c/EPA
EPA/635/R-14/312b
External Review 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
September 2014
NOTICE
This document is an External Review 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 KEYWORDS 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.1.3. Distribution D-3
D.1.4. Metabolism D-4
D.1.5. 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-14
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-28
D.4. ANIMAL STUDIES D-38
D.4.1. Oral Bioassays D-38
D.4.2. Inhalation Studies D-58
D.4.3. Dermal studies D-61
D.4.4. Reproductive and Developmental Toxicity Studies D-71
D.4.5. Inhalation D-87
D.5. OTHER PERTINENT TOXICITY INFORMATION D-91
D.5.1. Genotoxicity Information D-91
D.5.2. Tumor Promotion and Progression D-114
D.5.3. Benzo[a]pyrene Transcriptomic Microarray Analysis D-118
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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. Reference Dose (RfD) E-l
E.l.2. Reference Concentration (RfC) E-28
E.2. Cancer Endpoints E-31
E.2.1. Dose-Response Modeling for the Oral Slope Factor E-31
E.2.2. Dose-Response Modeling for the Inhalation Unit Risk E-66
E.2.3. Dose-Response Modeling for the Dermal Slope Factor E-75
APPENDIX F. DOCUMENTATION OF IMPLEMENTATION OF THE 2011 NATIONAL RESEARCH
COUNCIL RECOMMENDATIONS F-l
APPENDIX G. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND EPA'S
DISPOSITION G-l
REFERENCES FOR APPENDICES R-l
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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. Literature search strategy keywords for benzo[a]pyrene C-l
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-23
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-25
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-39
Table D-8. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene by gavage 5
days/week for 5 weeks D-43
Table D-9. Means ± SD for liver and thymus weights in Wistar rats exposed to benzo[a]pyrene
by gavage 5 days/week for 90 days D-45
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-47
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-51
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-53
Table D-13. Other oral exposure cancer bioassays in mice D-54
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) D-60
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-63
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-64
Table D-17. Tumor incidence in female Swiss mice dermally exposed to benzo[a]pyrene for up
to 93 weeks D-65
Table D-18. Skin tumor incidence in female NMRI and Swiss mice dermally exposed to
benzo[a]pyrene D-66
Table D-19. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene D-67
Table D-20. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene D-67
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Table D-21. Skin tumor incidence and time of appearance in female CFLP mice dermally
exposed to benzo[a]pyrene for 104 weeks D-68
Table D-22. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene for
life D-69
Table D-23. Skin tumor incidence in male C3H/HeJ mice dermally exposed to benzo[a]pyrene for
24 months D-70
Table D-24. Mortality and cervical histopathology incidences in female ICR mice exposed to
benzo[a]pyrene via gavage for 14 weeks D-74
Table D-25. Means ± SD for ovary weight in female Sprague-Dawley rats D-77
Table D-26. Reproductive effects in male and female CD-I F1 mice exposed in utero to
benzo[a]pyrene D-79
Table D-27. Effect of prenatal exposure to benzo[a]pyrene on indices of reproductive
performance in F1 female NMRI mice D-80
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-84
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-85
Table D-30. Pregnancy outcomes in female F344 rats treated with benzo[a]pyrene on GDs
11-21 by inhalation D-88
Table D-31. In vitro genotoxicity studies of benzo[a]pyrene in non-mammalian cells D-91
Table D-32. In vitro genotoxicity studies of benzo[a]pyrene in mammalian cells D-93
Table D-33. In vivo genotoxicity studies of benzo[a]pyrene D-98
Table D-34. Search terms and the number of studies retrieved from the gene expression
omnibus and array express microarray repositories D-118
Table D-35. Mapping of group numbers to time/dose groups D-121
Table E-l. Noncancer endpoints selected for dose-response modeling for benzo[a]pyrene: RfD E-2
Table E-2. 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-3
Table E-3. 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-7
Table E-4. 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-5. Summary of BMD modeling results for Morris water maze: escape latency in male
and female Sprague-Dawley rats exposed to benzo[a]pyrene by gavage for 90
days (Chen et al., 2012); BMR = 1 SD change from the control mean E-14
Table E-6. Summary of BMD modeling results for Morris water maze: time spent in quadrant for
in male and female Sprague-Dawley rats exposed to benzo[a]pyrene by gavage
for 90 days (Chen et al., 2012); BMR = 1 SD change from the control mean E-18
Table E-7. Summary of BMD modeling results for elevated plus maze: open arm entries for
females at PND 70 (Chen et al., 2012); BMR = 1 SD change from the control
mean E-21
Table E-8. 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 = 1 SD change from the control mean E-25
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Table E-9. 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-34
Table E-10. 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-37
Table E-ll. 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-40
Table E-12. Derivation of HEDs to use for BMD modeling of Wistar rat tumor incidence data
from Kroese et al. (2001) E-43
Table E-13. Derivation of HEDs for dose-response modeling of B6C3Fi female mouse tumor
incidence data from Beland and Culp (1998) E-43
Table E-14. 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-44
Table E-15. 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-63
Table E-16. 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-63
Table E-17. Individual pathology and tumor incidence data for male Syrian golden hamsters
exposed to benzo[a]pyrene via inhalation for lifetime—Thyssen et al. (1981) E-67
Table E-18. 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-70
Table E-19. Tumor incidence, with time to observation of tumor or death; CeH/HeJ male mice
exposed dermally to benzo[a]pyrene (Sivak et al., 1997; Arthur D Little, 1989) E-78
Table E-20. Skin tumor incidence, benign or malignant in female Swiss or NMRI mice dermally
exposed to benzo[a]pyrene; data from Roe et al. (1970), Schmidt et al. (1973),
Schmahl et al. (1977), Habs et al. (1980), Habs et al. (1984) E-79
Table E-21. Skin tumor incidence, benign or malignant, in C57L male mice dermally exposed to
benzo[a]pyrene; data from Poel (1959) E-80
Table E-22. Skin tumor incidence, benign or malignant, in female CFLP mice dermally exposed to
benzo[a]pyrene; data from Grimmer et al. (1983), Grimmer et al. (1984) E-80
Table E-23. Summary of BMD modeling results for best-fitting multistage-Weibull models, using
time-to-tumor data for male CeH/HeJ mice exposed dermally to benzo[a]pyrene
(Sivak et al., 1997; Arthur D Little, 1989) E-81
Table E-24. Summary of BMD model selection and modeling results using multistage models,
for multiple data sets of skin tumors in mice following lifetime dermal
benzo[a]pyrene exposure E-86
Table E-25. Alternative approaches to cross-species scaling E-115
Table F-l. The EPA's implementation of the National Research Council's recommendations in
the benzo[a]pyrene assessment F-2
Table F-2. National Research Council recommendations that the EPA is generally implementing
in the long term F-7
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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-115
Figure D-4. AhR pathway. 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-34). For instance, NRF2 is upregulated in
the 25 mg/kg D-122
Figure D-5. DNA damage pathway. Activation of transcriptional targets of p53, including p21
and GADD45, and upregulation of the downstream transcriptional target, PCNA,
suggests that p53 is activated D-123
Figure D-6. Nrf2 pathway. Nrf2 is upregulated by benzo[a]pyrene exposure, which results in the
upregulation of Phase II detoxifying enzymes. This appears to be a
compensatory response due to increased oxidative status within cells D-125
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-4
Figure E-2. Fit of linear model (constant variance) to data on decreased thymus weight in
female Wistar rats—90 days (Kroese et al., 2001) E-8
Figure E-3. Fit of linear/polynomial (1°) model to data on decreased ovary weight (Xu et al.,
2010) E-ll
Figure E-4. Fit of Hill model to data on Morris water maze test escape latency (Chen et al.,
2012) E-15
Figure E-5. Fit of Exponential 4 model to data on Morris water maze time spent in target
quadrant (Chen et al., 2012) E-18
Figure E-6. Fit of exponential model (4) to data on elevated plus maze open arm maze entries
(Chen et al., 2012) E-22
Figure E-7. Fit of log-logistic model to data on cervical epithelial hyperplasia (Gao et al., 2011) E-25
Figure E-8. Human fractional deposition E-29
Figure E-9. Rat fractional deposition E-30
Figure E-10. 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-46
Figure E-ll. Fit of multistage Weibull model to hepatocellular adenomas or carcinomas in male
rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-48
Figure E-12. Fit of multistage Weibull model to duodenum or jejunum adenocarcinomas in male
rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-50
Figure E-13. 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-52
Figure E-14. 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-54
Figure E-15. Fit of multistage Weibull model to kidney urothelial tumors of male rats exposed
orally to benzo[a]pyrene (Kroese et al., 2001) E-56
Figure E-16. 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-58
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Figure E-17. Fit of multistage Weibull model to hepatocellular adenomas or carcinomas in
female rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-60
Figure E-18. Fit of multistage Weibull model to duodenum or jejunum adenocarcinomas in
female rats exposed orally to benzo[a]pyrene (Kroese et al., 2001) E-62
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-65
Figure E-20. 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-72
Figure E-21. 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-74
Figure E-22. Fit of multistage Weibull model to skin carcinomas or papilloma for male CeH/HeJ
mice exposed dermally to benzo[a]pyrene (Sivak et al., 1997); BMR = 10% extra
risk E-83
Figure E-23. Fit of multistage Weibull model to skin carcinomas, keratoacanthoma or papilloma
for male CeH/HeJ mice exposed dermally to benzo[a]pyrene (Sivak et al., 1997);
BMR = 10% extra risk E-85
Figure E-24. Fit of multistage model to skin tumors in C57L mice exposed dermally to
benzo[a]pyrene (Poel, 1959) E-87
Figure E-25. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to
benzo[a]pyrene (Roe et al., 1970) E-90
Figure E-26. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Schmidt et al., 1973) E-92
Figure E-27. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to
benzo[a]pyrene (Schmidt et al., 1973) E-94
Figure E-28. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Schmahl et al., 1977) E-96
Figure E-29. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Habs et al., 1980) E-98
Figure E-30. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Habs et al., 1984) E-100
Figure E-31. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1983) E-102
Figure E-32. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984) E-104
Figure E-33. Fit of log-logistic model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984) E-106
Figure E-34. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984), highest dose dropped E-108
Figure E-35. Fit of multistage model to skin tumors in male CeH/HeJ mice exposed dermally to
benzo[a]pyrene (Sivak et al., 1997) E-110
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ABBREVIATIONS
1-OH-Py
1-hydroxypyrene
ETS
environmental tobacco smoke
AchE
acetylcholine esterase
EU
European Union
ADAF
age-dependent adjustment factor
Fe203
ferrous oxide
Ah
aryl hydrocarbon
FSH
follicle stimulating hormone
AHH
aryl hydrocarbon hydroxylase
GABA
gamma-aminobutyric acid
AhR
aryl hydrocarbon receptor
GD
gestational day
AIC
Akaike's Information Criterion
GI
gastrointestinal
AKR
aldo-keto reductase
GJIC
gap junctional intercellular
AMI
acute myocardial infarction
communication
ANOVA
analysis of variance
GSH
reduced glutathione
ARNT
Ah receptor nuclear translocator
GST
glutathione-S-transferase
AST
aspartate transaminase
GSTM1
glutathione-S-transferase Ml
ATSDR
Agency for Toxic Substances and
hCG
human chorionic gonadotropin
Disease Registry
HEC
human equivalent concentration
BMC
benchmark concentration
HED
human equivalent dose
BMCL
benchmark concentration lower
HERO
Health and Environmental Research
confidence limit
Online
BMD
benchmark dose
HFC
high-frequency cell
BMDL
benchmark dose, 95% lower bound
HPLC
high-performance liquid
BMDS
Benchmark Dose Software
chromatography
BMR
benchmark response
hprt
hypoxanthine guanine phosphoribosyl
BPDE
benzo[a]pyrene-7,8-diol-9,10-epoxide
transferase
BPQ
benzo [ajpyrene semiquinone
HR
hazard ratio
BrdU
bromodeoxyuridine
Hsp90
heat shock protein 90
BSM
benzene-soluble matter
i.p.
intraperitoneal
BUN
blood urea nitrogen
i.v.
intravenous
BW
body weight
Ig
immunoglobulin
CA
chromosomal aberration
IHD
ischemic heart disease
CAL/EPA
California Environmental Protection
IRIS
Integrated Risk Information System
Agency
LDH
lactate dehydrogenase
CASRN
Chemical Abstracts Service Registry
LH
luteinizing hormone
Number
LOAEL
lowest-observed-adverse-effect level
CERCLA
Comprehensive Environmental
MAP
mitogen-activated protein
Response, Compensation, and Liability
MCL
Maximum Contaminant Level
Act
MCLG
Maximum Contaminant Level Goal
CHO
Chinese hamster ovary
MIAME
Minimum Information About a
CI
confidence interval
Microarray Experiment
CYP
cytochrome
MLE
maximum likelihood estimate
CYP450
cytochrome P450
MMAD
mass median aerodynamic diameter
DAF
dosimetric adjustment factor
MN
micronucleus
dbcAMP
dibutyl cyclic adenosine
MPPD
Multi-Path Particle Deposition
monophosphate
mRNA
messenger ribonucleic acid
DMSO
dimethyl sulfoxide
MS
mass spectrometry
DNA
deoxyribonucleic acid
NCE
normochromatic erythrocyte
EC
European Commission
NCEA
National Center for Environmental
EH
epoxide hydrolase
Assessment
ELISA
enzyme-linked immunosorbent assay
NIOSH
National Institute for Occupational
EPA
Environmental Protection Agency
Safety and Health
EROD
7-ethoxyresorufin-0-deethylase
NK
natural-killer
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NMDA
N-methyl-D-aspartate
SHE
Syrian hamster embryo
NOAEL
no-observed-adverse-effect level
SIR
standardized incidence ratio
NPL
National Priorities List
SMR
standardized mortality ratio
NQO
NADPH:quinone oxidoreductase
SOAR
Systematic Omics Analysis Review
NRC
National Research Council
SOD
superoxide dismutase
NTP
National Toxicology Program
SRBC
sheep red blood cells
OECD
Organisation for Economic
SSB
single-strand break
Co-operation and Development
TCDD
2,3,7,8-tetrachlorodibenzo-p-dioxin
OR
odds ratio
TK
thymidine kinase
ORD
Office of Research and Development
ToxR
Toxicological Reliability Assessment
PAH
polycyclic aromatic hydrocarbon
TPA
12 -0 -tetr adecanoylphor bol-13 -acetate
PBMC
peripheral blood mononuclear cell
TUNEL
terminal deoxynucleotidyl transferase
PBPK
physiologically based pharmacokinetic
dUTP nick end labeling
PCA
Principal Components Analysis
TWA
time-weighted average
PCE
polychromatic erythrocyte
UCL
upper confidence limit
PCNA
proliferating cell nuclear antigen
UDP-UGT
uridine diphosphate-
PND
postnatal day
glucuronosyltransferase
POD
point of departure
UDS
unscheduled DNA synthesis
PUVA
psoralen plus ultraviolet-A
UF
uncertainty factor
RBC
red blood cell
UFa
interspecies uncertainty factor
RDDRer
regional deposited dose ratio for
UFd
database deficiencies uncertainty factor
extrarespiratory effects
UFh
intraspecies uncertainty factor
RfC
inhalation reference concentration
UFl
LOAEL-to-NOAEL uncertainty factor
RfD
oral reference dose
UFS
subchronic-to-chronic uncertainty
RNA
ribonucleic acid
factor
ROS
reactive oxygen species
UVA
ultraviolet-A
RR
relative risk
UVB
ultraviolet-B
s.c.
subcutaneous
WBC
white blood cell
see
squamous cell carcinoma
WESPOC
water escape pole climbing
SCE
sister chromatid exchange
WT
wild type
SCSA
sperm chromatin structure assay
WTC
World Trade Center
SD
standard deviation
XPA
xeroderma pigmentosum group A
SE
standard error
SEM
standard error of the mean
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1
2 APPENDIX A. CHEMICAL PROPERTIES AND
3 EXPOSURE INFORMATION
4 Benzo[a]pyrene is a five-ring polycyclic aromatic hydrocarbon (PAH) (Figure A-l). It is a
5 pale yellow crystalline solid with a faint aromatic odor. It is relatively insoluble in water and has
6 low volatility. Benzo[a]pyrene is released to the air from both natural and anthropogenic sources
7 and removed from the atmosphere by photochemical oxidation; reaction with nitrogen oxides,
8 hydroxy and hydroperoxy radicals, ozone, sulfur oxides, and peroxyacetyl nitrate; and wet and dry
9 deposition to land or water. In air, benzo[a]pyrene is predominantly adsorbed to particulates, but
10 may also exist as a vapor at high temperatures fHSDB. 20121. The half-lives for degradation of
11 benzo[a]pyrene in soil, air, water, and sediment are 229-309, 0.02-7, 39-71, and 196-2293 days,
12 respectively fHSDB. 2012: GLC. 20071.
13 The structural formula is presented in Figure A-l. The physical and chemical properties of
14 benzo[a]pyrene are shown in Table A-l.
Benzo[a]pyrene
16 Figure A-l. Structural formula of benzo[a]pyrene.
17
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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
O1 Neil etal. (2001)
Boiling point
310-312°C at 10 mm Hg
O1 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
O1 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 fPhillips. 1983: Cook et
al.. 19331. It is found ubiquitously in the environment, primarily as a result of incomplete
combustion emissions (Bostrom etal.. 2002). 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 (ATSDR. 1995). 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 fRowe 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. 1973). Chimney sweeping
can result in exposure to benzo[a]pyrene-contaminated soot (ATSDR. 1995). 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 fHSDB. 20121. An assessment of benzo[a]pyrene emissions in the Great Lakes
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Region in 2002 indicated thatthe 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 fATSDR. 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 et al. 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 (Naumova etal..
20021. Mitra and Wilson T19921 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. f20011 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. f20011 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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1 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
(Petrvetal. (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)
2
3 NA = not available.
4
5 Santodonato etal. (19811 estimated the adult daily intake from inhalation as 9-43 ng/day.
6 The European Commission (EC. 20021 reported benzo[a]pyrene air levels in Europe during the
7 1990s as 0.1-1 ng/m3 in rural areas and 0.5-3 ng/m3 in urban areas. The amount of
8 benzo[a]pyrene is reported to be 5-80 ng per cigarette in mainstream cigarette smoke, but
9 significantly higher, 25-200 ng per cigarette in sidestream smoke. Concentrations of
10 400-760,000 ng/m3 have been reported in a cigarette smoke-polluted environment (Cal/EPA.
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20101. The mean intake via inhalation for an adult nonsmoker was estimated as 20 ng/day.
Naumova etal. f2 0021 focused on nonsmoking residences and suggested that typical air exposures
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 fBostrom 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 fLietal.. 20091.
Kazerouni etal. f20011 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
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Food
Concentration (ng/g)
Milk
0.02
Fruit
0.01-0.17
Source: Kazerouni et al. (2001).
Kishikawa et al. f20031 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 adult person was estimated in the range of 0.05-0.29 ng/day (EC.
20021. 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%) (EC. 20021. The contribution from grilled foods appeared less important in Europe than in the
United States because grilled foods are consumed less often fEC. 20021. 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 flARC. 20101.
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 |ig/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. T19891 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.
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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
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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),
1996))
The guideline value for benzo[a]pyrene in drinking water of 0.7 |jg/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), 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 10-4,10~5,
and 10"6.)
Inhalation value
(WHO (2000),
1997))
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/mB 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.
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2 APPENDIX C. LITERATURE SEARCH STRATEGY
3 KEYWORDS
4 Table C-l. Literature search strategy keywords for benzo[a]pyrene
Database
Set#
Terms
Hits
Initial strategy
PubMed
Date range:
1950's to
2/14/2012
Search date:
2/14/2012
1A
("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
"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
5,184
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Database
Set#
Terms
Hits
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
Date range:
1960's-
2/14/2012
Search date:
2/14/2012
IB
(((50-32-8 [rn] OR "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") 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 inflammation OR
inflammatory OR interferon OR interferons OR interleukin OR
interleukins OR leukocyte OR leukocytes OR lymph OR lymphatic OR
25,621
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Database
Set#
Terms
Hits
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
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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.l.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
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r-7,t-8,9,c 10 tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-benzo[a]pyrene-tetrol, a
specific metabolite of benzo[a]pyrene) in the workers' urine by Wu etal. f20021. 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 etal.. 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 fGerde 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 fBevan and Ulman. 19911. Particle-bound
benzo[a]pyrene deposited in the respiratory tract is absorbed and cleared more slowly than the
neat compound (Gerde etal.. 2001).
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 fHechtetal.. 19791.
However, studies in laboratory animals demonstrate thatbenzo[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%
(Cavretetal.. 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,
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and high-fat diets, as compared with fiber- or protein-rich diets f O'Neill etal.. 1991: Kawamura et
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 fStavric and Klassen.
19941. The addition of wheat bran to the benzo[a]pyrene-containing diets increased fecal excretion
ofbenzo[a]pyrene fMirvishetal.. 19811.
Studies of benzo[a]pyrene metabolites or DNA adducts measured in humans exposed
dermally to benzo[a]pyrene-containing PAH mixtures demonstrate thatbenzo[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 fVanRooii 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 fVanRooii etal.. 19931.
Metabolism is also an important determinant of permeation, with very low rates observed in
nonviable skin (Kao etal.. 1985). 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 et al.. 1990: Kao etal.. 1985). 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 fKao 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 fourfold more extensive absorption than
benzo[a]pyrene in soil (Wester etal.. 1990: Yang etal.. 1989). 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.. 1989).
D.1.3. Distribution
No adequate quantitative studies of benzo[a]pyrene tissue distribution in exposed humans
were identified. Obana etal. f!9811 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)
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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
distributed systemically after administration via many routes of administration including
inhalation (or intratracheal instillation), oral, i.v., and dermal exposures fSaunders etal.. 2002: Moir
etal.. 1998: Neubert and Tapken. 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 (Schnizlein et al.. 1987). 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 fWevand 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) (Sun etal.. 1982). 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 (Withev etal.. 1993: Neubert and Tapken. 1988: Shendrikova and Aleksandrov. 1974).
The reactive metabolites of benzo[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. 1989).
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 et al.. 1997). demonstrating extensive metabolism of benzo[a]pyrene in
humans.
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l-OH BaP
9-OH BaP
BaP 2,3-oxide
3-OH BaP
BaP 1,2-oxide
BaP 9,10-transdiol
Benzo[a]pyrerie
BaP 7,8-oxide
BaP 7,8-transdio i
oh BaP 4,5-transdiol
BaP 4,5-oxkfe
6-OH BaP
\
6-oxo-BaP radical
7-OH BaP
BaP 7,8-diol-9,10-epoxide
BaP 1,6-hydroquinone BaP 1,6 semiquinone
[BaP 3,6 1
semiquinonel
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. 1980). 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) (IARC. 198311. The metabolism of
benzo[a]pyrene to phenols results in five phenol isomers (1-, 3-, 6-, 7, and 9-OH benzo[a]pyrene)
fPelkonen 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) (Grover. 1986: Deutsch etal.. 19791. myeloperoxidase
fMalletetal.. 19911. or prostaglandin h synthase (also known as cyclooxygenase) fMarnett. 19901.
and lipid peroxidation fBvczkowski and Kulkarni. 19901. The diolepoxides can react further by
spontaneously hydrolyzing to tetrols fHall 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
fDeutsch etal.. 19791.
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Epoxide Hydrolase hq"
(+)-BP-7R,8S-diol-9S,1 OR-epoxide
T( Mixed Function (+) anti BPDE
(+)-BP-7j8-oxide (-J-BP-7,8-diol
(-J-BP-7R ,8S-diol-9R ,1 OS-e poxide
(-) syn BPDE
Mixed Function
Oxidase System
Mixed Function
Oxidase System
(+)-BP-7S,8R-diol-9S,1 OR-epoxide
(+) syn BPDE
Epoxide Hydrolase |_|q
12 1
11
(-)-BP-7S,8R-diol-9Rl10S-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 fKleiner etal.. 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 fe.g.. Wormhoudtetal.. 19991. Recombinant CYP1A1 allelic variants
produced BPDE with generally lower catalytic activity and Km values than the WT allele (Schwarz
etal.. 2001). 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 H202, which
in turn can causes extensive DNA fragmentation (Penning 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 fEling 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 fOuinn 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 M-g/kg benzo[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 fBevan 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: Robertson etal.. 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 etal.. 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. 2004).
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. 1995). 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.. 1976).
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.. 2003) and lung cancer (Alexandrie etal.. 2004: Lin
etal.. 2003). Coke oven workers (who are exposed to PAHs, including benzo[a]pyrene)
homozygous at the 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 at the HI 13Y site of the EH
gene fLengetal.. 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 fWolffetal.. 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 (Wevand and Bevan. 1986). 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 fDahl 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.. 1980). 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.. 1976). 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 et al.. 2006) and total levels of benzo[a]pyrene-DNA adducts were
approximately 2-6 times greater in the lung slices than liver (Harrigan et al.. 2004).
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 fRamesh etal.. 2004: Ramesh etal.. 2001b: Yamazaki and
Kakiuchi. 1989). In rats administered a 100-nmol dose, >90% was recovered in portal blood as
metabolites (Bock etal.. 1979). Orally administered benzo[a]pyrene produced strong induction of
CYP1A1 in the intestine of mice (Brooks etal.. 1999). 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 fUno 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 that benzo[a]pyrene is
extensively metabolized by the intestinal Caco-2 cells and thatbenzo[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 etal.. 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 fNaufal etal.. 2010: Ruchirawatetal.. 2010: Suter
etal.. 2010: Mielzvriska etal.. 2006: Perera etal.. 2005a: Tang etal.. 1999: Whvatt et al.. 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: Neubertand Tapken. 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 (Ginsbergetal.. 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. Studies of benzo[a]pyrene elimination in animals
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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 fWangetal.. 2003: Likhachev etal.. 1992: Wolff etal..
1989: Yang etal.. 1989: Petridou-Fischer etal.. 1988: Wevand and Bevan. 1987: Sun etal.. 1982:
Hecht etal.. 19791. The metabolites in bile are primarily benzo[a]pyrene conjugates,
predominantly thioether conjugates of varying extent in different species fWevand and Bevan.
1987). 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 (Bevan 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 fMoir 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
fBevan 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 ng/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. 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
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 (Wevand and Bevan. 1987.1986).
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.
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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 fWiersma and Roth f!983al. 1983b}) 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
f!9991 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 etal. 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.
(20111 by optimizing against additional rodent data and altering partition coefficient derivation.
However, it still did not incorporate metabolites, and some tissues continued to exhibit poor model
fits.
An attempt to scale the Moir etal. f 19981 rodent PBPK model to humans, relevant to risk
assessment of oral exposures to benzo[a]pyrene, was presented by Zeilmaker et al. f!999al and
Zeilmaker et al. (1999b). The PBPK model for benzo[a]pyrene was derived from an earlier model
for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats (Zeilmaker and van Eijkeren. 1997). 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
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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
of benzo[a]pyrene (Zeilmaker etal.. 1999a).
Zeilmaker et al. (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 CYP450 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 (Zeilmaker et al.. 1999b).
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 et al. (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
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
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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
Burstvn etal. f20051 reported the association of death from cardiovascular disease with
benzo[a]pyrene exposure in a cohort of 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
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
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1 positively associated with IHD mortality, with a RR of approximately 1.6 in the highest exposure
2 quintile from both metrics, independent of total employment duration. Similar monotonic trends
3 were observed for all cardiovascular diseases (combined), although a dose-response relationship
4 was evident only for IHD and not hypertension or other individual heart disease categories. Similar
5 trends were also observed for coal tar exposure and IHD. Adjusting the RR to account for possible
6 confounding by smoking yields a RR of 1.39 under the assumptions mentioned above, and is still
7 elevated (1.21) if the contribution of smoking to cardiovascular disease etiology was greater than
8 the original assumptions. Furthermore, the RR for the high versus low exposure quintile is
9 1.24 even if the distribution of nonsmokers/former smokers/current smokers shifts to 0/30/70%,
10 using the original assumptions of cigarette smoke casual potency.
11 Table D-l. Exposure to benzo[a]pyrene and mortality from cardiovascular
12 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
IHD
Deaths
RR
95% CI
83
1.00
83
1.13
0.82-1.55
83
1.33
0.94-1.90
86
1.20
0.84-1.71
83
1.64
1.13-2.38
0.02
13
14 Reference category.
15
16 Source: Burstvn et al. (2005).
17
18 Friesenetal. (2010) examined the association between benzo[a]pyrene exposure and
19 deaths from chronic nonmalignant disease in a cohort of 6,423 male and 603 female Canadian
20 aluminum smelter workers (Table D-2). Inclusion criteria required at least 3 years of continuous
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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
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
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1 calculated the risk of death from IHD as 1.002 (95% CI 1.000-1.005) per |J.g/m3 from cumulative
2 benzo[a]pyrene exposure; however, visual inspection of the categorical relationships indicated that
3 the association is nonlinear, suggesting that this value may be an underestimate. Restricting the
4 cohort to only members who died within 3 0 days of active employment at the worksite, cumulative
5 benzo[a]pyrene exposure was not significantly associated with IHD or AMI, although the HR for the
6 highest exposure group was 2.39 (95% CI 0.95-6.05). Exposure-response relationships were
7 similarly examined in male smelter workers for chronic obstructive pulmonary disease and
8 cerebrovascular disease, but neither was significantly associated with cumulative benzo[a]pyrene
9 exposure in either internal or external comparisons.
10 Table D-2. Exposure to benzo[a]pyrene and mortality from cardiovascular
11 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
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
12
13 aTwo-sided test for trend using the person-year-weighted mean value for each category as a linear, continuous
14 variable.
15 bExposure variable was entered as a continuous, linear variable in the model.
