUE (SPERM OR SPERMAC? OR SPERMAG? OR SPERMATI? OR SPERMAS? OR SPERMATOB?
OR SPERMATOC? OR SPERMATOG?)
L52 QUE (SPERMATOI? OR SPERMATOL? OR SPERMATOR? OR SPERMATOX? OR SPERMATOZ?
ORSPERMATU?)
L53 QUE (SPERMI? ORSPERMO?)
L54 QUE (NEONAT? OR NEWBORN OR DEVELOPMENT OR DEVELOPMENTAL?)
L55 QUE ENDOCRIN? AND DISRUPT?
L56 QUE (ZYGOTE? OR CHILD OR CHILDREN OR ADOLESCEN? OR INFANT?)
L57 QUE (WEAN? OR OFFSPRING OR AGE(W)FACTOR?)
L58 QUE (DERMAL? OR DERMIS OR SKIN OR EPIDERM? OR CUTANEOUS?)
L59 QUE (CARCINOG? OR COCARCINOG? OR CANCER? OR PRECANCER? OR NEOPLAS?)
L60 QUE (TUMOR? OR TUMOUR? OR ONCOGEN? OR LYMPHOMA? OR CARCINOM?)
L61 QUE (GENETOX? OR GENOTOX? OR MUTAGEN?)
L62 QUE GENETIC(W)TOXIC?
L63 QUE L39 OR L40 OR L41 OR L42 OR L43 OR L44 OR L45 OR L46 OR L47 OR L48 OR L49 OR L50
OR L51
L64 QUE L52 OR L53 OR L54 OR L55 OR L56 OR L57 OR L58 OR L59 OR L60 OR L61 OR L62
L65 QUE L63 OR L64
L66 QUE NEPHROTOX? OR HEPATOTOX? OR ENDOCRIN? OR ESTROGEN? OR ANDROGEN? OR
HORMON?
L67 QUE L65 OR L66
L68 QUE RAT OR RATS OR MOUSE OR MICE OR GUINEA PIG OR MURIDAE OR DOG OR DOGS OR
RABBIT? OR HAMSTER? OR PIG OR PIGS OR SWINE OR PORCINE OR GOAT OR GOATS OR SHEEP OR
MONKEY? OR MACAQUE?
L69 QUE MARMOSET? OR FERRET? OR GERBIL? OR HAMSTER? OR RODENT? OR LAGOMORPHA
OR BABOON? OR BOVINE OR CANINE OR CAT OR CATS OR FELINE OR PIGEON?
L70 QUE OCCUPATION? OR WORKER? OR WORKPLACE? OR EPIDEM?
L71 QUE L67 OR L68 OR L69 OR L70
L72 S L38 AND L71
L73 S 50-32-8
L74 S L73 NOT PATENT/DT
L75 S L74 NOT TSCATS/FS
L76 S L75 AND ED>20080930
L77 S L75 AND PY>2007
L78 S L76 OR L77
L79 S L75 NOT L78
L80 S L79 AND (CANCER? OR CARCINOG? OR CARCINOM? OR COCARCINOG? OR LYMPHOMA?
OR NEOPLAS? OR ONCOGEN? OR PRECANCER? OR TUMOR? OR TUMOUR?)
L81 S L79 AND (AMES ASSAY OR AMES TEST OR ANEUPLOID? OR CHROMOSOM? OR
CLASTOGEN? OR CYTOGEN? OR DNA OR DOMINANT LETHAL OR GENETIC OR GENE? OR
GENOTOX? OR HYPERPLOID? OR MICRONUCLE? OR MITOTIC OR MUTAGEN? OR MUTAT? OR
RECESSIVE LETHAL OR SISTER CHROMATID)
L82 S L79 AND (BRAIN OR CEREBRAL OR COGNITION OR COGNITIVE OR ENCEPHAL? OR NERVE?
OR NERVOUS OR NEURAL OR NEUROLOG? OR NEURON? OR NEUROP? OR NEUROTOX? OR SPINAL
CORD)
L83 S L79 AND (ANTIBOD? OR ANTIGEN? OR AUTOIMMUN? OR CYTOKINE? OR GRANULOCYTE?
OR IMMUN? OR INFLAMM? OR INTERFERON? OR INTERLEUKIN? OR LEUKOCYTE? OR LYMPH? OR
LYMPHOCYT? OR MONOCYT?)
L84 S L79 AND (ABNORMAL? OR ABORT? OR CLEFT? OR DEVELOPMENT OR DEVELOPMENTAL OR
EMBRYO? OR ENDOCRINE OR FERTIL? OR FETAL? OR FETUS? OR FOETAL? OR FOETUS? OR
This document is a draft for review purposes only and does not constitute Agency policy.
C-7 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Database
Search Date
Query String
GESTATION? OR INFERTIL? OR MALFORM? OR NEONAT? OR NEWBORN? OR OVA OR OVARIES OR
OVARY OR OVUM )
L85 S L79 AND (PERINATAL? OR PLACENTA? OR POSTNATAL? OR PREGNAN? OR PRENATAL? OR
REPRODUC? OR SPERM? OR STERIL? OR TERATOGEN? OR WEAN? OR ZYGOTE?)
L86 S L80 OR L81 OR L82 OR L83 OR L84 OR L85
L87 S L72 AND L86
L88 S L87 AND PY>1999
L89 S L88 AND MEDLINE/FS
L90 S L88 AND BIOSIS/FS
L91 S L88 AND CAPLUS/FS
L92 S L88 AND IPA/FS
L93 DUP REM L89 L90 L92 L91
L98 S (L93) AND BIOSIS/FS
L106 S (L93) AND IPA/FS
Llll S (L93) AND CAPLUS/FS
L112 QUE RAT OR RATS OR MOUSE OR MICE OR GUINEA PIG OR MURIDAE OR DOG OR DOGS
OR RABBIT? OR HAMSTER? OR PIG OR PIGS OR SWINE OR PORCINE OR GOAT OR GOATS OR SHEEP
OR MONKEY? OR MACAQUE?
L113 QUE MARMOSET? OR PRIMATE? OR MAMMAL? OR FERRET? OR GERBIL? OR HAMSTER?
OR RODENT? OR LAGOMORPHA OR BABOON? OR BOVINE OR CANINE OR CAT OR CATS OR FELINE
OR PIGEON?
LI 14 QUE OCCUPATION? OR WORKER? OR EPIDEM?
L115 QUE HUMAN? OR HOMINIDAE OR MAMMAL?
L116 QUE SUBJECT? OR PATIENT?
L117 QUE GENOTOX? OR MUTAT? OR MUTAG?
L118 QUE L112 OR L113 OR L114 OR L115 OR L116 OR L117
L119 S Llll AND L118
L132 S(L93) AND MEDLINE/FS
L133 S L132 OR L98 OR L119 OR L106 OR L31
TSCATS 1
02/14/2012
50-32-8 Limit: Health Effects
TSCATS 2
08/08/2016
50-32-8
02/14/2012
50-32-8 Date limited, 2000 to date of search
TSCA 8e/FYI recent submissions
02/14/2012
Google: 91-20-3 (8e or fyi) tsca
TSCA 8E & FYI via CDAT1
08/08/2016
50-32-8
This document is a draft for review purposes only and does not constitute Agency policy.
C-8 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Database
Search Date
Query String
Secondary Refinement
02/14/2012
Additional terms applied within Endnote to pre-2008 search results only:
forestomach* OR tongue* OR (auditory AND canal*) OR (ear* AND canal*) OR esophagus* OR
esophageal* OR larynx* OR laryngeal* OR pharynx* OR pharyngeal* OR ((lung* OR pulmonary OR
skin*) AND (neoplasm* OR tumor* OR tumour* OR papilloma* OR carcinoma*)) OR leukemia* OR
leukaemia* OR sperm* OR testic* OR fertilit*OR infertilit* OR testosterone OR ((testis OR testes)
AND (weight* OR mass*)) OR epididymis* OR epididymal* OR seminiferous OR ((cervical* OR
cervix*) AND hyperplasia*) OR ovary OR ovaries OR ovarian OR primordial OR corpora lutea OR
corpus luteum OR estrous* OR estrus* OR thymus* OR spleen* OR spleno* OR immunoglobulin*
OR immunoglobin* OR ((immune OR immun*) AND (suppress* OR immunosuppress*)) OR
(functional AND observational AND battery) OR neurobehavioral*OR neurobehavioural* OR
rotarod* OR nerve* AND conduction* OR locomotor* OR neuromuscular* OR weight* OR
neurodevelopment* OR ((neuro* OR brain*) AND (development* OR developing)) OR intelligence*
OR cognition* OR cognitive* OR learn* OR memory OR righting*
1CDAT (Chemical Data Access Tool; http://java.epa.gov/oppt_chemical_search/)
Table C-2. Summary of detailed literature search strategies for
benzo(a)pyrene cardiovascular toxicity
Database
Search Date
Query String
PubMed
3/31/2016
((((("benzo(a)pyrene"[mh] OR ("benzo(a)pyrene"[tw] OR "Benzo a pyrene"[tw] OR "Benzo d, e, f
chrysene"[tw] OR "Benzo def chrysene"[tw] OR "3,4-Benzopyrene"[tw] OR "1,2-Benzpyrene"[tw]
OR "3,4-BP"[tw] OR "Benz(a)pyrene"[tw] OR "3,4-Benzpyren"[tw] OR "3,4-Benzpyrene"[tw] OR
"4,5-Benzpyrene"[tw] OR "6,7-Benzopyrene"[tw] OR Benzopirene[tw] OR
"benzo[alpha]pyrene"[tw] OR (("B(a)P"[tw] OR BaP[tw]) AND (pyrene*[tw] OR benzopyrene*[tw]
OR "pah"[tw] OR "pahs"[tw] OR "polycyclic aromatic hydrocarbon"[tw] OR "polycyclic aromatic
hydrocarbons"[tw])))) AND ("macrophages"[mh] OR "Cholesterol"[mh] OR "lschemia"[mh] OR
"Granulocytes"[mh] OR "Myocytes, Smooth Muscle"[mh] OR "Blood supply"[mh] OR
"Monocytes"[mh] OR "Lipoprotein"[mh] OR "Triglycerides"[mh] OR "Blood Vessels"[mh] OR
"Aorta, Thoracic"[mh] OR "Aorta"[mh] OR "Aortic Diseases"[mh] OR "Atherosclerosis"[mh] OR
"Cardiomegaly"[mh] OR "Cardiotoxicity"[mh] OR "Cardiovascular Diseases"[mh] OR
"Caveolae"[mh] OR "Endothelial Cells"[mh] OR "Endothelium, Vascular"[mh] OR "Heart Defects,
Congenital"[mh] OR "Heart"[mh] OR "Systole"[mh] OR "diastole"[mh] OR "Vascular Endothelial
Growth Factor A"[mh] OR "vasoconstriction"[mh] OR "benzo a pyrene/blood"[MeSH Terms] OR
"Angiogenesis"[tw] OR "Plaque"[tw] OR "plaques"[tw] OR "Myocardial"[tw] OR "Myocardia"[tw]
OR "Myocardiocyte"[tw] OR "Proatherogenic"[tw] OR "Systolic"[tw] OR "diastolic"[tw] OR
"Ventricle"[tw] OR "ventricular"[tw]))))) OR (((("benzo(a)pyrene"[tw] OR "Benzo a pyrene"[tw] OR
"Benzo d, e, f chrysene"[tw] OR "Benzo def chrysene"[tw] OR "3,4-Benzopyrene"[tw] OR "1,2-
Benzpyrene"[tw] OR "3,4-BP"[tw] OR "Benz(a)pyrene"[tw] OR "3,4-Benzpyren"[tw] OR "3,4-
Benzpyrene"[tw] OR "4,5-Benzpyrene"[tw] OR "6,7-Benzopyrene"[tw] OR Benzopirene[tw] OR
"benzo[alpha]pyrene"[tw] OR (("B(a)P"[tw] OR BaP[tw]) AND (pyrene*[tw] OR benzopyrene*[tw]
OR "pah"[tw] OR "pahs"[tw] OR "polycyclic aromatic hydrocarbon"[tw] OR "polycyclic aromatic
hydrocarbons"[tw]))) AND ("artery"[tw] OR "arteries"[tw] OR "arterial"[tw] OR
"atherogenesis"[tw] OR "angiogenesis"[tw] OR "plaque"[tw] OR "plaques"[tw] OR "thrombus"[tw]
OR "thrombosis"[tw] OR "myocardial"[tw] OR "myocardia"[tw] OR "myocardiocyte"[tw] OR
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"cholesterol"[tw] OR "ischemia"[tw] OR "cardiomyopathy"[tw] OR "lymphocyte"[tw] OR
"lymphocytes"[tw] OR "macrophage"[tw] OR "macrophages"[tw] OR "granulocyte"[tw] OR
"granulocytes"[tw] OR "smooth muscle cells"[tw] OR "proatherogenic"[tw] OR "hypertension"[tw]
OR "neutrophils"[tw] OR "systolic"[tw] OR "systole"[tw] OR "diastolic"[tw] OR "diastole"[tw] OR
"ventricle"[tw] OR "ventricles"[tw] OR "ventricular"[tw] OR "vasculature"[tw] OR "monocyte"[tw]
OR "monocytes"[tw] OR "lipoprotein"[tw] OR "triglyceride"[tw] OR "triglycerides"[tw])) NOT
medline[sb]))
1
2
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APPENDIX D. INFORMATION IN SUPPORT OF
HAZARD IDENTIFICATION AND DOSE-RESPONSE
ANALYSIS
D.l. TOXICOKINETICS
D.l.l. Overview
Benzo[a]pyrene is absorbed following exposure by oral, inhalation, and dermal routes. The
rate and extent of absorption are dependent upon the exposure medium. The presence of
benzo[a]pyrene in body fat, blood, liver, and kidney and the presence of benzo[a]pyrene
metabolites in serum and excreta demonstrate wide systemic tissue distribution. Benzo[a]pyrene
metabolism occurs in essentially all tissues, with high metabolic capacity in the liver and significant
metabolism in tissues at the portal of entry (lung skin, and gastrointestinal [GI] tract) and in
reproductive tissues. Stable metabolic products identified in body tissues and excreta are very
diverse and include phenols, quinones, and dihydrodiols. These classes of metabolites are typically
isolated as glucuronide or sulfate ester conjugates in the excreta, but can also include glutathione
conjugates formed from quinones or intermediary epoxides. The primary route of metabolite
elimination is in the feces via biliary excretion, particularly following exposure by the inhalation
route. To a lesser degree, benzo[a]pyrene metabolites are eliminated via urine. Overall,
benzo[a]pyrene is eliminated quickly with a biological half-life of several hours.
D.1.2. Absorption
The absorption of benzo[a]pyrene has been studied in humans and laboratory animals for
inhalation, ingestion, and dermal exposure. In the environment, human exposure to
benzo[a]pyrene predominantly occurs via contact with insoluble carbonaceous particles (e.g., soot,
diesel particles) to which organic compounds, such as polycyclic aromatic hydrocarbons (PAHs),
are adsorbed.
Studies of workers occupationally exposed to benzo[a]pyrene have qualitatively
demonstrated absorption via inhalation by correlating concentrations of benzo[a]pyrene in the air
and benzo[a]pyrene metabolites in the exposed workers' urine. Occupational exposures to
benzo[a]pyrene measured with personal air samplers were correlated to urine concentrations of
benzo[a]pyrene-9,10-dihydrodiol, a specific metabolite of benzo[a]pyrene, in 24-hour aggregate
urine samples by Grimmer et al. (1994). The amount of benzo[a]pyrene extracted from personal
air monitoring devices (a surrogate for ambient PAHs) of coke oven workers were correlated with
r-7,t-8,9,c 10 tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-benzo[a]pyrene-tetrol, a
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specific metabolite of benzo[a]pyrene) in the workers' urine by Wu etal. (2002). In both of these
studies, only a very small fraction (<1%) of the inhaled benzo[a]pyrene was recovered from urine,
consistent with studies in animals that find that urine is not a major route of elimination for
benzo[a]pyrene (as described in the excretion section below). These occupational studies cannot
be used to quantify absorption through inhalation-only exposure in humans because the
persistence of benzo[a]pyrene-contaminated particulate matter on surfaces and food may lead to
exposures via additional routes (Bostrom et al.. 2002). Nevertheless, the observation of
benzo[a]pyrene metabolites in excreta of exposed humans provides qualitative evidence for
benzo[a]pyrene absorption, at least some of which is likely to occur via inhalation. This conclusion
is supported by studies in experimental animals, which indicate thatbenzo[a]pyrene is readily
absorbed from carbonaceous particles following inhalation exposure (Gerde etal.. 2001: Hood et
al.. 20001.
Results from studies of animals following intratracheal instillation of benzo[a]pyrene
provide supporting, quantitative evidence that absorption by the respiratory tract is rapid (Gerde et
al.. 1993: Bevan and Ulman. 1991: Wevand and Bevan. 1987.1986). Following intratracheal
instillation of 1 [ig tritiated benzo[a]pyrene/kg dissolved in triethylene glycol to Sprague-Dawley
rats, radioactivity rapidly appeared in the liver (reaching a maximum of about 21% of the
administered dose within 10 minutes). Elimination of radioactivity from the lung was biphasic,
with elimination half-times of 5 and 116 minutes fWevand and Bevan. 19861. In bile-cannulated
rats, bile collected for 6 hours after instillation accounted for 74% of the administered radioactivity
(Wevand and Bevan. 1986). The results are consistent with rapid and extensive absorption by the
respiratory tract and rapid entry into hepatobiliary circulation following intratracheal instillation.
The respiratory tract absorption may also be affected by the vehicle, since higher amounts of
benzo[a]pyrene were excreted in bile when administered with hydrophilic triethylene glycol than
with lipophilic solvents ethyl laurate or tricaprylin (Bevan and Ulman. 1991). Particle-bound
benzo[a]pyrene deposited in the respiratory tract is absorbed and cleared more slowly than the
neat compound (Gerde etal.. 20011.
Studies conducted to assess levels of benzo[a]pyrene metabolites or benzo[a]pyrene-
deoxyribonucleic acid (DNA) adduct levels in humans exposed to benzo[a]pyrene by the oral route
are not adequate to develop quantitative estimates of oral bioavailability. The concentration of
benzo[a]pyrene was below detection limits (<0.1 ng/person) in the feces of eight volunteers who
had ingested broiled meat containing approximately 8.6 |ig of benzo[a]pyrene (Hechtetal.. 1979).
However, studies in laboratory animals demonstrate that benzo[a]pyrene is absorbed via ingestion.
Studies of rats and pigs measured the oral bioavailability of benzo[a]pyrene in the range of 10-40%
(Cavret etal.. 2003: Ramesh etal.. 2001b: Foth etal.. 1988: Hechtetal.. 1979). The absorption of
benzo[a]pyrene may depend on the vehicle. Intestinal absorption of benzo[a]pyrene was enhanced
in rats when the compound was solubilized in lipophilic compounds such as triolein, soybean oil,
and high-fat diets, as compared with fiber- or protein-rich diets fO'Neill etal.. 1991: Kawamura et
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al.. 19881. Aqueous vehicles, quercetin, chlorogenic acid, or carbon particles reduced biliary
excretion of benzo[a]pyrene, while lipid media such as corn oil increased it (Stavric and Klassen.
19941. The addition of wheat bran to the benzo[a]pyrene-containing diets increased fecal excretion
ofbenzo[a]pyrene (Mirvish etal.. 19811.
Studies of benzo[a]pyrene metabolites or DNA adducts measured in humans exposed
dermally to benzo[a]pyrene-containing PAH mixtures demonstrate that benzo[a]pyrene is
absorbed dermally. One study of dermal absorption in volunteers found absorption rate constants
ranging from 0.036 to 0.135/hour over a 45-minute exposure, suggesting that 20-56% of the dose
would be absorbed within 6 hours (VanRooii etal.. 19931. Dermal absorption rates varied 69%
between different anatomical sites (forehead, shoulder, volar forearm, palmar side of the hand,
groin, and ankle) and only 7% between different individual volunteers (VanRooii etal.. 19931.
Metabolism is also an important determinant of permeation, with very low rates observed in
nonviable skin (Kao etal.. 19851. The overall absorbed amount of benzo[a]pyrene in explanted
viable skin samples from tissue donors (maintained in short-term organ cultures) exposed for
24 hours ranged from 0.09 to 2.6% of the dose (Wester etal.. 1990: Kao etal.. 19851. Similar
amounts of penetration were measured in skin samples from other species including marmosets,
rats, and rabbits fKao etal.. 19851. Skin from mice allowed more of the dose to penetrate (>10%),
while that of guinea pig let only a negligible percentage of the dose penetrate (Kao etal.. 19851.
The vehicle for benzo[a]pyrene exposure is an important factor in skin penetration.
Exposure of female Sprague-Dawley rats and female rhesus monkeys topically to benzo[a]pyrene in
crude oil or acetone caused approximately 4-fold more extensive absorption than benzo[a]pyrene
in soil (Wester et al.. 1990: Yang etal.. 19891. The viscosity of oil product used as a vehicle also
changed skin penetration with increased uptake of benzo[a]pyrene for oils with decreased viscosity
fPotter etal.. 19991. Soil properties also greatly impact dermal absorption. Reduced absorption of
benzo[a]pyrene occurs with increasing organic carbon content of the soil and increased soil aging
(i.e., contact time between soil and chemical) fTurkall etal.. 2008: Roy and Singh. 2001: Yang etal..
19891.
D.1.3. Distribution
No adequate quantitative studies of benzo[a]pyrene tissue distribution in exposed humans
were identified. Obana etal. (19811 observed low levels of benzo[a]pyrene in liver and fat tissues
from autopsy samples. However, prior exposure histories were not available for the donors.
Nevertheless, the identification of benzo[a]pyrene metabolites or DNA adducts in tissues and
excreta of PAH-exposed populations suggest that benzo[a]pyrene is widely distributed.
Distribution of benzo[a]pyrene has been studied in laboratory animals for multiple routes
of exposure, including inhalation, ingestion, dermal, and intravenous (i.v.). Exposure to
benzo[a]pyrene in various species (Sprague-Dawley rats, Gunn rats, guinea pigs, and hamsters)
results in wide distribution throughout the body and rapid uptake into well-perfused tissues (i.e.,
lung, kidney, and liver) fWevand and Bevan. 1987.19861. Benzo[a]pyrene and its metabolites are
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distributed systemically after administration via many routes of administration including
inhalation (or intratracheal instillation), oral, i.v., and dermal exposures (Saunders etal.. 2002: Moir
etal.. 1998: NeubertandTapken. 1988: Wevand and Bevan. 1987.1986: Morse and Carlson. 19851.
Intratracheal instillation of radiolabeled benzo[a]pyrene in mice resulted in increased radioactivity
in lung-associated lymph nodes, suggesting distribution of benzo[a]pyrene or its metabolites via
the lymph fSchnizlein et al.. 19871. Rats with biliary cannulas had high excretion of benzo[a]pyrene
and benzo[a]pyrene metabolites in bile. The benzo[a]pyrene thioether and glucuronic acid-
conjugated metabolites in intestines indicated enterohepatic recirculation of benzo[a]pyrene and
benzo[a]pyrene metabolites (Wevand and Bevan. 19861. The vehicle for delivery of inhalated
benzo[a]pyrene impacts the distribution, with aerosolized benzo[a]pyrene more readily absorbed
directly in the respiratory tract than particle-adsorbed benzo[a]pyrene (which is cleared by the
mucociliary and then ingested) fSun etal.. 19821. The reactive metabolites ofbenzo[a]pyrene are
also transported in the blood and may be distributed to tissues incapable of benzo[a]pyrene
metabolism. Serum of benzo[a]pyrene-treated mice incubated with splenocytes or salmon sperm
DNA resulted in adduct formation, suggesting that reactive benzo[a]pyrene metabolites were
systemically distributed and available for interaction with target tissues (Ginsberg and Atherholt.
19891. Exposure of pregnant rats and mice to benzo[a]pyrene via inhalation and ingestion showed
a wide tissue distribution of benzo[a]pyrene, consistent with other studies, and demonstrated
placental transfer of benzo[a]pyrene and its metabolites fWithev etal.. 1993: Neubert and Tapken.
1988: Shendrikova and Aleksandrov. 19741. Data from lactating rats indicate that following
injection, distribution of 14C-labeled B[a]P in maternal blood is similar to levels in milk (Lavoie et
al.. 1987bl.
D.1.4. Metabolism
The metabolic pathways of benzo[a]pyrene (Figure D-l) and variation in species, strain,
organ system, age, and sex have been studied extensively with in vitro and in vivo experiments. In
addition, there have been numerous studies of exposed humans or animals with subsequent
detection of benzo[a]pyrene metabolites in tissues or excreta. For example, elevated frequency of a
detected urinary metabolite (7,8,9,10-tetrol) was observed in patients treated with coal tar
medication (Bowman etal.. 19971. demonstrating extensive metabolism of benzo[a]pyrene in
humans.
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l-OH BaP
9-OHBaP
3-OH BaP
BaP 1,2-oxide
BaP 9,10-transdiol
BaP 9,10-oxide
Benzo[a]pyrene
BaP 7,8-oxide
BaP 7,8-tr ansdio I
oh BaP 4,5-transdiol
BaP 4,5-oxide
6-OH BaP
7-OH BaP
\
6-oxo-BaP radical
BaP 7,8-diol-9,10-epoxide
BaP 1,6-hydroquinone BaP 1,6 semiquinone
[BaP 3,6 1
semiquinonel (r^T^I
BaP 1,6 quinone
BaP 6,12
semiquinone
BaP 6,12-hydroquinone
BaP 3,6-hydroquinone
BaP 6,12-quinone
Source: Miller and Ramos (2001).
Figure D-l. Metabolic pathways for benzo[a]pyrene.
Phase I metabolism results in a number of reactive metabolites such as epoxides,
dihydrodiols, phenols, quinones, and their various combinations that are likely to contribute to the
toxic effects of benzo[a]pyrene (e.g., phenols, dihydrodiols, epoxides, and quinones). Phase II
metabolism of benzo[a]pyrene metabolites protects the cells and tissues from the toxic effects of
benzo[a]pyrene phenols, dihydrodiols and epoxides by converting them to water soluble products
that are eliminated. In addition, Phase II metabolism of some benzo[a]pyrene dihydrodiols
prevents them from further bioactivation to reactive forms that bind to cellular macromolecules.
These metabolic process include glutathione conjugation of diol epoxides, sulfation and
glucuronidation of phenols, and reduction of quinones by NADPH:quinone oxidoreductase (NQO).
Numerous reviews on the metabolism of benzo[a]pyrene are available (Miller and Ramos. 2001:
IPCS. 1998: ATSDR. 1995: Connev etal.. 1994: Grover. 1986: Levin etal.. 1982: Gelboin. 19801. Key
concepts have been adapted largely from these reviews and supplemented with recent findings.
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Phase I Metabolism
Phase I reactions of benzo[a]pyrene are catalyzed primarily by cytochrome P450 (CYP450)
and produce metabolites including epoxides, dihydrodiols, phenols, and quinones (Figure D-2). The
first step of Phase I metabolism is the oxidation of benzo[a]pyrene that forms a series of epoxides,
the four major forms of which are the 2,3-, 4,5-, 7,8-, and 9,10-isomers fGelboin. 19801. Once
formed, these epoxides may undergo three different routes of metabolism: (1) spontaneous
rearrangement to phenols; (2) hydration to trans-dihydrodiols catalyzed by microsomal epoxide
hydrolase (EH); or (3) the Phase II detoxification of binding with glutathione (either spontaneously
or catalyzed by cytosolic glutathione-S-transferases (GSTs) flARC. 198311. The metabolism of
benzo[a]pyrene to phenols results in five phenol isomers (1-, 3-, 6-, 7, and 9-OH benzo[a]pyrene)
(Pelkonen and Nebert. 19821. Four benzo[a]pyrene epoxides (2,3-, 4,5-, 7,8-, and 9,10-) are
hydrated to trans-dihydrodiols. Benzo[a]pyrene-7,8-diol (formed from benzo[a]pyrene-7,8-oxide)
has been the focus of much of the study of benzo[a]pyrene metabolism. Benzo[a]pyrene-7,8-diol is
the metabolic precursor to the potent DNA-binding metabolite, benzo[a]pyrene-7,8-diol-
9,10-epoxide (BPDE). BPDE is formed from trans-benzo[a]pyrene 7,8-diol by multiple mechanisms
including catalysis by cytochromes (CYPs) fGrover. 1986: Deutsch etal.. 19791. myeloperoxidase
fMalletetal.. 19911. or prostaglandin h synthase (also known as cyclooxygenase) fMarnett. 19901.
and lipid peroxidation (Bvczkowski and Kulkarni. 19901. The diolepoxides can react further by
spontaneously hydrolyzing to tetrols (Hall and Grover. 19881.
The metabolism of benzo[a]pyrene proceeds with a high degree of stereoselectivity. Liver
microsomes from rats stereospecifically oxidize the 7,8-bond of benzo[a]pyrene to yield almost
exclusively the (+)-benzo[a]pyrene-(7,8)-oxide (see Figure D-2). Each enantiomer of
benzo[a]pyrene-7,8-oxide is stereospecifically converted by EH to a different stereoisomeric trans
dihydrodiol. The (+)-benzo[a]pyrene-7,8-oxide is preferentially hydrated to the (-)-trans-
benzo[a]pyrene-7,8-dihydrodiol enantiomer by rat CYP enzymes and the (-)-trans-
benzo[a]pyrene-7,8-dihydrodiol is preferentially oxidized by CYP enzymes to (+)-benzo[a]pyrene-
7R,8S-diol-9S,10R-epoxide [(+)-anti- BPDE], which is the most potent carcinogen among the four
stereoisomers (Figure D-2). Formation of these stereoisomers does not occur at equimolar ratios,
and the ratios differ between biological systems. For example, a study in rabbit livers
demonstrated that purified microsomes oxidized the (-)-benzo[a]pyrene-7,8-dihydrodiol to
isomeric diol epoxides in a ratio ranging from 1.8:1 to 11:1 in favor of the (+)-anti-BPDE isomer
(Deutsch etal.. 19791.
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(+)-BP-7R ,8S-d iol-9S,1 OR- epoxide
(+) anti BPDE
Mixed Function
Oxidase System
Epoxide Hydrolase |_jq
OH
(-J-BP-7,8-diol
(+)-BP-7i8-oxide
Mixed Function
2 Oxidase System
Mixed Function
Oxidase System
Epoxide Hydrolase ug
(-)- BP-7,8-oxide
+ -BP-7 jB-diol
(-)-BP-7R,8S-diol-9R,10S-epoxide
(-) syn BPDE
(+)-BP-7S,8R-diol-9S,1 OR-epoxide
(+) syn BPDE
(-)-BP-7S,8R-dio I-9R ,1 OS-epoxide
(-) anti BPDE
Source: Grover (1986).
Figure D-2. The stereospecific activation of benzo[a]pyrene.
Several studies have attempted to determine which CYP isozyme is predominantly
responsible for the metabolism of benzo[a]pyrene. Dermal administration of tritiated
benzo[a]pyrene to mice that have an aryl hydrocarbon (Ah) receptor (AhR) knock-out (AhR-/-)
had significantly decreased formation of (+)-anti-BPDE-DNA adducts compared to wild type (WT)
and 1B1-/- mice (Kleiner et al.. 20041. Gavage administration of benzo[a]pyrene in AhR knock-out
mice found that the AhR-/- mice (with lower levels of CYP1A1) had higher levels of protein
adducts and unmetabolized benzo[a]pyrene than the AhR+/+ or +/- mice fSagredo etal.. 20061.
Similarly CYP1A1 (-/-) knock-out mice administered benzo[a]pyrene in feed for 18 days had
higher steady-state blood levels of benzo[a]pyrene and benzo[a]pyrene-DNA adducts (Uno etal..
20061. These findings establish important roles in benzo[a]pyrene metabolism for CYP1A1, but the
relationship is not clear between the CYP enzymes and biological activation or detoxification.
Another important factor in evaluating variability in the metabolic activation of
benzo[a]pyrene by CYP450s is the effect of functional polymorphisms, which has been the subject
of numerous reviews (e.g.. Wormhoudt etal.. 19991. Recombinant CYP1A1 allelic variants
produced BPDE with generally lower catalytic activity and Km values than the WT allele (Schwarz
etal.. 20011. However, the formation of diol epoxides is stereospecific, with the allelic variants
producing about 3 times the amount of (±)-anti-BPDE isomers as compared to the stereoisomer,
(±)-syn-BPDE f Schwarz etal.. 20011. In a study of occupational exposures to benzo[a]pyrene, no
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relationship was observed between benzo[a]pyrene metabolite formation and the CYP1A1 Mspl
polymorphism fWu etal.. 20021.
Another pathway of benzo[a]pyrene metabolism is the conversion of benzo[a]pyrene to
6-OH benzo[a]pyrene, which can be further oxidized into quinones, primarily the 1,6-, 3,6-, and
6,12- isomers. Trans-benzo[a]pyrene-7,8-dihydrodiol can be converted by aldo-keto reductases
(AKR) to 7,8-dihydroxybenzo[a]pyrene (benzo[a]pyrene-7,8-catechol), which auto-oxidizes to
benzo[a]pyrene-7,8-quinone (BPQ). BPQ can undergo redox cycling in the presence of cellular
reducing equivalents. This reaction pathway produces reactive oxygen species (ROS), including
peroxide anion radicals, benzo[a]pyrene semiquinone radicals, hydroxyl radicals, and H2O2, which
in turn can causes extensive DNA fragmentation fPenning etal.. 1999: Flowers etal.. 1997: Flowers
etal.. 19961. 6-Hydroxybenzo[a]pyrene can be oxidized into 6-oxo-benzo[a]pyrene semi-quinone
radical and further metabolized into 1,6-, 3,6-, or 6,12-quinones spontaneously, or catalytically by
prostaglandin endoperoxide synthetase (Eling etal.. 19861. The CYP and AKR enzymes both can
metabolize trans-benzo[a]pyrene-7,8-dihydrodiol to different metabolites, BPDE and BPQ.
Reconstituted in vitro systems of human lung cells show that CYP enzymes have faster steady-state
reaction rate constants than AKR and basal expression of AKR is higher than CYP in lung cells,
suggesting that AKR and CYP enzymes compete for metabolism of trans-benzo[a]pyrene-
7,8-dihydrodiol (Ouinn and Penning. 20081.
Phase II Metabolism
The reactive products of Phase I metabolism are subject to the action of several Phase II
conjugation and detoxification enzyme systems that display preferential activity for specific
oxidation products of benzo[a]pyrene. These Phase II reactions play a critical role in protecting
cellular macromolecules from binding with reactive benzo[a]pyrene diolepoxides, radical cations,
or ROS. Therefore, the balance between Phase I activation of benzo[a]pyrene and its metabolites
and detoxification by Phase II processes is an important determinant of toxicity.
The diol epoxides formed from benzo[a]pyrene metabolism by Phase I reactions are not
usually found as urinary metabolites. Rather, they are detected as adducts of nucleic acids or
proteins or further metabolized by glutathione (GSH) conjugation, glucuronidation, and sulfation.
These metabolites make up a significant portion of total metabolites in excreta or tissues. For
example, the identified metabolites in bile 6 hours after a 2 |ig/kgbenzo[a]pyrene dose by
intratracheal instillation to male Sprague-Dawley rats were 49% glucuronides (quinol
diglucuronides or monglucuronides), 30.4% thioether conjugates, 6.2% sulfate conjugates, and
14.4% unconjugated metabolites (Bevan and Sadler. 19921.
Conjugation of benzo[a]pyrene with GSH is catalyzed by GSTs. Numerous studies using
human GSTs expressed in mammalian cell lines have demonstrated the ability of GST to metabolize
benzo[a]pyrene diol epoxides. Isolated human GSTs have significant catalytic activity toward
benzo[a]pyrene-derived diol epoxides and (±)anti-BPDE with variation in activity across GST
isoforms fDreii etal.. 2002: Roias etal.. 1998: Robertsonetal.. 19861. Benzo[a]pyrene quinones
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can also be conjugated with GSH (Agarwal etal.. 1991: IARC. 19831. This compelling evidence for a
role of GSTs in the metabolism of reactive benzo[a]pyrene metabolites has triggered several
molecular epidemiology studies. However, recent studies on the impact of polymorphism on
adduct levels in PAH-exposed human populations did not show a clear relationship between the
Phase I (CYP1A1, EH) or Phase II (GST) enzyme polymorphisms and the formation of DNA adducts
fHemminki et al.. 19971 or blood protein adducts fPastorelli et al.. 19981.
Conjugation with uridine diphosphate-glucuronide catalyzed by uridine diphosphate-
glucuronosyltransferase (UDP-UGT) enzymes is another important detoxification mechanism for
oxidative benzo[a]pyrene metabolites. UGT isoforms, as well as their allelic variants, are expressed,
and have glucuronidation activity toward, benzo[a]pyrene-derived phenols and diols in the
aerodigestive tract (tongue, tonsil, floor of the mouth, larynx, esophagus), but not in the lung or
liver fFang and Lazarus. 2004: Zheng etal.. 20021. UGT activity also shows significant
interindividual variability. Incubation of lymphocytes with benzo[a]pyrene resulted in covalent
binding to protein with a 143-fold interindividual variability and a statistically significant inverse
correlation between glucuronidation and protein binding (Hu and Wells. 20041.
Sulfotransferases can catalyze the formation of sulfates of benzo[a]pyrene metabolites. In
rat or mouse liver, cytosolic sulfotransferase (in the presence of 3'-phosphoadenosine 5'-phospho-
sulfate) catalyzes formation of sulfates of three benzo[a]pyrene metabolites: benzo[a]pyrene-
7,8,9,10-tetrahydro-7-ol, benzo[a]pyrene-7,8-dihydrodiol, and benzo[a]pyrene-7,8,9,10-tetrol. The
benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol-sulfate is able to form potentially damaging DNA adducts
(Surh andTannenbaum. 19951. In human lung tissue 3-hydroxybenzo[a]pyrene conjugation to
sulfate produces benzo[a]pyrene-3-yl-hydrogen sulfate, a very lipid soluble compound that would
not be readily excreted in the urine (Cohen etal.. 19761.
Although not specific for benzo[a]pyrene, there is now considerable evidence that genetic
polymorphisms of the GST, UGT, and EH genes impart an added risk to humans for developing
cancer. Of some significance to the assessment of benzo[a]pyrene may be that smoking, in
combination with genetic polymorphism at several gene loci, increases the risk for bladder cancer
(Moore etal.. 2004: Choi etal.. 2003: Park etal.. 20031 and lung cancer (Alexandrie etal.. 2004: Lin
etal.. 20031. Coke oven workers (who are exposed to PAHs, including benzo[a]pyrene)
homozygous atthe P187S site of the NQOl gene (an inhibitor of benzo[a]pyrene-quinone adducts
with DNA), or carrying the null variant of the glutathione-S-transferase Ml (GSTM1) gene, had a
significantly increased risk of chromosomal damage in peripheral blood lymphocytes. Meanwhile,
the risk was much lower than controls in subjects with a variant allele atthe H113Y site of the EH
gene (Lengetal.. 20041.
Tissue-Specific Metabolism
Benzo[a]pyrene metabolism has been demonstrated in vivo in laboratory animals for
various tissues via multiple routes including inhalation, ingestion, and dermal absorption.
Metabolism of benzo[a]pyrene at the site of administration such as in the respiratory tract, the GI
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tract, or the skin impact the amount of benzo[a]pyrene and its form as benzo[a]pyrene or one of the
metabolites that reach systemic circulation. Nasal instillation or inhalation of benzo[a]pyrene in
monkeys, dogs, rats, and hamsters resulted in the formation of dihydrodiols, phenols, quinones, and
tetrols in the nasal mucus and lung (Wolff etal.. 1989: Petridou-Fischer etal.. 1988: Wevand and
Lavoie. 1988: Wevand and Bevan. 1987.1986: Dahl etal.. 19851. In rats, the fractions of
metabolites in the lung at 6 hours after instillation were: 20% unmetabolized benzo[a]pyrene, 16%
conjugates or polyhydroxylated compounds, 10.7% 4,5-, 7,8-, and 9,10-dihydrodiols, 9.3% 1,6-, 3,6-,
and 6,12-quinone, and 6.9% 3- and 9-hydroxybenzo[a]pyrene fWevand and Bevan. 19861. In
hamsters, approximately 50% of the benzo[a]pyrene instilled was metabolized in the nose (nasal
tissues had the highest metabolic activity per-gram of the respiratory tract tissues), and the
metabolites produced were similar to other species (Dahl etal.. 19851.
In vitro studies of human and laboratory cells and cell lines provide further quantitative and
mechanistic details of the metabolism of benzo[a]pyrene in the cells of the respiratory tract, skin,
liver, and other tissues. Tracheobronchial tissues in culture of several species (including humans,
mice, rats, hamsters, and bovines) were all found to metabolize benzo[a]pyrene extensively to
phenols, diols, tetrols, quinones, and their conjugates (Autrup etal.. 19801. The results show a high
degree of interindividual variability (a 33-fold difference in human bronchus, a 5-fold variation in
human trachea, and a 3-fold difference in bovine bronchus), but minimal variation among
individuals of the laboratory animal species f Autrup etal.. 19801. Human bronchial epithelial and
lung tissue conjugated benzo[a]pyrene metabolites to glutathione and sulfates, but not with
glucuronide (Kiefer etal.. 1988: Autrup etal.. 1978: Cohen etal.. 19761. Lung tissue slices exposed
to benzo[a]pyrene induced expression of CYP1A1 and CYP1B1 at levels 10-20 times higher than in
the liver (Harrigan etal.. 20061 and total levels of benzo[a]pyrene-DNA adducts were
approximately 2-6 times greater in the lung slices than liver fHarrigan et al.. 20041.
Benzo[a]pyrene undergoes extensive metabolism in the GI tract and liver after oral
administration. In rats after administration of an oral dose, the majority of benzo[a]pyrene
detected in organs is as metabolites (Ramesh et al.. 2004: Ramesh etal.. 2001b: Yamazaki and
Kakiuchi. 19891. In rats administered a 100-nmol dose, >90% was recovered in portal blood as
metabolites (Bock etal.. 19791. Orally administered benzo[a]pyrene produced strong induction of
CYP1A1 in the intestine of mice (Brooks etal.. 19991. DNA post-labeling studies of mice
administered benzo[a]pyrene by gavage demonstrated higher benzo[a]pyrene-DNA adduct levels in
CYP1A1(-/-) than CYP1A1(+/+) mice in small intestines (Uno etal.. 20041. To compare the
relative roles of the liver and intestine in benzo[a]pyrene metabolism and absorption, a
multicompartment perfusion system was developed; it was found thatbenzo[a]pyrene is
extensively metabolized by the intestinal Caco-2 cells and that benzo[a]pyrene and its metabolites
are transported to the apical side of the Caco-2 cells away from the liver HepG2 cells (Choi etal..
20041.
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Dermal exposure in humans and animals resulted in benzo[a]pyrene metabolism, and the
permeation of benzo[a]pyrene in skin is linked to benzo[a]pyrene metabolism. Human skin
samples maintained in short-term organ culture (i.e., human epithelial tissue, samples from human
hair follicles, and melanocytes isolated from adult human skin) can metabolize benzo[a]pyrene into
dihydrodiols, phenols, quinones, and glucuronide and sulfate conjugates fAgarwal etal.. 1991:
Alexandrov etal.. 1990: Hall and Grover. 1988: Merk etal.. 19871. Nonviable skin is unable to
metabolize benzo[a]pyrene (the permeation into nonviable skin is lower than viable skin) as
measured in a range of species including humans, rats, mice, rabbits, and marmosets (Kao etal..
1985). Viable human skin samples treated with 2 ng/cm2 [14C]-benzo[a]pyrene in acetone and
incubated for 24 hours produced the following percentages of benzo[a]pyrene metabolites: 52%
water-soluble compounds, 8% polar compounds, 17% diols, 1% phenols, 2.5% quinones, and 18%
unmetabolizedbenzo[a]pyrene fKao etal.. 19851.
Benzo[a]pyrene that reaches systemic circulation is also metabolized by multiple tissues
that are targets of benzo[a]pyrene toxicity, including reproductive tissues such as prostate,
endometrium, cervical epithelial and stromal, and testes (Ramesh etal.. 2003: Bao etal.. 2002:
Williams etal.. 2000: Melikian et al.. 1999).
Age-Specific Metabolism
Metabolism of benzo[a]pyrene occurs in the developing fetus and in children, as indicated
by DNA or protein adducts or urinary metabolites (Naufal etal.. 2010: Ruchirawat et al.. 2 010: Suter
etal.. 2010: Mielzvriska etal.. 2006: Perera etal.. 2005a: Tang etal.. 1999: Whvattetal.. 1998).
Transport of benzo[a]pyrene and benzo[a]pyrene metabolites to fetal tissues including plasma,
liver, hippocampus, and cerebral cortex has been demonstrated in multiple studies (McCabe and
Flvnn. 1990: NeubertandTapken. 1988: Shendrikova and Aleksandrov. 1974). and benzo[a]pyrene
is metabolized by human fetal esophageal cell culture fChakradeo etal.. 19931. While expression of
CYP enzymes are lower in fetuses and infants, the liver to body mass ratio and increased blood flow
to liver in fetuses and infants may compensate for the decreased expression of CYP enzymes
(Ginsberg et al.. 2004). Prenatal exposure to benzo[a]pyrene upregulates CYP1A1 and may
increase the formation of benzo[a]pyrene-DNA adducts (Wu etal.. 2003a). Activity of Phase II
detoxifying enzymes in neonates and children is adequate for sulfation, but decreased for
glucuronidation and glutathione conjugation (Ginsberg et al.. 2004). The conjugation of
benzo[a]pyrene-4,5-oxide with glutathione was approximately one-third less in human fetal than
adult liver cytosol fPacifici et al.. 19881. The differential Phase I and II enzyme expression and
activity in the developing fetus and in children are consistent with an expectation that these
lifestages can be more susceptible to benzo[a]pyrene toxicity.
D.1.5. Elimination
Benzo[a]pyrene metabolites have been detected in the urine of exposed humans, but fecal
excretion has not been investigated in any detail. Benzo[a]pyrene and associated metabolites have
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also been detected in breast milk, especially in smokers, indicating some portion of dose is excreted
via lactation (Yu etal.. 2011: Zanieri etal.. 2007: Madhavan and Naidu. 19951.
Studies of benzo[a]pyrene elimination in animals following exposure via inhalation,
ingestion, and dermal routes have shown that benzo[a]pyrene is excreted preferentially in the feces
in multiple species of laboratory animals including rat, mice, hamsters, guinea pigs, monkeys, and
dogs fWang etal.. 2003: Likhachev etal.. 1992: Wolff etal.. 1989: Yang etal.. 1989: Petridou-Fischer
etal.. 1988: Wevand and Bevan. 1987: Sun etal.. 1982: Hechtetal.. 19791. The metabolites in bile
are primarily benzo[a]pyrene conjugates, predominantly thioether conjugates of varying extent in
different species (Wevand and Bevan. 19871. Six hours after a single intratracheal instillation of
benzo[a]pyrene (2 |ig/kg] to male Sprague-Dawley rats, relative metabolite levels were 31.2%
diglucuronides, 30.4% thioether conjugates, 17.8% monoglucuronides, 6.2% sulfate conjugates,
and 14.4% unconjugated metabolites fBevan and Sadler. 19921. Rats administered benzo[a]pyrene
via i.v. excrete a larger fraction in urine than via inhalation or oral exposure, suggesting an
important role for enterohepatic circulation of benzo[a]pyrene metabolite conjugates (Moir etal..
1998: Wevand and Bevan. 1986: Hirom etal.. 19831. The vehicle impacts the amount of
benzo[a]pyrene excreted and may, in part, be due to the elimination rate or to other factors such as
the absorption rate. For tritiated benzo[a]pyrene administered to Sprague-Dawley rats in
hydrophilic triethylene glycol, 70.5% of the dose was excreted into bile within 6 hours. When the
lipophilic solvents, ethyl laurate and tricaprylin, were used as vehicles, 58.4 and 56.2% of the dose
was excreted, respectively (Bevan and Ulman. 19911. In addition to benzo[a]pyrene and its
metabolites, adducts of benzo[a]pyrene with nucleotides have also been identified as a small
fraction of the administered dose in feces and urine of animals. The level of BPDE adducts with
guanine detected in urine of male Wistar rats was dose-dependent Forty-eight hours after dosing
with 100 M-g/kg tritiated benzo[a]pyrene, 0.15% of the administered benzo[a]pyrene dose was
excreted in the urine as an adduct with guanine (Autrup and Seremet. 19861. Benzo[a]pyrene is
also eliminated, to a limited extent, through milk in lactating animals. Levels of benzo[a]pyrene
eliminated via lactation after dietary administration constituted less than 0.003% of the maternal
dose in rabbits (West and Horton. 19761 and goats (Lapole etal.. 20071. whereas in sheep
lactational elimination represented about 0.014% of the total maternal dose (West and Horton.
19761.
Overall, the data in humans and laboratory animals are sufficient to describe
benzo[a]pyrene elimination qualitatively, but are limited in estimating quantitative rates of
elimination.
D.2. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS
Several toxicokinetic or pharmacokinetic models of benzo[a]pyrene have been developed
for rodents (rat and hamster). However, human models have only been developed via allometric
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scaling, and metabolic parameters in humans have not been calibrated against in vivo toxicokinetic
data or in vitro experiments.
Bevan and Wevand f!9881 performed compartmental pharmacokinetic analysis of
distribution of radioactivity in male Sprague-Dawley rats, following the intratracheal instillation of
benzo[a]pyrene to normal and bile duct-cannulated animals fWevand and Bevan. 1987.19861.
However, implicit simulation approaches were used, as opposed to physiologically-based
approaches. The model calculated linear rate constants among compartments, and assumed that
the kinetics of benzo[a]pyrene and its metabolites were the same.
Roth and Vinegar (19901 reviewed the capacity of the lung to impact the disposition of
chemicals and used benzo[a]pyrene as a case study. A PBPK model was presented based on data
from Wiersma and Roth (1983bl: Wiersma and Roth (1983al and was evaluated against tissue
concentration data from Schlede et al. f 19701. The model was structured with compartments for
arterial blood, venous blood, lung, liver, fat, and slowly and rapidly perfused tissues and an
adequate fit was obtained for some compartments; however, tissue-level data for calibration and
validation of this model were limited. Metabolism in liver and lung was estimated using kinetic
data from control rats and rats pretreated with 3-methylcholanthrene to induce benzo[a]pyrene
metabolism. In microsomal preparations from control and 3-methylcholanthrene induced rat livers
and lungs, benzo[a]pyrene hydroxylase activity was 1,000-fold greater in liver. In isolated rat
lungs, the clearance of benzo[a]pyrene was about one-sixth of the clearance in isolated rat livers
and in 3-methylcholanthrene-pretreated rats the clearance in lungs and livers were of similar
magnitude. The PBPK simulations model based on these data showed that for a bolus intravascular
injection of benzo[a]pyrene in rats, the majority of benzo[a]pyrene metabolism usually occurs in
the liver. Except for cases when rats are pretreated with enzyme-inducing agents or where the
exposure occurs via inhalation, the metabolic clearance in the lung is minor.
Moir etal. (19981 conducted a pharmacokinetic study on benzo[a]pyrene to obtain data for
model development Rats were injected with varying doses of [14C]-benzo[a]pyrene to 15 mg/kg,
and blood, liver, fat, and richly perfused tissue were sampled varying time points after dosing. Moir
(19991 then described a model for lung, liver, fat, richly and slowly perfused tissues, and venous
blood, with saturable metabolism occurring in the liver. The fat and richly perfused tissues were
modeled as diffusion-limited, while the other tissues were flow-limited. The model predicted the
blood benzo[a]pyrene concentrations well, although it overestimated the 6 mg/kg results at longer
times (>100 minutes). The model also produced a poor fit to the liver data. The model simulations
were also compared to data of Schlede et al. f!9701. who injected rats with 0.056 mg/kg body
weight of benzo[a]pyrene. The model predicted blood and fat benzo[a]pyrene concentrations well,
but still poorly predicted liver benzo[a]pyrene concentrations. The model included only one
saturable metabolic pathway, and only parent chemical concentrations were used to establish the
model. No metabolites were included in the model. This model was re-calibrated by Crowell et al.
f20111 by optimizing against additional rodent data and altering partition coefficient derivation.
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However, it still did not incorporate metabolites, and some tissues continued to exhibit poor model
fits.
An attempt to scale the Moir etal. T19981 rodent PBPK model to humans, relevant to risk
assessment of oral exposures to benzo[a]pyrene, was presented by Zeilmaker et al. (1999a) and
Zeilmaker etal. fl999bl. The PBPK model for benzo[a]pyrene was derived from an earlier model
for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats fZeilmaker and van Eiikeren. 19971. Most
compartments were perfusion-limited, and tissues modeled included blood, adipose (with diffusion
limitation), slowly and richly perfused tissues, and liver. However, there was no separate
compartment for the lung. The liver compartment featured the AhR-dependent CYP450 induction
mechanism and DNA adduct formation as a marker for formation of genotoxic benzo[a]pyrene
metabolites. It was assumed that DNA adduct formation and the bulk benzo[a]pyrene metabolism
were mediated by two different metabolic pathways. The model was experimentally calibrated in
rats with the data for 7-ethoxyresorufin-0-deethylase (EROD) and formation of DNA adducts in the
liver after i.v. administration of a single dose and per os administration of a single or repeated doses
ofbenzo[a]pyrene (Zeilmaker etal.. 1999a).
Zeilmaker etal. (1999b) assumed identical values for several parameters in rats and
humans (i.e., benzo[a]pyrene tissue partition coefficients, AhR concentration in liver, rate constant
for the decay of the benzo[a]pyrene-CYP450 complex, half-life of the CYP450 protein, fraction and
rate of GI absorption of benzo[a]pyrene, and rates of formation and repair of DNA adducts in liver).
The basal CYP45 0 activity in humans was assumed to be lower than that in rat liver. The
mechanism of AhR-dependent induction of CYP450 dominated the simulated benzo[a]pyrene-DNA
adduct formation in the liver. The results of PBPK model simulations indicated that the same dose
of benzo[a]pyrene administered to rats or humans might produce one order of magnitude higher
accumulation of DNA adducts in human liver when compared with the rat fZeilmaker etal.. 1999bl.
Even though the model of Zeilmaker et al. (1999b) represents a major improvement in
predictive modeling of benzo[a]pyrene toxicokinetics, the interspecies extrapolation introduces
significant uncertainties. As emphasized by the authors, the conversion of benzo[a]pyrene to its
mutagenic and carcinogenic metabolites could not be explicitly modeled in human liver because no
suitable experimental data were available. According to the authors, improvement of the model
would require direct measurements of basal activities of CYP1A1 and CYP1A2 and formation of
benzo[a]pyrene-DNA adducts in human liver. Metabolic clearance of benzo[a]pyrene in the lungs
was also not addressed. Additionally, the toxicokinetic modeling by Zeilmaker etal. (1999b)
addressed only one pathway of benzo[a]pyrene metabolic activation, a single target organ (the
liver), and one route of administration (oral). In order to model health outcomes of exposures to
benzo[a]pyrene, the PBPK model needs to simulate rate of accumulation of benzo[a]pyrene-DNA
adducts and/or the distribution and fate of benzo[a]pyrene metabolites (e.g., BPDE) that bind to
DNA and other macromolecules. Alternatively, stable toxic metabolites (e.g., trans-anti-tetrol-
benzo[a]pyrene) may be used as an internal dose surrogate. While the metabolic pattern of
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benzo[a]pyrene has been relatively well characterized qualitatively in animals, the quantitative
kinetic relationships between the more complex metabolic reactions in potential target organs are
not yet well defined.
D.2.1. Recommendations for the Use of PBPK Models in Toxicity Value Derivation
PBPK models for benzo[a]pyrene were evaluated to determine the capability to extrapolate
from rats to humans, or between oral and inhalation exposure routes. Due to significant
uncertainties with respect to the inter-species scaling of the metabolic parameters between rats
and humans, these models were not used for cross-species extrapolation. Furthermore, no
complete mechanistic PBPK model for the inhalation route was identified, nor was there a model
for humans that simulates the typical inhalation exposure to benzo[a]pyrene on poorly soluble
carbonaceous particles. This precluded the model's use for cross-route extrapolation to the
inhalation pathway.
D.3. HUMAN STUDIES
D.3.1. Noncancer Endpoints
Cardiovascular Endpoints
Burstvnetal. (2005) reported the association of death from cardiovascular disease with
benzo[a]pyrene exposure in a cohortof 12,367 male European asphalt workers (Table D-l). These
workers were first employed in asphalt paving between 1913 and 1999, and worked at least one
season. Average duration of follow-up was 17 ± 9 years (mean ± standard deviation [SD]),
encompassing 193,889 person-years of observation. Worker exposure to coal tar was estimated
using industrial process and hygiene information and modeling (presented in a previous report),
and coal tar exposure was found to be the strongest determinant of exposure to benzo[a]pyrene.
Benzo[a]pyrene exposure was assessed quantitatively using measurement-driven mixed effects
exposure models, using data collected from other asphalt industry workers, and this model was
constructed and validated previously. Due to limited data availability, only information regarding
the primary cause of death was collected, and this analysis was limited to diseases of the circulatory
system (ICD codes 390-459), specifically ischemic heart disease (IHD: ICD codes 410-414). Diesel
exhaust exposure was also assessed in this cohort, but varied little among the asphalt pavers, and
was not associated with risk of death from cardiovascular disease. Of the initial cohort, 0.25% was
lost to follow-up and 0.38% emigrated during the course of observation. Relative risks (RRs) and
associated 95% confidence intervals (CIs) were estimated using Poisson regression, and all models
included exposure index for agent of interest (coal tar or benzo[a]pyrene), age, calendar period of
exit from cohort, total duration of employment, and country, using the category of lowest exposure
as the reference. Confounding by tobacco smoke exposure was considered in relation to the
strength of its association with cardiovascular disease and the smoking prevalence in the
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population. The RR attributed to cigarette smoking in former and current smokers was assumed to
be 1.2 and 2, respectively, based upon literature reports. From analysis of smoking incidence in a
subcohort, the following smoking distribution was proposed: in the lowest exposure group, 40%
never-smokers, 30% former smokers, and 30% current smokers; and among the highest exposed,
the proportion shifted to 20/30/50%, respectively.
Exposed subjects were stratified into quintiles based upon IHD mortality, with
83-86 deaths per exposure category, composing approximately 2/3 of the 660 cardiovascular
disease-related deaths. Both cumulative and average exposure indices for benzo[a]pyrene were
positively associated with IHD mortality, with a RR of approximately 1.6 in the highest exposure
quintile from both metrics, independent of total employment duration. Similar monotonic trends
were observed for all cardiovascular diseases (combined), although a dose-response relationship
was evident only for IHD and not hypertension or other individual heart disease categories. Similar
trends were also observed for coal tar exposure and IHD. Adjusting the RR to account for possible
confounding by smoking yields a RR of 1.39 under the assumptions mentioned above, and is still
elevated (1.21) if the contribution of smoking to cardiovascular disease etiology was greater than
the original assumptions. Furthermore, the RR for the high versus low exposure quintile is
1.24 even if the distribution of nonsmokers/former smokers/current smokers shifts to 0/30/70%,
using the original assumptions of cigarette smoke casual potency.
Table D-l. Exposure to benzo[a]pyrene and mortality from cardiovascular
diseases in a European cohort of asphalt paving workers
Effect measured
Cumulative exposure (ng/m3-yrs)
p-value
for trend
0-1893
189-501
502-931
932-2,012
>2,013
Diseases of the circulatory system
Deaths
RR
95% CI
137
1.00
145
1.08
0.85-1.38
118
1.06
0.80-1.42
132
1.24
0.89-1.71
128
1.42
0.96-2.09
0.09
IHD
Deaths
RR
95% CI
83
1.00
83
0.99
0.72-1.36
84
1.22
0.86-1.74
83
1.24
0.82-1.85
85
1.58
0.98-2.55
0.06
Effect measured
Average exposure (ng/m3)
p-value
for trend
0-68a
68-105
106-146
147-272
>273
Diseases of the circulatory system
Deaths
RR
95% CI
128
1.00
142
1.30
1.01-1.67
143
1.55
1.18-2.05
139
1.45
1.09-1.93
108
1.58
1.16-2.15
<0.001
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Effect measured
Cumulative exposure (ng/m3-yrs)
p-value
for trend
0-1893
189-501
502-931
932-2,012
>2,013
IHD
Deaths
83
83
83
86
83
RR
1.00
1.13
1.33
1.20
1.64
0.02
95% CI
0.82-1.55
0.94-1.90
0.84-1.71
1.13-2.38
Reference category.
Source: Burstvn et al. (2005).
Friesenetal. (20101 examined the association between benzo[a]pyrene exposure and
deaths from chronic nonmalignant disease in a cohort of 6,423 male and 603 female Canadian
aluminum smelter workers (Table D-2). Inclusion criteria required at least 3 years of continuous
employment in either the smelter facility or power-generating station from 1954 to 1997, with
worker history collected up through 1999. This cohort was probabilistically linked to the Canadian
national mortality database for external comparison to the British Columbia population and
calculation of standardized mortality ratios (SMRs), which were adjusted for age, sex, and time
period. Ninety-five percent CIs were calculated for the SMRs assuming a Poisson distribution.
Internal comparisons were also made during the analysis of IHD mortality in male workers,
calculating hazard ratios (HRs) for IHD with or without acute myocardial infarction (AMI) after
1969, as AMI could not be differentiated from other IHD on death certificates issued previously.
HRs were calculated using Cox regression models, with age as a metamarker of time, also including
smoking status, time since first employed and work location status. Smoking information for 77%
of this updated cohort was collected by questionnaire, and workers were categorized as 75% ever-
smokers and 25% never-smokers. Quantitative exposure to coal tar pitch volatiles were estimated
by benzo[a]pyrene measurements, calculated by a job classification and time-based exposure
matrix, as described in a previous report; annual arithmetic mean values were calculated for
exposures from 1977 to 2000, while pre-1977 levels were backwards-extrapolated from 1977
values, incorporating major technological changes in time periods as appropriate.
Cumulative exposure metrics were highly skewed. Cumulative benzo[a]pyrene with a
5-year lag (past benzo[a]pyrene exposure) and cumulative benzo[a]pyrene in the most recent
5 years (recent benzo[a]pyrene exposure) were only slightly positively correlated (r = 0.10,
p < 0.001). Current benzo[a]pyrene exposure was highly correlated with cumulative exposure for
the most recent 5 years of exposure (r = 0.86, p < 0.001), but not with 5-year lagged cumulative
exposure (r = 0.03, p < 0.001). Lagged cumulative exposure metrics (0-10 years) were all highly
correlated with each other (r = 0.96, all p-values <0.001); lagged metrics for cumulative exposure
were used to distinguish between effects of current versus long-term exposure.
When exposed workers were pooled and compared externally to non-exposed referents, the
IHD and AMI SMRs were all <1.00 for males, and the only significant association in females was an
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SMR of 1.27 for AMI. For internal comparisons, exposed males were stratified into quintiles based
upon IHD mortality, with approximately 56 deaths per exposure category. Five-year lagged
cumulative benzo[a]pyrene exposure was significantly associated with elevated risk of IHD
mortality, HR = 1.62 (95% CI 1.06-2.46) in the highest exposure quintile, while no association was
observed between most recent (5 years) exposure and mortality. Restricting IHD events to only
AMI (1969 onward) resulted in similar monotonic trends, albeit of lower statistical significance. No
association was observed between benzo[a]pyrene exposure and non-AMI IHD. While there was
little difference in the exposure-response association among 0-, 2-, and 5-year lagged data, 10-year
lagged data resulted in a weaker association. All risk estimates were strengthened by the
incorporation of work status and time-since-hire to account for the healthy worker effect, as
evidenced by the SMR of 0.87 (95% CI 0.82-0.92) for all chronic nonmalignant diseases combined
in male exposed workers versus external referents. Using a continuous variable, the authors
calculated the risk of death from IHD as 1.002 (95% CI 1.000-1.005) per |ig/m3 from cumulative
benzo[a]pyrene exposure; however, visual inspection of the categorical relationships indicated that
the association is nonlinear, suggesting that this value may be an underestimate. Restricting the
cohort to only members who died within 30 days of active employment at the worksite, cumulative
benzo[a]pyrene exposure was not significantly associated with IHD or AMI, although the HR for the
highest exposure group was 2.39 (95% CI 0.95-6.05). Exposure-response relationships were
similarly examined in male smelter workers for chronic obstructive pulmonary disease and
cerebrovascular disease, but neither was significantly associated with cumulative benzo[a]pyrene
exposure in either internal or external comparisons.
Table D-2. Exposure to benzo[a]pyrene and mortality from cardiovascular
diseases in a Canadian cohort of male aluminum smelter workers
Effect measured
Categorical cumulative exposure with a 5-yr lag
(pg/m3-yr)
p-value
for
trend3
Continuous13
0
0-7.79
7.79-24.3
24.3-66.7
>66.7
All IHD (1957 onward)
Deaths
Person-years of follow-up
HR
95% CI
56
33,111
1
referent
56
37,581
1.11
0.76-1.62
57
34,838
1.48
1.01-2.17
56
31,533
1.28
0.86-1.91
56
13,688
1.62
1.06-2.46
0.053
281
150,751
1.002
1.000-1.005
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Effect measured
Categorical cumulative exposure with a 5-yr lag
(Hg/m3-yr)
p-value
for
trend3
Continuous13
0
0-7.79
7.79-24.3
24.3-66.7
>66.7
AMI (1969 onward)
0
0-7.51
7.51-27.7
27.7-67.4
>67.4
Deaths
Person-years of follow-up
HR
95% CI
35
25,071
1
referent
37
30,454
1.14
0.71-1.82
37
34,621
1.21
0.75-1.96
38
24,081
1.36
0.84-2.45
37
13,261
1.46
0.87-2.45
0.19
184
127,488
1.001
0.997-1.005
aTwo-sided test for trend using the person-year-weighted mean value for each category as a linear, continuous
variable.
bExposure variable was entered as a continuous, linear variable in the model.
Source: Friesen et al. (2010).
Reproductive and Developmental Endpoints
Wu etal. f20101 conducted a study of benzo[a]pyrene-DNA adduct levels in relation to risk
of fetal death in Tianjin, China. This case-control study included women who experienced a delayed
miscarriage before 14 weeks gestational age (i.e., a fetal death that remained in utero and therefore
required surgical intervention). Cases were matched by age and gravidity to controls (women
undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded
women who smoked, women with chronic disease and pregnancy complications, and women with
occupational exposures to PAHs. Residency within Tianjin for at least 1 year was also an eligibility
criterion. The participation rate was high: 81/84 eligible cases participated and 81/89 eligible
controls participated. Data pertaining to demographic characteristics, reproductive history, and
factors relating to potential PAH exposure were collected using a structured interview, and samples
from the aborted tissue were obtained. In two of the four hospitals used in the study, blood
samples from the women (n = 51 cases and 51 controls) were also collected. The presence of
benzo[a]pyrene-BPDE adducts was assessed in the blood and tissue samples using high-
performance liquid chromatography (HPLC). There was no correlation between blood and aborted
tissue levels of benzo[a]pyrene adducts (r = -0.12 for the 102 blood-tissue pairs, r = -0.02 for the
51 case pairs, and r = -0.21 for the 51 control pairs). (The authors noted that there was little
difference between women with and without blood samples in terms of the interview-based
measures collected or in terms of the DNA-adduct levels in aborted tissue.) Benzo[a]pyrene-adduct
levels were similar but slightly lower in the aborted tissue of cases compared with controls
(mean ± SD 4.8 ± 6.0 in cases and 6.0 ± 7.4 in controls, p = 0.29). In the blood samples, however,
benzo[a]pyrene-adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ± 2.2 in cases and controls,
respectively, p < 0.001). In logistic regression analyses using a continuous adduct measure, the
odds ratio (OR) was 1.35 (95% CI 1.11-1.64) per adduct/108 nucleotide. These results were
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adjusted for education, household income, and gestational age, but were very similar to the
unadjusted results. Categorizing exposure at the median value resulted in an adjusted OR of
4.27 (95% CI 1.41-12.99) in the high compared with low benzo[a]pyrene-adduct group. There was
no relation between benzo[a]pyrene-adduct levels in the aborted tissue and miscarriage in the
logistic regression analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93-1.02) or
dichotomous exposure measure (adjusted OR 0.76, 95% CI 0.37-1.54). Associations between
miscarriage and several interview-based measures of potential PAH exposure were also seen:
adjusted ORs of 3.07 (95% CI 1.31-7.16) for traffic congestion near residence, 3.52 (95% CI
1.44-8.57) for commuting by walking, 3.78 (95% CI 1.11-12.87) for routinely cooked during
pregnancy, and 3.21 (95% CI 0.98-10.48) for industrial site or stack near residence, but there was
no association with other types of commuting (e.g., by bike, car, or bus).