16
17 Source: Friesen et al. (2010).
18 Reproductive and Developmental Endpoints
19 Wu etal. f20101 conducted a study of benzo[a]pyrene-DNA adduct levels in relation to risk
20 of fetal death in Tianjin, China. This case-control study included women who experienced a delayed
21 miscarriage before 14 weeks gestational age (i.e., a fetal death that remained in utero and therefore
22 required surgical intervention). Cases were matched by age and gravidity to controls (women
23 undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded
24 women who smoked, women with chronic disease and pregnancy complications, and women with
25 occupational exposures to PAHs. Residency within Tianjin for at least 1 year was also an eligibility
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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
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).
Perera etal. (2005a) 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)-DNA adducts 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).
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Perera etal. (2005b). a reanalysis of Perera etal. (20041. 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. f20061 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,
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
adductlevels ((3 = -0.011, p = 0.057). Maternal adduct levels 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. 20081. 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. (20081 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
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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% below the detection limit in 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,
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 (deliveiy 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,
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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
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 at the 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
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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 fourfold 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.
Table D-3. Exposure-related effects in Chinese coke oven workers or
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
Source: Niu et al. (2010).
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1
2 Table D-4. Exposure-related effects in Chinese coke oven workers or
3 warehouse controls exposed to benzo[a]pyrene in the workplace, stratified by
4 urinary metabolite levels
Exposure group categorized by 1-OH-Py level
0-3.09 pmol/mol
3.09-3.90 pmol/mol
3.90-5.53 pmol/mol
Effect measured
creatinine
creatinine
creatinine
Number of subjects
33
72
36
p-value
Neurobehavioral function tests (mean ± SD)
Digit span
18.24 ±4.58
16.04 ± 4.24
15.78 ±3.71
0.003
Forward digit span
10.85 ±2.12
9.80 ±2.86
9.58 ±2.33
0.019
Backward digit span
7.20 ± 3.07
6.38 ±2.55
6.20 ±2.15
0.089
Right dotting
152.15 ±35.43
153.80 ±31.55
167.22 ±59.21
0.094
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
5
6 Source: Niu et al. (2010).
7 Immunotoxicity
8 Zhang etal. T20121 studied 129 Chinese coke-oven workers with elevated benzo[a]pyrene
9 exposure and compared them against 37 referents (workers in a supply warehouse), matched by
10 socioeconomic status, lifestyle, and health. Area benzo[a]pyrene levels were quantified in the
11 various work areas, and the primary endpoint was the level of early and late apoptosis in
12 peripheral blood mononuclear cells (PBMCs) isolated from each worker subgroup the morning
13 following an overnight fast The authors self-designed a study questionnaire to gather information
14 on worker education, vocational history, smoking and drinking habits, personal habits, and
15 personal and family medical history, as well as any current symptoms and medications used in the
16 previous several weeks. "Smoking" was defined as >10 cigarettes/day during the past year, with
17 "smoking index" defined as cigarettes/day x years smoking. Similarly, "drinking" was defined as
18 wine/beer/spirits consumed >3 times/week for the past 6 months, and "drinking index" defined as
19 grams of alcohol consumed/day x years drinking. Exposed workers were categorized by physical
20 worksite location and expected differences in benzo[a]pyrene exposure: 34 oven bottom workers,
21 48 oven side workers, and 47 oven top workers. Workplace environmental sampling stations were
22 established at each of the physical work locations, including the referent's warehouse, and dual
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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
(approximately twofold) for both early and late apoptosis. While self-reported smoking incidence
varied significantly among the worker groups, stratification by smoking years or smoking index did
not reveal any significant association with PBMC apoptosis. Multiple linear stepwise regression
analysis suggested that urine 1-OH-Py levels and years of coke oven operation were positively
associated with increased early and late PBMC apoptosis (Table D-5), and that years of ethanol
consumption was negatively associated with only early apoptosis. These associations were tested
by stratifying workers into three groups by urinary 1-OH-Py levels or coke oven operation years,
and in both cases, the groups with the highest urinary metabolite levels or longest oven operating
experience had statistically significantly higher levels of both early and late apoptotic PBMCs versus
the lowest or shortest duration groups, respectively. Likewise, when sorted into groups based
upon years of ethanol consumption, the highest ethanol "years of consumption" group had
statistically significantly lower early apoptosis rates when compared to the lowest ethanol
consuming group.
Table D-5. Background information on Chinese coke oven workers or
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
*p < 0.05 significantly different from control mean.
Source: Zhang et al. (2012).
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D.3.2. Cancer-related Endpoints
Benzo[a]pyrene-Induced Cytogenetic Damage
Many studies measure cytogenetic damage as biomarkers of early biological effects, which
also reflect exposure to genotoxic chemicals. Standard cytogenetic endpoints include chromosomal
aberration (CA), sister chromatid exchange (SCE), micronucleus (MN) formation, hypoxanthine
guanine phosphoribosyl transferase (hprt) mutation frequency, and glycophorin A mutation
frequency (Gvorffv et al.. 2008). These biomarkers are often incorporated in multi-endpoint
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.. 19931. In the first study, involving 48 workers, several biomarkers were
analyzed for dose-response and inter individual 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).
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In a second study, Perera etal. (19941 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 firstyear 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.
Kalina etal. (19981 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. (1998) 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.
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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 fTuntawiroon et al.. 20071.
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
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% (Wu 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-
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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 fLi 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
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.. 20081. 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 at9p21 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.. 20071. and a third involving 146 colorectal adenoma cases and
228 controls fSinha etal.. 20051. dietary intake of benzo[a]pyrene was estimated using food
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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.. 2007).
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.. 2004). 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 (Boffetta 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) (Armstrong et al.. 2004).
Spinelli et al. f20061 reported a 14-year update to a previously published historical cohort
study fSpinelli et al.. 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%
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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-
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 etal.. 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 30 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 et al.. 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
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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) fArmstrongetal.. 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)
fBosetti et al.. 20071. 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
lung cancer RR per 100 |ig/m3-years of cumulative benzo[a]pyrene exposure was 1.15 (95% CI
1.11-1.20) (Armstrong et al.. 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 et al.. 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. (19961 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
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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
indicator substance (Bostrom etal.. 2002). However, there are numerous studies of coke oven
worker cohorts that do not provide estimates of benzo[a]pyrene exposure. An overview of the
results of these and other studies can be obtained from the review of Boffetta et al. (1997).
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
f Burstvn et al.. 2 0 0 7: 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) (Bosettiet
al.. 2007). 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 etal..
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. 1983) are not included.
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1 Table D-6. Studies examining skin cancer risk in relation to therapeutic coal
2 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
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
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.
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 1.09 (0.69,1.72)
eczema
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.
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
No skin cancers observed
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Reference and study details
Results
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
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) [8 basal cell, 1
squamous cell, 2 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
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
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
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 weeks, <208 weeks), and Grenz rays or
x-rays for therapy (ever/never)
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 = 3.8(1.2,12.5)
24)
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Reference and study details
Results
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)
The U.S. Environmental Protection Agency (EPA) noted several limitations with respect to
study design and analysis in this literature, precluding the ability to provide a foundation for
evaluating the potential association between use of therapeutic coal tar treatment (particularly
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 fMitropoulos
and Norman. 2005): 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 etal. f20101 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 etal. f20101 included melanoma, in addition to squamous cell skin cancer,
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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.
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
(Torinuki and Tagami. 1988). 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 rates and
differences in disease ascertainment between cases and the reference population (Pittelkowetal..
1981: Maughan et al.. 19801. 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-StPaul, San Francisco-Oakland, Iowa, and Dallas-Fort Worth. Therefore, it is unclear
whether the reference population appropriately represents the case population. In addition, this
combination of UVB and coal tar makes it impossible to attribute risk to either individual
component This limitation effects the interpretation of the results of the PUVA trial studies (Stern
etal.. 1998: Stern and Laird. 1994: Stern etal.. 1980). 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 fLindelof and Sigurgeirsson f!99311 reported similar prevalence and risk
estimates for coal tar use and for UVB, reflecting the high correlation between these treatments.
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
(e.g.. Roelofzen etal.. 2010: Mitropoulos and Norman. 2005: Lindelof and Sigurgeirsson. 1993).
small size and short duration of follow-up (e.g.. Torinuki and Tagami. 1988). and choice of referent
rates and differences in disease ascertainment between cases and the reference population (e.g.,
Pittelkowetal.. 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 distinguish effects of
coal tar from the carcinogenic effects of UVB (e.g.. Stern et al.. 1998: Stern and Laird. 1994: Lindelof
and Sigurgeirsson. 1993: Stern etal.. 1980). 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. T19991 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,
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or 90 mg/kg-day (adjusted doses: 0, 2.14, 7.14, 21.4, and 64.3 mg/kg-day). Atthe end ofthe
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 Tongetal.. 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).
Table D-7. Exposure-related effects in male Wistar rats exposed to
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)
RBCs (109/L)
Hemoglobin (mmol/L)
Hematocrit (L/L)
14.96 ± 1.9
8.7 ±0.2
10.5 ±0.2
0.5 ±0.01
13.84 ±3.0
8.6 ±0.2
10.4 ±0.3
0.5 ±0.01
13.69 ± 1.8
8.3 ±0.2*
9.8 ±0.2*
0.47 ±0.01*
13.58 ±2.9
7.8 ±0.4*
9.5 ±0.4*
0.46 ±0.02*
8.53 ± 1.1*
7.1 ±0.4*
8.6 ±0.6*
0.43 ±0.02*
Serum Ig levels
(mean ± SD; n = 7-8)
IgM
IgG
IgA
100 ± 13
100 ± 40
100 ± 28
87 ± 16
141±106
73 ±29
86 ±31
104 ± 28
78 ±67
67 ± 16*
106 ± 19
72 ±22
81 ±26
99 ±29
39 ± 19*
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Dose (mg/kg-d)
Effect
0
3
10
30
90
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)
^Significantly (p < 0.05) different from control mean. For body weight and organ weight means, SDs were only
reported for thymus weights.
Source: De Jong et al. (1999).
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,
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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 long et al. f 19991. 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. f20011 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 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. f20011 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 fourfold increase in kidney casts in males compared
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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.
A significant, but not dose-dependent, increase in food consumption in males at
>1.5 mg/kg-day and a decrease in food consumption in females at >5 mg/kg-day was observed
(Kroese et al.. 2001). Water consumption was statistically significantly altered in males only: a
decrease at 1.5, 5, and 15 mg/kg-day and an increase at 50 mg/kg-day. Organ weights of lung,
spleen, kidneys, adrenals, and ovaries were not affected by treatment. There was a dose-related,
statistically significant decrease in thymus weight in males at 15 and 50 mg/kg-day (decreased by
28 and 33%, respectively) and a significant decrease in thymus weight in females at 50 mg/kg-day
(decreased by 17%) (Table D-8). In both sexes, liver weight was statistically significantly increased
only at 50 mg/kg-day by about 18% (Table D-8).
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Table D-8. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene
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*
^Significantly (p < 0.05) different from control mean; n = 10/sex/group.
Source: Kroese et al. (2001).
Hematological evaluation revealed only statistically nonsignificant, small, dose-related
decreases in hemoglobin in both sexes and RBC counts in males. Clinical chemistry analysis
showed a small, but statistically significant, increase in creatinine levels in males only at
1.5 mg/kg-day, but this effect was not dose-dependent A dose-dependent induction of liver
microsomal EROD activity was observed, with a 5-fold induction at 1.5 mg/kg-day compared to
controls, reaching 36-fold in males at 50 mg/kg-day; the fold induction in females at the top dose
was less than in males. At necropsy, significant, dose-dependent macroscopic findings were not
observed.
Histopathology examination revealed a statistically significant increase in basal cell
hyperplasia in the forestomach of females at doses >15 mg/kg-day fKroese et al.. 20011. The
induction of liver microsomal EROD was not accompanied by any adverse histopathologic findings
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. f20011 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.
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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 (Kroese 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 iny-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 et al. (2001) 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 fKroese etal.. 20011. 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 two- and three-fourfold 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 etal.. 20011. 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, butbenzo[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) fKroese etal.. 20011. 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'5
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
Carcinoma
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
Carcinoma
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. (2001) 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 fBrune etal.. 19811.
Other groups (32/sex/dose) were given gavage doses of 0.15 mgbenzo[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 et al. (1981)]. 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 thatthe 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.
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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
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 % 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 fBeland and Culp. 1998: Culp etal.. 19981. 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 25-ppm intake (about 21 ng/day), average daily doses for the three
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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.
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 (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.
Weyand et al.
(1995)
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Exposure
Results
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Reference
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 mg benzo[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 at the 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 et al. (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.
(1981) 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.
1990) revealed several discrepancies in the reported exposure protocol. The actual exposure
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Supplem en tal Information —Benzo[aJpyren e
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/weekonweek30; 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 two- to fivefold 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 f!9901 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. (19811. 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-13). 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 etal. (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-17 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 thatbenzo[a]pyrene is tumorigenic, but do
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not provide data useful for characterizing dose-response relationships because of their design
fKobavashi. 1975: Renzik-Schiiller and Mohr. 1974: Henry etal.. 1973: Mohr. 1971: Saffiotti et al..
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 mg benzo[a]pyrene once per week for 30-52 weeks) in four multiple-dose
studies (Feron and Kruvsse. 1978: Ketkar etal.. 1978: Feron etal.. 1973: Saffiotti etal.. 1972).
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 atportal-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
(Wevandetal.. 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 et al..
1978: Hoffmann et al.. 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 (Sivaketal.. 1997: Higginbotham etal.. 1993: Albert etal.. 1991: Grimmer etal..
1984: Habs etal.. 1984: Grimmer et al.. 1983: Habs etal.. 1980: Schmahl etal.. 1977: Schmidt et al..
1973: Roe etal.. 1970: Poel. 1963.19591. Early studies ofbenzo[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.. Emmettetal.. 19811 or only dose levels inducing 90-100%
incidence of mice with tumors (e.g., Wilson and Holland. 1988: Warshawskv and Barklev. 1987) 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 fBingham and Falk.
19691.
Poel T19591 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 of tumor appearance decreased (see Table D-15). Doses >3.8 |ig were 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 of tumor appearance decreased (Table D-16). The lowest dose level
18 did not induce an increased incidence of mice with skin tumors in any strain, but strain differences
19 in susceptibility were evident at higher dose levels. SWR and C3HeB mice showed skin tumors at
20 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 T19631 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. (19701 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. f 19731 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 etal. T19771 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 ng 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. fl9801 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. T19831 appliedbenzo[a]pvrene (in 0.1 mLofa
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. T19841 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 fHigginbotham et al.. 19931. 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 Sivaketal. (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. (2011) 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. Atthe 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 (Chungetal.. 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 (ANOVA) 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 atthe 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 0.1
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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 (~twofold 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. 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 fGao 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 Cervical 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 twofold higher than untreated or vehicle controls at the low dose, to
~sixfold 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-gene rational 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).
Arafaetal. 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 anticarcinogenie 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) fArafa etal.. 20091. 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. f20101 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. f20081 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 gestational 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 (1965) 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 fKristensenetal.. 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. (20121 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
pre weaning 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/kgbody 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 et al. 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) forelimb grip 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 at PNDs 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.
21
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 at PNDs 7-9. Only pups from the 20 mg/kg-day
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treatment group demonstrated significantly increased negative geotaxis latency, which was twofold
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 (threefold), 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 that premating 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. f20021 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 ng/m3 via
nose-only inhalation, 4 hours/day on GDs 11-20. Control animals were either sham-exposed to
carbon black or remained entirely unexposed. Results of particle size analysis of generated
aerosols were reported by several other reports from this laboratory (Invangetal.. 2003: Ramesh
etal.. 2001a: Hood etal.. 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 flnvangetal..
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. f20011 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 at the low dose. Biologically significant decreases in mean pup weights (expressed as
7 g per litter) of >5% were observed at doses >75 ng/m3 (14 and 16% decreases at 75 and
8 100 |J.g/m3, respectively, p < 0.05). Exposure to benzo[a]pyrene did not affect crown-rump length
9 (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 |J.g/m3 f Archibong etal.. 20021. 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 |J.g/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-17 p. 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,
Wormlevetal. (20041 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 fRamesh etal.. 2001a: Hood etal.. 20001. Particle size analysis of a 100-|ig/m:i
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. Dams were maintained to term and pups were weaned on
PND 30. Benzo[a]pyrene reduced the number of live pups to one-third of control values without
affecting the number of implantation sites. 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
distribution profile up to PND 15 and the hydroxy (3-OH-benzo[a]pyrene; 9-OH-benzo[a]pyrene)
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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. f20031 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 ng/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 (Invangetal.. 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
thought to 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 |J.g/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 thought to
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
reduction in the progressive motility of stored sperm and impairment of testicular function with
10 days of exposure at 75 ng/m3.
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Supplem en tal Information —Benzo[aJpyren e
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 [ig/m3 via nose-only inhalation,
4 hours/day for 60 days fArchibong et al.. 2008: Ramesh etal.. 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 |ig/m:i 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 regarding the genotoxicity of benzo [a]pyrene in in vitro and in vivo systems is
presented in Tables D-31, D-32, and D-33.
Table D-31. In vitro genotoxicity studies of benzo [a] pyrene in non-
mammalian cells
Result
Reference
+S9
-S9
Endpoint/test system: prokaryotic cells
Forward mutation
Salmonella typhimurium TM677
+
-
Rastetter et al. (1982)
S. typhimurium TM677
+
ND
Babson et al. (1986)
Reverse mutation
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —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
Gaoetal. (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)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
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
4 Table D-32. In vitro genotoxicity studies of benzo[a]pyrene in mammalian
5 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)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
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
0'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)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Assay/test system
Result
Reference
+S9
-S9
Human C-HC-4 and C-HC-20 hepatoma cells
ND
+
(Abe etal. (1983a), 1983b))
Human diploid fibroblast (TIG-II) cell line
+
+
Huh etal. (1982)
Human fibroblasts
ND
+
Juhl etal. (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 etal. (1983a), 1983b))
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 etal. (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 etal. (1983a), 1983b))
Rat esophageal tumor R1
ND
+
(Abe etal. (1983a), 1983b))
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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Supplem en tal Information —Benzo[aJpyren e
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.
D-97 DRAFT—DO NOT CITE OR QUOTE
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1 Table D-33. In vivo genotoxicity studies of benzo[a]pyrene
Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
Mutation
Human, blood
T lymphocytes (smokers
and nonsmokers); hprt
locus mutation assay
T-cells of lung cancer patients (smokers
and nonsmokers from lung cancer patients
and population controls with known
smoking status) analyzed for hprt locus
mutations.
+
Smokers and
nonsmokers
Splicing mutations, base-pair
substitutions, frameshift, and
deletion mutations observed.
Smokers and nonsmokers had
GC->TA transversions (13 and
6%, respectively) and GC->AT
transitions (24 and 35%,
respectively) in hprt gene
consistent with in vitro
mutagenicity of
benzo[a]pyrene.
Hackman
et al.
(2000)
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)
t>5
c
»¦»<
re
re
a
f
3
0
a
1
&3
re
a
§
3
a
re
This document is a draft for review purposes only and does not constitute Agency policy.
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
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)
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)
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D-99 DRAFT—DO NOT CITE OR QUOTE
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
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)
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)
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
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+I+ 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:
XPA1 > XPA*1*.
Bol et al.
(1998)
Mutation
Mouse, Cockayne
syndrome-deficient
(Csb^); heterozygous
(Csb+/~) and WT controls
(Csb+/+); hprt mutation
frequency assay
Csb'/'/lacZh/' 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, B6C3F!,
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)
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D-101 DRAFT—DO NOT CITE OR QUOTE
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
Mutation
Mouse, T-strain
Benzo[a]pyrene given to pregnant mice by
gavage in 0.5 mLcorn 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)
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)
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D-102 DRAFT—DO NOT CITE OR QUOTE
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
BPDE-DNA
adducts
Human, WBCs
96 people occupationally or medically
exposed to PAH mixtures (psoriatic
patients, coke oven workers, chimney
sweeps, and aluminum anode plant
workers); adducts measured by
HPLC/fluorescence analysis.
+
Percentages of subjects with
adduct levels greater than the
95th percentile control value
were 47% (7/15), 21% (4/19),
and 3% (1/34) in coke oven
workers, chimney sweeps, and
controls, respectively.
Pavanello
et al.
(1999)
BPDE-DNA
adducts
Human, WBCs
67 highly exposed coke oven workers
were tested for genetic factors that can
modulate individual responses to
carcinogenic PAHs; adducts measured by
HPLC/fluorescence analysis.
+
Levels of BPDE-DNA adducts
were significantly associated
with workplace PAH exposure
(as correlated with urinary
excretion of 1-pyrenol), lack of
GSTM1 activity, and low
nucleotide excision repair
capacity.
Pavanello
et al.
(2005)
BPDE-DNA
adducts
Human, peripheral
lymphocytes
585 Caucasian municipal workers (52%
males, 20-62 years old) from northeast
Italy environmentally exposed to PAH
mixtures were screened for adducts
measured by HPLC/fluorescence analysis.
+
Forty-two percent of the
participants had elevated anti-
BPDE-DNA adduct levels,
defined as >0.5 adducts/
108 nucleotides (mean,
1.28 ± 2.80 adducts/
108 nucleotides). Comparison
of adduct levels with
questionnaire responses
indicated that smoking,
frequent consumption of
PAH-rich meals (>52 versus
<52 times/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)
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
BPDE-DNA
adducts
Human, maternal and
umbilical cord blood
Maternal and umbilical cord blood
obtained following normal delivery from
329 nonsmoking pregnant women
exposed to emissions from fires during the
4 wks following the collapse of the WTC
building in New York City on 09/11/2001.
+
BPDE-DNA adduct levels in cord
and maternal blood were
highest in study participants
who lived within 1 mile of the
WTC, with inverse correlation
between cord blood levels and
distance from WTC.
Perera et
al. (2005b)
BPDE-DNA
adducts
Human, WBCs
Workers were exposed for 6-8 hrs/d for at
least 4-6 mo before blood collection;
leukocyte DNA isolated and digested, and
benzo[a]pyrene tetrols analyzed by HPLC
with fluorescent detection. Low, medium,
and high exposure groups correspond to
<0.15, 0.15-4, and >4 mg/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 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)
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)
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
BPDE-DNA
adducts
Mouse, (Ahr+'\ Ahf'~,
Ahf'~)
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
fourfold higher in Cyplal(-/-)
mice than Cyplal(+/-) mice.
Uno et al.
(2001)
BPDE-DNA
adducts
Mouse, B6C3F!
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 threefold higher
binding of benzo[a]pyrene than
that of forestomach.
Gangar et
al. (2006)
BPDE-DNA
adducts
Mouse, BALB/cAnN
(BALB), CBA/JN (CBA);
[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.
Reddv et al.
(1984)
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Endpoint
Test system
Test conditions
Results
Dose
Comment
Reference
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)
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
twofold higher than the levels
in liver; adduct profiles differed
between peripheral blood
lymphocytes and liver.
Willems et
al. (1991)
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Endpoint
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Test conditions
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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Test conditions
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
Test system
Test conditions
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
Test system
Test conditions
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),
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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Endpoint
Test system
Test conditions
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.
Roszinsky-
Kocher 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
Test conditions
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
Threefold enhancement in
lethals in treated versus
controls.
Vogel et al.
(1983)
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Endpoint
Test system
Test conditions
Results
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 fBurdick et al.. 20031. Benzo[a]pyrene is
metabolized to o-quinones, which are cytotoxic, and can generate ROS fBolton etal.. 2000: Penning
et al.. 1999). Benzo[a]pyrene o-quinones reduce the viability and survival of rat and human
hepatoma cells (Flowers-Geary et al.. 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 et al.. 2003).
Several studies have demonstrated that exposure to benzo[a]pyrene increases the
production of inflammatoiy cytokines, which may contribute to cancer progression. Garcon et al.
(2001a) and Garcon etal. (2001b) 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. f2 0041 also demonstrated a benzo[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.. 2006). The benzo[a]pyrene-induced increase in CCL1 mRNA was inhibited by the potent AhR
antagonist, 3'-methoxy-4'-nitroflavone.
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.. 2002). 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 Information —Benzo[aJpyren e
*
PAH
AHR
Hsp90
Hsp90
Increased
synthesis of
PAH metabolizing
enzymes
Increased
synthesis of
proteins that
regulate cell
differentiation and
proliferation
AHREdna = 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. (2006) also showed that
benzo[a]pyrene adduct levels in skin were reduced by 50% in CYP1A2 knock-out mice and by 90%
AHRE,
ARNT
nucleus
Hsp90
ARNT
ARNT
Hsp90
Enhanced
specific
mRNA
production
AHR
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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 f20071 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. f20061 showed that benzo[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 followingi.p. or oral exposure to benzo[a]pyrene (Sagredo etal.. 2006: Uno etal.. 2006:
Kondraganti etal.. 2003). These findings suggest that there may be alternative (i.e., non-AhR
mediated) mechanisms of benzo[a]pyrene activation in the mouse liver. Sagredo etal. (2006)
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. f20061 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 et al.. 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 etal.. 2007: Chung etal.. 2007: Chen etal.. 2003). Chung etal. (2007)
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.. 2007). Benzo[a]pyrene-
induced apoptosis was attenuated in cells transfected with a dominant-negative mutation of the
AhR.
Inhibition of gap junctional intercellular communication (GTIC)
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 et al.. 2006:
Yamasaki. 19901.
Blaha etal. (2002) 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 etal. f20061 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-34. 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-34. 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/kgbenzo[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
fhttp: //www.ncbi.nlm.nih.gov/geo/1 using the GEOquery package fDavis 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.. 2005).
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: Chuang etal.. 2007: Ein-Dor etal.. 2005: Lossos et al.. 2004: Fortunel et al.. 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-35. 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
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SS&Ts SjB 4J»e ' 4P*
Effect depends
on cell context
and concentration
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UGT1A7C
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Acetaminophen
metabolism
Phase I metabolic enzymes
Figure D-4. AhR pathway. 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-34). For instance, NRF2 is upregulated in
the 2 5 mg/kg.
AhR Signaling
The AhR regulates the transcription of several genes, including xenobiotic metabolism
genes (Figure D-4). It appears that benzo[a]pyrene is activating the AhR in these studies based on
the expression of many of its transcriptional targets. Relevant to further analysis and investigating
the mode of action, the c-Myc gene is upregulated at 4 and 24 hours in the time-course and at the
50 mg/kg dose in the dose-response, while Nrf2 is upregulated at the 4-hour time-point and at the
25 and 75 mg/kg doses, c-Myc has been shown to be upregulated following exposure to TCDD, and
a putative dioxin response element has been detected in the c-Myc promoter fDere etal.. 2011: Kim
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et al.. 20001. The AhR has been demonstrated to bind and regulate the Nrf2 promoter fDere et al..
2011: Lo etal.. 2011: Nair etal.. 20081.
DNA-damage-induced responses
_ o
Role of 14-3-3
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Figure D-5. DNA damage pathway. Activation of transcriptional targets of p53,
including p21 and GADD45, and upregulation of the downstream transcriptional
target, PCNA, suggests that p53 is activated.
DNA Damage Signaling
The strong upregulation 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
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
upregulated 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 MDM2-mediated ubiquitination. Coupled
with the upregulation of Cyclin D and PCNA at 75 mg/kg (among other conditions), this suggests a
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pro-mitotic shift may be occurring which could lead to cellular proliferation in the liver in the mice
exposed to 75 mg/kgper day.
Nrf2 Signaling
Nrf2 transcription may be upregulated by benzo[a]pyrene through activation of the AhR
(Figure D-4). The Nrf2 protein heterodimerizes with the MafF protein fSurh etal.. 2008: Marini et
al.. 2002: Kim etal.. 20001 to regulate the transcription of Phase II metabolism and anti-oxidative
enzymes (Figure D-6). Activated p53 competes with Nrf2 anti-oxidant signaling, perhaps to ensure
a large oxidative stress response is present in the cell to promote the induction of apoptosis
(Faraonio etal.. 20061. Upregulation of Cul3 at 4 hours and the 75 mg/kg dose in concert with the
upregulation of ubiquitin at the same time and dose suggests that repression of Nrf2 activity may
occur. This would support the p53-mediated pro-oxidant hypothesis, which is further
substantiated by the lack of upregulation of anti-oxidant genes at 75 mg/kg, with the exception of
GCL cat
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Supplem en tal Information—Benzo[aJpyren e
Chemical or
oxidative stress
Ptdlns(4,5)P2
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Protection of the cell from reactive oxygen
species and the products of peroxidation
2 Figure D-6. Nrf2 pathway. Nrf2 is up regulated by benzo[a]pyrene exposure,
3 which results in the upregulation of Phase II detoxifying enzymes. This appears to
4 be a compensatory response due to increased oxidative status within cells.
5 Pathway Analysis Summary
6 Activation of the AhR appears to be present based on the transcriptional data. This may
7 lead to formation of oxidative metabolites and radicals which may lead to oxidative damage and
8 DNA damage. Although the alterations to the Nrf2 pathway suggest cells are gearing up for a pro-
9 apoptotic environment, there is no transcriptional evidence that the apoptotic pathways are being
10 activated. Thus, there is significant uncertainty as to whether or not apoptosis may occur.
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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 upregulation 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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APPENDIX E. DOSE-RESPONSE MODELING FOR
THE DERIVATION OF REFERENCE VALUES FOR
EFFECTS OTHER THAN CANCER AND THE
DERIVATION OF CANCER RISK ESTIMATES
This appendix provides technical detail on dose-response evaluation and determination of
points of departure (PODs) for relevant toxicological endpoints, organized by risk value (reference
value or cancer risk value). Except where other software is noted, all endpoints were modeled
using the U.S. Environmental Protection Agency's (EPA's) Benchmark Dose Software (BMDS) fU.S.
EPA. 2012al: version 2.0 or later. The preambles for the cancer and noncancer parts below
describe the practices used in evaluating the model fit and selecting the appropriate model for
determining the POD, as outlined in the Benchmark Dose Technical Guidance fU.S. EPA. 2012bl
E.l. NONCANCER ENDPOINTS
E.l.l. Reference Dose (RfD)
Evaluation of Model Fit
For each dichotomous endpoint, BMDS dichotomous models were fitted to the data using
the maximum likelihood method. For the log-logistic and dichotomous Hill models, slope
parameters were restricted to be >1; for the gamma and Weibull models, power parameters were
restricted to be >1; and for the multistage models, betas were restricted to be non-negative (bi >0).
Each model was tested for goodness-of-fit using a chi-square goodness-of-fit test (x2 p-value <0.10
indicates lack of fit). Other factors were also used to assess model fit, such as scaled residuals,
visual fit, and adequacy of fit in the low-dose region and in the vicinity of the benchmark response
(BMR).
For each continuous endpoint, BMDS continuous models were fitted to the data using the
maximum likelihood method. For the polynomial models, betas were restricted to be non-negative (in
the case of increasing response) or non-positive (in the case of decreasing response data); and for
the Hill, power, and exponential models, power parameters were restricted to be >1. Model fit was
assessed by a series of tests as follows. For each model, first the homogeneity of the variances was
tested using a likelihood ratio test (BMDS Test 2). If Test 2 was not rejected (x2 p-value >0.10), 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
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1 either constant variance or modeled variance, models for the mean response were tested for
2 adequacy of fit using a likelihood ratio test (BMDS Test 4, withx2 p-value <0.10 indicating
3 inadequate fit). Other factors were also used to assess the model fit, such as scaled residuals, visual
4 fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.
5 Model selection
6 For each endpoint selected for modeling (see Table E-l), the BMDL estimate (95% lower
7 confidence limit on the benchmark dose [BMD], as estimated by the profile likelihood method) and
8 Akaike's Information Criterion (AIC) value were used to select a best-fit model from among the
9 models exhibiting adequate fit If the BMDL estimates were "sufficiently close," that is, differed by
10 at most threefold, then the model selected was the one that yielded the lowest AIC value. If the
11 BMDL estimates were not sufficiently close, then the lowest BMDL was selected as the POD.
12 Table E-l. Noncancer endpoints selected for dose-response modeling for
13 benzo[a]pyrene: RfD
Study
Endpoint
Species/sex
Doses and effect data
Kroese et
al. (2001)
Thymus
weight (mg)
Rat (Wistar)/
male
Dose (mg/kg-d)
0
3
10
30
Mean ±SDa
380 ± 60
380 ±110
330 ± 60
270 ± 40*
Kroese et
al. (2001)
Thymus
weight (mg)
Rat (Wistar)/
female
Dose (mg/kg-d)
0
3
10
30
Mean ±SDa
320 ± 60
310 ± 50
300 ± 40
230 ± 30*
Xu et al.
(2010)
Ovary
weight (mg)
Sprague-
Dawley/
female
Dose (mg/kg-d)b
0
2.5
5
Mean ± SD
0.160 ±
0.0146
0.143 ±
0.0098**
0.136 ±
0.0098**
Chen et
al. (2012)
Morris
water maze
Sprague-
Dawley/male
and female
Dose (mg/kg-d)
0
0.02
0.2
2.0
Escape latency
(sec); mean ± SD
9.89 ±5.76
12.5 ±5.10
19.1 ±5.85
33.5 ±
9.93
Time spent in
target quadrant
(sec); mean ± SD
33.6 ±8.92
31.9 ±8.63
16.6 ±5.74
11.1 ±
5.12
Elevated
plus maze
Sprague-
Dawley/
female
Number of open
arm entries
10.24 ±
1.905
10.36 ±
3.048
12.89 ±
2.667
16.39 ±
3.048
Gao et al.
(2011)
Cervical
epithelial
hyperplasia
ICR/female
Dose (mg/kg-d)c
0
0.71
1.4
2.9
Incidence
0/26
4/26
6/25
7/24
14
15 ^Significantly (p < 0.05) different from control mean; student t-test (unpaired, two-tailed); n = 10/sex/group.
16 **Statistically different (p < 0.05) from controls using one-way analysis of variance (ANOVA).
17 aReported as standard error (SE), but judged to be standard deviation (SD) (and confirmed by study authors).
18 bTime-weighted average (TWA) doses over the 60-day study period.
19 cDoses converted to mg/kg-day after adjustment for equivalent continuous dosing (2/7 days/week).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
1 Modeling results
2 Below are tables and figures summarizing the modeling results for the noncancer endpoints
3 modeled (see Tables E-2 through E-8 and Figures E-l through E-7).
4 Table E-2. Summary of BMD modeling results for decreased thymus weight in
5 male Wistar rats exposed to benzo[a]pyrene by gavage for 90 days (Kroese et
6 al.. 20011: 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), powerc
0.30
0.23
380.71
16.40
11.30
7
8 aValues <0.10 fail to meet conventional goodness-of-fit criteria.
9 bPower restricted to >1.
10
This document is a draft for review purposes only and does not constitute Agency policy.
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Linear Model with 0.95 Confidence Level
450
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Supplem en tal Information —Benzo[aJpyren e
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
lalpha = 8.56121
rho = 0
beta_0 = 380.763
beta 1 = -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
95.0% Wald Confidence Interval
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
Lower Conf. Limit
-37.9473
1.38062
346.558
-7 .11189
Upper Conf. Limit
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
66.6
37 . 9
0.0392
0.475
-0.591
0.0908
Model Descriptions for likelihoods calculated
Model A1: Yi
Var$$e(ij
Model A2: Yi
Var$$e(ij
j = Mu(i) + e(ij;
)} = Sigma^2
j = Mu(i) + e(ij;
)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var$$e(ij)} = exp(lalpha + rho*In(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var$$e(i)} = Sigma^2
Likelihoods of Interest
This document is a draft for review purposes only and does not constitute Agency policy.
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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
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.'
Test
2
Test
3
Test
4
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 adeguately 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
This document is a draft for review purposes only and does not constitute Agency policy.
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Table E-3. 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)c,d
0.17
0.77
350.80
13.29
7.77
Powerb
NA
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
Coefficients restricted to be negative.
dLowest 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Linear Model with 0.95 Confidence Level
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16:27 10/15 2009
Linear
BMDs and BMDLs indicated are associated with a change of 1SD from the control, and are in units of mg/kg-day.
Figure E-2. Fit of linear model (constant variance) to data on decreased
thymus weight in female Wistar rats—90 days (Kroese et al.. 2001).
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\female\durationadj usted\2Linkrolin
. (d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\90day\thymusweight\female\durationadjusted\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
This document is a draft for review purposes only and does not constitute Agency policy.
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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 1
alpha
1
2 . 4e-008
-2.3e-008
beta_0
2.4 e-008
1
-0. 68
beta_l
-2.3e-008
-0. 68
1
Parameter Estimates
Variable
alpha
beta_0
beta 1
Estimate
1954.92
322.144
-4.2018
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
437.134 1098.16 2811.69
9.48287 303.558 340.73
0.837537 -5.84334 -2.56026
Table of Data and Estimated Values of Interest
Obs Mean
Est Mean Obs Std Dev Est Std Dev Scaled Res.
0 10
2.1 10
7.1 10
21.4 10
320
310
300
230
322
313
292
232
60
50
40
30
44 . 2
44 . 2
44 . 2
44 . 2
-0.153
-0.237
0. 55
-0.159
Model Descriptions for likelihoods calculated
Model A1: Yi
Var$$e(ij
Model A2: Yi
Var$$e(ij
D = Mu(i) + e(ij;
)} = Sigma^2
j = Mu(i) + e(ij;
)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigma^2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var$$e(i)} = Sigma^2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
A1 -171.357252 5 352.714504
A2 -168.857234 8 353.714467
This document is a draft for review purposes only and does not constitute Agency policy.
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A3 -171.357252 5 352.714504
fitted -171.562118 3 349.124237
R -181.324151 2 366.648303
Explanation of Tests
Test 1:
Test
2
Test
3
Test
4
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately 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 24.9338 6 0.0003512
Test 2 5.00004 3 0.1718
Test 3 5.00004 3 0.1718
Test 4 0.409733 2 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 adeguately 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 = 7.64 037
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
1 Table E-4. 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 etal.. 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 (Xu et al.. 2010).
9 ====================================================================
10 Polynomial Model. (Version: 2.16; Date: 05/26/2010)
11 Input Data File:
12 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap AbsOvaryWeight/Xu2010 AbsOvaryWeight Linear lSD.(d)
13 Gnuplot Plotting File:
14 C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap AbsOvaryWeight/Xu2010 AbsOvaryWeight Linear lSD.plt
15 Tue Deo 14 13:51:32 2010
16 ====================================================================
17
This document is a draft for review purposes only and does not constitute Agency policy.
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The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*dose,A2 + ...
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
alpha =
rho =
beta_0 =
beta 1 =
Parameter Values
0. 000136
0 Specified
0.158333
-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 1
alpha
1
4e-010
-4 . 5e-010
beta_0
4e-010
1
-0.77
beta_l
-4 . 5e-010
-0.77
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
alpha 0.000118889 3.96296e-005 4.12162e-005 0.000196562
beta_0 0.158333 0.00406354 0.150369 0.166298
beta 1 -0.0048 0.00125904 -0.00726768 -0.00233232
Table of Data and Estimated Values of Interest
2 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
Model A1: Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigma/'2
Model A2: Yij = Mu(i) + e(ij)
This document is a draft for review purposes only and does not constitute Agency policy.
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Var$$e(ij)} = Sigma(i)/S2
Model A3: Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigma/S2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var$$e(i)} = Sigma/S2
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
Explanation of Tests
Test 1:
Test
2
Test
3
Test
4
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately 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 adeguately 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 Information —Benzo[aJpyren e
1 Table E-5. Summary of BMD modeling results for Morris water maze: escape
2 latency in male and female Sprague-Dawley rats exposed to benzo[a]pyrene
3 by gavage for 90 days (Chen etal.. 2012): BMR = 1 SD change from the control
4 mean
Model3
Goodness of fit
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
p-value
AIC
Hillb
0.515
386.3
0.106
0.061
Exponential 4, 5
0.466
386.4
0.115
0.071
Polynomial (2°)
0.423
386.6
0.123
0.083
Linear, power
0.002
396.7
0.543
0.421
Exponential 2, 3
<0.001
400.3
0.815
0.687
5
6 includes modeling of heterogeneous variances (BMDS Test 3, p = 0.313).
7 bPower parameter n was estimated to be 1 (boundary of parameter space).
8
9 Data from Morris water maze was presented graphically in Chen etal. f20121. but dose
10 group means and standard deviations (SDs) were provided upon request by the study authors,
11 which enabled modeling of this endpoint. In addition, the data for male and female rats were
12 combined for dose-response analysis because there was no substantive difference between males
13 and females for each dose group (supported by statistical testing using two-way analysis of
14 variance [ANOVA], and allowing for interactions), and because there was no rationale or
15 information available suggesting there would be sex-mediated differences for these
16 neurobehavioral tests.
17
This document is a draft for review purposes only and does not constitute Agency policy.
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Hill Model with 0.95 Confidence Level
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lalpha
rho
intercept
The model parameter(s) -n
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
lalpha
1
-0. 99
-0.27
0. 062
-0.11
rho
-0. 99
1
0.24
-0.063
0.12
intercept
-0.27
0.24
1
0. 017
0.47
v
0. 062
-0.063
0. 017
1
0.73
k
-0.11
0.12
0.47
0.73
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable
lalpha
rho
intercept
k
Estimate
0. 88775
0.998033
10.6545
28.7081
1
0.494812
Std. Err.
0. 974841
0.338845
0.914127
3.94381
NA
0.213359
Lower Conf. Limit
-1.0229
0.33391
8 .86283
20.9783
0. 0766351
Upper Conf. Limit
2.7984
1.66216
12 . 4461
36.4378
0. 912988
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
Obs Mean
Est Mean Obs Std Dev Est Std Dev Scaled Res.
0 20 9.89 10.7 5.76 5.08 -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 0.0952
2 20 33.5 33.7 9.93 9.01 -0.0706
Model Descriptions for likelihoods calculated
Model A1:
Model A2:
Model A3:
Yij = Mu(i) + e(ij;
Var$$e(ij)} = Sigma'" 2
j = Mu(i) + e(ij ;
Var$$e(ij)} = Sigma(i)'A2
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)} = Sigma'" 2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
A1 -192.799518 5 395.599036
A2 -186.795503 8 389.591006
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A3 -187.957975 6 387.915949
fitted -188.169983 5 386.339965
R -234.549118 2 473.098237
Explanation of Tests
Test 1:
Test
2
Test
3
Test
4
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately 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 95.5072 6 <.0001
Test 2 12.008 3 0.007356
Test 3 2.32494 2 0.3127
Test 4 0.424016 1 0.5149
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 adeguately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 0.106284
BMDL = 0.0609511
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Table E-6. Summary of BMD modeling results for Morris water maze: time
spent in quadrant for in male and female Sprague-Dawley rats exposed to
benzo[a]pyrene by gavage for 90 days (Chen etal.. 2012): BMR = 1 SD change
from the control mean
Model3
Goodness of fit
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
p-value
AIC
Exponential 4
0.576
395.4
0.065
0.043
Exponential 5
NAb
397.1
0.084
0.044
Hill
NAb
397.1
0.071
0.038
Linear, power, polynomial (1°, 2°, 3°)
<0.001
433.1
1.23
0.98
includes modeling of heterogenous variances (BMDS Test 3, p = 0.919).
bNA: insufficient degrees of freedom to evaluate x2-
Exponential Model 4 with 0.95 Confidence Level
40
Exponential
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BMDL BMD
0
0.5
1
1.5
2
dose
14:35 04/24 2012
Figure E-5. Fit of exponential 4 model to data on Morris water maze time
spent in target quadrant (Chen et al.. 2012).
Exponential Model. (Version: 1.7; Date: 12/10/2009)
Input Data File: C:\Documents and Settings\...\exp_Chen.FM.target_Exp-ModelVariance-
BMRlStd-Down.(d)
BMDS Model Run
The form of the response function by Model:
Model 2: Y[dose] = a * exp$$sign * b * dose}
Model 3: Y[dose] = a * exp$$sign * (b * dosej^d}
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Model 4: Y[dose] = a * [c-(c-l) * exp$$-b * dose}]
Model 5: Y[dose] = a * [c-(c-l) * exp$$-(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]))
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
MLE solution provided: Exact
Initial Parameter Values
Variable Model 4
lnalpha 0.666712
rho 1.047 99
a 35.3094
b 1.97191
c 0.300675
d 1
Parameter Estimates
Variable Model 4
lnalpha 0.601192
rho 1.05452
a 34.3199
b 7.26795
c 0.325841
d 1
NC = No Convergence
Table of Stats From Input Data
Dose N Obs Mean Obs Std Dev
0 20 33.63 8.924
0.02 20 31.94 8.633
0.2 20 16.56 5.744
2 20 11.15 5.117
Estimated Values of Interest
Dose Est Mean Est Std Scaled Residual
0 34.32 8.713 -0.3551
0.02 31.19 8.285 0.4069
0.2 16.59 5.939 -0.02044
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2 11.18 4.824 -0.03277
Other models for which likelihoods are calculated:
Model A1: Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigma/'2
Model A2 : Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigma(i)^2
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)} = Sigma/'2
Likelihoods of Interest
Model Log(likelihood)
A1 -197.0118
A2 -192.448
A3 -192.5331
R -238.8696
4 -192.6894
AIC
404.0235
400.896
397.0662
481.7393
395.3787
Additive constant for all log-likelihoods = -73.52. 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
Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R)
Test 2: Are Variances Homogeneous? (A2 vs. A1)
Test 3: Are variances adeguately modeled? (A2 vs. A3)
Test 6a: Does Model 4 fit the data? (A3 vs 4)
Test
Test 1
Test 2
Test 3
Test 6a
Tests of Interest
-2*log(Likelihood Ratio)
92 .84
9.127
0.1701
0.3126
p-value
< 0.0001
0.02764
0.9185
0.5761
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 6a is greater than .1. Model 4 seems
to adeguately describe the data.
Benchmark Dose Computations:
Specified Effect = 1.000000
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
1
2 Risk Type = Estimated standard deviations from coritro'l
3
4 Confidence Level = 0.950000
5
6 EMD = 0.0650194
7
8 E'.MDL = 0.0432761
9 Table E-7. Summary of BMD modeling results for elevated plus maze: open
10 arm entries for females at PND 70 (Chen et al.. 2012): BMR = 1 SD change from
11 the control mean
Goodness of fit
BMD1sd
(mg/kg-d)
BMDL1sd
(mg/kg-d)
Model3
p-valueb
AIC
Exponential (M2)
Exponential (M3)
0.107
125.93
1.086
0.845
Exponential (M4)
0.840
123.51
0.184
0.086
Exponential (M5)
NA
125.47
0.194
0.087
Hill
NA
125.47
0.193
0.066
Polynomial 1°
Polynomial 2°
Polynomial 3°
Power
0.129
125.57
0.964
0.713
12
13 aConstant variance models are presented (BMDS Test 2 p-value = 0.46), with the selected model in bold. Scaled
14 residuals for selected model for doses 0, 0.02,0.2, and 2 mg/kg-d were 0.13, -0.15, 0.03, and -0.003,
15 respectively.
16 For exponential model M3, parameter d = 1, reducing it to M2.
17 For the power model, the power parameter estimate was 1 (boundary of parameter space). For the polynomial
18 2° and 3° models, the b2 and b3 coefficient estimates were 0 (boundary of parameter space). Consequently,
19 these three models all reduced to the polynomial 1° model.
20 Exponential M5 and Hill model required four parameters and there are four dose groups, leaving no degrees of
21 freedom for the goodness-of-fit test. Therefore, these were not considered for model selection
22
23 For the elevated plus maze data, although effects of exposure were obseived across multiple
24 ages and both sexes, the results from female rats at PND 70 were chosen for dose-response
25 analyses as effects in females and older animals were of greater severity. While it is preferred that
26 elevated plus maze results be presented as percent of open arm entries or percent of time in the
27 open arms (as a function of total arm entries or time) in order to rule out potential differences in
28 motor activity or general exploration fHogg. 19961. the data provided in the study were sufficient to
29 rule out that total arm entries were affected by treatment.
30
This document is a draft for review purposes only and does not constitute Agency policy.
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Exponential Model 4 with 0.95 Confidence Level
Exponential
18
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BMDL
BMD
0
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dose
12:35 08/02 2012
Figure E-6. Fit of exponential model (4) to data on elevated plus maze open
arm maze entries (Chen etal.. 2012).
Exponential Model. (Version: 1.7; Date: 12/10/2009)
Input Data File: C:\Documents and Settings\jfox\My Documents\_CURRENTWORK\_CAST
plus\BaP\BMDS\exp_ChenF07 0_Exp-ConstantVariance-BMRlStd-Up.(d)
Gnuplot Plotting File: C:\Documents and Settings\jfox\My Documents\_CURRENTWORK\_CAST
plus\BaP\BMDS\exp_ChenF07 0_Exp-ConstantVariance-BMRlStd-Up.pit
Thu Aug 02 12:35:33 2012
BMDS Model Run
The form of the response function by Model:
Model 2
Model 3
Model 4
Model 5
Y[dose]
Y[dose]
Y[dose]
Y[dose]
exp{sign * b * dose}
exp{sign * (b * dose)^d}
[c-(c-l) * exp{-b * dose}]
[c-(c-l) * exp{-(b * dose)^d}
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 *In(Y[dose]))
rho is set to 0.
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
This document is a draft for review purposes only and does not constitute Agency policy.
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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
lnalpha
rho(S)
Model 4
1.88669
0
9.72892
1.12212
1.76842
1
(S)
Speci fied
Parameter Estimates
Variable
lnalpha
rho
Model 4
1. 8877
0
10.136
2.86365
1.61881
1
Table of Stats From Input Data
Obs Mean
Obs Std Dev
0
0. 02
0.2
2
10
10
10
10
10.24
10. 36
12 .89
16.39
1. 905
3. 048
2 . 667
3. 048
Estimated Values of Interest
Dose
0
0. 02
0.2
2
Est Mean
10.14
10.49
12 . 87
16.39
Est Std
2 . 57
2 . 57
2 . 57
2 . 57
Scaled Residual
0.1292
-0.1521
0.02563
-0.002716
Other models for which likelihoods are calculated:
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma/N2
Model A2 : Yij = Mu (i ) + e(ij)
Var{e(ij)} = Sigma(i)^2
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)} = Sigma/N2
Likelihoods of Interest
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Model Log(likeli
A1
-57.73371
A2
-56.43655
A3
-57.73371
R
-71.03323
4
-57.75397
DF AIC
5 125.4674
8 128.8731
5 125.4674
2 146.0665
4 123.5079
Additive constant for all log-likelihoods = -36.76. 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
Test
1:
Test
2 :
Test
3:
Does response and/or variances differ among Dose levels? (A2 vs. R)
Are Variances Homogeneous? (A2 vs. A1)
Are variances adeguately modeled? (A2 vs. A3)
Does Model 4 fit the data? (A3 vs 4)
Tests of Interest
Test -2*log(Likelihood Ratio) D. F. p-value
Test 1 29.19 6 < 0.0001
Test 2 2.594 3 0.4585
Test 3 2.594 3 0.4585
Test 6a 0.04053 1 0.8404
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 6a is greater than .1. Model 4 seems
to adeguately describe the data.
Benchmark Dose Computations:
Specified Effect = 1.000000
Risk Type = Estimated standard deviations from control
Confidence Level = 0.950000
BMD = 0.184087
BMDL = 0.0864691
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
1 Table E-8. 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 etal.. 2011): BMR = 1 SD change from the control
4 mean
Model
Goodness of fit
BMDisd
(mg/kg-d)
BMDLisd
(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
BMDL
BMD
0 0.5 1 1.5 2 2.5 3
dose
5 19:01 08/26 2011
6 Figure E-7. Fit of log-logistic model to data on cervical epithelial hyperplasia
7 fGaoetal.. 20111
This document is a draft for review purposes only and does not constitute Agency policy.
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Logistic Model. (Version: 2.13; Date: 10/28/2009)
Input Data File: C:\Users\hclynch\Documents\_Active Projects\_FA498 IRIS\xBaP\IASC Aug
2011\bmd modeling\lnl gao 2011 inflamm cells Opt.(d)
Gnuplot Plotting File: C:\Users\hclynch\Documents\ Active Projects\ FA498
IRIS\xBaP\IASC Aug 2011\bmd modeling\lnl_gao 2011 inflamm cells_Opt.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
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
background 0 * * *
intercept -1.6502 * * *
slope 1 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -39.4267 4
Fitted model -39.8502 1 0.847034 3 0.8382
Reduced model -45.7739 1 12.6945 3 0.005346
AIC: 81.7004
This document is a draft for review purposes only and does not constitute Agency policy.
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Goodness of Fit
Scaled
Dose Est. Prob. Expected Observed Size Residual
0.0000
0.0000
0. 000
0. 000
26
0. 000
0.7100
0.1200
3.119
4 . 000
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0.532
1.4000
0.2119
5.297
6. 000
25
0.344
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0.3577
8 . 584
7 . 000
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-0.675
.^2 = 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
This document is a draft for review purposes only and does not constitute Agency policy.
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E.1.2. Reference Concentration (RfC)
Candidate studies for the development of the RfC were not amenable to BMD modeling.
Dosimetry Modeling for Estimation of Human Equivalent Concentrations
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:
EDDRfr =—^x^xv 101/A
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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Region: Entire Lung
Wed, 03/17/2010, 02:07:20 PM EDT
0.750 I-
0.600
0.450
0.300
0.150
0.0
0.449
0.127
0.045
0.621
Head
TB
Region
Total
Species a Model Info:
Species/Ueometry: Human Limited
FRO Vblume: 3300.00 ml
Head Vblume: 50.00 ml
Breathing Route: nasal
Breathing Parameters:
Tidal Vtilume: 860.00 ml
Breathing Frequency: 16.00 1/min
Inspiratory Fraction: 0.50
Pause Fraction: 0.00
Particle Properties:
Diameter: IvyMAD: 1.70 pm
OSD: 1.00
Concentration: 4.20 pg/fri"3
Figure E-8. Human fractional deposition.
Species = humanlimited
FRC = 3300. 0
Head volume = 50.0
Density = 1.0
Number of particles calculated = single
Diameter = 1.7000000000000002 \im MMAD
Inhalability = yes
GSD =1.0
Breathing interval: One single breath
Concentration = 4.2
Breathing Freguency = 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 0.44 9
TB 0.045
P 0.127
Total 0.621
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Wed, 03/17/2010, 02:15:27 PM EOT
Region: Entire Lung
Species & Model Info:
Species/Geometry: Rat
FRC Volume: 4.00 ml
Head Volume: 0.42 ml
Breathing Route: nasal
Breathing Parameters:
Tidal Vblume: 1 .SO ml
Breathing Frequency: 102.00 1/friin
Inspiratory Fraction: 0.50
Pause Fraction: 0.00
Particle Properties:
Diameter: MV1AD: 1.70 pm
GSD: 1.00
Concentration: 4.20 pg/m"3
Figure E-9. Rat fractional deposition.
Species = rat
FRC =4.0
Head volume = 0.42
Density = 1.0
Number of particles calculated = single
Diameter = 1.7000000000000002 \im MMAD
Inhalability = yes
GSD =1.0
Breathing interval: One single breath
Concentration = 4.2
Breathing Freguency = 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
u.zuu
0.200
0.181
0
S 0.150
1
Ll_
C
o
3 0.100
0.072 0.068
III
Head TB P Total
Region
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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 + ... + qkdk) x (t ± t0)c],
where P(dt) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent
exposure in this case) and age t (in bioassay weeks); parameters q, > 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.
2010). 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
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the rat study of Kroese etal. (20011. tumors of the forestomach or liver were the principal cause of
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-9 (male rats), E-10
(female rats), and E-ll (female mice).
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. The dose estimates are provided in Tables E-12 (Kroese etal.. 2001) and E-13 (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. 2005) 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 (NRC. 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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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 f19941 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. 20101. 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 Information —Benzo[aJpyren e
1 Table E-9. 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 et al..
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 Information —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 Information —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.
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1 Table E-10. 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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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
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109
4
2
0
2
0
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Supplem en tal Information —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 Information —Benzo[aJpyren e
1 Table E-ll. 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 group
(ppm in diet)
Wk of death
Total examined
Number of animals with alimentary tract
squamous cell tumors
Fatal3
Incidental
0
31
l
0
0
74
l
0
0
89
2
0
0
91
1
0
0
93
2
0
0
94
2
0
0
97
2
0
0
98
2
0
0
99
1
0
0
100
2
0
0
101
2
0
0
104
1
0
0
105
29
0
1
5
25
1
0
0
55
1
0
0
83
1
0
0
86
1
0
0
87
2
0
0
88
2
0
0
90
1
0
0
94
1
0
0
95
2
0
0
96
1
0
0
97
2
0
0
98
2
0
0
101
2
0
0
102
2
0
0
105
27
0
3
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Supplem en tal Information —Benzo[aJpyren e
Dose group
(ppm in diet)
Wk of death
Total examined
Number of animals with alimentary tract
squamous cell tumors
25
44
l
1
0
47
l
0
0
64
l
0
0
70
l
l
0
77
l
l
0
80
l
0
0
81
l
1
0
84
1
1
85
l
1
0
86
l
1
0
88
l
1
0
89
l
0
0
90
4
4
0
93
3
2
1
94
2
2
0
96
3
0
2
97
1
1
0
98
1
1
0
99
2
1
1
100
1
1
0
101
1
0
0
102
2
2
0
104
1
1
0
105
13
0
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Supplem en tal Information —Benzo[aJpyren e
Dose group
(ppm in diet)
Wk of death
Total examined
Number of animals with alimentary tract
squamous cell tumors
100
39
l
1
0
40
l
1
0
42
l
1
0
47
2
0
49
l
0
0
50
l
1
0
53
l
0
0
55
3
0
56
l
1
0
57
l
1
0
58
l
1
0
59
3
3
0
60
1
1
0
61
3
3
0
62
5
5
0
63
4
4
0
64
3
3
0
65
2
2
0
66
3
3
0
68
1
1
0
69
2
2
0
70
2
2
0
71
1
1
0
72
1
1
0
73
1
1
0
74
1
1
0
79
1
1
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.
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Supplem en tal Information —Benzo[aJpyren e
1 Table E-12. Derivation of HEDs to use for BMD modeling of Wistar rat tumor
2 incidence data from Kroese etal. (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-13. 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.
15
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Supplem en tal Information —Benzo[aJpyren e
1 Dose-Response Modeling Results
2 Tables E-14 (male and female rats) and E-16 (female mice) summarize the modeling results
3 supporting the oral slope factor for benzo[a]pyrene. The model outputs and graphs following each
4 of these tables (Figures E-10 through E-19) provide more details for the best-fitting models in each
5 case.