Pereraetal. f2005al studied 329 nonsmoking pregnant women (30 ± 5 years old) possibly
exposed to PAHs from fires at the World Trade Center (WTC) during the 4 weeks after 09/11/2001.
Maternal and umbilical cord blood levels of benzo[a]pyrene (BPDE)-DNAadducts were highest in
study participants who lived within 1 mile of the WTC, with an inverse correlation between cord
blood levels and distance from the WTC. Neither cord blood adduct level nor environmental
tobacco smoke (ETS) alone was positively correlated with adverse birth outcomes. However, the
interaction between ETS exposure and cord blood adducts was significantly associated with
reduced birth weight and head circumference. Among babies exposed to ETS in utero, a doubling of
cord blood benzo[a]pyrene-DNA adducts was associated with an 8% decrease in birth weight
(p = 0.03) and a 3% decrease in head circumference (p = 0.04).
Pereraetal. (2005b). a reanalysis ofPerera etal. (2004). compared various exposures—
ETS, nutrition, pesticides, material hardship—with birth outcomes (length, head circumference,
cognitive development). ETS exposure and intake of PAH-rich foods by pregnant women were
determined by questionnaire. Levels of BPDE-DNA adducts were determined in umbilical cord
blood collected at delivery. The study population consisted of Dominican or African-American
nonsmoking pregnant women (n = 214;24±5 years old) free of diabetes, hypertension, HIV, and
drug or alcohol abuse. Benzo[a]pyrene adducts, ETS, and dietary PAHs were not significantly
correlated with each other. However, the interaction between benzo[a]pyrene-DNA adducts and
ETS exposure was significantly associated with reduced birth weights (-6.8%; p = 0.03) and
reduced head circumference (-2.9%; p = 0.04).
Tang etal. (2006) measured BPDE-DNA adducts in maternal and umbilical cord blood
obtained at delivery from a cohort of 150 nonsmoking women and their newborns in China.
Exposure assessment was related to the seasonal operation of a local, coal-fired power plant;
however, airborne PAH concentrations were not measured. Dietary PAH intake was not included as
a covariate because it did not significantly contribute to the final models, but ETS, sex, and maternal
height and weight were considered as covariates. DNA adduct levels were compared to several
birth outcomes and physical development parameters, such as gestational age at birth; infant sex,
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birth weight, length, head circumference, and malformations; maternal height and pregnancy
weight total weight gain; complications of pregnancy and delivery; and medications used during
pregnancy.
High cord blood adduct levels were significantly associated with reduced infant/child
weight at 18 months ((3 = -0.048, p = 0.03), 24 months ((3 = -0.041, p = 0.027), and 30 months of age
(P = -0.040, p = 0.049); decreased birth head circumference was marginally associated with DNA
adduct levels ((3 = -0.011, p = 0.057). Maternal adductlevels were correlated neither with cord
blood adduct levels nor with fetal and child growth. Among female infants, cord blood adduct levels
were significantly associated with smaller birth head circumference (p = 0.022) and with lower
weight at 18 months (p = 0.014), 24 months (p = 0.012), and 30 months of age (p = 0.033), and with
decreased body length at 18 months of age (p = 0.033). Among male infants, the corresponding
associations were also inverse, but were not statistically significant.
Considerable evidence of a deleterious effect of smoking on male and female fertility has
accumulated from epidemiological studies of time to pregnancy, ovulatory disorders, semen
quality, and spontaneous abortion (reviewed in Wavlen etal.. 2009: Cooper and Molev. 2008:
Spares and Melo. 2008). In addition, the effect of smoking, particularly during the time of the
perimenopausal transition, on acceleration of ovarian senescence (menopause) has also been
established (Midgette and Baron. 1990). More limited data are available pertaining specifically to
measures of benzo[a]pyrene and reproductive outcomes.
Neal etal. (2008) examined levels of benzo[a]pyrene and other PAHs in follicular fluid and
serum sample from 36 women undergoing in vitro fertilization at a clinic in Toronto, and compared
the successful conception rate in relation to benzo[a]pyrene levels. The women were classified by
smoking status, with 19 current cigarette smokers, 7 with passive or sidestream smoke exposure
(i.e., nonsmoker with a partner who smoked), and 10 nonsmokers exposed. An early follicular
phase blood sample and follicular fluid sample from the follicle at the time of ovum retrieval were
collected and analyzed for the presence of benzo[a]pyrene, acenapthelene, phenanthrene, pyrene,
and chrysene using gas chromatography/mass spectrometry (MS) (detection limit 5 pg/mL). The
frequency of nondetectable levels of serum benzo [a] pyrene was highest in the nonsmoking group
(60.0,14.3, and 21.0% below the detection limit in nonsmoking, sidestream smoke, and active
smoking groups, respectively). A similar pattern was seen with follicular fluid benzo[a]pyrene
(30.0,14.3, and 10.5% belowthe detection limitin nonsmoking, sidestream smoke, and active
smoking groups, respectively). In the analyses comparing mean values across groups, an assigned
value of 0 was used for nondetectable samples. Follicular fluid benzo[a]pyrene levels were higher
in the active smoking group (mean ± standard error [SE], 1.32 ± 0.68 ng/mL) than in the sidestream
(0.05 ± 0.01 ng/mL) or nonsmoking (0.03 ± 0.01 ng/mL) groups (p = 0.04). The between-group
differences in serum benzo [a]pyrene levels were not statistically significant (0.22 ± 0.15,
0.98 ± 0.56, and 0.40 ± 0.13 ng/mL in nonsmoking, sidestream smoke, and active smoking groups,
respectively), and there were no differences in relation to smoking status. Among active smokers,
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the number of cigarettes smoked per day was strongly correlated with follicular fluid
benzo[a]pyrene levels (r = 0.7, p < 0.01). Follicular fluid benzo[a]pyrene levels were significantly
higher among the women who did not conceive (1.79 ± 0.86 ng/mL) compared with women who
did getpregnant (mean approximately 0.10 ng/mL, as estimated from graph) (p < 0.001), but
serum levels of benzo[a]pyrene were not associated with successful conception.
A small case-control study conducted between August 2005 and February 2006 in Lucknow
city (Uttar Pradesh), India examined PAH concentrations in placental tissues (Singh etal.. 2008) in
relation to risk of preterm birth. The study included 29 cases (delivery between 28 and <36 weeks
of gestation) and 31 term delivery controls. Demographic data on smoking history, reproductive
history, and other information were collected by interview, and a 10-g sample of placental tissue
was collected from all participants. Concentration of specific PAHs in placental tissue was
determined using HPLC. In addition to benzo[a]pyrene, the PAHs assayed were naphthalene,
acenaphthylene, phenanthrene, fluorene, anthracene, benzo [a]anthracene, fluoranthene, pyrene,
benzo[k]fluoranthene, benzo[b]fluoranthene, benzo [g,h,i]perylene, and dibenzo[a,h]anthracene.
PAH exposure in this population was from environmental sources and from cooking. The age of
study participants ranged from 20 to 35 years. There was little difference in birth weight between
cases and controls (mean 2.77 and 2.75 kg in the case and control groups, respectively). Placental
benzo[a]pyrene levels were lower than the levels of the other PAHs detected (mean 8.83 ppb in
controls for benzo [a] pyrene compared with 25-30 ppb for anthracene, benzo [k] fluoranthene,
benzo [b] fluoranthene, and dibenzo [a, h] anthracene, 59 ppb for acenaphthylene, and 200-380 ppm
for naphthalene, phenanthrene, fluoranthene, and pyrene; nondetectable levels of fluorine,
benzo [a] anthracene, and benzo [g,h,i]perylene were found). There was little difference in
benzo[a]pyrene levels between cases (mean ± SE 13.85 ± 7.06 ppb) and controls (8.83 ± 5.84 ppb),
but elevated levels of fluoranthene (325.91 ± 45.14 and 208.6 ± 21.93 ppb in cases and controls,
respectively, p < 0.05) and benzo[b]fluoranthene (61.91 ± 12.43 and 23.84 ± 7.01 ppb in cases and
controls, respectively, p < 0.05) were seen.
Neurotoxicity
Niu etal. (2010) studied 176 Chinese coke-oven workers with elevated benzo[a]pyrene
exposure and compared them against 48 referents (workers in a supply warehouse), matched by
socioeconomic status, lifestyle, and health. Blood levels of monoamine, amino acid and chlorine
neurotransmitters were measured, and the World Health Organization Neurobehavioral Core Test
Battery was administered to assess emotional state, learning, memory, and hand-eye coordination.
The authors self-designed a study questionnaire to gather information on worker education,
vocational history, smoking and drinking habits, and personal habits, personal and family medical
history, as well as any current symptoms and medications used in the previous several weeks.
Workers were excluded from the study for any of the following criteria: if they reported feeling
depressed at any point during the previous 6 months; if they had taken medicine in the previous
2 weeks that could affect nervous system function; or if they reported undertaking vigorous
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exercise less than 48 hours previously. "Smoking" was defined as >10 cigarettes/day during the
past year. Similarly, "drinking" was defined as wine/beer/spirits consumed >3 times/week for the
past 6 months. Workplace environmental sampling stations were established at each of the
physical work locations, including the referent's warehouse, and dual automatic air sampling
pumps collected samples at personal breathing zone height for 6 hours/day, over 3 consecutive
days. Benzo[a]pyrene content was determined by HPLC, and relative exposure was compared to
post-shift urine levels of a benzo[a]pyrene metabolite, 1-hydroxypyrene (1-OH-Py). Blood was
collected in the morning before breakfast; monoamine (norepinephrine and dopamine) and amino
acid (glutamate, aspartate, glycine, and gamma-aminobutyric acid [GABA]) neurotransmitter levels
were determined by HPLC, acetylcholine levels determined by hydroxyamine chromometry, and
acetylcholine esterase (AchE) levels measured in lysed red blood cells (RBCs) using activity kits.
Benzo[a]pyrene mean concentrations were 19.56 ± 13.2,185.96 ± 38.6, and
1,623.56 ± 435.8 ng/m3 at the bottom, side, and top of the coke oven, respectively, all of which were
higher than the mean atthe referents' warehouse (10.26 ± 7.6 ng/m3). The authors did not report
stratified analysis by different levels of benzo[a]pyrene exposure, and reported only comparisons
between the referents and all exposed workers combined (Table D-3), or between workers grouped
by urinary benzo[a]pyrene metabolite 1-OH-Py levels (Table D-4). There were no significant
differences in age, education, or smoking or alcohol use between the coke oven and warehouse
workers. Urinary 1-OH-Py levels were 32% higher in coke oven workers compared to the referent
group, corresponding to the higher levels of benzo[a]pyrene detected in all coke oven workstation
compared to the supply warehouse. Performance in two neurobehavioral function tests, digit span
and forward digit span, were significantly decreased in the exposed oven workers versus the
control group; when stratified by urinary metabolite level, scores significantly decreased with
increasing 1-OH-Py levels. Of the neurotransmitters assessed, norepinephrine, dopamine,
aspartate, and GABA were significantly decreased in exposed versus control workers;
norepinephrine and aspartate were also significantly and inversely related with 1-OH-Py levels.
Dopamine levels appeared to decrease with increased urinary metabolite levels, although the
relationship was not statistically significant GABA levels were highly variable, and appeared to
increase with increasing 1-OH-Py levels, although this relationship was not statistically significant
Acetylcholine levels were 4-fold higher in coke oven workers compared to referents, and AchE
activity was 30% lower; both acetylcholine and AchE were significantly associated with urinary
benzo[a]pyrene metabolite levels, although acetylcholine increased and AchE activity decreased
with increasing 1-OH-Py. The authors reported the results of correlation analysis, indicating that
digit span scores correlated negatively with acetylcholine and positively with AchE (coefficients of
-0.230, -0.276 and 0.120, 0.170, respectively), although no indication of statistical significance was
given. No other associations were reported.
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1 Table D-3. Exposure-related effects in Chinese coke oven workers or
2 warehouse controls exposed to benzo[a]pyrene in the workplace
Exposure group
Effect measured
Controls (n = 48)
Exposed workers (n = 176)
p-value
Background information (mean ± SD, incidence or percent)
Age (yrs)
39.71 ±7.51
37.86 ±6.51
0.098
Education (junior/senior)
23/25
110/66
0.068
Smoking
11%
64%
0.093
Drinking
27%
39%
0.140
Urine benzo[a]pyrene metabolite (nmol/mol creatinine; mean ± SD)
1-OH-Py
2.77 ± 1.45
3.66 ±0.67
0.000
Neurobehavioral function tests (mean ± SD)
Simple reaction time
413.88 ± 95.40
437.39 ± 88.44
0.109
Digit span
17.31 ±4.54
15.47 ± 4.08
0.006
Forward digit span
10.65 ± 2.42
9.25 ±2.64
0.001
Neurotransmitter concentrations (mean ± SD)
Norepinephrine (ng/mL)
62.54 ± 58.07
40.62 ± 29.78
0.000
Dopamine (ng/mL)
1,566.28 ± 317.64
1,425.85 ± 422.66
0.029
Aspartate (ng/mL)
2.13 ± 1.66
1.58 ±0.99
0.004
Glutamate (ng/mL)
11.21 ±5.28
9.68 ±5.72
0.074
GABA (ng/mL)
2.52 ±5.16
1.01 ±2.21
0.004
Acetylcholine (ng/mL
172.60 ±67.19
704.00 ± 393.86
0.000
AchE activity (U/mg protein)
71.31 ±46.18
50.27 ± 34.02
0.012
3
4 Source: Niu et al. (2010).
5 Table D-4. Exposure-related effects in Chinese coke oven workers or
6 warehouse controls exposed to benzo[a]pyrene in the workplace, stratified by
7 urinary metabolite levels
Effect measured
Exposure group categorized by 1-OH-Py level
p-value
0-3.09 pmol/mol
creatinine
3.09-3.90 pmol/mol
creatinine
3.90-5.53 pmol/mol
creatinine
Number of subjects
33
72
36
Neurobehavioral function tests (mean ± SD)
Digit span
Forward digit span
Backward digit span
Right dotting
18.24 ±4.58
10.85 ±2.12
7.20 ± 3.07
152.15 ±35.43
16.04 ± 4.24
9.80 ± 2.86
6.38 ±2.55
153.80 ± 31.55
15.78 ±3.71
9.58 ±2.33
6.20 ±2.15
167.22 ±59.21
0.003
0.019
0.089
0.094
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Exposure group categorized by 1-OH-Py level
0-3.09 nmol/mol
3.09-3.90 nmol/mol
3.90-5.53 nmol/mol
Effect measured
creatinine
creatinine
creatinine
Number of subjects
33
72
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p-value
Neurotransmitter concentrations (mean ± SD)
Norepinephrine (ng/mL)
67.31 ±67.45
36.97 ±23.58
46.75 ±35.88
0.002
Dopamine (ng/mL)
1,614.45 ± 683.57
1,482.30 ± 323.66
1,405.06 ± 332.23
0.134
Aspartate (ng/mL)
2.29 ±2.13
1.61 ±0.71
1.47 ±0.58
0.001
Glutamate (ng/mL)
11.56 ±8.92
9.93 ±4.14
9.06 ± 3.30
0.070
GABA (ng/mL)
1.40 ±3.59
1.42 ± 3.44
1.56 ±3.24
0.964
Acetylcholine (ng/mL)
334.66 ± 83.75
483.71 ±57.87
665.85 ± 94.34
0.030
AchE activity (U/mg protein)
68.17 ±9.28
54.98 ±4.23
52.64 ±4.60
0.043
Source: Niu et al. (2010).
Immunotoxicity
Zhang etal. T20121 studied 129 Chinese coke-oven workers with elevated benzo[a]pyrene
exposure and compared them against 37 referents (workers in a supply warehouse), matched by
socioeconomic status, lifestyle, and health. Area benzo[a]pyrene levels were quantified in the
various work areas, and the primary endpoint was the level of early and late apoptosis in
peripheral blood mononuclear cells (PBMCs) isolated from each worker subgroup the morning
following an overnight fast. The authors self-designed a study questionnaire to gather information
on worker education, vocational history, smoking and drinking habits, personal habits, and
personal and family medical history, as well as any current symptoms and medications used in the
previous several weeks. "Smoking" was defined as >10 cigarettes/day during the pastyear, with
"smoking index" defined as cigarettes/day x years smoking. Similarly, "drinking" was defined as
wine/beer/spirits consumed >3 times/week for the past 6 months, and "drinking index" defined as
grams of alcohol consumed/day x years drinking. Exposed workers were categorized by physical
worksite location and expected differences in benzo[a]pyrene exposure: 34 oven bottom workers,
48 oven side workers, and 47 oven top workers. Workplace environmental sampling stations were
established at each of the physical work locations, including the referent's warehouse, and dual
automatic air sampling pumps collected samples at personal breathing zone height for 6 hours/day,
over 3 consecutive days. Benzo[a]pyrene content was determined by HPLC, and relative exposure
was compared to post-shift urine levels of a benzo[a]pyrene metabolite, 1-OH-Py. Collected and
purified PBMCs were incubated with Annexin-V and PI prior to analysis by flow cytometry; early
apoptotic cells were considered to be Annexin V+/PI-, while late apoptotic cells were considered
Annexin V+/PI+.
All apoptosis data were displayed graphically, and in all groupings, early:late apoptotic
PBMCs occurred at an approximate 2:1 frequency. PBMC apoptosis was similar in each of the three
coke oven worker groups, which were all statistically significantly higher than referents
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1 (approximately 2-fold) for both early and late apoptosis. While self-reported smoking incidence
2 varied significantly among the worker groups, stratification by smoking years or smoking index did
3 not reveal any significant association with PBMC apoptosis. Multiple linear stepwise regression
4 analysis suggested that urine 1-OH-Py levels and years of coke oven operation were positively
5 associated with increased early and late PBMC apoptosis (Table D-5), and thatyears of ethanol
6 consumption was negatively associated with only early apoptosis. These associations were tested
7 by stratifying workers into three groups by urinary 1-OH-Py levels or coke oven operation years,
8 and in both cases, the groups with the highest urinary metabolite levels or longest oven operating
9 experience had statistically significantly higher levels of both early and late apoptotic PBMCs versus
10 the lowest or shortest duration groups, respectively. Likewise, when sorted into groups based
11 upon years of ethanol consumption, the highest ethanol "years of consumption" group had
12 statistically significantly lower early apoptosis rates when compared to the lowest ethanol
13 consuming group.
14 Table D-5. Background information on Chinese coke oven workers or
15 warehouse controls exposed to benzo[a]pyrene in the workplace
Effect measured
Exposure group (ng/m3; mean ± SD)
p-value
10.2 ±7.6
19.5 ± 13.2
185.9 ± 38.6
1,623.5 ± 435.8
Number of subjects
37
34
48
47
Background information (mean ± SD or %)
Age (yrs)
Working years
Smoking
Drinking
37.16 ±6.00
17.35 ±7.19
62.2
24.3
39.09 ±5.53
18.58 ±7.23
64.7
41.2
36.98 ± 6.40
16.78 ±6.90
83.3
39.6
37.34 ±6.78
17.26 ± 7.44
53.2
44.7
0.451
0.742
0.017
0.259
Urine benzo[a]pyrene metabolite (nmol/mol creatinine; mean ± SD)
1-OH-Py
2.78 ± 1.04
3.22 ±0.81*
3.51 ±0.55*
3.66 ±0.58*
0.000
16
17 *p < 0.05 significantly different from control mean.
18
19 Source: Zhang et al. (2012).
20 D.3.2. Cancer-related Endpoints
21 Benzo[a]pyrene-Induced Cytogenetic Damage
22 Many studies measure cytogenetic damage as biomarkers of early biological effects, which
23 also reflect exposure to genotoxic chemicals. Standard cytogenetic endpoints include chromosomal
24 aberration (CA), sister chromatid exchange (SCE), micronucleus (MN) formation, hypoxanthine
25 guanine phosphoribosyl transferase (hprt) mutation frequency, and glycophorin A mutation
26 frequency (Gvorffy etal.. 20081. These biomarkers are often incorporated in multi-endpoint
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studies with other biomarkers of exposure. Because they indicate related but different endpoints,
there is often a lack of correlation between the different categories of biomarkers.
Merlo etal. f!9971 evaluated DNA adduct formation (measured by [32P]-postlabelling) and
MN in white blood cells (WBCs) of 94 traffic policemen versus 52 residents from the metropolitan
area of Genoa, Italy. All study subjects wore personal air samplers for 5 hours of one work shift,
and levels of benzo[a]pyrene and other PAHs were measured. Policemen were exposed to 4.55 ng
benzo[a]pyrene/m3 air, compared with urban residents who were exposed to 0.15 ng/m3. DNA
adduct levels in policemen were 35% higher than in urban residents (p = 0.007), but MN in urban
residents were 20% higher than in policemen (p = 0.02). Linear regressions of DNA adducts and
MN incidence, respectively, versus benzo[a]pyrene exposure levels did not reveal significant
correlations.
Perera and coworkers assessed DNA damage in Finnish iron foundry workers in two
separate studies and using three methodologies. Based on results from personal sampling and
stationary monitoring in both studies, three levels of benzo[a]pyrene air concentrations were
defined: low (<5 ng/m3 benzo[a]pyrene), medium (5-12 ng/m3), and high (>12 ng/m3) (Perera et
al.. 1994: Perera etal.. 1993). In the first study, involving 48 workers, several biomarkers were
analyzed for dose-response and interindividual variability fPerera etal.. 19931. PAH-DNA adducts
were determined in WBCs using an immunoassay and enzyme-linked immunosorbent assay
(ELISA) with fluorescence detection. Mutations at the hprt locus were also measured in WBC DNA.
The latter assay is based on the fact that each cell contains only one copy of the hprt gene, which is
located on the X-chromosome. While male cells have only one X-chromosome, female cells
inactivate one of the two X-chromosomes at random. The gene is highly sensitive to mutations such
that in the event of a crucial mutation in the gene, enzyme activity disappears completely from the
cell. In addition, mutations at the glycophorin A gene locus were measured in RBCs. The
glycophorin A mutation frequency was not correlated with either benzo[a]pyrene exposure or
PAH-DNA adduct formation. However, both PAH-DNA adduct levels and hprt mutation frequency
increased with increasing benzo[a]pyrene exposure. In addition, there was a highly significant
correlation between incidence of hprt mutations and PAH-DNA adduct levels (p = 0.004).
In a second study, Perera etal. (1994) surveyed 64 iron foundry workers with assessments
conducted in 2 successive years; 24 of the workers provided blood samples in both years. Exposure
to benzo[a]pyrene, collected by personal and area sampling in the first year of the study, ranged
from <5 to 60 ng/m3 and was estimated to have decreased by 40% in the second year. The levels of
PAH-DNA adducts were roughly 50% lower in the 2nd year, presumably reflecting decreased
exposure. The longer-lived hprt mutations were not as strongly influenced by the decreasing
exposure to benzo[a]pyrene. Study subjects who did not have detectable levels of DNA adducts
were excluded from the study. As in the previous study, a strong correlation between DNA adduct
levels and incidence of hprt mutations was observed fPerera etal.. 19931.
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Kalina etal. (1998) studied several cytogenetic markers in 64 coke oven workers and
34 controls employed at other locations within the same plant. Airborne benzo[a]pyrene and seven
other carcinogenic PAHs were collected by personal air samplers, which showed ambient
benzo[a]pyrene concentrations ranging widely from 0.002 to 50 |J.g/m3 in coke oven workers and
from 0.002 to 0.063 |J.g/m3 in controls. CAs, SCEs, high-frequency cells (HFCs), and SCE
heterogeneity index were all significantly increased with benzo[a]pyrene exposure. Except for
increases in HFCs, no effect of smoking was observed. Consistent with studies of PAH-DNA adduct
formation, reduced cytogenetic response at high exposure levels produced a nonlinear dose-
response relationship. The authors also evaluated the potential influence of polymorphisms in
enzymes involved in the metabolism of benzo[a]pyrene. GSTM1 and N-acetyl transferase-2
polymorphisms were studied and no evidence of the two gene polymorphisms having any influence
on the incidence of cytogenetic damage was found.
Motykiewicz etal. (19981 conducted a similar study of genotoxicity associated with
benzo[a]pyrene exposure in 67 female residents of a highly polluted industrial urban area of Upper
Silesia, Poland, and compared the results to those obtained from 72 female residents of another
urban but less polluted area in the same province of Poland. Urinary mutagenicity and 1-OH-Py
levels, PAH-DNA adducts in oral mucosa cells (detected by immunoperoxidase staining), SCEs,
HFCs, CAs, bleomycin sensitivity, and GSTM1 and CYP1A1 polymorphisms in blood lymphocytes
were investigated. High volume air samplers and gas chromatography were used to quantify
ambient benzo[a]pyrene levels, which were 3.7 ng/m3 in the polluted area and 0.6 ng/m3 in the
control area during the summer. During winter, levels rose to 43.4 and 7.2 ng/m3 in the two areas,
respectively. The cytogenetic biomarkers (CA and SCE/HFC), urinary mutagenicity, and urinary
1-OH-Py excretion were significantly increased in females from the polluted area, and differences
appeared to be more pronounced during winter time. PAH-DNA adduct levels were significantly
increased in the study population, when compared to the controls, only in the winter season. No
difference in sensitivity to bleomycin-induced lymphocyte chromatid breaks was seen between the
two populations. As with the study by Kalina etal. (1998). genetic polymorphisms assumed to
affect the metabolic transformation of benzo[a]pyrene were not associated with any difference in
the incidence of DNA damage.
In a study of Thai school boys in urban (Bangkok) and rural areas, bulky (including but not
limited to BPDE-type) DNA adduct levels were measured in lymphocytes along with DNA single-
strand breaks (SSBs), using the comet assay, and DNA repair capacity (Tuntawiroon etal.. 2007).
Ambient air and personal breathing zone measurements indicated that Bangkok school children
experienced significantly higher exposures to benzo[a]pyrene and total PAHs. A significantly
higher level of SSBs (tail length 1.93 ± 0.09 versus 1.28 ± 0.12 |im, +51%; p < 0.001) was observed
in Bangkok school children when compared with rural children, and this parameter was
significantly associated with DNA adduct levels. A significantly reduced DNA repair capacity
(0.45 ± 0.01 versus 0.26 ± 0.01 y-radiation-induced deletions per metaphase, -42%; p < 0.001) was
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also observed in the city school children, again significantly associated with DNA adduct levels. It
was not evident why higher environmental PAH exposure would be associated with lowered DNA
repair capacity. However, because the personal breathing zone PAH levels and DNA adduct levels
were not associated with each other, it is conceivable that the city school children had a priori
lower DNA repair capacities that contributed significantly to the high adduct levels. The authors
considered genetic differences between the two study populations as a possible reason for this
observation.
D.3.3. Epidemiologic Findings in Humans
The association between human cancer and contact with PAH-containing substances, such
as soot, coal tar, and pitch, has been widely recognized since the early 1900s fBostrom etal.. 20021.
Although numerous epidemiology studies establish an unequivocal association between PAH
exposure and human cancer, defining the causative role for benzo[a]pyrene and other specific PAHs
remains a challenge. In essentially all reported studies, either the benzo[a]pyrene exposure and/or
internal dose are not known, or the benzo[a]pyrene carcinogenic effect cannot be distinguished
from the effects of other PAH and non-PAH carcinogens. Nevertheless, three types of investigations
provide support for the involvement of benzo[a]pyrene in some human cancers: molecular
epidemiology studies; population- and hospital-based, case-control studies; and occupational
cohort studies. In some cohort studies, benzo[a]pyrene exposure concentrations were measured
and thus provide a means to link exposure intensity with observed cancer rates. In case-control
studies, by their nature, benzo[a]pyrene and total PAH doses can only be estimated.
Molecular Epidemiology and Case-Control Cancer Studies
Defective DNA repair capacity leading to genomic instability and, ultimately, increased
cancer risk is well documented fWu etal.. 2007: Wu etal.. 20051. Moreover, sensitivity to mutagen-
induced DNA damage is highly heritable and thus represents an important factor that determines
individual cancer susceptibility. Based on studies comparing monozygotic and dizygotic twins, the
genetic contribution to BPDE mutagenic sensitivity was estimated to be 48.0% fWu etal.. 20071.
BPDE has been used as an etiologically relevant mutagen in case-control studies to examine the
association between elevated lung and bladder cancer risk and individual sensitivity to BPDE-
induced DNA damage. Mutagen sensitivity is determined by quantifying chromatid breaks or DNA
adducts in phytohemagglutinin-stimulated peripheral blood lymphocytes as an indirect measure of
DNA repair capacity.
In a hospital-based, case-control study involving 221 lung cancer cases and 229 healthy
controls, DNA adducts were measured in stimulated peripheral blood lymphocytes after incubation
with BPDE in vitro (Li etal.. 20011. Lung cancer cases showed consistent statistically significant
elevations in induced BPDE-DNA adducts in lymphocytes, compared with controls, regardless of
subgroup by age, sex, ethnicity, smoking history, weight loss, or family history of cancer. The
lymphocyte BPDE-induced DNA adduct levels, when grouped by quartile using the levels in controls
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as cutoff points, were significantly dose-related with lung cancer risk (ORs 1.11,1.62, and 3.23;
trend test, p < 0.001). In a related hospital-based, case-control study involving 155 lung cancer
patients and 153 healthy controls, stimulated peripheral blood lymphocytes were exposed to BPDE
in vitro fWu etal.. 20051. DNA damage/repair was evaluated in lymphocytes using the comet assay,
and impacts on cell cycle checkpoints were measured using a fluorescence-activated cell-sorting
method. The lung cancer cases exhibited significantly higher levels of BPDE-induced DNA damage
than the controls (p < 0.001), with lung cancer risk positively associated with increasing levels of
lymphocyte DNA damage when grouped in quartiles (trend test, p < 0.001). In addition, lung cancer
patients demonstrated significantly shorter cell cycle delays in response to BPDE exposure to
lymphocytes, which correlated with increased DNA damage.
Sensitivity to BPDE-induced DNA damage in bladder cancer patients supports the results
observed in lung cancer cases. In a hospital-based, case-control study involving 203 bladder cancer
patients and 198 healthy controls, BPDE-induced DNA damage was specifically evaluated at the
chromosome 9p21 locus in stimulated peripheral blood lymphocytes (Gu etal.. 2008). Deletions of
9p21, which includes critical components of cell cycle control pathways, are associated with a
variety of cancers. After adjusting for age, sex, ethnicity, and smoking status, individuals with high
BPDE-induced damage at 9p21 were significantly associated with increased bladder cancer risk
(OR 5.28; 95% CI 3.26-8.59). Categorization of patients into tertiles for BPDE sensitivity relative to
controls demonstrated a dose-related association between BPDE-induced 9p21 damage and
bladder cancer risk. Collectively, the results of molecular epidemiology studies with lung and
bladder cancer patients indicate that individuals with a defective ability to repair BPDE-DNA
adducts are at increased risk for cancer and, moreover, that specific genes linked to tumorigenesis
pathways may be molecular targets for benzo[a]pyrene and other carcinogens.
Due to the importance of the diet as a benzo[a]pyrene exposure source, several population-
and hospital-based, case-control studies have investigated the implied association between dietary
intake of benzo[a]pyrene and risk for several tumor types. In a study involving 193 pancreatic
cancer cases and 674 controls (Anderson etal.. 2005). another involving 626 pancreatic cancer
cases and 530 controls (Li etal.. 2007). and a third involving 146 colorectal adenoma cases and
228 controls (Sinha etal.. 2005). dietary intake of benzo[a]pyrene was estimated using food
frequency questionnaires. In all studies, the primary focus was on estimated intake of
benzo[a]pyrene (and other carcinogens) derived from cooked meat. Overall, cases when compared
with controls, had higher intakes of benzo[a]pyrene and other food carcinogens, leading to the
conclusion thatbenzo[a]pyrene plays a role in the etiology of these tumors in humans. In a
supportive follow-up case-control study of colorectal adenomas, levels of leukocyte PAH-DNA
adducts were significantly higher in cases when compared with controls (p = 0.02), using a method
that recognizes BPDE and several other PAHs bound to DNA (Gunter etal.. 20071.
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Cohort Cancer Studies
Epidemiologic studies of workers in PAH-related occupations indicate increased human
cancer risks associated with iron and steel production, roofing, carbon black production, and
exposure to diesel exhaust fBosetti etal.. 20071. Exposure to benzo[a]pyrene is only one of
numerous contributors to the cancer risk from complex PAH-containing mixtures that occur in the
workplace. Although some occupational cohort studies report measured or estimated inhalation
exposure concentrations for benzo[a]pyrene, none report biomarkers of internal benzo[a]pyrene
dose in study subjects (reviewed in Bosetti etal.. 2007: Armstrong etal.. 20041. Several of these
cohort studies (summarized below) demonstrate a positive exposure-response relationship with
cumulative PAH exposure using benzo[a]pyrene—or a proxy such as benzene-soluble matter (BSM)
that can be converted to benzo[a]pyrene—as an indicator substance. These studies provide insight
and support for the causative role of benzo[a]pyrene in human cancer.
Cancer incidence in aluminum and electrode production plants
Exposure to benzo[a]pyrene and BSM in aluminum smelter workers is strongly associated
with bladder cancer and weakly associated with lung cancer fBoffetta etal.. 1997: Tremblav etal..
1995: Armstrong etal.. 1994: Gibbs. 1985: Theriaultetal.. 19841. In an analysis of pooled data from
nine cohorts of aluminum production workers, 688 respiratory tract cancer cases were observed
versus 674.1 expected (pooled RR 1.03; CI 0.96-1.11) fBosetti etal.. 20071. A total of 196 bladder
cancer cases were observed in eight of the cohorts, compared with 155.7 expected (pooled RR 1.29;
CI 1.12-1.49). Based on estimated airborne benzo[a]pyrene exposures from a meta-analysis of
eight cohort studies, the predicted lung cancer RR per 100 |ig/m3-years of cumulative
benzo[a]pyrene exposure was 1.16 (95% CI 1.05-1.28) f Armstrong etal.. 20041.
Spinelli et al. (20061 reported a 14-year update to a previously published historical cohort
study fSpinelli etal.. 19911 of Canadian aluminum reduction plant workers. The results confirmed
and extended the findings from the earlier epidemiology study. The study surveyed a total of
6,423 workers with >3 years of employment at an aluminum reduction plant in British Columbia,
Canada, between the years 1954 and 1997, and evaluated all types of cancers. The focus was on
cumulative exposure to coal tar pitch volatiles, measured as BSM and as benzo[a]pyrene.
Benzo[a]pyrene exposure categories were determined from the range of predicted exposures over
time from statistical exposure models. There were 662 cancer cases, of which approximately 98%
had confirmed diagnoses. The overall cancer mortality rate (SMR 0.97; CI 0.87-1.08) and cancer
incidence rate (standardized incidence ratio [SIR] 1.00; CI 0.92-1.08) were not different from that
of the British Columbia general population. However, this study identified significantly increased
incidence rates for cancers of the bladder (SIR 1.80; CI 1.45-2.21) and stomach (SIR 1.46; CI
1.01-2.04). The lung cancer incidence rate was only slightly higher than expected (SIR 1.10; CI
0.93-1.30). Significant dose-response associations with cumulative benzo[a]pyrene exposure were
seen for bladder cancer (p < 0.001), stomach cancer (p < 0.05), lung cancer (p < 0.001), non-
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Hodgkin lymphoma (p < 0.001), and kidney cancer (p < 0.01), although the overall incidence rates
for the latter three cancer types were not significantly elevated versus the general population.
Similar cancer risk results were obtained using BSM as the exposure measure; the cumulative
benzo[a]pyrene and BSM exposures were highly correlated (r = 0.94).
In several occupational cohort studies of workers in Norwegian aluminum production
plants, personal and stationary airborne PAH measurements were performed.
In a study covering 11,103 workers and 272,554 person x years of PAH exposure, cancer
incidence was evaluated in six Norwegian aluminum smelters (Romundstad etal.. 2000a) and
(Romundstad et al.. 2000b). Reported estimates of PAH exposure concentrations reached a
maximum of 3,400 |J.g/m3 PAH (680 |J.g/m3 benzo[a]pyrene). The overall number of cancers
observed in this study did not differ significantly from control values (SIR 1.03; CI 1.0-1.1). The
data from this study showed significantly increased incidences for cancer of the bladder (SIR 1.3;
CI 1.1-1.5) and elevated, but not significant, SIRs for larynx (SIR 1.3; CI 0.8-1.9), thyroid (SIR 1.4;
CI 0.7-2.5), and multiple myeloma (SIR 1.4; CI 0.9-1.9). Incidence rates for bladder, lung, pancreas,
and kidney cancer (the latter three with SIRs close to unity) were subjected to a cumulative
exposure-response analysis. The incidence rate for bladder cancer showed a trend with increasing
cumulative exposure and with increasing lag times (up to 3 0 years) at the highest exposure level.
The incidence of both lung and bladder cancers was greatly increased in smokers. The authors
reported that using local county rates rather than national cancer incidence rates as controls
increased the SIR for lung cancer (SIR 1.4; CI 1.2-1.6) to a statistically significant level.
Cancer incidence in coke oven, coal gasification, and iron and steel foundry workers
An increased risk of death from lung and bladder cancer is reported in some studies
involving coke oven, coal gasification, and iron and steel foundry workers (Bostrom etal.. 2002:
Boffetta etal.. 19971. An especially consistent risk of lung cancer across occupations is noted when
cumulative exposure is taken into consideration (e.g., RR of 1.16 per 100 unity-years for aluminum
smelter workers, 1.17 for coke oven workers, and 1.15 for coal gasification workers). In an analysis
of pooled data from 10 cohorts of coke production workers, 762 lung cancer cases were observed
versus 512.1 expected (pooled RR 1.58; CI 1.47-1.69) (Bosetti etal.. 2007). Significant variations in
risk estimates among the studies were reported, particularly in the large cohorts (RRs of 1.1,1.2,
2.0, and 2.6). There was no evidence for increased bladder cancer risk in the coke production
workers. Based on estimated airborne benzo[a]pyrene exposures from a meta-analysis of
10 cohort studies, the predicted lung cancer RR per 100 |ig/m3-years of cumulative benzo[a]pyrene
exposure was 1.17 (95% CI 1.12-1.22) f Armstrong etal.. 20041.
A meta-analysis of data from five cohorts of gasification workers reported 251 deaths from
respiratory tract cancer, compared with 104.7 expected (pooled RR 2.58; 95% CI 2.28-2.92)
(Bosetti et al.. 2007). Pooled data from three of the cohorts indicated 18 deaths from urinary tract
cancers, versus 6.0 expected (pooled RR 3.27; 95% CI 2.06-5.19). Based on estimated airborne
benzo[a]pyrene exposures from a meta-analysis of four gas worker cohort studies, the predicted
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lung cancer RR per 100 |ig/m3-years of cumulative benzo[a]pyrene exposure was 1.15 (95% CI
1.11-1.20) (Armstrong etal.. 20041.
Increased risks were reported in iron and steel foundry workers for cancers of the
respiratory tract, bladder, and kidney. In an analysis of pooled data from 10 cohorts,
1,004 respiratory tract cancer cases were observed versus 726.0 expected (pooled RR 1.40;
CI 1.31-1.49) fBosetti etal.. 20071. A total of 99 bladder cancer cases were observed in seven of the
cohorts, compared with 83.0 expected (pooled RR 1.29; CI 1.06-1.57). For kidney cancer, 40 cases
were observed compared with 31.0 expected based on four studies (pooled RR 1.30; 95% CI
0.95-1.77).
Xu etal. T19961 conducted a nested case-control study, surveying the cancer incidence
among 196,993 active or retired workers from the Anshan Chinese iron and steel production
complex. A large number of historical benzo[a]pyrene measurements (1956-1995) were available.
The study included 610 cases of lung cancer and 292 cases of stomach cancer, with 959 age- and
gender-matched controls from the workforce. After adjusting for nonoccupational risk factors such
as smoking and diet, significantly elevated risks for lung cancer and stomach cancer were identified
for subjects employed for >15 years, with ORs varying among job categories. For either type of
cancer, highest risks were seen among coke oven workers: lung cancer, OR = 3.4 (CI 1.4-8.5) and
stomach cancer, OR = 5.4 (CI 1.8-16.0).
There were significant trends for long-term, cumulative benzo[a]pyrene exposure versus
lung cancer (p = 0.004) or stomach cancer (p = 0.016) incidence. For cumulative total
benzo[a]pyrene exposures of <0.84, 0.85-1.96,1.97-3.2, and >3.2 |ig/m3-year, the ORs for lung
cancer were 1.1 (CI 0.8-1.7), 1.6 (CI 1.2-2.3), 1.6 (1.1-2.3), and 1.8 (CI 1.2-2.5), respectively. For
cumulative total benzo[a]pyrene exposures of <0.84, 0.85-1.96,1.97-3.2, and >3.2 |ig/m3-year, the
ORs for stomach cancer were 0.9 (CI 0.5-1.5), 1.7 (CI 1.1-2.6), 1.3 (0.8-2.1), and 1.7 (CI 1.1-2.7),
respectively. However, the investigators noted that additional workplace air contaminants were
measured, which might have influenced the outcome. Of these, asbestos, silica, quartz, and iron
oxide-containing dusts may have been confounders. For lung cancers, cumulative exposures to
total dust and silica dust both showed significant dose-response trends (p = 0.001 and 0.007,
respectively), while for stomach cancer, only cumulative total dust exposure showed a marginally
significant trend (p = 0.061). For cumulative total dust exposures of <69, 69-279, 280-882, and
>883 mg/m3, the ORs for lung cancer were 1.4 (CI 1.2-1.9), 1.2 (CI 1.0-2.19), 1.4 (CI 1.0-2.0), and
1.9 (CI 1.3-2.5), respectively. For cumulative silica dust exposures of <3.7, 3.7-10.39,10.4-27.71,
and >27.72 mg/m3, the ORs for lung cancer were 1.7 (CI 1.2-2.4), 1.5 (CI 1.0-2.1), 1.5 (CI 1.0-2.1),
and 1.8 (CI 1.2-2.5), respectively. For cumulative total dust exposures of <69, 69-279, 280-882,
and >883 mg/m3, ORs for stomach cancer were 1.3 (CI 0.8-2.1), 14 (CI 0.9-2.2), 12 (CI 0.8-1.9),
and 1.6 (CI 1.1-2.5), respectively.
Exposure-response data from studies of coke oven workers in the United States have often
been used to derive quantitative risk estimates for PAH mixtures, and for benzo[a]pyrene as an
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indicator substance (Bostrom etal.. 20021. However, there are numerous studies of coke oven
worker cohorts that do notprovide estimates of benzo[a]pyrene exposure. An overview of the
results of these and other studies can be obtained from the review of Boffetta etal. f!9971.
Cancer incidence in asphalt workers and roofers
These groups encompass different types of work (asphalt paving versus roofing) and also
different types of historical exposure that have changed from using PAH-rich coal tar pitch to the
use of bitumen or asphalt, both of which are rather low in PAHs due to their source (crude oil
refinery) and a special purification process. Increased risks for lung cancer were reported in large
cohorts of asphalt workers and roofers; evidence for increased bladder cancer risk is weak
fBurstvn etal.. 2007: Partanen and Boffetta. 1994: Chiazze etal.. 1991: Hansen. 1991.1989:
Hammond etal.. 19761. In an analysis of pooled data from two cohorts of asphalt workers, 822 lung
cancer cases were observed versus 730.7 expected (pooled RR 1.14; 95% CI 1.07-1.22) (Bosetti et
al.. 20071. In two cohorts of roofers, analysis of pooled data indicated that 138 lung cancer cases
were observed, compared with 91.9 expected (pooled RR 1.51; 95% CI 1.28-1.78) (Bosetti et al..
20071.
Epidemiology of patients treated with coal tar containing ointments
In addition to cohorts of workers occupationally exposed to PAH mixtures, another source
of potential exposure to benzo[a]pyrene is through topical coal tar formulations used for the
treatment of psoriasis, eczema, and dermatitis. Epidemiological studies examining skin cancer risk
in relation to various types of topical coal tar exposure are summarized below (see Table D-6); case
reports, reviews, and studies that did not include a measure of coal tar use (e.g.. Alderson and
Clarke. 19831 are not included.
Table D-6. Studies examining skin cancer risk in relation to therapeutic coal
tar
Reference and study details
Results
General population studies
Mitropoulos and Norman (2005) (United
States, Arizona)
Case-control study (Southeastern Arizona
Health Study-2), population-based;
n = 404 squamous cell skin cancer cases,
395 controls, 1992-1996, age >30 yrs;
controls selected using random digit dialing
(frequency matched by 5-yr age group and
gender); limited to whites; details regarding
participation rates not reported
Squamous cell carcinoma (SCC), coal tar/dandruff shampoo use:
Cases n Controls ORa ORb
(%) n (%) (95% CI) (95% CI)
101(25) 73(19) 1.50(1.05,2.14) 1.28(0.85,1.9)
aAdjusted for age and gender.
bAdjusted for age, gender, actinic keratosis, current number of arm
freckles, and reaction of skin to prolonged sun.
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Reference and study details
Results
Exposure: Interview, focusing on
occupational and other sources of sun
exposure, chemical exposures, and coal
tar/dandruff shampoo
Outcome: Incident squamous cell cancer
from regional skin cancer registry
Studies of patients with skin conditions
Roelofzen et al. (2010) (Netherlands)
Cohort (retrospective); total n = 13,200
(4,315 psoriasis 8,885 eczema patients),
identified through hospital records (manual).
Diagnosed 1960-1990 (>3 visits to
dermatologist); median age 28 yrs; follow-up
through 2003 (median follow-up 21 yrs)
Exposure: Coal tar treatment (pix lithantracis
and/or liquor carbonis detergens):
8,062 (39%); duration of use obtained from
1,100 users (14%), median = 6 mo
Outcome: Skin cancer diagnosis from
national cancer registry (operating since
1989) and cause of death registries, with
some supplemental questionnaire data from
61% of the cohort
Skin cancer (excluding basal cell carcinoma); includes melanoma
and squamous cell [number of cases = 145]
HR (95% CI) for use of coal tar; referent category = only used
dermatocorticosteroids:
Psoriasis 1.08 (0.43, 2.72)
Eczema 1.06 (0.62,1.83)
Psoriasis or eczema 1.09 (0.69,1.72)
Proportional hazards models, adjusted for age (continuous),
gender, severity (>10% of body area affected), interaction term of
coal tar and severity, calendar period, psoralen + ultraviolet-A
(PUVA) systemic therapy, and smoking (current and ever versus
never). Also examined skin type, history of sun exposure, and
alcohol consumption. Smoking data imputed for 58% of the
cohort.
Jemec and 0sterlind (1994) (Denmark)
Cohort (retrospective); n = 88 patients
hospitalized for atopic dermatitis/eczema
between 1917 and 1937; mean follow-up
38.5 yrs
Exposure: Extensive treatment with coal tar
was inferred based on the knowledge that
this was the recommended treatment at the
time of the patients' hospitalization
Outcome: Incident cancer diagnosis
between January 1943 and December, 1986
as determined by national cancer registry;
comparison with general population cancer
rates
No skin cancers observed.
Authors noted that non-melanoma skin cancers may have been
underreported in older records as there was no general record for
this endpoint in the registry.
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Reference and study details
Results
Jones et al. (1985) (Scotland)
Cohort (retrospective); n = 719 psoriasis
patients not treated with cytotoxic drugs,
ionizing radiation, or UV therapy; age range
from <15 to >64 yrs
Exposure: Past intermittent treatment with
tar for a 10-yr period between 1953 and
1973 as determined by clinic records;
median age at start of therapy 27 yrs (male)
and 23 yrs (female); exposure not quantified
Outcome: Incident skin cancer diagnosis
from regional cancer registry
Expected rates calculated from cancer registry data for group of
the same size and age as patient population (not further
described).
Skin cancer with coal tar usage:
Observed Expected
Males (n = 305) 3 0.9
Females (n = 414) 0 0.7
Bhate et al. (1993) (United Kingdom)
Prevalence study within cohort of
2,247 psoriasis patients; mean age 41 yrs
Exposure: Past treatment with tar and other
therapeutics determined from medical
records; exposure not quantified and
duration not provided
Outcome: Skin cancer diagnosis obtained
from patient records and confirmed by
medical examination
Skin cancer prevalence (percentage) among psoriatic patients
treated with coal tar:
Male 9/781 (1%)
Female 21/980 (2%)
Referent group not treated with coal tar was not included.
Coal tar use in studies with combined treatment with UVB therapy (Goeckerman regimen)
Hannuksela-Svahn et al. (2000) (Finland)
Nested case-control study within cohort of
5,687 patients hospitalized with a diagnosis
of psoriasis between 1973 and 1984;
n = 30 with squamous cell carcinoma and
n = 137 sex- and age-matched referents
without skin cancer; followed until 1995
Exposure: Prior treatment with Goeckerman
regimen or its modifications determined
from hospital files; magnitude and duration
of exposure not reported
Outcome: Squamous cell carcinoma
diagnosis determined from national cancer
registry and confirmed by review of hospital
records
Relative risk (95% CI) of skin cancer with Goeckerman treatment:
Squamous cell carcinoma 1.5 (0.3-7.3)
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Reference and study details
Results
Torinuki and Tagami (1988) (Japan)
Cohort (prospective); total n = 151 psoriasis
patients including 43 treated with
Goeckerman regimen without PUVA
treatment, mean age 43 yrs; patients
treated between 1976-1986; follow-up:
5/43 Goeckerman patients followed for
>6 yrs
Exposure: Goeckerman regimen without
PUVA treatment; duration of use not
reported
Outcome: Skin cancer diagnosis from case
records
No skin cancers observed
Maughan et al. (1980) (United States, Mavo
Clinic)
Cohort (retrospective); n = 426 atopic
dermatitis or neurodermatitis patients,
treated with Goeckerman regimen between
1950-1954; follow-up: 305 (72%) followed
to approximately 1980 (25 yrs)
Exposure: Goeckerman regimen (ultraviolet-
B [UVB] + coal tar treatments) at hospital;
follow-up questionnaire inquired about
other treatment (including coal tar
treatment) after hospitalization; coal tar use
ranged from none to every day for 26 yrs
Outcome: Skin cancer diagnosis by self-
report (follow-up questionnaire) with
confirmation through histology specimens;
9 of 11 nonmelanoma skin cancers
confirmed
Eleven nonmelanoma skin cancer cases (observed) [eight basal
cell, one squamous cell, two unknown]
Expected rates from Third National Cancer Survey:
Observed/Expected Expected
Minneapolis-St Paul 6.7 1.64
San Francisco-Oakland 9.4 1.17
Iowa 5.3 2.08
Dallas-Fort Worth 18.8 0.59
No difference in duration of coal tar use after hospitalization in
skin cancer patients compared to those who did not develop skin
cancer.
Pittelkow et al. (1981) (United States, Mavo
Clinic)
Cohort (retrospective); n = 280 psoriasis
patients, hospitalized 1950-1954 at Mayo
Clinic; 260 (92%) followed to 1978 (25 yrs)
Exposure: Goeckerman regimen (UVB + coal
tar treatments) at hospital; other treatment
(including coal tar treatment) recorded from
clinical records. Median duration use
Among patients reporting coal tar therapy use:
n = 19 nonmelanoma squamous cell or basal cell (or unknown) skin
cancer cases(observed)
Expected rates from Third National Cancer Survey:
Observed/Expected Expected
Minneapolis-St Paul 18.7 1.01
San Francisco-Oakland 23.1 0.82
Iowa 15.5 1.22
Dallas-Fort Worth 49.2 0.39
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Reference and study details
Results
approximately 15 d in 1951-1955 and 21 d
in 1956-1960
Outcome: Skin cancer diagnosis by self-
report (follow-up questionnaire) with
confirmation through histology specimens;
20 of 22 confirmed
Coal tar use in studies with combined treatment ofPUVA therapy
Stern et al. (1998): Stern and Laird (1994)
(United States, 16 centers)
Cohort (prospective); total n = 1,380
psoriasis patients, enrolled between 1975
and 1976 in the PUVA cohort study; mean
age 44 yrs; follow-up at 12-15-mo intervals
through 1996 (approximately 20 years);
1,049 (91%) patients interviewed at final
follow-up
Exposure: Non-PUVA treatments (including
topical coal tar, ultraviolet B, methotrexate,
and ionizing radiation) were collected at
start of PUVA treatment and during follow-
up; coal tar use was noted to be highly
correlated with UVB therapy and thus
reported as a single parameter; 'high use'
defined as >45 mo topical tar therapy or
>300 UVB treatments
Outcome: Skin cancer diagnosis reported at
follow-up, confirmed by histopathology
From 1996 follow-up (limited to first occurrence 1986-1996):
Cancer type OR (95% CI) [n cases]
Squamous 1.4 (1.0, 2.0) [1,047]
Basal cell 1.5 (1.1, 2.0) [821]
OR compares 'high' exposure to UVB/tar to 'low' exposure to
UVB/tar, adjusted for age, sex, geographic area, anatomic site
(head and neck, other), PUVA treatments through 1985 (five
categories from <100 to >336), PUVA treatments after 1985 (>50,
<50), methotrexate (>208 wks, <208 wks), and Grenz rays or x-rays
for therapy (ever/never)
Maier et al. (1996) (Austria)
Cohort (retrospective); n = 496 psoriasis
patients with more than 5 PUVA treatments
and first treatment before 1987; median age
50 yrs; median follow-up was 82 mo
Exposure: Non-PUVA treatments (arsenic,
x-rays, tar, UVB, and methotrexate) were
determined by interview
Outcome: Skin cancer diagnosis determined
by interview or biopsy at time of follow-up
Relative risk (p-value) of skin carcinoma with coal tar usage and
more than 5 PUVA treatments (partial analysis):
Basal cell and squamous cell 3.83 (0.04)
Squamous cell 7.85(0.061)
Multivariate partial analysis considered sex, age, skin type,
cumulative UVA dose, and exposure to arsenic, x-rays, UVB, and
methotrexate.
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Reference and study details
Results
Stern et al. (1980) (United States,
16 centers)
Nested case-control study based on a study
following 1,373 PUVA-treated patients
(34 incident cases, 24 prevalent cases;
126 controls); matched by age (within 5 yrs),
sex, skin type, geographic area, and ionizing
radiation; incident cases also matched for
number of PUVA treatments; average
follow-up 2.7 yrs
Exposure: Exposure to coal tar therapy
and/or ultraviolet radiation based on follow-
up interview; includes exposures before
PUVA trial began; coal tar use quantified as
number of months in which crude coal tar
preparations was used at least weekly; high
coal tar exposure defined as >90 mo of use;
high ultraviolet radiation exposure defined
as >300 sunlamp treatments; assumption
made that coal tar and ultraviolet radiation
have the same quantitative effect on risk of
skin cancer
Outcome: Skin cancer, prevalent cases
occurred before PUVA trial started; incident
cases occurred during follow-up period
RR (95% CI) of skin cancer (skin cancer type not specified) among
high exposure (>90 mo of tar use or >300 sunlamp treatments)
Matched analysis:
All cases (n = 58) 4.7 (2.2,10.0)
Incident cases (n = 34) 5.6 (1.9,16.2)
Prevalent cases (n = 24) 3.8 (1.2,12.5)
Lindelof and Sigurgeirsson (1993) (Sweden)
Nested case-control study based on a study
following 4,799 PUVA-treated patients
(24 cases, 96 controls); matched by gender,
age, diagnosis, PUVA dose, number of
treatments, type of psoralen regimen, site of
treatment, and skin type; clinic location
matching utilized when possible; mean age
52 yrs
Exposure: Non-PUVA treatments (including
tar, topical corticosteroids, UVB, and
anthralin) collected by questionnaire;
exposure not quantified and duration not
provided
Outcome: Skin cancer diagnosis obtained
from Swedish cancer registry
SCC with coal tar usage:
Cases Controls
n (%) n (%) OR (95% CI)
17 (70) 62 (64) 1.3 (0.5,3.5)
(Similar results were seen for UVB exposure [OR 1.3, 95% CI 0.5,
3.5], reflecting the high correlation between these treatments)
1
2 The U.S. Environmental Protection Agency (EPA) noted several limitations with respect to
3 study design and analysis in this literature, precluding the ability to provide a foundation for
4 evaluating the potential association between use of therapeutic coal tar treatment (particularly
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long-term treatment) and risk of skin cancer. A primary limitation concerns the quality of the
exposure assessment Only one population-based, case-control study was identified (Mitropoulos
and Norman. 20051: this study examined self-reported use of coal tar/dandruff shampoo and
incidence of squamous cell cancer in a population in Arizona (adjusted OR 1.28, 95% CI 0.85,1.9).
This exposure measure is likely to be highly susceptible to misclassification bias. EPA considered
the likelihood of non-differential misclassification to be high; differential misclassification was also
considered to be possible, but of lower likelihood. Non-differential misclassification would arise
from lack of awareness of the content of shampoos, inability to recall use of individual shampoos,
and the lack of specificity of this particular question. Differential misclassification would arise from
differential reporting based on disease status. EPA noted similar concerns regarding exposure
quality in the nested case-control study conducted among patients receiving psoralen plus
ultraviolet-A (PUVA) treatment (in addition to a variety of other treatments, including coal tar
treatments and ultraviolet-B [UVB]) by Lindelof and Sigurgeirsson (1993). Use of coal tar was
collected through a mailed questionnaire, with no information on duration of use and no
verification with medical records. A large study of psoriasis and eczema patients
(n = 13,200 patients) by Roelofzen et al. (2010) with a 21-year follow-up period obtained data on
coal tar treatment through manual chart review; this chart review was conducted in 2003 on
medical records going back to 1960. Duration of use (median 6 months) was available for only 14%
of the patients who had an indication of use. Thus, considerable non-differential misclassification
of exposure (coal tar use) is likely, and the limited exposure data did not allow examination of
variation in exposure level. Misclassification of disease was also noted to be a limitation of this
study in that Roelofzen et al. (2010) included melanoma, in addition to squamous cell skin cancer,
which introduces a lack of specificity of outcome into the analysis as melanoma is not thought to be
associated with PAH exposure. Given these issues of exposure and disease misclassification, the
RRs from these studies do not provide a sound basis for interpretation as no risk, and would be
expected to diminish effect estimates.
Potential misclassifications of both exposure and outcome were also important limitations
of the study by Temec and Osterlind (1994). In this study, coal tar treatment was inferred (not
established based on medical records or patient recall) based on the widespread use of this
treatment between 1917 and 1937; in addition, the authors noted that nonmelanoma skin cancers
were likely underreported in the early years of the cancer registry used to identify cases (Temec and
Osterlind. 1994). While Bhate etal. (1993) used patient medical records to determine exposure
and skin cancer diagnosis, this study reported only the prevalence of skin cancer in psoriasis
patients treated with coal tar; a referent group of patients not treated with coal tar was not
included for comparison. Similarly, Tones etal. (1985) compared the skin cancer incidences in
psoriatic patients treated with coal tar with cancer rates estimated from regional cancer registry
data for a group of the same size and age as the patient population. Because the referent group did
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not consist exclusively of psoriasis patients, the influence of coal tar treatment on skin cancer risk
cannot be distinguished from the role of psoriasis in development of skin cancer.
A common regimen for treatment of psoriasis and other skin conditions combines coal tar
treatment with UVB radiation (referred to as the Goeckerman regimen). One study of this regimen
was very small (n = 43 patients) with only 5 of the patients followed for more than 6 years
fTorinuki and Tagami. 19881. Two larger Goeckerman treatment studies (280-426 patients) had a
longer follow-up period (25 years), but were limited in terms of the choice of referent groups and
differences in disease ascertainment between cases and the reference population fPittelkowetal..
1981: Maughan et al.. 1980). Specifically, dermatology patients were seen at the Mayo Clinic in
Rochester, Minnesota, but the reference rates for cancer were obtained from survey data from
Minneapolis-St Paul, San Francisco-Oakland, Iowa, and Dallas-Fort Worth. Therefore, it is unclear
whether the reference population appropriately represents the case population. In a nested case-
control study examining skin cancer and treatment with the Goeckerman regimen, disease
ascertainment was accomplished using both a national cancer registry and review of patient files
fHannuksela-Svahn etal.. 20001. However, this study is limited by potential misclassification of
exposure, because exposure information was obtained only from hospital records, so coal tar
treatment in an outpatient setting was not considered. In addition, the combination of UVB and
coal tar in the Goeckerman regimen makes it impossible to attribute risk to either individual
component. This limitation also affects the interpretation of the results of PUVA trial studies (Stern
etal.. 1998: Stern and Laird. 1994: Stern etal.. 19801 in which the analysis was conducted using a
definition of "high" exposure as >4 months of topical tar therapy or >300 UVB treatments.
Similarly, the study by Lindelof and Sigurgeirsson T19931 reported similar prevalence and risk
estimates for coal tar use and for UVB, reflecting the high correlation between these treatments.
Another study of skin cancer risk in psoriatic patients treated more than 5 times with PUVA did not
report similar risk estimates for coal tar and UVB (Maier etal.. 19961: however, both exposure to
non-PUVA treatments and skin cancer diagnosis were determined by patient recall, a method that is
susceptible to both exposure and outcome misclassification.
In summary, the available studies examining therapeutic topical coal tar use and risk of skin
cancer were limited by low-quality exposure data with high potential of exposure misclassification
fe.g.. Roelofzen etal.. 2010: Mitropoulos and Norman. 2005: Hannuksela-Svahn et al.. 2000: Maier
etal.. 1996: Temec and Osterlind. 1994: Lindelof and Sigurgeirsson. 1993): significant potential for
outcome misclassification (e.g.. Temec and Osterlind. 1994): small size (e.g., Temec and Osterlind.
1994: Torinuki andTagami. 1988): short duration of follow-up (e.g.. Torinuki andTagami. 1988):
choice of referent group (e.g., Bhate etal.. 1993: Tones etal.. 1985: Pittelkow etal.. 1981: Maughan
etal.. 1980): and/or differences in disease ascertainment between cases and the reference
population (e.g., Pittelkow etal.. 1981: Maughan et al.. 19801. In addition, clinic-based studies
focused on the commonly used regimen of coal tar in conjunction with UVB therapy cannot
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distinguish effects of coal tar from the carcinogenic effects of UVB (e.g., Hannuksela-Svahn et al..
2000: Torinuki andTagami. 1988: Pittelkowetal.. 1981: Maughan et al.. 19801. Likewise, clinic-
based studies of coal tar use among patients also treated with PUVA cannot discern the effects of
coal tar from those of PUVA (e.g.. Stern et al.. 1998: Maier etal.. 1996: Stern and Laird. 1994:
Lindelof and Sigurgeirsson. 1993: Stern etal.. 19801. Therefore, the available studies do not
provide an adequate basis for examining the potential association between coal tar treated patients
and skin cancer.
D.4. ANIMAL STUDIES
D.4.1. Oral Bioassays
Subchronic Studies
De long etal. (1999) treated male Wistar rats (eight/dose group) with benzo[a]pyrene
(98.6% purity) dissolved in soybean oil by gavage 5 days/week for 35 days at doses of 0, 3,10, 30,
or 90 mg/kg-day (adjusted doses: 0, 2.14, 7.14, 21.4, and 64.3 mg/kg-day). At the end of the
exposure period, rats were necropsied, organ weights were determined, and major organs and
tissues were prepared for histological examination (adrenals, brain, bone marrow, colon, caecum,
jejunum, heart, kidney, liver, lung, lymph nodes, esophagus, pituitary, spleen, stomach, testis, and
thymus). Blood was collected for examination of hematological endpoints, but there was no
indication that serum biochemical parameters were analyzed. Immune parameters included
determinations of serum immunoglobulin (Ig) levels (IgG, IgM, IgE, and IgA), relative spleen cell
distribution, and spontaneous cytotoxicity of spleen cell populations determined in a natural-killer
(NK) cell assay.
Body weight gain was decreased beginning at week 2 at the high dose of 90 mg/kg-day;
there was no effect at lower doses fDe long et al.. 19991. Hematology revealed a dose-related
decrease in RBC count, hemoglobin, and hematocrit at >10 mg/kg-day (Table D-7). A minimal but
significant increase in mean cell volume and a decrease in mean cell hemoglobin concentration
were noted at 90 mg/kg-day, and may indicate dose-related toxicity for the RBCs and/or RBC
precursors in the bone marrow. A decrease in WBCs, attributed to a decrease in the number of
lymphocytes (approximately 50%) and eosinophils (approximately 90%), was observed at
90 mg/kg-day; however, there was no effect on the number of neutrophils or monocytes. A
decrease in the cell number in the bone marrow observed in the 90 mg/kg-day dose group was
consistent with the observed decrease in the RBC and WBC counts at this dose level. In the
90 mg/kg-day dose group, brain, heart, kidney, and lymph node weights were decreased and liver
weight was increased (Table D-7). Decreases in heart weight at 3 mg/kg-day and in kidney weight
at 3 and 30 mg/kg-day were also observed, but these changes did not show dose-dependent
responses. Dose-related decreases in thymus weight were statistically significant at
>10 mg/kg-day (Table D-7).
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1 Table D-7. Exposure-related effects in male Wistar rats exposed to
2 benzo[a]pyrene by gavage 5 days/week for 5 weeks
Dose (mg/kg-d)
Effect
0
3
10
30
90
Hematologic effects
(mean ± SD; n = 7-8)
WBCs (109/L)
14.96 ± 1.9
13.84 ± 3.0
13.69 ± 1.8
13.58 ±2.9
8.53 ± 1.1*
RBCs (109/L)
8.7 ±0.2
8.6 ±0.2
8.3 ±0.2*
7.8 ±0.4*
7.1 ±0.4*
Hemoglobin (mmol/L)
10.5 ± 0.2
10.4 ± 0.3
9.8 ±0.2*
9.5 ±0.4*
8.6 ±0.6*
Hematocrit (L/L)
0.5 ±0.01
0.5 ±0.01
0.47 ±0.01*
0.46 ± 0.02*
0.43 ±0.02*
Serum Ig levels
(mean ± SD; n = 7-8)
IgM
100 ± 13
87 ± 16
86 ±31
67 ± 16*
81 ±26
IgG
100 ± 40
141±106
104 ± 28
106 ± 19
99 ±29
IgA
100 ± 28
73 ±29
78 ±67
72 ±22
39 ± 19*
IgE
100 ± 65
50 ±20
228 ±351
145 ±176
75 ±55
Cellularity (mean ± SD; n = 7-8)
Spleen (cell number x 107)
59 ± 15
71 ± 14
59 ± 13
63 ± 10
41 ± 10*
Bone marrow (G/L)
31 ±7
36 ±5
31 ±8
27 ±8
19 ±4*
Spleen cell distribution (%)
B cells
39± 4
36 ±2
34 ±3*
32 ±4*
23 ±4*
T cells
40 ±9
48 ± 12
40 ±9
36 ±2
44 ±6
Th cells
23 ±7
26 ±7
24 ±5
22 ±4
26 ±4
Ts cells
24 ±5
26 ±6
24 ±7
19 ±2
27 ±5
Body (g) and organ (mg) weights
(means; n = 7-8)
Body weight
305
282*
300
293
250*
Brain
1,858
1,864
1,859
1,784
1,743*
Heart
1,030
934*
1,000
967
863*
Kidney
1,986
1,761*
1,899
1,790*
1,626*
Liver
10,565
9,567
11,250
11,118
12,107*
Thymus
517 ± 47
472 ± 90
438 ± 64*
388 ± 71*
198 ±65*
Spleen
551
590
538
596
505
Mandibular lymph nodes
152
123
160
141
89*
Mesenteric lymph nodes
165
148
130*
158
107*
Popliteal lymph nodes
19
18
19
17
10*
Thymus cortex surface area
77.9 ±3.8
74.4 ± 2.2
79.2 ±5.9
75.8 ±4.0
68.9 ±5.2*
(% of total surface area of thymus;
mean ± SD; n = 6-8)
3
4 ^Significantly (p < 0.05) different from control mean. For body weight and organ weight means, SDs were only
5 reported for thymus weights.
6
7 Source: De Jong et al. (1999).
8
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Statistically significant reductions were also observed in the relative cortex surface area of
the thymus and thymic medullar weight at 90 mg/kg-day, but there was no difference in cell
proliferation between treated and control animals using the proliferating cell nuclear antigen
(PCNA) technique. Changes in the following immune parameters were noted: dose-related and
statistically significant decrease in the relative number of B cells in the spleen at 10 (13%),
30 (18%), and 90 mg/kg-day (41%); significant decreases in absolute number of cells harvested in
the spleen (31%), in the number of B cells in the spleen (61%), and NK cell activity in the spleen
(E:T ratio was 40.9 ± 28.4% that of the controls) at 90 mg/kg-day; and a decrease in serum IgM
(33%) and IgA (61%) in rats treated with 30 and 90 mg/kg-day, respectively. The decrease in the
spleen cell count was attributed by the study authors to the decreased B cells and suggested a
possible selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The study
authors considered the decrease in IgA and IgM to be due to impaired production of antibodies,
suggesting a role of thymus toxicity in the decreased (T-cell dependent) antibody production. In
addition to the effects on the thymus and spleen, histopathologic examination revealed treatment-
related lesions only in the liver and forestomach at the two highest dose levels, but the incidence
data for these lesions were not reported by De Tongetal. (1999). Increased incidence for
forestomach basal cell hyperplasia (p < 0.05 by Fisher's exact test) was reported at 30 and
90 mg/kg-day, and increased incidence for oval cell hyperplasia in the liver was reported at
90 mg/kg-day (p < 0.01, Fisher's exact test). The results indicate that 3 mg/kg-day was a no-
observed-adverse-effect level (NOAEL) for effects on hematological parameters (decreased RBC
count, hemoglobin, and hematocrit) and immune parameters (decreased thymus weight and
percent of B cells in the spleen) noted in Wistar rats at 10 mg/kg-day (the lowest-observed-
adverse-effect level [LOAEL]) and above. Lesions of the liver (oval cell hyperplasia) and
forestomach (basal cell hyperplasia) occurred at doses >30 mg/kg-day.
Knuckles etal. (2001) exposed male and female F344 rats (20/sex/dose group) to
benzo[a]pyrene (98% purity) at doses of 0, 5, 50, or 100 mg/kg-day in the diet for 90 days. Food
consumption and body weight were monitored, and the concentration of benzo[a]pyrene in the
food was adjusted every 3-4 days to maintain the target dose. The authors indicated that the actual
intake of benzo[a]pyrene by the rats was within 10% of the calculated intake, and the nominal
doses were not corrected to actual doses. Hematology and serum chemistry parameters were
evaluated. Urinalysis was also performed. Animals were examined for gross pathology, and
histopathology was performed on selected organs (stomach, liver, kidney, testes, and ovaries).
Statistically significant decreases in RBC counts and hematocrit level (decreases as much as 10 and
12%, respectively) were observed in males at doses >50 mg/kg-day and in females at
100 mg/kg-day. A maximum 12% decrease (statistically significant) in hemoglobin level was noted
in both sexes at 100 mg/kg-day. Blood chemistry analysis showed a significant increase in blood
urea nitrogen (BUN) only in high-dose (100 mg/kg-day) males. Histopathology examination
revealed an apparent increase in the incidence of abnormal tubular casts in the kidney in males at
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5 mg/kg-day (40%), 50 mg/kg-day (80%), and 100 mg/kg-day (100%), compared to 10% in the
controls. Only 10% of the females showed significant kidney tubular changes at the two high-dose
levels compared to zero animals in the female control group. The casts were described as molds of
distal nephron lumen and were considered by the study authors to be indicative of renal
dysfunction. From this study, male F344 rats appeared to be affected more severely by
benzo[a]pyrene treatment than the female rats. However, the statistical significance of the kidney
lesions is unclear. Several reporting gaps and inconsistencies regarding the reporting of kidney
abnormalities in Knuckles et al. (2001) make interpretation of the results difficult. Results of
histopathological kidney abnormalities (characterized primarily as kidney casts) were presented
graphically and the data were not presented numerically in this report No indication was given in
the graph that any groups were statistically different than controls, although visual examination of
the magnitude of response and error bars appears to indicate a 4-fold increase in kidney casts in
males compared to the control group (40 compared to 10%). The figure legend reported the data
as "percentage incidence of abnormal kidney tissues" and reported values as mean ± SD. However,
the text under the materials and methods section stated that Fisher's exact test was used for
histopathological data, which would involve the pairwise comparison of incidence and not means.
There are additional internal inconsistencies in the data presented. The data appeared to indicate
that incidences for males were as follows: control, 10%; 5 mg/kg-day, 40%; 50 mg/kg-day, 80%;
and 100 mg/kg-day, 100%; however, these incidences are inconsistent with the size of the study
groups, which were reported as 6-8 animals per group. The study authors were contacted, but did
not respond to EPA's request for clarification of study design and/or results. Due to issues of data
reporting, a LOAEL could not be established for the increased incidence of kidney lesions. Based on
the statistically significant hematological effects including decreases in RBC counts, hematocrit, and
BUN, the NOAEL in males was 5 mg/kg-day and the LOAEL was 50 mg/kg-day, based on in F344
rats. No exposure-related histological lesions were identified in the stomach, liver, testes, or
ovaries in this study.
In a range-finding study, Wistar (specific pathogen-free Riv:TOX) rats (10/sex/dose group)
were administered benzo[a]pyrene (97.7% purity) dissolved in soybean oil by gavage at dose levels
of 0,1.5, 5,15, or 50 mg/kg body weight-day, 5 days/week for 5 weeks (Kroese etal.. 2001).
Behavior, clinical symptoms, body weight, and food and water consumption were monitored. None
of the animals died during the treatment period. Animals were sacrificed 24 hours after the last
dose. Urine and blood were collected for standard urinalysis and hematology and clinical chemistry
evaluation. Liver enzyme induction was monitored based on EROD activity in plasma. Animals
were subjected to macroscopic examination, and organ weights were recorded. The esophagus,
stomach, duodenum, liver, kidneys, spleen, thymus, lung, and mammary gland (females only) from
the highest-dose and control animals were evaluated for histopathology. Intermediate-dose groups
were examined if abnormalities were observed in the higher-dose groups.
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1 A significant, but not dose-dependent, increase in food consumption in males at
2 >1.5 mg/kg-day and a decrease in food consumption in females at >5 mg/kg-day was observed
3 fKroese etal.. 20011. Water consumption was statistically significantly altered in males only: a
4 decrease at 1.5, 5, and 15 mg/kg-day and an increase at 50 mg/kg-day. Organ weights of lung,
5 spleen, kidneys, adrenals, and ovaries were not affected by treatment There was a dose-related,
6 statistically significant decrease in thymus weight in males at 15 and 50 mg/kg-day (decreased by
7 28 and 33%, respectively) and a significant decrease in thymus weight in females at 50 mg/kg-day
8 (decreased by 17%) (Table D-8). In both sexes, liver weight was statistically significantly increased
9 only at 50 mg/kg-day by about 18% (Table D-8).
10 Table D-8. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene
11 by gavage 5 days/week for 5 weeks
Organ
Dose (mg/kg-d)
0
1.5
5
15
50
Liver weight (g; mean ± SD)
Males
Females
6.10 ±0.26
4.28 ±0.11
6.19 ±0.19
4.40 ± 0.73
6.13 ±0.10
4.37 ±0.11
6.30 ±0.14
4.67 ±0.17
7.20 ±0.18*
5.03 ±0.15*
Thymus weight (mg; mean ± SD)
Males
Females
471 ± 19
326 ± 12
434 ± 20
367 ± 23
418 ± 26
351 ±25
342 ± 20*
317 ± 30
317 ±21*
271±16*
Basal cell hyperplasia of the
forestomach (incidence with slight
severity)
Males
Females
1/10
0/10
1/10
1/10
4/10
1/10
3/10
3/10*
7/10
7/10*
12
13 ^Significantly (p < 0.05) different from control mean; n = 10/sex/group.
14
15 Source: Kroese et al. (2001).
16
17 Hematological evaluation revealed only statistically nonsignificant, small, dose-related
18 decreases in hemoglobin in both sexes and RBC counts in males. Clinical chemistry analysis
19 showed a small, but statistically significant, increase in creatinine levels in males only at
20 1.5 mg/kg-day, but this effect was not dose-dependent. A dose-dependent induction of liver
21 microsomal EROD activity was observed, with a 5-fold induction at 1.5 mg/kg-day compared to
22 controls, reaching 36-fold in males at 50 mg/kg-day; the fold induction in females at the top dose
23 was less than in males. At necropsy, significant, dose-dependent macroscopic findings were not
24 observed.
25 Histopathology examination revealed a statistically significant increase in basal cell
26 hyperplasia in the forestomach of females at doses >15 mg/kg-day fKroese et al.. 2001). The
27 induction of liver microsomal EROD was not accompanied by any adverse histopathologic findings
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in the liver at the highest dose, 50 mg/kg-day, so the livers from intermediate-dose groups were,
therefore, not examined. An increased incidence of brown pigmentation of red pulp (hemosiderin)
in the thymus was observed in treated animals of both sexes. However, this tissue was not
examined in intermediate-dose groups. This range-finding, 5-week study identified a NOAEL of
5 mg/kg-day and a LOAEL of 15 mg/kg-day, based on decreased thymus weight and forestomach
hyperplasia in Wistar rats.
Kroese etal. (20011 exposed Wistar (Riv:T0X) rats (10/sex/dose group) to benzo[a]pyrene
(98.6% purity, dissolved in soybean oil) by gavage at 0, 3,10, or 30 mg/kg body weight-day,
5 days/week for 90 days. The rats were examined daily for behavior and clinical symptoms and by
palpation. Food and water consumption, body weights, morbidity, and mortality were monitored.
At the end of the exposure period, rats were subjected to macroscopic examination and organ
weights were recorded. Blood was collected for hematology and serum chemistry evaluation, and
urine was collected for urinalysis. All gross abnormalities, particularly masses and lesions
suspected of being tumors, were evaluated. The liver, stomach, esophagus, thymus, lung, spleen,
and mesenteric lymph node were examined histopathologically. In addition, cell proliferation in
forestomach epithelium was measured as the prevalence of S-phase epithelial cells displaying
bromodeoxyuridine (BrdU) incorporation.
There were no obvious effects on behavior of the animals, and no difference was observed
in survival or food consumption between exposed animals and controls fKroese etal.. 20011.
Higher water consumption and slightly lower body weights than the controls were observed in
males, but not females, at the high dose of 30 mg/kg-day. Hematological investigations showed
only nonsignificant, small dose-related decreases in RBC count and hemoglobin level in both sexes.
Clinical chemistry evaluation did not show any treatment-related group differences or dose-
response relationships for alanine aminotransferase, serum aspartate transaminase (AST), lactate
dehydrogenase (LDH), or creatinine, but a small dose-related decrease in y-glutamyl transferase
activity was observed in males only. Urinalysis revealed an increase in urine volume in males at
30 mg/kg-day, which was not dose related. At the highest dose, both sexes showed increased levels
of urinary creatinine and a dose-related increase in urinary protein. However, no further
investigation was conducted to determine the underlying mechanisms for these changes. At
necropsy, reddish to brown/gray discoloration of the mandibular lymph nodes was consistently
noted in most rats; occasional discoloration was also observed in other regional lymph nodes
(axillary). Statistically significant increases in liver weight were observed at 10 and 30 mg/kg-day
in males (15 and 29%) and at 30 mg/kg-day in females (17%). A decrease in thymus weight was
seen in both sexes at 30 mg/kg-day (17 and 33% decrease in females and males, respectively,
compared with controls) (Table D-9). At 10 mg/kg-day, thymus weight in males was decreased by
15%, but the decrease did not reach statistical significance.
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Table D-9. Means ± SDa for liver and thymus weights in Wistar rats exposed to
benzo[a]pyrene by gavage 5 days/week for 90 days
Dose (mg/kg-d)
Organ
0
3
10
30
Liver weight (g)
Males
7.49 ±0.97
8.00 ± 0.85
8.62 ± 1.30*
9.67 ± 1.17*
Females
5.54 ±0.70
5.42 ±0.76
5.76 ±0.71
6.48 ±0.78*
Thymus weight (mg)
Males
380 ± 60
380 ±110
330 ± 60
270 ± 40*
Females
320 ± 60
310 ± 50
300 ± 40
230 ± 30*
^Significantly (p < 0.05) different from control mean; student t-test (unpaired, two-tailed); n = 10/sex/group.
aReported as SE, but judged to be SD (and confirmed by study authors).
Source: Kroese et al. (2001).
Histopathologic examination revealed what was characterized by Kroese etal. (20011 as
basal cell disturbance in the epithelium of the forestomach in males (p < 0.05) and females
(p < 0.01) at 30 mg/kg-day. The basal cell disturbance was characterized by increased number of
basal cells, mitotic figures, and remnants of necrotic cells; occasional early nodule development;
infiltration by inflammatory cells (mainly histiocytes); and capillary hyperemia, often in
combination with the previous changes (Kroese etal.. 2001). Incidences for these lesions (also
described as "slight basal cell hyperplasia") in the 0, 3,10, and 30-mg/kg-day groups were 0/10,
2/10, 3/10, and 7/10, respectively, in female rats and 2/10, 0/10, 6/10, and 7/10, respectively, in
male rats. Nodular hyperplasia was noted in one animal of each sex at 30 mg/kg-day. A significant
(p <0.05) increase in proliferation of forestomach epithelial cells was detected at doses
>10 mg/kg-day by morphometric of analysis of nuclei with BrdU incorporation. The mean numbers
of BrdU-staining nuclei per unit surface area of the underlying lamina muscularis mucosa were
increased by about 2- and 3-4-fold at 10 and 30 mg/kg-day, respectively, compared with controls.
A reduction of thymus weight and increase in the incidence of thymus atrophy (the report
described the atrophy as slight, but did not specify the full severity scale used in the pathology
examination) was observed in males only at 30 mg/kg-day (p < 0.01 compared with controls).
Respective incidences for thymus atrophy for the control through high-dose groups were 0/10,
0/10, 0/10, and 3/10 for females and 0/10, 2/10,1/10, and 6/10 for males. No significant
differences were observed in the lungs of control and treated animals. In the esophagus,
degeneration and regeneration of muscle fibers and focal inflammation of the muscular wall were
judged to be a result of the gavage dosing rather than of benzo[a]pyrene treatment
The target organs of benzo[a]pyrene toxicity in this 90-day dietary study of Wistar rats
were the forestomach, thymus, and liver. The LOAEL for forestomach hyperplasia, decreased
thymus weight, and thymus atrophy was 30 mg/kg-day and the NOAEL was 10 mg/kg-day.
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Chronic Studies and Cancer Bioassays
Kroese etal. f20011 exposed Wistar (Riv:T0X) rats (52/sex/dose group) to benzo[a]pyrene
(98.6% purity) in soybean oil by gavage at nominal doses of 0, 3,10, or 30 mg/kg-day, 5 days/week,
for 104 weeks. Mean achieved dose levels were 0, 2.9, 9.6, and 29 mg/kg-day. Additional rats
(6/sex/group) were sacrificed after 4 and 5 months of exposure for analysis of DNA adduct
formation in blood and major organs and tissues. The rats were 6 weeks old at the start of
exposure. The rats were examined daily for behavior and clinical symptoms and by palpation.
Food and water consumption, body weights, morbidity, and mortality were monitored during the
study. Complete necropsy was performed on all animals that died during the course of the study,
that were found moribund, or at terminal sacrifice forgan weight measurement was not mentioned
in the report by Kroese et al.. 2001). The organs and tissues collected and prepared for microscopic
examination included brain, pituitary, heart, thyroid, salivary glands, lungs, stomach, esophagus,
duodenum, jejunum, ileum, caecum, colon, rectum, thymus, kidneys, urinary bladder, spleen, lymph
nodes, liver pancreas, adrenals, sciatic nerve, nasal cavity, femur, skin including mammary tissue,
ovaries/uterus, and testis/accessory sex glands. Some of these tissues were examined only when
gross abnormalities were detected. All gross abnormalities, particularly masses and lesions that
appeared to be tumors, were also examined.
At 104 weeks, survival in the control group was 65% (males) and 50% (females), whereas
mortality in the 30 mg/kg-day dose group was 100% after about week 70. At 80 weeks, survival
percentages were about 90, 85, and 75% in female rats in the 0, 3, and 10 mg/kg-day groups,
respectively; in males, respective survival percentages were ~95, 90, and 85% at 80 weeks.
Survival of 50% of animals occurred at 104,104, ~90, and 60 weeks for control through high-dose
females; for males, the respective times associated with 65% survival were 104,104,104, and
~60 weeks. The high mortality rate in high-dose rats was attributed to liver or forestomach tumor
development, not to noncancer systemic effects. After 20 weeks, body weight was decreased
(compared with controls by >10%) in 30-mg/kg-day males, but not in females. This decrease was
accompanied by a decrease in food consumption. Body weights and food consumption were not
adversely affected in the other dose groups compared to controls. In males, there was a dose-
dependent increase in water consumption starting at week 13, but benzo[a]pyrene treatment had
no significant effects on water consumption in females.
Tumors were detected at significantly elevated incidences at several tissue sites in female
and male rats at doses >10 and >3 mg/kg-day, respectively (Table D-10) (Kroese etal.. 2001). The
tissue sites with the highest incidences of tumors were the liver (hepatocellular adenoma and
carcinoma) and forestomach (squamous cell papilloma and carcinoma) in both sexes (Table D-10).
The first liver tumors were detected in week 35 in high-dose male rats. Liver tumors were
described as complex, with a considerable proportion (59/150 tumors) metastasizing to the lungs.
At the highest dose level, 95% of rats with liver tumors had malignant carcinomas (95/100;
Table D-10). Forestomach tumors were associated with the basal cell proliferation observed
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(without diffuse hyperplasia) in the forestomach of rats in the preliminary range-finding and
90-day exposure studies. At the highest dose level, 59% of rats with forestomach tumors had
malignant carcinomas (60/102; Table D-10). Other tissue sites with significantly elevated
incidences of tumors in the 30 mg/kg-day dose group included the oral cavity (papilloma and
squamous cell carcinoma [SCC]) in both sexes, and the jejunum (adenocarcinoma), kidney (cortical
adenoma), and skin (basal cell adenoma and carcinoma) in male rats (Table D-10). In addition,
auditory canal tumors (carcinoma or squamous cell papilloma originating from pilo-sebaceous
units including the Zymbal's gland) were also detected in both sexes at 30 mg/kg-day, but auditory
canal tissue was not histologically examined in the lower dose groups and the controls
(Table D-10). Gross examination revealed auditory canal tumors only in the high-dose group.
Table D-10. Incidences of exposure-related neoplasms in Wistar rats treated
by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks
Dose (mg/kg-d)
0
3
10
30a
Site
Females'3
Oral cavity
Papilloma
0/19
0/21
0/9
9/31*
SCC
1/19
0/21
0/9
9/31*
Basal cell adenoma
0/19
0/21
1/9
4/31
Sebaceous cell carcinoma
0/19
0/21
0/9
1/31
Esophagus
Sarcoma undifferentiated
0/52
0/52
2/52
0/52
Rhabdomyosarcoma
0/52
1/52
4/52
0/52
Fibrosarcoma
0/52
0/52
3/52
0/52
Forestomach
Squamous cell papilloma
1/52
3/51
20/51*
25/52*
SCC
0/52
3/51
10/51*
25/52*
Liver
Hepatocellular adenoma
0/52
2/52
7/52*
1/52
Hepatocellular carcinoma
0/52
0/52
32/52*
50/52*
Cholangiocarcinoma
0/52
0/52
1/52
0/52
Anaplastic carcinoma
0/52
0/52
1/52
0/52
Auditory canal
Benign tumor
0/0
0/0
0/0
1/20
Squamous cell papilloma
0/0
0/1
0/0
1/20
Ca rcinoma
0/0
0/1
0/0
13/20*
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Dose (mg/kg-d)
0
3
10
30a
Site
Males'3
Oral cavity
Papilloma
0/24
0/24
2/37
10/38*
see
1/24
0/24
5/37
11/38*
Basal cell adenoma
0/24
0/24
0/37
2/38
Sebaceous cell carcinoma
0/24
0/24
0/37
2/38
Forestomach
Squamous cell papilloma
0/52
7/52*
18/52*
17/52*
see
0/52
1/52
25/52*
35/52*
Jejunum
Adenocarcinoma
0/51
0/50
1/51
8/49*
Liver
Hepatocellular adenoma
0/52
3/52
15/52*
4/52
Hepatocellular carcinoma
0/52
1/52
23/52*
45/52*
Cholangiocarcinoma
0/52
0/52
0/52
1/52
Kidney
Cortical adenoma
0/52
0/52
7/52*
8/52*
Adenocarcinoma
0/52
0/52
2/52
0/52
Urothelial carcinoma
0/52
0/52
0/52
3/52
Auditory canal
Benign
0/1
0/0
1/7
0/33
Squamous cell papilloma
0/1
0/0
0/7
4/33
Ca rcinoma
0/1
0/0
2/7
19/33*
Sebaceous cell adenoma
0/1
0/0
0/7
1/33
Skin and mammary
Basal cell adenoma
2/52
0/52
1/52
10/51*
Basal cell carcinoma
1/52
1/52
0/52
4/51
see
0/52
1/52
1/52
5/51
Keratoacanthoma
1/52
0/52
1/52
4/51
Trichoepithelioma
0/52
1/52
2/52
8/51*
Fibrosarcoma
0/52
3/52
5/52
0/51
Fibrous histiocytoma (malignant)
0/52
0/52
1/52
1/52
^Statistically significant difference (p < 0.01), Fisher's exact test; analysis of auditory canal tumor incidence was
based on assumption of n = 52 and no tumors in the controls.
aThis group had significantly decreased survival.
incidences are for number of rats with tumors compared with number of tissues examined histologically.
Auditory canal and oral cavity tissues were only examined histologically when abnormalities were observed upon
macroscopic examination.
Source: Kroese et al. (2001).
Kroese etal. (20011 did not systematically investigate nonneoplastic lesions detected in rats
sacrificed during the 2-year study because the focus was to identify and quantitate tumor
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occurrence. However, incidences were reported for nonneoplastic lesions in tissues or organs in
which tumors were detected (i.e., oral cavity, esophagus, forestomach, jejunum, liver, kidney, skin,
mammary, and auditory canal). The reported nonneoplastic lesions associated with exposure were
the forestomach basal cell hyperplasia and clear cell foci of cellular alteration in the liver.
Incidences for forestomach basal cell hyperplasia in the control through high-dose groups were
1/52, 8/51,13/51, and 2/52 for females and 2/50, 8/52, 8/52, and 0/52 for males. Incidences for
hepatic clear cell foci of cellular alteration were 22/52, 33/52, 4/52, and 2/52 for females and
8/52, 22/52,1/52, and 1/52 for males. These results indicate that the lowest dose group,
3 mg/kg-day, was a LOAEL for increased incidence of forestomach hyperplasia and hepatic
histological changes in male and female Wistar rats exposed by gavage to benzo[a]pyrene for up to
104 weeks (see Table D-10). The lack of an increase in incidence of these nonneoplastic lesions in
the forestomach and liver at the intermediate and high doses (compared with controls) was
associated with increased incidences of forestomach and liver tumors at these dose levels. The
authors of this study noted that nonneoplastic effects were not quantified in organs with tumors.
As an adjunct study to the 2-year gavage study with Wistar rats, Kroese etal. (2001)
sacrificed additional rats (6/sex/group) after 4 and 5 months of exposure (0,1, 3,10, or
30 mg/kg-day) for analysis of DNA adduct formation in WBCs and major organs and tissues.
Additional rats (6/sex/time period) were exposed to 0.1 mg/kg-day benzo[a]pyrene for 4 and
5 months for analysis of DNA adduct formation. Using the [32P]-postlabeling technique, five
benzo[a]pyrene-DNA adducts were identified in all of the examined tissues at 4 months (WBCs,
liver, kidney, heart, lung, skin, forestomach, glandular stomach, brain). Only one of these adducts
(adduct 2) was identified based on co-chromatography with a standard. This adduct, identified as
10p-(deoxyguanosin-N2-yl)-7p,8a,9a-trihydroxy-7,8,9,10 tetrahydro-benzo[a]pyrene, was the
predominant adduct in all organs of female rats exposed to 10 mg/kg-day, except the liver and
kidney, in which another adduct (unidentified adduct 4) was predominant Levels of total adducts
(number of benzo[a]pyrene-DNA adducts per 1010 nucleotides) in examined tissues (from the
single 10 mg/kg-day female rat) showed the following order: liver > heart > kidney > lung > skin >
forestomach * WBCs > brain. Mean values for female levels of total benzo[a]pyrene-DNA adducts
(number per 1010 nucleotides) in four organs showed the same order, regardless of exposure
group: liver > lung > forestomach * WBCs; comparable data for males were not reported. Mean
total benzo[a]pyrene-DNA adduct levels in livers increased in both sexes from about 100 adducts
per 1010 nucleotides at 0.1 mg/kg-day to about 70,000 adducts per 1010 nucleotides at
30 mg/kg-day. In summary, these results suggest that total benzo[a]pyrene-DNA adduct levels in
tissues at 4 months were not independently associated with the carcinogenic responses noted after
2 years of exposure to benzo[a]pyrene. The liver showed the highest total DNA adduct levels and a
carcinogenic response, but total DNA adduct levels in heart, kidney, and lung (in which no
carcinogenic responses were detected) were higher than levels in forestomach and skin (in which
carcinogenic responses were detected).
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Groups of Sprague-Dawley rats (32/sex/dose) were fed diets delivering a daily dose of
0.15 mg benzo[a]pyrene/kg body weight every ninth day or 5 times/week (Brune etal.. 19811.
Other groups (32/sex/dose) were given gavage doses of 0.15 mg benzo[a]pyrene (in aqueous 1.5%
caffeine solution)/kg every ninth day, every third day, or 5 times/week. The study included an
untreated control group (to compare with the dietary exposed groups) and a gavage vehicle control
group (each with 32 rats/sex). Rats were treated until moribundity or death occurred, with
average annual doses reported in Table D-ll [mg/kg-year, calculated by Brune etal. f 19811]. The
following tissues were prepared for histopathological examination: tongue, larynx, lung, heart,
trachea, esophagus, stomach, small intestine, colon, rectum, spleen, liver, urinary bladder, kidney,
adrenal gland, and any tissues showing tumors or other gross changes. Survival was similar among
the groups, with the exception that the highest gavage-exposure group showed a decreased median
time of survival (Table D-ll). Significantly increased incidences of portal-of-entry tumors
(forestomach, esophagus, and larynx) were observed in all of the gavage-exposed groups and in the
highest dietary exposure group (Table D-ll). Following dietary administration, all observed
tumors were papillomas. Following gavage administration, two malignant forestomach tumors
were found (one each in the mid- and high-dose groups) and the remaining tumors were benign.
The data in Table D-ll show that the carcinogenic response to benzo[a]pyrene was stronger with
the gavage protocol compared with dietary exposure, and that no distinct difference in response
was apparent between the sexes. Tumors at distant sites (mammary gland, kidney, pancreas, lung,
urinary bladder, testes, hematopoietic, and soft tissue) were not considered treatment-related as
they were also observed at similar rates in the control group (data not provided). The study report
did not address noncancer systemic effects.
Table D-ll. Incidences of alimentary tract tumors in Sprague-Dawley rats
chronically exposed to benzo[a]pyrene in the diet or by gavage in caffeine
solution
Average annual
dose (mg/kg-yr)
Estimated average
daily dosea
(mg/kg-d)
Forestomach tumorsb
Total alimentary tract
tumors0 (larynx,
esophagus,
forestomach)
Median
survival time
(wks)
Benzo[a]pyrene by gavage in 1.5% caffeine solution
0
0
3/64 (4.7%)
6/64 (9.4%)
102
6
0.016
12/64 (18.8%)*
13/64 (20.3%)
112
18
0.049
26/64 (40.1%)**
26/64 (40.6%)
113
39
0.107
14/64 (21.9%)**
14/64 (21.9%)
87
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Average annual
dose (mg/kg-yr)
Estimated average
daily dosea
(mg/kg-d)
Forestomach tumorsb
Total alimentary tract
tumors0 (larynx,
esophagus,
forestomach)
Median
survival time
(wks)
Benzo[a]pyrene in diet
0
0
2/64 (3.1%)
3/64 (4.7%)
129
6
0.016
1/64 (1.6%)
3/64 (4.7%)
128
39
0.107
9/64 (14.1%)*
10/64 (15.6%)
131
^Significantly (p < 0.1) different from control using a modified %2test that accounted for group differences in
survival time.
**Significantly (p < 0.05) different from control using a modified %2 test that accounted for group differences in
survival time.
aAverage annual dose divided by 365 days.
bNo sex-specific forestomach tumor incidence data were reported by Brune et al. (1981).
°Sex-specific incidences for total alimentary tract tumors were reported as follows:
Gavage (control, high dose): Male: 6/32, 7/32,15/32, 8/32
Female: 0/32, 6/32,11/32, 6/32
Diet (control, high dose): Male: 3/32,3/32,8/32
Female: 0/32, 0/32, 2/32
Source: Brune et al. (1981).
In the other modern cancer bioassay with benzo[a]pyrene, female B6C3Fi mice (48/dose
group) were administered benzo[a]pyrene (98.5% purity) at concentrations of 0 (acetone vehicle),
5, 25, or 100 ppm in the diet for 2 years (Beland and Culp. 1998: Culp etal.. 1998). This study was
designed to compare the carcinogenicity of coal tar mixtures with that of benzo[a]pyrene and it
included groups of mice fed diets containing one of several concentrations of two coal tar mixtures.
Benzo[a]pyrene was dissolved in acetone before mixing with the feed. Control mice received only
acetone-treated feed. Female mice were chosen because they have a lower background incidence of
lung tumors than male B6C3Fi mice. Culp etal. (1998) reported that the average daily intakes of
benzo[a]pyrene in the 25- and 100-ppm groups were 104 and 430 ng/day, but did not report the
intake for the 5-ppm group. Based on the assumption that daily benzo[a]pyrene intake at 5 ppm
was one-fifth of the 2 5-ppm intake (about 21 |ig/day), average daily doses for the three
benzo[a]pyrene groups are estimated as 0.7, 3.3, and 16.5 mg/kg-day. Estimated doses were
calculated using time-weighted average (TWA) body weights of 0.032 kg for the control, 5- and
25-ppm groups and 0.026 kg for the 100-ppm group (estimated from graphically presented data).
Food consumption, body weights, morbidity, and mortality were monitored at intervals, and lung,
kidneys, and liver were weighed at sacrifice. Necropsy was performed on all mice that died during
the experiment or survived to the end of the study period. Limited histopathologic examinations
(liver, lung, small intestine, stomach, tongue, esophagus) were performed on all control and high-
dose mice and on all mice that died during the experimental period, regardless of treatment group.
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In addition, all gross lesions found in mice of the low- and mid-dose groups were examined
histopathologically.
None of the mice administered 100 ppm benzo[a]pyrene survived to the end of the study,
and morbidity/mortality was 100% by week 78. Decreased survival was also observed at 25 ppm
with only 27% survival at 104 weeks, compared with 56 and 60%, in the 5-ppm and control groups,
respectively. In the mid- and high-dose groups, 60% of mice were alive at about 90 and 60 weeks,
respectively. Early deaths in exposed mice were attributed to tumor formation rather than other
causes of systemic toxicity. Food consumption was not statistically different in benzo[a]pyrene-
exposed and control mice. Body weights of mice fed 100 ppm were similar to those of the other
treated and control groups up to week 46, and after approximately 52 weeks, body weights were
reduced in 100-ppm mice compared with controls. Body weights for the 5- and 25-ppm groups
were similar to controls throughout the treatment period. Compared with the control group, no
differences in liver, kidney, or lung weights were evident in any of the treated groups (other organ
weights were not measured).
Papillomas and/or carcinomas of the forestomach, esophagus, tongue, and larynx at
elevated incidences occurred in groups of mice exposed to 25 or 100 ppm, but no exposure-related
tumors occurred in the liver or lung fBeland and Culp. 1998: Culp etal.. 19981. The forestomach
was the most sensitive tissue, demonstrated the highest tumor incidence among the examined
tissues, and was the only tissue with an elevated incidence of tumors at 25 ppm (Table D-12). In
addition, most of the forestomach tumors in the exposed groups were carcinomas, as 1, 31, and
45 mice had forestomach carcinomas in the 5-, 25-, and 100-ppm groups, respectively.
Nonneoplastic lesions were also found in the forestomach at significantly (p < 0.05) elevated
incidences: hyperplasia at >25 ppm and hyperkeratosis at >25 ppm (Table D-12). The esophagus
was the only other examined tissue showing elevated incidence of a nonneoplastic lesion (basal cell
hyperplasia, see Table D-12). Tumors (papillomas and carcinomas) were also significantly elevated
in the esophagus and tongue at 100 ppm (Table D-12). Esophageal carcinomas were detected in
1 mouse at 25 ppm and 11 mice at 100 ppm. Tongue carcinomas were detected in seven 100-ppm
mice; the remaining tongue tumors were papillomas. Although incidences of tumors of the larynx
were not significantly elevated in any of the exposed groups, a significant dose-related trend was
apparent (Table D-12).
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1 Table D-12. Incidence of nonneoplastic and neoplastic lesions in female
2 B6C3Fi mice fed benzo[a]pyrene in the diet for up to 2 years
Tissue and lesion
Incidence (%)
Benzo[a]pyrene concentration (ppm) in diet
0
5
25
100
Average daily doses (mg/kg-d)
0
0.7
3.3
16.5
Liver (hepatocellular adenoma)
2/48 (2)
7/48 (15)
5/47 (11)
0/45 (0)
Lung (alveolar/bronchiolar adenoma and/or carcinoma)
5/48 (10)
0/48 (0)
4/45 (9)
0/48 (0)
Forestomach (papilloma and/or carcinoma)
l/48a (2)
3/47 (6)
36/46* (78)
46/47* (98)
Forestomach (hyperplasia)
13/48a (27)
23/47 (49)
33/46* (72)
37/47* (79)
Forestomach (hyperkeratosis)
13/48a (27)
22/47 (47)
33/46* (72)
38/47* (81)
Esophagus (papilloma and/or carcinoma)
0/48a (0)
0/48 (0)
2/45 (0)
27/46* (59)
Esophagus (basal cell hyperplasia)
l/48a (2)
0/48 (0)
5/45 (11)
30/46* (65)
Tongue (papilloma and/or carcinoma)
0/49a (0)
0/48 (0)
2/46 (4)
23/48* (48)
Larynx (papilloma and/or carcinoma)
0/35a (0)
0/35 (0)
3/34 (9)
5/38 (13)
3
4 ^Significantly different from control incidence (p < 0.05); using a modified Bonferonni procedure for multiple
5 comparisons to the same control.
6 Significant (p < 0.05) dose-related trend calculated for incidences of these lesions.
7
8 Sources: Beland and Culp (1998): Culp et al. (1998).
9
10 Neal and Rigdon (19671 fed benzo[a]pyrene (purity not reported) at concentrations of 0,1,
11 10, 20, 30, 40, 45, 50,100, and 250 ppm to male and female CFW-Swiss mice in the diet.
12 Corresponding doses (in mg/kg-day) were calculated1 as 0, 0.2,1.8, 3.6, 5.3, 7.1, 8, 8.9,17.8, and
13 44.4 mg/kg-day. The age of the mice ranged from 17 to 180 days old and the treatment time was
14 from 1 to 197 days; the size of the treated groups ranged from 9 to 73. There were 289 mice
15 (number of mice/sex not stated) in the control group. No forestomach tumors were reported at 0,
16 0.2, or 1.8 mg/kg-day. The incidences of forestomach tumors at 20, 30, 40, 45, 50,100, and
17 250 ppm dose groups (3.6, 5.3, 7.1, 8, 8.9,17.8, and 44.4 mg/kg-day) were 1/23, 0/37,1/40, 4/40,
18 23/34,19/23, and 66/73, respectively.
'Calculation: mg/kg-day = (ppm in feed x kg food/day)/kg body weight. Reference food consumption rates of
0.0062 kg/day (males) and 0.0056 kg/day (females) and reference body weights of 0.0356 kg (males) and
0.0305 kg (females) were used by the U.S. EPA (1988) and resulting doses were averaged between males and
females.
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1 Other Oral Exposure Cancer Bioassays in Mice
2 Numerous other oral exposure cancer bioassays in mice have limitations that restrict their
3 usefulness for characterizing dose-response relationships between chronic-duration oral exposure
4 to benzo[a]pyrene and noncancer effects or cancer, but collectively, they provide strong evidence
5 that oral exposure to benzo[a]pyrene can cause portal-of-entry site tumors (see Table D-13 for
6 references).
7 Table D-13. Other oral exposure cancer bioassays in mice
Species/strain
Exposure
Results
Comments
Reference
Rat/Sprague-
Groups of rats (32/sex/dose)
Larynx, esophagus, and
Doses are
Brune et al.
Dawley
were fed diets delivering a
forestomach tumors
annual
(1981)
daily dose of 0.15 mg
averages.
benzo[a]pyrene/kg body
Dose
Nonstandard
weight every 9th d or
(gavage)
treatment
5 times/wk (Brune et al..
0
0.016
0.049
0.107
6/64
13/64
26/64
14/64
protocol
1981). Other groups
involved
(32/sex/dose) were given
animals being
gavage doses of 0.15 mg
treated for
benzo[a]pyrene (in aqueous
<5 d/wk;
1.5% caffeine solution)/kg
Dose
(diet)
0
0.016
0.107
relatively high
every 9th d, every 3rd d, or
control
5 times/wk.
3/64
3/64
10/64
incidence
compared to
other gavage
studies.
Mouse/HalCR
Groups of 12-20 mice
Incidence with
Less-than-
Wattenberg
(10 wks old) were fed
forestomach tumors:
lifetime
(1972)
benzo[a]pyrene in the diet
Low, 11/20 (18 wks)
exposure
(0.1, 0.3, or 1.0 mg/g diet) for
Mid, 13/19 (20 wks)
duration; only
12-20 wks. Estimated doses
High, 12/12 (12 wks)
stomachs were
were 14.3, 42.0, or
examined for
192 mg/kg-d.
tumors; tumors
found only in
forestomach.
Mouse/HalCR
Groups of nine mice (9 wks
Incidence with
Less-than-
Triolo et al.
old) were fed benzo[a]pyrene
forestomach tumors:
lifetime
(1977)
in the diet (0, 0.2, or 0.3 mg/g
Control, 0/9
exposure
diet) for 12 wks and
Low, 6/9
duration;
sacrificed. Estimated doses
High, 9/9
glandular
were 0, 27.3, or 41 mg/kg-d.
stomach, lung,
and livers from
control and
exposed mice
showed no
tumors.
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Species/strain
Exposure
Results
Comments
Reference
Mouse/HalCR
20 mice (9 wks old) were
given benzo[a]pyrene in the
diet (0.3 mg
benzo[a]pyrene/g diet) for
6 wks and sacrificed after
20 wks in the study.
8/20 exposed mice had
forestomach tumors
Less-than-
lifetime
exposure
duration; only
stomachs were
examined for
tumors; tumors
found only in
forestomach; no
nonexposed
controls were
mentioned.
Wattenberg
(1974)
Mouse/CD-I
20 female mice (9 wks old)
were given 1 mg
benzo[a]pyrene by gavage
2 times/wk for 4 wks and
observed for 19 wks.
Estimated dose was
33 mg/kg-d, using an average
body weight of 0.030 kg from
reported data.
Incidence with
forestomach tumors:
Exposed, 17/20 (85%)
Controls, 0/24
Less-than-
lifetime
exposure
duration; only
stomach were
examined for
tumors; tumors
found only in
forestomach.
El-Bavoumv
(1985)
Mouse/BALB
25 mice (8 wks old) were
given 0.5 mg benzo[a]pyrene
2 times/wk for 15 wks.
5/25 mice had squamous
carcinomas of the
forestomach; tumors were
detected 28-65 wks after
treatment
Less-than-
lifetime
exposure
duration; the
following details
were not
reported:
inclusion of
controls,
methods for
detecting
tumors, and
body weight
data.
Biancifiori et
al. (1967)
Mouse/C3H
19 mice (about 3 mo old)
were given 0.3 mL of 0.5%
benzo[a]pyrene in
polyethylene glycol-400 by
gavage, once/d for 3 d.
By 30 wks, 7/10 mice had
papillomas; no carcinomas
were evident
Less-than-
lifetime
exposure
duration.
Berenblum
and Haran
(1955)
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Species/strain
Exposure
Results
Comments
Reference
Mouse/albino
Groups of 17-18 mice were
Incidence of mice (that
Less-than-
Field and Roe
given single doses of
survived at least to 60 d)
lifetime
(1965)
benzo[a]pyrene and allowed
with forestomach
exposure
to survive until terminal
papillomas:
duration; Gl
sacrifice at 569 d.
Incidence
(Experiment 1) Dose (ng)
(Experiment 2)
Control 0/17
0/18
12.5 3/17
2/18
50 0/17
1/17
200 8/17
Not evaluated
tract examined
for tumors with
hand lens; body
weight data not
reported.
Mouse/albino
Groups of about 160 female
Gastric tumors were
Close to lifetime
Chouroulinkov
mice (70 d of age; strain
observed at the following
exposure
etal. (1967)
unknown) were given 0 or
incidence:
duration; daily
8 mg benzo[a]pyrene mixed
Control, 0/158
dose levels and
in the diet over a period of
8 mg benzo[a]pyrene total,
methods of
14 mo.
13/160
detecting
tumors were
not clearly
reported.
Mouse/CFW
Groups of mice (mixed sex)
Fore-
Less-than-
Neal and
were fed benzo[a]pyrene in
stomach
lifetime
Rigdon (1967)
the diet (dissolved in benzene
Exposure tumor
exposure
and mixed with diet) at 0, 1,
ppm (d) incidence
duration; no
10, 20, 30, 40, 45, 50, 100, or
1 110 0/25
10 110 0/24
20 110 1/23
vehicle control
250 ppm in the diet.
group; animals
ranged from
3 wks to 6 mo
old at the start
of dosing; only
alimentary tract
was examined
for tumors.
30 110 0/37
40 110 1/40
45 110 4/40
50 152 24/34
100 110 19/23
250 118 66/73
Mouse/Swiss
Groups of mice (9-14 wks
Forestomach tumor
Less-than-
Roe et al.
albino
old) were given single doses
incidence:
lifetime
(1970)
of 0 or 0.05 mg
Carcinoma
Dose (ng) papilloma
0 0/65
2/65
50 1/61
20/61
duration of
benzo[a]pyrene in
polyethylene glycol-400 by
exposure;
exposure-
gavage. Surviving mice were
related tumors
killed at 18 mo of age and
only found in
examined for macroscopic
tumors.
forestomach.
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Species/strain
Exposure
Results
Comments
Reference
Mouse/ICR
Groups of 20 or 24 mice (71 d
old) were given 1.5 mg
benzo[a]pyrene by gavage
2 times/wk for 4 wks;
terminal sacrifice was at
211 d of age. Estimated dose
was about 50 mg
benzo[a]pyrene/kg, using an
average body weight of
0.03 kg during exposure from
reported data.
Incidence of mice with
forestomach neoplasms
Experiment 1, 23/24
Experiment 2,19/20
Less-than-
lifetime
duration of
exposure; only
stomachs were
examined for
tumors; tumors
found only in
forestomach;
nonexposed
controls were
not mentioned.
Beniamin et al.
(1988)
Mouse/white
Groups of 16-30 mice were
given benzo[a]pyrene in
triethylene glycol
(0.001-10 mg) weekly for
10 wks and observed until
19 mo.
Tumors in stomach antrum
Carcinoma
Dose (mg) papilloma
0.001 0/16
0/16
0.01 0/26
2/26
0.1 0/24
5/24
1.0 11/30
12/30
10 16/27
7/27
Less-than-
lifetime
exposure
duration.
Fedorenko and
Yansheva
(1967); as
cited in U.S.
EPA (1991a)
Mouse/A/HeJ
12 female mice (9 wks old)
were given standard diet for
25 d, and 3 mg
benzo[a]pyrene by gastric
intubation on d 7 and 21 of
the study. Mice were killed
at 31 wks of age and
examined for lung tumors.
12/12 exposed mice had
lung tumors
Less-than-
lifetime
exposure
duration; only
lungs examined
for tumors; no
nonexposed
controls were
mentioned.
Wattenberg
(1974)
Mouse/A/J
Groups of female mice were
fed benzo[a]pyrene in the
diet at 0,16, or 98 ppm for
260 d. Average intakes of
benzo[a]pyrene were 0, 40.6,
and 256.6 ng/mouse/d.
Estimated doses were 0,1.6,
and 9.9 mg/kg-d using a
chronic reference body
weight value of 0.026 kg (U.S.
EPA, 1988).
Incidence of mice surviving
to 260 d:
Lung tumors
Control, 4/21
16 ppm, 9/25
98 ppm, 14/27
Forestomach tumors
Control, 0/21
16 ppm, 5/25
98 ppm, 27/27
Close to lifetime
exposure
duration; A/J
strain of mice
particularly
sensitive to
chemically
induced cancer;
only lungs and
stomachs were
examined for
tumors.
Wevand et al.
(1995)
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Species/strain
Exposure
Results
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Mouse/A/J
Groups 40 female mice (8
wks old) were given 0 or
0.25 mg benzo[a]pyrene (in
2% emulphor) by gavage
3 times/wk for 8 wks. Mice
were killed at 9 mo of age
and examined for lung or
forestomach tumors.
Incidence for mice
surviving at 9 mo of age:
Lung tumors
Control, 11/38
Exposed, 22/36
Forestomach tumors
Control, 0/38
Exposed, 33/36
Less-than-
lifetime
duration of
exposure; only
lungs and Gl
tract were
examined for
tumors.
Robinson et al.
(1987)
D.4.2. Inhalation Studies
Short-Term andSubchronicStudies
Wolff etal. (19891 exposed groups of 40 male and 40 female F344/Crl rats, via nose only, to
7.5 mgbenzo[a]pyrene/m3 for 2 hours/day, 5 days/week for 4 weeks (corresponding to a TWA of
0.45 mg/m3]. Rats were 10-11 weeks old atthe beginning of the experiment. Benzo[a]pyrene
(>98% pure) aerosols were formed by heating and then condensing the vaporized benzo[a]pyrene.
The particle mass median aerodynamic diameter (MMAD) was 0.21 |im. Subgroups of these
animals (six/sex/dose) were exposed for 4 days or 6 months after the end of the 4-week exposure
to radiolabeled aluminosilicate particles. Lung injury was assessed by analyzing clearance of
radiolabeled aluminosilicate particles and via histopathologic evaluations. Body and lung weights,
measured in subgroups from 1 day to 12 months after the exposure did not differ between controls
and treated animals. Radiolabeled particle clearance did not differ between the control and treated
groups, and there were no significant lung lesions. This study identified a NOAEL for lung effects of
0.45 mg/m3 for a short-term exposure.
Chronic Studies and Cancer Bioassays
Thvssen etal. (19811 conducted an inhalation study in which male Syrian golden hamsters
were exposed to benzo[a]pyrene for their natural lifetime. Groups of 24 animals (8 weeks old)
were exposed by nose-only inhalation to NaCl aerosols (controls; 240 |ig NaCl/m3) or
benzo[a]pyrene condensed onto NaCl aerosols at three target concentrations of 2,10, or 50 mg
benzo[a]pyrene/m3 for 3-4.5 hours/day, 5 days/week for 1-41 weeks, followed by 3 hours/day,
7 days/week for the remainder of study (until hamsters died or became moribund). Thvssen et al.
(19811 reported average measured benzo[a]pyrene concentrations to be 0, 2.2, 9.5, or 46.5 mg/m3.
More than 99% of the particles were between 0.2 and 0.5 |im in diameter, and over 80% had
diameters between 0.2 and 0.3 |im. The particle analysis of the aerosols was not reported to
modern standards (MMAD and geometric SD were not reported). Final overall group sizes were
larger as animals dying during the first 12 months of the study were replaced.
Review of the individual animal data (including individual animal pathology reports, time-
to-death data, and exposure chamber monitoring data) provided by Thyssen et al. to EPA (U.S. EPA.
1990a) revealed several discrepancies in the reported exposure protocol. The actual exposure
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protocol was as follows: 4.5 hours/day, 5 days/week on weeks 1-12; 3 hours/day, 5 days/week on
weeks 13-29; 3.7 hours/day, 5 days/week on week 30; 3 hours/day, 5 days/week on weeks 31-41;
and 3 hours/day, 7 days/week for the reminder of the experiment
Analytical chamber monitoring data were generally recorded about once or twice per week,
with some exceptions ranging from no measurements for a 3-week period to as many as five
measurements in 1 week. Individual measurements (in mg/m3) were 0.2-4.52,1.16-19.2, and
0.96-118.6 in the 2,10, and 50 mg/m3 target concentration groups, respectively. Overall, weekly
average exposure concentrations varied 2-5-fold from the overall average for each group over the
course of the study, with no particular trends over time (data not shown). The 95% confidence
limits for the average exposure level over time in each group varied within 4-7% of the averages.
Because some animals were started at different times and the exposure protocol changed over time,
each individual animal had an exposure history somewhat different than others in the same
exposure group. In order to address this variability, U.S. EPA (1990a) used the individual animal
data and the chamber monitoring data to calculate a lifetime average continuous exposure for each
individual hamster. Group averages of these individual TWA concentrations were 0, 0.25,1.01, and
4.29 mg/m3 for the control through high-exposure groups.
Statistical analysis of outcomes was not reported by Thvssenetal. fl9811. Survival was
similar in the control, low-, and mid-exposure groups, but was decreased about 40% in the high-
exposure group. Average survival times in the control, low-, mid-, and high-exposure groups were
96.4 ± 27.6, 95.2 ± 29.1, 96.4 ± 27.8, and 59.5 ± 15.2 weeks, respectively. After the 60th week, body
weights decreased and mortality increased steeply in the highest exposure group. Histologic
examination of organs2 revealed an exposure-related increase in the mid- and high-exposure
groups of benign and malignant tumors of the upper respiratory tract, including the nasal cavity,
larynx, and trachea, and of the upper digestive tract, including the pharynx, esophagus, and
forestomach (Table D-14). No lung tumors were observed. Tumors were detected in other sites,
but none of these appeared to be related to exposure.
2Thvssen et al. f 19811 did not report a complete list of organs examined histologically. The individual animal
pathology reports documented examination of brain, pituitary, eyes, salivary gland, larynx, pharynx, thyroid,
trachea, esophagus, thymus, heart lung, stomach, liver, spleen, pancreas, duodenum, jejunum and ileum,
cecum, colon and rectum, kidneys, adrenals, bladder, testicle, epididymides, prostate, submandibular and
mesenterial lymph nodes, aorta, sternum, bone, and muscle.
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1 Table D-14. Tumor incidence in the respiratory tract and upper digestive
2 tract for male Syrian golden hamsters exposed to benzo[a]pyrene via
3 inhalation for lifetime—Thvssen et al. (1981)a
Target exposure
concentration
and (lifetime
average
continuous
exposure)15,
mg/m3
Papillomas, polyps, papillary
polyps, or carcinomas (total malignant tumors)
Respiratory tract
Upper digestive tract
Incidence of
pharynx or
respiratory
tract tumors0
Larynx
Trachea
Nasal
cavity
Pharynx
Esophagus
Forestomach
0
0/23d
0/24
0/23
0/21
0/24
0/24
0/2 le
2 (0.25)
0/19
0/20
0/20
0/18
0/20
0/20
0/18
10 (1.01)
11/23 (8)f
2/23 (0)
4/23 (1)
9/19 (7)
0/23 (0)
1/23 (1)
17/22 (ll)f
50 (4.29)
11/23 (8)
3/23 (1)
1/23 (0)
18/22 (17)
2/23 (0)
2/23 (0)
18/22 (17)
4
5 aHistopathology incidence data from the raw data obtained from the Thyssen study (Clement Associates, 1990),
6 adjusted to show animals only on study long enough to be at risk of tumor development: at least 1 year (0, 2, or
7 10 mg/m3 groups) or until the first tumor occurrence (week 40 in the 50 mg/m3 group). See Table E-30 for a list of
8 all animals with histopathology results.
9 bSee text.
10 Excludes animals with unexamined tissues, unless a tumor was diagnosed in the tissues that were examined.
11 fractions represent the number of animals diagnosed with at least one of the specified tumors, among the
12 animals examined for each tissue.
13 Statistically significant trends by Cochran-Armitage trend test, conducted by EPA: all tumors: p < 0.0001,
14 malignant tumors only: p < 0.0001.
15 'Includes one animal with an in situ carcinoma in the larynx.
16
17 The tumor types observed in the upper respiratory and upper digestive tract were very
18 similar, characterized as polyps, papillomas, papillary polyps, and squamous carcinomas, with the
19 exceptions of one in situ carcinoma and one adenocarcinoma (both in the mid-exposure group),
20 reflecting similar cell types. Consequently, evaluation of the overall cancer hazard included
21 consideration of the joint incidence of these tumor types. The pharynx and larynx (including the
22 epiglottis), clearly the main cancer targets, can be difficult to distinguish given their close proximity.
23 There were a few instances of nasal cavity or trachea tumors among animals without larynx or
24 pharynx tumors. Tumors of the upper digestive tract may have been a consequence of mucociliary
25 particle clearance (Thyssen etal.. 1981). but the tumors in the esophagus and forestomach
26 observed in the mid- and high-exposure groups all occurred in animals that also had pharynx or
27 respiratory tract tumors. Overall, there were increasing trends in tumor incidence with increasing
28 exposure, both for the combined incidence of benign or malignant tumors, or for only malignant
29 tumors (Table D-14), and earlier occurrence of tumors with increasing exposure levels. Several
30 studies have investigated the carcinogenicity of benzo[a]pyrene in hamsters exposed by
31 intratracheal instillation. Single-dose studies verified that benzo[a]pyrene is tumorigenic, but do
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not provide data useful for characterizing dose-response relationships because of their design
(Kobavashi. 1975: Renzik-Schiiller and Mohr. 1974: Henry etal.. 1973: Mohr. 1971: Saffiotti etal..
1968: Gross etal.. 1965: Herrold and Dunham. 19621. One multiple-dose study, which utilized very
low doses (0.005, 0.02, and 0.04 mg once every 2 weeks), failed to find any tumorigenic response
fKunstler. 19831. Tumorigenic responses (mostly in the respiratory tract) were found at higher
dosage levels (0.25-2 mgbenzo[a]pyrene once per week for 30-52 weeks) in four multiple-dose
studies (Feronand Kruvsse. 1978: Ketkar etal.. 1978: Feronetal.. 1973: Saffiotti etal.. 19721.
These studies identify the respiratory tract as a cancer target with exposure to benzo[a]pyrene by
intratracheal instillation and provide supporting evidence for the carcinogenicity of
benzo[a]pyrene at portal-of-entry sites.
D.4.3. Dermal studies
Skin-Tumor Initiation-Promotion Assays
Results from numerous studies indicate that acute dermal exposure to benzo[a]pyrene
induces skin tumors in mice when followed by repeated exposure to a potent tumor promoter
(Wevand etal.. 1992: Cavalieri etal.. 1991: Rice etal.. 1985: El-Bavoumvetal.. 1982: LaVoie etal..
1982: Raveh etal.. 1982: Cavalieri etal.. 1981: Slaga etal.. 1980: Wood etal.. 1980: Slaga etal..
1978: Hoffmann etal.. 19721. The typical exposure protocol in these studies involved the
application of a single dose of benzo[a]pyrene (typically >20 nmol per mouse) to dorsal skin of mice
followed by repeated exposure to a potent tumor promoter, such as 12-O-tetradecanoylphorbol-
13-acetate (TPA).
Carcinogenicity Bioassays
Repeated application of benzo[a]pyrene to skin (in the absence of exogenous promoters)
has been variously demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs
flARC. 2010: IPCS. 1998: ATSDR. 1995: IARC. 1983.19731. Mice have been most extensively
studied, presumably because of early evidence that they may be more sensitive than other animal
species, but comprehensive comparison of species differences in sensitivity to lifetime dermal
exposure are not available. Early studies of complete dermal carcinogenicity in other species (rats,
hamsters, guinea pigs, and rabbits) have several limitations that make them not useful for dose-
response analysis [see IARC T19731 for descriptions of studies]. The limitations in these studies
include inadequate reporting of the amount of benzo[a]pyrene applied, use of the carcinogen
benzene as a vehicle, and less-than-lifetime exposure duration.
This section discusses complete carcinogenicity bioassays in mice that provide the best
available dose-response data for skin tumors caused by repeated dermal exposure to
benzo[a]pyrene (Sivak etal.. 1997: Higginbotham etal.. 1993: Albert etal.. 1991: Grimmer etal..
1984: Habs etal.. 1984: Grimmer etal.. 1983: Habs etal.. 1980: Schmahl etal.. 1977: Schmidt etal..
1973: Roe etal.. 1970: Poel. 1963.19591. Early studies of benzo[a]pyrene complete carcinogenicity
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in mouse skin (Wvnder and Hoffmann. 1959: Wvnder et al.. 19571 are not further described herein,
because the investigators applied solutions of benzo[a]pyrene at varying concentrations on the
skin, but did not report volumes applied. As such, applied doses in these studies cannot be
determined. Other complete carcinogenicity mouse skin tumor bioassays with benzo[a]pyrene are
available, but these are not described further in this review, because: (1) they only included one
benzo[a]pyrene dose level fe.g.. Emmett et al.. 19811 or only dose levels inducing 90-100%
incidence of mice with tumors (e.g., Wilson and Holland. 1988: Warshawskv and Barklev. 19871 and
thus provide no information about the shape of the dose-response relationship; (2) they used a
1-time/week (e.g.. Nesnow et al.. 19831 or 1-time every 2 weeks (e.g.. Levin et al.. 19771 exposure
protocol, which is less useful for extrapolating to daily human exposure; or (3) they used a vehicle
demonstrated to interact with or enhance benzo[a]pyrene carcinogenicity (Bingham and Falk.
19691.
Poel (19591 applied benzo[a]pyrene in toluene to shaved interscapular skin of groups of
13-56 male C57L mice at doses of 0, 0.15, 0.38, 0.75, 3.8,19, 94,188, 376, or 752 |ig, 3 times/week
for up to 103 weeks or until the appearance of a tumor by gross examination (3 times weekly).
Some organs (not further specified) and interscapular skin in sacrificed mice were examined
histologically. With increasing dose level, the incidence of mice with skin tumors increased and the
time oftumor appearance decreased (see Table D-15). Doses >3.8 [igwere associated with 100%
mortality after increasingly shorter exposure periods, none greater than 44 weeks. Poel T19591 did
not mention the appearance of exposure-related tumors in tissues other than interscapular skin.
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1 Table D-15. Skin tumor incidence and time of appearance in male C57L mice
2 dermally exposed to benzo[a]pyrene for up to 103 weeks
Dose (pg)a
Incidence of mice with
gross skin tumors
Time o first tumor
appearance (wks)
Incidence of mice
with epidermoid
carcinoma13
Length of exposure
period (wks)
0 (toluene)
0/33 (0%)
-
0/33 (0%)
92
0.15
5/55 (9%)
42-44°
0/55 (0%)
98
0.38
11/55 (20%)
24
2/55 (4%)
103
0.75
7/56(13%)
36
4/56 (7%)
94
3.8
41/49 (84%)
21-25
32/49 (65%)
82
19
38/38(100%)
11-21
37/38 (97%)
25-44°
94
35/35(100%)
8-19
35/35 (100%)
22-43
188
12/14 (86%)
9-18
10/14 (71%)
20-35
376
14/14(100%)
4-15
12/14 (86%)
19-35
752
13/13(100%)
5-13
13/13 (100%)
19-30
3
4 indicated doses were applied to interscapular skin 3 times/week for up to 103 weeks or until time of appearance
5 of a grossly detected skin tumor.
6 bCarcinomas were histologically confirmed.
7 cRanges reflect differing information in Tables 4 and 6 of Poel (1959).
8
9 Source: Poel (1959).
10
11 Poel (19631 applied benzo[a]pyrene in a toluene vehicle to shaved interscapular skin of
12 groups of 14-25 male SWR, C3HeE>, or A/He mice 3 times/week at doses of 0, 0.15, 0.38, 0.75, 3.8,
13 19.0, 94.0, or 470 |igbenzo[a]pyrene per application, until mice died or a skin tumor was observed.
14 Time ranges for tumor observations were provided, but not times of death for mice without tumors,
15 so it was not possible to evaluate differential mortality among all dose groups or the length of
16 exposure for mice without tumors. With increasing dose level, the incidence of mice with skin
17 tumors increased and the time oftumor appearance decreased (Table D-16). The lowestdose level
18 did not induce an increased incidence of mice with skin tumors in any strain, but strain differences
19 in susceptibility were evident at higher dose levels. SWR and C3HeB mice showed skin tumors at
20 doses >0.38 |igbenzo[a]pyrene, whereas AH/e mice showed tumors at doses >19 |ig
21 benzo[a]pyrene (Table D-16). Except for metastases of the skin tumors to lymph nodes and lung,
22 Poel (19631 did not mention the appearance of exposure-related tumors in tissues other than
23 interscapular skin.
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1 Table D-16. Skin tumor incidence and time of appearance in male SWR,
2 C3HeB, and A/He mice dermally exposed to benzo[a]pyrene for life or until a
3 skin tumor was detected
Dose (pg)a
SWR Mice
C3HeB Mice
A/He Mice
Tumor
incidence13
Time of
tumor
appearance
(wks)
Tumor
incidence13
Time of
tumor
appearance
(wks)
Tumor
incidence13
Time of
tumor
appearance
(wks)
0 (toluene)
0/20 (0%)
-
0/17 (0%)
-
0/17 (0%)
-
0.15
0/25 (0%)
-
0/19 (0%)
-
0/18 (0%)
-
0.38
2/22 (9%)
55
3/17 (18%)
81-93
0/19 (0%)
-
0.75
15/18 (83%)
25-72
4/17 (24%)
51-93
0/17 (0%)
-
3.8
12/17 (70%)
25-51
11/18(61%)
35-73
0/17 (0%)
-
19.0
16/16 (100%)
12-28
17/17 (100%)
13-32
21/23 (91%)
21-40
94.0
16/17 (94%)
9-17
18/18 (100%)
10-22
11/16 (69%)
14-31
470.0
14/14(100%)
5-11
17/17 (100%)
4-19
17/17(100%)
4-21
4
5 indicated doses were applied 3 times/week for life or until a skin tumor was detected. Mice were 10-14 weeks
6 old at initial exposure.
7 incidence of mice exposed >10 weeks with a skin tumor.
8
9 Source: Poel (1963).
10
11 Roe etal. fl9701 treated groups of 50 female Swiss mice with 0 (acetone vehicle), 0.1, 0.3,1,
12 3, or 9 ng benzo[a]pyrene applied to the shaved dorsal skin 3 times/week for up to 93 weeks; all
13 surviving mice were killed and examined for tumors during the following 3 weeks. The dorsal skin
14 of an additional control group was shaved periodically but was not treated with the vehicle. Mice
15 were examined every 2 weeks for the development of skin tumors at the site of application.
16 Histologic examinations included: (1) all skin tumors thought to be possibly malignant; (2) lesions
17 of other tissues thought to be neoplastic; and (3) limited nonneoplastic lesions in other tissues. As
18 shown in Table D-17, markedly elevated incidences of mice with skin tumors were only found in
19 the two highest dose groups (3 and 9 |ig), compared with no skin tumors in the control groups.
20 Malignant skin tumors (defined as tumors with invasion or penetration of the panniculus carnosus
21 muscle) were detected in 4/41 and 31/40 mice in the 3- and 9-|ig groups, respectively, surviving to
22 at least 300 days. Malignant lymphomas were detected in all groups, but the numbers of cases were
23 not elevated compared with expected numbers after adjustment for survival differences. Lung
24 tumors were likewise detected in control and exposed groups at incidences that were not
25 statistically different
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1 Table D-17. Tumor incidence in female Swiss mice dermally exposed to
2 benzo[a]pyrene for up to 93 weeks
Dose (ng)a
Cumulative number of mice with skin
tumor/survivors
Skin tumor
incidence13
Malignant
lymphoma
incidence0
Lung tumor
incidence0
200 d
300 d
400 d
500 d
600 d
700 d
No treatment
0/48
0/43
0/40
0/31
0/21
0/0
0/43 (0%)
19/44 (43%)
12/41 (29%)
Acetone
0/49
0/47
0/45
0/37
0/23
0/0
0/47 (0%)
12/47 (26%)
10/46 (22%)
0.1
0/45
1/42
1/35
1/31
1/22
1/0
1/42 (2%)
11/43 (26%)
10/40 (25%)
0.3
0/46
0/42
0/37
0/30
0/19
0/0
0/42 (0%)
10/43 (23%)
13/43 (30%)
1
0/48
0/43
0/37
1/30
1/18
1/0
1/43 (2%)
16/44 (36%)
15/43 (35%)
3
0/47
0/41
1/37
7/35
8/24
8/0
8/41 (20%)
23/42 (55%)
12/40 (30%)
9
0/46
4/40
21/32
28/21
33/8
34/0
34/46 (74%)
9/40 (23%)
5/40 (13%)
3
4 aDoses were applied 3 times/week for up to 93 weeks to shaved dorsal skin.
5 bNumerator: number of mice detected with a skin tumor. Denominator: number of mice surviving to 300 days for
6 all groups except the highest dose group. For the highest dose group (in which skin tumors were first detected
7 between 200 and 300 days), the number of mice surviving to 200 days was used as the denominator.
8 Numerator: number of mice detected with specified tumor. Denominator: number of mice surviving to 300 days
9 unless a tumor was detected earlier, in which case, the number dying before 300 days without a tumor was
10 subtracted from the number of animals reported to have been examined.
11
12 Source: Roe et al. (1970).
13
14 Schmidt etal. T19731 dermally administered benzo[a]pyrene in acetone to female NMRI
15 mice (100/group) and female Swiss mice. Benzo[a]pyrene was applied to the shaved dorsal skin
16 twice weekly at doses of 0, 0.05, 0.2, 0.8, or 2 |ig until spontaneous death occurred or until an
17 advanced carcinoma was observed. Skin carcinomas were identified by the presence of crater-
18 shaped ulcerations, infiltrative growth, and the beginning of physical wasting (i.e., cachexia).
19 Necropsy was performed for all animals, and histopathological examination of the dermal site of
20 application and any other tissues with gross abnormalities was conducted. Skin tumors were
21 observed at the two highest doses in both strains of female mice (see Table D-18), with induction
22 periods of 53.0 and 75.8 weeks for the 0.8 and 2.0 |ig NMRI mice and 57.8 and 60.7 weeks for the
23 Swiss mice, respectively. The authors indicated that the latency period for tumor formation was
24 highly variable, and significant differences among exposure groups could not be identified, but no
25 further timing information was available, including overall survival. Carcinoma was the primary
26 tumor type seen after lifetime application of benzo[a]pyrene to mouse skin.
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1 Table D-18. Skin tumor incidence in female NMRI and Swiss mice dermally
2 exposed to benzo[a]pyrene
Dose (pg)a
Skin tumor incidence (all
types)
Incidence of papilloma
Incidence of carcinoma
Female NMRI mice
0 (acetone)
0/100 (0%)
0/100 (0%)
0/100 (0%)
0.05
0/100 (0%)
0/100 (0%)
0/100 (0%)
0.2
0/100 (0%)
0/100 (0%)
0/100 (0%)
0.8
2/100 (2%)
0/100 (0%)
2/100 (2%)
2
30/100 (30%)
2/100 (2%)
28/100 (28%)
Female Swiss mice
0 (acetone)
0/80 (0%)
0/80 (0%)
0/80 (0%)
0.05
0/80 (0%)
0/80 (0%)
0/80 (0%)
0.2
0/80 (0%)
0/80 (0%)
0/80 (0%)
0.8
5/80 (6%)
0/80 (0%)
5/80 (6%)
2
45/80 (56%)
3/80 (4%)
42/80 (52%)
3
4 aMice were exposed until natural death or until they developed a carcinoma at the site of application; indicated
5 doses were applied 2 times/week to shaved skin of the back.