6 Table E-14. Summary of BMD modeling results for best-fitting multistage-
7 Weibull models, using time-to-tumor data for Wistar rats exposed to
8 benzo[a]pyrene via gavage for 104 weeks fKroese et al.. 20011: BMR = 10%
9 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
10
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Supplem en tal Information —Benzo[aJpyren e
Male Rat (Kroese et al.. 2001): 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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
beta_l
beta 3
-0. 53
-0. 93
-0. 99
1
0.47
0. 57
0.47
1
0. 9
0. 57
0. 9
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 3.74559 0.447309 2.86888 4.6223
t_0 41.4581 2.14975 37.2447 45.6716
beta_0 0 NA
beta_l 4.37 816e-00 9 1.07528e-008 -1.6697e-008 2.54533e-008
beta_2 0 NA
beta_3 1.01904e-008 1.94164e-008 -2.78651e-008 4.82458e-008
NA - Indicates that this parameter has hit a bound implied by some ineguality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -108.512 6 229.024
Data Summary
CONTEXT
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Supplem en tal Inform ation —Benzo[aJpyren e
DOSE
U Total Expected Response
0
52
0
0
0
52
o
o
o
0. 54
44
0
8
0
52
6.77
i-1
CO
7
0
45
0
52
41. 69
5.2
0
9
43
0
52
49. 97
Minimum observation time for F tumor context = 4 4
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect =
0
1
o
o
i-1
0. 001
BMD =
0 .
,453
4 7
1
0.0633681
0.00636659
BMDL =
0 .
,281
0 4
4
0.0286649
0.00285563
BMDU =
0 .
,612
4 6
2
0.248377
> 0.050932
Incidental Risk: OralForstKroeseM3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00 Dose = 0.54
Figure E-10. 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 etal.. 2001).
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Supplem en tal Information —Benzo[aJpyren e
Male Rat (Kroese et al.. 2001): 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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 = 2.7 3535e-00 9
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
c 1 -0.84 -0.88 -1
t_0
beta_l
beta 3
-0.84
-0.88
-1
1
0.71
0.86
0.71
1
0.86
OA
OA
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 3.49582 0.629257 2.26249 4.72914
t_0 40.2211 5.65421 29.1391 51.3032
beta_0 0 NA
beta_l 4.4 3 906e-00 9 1.76051e-008 -3.00664e-008 3.89445e-008
beta_2 0 NA
beta_3 2.35065e-008 6.47999e-008 -1.03499e-007 1.50512e-007
NA - Indicates that this parameter has hit a bound implied by some ineguality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -138.544 6 289.088
Data Summary
CONTEXT
C F I U Total Expected Response
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
DOSE
0
52
0
0
0
52
o
o
o
0. 54
48
0
4
0
52
3. 38
i-1
CO
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 0.01 0.001
BMD = 0.6507 0.173556 0.0199908
BMDL = 0.44868 0.0530469 0.00530386
BMDU = 0.772467 0.352684 > 0.159927
Incidental Risk: Hepatocellular_Kroese_M3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00
Dose = 0.54
-Q
05
-Q
O
CO
O
O
O
O
i 1 1 1 1 T
0 20 40 60 80 100
-Q
05
-Q
O
00
O
O
O
O
n 1 1 t t r
0 20 40 60 80 100
Time
Time
Dose = 1.81
-Q
05
-Q
O
00
O
O
O
O
paoaoQ.o
i 1 1 t r
0 20 40 60 80 100
Time
Dose = 5.17
-Q
05
-Q
O
00
O
o
o
o
"i 1 1 1 r
20 40 60 80 100
Time
Figure E-ll. Fit of multistage Weibull model to hepatocellular adenomas or
carcinomas in male rats exposed orally to benzo[a]pyrene (Kroese etal..
2001V
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Male Rat (Kroese et al.. 2001): 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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 = 2.9634 7 e-025
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
Parameter Estimates
Variable
c
beta_0
beta_l
beta_2
beta 3
Estimate
1.77722
0
0
0
32 635e-007
Std. Err.
2.03042
NA
NA
NA
5. 29355e-006
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-2.20233 5.75677
-1.527 2 4 e-005
1.72377e-005
NA - Indicates that this parameter has hit
and thus has no standard error.
a bound implied by some ineguality constraint
Log(likelihood) # Param AIC
Fitted Model -28.4387 5 66.8773
Data Summary
CONTEXT
F I U Total Expected Response
DOSE
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Supplem en tal Information —Benzo[aJpyren e
o
0.54
1. 8
5.2
52
52
51
43
52
52
52
52
0. 0 0
0. 03
1. 04
8 . 96
Benchmark Dose Computation
Risk Response
Risk Type
Specified effect
Confidence level
Time
Incidental
Extra
0.1
0. 9
104
Specified effect
BMD
BMDL
BMDU
1
0 3 2 9 1
3 7 7 8 2
8 7 18 3
0. 01
1.38578
0. 418285
1.76166
0. 001
0.642252
0.0420835
0. 811476
Incidental Risk: DuoJej_Kroese_M3
Dose = 0.00
Dose = 0.54
-Q
-Q
o
i i r
20 40 60 80 100
-Q
-Q
o
0 20 40 60 80 100
Time
Time
Dose =1.81
Dose = 5.17
-Q
-Q
o
-Q
-Q
o
20 40 60 80 100
"I—I—T
20 40 60 80 100
Time
Time
Figure E-12. Fit of multistage Weibull model to duodenum or jejunum
adenocarcinomas in male rats exposed orally to benzo[a]pyrene (Kroese etal..
20011.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Male Rat (Kroese et al.. 2001): 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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 = 1.06194 e-02 8
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
beta 3
-1
-1
1 0.99
0.99 1
Variable
c
beta_0
beta_l
beta_2
beta 3
Estimate
1. 47227
2.54 7 8 6e-005
0
0
4 . 81611e-006
Parameter Estimates
Std. Err.
1.76686
0.000211261
NA
NA
3.49e-005
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-1.9907 4.93525
-0.000388585 0.000439542
-6.358
5e-005
7.3218 8e-005
NA - Indicates that this parameter has hit
and thus has no standard error.
a bound implied by some ineguality constraint
Log(likelihood) # Param AIC
Fitted Model -47.3623 5 104.725
Data Summary
CONTEXT
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
C F I U Total Expected Response
DOSE
0
50
0
2
0
52
i-1
i-1
CO
0. 54
51
0
1
0
52
1. 22
i-1
CO
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
Incidental Risk: Skin Mam Basal Kroese M3
Dose = 0.54
Dose = 1.81
_Q
CT5
_Q
O
CO
O
O
O
O
_Q
CT5
_Q
O
CO
O
O
O
O
0 20 40 60 80
0 20 40 60 80
Time
Time
Dose = 5.17
_Q
CT5
_Q
O
CO
O
O
O
O
n 1 T
0 20 40 60 80
Time
Figure E-13. Fit of multistage Weibull model to skin or mammary gland basal
cell tumors of male rats exposed orally to benzo[a]pyrene f Kroese et al..
2001).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Male Rat (Kroese et al.. 2001): 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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
c 1 -0.99 -1
beta_l -0.99 1 0.99
beta_3 -1 0.99 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 2.96213 2.591 -2.11613 8.04039
beta_0 0 NA
beta_l 1.50104e-008 1.86972e-007 -3.51447e-007 3.81468e-007
beta_2 0 NA
beta_3 3.9084e-009 4.15374e-008 -7.75033e-008 8.53201e-008
NA - Indicates that this parameter has hit a bound implied by some ineguality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -27.652 5 65.304
Data Summary
CONTEXT
C F I U Total Expected Response
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
DOSE
0
52
0
0
0
52
o
o
o
0. 54
51
0
1
0
52
0.42
i-1
CO
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: OralForstKroeseM3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00 Dose = 0.54
GO
C=i
CO
CD
CD
r-j
CD
CD
CD
~_
CO
CD
CO
CD
CD
r-j
CD
o
o
Time
Time
Dose = 1.81
Figure E-14. 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).
This document is a draft for review purposes only and does not constitute Agency policy.
E-54 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Information —Benzo[aJpyren e
Male Rat (Kroese et al.. 2001): 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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 = 1.5 927 8e-027
beta_2 = 2.718e-024
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
beta 3
1
-1
-1
1
Variable
c
beta_0
beta_l
beta_2
beta 3
3
Estimate
1.74897
0
0
0
11107 e-007
Parameter Estimates
Std. Err.
3.79403
NA
NA
NA
4.90313e-006
95.0% Wald Confidence Interval
Lower Conf.
-5.6i
Limit
1719
Upper Conf.
9. li
Limit
512
-9.2 98 85e-006
9.92107 e-006
NA - Indicates that this parameter has hit a
bound implied by some ineguality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -11.3978 5 32.7956
Data Summary
CONTEXT
C F I
U Total Expected Response
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
DOSE
0
52
0
0
0
52
o
o
o
0. 54
52
0
0
0
52
o
o
I-1
i-1
CO
52
0
0
0
52
0.29
5.2
49
0
3
0
52
2 .71
Benchmark Dose
Risk Response
Risk Type
Confidence level :
Time
Computation
Incidental
Extra
0. 9
104
Specified effect =
0
1
o
o
i-1
0. 001
BMD =
4 .
. 6 4
8 8 6
2 .12413
0.984449
BMDL =
2 .
. 4 9
9 7 2
0. 734665
0.0748097
BMDU =
9 .
. 0 1
0 2 3
3.49311
1.61892
Incidental Risk: Kidney_Kroese_M3
Dose = 0.00
Dose = 0.54
(H
-Q
2
Q_
(0
-Q
2
Q_
Figure E-15. Fit of multistage Weibull model to kidney urothelial tumors of
male rats exposed orally to benzo[a]pyrene (Kroese etal.. 2001).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Female Rat (Kroese et al.. 2001): 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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 = 1.1164 5e-00 9
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
1 -0.79 -0.92 -0.93 -1
t_0 -0.7 9
beta_0 -0.92
beta_l -0.93
beta 3 -1
1 0.73
0.73 1
0.72 0.79
0.8 0.92
0.72 0.8
0.79 0.92
1 0. 91
0. 91 1
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
Estimate
3.52871
46.553
1.5358 9e-00 9
7.57 004 e-00 9
0
2.5312 6e-008
Parameter Estimates
Std. Err.
0.701117
5.93306
5 . 4 0523e-00 9
2.9647e-008
NA
7.66404e-008
95.0% Wald Confidence Interval
Lower Conf. Limit
2.15454
34.9244
-9.05817e-009
-5.0536 9e-008
-1.249e-007
Upper Conf. Limit
4.90287
58 .1816
1.212 9 9e-008
6.567 7 e-008
1.75525e-007
NA - Indicates that this parameter has hit
and thus has no standard error.
a bound implied by some ineguality constraint
Log(likelihood) # Param AIC
Fitted Model -94.5119 6 201.024
Data Summary
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
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
=
Benchmark
Dose
Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect =0 . 1 0.01 0.001
BMD = 0.538801 0.0981283 0.0100797
BMDL = 0.328135 0.0345104 0.00344714
BMDU = 0.717127 0.325909 > 0.0806373
Incidental Risk: OralForstKroeseF3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00 Dose = 0.49
-Q
03
-Q
O
CO
o
"3"
o
o
o
i 1 1 1 r
20 40 60 80 100
-Q
03
-Q
O
CO
o
"3"
o
o
o
i 1 1 1 r
20 40 60 80 100
Time
Time
Dose = 1.62
-Q
03
-Q
O
CO
o
-<3-
o
° "i i r
0 20 40 60 80 100
Time
Dose = 4.58
-Q
03
-Q
O
CO
o
o
o
o
i i i i r
20 40 60 80 100
Time
Figure E-16. 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).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Female Rat (Kroese et al.. 2001V. 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
Timer to Tumor Model, Liver Hepatocellular Tumors, Kroese et al, Female
The form of the probability function is:
P[response] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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 = 31.7778
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
c 1 -0.9 -1
t_0
beta 3
-0.9 1 0.92
-1 0.92 1
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
Estimate
3.11076
38.6965
0
0
0
2.94 354 e-007
Parameter Estimates
Std. Err.
0.549208
5. 21028
NA
NA
NA
7.19418e-007
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
2.03434 4.18719
28.4846 48.9085
-1.11568e-00e
1.70439e-006
NA - Indicates that this parameter has hit
and thus has no standard error.
a bound implied by some ineguality constraint
Log(likelihood) # Param AIC
Fitted Model -228.17 6 468.34
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Data Summary
CONTEXT
C
F
I
U
Total
Expected
DOSE
0
52
0
0
0
52
0. 00
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
Risk Type
Confidence level
Time
Incidental
Extra
0. 9
104
Specified effect
BMD
BMDL
BMDU
1 0.01
5 7 5 12 7 0.262783
5 0 6 6 3 3 0.134213
6 2 9 8 0 6 0.287232
0. 001
0.12179
0.0152934
0.133064
Incidental Risk: Hepatocellular_Kroese_F3
points show rionparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00
Dose = 0.49
-Q
CT3
-Q
O
i 1 1 1 r
20 40 60 80 100
-Q
CT3
-Q
O
CO
o
o
o
i 1 1 1 1 r
0 20 40 60 80 100
Time
Time
Dose = 1.62
Dose = 4.58
-Q
CT3
-Q
O
80 100
-Q
CT3
-Q
O
CO
o
o
o
i 1 1 1 r
0 20 40 60 80 100
Time
Time
Figure E-17. 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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Supplem en tal Information —Benzo[aJpyren e
Female Rat (Kroese et al.. 2001V. 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] = l-EXP$$-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2+beta_3*dose/s3) }
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
c 1-1
beta_3 -1 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 2.32531 3.58729 -4.70565 9.35626
beta_0 0 NA
beta_l 0 NA
beta_2 0 NA
beta_3 5.32209e-008 7.98487e-007 -1.51178e-006 1.61823e-006
NA - Indicates that this parameter has hit a bound implied by some ineguality constraint
and thus has no standard error.
Log(likelihood) # Param AIC
Fitted Model -13.8784 5 37.7569
Data Summary
CONTEXT
C F I U Total Expected Response
DOSE
0 52 0 0 0 52 0.00
0.49 52 0 0 0 52 0.01
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i. t
4 . <
52
48
52
52
0.44
3.57
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect =
0
1
o
o
i-1
0. 001
BMD =
3 .
, 4
3
1
2
9
1.56781
0.726615
BMDL =
1 .
, 9
4
7
4
5
0.560867
0.0584891
BMDU =
5 .
. 7
0
1
0
8
2.61447
1.21046
Incidental Risk: DuoJej_Kroese_F3
Dose = 0.00 Dose = 0.49
JD
CO
JD
O
Time
Time
Dose = 1.62
Dose = 4.58
Figure E-18. Fit of multistage Weibull model to duodenum or jejunum
adenocarcinomas in female rats exposed orally to benzo[a]pyrene (Kroese et
al.. 20011.
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Supplem en tal Information —Benzo[aJpyren e
1 Table E-l 5. 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 (Kroese etal.. 2001)
Risk value3 at
Proportion
Data set
Tumor site
BMDooi
BMDLqoi
BMDqoi
BMDLqoi
SD
SD2
of total
variance
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/
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
jejunum
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 BMD00i:
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/
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
jejunum
Sum, risk values at BMD00i:
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/BMDLqoi-
6 bOverall SD = (sum, SD2)0'5.
7 cUpper bound on the overall risk estimate = sum of BMD00i risk values + 1.645 x overall SD.
8 Table E-16. Summary of BMD model selection among multistage-Weibull
9 models fit to alimentary tract tumor data for female B6C3Fi mice exposed to
10 benzo[a]pyrene for 2 years (Beland and Culp. 1998)
Model
stages
AIC
BMD10a
BMDL10-BMDUioa
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
11
12 Evaluated at 104 weeks
13
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Female Mice (Beland and Culp. 19981: 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_FemaleSguamF3i.(d)
The form of the probability function is:
P[response] = l-EXP$$-(t - t_0)^c *
(beta_0+beta_l*dose/sl+beta_2*dose/s2+beta_3*dose/s3) }
The parameter betas are restricted to be posi'
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
t_0
beta_0
beta_l
beta_2
beta 3
2
15
1.6e-014
0
5.5e-012
4.4 e-012
Asymptotic Correlation Matrix of Parameter Estimates
beta 1
c
t_0
beta_0
beta_l
beta_2
beta 3
1
-0.78
-0. 97
-0.42
-0. 99
-0. 99
t_0
-0.78
1
0.76
0.39
0.74
0.84
beta_0
-0. 97
0.76
1
0. 33
0. 97
0. 96
-0.42
0.39
0. 33
1
0. 31
0.46
beta_2
-0. 99
0.74
0. 97
0. 31
1
0. 97
beta_3
-0. 99
0.84
0. 96
0.46
0. 97
1
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
Estimate
6.92317
13.9429
2.46916e-016
0
5.854 52e-014
9.7 654 2e-014
Parameter Estimates
Std. Err.
1.33874
4.9664 6
1.4 7 619e-015
1.30525e-014
3.7 514 4 e-013
5.62 017 e-013
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
4.29929
4.20881
-2.64 636e-015
-2 . 55825e-014
-6.7 67 23e-013
-1.0038 8e-012
9.54705
23.677
3.14019e-015
2.55825e-014
7.93813e-013
1.19919e-012
Log(likelihood) # Param AIC
Fitted Model -306.265 6 624.53
Data Summary
Class
C F I
U Total Expected Response
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Dose
0
47
0
1
0
48
co
cn
o
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
Risk Response
Risk Type
Specified effect :
Confidence level :
Time
Computation
Incidental
Extra
0.1
0. 9
104
BMD = 0.126983
BMDL = 0.0706103
BMDU = 0.179419
Incidental Risk: BaP_FemaleSquamF3i
points show nonparam. est for Incidental (unfilled) and Fatal (filled)
Dose= 0.00 Dose= 0.10
Dose = 0.48
Dose= 2.32
Figure E-19. Fit of multistage Weibull model to duodenum or jejunum
adenocarcinomas in male rats exposed orally to benzo[a]pyrene (Kroese etal..
2001V
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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 etal.. 1981: see Appendix D for
study details). The computer software program Multistage Weibull (U.S. EPA. 2010) 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 (U.S. EPA. 1990) 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 et al. (1981) did not determine cause of death for any of the animals. Since the
investigators for the oral bioassays considered the same tumors to be fatal at least some of the time,
bounding estimates for the Thvssen etal. fl9811 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-17. 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 fU.S. EPA. 19901.
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Supplem en tal Information —Benzo[aJpyren e
1 Table E-17. Individual pathology and tumor incidence data for male Syrian
2 golden hamsters exposed to benzo[a]pyrene via inhalation for lifetime—
3 Thvssen etal. (1981)a
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
(d)
Larynx
Pharynx
Trachea
cavity
s
stomach
tumors
0
112
0
c
0
0
0
0
(0)
270
0
0
0
0
0
0
0
314
0
0
0
0
0
0
0
553
0
0
0
0
0
0
0
577
0
0
0
0
0
594
0
0
0
0
0
596
0
0
0
0
0
0
0
611
0
0
0
0
0
0
0
611
0
0
0
0
0
0
0
616
0
0
0
0
0
0
0
619
0
0
0
0
0
0
0
623
0
0
0
0
0
0
0
704
0
0
0
0
0
0
0
710
0
0
0
0
0
0
0
721
0
0
0
0
0
0
0
739
0
0
0
0
0
0
0
751
0
0
0
0
0
0
0
762
0
0
0
0
0
0
0
779
0
0
0
0
0
0
0
800
0
0
0
0
0
0
0
808
0
0
0
0
0
847
0
0
0
0
0
0
0
857
0
0
0
0
0
0
0
867
0
0
0
0
0
868
0
0
0
0
0
0
0
885
0
0
0
0
0
917
0
0
0
0
0
0
0
2
93
0
0
0
(0.25)
247
0
0
0
0
0
0
0
370
0
0
0
0
0
0
0
407
0
0
0
0
0
0
0
489
0
0
0
0
0
0
0
539
0
0
0
0
0
0
0
554
0
0
0
0
0
0
0
591
0
0
0
0
0
0
0
612
0
0
0
0
0
0
0
650
0
0
0
0
0
0
0
682
0
0
0
0
0
690
0
0
0
0
0
0
0
717
0
0
0
0
0
0
0
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Supplem en tal Information —Benzo[aJpyren e
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
(d)
Larynx
Pharynx
Trachea
cavity
s
stomach
tumors
717
0
0
0
0
0
0
0
755
0
0
0
0
0
0
0
788
0
0
0
0
0
0
0
795
0
0
0
0
0
0
0
802
0
0
0
0
0
0
0
808
0
0
0
0
0
0
0
836
0
0
0
0
0
0
0
848
0
0
0
0
925
0
0
0
0
0
0
0
10
212
0
0
0
0
0
0
0
(1.01)
227
0
0
0
0
0
0
0
357
0
0
0
0
0
0
0
465
0
0
0
0
0
0
0
509
0
0
0
0
0
0
0
530
0
1(1)
0
0
0
0
1
531
0
1(1)
0
0
0
0
1
557
1 (l)d
0
0
0
0
0
0
597
0
0
0
0
0
0
0
653
1(1)
0
0
0
0
0
1
695
0
0
0
0
0
0
0
712
0
1(0)
0
0
0
0
1
732
1(1)
1(1)
0
0
0
0
1
773
0
1(1)
0
0
0
0
1
788
0
1(0)
0
0
0
0
1
796
1(1)
1(1)
0
0
0
0
1
803
1(1)
—
1(0)
1(0)
0
0
1
808
0
—
1(0)
d)
1
1
0
0
1
812
1(0)
—
0
0
0
0
1
822
1(1)
1(1)
0
0
0
0
1
826
1(0)
0
0
0
0
0
1
826
0
0
0
0
0
826
1(1)
0
0
1(0)
0
1(1)
1
848
1(0)
0
0
0
0
0
1
867
1(1)
1(1)
0
0
0
0
1
868
0
0
0
1(0)
0
0
1
50
144
—
—
—
0
0
0
—
(4.29)
151
—
—
—
0
0
0
—
178
—
0
0
0
—
210
—
0
0
0
—
211
0
—
0
0
0
0
—
213
0
0
0
242
0
0
0
253
0
0
0
0
0
0
0
255
0
0
0
263
0
0
0
0
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Supplem en tal Information —Benzo[aJpyren e
Exposure
concentration:
target
(lifetime
average
continuous
exposure)15,
mg/m3
Time of
tumor
observed
(d)
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
281
of
i(i)
1(0)
0
0
0
1
284
0
0
0
0
0
0
0
284
0
0
0
294
0
0
0
0
0
0
0
296
0
0
0
0
0
0
0
324
1(1)
1(1)
0
0
0
0
1
329
0
1(1)
0
0
0
0
1
371
0
—
0
0
0
0
388
1(1)
1(1)
0
0
0
0
1
395
0
1(1)
0
0
0
0
1
421
0
1(1)
0
0
0
0
1
436
0
0
0
0
0
0
442
0
1(0)
0
0
0
1(0)
1
462
1(1)
1(1)
0
0
1
471
0
1(1)
0
0
0
0
1
486
1(0)
1(1)
0
0
1(0)
0
1
494
1(1)
1(1)
1(1)
0
0
0
1
498
1(0)
1(1)
0
0
0
1(0)
1
504
1(1)
1(1)
0
0
0
0
1
506
1(1)
1(1)
0
0
0
0
1
572
0
1(1)
0
0
0
0
1
575
1(1)
1(1)
0
0
1(0)
0
1
578
1(0)
1(1)
0
0
0
0
1
717s
1(1)
1(1)
1(0)
1(0)
0
0
1
1
2 aHistopathology incidence from (Clement Associates (1990); U.S. EPA (1990)).
3 bSee Section D.4.2.
4 cTissue 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 8 Necropsy occurred 24 weeks after 79 weeks of exposure.
9
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Dose-Response Modeling Results
Table E-18 summarizes the modeling results supporting the derivation of an inhalation unit
risk value for benzo[a]pyrene. The model outputs and graphs (Figures E-20 and E-21) following
Table E-18 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.
Table E-18. 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
BMD10a
BMDLio"
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 (BMDU10 = 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 (BMDU10 = 0.544)
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: ThyssenInc2sLl0 4noUB70.(d)
Tue Mar 11 12:58:28 2014
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)^c *
(beta_0+beta_l*dose/'l+beta_2*dose/'2 ) }
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 = 1.3217 6e-037
beta_l = 3.02455e-036
beta 2 = 2.03765e-013
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Asymptotic Correlation Matrix of Parameter Estimates
( The model parameter(s) -t 0 -beta 0 -beta 1
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
beta 2
1
-1
beta_2
-1
1
Variable
c
beta_0
beta_l
beta 2
Estimate
4 .71714
0
0
5.16891e-014
Parameter Estimates
Std. Err.
0.957627
NA
NA
3 .13384e-013
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
2.84023 6.59406
-5.62533e-013
j.65911e-013
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log i
[likelihood) i
f Param
AIC
Fitted
Model
-16.
.2088
4
40.4176
Data Summary
CONTEXT
C
F
I
U
Total
Expected Response
DOSE
0
21
0
0
0
21
0. 00
0.25
19
0
0
0
19
1. 63
1
8
0
17
0
25
16.27
4 . 3
5
0
18
0
23
17 .75
Benchmark
Dose
Computation
Risk Response
=
Incidental
Risk Type
=
Extra
Specified effect
Confidence level
0.1
0. 9
Time
72£
BMD
BMDL
BMDU
0.253569
0.163052
0.323781
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Incidental Risk: Thyssenlnc2sL104noll
Dose = 0.00 Dose = 0.25
oo
ci
ci
o
ci
"i 1 1 T
200 400 600 800
.Q
CO
_Q
O
OO
ci
o
o
ci
1 1 1 r
200 400 600 800
Time
Time
Dose = 1.01 Dose = 4.29
Time
Time
200 400 600
800
I
200
400 600
~~I
800
Figure E-20. Fit of multistage Weibull model to respiratory tract tumors in
male hamsters exposed via inhalation to benzo[a]pyrene (Thvssen et al..
1981): tumors treated as incidental to death.
Output for Respiratory Tract Tumors: All Tumors Considered to be Cause Of Death
Time of tumor observation was converted to weeks in order to run this form of the multistage-
Weibull model.
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: ThyssenF2sLl04noU.(d)
Thu Mar 13 14:30:45 2014
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)/Nc *
(beta_0+beta_l*dose/sl+beta_2*dose/N2 ) }
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
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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_0 = 2.0496e-036
beta~l = 4 .12988e-014
beta 2 = 3.37033e-013
Asymptotic Correlation Matrix of Parameter Estimates
( The model parameter(s) -t 0 -beta 0 -beta 1
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
c 1-1
beta 2-11
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 6.61992 0.915036 4.82649 8.41336
beta_0 0 NA
beta_l 0 NA
beta~2 2.13816e-014 8.96466e-014 -1.54323e-013 1.97086e-013
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 -147.66 4 303.319
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
CO
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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Supplem en tal Information —Benzo[aJpyren e
Fatal Risk:
Dose = 0.00
Dose = 0.25
.Q
CO
.Q
O
00
d
CO
d
d
CM
d
o
d
0 20 40 60 80 100 120
"i 1 1 r
60 80 100 120
Time
Time
Dose = 1.01
Time
Dose = 4.29
Time
60 80
100 120
Figure E-21. Fit of multistage Weibull model to respiratory tract tumors in
male hamsters exposed via inhalation to benzo[a]pyrene (Thvssen et al..
1981): tumors treated as cause of death.
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E.2.3. Dose-Response Modeling for the Dermal Slope Factor
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) and the inhalation unit risk (see Section E.2.2, Dose Response-
modeling for the Inhalation Unit Risk), there was earlier occurrence of tumors with increasing
exposure, as shown in the individual animal data in the technical report for the National Institute
for Occupational Safety and Health (NIOSH) study (Sivak etal.. 1997: Arthur D Little. 19891. The
computer software program Multistage Weibull (U.S. EPA. 20101 was used for the analysis of the
dermal carcinogenicity data. See Section E.2.1 for details of the modeling approach, including
evaluation of model fit and model selection. Tumors were classified as incidental because the
appearance of tumors generally preceded death by weeks in all cases, and time of first appearance
of tumors or time on study without tumors were used for the time input.
For the other supporting studies identified in Section 2.5.1, multistage models [BMDS; (U.S.
EPA. 2012a): v 2.1] were used. See Section E.l.l for details of fitting the multistage model. The
BMDL estimate (95% lower confidence limit on the BMD, as estimated by the profile likelihood
method) and AIC value were used to select a best-fit model from among the models exhibiting
adequate fit. The data modeled are summarized in Tables E-19 through E-23.
Data Adjustments Prior to Modeling
For time-to-tumor modeling, no adjustment other than an estimate of daily exposure was
used. The data modeled are provided in Table E-19.
For the remaining studies, two types of adjustments were considered: (1) for study groups
that were reported to end before 104 weeks, but well after 1 year of exposure, it was judged
reasonable to assume that the tumor incidence observed at the time of early termination could have
been realized in a full lifetime study using a lower dose; and (2) reductions of the group sizes when
there was mortality prior to the first appearance of tumors, in order to estimate the effective
number at risk. Equivalent lifetime doses were estimated by multiplying the relevant average daily
doses by (Le/104)3, where Le is the length of exposure, based on observations that tumor incidence
tends to increase with age (Doll. 19711. Note that exposure periods <52 weeks would lead to a
relatively large adjustment [i.e., (52/104)3 = 0.125, or an eightfold lower dose than administered],
reflecting considerable uncertainty in lifetime equivalent dose estimates generated from relatively
short studies. This adjustment was applied to all dose groups in Poel T19591 and Roe etal. f!9701.
and the highest dose group in Habs etal. (1980) and the grouped data reported by Sivak et al.
(1997). The following discussion summarizes how each adjustment was carried out when relevant.
Roe etal. (1970) applied benzo[a]pyrene dermally for 93 weeks or until natural death; with
the exception of the highest dose group, each group had approximately 20 animals (or ~40%
survival) at 600 days (see Table D-17). The tumors were first observed in the lowest and highest
dose groups during the interval of days 200-300. Mice dying before day 200 were likely not at risk
long enough for tumor development However, because tumor incidence and mortality were
reported in 100-day intervals, mice that had not been on study long enough to develop tumors were
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not easily identifiable. Incidence denominators reflect the number of animals alive at day 200, and
may thus lead to underestimates of tumor risk if the number of animals at risk has been
overestimated. Table E-20 summarizes the dose adjustments to estimate equivalent 104-week
exposures, as well as incidence data adjusted for mortality prior to skin tumor appearance.
Schmidt etal. f 19731 did not report survival information; instead, the authors provided
incidences based on the numbers of mice initially included in each dose group at the start of the
study. Overall latency was reported for the two high-dose groups in each series, but these data only
describe the survival of mice with tumors (animals were removed from study when a tumor
appeared). It is not clear how long exposures lasted overall in each dose group, or whether some
mice may have died on study from other causes before tumors appeared. While it is possible that
no mice died during the study, all of the other studies considered here demonstrate mortality.
However, the data were modeled as reported, with no adjustments, recognizing the possibility of
underestimating risk associated with incidences reported and lack of duration of exposure (see
Table E-20).
Schmahl etal. (1977) reported that reduced numbers of animals at risk (77-88 mice per
dose group compared with the initial group sizes of 100) resulted from varying rates of autolysis.
No other survival or latency information was provided, so all exposures were assumed to have
lasted for 104 weeks and were modeled as reported. Given the results of the other studies, it seems
possible that the numbers at risk in each group may be overestimated, which could lead to an
underestimate of lifetime risk (see Table E-20).
Habs etal. (1980) reported age-standardized skin tumor incidence rates, indicating earlier
mortality in the two highest dose groups (2.8 and 4.6 ng/application). These rates were used to
estimate the number at risk for dose-response modeling, by dividing the number of mice with
tumors by the age-standardized rates. Exposure lasted longer than 104 weeks in the two lower
exposure groups, at about 120 and 112 weeks, and until about 88 weeks in the highest exposure
group. Incidence in the two lower exposure groups may be higher than if the exposure had lasted
just 104 weeks. There was mortality in the first 52 weeks of exposure, about 10-15% in the three
exposure groups, but because there was no information concerning when tumors first appeared, it
is not possible to determine how much the early mortality may have impacted the number of mice
at risk in each group (see Table E-20).
Habs etal. T19841 reported mean survival times (with 95% CIs) for each dose group. The
CIs supported the judgment that the control and lower dose groups were treated for 104 weeks.
The higher dose group (4 ng/application) was probably treated for <104 weeks, because the upper
95% confidence limit for the mean survival was approximately 79 weeks. However, since it was
not possible to estimate a more realistic duration for this group, an estimate of 104 weeks was used
(see Table E-20).
The Poel (1959) study was conducted in male mice and used toluene as the vehicle. In
addition to a control group, there were nine dose groups. All C57L mice in dose groups with
>3.8 ng/application died by week 44 of the study. Therefore, these five dose groups were omitted
prior to dose-response modeling because of the relatively large uncertainty in extrapolating cancer
risk as a result of lifetime exposure. Four dose groups in addition to control remained. Among
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these groups, mice survived and were exposed until weeks 83-103. According to the lifespan
ranges provided, at least one mouse in each dose group died before the first appearance of tumor,
but insufficient information was available to determine how many; consequently, the incidence
denominators were not adjusted. Although mice in the lowest exposure group were treated up to
103 weeks, the control group demonstrated a median survival time of 60 weeks and concluded at
week 92, suggesting a lifetime <104 weeks is more relevant for this mouse strain. Doses were
adjusted to approximate exposures resulting in the same responses at 104 weeks of exposure for
comparability among these lifetime studies. This adjustment had little impact on the lowest
exposure level, which had an observed response of 9%, close to the BMR of 10% extra risk; in this
case, the adjustment is not expected to affect the BMDio significantly. The dose-response data are
summarized in Table E-21.
Grimmer etal. f19841 and Grimmer etal. T19831 studied female CFLP mice, using
acetone:dimethyl sulfoxide (DMSO) (1:3) as the vehicle. Mean or median latency times were
reported (as well as measures of variability), but no information concerning overall survival was
included in the results. The total of tumor-bearing mice and the reported percentages of mice with
any skin tumors was reported, and varied at most one animal from the number of animals initially
placed on study. The decreasing latency and variability and increasing tumor incidence with
increasing benzo[a]pyrene exposure suggests that exposure probably did not last for the full
scheduled 104 weeks in at least the high-dose group. The data reported were modeled under the
assumption that at least some animals in each group were treated and survived until week 104 (see
Table E-22).
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1 Table E-19. Tumor incidence, with time to observation of tumor or death;
2 CeH/HeJ male mice exposed dermally to benzo[a]pyrenea (Sivak et al.. 1997:
3 Arthur D Little. 1989)
Time of first
Number of
Time of first
Average
appearance
animals with
Average
appearance
Number of
daily
of tumor, or
papillomas
daily
of tumor, or
animals with
dose
time on
Total
or
dose
time on
Total
papillomas or
(Hg/d)
study (d)b
examined
carcinomas
(Hg/d)
study (d)b
examined
carcinomas
0
104
l
0
0.14
354
l
0
237
l
0
453
l
l
239
l
0
460
l
0
444
l
0
509
l
0
483
l
0
525
l
0
504
l
0
537
l
0
509
l
0
560
l
1
537
l
0
573
l
1
545
l
0
601
l
0
590
l
0
663
l
lb
609
l
0
669
l
0
613
l
0
677
l
0
615
0
685
l
0
629
l
0
701
l
0
630
l
0
727
l
1
658
l
0
731
l
0
699
l
0
732
l
0
719
l
0
734
13
0
748
11
0
Totals
30
0
Totals
30
5
0.014
235
1
0
1.4
233
1
0
238
1
0
234
1
0
430
1
0
235
1
0
446
1
0
307
1
1
456
1
0
348
2
2
463
1
0
355
2
2
470
1
0
362
2
2
503
1
0
369
3
3
511
1
0
376
1
1
590
1
0
383
1
1
604
1
0
390
1
1
663
1
0
397
3
3
680
1
0
404
1
1
684
1
0
411
5
5
727
1
0
418
1
1
747
15
0
440
2
2
446
1
1
474
1
1
Totals
30
0
Totals
30
27
4
5 aDoses were applied twice/weekly to shaved dorsal skin. Vehicle for all groups was 1:1 cyclohexanone/acetone.
6 bFor animals with skin tumors, the time of the tumor; if no skin tumor was observed, the time is the time of death
7 or sacrifice.
8 cThe tumor diagnosis was keratoacanthoma.
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Table E-20. Skin tumor incidence, benign or malignant in female Swiss or
NMRI mice dermally exposed to benzo[a]pyrene; data from Roe etal. (1970).
Schmidt etal. (1973). Schmahl etal. (1977). Hahs etal. (1980). Hahs etal.
f19841
Average
First
Lifetime
daily
appearance
Length of
average
Skin tumor
Mouse
dose
of tumor
exposure
daily dose
incidence (all
Study
strain
Dose(pg)
(Hg/d)
(wks)
(wks)
(Hg/d)
types)
Roe et al.
Swiss
0 (acetone)
0
-
93
0.00
0/49 (0%)
(1970)ab
0.1
0.04
29-43
93
0.03
1/45 (2%)
0.3
0.13
-
93
0.09
0/46 (0%)
1
0.43
57-71
93
0.31
1/48 (2%)
3
1.29
43-57
93
0.92
8/47 (20%)
9
3.86
29-43
93
2.76
34/46 (74%)
Schmidt et
NMRI
0 (acetone)
0
-
104d
0
0/100 (0%)
al. (1973)°
0.05
0.01
-
104
0.01
0/100 (0%)
0.2
0.06
-
104
0.06
0/100 (0%)
0.8
0.23
53e
104
0.23
2/100 (2%)
2
0.57
76e
104
0.57
30/100(30%)
Swiss
0 (acetone)
0
-
104
0
0/80 (0%)
0.05
0.01
-
104
0.01
0/80 (0%)
0.2
0.06
-
104
0.06
0/80 (0%)
0.8
0.23
d)
00
LO
104
0.23
5/80 (6%)
2
0.57
d)
1
ID
104
0.57
45/80 (56%)
Schmahl et
NMRI
0 (acetone)
0
-
104
0
1/81 (1%)
al. (1977)°
1
0.29
NR
104
0.29
11/77 (14%)
1.7
0.49
NR
104
0.49
25/88 (28%)
3
0.86
NR
104
0.86
45/81 (56%)
Habs et al.
NMRI
0 (acetone)
0
-
128
0
0/35 (0%)
(1980)cf
1.7
0.49
NR
120
0.49
8/34 (24.8%)
2.6
0.74
NR
112
0.74
24/27 (89.3%)
4.6
1.31
NR
88
0.80
22/24 91.7%)
Habs et al.
NMRI
0 (acetone)
0
-
104
0
0/20 (0%)
(1984)°
2
0.57
NR
104
0.57
9/20 (45%)
4
1.14
NR
104
1.14
17/20 (85%)
aDoses were applied 3 times/week for up to 93 weeks to shaved dorsal skin.
bNumerator: number of mice detected with a skin tumor. Tumors were thought to be malignant based on
invasion or penetration of the panniculus carnosus muscle. Denominator: number of mice surviving to 29 weeks
(200 days).
cDoses were applied 2 times/week to shaved skin of the back. Mice were exposed until natural death or until they
developed a carcinoma at the site of application.
dExposure periods not reported were assumed to be 104 weeks; indicated in italics.
6Central tendency estimates; range or other variability measure not reported.
fThe percentages were reported by the authors as age-standardized incidences of animals with local tumors,
derived using mortality data from the entire study population. The incidences reflect reported counts of tumor-
bearing animals and denominators estimated from the reported age-standardized rates. The authors did not
report the percentages of local tumors which were carcinomas or papillomas.
NR = not reported.
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Supplem en tal Information —Benzo[aJpyren e
1 Table E-21. Skin tumor incidence, benign or malignant, in C57L male mice
2 dermally exposed to benzo[a]pyrene; data from Poel (1959)
Average
First
daily
appearance
Length of
Lifetime
Skin tumor
Mouse
dose
of tumor
exposure
average daily
incidence (all
Study
strain
Dose (pg)a
(Hg/d)
(wks)
(wks)
doseb
types)0
Poel (1959)
C57L
0 (toluene)
0
-
92
0.00
0/33 (0%)
0.15
0.06
42
98
0.05
5/55 (9%)
0.38
0.16
24
103
0.16
11/55 (20%)
0.75
0.32
36
94
0.24
7/56 (13%)
3.8
1.63
21-25
82
0.80
41/49 (84%)
3
4 aDoses were applied to interscapular skin 3 times/week for up to 103 weeks or until time of appearance of a
5 grossly detected skin tumor. See Table E-15 for data of five highest dose groups (19-752 ng) in which all mice
6 died by week 44. These groups were not considered for dose-response modeling.
7 bSee text in this section for discussion of extrapolation to lifetime average daily doses.
8 cTumors were histologically confirmed as epidermoid carcinomas.
9 Table E-22. Skin tumor incidence, benign or malignant, in female CFLP mice
10 dermally exposed to benzo[a]pyrene; data from Grimmer et al. f19831.
11 Grimmer et al. (1984)
Mean or
median
Average
time of
Lifetime
daily
tumor
Length of
average daily
Skin tumor
dose
appearance
exposure
dose
incidence (all
Study
Dose (pg)a
(Hg/d)
(wks)
(wks)b
(Hg/d)
types)0
Grimmer et
0 (1:3 acetone:DMSO)
0
-
104
0
0/80 (0%)
al. (1983)
3.9
l.i
74.6 ± 16.8d
104
l.i
22/65 (34%)
7.7
2.2
60.9 ± 13.9
104
2.2
39/64 (61%)
15.4
4.4
44.1 ±7.7
104
4.4
56/64 (88%)
Grimmer et
0 (1:3 acetone:DMSO)
0
-
104
0
0/80 (0%)
al. (1984)
3.4
0.97
61 (53—65)e
104
0.97
43/64 (67%)
6.7
1.9
47 (43-50)
104
1.9
53/65 (82%)
13.5
3.9
35 (32-36)
104
3.9
57/65 (88%)
12
13 indicated doses were applied twice/week to shaved skin of the back for up to 104 weeks.
14 bAssumed exposure period is indicated in italics.
15 "incidence denominators were calculated from reported tumor-bearing animals and reported percentages.
16 dMean±SD.
17 eMedian and 95% confidence limit.
18 Dose-Response Modeling Results
19 The modeling results are summarized in Tables E-23 (time-to-tumor modeling of individual
20 data) and E-24 (multistage modeling of group incidence data). The modeling details are provided
21 with Figures E-22 through E-33.
22 Adequate model fits for the supporting studies were found using the multistage model for
23 all but one of the mouse skin tumor incidence data sets (Table E-24). The data from Grimmer et al.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
(19841 could not be adequately fit by the multistage model initially, and the other dichotomous
models available in BMDS were used. Due to the supralinear shape of the dose-response data, only
the log-logistic and dichotomous Hill models provided adequate fits. Also due to the supralinear
dose-response shape, the POD for slope factor derivation was identified near the lowest response of
~70%, because of the lack of data to inform the dose-response relationship at lower doses. Overall,
model fits demonstrated typical statistical variability at the PODs, with BMDLs generally less than
twofold lower than corresponding BMDs.
A comparison of model types was feasible for the NIOSH study data (Arthur D Little. 1989).
which included the keratoacanthoma in the mid-dose group. The multistage model fit of the
grouped data reported by (Sivak etal.. 19971 yielded BMDio and BMDLio values of 0.109 and
0.058 |ig/day, respectively, while the multistage-Weibull fit of the corresponding individual data
yielded BMDio and BMDLio values of 0.0890 and 0.0514 ng/day, respectively. Use of the
multistage-Weibull model was associated with a 10-20% lower POD for these data.
Table E-23. Summary of BMD modeling results for best-fitting multistage-
Weibull models, using time-to-tumor data for male CeH/HeJ mice exposed
dermally to benzo[a]pyrene (Sivak et al.. 1997: Arthur D Little. 1989)
Model
BMDio
BMDL10-BMDUio
Endpoints
stages
AIC
(Hg/d)
(Hg/d)
Basis for model selection
Skin papilloma or
l
51.9
0.0410
carcinoma
2
42.9
0.0985
0.0600-0.1466
Lowest AIC, best fit to low dose data
3
44.3
0.1047
Skin papilloma,
1
53.7
0.0391
keratoacanthoma or
2
46.2
0.0890
0.0514-0.1303
Lowest AIC, best fit to low dose data
carcinoma
3
47.9
0.0919
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Supplem en tal Information —Benzo[aJpyren e
Male Mice (Sivak et al.. 1997: Arthur D Little. 1989): Skin 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: SivakCarPapM2.(d)
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)^c *
(beta_0+beta_l*dose/'l+beta_2*dose/'2 ) }
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 120
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 = 2.57143
t_0 = 0 Specified
beta_0 = 0
beta~l = 5.45626e-028
beta 2=3.97261e-007
Asymptotic Correlation Matrix of Parameter Estimates
( The model parameter(s) -t 0 -beta 0 -beta 1
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
beta 2
1
-1
beta_2
-1
1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 2.75512 0.94014 0.912478 4.59776
beta_0 0 NA
beta_l 0 NA
beta~2 1.31225e-007 7.47141e-007 -1.33315e-006 1.5956e-006
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Log i
! likelihood)
# Param
AIC
Fitted
Model
-17 .4278
4
42.8557
Data Summary
CONTEXT
C
F I
U
Total
Expected Response
DOSE
0
30
0 0
0
30
0. 00
0. 014
30
0 0
0
30
0. 05
0.14
26
0 4
0
30
4 . 38
1. 4
3
0 27
0
30
27 .72
Benchmark Dose
Risk Response
Risk Type
Confidence level :
Time
Computation
Incidental
Extra
0. 9
748
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Supplem en tal Information —Benzo[aJpyren e
Specified effect
BMD
BMDL
BMDU
0.1
0.0984828
0.0599711
0.14663
0. 01
0.0304167
0.00728535
0.0434989
0. 001
0.00959688
0.000745583
0.0137323
Incidental Risk: SivakCarPapM2
Dose = 0.00
Dose = 0.014
-Q
03
-Q
O
CO
o
o
o
200 400 600
-Q
03
-Q
O
CO
o
o
o
0 200 400 600
Time
Time
Dose =0.14
Dose = 1.40
-Q
03
-Q
O
CO
o
o
o
200 400 600
-Q
03
-Q
O
CO
o
o
o
i i i r
0 200 400 600
Time
Time
Figure E-22. Fit of multistage Weibull model to skin carcinomas or papilloma
for male CeH/HeJ mice exposed dermally to benzo[a]pyrene fSivak et al..
1997): BMR = 10% extra risk.
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Supplem en tal Information —Benzo[aJpyren e
Male Mice fSivak etal.. 19971: Skin papilloma, Keratoacanthoma, 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: SivakCarPapKerM2.(d)
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)^c *
(beta_0+beta_l*dose/'l+beta_2*dose/'2 ) }
The parameter betas are restricted to be positive
Dependent variable = CONTEXT
Independent variables = DOSE, TIME
Total number of observations = 120
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 = 3
t_0 = 0 Specified
beta_0 = 4 . 4 6003e-033
beta_l = 0
beta~2 = 3.21731e-008
Asymptotic Correlation Matrix of Parameter Estimates
( The model parameter(s) -t 0 -beta 0 -beta 1
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
beta 2
1
-1
beta_2
-1
1
Parameter Estimates
Variable
c
beta_0
beta_l
beta 2
Estimate
2.97877
0
0
3 . 6606e-008
Std.
0.
2 . 03£
Err.
91338
NA
NA
e-007
95.0% Wald Confidence Interval
Lower Conf. Limit
1.18857
-3.62561e-007
Upper Conf. Limit
4.76896
4.35773e-007
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Log i
! likelihood)
# Param
AIC
Fitted
Model
-19.076
4
46.1521
Data
Summary
CONTEXT
C
F
I
U
Total
Expected Response
DOSE
0
30
0
0
0
30
0. 00
0. 014
30
0
0
0
30
0. 05
0.14
25
0
5
0
30
5.12
1. 4
3
0
27
0
30
27.79
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 748
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
2 Specified effect = 0.1 0.01 0.001
3 BMD = 0.0889653 0.0274772 0.00866942
4 BMDL = 0.0514113 0.00585974 0.000598209
5 BMDU = 0.130325 0.0401556 0.012538
Incidental Risk: SivakCarPapKerM2
11
Dose = 0.00
Dose = 0.014
-Q
03
-Q
O
CO
o
o
o
i I r
0 200 400
600
-Q
03
-Q
O
CO
o
o
o
i I r
0 200 400
600
Time
Time
Dose =0.14
Dose = 1.40
-Q
03
-Q
O
CO
o
o
o
200 400 600
-Q
03
-Q
O
CO
o
o
o
0 200 400 600
6
7
Time
Time
8
9
10
Figure E-23. Fit of multistage Weibull model to skin carcinomas,
keratoacanthoma or papilloma for male CeH/HeJ mice exposed dermally to
benzo[a]pyrene (Sivak et al.. 1997): BMR = 10% extra risk.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
1 Table E-24. Summary of BMD model selection and modeling results using
2 multistage models, for multiple data sets of skin tumors in mice following
3 lifetime dermal benzo[a]pyrene exposure
Goodness-of-
fit
BMD!
(Hg/d)
Data set
Model
P"
value
AIC
BMDL10
(Hg/d)
Basis for model selection3
Figu re
number
Poel (1959)
Male C57L
Mult
Mult
Mult
Mult
stage 1°
stage 2°
stage 3°
stage 4°
0.011
0.027
0.053
0.068
191.5
188.6
186.9
186.2
0.070
0.134
0.127
0.123
0.057
0.078
0.078
0.077
No significant improvement in model
fit with higher stage
E-24
Roe et al. (1970)
Mult
stage 1°
0.110
131.1
0.318
0.249
Female Swiss
Mult
Mult
stage 2°
stage 3°
0.485
0.485
123.6
123.6
0.748
0.748
0.480
0.480
No significant improvement in model
fit with higher stages
E-25
Schmidt et al.
(1973)
Female NMRI
Mult
Mult
Mult
stage 1°
stage 2°
stage 3°
0.008
0.609
0.999
162.7
147.4
143.9
0.256
0.329
0.381
0.194
0.287
0.326
No significant improvement in model
fit with higher stages
E-26
Schmidt et al.
(1973)
Female Swiss
Mult
Mult
Mult
Mult
stage 1°
stage 2°
stage 3°
stage 4°
<0.01
0.514
0.983
0.983
178.0
153.3
151.3
151.3
0.116
0.216
0.282
0.282
0.093
0.192
0.223
0.223
No significant improvement in model
fit with higher stage
E-27
Schmahl et al.
(1977)
Female NMRI
Mult
Mult
Mult
stage 1°
stage 2°
stage 3°
0.136
0.939
0.939
298.4
296.3
296.3
0.140
0.233
0.233
0.117
0.149
0.143
No significant improvement in model
fit with higher stage
E-28
Habs et al.
(1980)
Female NMRI
Mult
Mult
Mult
stage 1°
stage 2°
stage 3°
0.0
0.009
0.207
96.5
84.4
76.7
0.063
0.198
0.294
0.050
0.143
0.215
Only model with adequate fit
E-29
Habs et al.
(1984)
Female NMRI
Mult
Mult
stage 1°
stage 2°
0.577
1.000
48.4
47.6
0.078
0.171
0.056
0.060
No significant improvement in model
fit with higher stage
E-30
Grimmer et al.
(1983)
Female CFLP
Multistage 1°
Multistage 2°
Multistage 3°
0.850
0.972
0.972
219.9
221.1
221.1
0.245
0.292
0.292
0.208
0.213
0.213
No significant improvement in model
fit with higher stages
E-31
Grimmer et al.
(1984)b
Female CFLP
Multistage 1°
LogLogistic
Dichotomous-
Hill
LogProbit
Gamma,
Weibull
Logistic
Probit
0.003
0.919
1.000
0.047
0.003
0.0
0.0
205.3
195.8
197.7
200.2
205.3
250.5
255.4
0.132
1.07
0.902
1.33
0.132
2.03
2.29
0.113
0.479
0.533
1.11
0.113
1.76
2.03
(Higher stages did not provide better
fit)
Lowest AIC among adequately fitting
models.
(Same as Multistage 1°)
E-32
E-33
Multistage 1°,
high dose
dropped
0.499
1.21
1.01
E-34
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Supplem en tal Information —Benzo[aJpyren e
Data set
Model
Goodness-of-
fit
BMD!
(Hg/d)
BMDL10
(Hg/d)
Basis for model selection3
Figu re
number
P"
value
AIC
Sivak et al.
(1997)
Male CeH/HeJ
Multistage 1°
Multistage 2°
Multistage 3°
0.059
0.998
0.998
57.8
48.6
48.6
0.036
0.109
0.109
0.026
0.058
0.052
No significant improvement in model
fit with higher stage
E-35
aAdequate fit: goodness-of-fit p > 0.05, scaled residuals <2.0, good fit near BMR, lack of extreme curvature not
reflected in the observed data.
bThe POD forGrimmer et al. (1984), using a BMR of 70% (near response at the lowest dose), was based on the
LogLogistic model. For comparison purposes, the multistage model was it fit to the Grimmer et al. (1984) data
with the highest dose dropped (AIC not provided because it is not comparable to fits of the full dataset).
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
BMDL
3MD
j.
j.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
dose
Figure E-24. Fit of multistage model to skin tumors in C57L mice exposed
dermally to benzo[a]pyrene fPoel. 1959).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_l959_MultiCanc3_0.1.(d)
Gnuplot Plotting File:
C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_l959_MultiCanc3_0.1.pit
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/N2-beta3*dose/N3) ]
The parameter betas are restricted to be positive
Dependent variable = NumAff
Independent variable = LADD
This document is a draft for review purposes only and does not constitute Agency policy.
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Total number of observations = 5
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
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
Background = 0.0449589
Beta(1) = 0.490451
Beta(2) = 0
Beta(3) = 2.68146
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 )
Background Beta(l) Beta(3)
Background 1 -0.87 0.74
Beta(1) -0.87 1 -0.92
Beta(3) 0.74 -0.92 1
Parameter Estimates
Variable
Background
Beta(1)
Beta(2)
Beta(3)
Estimate
0. 0176699
0.79766
0
2 .17146
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)
-87.1835
-90.4265
-141.614
Param's
5
3
1
Deviance Test d.f.
P-value
5.48606
108.86
0.03905
<.0001
AIC:
186.853
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
0.0177
0.583
0.
000
33
-0.770
0.0500
0.0563
3. 098
5.
000
55
1.112
0.1600
0.1430
7 .866
11.
000
55
1.207
0.2400
0.2128
11.917
7 .
000
56
-1.605
0.8000
0.8293
40.635
41.
000
49
0.139
ho
II
(jn
CD
CO
d.f.
= 2 P
-value
= 0.0528
Benchmark Dose Computation
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
0.1
Extra risk
0. 95
0.126567
0. 0777875
0.272961
Taken together, (0.0777875, 0.272961) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor
1.28555
This document is a draft for review purposes only and does not constitute Agency policy.
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Multistage Cancer Model with 0.95 Confidence Level
BMDL
0 0.5 1 1.5 2 2.5
dose
Multistage Cancer
Linear extrapolation
Figure E-25. Fit of multistage model to skin tumors in female Swiss mice
exposed dermally to benzo[a]pyrene (Roe etal.. 1970).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Roe_l970_Setting.(d)
Gnuplot Plotting File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Roe_l970_Setting.pit
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/N2-beta3*dose/s3-beta4*doseA4-beta5*dose^5 ) ]
The parameter betas are restricted to be positive
Dependent variable = tumors
Independent variable = LADD
Total number of observations = 6
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 0
Degree of polynomial = 5
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
Background = 0
Beta(1) = 0.0962491
This document is a draft for review purposes only and does not constitute Agency policy.
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Beta(2) = 0.141689
Beta(3) = 0
Beta(4) = 0
Beta(5) = 0
Asymptotic Correlation Matrix of Parameter Estimates
Background
Beta(1)
Beta(2)
The model parameter(s) -Beta(3) -Beta(4) -Beta(5)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background
1
-0.57
0.45
Beta(1)
-0.57
1
-0. 94
Beta(2)
0.45
-0. 94
1
Parameter Estimates
Variable
Background
Beta(1)
Beta(2)
Beta(3)
Beta(4)
Beta(5)
Estimate
0. 00584893
0. 0379152
0.166839
0
0
0
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
- Indicates that this value is not calculated.
Analysis of Deviance Table
Param's Deviance Test d.f.
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-56.1835
-57.5694
-118.948
121.139
2 .77176
125.529
P-value
0. 4282
<.0001
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.
0000
0
0058
0.275
0.
000
47
-0.526
0.
0300
0
0071
0.321
1.
000
45
1.204
0.
0900
0
0106
0.444
0.
000
42
-0.670
0.
3100
0
0331
1.423
1.
000
43
-0.361
0.
9200
0
1664
6. 821
8 .
000
41
0. 494
2 .
7600
0
7488
34.444
34 .
000
46
-0.151
^2
= 2.57
d.f.
= 3 P
-value
= 0.4626
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
0.1
Extra risk
0. 95
0. 689131
0.393806
0.952365
Taken together, (0.393806, 0.952365) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor
0.253932
This document is a draft for review purposes only and does not constitute Agency policy.
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
BMDL
BMD
0 0.1 0.2 0.3 0.4 0.5
dose
Figure E-26. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Schmidt et al.. 1973).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973femaleNMRI\2MulSchMS_.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973femaleNMRI\2MulSchMS_.pit
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2 ) ]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 5
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
We are sorry but Relative Function and Parameter Convergence
are currently unavailable in this model. Please keep checking
the web sight for model updates which will eventually
incorporate these convergence criterion. Default values used.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Default Initial Parameter Values
Background = 0
Beta(1) = 0
Beta(2) = 1.11271
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(2)
Beta(2) 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 * * *
Beta(1) 0 * * *
Beta (2) 0.970648 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-70.8903
-72.6831
-118.917
Param's Deviance Test d.f. P-value
5
1 3.58562 4 0.465
1 96.054 4 <.0001
AIC:
147.366
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
0.0000
0. 000
0.
000
100
0
000
0.0100
0.0001
0. 010
0.
000
100
-0
099
0.0600
0.0035
0.349
0.
000
100
-0
592
0.2300
0.0501
5. 005
2 .
000
100
-1
378
0.5700
0.2705
27.048
30.
000
100
0
665
^2 = 2.70
d.f.
= 4
P
-value
= 0.6091
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.329464
BMDL = 0.286624
BMDU = 0.384046
Taken together, (0.286624, 0.384046) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.348889
This document is a draft for review purposes only and does not constitute Agency policy.
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.7
0.6
0.5
0.4
0.3
0.2
1
0
BMD
BMDL
0
0.1
0.2
0.3
0.4
0.5
dose
Figure E-27. Fit of multistage model to skin tumors in female Swiss mice
exposed dermally to benzo[a]pyrene (Schmidt et al.. 1973).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973swissmice\3MulSchMS_.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidt1973swissmice\3MulSchMS_.pit
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/N2-beta3*dose/N3) ]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 5
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
We are sorry but Relative Function and Parameter Convergence
are currently unavailable in this model. Please keep checking
the web sight for model updates which will eventually
incorporate these convergence criterion. Default values used.
This document is a draft for review purposes only and does not constitute Agency policy.
E-94 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Information —Benzo[aJpyren e
Default Initial Parameter Values
Background = 0
Beta(1) = 0
Beta(2) = 0.338951
Beta(3) = 3.8728
Asymptotic Correlation Matrix of Parameter Estimates
( The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(2) Beta(3)
Beta(2) 1 -0.99
Beta(3) -0.99 1
Parameter Estimates
Variable
Background
Beta(1)
Beta(2)
Beta(3)
Estimate
0
0
0.108125
4.31441
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
- Indicates that this value is not calculated.