6
7 Source: Schmidt et al. (1973).
8
9 Schmahl et al. (19771 applied benzo[a]pyrene 2 times/week to the shaved dorsal skin of
10 female NMRI mice (100/group) at doses of 0,1,1.7, or 3 |ig in 20 |j.L acetone. The authors reported
11 that animals were observed until natural death or until they developed a carcinoma at the site of
12 application. The effective numbers of animals at risk was about 80% of the nominal group sizes,
13 which the authors attributed to autolysis; no information was provided concerning when tumors
14 appeared in the relevant groups, how long treatment lasted in each group, or any times of death.
15 Necropsy was performed on all mice and the skin of the back, as well as any organs that exhibited
16 macroscopic changes, were examined histopathologically. The incidence of all types of skin tumors
17 was increased in a dose-related manner compared to controls (see Table D-19). Carcinoma was the
18 primary tumor type observed following chronic dermal exposure to benzo[a]pyrene, and skin
19 papillomas occurred infrequently. Dermal sarcoma was not observed.
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1 Table D-19. Skin tumor incidence in female NMRI mice dermally exposed to
2 benzo[a]pyrene
Dose (pg)a
Skin tumor incidence (all types)
Incidence of papilloma
Incidence of carcinoma
0
1/81 (l%)b
0/81 (0%)
0/81 (0%)
l
11/77 (14%)
1/77 (1%)
10/77 (13%)
1.7
25/88 (28%)
0/88 (0%)
25/88(28%)
3
45/81 (56%)
2/81 (3%)
43/81 (53%)
3
4 aMice were exposed until natural death or until they developed a carcinoma at the site of application; indicated
5 doses were applied 2 times/week to shaved skin of the back.
6 bSarcoma.
7
8 Source: Schmahl et al. (1977).
9
10 Habs etal. f 19801 applied benzo[a]pyrene to the shaved interscapular skin of female NMRI
11 mice (40/group) at doses of 0,1.7, 2.8, or 4.6 |ig in 20 |j.L acetone twice weekly, from 10 weeks of
12 age until natural death or gross observation of infiltrative tumor growth. Latency of tumors, either
13 as time of first appearance or as average time of appearance of tumors, was not reported. Necropsy
14 was performed on all animals, and the dorsal skin, as well as any organs showing gross alterations
15 at autopsy, was prepared for histopathological examination. Age-standardized mortality rates,
16 using the total population of the experiment as the standard population, were used to adjust tumor
17 incidence findings in the study. Benzo[a]pyrene application was associated with a statistically
18 significant increase in the incidence of skin tumors at each dose level (see Table D-20).
19 Table D-20. Skin tumor incidence in female NMRI mice dermally exposed to
20 benzo[a]pyrene
Dose (pg)a
Skin tumor incidence
Age-standardized tumor incidence13
0 (acetone)
0/35 (0%)
0%
1.7
8/34 (24%)
24.8%
2.8
24/35(68%)
89.3%
4.6
22/36 (61%)
91.7%
21
22 aMice were exposed until natural death or until they developed a carcinoma at the site of application; indicated
23 doses were applied 2 times/week to shaved skin of the back.
24 bMortality data of the total study population were used to derive the age-standardized tumor incidence.
25
26 Source: Habs et al. (1980).
27
28 Grimmer etal. T19841 and Grimmer etal. f 19831 applied benzo[a]pyrene (in 0.1 mL of a
29 1:3 solution of acetone:dimethyl sulfoxide [DMSO]) to the interscapular skin of female CFLP mice
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1 (65-80/group) 2 times/week for 104 weeks. Doses were 0, 3.9, 7.7, and 15.4 |ig in the 1983
2 experiment, and 0, 3.4, 6.7, and 13.5 |ig in the 1984 experiment. Mice were observed until
3 spontaneous death, unless an advanced tumor was observed or if animals were found moribund.
4 Survival information was not provided; incidences reflect the number of animals placed on study.
5 Necropsy was performed on all mice. Histopathological examination of the skin and any other
6 organ showing gross abnormalities was performed. Chronic dermal exposure to benzo[a]pyrene
7 produced a dose-related increase in skin tumor incidence and a decrease in tumor latency (see
8 Table D-21). Carcinoma was the primary tumor type observed and a dose-response relationship
9 was evident for carcinoma formation and incidence of all types of skin tumors.
10 Table D-21. Skin tumor incidence and time of appearance in female CFLP mice
11 dermally exposed to benzo[a]pyrene for 104 weeks
Dose (pg)a
Skin tumor incidence
(all types)
Incidence of
papilloma
Incidence of
carcinoma
Tumor appearance
(Wks)
Grimmer et al. 11983)
0 (1:3 Solution of
acetone:DMSO)
0/80 (0%)
0/80 (0%)
0/80 (0%)
-
3.9
22/65 (34%)
7/65 (11%)
15/65 (23%)
74.6 ± 16.78b
7.7
39/64 (61%)
5/64 (8%)
34/64 (53%)
60.9 ± 13.90
15.4
56/64 (88%)
2/64 (3%)
54/64 (84%)
44.1 ±7.66
Grimmer et al. 11984)
0 (1:3 Solution of
acetone:DMSO)
0/65 (0%)
0/65 (0%)
0/65 (0%)
-
3.4
43/64 (67%)
6/64 (9%)
37/64 (58%)
61 (53—65)c
6.7
53/65 (82%)
8/65 (12%)
45/65 (69%)
47 (43-50)
13.5
57/65 (88%)
4/65 (6%)
53/65 (82%)
35 (32-36)
12
13 indicated doses were applied twice/week to shaved skin of the back.
14 bMean±SD.
15 cMedian with 95% CI.
16
17 Sources: Grimmer et al. (1984) and Grimmer et al. (1983).
18
19 Habs etal. f19841 applied benzo[a]pyrene (in 0.01 mL acetone) to the shaved interscapular
20 skin of female NMRI mice at doses of 0, 2, or 4 [ig, 2 times/week for life. Animals were observed
21 twice daily until spontaneous death, unless an invasive tumor was observed. All animals were
22 necropsied and histopathological examination was performed on the dorsal skin and any other
23 organ with gross abnormalities. Chronic dermal exposure to benzo[a]pyrene did not affect body
24 weight gain, but appeared to reduce survival at the highest dose with mean survival times of 691,
25 648, and 528 days for the 0, 2, and 4 ng/day groups, respectively. The total length of exposure for
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1 each group was not reported, but can be inferred from the survival data. Latency also was not
2 reported. Benzo[a]pyrene application resulted in a dose-related increase the incidence of total skin
3 tumors and skin carcinomas (see Table D-22). Hematopoietic tumors (at 6/20, 3/20, and 3/20)
4 and lung adenomas (at 2/20,1/20, and 0/20) were observed in the controls and in the
5 benzo[a]pyrene treatment groups, but did not appear to be treatment related according to the
6 study authors.
7 Table D-22. Skin tumor incidence in female NMRI mice dermally exposed to
8 benzo [a] pyrene for life
Dose (pg)a
Skin tumor
incidence (all
types)
Incidence of
papilloma
Incidence of
carcinoma
Mean survival
time, days (95% CI)
0 (Acetone)
0/20 (0%)
0/20 (0%)
0/20 (0%)
691 (600-763)
2
9/20 (45%)
2/20(10%)
7/20 (35%)
648 (440-729)
4
17/20 (85%)
0/20 (0%)
17/20 (85%)
528 (480-555)
9
10 aMice were exposed until natural death or until they developed an invasive tumor at the site of application;
11 indicated doses were applied 2 times/week to shaved interscapular skin.
12
13 Source: Habs et al. (1984).
14
15 Groups of 23-27 female Ah-receptor-responsive Swiss mice were treated on a shaved area
16 of dorsal skin with 0,1, 4, or 8 nmol (0, 0.25,1, or 2 ng/treatment) benzo [a]pyrene (>99% pure) in
17 acetone 2 times weekly for 40 weeks (Higginbotham etal.. 1993). Surviving animals were
18 sacrificed 8 weeks later. Complete necropsies were performed, and tissues from the treated area,
19 lung, liver, kidney, spleen, urinary bladder, ovary, and uterus were harvested for histopathologic
20 examination. Histopathologic examination was performed on tissues from the treated area, lungs,
21 liver, kidneys, spleen, urinary bladder, uterus, and ovaries, as well as any other grossly abnormal
22 tissue. Lung adenomas occurred in each group (1/27, 2/24,1/23,1/23), and other tumors were
23 noted in isolated mice (i.e., malignant lymphoma [spleen] in one low-dose and one mid-dose mouse;
24 malignant lymphoma with middle organ involvement in one high-dose mouse; and hemangioma
25 [liver] in one mid-dose mouse) and were not considered dose related. In addition, benzo[a]pyrene
26 showed no skin tumors under the conditions of this bioassay.
27 Sivak etal. (1997) designed a study to compare the carcinogenicity of condensed asphalt
28 fumes (including benzo[a]pyrene and other PAHs) with several doses of benzo [a]pyrene alone. For
29 the purposes of this assessment, the exposure groups exposed to PAH mixtures are not discussed.
30 Groups of 30 male C3H/HeJ mice were treated dermally twice/week to 0, 0.0001, 0.001, or 0.01%
31 (0, 0.05, 0.5, or 5 |ig) benzo[a]pyrene in a 50 [J.L volume of cyclohexanone/acetone (1:1) for
32 104 weeks beginning at 8 weeks of age. Mice dying during the exposure period or sacrificed at the
33 24-month termination were necropsied; mice with skin tumors that persisted for 4 consecutive
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1 weeks with diameters >3 cm were sacrificed before the study termination and also necropsied.
2 Skin samples and any grossly observed lesions were subjected to histopathological examination.
3 Carcinomas and sarcomas were referred to as carcinomas, whereas papillomas, keratoacanthomas,
4 and fibromas were referred to as papillomas. The incidences of mice with skin tumors and mean
5 survival times for each group are shown in Table D-23. All high-dose mice died before the final
6 sacrifice, and 80% showed scabs and sores at the site of application. The time of first tumor
7 appearance was not reported for the tumor-inducing groups, but from a plot of the tumor incidence
8 in the high-dose group versus treatment days, an estimate of ~320 days (~43 weeks) is obtained
9 for this group. The extent of deaths prior to 1 year in each group was not provided, so the reported
10 incidence may underestimate the tumor rate of animals exposed long enough to develop tumors.
11 However, the crude skin tumor rates show an increasing trend in incidence.
12 Table D-23. Skin tumor incidence in male C3H/HeJ mice dermally exposed to
13 benzo[a]pyrene for 24 months
Dose (pg)a
Skin tumor incidence
(all types)b
Number of mice that
died before final
sacrifice
Mean survival time
(days)
0 cyclohexanone/acetone (1:1)
0/30 (0%)
19
607
0.05
0/30 (0%)
15
630
0.5
5/30 (20%)
15
666
5.0
27/30 (90%)
30
449
14
15 indicated doses were applied twice/week to shaved dorsal skin.
16 bNumber of skin tumor-bearing mice. In the high-dose group, 1 papilloma and 28 carcinomas were detected; in
17 the 0.5 ng group, 2 papillomas and 3 carcinomas were detected.
18
19 Source: Sivak et al. (1997).
20
21 To examine dose-response relationships and the time course of benzo[a]pyrene-induced
22 skin damage, DNA adduct formation, and tumor formation, groups of 43-85 female Harlan mice
23 were treated dermally with 0,16, 32, or 64 |ig of benzo[a]pyrene in 50 |j.L of acetone once per week
24 for 29 weeks f Albert etal.. 19911. Interscapular skin of each mouse was clipped 3 days before the
25 first application and every 2 weeks thereafter. Additional groups of mice were treated for 9 weeks
26 with 0, 8,16, 32, or 64 |ig radiolabeled benzo[a]pyrene to determine BPDE-DNA adduct formation
27 in the epidermis at several time points (1, 2, 4, and 9 weeks). Tumor formation was monitored only
28 in the skin.
29 No tumors were present in vehicle-treated or untreated control mice. In exposed groups,
30 incidences of mice with skin tumors were not reported, but time-course data for cumulative
31 number of tumors per mouse, corrected for deaths from nontumor causes, were reported. Tumors
32 began appearing after 12-14 weeks of exposure for the mid- and high-dose groups and at 18 weeks
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for the low-dose group. At study termination (35 weeks after start of exposure), the mean number
of tumors per mouse was approximately one per mouse in the low- and mid-dose groups and eight
per mouse in the high-dose group, indicating that most, if not all, mice in each exposure group
developed skin tumors and that the tumorigenic response was greatest in the highest dose group.
The majority of tumors were initially benign, with an average time of 8 weeks for progression from
benign papillomas to malignant carcinomas. Epidermal damage occurred in a dose-related manner
(more severe in the high-dose group than in the low- and mid-dose groups) and included
statistically significant increases (compared with controls) in: [3H]-thymidine labeling and mitotic
indices; incidence of pyknotic and dark cells (signs of apoptosis); and epidermal thickness. Only a
minor expansion of the epidermal cell population was observed. In the high-dose group, indices of
epidermal damage increased to a plateau by 2 weeks of exposure. The early time course of
epidermal damage indices was not described in the low- or mid-dose groups, since data for these
endpoints were only collected at 20, 24, and 30 weeks of exposure. An increased level of BPDE-
DNA adducts, compared with controls, was apparent in all exposed groups after 4 weeks of
exposure in the following order: 64>32>16>8 ng/week. The time-course data indicate that
benzo[a]pyrene-induced increases in epidermal damage indices and BPDE-DNA adducts preceded
the appearance of skin tumors.
D.4.4. Reproductive and Developmental Toxicity Studies
Oral
In a study evaluating the combined effects of dibutyl phthalate and benzo[a]pyrene on the
male reproductive tract, Chen etal. (2011) administered benzo[a]pyrene alone in corn oil via daily
gavage at 5 mg/kg-day to 30 male Sprague-Dawley rats (28-30 days old); a group of 30 rats
received only vehicle. Body weight was measured weekly. Groups of 10 rats per group were
sacrificed after 4, 8, and 12 weeks of exposure. At sacrifice, blood was collected for analysis of
serum testosterone levels by radioimmunoassay. The testes and epididymides were weighed, and
the right testis and epididymis were examined microscopically. The left epididymis was used for
evaluation of sperm parameters (sperm count and morphology). Oxidative stress, as measured by
superoxide dismutase (SOD), glutathione peroxidase, and catalase activity and malondialdehyde
levels, was evaluated in the left testis of each rat Exposure to benzo[a]pyrene did not affect body
weight, and no signs of toxicity were seen. Testes and epididymides weights of exposed rats were
similar to controls at all time points. Sperm counts and percent abnormal sperm were also similar
to controls at 4 and 8 weeks of exposure, but were significantly (p < 0.05) different from controls
after 12 weeks of exposure to benzo[a]pyrene (29% decrease in sperm count and 54% increase in
percent abnormal sperm). Serum testosterone levels were significantly increased relative to
controls after 4 weeks (>2-fold higher) and 8 weeks (~1.5-fold higher) of benzo[a]pyrene exposure,
but were comparable to controls after 12 weeks. Histopathology evaluation of the testes revealed
irregular and disordered arrangement of germ cells in the seminiferous tubules of treated rats; the
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authors did not report incidence or severity of these changes. Among measures of testicular
oxidative stress, only catalase activity was significantly affected by benzo[a]pyrene exposure,
showing an increase of ~50% after 12 weeks of exposure. These data suggest a LOAEL of 5 mg/kg-
day (the only dose tested) for decreased sperm count, increased percentage of abnormal sperm,
altered testosterone levels, and histopathology changes in the testes following 13 weeks of
exposure.
Chung etal. (20111 evaluated the effects of low-dose benzo[a]pyrene exposure on
spermatogenesis and the role of altered steroidogenesis on the sperm effects. Groups of
20-25 male Sprague-Dawley rats (8 weeks old) were given daily gavage doses of 0, 0.001, 0.01, or
0.1 mg/kg-day benzo[a]pyrene in DMSO for 90 consecutive days. At the end of exposure, the
animals were sacrificed for removal of the pituitary, testes, and epididymides, and collection of
serum and testicular interstitial fluid. Subgroups of each exposure group were used for various
analyses. Serum levels of testosterone and luteinizing hormone (LH) were measured, as was
testosterone concentration in the interstitial fluid (ELISA). Body and testes weights were recorded.
Sections of the testis were analyzed for apoptotic germ cells using the terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) assay. Evaluation of the epididymis included
histopathology as well as measurement of caput and caudal epididymal tubule diameters. In
addition, sperm were isolated from the cauda epididymis for analysis of sperm number and
motility, acrosomal integrity, and immunocytochemistry for ADAM3 (a disintegrin and
metallopeptidase domain 3; a sperm surface protein associated with fertilization).
Leydig cells were isolated from the right testis of animals from each dose group and
cultured with or without human chorionic gonadotropin (hCG) or dibutyl cyclic adenosine
monophosphate (dbcAMP) to evaluate testosterone production (Chung etal.. 2011). Cultured
Leydig cells were also subjected to western blot and immunocytochemistry analyses to evaluate
changes in the expression of genes involved in steroidogenesis (steroidogenic acute regulatory
protein, p450 side-chain cleavage, and 3(3-hydroxysteroid dehydrogenase isomerase). Finally,
pituitary gland extracts were evaluated for LH protein content using immunohistochemistry. Data
were reported graphically and analyzed by analysis of variance (AN OVA) followed by Duncan's post
hoc test, using a p-value cutoff of 0.05 for significant difference.
At termination of exposure, body weights of treated animals were similar to controls, as
were absolute testes weights fChung etal.. 20111. Testosterone concentrations in both serum and
testicular interstitial fluid were significantly reduced at the high dose of benzo[a]pyrene
(0.1 mg/kg-day); based on visual inspection of the data, the mean serum concentration in this
group was ~20% of the control and the mean interstitial fluid concentration was ~60% of the
control (n = 9 animals/dose for these evaluations). In addition, baseline production of testosterone
by cultured Leydig cells was significantly decreased (~50% based on data shown graphically) at
0.1 mg/kg-day. Both hCG- and dbcAMP-stimulated testosterone production measurements were
lower (~60% lower than controls) in Leydig cells from rats exposed to either 0.01 or
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0.1 mg/kg-day. Serum LH was significantly increased at both 0.01 and 0.1 mg/kg-day (~65-75%
higher than controls based on visual inspection of graphs); concordant increases in the intensity of
LH immunoreactivity were evident in pituitary extracts from exposed rats.
Dose-related increases in the number of apoptotic germ cells, primarily spermatogonia,
were demonstrated both via TUNEL assay and caspase-3 staining; the number per tubule was
significantly increased over control at all doses f Chung etal.. 20111. Numbers of sperm were lower
in the treatment groups, but did not differ significantly from the control group. However, sperm
motility was significantly reduced in exposed groups compared with controls. The authors did not
report sperm motility for all dose groups, but showed only the significant decrease in the
0.01 mg/kg-day mid-dose group (~30% lower than controls based on visual inspection of graph).
Acrosomal integrity (measured by LysoTracker staining) was diminished in sperm heads from
exposed rats; likewise, the expression of ADAM3 protein was downregulated by exposure to
benzo[a]pyrene; the authors reported a significant decrease in the 0.01 mg/kg-day group, but did
not provide details of the analysis of other exposure groups. Histopathology examination of the
caput and cauda epididymides revealed dose-related decreases in both cauda and caput tubule
diameters that were statistically significantly lower than controls at all doses (~10-30% smaller
mean diameter than control based on measurements of 175 tubules collected from five samples in
each group; data reported graphically).
Statistically significant effects observed at the lowest dose (0.001 mg/kg-day) of
benzo[a]pyrene in this study included decreased caput and cauda epididymal tubule diameters
(~10-15% lower than controls) and increased numbers of apoptotic germ cells (~2-fold higher
than controls) by TUNEL assay (Chung etal.. 2011). The authors reported that "sperm motility was
significantly reduced in the benzo[a]pyrene-exposed groups in comparison to that of the control"
but provided quantitative data only for the middle dose group, which exhibited a ~30% decrease in
percent motile sperm. No statistically significant decrease in sperm count was reported at any
dose. The middle dose (0.01 mg/kg-day) is considered to be a LOAEL based on reduced sperm
motility.
Gao etal. (2011) examined effects of benzo[a]pyrene exposure via on cervical cell
morphology within the uterus. Female ICR mice (18-22 g) were exposed to doses of 0, 2.5, 5, or 10
mg/kg twice per week for 14 weeks, either by gavage or by intraperitoneal (i.p.) injection (for this
review, only oral results are reported). After adjustment for equivalent continuous dosing (2/7
days/week), the equivalent daily doses are estimated to be 0.7,1.4, and 2.9 mg/kg-day. Both
vehicle (sesame oil) and untreated control groups were maintained. Body weights were
determined weekly. Groups of 26 mice per dose per exposure route were sacrificed at the end of
exposure for evaluation of cervical weight and histopathology. Additional groups of 10 mice were
exposed for 14 weeks and used for determination of lipid peroxidation (malondialdehyde and
glutathione-S-transferase levels) and CYP1A1 activity (EROD) in both liver and cervix, as well as
creatine kinase activity, AST activity, and IL-6 levels in cervix and serum.
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1 Mortality was observed in all exposure groups with the exception of the low-dose oral
2 exposure group; the authors did not indicate the timing or causes of death (Gao etal.. 20111. There
3 were no control deaths. Mortality incidences in the oral exposure groups (low to high dose) were
4 0/26 (untreated control), 0/26 (vehicle control), 0/26,1/36, and 2/26. Benzo[a]pyrene treatment
5 resulted in dose-dependent decreases in body weight gain. In the high-dose group of both
6 treatments, body weight began to decline after ~7 weeks of exposure. Based on visual examination
7 of data presented graphically, mean terminal body weights in the low-, mid-, and high-dose oral
8 exposure groups were ~10,15, and 30% lower (respectively) than the vehicle control mean. The
9 untreated control mean body weight for the oral exposure group was similar to the vehicle control
10 mean body weight. Uterine weight as a function of body weight was not affected by oral
11 benzo[a]pyrene exposure. Microscopic examination of the cervix revealed increased incidences of
12 epithelial hyperplasia and inflammatory cells in the cervix of all groups of exposed mice, and
13 atypical hyperplasia of the cervix in mice exposed to 1.4 or 2.9 mg/kg -day benzo[a]pyrene.
14 Statistical analysis of the findings was conducted, but was poorly reported in the publication.
15 Table D-24 shows the incidences in the oral exposure groups, along with the results of Fisher's
16 exact tests performed for this review.
17 Table D-24. Mortality and cervical histopathology incidences in female ICR
18 mice exposed to benzo[a]pyrene via gavage for 14 weeks
Endpoint
Dose (mg/kg-d)
Untreated
control
Vehicle
control
0.7
1.4
2.9
Mortality
0/26
0/26
0/26
1/26
2/26
Cervical epithelial hyperplasia
0/26
0/26
4/26
6/25*
7/24*
Atypical hyperplasia of cervix
0/26
0/26
0/26
2/25
4/24*
Inflammatory cells in cervix
2/26
3/26
10/26*
12/25*
18/24*
19
20 ^Significantly different from vehicle control by Fisher's exact test performed for this review (one-sided p < 0.05).
21
22 Source: Gao et al. (2011).
23
24 Levels of malondialdehyde in both the cervix and liver were significantly higher than
25 controls in all dose groups of animals treated by either oral (1.5-2-fold higher in the cervix and
26 ~3-7-fold higher in the liver after oral exposure, p < 0.05) or i.p. exposure. Concomitant decreases
27 in GST activity (~15-50% lower than controls in the cervix and ~30-60% lower in the liver after
28 oral exposure, p < 0.05) were also observed at all doses and in both organs and both treatments.
29 EROD activity was increased in the cervix (~4—12-fold) and liver (~12—35-fold) of all exposure
30 groups. Measurement of creatine kinase and AST activity in the cervix and serum also showed
31 significant increases at all doses and after both exposures (~1.5-2-fold in the cervix, and ~20-50%
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higher than controls in the liver after oral exposure). Finally, levels of the inflammatory cytokine
IL-6 were significantly (p < 0.05) increased in the cervix of all treated mice, and were markedly
increased (from more than 2-fold higher than untreated or vehicle controls at the low dose, to
~6-fold higher at the high dose) in the serum of treated mice.
Based on the observations of decreased body weight and increased cervical epithelial
inflammation and hyperplasia, a LOAEL of 0.7 mg/kg-day (the lowest dose tested) is identified for
this study.
Mohamedetal. (2010) investigated multi-generational effects in male mice following
exposure of 6-week-old C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day
benzo[a]pyrene for 6 weeks by gavage. Following final treatment, male mice were allowed to
stabilize for 1 week prior to being mated with two untreated female mice to produce an
F1 generation. Male mice were sacrificed 1 week after mating. F1 males were also mated with
untreated female mice, as were F2 males. The mice of the Fl, F2, and F3 generations were not
exposed to benzo[a]pyrene. The F0, Fl, F2, and F3 mice were all sacrificed at the same age
(14 weeks) and endpoints including testis histology, sperm count, sperm motility, and in vitro
sperm penetration (of hamster oocytes) were evaluated. These endpoints were analyzed
statistically using ANOVA and Tukey's honest significance test and results were reported
graphically as means ± SD.
Testicular atrophy was observed in the benzo[a]pyrene treatment groups, but was not
statistically different than controls. Statistically significant reductions were observed in epididymal
sperm counts of F0 and Fl generations treated with the high or low dose of benzo[a]pyrene. For F0
and Fl generations, epididymal sperm counts were reduced approximately 50 and 70%,
respectively, in the low- and high-dose groups. Additionally, sperm motility was statistically
significantly decreased at the high dose in the F0 and Fl generations. Sperm parameters of the F3
generation were not statistically different from controls. An in vitro sperm penetration assay
revealed statistically significantly reduced fertilization in F0 and Fl generations of the low- and
high-dose groups. However, the value of this in vitro test is limited as it bypasses essential
components of the intact animal system (U.S. EPA. 1996). Based on decreased epididymal sperm
counts of F0 and Fl generations, a LOAEL of 1 mg/kg-day was established from this study (no
NOAEL was identified).
Arafa etal. f20091 exposed groups of 12 male Swiss albino rats to benzo[a]pyrene in olive
oil (0 or 50 mg/kg-day via gavage) for 10 consecutive days, either alone or after similar treatment
with 200 mg/kg-day of the flavonoid hesperidin, which has been shown to exert anti-inflammatory,
antioxidant, and anticarcinogenic activity. One day after the final dose, the animals were sacrificed
for removal of the cauda epididymides and testes. Epididymal sperm count and motility were
assessed, as was daily sperm production in the testes. The study authors also investigated the
testicular activity of LDH, SOD, and GST, as well as GSH, malondialdehyde, and protein content The
testes were examined under light microscope.
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Relative testes weights (normalized to body weight) of benzo[a]pyrene exposed-animals
were significantly decreased compared with controls (35% lower, p < 0.05) (Arafa etal.. 2009). In
addition, exposure to benzo[a]pyrene alone resulted in significantly decreased sperm count,
numbers of motile sperm, and daily sperm production (~40% decrease from control in each
parameter, p < 0.05). Effects on sperm count and production were abolished by hesperidin
pretreatment, but the number of motile sperm remained significantly depressed (compared with
the control group) in the group exposed to both benzo[a]pyrene and hesperidin. Measures of
antioxidant enzymes and lipid peroxidation showed statistically significant induction of oxidative
stress in the testes of benzo[a]pyrene-exposed rats. With the exception of the decrease in testicular
GSH content (which was partially mitigated), pretreatment with hesperidin eliminated the effects of
benzo[a]pyrene on lipid peroxidation and antioxidant enzymes.
Xu etal. f 20101 treated female Sprague-Dawley rats (6/group) to 0 (corn oil only), 5, or
10 mg/kg-day benzo[a]pyrene by gavage every other day for a duration of 60 days. This resulted in
TWA doses of 0, 2.5, and 5 mg/kg-day over the study period of 60 days. Endpoints examined
included ovary weight, estrous cycle, 17B-estradiol blood level, and ovarian follicle populations
(including primordial, primary, secondary, atretic, and corpora lutea). Animals were observed daily
for any clinical signs of toxicity and following sacrifice, gross pathological examinations were made
and any findings were recorded. All animals survived to necropsy. A difference in clinical signs was
not observed for the treated groups and body weights were not statistically different in treated
animals (although they appear to be depressed 6% at the high dose). Absolute ovary weight was
statistically significantly reduced in both the low- and high-dose groups (11 and 15%, respectively)
(see Table D-25). Animals treated with the high dose were noted to have a statistically significantly
prolonged duration of the estrous cycle and nonestrus phase compared to controls. Animals in the
high-dose group also had statistically significantly depressed levels of estradiol (by approximately
25%) and decreased numbers of primordial follicles (by approximately 20%). This study also
indicated a strong apoptotic response of ovarian granulosa cells as visualized through TUNEL
labeling; however, the strongest response was seen at the low dose; decreased apoptosis was also
observed at the high dose. Based on decreased ovary weight following a 60-day oral exposure to
benzo[a]pyrene, a LOAEL of 2.5 mg/kg-day was established from this study (no NOAEL was
identified).
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Table D-25. Means ± SD for ovary weight in female Sprague-Dawley rats
Dose (mg/kg-d)a
0
2.5
5
Ovary weight (g)
0.160 ±0.0146
0.143 ±0.0098*
0.136 ±0.0098*
Body weight (g)
261.67 ± 12.0
249.17 ± 11.2
247.25 ± 11.2
^Statistically different from controls (p < 0.05) using one-way ANOVA.
aTWA doses over the 60-day study period.
Source: Xu et al. (2010).
Zheng etal. (20101 treated male Sprague-Dawley rats to 0 (corn oil only), 1, or 5 mg/kg-day
benzo[a]pyrene by daily gavage for a duration of 30 (8/group) or 90 days (8/group). At necropsy,
the left testis of each animal was collected and weighed. Testes testosterone concentrations were
determined by radioimmunoassay and results were expressed as ng/g testis and reported
graphically. Testicular testosterone was statistically significantly decreased in the high-dose group
approximately 15% following 90 days of exposure. The low-dose group also appeared to have a
similar average depression of testosterone levels; however, the change did not reach statistical
significance. Testosterone levels measured in animals sacrificed following 30 days of
benzo[a]pyrene exposure were not statistically different than controls. Based on decreased
testicular testosterone levels following a 90-day oral exposure to benzo[a]pyrene, a LOAEL of
5 mg/kg-day and a NOAEL of 1 mg/kg-day were identified.
McCallister et al. (20081 administered 0 or 300 ng/kg-day benzo[a]pyrene by gavage in
peanut oil to pregnant Long-Evans rats (n = 5 or 6) on gestation days (GDs) 14-17. At this
exposure level, no significant changes were see in number of pups per litter, pup growth, or liver to
body weight ratios in control compared to benzo[a]pyrene exposed offspring. Treatment-related
differences in brain to body weight ratios were observed only on postnatal days (PNDs) 15 and 30.
Decreases in cerebrocortical messenger ribonucleic acid (mRNA) expression of the glutamatergic
N-methyl-D-aspartate (NMDA) receptor subunit was significantly reduced (50%) in treated
offspring compared to controls. In addition, in utero exposed offspring exhibited decreased evoked
cortical neuronal activity in the barrel field cortex when tested at PNDs 90-120.
Rigdon and Neal (19651 administered diets containing 1,000 ppm benzo[a]pyrene to
pregnant mice (nine/group) on GDs 10-21 or 5-21. The pups were reported as appearing
generally normal at birth, but cannibalism was elevated in the exposed groups. These results are in
contrast with an earlier study fRigdon and Rennels. 19641 in which rats (strain not specified) were
fed diets containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and
during gestation. In the earlier study, five of eight treated females mated with untreated males
became pregnant, but only one delivered live young. The treated dam that delivered had two live
and two stillborn pups; one dead pup was grossly malformed. In the remaining treated females,
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vaginal bleeding was observed on GDs 23 or 24. In the inverse experimental design, three of six
controls mated to benzo[a]pyrene-treated males became pregnant and delivered live young.
Visceral and skeletal examinations of the pups were not conducted. These studies were limited by
the small numbers of animals, minimal evaluation of the pups, lack of details on days of treatment
(food consumption, weight gain), and occurrence of cannibalism.
Reproductive Effects of In Utero Exposure Via Oral Route
Mackenzie and Angevine (1981) conducted a two-generation reproductive and
developmental toxicity study for benzo[a]pyrene in CD-I mice. Benzo[a]pyrene was administered
by gavage in 0.2 mL of corn oil to groups of 30 or 60 pregnant (the F0 generation) mice at doses of
0,10, 40, or 160 mg/kg-day on GDs 7-16 only. Therefore, unlike the standard two-generation
study, F1 animals were exposed only in utero. F1 offspring were evaluated for postnatal
development and reproductive function as follows. F1 pups (four/sex when possible) were allowed
to remain with their mothers until weaning on PND 20. Crossover mating studies were then
conducted. Beginning at 7 weeks of age, each F1 male mouse (n = 20-45/group) was allowed to
mate with two untreated virgin females for 5-day periods for 25 days (for a total exposure of
10 untreated females/Fl male), after which time the males were separated from the females.
Fourteen days after separation from the males (i.e., on days 14-19 of gestation), the females were
sacrificed and the numbers of implants, fetuses, and resorptions were recorded. The F2 fetuses
were then examined for gross abnormalities. Similarly, each F1 female mouse (n = 20-55/group),
beginning at 6 weeks of age, was paired with an untreated male for a period of 6 months. Males
were replaced if the females failed to produce a litter during the first 30-day period. All F2 young
were examined for gross abnormalities on day 1 of life and their weights were recorded on day 4.
This F2 group was sacrificed on day 20 postpartum, while the F1 female was left with a male until
the conclusion of the study. At 6 weeks of age, gonads of groups of 10 male and 10 female F1 mice
exposed to 0,10, or 40 mg/kg-day benzo[a]pyrene in utero were subjected to gross pathology and
histologic examinations.
No maternal toxicity was observed. The number of F0 females with viable litters at
parturition at the highest dose was statistically significantly reduced by about 35% (Table D-26),
but progeny were normal by gross observation. Parturition rates of the low- and mid-dose groups
were unaffected by treatment, and litter sizes of all treated groups were similar to the control group
throughout lactation. However, body weights of the F1 pups in the mid- and high-dose groups were
statistically significantly decreased on PND 20, by 7 and 13%, respectively, and in all treated pups
on PND 42, 6, 6, and 10% for the low, mid, and high dose, respectively (Table D-26). The number of
F1 pups surviving to PNDs 20 and 42 was significantly reduced at the high dose (p < 0.01), by 8 and
16%, respectively. When F1 males were bred to untreated females and F1 females were mated
with untreated males, a marked dose-related decrease in fertility of >30% was observed in both
sexes, starting at the lowest exposure. There were no treatment-associated gross abnormalities or
differences in body weights in the F2 offspring.
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1 Table D-26. Reproductive effects in male and female CD-I F1 mice exposed in
2 utero to benzo[a]pyrene
Effect
Dose (mg/kg-d)a
0
10
40
160
F0 mice with viable litters at parturition
46/60 (77%)
21/30 (70%)
44/60 (73%)
13/30 (43%)*
Mean ± SEM pup weight (g) at PND 20
11.2 ±0.1
11.6 ±0.1
10.4 ±0.1*
9.7 ±0.2*
Mean ± SEM pup weight (g) at PND 42
29.9 ±0.2
28.2 ±0.3*
28.0 ±0.2*
26.8 ±0.4*
F1 male fertility indexb
80.4
52.0*
4.7*
0.0*
F1 female fertility index0
100.0
65.7*
0.0*
0.0*
3
4 ^Significantly (p < 0.05) different from control by unspecified tests.
5 aPregnant F0 mice were administered daily doses of benzo[a]pyrene in corn oil on GDs 7-16.
6 bBeginning at 7 weeks of age, each F1 male mouse (20-45/group) was exposed to 10 untreated females over a
7 period of 25 days. Index = (females pregnant/females exposed to males) x 100.
8 beginning at 6 weeks of age, each F1 female mouse (20-55/group) was cohabitated with an untreated male for a
9 period of 6 months.
10
11 SEM = standard error of the mean.
12
13 Source: Mackenzie and Angevine (1981).
14
15 Exposure to benzo[a]pyrene caused a marked dose-related decrease in the size of the
16 gonads. In F1 males, testes weights were statistically significantly reduced. Testes from animals
17 exposed in utero to 10 and 40 mg/kg-day weighed approximately 42 and 82%, respectively, of the
18 weight of testes from the control animals (no F2 offspring were produced in the high-dose group).
19 This was confirmed by histopathologic observation of atrophic seminiferous tubules in the
20 40 mg/kg-day group that were smaller than those of controls and were empty except for a basal
21 layer of cells. The number of interstitial cells in the testes was also increased in this group. Males
22 from the 10 mg/kg-day group showed limited testicular damage; although all exhibited evidence of
23 tubular injury, each animal had some seminiferous tubules that displayed active spermatogenesis.
24 Ovarian tissue was absent or reduced in F1 females such that organ weights were not possible to
25 obtain. Examination of available tissue in these females revealed hypoplastic ovaries with few
26 follicles and corpora lutea (10 mg/kg-day) or with no evidence of folliculogenesis (40 mg/kg-day).
27 Ovarian tissue was not examined in highest-dose females.
28 The LOAEL in this study was 10 mg/kg-day based on decreases in mean pup weight (<5%)
29 at PND 42 of F1 offspring of dams treated with 10, 40, or 160 mg/kg-day benzo[a]pyrene, marked
30 decreases in the reproductive capacity (as measured by fertility index) of both male and female F1
31 offspring exposed at all three treatment levels of benzo[a]pyrene (by approximately 30% in males
32 and females), decreased litter size (by about 20%) in offspring of F1 dams, and the dramatic
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1 decrease in size and alteration in anatomy of the gonads of both male and female F1 mice exposed
2 to 10 and 40 mg/kg-day benzo[a]pyrene in utero. A NOAEL was not identified.
3 In another reproductive and developmental toxicity study, benzo[a]pyrene was
4 administered by gavage in corn oil to nine female NMRI mice at a dose of 10 mg/kg-day on
5 GDs 7-16; a group of nine controls received corn oil fKristensen etal.. 19951. Body weights were
6 monitored. F0 females were kept with their offspring until after weaning (21 days after delivery).
7 At 6 weeks of age, one F1 female from each litter (n = 9) was caged with an untreated male. The
8 F2 offspring were inspected for gross deformities at birth, weight and sex were recorded 2 days
9 after birth, and the pups were sacrificed. The F1 females were sacrificed after 6 months of
10 continuous breeding. The effects of benzo[a]pyrene treatment on fertility, ovary weights, follicles,
11 and corpora lutea were evaluated. F0 females showed no signs of general toxicity, and there was no
12 effect on fertility. F1 females had statistically significantly lower median numbers of offspring,
13 number of litters, and litter sizes and a statistically significantly greater median number of days
14 between litters as compared with the controls (Table D-27). At necropsy, the F1 females from
15 treated F0 females had statistically significantly reduced ovary weights; histologic examination of
16 the ovaries revealed decreased numbers of small, medium, or large follicles and corpora lutea
17 (Table D-27). Only one dose group was used in this study, with decreased F1 female fertility
18 observed following in utero exposure at the LOAEL of 10 mg/kg-day; no NOAEL was identified.
19 Table D-27. Effect of prenatal exposure to benzo[a]pyrene on indices of
20 reproductive performance in F1 female NMRI mice
Endpoint (median with range in
parentheses)
Control3
Benzo[a]pyrene exposed3 (10 mg/kg-d)
Number of F2 offspring
92 (26-121)
22* (0-86)
Number of F2 litters
8 (3-8)
3* (0-8)
F2 litter size (number of pups per litter)
11.5 (6-15)
8* (3-11)
Number of d between F2 litters
20.5 (20-21)
21* (20-23)
F1 ovary weight (mg)
13 (13-20)
9* (7-13)
Number of small follicles
44 (1-137)
0* (0-68)
Number of medium follicles
9 (5-25)
0* (0-57)
Number of large follicles
14 (6-23)
0* (0-19)
Number of corpora lutea
16 (6-35)
0* (0-14)
21
22 ^Significantly (p < 0.05) different from control group by Wilcoxon rank sum test or Kruskall-Wallis two-tailed test.
23 aGroups of nine female NMRI F0 mice were administered 0 or 10 mg benzo[a]pyrene/kg-day by gavage in corn oil
24 on GDs 7-16. One F1 female from each litter was continuously bred with an untreated male for 6 months.
25
26 Source: Kristensen et al. (1995).
27
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Chen etal. (2012) treated male and female neonatal Sprague-Dawley rats (10/sex/group)
with benzo[a]pyrene (unspecified purity) 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 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 and female 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
preweaning period 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" period from 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 assigned for 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,
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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 benzo[a]pyrene treatment x sex) indicated that effects of
benzo[a]pyrene 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 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 benzo[a]pyrene 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 benzo[a]pyrene, 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 benzo[a]pyrene 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 and females 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 benzo[a]pyrene 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)
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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 benzo[a]pyrene 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.
Tules etal. f20121 treated pregnant Long-Evans Hooded rats with benzo[a]pyrene
(unspecified purity) dissolved in 0.875 mL peanut oil by gavage daily on GDs 14-17, at doses of
150, 300, 600, and 1,200 [igbenzo[a]pyrene/kg body weight, with animals weighed daily. Cage-
side observations were performed until pup weaning, and litter size was evaluated for each
treatment group. Pups from four to five individual litters were analyzed for each endpoint, which
was independently repeated for a total of three replicates. Delivery was designated PND 0, and
pups were harvested on PNDs 0-15 for benzo[a]pyrene metabolite identification, or for other
endpoints as young adults at PND 53. Systolic/diastolic blood pressure and heart rate was
recorded by a volume pressure recording sensor and occlusion tail-cuff applied to conscious, non-
anesthetized animals. Animals were preconditioned to the restraint device and tail-cuff by daily
acclimatization sessions during PNDs 46-50, to minimize stress effects during data collection.
Cardiac function values were averaged from 15 readings each collected over a 1-minute interval
every other minute for 30 minutes on PND 53.
No significant differences in litter size or pup weight gain from PND 0 to 15 were reported
in any treatment group, and no convulsions, tremors, or abnormal movements were reproducibly
observed. Most analytical data were reported graphically, as mean ± standard error of the mean
(SEM) of three replicates of 3-5 offspring measured/group. Plasma and heart tissue total
benzo[a]pyrene metabolite levels were maximal at PND 0 (the first time point sampled) and
progressively decreased from PNDs 0 to 13. Compared to the low-dose group (150 [ig/kg), plasma
metabolite levels were significantly elevated in the 600 and 1,200 [ig/kg-day benzo[a]pyrene
groups through PND 13, while heart metabolite levels were significantly increased through PND 11.
Metabolites in mid-dose group, 300 [ig/kg-day, trended between the 150 and 600 [ig/kg-day group
levels from PND 0 to 7, while not achieving statistically significant differences in pair-wise
comparisons. Three principal groups of benzo[a]pyrene metabolites were identified. More than
70% of the total heart metabolite burden was composed of diol metabolites through PND 13, while
the more reactive hydroxyl metabolites increased in relative composition from PND 9 to 13, and the
dione population remained constant at <5%.
Cardiovascular function was evaluated in pups exposed in utero to 600 or 1,200 [ig/kg-day
benzo[a]pyrene versus controls (see Table D-28). A dose-related and statistically significant
increase in both systolic (20, 50%) and diastolic pressure (30, 80%) was observed in mid- and
high-dose pups, respectively. Heart rate was also significantly altered; a 10% increased heart rate
was reported in the 600 [ig/kg-day benzo[a]pyrene group, while the average heart rate of the
1,200 [ig/kg-day benzo[a]pyrene groups decreased 8%.
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Table D-28. Exposure-related effects in Long-Evans Hooded rats exposed to
benzo[a]pyrene by gavage daily in utero from GD 14 to 17
Effect measured
Dose (mg/kg-d)
0
0.600
1.20
Heart rate (bpm; mean ± SEM)
504.6 ± 15.7
554.6 ±26.2*
466.3 ± 16.9*
Blood pressure measured by tail cuff (mmHg; mean ± SEM)
Systolic pressure
131.6 ± 1.2
151.6 ±45*
200.4 ± 2.4*
Diastolic pressure
85.0 ±4.2
113.0 ±3.3*
155.6 ±3.2*
^Significantly (p < 0.05) different from control mean; n = 4-5/replicate, 3 replicates performed.
Source: Jules et al. (2012).
Bouaved etal. f2009al treated nursing female Swiss Albino 0F1 mice (5/dose group) with
benzo[a]pyrene (unspecified purity) dissolved in avocado oil by gavage daily while nursing pups
from PND 1 to 14 at 0, 2, or 20 mg/kg-day in 10 mL/kg body weight, individually determined each
day. Prior to benzo[a]pyrene treatment, litters were culled to 10 pups (5/sex when possible), and
nurturing females were assigned to litters that were stratified randomly to achieve equivalent
mean pup litter body weights across the designated treatment groups. As the effects of
benzo[a]pyrene on maternal nurturing behavior was unknown, dam behavior was visually
monitored daily until weaning. Furthermore, maternal nurturing performance from PND 0 to 21
was assessed by two methods: a nest-building test administered twice a day where nest quality/
complexity was scored 15 minutes after cotton material was supplied; and pup retrieval, in which
latency to return the displaced pup to the nest was measured twice and averaged, was evaluated
once daily At the indicated times, two mice/sex/litter were randomly selected and weighed, and
their brains were resected for later mRNA expression analysis (n = 20/group).
Pup neuromotor maturation and behavior was assessed during pre-weaning by four
standard methods (administered between 10 am and 1 pm on testing days, and in temporal order
as indicated): (1) righting reflex test, maximum duration of 120 seconds, administered on PNDs 3, 5,
7, and 9; (2) negativegeotaxis test, maximum duration of 120 seconds, administered on PNDs 5, 7,
9, and 11; (3)forelimbgrip test, duration until failure, administered on PNDs 9 and 11; and (4) open
field test, 6-minute evaluation of locomotor activity and rearing following a 1-minute habituation
period, administered on PND 15. Adolescent function was evaluated by three methods: water
escape pole climbing (WESPOC) test, administered at PND 20, in which the time to find the pole, time
to climb the pole, and the time to reach the safety platform were reported; elevated plus maze,
administered at PND 32 for 5 minutes, in which the latency time to first open arm entry, number of
entries into open arms, total number of entries, percent of time spent in open arms, and percent of
entries into open arms was determined; and Y-maze spontaneous alternation test, administered at
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1 PND 40 for 5 minutes, in which the percentage of spontaneous alternation was calculated by: [(the
2 number of successful overlapping triplets)/(total number of arm entries - 2) x 100%].
3 Benzo[a]pyrene treatment did not significantly affect the body weight of nursing mothers
4 during the 2-week treatment period. Since 3-way ANOVA indicated that changes in pup weight as a
5 result of benzo[a]pyrene treatment were not sex-dependent, data from male and female pups were
6 combined. Benzo[a]pyrene treatment of nursing mothers was associated with a 8-9% weight gain
7 in pups nursing from the 2 mg/kg-day group and a 10-12% weight gain in pups from the 20
8 mg/kg-day group atPNDs 12-20 (see Table D-29). While not significantly different from PND 26 to
9 40, pup weight in the 20 mg/kg-day group was continuously higher than either the 2 mg/kg-day
10 group or vehicle-treated controls. There were no significant differences in pup brain weight or eye
11 opening observed. Likewise, benzo[a]pyrene treatment of nursing mothers did not affect nest-
12 building interest or quality, and while not significantly impacting pup retrieval time, the retrieval
13 latency period was observed to increase with increasing treatment duration in both
14 benzo[a]pyrene groups versus controls.
15 Table D-29. Exposure-related pup body weight effects in Swiss Albino OF1
16 mice exposed as pups to benzo[a]pyrene in breast milk from dams treated by
17 gavage daily from PND 1 to 14
Pup body weight (g; mean ± SEM,
n = 20)
Dose (mg/kg-d)
0
2
20
PND 0
1.70 ± 0.02
1.73 ±0.02
1.74 ± 0.02
PND 4
3.01 ±0.08
3.08 ± 0.06
3.16 ±0.04
PND 8
5.08 ±0.1
5.26 ±0.09
5.30 ±0.08
PND 12
6.57 ±0.12
7.16 ±0.06*
7.39 ±0.05*
PND 20
12.51 ±0.24
13.55 ±0.25**
13.79 ±0.14*
PND 26
17.71 ±0.49
18.60 ±0.36
18.35 ±0.34
PND 32
24.47 ± 0.55
25.59 ±0.57
25.38 ±0.54
PND 40
30.55 ±0.94
30.90 ±0.93
31.78 ±0.97
18
19 *p < 0.001 significantly different from control mean.
20 **p<0.01.
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22 Source: Bouaved et al. (2009a).
23
24 Behavioral test data was reported graphically, as mean ± SEM of n = 20/group. For the pre-
25 weaning neuromotor developmental tests, benzo[a]pyrene treatment was found to not depend on
26 sex; therefore, data from male and female pups were combined. Pups nursing from mothers
27 administered 2 or 20 mg/kg-day benzo[a]pyrene had significantly elevated righting reflex times at
28 PNDs 3-5, which decreased to control times atPNDs 7-9. Only pups from the 20 mg/kg-day
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treatment group demonstrated significantly increased negative geotaxis latency, which was 2-fold
greater than controls at PNDs 5, 7, and 9, but returned to control levels at PND 11. Interestingly,
mice in the 20 mg/kg-day group had increased forelimb grip strength, which was significantly
greater than control mice at PNDs 9 and 11, corresponding to increased body weight in the
benzo[a]pyrene-treated mice versus controls. Mice in the 2 mg/kg-day group also performed
better than controls at PND 9, but were equivalent at PND 11. No treatment or sex-related effects
were reported on locomotion or rearing activity during the open field test Sex-dependency on test
performance became evident during the analysis of the WESPOC test data: female pups were not
significantly affected using any metric, while males in the 20 mg/kg-day group demonstrated a
statistically significantly longer pole-grasping latency (3-fold), and took 13 times longer to escape
the pole and board the safety platform versus vehicle controls. While performance of male pups
from the 2 mg/kg-day group was not statistically significantly worse than vehicle controls by pair-
wise comparison, latency for both pole-grasping and escape in this treatment group contributed to
a significant trend for treatment dose and these effects. In the evaluation of the elevated plus maze,
treatment effects did not appear to depend upon sex, so both male and female performance was
analyzed together. Mice in both benzo[a]pyrene treatment groups demonstrated decreased latency
time to first entering an open arm (30-50%), as well as entered open arms 2 times more frequently
and spent twice as much time there versus vehicle controls. While mice in the 2 mg/kg-day
treatment group entered into closed arms 20% less frequently than controls, mice in the
20 mg/kg-day group were not significantly different Likewise, mice nursing from mothers treated
with 2 mg/kg-day benzo[a]pyrene performed 15% more spontaneous alternations in the Y-maze
spontaneous alternation test compared to controls, while mice in the high-dose group were not
significantly different. The brains of pups nursing from the 20 mg/kg-day group expressed
approximately 50% lower levels of 5-hydroxytryptamine (serotonin) 1A (5HT1A), and mu 1-opioid
(MORI) mRNA, and a trend was observed in the low-dose group as well. No significant changes in
alpha-ID adrenergic or GABA-A mRNA levels were detected.
Reproductive Effects in Adults and Repeated Oral Exposure
Rigdon and Neal (1965) conducted a series of experiments to assess the reproductive
effects of orally administered benzo[a]pyrene to Ah-responsive white Swiss mice. Female animals
(number not stated) were administered benzo[a]pyrene at 250, 500, or 1,000 ppm in the feed
before or during a 5-day mating period. Based on the initial body weight, the doses can be
estimated as 32, 56, and 122 mg/kg-day, respectively. No effect on fertility was observed at any
treatment dose, even when animals were fed 1,000 ppm benzo[a]pyrene for 20 days prior to
mating, but interpretation of this finding was marred by large variability in numbers of pregnant
females and litter sizes for both treated and control mice. In separate experiments, the fertility of
five male mice/group was not affected by exposure to 1,000 ppm in food for up to 30 days prior to
mating with untreated females. Histologic examinations showed that male mice fed 500 ppm
benzo[a]pyrene for 30 days had spermatozoa present in their testes; further details were not
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provided. The only treatment-related effect was a lack of weight gain related to feed unpalatability.
While this study suggests thatpremating exposure of male or female mice to doses up to
122 mg/kg-day for 20 days may not affect fertility, the sample sizes were too small and the study
designs were too inconsistent to provide reliable NOAELs and LOAELs for reproductive/
developmental toxicity.
In an earlier study fRigdon and Rennels. 19641. rats (strain not specified) were fed diets
containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and during
gestation. In this study, five of eight treated females mated with untreated males became pregnant,
but only one delivered live young. The treated dam that delivered had two live and two stillborn
pups; one dead pup was grossly malformed. In the remaining treated females, vaginal bleeding was
observed on GDs 23 or 24. In the inverse experimental design, three of six controls mated to
benzo[a]pyrene-treated males became pregnant and delivered live young. Visceral and skeletal
examinations of the pups were not conducted. These studies are insufficiently reported and of
insufficient design (e.g., inadequate numbers of animals for statistical analysis) to provide reliable
NOAELs or LOAELs for reproductive effects from repeated oral exposure to benzo[a]pyrene.
D.4.5. Inhalation
Reproductive Toxicity and In Utero Exposure via Inhalation
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 had four 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 of 25, 75, or 100 |J.g/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 of particle size analysis of generated
aerosols were reported by several other reports from this laboratory (Invangetal.. 2003: Ramesh
etal.. 2001a: Hoodetal.. 2000). Aerosols showed a trimodal distribution (average of cumulative
mass, diameter) <95%, 15.85 |im; 89%, <10 |im; 55%, <2.5 |im; and 38%, <1 |im (Invangetal..
20031. Ramesh etal. f2001al reported that the MMAD (± geometric SD) for the 55% mass fraction
with diameters <2.5 |im was 1.7 ± 0.085. Progesterone, estradiol-17(3, 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
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1 two control groups; fetal survival rates were 78.3, 38.0, and 33.8% per litter at 25, 75, and
2 100 H-g/m3, respectively, and 96.7% with carbon black or 98.8% per litter in untreated controls (see
3 Table D-30). Consequently, the number of pups per litter was also decreased in a concentration-
4 dependent manner. The decrease was ~50% at 75 |J.g/m3 and ~65% at 100 |J.g/m3, compared with
5 sham-exposed and unexposed control groups. No effects on hormone levels were observed on
6 GDs 15 or 17 atthe low dose. Biologically significant decreases in mean pup weights (expressed as
7 g per litter) of >5% relative to the untreated control group were observed at doses >75 |J.g/m3
8 (14 and 16% decreases at 75 and 100 |J.g/m3, respectively, p < 0.05). There were no statistically
9 significant differences from the control groups in crown-rump length (see Table D-30).
10 Table D-30. Pregnancy outcomes in female F344 rats treated with
11 benzo[a]pyrene on GDs 11-21 by inhalation
Parameter3
Administered concentration of benzo[a]pyrene (pg/m3)
0 (unexposed control)
0
(carbon
black)
25
75
100
Implantation sites
8.6 ±0.2
8.8 ±0.1
8.8 ±0.5
9.0 ±0.2
8.8 ±0.1
Pups per litter
8.5 ±0.2
8.7 ±0.2
7.4 ±0.5*
4.2 ±0.1*
3.0 ±0.2*
Survival (litter %)
98.9 ± 1.1
96.7 ± 1.7
78.3 ±4.1*
38.0 ±2.1*
33.8 ± 1.3*
Pup weight (g/litter)
10.6 ±0.1
8.8 ±0.1
10.5 ± 0.2
9.1 ±0.2*
8.9 ±0.1*
Crown-rump length (mm/litter)
29.4 ±0.6
29.3 ±0.5
28.0 ±0.6
27.3 ±0.7
27.9 ±0.7
12
13 ^Significantly different from controls at p < 0.05 by one-tailed post-hoc t-testing following ANOVA.
14 aValues presented as means ± SEM.
15
16 Source: Archibong et al. (2002).
17
18 Benzo[a]pyrene exposure at 75 |J.g/m3 caused a statistically significant decrease in plasma
19 progesterone, estradiol, and prolactin on GD 17; these levels were not determined in the rats
20 exposed to 100 ng/m3 (Archibong etal.. 2002). Plasma prolactin is an indirect measure of the
21 activity of decidual luteotropin, a prolactin-like hormone whose activity is necessary for luteal
22 maintenance during pregnancy in rats. Control levels of prolactin increased from GD 15 to 17, but
23 this increase did not occur in the rats exposed to 75 |J.g/m3. Although the progesterone
24 concentration at 75 |ig/m3 was significantly lower than in controls on GD 17, the authors thought
25 that the circulating levels should have been sufficient to maintain pregnancy; thus, the increased
26 loss of fetuses was thought to be caused by the lower prolactin levels rather than progesterone
27 deficiency. The reduced circulating levels of progesterone and estradiol-17(3 among
28 benzo[a]pyrene-treated rats were thought to be a result of limited decidual luteotropic support for
29 the corpora lutea. The authors proposed the following mechanism for the effects of benzo[a]pyrene
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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-17(3 may decrease uterine levels of
progesterone receptors, thereby resulting in fetal mortality. Based on biologically and statistically
significant decreases in pups/litter and percent fetal survival/per litter, the LOAEL was 25 |ig/m:i;
no NOAEL was identified.
Neurotoxicity and In Utero Exposure via Inhalation
To evaluate the effects of benzo[a]pyrene on the developing central nervous system,
Wormlev etal. (20041 studied rat offspring from those produced by the Archibong et al. (20021
investigation (personal communication, D. Hood to K. Hogan, 5/11/2016), in which exposed timed-
pregnant F344 rats (10/group) to benzo[a]pyrene:carbon black aerosols by nose-only inhalation on
GDs 11-21 for 4 hours/day at a concentration of 100 ng/m3. Results of particle size analysis of
generated aerosols were reported by other reports from this laboratory (Ramesh etal.. 2001a:
Hood etal.. 20001. Particle size analysis of a 100-ng/m3 aerosol showed a trimodal distribution
(average of cumulative mass, diameter): <95%, 15.85 |im; 90%, <10 |im; 67.5%, <2.5 |im; and
66.2%, <1 |im; the MMAD ± geometric SD for the latter fraction was 0.4 ± 0.02 |im (Hood etal..
20001. As noted by Archibong et al. f20021. benzo[a]pvrene reduced the number of live pups at this
exposure level to one-third of control values. During PNDs 60-70, electrical stimulation and
evoked field potential responses were recorded via electrodes implanted into the brains of the
animals. Direct stimulation of perforant paths in the entorhinal region revealed a diminution in
long-term potentiation of population spikes across the perforant path-granular cell synapses in the
dentate gyrus of the hippocampus of F1 generation benzo[a]pyrene-exposed animals; responses in
exposed offspring were about 25% weaker than in control offspring. Additionally, NMDA receptor
subunit 1 protein (important for synaptic functioning) was down-regulated in the hippocampus of
benzo[a]pyrene-exposed F1 pups. The authors interpreted their results as suggesting that
gestational exposure to benzo[a]pyrene inhalation attenuates the capacity for long-term
potentiation (a cellular correlate of learning and memory) in the F1 generation.
In another study by this same group of investigators, Wu etal. (2003a) evaluated the
generation of benzo[a]pyrene metabolites in F1 generation pups, as well as the developmental
profile for AhR and mRNA. In this study, confirmed-pregnant F344 rats were exposed to
benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 |J.g/m3 via nose-only inhalation,
4 hours/day, for 10 days (GDs 11-21). Control animals either were exposed to carbon black
(sham) to control for inert carrier effects or remained untreated. Each benzo[a]pyrene
concentration had its own set of controls (carbon black and untreated). Two randomly selected
pups were sacrificed on each of PNDs 0, 3, 5,10,15, 20, and 30. Body, brain, and liver weights were
recorded. Benzo[a]pyrene metabolites were analyzed in the cerebral cortex, hippocampus, liver,
and plasma. A dose-related increase in plasma and cortex benzo[a]pyrene metabolite
concentrations in pups was observed. Dihydrodiols (4,5-; 7,8-; 9,10-) dominated the metabolite
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distribution profile up to PND 15 and the hydroxy (3-0H-benzo[a]pyrene; 9-0H-benzo[a]pyrene)
metabolites after PND 15 at 100 |J.g/m3 (the only exposure concentration reported). Results
indicated a dose-related decrease in the ratio of the total number of pups born per litter to the total
number of implantation sites per litter. The number of resorptions at 75 and 100 |J.g/m3, but not at
25 |ig/m:i, was statistically significantly increased compared with controls.
Adult Male Reproductive Effects and Repeated Inhalation Exposure
Invangetal. (2003) evaluated the effect of subacute exposure to inhaled benzo[a]pyrene on
testicular steroidogenesis and epididymal function in rats. Male F344 rats (10/group), 13 weeks of
age, were exposed to benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 |J.g/m3 via nose-only
inhalation, 4 hours/day for 10 days. Control animals either were exposed to carbon black (sham) to
control for exposure to the inert carrier or remained untreated. Each benzo[a]pyrene
concentration had its own set of controls (carbon black and untreated). Aerosols showed a
trimodal distribution (average of cumulative mass, diameter): 95%, <15.85 |im; 89%, <10 |im; 55%,
<2.5 |im; and 38%, <1 |im (Invang et al.. 2003): an earlier report from this laboratory indicated that
the 55% mass fraction had a MMAD ± geometric SD of 1.7 ± 0.085 (Ramesh etal.. 2001a). Blood
samples were collected at 0, 24, 48, and 72 hours after cessation of exposure to assess the effect of
benzo[a]pyrene on systemic concentrations of testosterone and LH, hormones that regulate
testosterone synthesis. Reproductive endpoints such as testis weight and motility and density of
stored (epididymal) sperm were evaluated.
Regardless of the exposure concentration, inhaled benzo[a]pyrene did not affect testis
weight or the density of stored sperm compared with controls. However, inhaled benzo[a]pyrene
caused a concentration-dependent reduction in the progressive motility of stored sperm.
Progressive motility was similar at 75 and 100 |ig/m:i, but these values were significantly lower
(p < 0.05) than in any other group. The reduction in sperm motility postcessation of exposure was
thoughtto be the result of benzo[a]pyrene limiting epididymal function. Benzo[a]pyrene exposure
to 75 |J.g/m3 caused a decrease in circulating concentrations of testosterone compared with controls
from the time of cessation of exposure (time 0) to 48 hours posttermination of exposure (p < 0.05).
However, the decrease was followed by a compensatory increase in testosterone concentration at
72 hours postcessation of exposure. Exposure to 75 ng/m3 caused a nonsignificant increase in
plasma LH concentrations at the end of exposure compared with controls, which increased further
and turned significant (p < 0.05) for the remaining time of the study period. The decreased plasma
concentration of testosterone, accompanied by an increased plasma LH level, was thoughtto
indicate that benzo[a]pyrene did not have a direct effect on LH. The authors also noted that the
decreased circulating testosterone may have been secondary to induction of liver CYP450 enzymes
by benzo[a]pyrene. The authors concluded that subacute exposure to benzo[a]pyrene contributed
to impaired testicular endocrine function that ultimately impaired epididymal function. For this
study, the NOAEL was 25 |ig/m3 and the LOAEL was 75 |ig/m3, based on a statistically significant
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reduction in the progressive motility of stored sperm and impairment of testicular function with
10 days of exposure at 75 |J.g/m3.
In a follow-up study with longer exposure duration, adult male F344 rats (10 per group)
were exposed to benzo[a]pyrene:carbon black aerosols at 75 ng/m3 via nose-only inhalation,
4 hours/day for 60 days fArchibongetal.. 2008: Ramesh et al.. 20081. Rats in the control group
were subjected to the nose-only restraint, but were not exposed to the carbon black carrier. Blood
samples were collected at 0, 24, 48, and 72 hours after exposure terminated, and the animals were
sacrificed for tissue analyses following the last blood sampling. Data were analyzed statistically for
benzo[a]pyrene effects on weekly body weights, total plasma testosterone and LH concentrations,
testis weights, density of stored spermatozoa, sperm morphological forms and motility,
benzo[a]pyrene metabolite concentrations and aryl hydrocarbon hydroxylase (AHH) activity, and
morphometric assessments of testicular histologies. Relative to controls, the results indicated 34%
reduced testis weight (p < 0.025), reduced daily sperm production (p < 0.025), and reduced
intratesticular testosterone concentrations (p < 0.025). Plasma testosterone concentrations were
reduced to about one-third of the level in controls on the last day of exposure (day 60) and at 24,
48, and 72 hours later (p < 0.05). However, plasma LH concentrations in benzo[a]pyrene-exposed
rats were elevated throughout the blood sampling time periods compared with controls (p < 0.05).
In testis, lung, and liver, AHH activity and benzo[a]pyrene-7,8-dihydrodiol (precursor to the
DNA-reactive BPDE) and benzo[a]pyrene-3,6-dione metabolites were significantly (p < 0.05)
elevated relative to controls. Progressive motility and mean density of stored spermatozoa were
significantly reduced (p < 0.05). Weekly body weight gains were not affected by benzo[a]pyrene
exposure. These results indicate that a 60-day exposure of adult male rats to benzo[a]pyrene:
carbon black aerosols at 75 ng/m3 produced decreased testis weight; decreased intratesticular and
plasma testosterone concentrations; and decreased sperm production, motility, and density.
D.5. OTHER PERTINENT TOXICITY INFORMATION
D.5.1. Genotoxicity Information
Information summarizing methods commonly used to detect DNA adducts following PAH or
benzo[a]pyrene exposure is presented in Table D-31. Information regarding the genotoxicity of
benzo[a]pyrene in in vitro and in vivo systems is presented in Tables D-32, D-33, D-34, and D-35.