Analysis of Deviance Table
Deviance Test d.f.
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-73.5285
-73.6628
-150.708
Param's
5
2
1
0.268637
154.359
P-value
0.9658
<.0001
AIC:
151.326
Dose
Est. Prob.
Goodness of Fit
Expected
Observed
Size
Scaled
Residual
0.
0000
0.
0000 0.000
0.
000
80
0. 000
0.
0100
0.
0000 0.001
0.
000
80
-0.035
0.
0600
0.
0013 0.106
0.
000
80
-0.325
0.
2300
0.
0566 4.524
5.
000
80
0.230
0.
5700
0.
5657 45.260
45.
000
80
-0.059
Chi ^2
= 0.16
d.f. = 3 P
-value
= 0.9833
Benchmark
Dose
Computation
Specified effect
Risk Type
Confidence level
0.1
Extra risk
0. 95
BMD
BMDL
BMDU
0.282007
0.223401
0.309888
Taken together, (0.223401, 0.309888) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor
0. 447626
This document is a draft for review purposes only and does not constitute Agency policy.
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BMDL
Multistage Cancer Model with 0.95 Confidence Level
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
dose
Figure E-28. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Schmahl et al.. 1977).
Multistage Cancer
Linear extrapolation
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahl1977 femaleNMRI\2MulschMS_. (d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahl1977 femaleNMRI\2MulschMS_.pit
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/N2 ) ]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
¦*** We are sorry but Relative Function and Parameter Convergence
¦*** are currently unavailable in this model. Please keep checking
This document is a draft for review purposes only and does not constitute Agency policy.
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the web sight for model updates which will eventually
incorporate these convergence criterion. Default values used.
Default Initial Parameter Values
Background = 0.0115034
Beta (1) = 0.284955
Beta ( 2) = 0.750235
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l) Beta(2)
Background
Beta(1)
Beta(2)
1
-0. 67
0.47
-0. 67
1
-0. 94
0.47
-0. 94
1
Parameter Estimates
Variable
Background
Beta(1)
Beta(2)
Estimate
0. 0123066
0.274413
0.764244
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
- Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param'
-145.127 4
-145.13 3
-184.158 1
Deviance Test d.f.
0.00579898
78.0608
P-value
0. 9393
<.0001
AIC:
296.261
Dose
Goodness of Fit
Est. Prob.
Expected
Observed
Size
Scaled
Residual
0.0000
0.2900
0.4900
0.8600
0.0123
0.1446
0.2813
0.5567
0. 997
11.137
24.756
45.096
1. 000
11.000
25.000
45.000
81
77
81
0. 003
-0.045
0. 058
-0.022
Chi/S2
0. 01
d.f.
P-value
0.9393
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
0.1
Extra risk
0. 95
BMD
BMDL
BMDU
0.232893
0.148895
0.320396
Taken together, (0.148895, 0.320396) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor
0.671616
This document is a draft for review purposes only and does not constitute Agency policy.
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Multistage Cancer Model with 0.95 Confidence Level
1
Multistage Cancer
Linear extrapolation
0
BMDL
gMD
0
0.1
0.2
0.3
0.4
dose
0.5
0.6
0.7
0.8
Figure E-29. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Hahs etal.. 1980).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: M:\_BMDS\msc_BAP_HABSl980_MultiCanc3_0.1.(d)
Gnuplot Plotting File: M:\_BMDS\msc_BAP_HABSl980_MultiCanc3_0.1.pit
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
The parameter betas are restricted to be positive
Dependent variable = NumAff
Independent variable = LADD
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
-betal*dose/sl-beta2*dose/s2-beta3*dose/s3) ]
Default Initial Parameter Values
Background
0
0
Beta(1)
Beta(2)
Beta(3)
4.23649
0
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplem en tal Information —Benzo[aJpyren e
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -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 )
Beta(3)
Beta(3) 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 * * *
Beta(1) 0 * * *
Beta(2) 0 * * *
Beta(3) 4.1289 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -34.8527 4
Fitted model -37.3373 1 4.96903 3 0.1741
Reduced model -82.5767 1 95.4478 3 <.0001
AIC: 7 6.6745
Goodness of Fit
Scaled
Dose Est. Prob. Expected Observed Size Residual
0.0000
0.0000
0. 000
0. 000
35
0. 000
0.4900
0.3848
13.082
8 . 000
34
-1.791
0.7400
0.8123
21.933
24.000
27
1. 019
0.8000
0.8792
21.102
22.000
24
0.563
Chi'" 2 = 4.56 d.f. = 3 P-value = 0.2067
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.294407
BMDL = 0.215151
BMDU = 0.320955
Taken together, (0.215151, 0.320955) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.46479
This document is a draft for review purposes only and does not constitute Agency policy.
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Multistage Cancer Model with 0.95 Confidence Level
1
0.8
0
1
0.6
0.4
0.2
0
Multistage Cancer
Linear extrapolation
BMDL BMD
0 0.2 0.4 0.6 0.8 1
dose
Figure E-30. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Habs et al.. 1984).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\Usepa\BMDS21\mscDax_Setting.(d)
Gnuplot Plotting File: C:\Usepa\BMDS21\mscDax_Setting.plt
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l)]
The parameter betas are restricted to be positive
Dependent variable = tumors
Independent variable = LADD
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
This document is a draft for review purposes only and does not constitute Agency policy.
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Default Initial Parameter Values
Background = 0
Beta(1) = 1.66414
Asymptotic Correlation Matrix of Parameter Estimates
( The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(1)
Beta(1) 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 * * *
Beta(1) 1.35264 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -22.217 3
Fitted model -22.7878 1 1.14175 2 0.565
Reduced model -41.0539 1 37.6739 2 <.0001
AIC: 47.5757
Goodness of Fit
Scaled
Dose Est. Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 20 0.000
0.5700 0.5375 10.749 9.000 20 -0.784
1.1400 0.7860 15.721 17.000 20 0.697
Chi ^2 = 1.10 d.f. = 2 P-value = 0.5765
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.0778926
BMDL = 0.0558466
BMDU = 0.111853
Taken together, (0.0558466, 0.111853) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 1.79062
This document is a draft for review purposes only and does not constitute Agency policy.
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J3MDL
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
dose
Figure E-31. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al.. 1983).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmer1983CFLPmice\lMulGriMS_.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmer1983CFLPmice\lMulGriMS_.pit
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
¦*** We are sorry but Relative Function and Parameter Convergence
¦*** are currently unavailable in this model. Please keep checking
This document is a draft for review purposes only and does not constitute Agency policy.
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the web sight for model updates which will eventually
incorporate these convergence criterion. Default values used.
Default Initial Parameter Values
Background = 0
Beta(1) = 0.478645
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(1)
Beta(1) 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 * * *
Beta(1) 0.430366 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -108.532 4
Fitted model -108.943 1 0.823537 3 0.8438
Reduced model -186.434 1 155.805 3 <.0001
AIC: 219.887
Goodness of Fit
Scaled
Dose Est. Prob. Expected Observed Size Residual
0.0000
0.0000
0. 000
0. 000
80
-0.000
1.1100
0.3798
24.687
22.000
65
-0.687
2 .2000
0.6120
39.169
39.000
64
-0.043
4 .4000
0.8495
54.366
56.000
64
0.571
Chi/S2 = 0.80 d.f. = 3 P-value = 0.84 96
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.244816
BMDL = 0.208269
BMDU = 0.289606
Taken together, (0.208269, 0.289606) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.480148
This document is a draft for review purposes only and does not constitute Agency policy.
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"O
(D
O
(D
<
C
o
o
ro
ll
Figure E-32. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al.. 1984).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmerl984_MultiCancl_0.1.(d)
Gnuplot Plotting File:
C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Grimmerl984_MultiCancl_0.1.pit
Wed Apr 27 17:11:28 2011
[add notes here]
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl)]
The parameter betas are restricted to be positive
Dependent variable = NumAff
Independent variable = LADD
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
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
Background = 0.311241
Beta(1) = 0.502556
Asymptotic Correlation Matrix of Parameter Estimates
Multistage Cancer Model with 0.95 Confidence Level
1 » Multistage Cancer
Linear extrapolation
qjVIDL, pMD . ^^ .
0 0.5 1 1.5 2 2.5 3 3.5 4
dose
This document is a draft for review purposes only and does not constitute Agency policy.
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( *** xhe model parameter(s) -Background
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(1)
Beta(1) 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 * * *
Beta(1) 0.796546 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-95.8385
-101.643
-175.237
# Param's
4
1
1
Deviance Test d.f.
11. 61
158.797
P-value
0.008846
<.0001
AIC:
205.287
Dose
Est. Prob.
Goodness of Fit
Expected
Observed
Size
Scaled
Residual
0000
9700
9100
9000
0000
5382
7816
9552
0.000
34.446
50.804
62.091
0.000
43.000
53.000
57.000
65
64
65
65
0.000
2.145
0.659
-3.054
ChiA2
14.36
d.f.
P-value
0.0025
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.132272
BMDL = 0.113427
BMDU = 0.154848
Taken together, (0.113427, 0.154848) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.881621
This document is a draft for review purposes only and does not constitute Agency policy.
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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMDL
BMD
1.5
2
2.5
3
3.5
4
0
0.5
1
dose
Figure E-33. Fit of log-logistic model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al.. 1984).
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File:
C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmerl984_Grimmerl984_0.7 Ou. (d)
Gnuplot Plotting File:
C:\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmerl984_Grimmerl984_0.7 Ou.pit
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]
Dependent variable = NumAff
Independent variable = LADD
Slope parameter is not restricted
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 = 0.799142
slope = 0.8 9412 9
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter (s) -background
This document is a draft for review purposes only and does not constitute Agency policy.
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have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
intercept slope
intercept 1 -0.68
slope -0.68 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
background 0 * * *
intercept 0.783559 * * *
slope 0.922655 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -95.8385 4
Fitted model -95.9236 2 0.17031 2 0.9184
Reduced model -175.237 1 158.797 3 <.0001
AIC: 195.847
Goodness of Fit
Scaled
Dose Est. Prob. Expected Observed Size Residual
0.0000
0.0000
0. 000
0. 000
65
0. 000
0.9700
0.6804
43.543
43.000
64
-0.146
1.9100
0.7991
51.941
53.000
65
0.328
3.9000
0.8849
57.516
57.000
65
-0.200
Chi/S2 = 0.17 d.f. = 2 P-value = 0.9190
Benchmark Dose Computation
Specified effect = 0.7
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.07152
BMDL = 0.478669
This document is a draft for review purposes only and does not constitute Agency policy.
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Muitistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.6
0.4
0.2
0
BMDL
BMD
0
0.5
1
1.5
2
dose
Figure E-34. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene fGrimmer et al.. 19841. highest dose
dropped.
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File: C:/Usepa/_BaP/msc_BaP_Grimmerl984_drophidose_MultiCancl_0.7.(d)
Gnuplot Plotting File: C:/Usepa/_BaP/msc_BaP_Grimmerl984_drophidose_MultiCancl_0.7.pit
[add_notes_here]
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l)]
The parameter betas are restricted to be positive
Dependent variable = NumAff
Independent variable = LADD
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
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
Background = 0.0806622
Beta(1) = 0.88595
Asymptotic Correlation Matrix of Parameter Estimates
This document is a draft for review purposes only and does not constitute Agency policy.
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The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(1)
Beta(1) 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 * * *
Beta(1) 0.997117 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -71.5928 3
Fitted model -72.2756 1 1.36568 2 0.5052
Reduced model -134.46 1 125.735 2 <.0001
AIC: 146.551
Goodness of Fit
Scaled
Dose Est. Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 65 0.000
0.9700 0.6199 39.671 43.000 64 0.857
1.9100 0.8511 55.322 53.000 65 -0.809
Chi/S2 = 1.39 d.f. = 2 P-value = 0.4 992
Benchmark Dose Computation
Specified effect = 0.7
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.20745
BMDL = 1 . 00734
BMDU = 1 . 457 8 9
Taken together, (1.00734, 1.45789) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.6949
This document is a draft for review purposes only and does not constitute Agency policy.
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Multistage Cancer Model with 0.95 Confidence Level
1
0.8
0.6
0.4
0.2
0
Multistage Cancer
Linear extrapolation
BMDL
BMD
0 0.1 0.2 0.3 0.4 0.5
dose
Figure E-35. Fit of multistage model to skin tumors in male CeH/HeJ mice
exposed dermally to benzo[a]pyrene (Sivak etal.. 1997).
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivakl993_MultiCanc2_0.1.(d)
Gnuplot Plotting File:
C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivakl993_MultiCanc2_0.1.pit
[add notes here]
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/N2 ) ]
The parameter betas are restricted to be positive
Dependent variable = NumAff
Independent variable = LADD
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
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
Background = 0
Beta(1) = 0.0936505
Beta(2) = 8.67239
This document is a draft for review purposes only and does not constitute Agency policy.
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Asymptotic Correlation Matrix of Parameter Estimates
( The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(2)
Beta(2) 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 * * *
Beta(1) 0 * * *
Beta(2) 8.9375 * * *
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -23.2693 4
Fitted model -23.3009 1 0.0631003 3 0.9959
Reduced model -69.5898 1 92.641 3 <.0001
AIC: 48.6018
Goodness of Fit
Scaled
Dose Est. Prob. Expected Observed Size Residual
0.0000
0.0000
0. 000
0. 000
30
0. 000
0.0100
0.0009
0. 027
0. 000
30
-0.164
0.1400
0.1607
4 . 821
5. 000
30
0. 089
0.5100
0.9022
27.065
27.000
30
-0.040
Chi'A2 = 0.04 d.f. = 3 P-value = 0.9982
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.108575
BMDL = 0.058484
BMDU = 0.129641
Taken together, (0.058484, 0.129641) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 1.70987
This document is a draft for review purposes only and does not constitute Agency policy.
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Alternative Approaches for Cross-Species Scaling of the Dermal Slope Factor
Several publications that develop a dermal slope factor for benzo[a]pyrene are available in
the peer-reviewed literature fKnafla etal.. 2011: Knafla etal.. 2006: Hussain etal.. 1998: Lagov and
Quirk. 1994: Sullivan etal.. 19911. With the exception of Knafla etal. f20111. none of these
approaches applied quantitative adjustments to account for interspecies differences, although the
proposed slope factors were developed to account for human risk. Knafla etal. f20111 qualitatively
discuss processes that could affect the extrapolation between mice and humans, including skin
metabolic activity adduct formation, stratum corneum thickness, epidermal thickness, etc.
Ultimately, the authors apply an adjustment based on the increased epidermal thickness of human
skin on the arms and hands compared to mouse interscapular epidermal thickness. They
hypothesize that the carcinogenic potential of benzo[a]pyrene may be related to the thickness of
the epidermal layer.
Because there is no established methodology for cross-species extrapolation of dermal
toxicity, several alternative approaches were evaluated. Each approach begins with the POD of
0.066 [ig/day that was based on a 10% extra risk for skin tumors in male mice. Based on the
assumptions of each approach, a dermal slope factor for humans is calculated. The discussion of
these approaches uses the following abbreviations:
DSF = dermal slope factor
PODm = point of departure (for 10% extra risk) from mouse bioassay, in [ig/day
BWm = mouse body weight = 0.035 kg (assumed)
BWh = human body weight = 70 kg (assumed)
SAh = total human surface area = 19,000 cm2 (assumed)
SAm = total mouse surface area =100 cm2 (assumed)
Approach 1. No Interspecies Adjustment to Daily Applied Dose (POD) in Mouse Model
Under this approach, a given mass of benzo[a]pyrene, applied daily, would pose the same
risk in an animal or in humans, regardless of whether it is applied to a small surface area or to a
larger surface area at a proportionately lower concentration.
DSF = 0.1/P0Dm
DSF = 0.1/0.060 [ig/day = 1.7 (ng/day)i
Assumptions: The same mass of benzo[a]pyrene, applied daily, would have same potency in
mice as in human skin regardless of treatment area.
Approach 2. Cross-Species Adjustment Based on Whole-Body Surface-Area Scaling
Under this approach, animals and humans are assumed to have equal lifetime cancer risk
with equal average whole-body exposures in loading units ([ig/cm2-day). As long as doses are low
This document is a draft for review purposes only and does not constitute Agency policy.
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enough that risk is proportional to the mass of applied compound, the daily dermal dose of
benzo[a]pyrene can be normalized over the total surface area.
POD (|ig/cm2-day) = P0DM/sa (|ig/cm2-day) = P0DM (|ig/day) / SAM (cm2)
POD = (0.060 [ig/day) / 100 cm2 = 0.00060 [ig/cm2-day
DSF = 0.1/(0.00060 |ig/cm2-day) « 170 (ng/cm2-day)-1
Assumptions: Mouse and human slope factors are equipotent if total dermal dose is
averaged over equal fractions of the entire surface area. Tumor potency of benzo[a]pyrene is
assumed to be related to overall dose and not dose per unit area. For example, a human exposed to
0.01 [ig/day on 10 cm2 would be assumed to have the same potential to form a skin tumor as
someone treated with 0.01 [ig/day over 19,000 cm2 (assumed human surface area).
Approach 3. Cross-Species Adjustment Based on Body Weight
Under this approach, a given mass of benzo[a]pyrene is normalized relative to the body
weight of the animal or human.
PODm/ BWm= 0.060 [ig/0.035 kg-day = 1.7 [ig/kg-day
DSF= 0.1/1.7 ng/kg-day = 0.058 (ng/kg-day)_1
Assumptions: The potency of point of contact skin tumors is related to body weight, and
humans and mice would have an equal likelihood of developing skin tumors based on a dermal dose
per kg basis.
Issues: Skin cancer following benzo[a]pyrene exposure is a local effect and not likely
dependent on body weight.
Approach 4. Cross-Species Adjustment Based on Allometric Scaling Using Body Weight to the
3/4 Power
Under this approach, rodents and humans exposed to the same daily dose of a carcinogen,
adjusted for BW3/4, would be expected to have equal lifetime risks of cancer. That is, a lifetime dose
expressed as [ig/kg3/4-day would lead to an equal risk in rodents and humans. This scaling reflects
the empirically observed phenomena of more rapid distribution, metabolism, and clearance in
smaller animals. The metabolism of benzo[a]pyrene to reactive intermediates is a critical step in
the carcinogenicity of benzo[a]pyrene, and this metabolism occurs in the skin.
POD (jig/day) = P0DM (|ig/day) x (BWH / BWM)3/4
POD ([ig/day) = 0.060 [ig/day x (70 kg / 0.035 kg)3/4 = 17.9 [ig/day
DSF = 0.1/(17.9 [ig/day) * 0.0056 (ng/day)"1
Assumptions: Risk at low doses of benzo[a]pyrene is dependent on absolute dermal dose
and not dose per unit of skin, meaning that a higher exposure concentration of benzo[a]pyrene
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contacting a smaller area of exposed skin could carry the same risk of skin tumors as a lower
exposure concentration of benzo[a]pyrene that contacts a larger area of skin.
Issues: It is unclear if scaling of doses based on body weight ratios will correspond to
differences in metabolic processes in the skin of mice and humans.
Synthesis of the Alternative Approaches to Cross-Species Scaling
A comparison of the above approaches is provided in Table E-25. The lifetime risk from a
nominal human dermal exposure to benzo[a]pyrene over a 5% area of exposed skin (approximately
950 cm2), estimated at 1 x 10"4 [ig/day, is calculated for each of the approaches in order to judge
whether the method yields risk estimates that are unrealistically high.
Other Potential Interspecies Adjustments
The above discussion presents several mathematical approaches that result from varying
assumptions about what is the relevant dose metric for determining equivalence across species.
Biological information (that is not presently comprehensive or detailed enough to develop robust
models) that could be used in future biologically based models for cross-species extrapolation
include:
a. Quantitative information on interspecies differences in partitioning from exposure medium
to the skin and absorption through the skin
b. Thickness of the stratum corneum between anatomical sites and between species
c. Thickness of epidermal layer
d. Skin permeability
e. Metabolic activity of skin
f. Formation of DNA adducts in skin
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Table E-25. Alternative approaches to cross-species scaling
Approach
Assumptions
Dose metric
Dermal
slope factor
Risk at nominal
exposure
(0.0004 pg/day)a
1. Mass-per-
day scaling (no
adjustment)
Equal mass per day (ng/d), if applied to equal areas of skin (cm2), will affect similar
numbers of cells across species. Cancer risk is proportional to the area (cm2)
exposed if the loading rate (ng/cm2-d) is the same. This approach assumes that risk
is proportional to dose expressed as mass per day. This approach implies that any
combination of loading rate (ng/cm2-day) and skin area exposed (cm2) that have the
same product when multiplied will result in the same risk.
Hg/day
1.7 per ng/d
7 x 10"4
2. Surface-
area scaling
Eaual mass per dav (ue/d), if applied to eaual fractions of total skin surface (cm2) will
have similar cancer risks. That is, lifetime exposure normalized over the whole body
(e.g., 5%-of-the-body lifetime exposure) at the same loading rate (ng/cm2-d) gives
similar cancer risks across species. This approach assumes that risk is proportional to
dose expressed as mass per area per day. This approach implies that risk does not
increase with area exposed as long as dose per area remains constant.
Hg/cm2-day
170 per
Hg/cm2-d
4 x 10"6
3. Body-
weight scaling
The skin is an organ with thickness and volume; benzo[a]pyrene is distributed within
this volume of skin. Cancer risk is proportional to the concentration of
benzo[a]pyrene in the exposed volume of skin. Equal mass per day (ng/d), if
distributed within equal fractions of total body skin will have similar cancer risks.
That is, whole-body lifetime exposure (e.g., 5%-of-the-body lifetime exposure) at the
same loading rate (ng/cm2-d) gives similar cancer risks across species. This approach
assumes that risk is proportional to dose expressed as mass per kg body weight per
day. This approach implies that any combination of dose (ng/d) and body weight
(kg) that have the same result when divided will result in the same risk.
Hg/kg-day
0.058 per
Hg/kg-d
3 x 10"7
4. Allometric
scaling (BW3/4)
Same as for body-weight scaling, except that benzo[a]pyrene distribution and
metabolism takes place within this volume of skin. Allometric scaling is generally
regarded as describing the relative rate of toxicokinetic processes across species.
This approach also is used by EPA to scale oral exposures.
Hg/day
0.0056 per
Mg/d
2 x 10"6
Calculated as a central tendency exposure using an average benzo[a]pyrene soil concentration of 100 ppb, rounded to one significant figure (see Appendix A,
Table A-4) and standard exposure assumptions from U.S. EPA (2004).
3
a
re
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APPENDIX F. DOCUMENTATION OF
IMPLEMENTATION OF THE 2011 NATIONAL
RESEARCH COUNCIL RECOMMENDATIONS
Background: On December 23, 2011, The Consolidated Appropriations Act, 2012, was
signed into law3. The report language included direction to the U.S. Environmental Protection
Agency (EPA) for the Integrated Risk Information System (IRIS) Program related to
recommendations provided by the National Research Council (NRC) in their review of EPA's draft
IRIS assessment of formaldehyde4. The report language included the following:
The Agency shall incorporate, as appropriate, based on chemical-specific datasets
and biological effects, the recommendations of Chapter 7 of the National Research
Council's Review of the Environmental Protection Agency's Draft IRIS Assessment of
Formaldehyde into the IRIS process... For draft assessments released in fiscal year
2012, the Agency shall include documentation describing how the Chapter 7
recommendations of the National Academy of Sciences (NAS) have been
implemented or addressed, including an explanation for why certain
recommendations were not incorporated.
The NRC's recommendations, provided in Chapter 7 of their review report, offered
suggestions to EPA for improving the development of IRIS assessments. Consistent with the
direction provided by Congress, documentation of how the recommendations from Chapter 7 of the
NRC report have been implemented in this assessment is provided in the table below. Where
necessary, the documentation includes an explanation for why certain recommendations were not
incorporated.
The IRIS Program's implementation of the NRC recommendations is following a phased
approach that is consistent with the NRC's "Roadmap for Revision" as described in Chapter 7 of the
formaldehyde review report. The NRC stated that "the committee recognizes that the changes
suggested would involve a multi-year process and extensive effort by the staff at the National
Center for Environmental Assessment and input and review by the EPA Science Advisory Board and
others."
Phase 1 of implementation has focused on a subset of the short-term recommendations,
such as editing and streamlining documents, increasing transparency and clarity, and using more
tables, figures, and appendices to present information and data in assessments. Phase 1 also
3Pub. L. No. 112-74, Consolidated Appropriations Act, 2012.
4f ENREF 3511. Review of the Environmental Protection Agency's Draft IRIS Assessment of Formaldehyde.
This document is a draft for review purposes only and does not constitute Agency policy.
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focused on assessments near the end of the development process and close to final posting. The
IRIS benzo[a]pyrene assessment is in Phase 2 and represents a significant advancement in
implementing the NRC recommendations shown in Table F-l below. The Program is implementing
all of these recommendations, but recognizes that achieving full and robust implementation of
certain recommendations will be an evolving process with input and feedback from the public,
stakeholders, and external peer review committees. Phase 3 of implementation will incorporate
the longer-term recommendations made by the NRC as outlined below in Table F-2, including the
development of a standardized approach to describe the strength of evidence for noncancer effects.
In May 2014, the NRC released their report reviewing the IRIS assessment development process.
As part of this review, the NRC reviewed current methods for evidence-based reviews and made
several recommendations with respect to integrating scientific evidence for chemical hazard and
dose-response assessments. In their report, the NRC states that EPA should continue to improve its
evidence-integration process incrementally and enhance the transparency of its process. The
committee did not offer a preference but suggests that EPA consider which approach best fits its
plans for the IRIS process. The NRC recommendations will inform the IRIS Program's efforts in this
area going forward.
Table F-l. The EPA's implementation of the National Research Council's
recommendations in the benzo[a]pyrene assessment
National Research Council recommendations
that EPA is implementing in the short term
Implementation status
General recommendations for completing the IRIS formaldehyde assessment that EPA will adopt for all IRIS
assessments (p. 152 of the NRC report)
1. To enhance the clarity of the document, the draft
IRIS assessment needs rigorous editing to reduce the
volume of text substantially and address
redundancies and inconsistencies. Long descriptions
of particular studies should be replaced with
informative evidence tables. When study details are
appropriate, they could be provided in appendices.
Implemented. The overall document structure has been
revised in consideration of this NRC recommendation.
The new structure includes a concise Executive Summary
and an explanation of the literature review search
strategy, study selection criteria, and methods used to
develop the assessment. The main body of the
assessment has been reorganized into two sections,
Hazard Identification and Dose-Response Analysis, to
help reduce the volume of text and redundancies that
were a part of the previous document structure.
Section 1 provides evidence tables and a concise
synthesis of hazard information organized by health
effect. The Supplemental Information provides more
detailed summaries of the most pertinent epidemiology
and experimental animal studies (Appendix D), as well as
information on chemical and physical properties and
toxicokinetics (Appendix D). The main text of the
Toxicological Review is approximately 130 pages, which is
a major reduction from previous IRIS assessments.
Technical and scientific edits were performed to
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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
eliminate any redundancies or inconsistencies.
2. Chapter 1 needs to be expanded to describe more
fully the methods of the assessment, including a
description of search strategies used to identify
studies with the exclusion and inclusion criteria
articulated and a better description of the outcomes
of the searches and clear descriptions of the weight-
of-evidence approaches used for the various
noncancer outcomes. The committee emphasizes
that it is not recommending the addition of long
descriptions of EPA guidelines to the introduction, but
rather clear concise statements of criteria used to
exclude, include, and advance studies for derivation
of the reference concentrations (RfCs) and unit risk
estimates.
Implemented. Chapter 1 has been replaced with a
Preamble that describes the application of existing EPA
guidance and the methods and criteria used in
developing the assessment. The term "Preamble" was
chosen to emphasize that these methods and criteria are
being applied consistently across IRIS assessments. The
new Preamble includes information on identifying and
selecting pertinent studies, evaluating the quality of
individual studies, weighing the overall evidence of each
effect, selecting studies for derivation of toxicity values,
and deriving toxicity values. These topics correspond
directly to the five steps that the NRC identified in
Figure 7-2 of their 2011 report.
A new section, Literature Search Strategy and Study
Selection, provides detailed information on the search
strategy used to identify health effect studies, search
outcomes, and selection of studies for hazard
identification. This information is chemical-specific and
has been designed to provide enough information that an
independent literature search would be able to replicate
the results. This section also includes information on
how studies were selected to be included in the
document and provides a link to EPA's Health and
Environmental Research Online (HERO) database
(www.epa.gov/hero) that contains the references that
were cited in the document, along with those that were
considered but not cited.
3. Standardized evidence tables for all health
outcomes need to be developed. If there were
appropriates tables, long text descriptions of studies
could be moved to an appendix.
Implemented. In the new document template,
standardized evidence tables that present key study
findings that support how toxicological hazards are
identified for all major health effects are provided in
Section 1.1. More detailed summaries of the most
pertinent epidemiology and experimental animal studies
are provided in the Supplemental Information
(Appendix D).
4. All critical studies need to be thoroughly evaluated
with standardized approaches that are clearly
formulated and based on the type of research, for
example, observational epidemiologic or animal
bioassays. The findings of the reviews might be
presented in tables to ensure transparency.
Partially Implemented. Information in Section 4 of the
Preamble provides an overview of the approach used to
evaluate the quality of individual studies. Critical
evaluation of the epidemiologic and experimental animal
studies is included in the evidence tables in Section 1.1.
As more rigorous systematic review processes are
developed, they will be utilized in future assessments.