Table D-31. Select PAH-DNA adduct detection methods3
Adduct detection method
Adduct
detection limit
(nucleotides)
Quantitation
Adduct identification
Radiolabeled compounds
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Adduct
detection limit
Adduct detection method
(nucleotides)
Quantitation
Adduct identification
Accelerator mass spectroscopy
-------
Supplem en tal Inform ation —Benzo[aJpyren e
Result
Reference
+S9
-S9
S. typhimurium TA98, TA1538
+
ND
Ames et al. (1975)
S. typhimurium TA98, TAIOO, TA1538
+
ND
Mccann et al. (1975)
S. typhimurium TA1538, TA98
+
-
Wood et al. (1976)
S. typhimurium TA98, TAIOO, TA1537
+
-
Epler et al. (1977)
S. typhimurium TA98, TAIOO
+
-
Obermeier and Frohberg (1977)
S. typhimurium TA98
+
-
Pitts et al. (1978)
S. typhimurium TA98, TAIOO
+
ND
Lavoie et al. (1979)
S. typhimurium TA98, TAIOO
+
-
Simmon (1979a)
S. typhimurium TA98
+
ND
Hermann (1981)
S. typhimurium TA98, TAIOO
+
ND
Alfheim and Ramdahl (1984)
S. typhimurium TA98, TAIOO, TA1538
ND
-
Glatt et al. (1985)
S. typhimurium TA97, TA98, TAIOO
+
-
Sakai et al. (1985)
S. typhimurium TA97, TA98, TAIOO, TA1537
+
-
Glatt et al. (1987)
S. typhimurium TA97, TA98, TAIOO
+
ND
Marino (1987)
S. typhimurium TA98
+
-
Alzieu et al. (1987)
S. typhimurium TA98, TAIOO
+
-
Prasanna et al. (1987)
S. typhimurium TA98
+
ND
Ampv et al. (1988)
S. typhimurium TA98, TAIOO
+
ND
Bos et al. (1988)
S. typhimurium TA98
+
ND
Lee and Lin (1988)
S. typhimurium TA98
+
ND
Antignac et al. (1990)
S. typhimurium TA98
-
ND
Gao et al. (1991)
S. typhimurium TA98
+
ND
Balanskv et al. (1994)
S. typhimurium TAIOO
+
ND
Norpoth et al. (1984)
S. typhimurium TAIOO
+
-
Carver et al. (1986)
S. typhimurium TAIOO
+
ND
Pahlman and Pelkonen (1987)
S. typhimurium TAIOO
+
ND
Tang and Friedman (1977)
S. typhimurium TAIOO
+
ND
Bruce and Heddle (1979)
S. typhimurium TAIOO
+
ND
Phillipson and loannides (1989)
S. typhimurium TAIOO
-
ND
Balanskv et al. (1994)
S. typhimurium TA1537, TA1538
+
-
Ames et al. (1973)
S. typhimurium TA1537, TA1538
+
-
Glatt et al. (1975)
S. typhimurium TA1537
+
ND
Oesch et al. (1976)
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Result
Reference
+S9
-S9
5. typhimurium TA1538
+
ND
Egert and Greim (1976)
S. typhimurium TA1538
+
-
Rosenkranz and Poirier (1979)
S. typhimurium TA1535
-
-
Ames et al. (1973)
S. typhimurium TA 1535
-
-
Glatt et al. (1975)
S. typhimurium TA 1535
-
ND
Mccann et al. (1975)
S. typhimurium TA1535
-
-
Epler et al. (1977)
DNA damage
Escherichia coli/pol A
+
-
Rosenkranz and Poirier (1979)
E. coti/differentiat killing test
+
-
Tweats (1981)
E. coli WP2-WP100/rec-assay
+
ND
Mamber et al. (1983)
E. coli/SOS chromotest Pq37
+
-
Mersch-Sundermann et al.
(1992)
Endpoint/test system: nonmammalian eukaryotes
Mitotic recombination
Saccharomyces cerevisiae D4-RDII
ND
-
Siebert et al. (1981)
S. cerevisiae D3
-
-
Simmon (1979b)
1
2 + = positive; - = negative; ND = not determined.
3 Table D-33. In vitro genotoxicity studies of benzo[a]pyrene in mammalian
4 cells
Assay/test system
Result
Reference
+S9
-S9
Forward mutation
Human AHH-1 lymphoblastoid cells
ND
+
Danheiser et al. (1989)
Human lymphoblast (AHH-1) cells (hprt)
ND
+
Crespi et al. (1985)
Human lymphoblastoid (AHH-1) cell line
ND
+
Chen et al. (1996)
Human fibroblast (MRC5CV1) cell line (hprt)
-
ND
Hanelt et al. (1997)
Human lymphoblast (TK) cells
ND
+
Barfknecht et al. (1982)
Human lymphoblast (TK6) cells
+
ND
Crespi et al. (1985)
Human embryonic epithelial (EUE) cells
ND
+
Rocchi et al. (1980)
Human HSC172 lung fibroblasts
+
-
Gupta and Goldstein (1981)
Human Q3-wp normal lung keratinocytes
+
ND
Allen-Hoffmann and Rheinwald (1984)
Human SCC-13Y lung keratinocytes
ND
+
Allen-Hoffmann and Rheinwald (1984)
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Assay/test system
Result
Reference
+S9
-S9
Mouse lacZtransgenic Muta™Mouse primary
hepatocytes
ND
+
Chen et al. (2010)
Mouse L5178Y/HGPRT
+
-
Clive et al. (1979)
Mouse lymphoma (L5178Y/TK+/-) cells
+
-
Clive et al. (1979)
Mouse lymphoma (L5178Y/TK+/-) cells
+
ND
Amacher et al. (1980); Amacher and
Turner (1980)
Mouse lymphoma (L5178Y/TK+/-) cells
+
-
Amacher and Paillet (1983)
Mouse lymphoma (L5178Y/TK+/-) cells
+
ND
Arce et al. (1987)
Chinese hamster ovary (CHO) cells (aprt)
+
ND
Yang et al. (1999)
CHO cells (5 marker loci)
+
+
Gupta and Singh (1982)
Chinese hamster V79 cells (co-cultured with
irradiated HepG2 cells)
+
ND
Diamond et al. (1980)
Chinese hamster V79 lung epithelial cells
+
ND
Huberman et al. (1976)
Chinese hamster V79 lung epithelial cells
+
ND
Arce et al. (1987)
Chinese hamster V79 lung epithelial cells
+
ND
O'Donovan (1990)
Rat/Fischer, embryo cells/OuaR
ND
+
Mishra et al. (1978)
DNA damage
DNA adducts
Human peripheral blood lymphocytes
ND
+
Wiencke et al. (1990)
Human peripheral blood lymphocytes
ND
+
Li et al. (2001)
Human peripheral blood lymphocytes
ND
+
Wu et al. (2005)
Human peripheral blood lymphocytes
ND
+
Gu et al. (2008)
Human fibroblast (MRC5CV1) cell line
+
ND
Hanelt et al. (1997)
Human hepatoma (HepG2) cell line
ND
+
Tarantini et al. (2009)
Hamster tracheal cells
ND
+
Roggeband et al. (1994)
Chinese hamster V79 lung epithelial cells
+
ND
Arce et al. (1987)
Virus transformed SHE and mouse C3H10T1/2
cells
ND
+
Arce et al. (1987)
Mouse lymphoma (L5178Y/TK+/-) cells
+
ND
Arce et al. (1987)
Rat tracheal cells
ND
+
Roggeband et al. (1994)
Unscheduled DNA synthesis
HeLa cells
+
ND
Martin et al. (1978)
Human fibroblasts
+
ND
Agrelo and Amos (1981)
Human fibroblasts
+
-
Robinson and Mitchell (1981)
Human HepG2
ND
+
Valentin-Severin et al. (2004)
Hamster primary embryo cells
ND
+
Casto et al. (1976)
Hamster tracheal cells
ND
+
Roggeband et al. (1994)
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Assay/test system
Result
Reference
+S9
-S9
Rat hepatocytes
ND
+
Michalopoulos et al. (1978)
Rat tracheal cells
ND
-
Roggeband et al. (1994)
DNA repair
Human mammary epithelial cells
ND
+
Leadon et al. (1988)
Human skin fibroblasts
ND
+
Milo et al. (1978)
Baby hamster kidney (BHK21/cl3) cells
ND
+
Feldman et al. (1978)
secondary mouse embryo fibroblasts (C57BL/6)
and human lymphocytes
ND
+
Shinohara and Cerutti (1977)
Rat/F344 hepatocytes
ND
+
Williams et al. (1982)
Cytogenetic damage
CAs
Human blood cells
ND
+
Salama et al. (2001)
Human WI38 fibroblasts
+
-
Weinstein et al. (1977)
Chinese hamster lung cells
+
-
Matsuoka et al. (1979)
Chinese hamster V79-4 lung epithelial cells
-
-
Popescu et al. (1977)
Mouse lymphoma (L5178Y/TK+/-) cells
+
ND
Arce et al. (1987)
Rat Liver RL1 cells
+
ND
Dean (1981)
MN
Human AHH-1 lymphoblastoid cells
ND
+
Crofton-Sleigh et al. (1993)
Human HepG2 liver cells
ND
+
Wu et al. (2003a)
Human lymphoblastoid (TK) cells
ND
+
Fowler et al. (2010)
Human MCL-5 lymphoblastoid cells
ND
+
Crofton-Sleigh et al. (1993)
Human peripheral blood lymphocytes
+
ND
Lo Jacono et al. (1992)
Chinese hamster V79 cells
ND
+
Whitwell et al. (2010)
Chinese hamster V79-MZ cells
ND
+
Matsuoka et al. (1999)
DNA strand breaks
Human sperm
+
+
Sipinen et al. (2010)
Human peripheral blood lymphocytes
+
+
Rodriguez-Romero et al. (2012)
Human fibroblast (MRC5CV1) cell line
+
ND
Hanelt et al. (1997)
Human hepatoma (HepG2) cell line
ND
+
Tarantini et al. (2009)
Human prostrate carcinoma (DU145) cell line
ND
+
Nwagbara et al. (2007)
Mouse embryo fibroblast (C3H/10T1/2 CL 8) cells
ND
+
Lubet et al. (1983)
Rat C18 trachea epithelial cells
ND
+
Cosma and Marchok (1988); Cosma et
al. (1988)
Rat lymphocytes
ND
+
Gao et al. (1991)
SCEs
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Assay/test system
Result
Reference
+S9
-S9
Human C-HC-4 and C-HC-20 hepatoma cells
ND
+
Abe et al. (1983b); Abe et al. (1983a)
Human diploid fibroblast (TIG-II) cell line
+
+
Huh et al. (1982)
Human fibroblasts
ND
+
Juhletal. (1978)
Human blood cells
ND
+
Salama et al. (2001)
Human peripheral blood lymphocytes
ND
+
Rudiger et al. (1976)
Human peripheral blood lymphocytes
ND
+
Craig-Holmes and Shaw (1977)
Human peripheral blood lymphocytes
ND
+
Schonwald et al. (1977)
Human peripheral blood lymphocytes
ND
+
Wiencke et al. (1990)
Human peripheral blood lymphocytes
+
-
Tohda et al. (1980)
Human peripheral blood lymphocytes
+
ND
Lo Jacono et al. (1992)
Chinese hamster Don-6 cells
ND
+
Abe et al. (1983b); Abe et al. (1983a)
Chinese hamster V79 lung epithelial cells
+
-
Popescu et al. (1977)
Chinese hamster V79 lung epithelial cells
+
ND
Mane et al. (1990)
Chinese hamster V79 lung epithelial cells
+
ND
Woiciechowski et al. (1981)
Chinese hamster V79 lung epithelial cells
+
ND
Arce et al. (1987)
Chinese hamster V79 lung epithelial cells
ND
+
Kulka et al. (1993a)
CHO cells
+
-
de Raat (1979)
CHO cells
+
-
Husgafvel-Pursiainen et al. (1986)
CHO cells
ND
+
Wolff and Takehisa (1977)
CHO cells
ND
+
Pal et al. (1978)
Chinese hamster lung cells
ND
+
Shimizu et al. (1984)
Rabbit peripheral blood lymphocytes
ND
+
Takehisa and Wolff (1978)
Rat ascites hepatoma AH66-B
ND
+
Abe et al. (1983b); Abe et al. (1983a)
Rat esophageal tumor R1
ND
+
Abe et al. (1983b); Abe et al. (1983a)
Rat hepatocyte (immortalized) cell lines (NRL cl-B,
NRLcl-C, andARL)
+
ND
Kulka et al. (1993b)
Rat hepatoma (Reuber H4-II-E) cells
ND
+
Dean et al. (1983)
Rat liver cell line ARL18
ND
+
Tong et al. (1981)
Rat pleural mesothelial cells
ND
+
Achard et al. (1987)
Aneuploidy
Chinese hamster V79-MZ cells
ND
+
Matsuoka et al. (1998)
Cell transformation
Human BEAS-2B lung cells
ND
+
van Agen et al. (1997)
Human breast epithelial (MCF-10F, MCF-7, T24)
cell lines
ND
+
Calaf and Russo (1993)
Baby hamster kidney (BHK21/cl3) cells
+
ND
Greb et al. (1980)
This document is a draft for review purposes only and does not constitute Agency policy.
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Assay/test system
Result
Reference
+S9
-S9
Golden hamster embryo cells
+
ND
Mager et al. (1977)
Syrian hamster embryo (SHE) cells
ND
+
Dipaolo et al. (1971); Dipaolo et al.
(1969)
SHE cells
ND
+
Dunkel et al. (1981)
SHE cells
ND
+
Leboeuf et al. (1990)
SHE cells/focus assay
ND
+
Casto et al. (1977)
Fetal Syrian hamster lung cells
ND
+
Emura et al. (1987); Emura et al. (1980)
Virus infected rat embryo RLV/RE and RAT cells;
mouse embryo AKR/Me cells; Syrian hamster
embryo cells
ND
+
Heidelberger et al. (1983)
Virus transformed SHE and mouse C3H10T1/2
cells
ND
+
Arce et al. (1987)
Mouse C3H/10T1/2 embryo fibroblasts
ND
+
Nesnow et al. (2002); Nesnow et al.
(1997)
Mouse embryo fibroblast (C3H/10T1/2 CL 8) cells
ND
+
Peterson et al. (1981)
Mouse embryo fibroblast (C3H/10T1/2 CL 8) cells
ND
+
Lubet et al. (1983)
Mouse SHE cells; BALB/c-3T3 cells; C3H/10T1/2
cells; prostate cells
ND
+
Heidelberger et al. (1983)
Mouse BALB/c-3T3 cells
ND
+
Dunkel et al. (1981)
Mouse BALB/c-3T3 cells
ND
+
Matthews (1993)
Mouse BALB/c-3T3 clone A31-1-1
ND
+
Little and Vetrovs (1988)
Rat/Fischer, embryo cells (leukemia virus
transformed)
ND
+
Dunkel et al. (1981)
Rat/Fischer, embryo cells/OuaR
ND
+
Mishra et al. (1978)
1
2 + = positive; - = negative; CHO = Chinese hamster ovary; ND = not determined; SHE = Syrian hamster embryo;
3 TK = thymidine kinase.
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Table D-34. Studies of benzo[a]pyrene-induced genotoxicity in humans exposed to PAHs
Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
Mutation
Human, hprt locus
mutation assay in
T lymphocytes
T-cells of lung cancer patients (smokers
and nonsmokers from lung cancer patients
and population controls with known
smoking status) analyzed for hprt locus
mutations.
+
Splicing mutations, base-pair
substitutions, frameshift, and
deletion mutations observed.
Smokers and nonsmokers had
GC->TA transversions (13 and
6%, respectively) and GC->AT
transitions (24 and 35%,
respectively) in hprt gene
consistent with in vitro
mutagenicity of
benzo[a]pyrene.
Hackman et
al. (2000)
Mutation
Human, K-ras and p53
mutations in tumor
tissues
Lung tumors from 24 nonsmoking women
who used smoky coal in their homes in
Xuan Wei County, Yunnan Province, China.
Mutations determined by multiplex PCR
amplification and cycle-sequencing.
+
86% of KRAS mutations and
76% of TP53 mutations were
G->T transversions.
Demarini et
al. (2001)
Mutation
Human, K-ras mutations
in tumor tissues
Comparison of lung tumors or sputum
samples from 102 lung cancer patients (41
nonsmoking women and 61 smoking men)
who used smoky coal in their homes in
Xuan Wei County, Yunnan Province, China,
and 50 lung cancer patients (14
nonsmoking women, 33 smoking men, 3
nonsmoking men) from Beijing and Henan
using natural gas in the home.
+
The frequency of nonsmoking
women in Xuan Wei county
with mutations (21.9%) and
G->T transversions (66.7%)
were similar to that of smoking
men in Beijing and Henan
(16.7% and 66.7%,
respectively).
Keohavong
et al. (2003)
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BPDE-DNA
adducts
Human, WBCs
96 people occupationally or medically
exposed to PAH mixtures (psoriatic
patients, coke oven workers, chimney
sweeps, and aluminum anode plant
workers); anti-BPDE-DNA adducts in
lymphocyte plus monocyte fraction (LMF)
measured by HPLC/fluorescence analysis.
+
Percentages of subjects with
BPDE-DNA adduct levels
greater than the 95th
percentile control value were
47% (7/15) in coke oven
workers and 21% (4/19) in
chimney sweeps, compared to
3% (1/34) in controls.
Pavanello et
al. (1999)
BPDE-DNA
adducts
Human, WBCs
95 male coke-oven workers from two
plants were tested for GSTM1
polymorphisms and anti-BPDE-DNA
adducts in lymphocyte plus monocyte
fraction (LMF) measured by
HPLC/fluorescence analysis.
+
Compared to GSTMl-active,
GSTMl*0/*0 workers had
significantly higher BPDE-DNA
adducts (p=0.011); these were
significantly related to
exposures to PAHs (p<0.01)
and to lack of GSTM1 (p<0.001)
and not to other sources of
exposure.
Pavanello et
al. (2004)
BPDE-DNA
adducts
Human, WBCs
67 highly exposed coke oven workers
were tested for genetic factors that can
modulate individual responses to
carcinogenic PAHs; adducts measured by
HPLC/fluorescence analysis.
+
Levels of BPDE-DNA adducts
were significantly associated
with workplace PAH exposure
(as correlated with urinary
excretion of 1-pyrenol), lack of
GSTM1 activity, and low
nucleotide excision repair
capacity.
Pavanello et
al. (2005)
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BPDE-DNA
adducts
Human, WBCs
585 Caucasian municipal workers (52%
males, 20-62 years old) from northeast
Italy environmentally exposed to PAH
mixtures were screened for anti-BPDE-
DNA adducts in lymphocyte plus
monocyte fraction (LMF) measured by
HPLC/fluorescence analysis.
+
42% of the participants had
elevated anti-BPDE-DNA
adduct levels, defined as >0.5
adducts/108 nucleotides
(mean, 1.28 ± 2.80 adducts/
108 nucleotides). Comparison
of adduct levels with
questionnaire responses
indicated that smoking,
frequent consumption of
PAH-rich meals (>52 versus
<52 times/yr), and long time
periods spent outdoors
(>4 versus <4 hrs/d) were risk
factors as all increased BPDE-
DNA adduct levels significantly.
Pavanello et
al. (2006)
BPDE-DNA
adducts
Human, WBCs
39 male coke oven workers and 39
matched controls, smokers and non-
smokers, exposed to PAHs for 6-8 hrs/d
for at least 4-6 mo before blood
collection; leukocyte DNA isolated and
digested, and benzo[a]pyrene tetrols
analyzed by HPLC with fluorescent
detection. Low, medium, and high
exposure groups correspond to <0.15,
0.15-4, and >4 mg/m3 of benzo[a]pyrene,
respectively.
+
<0.15, 0.15-4,
or >4 ng/m3 of
benzo[a]pyrene
Anti-BPDE-DNA adducts
detected in 51% of coke oven
workers (mean 15.7±37.8/108
nucleotides) vs. 18% non-
exposed (mean 2.0±8.7/108
nucleotides). Interindividual
variation of adduct levels was
100-fold in workers and 50-fold
in control; smokers had 3.5-
fold more adducts than non-
smokers.
Roias et al.
(1995)
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BPDE-DNA
adducts
Human, WBCs
20 male coke oven workers, all smokers,
were selected from workers studied in
Rojas et al. (1995); workers were exposed
to PAHs for 6-8 hrs/d for at least 4-6 mo
before blood collection; leukocyte DNA
isolated and digested, and benzo[a]pyrene
tetrols analyzed by HPLC with fluorescent
detection. Low, medium, and high
exposure groups correspond to <0.15,
0.15-4, and >4 mg/m3 of benzo[a]pyrene,
respectively.
+
<0.15, 0.15-4,
or >4 ng/m3 of
benzo[a]pyrene
Levels of anti-BPDE-DNA
adducts significantly correlated
with genotype: GSTMl*0/*0 +
CYP1A1*2A/*2A or *2A/*2B »
GSTMl*0/*0 + CYP1A1*1/*1 or
*1/*2A or *1/*2B » GSTM1-
active (no detectable adducts).
Results correlated with adduct
levels in non-tumorous lung
tissues from 20 lung cancer
patients.
Roias et al.
(1998)
BPDE-DNA
adducts
Human, WBCs
89 male coke oven workers and 44 power
plant workers were exposed to PAHs for
6-8 hrs/d for at least 4-6 mo before blood
collection; leukocyte DNA isolated and
digested, and benzo[a]pyrene tetrols
analyzed by HPLC with fluorescent
detection. Low, medium, and high
exposure groups correspond to <0.15,
0.15-4, and >4 mg/m3 of benzo[a]pyrene,
respectively.
+
<0.15, 0.15-4,
or >4 ng/m3 of
benzo[a]pyrene
PAH exposure, CYP1A1 status
and smoking significantly
affected DNA adduct levels,
i.e., CYPlAl(*l/*2 or *2A/*2a)
> CYP1A1*1/*1; occupational >
environmental exposure;
smokers > nonsmokers;
adducts increased with dose
and duration of smoking.
Roias et al.
(2000)
BPDE-DNA
adducts
Human, WBCs
Coke oven workers were exposed to PAHs
and benzo[a]pyrene-WBC DNA analyzed
by HPLC-fluorescence detection for BPDE-
DNA adducts.
+
0.14 ng/m3
Median detectable BPDE-DNA
adducts in workers versus
controls not significant (p=0.51)
due to low number of subjects
(9 workers, 26 controls);
4/9 workers had adducts
substantially higher than all
controls. No significant
difference between smokers
and nonsmokers; no
correlation with air
benzo[a]pyrene levels and
adduct levels.
Mensing et
al. (2005)
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BPDE-DNA
adducts
Human, WBCs
35 iron foundry workers (12 nonsmokers
and 23 smokers) and 10 matched controls
(6 nonsmokers and 4 smokers) between
August 1985 and May 1986; workers
stratified according to job title and
assigned exposure category. BPDE-DNA
adducts measured by ELISA
(immunoassay).
+
<0.05, 0.05-0.2,
or >0.2 ng/m3
of
benzo[a]pyrene
Benzo[a]pyrene exposures
significantly associated with
adduct formation (p=0.0001).
Low, medium, and high
exposure groups all
significantly elevated compared
to controls; low group
significantly higher than
medium or high categories.
Perera et al.
(1988)
BPDE-DNA
adducts
Human, WBCs from
maternal and umbilical
cord blood
Cohort study of 329 nonsmoking pregnant
women exposed to emissions from fires
during the 4 wks following the collapse of
the WTC building in New York City on
09/11/2001; BPDE-DNA adducts measured
by HPLC/fluorescence analysis.
+
BPDE-DNA adduct levels in cord
and maternal blood were
highest in study participants
who lived within 1 mile of the
WTC, with inverse correlation
between cord blood levels and
distance from WTC.
Perera et al.
(2005b);
Perera et al.
(2004)
BPDE-DNA
adducts
Human, WBCs from
umbilical cord blood
164 pregnant women in NYC wearing
personal air monitors during the third
trimester; umbilical cord blood was
screened for BPDE-DNA adducts and
global DNA methylation levels using
HPLC/fluorescence analysis.
+
50% above and
50% below
median of
5.314 ng/m3
(all PAHs
including
pyrene)
BPDE-DNA adducts were not
significantly associated with
individual PAH exposures, but
did correlate with increased
global DNA methylation.
Herbstman
et al. (2012)
BPDE-DNA
adducts
Human, placenta
28 smoking (15) and nonsmoking (13)
pregnant women with uncomplicated
pregnancies; placental nuclei analyzed by
immunoaffinity chromatography,
HPLC/SFS and GC/MS to identify BPDE-
DNA adducts.
+
BPDE-DNA adducts detected;
no correlation with smoking
history.
Manchester
et al. (1988)
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BPDE-DNA
adducts
Human, skin punch
biopsies
10 eczema patients (3 males and 7
females) treated with coal tar ointment
2 times/day for 7-33 days were biopsied
and DNAfrom skin analyzed by HPLC-
fluorescence detection for BPDE-DNA
adducts.
+
3-10% coal tar
ointment
Presence of BPDE-DNA adducts
significantly correlated with
normal myeloperoxidase levels
(wild-type MPO-463GG)
compared to reduced levels in
patients with the MPO-
463AA/AG polymorphism
Roias et al.
(2001)
BPDE-DNA
adducts
Human, lung parenchyma
13 lung cancer patients (11 smokers, 2
exsmokers); nontumorous lung
parenchyma analyzed by HPLC-
fluorescence detection for anti- and syn-
BPDE-DNA adducts.
+
Anti- and syn-BPDE-DNA
adducts detected in 9 of 11
smokers and 2 of 2 exsmokers.
Alexandrov
et al., 1992
BPDE-DNA
adducts
Human, lung tissues
39 lung cancer patients (26 smokers, 11
exsmokers, 2 nonsmokers); tumor and
nontumor tissues (not specified) analyzed
by 32P-postlabelling and synchronous
fluorescence spectrophotometry after
immunoaffinity chromatography and HPLC
to detect BPDE-DNA adducts.
+
Detectable adducts in 33/39 by
postlabelling, 11/39 by
SFS+IAC, and 6 of these 11
when adding HPLC.
Significantly higher levels of
adducts in heavy smokers;
weak association between
adducts and TP53 mutations.
Andreassen
et al. (1996)
BPDE-DNA
adducts
Human, lung tissues
24 lung cancer patients (13 smokers, 11
nonsmokers); nontumorous lung tissues
adjacent to tumor tissue analyzed for PAH-
DNA adducts by 32P-postlabeling and
chromatographic co-migration with BPDE
standard.
+
Putative BPDE-DNA adducts
were significantly higher in
smokers (1.5±1.0/108
nucleotides) than nonsmokers
(0.2±0.2/108 nucleotides)
(p<0.001); may be
overestimation due to co-
migration of other PAH
adducts.
Godschalk et
al. (2002)
1
2 Table D-35. Non-human in vivo genotoxicity studies of benzo[a]pyrene
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Mutation,
germline
Mouse, T-stock, (SEC x
C57BL)F1, (C3H x 101)F1,
or (C3H x C57BL)F1 for
females; (101 x C3H)F1 or
(C3H x 101)F1 for males;
dominant-lethal mutation
assay
12-wk-old males dosed with
benzo[a]pyrene i.p. and mated 3.5-6.5 d
posttreatment with 12-wk-old females
from different stocks; sacrificed on
d 12-15 after vaginal plug was observed;
females kept in a 5-hr dark phase to
synchronize ovulation 5 wks before the
start of the experiment; fertilized eggs
collected 9-11 hrs after mating and first-
cleavage metaphase chromosomes
prepared 20 hrs after mating.
+
500 mg/kg
The percent of dominant lethal
mutations were in the order of
T-stock = (C3H x 101)F1 >
(SEC x C57BL)F1 >
(C3H x C57BL)F1.
Generoso
et al.
(1979)
Mutation,
germline
Mouse, male stocks: (101
x C3H)F1; female stocks
(A): (101 x C3H)F1, (B):
(C3H x 101)F1, (C): (C3H
x C57BL)F1, (D):(SECx
C57BL)F1, (E):T-stock
females; dominant lethal
mutations
In dominant lethal assay, 12-wk-old males
dosed i.p. with benzo[a]pyrene and mated
with 10-12-wk-old (#1) stock A females; or
(#2) stock B females on the day of dosing;
or with (#3a) with stocks B, C, and D
females 3.5-7.5 d postdosing, or with
(#3b) with stocks B, C, D, and E females
3.5-6.5 d postdosing. Control group
mated at time corresponding to 1.5-4.5 d
posttreatment in the test groups.
+
500 mg/kg
Dominant lethal effects were
observed in early to middle
(4.5-5.5 and 6.5-7.5 d
posttreatment, respectively)
spermatozoa and in
preleptotene spermatocytes
(32.5-33.5 and 34.5-35.5 d
posttreatment).
Generoso
et al.
(1982)
Mutation,
germline
Mouse, male stocks:
(101 xC3H)Fl; female
stocks (A): (101 x C3H)F1,
(B): (C3H x 101)F1,
(C): (C3H x C57BL)F1,
(D): (SEC x C57BL)F1,
(E): T-stock females;
heritable translocations
For heritable translocation assay, males
were mated with stocks B and D females
3.5-7.7 d post-benzo[a]pyrene treatment
and male progeny screened for
translocation heterozygosity.
500 mg/kg
No significant differences were
observed between treated and
control progeny.
Generoso
et al.
(1982)
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Mutations
and BPDE-
DNA
adducts,
germline
Mouse, C57BL/6, ell
transgenic (Big Blue®)
Benzo[a]pyrene administered i.p. in corn
oil on d 0,1, and 2; sacrificed at d 4,16,
30, 44, or 119. Caput and cauda
epididymal spermatozoa analyzed for ell
mutation frequency, and DNA adducts
analyzed in testis by liquid
chromatography-MS/MS selected reaction
monitoring with 15N-deoxyguanosine
labeling.
+
50 mg/kg
Exposed spermatocytes
acquired persistent BPDE-DNA
adducts; exposed
spermatogonia gave rise to
spermatocytes with mutations
consistent with a
benzo[a]pyrene spectrum
(GC>TA transversions).
Olsen et al.
(2010)
Mutations
and BPDE-
DNA
adducts,
germline
Mouse, C57BL/6 males,
WT and Xpc_/" with
pUR288 lacZ reporter
gene
Benzo[a]pyrene given via gavage in
sunflower oil 3 times/wk for 1, 4, or 6 wks
(Xpc-/") or 6 wks (WT). Spleen, testis, and
sperm cells analyzed for lacZ mutation
frequency, and DNA adducts analyzed in
testis by [32P]-postlabeling.
+
13 mg/kg
Statistically significant
increases in lacZ mutation
frequencies in Xpc_/" spleen at 4
and 6 wks (dose dependent)
and in WT spleen and sperm at
6 wks; DNA adducts were
statistically significant in testis
in all exposed groups.
Verhofstad
et al.
(2011)
Mutations
and BPDE-
DNA
adducts
Mouse, C57BL/6 lacZ
transgenic
Mice dosed with single i.p. injection of
benzo[a]pyrene in DMSO; sacrificed 1, 3,
5, 7,14, 21, and 28 d posttreatment;
spleen, lung, liver, kidney, and brain
collected, DNA isolated and analyzed for
mutations in lacZ reporter gene in E. coli
and adducts by [32P]-postlabeling assay.
+
50 mg/kg
BPDE-dG adduct levels peaked
between 5 and 7 d
posttreatment, followed by
gradual decline; rate of
removal highest in lung, liver,
and spleen and lowest in
kidney and brain; mutant
frequencies peaked between 7
and 14 d in lung, spleen, liver,
and kidney; brain was not
significant at any time point.
Boerrigter
(1999)
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Mutation
Mouse, C57BL female x
T-strain male; somatic
mutation assay
Mice mated for a 5-d period; 10.25 d post-
appearance of vaginal plug, females
injected i.p. with benzo[a]pyrene or
vehicle; offspring (pups) scored for
survival, morphology, and presence of
white near-midline ventral spots and
recessive spots.
+
100 or
500 mg/kg
Induced coat color mosaics
represent genetic changes
(e.g., point mutations) in
somatic cells. White near-
midline ventral spots and
recessive spots represent
melanocyte cell killing and
mutagenicity, respectively.
Benzo[a]pyrene caused high
incidence of recessive spots but
did not correlate with white
near-midline ventral spots.
Russell
(1977)
Mutation
Mouse, lacZ transgenic
(Muta™Mouse)
Benzo[a]pyrene given via gavage in olive
oil daily for 28 consecutive d; sacrificed 3 d
after last dosing; four organs analyzed for
lacZ mutation frequency.
+
25, 50, and
75 mg/kg-d
Highest lacZ mutation
frequency observed in small
intestine, followed by bone
marrow, glandular stomach,
and liver.
Lemieux et
al. (2011)
Mutation
Mouse, lacZ transgenic
(Muta™Mouse)
Benzo[a]pyrene given orally in corn oil for
5 consecutive d; sacrificed 14 d after last
dosing; 11 organs analyzed for lacZ
mutation frequency.
+
125 mg/kg-d
Highest mutation frequency
observed in colon followed by
ileum > forestomach > bone
marrow = spleen > glandular
stomach > liver = lung >
kidney = heart.
Hakura et
al. (1998)
Mutation
Mouse, C57BL/6J Dlb-1
congenic; Dlb-1 locus
assay
Animals dosed: (1) i.p. with vehicle or
benzo[a]pyrene two, four, or six doses at
96-hr intervals; or (2) single dose of
benzo[a]pyrene given i.p. or orally alone
or 96 hrs following a single i.p. dosing with
10 ng/kg TCDD.
+
40 mg/kg
Benzo[a]pyrene caused a dose-
dependent increase in mutant
frequency; i.p. route showed
higher mutant frequency than
oral route; induction of
mutations were associated
with Ah-responsiveness.
Brooks et
al. (1999)
Mutation
Mouse, C57BL/6 (lacZ
negative and XPA+/+ and
XPA'1')-, hprt mutations in
T lymphocytes
Gavage in corn oil 3 times/wk for 0,1, 5, 9,
or 13 wks; sacrificed 7 wks after last
treatment.
+
13 mg/kg
Mutation sensitivity:
XPA ' > XPA*1*.
Bol et al.
(1998)
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Mutation
Mouse, Cockayne
syndrome-deficient
(Csb'/); heterozygous
(Csb+/~) and WT controls
(Csb+/+); hprt mutation
frequency assay
Csb~/~/lacZ*/~ and Csb+/~/lacZ*/~ mice were
dosed i.p. with benzo[a]pyrene 3 times/wk
for 5, 9, or 13 wks; for hprt mutation
frequency analysis mice were sacrificed
3 wks after last treatment; splenocytes
collected; for lacZ mutation frequency
analysis, mice were sacrificed 3 d after last
treatment and liver, lung, and spleen were
collected.
+
13 mg/kg
lacZ mutation frequency
detected in all tissues but no
differences between WT and
Csb'/~ mice; hprt mutations
significantly higher in Csb~/~
mice than control mice. BPDE-
dGuo adducts in hprt gene are
preferentially removed in WT
mice than Csb_/~ mice.
Wiinhoven
et al.
(2000)
Mutation
Mouse, B6C3Fi,
forestomach H-ras, K-ras,
and p53 mutations
Benzo[a]pyrene given in feed in a 2-yr
chronic feeding study.
+
5, 25, or
100 ppm
68% K-ras (codons 12,13), 10%
H-ras (codon 13), 10% p53
mutations; all G->T
transversions.
Culp et al.
(2000)
Mutation
Mouse, lacZ/galE (Muta™
Mouse); skin painting
study
Mice topically treated with a single dose
or in five divided doses daily; sacrificed 7
or 21 d after the single or final treatment;
DNAfrom skin, liver, and lung analyzed for
mutations.
+sk or
_Li,Lu
1.25 or
2.5 mg/kg (25 or
50 ng/mouse)
Skin showed significant dose-
and time-dependent increase
in mutation frequency; liver
and lung showed no mutations;
mutation frequency for single-
or multiple-dose regimens was
similar.
Dean et al.
(1998)
Mutation
Mouse, T-strain
Benzo[a]pyrene given to pregnant mice by
gavage in 0.5 mL corn oil on GDs 5-10.
+
10 mg/mouse
(5x2 mg)
Davidson
and
Dawson
(1976)
Mutation
Mouse, 129/Ola (WT);
hprt mutations in splenic
T lymphocytes
Single i.p. injection followed by sacrifice
7 wks posttreatment.
+
0, 50, 100, 200,
or 400 mg/kg
Dose-dependent increase in
hprt mutation frequency.
Bol et al.
(1998)
Mutation
Mouse, A/J, male
Single i.p. injection followed by sacrifice
28 days posttreatment.
+
0, 0.05, 0.5, 5, or
50 mg/kg
Dose-dependent increase in
lung tissue K-ras codon 12 G->T
mutation frequency.
Meng et al.
(2010)
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Mutation
Mouse, CD-I; skin
papillomas (Ha-ras
mutations)
Female mice were initiated topically with
a single dose of benzo[a]pyrene and 1 wk
after initiation promoted twice weekly
with 5 nmol TPA for 14 wks. One month
after stopping TPA application, papillomas
were collected and DNA from 10 individual
papillomas was analyzed for Ha-ras
mutations by polymerase chain reaction
and direct sequencing.
+
600 nmol/mouse
About 90% of papillomas
contained Ha-ras mutations, all
of them being transversions at
codons 12 (20% GGA->GTA),
13 (50% GGC->GTC), and 61
(20% CAA->CTA).
Colapietro
et al.
(1993)
Mutation
Rat, Wistar
Single dose by gavage; urine and feces
collected 0-24, 24-48, and 48-72 hrs
posttreatment; urine and extracts of feces
tested in 5. typhimurium TA100 strain with
or without S9 mix and p-glucuronidase.
+
0,1, 5,10, or
100 mg/kg
Fecal extracts and urine
showed mutagenicity >1 and
10 mg/kg body weight
benzo[a]pyrene, respectively.
Highest mutagenic activity
observed for 0-24 hrs
posttreatment for feces and
24-48 hrs posttreatment for
urine with p-glucuronidase ± S9
mix.
Willems et
al. (1991)
BPDE-DNA
adducts
Mouse, lacZ transgenic
(Muta™Mouse)
Benzo[a]pyrene given via gavage in olive
oil daily for 28 consecutive d; sacrificed 3 d
after last dosing; four organs analyzed for
DNA adducts using [32P]-postlabeling with
nuclease PI digestion enrichment.
+
25, 50, and
75 mg/kg-day
Highest adduct levels observed
in liver, followed by glandular
stomach, small intestine, and
bone marrow.
Lemieux et
al. (2011)
BPDE-DNA
adducts
Mouse, (Ahr+/+, Ahr+/-,
Ahr-/-)
Gavage; sacrificed 24 hrs posttreatment.
+
100 mg/kg
No induction of CYP in Ahr-/-,
but all alleles positive for
adduct formation.
Sagredo et
al. (2006)
BPDE-DNA
adducts
Mouse, C57BL/6J
Cyplal(+/-) and Cyplal
(-/-)
Single i.p. injection; sacrificed 24 hrs
posttreatment; liver DNA analyzed by
[32P]-postlabeling assay.
+
500 mg/kg
BPDE-DNA adduct levels 4-fold
higher in Cyplal(-/-) mice than
Cypla!(+/-) mice.
Uno et al.
(2001)
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Reference
BPDE-DNA
adducts
Mouse, B6C3Fi
Benzo[a]pyrene fed in diet for 4 wks
(100 ppm) or for 1, 2, 8,16, and 32 wks
(5 ppm); sacrificed and liver, lungs,
forestomach, and small intestine
collected; DNA analyzed by
[32P]-postlabeling assay.
+
5 ppm (32 wks)
and 100 ppm
(4 wks)
Linear dose-response in 4-wk
study; the 5 ppm groups
showed a plateau after 4 wks
of feeding.
Culp et al.
(2000)
BPDE-DNA
adducts
Mouse, BALB/c
Single i.p. injection; sacrificed 12 hrs
postinjection; liver and forestomach
collected; DNA binding of [3H]-benzo[a]-
pyrene analyzed by scintillation counting.
+
140 nCi/100 g
body weight
Liver DNA had 3-fold higher
binding of benzo[a]pyrene than
that of forestomach.
Gangar et
al. (2006)
BPDE-DNA
adducts
Mouse, BALB/cAnN
(BALB), CBA/JN (CBA);
[32P]-postlabeling assay
Animals dosed i.p. with or without 24-hr
pretreatment with TCDD.
+
50 and
200 mg/kg
Adduct levels similar in both
strains dosed with
benzo[a]pyrene alone. TCDD
pretreatment had a greater
suppressive effect on adduct
formation in BALB relative to
CBA mice at low dose but
resulted in no significant
difference in adduct levels at
high dose.
Wu et al.
(2008)
BPDE-DNA
adducts
Mouse, BALB/c, skin
Four doses of benzo[a]pyrene topically
applied to the shaved backs of animals at
0, 6, 30, and 54 hrs; sacrificed 1 d after last
treatment; DNA analyzed by
[32P]-postlabeling assay.
+
4 x 1.2 nmol/
animal
Five adducts spots detected.
Reddy et al.
(1984)
BPDE-DNA
adducts
Mouse, Swiss, epidermal
and dermal skin
Single topical application on shaved backs;
sacrificed 1, 3, and 7 d posttreatment;
epidermal and dermal cells separated;
DNA isolated, digested with DNAsel, and
estimated DNA binding; adducts separated
by HPLC.
+
250 nmol in
150 nL acetone
Both cells positive for
benzo[a]pyrene adducts;
epidermis > dermis; adducts
persisted up to 7 d with a
gradual decline in levels.
Oueslati et
al. (1992)
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Dose
Comment
Reference
BPDE-DNA
adducts
Rat, CD, peripheral blood
lymphocytes, lungs, and
liver
Single i.p. injection; sacrificed 3 d
posttreatment; DNA analyzed by Nuclease
Pl-enhanced [32P]-postlabeling assay.
+
2.5 mg/animal
BPDE-dG as major adducts and
several minor adducts detected
in all tissues.
Ross et al.
(1991)
BPDE-DNA
adducts
Rat, Sprague-Dawley, liver
Single i.p. injection followed by sacrifice at
4 hrs posttreatment; liver DNA isolated
and analyzed by [32P]-postlabeling assay.
+
100 mg/kg
Two adduct spots detected.
Reddv et al.
(1984)
BPDE-DNA
adducts
Rat, Lewis, lung and liver
Animals received a single oral dose of
benzo[a]pyrene in tricaprylin; sacrificed 1,
2, 4,11, and 21 d postdosing; analyzed
liver and lung DNA for BP-DNA adducts by
[32P]-postlabeling assay and urine for
8-oxo-7,8-dihydro-2'-deoxyguanosine
adducts by HPLC-electrochemical
detection.
+
10 mg/kg
BPDE-dG levels peaked 2 d
after exposure in both tissues,
higher in lungs than liver at all
time points, decline faster in
liver than lung; Increased
8-oxo-7,8-dihydro-
2'-deoxyguanosine levels in
urine and decreased levels in
liver and lung.
Briede et
al. (2004)
BPDE-DNA
adducts
Rat, F344;
[32P]-postlabeling assay
Benzo[a]pyrene given in the diet for 30,
60, or 90 d; animals sacrificed and liver
and lung isolated and DNA extracted and
analyzed for adducts.
+
0, 5, 50, or
100 mg/kg
Adduct levels linear at low and
intermediate doses, nonlinear
at high dose.
Ramesh
and
Knuckles
(2006)
BPDE-DNA
adducts
Rat, Wistar; liver and
peripheral blood
lymphocyte adducts
Single dose by gavage; sacrificed 24 hrs
postdosing; peripheral blood lymphocytes
and liver DNA analyzed by
[32P]-postlabeling for BPDE-DNA adducts.
+
0,10, or
100 mg/kg
At 100 mg/kg dose, total
adduct levels in peripheral
blood lymphocytes were 2-fold
higher than the levels in liver;
adduct profiles differed
between peripheral blood
lymphocytes and liver.
Willems et
al. (1991)
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Endpoint
Test system
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Results
Dose
Comment
Reference
CAs
Mouse, C57 (high AHH
inducible) and DBA (low
AHH inducible) strains;
11-d-old embryos; adult
bone marrows
Study used four matings (female x male):
C57 x C57; DBA x DBA; C57 x DBA; and
DBA x C57; pregnant mice treated orally
on GD 11 with benzo[a]pyrene; sacrificed
15 hrs posttreatment; material liver, bone
marrow and placenta and embryos
collected; male mice dosed similarly and
bone marrows collected; individual
embryo cell suspensions and bone marrow
preparations scored for CAs. Tissue AHH
activity measured.
+
150 mg/kg
Levels of CAs: hybrid embryos
> homozygous DBA embryos >
homozygous C57 embryos;
tissue AHH activity: C57
mothers and their embryos >
DBA females and their
homozygous embryos. No
quantitative correlation
between benzo[a]pyrene-
induced CAs and AHH
inducibility. No differences in
bone marrow mitotic index of
males of different strains
between control and treatment
groups.
Adler et al.
(1989)
CAs
Mouse, 1C3F1 hybrid
(101/E1 x C31 x E1)F1;
CAs in bone marrow
Single dose by gavage; sacrificed 30 hrs
postdosing; bone marrow from femur
isolated and analyzed for CAs.
+
63 mg/kg
Significant increase in CAs in
benzo[a]pyrene-treated
animals compared to controls.
Adler and
Ingwersen
(1989)
CAs
Rat, Wistar; peripheral
blood lymphocytes
Single dose by gavage; sacrificed 6, 24,
and 48 hrs posttreatment; blood from
abdominal aorta collected, whole blood
cultures set up, CAs scored in 100 first-
division peripheral blood lymphocytes per
animal.
0,10,100, or
200 mg/kg
No difference between control
and treatment groups at any
dose or at any sampling time
observed.
Willems et
al. (1991)
CAs
Hamster; bone marrow
Single, i.p. injection of benzo[a]pyrene
dissolved in tricapryline; animals sacrificed
24 hrs post-exposure.
+
25, 50, or
100 mg/kg
Benzo[a]pyrene induced CAs at
50 mg/kg body weight only,
with negative responses at the
low and high dose.
Baver
(1978)
MN
Mouse, lacZ transgenic
(Muta™Mouse)
Benzo[a]pyrene given via gavage in olive
oil daily for 28 consecutive d; blood
samples were collected 48 h after last
dose; percent of PCEs and NCEs reported.
+
25, 50, and
75 mg/kg-d
Statistically significant, dose-
dependent increases in percent
of PCEs and NCEs at all doses.
Lemieux et
al. (2011)
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Endpoint
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Results
Dose
Comment
Reference
MN
Mouse, CD-I and BDF1;
bone marrow
Dosed orally once, twice, or thrice at 24-hr
intervals; sacrificed 24 hrs after last
treatment.
+
250, 500, 1,000,
or 2,000 mg/kg
Significant increase at all doses;
no dose-response; double
dosing at 500 mg/kg dose gave
best response.
Shimada et
al. (1990)
MN
Mouse, CD-I and BDF1,
peripheral blood
reticulocytes
Given single i.p injection; tail blood
collected at 24-hr intervals from 0 to
72 hrs.
+
62.5, 125, 250,
or 500 mg/kg
Maximum response seen at
48 hrs posttreatment.
Shimada et
al. (1992)
MN
Mouse, ICR [Hsd: (ICR)Br]
Benzo[a]pyrene was heated in olive oil
and given orally as a single dose; males,
females, and pregnant mothers used;
pregnant mice dosed on GDs 16-17 and
sacrificed on GDs 17-18; micronuclei
evaluated in adult bone marrow and fetal
liver.
+
150 mg/kg
All groups significantly higher
than controls for MN; fetal liver
more sensitive than any other
group.
Harper et
al. (1989)
MN
Mouse, Swiss albino;
bone marrow
Given orally in corn oil; sacrificed 24 hrs
post-exposure.
+
75 mg/kg
Koratkar et
al. (1993)
MN
Mouse, Swiss; bone
marrow polychromatic
erythrocytes
Given by gavage and sacrificed 36 hrs
posttreatment.
+
75 mg/kg
Rao and
Nandan
(1990)
MN
Mouse, CD-I and MS/Ae
strains
i.p. and oral administration.
+
62.5, 125, 250,
or 500 mg/kg
Good dose-response by both
routes, strains; i.p. better than
oral; MS/Ae strain more
sensitive than CD-I strain.
Awogi and
Sato (1989)
MN
Mouse, BDF1, bone
marrow
Male and female mice aged 12-15 wks
given single i.p. injection of
benzo[a]pyrene or corn oil; sacrificed 24,
48, and 72 hrs posttreatment; bone
marrow smears prepared, stained with
May-Grunwald-Giemsa technique and
scored for MN PCEs.
+
0, 25, 50, or
60 mg/kg
Positive at all doses, time
points, and sexes tested. Dose-
dependent increase in MN
observed in both sexes; males
responded better than females;
highest positive response
observed at 72 hrs
postinjection.
Balanskv et
al. (1994)
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Endpoint
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Results
Dose
Comment
Reference
MN
Mouse, HRA/Skh hairless,
keratinocytes
Single topical application.
+
0.5, 5, 50, 100,
or
500 mg/mouse
He and
Baker
(1991)
MN
Mouse, HOS:HR-l,
hairless; skin micronuclei
Topical application once daily for 3 d;
sacrificed 24 hrs after last treatment.
+
0.4,1, 2, or 4 mg
Nishikawa
et al.
(2005)
MN
Mouse, HR-1 hairless, skin
(benzo[a]pyrene with
slight radiation)
+
Exposure to sunlight simulator
to evaluate photogenotoxicity
and chemical exposure.
Hara et al.
(2007)
MN
Rat, Sprague-Dawley,
peripheral blood
reticulocytes
Given single i.p injection; tail blood
collected at 24-hr intervals from 0 to
96 hrs.
+
62.5, 125, 250,
500, or
1,000 mg/kg
Maximum response seen at
72 hrs posttreatment.
Shimada et
al. (1992)
MN
Rat, Sprague-Dawley,
pulmonary alveolar
macrophages
Intratracheal instillation, once/day for 3 d.
+
25 mg/kg
De Flora et
al. (1991)
MN
Rat, Sprague-Dawley,
bone marrow cells
Intratracheal instillation, once/day for 3 d.
-
25 mg/kg
De Flora et
al. (1991)
MN
Hamster; bone marrow
Single, i.p. injection of benzo[a]pyrene
dissolved in tricaprylin; animals sacrificed
30 hrs post-exposure.
100, 300, or
500 mg/kg
Baver
(1978)
MN
Fish (carp, rainbow trout,
clams); blood and
hemolymph
+
0.05, 0.25, 0.5,
orl ppm
Kim and
Hvun
(2006)
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Endpoint
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Results
Dose
Comment
Reference
DNA
strand
breaks
Rat, Sprague-Dawley;
comet assay
Instilled intratracheal^ with: (1) single
dose of benzo[a]pyrene in aqueous
suspension; sacrificed at 3, 24, and 48 hrs
posttreatment; alveolar macrophages,
lung cells, lymphocytes, and hepatocytes
collected or (2) dose-response study and
sacrificed at 24 hrs posttreatment; lungs
collected; controls received normal saline
instillation; all cells analyzed by comet
assay.
+
Experiment #1:
3 mg of
benzo[a]pyrene;
Experiment #2:
dose-response
study with 0.75,
1.5, or 3 mg
benzo[a]pyrene
All time points showed
significant increase in SSBs
(Experiment #1); a dose-
response in SSBs was observed
(Experiment #2).
Garrv et al.
(2003a);
Garrv et al.
(2003b)
DNA
strand
breaks
Aquatic organisms: carp
(Cyprinus carpio), rainbow
trout (Oncorhynchus
mykiss), and clams
(Spisula sachalinensis);
Comet assay
All organisms acclimatized in tanks for 2 d,
water changed every 24 hrs; exposed to
benzo[a]pyrene in DMSO in a tank; one-
third volume of tank contents changed
every 12 hrs; organisms sacrificed at 24,
48, 72, and 96 hrs posttreatment; cell
suspensions prepared from liver (carp and
trout) or digestive gland (clam) for comet
assay.
+
0.05, 0.25, 0.5,
and 1 ppm
Significant dose-response for
strand breaks observed; carp
and trout liver showed highest
response at 48 hrs and clam
digestive gland showed time-
dependent increase at highest
concentration.
Kim and
Hvun
(2006)
DNA
strand
breaks
Rat, Brown Norway
UDS determined after 5 and 18 hrs of a
single intragastric dosing.
62.5 mg/kg
Negative at both time points.
Mullaart et
al. (1989)
UDS
Rat, F344
Single i.p. injection of benzo[a]pyrene or
DMSO; sacrificed at 2 or 12 hrs post-
exposure; liver isolated, hepatocyte
cultures were set up and incubated with
10 mCi/mL [3H]-thymidine for 4 hrs;
washed and autoradiography performed.
100 mg/kg
Benzo[a]pyrene was negative
at both time points.
Mirsalis et
al. (1982)
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Results
Dose
Comment
Reference
UDS
Mouse, HOS:HR-l
hairless; skin
Single topical application on two spots on
the backs after stripping stratum corneum
with adhesive tape to enhance
penetration; sacrificed 24 hrs
posttreatment, skin isolated
[3H]-thymidine; cultured; epidermal UDS
measured.
+
0, 0.25, 0.5, and
1% (w/v) in
acetone
UDS index showed a dose-
dependent increase up to 0.5%
benzo[a]pyrene dose and then
plateaued.
Mori et al.
(1999)
UDS
Rat, Brown Norway; liver
Single intragastric injection; sacrificed at
5 and 18 hrs post-injection.
-
62.5 mg/kg
Benzo[a]pyrene was negative
at both time points.
Mullaart et
al. (1989)
UDS
Mouse, (C3Hf x 101)F1
hybrid, germ cells
i.p. injection of benzo[a]pyrene;
[3H]-thymidine injection later.
-
0.3 mL
Concentration not specified.
Sega (1979)
UDS
Mouse, early spermatid
i.p. injection.
250-500 mg/kg
Reviewed bv Sotomavor and
Sega (2000).
Sega (1982)
SCEs
Hamster; SCEs in bone
marrow
8-12-wk-old animals dosed with two i.p.
injections of benzo[a]pyrene given 24 hrs
apart; animals sacrificed 24 hrs after last
treatment; bone marrow from femur
isolated and metaphases analyzed.
+
450 mg/kg
Significant increase in
metaphase SCEs in
benzo[a]pyrene-treated
animals compared to vehicle-
treated controls.
Roszinskv-
Koecher et
al. (1979)
SCEs
Hamster
Animals implanted subcutaneously (s.c.)
with BrdU tablet; 2 hrs later given
phorone (125 or 250 mg/kg) i.p.; another
2 hrs later dosed i.p. with benzo[a]pyrene;
24 hrs post-BrdU dosing, animals injected
with colchicine 10 mg/kg body weight,
sacrificed 2 hrs later; bone marrow from
femur prepared for SCE assay.
+
50 or 100 mg/kg
SCEs increased with low dose
of phorone significantly.
Baver et al.
(1981)
SCEs
Hamster; fetal liver
i.p. injection to pregnant animals on
GDs 11,13, or 15; fetal liver SCEs were
analyzed.
+
50 and
125 mg/kg
Produced doubling of SCE
frequency.
Pereira et
al. (1982)
SCEs
Hamster; bone marrow
Not available
+
2.5, 25, 40, 50,
75, or 100 mg/kg
Frequency of SCEs increased
>40 mg/kg body weight.
Baver
(1978)
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Endpoint
Test system
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Results
Dose
Comment
Reference
SCEs
Mouse, DBA/2 and
C57BL/6, bone marrow
cells
Two intragastric injections given; mice
implanted with Brdll tablets, sacrificed on
d 5, SCEs estimated.
+
10 or 100 mg/kg
SCEs and benzo[a]pyrene-DNA
adducts in the order of C57BI/6
(AHH-inducible) < DBA/2
(AHH-noninducible).
Wielgosz et
al. (1991)
SCEs
Mouse, DBA/2 and
C57BL/6, splenic
lymphocytes
Two intragastric injections given; mice
killed on 5th day and cells cultured for
48 hrs with Brdll.
+
10 or 100 mg/kg
SCEs and benzo[a]pyrene-DNA
adducts in the order of C57BI/6
(AHH-inducible) < DBA/2
(AHH-noninducible).
Wielgosz et
al. (1991)
SCEs
Rat, Wistar; peripheral
blood lymphocytes
Single dose by gavage; sacrificed 6, 24,
and 48 hrs posttreatment; blood from
abdominal aorta collected, whole blood
cultures set up, SCEs scored in 50 second-
division metaphases in peripheral blood
lymphocytes per animal.
+
0,10,100, or
200 mg/kg
Linear dose-response at any
sampling time; however,
significant at the highest dose
only; no interaction between
dose and sampling time.
Willems et
al. (1991)
Mutation
Drosophila melanogaster,
sex-linked recessive lethal
test
Base males exposed to benzo[a]pyrene
were mated with virgin females of Berlin K
or mei-9L1 strains.
+
10 mM
Data inconclusive due to low
fertility rates of mei-911
females.
Vogel et al.
(1983)
Mutation
D. melanogaster, sex-
linked recessive lethal
test
Adult Berlin males treated orally with
benzo[a]pyrene.
+
5 or 7.5 mM
Low mutagenic activity.
Vogel et al.
(1983)
Mutation
D. melanogaster, Berlin-K
and Oregon-K strains; sex-
linked recessive lethal
test
Benzo[a]pyrene dissolved in special fat
and injected into the abdomen of flies.
2 or 5 mM
Negative at both doses.
Ziilstra and
Vogel
(1984)
Mutation
D. melanogaster, sex-
linked recessive lethal
test
Male Berlin K larvae treated with
benzo[a]pyrene for 9-11 d.
+
0.1-4 mM
3-Fold enhancement in lethals
in treated versus controls.
Vogel et al.
(1983)
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Dose
Comment
Reference
Mutation
D. melanogaster, Canton-
S (WT) males, FM6
(homozygous for an
X-chromosome) females;
sex-linked recessive lethal
test
Adult male flies were fed on filters soaked
in benzo[a]pyrene for 48 or 72 hrs; treated
and control males mated with FM6a
females, males transferred to new groups
of females at intervals of 3, 2, 2, and 3 d;
four broods obtained; a group of 100
daughters of each male were mated again;
scored for percent lethal.
250 or 500 ppm
Authors report incomplete
dissolution of benzo[a]pyrene
in DMSO as a possible cause of
negative result.
Valencia
and
Houtchens
(1981)
Mutation
D. melanogaster; somatic
mutation, eye color
mosaicism
Fifty females and 20 females were mated
in a culture bottle for 48 hrs allowing
females to oviposit; adults were then
discarded and the eggs were allowed to
hatch; larvae fed on benzo[a]pyrene
deposited on food surface and the
emerging adult males were scored for
mosaic eye sectors.
+
1, 2, or 3 mM
Benzo[a]pyrene was effective
as a mutagen; no dose-
response observed.
Fahmv and
Fahmv
(1980)
Cell trans-
formation
Hamster, LVG:LAK strain
(virus free);
transplacental host-
mediated assay
Pregnant animals dosed i.p. with
benzo[a]pyrene on GD 10; sacrificed on
GD 13, fetal cell cultures prepared,
10 x 10s cells/plate; 5 d post-culture
trypsinized; subcultured every 4-6 d
thereafter and scored for plating efficiency
and transformation.
+
3 mg/100 g body
weight
Quarles et
al. (1979)
aFM6 = First Multiple No. 6 is an X-chromosome with a complex of inversions (to suppress cross-over) and visible markers such as yellow body and white and
narrow eyes.
NCE = normochromatic erythrocyte; PCE = polychromatic erythrocyte; UDS = unscheduled DNA synthesis; XPA = xeroderma pigmentosum group A.
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D.5.2. Tumor Promotion and Progression
Cytotoxicity and Inflammatory Response
The cytotoxicity of benzo[a]pyrene metabolites may contribute to tumor promotion via
inflammatory responses leading to cell proliferation (Burdick et al.. 20031. Benzo[a]pyrene is
metabolized to o-quinones, which are cytotoxic, and can generate ROS (Bolton etal.. 2000: Penning
et al.. 19991. Benzo[a]pyrene o-quinones reduce the viability and survival of rat and human
hepatoma cells (Flowers-Geary etal.. 1996: Flowers-Geary etal.. 19931. Cytotoxicity was also
induced by benzo[a]pyrene and BPDE in a human prostate carcinoma cell line (Nwagbara etal..
20071. Inflammatory responses to cytotoxicity may contribute to the tumor promotion process.
For example, benzo[a]pyrene quinones (1,6-, 3,6-, and 6,12-benzo[a]pyrene-quinone) generated
ROS and increased cell proliferation by enhancing the epidermal growth factor receptor pathway in
cultured breast epithelial cells (Burdick etal.. 20031.
Several studies have demonstrated that exposure to benzo[a]pyrene increases the
production of inflammatory cytokines, which may contribute to cancer progression. Garcon et al.
(2001al and Garcon etal. (2001bl exposed Sprague-Dawley rats by inhalation to benzo[a]pyrene
with or without ferrous oxide (Fe2C>3) particles. They found that benzo[a]pyrene alone or in
combination with Fe2C>3 particles elicited mRNA and protein synthesis of the inflammatory
cytokine, IL-1. Tamaki etal. (20041 also demonstrated abenzo[a]pyrene-induced increase in IL-1
expression in a human fibroblast-like synoviocyte cell line (MH7A). Benzo[a]pyrene increases the
expression of the mRNA for CCL1, an inflammatory chemokine, in human macrophages (N'Diave et
al.. 20061. The benzo[a]pyrene-induced increase in CCL1 mRNA was inhibited by the potent AhR
antago nist, 3'- methoxy- 4'- nitro flavo ne.
AhR-Mediated Effects
The promotional effects of benzo[a]pyrene may also be related to AhR affinity and the
upregulation of genes related to biotransformation (i.e., induction of CYP1A1), growth, and
differentiation (Bostrom etal.. 20021. Figure D-3 illustrates the function of the AhR and depicts the
genes regulated by this receptor as belonging to two major functional groups (i.e., induction of
metabolism or regulation cell differentiation and proliferation). PAHs bind to the cytosolic AhR in
complex with heat shock protein 90 (Hsp90). The ligand-bound receptor is then transported to
nucleus in complex with the Ah receptor nuclear translocator. The AhR complex interacts with the
Ah responsive elements of the DNA to increase the transcription of proteins associated with
induction of metabolism and regulation of cell differentiation and proliferation.
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Supplem en tal Inform ation —Benzo[aJpyren e
PAH
AHR
Hsp90
Hsp90
ARNT
ARNT
AHRE
Enhanced
specific
mRNA
production
ARNT
nuc eus
Increased
synthesis of
PAH metabolizing
enzymes
Increased
synthesis of
proteins that
regulate cell
differentiation and
proliferation
AHRE dna = Ah-responsive elements of DNA; ARNT = Ah receptor nuclear translocator.
Source: Okev et al. (1994).
Figure D-3. Interaction of PAHs with the AhR.
Binding to the AhR induces enzymes that increase the formation of reactive metabolites,
resulting in DNA binding and, eventually, tumor initiation. In addition, with persistent exposure,
the ligand-activated AhR triggers epithelial hyperplasia, which provides the second step leading
from tumor initiation to promotion and progression fNebertetal.. 19931. Ma and Lu f20071
reviewed several studies of benzo[a]pyrene toxicity and tumorigenicity in mouse strains with high
and low affinity AhRs. Disparities were observed in the tumor pattern and toxicity of
Ah-responsive (+/+ and +/-) and Ah-nonresponsive (-/-) mice. Ah-responsive mice were more
susceptible to toxicity and tumorigenicity in proximal target tissues such as the liver, lung, and skin.
For example, Shimizu etal. (20001 reported that AhR knock-out mice (-/-), treated with
benzo[a]pyrene by s.c. injection or dermal painting, did not develop skin cancers at the treatment
site, while AhR-responsive (+/+) or heterozygous (+/-) mice developed tumors within
18-25 weeks after treatment Benzo[a]pyrene treatment increased CYP1A1 expression in the skin
and liver of AhR-positive mice (+/- or +/+), but CYP1A1 expression was not altered by
benzo[a]pyrene treatment in AhR knock-out mice (-/-). Talaska etal. f2 0061 also showed that
benzo[a]pyrene adduct levels in skin were reduced by 50% in CYP1A2 knock-out mice and by 90%
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in AhR knock-out mice compared with WT C57B16/J mice following a single dermal application of
33 mg/kg benzo[a]pyrene for 24 hours. Ma and Lu (20071 further noted that Ah-nonresponsive
mice were at greater risk of toxicity and tumorigenicity in remote organs, distant from the site of
exposure (i.e., bone marrow). As an example, Uno etal. (20061 showed thatbenzo[a]pyrene
(125 mg/kg-day, orally for 18 days) caused marked wasting, immunosuppression, and bone
marrow hypocellularity in CYP1A1 knock-out mice, but not in WT mice.
Some studies have demonstrated the formation of DNA adducts in the liver of AhR knock-
out mice following i.p. or oral exposure to benzo[a]pyrene (Sagredo etal.. 2006: Uno etal.. 2006:
Kondraganti etal.. 20031. These findings suggest that there maybe alternative (i.e., no n-AhR
mediated) mechanisms ofbenzo[a]pyrene activation in the mouse liver. Sagredo etal. f20061
studied the relationship between the AhR genotype and CYP metabolism in different organs of the
mouse. AhR+/+, +/-, and -/- mice were treated once with 100 mg/kg benzo[a]pyrene by gavage.
CYP1A1, CYP1B1, and AhR expression was evaluated in the lung, liver, spleen, kidney, heart, and
blood, via real-time or reverse transcriptase polymerase chain reaction, 24 hours after treatment
CYP1A1 RNA was increased in the lung and liver and CYP1B1 RNA was increased in the lung
following benzo[a]pyrene treatment in AhR+/+ and +/- mice (generally higher in heterozygotes).
Benzo[a]pyrene treatment did not induce CYP1A1 or CYP1B1 enzymes in AhR-/- mice. The
expression of CYP1A1 RNA, as standardized to (3-actin expression, was generally about 40 times
that of CYP1B1. The concentration of benzo[a]pyrene metabolites and the levels of DNA and
protein adducts were increased in mice lacking the AhR, suggesting that there may be an
AhR-independent pathway for benzo[a]pyrene metabolism and activation. The high levels of
benzo[a]pyrene DNA adducts in organs other than the liver of AhR-/- mice may be the result of
slow detoxification of benzo[a]pyrene in the liver, allowing high concentrations of the parent
compound to reach distant tissues.
Uno etal. (20061 also demonstrated a paradoxical increase in liver DNA adducts in AhR
knock-out mice following oral exposure to benzo[a]pyrene. WT C57BL/6 mice and several knock-
outmouse strains (CYP1A2-/- and CYP1B1-/- single knock-out, CYP1A1/1B1-/- and
CYP1A2/1B1-/- double knock-out) were studied. Benzo[a]pyrene was administered in the feed at
1.25,12.5, or 125 mg/kg for 18 days (this dose is well-tolerated by WT C57BL/6 mice for 1 year,
but lethal within 30 days to the CYP1A1-/- mice). Steady-state blood levels of benzo[a]pyrene,
reached within 5 days of treatment, were ~25 times higher in CYP1A1-/- and ~75 times higher in
CYP1A1 /1B1-/- than in WT mice, while clearance was similar to WT mice in the other knock-out
mouse strains. DNA adduct levels, measured by [32P]-postlabeling in liver, spleen, and bone
marrow, were highest in the CYP1A1-/- mice at the two higher doses, and in the CYP1A1 /1B1-/-
mice at the mid dose only. Adduct patterns, as revealed by 2-dimensional chromatography, differed
substantially between organs in the various knock-out types.
AhR signaling may play a role in cytogenetic damage caused by benzo[a]pyrene (Dertinger
etal.. 2001: Dertinger etal.. 20001. The in vivo formation of MN in peripheral blood reticulocytes of
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C57B1/6J mice induced by a single i.p. injection of benzo[a]pyrene (150 mg/kg) was eliminated by
prior treatment with the potent AhR antagonist 3 '-methoxy-4'-nitroflavone. This antagonist also
protected AhR-null allele mice from benzo[a]pyrene-induced increases in MN formation, suggesting
that 3'-methoxy-4'-nitroflavone may also act through a mechanism independent of the AhR
fDertinger etal.. 20001.
Several in vitro studies have suggested that the AhR plays a role in the disruption of cell
cycle control, possibly leading to cell proliferation and tumor promotion following exposure to
benzo[a]pyrene (Andrvsik et al.. 2007: Chung etal.. 2007: Chen etal.. 20031. Chung etal. (20071
showed thatbenzo[a]pyrene-induced cytotoxicity and apoptosis in mouse hepatoma (Hepalclc7)
cells occurred through a p53 and caspase-dependent process requiring the AhR. An accumulation
of cells in the S-phase of the cell cycle (i.e., DNA synthesis and replication) was also observed,
suggesting that this process may be related to cell proliferation. Chen etal. f20031 also
demonstrated the importance of the AhR in benzo[a]pyrene-7,8-dihydrodiol- and BPDE-induced
apoptosis in human HepG2 cells. Both the dihydrodiol and BPDE affected Bcl2 (a member of a
family of apoptosis suppressors) and activated caspase and p38 mitogen-activated protein (MAP)
kinases, both enzymes that promote apoptosis. When the experiments were conducted in a cell line
that does not contain Ah receptor nuclear translocator (see Figure D-3), the dihydrodiol was not
able to initiate apoptotic event sequences, indicating that activation to BPDE by CYP1A1 was
required. BPDE did not induce apoptosis-related events in a p38-defective cell line, illustrating the
importance of MAP kinases in this process. In rat liver epithelial cells (WB-F344 cells), in vitro
exposure to benzo[a]pyrene resulted in apoptosis, a decrease in cell number, an increase in the
percentage of cells in S-phase (comparable to a proliferating population of WB-F334 cells), and
increased expression of cell cycle proteins (e.g., cyclin A) (Andrvsik et al.. 20071. Benzo[a]pyrene-
induced apoptosis was attenuated in cells transfected with a dominant-negative mutation of the
AhR.
Inhibition of gap junctional intercellular communication (GTIC1
Gap junctions are channels between cells that allow substances of a molecular weight up to
roughly 1 kDa to pass from one cell to the other. This process of metabolic cooperation is crucial
for differentiation, proliferation, apoptosis, and cell death and consequently for the two epigenetic
steps of tumor formation, promotion, and progression. Chronic exposure to many toxicants results
in down-regulation of gap junctions. For tumor promoters, such as TPA or TCDD, inhibition of
intercellular communication is correlated with their promoting potency fSharovskava etal.. 2006:
Yamasaki. 19901.
Blaha etal. (20021 surveyed the potency of 35 PAHs, including benzo[a]pyrene, to inhibit
GJIC. The scrape loading/dye transfer assay was employed using a rat liver epithelial cell line that
was incubated in vitro for 15, 30, or 60 minutes with 50 |iM benzo[a]pyrene. After incubation, cells
were washed, and then a line was scraped through the cells with a surgical blade. Cells were
exposed to the fluorescent dye lucifer yellow for 4 minutes and then fixed with formalin. Spread of
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the dye from the scrape line into cells remote from the scrape was estimated under a fluorescence
microscope. Benzo[a]pyrene reduced spread of the dye after 30 minutes of exposure
(approximately 50% of control). Recovery of GJIC was observed 60 minutes after exposure.
Sharovskava et al. (20061 studied the effects of carcinogenic and noncarcinogenic PAHs on
GJIC in HepG2 cells. Individual carcinogenic PAHs inhibited GJIC in a temporary fashion (70-100%
within 24 hours), but removal of the PAH from culture reversed the effect Noncarcinogenic PAHs
had very little effect on GJIC. Benzo[a]pyrene at 20 |j.M inhibited GJIC completely within 24 hours,
while its noncarcinogenic homolog, benzo[e]pyrene, produced <20% inhibition. The effect was not
AhR-dependent, because benzo[a]pyrene inhibited GJIC in HepG2 cells to the same extent as in
hepatoma G27 cells, which express neither CYP1A1 nor AhR. The authors concluded that the
effects of benzo[a]pyrene and benzo[e]pyrene on GJIC were direct (i.e., not caused by metabolites).
D.5.3. Benzo[a]pyrene Transcriptomic Microarray Analysis
The objective of this analysis was to use transcriptomic microarray analysis to help inform
the cancer mode of action for benzo[a]pyrene. A systematic review and meta-analysis approach
was used to: (1) identify studies; (2) analyze the raw data; (3) assess data quality; and (4) combine
evidence from multiple studies to identify genes that were reproducibly active across all of the
studies.
The Gene Expression Omnibus and Array Express microarray repositories were searched
for studies that used benzo[a]pyrene as a test chemical and raw data were available. The search
terms used and the number of studies retrieved are listed in Table D-36. Many of the search terms
included terms for specific PAH mixtures, as benzo[a]pyrene is commonly used as a reference
chemical in PAH mixture studies, to ensure the available and usable benzo[a]pyrene microarray
data were identified.
Table D-36. Search terms and the number of studies retrieved from the gene
expression omnibus and array express microarray repositories
Search term
Number of microarray studies retrieved
Coal tar
2
Polycyclic aromatic hydrocarbons
13
B[a]P
52
Diesel
11
Smoke NOT cigarette
16
Benzo[a]pyrene
53
Fuel oil
1
Cigarette smoke
63
Tobacco smoke
16
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Forty responsive gene expression datasets were identified, representing 26 peer-reviewed
publications. These datasets were further culled for analysis by focusing on publicly available
results and species and organs represented by more than one available dataset on the same
microarray platform. Crossing microarray platforms and species boundaries adds significant
uncertainty to the interpretation with respect to comparisons of the probes being measured, how
those different probes align to the genome and are mapped to specific genes, and creates an open
question regarding the discovery and mapping of orthologous genes across species. Thus, the
analysis included two studies that focused on mouse in vivo transcriptomic studies of the liver
(Gene Expression Omnibus accessions: GSE24907 and GSE18789).
The first study (Malik etal.. 20121. GSE24907, exposed five male Muta mice (a LacZ
transgenic mouse line) per group to 25, 50, or 75 mg/kg benzo[a]pyrene or olive oil vehicle for
28 days by gavage. The second study fYauk etal.. 20111. GSE18789, exposed 27-30-day-old male
B6C3Fi mice to 150 mg/kg benzo[a]pyrene by gavage for 3 days and sacrificed 4 or 24 hours after
the final dose. Both studies were subjected to study quality evaluation by the Systematic Omics
Analysis Review (SOAR) tool.
SOAR was developed to assist in the quick and transparent identification of studies that are
suitable for hazard assessment development. SOAR consists of a series of objective questions that
examine the overall study quality of a transcriptomic microarray study. SOAR combines questions
from the Toxicological Reliability Assessment (ToxR) Tool, the Minimum Information About a
Microarray Experiment (MIAME) standard, and the Checklist for Exchange and Interpretation of
Data from a Toxicology Study. Both studies were determined to be relevant and suitable for hazard
assessment development using SOAR.
Data Analysis Overview
Raw data for both studies were obtained from the Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gOv/geo/l using the GEOquery package (Davis and Meltzer. 20071 in
Bioconductor (a bioinformatics software repository for packages that may be used in the
R statistical environment). Each study was pre-processed, normalized, subjected to quality control
analysis (see below) and analyzed independently to determine the number of active genes using a
fold-change cut-off, and then a subsequent p-value cut-off.
Pre-processing involves the acquisition of data, background subtraction (not performed
here), and synthesis of gene expression data across multiple probesets (only for Affymetrix data,
and only if analysis is performed on a probeset basis). Normalization is the mathematical
adjustment of data to correct. Data were normalized using fastlo within-groups to control for
technical variance (Eckel etal.. 20051.
The raw microarray data from both studies were analyzed for quality using Principal
Components Analysis (PCA) and boxplot analysis. PCA is commonly used for cluster analysis based
on the variance within the dataset The PCA algorithm (in this case, singular value decomposition
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was used) can be thought of projecting the data into a multidimensional space, and drawing an axis
through the data cloud to explain the largest amount of variance. The next axis is drawn through
the cloud to explain the next largest amount of variance while also being orthogonal to the first axis
(e.g., the Y-axis is orthogonal to the X-axis in a Cartesian plane). The idea is that samples will
naturally cluster in a way that is easily visualized in a simple 2-dimensional plot, where the axis
representing the largest variance is the X-axis. For quality control purposes, observation of
samples from the same biological grouping (e.g., all of the controls, or all of the samples treated the
same way for the same duration) clustered in the X-Y plane is preferable. The samples in
GSE24907 separated mostly by group when the normalized data were visualized by PCA. The
boxplots exhibited a somewhat compressed interquartile range. Overall, the data were deemed to
be of high enough quality to continue analysis, although the compressed interquartile range could
manifest data compression issues which may decrease the overall statistical power.
The normalized samples in GSE18709 also separated mostly by group; however, one
benzo[a]pyrene treated 24-hour sample and one 4-hour control sample clustered distantly from the
rest of their groups. This raises concerns that there remains a significant amount of variance in the
data that the normalization could not overcome. This variance may decrease the overall statistical
power of the meta-analysis. The boxplots of normalized data for this study were more compressed
than that for GSE24907.
Data were analyzed using limma and an empirical Bayes moderated t-test fSmvth. 20041.
Following analysis, active genes were identified. A gene was considered active if it exhibited a
1.5-fold-change and a p-value <0.1 in at least one condition or group (e.g., time-point or dose).
A data mining/pathway analysis approach was undertaken using the GeneGo Metacore
software and using the active gene lists. This approach compares the pathways identified from
bioinformatics analyses of the active gene lists from both studies. The active gene lists from both
studies were analyzed using the GeneGo Metacore software. The data were mined to identify
GeneGo Metacore pathways that represent a large number of genes from both datasets. Gene
expression data were overlaid only for those conditions where the gene was at least 1.5-fold up- or
down-regulated. The GeneGo pathways were analyzed for relevance to the hypothesized mode of
action for benzo[a]pyrene, and for pathways that may illustrate new modes of action. This analysis
is strictly an exploratory pathway analysis to help inform the interpretation of the transcriptomics
data.
The pathway analysis is a powerful method for comparing study results and identifying
consistency than a direct comparison of the active gene list For instance, differentially expressed
gene lists reported in the peer-reviewed literature are not reproducible across similar studies (Shi
etal.. 2008: Chuangetal.. 2007: Ein-Dor etal.. 2005: Lossos etal.. 2004: Fortunel etal.. 2003). In
one example, three different studies aimed at identifying genes that confer "sternness" (i.e., genes
which are responsible for conferring stem-cell like capabilities) each yielded 230, 283, and
385 active genes, yet the overlap between them was only one gene fFortunel etal.. 20031. This
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1 demonstrates that the use of simple Venn diagrams to show the overlap of genes across studies are
2 not as informative as pathway analysis, and are less likely to provide support to potential mode-of-
3 action hypotheses.
4 Three candidate pathways were identified. These are:
5 • AhR signaling
6 • DNA damage regulation of the Gl/S phase transition
7 • Nrf2 regulation of oxidative stress
8 Gene differential expression is represented on the pathway map as a "thermometer" next to
9 the protein symbol. Upregulation is symbolized by an upward pointing thermometer, where the
10 length of the red bar represents a relative log2 fold-change. Downregulation is symbolized by a
11 downward pointing thermometer, where the length of the blue bar represents a relative log2 fold-
12 change. A red line connecting proteins represents inhibition. A green line connecting proteins
13 represents activation. A symbol legend accompanies this report
14 Table D-37. Mapping of group numbers to time/dose groups
Number under Thermometer
in Figures D-4-D-6
Dose
Time point
Reference
2
150 mg/kg
3-d exposure (sacrificed 4 hrs after final dose)
Yauketal. (2011)
3
150 mg/kg
3-d exposure (sacrificed 24 hrs after final dose)
Yauketal. (2011)
4
75 mg/kg
28-d exposure
Malik etal. (2012)
5
50 mg/kg
28-d exposure
Malik etal. (2012)
6
25 mg/kg
28-d exposure
Malik etal. (2012)
15 AhR Signaling
16 The AhR regulates the transcription of several genes, including xenobiotic metabolism
17 genes (Figure D-4). It appears that benzo[a]pyrene is activating the AhR in these studies based on
18 the expression of many of its transcriptional targets. Relevant to further analysis and investigating
19 the mode of action, the c-Myc gene is upregulated at 4 and 24 hours in the time-course and at the
20 50 mg/kg dose in the dose-response, while Nrf2 is upregulated at the 4-hour time-point and at the
21 25 and 75 mg/kg doses. c-Myc has been shown to be upregulated following exposure to TCDD, and
22 a putative dioxin response element has been detected in the c-Myc promoter fDere etal.. 2011: Kim
23 etal.. 20001. The AhR has been demonstrated to bind and regulate the Nrf2 promoter fDere etal..
24 2011: Lo etal.. 2011: Nair etal.. 20081.
25
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Sup pi em en tal Inform ation—Benzo[aJpyren e
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For Figures D-4-D-6, the "thermometers" display the fold change gene expression. The numbers under the
thermometer represent the group within the two studies (see Table D-37). For instance, NRF2 is upregulated in
the 25 mg/kg group.
Figure D-4. AhR pathway.
DNA Damage Signaling
The strong up regulation of p21 and MDM2 at 4 hours and 75 mg/kg suggests that p53 is
activated following exposure to benzo[a"|pyrene, suggesting that benzo[a]pyrene induces DNA
damage as early as the 4-hour time-point, and at 75 mg/kg in mice (Figure D-5). MDM2 is a target
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gene of p53, and also negatively feedback inhibits p53 signaling through ubiquitination. Ubiquitin is
also upregulated at 4 hours and 75 mg/kg further suggesting that that p53 may initially be
up regulated at times prior to 4 hours and prior to sacrifice in the 75 mg/kg groups, and that at the
time of sacrifice, the p53 signal may be degraded due to MI)M2-mediated ubiquitination. Coupled
with the upregulation of Cyclin D and PCNA at 75 mg/kg (among other conditions), this suggests a
pro-mitotic shift may be occurring which could lead to cellular proliferation in the liver in the mice
exposed to 75 mg/kg per day.
DNA-damage-induced responses
Role of 14-3-3
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Sup pi em en tal Inform ation—Benzo[aJpyren e
1 a large oxidative stress response is present in the cell to promote the induction of apoptosis
2 fFaraonio et al.. 20061. Upregulation of Cul3 at 4 hours and the 75 mg/kg dose in concert with the
3 upregulation of ubiquitin at the same time and dose suggests that repression of Nrf2 activity may
4 occur. This would support the p53-mediated pro-oxidant hypothesis, which is further
5 substantiated by the lack of upregulation of anti-oxidant genes at 75 mg/kg with the exception of
6 GCL cat.
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8 Nrf2 is upregulated by benzo[a]pyrene exposure, which results in the upregulation of Phase il detoxifying
9 enzymes. This appears to be a compensatory response due to increased oxidative status within cells.
10 Figure D-6. Nrf2 pathway.
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Pathway Analysis Summary
Activation of the AhR appears to be present based on the transcriptional data. This may
lead to formation of oxidative metabolites and radicals which may lead to oxidative damage and
DNA damage. Although the alterations to the Nrf2 pathway suggest cells are gearing up for a pro-
apoptotic environment, there is no transcriptional evidence that the apoptotic pathways are being
activated. Thus, there is significant uncertainty as to whether or not apoptosis may occur.
The transcriptomics data support a potential mutagenic and cellular proliferation mode of
action. The transcriptomics data support the hypothesis that DNA damage is occurring at 4 hours
following three daily doses of 150 mg/kg-day of benzo[a]pyrene and 75 mg/kg-day for 28 days.
This is supported by the transcriptional activation of p53 target genes, including p21 and MDM2.
The transcriptional data further suggest that p53 signaling may be waning under these conditions,
as ubiquitin and MDM2 are both upregulated, and work together to degrade p53. Furthermore, the
transcriptional up regulation of Cyclin D in the 75 mg/kg-day exposure may result in enough Cyclin
D protein to overcome the p21 inhibitory competition for CDK4, allowing for Gl/S phase transition
to occur. In addition, the upregulation of PCNA in the 75 mg/kg-day exposure group together with
upregulation of ubiquitin further supports the argument that cells are moving towards a more
Gl/S phase transition friendly environment Translesion synthesis (i.e., a DNA repair/bypass
mechanism, whereby DNA adducts are allowed to remain in newly synthesized DNA, so as to allow
the cell to continue with DNA synthesis and complete the cell cycle) by ubiquitinated PCNA may
favor mutagenesis if the Gl/S phase transition occurs by allowing DNA adducts to persist in
daughter cells.
There are a number of areas of uncertainty within the transcriptomics data that require
additional research. For instance, transcriptomics data only measure changes in gene expression;
these studies did not monitor changes in protein or metabolite expression, which would be more
indicative of an actual cellular state change. Inferences of protein activation and changes in protein
activity and cellular signaling are made based on the transcriptomics data. Further research is
required at the molecular level to demonstrate that the cellular signaling events being inferred are
actually taking place, and that these events result in phenotypic changes, consistent with the overall
mode of action. The studies also have inherent uncertainty with respect to extrapolation from short
term, high dose studies to low dose exposures across a lifetime. In addition, this work uses a
hypothesized mode of action in the liver to support an overall mode of action.
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1
2 APPENDIX E. DOSE-RESPONSE MODELING FOR
3 THE DERIVATION OF REFERENCE VALUES FOR
4 EFFECTS OTHER THAN CANCER AND THE
s DERIVATION OF CANCER RISK ESTIMATES
6 This appendix provides technical detail on dose-response evaluation and determination of
7 points of departure (PODs) for relevant toxicological endpoints, organized by risk value (reference
8 value or cancer risk value). Except where other software is noted, all endpoints were modeled
9 using the U.S. Environmental Protection Agency's (EPA's) Benchmark Dose Software (BMDS) fU.S.
10 EPA. 2012al: version 2.0 or later. The preambles for the cancer and noncancer parts below
11 describe the practices used in evaluating the model fit and selecting the appropriate model for
12 determining the POD, as outlined in the Benchmark Dose Technical Guidance (U.S. EPA. 2012b).
13 E.l. NONCANCER ENDPOINTS
14 E.l.l. Data Sets
15 The noncancer endpoints that were considered for dose-response modeling are presented
16 in Tables E-l (for the RfD, from oral exposure) and E-2 (for the RfC, from inhalation exposure). For
17 each endpoint, the exposures and response data used for the modeling are presented. See Sections
18 2.1 and 2.2 for discussion of selecting these particular data sets. Further details for some data
19 sets—e.g., regarding data transformations or digitization from figures, highlighting particular
20 subsets or combining similar subsets of data from an investigation—are provided below.
21 All data reported by Chenetal. f20121 were presented graphically; dose group means and
22 standard deviations (SDs) were digitized from the publication. For the Morris water maze data,
23 individual animal data for PND 74 were provided upon request by the study authors. For the
24 elevated plus maze data Chen etal. (2012). the results from female rats at PND 70 were chosen for
25 dose-response analyses, as effects in females and older animals were greater relative to control
26 than in males or at PND 35. For the other outcomes from this study considered for dose-response
27 analysis, data for male and female rats were combined because there was no substantive difference
28 between males and females for each dose group (supported by the authors' statistical testing using
29 two-way analysis of variance [ANOVA], and allowing for interactions), and because there was no
30 rationale or information available suggesting there would be sex-mediated differences for these
31 tests. However, although there were then 20 rats in each dose group, there were 10 litters, with 1
32 male and 1 female from each litter who were not technically independent due to intralitter
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1 correlation. These analyses were carried out using N = 20/group, then repeated using
2 N = 10/group, under a bounding assumption of 100% intralitter correlation.
3 Table E-l. Noncancer endpoints selected for dose-response modeling for
4 benzo[a]pyrene: RfD
Study, Species (strain),
Endpoint
Doses (mg/kg-d) and effect data
Kroese et al. (2001); Rat (Wistar)
Dose
0
3
10
30
N
10
10
10
10
Thymus weight (mg), Male
Thymus weight (mg), Female
Mean ± SDa
Mean ± SDa
380 ± 60
320 ± 60
380 ±110
310 ± 50
330 ± 60
300 ± 40
270 ± 40
230 ± 30
Xu et al. (2010); Rat (Sprague-Dawlev)/
Doseb
0
2.5
5
Female
N
6
6
6
Ovary weight (mg)
Primordial follicles (count)
Mean ± SD
Mean ± SD
0.160 ±0.0146
147 ± 13.8
0.143 ±0.0098
138 ± 23.0
0.136 ±0.0098
115 ± 12.3
Chen et al. (2012); Rat (Sprague-Dawlev)
Dose
0
0.02
0.2
2.0
Open field, number of crossed
squares, M+F, PND 69
Mean ± SD
N
68.1 ± 16.2
(20)
68.4 ± 13.2
(20)
82.5 ± 19.3
(20)
94.5 ± 17.1
(20)
Elevated plus maze—Number of open
arm entries, F—PND 70
Mean ± SD
N
10.24 ± 1.9
(10)
10.36 ± 3.0
(10)
12.89 ±2.7
(10)
16.39 ±3.0
(10)
Morris water maze, M+F:
N
(20)
(20)
(20)
(20)
Escape latency (sec), PND 71
PND 72
PND 73
PND 74
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
33.1 ± 11.4
24.4 ± 9.9
18.0 ± 9.9
9.9 ±5.8
35.8 ± 11.6
26.5 ±7.9
19.7 ± 10.1
12.5 ±5.1
38.6 ±9.9
31.0 ±8.4
25.5 ±7.2
19.1 ±5.9
50.8 ±9.3
47.8 ± 8.4
39.7 ± 11.3
33.5 ±9.9
Gao et al. (2011); Mouse (ICR)/female
Dose0
0
0.71
1.4
2.9
N
26
26
25
24
Cervical epithelial hyperplasia
Incidence
0/26
4/26
6/25
7/24
5
6 aReported as standard error (SE), but confirmed to be standard deviation (SD) by study authors.
7 bTime-weighted average (TWA) doses corresponding to dosing every other day.
8 TWA doses corresponding to dosing twice per week (2/7 days/week).
9
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1 Table E-2. Noncancer endpoints selected for dose-response modeling for
2 benzo[a]pyrene: RfC
Study, Species (strain),
Endpoint
Doses (mg/kg-d) and effect data
Archibong et al. (2002), Female Rats
(F344)
Exposure
level, ng/m3
0
(Carbon black)
25
75
100
N
10
10
10
10
Fetal Survival (litter %)
Mean ± SEa
96.7 ± 1.7
78.3 ±4.1
38.0 ±2.1
33.8 ± 1.3
Archibong et al. (2012), Female Rats
(F344)
Exposure
level, ng/m3
0
50
75
100
N
5
5
5
5
Ovary weight (g)
Ovulation rate (eggs/dam)
Mean ± SEa
Mean ± SEa
0.68 ± 0.004
15.3 ±2.0
0.61 ±0.003
13.9 ±3.0
0.59 ± 0.002
12.8 ±2.5
0.60 ± 0.003
8.3 ± 1.0
3
4 aSE reported in source, converted to SD for modeling using SD = SE x N1/2.
5
6 While the preferred measure for elevated plus maze results is percent of open arm entries
7 or percent of time in the open arms, as a function of total arm entries or time, in order to rule out
8 potential differences in motor activity or general exploration (Hogg. 19961. the data reported by
9 Chen etal. (20121 were not normalized by either quantity. However, since sufficient information
10 was reported to rule out an impact of treatment on total arm entries, the number of open arm
11 entries was considered a suitable measure for dose-response analysis.
12 E.1.2. Dose Response Modeling for Noncancer Endpoints
13 E.1.2.1. Models and Evaluation of Model Fit
14 For each dichotomous endpoint, BMDS dichotomous models were fitted to the data using
15 the maximum likelihood method. For the log-logistic and dichotomous Hill models, slope
16 parameters were restricted to be >1; for the gamma and Weibull models, power parameters were
17 restricted to be >1; and for the multistage models, betas were restricted to be non-negative (bi >0).
18 Each model was tested for goodness-of-fit using a chi-square goodness-of-fit test (x2 p-value <0.10
19 indicates lack of fit). Other factors were also used to assess model fit, such as scaled residuals,
20 visual fit, and adequacy of fit in the low-dose region and in the vicinity of the benchmark response
21 (BMR).
22 For each continuous endpoint, BMDS continuous models were fitted to the data using the
23 maximum likelihood method. For the polynomial models, betas were restricted to be non-negative
24 (in the case of increasing response) or non-positive (in the case of decreasing response data); and
25 for the Hill, power, and exponential models, power parameters were restricted to be >1. Model fit
26 was assessed by a series of tests as follows. For each model, first the homogeneity of the variances
27 was tested using a likelihood ratio test (BMDS Test 2). If Test 2 was not rejected (x2 p-value >0.10),
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then the model was fitted to the data assuming constant variance. If Test 2 was rejected (x2 p-value
<0.10), then the variance was modeled as a power function of the mean, and the variance model
was tested for adequacy of fit using a likelihood ratio test (BMDS Test 3). For fitting models using
either constant variance or modeled variance, models for the mean response were tested for
adequacy of fit using a likelihood ratio test (BMDS Test 4, with x2 p-value <0.10 indicating
inadequate fit). Other factors were also used to assess the model fit, such as scaled residuals, visual
fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.
E.l.2.2. Model selection
For each endpoint selected for modeling, the BMDL estimate (95% lower confidence limit
on the benchmark dose [BMD], as estimated by the profile likelihood method) and Akaike's
Information Criterion (AIC) value were used to select a best-fit model from among the models
exhibiting adequate fit If the BMDL estimates were "sufficiently close," that is, differed by at most
3-fold, then the model selected was the one that yielded the lowest AIC value. If the BMDL
estimates were not sufficiently close, then the lowest BMDL was selected as the POD.
E.l.2.3. Modeling results
The following tables and figures summarize the modeling results for the noncancer
endpoints modeled (RfD: Tables E-3 through E-14, Figures E-l through E-12; RfC: Tables E-15
through E-18, Figures E-13, E-14).
For the dose-response analyses of the Chen etal. (2012) outcomes involving combined male
and female responses, the alternate analyses allowing for 100% intralitter correlation yielded
BMDLs up to 30% lower than assuming complete independence of the pups (analyses not shown).
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1 Table E-3. Summary of BMD modeling results for decreased thymus weight in
2 male Wistar rats exposed to benzo[a]pyrene by gavage for 90 days (Kroese et
3 al.. 2001): BMR = 1 SD change from the control mean
Model
Variance
p-valuea
Goodness of fit
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
p-value
AIC
Constant variance
Linear
0.01
0.74
384.84
12.97
8.97
Nonconstant variance
Hillb
Insufficient degrees of freedom
Linear, polynomial (2-degree), power
0.30
0.23
380.71
16.40
11.30
4
5 aValues <0.10 fail to meet conventional goodness-of-fit criteria.
6 bPower restricted to >1.
Linear Model with 0.95 Confidence Level
Linear
450
400
350
300
250
BMDL
BMD
0
5
10
15
20
dose
7 15:33 10/15 2009
8 BMDs and BMDLs indicated are associated with a change of 1 SD from the control, and are in units of mg/kg-day.
9 Figure E-l. Fit of linear model (nonconstant variance) to data on decreased
10 thymus weight in male Wistar rats—90 days (Kroese et al.. 2001).
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Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2 0 01\90day\thymusweight\male\durationadj usted\2Linkrolin. (
d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2 001\90day\thymusweight\male\durationadjusted\2Linkrolin.p
It
BMDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
Dependent variable = mean
Independent variable = dose
The polynomial coefficients are restricted to be negative
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial
lalpha =
rho =
beta_0 =
beta 1 =
Parameter Values
8.56121
0
380.763
-5.3285
Asymptotic Correlation Matrix of Parameter Estimates
lalpha
rho
beta_0
beta 1
lalpha
1
-1
0.048
-0.061
rho
-1
1
-0.048
0.061
beta_0
0.048
-0.048
1
-0 .84
beta_l
-0.061
0.061
-0 .84
1
Parameter Estimates
Variable
lalpha
rho
beta_0
beta 1
Estimate
-18.8293
4.66515
378.954
-5.14219
Std. Err.
9.75429
1.67581
16.5291
1.00497
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-37.9473
1.38062
346.558
-7.11189
0.288754
7.94967
411.351
-3.17249
Table of Data and Estimated Values of Interest
Dose
Obs Mean
Est Mean Obs Std Dev Est Std Dev Scaled Res.
0 10
2.1 10
7.1 10
21.4 10
380
380
330
270
379
368
342
269
60
110
60
40
84 . 3
78.8
6 6.6
37 . 9
0.0392
0 .475
-0.591
0.0908
Model Descriptions for likelihoods calculated
Model A1: Yij = Mu(i) + e(ij)
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Var$$e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigrna(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var$$e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var$$e(i)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
A1 -189.116991 5 388.233982
A2 -183.673279 8 383.346558
A3 -184.883626 6 381.767253
fitted -186.353541 4 380.707081
R -196.353362 2 396.706723
Test 1:
Test
Test
Test
Explanation of Tests
and/or variances differ among Dose levels?
Do responses
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adequately modeled? (A2 vs
Does the Model for the Mean Fit? (A3 vs.
A3)
fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df p-value
Test 1 25.3602 6 0.0002928
Test 2 10.8874 3 0.01235
Test 3 2.42069 2 0.2981
Test 4 2.93983 2 0.2299
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 16.4008
BMDL = 11.2965
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Table E-4. Summary of BMD modeling results for decreased thymus weight in
female Wistar rats exposed to benzo[a]pyrene by gavage for 90 days (Kroese
etal.. 2001): BMR = 1 SD change from the control mean
Model (constant variance)
Goodness of fit
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
Variance
p-valuea
Mean
p-valuea
AIC
Hillb
NA
Linearc
0.17
0.81
349.12
10.52
7.64
Polynomial (2-degree)b
0.17
0.77
350.80
13.29
7.77
Power
NA
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bLowest degree polynomial with an adequate fit is reported.
BMD/BMC = maximum likelihood estimate (MLE) of the dose/concentration associated with the selected BMR;
NA = not applicable; model failed to generate.
Linear Model with 0.95 Confidence Level
Linear
360
340
320
300
280
260
240
220
BMDL
BMD
200
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5
10
15
20
dose
16:27 10/15 2009
BMDs and BMDLs indicated are associated with a change of 1 SD from the control, and are in units of mg/kg-day.
Figure E-2. Fit of linear model (constant variance) to decreased thymus
weight in female Wistar rats exposed to benzo[a]pyrene by gavage for 90 days
(Kroese et al.. 20011.
This document is a draft for review purposes only and does not constitute Agency policy.
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Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2 0 01\90day\thymusweight\female\durationadj usted\2Linkrolin
. (d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2 0 01\90day\thymusweight\female\durationadj usted\2Linkrolin
.pit Thu Oct 15 16:27:44 2009
BMDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*dose^2 + ...
Dependent variable = mean
Independent variable = dose
rho is set to 0
The polynomial coefficients are restricted to be negative
A constant variance model is fit
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 1
rho = 0 Specified
beta_0 = 322.144
beta 1 = -4.2018
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
alpha beta_0 beta_l
alpha 1 2.4e-008 -2.3e-008
beta_0 2.4e-008 1 -0.68
beta 1 -2.3e-008 -0.68 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
alpha 1954.92 437.134 1098.16 2811.69
beta_0 322.144 9.48287 303.558 340.73
beta 1 -4.2018 0.837537 -5.84334 -2.56026
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
0
10
320
322
60
44.2
-0.153
2 . 1
10
310
313
50
44.2
-0.237
7 . 1
10
300
292
40
44.2
0 . 55
21.4
10
230
232
30
44.2
-0.159
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Model Descriptions for likelihoods calculated
Model A1: Yij = Mu(i) + e(ij)
Var$$e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) +e(ij)
Var$$e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) +e(ij)
Var$$e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var$$e(i)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
A1 -171.357252 5 352.714504
A2 -168.857234 8 353.714467
A3 -171.357252 5 352.714504
fitted -171.562118 3 349.124237
R -181.324151 2 366.648303
Test 1:
Test 2
Test 3
Test 4
(Note:
Explanation of Tests
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adequately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
When rho=0 the results of Test 3 and Test 2 will be the same.)
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio) Test df
24.9338
5.00004
5.00004
0.409733
p-value
0.0003512
0.1718
0.1718
0.8148
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 10.5228
BMDL = 7.64037
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-5. Summary of BMD modeling results for decreased ovary weight in
2 female Sprague-Dawley rats exposed to benzo[a]pyrene by gavage for 60 days
3 (Xu et al.. 2010): BMR = 1 SD change from the control mean
Model
Goodness of fit
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
p-value
AIC
Power
NAa
Linear, polynomial (1°)
0.39
-138.67
2.27
1.49
4
5 aNA = not applicable; model failed to generate.
Linear Model with 0.95 Confidence Level
0.18
Linear
0.17
0.16
0.15
0.14
0.13
BMDL
BMD
0
1
2
3
4
5
dose
6 16:03 12/14 2010
7 Figure E-3. Fit of linear/polynomial (1°) model to data on decreased ovary
8 weight in female Sprague-Dawley rats exposed to benzo[a]pyrene by gavage
9 for 60 days (Xu et al.. 2010).
10
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Polynomial Model. (Version: 2.16; Date: 05/26/2010)
Input Data File:
C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2GlG_AbsOvaryWeight_Linear_lSD.(d)
Gnuplot Plotting File:
C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_lSD.pit
Tue Dec 14 13:51:32 2010
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*dose^2 + ...
Dependent variable = Mean
Independent variable = Dose
rho is set to 0
Signs of the polynomial coefficients are not restricted
A constant variance model is fit
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 0.000136
rho = 0 Specified
beta_G = 0.158333
beta 1 = -0.0048
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
alpha beta_0 beta_l
alpha 1 4e-010 -4.5e-G10
beta_0 4e-010 1 -0.77
beta 1 -4.5e-010 -0.77 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable
alpha
beta_0
beta 1
Estimate
0.000118889
0.158333
-0.0048
Std. Err.
. 96296e-005
0.00406354
0.00125904
Lower Conf. Limit
4 . 12162e-005
0.150369
-0.00726768
Upper Conf. Limit
0.000196562
0.166298
-0.00233232
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
0 6 0.16 0.158 0.0147 0.0109 0.374
2.5 6 0.143 0.146 0.0098 0.0109 -0.749
5 6 0.136 0.134 0.0098 0.0109 0.374
Model Descriptions for likelihoods calculated
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Model A1: Yij = Mu(i) + e(ij)
Var$$e(ij)} = SigmaA2
Model A2: Yij = Mu(i) +e(ij)
Var$$e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) +e(ij)
Var$$e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var$$e(i)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
A1 72.766595 4 -137.533190
A2 73.468565 6 -134.937129
A3 72.766595 4 -137.533190
fitted 72.335891 3 -138.671782
R 67.008505 2 -130.017010
Test
1:
Test
2 :
Test
3 :
Test
4 :
(Note:
Explanation of Tests
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adequately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df p-value
Test 1 12.9201 4 0.01167
Test 2 1.40394 2 0.4956
Test 3 1.40394 2 0.4956
Test 4 0.861408 1 0.3533
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 2.27159
BMDL = 1.49968
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-6. Summary of BMD modeling results for decreased primordial
2 follicles in female Sprague-Dawley rats exposed to benzo[a]pyrene by gavage
3 for 60 days (Xu et al.. 2010): BMR = 1 SD change from the control mean
Model
Goodness of fit
BMDisd
mg/kg-d
BMDLisd
mg/kg-d
Basis for model selection
p-value
AIC
Constant variance
Exponential (model 2)e
0.31
123.82
2.40
1.47
Among adequately fitting
models, with narrow range of
BMDLs, Linear model had
lowest AIC.
Exponential (model 3)e
NA
124.80
3.35
1.60
Exponential (model 4)e
0.31
123.82
2.40
1.24
Power6
NA
124.80
3.37
1.70
BMDiord
mg/kg-d
BMDLiord
mg/kg-d
Polynomial (2°)d
NA
124.80
3.39
1.70
Linear, polynomial (1°)
0.37
123.59
2.48
1.60
2.33
1.64
4
5
c
o
CL
0
CtL
c
ro
0
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDI
160
150
140
130
120
110
100
14:07 04/20 2016
Linear
BMDL
dose
7
8
9
Figure E-4. Fit of linear/polynomial (1°) model to primordial follicle count
data for female Sprague-Dawley rats exposed to benzo[a]pyrene by gavage for
60 days (Xu etal.. 2010).
10
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Exponential Model. (Version: 1.9; Date: 01/29/2013)
Input Data File:
C:/BMDS250_2 014/Data/BenzoaPyrene_iris2 016/exp_IRIS_BaP_ovafollicles_adj_Exp-ConstantVariance-
BMRlStd-Down.(d)
Gnuplot Plotting File:
Wed Apr 20 13:50:20 2016
The form of the response function by Model:
Model 2
Model 3
Model 4
Model 5
Y[dose]
Y[dose]
Y[dose]
Y[dose]
a * expfsign
a * expfsign
a * [c-(c-1)
a * [c-(c-1)
b * dose}
(b * dose)Ad}
exp{-b * dose}]
exp{-(b * dose)Ad}]
Note: Y[dose] is the median response for exposure = dose;
sign = +1 for increasing trend in data;
sign = -1 for decreasing trend.
Model 2 is nested within Models 3 and 4.
Model 3 is nested within Model 5.
Model 4 is nested within Model 5.
Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
rho is set to 0.
A constant variance model is fit.
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 500
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
MLE solution provided: Exact
Initial Parameter Values
Variable Model 2
lnalpha 5.48899
rho(S) 0
a 117.306
b 0.0491001
c 0
d 1
(S) = Specified
Parameter Estimates
Variable Model 2
lnalpha 5.54529
rho 0
a 149.342
b 0.0471702
c 0
d 1
NC = No Convergence
This document is a draft for review purposes only and does not constitute Agency policy.
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Table of Stats From Input Data
Dose N Obs Mean Obs Std Dev
0
6
147
13 .81
2 . 5
6
138
23 . 02
5
6
115
12 .28
Estimated Values of Interest
Dose Est Mean Est Std Scaled Residual
0 149.3 16 -0.3585
2.5 132.7 16 0.8068
5 118 16 -0.4539
Other models for which likelihoods are calculated:
Model R1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) +e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) +e(ij)
Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)
Model R: Yij = Mu + e(i)
Var{e(ij)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood) DF AIC
A1 -58.40088 4 124.8018
A2 -56.97516 6 125.9503
A3 -58.40088 4 124.8018
R -63.43841 2 130.8768
2 -58.90764 3 123.8153
Additive constant for all log-likelihoods = -16.54. This constant added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Does response and/or variances differ among Dose levels? (A2 vs. R)
Are Variances Homogeneous? (A2 vs. Al)
Are variances adequately modeled? (A2 vs. A3)
Does Model 2 fit the data? (A3 vs. 2)
Tests of Interest
Test -2*log(Likelihood Ratio) D. F. p-value
Test 1 12.93 4 0.01164
Test 2 2.851 2 0.2403
Test 3 2.851 2 0.2403
Test 4 1.014 1 0.3141
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose
levels, it seems appropriate to model the data.
The p-value for Test 2 is greater than .1. A homogeneous
variance model appears to be appropriate here.
This document is a draft for review purposes only and does not constitute Agency policy.
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The p-value for Test 3 is greater than .1. The modeled
variance appears to be appropriate here.
The p-value for Test 4 is greater than .1. Model 2 seems
to adequately describe the data.
Benchmark Dose Computations:
Specified Effect = 1.000000
Risk Type = Estimated standard deviations from control
Confidence Level = 0.950000
BMD = 2 .40255
BMDL = 1.4 6958
This document is a draft for review purposes only and does not constitute Agency policy.
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Table E-7. Summary of BMD modeling results for mean number of squares
crossed on PND 69 by male and female Sprague Dawley rats exposed to
benzo[a]pyrene by gavage, PNDs 5-11 (Chen etal.. 2012): BMR = 1 SD change
from control mean
Model3
Goodness of fit
BMDisd
(mg/kg)
BMDLisd
(mg/kg)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.0244
538.60
1.52
1.18
One model provided an adequate
fit and a valid BMDL estimate—
the Exponential M4 CV model
was selected/
Exponential (M4)
0.727
533.30
0.225
0.105
Exponential (M5)
N/Ac
535.18
0.221
0.107
Hill
N/Ac
535.18
0.229
0.0839
Linear, Powerd
Polynomial 2°, 3°d
0.0285
538.29
1.44
1.08
aConstant variance case presented (BMDS Test 2 p-value = 0.404), selected model in bold; scaled residuals for
selected model for doses 0, 0.02, 0.2, and 2 mg/kg were 0.22, -0.27, 0.05, and -0.01, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary); this model reduced to the Exponential (M2)
model.
cNo available degrees of freedom to calculate a goodness of fit value.
dFor the Power model, the power parameter estimate was 1; f or the Polynomial 2° and 3° models, the coefficient
estimates of higher order than bl were 0 (boundary of parameter space). These models reduced to the Linear
model.
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Exponential 4
100
90
80
70
-
60
0
0.5
1
1.5
2
Figure E-5. Plot of mean squares crossed on PND 69 by male and female
Sprague Dawley rats exposed to benzo[a]pyrene by gavage on PNDs 5-11, by
dose, with fitted curve for Exponential (M4) model with constant variance
(Chen et al.. 20121: BMR = 1 SD change from control mean; dose shown in
mg/kg.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Exponential Model (Version: 1.10; Date: 01/12/2015)
2 The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
3 A constant variance model is fit
4
5 Benchmark Dose Computation.
6 BMR = 1.0000 Estimated standard deviations from control
7 BMD = 0.224896
8 BMDL atthe 95% confidence level = 0.104872
9
10 Parameter Estimates
Variable
Estimate
Default initial parameter values
Inalpha
5.56624
5.56471
rho
N/A
0
a
67.303
64.695
b
4.00574
1.02094
c
1.4046
1.53357
d
N/A
1
11
Table ol
Data and Estimated Va
ues of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
20
68.1
67.3
16.17
16.17
0.2204
0.02
20
68.44
69.4
13.15
16.17
-0.2654
0.2
20
82.51
82.31
19.27
16.17
0.05465
2
20
94.49
94.53
17.13
16.17
-0.009693
13
14 Likelihoods of Interest
Model
Log(likelihood)
Number of parameters
AIC
A1
-262.5886
5
535.1772
A2
-261.1275
8
538.2549
A3
-262.5886
5
535.1772
R
-277.7454
2
559.4908
4
-262.6497
4
533.2994
15
16 Tests of Interest
Test
-2*log(likelihood ratio)
Test df
p-value
Test 1
33.24
6
<0.0001
Test 2
2.922
3
0.4038
Test 3
2.922
3
0.4038
Test 6a
0.1222
1
0.7267
17
18
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-8. Summary of BMD modeling results for elevated plus maze: open
2 arm entries at PND 70 for female Sprague Dawley rats exposed to
3 benzo[a]pyrene by gavage on PNDs 5-11 (Chen etal.. 2012): BMR = 1 SD
Model3
Goodness of fit
BMDisd
(mg/kg)
BMDLisd
(mg/kg)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.154
132.71
1.17
0.898
Among adequately fitting
models, the BMDLs covered a
tenfold range. The Exponential
4 model had the lowest BMDL
(and the lowest AIC).
Exponential (M4)
0.848
131.00
0.208
0.0917
Exponential (M5)
N/Ac
132.96
0.212
0.0921
Hill
N/Ac
132.96
0.214
0.0692
Linear; Power
Polynomial 2°, 3°d
0.180
132.39
1.04
0.759
a Constant variance case presented (BMDS Tests 2 and 3 p-value = 0.719).
b For the Exponential (M3) model, the estimate of d was 1 (boundary). This model reduced to the Exponential (M2) model.
c No available degrees of freedom to calculate a goodness of fit value.
d For the Power model, the power parameter estimate was 1; for the Polynomial 2° and 3° models, the coefficient estimates
of higher order than bl were 0 (boundary of parameter space). All models in this row reduced to the Linear model.
4
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Exponential 4
18
16
14
12
10
8
BMDL
BMD
0
0.5
1
1.5
2
5 15:31 09/28 2016
6
7 Figure E-6. Fit of exponential 4 model for elevated plus maze, open arm maze
8 entries on PND 70 for female Sprague Dawley rats exposed to BaP by oral
9 gavage PNDs 5 - PND 11 (Chen etal.. 2012): BMR = 1 SD.
This document is a draft for review purposes only and does not constitute Agency policy.
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Exponential Model. (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation.
BMR = 1.0000 Estimated standard deviations from control
BMD = 0.208365
BMDL at the 95% confidence level = 0.0916703
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Inalpha
2.07497
2.07406
rho
n/a
0
a
10.0002
9.6045
b
2.84307
1.12639
c
1.63133
1.78088
d
n/a
1
Table of Data and Estimated Values of
nterest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
10
10.11
10
2.31
2.82
0.123
0.02
10
10.22
10.35
3.16
2.82
-0.1448
0.2
10
12.76
12.74
3.1
2.82
0.0243
2
10
16.29
16.29
3.23
2.82
-0.002547
Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
-61.48113
5
132.9623
A2
-60.80983
8
137.6197
A3
-61.48113
5
132.9623
R
-73.16117
2
150.3223
4
-61.49948
4
130.999
Tests of Interest
Test
-2*log(Likelihood Ratio)
Test df
p-value
Test 1
24.7
6
0.0003876
Test 2
1.343
3
0.719
Test 3
1.343
3
0.719
Test 6a
0.0367
1
0.8481
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Table E-9. Summary of BMD Modeling Results for escape latency of male and
female Sprague-Dawley rats at PND 71 exposed to benzo[a]pyrene by gavage
on PNDs 5-11, fChen etal.. 20121: BMR = 1 SDa change from the control mean
Modelb
Goodness of fit
BMDisd
(mg/kg)
BMDLisd
(mg/kg)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)c
0.433
461.23
1.24
1.01
Among adequately fitting models,
BMDLs ranged up to ~6-fold
above that of the Hill model; Hill
model selected for POD
derivation
Exponential (M4)
Exponential (M5)c
0.503
462.01
0.466
0.178
Hill
0.51
461.99
0.494
0.163
Linear, Power
Polynomial 2°, 3°d
0.474
461.05
1.14
0.883
aA common estimate of SD across all trial days for escape latency, PNDs 71-74, yielded a SD of 9 seconds. In order
to implement this value as a BMR across all trial days, the value was treated equivalently as an absolute deviation
of 9 seconds. Also see Section 2.1.2.
bConstant variance case presented (BMDS Test 2 p-value = 0.711, BMDS Test 3 p-value = 0.711).
Tor the Exponential (M3) and (M5) models, the estimate of d was 1 (boundary); these models reduced to the (M2)
and (M4) models, respectively.
dFor the Power model, the power parameter estimate was 1. For the Polynomial 2° and 3° models, the coefficient
estimates of higher order than bl were 0 (boundary of parameter space). The models in this row reduced to the
Linear model.
Hill Model, with BMR of 9 Abs. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
55
50
45
40
35
30
25
0
0.5
1
1.5
2
13:51 07/25 2016
Figure E-7. Plot of escape latency at PND 71 by dose, with fitted curve for Hill
model using constant variance, for male and female Sprague-Dawley rats
exposed to benzo[a]pyrene by gavage on PNDs 5-11 fChen et al.. 20121: BMR =
1 SD from control mean; dose shown in mg/kg.
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Hill Model (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = intercept + v*doseAn/(kAn + doseAn)
3 A constant variance model is fit
4
5 Benchmark Dose Computation
6 BMR = 9 seconds, Absolute deviation
7 BMD = 0.494368
8 BMDL atthe 95% confidence level = 0.162618
9
10 Parameter Estimates
Variable
Estimate
Default Initial Parameter Values
alpha
107.223
112.255
rho
N/A
0
intercept
34.087
33.11
V
23.2217
17.69
n
1
0.308231
k
0.781197
3.30822
11
12 Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
20
33.1
34.1
11.4
10.4
-0.422
0.02
20
35.8
34.7
11.6
10.4
0.498
0.2
20
38.6
38.8
9.9
10.4
-0.0822
2
20
50.8
50.8
9.3
10.4
0.00601
13
14 Likelihoods of Interest
Model
Log(likelihood)
Number of parameters
AIC
A1
-226.779191
5
463.558382
A2
-226.09162
8
468.183241
A3
-226.779191
5
463.558382
fitted
-226.996255
4
461.99251
R
-241.044463
2
486.088927
15
16 Tests of Interest
Test
-2*log(likelihood ratio)
Test df
p-value
Test 1
29.9057
6
<0.0001
Test 2
1.37514
3
0.7114
Test 3
1.37514
3
0.7114
Test 4
0.434128
1
0.51
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Table E-10. Summary of BMD Modeling Results for escape latency of male and
female Sprague-Dawley rats at PND 72 exposed to benzo[a]pyrene by gavage
on PNDs 5-11 (Chen et al.. 2012): BMR = 1 SDa from control mean
Modelb
Goodness of fit
BMD9AD
(mg/kg)
BMD L9AD
(mg/kg)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)c
0.170
430.81
0.991
0.883
Among adequately fitting models,
BMDLs ranged up to ~5-fold that
of the Hill model; Hill selected for
POD derivation
Exponential (M4)
Exponential (M5)c
0.587
429.56
0.322
0.170
Hill
0.598
429.54
0.329
0.162
Linear, Power
Polynomial 2°, 3°d
0.244
430.08
0.833
0.708
aA common estimate of SD across all trial days for escape latency, PNDs 71-74, yielded a SD of 9 seconds. In order
to implement this value as a BMR across all trial days, the value was treated equivalently as an absolute deviation
of 9 seconds. Also see Section 2.1.2.
bConstant variance case presented (BMDS Test 2 p-value = 0.751, BMDS Test 3 p-value = 0.751).
Tor the Exponential (M3) and (M5) models, the estimate of d was 1 (boundary); these models reduced to the (M2)
and (M4) models, respectively.
dFor the Power model, the power parameter estimate was 1. For the Polynomial 2° and 3° models, the coefficient
estimates of higher order than bl were 0 (boundary of parameter space) The models in this row reduced to the
Linear model.
Hill Model, with BMR of 9 Abs. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
50
45
40
35
30
25
20
0
0.5
1
1.5
2
Figure E-8. Plot of mean escape latency at PND 72 by dose, with fitted curve
for Hill model with constant variance for male and female Sprague-Dawley
rats exposed to benzo[a]pyrene by gavage on PNDs 5-11 (Chen et al.. 2012):
BMR = 1 SD from control mean; dose shown in mg/kg.
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Hill Model (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = intercept + v*doseAn/(kAn + doseAn)
3 A constant variance model is fit
4
5 Benchmark Dose Computation
6 BMR = 9 Absolute deviation
7 BMD = 0.329352
8 BMDL atthe 95% confidence level = 0.162043
9
10 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
71.4666
74.9675
rho
N/A
0
intercept
24.9901
24.35
V
32.6091
23.42
n
1
0.391771
k
0.863966
3.25689
11
12 Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
20
24.4
25
9.9
8.45
-0.339
0.02
20
26.5
25.7
7.9
8.45
0.398
0.2
20
31
31.1
8.4
8.45
-0.0634
2
20
47.8
47.8
8.3
8.45
0.00418
13
14 Likelihoods of Interest
Model
Log(likelihood)
Number of parameters
AIC
A1
-210.630456
5
431.260911
A2
-210.025963
8
436.051926
A3
-210.630456
5
431.260911
fitted
-210.769197
4
429.538393
R
-241.925097
2
487.850194
15
16 Tests of Interest
Test
-2*log(likelihood ratio)
Test df
p-value
Test 1
63.7983
6
<0.0001
Test 2
1.20899
3
0.7509
Test 3
1.20899
3
0.7509
Test 4
0.277482
1
0.5984
17
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Table E-ll. Summary of BMD Modeling Results for escape latency of male and
female Sprague-Dawley rats at PND 73 exposed to benzo[a]pyrene by gavage
on PNDs 5-11 (Chen et al.. 2012): BMR = 1 SDa change from control mean
Model3
Goodness of fit
BMD9AD
(mg/kg)
BMD L9AD
(mg/kg)
Basis for model selection
p-value
AIC
Exponential (M2),
Exponential (M3)c
0.113
450.51
1.09
0.956
Among adequately fitting models,
BMDLs ranged ~8-fold from that
of the Hill model; Hill selected for
POD derivation
Exponential (M4)
Exponential (M5)c
0.762
448.24
0.266
0.137
Hill
0.786
448.22
0.272
0.122
Linear, Power
Polynomial 2°, 3°d
0.166
449.74
0.909
0.747
aA common estimate of SD across all trial days for escape latency, PNDs 71-74, yielded a SD of 9 seconds. In
order to implement this value as a BMR across all trial days, the value was treated equivalently as an absolute
deviation of 9 seconds. Also see Section 2.1.2.
bConstant variance case presented (BMDS Test 2 p-value = 0.262, BMDS Test 3 p-value = 0.262), no model was
selected as a best-fitting model.
Tor the Exponential (M3) and (M5) models, the estimate of d was 1 (boundary); these models reduced to the (M2)
and (M4) models, respectively.
dFor the Power model, the power parameter estimate was 1. For the Polynomial 2° and 3° models, the coefficient
estimates of higher order than bl were 0 (boundary of parameter space) The models in this row reduced to the
Linear model.
Hill Model, with BMR of 9 Abs. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
45
40
35
30
25
20
15
0
0.5
1
1.5
2
14:03 07/25 2016
Figure E-9. Plot of mean escape latency at PND 73 by dose, with fitted curve
for Hill model with constant variance, for male and female Sprague-Dawley
rats exposed to benzo[a]pyrene by gavage PNDs 5-11 fChen et al.. 20121:
BMR = 1 SD change from control mean; dose shown in mg/kg.
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Supplem en tal Inform ation —Benzo[aJpyren e
1
2 Hill Model. (Version: 2.17; Date: 01/28/2013)
3 The form of the response function is: Y[dose] = intercept + v*doseAn/(kAn + doseAn)
4 A constant variance model is fit
5
6 Benchmark Dose Computation
7 BMR = 9 Absolute deviation
8 BMD = 0.271642
9 BMDL atthe 95% confidence level = 0.121722
10
11 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
90.2658
94.9287
rho
N/A
0
intercept
18.3451
17.98
V
27.2509
21.74
n
1
0.348791
k
0.550858
3.37789
12
13 Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
20
18
18.3
9.91
9.5
-0.172
0.02
20
19.7
19.3
10.1
9.5
0.207
0.2
20
25.5
25.6
7.21
9.5
-0.0394
2
20
39.7
39.7
11.3
9.5
0.00413
14
15 Likelihoods of Interest
Model
Log(likelihood)
Number of parameters
AIC
A1
-220.073327
5
450.146655
A2
-218.073516
8
452.147032
A3
-220.073327
5
450.146655
fitted
-220.11036
4
448.220721
R
-243.776723
2
491.553446
16
17 Tests of Interest
Test
-2*log(likelihood ratio)
Test df
p-value
Test 1
51.4064
6
<0.0001
Test 2
3.99962
3
0.2615
Test 3
3.99962
3
0.2615
Test 4
0.0740662
1
0.7855
18
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Table E-12. Summary of BMD modeling results for escape latency at PND 74
for male and female Sprague-Dawley rats exposed to benzo[a]pyrene by
gavage PNDs 5-11 (Chen etal.. 2012): BMR = 1 SDa change from control mean
Modelb
Goodness of fit
BMD9AD
(mg/kg)
BMD L9AD
(mg/kg)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)c
2.80E-04
400.22
1.10
0.988
Among adequately fitting models,
BMDLs ranged up to ~7-fold
above that of the Hill model; Hill
model selected for POD
derivation
Exponential (M4)
Exponential (M5)c
0.466
386.39
0.227
0.147
Hill
0.515
386.28
0.226
0.134
Linear, Power
Polynomial 2°, 3°d
0.00166
396.66
0.825
0.689
aA common estimate of SD across all trial days for escape latency, PNDs 71-74, yielded a SD of 9 seconds. In order
to implement this value as a BMR across all trial days, the value was treated equivalently as an absolute deviation
of 9 seconds. Also see Section 2.1.2.
bModeled variance case presented (BMDS Test 2 p-value = 0.00736, BMDS Test 3 p-value = 0.314), no model was
selected as a best-fitting model.
Tor the Exponential (M3) and (M5) models, the estimate of d was 1 (boundary); these models reduced to the (M2)
and (M4) models, respectively.
dFor the Power model, the power parameter estimate was 1. For the Polynomial 2° and 3° models, the coefficient
estimates of higher order than bl were 0 (boundary of parameter space) The models in this row reduced to the
Linear model.
Hill Model, with BMR of 9 Abs. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
40
35
30
25
20
15
10
, BMDL
5
0
0.5
1
1.5
2
Figure E-10. Plot of mean response by dose with fitted curve for Hill model
with modeled variance for escape latency of male and female Sprague-Dawley
rats at PND 74 exposed to benzo[a]pyrene by gavage on PNDs 5-11 (Chenet
al.. 2012): BMR = 9 absolute deviation from control mean; dose shown in
mg/kg.
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Supplem en tal Inform ation —Benzo[aJpyren e
1
2 Hill Model (Version: 2.17; Date: 01/28/2013)
3 The form of the response function is: Y[dose] = intercept + v*doseAn/(kAn + doseAn)
4 A modeled variance is fit
5
6 Benchmark Dose Computation
7 BMR = 9 Absolute deviation
8 BMD = 0.225851
9 BMDL atthe 95% confidence level = 0.134475
10
11 Parameter Estimates
Variable
Estimate
Default initial parameter values
lalpha
0.885005
3.87067
rho
0.998715
0
intercept
10.6552
9.89
V
28.6997
23.635
n
1
0.28055
k
0.494355
3.47106
12
13 Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
20
9.89
10.7
5.75
5.07
-0.675
0.02
20
12.5
11.8
5.1
5.33
0.641
0.2
20
19.1
18.9
5.85
6.76
0.0948
2
20
33.5
33.7
9.93
9.01
-0.0704
14
15 Likelihoods of Interest
Model
Log(likelihood)
Number of parameters
AIC
A1
-192.775022
5
395.550043
A2
-186.77088
8
389.54176
A3
-187.928576
6
387.857153
fitted
-188.14043
5
386.280859
R
-234.533996
2
473.067991
16
17 Tests of Interest
Test
-2*log(likelihood ratio)
Test df
p-value
Test 1
95.5262
6
<0.0001
Test 2
12.0083
3
0.007355
Test 3
2.31539
2
0.3142
Test 4
0.423706
1
0.5151
18
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1 Table E-13. Summary of BMD modeling results for incidence of cervical
2 epithelial hyperplasia in female ICR mice exposed to benzo[a]pyrene by oral
3 exposure for 98 days (Gao et al.. 2011): BMR = 10% extra risk
Model
Goodness of fit
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
p-value
AIC
Gamma
0.6874
82.2821
0.659
0.452
Logistic
0.1422
88.4607
1.422
1.052
Log-logistic
0.8360
81.7004
0.578
0.369
Probit
0.1544
88.1151
1.326
0.979
Log-probit
0.0775
88.2004
1.012
0.686
Multistage
0.6874
82.2821
0.659
0.452
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
0.5
0.4
0.3
0.2
1
0
BMDL
BMD
0
0.5
1
1.5
2
2.5
3
dose
4 19:01 08/26 2011
5 Figure E-ll. Fit of log-logistic model to data on cervical epithelial hyperplasia
6 fGao et al.. 20111
7 ====================================================================
8 Logistic Model. (Version: 2.13; Date: 10/28/2009)
9 Input Data File: C:\Users\hclynch\Documents\_Active Proj ects\_FA4 58 IRISVcBaPMASC Aug
10 2011\bmd modeling\lnl_gao 2011 inflamm cells_Opt.(d)
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Gnuplot Plotting File: C:\Users\hclynch\Documents\_Active Proj ects\_FA4 98 IRIS\xBaP\IASC
Aug 2011\bmd modeling\lnl_gao 2011 inflamm cells_0pt.pit
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose) ) ]
Dependent variable = Col3
Independent variable = Coll
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0
intercept = -1.60901
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background -slope
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
intercept
intercept 1
Parameter Estimates
Variable
background
intercept
slope
Estimate
0
-1.6502
1
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-39.4267
-39.8502
-45.7739
# Param'
4
1
1
Deviance Test d.f.
0.847034
12.6945
P-value
0.8382
0.005346
AIC:
31.7004
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
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0.0000
0.0000
0 .000
0 .000
26
0 .000
0.7100
0.1200
3 . 119
4 .000
26
0 . 532
1.4000
0.2119
5.297
6.000
25
0.344
2.9000
0.3577
8 . 584
7 .000
24
-0.675
ChiA2 = 0.86 d.f. = 3 P-value = 0.8360
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.578668
BMDL = 0.368701
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-14. Summary of BMD modeling results of embryo/fetal survival for
2 female F344 rats exposed to benzo[a]pyrene via inhalation on GDs 11-20
3 (Archibong et al.. 2002): BMR = 10 percentage points absolute deviation from
4 control mean
Model3
Goodness of fit
BMDioad
(Hg/m3)
BMDLioad
(Hg/m3)
Basis for model selection
p-value
AIC
Constant variances assumed3
No adequate fit: variances for
the variability among litter mean
percentages could not be fit as a
function of exposure; no model
selected.
Exponential (M2)
0.0382
214.98
9.49
8.40
Exponential (M3)
0.0239
215.55
12.6
8.73
Exponential (M4)
0.0382
214.98
9.49
7.76
Exponential (M5)
N/Ab
212.45
17.1
12.3
Hill
N/Ab
212.45
18.7
13.5
Linear, Power
Polynomial 2°, 3°c
0.00240
220.52
15.1
13.8
Variances modeled as a function of exposure3
Exponential (M2)
0.0148
213.79
9.42
8.32
Exponential (M3)
0.00515
215.18
11.7
8.44
Exponential (M4)
0.00376
215.75
9.12
6.68
Exponential (M5)
N/Ab
209.36
17.9
12.4
Hill
N/Ab
209.36
19.3
14.0
Linear, Power,
Polynomial 2°, 3°c
2.61E-04
221.86
15.4
14.0
5
6 aUnder constant variance assumption (BMDS Test 2 p-value = 0.000134) and modeled variances (BMDS Test 3
7 p-value = 0.00512), no model was selected as a best-fitting model.
8 bNo available degrees of freedom to calculate a goodness of fit value.
9 Tor the Power model, the power parameter estimate was 1. For the Polynomial 2° and 3° models, the coefficients
10 of higher order than bl were estimated to be 0 (boundary of parameters space). These models reduced to the
11 Linear model.
12
13 As detailed in Table E-14, continuous dose-response models were not successful in fitting
14 the percentage survival data from Archibong et al. f20021 due to non-monotonic variances.
15 Continuous models rely on assuming that a normal distribution can adequately characterize the
16 observed data. However, for dichotomous responses that cover a broad range of responses, as
17 here, the variances may not be straightforward to address. Consequently, characterizing the data in
18 terms of the underlying binomial responses, in order to apply binomial models, was considered.
19 However, the individual animal data needed for applying a nested model were not available, and an
20 approximation of the proportions affected, adjusted for litter effect (the tendency of littermates to
21 respond more like each other than those in other similarly treated litters), was used.
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1 To approximate the underlying incidence data from data reported as the mean of litter
2 percentages, the following steps were taken:
3 • Total number of embryos/fetuses in each group—this was estimated from the number of
4 litters in each group and the mean number of implantations (see Table E-16).
5 • Total number of affected embryos/fetuses in each group—The mean of litter-specific
6 survival percentages was taken to be the overall estimate of surviving embryos/fetuses for
7 each exposure group. As BMDS dichotomous models only address increasing responses,
8 these percentages were converted to the equivalent percentage not surviving (see Table E-
9 16).
10 • Allowance for litter effect, or intralitter correlation—Although the approach of applying
11 continuous models to means and standard deviations appropriately gave equal weight to
12 each litter as the experimental unit, the proportion of affected embryos/fetuses among all in
13 a dose group does not. Consequently, this transformation of the reported data to total
14 affected embryos/fetuses among total exposed embryos/fetuses needed to address litter
15 effect A data adjustment has been developed that reduces the total numbers of fetuses to
16 account for litter effect (Fox etal.. 20161. The adjustment reduces the sample size, here
17 total number of implantations, as a means of addressing litter effect.
18 Dose-response modeling of the adjusted data by BMDS dichotomous models was carried out
19 using the percentage affected in the "% Positive" option, which calculates incidence from inputs of
20 percentage and sample size for each group. The data inputs are bolded in Table E-16.
21 Table E-15. Derivation of incidence data adjusted for design effect, for
22 embryo/fetal resorption data in Archibong et al. (2002)
Endpoint
Exposures and effect data
Embryo/fetus
survival (%)
Exposure (ng/m3), continuous
equivalent
0
(carbon black)
4.6
13.8
18.4
Mean ± SE,
Number of litters (Nl)
Mean number of implantations
96.7 ± 1.7
(10)
8.8
78.3 ±4.1
(10)
8.8
38.0 ±2.1
(10)
9.0
33.8 ± 1.3
(10)
8.8
Embryo/fetus
resoptions3 (%)
Estimated percentage, Pf Number
of embryos/fetuses, (Nf)
3.3
(88)
21.7
(88)
62.0
(90)
66.2
(88)
Design effect Db
1.87
3.61
5.22
5.34
Adjusted N (Nf/D)
47.2
24.4
17.3
16.5
23
24 aEmbryo/fetus resorptions was calculated by subtracting the reported percentage survival from 100%.
25 bThe design effect was estimated using the average of two estimates of the relationship between fetal proportions
26 and design effect developed from historical data from NTP developmental studies of rats (Fox et al., 2016). Using
27 the model Di = exp(a + b*log(PF) + 0.5* Ores2): Dls used the parameter values a=1.6852, b=0.3310, and ares2=0.1248;
28 Dor used the parameter values a=1.8327, b=0.3690, and ares2=0.1090.
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Table E-16. Summary of BMD modeling results for estimated incidence of
embryo/fetal resorptions (Archibong et al.. 2002). adjusted for design effect;
BMR=1, 5, or 20% extra risk3
Modelb
Goodness of fit
BMDi%
(Hg/m3)
BMDLi%
(Hg/m3)
BMDs%
(Hg/m3)
BMDI.5%
(Hg/m3)
BMD2o%
(Hg/m3)
BMDI.20%
(Hg/m3)
p-value
AIC
Gamma
0.634
89.529
2.17
0.681
7.76
3.47
25.2
15.1
Dichotomous-Hill
N/A
91.303
7.35
0.430
13.8
2.26
25.7
18.7
Logistic
0.254
90.102
3.82
2.53
15.7
11.3
41.9
33.7
LogLogistic
0.716
89.436
3.23
0.462
9.34
2.41
25.4
11.4
Probit
0.320
89.593
3.41
2.31
14.2
10.4
39.6
32.2
LogProbit
0.721
89.431
5.34
0.427
10.9
1.85
25.4
10.0
Weibull
0.621
89.548
1.81
0.680
7.19
3.47
24.9
15.1
Multistage 2, 3°c
0.583
89.605
1.18
0.677
5.93
3.46
24.2
15.0
Quantal-Linear
0.805
87.748
0.934
0.672
4.77
3.43
20.7
14.9
N/A=not available
aMultiple BMRs provided in order to inform low-dose extrapolation; the BMR of 20% provides the basis for judging
fit to the observed data. Note that the BMDLs do not reflect allowance for simultaneous predictions; only one
BMDL from the selected model is used to derive a reference value, depending on the degree of low-dose
extrapolation that is justified.
bBasis for Model Selection: Among the adequately fitting models, BMDL20S ranged close to 3-fold, and the quantal-
linear model had the lowest AIC. However, in the response range where a POD is needed, model uncertainty
increases, as shown by increasing range of BMDLs and greater BMD/BMDL range for several models.
Tor Multstage 3°, the b3 parameter was estimated at the boundary of the parameter space (0), and the model
reduced to Multstage 2°.
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Quantal Linear Model, with BMR of 20% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Quantal Linear
0.8
0
BMDL
3MD
0
20
40
60
80
100
dose
13:09 07/28 2016
Figure E-12. Plot of incidence of embryo/fetal resorptions by dose, with fitted
curve for Quantal-Linear model, for F344 female rats exposed to
benzo[a]pyrene by inhalation on GDs 11-20 (Archibong et al.. 2012): dose
shown in ng/m3
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1
2 Quantal Linear Model using Weibull Model (Version: 2.16; Date: 2/28/2013)
3 The form of the probability function is: P [response] = background +
4 (l-background)*[l-EXP(-slope*dose)]
5
6 Benchmark Dose Computation
7 BMR = 20% Extra risk
8 BMD = 20.7381
9 BMDL atthe 95% confidence level = 14.9171
10
11 Parameter Estimates
Variable
Estimate
Default initial parameter values
Background
0.0312684
0.0519837
Slope
0.0107601
0.00980808
Power
N/A
1
12
13 Analysis of Deviance Table
Model
Log(likelihood)
Number of parameters
Deviance
Test df
p-value
Full model
-41.65
4
Fitted model
-41.87
2
0.444722
2
0.8
Reduced model
-61.52
1
39.734
3
<0.0001
14
15 AIC: = 87.7479
16
17 Goodness-of-Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled residuals
0
0.0313
1.476
1.558
47.2
0.07
25
0.2598
6.338
5.295
24.4
-0.48
75
0.5678
9.822
10.726
17.3
0.44
100
0.6697
11.05
10.923
16.5
-0.07
18
19 ChiA2 = 0.43 df=2 p-value = 0.8052
20
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Table E-17. Summary of BMD Modeling Results for ovarian weight in F344
rats exposed to benzo[a]pyrene via inhalation for 14 days prior to mating
(Archibong et al.. 2012): BMR = 10% relative deviation from control mean
Model3
Goodness of fit
BMDiord
(Hg/m3)
BMDLiord
(Hg/m3)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
<0.0001
-140.91
74.7
63.3
No adequate fit; Exponential
(M4) fit shown as illustration
only. Dropping the high dose
group reduced the number of
applicable models; no adequate
fits. (Equivalent coefficients of
variation were <1%)
Exponential (M4)
0.00117
-165.02
41.0
28.6
Exponential (M5)
N/Ac
-167.87
49.4
42.9
Hill
0.0170
-169.86
49.6
44.9
Power, Polynomial
2°, 3°, Lineard
<0.0001
-139.36
77.0
65.6
aConstant variance case presented (BMDS Test 2 p-value = 0.520, BMDS Test 3 p-value = 0.520), no model was
selected as a best-fitting model.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary); this model reduced to the Exponential (M2)
model.
cNo available degrees of freedom to calculate a goodness of fit value.
dFor the Power model, the power parameter estimate was 1. For the Polynomial 2° and 3° models, the coefficient
estimates of higher order than bl were 0 (boundary of parameters space). The models in this row reduced to the
Linear model.