5. The rationales for the selection of the studies that
are advanced for consideration in calculating the RfCs
and unit risks need to be expanded. All candidate
Implemented. The Dose-Response Analysis section of
the new document structure provides a clear explanation
of the rationale used to select and advance studies that
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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
RfCs should be evaluated together with the aid of
graphic displays that incorporate selected information
on attributes relevant to the database.
were considered for calculating toxicity values.
Rationales for the selection of studies advanced for
reference value derivation are informed by the weight-of-
evidence for hazard identification as discussed in Section
1.2. In support of the derivation of reference values for
benzo[a]pyrene, exposure-response arrays are included
that compare effect levels for several toxicological effects
(Section 2.1, Figure 2-1; Section 2.2, Figure 2.2). The
exposure-response array provides a visual representation
of points of departure (PODs) for various effects resulting
from exposure to benzo[a]pyrene. The array informs the
identification of doses associated with specific effects,
and the choice of principal study and critical effects. In
the case of the benzo[a]pyrene RfD, the database
supported the development of multiple organ/system-
specific RfDs. Such values have been developed
previously on a case-by-case basis and will be developed
in future assessments, where the data allow.
6. Strengthened, more integrative, and more
transparent discussions of weight-of-evidence are
needed. The discussions would benefit from more
rigorous and systematic coverage of the various
determinants of weight-of-evidence, such as
consistency.
Partially implemented. The new Hazard Identification
(Section 1) provides a more strengthened, integrated,
and transparent discussion of the weight of the available
evidence. This section includes standardized evidence
tables to present the key study findings that support how
potential toxicological hazards are identified and
exposure-response arrays for each potential toxicological
effect. Summary discussions are provided as a statement
of hazard for each major effect (Section 1.1.1—
developmental toxicity, Section 1.1.2—reproductive
toxicity, Section 1.1.3—immunotoxicity, Section 1.1.4—
other toxicological effects, and Section 1.1.5 —
carcinogenicity) as well as a general weight-of-evidence
discussion for effects other than cancer (Section 1.2.1)
and cancer (Section 1.2.2). A more rigorous and
formalized approach for characterizing the weight-of-
evidence will be developed as a part of Phase 3 of the
implementation process.
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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
Other specific recommendations (p. # in NRC report)
General Guidance for the Overall Process (p. 164)
7. Elaborate an overall, documented, and quality-
controlled process for IRIS assessments.
Implemented. EPA has created Chemical Assessment
Support Teams to formalize an internal process to
provide additional overall quality control for the
8. Ensure standardization of review and evaluation
approaches among contributors and teams of
contributors; for example, include standard
approaches for reviews of various types of studies to
ensure uniformity.
development of IRIS assessments. This initiative uses a
team approach to making timely, consistent decisions
about the development of IRIS assessments across the
Program. This team approach has been utilized for the
development of the benzo[a]pyrene assessment.
Additional objectives of the teams are to help ensure
that the necessary disciplinary expertise is available for
assessment development and review, to provide a forum
for identifying and addressing key issues prior to external
peer review, and to monitor progress in implementing
the NRC recommendations.
9. Assess disciplinary structure of teams needed to
conduct the assessments.
Evidence Identification: Literature Collection and
Collation Phase (p. 164)
10. Select outcomes on the basis of available evidence
and understanding of mode of action.
Partially Implemented. A new section, Literature Search
Strategy and Study Selection, contains detailed
information on the search strategy used for the
benzo[a]pyrene assessment, including key words used to
identify relevant health effect studies. Figure LS-1 depicts
the study selection strategy and the number of
references obtained at each stage of literature screening.
This section also includes information on how studies
were selected to be included in the document and
provides a link to an external database
(www.eDa.gov/hero) that contains the references that
were cited in the document, along with those that were
considered but not cited. Each citation in the
Toxicological Review is linked to HERO such that the
public can access the references and abstracts to the
scientific studies used in the assessment. The
implementation of these NRC recommendations in the
benzo[a]pyrene assessment represents a major advance
in the standardization and transparency of evidence
identification. Section 3 of the Preamble summarizes the
standard protocols for evidence identification that are
provided in EPA guidance. For each potential
toxicological effect identified for benzo[a]pyrene, the
available evidence is informed by the mode-of-action
information as discussed in Section 1.1. As more rigorous
systematic review processes are developed, they will be
utilized in future assessments.
11. Establish standard protocols for evidence
identification.
12. Develop a template for description of the search
approach.
13. Use a database, such as the HERO database, to
capture study information and relevant quantitative
data.
Evidence Evaluation: Hazard Identification and Dose-
Response Modeling (p. 165)
14. Standardize the presentation of reviewed studies
in tabular or graphic form to capture the key
dimensions of study characteristics, weight-of-
Implemented. Standardized tables have been developed
that provide summaries of key study design information
and results by health effect. The inclusion of all positive
and negative findings in each health effect-specific
evidence table supports a weight-of-evidence analysis. In
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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
evidence, and utility as a basis for deriving reference
values and unit risks.
addition, exposure-response arrays are utilized in the
assessment to provide a graphical representation of PODs
for various effects resulting from exposure to
benzo[a]pyrene. The exposure-response arrays inform
the identification of doses associated with specific effects
and the weight-of-evidence for those effects.
15. Develop templates for evidence tables, forest
plots, or other displays.
Implemented. Templates for evidence tables and
exposure-response arrays have been developed and are
utilized in Section 1.1.
16. Establish protocols for review of major types of
studies, such as epidemiologic and bioassay.
Partially Implemented. General principles for reviewing
epidemiologic and experimental animal studies are
described in Section 4 of the Preamble. The development
of standardized protocols for systematic review of
evidence is an ongoing process.
Selection of Studies for Derivation of Reference
Values and Unit Risks (p. 165)
17. Establish clear guidelines for study selection.
Implemented. EPA guidelines for study selection,
including balancing strengths and weaknesses and
weighing human vs. experimental evidence, are
described in the Preamble (Sections 3-6). These
guidelines have been applied in Section 2 of the
benzo[a]pyrene assessment to evaluate the strengths and
weaknesses of individual studies considered for reference
value derivation.
In the case of benzo[a]pyrene, the database did not
support the combination of estimates across studies. In
future assessments, combining estimates across studies
will be routinely considered.
a) Balance strengths and weaknesses.
b) Weigh human vs. experimental evidence.
c) Determine whether combining estimates among
studies is warranted.
Calculation of Reference Values and Unit Risks
(pp. 165-166)
18. Describe and justify assumptions and models
used. This step includes review of dosimetry models
and the implications of the models for uncertainty
factors; determination of appropriate PODs (such as
benchmark dose [BMD], NOAEL, and LOAEL), and
assessment of the analyses that underlie the PODs.
Implemented. The rationale for the selection of the
PODs for the organ/system-specific oral reference values
is provided in Section 2.1. The rationale for the selection
of the POD and the inhalation dosimetry modeling (for
the approximation of a HEC) for the derivation of the
inhalation reference value is transparently described in
Section 2.2. The BMD modeling for candidate reference
values is transparently described in the Supplemental
Information (Appendix E).
19. Provide explanation of the risk-estimation
modeling processes (for example, a statistical or
biologic model fit to the data) that are used to
develop a unit risk estimate.
Implemented. The risk-estimation modeling processes
used to develop cancer risk estimates for benzo[a]pyrene
are described in Section 2 of the Toxicological Review and
in the Supplemental Information (Appendix E).
20. Provide adequate documentation for conclusions
and estimation of reference values and unit risks. As
noted by the committee throughout the present
report, sufficient support for conclusions in the
formaldehyde draft IRIS assessment is often lacking.
Given that the development of specific IRIS
Implemented. The new template structure that has been
developed in response to the NRC recommendations
provides a clear explanation of the literature search
strategy, study selection criteria, and methods used to
develop the benzo[a]pyrene assessment. It provides for a
clear description of the decisions made in developing the
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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
assessments and their conclusions are of interest to
many stakeholders, it is important that they provide
sufficient references and supporting documentation
for their conclusions. Detailed appendixes, which
might be made available only electronically, should be
provided when appropriate.
hazard identification and dose-response analysis.
Information contained in the Preamble and throughout
the document reflects the guidance that has been utilized
in developing the assessment. As recommended,
supplementary information is provided in the
accompanying appendices. Detailed modeling analyses
are presented in the appendices.
Table F-2. National Research Council recommendations that the EPA is
generally implementing in the long term
National Research Council recommendations
that EPA is generally implementing in the long-
term (p. # in NRC report)
Implementation status
Weight-of-Evidence Evaluation: Synthesis of
Evidence for Hazard Identification (p. 165)
1. Review use of existing weight-of-evidence
guidelines.
2. Standardize approach to using weight-of-evidence
guidelines.
3. Conduct agency workshops on approaches to
implementing weight-of-evidence guidelines.
4. Develop uniform language to describe strength of
evidence on noncancer effects.
5. Expand and harmonize the approach for
characterizing uncertainty and variability.
6. To the extent possible, unify consideration of
outcomes around common modes of action rather
than considering multiple outcomes separately.
As indicated above, Phase 3 of EPA's implementation plan
will incorporate the longer-term recommendations made
by the NRC, including the development of a standardized
approach to describe the strength of evidence for
noncancer effects. On May 16, 2012, EPA announced6 that
as a part of a review of the IRIS Program's assessment
development process, the NRC will also review current
methods for weight-of-evidence analyses and recommend
approaches for weighing scientific evidence for chemical
hazard identification. In addition, EPA will hold a
workshop on August 26, 2013 on issues related to weight-
of-evidence.
Calculation of Reference Values and Unit Risks
(pp. 165-166)
7. Assess the sensitivity of derived estimates to model
assumptions and endpoints selected. This step should
include appropriate tabular and graphic displays to
illustrate the range of the estimates and the effect of
uncertainty factors on the estimates.
Multiple, endpoint-specific reference values were derived
for benzo[a]pyrene (RfD: Table 2-3 and Figure 2-1; RfC:
Table 2-5 and Figure 2-2) and demonstrate the sensitivity
of the overall reference values depending on the selection
of the overall endpoints.
6EPA Announces NAS' Review of IRIS Assessment Development Process (www.epa.gov/iris).
This document is a draft for review purposes only and does not constitute Agency policy.
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APPENDIX G. SUMMARY OF EXTERNAL PEER
REVIEW AND PUBLIC COMMENTS AND EPA'S
DISPOSITION
EPA's Response to Selected Major Public Comments on the Public Comment Draft
(August 2013) of the IRIS Toxicological Review of Benzo[a]pyrene
Purpose: The Integrated Risk Information System (IRIS) assessment development process of May
2009, includes release of the draft IRIS assessment for public review and comment and
independent expert peer review (Step 4). During this step, EPA holds a public meeting to discuss
the draft assessment and draft peer review charge. As part of enhancements to the IRIS assessment
development process announced in July 2013, in some cases, the IRIS Program may decide to revise
the draft assessment and peer review charge after hearing the public's comments about these
materials. For a complete description of the IRIS process, visit the IRIS website at
www, epa. gov/iris.
The following are EPA's responses to the scientific issues raised in the public comments received on
the draft IRIS Toxicological Review of Benzo[a]pyrene (dated August 2013). The comments have
been synthesized and paraphrased, and are organized to follow the order of the Toxicological
Review. Editorial changes were incorporated in the document as appropriate and are not discussed
further. All public comments provided were taken into consideration in revising the draft
assessment prior to posting for external peer review. The complete set of public comments are
available on the docket at http: //www.regulations.gov (Docket ID No. EPA-HQ-ORD-2011-0391).
Background: The Toxicological Review of Benzo[a]pyrene was released for a 60-day public
comment period on August 21, 2013. The comment period was subsequently extended to 90 days,
ending on November 21, 2013. During this period, public comments on the assessment were
submitted to EPA by the Utility Solid Waste Activities Group (USWAG), CDM Smith Inc, Pavement
Coating Technology Council, Electric Power Research Institute (EPRI), Duke Energy, CH2M Hill,
Gradient, American Chemistry Council (ACC), Agnes Francisco, Melanie Nembhard, and by Arcadis
on behalf of American Coke and Coal Chemicals Institute, American Fuels and Petrochemical
Manufacturers, American Petroleum Institute, Asphalt Institute, Association of American Railroads,
Beazer East, Inc. and Pavement Coatings Technology Council. In addition, a public meeting was
held in December 2013 to provide the public an opportunity to engage in early discussions on the
draft IRIS Toxicological Review of Benzo[a]pyrene and the draft charge to the peer review panel
prior to release for external peer review.
RESOLUTION OF PUBLIC COMMENTS ON THE DRAFT TOXICOLOGICAL REVIEW (dated August
2013)
This document is a draft for review purposes only and does not constitute Agency policy.
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CONSIDERATION OF ADDITIONAL LITERATURE
Comment: Inclusion of supporting data for critical study used to derive the dermal slope factor. The
August 2013 public comment draft derived the dermal slope factor from points of departure from
two lifetime dermal cancer bioassays that were deemed to be of similar quality fSivak etal.. 1997:
Poel. 19591. Gradient identified a report f Arthur D Little. 19891 that presents the original results,
including the individual animal data, for the NIOSH dermal study publication (Sivak etal.. 1997). In
addition, Gradient also provided an additional reference, Clark etal. (20111. for consideration in the
development of the dermal slope factor.
EPA Response: EPA has revised the dose response analysis for the derivation of the dermal slope
factor to incorporate the individual animal survival data identified by the public commenter
(Section 2.5.2 of the Toxicological Review and Appendix E.2.3 of the Supplemental Information).
This allowed EPA to utilize the MultiStage-Weibull model, a model that incorporates dose and the
time at which death with tumor occurred. Use of this model accounts for competing risks
associated with decreased survival times and other causes of death, including other tumors.
Previously, the PODs from the Sivak etal. (1997) and Poel (1959) studies were considered to be of
similar quality and thus were averaged to derive the dermal slope factor. In consideration of these
additional supporting data, EPA selected the NIOSH dermal study fSivak etal.. 1997: Arthur D Little.
19891 as the best available study for dose-response analysis and extrapolation to lifetime cancer
risk following dermal exposure to benzo[a]pyrene.
The publication by Clark etal. f20111. reported results of a lifetime dermal cancer bioassay which
primarily tested polycyclic aromatic hydrocarbon (PAH) mixtures, and included one high dose
group of benzo[a]pyrene as a positive control. EPA considered this study, but did not present Clark
etal. f20111 in the revised assessment due to the availability of several dermal lifetime cancer
bioassays for benzo[a]pyrene with multiple doses which enable greater characterization of the
dose-response relationship, especially in the low dose range.
Comment: Further consideration of human skin graft mouse bioassay studies. Arcadis, EPRI and
CDM Smith recommended increased consideration of studies of PAH exposure in murine models
with human skin xenografts (Atillasov etal.. 1997: Soballe etal.. 1996: Urano etal.. 1995: Graem.
1986). These studies transplanted human skin onto the backs of immunodeficient mice and after
grafts had been established, treated the skin with carcinogens, including PAHs, and mice did not
develop tumors. The commenters stated that these papers demonstrate that human skin is
resistant to the skin tumorigenesis that is seen in mouse skin with benzo[a]pyrene and other PAHs.
EPA Response: Several studies identified by the commenters used the potent carcinogen 7,12-
dimethylbenz(a)anthracene (DMBA) (Soballe etal.. 1996: Urano etal.. 1995: Graem. 1986) and a
single study tested benzo[a]pyrene (Urano etal.. 1995). In the studies using benzo[a]pyrene or
DMBA alone, tumors were identified at the graft border, and judged to be of mouse skin origin, but
no tumors were identified as originating from the human skin graft The ability of this model
system to predict hazard for human skin cancer risk from dermally active procarcinogens is
unclear. Though some studies indicate that the skin grafts maintain some metabolic function (Das
etal.. 19861. it is unclear whether the human skin grafts (some obtained from cadavers) maintain
the same viability, vascularization, and full metabolic capacity as human skin in vivo fKappes etal..
2004). Potent mutagenic carcinogens such as DMBA, methylcholanthrene,
methylnitronitrosoguanidine also fail to produce skin tumors in this model system (Soballe etal..
This document is a draft for review purposes only and does not constitute Agency policy.
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1996: Urano etal.. 1995: Graem. 19861. In addition, the treatment time and the period of
observation was quite short in all identified studies. The mice with the benzo[a]pyrene-treated
human skin grafts in Urano etal. (19951 died within six months after the initial benzo[a]pyrene
treatment In addition, the PAH-treated skin graft mice in Graem f!9861 and Soballe etal. f!9961
were only followed for an average of 4-7 months from the start of treatment. While six months is
generally sufficient for the development of skin tumors in mouse skin (depending on dose level),
human latency for squamous cell carcinoma in PAH-exposed occupational cohorts is thought to be
greater than 20 years (Young etal.. 2012: Voelter-Mahlknecht etal.. 2007: Everall and Dowd. 19781.
Therefore, the single available study fUrano etal.. 19951 evaluating the carcinogenicity of
benzo[a]pyrene in this model system was considered in the Toxicological Review, but was regarded
with low confidence.
Additional text to clarify these points has been added to Section 1.1.5 of the Toxicological Review.
Comment: Inclusion of studies of patients therapeutically treated with coal tar. Arcadis, EPRI, and
CDM Smith suggested the inclusion of epidemiologic studies of skin cancer risk in eczema and
psoriasis patients treated therapeutically with a dermatological formulations containing coal tar (a
PAH mixture). These commenters suggested that the available epidemiological studies of coal tar
treated patients clearly demonstrate that benzo[a]pyrene does not cause skin cancer in humans.
EPA Response: In addition to the comments described above, EPA received comments from the
American Petroleum Institute in March 2013 listing references related to therapeutic coal tar use.
EPA reviewed the references noted in these comments, as well as those identified through
additional review of citations contained in the identified studies and related reviews. Studies that
included a measure of coal tar exposure in relation to a measure of skin cancer risk were
considered and discussed in the Toxicological Review and Supplemental Information drafts dated
August 2013. Specifically, Table D-6 of the Supplemental Information presents a summary of the
methodological features as well as the results of these studies. A discussion of this database was
also included in Section 1.1.5 of the Toxicological Review and Appendix D.3.3 of the Supplemental
Information. Case reports and studies that did not include a measure of coal tar exposure were not
included in the August 2013 draft assessment.
EPA noted considerable limitations to this body of literature, particularly relating to the level of
detail pertaining to exposure measures, length of follow-up, and ability to address effects
attributable to other types of therapies. A single population-based case-control study was
identified fMitropoulos and Norman. 20051: this study examined self-reported use of coal
tar/dandruff shampoo and the association with increased incidence of dermal squamous cell
carcinomas. EPA considered this exposure measure to be highly susceptible to misclassification.
Other epidemiological studies of patients with specific types of skin conditions (e.g., psoriasis,
eczema) were limited by the quality of the exposure data and inability to examine variation in
exposure level (i.e., duration of use), sample size and duration of follow-up, and choice of referent
rates and differences in disease ascertainment between cases and the reference population. In
addition, clinic-based studies focused on the commonly used regimen of coal tar in conjunction with
ultraviolet (UV) B therapy (e.g., the Goeckerman treatment), and could not distinguish effects of
coal tar alone.
EPA does not consider the identified studies to adequately address the question of the potential
association between coal tar treatments and skin cancer due to limitations in design and conduct
This document is a draft for review purposes only and does not constitute Agency policy.
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Thus, EPA disagrees with the commenters' view that these studies demonstrate that
benzo[a]pyrene does not cause skin cancer in humans.
Although EPA does not consider the available studies sufficient to evaluate the potential association
between use of coal tar therapies and risk of skin cancer, acute studies of coal tar treated patients
provide in vivo evidence of benzo[a]pyrene-specific genotoxicity (increased BPDE-DNA adducts) in
human skin (Godschalk etal.. 2001: Rojas etal.. 2001: Zhang etal.. 1990). an early key event in the
carcinogenic mode of action of benzo[a]pyrene (Figure 1-6 of Section 1.1.5 of the Toxicological
Review).
COMMENTS ON THE WEIGHT OF EVIDENCE AND MODE OF ACTION FOR CANCER
Comment: Use of epidemiological studies to support the weight of evidence for cancer. Arcadis and
EPRI commented that the draft benzo[a]pyrene assessment has mischaracterized the evidence
supporting an association between benzo[a]pyrene exposure and lung and skin cancers in humans.
They further state that the human studies that have been presented are studies of worker groups
who were exposed to complex mixtures and it is not possible to attribute the effects of the mixture
to one component
EPA Response: EPA agrees that benzo[a]pyrene exposure in the environment occurs as a complex
mixture with many components including other PAHs, and notes in Section 1.1 of the Toxicological
Review that accordingly, there are few epidemiologic studies designed to investigate the effects of
benzo[a]pyrene, though there are many that have investigated the effects of exposure to PAH
mixtures as a whole. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005) emphasize
the importance of weighing all of the evidence in reaching conclusions about human carcinogenic
potential. Specifically, the guidelines describe that the descriptor of "carcinogenic to humans" can
be used when the following conditions are met: (a) there is strong evidence of an association
between human exposure and either cancer or the key precursor events of the agent's mode of
action but not enough for a causal association, (b) there is extensive evidence of carcinogenicity in
animals, (c) the mode or modes of carcinogenic action and associated key precursor events have
been identified in animals, and (d) there is strong evidence that the key precursor events that
precede the cancer response in animals are anticipated to occur in humans and progress to tumors,
based on available biological information. In Section 1.2.2 and Table 1-18 of the Toxicological
Review the data for benzo[a]pyrene supporting these four conditions are presented.
Extensive evidence of carcinogenicity in animal bioassays along with strong and consistent
mechanistic data for a mutagenic mode of action support conditions (b) and (c) above. Numerous
studies demonstrate the carcinogenicity of benzo[a]pyrene in multiple species by all tested routes
of administration. In addition, mechanistic studies provide strong evidence that links the
metabolism of benzo[a]pyrene to DNA reactive agents with key mutational events in genes that can
lead to tumor development. These events include the formation of specific DNA adducts and
characteristic mutations in oncogenes and tumor suppressor genes.
Several human exposure studies are available supporting both conditions (a) and (d) above.
Specifically, epidemiological studies evaluating exposure to PAH mixtures (both occupational
exposures and tobacco smoke) containing benzo[a]pyrene demonstrate an association with cancer.
Furthermore, multiple studies have reported benzo[a]pyrene-specific DNA adducts as well as
This document is a draft for review purposes only and does not constitute Agency policy.
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characteristic mutations in oncogenes and tumor suppressor genes in humans exposed to PAH
mixtures.
EPA recognizes thatbenzo[a]pyrene is one of many PAHs that could contribute to the observed
increases in cancer in humans exposed to PAH mixtures. However, the combination of strong and
consistent human, animal, and mechanistic evidence for the carcinogenicity of benzo[a]pyrene
provides the basis for characterizing benzo[a]pyrene as "carcinogenic to humans".
COMMENTS ON THE DERIVATION OF THE REFERENCE DOSE AND REFERENCE CONCENTRATION
Comment: Metric used to characterize results in the elevated plus maze. Dose-related differences in
number of open arm entries, increased time spent in the open arms, and decreased closed arm
entries were observed in the study by Chen etal. (2012). The number of entries into the open arms
was used for benchmark dose modeling to extrapolate a POD for the RfD. Arcadis commented that
according to Hogg T1996I the preferred way to express results from the elevated plus maze is as
percentage of open arm entries (among total arm entries) or percentage of time spent in open arms,
to correct for overall changes in exploration and reduce activity-induced artifacts.
EPA Response: EPA agrees that the optimal way to express elevated plus maze data is in relation
to the total number of arm entries or to the total amount of time spent in the arms of the maze. The
reason for presenting the information in such a way is to account for potential differences due to
changes in general locomotor or exploratory behaviors, rather than changes in anxiety (which is the
critical effect). While Chen etal. f20121 did not present the data in this manner (EPA's attempts to
obtain the raw data have been unsuccessful), the authors did present enough information for EPA
to arrive at the conclusion that general locomotor or exploratory behaviors were not affected by
exposure. Specifically, as a measure of total locomotor activity and exploration in the elevated plus
maze test, the total number of arm entries between control and benzo[a]pyrene-treated animals
were calculated from the graphically reported results provided for open and closed arm entries;
total arm entries were unchanged with treatment (e.g., the number of closed arm entries was
decreased with exposure). Therefore, it is unlikely that the results observed were confounded by a
general increase in locomotor activity in the benzo[a]pyrene treated animals.
Two additional studies which tested the effects of benzo[a]pyrene exposure in the elevated plus
maze fBouaved et al.. 2009a: Grova etal.. 20081 reported findings consistent with Chen et al.
f20121. and similarly did not observe an increase in general locomotor activity with treatment
Although they tested higher doses of benzo[a]pyrene than Chen etal. (2012). both Bouaved et al.
(2009a) and Grova etal. (2008) observed statistically significant effects of exposure on percentage
of open arm entries and no effects on the total number of arm entries, the former following oral
exposure during lactation and the latter following i.p. injection in adulthood. See Section 1.1.1 and
Section 2.1.1 for discussion of the consistency of the decreased anxiety-like effects in the evidence
database.
Although EPA concluded that the elevated plus maze data presented by Chen etal. (2012) is
appropriate for use in dose-response modeling, EPA also conducted a sensitivity analysis using the
metric proposed by Hogg f!9961. expressing the data as a fraction of the open arm entries over the
total arm entries. The data for this endpoint were only available as group means and standard
deviations for the open and closed arm entries (separately), presented graphically; the group
means and standard deviations for total arm entries were not reported, and would ideally be
This document is a draft for review purposes only and does not constitute Agency policy.
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calculated from individual animal data. However, it was feasible to derive inputs for dose-response
analysis by summing the group means for open and closed arm entries to yield total arm entries,
and by using Monte Carlo simulations (assuming a normal distribution) to estimate the variance of
the ratio of open arm entries to total arm entries. The resulting BMD and BMDL for a one standard
deviation decrease in mean percentage of open arm entries among total entries were 0.09 and 0.05
mg/kg-day, respectively. Compared to the BMD and BMDL (POD) of 0.16 and 0.09 mg/kg-day
based on the Chen etal. (2012) data, the analysis incorporating total arm entries suggests a lower
POD for this effect; however, EPA notes that the two sets of results overlap substantially. EPA
considers the results corroborative of the POD for this effect based on the published data.
Comment: Use of decreased anxiety-like effects as a critical effect. Arcadis and ACC questioned why
the behavioral effect of "decreased anxiety" observed in rodents tested in the elevated plus maze
was regarded as an adverse effect given that this test is used in pharmacology to evaluate the
efficacy of anxiety reducing drugs.
EPA Response: A normal level of anxiety is a protective function of the nervous system. A decrease
in anxiety, a clear change in nervous system function, can impair an organism's ability to react to a
potentially harmful situation. The decreased ability of an organism to adapt to the environment is
considered to be an adverse effect (U.S. EPA. 1998). In addition, any functional alteration resulting
from developmental exposure is considered biologically significant fU.S. EPA. 1991cl. Additional
discussion of the significance of this endpointhas been added in Section 2.1.1 of the Toxicological
Review.
In contrast to environmental exposure, exposure to drugs is intended to occur at a controlled
dosage and with a defined periodicity and duration, and drugs are only administered to a subset of
the population with an underlying, clinically-identified neurological dysfunction that requires
treatment
Comment: Consideration of additional studies for the derivation of the RfC. Arcadis commented that
the RfC should quantitatively consider the NOAELs and LOAELs from Archibong et al. (2002) and
Wu etal. f2003al to develop the RfC based on decreased pup survival. They state that the
publication by {Archibong, 1007602} reported a LOAEL for this effect as 25 |ig/m3, but that another
publication, from the same laboratory, Wu etal. (2003a). reported that this exposure concentration
was a NOAEL. They suggest that both studies can be used quantitatively to establish the point of
departure for the RfC.
EPA Response: The publications by Archibong etal. (2002) and Wu etal. (2003a) were generated
by the same laboratory and used identical exposure methods. As both publications reported
similar results for fetal survival, it appears possible that both reported effects on the same group of
exposed dams. EPA notes that reporting for the endpoint of decreased fetal survival met a higher
standard in Archibong etal. (2002) compared with Wu etal. (2003a). with Archibong et al. (2002)
reporting means and variances for implantation sites, pups per litter, and % litter survival and Wu
etal. f2003al reporting decreased survival graphically. Although the focus of this study was on
metabolic activation in the liver and brain of exposed offspring, Wu etal. f2003al did report that
"the number of resorptions was more at 75 and 100 |ig/m3 compared to 25 |ig/m3 and was
statistically significant," but did not report whether the apparent decrease in birth index at 25
[ig/m3 was statistically significant compared to the vehicle control group. This decrease in fetal
survival at 25 |ig/m3 is consistent with the lowest exposure level being a LOAEL rather than a
NOAEL as suggested by the commenters. Due to incomplete reporting on this endpoint, the dataset
reported by Wu etal. f2003al was inadequate for the derivation of a candidate RfC. These points
This document is a draft for review purposes only and does not constitute Agency policy.
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have been clarified in the discussion ofWu etal. f2003al in Section 1.1.1 and 2.2.1 of the
T oxicological Review.
GENERAL COMMENTS ON THE QUANTITATIVE CANCER ASSESSMENT
Comment: Apparent threshold in animal cancer bioassays. Arcadis, CDM Smith, and USWAG
commented that the animal carcinogenicity studies of benzo[a]pyrene used as the bases of the oral
slope factor fBeland and Culp. 19981. inhalation unit risk fThvssenetal.. 19811. and dermal slope
factor (Sivak etal.. 1997: Poel. 1959). demonstrate threshold exposures for benzo[a]pyrene. They
state that plots resulting from EPA's Benchmark Dose Modeling Software show that the bioassay
data exhibit threshold responses near the origin and that the tumor incidence of 0% at the low
doses demonstrate evidence of a threshold below which no cancer effects are seen.