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
0.7
Exponential 4
0.68
0.66
0.64
0.62
0.6
0.58
O
20
40
60
80
100
17:20 07/14 2016
Figure E-13. Plot of mean ovarian weight by dose, with fitted curve for
Exponential (M4) model with constant variance for female F344 rats exposed
to benzo[a]pyrene for 14 days prior to mating fArchibong et al.. 20121:
BMR = 10% relative deviation from control mean; dose shown in ng/m3.
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Table E-18. Summary of BMD modeling results for ovulation rate (ovulated
oocytes/dam) in female F344 rats following inhalation exposure to
benzo[a]pyrene for 14 days (Archibong et al.. 2012): BMR = 1 or 10% relative
deviation from control mean
Model3
Goodness of fit
BMDird
(Hg/m3)
BMDLird
(Hg/m3)
BMDiord
(Hg/m3)
BMDLiord
(Hg/m3)
Basis for model
selection
p-value
AIC
Exponential (M2)
0.423
87.885
2.22
1.24
23.3
13.0
Among adequately fitting
models at BMR=10%
(omitting Exp. M4C), the
BMDLs covered a 3-fold
range; Polynomial 2° had
the lowest AIC. However,
the BMDL at 1% was
excessively low. No model
was selected.
Exponential (M3)
0.721
88.291
36.9
1.49
65.8
15.6
Exponential (M4)c
0.423
87.885
2.22
0.011
23.3
0.190
Exponential (M5)
0.721
88.291
36.9
1.20
65.8
13.5
Hill
N/Ab
90.263
32.3
1.82
64.1
18.3
Power
0.753
88.262
32.2
1.84
64.1
18.4
Polynomial 3°
0.742
88.271
26.3
1.84
60.2
18.4
Polynomial 2°
0.845
86.500
15.1
1.80
47.9
39.0
Linear
0.500
87.551
2.56
1.67
25.6
16.7
aConstant variance case presented (BMDS Test 2 p-value = 0.148, BMDS Test 3 p-value = 0.148), no model was
selected as a best-fitting model.
bNo available degrees of freedom to calculate a goodness of fit value.
Exponential (M4) parameters were identical to Exponential (M2).
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Polynomial
20
15
10
5
0
20
40
60
80
100
Figure E-14. Plot of mean ovulation rate by dose, with fitted curve for
Polynomial 2° model with constant variance, for female F344 rats following
inhalation exposure to benzo[a]pyrene for 14 days fArchibong et al.. 20121:
BMR = 10% relative deviation from control mean; dose shown in ng/m3.
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1 Polynomial Model (Version: 2.20; Date: 10/22/2014)
2 The form of the response function is: Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
3 A constant variance model is fit
4
5 Benchmark Dose Computation
6 BMR = 10% Relative deviation
7 BMD = 47.8549
8 BMDL atthe 95% confidence level = 39.0362
9
10 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
20.5942
25.3125
rho
N/A
0
beta_0
15.6769
15.2145
beta_l
-1.37569E-24
0
beta_2
-0.000684553
-0.00101091
11
12 Table of Data and Estimated Values of Interest
Dose
N
Oberved mean
Estimated mean
Observed SD
Estimated SD
Scaled Residual
0
5
15.3
15.7
4.47
4.54
-0.186
50
5
13.9
14
6.71
4.54
-0.0323
75
5
12.8
11.8
5.59
4.54
0.48
100
5
8.3
8.83
2.24
4.54
-0.262
13
14 Likelihoods of Interest
Model
Log(likelihood)
Number of parameters
AIC
A1
-40.081548
5
90.163096
A2
-37.403195
8
90.806389
A3
-40.081548
5
90.163096
fitted
-40.250096
3
86.500193
R
-43.005249
2
90.010499
15
16 Tests of Interest
Test
-2*log(likelihood ratio)
Test df
p-value
Test 1
11.2041
6
0.08227
Test 2
5.35671
3
0.1475
Test 3
5.35671
3
0.1475
Test 4
0.337097
2
0.8449
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E.1.3. Dosimetry Modeling for Estimation of Human Equivalent Concentrations for
Reference Concentration (RfC)
As discussed in Section 2.2.2, the human equivalent concentration (HEC) was calculated
from the PODadj by multiplying by a dosimetric adjustment factor (DAF), which, in this case, was the
regional deposited dose ratio (RDDRer) for extrarespiratory (i.e., systemic) effects. The observed
developmental effects are considered systemic in nature (i.e., extrarespiratory) and the normalizing
factor for extrarespiratory effects of particles is body weight. The RDDRer was calculated as
follows:
RDDRer =^3lx IMl x CWa
BWa (Ve)h (ftot)h
where:
BW = body weight (kg)
Ve = ventilation rate (L/minute)
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 and clearance (MPPD; Version 2.0 © 2006, publicly
available through the Hamner Institute). See model output below.
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Wed. 03/17/2010. 02:07:20 PM EDT
Region: Entire Lung
o
0.750
0.600
0.450
0.300
0.150
0.0
0.449
0.621
0.127
0.045
Region
Species & Model Info:
Species/Geometry: Human Limited
FRC Vblume: 3300.00 ml
Head Vblume: 50.00 ml
Breathing Route: nasal
Breathing Parameters:
Tidal 'vblume: 860.00 ml
Breathing Frequency: 16.00 1/min
Inspiratory Fraction: 0.50
Pause Fraction: 0.00
Particle Properties:
Diameter: MMAD: 1.70 ^m
GSD: 1.00
Concentration: 4.20 pgAn"3
Figure E-15. Human fractional deposition.
Species = humanlimited
FRC = 33 0 0.0
Head volume = 50.0
Density = 1.0
Number of particles calculated = single
Diameter = 1.7000000000000002 yim MMAD
Inhalability = yes
GSD =1.0
Breathing interval: One single breath
Concentration = 4.2
Breathing Frequency = 16.0
Tidal Volume = 860.0
Inspiratory Fraction = 0.5
Pause Fraction = 0.0
Breathing Route = nasal
Region: Entire Lung
Region: Entire Lung
Region Deposition Fraction
Head 0.449
TB 0.045
P 0.127
Total 0.621
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Region: Entire Lung
Wed, 03/17/2010, 02:15:27 FM EDT
0.250
0.200
o
0.150
c
o
to
o
GL
m
Q
0.100
0.050
0.0
0.181
0.068
Region
Species & Model Info:
Species/Geometry: Rat
FRC Vblurrre: 4.00 ml
Head Vblume: 0.42 ml
Breathing Route: nasal
Breathing Parameters:
Tidal Mslume: 1.80 ml
Breathing Frequency: 102.00 1/tTiin
Inspiratory Fraction: 0.50
Pause Fraction: 0.D0
Particle Properties:
Diameter: MV1AD: 1.70 ^jm
GSD: 1.00
Concentration: 4.20 (jg/m"3
Figure E-16. Rat fractional deposition.
Species = rat
FRC =4.0
Head volume = 0.42
Density = 1.0
Number of particles calculated = single
Diameter = 1.7000000000000002 yim MMAD
Inhalability = yes
GSD =1.0
Breathing interval: One single breath
Concentration = 4.2
Breathing Frequency = 102.0
Tidal Volume =1.8
Inspiratory Fraction = 0.5
Pause Fraction = 0.0
Breathing Route = nasal
Region: Entire Lung
Region: Entire Lung
Region Deposition Fraction
Head 0.072
TB 0.041
P 0.068
Total 0.181
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E.2. Cancer Endpoints
E.2.1. Dose-Response Modeling for the Oral Slope Factor
Dose-Response Models
Due to the occurrence of multiple tumor types, earlier occurrence with increasing exposure,
and early termination of the high-dose group in the oral carcinogenicity studies (see Appendix D for
study details), methods that can reflect the influence of competing risks and intercurrent mortality
on site-specific tumor incidence rates are preferred. EPA has generally used a model that
incorporates the time at which death-with-tumor occurred as well as the dose; the multistage-
Weibull model is multistage in dose and Weibull in time, and has the form:
P(d, t] = 1 - exp[-[q0 + qid + q2d2 + ... + qi 0, for / = 0,1,..., k; t is the time
at which the tumor was observed; and c is a parameter which characterizes the change in response
with age. The parameter to represents the time between when a potentially fatal tumor becomes
observable and when it causes death, and is generally set to 0 either when all tumors are
considered incidental or because of a lack of data to estimate the time reliably. The dose-response
analyses were conducted using the computer software program MultiStage-Weibull (U.S. EPA.
2010b). which is based on Weibull models drawn from Krewski et al. (1983). Parameters were
estimated using the method of maximum likelihood. From specific model fits using stages up to
n -1, where n is the number of dose groups, the model fit with the lowest AIC was selected.
Data Adjustments Prior to Modeling
Two general characteristics of the observed tumor types were considered prior to
modeling: allowance for different, although unidentified modes of action, and allowance for relative
severity of tumor types. First, etiologically different tumor types were not combined across sites
prior to modeling (i.e., overall counts of tumor-bearing animals were not tabulated) in order to
allow for the possibility that different tumor types could have different dose-response relationships
due to different underlying mechanisms or factors, such as latency. Consequently, all of the tumor
types were also modeled separately.
Additionally, the multistage-Weibull model can address relative severity of tumor types to
some extent by distinguishing between tumors as being either fatal or incidental to the death of an
animal in order to adjust partially for competing risks. In contrast to fatal tumors, incidental
tumors are those tumors thought not to have caused the death of an animal. Cause-of-death
information for most early animal deaths was provided by the investigators of both bioassays. In
the rat study of Kroese etal. (2001). tumors of the forestomach or liver were the principal cause of
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death for most animals dying or sacrificed (due to moribundity) before the end of the study, while
tumors of the forestomach were the most common cause of early deaths in the mouse study of
Beland and Culp T1998I The incidence data modeled are listed in Tables E-19 (male rats), E-20
(female rats), and E-21 (female mice).
Consistent with EPA's Recommended use of body weight 3/4 as the default method in
derivation of the oral reference dose, human-equivalent dose (HED) estimates used for dose-
response modeling were based on scaling by body weight3/4, as there were no pharmacokinetic
models or data to inform another approach (U.S. EPA. 20111. The dose estimates are provided in
Tables E-22 (Kroese etal.. 2001) andE-23 (Beland and Culp. 1998).
Evaluation of Model Fit and Model Selection
Each model was examined for adequacy of fit in the low-dose region and in the vicinity of
the BMR of 10% extra risk. In general, the model fit with the lowest AIC was selected, except when
model fit near the BMR and in the low-dose region was improved by including an additional stage
(parameter) in the model.
PODs for estimating low-dose risk were identified at doses at the lower end of the observed
data, generally corresponding to 10% extra risk, where extra risk is defined as [P(d) - P(0)]/
[1 - P(0)]. The lifetime oral cancer slope factor for humans is defined as the slope of the line from
the lower 95% bound on the exposure at the POD to the control response (slope factor =
0.1/BMDLio). This slope, a 95% upper confidence limit (UCL), represents a plausible upper bound
on the true risk.
Overall Risk
Although the time-to-tumor modeling helps account for competing risks associated with
decreased survival times and other tumors, considering the tumor sites individually still does not
convey the total amount of risk potentially arising from the sensitivity of multiple sites (i.e., the risk
of developing any combination of the increased tumor types, not just the risk of developing all
simultaneously). One approach suggested in the Guidelines for Carcinogen Risk Assessment (U.S.
EPA. 2005a) would be to estimate cancer risk from tumor-bearing animals. EPA traditionally used
this approach until the National Research Council (NRC) document Science and Judgment in Risk
Assessment fNRC. 1994] made a case that this approach would tend to underestimate overall risk
when tumor types occur in a statistically independent manner. In addition, application of one
model to a composite data set does not accommodate biologically relevant information that may
vary across sites or may only be available for a subset of sites. For instance, the time courses of the
multiple tumor types evaluated varied, as is suggested by the variation in estimates of c, from
1.5 (e.g., male rat skin or mammary gland basal cell tumors), indicating relatively little effect of age
on tumor incidence, to 3.7 (e.g., male mouse alimentary tract tumors), indicating a more rapidly
increasing response with increasing age (in addition to exposure level). The result of fitting a
model with parameters that can reflect underlying mechanisms, such as z in the multistage-Weibull
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Supplem en tal Inform ation —Benzo[aJpyren e
model, would be difficult to interpret with composite data (i.e., counts of tumor-bearing animals). A
simpler model, such as the multistage model, could be used for the composite data, but relevant
biological information would then be ignored.
Following the recommendations of the NRC (19941 regarding combining risk estimates,
statistical methods that can accommodate the underlying distribution of slope factors are optimal,
such as through maximum likelihood estimation or through bootstrapping or Bayesian analysis.
However, these methods have not yet been extended to models such as the multistage-Weibull
model. A method involving the assumption that the variability in the slope factors could be
characterized by a normal distribution is detailed below (U.S. EPA. 2010b). Using the results in
female rats to illustrate, the overall risk estimate involved the following steps:
1) It was assumed that the tumor groupings modeled above were statistically independent
(i.e., that the occurrence of a liver tumor was not dependent upon whether there was a
forestomach tumor). This assumption cannot currently be verified, and if not correct, could
lead to an overestimate of risk from summing across tumor sites. However, NRC T19941
argued that a general assumption of statistical independence of tumor-type occurrences
within animals was not likely to introduce substantial error in assessing carcinogenic
potency from rodent bioassay data.
2) The models previously fitted to estimate the BMDs and BMDLs were used to extrapolate to a
lower level of risk (R), in order to reach the region of each estimated dose-response
function where the slope was reasonably constant and upper bound estimation was still
numerically stable. For these data, a 10-3 risk was generally the lowest risk necessary. The
oral slope factor for each site was then estimated by R/BMDLr, as for the estimates for each
tumor site above.
3) The maximum likelihood estimates (MLE) of unit potency (i.e., risk per unit of exposure)
estimated by R/BMDr, were summed across the alimentary tract, liver, and jejunum/
duodenum in female rats.
4) An estimate of the 95% (one-sided) upper bound on the summed oral slope factor was
calculated by assuming a normal distribution for the individual risk estimates, and deriving
the variance of the risk estimate for each tumor site from its 95% UCL according to the
formula:
95% UCL = MLE + 1.645 x SD,
rearranged to:
SD = (UCL - MLE) / 1.645,
where 1.645 is the t-statistic corresponding to a one-sided 95% confidence interval (CI) and
>120 degrees of freedom, and the SD is the square root of the variance of the MLE. The variances
(variance = SD2) for each site-specific estimate were summed across tumor sites to obtain the
variance of the sum of the MLEs. The 95% UCL on the sum of MLEs was calculated from the
expression above for the UCL, using the variance of the sum of the MLE to obtain the relevant SD
(SD = variance1/2).
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-19. Tumor incidence data, with time to death with tumor for male
2 Wistar rats exposed by gavage to benzo[a]pyrene for 104 weeks (Kroese etal..
3 20011
Numbers of animals with:
Skin or mammary
gland
Oral cavity or
forestomach
Duodenum
or jejunum
Basal cell
Squamous
cell
Kidney
urothelial
Dose
Wkof
Total
tumors
Liver tumors
tumors
tumors
tumors
carcinoma
(mg/kg-d)
death
examined
Incidental3
Fatal3
Incidental
Fatal
Incidental
Incidental
Incidental
Incidental
0
44
1
0
0
0
0
0
1
0
0
80
1
0
0
0
0
0
0
0
0
82
1
0
0
0
0
0
0
0
0
84
1
0
0
0
0
0
0
0
0
89
1
0
0
0
0
0
0
0
0
90
0
0
0
0
0
0
0
0
91
1
0
0
0
0
0
0
0
0
92
1
0
0
0
0
0
0
0
0
93
1
0
0
0
0
0
0
0
0
94
1
0
0
0
0
0
0
0
0
95
0
0
0
0
0
0
0
0
96
0
0
0
0
0
0
0
0
97
1
0
0
0
0
0
0
0
0
98
1
0
0
0
0
0
0
0
0
100
0
0
0
0
0
1
0
0
104
1
0
0
0
0
0
0
0
0
105
1
0
0
0
0
0
0
0
0
108
7
0
0
0
0
0
0
0
0
109
22
0
0
0
0
0
0
0
0
3
29
1
0
0
0
0
0
0
0
0
40
1
1
0
0
0
0
0
0
0
74
1
0
0
0
0
0
0
0
0
76
1
0
0
0
0
0
0
0
0
79
1
0
0
0
0
0
0
0
0
82
1
0
0
0
0
0
0
0
0
92
0
0
0
0
0
0
0
0
93
1
0
0
0
0
0
0
0
0
94
1
0
0
0
0
0
0
0
0
95
0
0
0
0
0
0
0
0
98
1
0
0
0
0
0
0
0
0
107
10
4
0
1
0
0
0
0
0
108
15
2
0
3
0
0
1
1
0
109
14
1
0
0
0
0
0
0
0
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Supplem en tal Inform ation —Benzo[aJpyren e
Dose
(mg/kg-d)
Wkof
death
Total
examined
Numbers of animals with:
Oral cavity or
forestomach
tumors
Liver tumors
Duodenum
or jejunum
tumors
Skin or mammary
gland
Kidney
urothelial
carcinoma
Basal cell
tumors
Squamous
cell
tumors
Incidental3
Fatal3
Incidental
Fatal
Incidental
Incidental
Incidental
Incidental
10
39
1
0
0
0
0
0
0
0
0
47
2
0
0
0
0
0
0
0
0
63
1
1
0
0
0
0
0
0
0
68
2
2
0
0
0
0
0
0
0
69
1
1
0
0
0
0
0
0
0
77
1
0
0
1
0
0
0
0
0
80
1
0
0
1
0
0
0
0
0
81
1
1
0
0
0
1
0
0
0
84
1
1
0
0
1
0
0
0
0
86
1
0
0
1
0
0
0
0
0
90
1
1
0
0
0
0
0
0
0
95
3
3
0
2
0
0
0
0
0
97
1
1
0
0
1
0
0
0
0
100
1
1
0
1
0
0
0
0
0
102
1
1
0
1
0
0
0
0
0
103
1
1
0
1
0
0
0
0
0
104
3
3
0
3
0
0
0
0
0
107
12
12
0
11
0
0
0
1
0
108
11
11
0
11
0
0
1
0
0
109
6
5
0
3
0
0
0
0
0
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Supplem en tal Inform ation —Benzo[aJpyren e
Dose
(mg/kg-d)
Wkof
death
Total
examined
Numbers of animals with:
Oral cavity or
forestomach
tumors
Liver tumors
Duodenum
or jejunum
tumors
Skin or mammary
gland
Kidney
urothelial
carcinoma
Basal cell
tumors
Squamous
cell
tumors
Incidental3
Fatal3
Incidental
Fatal
Incidental
Incidental
Incidental
Incidental
30
32
1
1
0
0
0
0
0
0
0
35
1
1
0
1
0
0
0
0
0
37
1
1
0
0
0
0
0
0
0
44
1
0
1
1
0
0
0
0
0
45
2
0
2
0
0
0
0
0
47
1
1
0
1
0
0
0
0
0
48
1
1
0
1
0
0
0
0
0
49
1
1
0
1
0
0
0
0
0
50
1
1
0
1
0
0
0
0
0
51
1
1
0
1
0
1
0
0
0
52
4
3
1
3
1
0
1
1
0
53
1
1
0
1
0
0
1
0
0
56
2
1
1
1
1
0
0
0
0
58
2
2
0
2
0
0
1
0
0
59
2
2
0
2
0
0
0
0
0
60
2
1
1
1
1
1
0
0
0
61
3
2
1
1
2
1
0
0
0
62
5
5
0
0
4
3
0
0
0
63
5
5
0
4
1
1
2
1
2
64
2
2
0
1
1
0
0
0
1
65
3
2
1
1
2
0
3
2
0
66
1
1
0
0
1
0
0
0
0
67
3
1
2
2
1
1
1
1
0
68
1
1
0
1
0
0
0
0
0
70
2
2
0
1
1
1
1
0
0
71
1
1
0
1
0
0
1
1
0
73
1
0
1
1
0
0
1
0
0
76
1
1
0
0
1
0
1
0
0
1
2 a"lncidental" denotes presence of tumors not known to have caused death of particular animals. "Fatal" denotes
3 incidence of tumors reported by the study investigators to have caused death of particular animals.
This document is a draft for review purposes only and does not constitute Agency policy.
E-49 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-20. Tumor incidence data, with time to death with tumor for female
2 Wistar rats exposed by gavage to benzo[a]pyrene for 104 weeks (Kroese et al..
3 20011
Numbers of animals with:
Duodenum or
Oral cavity or forestomach
jejunum
Dose
Wkof
Total
tumors
Liver tumors
tumors
(mg/kg-d)
death
examined
Incidental3
Fatal3
Incidental
Fatal
Incidental
0
64
1
0
0
0
0
0
69
1
0
0
0
0
0
75
1
0
0
0
0
0
104
1
0
0
0
0
0
106
0
0
0
0
0
107
7
0
0
0
0
0
108
7
0
0
0
0
0
109
30
1
0
0
0
0
3
8
1
0
0
0
0
0
47
1
0
0
0
0
0
52
1
0
0
0
0
0
60
1
0
0
0
0
0
65
1
0
0
0
0
0
76
1
0
0
0
0
0
77
1
0
0
0
0
0
83
0
0
0
0
0
85
1
0
0
0
0
0
86
1
0
0
0
0
0
88
1
0
0
0
0
0
93
0
0
0
0
0
94
1
0
0
0
0
0
97
1
1
0
0
0
0
107
6
2
0
1
0
0
108
9
2
0
0
0
0
109
21
1
0
0
0
0
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Supplem en tal Inform ation —Benzo[aJpyren e
Dose
(mg/kg-d)
Wkof
death
Total
examined
Numbers of animals with:
Oral cavity or forestomach
tumors
Liver tumors
Duodenum or
jejunum
tumors
Incidental3
Fatal3
Incidental
Fatal
Incidental
10
42
1
0
0
0
0
0
43
1
0
0
0
0
0
44
1
0
0
0
0
0
45
1
0
0
0
0
0
48
1
0
0
0
0
0
55
1
0
0
1
0
0
59
1
0
0
0
0
0
75
1
0
0
1
0
0
76
0
0
1
0
0
77
0
0
0
0
0
80
1
1
0
1
0
0
81
1
1
0
0
1
0
82
1
1
0
1
0
0
83
1
0
1
0
0
85
1
0
1
1
0
86
1
1
0
0
1
0
87
1
0
1
0
0
88
1
0
1
1
0
89
1
1
0
0
1
0
91
1
0
0
0
1
0
95
1
0
0
0
0
0
96
1
0
0
0
0
0
98
2
0
1
1
0
99
3
0
1
2
0
102
1
1
0
0
1
0
104
1
1
0
1
0
0
105
1
0
1
1
0
106
1
1
0
0
1
0
107
5
0
5
0
0
108
7
7
0
7
0
0
109
4
2
0
2
0
0
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Supplem en tal Inform ation —Benzo[aJpyren e
Dose
(mg/kg-d)
Wkof
death
Total
examined
Numbers of animals with:
Oral cavity or forestomach
tumors
Liver tumors
Duodenum or
jejunum
tumors
Incidental3
Fatal3
Incidental
Fatal
Incidental
30
26
1
0
0
0
0
0
44
4
4
0
3
1
0
47
3
3
0
2
1
0
48
1
1
0
0
1
0
54
1
0
0
1
0
0
55
3
3
0
1
2
0
56
2
2
0
0
2
0
57
2
2
0
2
0
0
58
4
3
1
0
4
0
59
2
1
1
0
2
0
60
1
0
1
1
0
0
61
2
2
0
0
2
0
62
2
2
0
1
1
0
63
3
3
0
0
3
0
64
5
5
0
0
5
3
66
3
3
0
0
3
0
67
2
1
1
0
2
0
68
1
1
0
0
1
0
69
4
3
1
1
3
1
71
4
3
1
1
3
0
72
2
1
1
0
2
0
1
2 a"lncidental" denotes presence of tumors not known to have caused death of particular animals. "Fatal" denotes
3 incidence of tumors indicated by the study investigators to have caused death of particular animals.
4
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-21. Tumor incidence, with time to death with tumor; B6C3Fifemale
2 mice exposed to benzo[a]pyrene via diet for 2 years (Beland and Culp. 1998)
Dose
Number of animals with
Dose
Number of animals with
group
alimentary tract
group
alimentary tract
(ppm in
Wkof
Total
squamous cell tumors
(ppm in
Wk of
Total
squamous cell tumors
diet)
death
examined
Fatal3
Incidental
diet)
death
examined
Fatal3
Incidental
0
31
1
0
0
5
25
1
0
0
74
1
0
0
55
1
0
0
89
2
0
0
83
1
0
0
91
1
0
0
86
1
0
0
93
2
0
0
87
2
0
0
94
2
0
0
88
2
0
0
97
2
0
0
90
1
0
0
98
2
0
0
94
1
0
0
99
1
0
0
95
2
0
0
100
2
0
0
96
1
0
0
101
2
0
0
97
2
0
0
104
1
0
0
98
2
0
0
105
29
0
1
101
2
0
0
102
2
0
0
105
27
0
3
25
44
1
1
0
100
39
1
1
0
47
1
0
0
40
1
1
0
64
lb
0
0
42
1
1
0
70
1
1
0
47
2
0
77
1
1
0
49
1
0
0
80
1
0
0
50
1
1
0
81
1
1
0
53
lb
0
0
84
2
1
1
55
3
0
85
1
1
0
56
1
1
0
86
1
1
0
57
1
1
0
88
1
1
0
58
1
1
0
89
1
0
0
59
3
3
0
90
4
4
0
60
1
1
0
93
3
2
1
61
3
3
0
94
2
2
0
62
5
5
0
96
3
0
2
63
4
4
0
97
1
1
0
64
3
3
0
98
1
1
0
65
2
2
0
99
2
1
1
66
3
3
0
100
1
1
0
68
1
1
0
101
1
0
0
69
2
2
0
102
2
2
0
70
2
2
0
104
1
1
0
71
1
1
0
105
13
0
10
72
1
1
0
73
1
1
0
74
1
1
0
79
1
1
0
3
4 a"lncidental" denotes presence of tumors not known to have caused death of particular animals. "Fatal" denotes
5 incidence of tumors indicated by the study investigators to have caused death of particular animals.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-22. Derivation of HEDs to use for BMD modeling of Wistar rat tumor
2 incidence data from Kroese et al. (2001)
Benzo[a]pyrene dose (mg/kg-d)
TWA body weight (kg)
Interspecies
scaling factor3
HEDb (mg/kg-d)
Male
3
0.349
0.27
0.54
10
0.349
0.27
1.81
30
0.288
0.25
5.17
Female
3
0.222
0.24
0.49
10
0.222
0.24
1.62
30
0.222
0.24
4.85
3
4 aScaling factors were calculated using U.S. EPA (1988) reference body weights for humans (70 kg), and the TWA
5 body weights for each dose group: rat-to-human = (TWA body weight/70)0 25 = scaling factor.
6 bHED = administered dose x scaling factor.
7 Table E-23. Derivation of HEDs for dose-response modeling of B6C3Fi female
8 mouse tumor incidence data from Beland and Culp (1998)
Benzo[a]pyrene
dose in diet
(ppm)
Intake (pg/d)
TWA body
weight average
(kg)
Administered
dose3 (mg/kg-d)
Scaling factorb
HEDC (mg/kg-d)
5
21
0.032
0.7
0.15
0.10
25
104
0.032
3.3
0.15
0.48
100
430
0.027
16.5
0.14
2.32
9
10 Administered doses in mg/kg-day were calculated from dietary concentrations of benzo[a]pyrene using the TWA
11 body weight and reported food intakes for mice.
12 bScaling factors were calculated using U.S. EPA (1988) reference body weights for humans (70 kg), and the TWA
13 body weights for each dose group: mouse-to-human = (TWA body weight/70)0 25 = scaling factor.
14 CHED = administered dose x scaling factor.
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
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E.2.1.5. Sensitivity Analyses
Alternative dose-response models were also considered, limited to the most sensitive sites
for male and female rats (alimentary system tumors) and the overall incidence of alimentary
system tumors for female mice. Tumor incidences were adjusted for early mortality using the poly-
3 procedure (Bailer and Portier, 1988); the adjusted incidences are provided in Tables E-25 (male
rats), E-26 (female rats) and E-29 (female mice). Adjusted incidences were fit using dichotomous
models in BMDS (see Section E.1.2. for model fitting methods).
Dose-Response Modeling Results
Tables E-24 (male and female rats), and E-28 (female mice) summarize the multistage-
Weibull modeling results supporting the oral slope factor for benzo[a]pyrene. The model outputs
and graphs following each of these tables (male rats: Figures E-17 through E-22; female rats:
Figures E-23 through E-25; female mice: Figure E-26) provide more details for the best-fitting
models in each case.
Derivations of overall risk estimates for male and female rats are summarized in Table E-27.
Alternative dose-response modeling results are provided in Tables E-25 (male rats), E-26
(female rats), and E-29 (female mice).
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1 Table E-24. Summary of BMD modeling results for best-fitting multistage-
2 Weibull models, using time-to-tumor data for Wistar rats exposed to
3 benzo[a]pyrene via gavage for 104 weeks (Kroese et al.. 2001): BMR = 10%
4 extra risk
Model
BMDLio -
Endpoints
stages
AIC
BMDio
BMDUio
Basis for model selection
Male
Oral cavity and
l
577.8
0.104
rats
forestomach:
2
407.6
0.678
squamous cell
3
229.0
0.453
0.281-0.612
Lowest AIC, best fit to low dose data
tumors
Hepatocellular
1
367.3
0.181
tumors
2
301.5
0.472
3
289.1
0.651
0.449-0.772
Lowest AIC, best fit to low dose data
Duodenum and
1
69.6
2.64
jejunum tumors
2
65.9
3.04
3
66.9
3.03
2.38-3.87
Best fit to data
Kidney: uroethelial
1
31.9
9.16
carcinoma
2
31.7
5.71
3
32.8
4.65
2.50-9.01
Best fit to data
Skin and mammary
1
110.6
1.88
gland: basal cell
2
105.1
2.58
tumors
3
104.7
2.86
2.35-3.62
Lowest AIC, best fit to low dose data
Skin and mammary
1
63.5
3.36
gland: squamous
2
64.3
2.75
cell tumors
3
65.3
2.64
1.77-4.42
Best fit to low dose data
Female
Oral cavity and
1
277.1
0.245
rats
forestomach:
2
211.6
0.428
squamous cell
3
201.0
0.539
0.328-0.717
Lowest AIC, best fit to low dose data
tumors
Hepatocellular
1
595.5
0.146
tumors
2
774.9
0.370
3
468.3
0.575
0.507-0.630
Lowest AIC, best fit to low dose data
Duodenum and
1
37.9
6.00
jejunum tumors
2
37.0
4.33
3
37.8
3.43
1.95-5.70
Best fit to low dose data
5
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Male Rat (Kroese etal. 20011: Squamous Cell Papilloma or Carcinoma in Oral Cavity or
Forestomach
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: OralForstKroeseM3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 3.6
t_0 = 39.1111
beta_0 = 0
beta_l = 8.8911e-009
beta_2 = 1.60475e-031
beta 3=1.95818e-008
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -beta_0 -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c t_0 beta_l beta_3
c 1 -0.53 -0.93 -0.99
t_0 -0.53 1 0.47 0.57
beta_l -0.93 0.47 1 0.9
beta 3 -0.99 0.57 0.9 1
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
Estimate
3 .74559
41.4581
0
4 . 37816e-009
0
1.01904e-008
Parameter Estimates
Std. Err.
0.447309
2.14975
NA
1.07528e-008
NA
1.94164e-008
95.0% Wald Confidence Interval
Lower Conf. Limit
2.86888
37.2447
-1.6697e-008
-2.78651e-008
Upper Conf. Limit
4.6223
45.6716
2.54533e-008
4.82458e-008
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -108.512 6
AIC
229.024
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Data Summary
CONTEXT
C F I U Total Expected Response
DOSE
0
52
0
0
0
52
o
o
o
0 . 54
44
0
8
0
52
6.77
1.8
7
0
45
0
52
41.69
5.2
0
9
43
0
52
49. 97
Minimum observation time for F tumor context = 44
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect = 0.1 0.01 0.001
BMD = 0.453471 0.0633681 0.00636659
BMDL = 0.281044 0.0286649 0.00285563
BMDU = 0.612462 0.248377 > 0.0509326
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Incidental Risk: OralFoistKioeseM3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00 Dose = 0.54
Time
Dose = 1.81
Dose = 5.17
t 1 1 1 r
20 40 60 80 100
1 1—
20 40 60 80 100
Time
Time
Figure E-17. Fit of multistage Weibull model to squamous cell papillomas or
carcinomas in oral cavity or forestomach of male rats exposed orally to
benzo[a]pyrene fKroese et al.. 20011.
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1 Table E-25. Summary of alternate BMD modeling results for squamous cell
2 papillomas or carcinomas in oral cavity or forestomach of male rats exposed
3 orally to benzo[a]pyrene (Kroese et al.. 20011: poly-3 incidences a
4
Model
Goodness of fit
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Comments
p-value
AIC
Multistage 3°b
1.000
62.662
0.406
0.200
Among multistage models,
two-stage model provided the
most parsimonious fit.
Multistage 2°
0.861
61.467
0.349
0.243
Quantal-Linear
0.0012
79.862
0.106
0.0838
Gamma
1.000
62.662
0.439
0.323
Other dichotomous models
yielded BMDi0s ranging
0.412-0.470 and BMDLi0s
ranging 0.287-0.367
Dichotomous-Hill
LogLogistic
0.980
62.741
0.455
0.364
Logistic
0.539
64.750
0.470
0.357
Probit
0.657
64.053
0.454
0.343
LogProbit
0.999
62.665
0.459
0.367
Weibull
1.000
62.662
0.412
0.287
a Dose: 0 mg/kg-d 0/43
0.54 8/45
1.8 45/47
5.2 52/52
b Coefficients b0, bi fit at boundary of permitted values (0).
5
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Male Rat (Kroese etal. 20011: Hepatocellular Adenoma or Carcinoma
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: LiverKroeseM3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 3.6
t_0 = 34.6667
beta_0 = 0
beta_l = 2.73535e-009
beta_2 = 8.116e-028
beta 3 = 1.43532e-008
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -beta_0 -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c
t_0
beta_l
beta 3
1
-0 .84
-0 .88
-1
t_0
-0 .84
1
0.71
0.86
beta_l
-0 .88
0.71
1
0.86
beta_3
-1
0.86
0.86
1
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
Estimate
3.49582
40.2211
0
4.43906e-009
0
2.35065e-008
Parameter Estimates
Std. Err.
0.629257
5.65421
NA
1.7 6051e-008
NA
6.47 999e-008
95.0% Wald Confidence Interval
Lower Conf. Limit
2.26249
29.1391
-3.00664e-008
-1.03499e-007
Upper Conf. Limit
4.72914
51.3032
3.89445e-008
1.50512e-007
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -138.544 6
AIC
289.088
Data Summary
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CONTEXT
C
F
I
U
Total
Expected
DOSE
0
52
0
0
0
52
0 .00
0 . 54
48
0
4
0
52
3 .38
1.8
14
2
36
0
52
36.81
5.2
3
17
32
0
52
49.55
Minimum observation time for F tumor context
52
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect =
0 ,
. 1
BHD =
0 ,
. 6507
BMDL =
0 ,
.44868
BMDU =
0 ,
.772467
0.01 0.001
0.173556 0.0199908
0.0530469 0.00530386
0.352684 > 0.159927
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Dose = 0.00
Incidental Risk: Hepatocellular_Kroese_M3
points show nonparam. est. for Incidental (unfillec
Dose = 0.54
JD
CD
JD
O
i i i r
0 20 40 60 80 100
JD
CD
JD
O
i i i i i r
0 20 40 60 80 100
Time
Time
Dose = 1.81
Dose = 5.17
JD
CD
JD
O
00X0 0,6
i 1 1 1 1 r
0 20 40 60 80 100
JD
CD
JD
O
i 1 1 1 1 r
0 20 40 60 80 100
Time
Time
Figure E-18. Fit of multistage Weibull model to hepatocellular adenomas or
carcinomas in male rats exposed orally to benzo[a]pyrene fKroese et al..
20011.
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Male Rat (Kroese et al.. 20011: Duodenum or Jejunum Adenocarcinoma
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: DuoJejKroeseM3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 1
Degree of polynomial = 3
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 1.63636
t_0 = 0 Specified
beta_0 = 4 . 31119e-027
beta_l = 2.96347e-G25
beta_2 = 0
beta 3 = 1.76198e-006
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) ~t_0 -beta_0 -beta_l -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c beta_3
1 -1
-1 1
c
beta 3
Variable
c
beta_0
beta_l
beta_2
beta 3
Estimate
1.77722
0
0
0
9.82 635e-0 07
Parameter Estimates
Std. Err.
2.03042
NA
NA
NA
8 . 29355e-006
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-2.20233 5.75677
-1.52724e-005
1.72377e-005
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log (likelihood) # Param
Fitted Model -28.4387 5
AIC
66.8773
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Data Summary
CONTEXT
C F I U Total Expected Response
DOSE
0
52
0
0
0
52
o
o
o
0 . 54
52
0
0
0
52
0 . 03
1.8
51
0
1
0
52
1.04
5.2
43
0
9
0
52
8 . 96
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Specified effect = 0.1
Confidence level = 0.9
Time = 104
Specified effect = 0.1 0.01 0.001
BMD = 3.03291 1.38578 0.642252
BMDL = 2.37782 0.418285 0.0420835
BMDU = 3.87183 1.76166 0.811476
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Incidental Risk: DuoJej_Kroese_M3
Dose = 0.00
Dose = 0.54
-Q
-Q
o
1 1 1 1 1 r
0 20 40 60 80 100
-Q
-Q
o
20 40 60 80 100
Time
Time
Dose = 1.81
Dose = 5.17
-Q
-Q
o
1—i—i—i—i—r
0 20 40 60 80 100
-Q
o
20 40 60 80 100
Time
Time
1
2 Figure E-19. Fit of multistage Weibull model to duodenum or jejunum
3 adenocarcinomas in male rats exposed orally to benzo[a]pyrene fKroese et al..
4 20011.
5
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Male Rat (Kroese etal. 20011: Skin or Mammary Gland Basal Cell Tumors
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: SKinMamBasalKroeseM3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 1
Degree of polynomial = 3
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 1.38462
t_0 = 0 Specified
beta_0 = 3.84298e-005
beta_l = 1.06194e-028
beta_2 = 0
beta 3 = 6.84718e-006
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -t_0 -beta_l -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c beta_0 beta_3
c 1-1-1
beta_0 -1 1 0.99
beta 3 -1 0.99 1
Variable
c
beta_0
beta_l
beta_2
beta 3
Estimate
1.47227
2 . 54786e-005
0
0
4.81611e-006
Parameter Estimates
Std. Err.
1.76686
0.000211261
NA
NA
3.4 9e-005
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-1.9907 4.93525
-0.000388585 0.000439542
-6.35866e-005
7.32188e-005
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -47.3623 5
AIC
104.725
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Data Summary
CONTEXT
C
F
I
U
Total
Expectei
DOSE
0
50
0
2
0
52
1.18
0 . 54
51
0
1
0
52
1.22
1.8
51
0
1
0
52
2 . 32
5.2
39
0
13
0
52
12 . 54
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect = 0.1 0.01 0.001
BMD = 2.86276 1.30804 0.606222
BMDL = 2.35118 0.415897 0.0424277
BMDU = 3.62258 1.69571 0.761447
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Incidental Risk: Skin Mam Basal Kroese M3
Dose = 0.54
.Q
CO
.Q
O
"l 1 1 r
20 40 60 80
Time
Dose = 1.81
.Q
CO
.Q
O
"i 1 1 1 T
20 40 60 80
Time
Dose = 5.17
CO
d
T
cb
o
d
0 20 40 60 80
Time
1
2 Figure E-20. Fit of multistage Weibull model to skin or mammary gland basal
3 cell tumors of male rats exposed orally to benzo[a]pyrene (Kroese et al..
4 20011.
5
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Male Rat (Kroese etal. 20011: Skin or Mammary Gland Squamous Cell Tumors
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: SKinMamSCCKroeseM3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 1
Degree of polynomial = 3
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 3
t_0 = 0 Specified
beta_0 = 0
beta_l = 1.25256e-008
beta_2 = 1.25627e-030
beta 3 = 3.34696e-009
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -t_0 -beta_0 -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c beta_l beta_3
1 -0.99 -1
-0.99 1 0.99
-1 0.99 1
c
beta_l
beta 3
Variable
c
beta_0
beta_l
beta_2
beta 3
Estimate
2.96213
0
1.50104e-008
0
3.9084e-009
Parameter Estimates
Std. Err.
2 .591
NA
1.86972e-007
NA
4.1537 4e-008
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-2.11613 8.04039
-3.51447e-007
-7.75033e-008
3.814 68e-007
8.53201e-008
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -27.652 5
AIC
65.304
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Data Summary
CONTEXT
C F I U Total Expected Response
DOSE
0
52
0
0
0
52
o
o
o
0 . 54
51
0
1
0
52
0 .42
CO
1—1
51
0
1
0
52
2 . 12
5.2
46
0
6
0
52
5.51
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect = 0.1 0.01 0.001
BMD = 2.6414 0.64109 0.070558
BMDL = 1.76931 0.211043 0.0210552
BMDU = 4.42145 2.03605 >0.564463
Incidental Risk: Skin Mam SCC Kroese M3
Dose = 0.00
Dose = 0.54
"I 1 1 1 T
20 40 60 80 100
Time
Figure E-21. Fit of multistage Weibull model to skin or mammary gland
squamous cell tumors of male rats exposed orally to benzo[a]pyrene (Kroese
etal.. 20011.
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Male Rat (Kroese etal. 20011: Kidney Urothelial Carcinomas
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: KidneyUrothelialCarKroeseM3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 1
Degree of polynomial = 3
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 1.63636
t_0 = 0 Specified
beta_0 = 3.78734e-027
beta_l = 1.59278e-G27
beta_2 = 2.718e-G24
beta 3 = 4.96063e-007
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) ~t_0 -beta_0 -beta_l -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c beta_3
1 -1
-1 1
c
beta 3
Variable
c
beta_0
beta_l
beta_2
beta 3
Estimate
1.74897
0
0
0
3 .11107e-007
Parameter Estimates
Std. Err.
3.79403
NA
NA
NA
4.90313e-006
95.0% Wald Confidence Interval
Lower Conf. Limit
-5.68719
- 9.2 98 85e-0 0 6
Upper Conf. Limit
9.18512
9.92107e-006
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -11.3978 5
AIC
32.7956
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Data Summary
CONTEXT
C F I U Total Expected Response
DOSE
0
52
0
0
0
52
o
o
o
0 . 54
52
0
0
0
52
0 .01
CO
1—1
52
0
0
0
52
0.29
5.2
49
0
3
0
52
2.71
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect =
0 . 1
0 .01
0
BMD =
4.64886
2.12413
0
BMDL =
2.49972
0.734665
0
BMDU =
9.01023
3.49311
1
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Incidental Risk: Kidney_Kroese_M3
Dose = 0.00
Dose = 0.54
in
o
"i r
20 40 60 80 100
Time
in
o
1
2 Figure E-22. Fit of multistage Weibull model to kidney urothelial tumors of
3 male rats exposed orally to benzo[a]pyrene (Kroese et al.. 2001).
4
This document is a draft for review purposes only and does not constitute Agency policy.
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Female Rat (Kroese et al.. 20011: Oral Cavity or Forestomach, Squamous Cell Papilloma or
Carcinoma
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: OralForstKroeseF3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 3.6
t_0 = 45.1111
beta_0 = 1.11645e-009
beta_l = 4.85388e-009
beta_2 = 0
beta_3 = 1.95655e-008
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c t_0 beta_0 beta_l beta_3
c 1 -0.79 -0.92 -0.93 -1
t_0 -0.79 1 0.73 0.72 0.8
beta_0 -0.92 0.73 1 0.79 0.92
beta_l -0.93 0.72 0.79 1 0.91
beta 3 -1 0.8 0.92 0.91 1
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
Estimate
3 . 52871
46.553
1.53589e-009
7.57004e-009
0
2.53126e-008
Parameter Estimates
Std. Err.
0.701117
5.93306
5.40523e-009
2.9647e-008
NA
7.66404e-008
95.0% Wald Confidence Interval
Lower Conf. Limit
2.15454
34.9244
-9.05817e-009
-5.05369e-008
-1.24 9e-007
Upper Conf. Limit
4.90287
58.1816
1.21299e-008
6.5677e-008
1.75525e-007
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood)
# Param
AIC
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Fitted Model
94.5119
6
201.024
Data
Summary
CONTEXT
C
F
I
U
Total
Expected Response
DOSE
0
51
0
1
0
52
1.14
0.49
46
0
6
0
52
4 . 90
1. 6
22
0
30
0
52
31.81
4 . 6
2
7
43
0
52
49.43
Minimum
observation
time for F
tumor context = 58
Benchmark
Dose
Computation
Risk Response
Risk Type
Confidence level
Time
Incidental
Extra
0. 9
104
Specified effect = 0.1 0.01 0.001
BMD = 0.538801 0.0981283 0.0100797
BMDL = 0.328135 0.0345104 0.00344714
BMDU = 0.717127 0.325909 > 0.0806373
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Dose = 0.00
Incidental Risk: OralForstKroeseF3
points show nonparam. est. for Incidental (unfillec
Dose = 0.49
CO
_Q
O
_Q
CO
_Q
O
20 40 60 80 100
20 40 60 80 100
Time
Time
Dose = 1.62
Dose = 4.58
CO
_Q
O
i 1 1 1 r
20 40 60 80 100
Time
TO
_Q
O
0 20 40 60 80 100
Time
1
2 Figure E-23. Fit of multistage Weibull model to squamous cell papillomas or
3 carcinomas in oral cavity or forestomach of female rats exposed orally to
4 benzo[a]pyrene (Kroese et al.. 20011.
5
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1 Table E-26. Summary of alternative BMD modeling results for squamous cell
2 papillomas or carcinomas in oral cavity or forestomach of female rats exposed
3 orally to benzo[a]pyrene (Kroese et al.. 20011: poly-3 adjusted incidences3
4
Model
Goodness of fit
BMDioPct
(mg/kg-d)
BMDLioPct
(mg/kg-d)
Comments
p-value
AIC
Multistage 3°
0
63912
4.92E-07
4.92E-07
Among multistage models,
only the two-stage model
provided an acceptable fit.
Multistage 2°
0.991
92.349
0.435
0.228
Quantal-Linear
0.0174
100.65
0.139
0.110
Gamma
0.873
92.397
0.446
0.279
Among other dichotomous
models, BMDi0s ranged
0.435-0.516 and BMDLi0s
ranged 0.258-0.395.
Dichotomous-Hill
LogLogistic
0.369
93.694
0.474
0.333
Logistic
0.804
90.817
0.516
0.395
Probit
0.938
90.482
0.471
0.364
LogProbit
0.559
92.913
0.466
0.338
Weibull
0.991
92.349
0.435
0.258
a Dose: 0 mg/kg-d 1/49
0.49 6/42
1.6 30/39
4.6 50/50
5
6
This document is a draft for review purposes only and does not constitute Agency policy.
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Female Rat (Kroese etal. 20011: Hepatocellular Adenoma or Carcinoma
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: LiverKroeseF3.(d)
Fri Apr 16 09:08:03 2010
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3) }
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 3.6
t_0 = 31.777 8
beta_0 = 0
beta_l = 4 . 9104e-031
beta_2 = 5.45766e-030
beta 3 = 3.44704e-008
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -beta_0 -beta_l -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c t_0 beta_3
1 -0.9 -1
-0.9 1 0.92
-1 0.92 1
c
t_0
beta 3
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 3.11076 0.549208 2.03434 4.18719
t_0 38.6965 5.21028 28.4846 48.9085
beta_0 0 NA
beta_l 0 NA
beta_2 0 NA
beta_3 2.94354e-007 7.19418e-007 -1.11568e-006 1.70439e-006
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -228.17 6 468.34
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Data Summary
CONTEXT
C F I U Total Expected Response
DOSE
0
52
0
0
0
52
o
o
o
0.49
51
0
1
0
52
3 . 02
1. 6
13
12
27
0
52
38.36
4 . 6
1
38
13
0
52
51.36
Minimum observation time for F tumor context = 44
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect = 0.1 0.01 0.001
BMD = 0.575127 0.262783 0.12179
BMDL = 0.506633 0.134213 0.0152934
BMDU = 0.629806 0.287232 0.133064
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Dose = 0.00
Incidental Risk: Hepatocellular_Kroese_F3
points show nonparam. est. for Incidental (unfillec
Dose = 0.49
-Q
CT3
-Q
O
CO
O
O
o
i r
0 20 40 60 80 100
-Q
CT3
-Q
O
CO
o
o
o
20 40 60 80 100
Time
Time
Dose = 1.62
Dose = 4.58
-Q
OS
JZJ
O
CO
o
o
o
n 1 1 1 r
20 40 60 80 100
-Q
OS
JZJ
O
CO
o
o
o
n 1 1 1 1 r
0 20 40 60 80 100
Time
Time
Figure E-24. Fit of multistage Weibull model to hepatocellular adenomas or
carcinomas in female rats exposed orally to benzo[a]pyrene (Kroese et al..
2001).
This document is a draft for review purposes only and does not constitute Agency policy.
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Female Rat (Kroese etal. 20011: Duodenum or Jejunum Adenocarcinoma
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: DuoJejKroeseF3.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 208
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 1
Degree of polynomial = 3
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 2.25
t_0 = 0 Specified
beta_0 = 0
beta_l = 0
beta_2 = 0
beta 3 = 7.289e-008
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) ~t_0 -beta_0 -beta_l -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c beta_3
1 -1
-1 1
c
beta 3
Variable
c
beta_G
beta_l
beta_2
beta 3
Estimate
2.32531
0
0
0
5 . 3220 9e-008
Parameter Estimates
Std. Err.
3.58729
NA
NA
NA
7.98487e-007
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-4.70565 9.35626
-1.5117 8e-0 0 6
1.61823e-006
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -13.8784 5 37.7569
Data Summary
CONTEXT
This document is a draft for review purposes only and does not constitute Agency policy.
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C F I U Total Expected Response
DOSE
0
52
0
0
0
52
o
o
o
0.49
52
0
0
0
52
0 .01
1. 6
52
0
0
0
52
0.44
4 . 6
48
0
4
0
52
3 . 57
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect = 0.1 0.01 0.001
BMD = 3.43129 1.56781 0.726615
BMDL = 1.94745 0.560867 0.0584891
BMDU = 5.70108 2.61447 1.21046
This document is a draft for review purposes only and does not constitute Agency policy.
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Incidental Risk: DuoJej_Kroese_F3
Dose = 0.00 Dose = 0.49
20 40
60
Time
80
100
JD
CO
JD
O
LT>
O
O
O
Figure E-25. Fit of multistage Weibull model to duodenum or jejunum
adenocarcinomas in female rats exposed orally to benzo[a]pyrene (Kroese et
al.. 20011.
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Table E-27. Summary of human equivalent overall oral slope factors, based on
2 tumor incidence in male and female Wistar rats exposed to benzo[a]pyrene by
3 gavage for 104 weeks fKroese et al.. 20011
Data set
Tumor site
BMDooi
BMDLooi
Risk value3 at
SD
SD2
Proportion
of total
variance
BMDooi
BMDLooi
Males
Oral cavity/
forestomach
6.37 x 10"3
2.86 x 10"3
1.57 x 10"1
3.50 x 10"1
1.17 x 10"1
1.38 x 10"2
0.64
Liver
2.00 x 10"2
5.30 x 10"3
5.00 x 10"2
1.89 x 10"1
8.42 x 10"2
7.09 x 10"3
0.33
Duodenum/
jejunum
6.42 x 10"1
4.21 x 10"2
1.56 x 10"3
2.38 x 10"2
1.35 x 10"2
1.82 x 10"4
0.01
Skin/mammary
gland: basal cell
6.06 x 10"1
4.24 x 10"2
1.65 x 10"3
2.36 x 10"2
1.33 x 10"2
1.78 x 10"4
0.01
Skin/mammary
gland: squamous
cell
7.06 x 10"2
2.11 x 10"2
1.42 x 10"2
4.75 x 10"2
2.03 x 10"2
4.10 x 10"4
0.02
Kidney
9.84 x 10"1
7.48 x 10"2
1.02 x 10"3
1.34 x 10"2
7.51 x 10"3
5.64 x 10"5
0.00
Sum, risk values at BMDooi:
2.25 x 10"1
Sum, SD2:
2.17 x 10"2
Overall SDb:
1.47 x 10"1
Upper bound on sum of risk estimates0:
4.68 x 10"1
Females
Oral cavity/
forestomach
3.45 x 10"3
1.01 x 10"2
2.90 x 10"1
9.92 x 10"2
1.16 x 10"1
1.35 x 10"2
0.91
Liver
1.53 x 10"2
1.22 x 10"1
6.54 x 10"2
8.21 x 10"3
3.48 x 10"2
1.21 x 10"3
0.08
Duodenum/
jejunum
5.85 x 10"2
7.27 x 10"1
1.71 x 10"2
1.38 x 10"3
9.56 x 10"3
9.13 x 10"5
0.01
Sum, risk values at BMDooi:
1.09 x 10"1
Sum, SD2:
1.48 x 10"2
Overall SD:
1.22 x 10"1
Upper bound on sum of risk estimates0:
3.09 x 10"1
4
5 aRisk value = 0.001/BMDLooi.
6 "Overall SD = (sum, SD2)05.
7 cUpper bound on the overall risk estimate = sum of BMDooi risk values + 1.645 x overall SD.
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Table E-28. Summary of BMD model selection among multistage-Weibull
models fit to alimentary tract tumor data for female B6C3Fi mice exposed to
benzo[a]pyrene for 2 years fBeland and Culp. 19981
Model
stages
AIC
BMDio3
BMDLio-BMDUio3
Basis for model selection
l
688.5
0.104
2
629.2
0.102
3
624.5
0.127
0.071-0.179
Lowest AIC, best fit to low dose data
a Corresponding to lifetime exposure (104 weeks).
Female Mice fBeland and Culp. 1998): Alimentary Tract Squamous Cell Tumors
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: C:\mswl0-09\benzo[a]pyrene_FemaleSquamF3i.(d)
The form of the probability function is:
P[response] = 1-EXP$$-(t - t_G)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2+beta_3*doseA3)}
The parameter betas are restricted to be positive
Dependent variable = Class
Independent variables = Dose, time
Total number of observations = 191
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
User Inputs Initial Parameter Values
c 2
t_0 = 15
beta_G = 1.6e-G14
beta_l = 0
beta_2 = 5.5e-G12
beta 3 = 4 . 4e-012
Asymptotic Correlation Matrix of Parameter Estimates
c t_0 beta_0 beta_l beta_2 beta_3
c 1 -0.78 -0.97 -0.42 -0.99 -0.99
t 0 -0.78 1 0.76 0.39 0.74 0.84
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beta_0
beta_l
beta_2
beta 3
-0 . 97
-0 .42
-0 . 99
-0 . 99
0.76
0.39
0.74
0 .84
1
0 . 33
0 . 97
0 . 96
0 . 33
1
0 .31
0.46
0 . 97
0 .31
1
0 . 97
0 . 96
0.46
0 . 97
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable
Estimate
Std. Err.
Lower Conf. Limit
Upper Conf. Limit
c
6.92317
1.33874
4.29929
9.54705
t 0
13.9429
4.9664 6
4.20881
23.677
beta 0
2.4 6916e-016
1.47619e-015
-2.64636e-015
3.14019e-015
beta 1
0
1.30525e-014
-2.55825e-014
beta 2
5.85452e-014
3.75144e-013
-6.76723e-013
7.93813e-013
beta 3
9.7 6542e-014
5.62017e-013
-1.00388e-012
1.19919e-012
Log(likelihood)
# Param
AIC
Fitted Model
-306.265
6
624.53
Data Summary
Class
C
F
I
U
Total
Expectei
Dose
0
47
0
1
0
48
0 . 93
0 .1
45
0
3
0
48
3 .21
0.48
8
23
15
1
47
30 . 82
2 . 3
1
46
0
1
48
41. 91
Minimum observation time for F tumor context
39
Benchmark Dose Computation
Risk Response =
Incidental
Risk Type =
Extra
Specified effect =
0 .1
Confidence level =
0 . 9
Time
104
BMD =
0.126983
BMDL =
0 . 0706103
BMDU =
0.179419
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Incidental Risk: BaP_FemaleSquamF3i
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose= 0.00 Dose= 0.10
20 40 60 80 100
Time
00
Cli
CD
di
-=T
O
CM
o
o
o
Dose = 0.48
Dose = 2.32
Figure E-26. Fit of multistage Weibull model to duodenum or jejunum
adenocarcinomas in male rats exposed orally to benzo[a]pyrene fKroese et al..
20011.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Inform ation —Benzo[aJpyren e
1 Table E-29. Summary of alternative BMD modeling results for alimentary tract
2 squamous cell tumors in female B6C3Fi mice exposed to benzo[a]pyrene for 2 years
3 fBeland and Culp. 19981: poly-3 adjusted incidences3
Model3
Goodness of fit
BMDio
(mg/kg/d)
BMDLio
(mg/kg/d)
Comments
p-value
AIC
Multistage 3°
1.000
72.015
0.138
0.0712
Among multistage models, 2-
stage model provided most
parsimonious fit
Multistage 2°
0.845
70.371
0.113
0.0730
Quantal-Linear
0.0049
83.200
0.0358
0.0274
Gamma
1.000
72.015
0.129
0.0815
Other dichotomous models
provided BMDi0s ranging
0.123-0.150, and BMDLi0s
ranging 0.079-0.110.
Dichotomous-Hill
LogLogistic
0.803
72.133
0.129
0.0857
Logistic
0.999
70.016
0.150
0.110
Probit
0.972
70.070
0.134
0.101
LogProbit
0.956
72.021
0.123
0.0859
Weibull
1.000
72.015
0.135
0.0793
4 a Dose: 0 mg/kg-d 1/43
5 0.1 3/41
6 0.48 38/44
7 2.3 46/46
8
9
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E.2.2. Dose-Response Modeling for the Inhalation Unit Risk
Modeling Methods
As with the tumor data used for the oral slope factor (see Section E.2.1, Dose Response-
modeling for the Oral Slope Factor), there was earlier occurrence of tumors with increasing
exposure, and early termination of the high-dose group fThvssen et al.. 1981: see Appendix D for
study details). The software program Multistage Weibull (U.S. EPA. 2010b) was used as described
in the analysis of the oral carcinogenicity data. See Section E.2.1 for details of the modeling
methods. A previous time-to-tumor analysis fU.S. EPA. 1990al was not used because of several
discrepancies between the summarized dose-response data and the individual pathology reports,
because the use of age at necropsy rather than the time since first exposure, and because multistage
Weibull provides a corrected estimate of the confidence bounds on the BMD.
Data Adjustments Prior to Modeling
As with the oral slope factor (see Section E.2.1, Dose Response-modeling for the Oral Slope
Factor), etiologically similar tumor types (i.e., benign and malignant tumors of the same cell type)
were combined for dose-response modeling. Here the benign tumors (papillomas, polyps, and
papillary polyps) were judged to be of the same cell type as the squamous cell carcinomas (SCCs).
As described in Section 2.4.2, the overall incidences of benign or malignant tumors in the
respiratory tract (larynx, trachea, and nasal cavity) and pharynx were used for dose-response
modeling.
Thvssen etal. f 19811 did not determine cause of death for any of the animals. Although the
fU.S. EPA. 1990al analysis made use of judgments from an independent toxicologist about the likely
causes of death for each animal, these judgments were not available for this assessment._Since the
investigators for the oral bioassays considered the same tumor types to be fatal at least some of the
time, bounding estimates for the Thvssen etal. (1981) data were developed by treating the tumors
alternately as either all incidental or all fatal. In either case, therefore, an estimate of to (the time
between a tumor first becoming observable and causing death) could not be estimated and was set
to 0. The data analyzed are summarized in Table E-30. Animals without confirmation of one or
more of the pharynx or respiratory tract tissues being examined were not included in the
incidences, unless a tumor was diagnosed in those that were examined. Group average TWA
continuous exposures, based on chamber air monitoring data and individual hamsters' time on
study, of 0, 0.25,1.01, and 4.29 mg/m3 corresponded to the 0, 2,10, and 50 mg/m3 nominal study
concentrations, respectively (U.S. EPA. 1990a).
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1 Table E-30. Individual pathology and tumor incidence data for male Syrian
2 golden hamsters exposed to benzo[a]pyrene via inhalation for lifetime—
3 Thvssen et al. f 198113
Exposure
Incidence of papillomas, polyps, papillary polyps, or
concentration:
carcinomas
target
(total malignant tumors)
Incidence of
(lifetime
average
Time of
respiratory
continuous
tumor
tract or
exposure)15,
observed
Nasal
Esophagu
Fore-
pharynx
mg/m3
(week)
Larynx
Pharynx
Trachea
cavity
s
stomach
tumors
0
16
0
c
0
0
0
0
(0)
39
0
0
0
0
0
0
0
45
0
0
0
0
0
0
0
79
0
0
0
0
0
0
0
82
0
0
0
0
0
85
0
0
0
0
0
85
0
0
0
0
0
0
0
87
0
0
0
0
0
0
0
87
0
0
0
0
0
0
0
88
0
0
0
0
0
0
0
88
0
0
0
0
0
0
0
89
0
0
0
0
0
0
0
101
0
0
0
0
0
0
0
101
0
0
0
0
0
0
0
103
0
0
0
0
0
0
0
106
0
0
0
0
0
0
0
107
0
0
0
0
0
0
0
109
0
0
0
0
0
0
0
111
0
0
0
0
0
0
0
114
0
0
0
0
0
0
0
115
0
0
0
0
0
121
0
0
0
0
0
0
0
122
0
0
0
0
0
0
0
124
0
0
0
0
0
124
0
0
0
0
0
0
0
126
0
0
0
0
0
131
0
0
0
0
0
0
0
2
13
0
0
0
(0.25)
35
0
0
0
0
0
0
0
53
0
0
0
0
0
0
0
58
0
0
0
0
0
0
0
70
0
0
0
0
0
0
0
77
0
0
0
0
0
0
0
79
0
0
0
0
0
0
0
84
0
0
0
0
0
0
0
87
0
0
0
0
0
0
0
93
0
0
0
0
0
0
0
97
0
0
0
0
0
—
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Exposure
Incidence of papillomas, polyps, papillary polyps, or
concentration:
carcinomas
target
(total malignant tumors)
Incidence of
(lifetime
average
Time of
respiratory
continuous
tumor
tract or
exposure)15,
observed
Nasal
Esophagu
Fore-
pharynx
mg/m3
(week)
Larynx
Pharynx
Trachea
cavity
s
stomach
tumors
99
0
0
0
0
0
0
0
102
0
0
0
0
0
0
0
102
0
0
0
0
0
0
0
108
0
0
0
0
0
0
0
113
0
0
0
0
0
0
0
114
0
0
0
0
0
0
0
115
0
0
0
0
0
0
0
115
0
0
0
0
0
0
0
119
0
0
0
0
0
0
0
121
0
0
0
0
132
0
0
0
0
0
0
0
10
30
0
0
0
0
0
0
0
(1.01)
32
0
0
0
0
0
0
0
51
0
0
0
0
0
0
0
66
0
0
0
0
0
0
0
73
0
0
0
0
0
0
0
76
0
1(1)
0
0
0
0
1
76
0
1(1)
0
0
0
0
1
80
l(l)d
0
0
0
0
0
0
85
0
0
0
0
0
0
0
93
1(1)
0
0
0
0
0
1
99
0
0
0
0
0
0
0
102
0
1(0)
0
0
0
0
1
105
1(1)
1(1)
0
0
0
0
1
110
0
1(1)
0
0
0
0
1
113
0
1(0)
0
0
0
0
1
114
1(1)
1(1)
0
0
0
0
1
115
1(1)
—
1(0)
1(0)
0
0
1
115
0
—
1(0)
1 (l)e
0
0
1
116
1(0)
—
0
0
0
0
1
117
1(1)
1(1)
0
0
0
0
1
118
1(0)
0
0
0
0
0
1
118
0
0
0
0
0
118
1(1)
0
0
1(0)
0
1(1)
1
121
1(0)
0
0
0
0
0
1
124
1(1)
1(1)
0
0
0
0
1
124
0
0
0
1(0)
0
0
1
50
21
—
—
—
0
0
0
—
(4.29)
22
—
—
—
0
0
0
—
25
0
0
0
—
30
0
0
0
—
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Exposure
concentration:
target
(lifetime
average
continuous
exposure)15,
mg/m3
Time of
tumor
observed
(week)
Incidence of papillomas, polyps, papillary polyps, or
carcinomas
(total malignant tumors)
Incidence of
respiratory
tract or
pharynx
tumors
Larynx
Pharynx
Trachea
Nasal
cavity
Esophagu
s
Fore-
stomach
30
0
—
0
0
0
0
—
30
0
0
0
35
0
0
0
36
0
0
0
0
0
0
0
36
—
—
—
0
0
0
38
—
—
—
0
0
0
0
40
of
1(1)
1(0)
0
0
0
1
41
0
0
0
0
0
0
0
41
0
0
0
42
0
0
0
0
0
0
0
42
0
0
0
0
0
0
0
46
1(1)
1(1)
0
0
0
0
1
47
0
1(1)
0
0
0
0
1
53
0
—
0
0
0
0
55
1(1)
1(1)
0
0
0
0
1
56
0
1(1)
0
0
0
0
1
60
0
1(1)
0
0
0
0
1
62
0
0
0
0
0
0
63
0
1(0)
0
0
0
1(0)
1
66
1(1)
1(1)
0
0
1
67
0
1(1)
0
0
0
0
1
69
1(0)
1(1)
0
0
1(0)
0
1
71
1(1)
1(1)
1(1)
0
0
0
1
71
1(0)
1(1)
0
0
0
1(0)
1
72
1(1)
1(1)
0
0
0
0
1
72
1(1)
1(1)
0
0
0
0
1
82
0
1(1)
0
0
0
0
1
82
1(1)
1(1)
0
0
1(0)
0
1
83
1(0)
1(1)
0
0
0
0
1
102g
1(1)
1(1)
1(0)
1(0)
0
0
1
1
2 aHistopathology incidence from U.S. EPA (1990a); Clement Associates (1990).
3 bSee Section D.4.2.
4 Tissue was not examined.
5 dln situ carcinoma; not included in overall tumor incidence.
6 Adenocarcinoma; not included in overall tumor incidence.
7 'Metastasis from pharynx not shown.
8 g Necropsy occurred 24 weeks after 79 weeks of exposure.
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E.2.2.3 Sensitivity Analyses
Alternative dose-response models and alternative assumptions regarding missing
observations and latency estimates were also conducted. First, alternative dose-response models
were considered through applying dichotomous models in BMDS to summary incidence data for
each exposure group, adjusted for early mortality using the poly-3 technique (Bailer and Portier,
1988).
Dose-Response Modeling Results
Table E-31 summarizes the modeling results supporting the derivation of an inhalation unit
risk value for benzo[a]pyrene. The model outputs and graphs (Figures E-27 and E-28) following
Table E-31 provide more details for the best-fitting models under the conditions of taking all
tumors to be incidental to the cause of death, or to be the cause of death, respectively.
The sensitivity analyses of Section E.2.2.3 are summarized in Table E-32.
Table E-31. Summary of BMD model selection among multistage-Weibull
models fit to tumor data for male Syrian golden hamsters exposed to
benzo[a]pyrene via inhalation for lifetime (Thvssen et al.. 1981)
Tumor context
Model
stages
AIC
BMDio3
BMDLio3
Basis for model selection
All tumors considered
incidental to cause of
death
1
2
50.5
40.4
0.076
0.254
0.052
0.163
Lowest AIC, best fit to data (BMDUio = 0.324)
All tumors considered to
be cause of death
1
2
315.0
302.9
0.135
0.468
0.104
0.256
Lowest AIC; best fit to data (BMDUio = 0.544)
a Corresponding to lifetime exposure (104 weeks).
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Output for Squamous Cell Neoplasia Following Inhalation Exposure to Benzo[a]pyrene: All
Tumors Considered Incidental to Cause of Death
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: ThyssenI2sL104noUw.(d)
Fri Oct 14 10:23:57 2016
The form of the probability function is:
P[response] = l-EXP{-(t - t_G)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2) }
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 88
Total number of records with missing values = 0
Total number of parameters in model = 5
Total number of specified parameters = 1
Degree of polynomial = 2
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 4.5
t_0 = 0 Specified
beta_0 = 8.02969e-034
beta_l = 5.12551e-032
beta 2 = 1.30309e-009
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) ~t_0 -beta_0 -beta_l
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c beta_2
1 -1
-1 1
c
beta 2
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 4.70606 0.953708 2.83682 6.57529
beta_G 0 NA
beta_l 0 NA
beta_2 5.296G9e-GlG 2.21617e-QG9 -3. 8140le-009 4.87323e-GG9
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -16.18 4 40.36
Data Summary
CONTEXT
This document is a draft for review purposes only and does not constitute Agency policy.
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C F I U Total
DOSE
0
21
0
0
0
21
0 . 25
19
0
0
0
19
1
8
0
17
0
25
4 . 3
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Benchmark Dose Computation
Risk Response
Risk Type
Specified effect
Confidence level
Incidental
Extra
0.1
0. 9
Time
104
BMD
BMDL
BMDU
0.253061
0.163183
0.318982
Incidental Risk: Thyssenlnc2sL104noll
Dose = 0.00
Dose = 0.25
CO
_Q
O
1 1 1 1 r
0 200 400 600 800
CO
_Q
O
"l 1 1 r
200 400 600 800
Time
Time
Dose = 1.01
CO
_Q
O
i 1 1 r
200 400 600 800
Time
Dose = 4.29
CO
_Q
O
"l 1 1 r
200 400 600 800
Time
Figure E-27. Fit of multistage Weibull model to respiratory tract tumors in
male hamsters exposed via inhalation to benzo[a]pyrene fThvssen et al..
1981): tumors treated as incidental to death.
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Output for Respiratory Tract Tumors: All Tumors Considered to be Cause Of Death
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: ThyssenF2sL104noU.(d)
Thu Mar 13 14:30:45 2014
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)Ac *
(beta_0+beta_l*doseAl+beta_2*doseA2) }
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 88
Total number of records with missing values = 0
Total number of parameters in model = 5
Total number of specified parameters = 1
Degree of polynomial = 2
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 64
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
Default Initial Parameter Values
c = 6
t_0 = 0 Specified
beta_G = 2.0496e-G36
beta_l = 4.12988e-G14
beta 2 = 3.37033e-013
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) ~t_0 -beta_0 -beta_l
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c beta_2
1 -1
-1 1
c
beta 2
Variable
c
beta_0
beta_l
beta 2
Estimate
6.61992
0
0
2 .13816e-014
Parameter Estimates
Std. Err.
0. 915036
NA
NA
8.964 66e-014
95.0% Wald Confidence Interval
Lower Conf. Limit
4 . 8264 9
-1.54323e-013
Upper Conf. Limit
8.41336
1. 97086e-013
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Fitted Model
Log(likelihood)
-147.66
# Param
AIC
303.319
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Data Summary
CONTEXT
C
F
I
u
Total
DOSE
0
21
0
0
0
21
0 . 25
19
0
0
0
19
1
8
17
0
0
25
4 . 3
5
18
0
0
23
Minimum observation time for F tumor context = 40
Benchmark Dose Computation
Risk Response =
Fatal
Risk Type =
Extra
Specified effect =
0 .1
Confidence level =
0 . 9
Time
104
BMD =
0.467752
BMDL =
0.256206
BMDU =
0.543965
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Fatal Risk:
Dose = 0.00
Dose = 0.25
i 1 1 1 1 1 r
0 20 40 60 80 100 120
-Q
03
-Q
O
00
O
CD
O
O
CM
O
o
o
i 1 1 1 1 1 r
0 20 40 60 80 100 120
Time
Time
Figure E-28. Fit of multistage Weibull model to respiratory tract tumors in
male hamsters exposed via inhalation to benzo[a]pyrene fThvssen et al..
1981): tumors treated as cause of death.
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1 Table E-32. Summary of alternative dose-response modeling results for
2 respiratory tumors in male Syrian golden hamsters exposed to
3 benzo[a]pyrene via inhalation for lifetime fThvssen et al.. 19811
Model
Goodness of fit
BMDio
(mg/m3)
BMDLio
(mg/m3)
Comments
p-value
AIC
Dichotomous models applied to poly-3 adjusted group incidences3
Gamma
0.0057
52.800
0.175
0.0990
Excepting the dichotomous Hill
model, no models fit adequately.
The dichotomous Hill model fit 4
parameters to the observed data,
with the exponent set at the lower
allowable limit (1), and yielded an
implausible shape in the region
near the BMR (<10% extra risk).
Dichotomous-Hill
1.000
42.606
0.784
0.635
Logistic
0
69.973
0.395
0.278
LogLogistic
0.0374
46.943
0.291
0.149
Probit
0
71.796
0.465
0.343
LogProbit
0.0334
47.964
0.276
0.142
Weibullb, Multistage 2°, 1°
0.0246
50.980
0.136
0.0979
Multistage 3°c
0.0246
50.980
0.136
0.0979
Multistage 2°: highest dose
group dropped
0.449
29.98
0.290
0.186
Adequate fit
Multistage Weibull model, with alternative assumptions
Parameter estimates6
tO (wks)
c
b2
Benign tumors->incidental,
malignant tumors->fatal
NAd
281.108
0.337
0.198
14.5f
5.4
1.4 x 1011
All tumors fatal8
NA
302.9
0.468
0.256
0
6.6
2.1 x 1014
All tumors considered
incidental, latency fixed6
NA
NA
NA
NA
191.29
115.39
51.62
40.45
0.431
0.364
0.261
0.252
0.245
0.238
0.184
0.164
5
15
45
90
6.5
6.2
5.4
4.7
5.4 x 10 14
2.3 x 10 13
1.8 x 10 11
4.8 x 10 10
Incidental tumors; missing
diagnoses assumed negative
NA
50.68
0.286
0.190
0
4.8
2.3 x 10 14
All tumors incidental8
NA
40.36
0.254
0.163
0
4.7
5.2 x 10 14
a Exposure (mg/m3) Polv-3 adjusted Incidence (denominators address all animals with missing diagnoses)
0 0/24
0.25 0/18
1.01 17/23
4.29 18/20
b For the Weibull model, the power parameter estimate was 1 (boundary of parameter space). The models in this row all
reduced to the Quantal-Linear model.
c The multistage 3° model differs from the models in the line just above (Weibull etc.) in additional digits not displayed in the
table.
d NA—Goodness of fit tests are not available for the multistage Weibull model.
e Models estimated bO and bl at 0; latency (tO) fixed at listed values unless noted otherwise.
f Maximum likelihood estimate,
s Repeated from Table E-31, for comparison.
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APPENDIX F. SUMMARY OF SAB PEER REVIEW
COMMENTS AND EPA'S DISPOSITION
The draft Toxicological Review of Benzo[a]pyrene, dated September 2014, underwent a
formal external peer review in accordance with EPA guidance on peer review (U.S. EPA. 20061. This
peer review was conducted by the Chemical Assessment Advisory Committee (CAAC) Augmented for
the IRIS benzo[a]pyrene assessment (CAAC benzo[a]pyrene panel) of EPA's Science Advisory Board
(SAB). An external peer review workshop was held on April 15-17, 2015. Public teleconferences of
the SAB-CAAC benzo[a]pyrene panel were held on March 4, August 21, and September 2, 2015. The
SAB held a public teleconference on January 26, 2016 to conduct a quality review of the draft peer
review report. The final report of the SAB was released in April 5, 2016.
The SAB was tasked with providing feedback in response to charge questions that
addressed scientific issues related to the hazard identification and dose-response assessment of
benzo[a]pyrene, as well as EPA's disposition of major public comments. A summary of major
recommendations of the SAB and EPA's responses to these recommendations, organized by charge
question, follow.
Charge Question 1. The process for identifying and selecting pertinent studies for
consideration in developing the assessment is detailed in the Literature Search
Strategy/Study Selection and Evaluation section. Please comment on whether the literature
search approach, screening, evaluation, and selection of studies for inclusion in the
assessment are clearly described and supported. Please comment on whether EPA has
clearly identified the criteria (e.g. study quality, risk of bias) used for selection of studies to
review and for the selection of key studies to include in the assessment. Please identify any
additional peer-reviewed studies from the primary literature that should be considered in
the assessment of noncancer and cancer health effects of benzo[a]pyrene.
Comment: The EPA should specify whether the literature search strategy included a review of the
references in the primary and secondary literature as a means to identify potentially relevant
articles not identified through the systematic searching and manual screening processes, and EPA
should conduct secondary literature searches as evidence for additional effects (e.g., cardio) or
specific data gaps (e.g., mechanistic, in vitro studies) that emerged.
Response: Comprehensive literature searches of several databases were performed for
benzo[a]pyrene in 2008 and 2012 (see Table LS-1 in the Toxicological Review). In addition to
EPA's search of online databases, secondary references, primarily assessments by other health
agencies, were consulted to ensure that critical studies were not missed by the literature search.
The database literature searches performed for benzo[a]pyrene were designed to search for all
possible health outcomes of benzo[a]pyrene exposure, and as such did not include terms for
This document is a draft for review purposes only and does not constitute Agency policy.
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specific organs or endpoints. Instead, the literature search strategy used for benzo[a]pyrene was
designed to use fewer, more comprehensive terms that capture many health outcomes, such as
"benzo[a]pyrene", "toxicity" and "adverse effect". The use of these broad terms captures the vast
majority of studies, likely more than would be identified with a more targeted literature search.
Many of the cardiovascular studies identified by the SAB as missing from the assessment were
identified in early literature searches. However, these studies were not included because the
assessment focused on endpoints that were established in subchronic or chronic oral and
inhalation studies, rather than in vitro studies, studies using less environmentally-relevant routes
of exposure, and studies in genetically modified animals or non-mammalian species.
In addition to the comprehensive search, iterative literature searches were conducted during the
draft development process. For example, specialized searches were conducted during draft
development to provide additional context for potential mechanisms of hazards identified from in
vivo subchronic, chronic, and developmental studies. These additional searches of PubMed were
conducted to fill data gaps and to help address peer review comments.
The assessment section entitled "Literature Search Strategy/Study Selection has been updated to
clarify these aspects of the literature search strategy.
Comment: The EPA should provide sufficiently detailed criteria for each step of the process leading
to the selection of key studies for the point of departure (POD) assessment while the handbook
which will outline the tools and processes is being developed.
Response: General considerations for the identification of pertinent studies, credible health
hazards, and informative studies for dose-response analysis are discussed in the IRIS preamble
which is included in the front matter of the IRIS Toxicological Review of Benzo[a]pyrene. Sections
especially pertinent to the SAB comment include: Section 3. Identifying and Selecting Pertinent
Studies, Section 4. Evaluating Study Methods and Quality, Section 6, Selecting Studies for Derivation
of Toxicity Values, and Section 7, Deriving Toxicity Values.
Rationales specific to the benzo[a]pyrene database, which lead to the selection of key studies and
the points of departure, are discussed throughout the document starting with considerations for
literature screening and evaluation in the Literature Search Strategy/Study Selection section of the
document. Considerations for the selection of studies for dose-response analysis specific to the
benzo[a]pyrene database are discussed in Sections 2.1.1 (for the oral database) and 2.2.1 (for the
inhalation database) of the Toxicological Review.
Charge Question 2a. The draft assessment concludes that developmental toxicity and
developmental neurotoxicity are human hazards of benzo[a]pyrene exposure. Do the
available human and animal studies support this conclusion?
The SAB concurred that the available human studies support the conclusion thatbenzo[a]pyrene
exposure contributes to human developmental toxicity and that the available animal studies
support this conclusion. The SAB subdivided this Charge Question into two parts: developmental
This document is a draft for review purposes only and does not constitute Agency policy.
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neurotoxicity and developmental toxicity other than neurodevelopment. The SAB had the following
specific recommendations:
Developmental neurotoxicity
Comment: The SAB recommended that rather than relying only on the elevated plus maze data and
dismissing the Morris water maze data, all the data in Chen etal. (20121 should be considered
collectively, and viewed in their totality as evidence of a developmental neurobehavioral effect of
neonatal benzo[a]pyrene exposure. The SAB also commented that the Least Significant Difference
(LSD) test may have been inappropriate for establishing the weight of evidence for developmental
neurobehavioral effects.
Response: EPA agrees with this recommendation and the revised assessment gives further
consideration to all of the behavioral outcomes reported in Chen etal. (20121 for use in hazard
identification and dose-response analyses. Specifically, text within Section 1.1.1 (e.g.
"Neurodevelopmental Effects" and "Summary of Developmental Effects") of the revised assessment
provides increased consideration of the following endpoints as collectively providing evidence of a
neurodevelopmental effect: surface righting, negative geotaxis, open field activity, elevated plus
maze, and Morris water maze.
Concerning the LSD test, EPA agrees that this test can over-emphasize differences as significant that
may not be (i.e., by underestimating p-values). Statistical significance testing was one of several
factors in evaluating the weight of evidence, including evaluating magnitudes of effect, overall
biological significance across the various time points evaluated, and consistency of the effects
across similar protocols. Clarification to this effect was added to Table 1-4.
Ultimately, the revised assessment emphasized the totality of the evidence for behavioral effects
assessed by Chen etal. (20121 for dose-response analyses. (See also response to Charge Question
3a).
Comment: EPA should consider the significant exposure gaps in brain development in existing
studies in the overall evaluation of benzo[a]pyrene developmental neurotoxicity.
Response: The EPA agrees that this is an important point. In the revised assessment, a figure
arraying the exposure paradigms used across the available studies evaluating developmental
neurotoxicity has been added to Section 1.1.1. This figure provides a visual representation of
exposure gaps across the available developmental neurotoxicity studies.
Neurodevelopmental exposure gaps identified are now summarized in the Summary of Section
1.1.1 and considered more carefully in Section 2. Overall, the exposure gaps indicate that the
available benzo[a]pyrene studies do not comprehensively cover the exposure periods pertinent to
assessing the potential vulnerability of the developing nervous system to toxic insult, namely from
implantation through adolescence.
This document is a draft for review purposes only and does not constitute Agency policy.
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Furthermore, since developmental neurotoxicity can be expressed differentially depending on both
the timing of exposure and the endpoint measures assessed (noting that the mode-of-action for
benzo[a]pyrene-induced neurotoxicity remains unknown), and because many studies in the
database did not evaluate multiple parameters of nervous system structure and function, it is likely
that at least some of the exposure periods examined were not adequately assessed. However, the
available studies include a detailed evaluation of exposure during several developmental ages
known to be sensitive for detecting developmental neurotoxicity. These include late gestation and
the early neonatal period (although exposures combining these periods were not evaluated), during
which substantial brain region-specific changes in proliferation, synaptogenesis, and perhaps most
noticeably, growth, occur. These exposure gaps during sensitive periods of brain development
were considered in the application of a database UF to help address residual uncertainty associated
with the potential for neurodevelopmental effects at lower doses (see also response to Charge
Question 3a).
Developmental toxicity other than neurodevelopment
Comment: The SAB recommended EPA conduct a more complete literature search on
developmental toxicity of benzo[a]pyrene to characterize benzo[a]pyrene-mediated developmental
toxicity. Specifically, several older teratology studies were suggested for inclusion (Shum etal..
1979: Nebertetal.. 1977: Rigdon and Rennels. 19641. In addition, the SAB recommended
consideration of a publication by Thakur etal. (2014). evaluating fetal benzo [a] pyrene-related
effects on fetal lung growth and function.
Response: Several teratology studies were suggested for inclusion by the SAB fShum etal.. 1979:
Nebert etal.. 1977: Rigdon and Rennels. 1964). Two oral, high dose teratology studies in rats
fRigdon and Rennels. 19641 and mice fRigdon and Neal. 19651were identified in the original
comprehensive literature search for benzo[a]pyrene and were discussed in the supplementary
material in Appendix D. However, these older, high dose teratology studies were generally limited
in terms of study design, documentation of methods, and reporting of results (see Appendix D for
details).
Two additional studies recommended by the SAB (Shum etal.. 1979: Nebertetal.. 1977).
were considered to provide mechanistic information. Shum etal. (1979). a high dose IP study (200
mg/kg), suggests that developmental effects of benzo[a]pyrene may occur via the AhR pathway.
Similar developmental findings were reported in Nebertetal. (1977) which looked at
developmental toxicity of two PAHs (3-methylcholanthrene and 7,12-dimethylbenz [a] anthracene)
in AhR responsive and non-responsive mice. These studies have been included in Section 1.1.1,
Mode of Action Analysis—Developmental Toxicity and Developmental Neurotoxicity.
Regarding the study by Thakur etal. f20141 (also discussed under Charge Question 2e), this
study reported increased susceptibility to lung injury in offspring of rat dams treated with high
dose intraperitoneal exposure (25 mg/kg) to benzo[a]pyrene on GDs 18-20 and subsequent
challenge with hyperoxic air (85% O2). However, interpretation of this study is complicated due to
the route of exposure and the high doses employed, which were one to two magnitudes greater
than doses at which effects were observed in the oral developmental database. Discussion of this
study's implications regarding increased susceptibility to oxidative stress subsequent to
This document is a draft for review purposes only and does not constitute Agency policy.
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benzo[a]pyrene exposure has been added to Section 1.1.1, Mode of Action Analysis—Developmental
Toxicity and Developmental Neurotoxicity.
Comment: Adverse outcomes resulting from benzo[a]pyrene exposure should take into context the
susceptible window of exposure [i.e., whether exposure occurs in early gestation, late gestation (GD
6-12/15), or postnatal exposure].
Response: EPA agrees that the timing of developmental exposures can be a critical determinant of
health effects observed in a particular study. The available developmental studies in the
benzo[a]pyrene database often exposed animals during different windows of development. Specific
durations of exposure are listed in the relevant evidence tables (See Tables 1-2 and 1-4) and
discussed in the text. However, conclusions regarding the windows of development most relevant
to benzo[a]pyrene-induced developmental effects cannot be made due to varying study design
across studies. Increased discussion regarding the exposure timing of developmental studies has
been added to the document in Section 1.1.1 and Section 2.1.3.
Comment: The EPA should consider including mechanistic studies that provide perspectives on the
likely mode of action leading to benzo[a]pyrene-related developmental toxicity. Specifically, the
SAB recommended the addition of studies investigating the role of mechanisms such as
genotoxicity and oxidative stress.
Response: Mechanistic information potentially informative of benzo[a]pyrene-related
developmental effects is included in Section 1.1.1. "Developmental Toxicity" under the subsection
"Mode of Action Analysis—Developmental Toxicity and Neurodevelopmental Toxicity". Additional
consideration has been given to the studies suggested for consideration. This section has been
expanded to acknowledge potential developmental effects subsequent to genotoxic and mutagenic
mechanisms in germline and fetal cells, as well as changes in oxidative stress as a possible
contributing mechanism to developmental toxicity. Mechanistic references suggested by the peer
reviewers have been considered and incorporated where relevant
Comment: Toxicokinetic information regarding fetal exposures and lactational transfer should be
included in the consideration of developmental hazard.
Response: Information regarding the potential for lactational transfer of benzo[a]pyrene has been
added to the toxicokinetic information in Section D.l of the Supplemental Information. In addition,
a concise discussion of this information, as well as information on fetal distribution has been added
to Section 1.1.1 Developmental Toxicity.
Charge Question 2b. The draft assessment concludes that male and female reproductive
effects are a human hazard of benzo[a]pyrene exposure. Do the available human, animal and
mechanistic studies support this conclusion?
The SAB agreed that the data support the conclusion thatbenzo[a]pyrene is a male and female
reproductive toxicant, with rodent data demonstrating convincingly that benzo[a]pyrene affects
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fertility and fecundity. The SAB had the following specific recommendations:
Comment: The SAB recommends that the EPA consider additional female reproductive endpoints
for point of departure/BMD analyses and RfD derivation. The SAB suggested that decreased
follicular counts be considered as well as uterine hyperplasia and inflammation observed in the Gao
et al. (2011) study. The SAB recommends that the EPA either include these endpoints, or provide
appropriate justification as to why that they are not suitable for RfD determination.
Response: In response to the SAB recommendation to consider ovarian follicular counts further,
decreased primordial follicles reported by Xu etal. (20101 were considered supportive of
reproductive toxicity, as a depletion of follicles can result in shortening of a woman's reproductive
lifespan (U.S. EPA. 19961. Means and standard deviation were obtained from this graphically
reported endpoint, modeled, and included for candidate value derivation in Section 2 (also see
Appendix E.l).
A single study fGao etal.. 20111 reported increased inflammatory cells in the uterine cervix as well
as hyperplasia at higher doses. Effects in the uterus were not evaluated in other noncancer or
cancer bioassays in the database, except perhaps grossly in the cancer bioassay by Kroese et al.
(2001). Furthermore, it is unclear that the observed histological changes in the cervix are
associated with impaired reproductive function. This study also observed a depression of
bodyweight (10,15, and 30%) and elevated mortality in the two higher dose groups (4 and 8%),
suggesting general systemic toxicity. Overall, benzo[a]pyrene-related effects in the uterus are less
supported than ovarian/oocyte effects reported in subchronic and gestational studies (Xu etal..
2010: Kristensen etal.. 1995: Mackenzie and Angevine. 19811 and supported by a large body of
studies by other routes of exposure (IP) as well as in vitro mechanistic data (see Section 1.1.2,
"Mode-of-action analysis—female reproductive effects"). Therefore, although uterine hyperplasia
reported by Gao etal. (20111. was modeled and considered as a candidate toxicity value,
ovotoxicity, including decreased ovarian weight and decreased ovarian follicles, was deemed to be
more representative of the current body of evidence regarding benzo[a]pyrene-induced female
reproductive toxicity.
Sections 1.1.2 and 2.1 have been clarified to reflect the above considerations.
Comment: The SAB recommended that the EPA consider other male reproductive endpoints in
addition to the classical reproductive hazard endpoints included in the draft assessment. The SAB
specifically recommended considering germline mutagenesis as an endpoint
Response: Studies which evaluated germ cell mutagenesis in experimental animals following oral
exposure were not identified in the benzo[a]pyrene database. However, discussion of increased
male germ cell mutation in transgenic lac I mice treated with high dose intraperitoneal doses (Xu et
al.. 20141 has been added to the document in Section 1.1.2 Mode-of-action analysis—male
reproductive effects). The derivation of candidate toxicity values based on other effects in male
germ cells, such as decreased sperm count and mobility, which may be related to genotoxic
mechanisms of benzo[a]pyrene (also reviewed in Section 1.1.2 Mode-of-action analysis—male
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reproductive effects] were considered in the assessment (see Section 2.1.1). The stronger of the
available studies reporting these sperm endpoints, Mohamed etal. f201Cn. still involved too much
overall uncertainty as reflected in the composite uncertainty factor (see Table 2-2), therefore a
candidate value to represent effects on male germ cells could not be derived.
Comment: For male reproductive studies, the SAB recommends considering the recovery time after
treatment prior to endpoint measurement since the testis is proliferative and new rounds of
spermatogenesis could change the outcome. The SAB also noted that because the testis matures
after birth, additional consideration be given to the life stage at which the animals are exposed to
benzo[a]pyrene. The SAB specifically recommended consideration of studies demonstrating that
exposure at different life stages (e.g., pre-adult vs. adult), can have differential effects on reproductive
health.
Response: The discussion of studies which evaluated reproductive endpoints in male rodents has
been clarified to note the age of the animals at treatment (see Table 1-5). For the male
reproductive studies evaluated in Section 1.1.2, all but two of the studies f Mohamed et al.. 2 010:
Archibongetal.. 2008) evaluated endpoints directly following the exposure period. A footnote has
been added to the evidence table to clarify that endpoints were assessed directly following the
exposure period unless otherwise indicated.
Furthermore, additional discussion of studies indicating differential effects on male reproductive
endpoints following early life exposure (Xu etal.. 2014: Liang etal.. 2012)has been added to Section
1.1.2 in the subsection Susceptible Populations and Lifestages.
Comment: The SAB recommends that genotoxic and mutagenic aspects of reproductive hazard be
addressed, especially as they provide perspective on likely mode of action.
Response: Additional discussion of genotoxic and mutagenic properties of benzo[a]pyrene and the
corresponding endpoint of germline mutagenesis and its potential impact on reproductive hazard
has been added to the mode of action analysis sections for male and female reproductive effects
(see Section 1.1.2).
Comment: Several publications were recommended regarding inform sperm effects fleng etal..
20131. ovarian effects fKummer et al.. 2013: Sadeu and Foster. 2011: Mattison and Nightingale.
1980: Mattison. 1980). and the mode of action for female reproductive effects (Young etal.. 2014:
Sadeu and Foster. 2013).
Response: Two of these studies were already discussed in the assessment fSadeu and Foster. 2011:
Mattison and Nightingale. 1980). Of the other suggested studies, Tengetal. (2013). was identified as
a new subchronic study. The sperm effects observed in this study were supportive of the existing
characterization of benzo[a]pyrene as a male reproductive hazard, butwere seen at higher doses
than other studies investigating sperm parameters. This study has been added to the text and
evidence table informing male reproductive effects (see Section 1.1.2 and Table 1-5). The
additional studies informing potential mechanisms of ovarian follicle toxicity studies suggested by
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the SAB (Kummer etal.. 2013: Sadeu and Foster. 2011: Mattison. 19801supportthe hazard
conclusions in the assessment and support suspected mechanistic pathways of benzo[a]pyrene and
have been added to Section 1.1.2 Mode-of-action analysis—female reproductive effects. Young et al.
(20141 was not considered, as it was available as an abstract only.
Charge Question 2c. The draft assessment concludes that immunotoxicity is a potential
human hazard of benzo[a]pyrene exposure. Do the available human, animal, and
mechanistic studies support this conclusion?
The SAB agreed that the available immunotoxicity data from animal models and humans exposed to
complex PAH mixtures exposures support the claim that benzo[a]pyrene is a human hazard for the
immune system. The SAB listed several recommendations:
Comment: The SAB noted concerns that sensitive immune function endpoints (e.g. functional
immune tests) are not available to permit proper evaluation of benzo[a]pyrene immunotoxicity in
animal models, especially in developing animals. In addition, potential gender differences in
immunotoxicity were not addressed. The SAB recommended that these data gaps be acknowledged
in the draft assessment
Response: The available benzo[a]pyrene animal and mechanistic studies, as well as supportive data
from PAH mixture exposures in humans indicate that immune toxicity is a hazard of
benzo[a]pyrene exposure. However as pointed out by the SAB, data gaps exist in the assessment of
immune hazard from benzo[a]pyrene. Discussion of the lack of functional endpoints to assess
immunotoxicity of benzo[a]pyrene following subchronic or chronic exposure has been added in the
assessment in Section 1.1.3 and 2.1.1. In addition, the lack of studies evaluating functional changes
in the immune system following developmental exposure is discussed in Section 1.1.3 of the
Toxicological Review under "Susceptible Populations and Lifestages".
Scarce data is available to inform gender differences in immunotoxicity of benzo[a]pyrene.
However, increased discussion of the available studies has been added in Section 1.1.3 under
"Susceptible Populations and Lifestages".
Comment: The SAB recommended that the EPA consider developing guidelines for immunotoxicity
risk assessment, as has been done by the WHO (20121.
Response: The development of EPA Immunotoxicity guidelines would be helpful in the
consideration of immunotoxicity data, however, such an effort is outside of the scope of the
benzo[a]pyrene IRIS Toxicological Review.
Comment: The SAB recommended the consideration of additional studies including in vitro studies
in human peripheral blood mononuclear cells (no specific references were suggested) which may
inform mode of action, as well as three epidemiological studies which have investigated the
association of benzo[a]pyrene-adducts and immune endpoints (lung etal.. 2015: Tang etal.. 2012:
Tedrvchowskietal.. 20111.
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Response: Additional literature was considered and incorporated into the assessment where
relevant.
Charge Question 2d. The draft assessment concludes that benzo[a]pyrene is "carcinogenic to
humans" by all routes of exposure. Do the available human, animal, and mechanistic studies
support this conclusion?
The SAB concurred that the EPA has demonstrated that benzo[a]pyrene is a human carcinogen in
accordance with the Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005al. The SAB had the
following specific recommendations:
Comment: The Supplemental Material document contains only 6 papers in which DNA adduct
formation has been measured in humans. There are many more such papers in the literature and
this draft assessment would be more balanced if at least 20 of the most significant papers could be
included.
Response: In Section D.5.I., "Genotoxicity Information," of the Supplemental Information document,
Table D-33, "In vivo genotoxicity studies of benzo[a]pyrene," has been split into two tables: Table
D-34, "Studies of benzo[a]pyrene-induced genotoxicity in humans exposed to PAHs," and Table D-
35, "Non-human in vivo genotoxicity studies of benzo[a]pyrene." The previous table contained a
selection of studies in humans; the new Table D-34 contains all studies measuring BPDE-DNA
adduct formation in humans exposed to PAHs, along with the methods used, that are cited in the
mode of action for carcinogenicity in Section 1.1.5.
Comment: The current version of the draft assessment does not make a clear case for the pathway
of benzo[a]pyrene biotransformation that results in a mutagenic MOA. A series of the classical
critical papers, and their findings, have been listed as bullet points (under the discussion of EPA
Criterion 2), and this material should be included in the final benzo[a]pyrene document
Response: EPA has revised the "Mode of Action Analysis—Carcinogenicity" in Section 1.1.5 to more
clearly describe the sequence of key events leading to cancer following benzo[a]pyrene exposure
and to include the following references suggested in the bullet points in the SAB comments on EPA
Criterion 2 (Hussain etal.. 2001: Sticha etal.. 2000: Beland and Culp. 1998: Culp etal.. 1998: Wei et
al.. 1995: Manchester etal.. 1988: Marshall etal.. 1984: Grover etal.. 1976: Teffrev etal.. 1976: King
etal.. 1976: Osborne etal.. 1976: Daudel etal.. 1975: Sims etal.. 19741. Two references were not
added: Bovsen and Hecht (20031 is a review of methods for analyzing DNA adducts, and Pratt et al.
f20111 utilized an immunoassay for detecting PAH-DNA adducts that was not specific to
benzo[a]pyrene.
Comment: There is evidence of a strong association (Relative Risk or Odds Ratio) between
increased human cancer risk in particular organs, such as lung fTang etal.. 19951 and colon (Gunter
etal.. 20071 and high levels of BPdG or PAH-DNA adduct formation in human nucleated blood cells.
It would be useful to have these mentioned in a paragraph.
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Response: EPA recognizes the importance of correlations between levels of PAH-DNA adducts in
humans and cancer risk. However, for the assessment of benzo[a]pyrene, more weighthas been
given to studies specifically detecting BPDE-DNA adducts (and primarily BPdG adducts) that
strengthen the causal relationship between benzo[a]pyrene exposure and cancer risk. Tangetal.
(19951 and Gunter etal. (20071 utilized methods of detecting PAH-DNA adducts that were not
specific to benzo[a]pyrene. Therefore, these studies were not added to the Mode of Action
discussion.
Comment: A table describing the nomenclature, characteristics, specificity, sensitivity range, and
detection limit for the various methodologies used for human BPdG and PAH-DNA adduct
measurements could be easily assembled.
Response: EPA added Table D-31 to Section D.5.I., "Genotoxicity Information," in the Supplemental
Information document, which summarizes the nomenclature, characteristics, specificity, sensitivity
range, and detection limit on the various methodologies for adduct detection.
Charge Question 2e. The draft assessment concludes that the evidence does not support
other types of noncancer toxicity as a potential human hazard. Are there other types of
noncancer toxicity that can be credibly associated with benzo[a]pyrene exposure?
With respect to the health endpoints discussed in Section 1.1.4, "Other Toxicity", the SAB concurs
with the conclusion that the evidence presented does not support liver, kidney, and hematological
effects as human hazards. However, the SAB requested additional clarification on the hazard
conclusions regarding additional endpoints (such as forestomach toxicity, cardiovascular toxicity,
and adult nervous system effects) as discussed in the following comments:
Comment: The EPA should evaluate the missing references identified by the SAB on cardiovascular,
pulmonary, and kidney toxicity of benzo[a]pyrene. The SAB suggested several specific references
and opined that the literature search and study selection process may not have been sufficiently
comprehensive to identify all potential hazards credibly associated with benzo[a]pyrene exposure.
Response: The literature search performed for benzo[a]pyrene was designed to search for all
possible health outcomes of benzo[a]pyrene exposure, and as such did not include individual terms
for all organs or endpoints. (See discussion under Charge Question 1 - Literature Search, Study
Selection and Evaluation.). For example, hazard identification for chronic health effects, such as
cardiovascular toxicity, gave preference to studies that examined animal models translatable to
humans fe.g. Tules et al.. 20121. rather than on studies of genetically modified animals with
heightened disease susceptibility fKnaapen etal.. 2007: Curfs etal.. 2005: Curfs etal.. 20041 or non-
mammalian species (Hough etal.. 1993: Albert etal.. 19771.
In other comments made by the SAB in response to this charge question, adult and developmental
pulmonary toxicity were proposed as additional noncancer endpoints potentially associated with
benzo[a]pyrene exposure. However, little data exist to evaluate noncancer pulmonary effects in
This document is a draft for review purposes only and does not constitute Agency policy.
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adult or developing animals. As noted in the assessment, a 4-week inhalation study in adult rats is
available that investigated, but did not detect, lung injury fWolffetal.. 19891. Regarding pulmonary
effects in developing animals, a recent developmental study (highlighted for consideration by the
SAB) suggests pulmonary effects with high dose (25 mg/kg), intraperitoneal exposure to
benzo[a]pyrene and subsequent challenge with hyperoxic air (Thakur etal.. 20141. However,
interpretation of this study is complicated due to the route of exposure and the high doses
employed, which were one to two orders of magnitudes greater than doses at which effects were
observed in the oral developmental database. Therefore, the evidence available for pulmonary
noncancer effects was judged too sparse to make a hazard determination. The document has been
clarified to reflect these points in Section 1.1.4 and 1.2.1.
Additional references suggested by the SAB regarding cardiovascular, pulmonary, and kidney
toxicity of benzo[a]pyrene were reviewed and incorporated in the document where relevant
Comment: The EPA should be explicit as to the rationale for concluding that the available evidence
either does or does not support adult nervous system effects as a potential human hazard. The SAB
also states that the basis for arriving at the hazard conclusions for the other endpoints identified in
Section 1.1.4 "Other Toxicities", be expanded (e.g. for hematological toxicity, liver toxicity, kidney
toxicity, and cardiovascular toxicity). They state that the current text does not provide an adequate
rationale for the characterization (in Section 1.2.1) thatthe evidence does not support these
noncancer effects as potential human hazards. The SAB suggested additional clarification be
provided as to whether this conclusion is due to insufficient data, inconsistent data, or sufficient
data to conclude that these health endpoints are not sensitive endpoints.
Response: The characterizations of hazard summarized in Section 1.2.1. "Weight of Evidence for
Effects Other than Cancer" have been expanded for organ/systems discussed in Section 1.1.4 to
further clarify the overall hazard characterization.
• Specifically regarding the potential for benzo[a]pyrene exposure to cause adult nervous
system toxicity, this evidence is now more explicitly considered in the context of the totality
of the evidence available for potential nervous system effects of benzo[a]pyrene exposure in
Sections 1.1.4 and 1.2.1. As a result, while the adult neurotoxicity data are discussed as
consistent with the developmental neurotoxicity endpoints and indicated as suggestive of a
potential hazard in themselves, these data were comparably less robust than the studies
and data supporting developmental neurotoxicity as a hazard, and additional studies are
needed to draw a stronger conclusion regarding the identification of adult neurotoxicity as
a human hazard.
• Regarding the hazard characterization of forestomach toxicity (specifically forestomach
hyperplasia), EPA agrees with SAB that forestomach toxicity in animal models is credibly
associated with benzo[a]pyrene exposure and that it likely reflects early events in
benzo[a]pyrene-induced carcinogenicity. As benzo[a]pyrene-induced forestomach
hyperplasia was determined to be a preneoplastic lesion, it was relocated from Section 1.1.4
This document is a draft for review purposes only and does not constitute Agency policy.
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"Other Toxicities" to the discussion of forestomach tumors and related lesions in Section
1.1.5 "Carcinogenicity".
• Regarding the hazard characterization for cardiovascular effects of benzo[a]pyrene, the
interpretation of hazard is complicated by issues of co-exposure in human studies of
cardiovascular effects in populations highly exposed to benzo[a]pyrene as a component of a
complex PAH mixtures as well as the paucity of studies examining cardiovascular endpoints
in wild-type laboratory animals exposed by environmentally relevant routes for subchronic
or chronic durations. Short duration animal studies and studies by other routes of exposure
(e.g. IP and installation), as well as studies in genetically modified, highly susceptible animal
strains (e.g. APOE-/- mice), contribute to the plausibility of cardiovascular effects providing
suggestive evidence of cardiovascular toxicity due to benzo[a]pyrene exposure. The
cardiovascular endpoints were not considered for dose-response due to the relatively lower
confidence in this hazard. The discussion of cardiovascular hazard in Section 1.1.4 and
Section 1.2.1 has been expanded and clarified.
• In addition, as hematological effects can inform the weight of evidence for immunotoxicity
(WHO 2012), the tables and discussion regarding hematological effects observed in
subchronic and chronic studies have been relocated from Section 1.1.4 "Other Toxicity" to
Section 1.1.3 "Immune Toxicity". Hematological changes are therefore considered within
the context of the overall body of immune system changes.
Charge Question 3a. The draft assessment proposes an overall reference dose of 3x10-4
mg/kg-d based on developmental toxicity during a critical window of development. Is this
value scientifically supported, giving due consideration to the intermediate steps of
selecting studies appropriate for dose-response analysis, calculating points of departure,
and applying uncertainty factors? Does the discussion of exposure scenarios (section 2.1.5)
reflect the scientific considerations that are inherent for exposures during a critical window
of development?
Comment: The EPA should specifically consider the overall picture of neurodevelopmental impact
from all of the neurodevelopmental endpoints in Chen etal. f20121. including plus maze, reflex,
locomotor activity and water maze to justify and support the choice of the critical endpoint. In
particular, the SAB suggests that the EPA reconsider or provide stronger justification for not using
escape latency from the Morris water maze.
Response: As summarized in EPA's response to Charge Question 2a, EPA has further evaluated the
collection of neurodevelopmental behavioral effects reported by Chen etal. (2012). rather than
relying on the elevated plus maze alone. In the revised assessment, modeling results in PND 69-74
rats for open arm entries in the elevated plus maze (female rats), locomotor activity in the open
field (both sexes), and escape latency in the Morris water maze (both sexes) are used to define the
overall effect on behavior. Together, these results represent the most reliable and persistent
behavioral effects of benzo[a]pyrene exposure detected by Chen etal. (2012).
This document is a draft for review purposes only and does not constitute Agency policy.
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Comment: The EPA should explain how the BMD was calculated for escape latency in the Morris
water maze.
Response: The external peer review draft was not clear that escape latency for males and females
combined at PND 74 was used to calculate the BMD. Following the SAB's recommendation to
consider the overall impact on neurodevelopmental effects, and in considering the lack of
differences in escape latency across sexes as a result of changes in learning (as inferred by the EPA
and corroborated by the SAB), the revised assessment considers all four trial days. A more
transparent description of the BMD calculation for escape latency is provided (see Section 2.1.2 and
Appendix E.l). Specifically, EPA performed BMD modeling for escape latency at each of the four
trial days, PNDs 71-74, for males and females combined. EPA interpreted the trial day results to
equally represent the observed behavioral effect (although the underlying behavior affected
remains unidentified), and the revised assessment presents the ranges of the BMD and BMDL
values to characterize this effect
Comment: EPA should consider data on reproductive outcomes, including cervical hyperplasia and
inflammation from Gao etal. f20111 and clearly articulate the rationale for a candidate RfD based
on an ovarian effect
Response: See response to Charge Question 2b, which summarizes the evidence for hazard among
the reproductive outcomes. The revised assessment provides candidate RfDs for uterine
hyperplasia of the cervix, reduced ovarian weight, and reduced ovarian follicle count.
Comment: The EPA should consider application of a BW3/4 adjustment for extrapolation from
neonatal animal to neonatal human.
Response: The peer review draft benzo[a]pyrene assessment did not perform allometric scaling in
the calculation of an RfD based on animals directly dosed on PND 5-11 (Chen etal.. 2012). This was
due to several areas of uncertainty. The first issue was whether allometric (i.e., body weightA3/4)
scaling, originally derived from data in adult animals and adult humans, holds when extrapolating
from doses in neonatal animals to neonatal humans. This uncertainty arises because of the absence
of quantitative information to characterize the toxicokinetic and toxicodynamic differences
between animals and humans in early life stages fU.S. EPA. 20111. In addition, interspecies
extrapolation across early life stages is complicated by differences in temporal patterns of
development across species. U.S. EPA f20111. Recommended Use of Body Weight 3/4 as the Default
Method in Derivation of the Oral Reference Dose, states that when such an extrapolation is
considered, key developmental processes need to be matched in a species-dependent manner,
because the temporal pattern of development differs across species. In the study at issue, Chen et
al. f20121 Chen et al (2012), neurobehavioral changes were observed in adult rats after dosing on
PND 5-11. This postnatal period of brain development in rats is believed to be more akin to human
brain development occurring in the third trimester of pregnancy (Dobbingand Sands. 1979.1973).
thus challenging the suitability of extrapolating exposure doses from rats directly exposed through
gavage on PND 5-11 to the equivalent developmental period in third trimester humans (where
exposure would occur transplacental^).
This document is a draft for review purposes only and does not constitute Agency policy.
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Therefore, due to several associated uncertainties, EPA did not apply a BW3/4 adjustment for
extrapolation from neonatal animal to neonatal human. Additional clarification of these
considerations has been added to the assessment in Section 2.1.2.
Comment: The SAB recommended EPA further justify whether the application of a UF of 3 for
database deficiency is adequate. The SAB specifically highlighted endpoints which may
qualitatively support a hazard, but lack dose-response data sufficient for developing toxicity values
(such as cardiovascular effects and developmental immunotoxicity). The SAB also requested
additional consideration of the database UF in the context of potential effects such as miscarriage,
birth defects, and genetic disease.
Response: The database UF is intended to account for the potential for deriving an under protective
reference value as a result of an incomplete characterization of the chemical's toxicity (U.S. EPA.
20021. In addition to identifying toxicity information that is lacking, existing data may also suggest
that a lower reference value might result if additional data were available. When applying this
uncertainty factor, both the data lacking and the data available are considered. For benzo[a]pyrene,
a database uncertainty factor, UFd, of 3 was applied to account for database deficiencies, including
the lack of a standard multigenerational study or extended 1-generation study that includes
exposure from premating through lactation. These types of studies would be useful to
understanding the full potential for benzo[a]pyrene exposure to cause reproductive and
neurodevelopmental effects. Considering that benzo[a]pyrene has been shown to affect fertility in
adult male and female animals by multiple routes of exposure and that decreased fertility in adult
male and female mice is observed both following premating exposure and following gestational
exposure (see Section 1.1.2), it is plausible that exposure occurring over this more comprehensive
period of development or over multiple generations could result in a more sensitive POD, than the
POD selected for developmental neurotoxicity.
Some additional uncertainties exist in the benzo[a]pyrene database, including the paucity of
sensitive studies evaluating endpoints of immune and cardiovascular toxicity. The lack of
developmental immune toxicity studies, especially those examining functional endpoints, is a
notable uncertainty in the benzo[a]pyrene database. Some consideration was given to
cardiovascular effects through the candidate value derived for developmental effects of the
cardiovascular system flules etal.. 20121. Although this candidate value was not as sensitive as the
candidate value derived from the neurodevelopmental study selected as the basis of the overall RfD.
As the SAB suggests, genotoxic effects of benzo[a]pyrene could potentially manifest through
miscarriage, birth defects, and genetic disease. However, several developmental studies are
available which do not report birth defects at doses much higher than the POD used for the RfD
(Kristensen etal.. 1995: Mackenzie and Angevine. 19811. A decrease in pups per litter/decreased
fetal survival was observed in F0 dams at 60 mg/kg-day (Mackenzie and Angevine. 19811. but has
not be observed in oral exposure studies at lower doses closer to the POD used for the RfD (see
Table 1-2). While consequences of genotoxic aspects of reproductive hazard are plausible due to
the genotoxic and mutagenic MOA of benzo[a]pyrene, endpoints of fetal survival and birth defects,
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while affected at high doses, have not been detected at studies near the POD for
neurodevelopmental changes.
The POD for the overall RfD was based on several sensitive neurobehavioral endpoints observed
following treatment during a sensitive period of brain development and were among the lowest
effect levels observed in the benzo[a]pyrene database, even among other developmental studies
utilizing low doses of benzo[a]pyrene (such as Tules et al.. 20121: thus, application of a full database
UF of 10 was not judged to be warranted. However, because studies following a more
comprehensive period of developmental exposure (i.e., early gestation through lactation, if not
through adolescence) were not available, a database UF of 3 was applied to address residual
uncertainty associated with the potential for effects at lower doses (see also response to Charge
Question 2a).
Additional justification of the database uncertainty factor has been added to Section 2.1.3.
Charge Question 3b. The draft assessment proposes an overall reference concentration of 2 x
10"6 mg/m3 based on decreased fetal survival during a critical window of development. Is
this value scientifically supported, giving due consideration to the intermediate steps of
selecting studies appropriate for dose-response analysis, calculating points of departure,
and applying uncertainty factors? Does the discussion of exposure scenarios (section 2.2.5)
reflect the scientific considerations that are inherent for exposures during a critical window
of development?
Comment: The study used to derive the overall RfC (Archibongetal.. 2002) reported decreases in
fetal survival that occurred at all concentrations. EPA reported that the data for this endpoint were
not amenable to dose-response modeling, therefore the point of departure for this endpoint was
derived from the LOAEL for decreased fetal survival (a 19% response relative to control). The peer
reviewers noted that the rationale for not employing benchmark dose modeling was unclear and
that the LOAEL provides a weaker basis than a NOAEL for the derivation of the RfC.
Response: EPA agrees that the rationale for not employing benchmark dose modeling for
decreased fetal survival was unclear, and that a LOAEL is a less desirable POD. Since the release of
the External Peer Review draft, a recently published approach by Fox et al. (2016) has facilitated
the dose-response modeling of the above embryo/fetal survival data using dichotomous BMDS
models. In the revised assessment, points of departure derived from this newer approach, including
derivation of adjustment factors for each exposure group, are provided for comparison. Further
details of the modeling calculations are provided in the Supplemental Information (see Appendix
E.1.2).
To clarify, in the External Peer Review draft, benchmark dose modeling was attempted by applying
continuous dose-response models because the data for this endpoint were reported as means and
standard deviations of litter-specific percentages of fetuses surviving at birth. These models relied
on the assumption that a normal approximation for binomial responses was adequate to
characterize the underlying (dichotomous) survival incidence data. However, the non-
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monotonicity of the observed variances, which showed maximum variability near 50% response
levels, typical of binomial variability, could not be addressed, and there were no adequate fits.
These issues have been clarified in Appendix E.1.2.
Accordingly, a LOAEL was judged to be the only feasible approach for this data set at the time. EPA
agrees that a LOAEL is a less desirable POD than a NOAEL, but the study has no NOAEL. However,
an approach for approximating the underlying dichotomous data from reported percentages has
been developed, thus facilitating application of more relevant dichotomous dose-response models
(Fox etal.. 20161. The approach, which is included in Appendix E.1.2 for comparative purposes, is
based on the work of Rao and Scott f!9921. which relies on the incidence among total offspring in
each group. While this measure is known to estimate means of effect adequately, it also
underestimates variability by overestimating effective sample sizes. Rao and Scott f!9921
developed a data transformation that relies on individual litter data to correct for this
overestimation through estimating "design effect." fFoxetal.. 20161 extended this approach for
data sets without individual litter data, through analysis of historical data sets of developmental
toxicity.
Dose-response modeling of the adjusted data yielded adequate fits to the observed data with all but
one of the dichotomous BMDS models. Further details are provided in the Supplemental
Information (see Appendix E.1.2), including derivation of the adjustment factors for each exposure
group.
Comment: The RfC was based on one outcome in one study (decreased fetal survival noted
following gestational exposure to rat dams). Peer reviewers suggested two additional studies, Wu
etal. (2003a) and Archibong etal. (2012). as potentially useful in developing a more
comprehensive dose-response relationship for the RfC and suggested consideration of these
endpoints for BMD analysis, potentially increasing confidence in the RfC.
Response: The gestational exposure study by Wu etal. (2003a) followed a protocol similar to that
of an associated group of collaborators (Archibong etal.. 2002). including using the same strain of
rats (Sprague-Dawley). However the publication omitted the numbers of dams treated in each
group as well as the resulting number of offspring. Following exposure on GDs 11-20, Wu et al.
reported statistically significant decreases in fetal survival at 75 and 100 |ig/m:i benzo[a]pyrene,
and an apparent decrease in fetal survival of approximately 9% (relative to the pooled carbon black
control groups) at 25 ng/m3. If it can be assumed that there were 10 dams per group, as used by
Archibong et al. f20021. there would be a statistically significant trend in decreased survival
(p=0.0004) across the pooled carbon black control groups and the exposure levels tested.
Therefore, Wu etal. (2003a) showed very similar fetal mortality to that observed by Archibong et
al. (2002) and adds to the weight of evidence for this outcome, but was not considered for dose-
response modeling due to incomplete reporting of data.
The 14-day premating study of F344 rats exposed to 50, 75,100 ug/m3 nose-only inhalation for 4
hours/day (Archibong etal.. 2012) showed reductions in ovarian function (ovulation rate), ovarian
weight, mean numbers of pups born, and fetal survival with increasing exposure concentration
(100 ug/m3), and has been added to the Table 1-7 pertaining to female reproductive effects. This
study covered an exposure period distinct from the developmental period covered in Archibong et
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al. (2002) (GDs 11-20), and suggests that exposure prior to mating could result in a decreased
number of pups per litter distinct from that observed following gestation-only exposure.
The assessment has been clarified to reflect these considerations [see Section 1.1.1] and outcomes
reported by Archibong et al. (2012) are considered for RfC derivation in Section 2.2.1.
Comment: The SAB specifically commented on the use of 3 instead of 10 for interspecies
extrapolation. The SAB noted that the UF of 3 to address residual uncertainty for interspecies
extrapolation in the inhalation reference concentration may be too low as the rat to human
dosimetric adjustment may not completely account for systemic toxicokinetics leading to a non-
respiratory effect of decreased fetal survival following an inhalation exposure. Furthermore, the
SAB expressed concern that the dosimetric adjustment used by EPA inadequately accounts for
interspecies differences in filtration of the aerosol (based on particle size) by the upper respiratory
tract
Response: EPA agrees that there is uncertainty in the dosimetric adjustment. Since the mode of
action leading to decreased fetal survival is not known, it appears reasonable to consider the dose
to the entire respiratory tract in either species instead of, for instance, the dose only to the deep
lung (in which case the more efficient filtration of 2.5 |a,m particles by the rat nose compared to the
human nose would have to be accounted for in the extrapolation.) Secondly, data for modeling
species differences in clearance and metabolism of the deposited particles are not available.
Therefore, given these uncertainties, EPA assumes the relevant dose metric to be the mass of
benzo[a]pyrene deposited per day in the entire respiratory tract normalized by the body weight
This metric would be more accurate than using exposure concentration as the default even if it does
not fully account for the toxicokinetics. Accordingly, as per EPA policy, an uncertainty factor of 3 is
used to account for species differences in toxicodynamics and residual differences in toxicokinetics
not accounted for in the dosimetric adjustment. Consideration of the above uncertainties in the
interspecies adjustment for the RfC has been added to Section 2.2.3, Derivation of Candidate Values.
Comment: SAB recommends that the EPA include a brief discussion of the rationale for selection of
the allometric scaling factor in the context of inhalation exposure to benzo[a]pyrene leading to
decreased fetal survival. In particular, the SAB highlighted text from EPA's RfC methodology (US
EPA, 1994) that suggests that EPA used BWi-scaling for this outcome, rather than the BW3/4-scaling
used for the oral toxicity values.
Response: EPA's RfC methodology for estimating human equivalent doses resulting from particle
exposure distinguishes between portal of entry effects, for which the mass of chemical deposited in
the respiratory tract is normalized by the surface area of the affected region, from remote effects,
for which body weight is the normalizing factor (analogously to mg/kg-day for oral exposure). The
overall dosimetric adjustment factor also involves estimating the mass of particles deposited from
minute volume (mL/min) and the fraction of inhaled dose that is deposited in the respiratory tract,
which in turn relies on functional residual capacity and upper respiratory tract volume (mL); all of
these considerations incorporate allometric differences between humans and the experimental
animals.
This document is a draft for review purposes only and does not constitute Agency policy.
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The dosimetric adjustment factor for extrarespiratory effects observed in rats was estimated to be
1.1, which is nearly equivalent to assuming that concentrations in air are equipotent across species.
Since intakes scale by BW3/4, this allometric scaling factor was consistent with the BW3/4-scaling
used for the oral toxicity values. Additional clarification has been added to the assessment (see
Sections 2.2.2 and 2.2.8).
Charge Question 3c. The draft assessment proposes an oral slope factor of 1 per mg/kg-d
based on alimentary tract tumors in mice. Is this value scientifically supported, giving due
consideration to the intermediate steps of selecting studies appropriate for dose-response
analysis and calculating points of departure?
Comment: The SAB noted that if no biological basis exists for concluding that the mouse study is
more representative of human response than the rat study, the EPA should consider averaging over
both studies to derive the oral slope factor for benzo[a]pyrene.
Response: EPA is not aware of a biological basis for concluding that the mouse study is more
representative of human response than the rat study. The three estimated slope factors fall within
a fivefold range (before rounding to one significant digit). Under the assumption that the three data
sets have equal relevance for extrapolating to humans, a geometric mean of the three slope factors
is 0.60 per mg/kg-day, and a geometric mean that gives equal weight to rats and mice is 0.74 per
mg/kg-day, about 50% of the highest slope factor (1.4 per mg/kg-day).
EPA notes that slope factors are intended to provide an upper bound on the cancer risk of a
randomly selected individual fU.S. EPA. 2005al EPA's approach to quantifying low-dose cancer risk
relies on a 95% upper bound on the cancer risk that typically addresses only the experimental
variability in homogeneous laboratory animals. The NRC (2009) has pointed out that when cancer
risk is expected to be linear at low exposures, as with benzo[a]pyrene, EPA's cancer risk values tend
not to address human variability and susceptibility adequately. Concern for sensitive
subpopulations supports use of the higher value here as an overall upper bound, twofold higher
than the geometric mean slope factor.
Comment: The SAB recommended that EPA should compare oral slope factors derived from fitting a
range of models to dose-response data.
Response: Given that low-dose linearity is expected for benzo[a]pyrene carcinogenicity due to its
mutagenic mode of action, the multistage-Weibull model is preferred because it can incorporate the
individual animal data that were available for time and cause of death. For comparison purposes,
however, an approximate survival adjustment was applied to summary incidence data using the
poly-3 technique (Bailer and Portier. 1988). in order to take into account reductions in animals at
risk while using a range of dichotomous models. The incidence data for the alimentary system
tumors only were used as basis of comparison across the three data sources. All of the
dichotomous BMDS models fit the adjusted summary incidence data well. For each data set, the
ranges of BMDioS and BMDLioS derived from these models, including models that allow low-dose
nonlinearity, were found to include the time-to-tumor estimates and varied less than 1.5-fold from
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the time-to-tumor estimates. These analyses are summarized in Section 2.3.3 and Appendix E.2.1
(Cancer Endpoints).
Comment: The SAB recommends that the EPA provide an explanation of the rationale for its
selection of an allometric scaling factor for the benzo[a]pyrene oral cancer slope factor given what
is known about the benzo[a]pyrene mode of action for carcinogenicity, reaction rates, and
toxicokinetics, and specifically, how the selection of the allometric scaling factor applies when there
is a portal of entry effect
Response: Despite extensive research into benzo[a]pyrene toxicokinetics, very little information
directly informs estimates of human-equivalent benzo[a]pyrene doses. Itis understood that
benzo[a]pyrene carcinogenicity involves a mutagenic MOA mediated by DNA-reactive metabolites
in the tissues where tumors appear, both at the portal of entry and those involving systemic
distribution (i.e., liver, kidney, and skin in rats). While the metabolites are highly reactive (likely
not limited by processes consistent with BW3/4 proportionality), distribution of benzo[a]pyrene to
these tissues and formation of the metabolites are limited by processes likely to be consistent with
BW3/4 proportionality.
EPA guidance (U.S. EPA. 2011) observed that because a "BW3/4 relationship exists among species in
studies where dose is administered in food, because interspecies food consumption follows a BW3/4
relationship ... it is reasonable to apply the BW3/4 approach for gastrointestinally related, portal-of-
entry effects." Beland and Culp (1998) administered benzo[a]pyrene in the diet, while Kroese et al.
(2001) administered benzo[a]pyrene via gavage. It is not clear what impact this difference in
administration has on estimating human equivalent doses of benzo[a]pyrene. This issue has been
clarified in the assessment (see Section 2.3.2.).
EPA's guidance also emphasizes that for a portal-of-entry scenario, "the most appropriate dose
metric would likely be mass of agent per surface area, e.g., mg/cm2," but acknowledges that
implementation of this approach involves such issues as "the lack of a human anatomical parallel to
the rodent forestomach," surface areas of the GI tract in rodents and humans, rates and scenarios of
ingestion, and diffusion rates (US EPA, 2011). These considerations have yet to be developed;
therefore, EPA has utilized BW3/4 for interspecies extrapolation, as recommended by the guidance
document. Section 2.3.2 includes clarification of this issue.
Charge Question 3d. The draft assessment proposes an inhalation unit risk of 0.6 per mg/m3
based on a combination of several types of benign and malignant tumors in hamsters. Is this
value scientifically supported, giving due consideration to the intermediate steps of
selecting studies appropriate for dose-response analysis and calculating points of
departure?
Comment: The SAB noted that EPA should conduct supplemental sensitivity analyses using other
dose-response models, alternative assumptions, and not eliminating from the analysis all animals
without confirmed examination of one or more of the pharynx or respiratory tract tissues.
This document is a draft for review purposes only and does not constitute Agency policy.
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Response: EPA agrees that supplemental sensitivity analyses provide useful perspective for the
estimated unit risk and has included a summary of analyses addressing the issues that the SAB
identified (see Appendix E.3.2, Table E-32).
First, concerning the incidence of animals without confirmed examination of one or more of the
pharynx or respiratory tract tissues, the MSW software does not take into account the durations of
exposure corresponding to unknown tumor status. An alternative analysis was conducted to
evaluate the impact of assuming that each animal with unknown tumor status had no tumors, a
possible underestimate of the true situation; the resulting unit risk was about 20% lower than that
based on complete data. The other extreme, assuming that all animals with unknown dispositions
actually had tumors, was not considered due to its higher implausibility.
Concerning other dose-response models, BMDS dichotomous dose-response models were applied
to poly-3 adjusted incidence data to address intercurrent mortality. These adjusted estimates also
considered the length of time on study for the animals with incomplete histopathology. Only one
model, a two-degree multistage model applied after dropping the high-exposure group, provided an
adequate fit
Concerning latency in the time-to-tumor model, the lack of information in the scientific literature
for respiratory tumors and lack of cause-of-death information in the Thvssenetal. fl9811 data set
limited the options for sensitivity analyses. One approach involved making assumptions about
which tumors were or were not the cause death, and yielded a latency estimate of 14.5 weeks. In
another approach, latency was fixed at several values in the range 2-90 weeks; the best-fitting
model, as judged by AICs, assumed a latency of 90 weeks yetyielded a BMDio and BMDLio very
similar to those for the recommended unit risk. These results suggest some insensitivity to latency,
or possibly that a constant value for latency across exposure levels is not supported.
Alternatives for cross-species equivalence of exposures were considered independently of
additional modeling, because there is no information to suggest that this equivalence changes with
exposure level. The recommended unit risk relies on the assumption that the amount inhaled,
normalized by body weight, leads to comparable cancer risk across species. The alternatives
comprised consideration of scaling inhaled doses, in mg/kg-day units, by bodyweight3/4
(highlighting allometric differences in metabolism and clearance rates over their lifetimes) and by
bodyweight2/3 (highlighting species differences proportional to relative surface areas). Both
considerations suggest higher risks to humans than to hamsters at the same exposure level, by
about fivefold and eightfold, respectively.
Comment: The SAB recommended that EPA give further consideration to occupational studies,
specifically, studies of lung cancer with airborne inhalation exposures to PAHs in coke oven and
aluminum smelter workers (or meta-analysis of occupational studies), to develop unit risk
estimate(s) for inclusion in Table 2-9 alongside the benzo[a]pyrene unit risk estimates calculated
from the chronic inhalation cancer bioassay in hamsters fThvssen et al.. 19811. The panel
acknowledged that interpretation of the epidemiological evidence is challenging given that
exposures are to mixtures of PAHs with poorly understood interactions, but suggest that a model
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using relative potency factors and an assumption of dose additivity could be considered for
adjustment of epidemiological results in estimation of the unit risk attributable to benzo[a]pyrene
alone.
Response: EPA agrees that some occupational studies of benzo[a]pyrene-containing PAH mixtures
may support development of unit risk estimates, given suitable information to characterize the
contributions of other chemicals in the mixtures. Although the SAB suggested a framework for this
analysis fU.S. EPA. 1990b! EPA is currently revising its relative potency factor (RPF) approach for
PAH mixtures, and will defer any indirect benzo[a]pyrene unit risk derivation from human studies
of PAH mixtures until revised estimates, currently underway, are available.
EPA agrees that studies of cancer in occupations that are highly exposed to PAHs could be used
quantitatively to develop unit risk estimates. However, the resulting cancer risk estimate would
represent the carcinogenic potential of the entire mixture including a spectrum of PAHs as well as
other potentially carcinogenic components, and would not be representative of benzo[a]pyrene
alone. The establishment of an IUR for benzo[a]pyrene alone is important as it serves as the index
chemical for the EPA's relative potency factor approach for assessing the carcinogenic potential of
PAH mixtures (U.S. EPA. 1990b) which allows for the estimation of carcinogenic potential of PAH
mixtures when unit risk estimates for the whole mixture is not available. While the exercise
suggested by the SAB might provide interesting comparisons that could roughly inform the
plausibility of the IUR calculated from the available animal data, the results would likely be highly
uncertain and inconclusive.
Comment: The SAB also suggests the inclusion of an explicit conclusion statement regarding overall
uncertainty of the unit risk value, and a brief discussion of the applicability of this value to typical
environmental exposures (especially for sensitive populations).
Response: Criteria to weigh the confidence in cancer risk values have not yet been developed for
IRIS Toxicological Reviews, thus explicit conclusion statements regarding the overall confidence in
cancer risk values were not added to this assessment. However, specific uncertainties in the unit
risk value for benzo[a]pyrene are discussed in Section 2.4.4 and outlined in Table 2-10.
Regarding the applicability of the IUR to typical environmental exposures including those in early
life, statements have been added in Section 2.3.3 to clarify the intended use of this value.
Specifically, because cancer risk values calculated for benzo[a]pyrene were derived from adult
animal exposures, and because benzo[a]pyrene carcinogenicity occurs via a mutagenic MOA,
exposures which occur during early life would require the application of age-dependent-
adjustment-factors (see Section 2.6). In addition, the IUR for benzo[a]pyrene is derived with the
intention that it will be paired with EPA's relative potency factors (RPFs) for the assessment of the
carcinogenicity of PAH mixtures.
This document is a draft for review purposes only and does not constitute Agency policy.
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Charge Question 3e. The draft assessment proposes a dermal slope factor of 0.006 per
|ig/day based on skin tumors in mice. Is this value scientifically supported, giving due
consideration to the intermediate steps of selecting studies appropriate for dose-response
analysis, calculating points of departure, and scaling from mice to humans? Does the method
for cross-species scaling (section 2.5.4 and appendix E) reflect the appropriate scientific
considerations?
Comment: The SAB commended the EPA's efforts to derive the IRIS Program's first dermal slope
factor. However, they noted that the proposed dermal slope factor and the proposed method for
cross-species scaling was not sufficiently supported. The SAB did not have a specific
recommendation as to dose metric, except to note that it should be based on absorbed dose. They
went on to recommend that in the absence of empirical data, the decision be based upon a clearly
articulated, logical, scientific structure that includes what is known about the dermal absorption of
benzo[a]pyrene under in laboratory animal bioassays and anticipated human exposures.
Response: EPA is reviewing the SAB panel's specific advice and is initiating further scientific
discussions to gather a broad range of scientific perspectives in order to further refine EPA's
approach for deriving a benzo[a]pyrene dermal slope factor. In the interest of timeliness and in
consideration of the support for the cancer characterization and the other toxicity values within the
benzo[a]pyrene assessment, the continuing efforts to refine the dermal slope factor methodology
will be addressed in a separate assessment.
Charge Question 3f. The draft assessment proposes the application of age-dependent
adjustment factors based on a determination that benzo(a)pyrene induces cancer through a
mutagenic mode of action. Do the available mechanistic studies in humans and animals
support a mutagenic mode of action for cancer induced by benzo(a)pyrene?
The SAB agreed that the available mechanistic studies in humans and animals support a mutagenic
mode of action for benzo[a]pyrene-induced cancers. They also supported the proposed use of age-
dependent adjustment factors (ADAFs), as established in EPA's Supplemental Guidance for
Assessing Susceptibility from Early-Life Exposures to Carcinogens fU.S. EPA. 2005b). for the
adjustment of tumor risk from childhood exposures to carcinogens with a mutagenic mode of
action.
Charge Question 4. Does the executive summary clearly and appropriately present the major
conclusions of the assessment?
The SAB found that the major conclusions of the draft assessment were clearly and appropriately
presented in the Executive Summary.
Charge Question 5. In August 2013, EPA asked for public comments on an earlier draft of this
assessment. Appendix G summarizes the public comments and this assessment's responses
to them. Please comment on EPA's responses to the scientific issues raised in the public
comments. Please consider in your review whether there are scientific issues that were
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raised by the public as described in Appendix G that may not have been adequately
addressed by EPA.
The SAB found that most of the scientific issues raised by the public, as described in Appendix G of
the peer review draft Supplemental Information document, were adequately addressed by EPA.
However, there were some issues for which the SAB provided additional discussion in the report
under Charge Questions 2e and 3e, and EPA responded accordingly.
Comment: The SAB recommended that major science issues pointed out by public commenters
should be included in the relevant charge questions, allowing the SAB to weigh in on EPA's
approach. The SAB recommended that in the future, they should not be asked if EPA has
adequately addressed all public comments.
Response: Going forward, EPA will capture major science issues expressed in the public comments
within the body of related charge questions.
This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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