EPA Response: EPA disagrees that the cited studies establish thresholds. Although animal
bioassays may seem to suggest thresholds, even the largest studies that are feasible to conduct have
insufficient power to detect risk levels of concern for public health. For example, a response of
1/50, or 2%, is the lowest response that can be observed in typical carcinogenicity bioassays. Such
a study cannot demonstrate a response of 0.1% (1/1000), say, often considered a high level of
cancer risk in a human population. If the true cancer rate at a particular exposure level is 0.1%, the
experimental outcome in a group of 50 will be 0% about 95% of the time.
Evidence for thresholds is more solidly determined through consideration of modes of action and
toxicokinetic pathways. Without consideration of such information, lack of response at low
exposures in animal bioassays cannot be distinguished from lack of statistical power.
COMMENTS ON THE INHALATION UNIT RISK
Comment: Availability of an additional benzo[a]pyrene inhalation study. Arcadis and EPRI
commented that an abstract for an unpublished study by Pauluhn et al. f!9851 contradicts the
findings of carcinogenicity in Thvssenetal. f 19811. and recommended that EPA acquire the raw
data.
EPA Response: The study referred to by the commenters, "Long-term inhalation study with
benzo[a]pyrene and SO2 in Syrian golden hamsters," assesses the effects of combined SO2 and
benzo[a]pyrene (abbreviated as BP in the abstract) exposure in hamsters. It is unclear how this
study provides negative or contradictory evidence because the abstract indicates a carcinogenic
response: "[i]n hamsters which were exposed to 2 or 10 mg BP/m3 air without SO2, a few
neoplastic alterations were found". While it would be useful to have another study of inhalation
exposure, no further information is available in the published literature, and EPA's attempts to
obtain the raw data have been unsuccessful.
Comment: Exceedance of maximum tolerated dose in Inhalation Unit Risk study. In comments
submitted by Arcadis and EPRI, it was stated that the exposure concentrations in the inhalation
study by Thvssenetal. f 19811 likely caused particle overload, exceeding the maximum tolerated
dose (MTD), and according to U.S. EPA f20051. data that exceed the MTD should not be used for
quantitative risk assessment purposes.
This document is a draft for review purposes only and does not constitute Agency policy.
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EPA Response: In Thvssenetal. f 19811. hamsters received time-weighted, average daily
concentrations of 0.25,1.01, and 4.29 mg/m3 benzo[a]pyrene condensed onto sodium chloride
particles (calculated based on weekly exposure chamber measurements). Animals in the low and
mid benzo[a]pyrene concentration groups had comparable survival to the vehicle control group.
The study authors reported that decreased survival in the high concentration group was associated
with increased incidence of tumors in the larynx and pharynx (affecting approximately half of the
high dose group). The study authors also concluded that particle clearance mechanisms of the
respiratory epithelium remained intact since the tumors of the upper respiratory and digestive
tract occurred relatively late in life.
EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005) distinguish between cancer and
other effects when assessing toxicity, e.g.: "If adequate data demonstrate that the effects are solely
the result of excessive toxicity rather than carcinogenicity [emphasis added] of the tested agent per
se, then the effects may be regarded as not appropriate to include in assessment of the potential for
human carcinogenicity of the agent." No evidence supports that the tumors noted by Thvssen et al.
f!9811 in the upper respiratory and digestive tract were solely the result of excessive toxicity
rather than carcinogenicity. The decreased survival observed in the high dose group appears to be
a direct effect of the early occurrence of tumors in this group. Therefore, the Thvssenetal. (1981)
study was judged to support the derivation of a unit risk.
Comment: Apparent discrepancies in cancer incidence data used in the derivation of the inhalation
unit risk. In comments submitted by Arcadis and EPRI, it was noted that the total number of
animals at risk and the total number of tumors observed in each treatment group from the Thvssen
etal. fl9811 study varied between that publication, a secondary analysis fU.S. EPA. 19901. and
study design descriptions and tables (D-13, D-14, E-17, and dose-response model output
summaries) presented in the Toxicological Review.
EPA Response: Concerning the number of tumor-bearing animals, Thvssenetal. f!9811
summarized only the animals with malignant tumors without identifying them as such; EPA's
review (U.S. EPA. 1990) of the individual animal pathology reports provided by the investigators
f Clement Associates. 19901 showed that totals reported in the publication matched the incidence of
malignant tumors, and that there were benign tumors as well. Table D-13 of the Supplemental
Information has been revised to reflect the incidence of benign and malignant tumors.
Concerning the numbers of animals at risk, the totals in Thvssenetal. f!9811. U.S. EPA f!9901. and
the draft Toxicological Review differed because the number of animals examined for histopathology
varied for different tissues:
• Thvssen etal. T19811 reported the overall number of animals evaluated for histopathology,
but did not clarify when individual tissues were not available for examination.
• The draft Toxicological Review (August 2013) relied on the summaries in U.S. EPA f!9901
augmented by details from the histopathology reports (Clement Associates. 1990). such as
clarifications that some tumors were metastases or types not likely related to squamous
neoplasia. Upon re-review of the histopathology reports, EPA determined that results
were not available for five low-exposure animals included in the U.S. EPA (1990) analysis.
EPA has revised the incidence summaries and the dose-response modeling and has
omitted data for these animals.
This document is a draft for review purposes only and does not constitute Agency policy.
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Comment: Exposure variability in the study used to derive inhalation unit risk. Arcadis and EPRI
stated thatThvssen etal. (1981) is inappropriate for dose response modeling given concerns
regarding variability in exposure concentrations.
EPA Response: As discussed in Sections 2.4.1 and 2.4.4 of the Toxicological Review, the lifetime
inhalation bioassay by Thvssen etal. f!9811. reported weekly averages of chamber concentrations
of benzo[a]pyrene which varied two- to fivefold from the overall average for each group, which
exceeds the limit for exposure variability of <20% for aerosols recommended by OECD (20091.
Continuous time weighted group average concentrations were calculated for use in dose response
modeling under the assumption that equal cumulative exposures are expected to lead to similar
outcomes. For risk assessment purposes, EPA generally assumes that cancer risk is proportional to
cumulative exposure, and therefore to lifetime average exposure as estimated here, when there is
no information to the contrary. Under this assumption, the variability of the chamber
concentrations has little impact on the estimated exposure-response relationship.
COMMENTS ON THE DERMAL SLOPE FACTOR
Comment: Consideration of nonlinear MOAsfor dermal carcinogenicity. USWAG commented that
EPA failed to consider nonlinear dose-response models to extrapolate from dermal exposure
studies to predict cancer risk. USWAG cited the availability of a dermal exposure study in mice (and
follow up study) that measured DNA adducts, necrosis, and inflammation after 5 weeks of exposure
and observed the tumor response at approximately 8 months of treatment (Albert etal.. 1996:
Albert etal.. 1991). This commenter stated that the observed tumor response in this study was
"remarkably nonlinear, with pronounced tumor formation at 35 weeks observed only in the high
dose group" and that this study "demonstrated definitively, and quantitatively, that pervasive
dermal tissue injury was induced by all benzo[a]pyrene dose levels investigated in this study." They
also go on to state that" [t] he studies by f Albert etal. f!9961: Albert etal. f 199111 demonstrate that
benzo[a]pyrene-induced inflammation, cell killing, and cell replication was highly likely to have
occurred at tumorigenic benzo[a]pyrene dose levels used in every bioassay that EPA relied on to
calculate a DSF."
EPA Response: EPA agrees that the studies by f Albert etal. f 19961: Albert etal. f!99111 appear to
indicate that at high doses of benzo[a]pyrene, inflammation promotes the formation of tumors.
Albert etal. (1991) treated animals with 16, 32, or 64 [ig of benzo[a]pyrene once per week and
reported the number of tumors per mouse after 8 months (current standardized rodent cancer
bioassays treat animals for 18 to 24 months fU.S. EPA. 200511. The mice in the 16 and 32 [ig dose
groups had an average of 1 tumor/animal, whereas the animals in the highest dose group had
approximately 8 tumors/animal. A follow up to this study, by the same authors, measured DNA
adducts, necrosis, and inflammation in treated mice (at the site of exposure) after 5 weeks of
dermal exposure. In the 64 [ig/week dose group, statistically elevated levels of DNA adducts,
inflammation, and necrosis were reported; however, in the lower dose group (16 [ig/week), DNA
adducts were statistically significantly elevated without increases in inflammation and necrosis.
In comparison, the lifetime dermal cancer bioassay in mice used for the derivation of the dermal
slope factor, Sivak etal. (1997). used much lower exposure concentrations of benzo[a]pyrene (0.1,
1 or 10 [ig/week). Even so, after two years of exposure, animals in highest dose group had a tumor
incidence of 27/30 with dermal scabs and sores reported in 80% of animals. However, the next
dose level down reported tumors in 5/30 animals with no elevation of cytotoxic lesions.
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Cytotoxicity and mutation are not mutually exclusive modes of action; some observed effects can be
consistent with more than one mode of action. A mutagen at high doses can cause cytotoxicity and
regenerative proliferation that is a secondary response to massive DNA damage. As discussed in
Section 1.1.5 of the Toxicological Review, benzo[a]pyrene is a complete carcinogen; the
contributing roles of other processes involved in the promotion and progression of
benzo[a]pyrene-induced tumors, including cytotoxicity, inflammation, and regenerative cell
proliferation, are acknowledged within the MOA discussion. However, there is insufficient evidence
that these mechanisms act independently of DNA damage and mutation to produce
benzo[a]pyrene-induced tumors.
EPA has revised the assessment to provide additional discussion of the (Albert etal. (19961: Albert
etal. (199111 studies in the cancer MOA section with particular attention to the observed
quantitative and temporal relationship between DNA adducts and indicators of cytotoxicity
(including inflammation and necrosis).
Comment: Potential for different mode of action for benzo[a]pyrene-induced tumors in mouse skin
versus human skin. Arcadis and EPRI commented that PAH-induced mouse skin tumors have a
different genetic signature than tumors in human skin. They cited Balmain and Harris (2000) as
support for their hypothesis that PAH-induced tumors in mouse skin have an H-ras mutation
signature whereas human skin cancers have a p53 mutation signature, therefore showing that
PAHs are not causally related to human skin cancers.
EPA Response: The review article cited above (Balmain and Harris. 2000) does not discuss PAH-
induced skin tumors in mouse skin or human skin. This article reviews the association between
benzo[a]pyrene exposure from tobacco smoke and p53 mutations in human lung cancer, the
association between sun exposure and p53 mutations in human skin cancer, and the association
between dietary aflatoxin B1 exposure and p53 mutations in human liver cancer.
Benzo[a]pyrene has been shown to be a complete carcinogen in multiple animal species. Skin
tumors in mice, rats, rabbits, and guinea pigs have been associated with repeated application of
benzo[a]pyrene to skin in the absence of exogenous promoters fSivak etal.. 1997: Grimmer et al..
1984: Habs etal.. 1984: Grimmer et al.. 1983: Habs etal.. 1980: Schmahl etal.. 1977: Schmidt et al..
1973: Roe etal.. 1970: Poel. 1959). The proposed mutagenic MOA for benzo[a]pyrene involves the
bioactivation of benzo[a]pyrene to DNA-reactive metabolites, direct DNA damage by reactive
metabolites, formation and fixation of DNA mutations, and clonal expansion of mutated cells. These
key events have been observed in animals and humans and by multiple routes of exposure (see
Table 1-17 of the Toxicological Review). Benzo[a]pyrene specific skin adducts have been observed
in vivo in both benzo[a]pyrene-treated mouse skin and human skin exposed to PAH-mixtures
fGodschalk etal.. 2001: Roias etal.. 2001: Zhang etal.. 19901. In addition, studies of dermal
benzo[a]pyrene exposure in mice have shown increased mutations in several gene targets including
the tumor suppressor p53 (Ruggeri et al.. 1993). the proto-oncogene H-Ras (Wei etal.. 1999:
Chakravarti etal.. 19951 and the lacz transgene fMiller etal.. 20001. Studies which examine
mutational spectra in human skin tumors thought to be related to PAH exposure are not available in
the literature.
The lack of specific H-ras mutational evidence in humans (due to lack of human studies) or
suggestive evidence of p53 mutations in UV-induced human skin tumors does not preclude the fact
that benzo[a]pyrene can initiate DNA damage via DNA adducts. If not adequately repaired, this
damage can form mutations and mutagenesis is a well-established cause of carcinogenesis.
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Text in the mode of action analysis for carcinogenicity (in Section 1.1.5) has been modified to
include studies observing additional target gene mutations (in addition to H-ras) in dermally
exposed mice fMiller etal.. 2000: Ruggeri et al.. 19931.
Comment: Exceedance of maximum tolerated dose in studies used to derive the dermal slope factor.
Arcadis and EPRI disagreed with the study that EPA selected for characterizing the dermal slope
factor, Sivak etal. (1997). stating that this study included exposure levels that exceeded the
maximum tolerated dose (MTD) by causing significant skin toxicity and excessive mortality, and
therefore is not suitable for the derivation of the dermal slope factor.
EPA Response: While lower lifetime exposures are preferred for cancer risk estimation, EPA
disagrees that studies demonstrating carcinogenicity, with or without causing mortality, should
automatically be excluded from cancer risk assessment EPA's Guidelines for Carcinogen Risk
Assessment (U.S. EPA. 2005) distinguish between cancer and other effects when assessing toxicity
at exposures at or above the maximum tolerated dose, e.g.: "If adequate data demonstrate that the
effects are solely the result of excessive toxicity rather than carcinogenicity [emphasis added] of the
tested agent perse, then the effects may be regarded as not appropriate to include in assessment of
the potential for human carcinogenicity of the agent" Therefore, EPA concluded it is justified in
excluding individual dose groups with exposure above the MTD, but not necessarily the entire study.
Some skin toxicity was noted in the highest dose group with an 80% incidence of scabs and sores.
However, the next lower dose group did not produce notable non-cancer skin lesions. An analysis
excluding the high dose group from Sivak etal. f 19971 (not shown in the assessment) showed no
impact on the resulting dermal slope factor (within one significant figure), because the slope factor
reflects the dose-response relationship at the lower exposure levels. When reported by study
authors, text to clarify incidence of any non-cancerous skin lesions has been added to Section 1.1.5.
Comment: Cross-species extrapolation of dermal slope factor. CDM Smith, Arcadis and EPRI
commented that differences between mouse and human skin should be recognized and accounted
for in the calculation of the dermal slope factor for skin cancer. Specifically, the commenters stated
mouse skin is thinner and more permeable to benzo[a]pyrene and produces more benzo[a]pyrene-
diol epoxide metabolites than humans. They also stated that aryl hydrocarbon hydroxylase
inducibility is higher in mice than humans and that DNA-adducts are formed at higher rates in
mouse skin and repaired at lower rates relative to human skin. Another commenter, CH2M Hill
recommended increased discussion of the uncertainty regarding the method for interspecies
scaling of the dermal slope factor.
EPA Response: The key events in the mutagenic mode of action of benzo[a]pyrene are well
conserved between species and tissues (see Section 1.1.5 and Table 1-17 of the Toxicological
Review). However, the quantitative differences in processes that may presumably affect the
carcinogenicity of benzo[a]pyrene are not well known. These processes likely include species (and
inter-individual) differences in rates of absorption, metabolism into reactive metabolites, de-
activation of reactive metabolites, DNA repair, and DNA replication. As mentioned in Section E of
the Supplemental Information, biological information is not currently comprehensive or detailed
enough to develop robust models for cross-species extrapolation. This assessment evaluated
several alternative approaches in Appendix E of the Supplemental Information. Of these potential
approaches, allometric scaling using body weight to the % power was selected based on observed
species differences in the rate of dermal absorption and metabolism of benzo[a]pyrene. Using this
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approach, rodents and humans exposed to the same daily dose of a carcinogen, adjusted for BW3/4,
would be expected to have equal lifetime risks of cancer. Several assumptions are made in the use
of this scaling method. First, it is assumed that the toxicokinetic processes in the skin will scale
similarly to interspecies differences in whole-body toxicokinetics. Secondly, it is assumed that the
risk at low doses of benzo[a]pyrene is linear. A charge question on the method used for
interspecies scaling of the dermal slope factor has been included in the charge to the external peer
reviewers.
Comment: Uncertainties regarding the implementation of the dermal slope factor. CH2M Hill
suggested that the IRIS assessment for benzo[a]pyrene could be strengthened with increased
discussion of uncertainties in assessing risk from exposure to benzo[a]pyrene in soil. In addition,
they noted that EPA's Risk Assessment Guidelines for Superfund part E fU.S. EPA. 20041
recommend an absorption factor of 13% for benzo[a]pyrene exposure through soil, but that this is
for systemic absorption and may not be consistent with the mode of action of benzo[a]pyrene in the
skin. Another commenter, USWAG, stated that expressing skin cancer risk for benzo[a]pyrene in
terms of (ug/d)-1 is not appropriate because of the implicit assumption that the surface area over
which exposure occurs is irrelevant
EPA Response: The lifetime dermal cancer bioassays available for benzo[a]pyrene reported the
total dose applied to skin and reported the general area treated (i.e. dorsal or interscapular area),
but did not quantify the actual cm2 of skin treated. For this reason, the draft dermal slope factor
expresses risk of skin tumors from benzo[a]pyrene dermal exposure as risk per ug/d. The
assumption of this dose metric is that risk at low doses of benzo[a]pyrene is dependent on absolute
dermal dose and not dose per unit of skin, meaning that a higher exposure concentration of
benzo[a]pyrene contacting a smaller area of exposed skin could carry the same risk of skin tumors
as a lower exposure concentration of benzo[a]pyrene that contacts a larger area of skin. The skin
surface area exposed to benzo[a]pyrene contaminated media is an important variable in the
exposure assessment calculation of the absolute dermal dose of benzo[a]pyrene. An increase in
skin area exposed to benzo[a]pyrene contaminated media would result in an increased absolute
dermal dose, and therefore an associated increase in risk. Example equations for calculating the
absolute dermal dose of benzo[a]pyrene are included below.
The dermal slope factor is based on applied dose of benzo[a]pyrene in solvent As environmental
dermal exposure is assumed to be predominantly through contaminated soil, it is recommended
that exposure assessment include an adjustment for exposure through soil. In the attached
example exposure calculations for exposure to benzo[a]pyrene through soil, a soil to skin transfer
coefficient (Ksoil) of 0.25 was estimated based on a study in monkeys (Wester et al.. 1990) which
measured dermal absorption of benzo[a]pyrene from soil as 13% and from acetone as 51%. In the
acetone vehicle, all of the benzo[a]pyrene contacts the skin after the acetone evaporates, implying
that 51% of the benzo[a]pyrene contacting the skin is absorbed. The soil experiment found that
13% was absorbed, so dividing this by 0.51 indicates that 25% of the benzo[a]pyrene in soil must
have contacted the skin. This may be a high estimate because Wester etal. fl9901 used a low
carbon soil (0.9%).
Comment: "Real world" validation of dermal slope factor. Arcadis and EPRI recommended that EPA
perform calculations to determine if the proposed dermal slope factor is scientifically supportable.
These commenters stated that based on their calculations (details not provided to EPA) the current
dermal slope factor would indicate thatbenzo[a]pyrene and benzo[a]pyrene-toxic equivalents in
soil throughout the United States are the cause of 30% of all human skin cancers and that 100% of
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users of pharmaceutical coal tar products should develop skin cancer due to exposure to
benzo[a]pyrene. The commenters recommended that EPA not derive a dermal slope factor in the
benzo[a]pyrene assessment, but stated that if a dermal slope factor was included a real world
validation should be included to determine if the value is scientifically supportable.
EPA Response: The commenters did not provide the exposure equation, benzo[a]pyrene soil
concentration, or assumptions used in their calculation of a 30% risk estimate. Without these
details, EPA could not reproduce the exposure and associated risk estimates presented in the
written comments.
EPA has used the proposed dermal slope factor to calculate average daily dermal doses and
associated risks at those dermal doses. A modified exposure equation, similar to equation 3.11 (for
dermal absorbed dose upon soil contact) in EPA's Risk Assessment Guidelines for Superfund part E
(U.S. EPA. 2004). was used to calculate the average-daily dose of benzo[a]pyrene absorbed into the
skin (not absorbed systemically, which is the original intent of the equation). Central tendency
exposures using benzo[a]pyrene soil concentrations of 100 ppb (a central estimate of several
published measurements of uncontaminated sites) and default exposure assumptions result in risk
estimates of approximately 6 x 10 6 for average lifetime exposure that occurs during childhood, and
1 x 10"6 for average lifetime exposure that occurs during adulthood.
Example exposure calculations for risk associated with a central tendency exposure to
benzo[a]pyrene throughout a lifetime (including childhood) are included below.
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Supplem en tal Information —Benzo[aJpyren e
Example Calculations: Estimated Dermal Dose and Risk
from Exposure to Benzo[a]pyrene in Soil
Ccmi x CF x SA x AF x ED x EF x K«mi
LAD Ddermal
=
AT
Riskdermal
LADDdenn.iI ^ SFdermal ^ ADAF
2
3
LADDdermai = Lifetime Average Daily Dose (ug/day)
CSoii = Soil Concentration (ug/kg)
CF = Conversion Factor (kg/ug)
SA = Surface Area of Skin Exposed (cm2)
AF = Soil Adherence Factor (ug/cm2-day)
AT = averaging time, the period over which the
exposure is averaged (days)
ED = Exposure Duration (years)
EF = Exposure Frequency (days/year)
KSoii = Soil to skin Transfer Coefficient for benzo[a]pyrene
(unitless)
SFdermai = Dermal Slope Factor for benzo[a]pyrene (ug/day)1
ADAF = Age Dependent Adjustment Factor (unitless)
Central Tendency Exposure (CTE) Dose: Dermal Contact with
benzo|a]pyrene in Soil - Child age
-<2 years
LAD Ddermal
Csoil
CF
SAchild l-<6
AF child l-<6
EDchild l-<6
EF
Ksoil
AT
ug/day
ug/kg
kg/ug
cm2
ug/cm2-day
years
days/year
unitless
days
3.84E-05
100
1.00E-09
2800
40
1
350
0.25
25550
Riskdermal
LADD
SFdermal
ADAF
unitless
ug/day
(ug/day)1
2.30E-06
3.84E-05
0.006
10
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Supplem en tal Information —Benzo[aJpyren e
Central Tendency Exposure (CTE) Dose: Dermal Contact with benzo[a]pyrene in
Soil - Child age 2-<6 years
LAD Ddermal
ug/day
Csoil
ug/kg
CF
kg/ug
SAchild l-<6
cm2
AFchild l-<6
ug/cm2-day
EDchild l-<6
years
EF
days/year
Ksoil
unitless
AT
days
1.53E-04
100
1.00E-09
2800
40
4
350
0.25
25550
Riskderm.il
LADD
SFdermal
ADAF
unitless
ug/day
fug/day]1
2.76E-06
1.53E-04
0.006
3
Central Tendency Exposure (CTE) Dose: Dermal Contact with benzo[a]pyrene in
Soil - Child age 6-<10 years
LAD Ddermal
ug/day
Csoil
ug/kg
CF
kg/ug
SAchild 6-<10
cm2
AF child 6-<10
ug/cm2-day
EDchild 6-<10
years
EF
days/year
Ksoil
unitless
AT
days
7.81E-05
100
1.00E-09
5700
10
4
350
0.25
25550
Riskdermal
LADD
SFdermal
ADAF
unitless
ug/day
fug/day]1
1.41E-06
7.81E-05
0.006
3
Total CTE
Riskchiid
6.47E-06
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Supplem en tal Information —Benzo[aJpyren e
Central Tendency Exposure (CTE) Dose: Dermal Contact with
benzo[a]pyrene in Soil - Adult
LAD D(ie I'm.) i
Csoil
CF
SAadult
AF adult
ED adult
EF
Ksoil
AT
ug/day
ug/kg
kg/ug
cm2
ug/cm2-day
years
days/year
unitless
days
1.76E-04
100
1.00E-09
5700
10
9
350
0.25
25550
Riskderm.,1
LADD
SFdermal
ADAF
unitless
ug/day
(ug/day)1
1.05E-06
1.76E-04
0.006
1
CTE Assumptions:
Csoii = an assumed concentration of 0.1 ppm or 100 ug/kg; central tendency value based on average of data for all
sites (to 1 significant figure) in Table A-4 of Supplemental Information of the Public Comment Draft (dated August
2013) except the 3 high-end urban sites.
SAchiid i-<6 years = 2800 cm2 for child residents; average of the 50th percentile values for males and females 1 to <6
years of age (U.S. EPA. 2004. Exhibit 3-5): the child resident was assumed to wear a short-sleeved shirt, and shorts
(no shoes); therefore, the exposed skin surface is limited to the head, hands, forearms, lower legs, and feet.
SAchiid 6-< 10 years — 5700 cm2; adult value assumed based on (U.S. EPA. 2004. Page 3-19).
SAaduit = 5700 cm2 for adults; average of the 50th percentile values for males and females greater than 18 years of age
fU.S. EPA. 2004. Exhibit 3-51: assumed to wear a short-sleeved shirt, short pants and shoes; therefore, the exposed
skin surface is limited to the head, hands, lower legs, and forearms.
AFchiid i-<6 years = 40 ug/cm2-day for children; based on the 50th percentile weighted AF for children playing at a
daycare center (U.S. EPA. 2004. Exhibit 3-3 and 3-5): weighted based on body parts exposed.
AFchiid 6-< 10years = 10 ug/cm2-day; adult value assumed based on (U.S. EPA. 2004. Page 3-19).
AFaduit = 10 ug/cm2-day for adult residents; based on the geometric mean weighted AFs for groundskeepers fU.S. EPA.
2004. Exhibit 3-3 and 3-51: weighted based on body parts exposed.
AT = averaging time (the period over which the exposure is averaged).
EDChiidi-<2 years = 1 year for a child resident.
EDChiid2-<6years = 4 years for a child resident.
EDChiid6-< 10years = 4 years for a child resident based on a central tendency residency time of 9 years minus 5 years as a
child age l-<6 years at that residence.
EDaduit = 9 years for adults based on a central tendency residency time of 9 years.
EF = 350 days per year; assumes 2 weeks away from the contaminated area each year for vacation.
Ksoii = 0.25; This estimate was based on a study in monkeys (Wester et al„ 1990) which measured dermal absorption
of benzo[a]pyrene from soil as 13% and from acetone as 51%. In the acetone vehicle, all of the benzo [ajpyrene
contacts the skin after the acetone evaporates, implying that 51% of the benzo [ajpyrene contacting the skin is
absorbed. The soil experiment found that 13% was absorbed, so dividing this by 0.51 indicates that 25% must have
contacted the skin.
AT = assumed to be 70 years converted to days: 25550 days.
SFdermal = 0.006 (ug/day)-1
ADAF = 10 for children l-<2 years; 3 for children 2-<10 years.
1
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REFERENCES FOR APPENDICES
Abe. S; Nemoto. N; Sasaki. M. (1983a). Comparison of aryl hydrocarbon hydroxylase activity and
inducibility of sister-chromatid exchanges by polycyclic aromatic hydrocarbons in
mammalian cell lines. Mutat Res 122: 47-51. http://dx.doi.org/10.1016/Q165-
7992(83)90141-0
Abe. S; Nemoto. IN; Sasaki. M. (1983b). Sister-chromatid exchange induction by indirect
mutagens/carcinogens, aryl hydrocarbon hydroxylase activity and benzo[alpha]pyrene
metabolism in cultured human hepatoma cells. Mutat Res 109: 83-90.
http://dx.doi.org/10.1016/0027-5107(83)90097-0
Achard. S; Perderiset. M; Jaurand. MC. (1987). Sister chromatid exchanges in rat pleural
mesothelial cells treated with crocidolite, attapulgite, or benzo 3-4 pyrene. Br J Ind Med
44: 281-283.
Adler. ID: Ingwersen. I. (1989). Evaluation of chromosomal aberrations in bone marrow of
1C3F1 mice. Mutat Res 224: 343-345. http://dx.doi.org/10.1016/0165-1218(89)90176-6
Adler. ID: Kliesch. U; Kiefer. F. (1989). Clastogenic effects of benzo[a]pyrene in postimplantation
embryos with different genetic background. Teratog Carcinog Mutagen 9: 383-392.
http://dx.doi.org/10.1002/tcm.17700906Q6
Agarwal, R; Medrano, EE; Khan. IU; Nordlund.JJ; Mukhtar, H. (1991). Metabolism of
benzo[a]pyrene by human melanocytes in culture. Carcinogenesis 12: 1963-1966.
http://dx.doi.org/10.1093/carcin/12.10.1963
Agrelo, C; Amos. H. (1981). DNA repair in human fibroblasts. In FJ de Serres; J Ashby (Eds.),
Evaluation of short-term tests for carcinogens: Report of the International Collaborative
Program (pp. 528-532). New York, NY: Elsevier/North-Holland.
Albert. RE: Miller. ML: Cody. T; Andringa. A: Shukla. R; Baxter. CS. (1991). Benzo[a]pyrene-
induced skin damage and tumor promotion in the mouse. Carcinogenesis 12:1273-
1280. http://dx.doi.Org/10.1093/carcin/12.7.1273
Albert. RE: Miller. ML: Cody. TE; Talaska. G; Underwood. P; Andringa. A. (1996). Epidermal
cytokinetics, DNA adducts, and dermal inflammation in the mouse skin in response to
repeated benzo[a]pyrene exposures. Toxicol Appl Pharmacol 136: 67-74.
Alderson. MR: Clarke. JA. (1983). Cancer incidence in patients with psoriasis. Br J Cancer 47:
857-859.
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