/EPA
EPA/635/R-13/138b
Public Comment 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
August 2013
NOTICE
This document is a Public Comment 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
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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.I. TOXICOKINETICS D-l
D.I.I. Overview D-l
D.I.2. Absorption D-l
D.I.3. Distribution D-3
D.1.4. Metabolism D-4
D.I.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. Non-Cancer Endpoints D-15
D.3.2. Cancer-related Endpoints D-27
D.3.3. Epidemiologic Findings in Humans D-29
D.4. ANIMAL STUDIES D-34
D.4.1. Oral Bioassays D-34
D.4.2. Inhalation Studies D-54
D.4.3. Dermal studies D-58
D.4.4. Reproductive and Developmental Toxicity Studies D-67
D.4.5. Inhalation D-84
D.5. OTHER PERTINENT TOXICITY INFORMATION D-88
D.5.1. Genotoxicity Information D-88
D.5.2. Tumor Promotion and Progression D-lll
D.5.3. Benzo[a]pyrene Transcriptomic Microarray Analysis D-115
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APPENDIX E.
APPENDIX F.
APPENDIX G.
DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES FOR
EFFECTS OTHER THAN CANCER AND THE DERIVATION OF CANCER RISK
ESTIMATES E-l
E.I. NON-CANCER ENDPOINTS E-l
E.I.I. Reference Dose (RfD) E-l
E.2. Cancer Endpoints E-31
E.2.1. Dose-Response Modeling for the Oral Slope Factor E-31
E.2.2. Data Adjustments Prior to Modeling E-31
E.2.3. Dose-Response Modeling for the Inhalation Unit Risk E-69
E.2.4. Dose-Response Modeling for the Dermal Slope Factor E-76
DOCUMENTATION OF IMPLEMENTATION OF THE 2011 NATIONAL RESEARCH
COUNCIL RECOMMENDATIONS
. F-l
SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND EPA'S
DISPOSITION G-9
REFERENCES FOR APPENDICES.
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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-24
Table D-4. Exposure-related effects in Chinese coke oven workers or warehouse controls
exposed to benzo[a]pyrene in the workplace, stratified by urinary metabolite
levels D-25
Table D-5. Background information on Chinese coke oven workers or warehouse controls
exposed to benzo[a]pyrene in the workplace D-26
Table D-6. Exposure-related effects in male Wistar rats exposed to benzo[a]pyrene by gavage 5
days/week for 5 weeks D-36
Table D-7. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene by gavage 5
days/week for 5 weeks D-39
Table D-8. Means ± SDa for liver and thymus weights in Wistar rats exposed to benzo[a]pyrene
by gavage 5 days/week for 90 days D-41
Table D-9. Incidences of exposure-related neoplasms in Wistar rats treated by gavage with
benzo[a]pyrene, 5 days/week, for 104 weeks D-43
Table D-10. 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-47
Table D-ll. Incidence of nonneoplastic and neoplastic lesions in female B6C3F! mice fed
benzo[a]pyrene in the diet for up to 2 years D-49
Table D-12. Other oral exposure cancer bioassays in mice D-50
Table D-13. Incidence of respiratory and upper digestive tract tumors in male hamsters treated
for life with benzo[a]pyrene by inhalation D-56
Table D-14. Number of animals with pharynx and larynx tumors in male hamsters exposed by
inhalation to benzo[a]pyrene for life D-57
Table D-15. Skin tumor incidence and time of appearance in male C57L mice dermally exposed
to benzo[a]pyrenefor up to 103 weeks D-59
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-60
Table D-17. Tumor incidence in female Swiss mice dermally exposed to benzo[a]pyrene for up
to 93 weeks D-61
Table D-18. Skin tumor incidence in female NMRI and Swiss mice dermally exposed to
benzo[a]pyrene D-62
Table D-19. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene D-63
Table D-20. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene D-63
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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-64
Table D-22. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene for
life D-65
Table D-23. Skin tumor incidence in male C3H/HeJ mice dermally exposed to benzo[a]pyrene for
24 months D-66
Table D-24. Mortality and cervical histopathology incidences in female ICR mice exposed to
benzo[a]pyrene via gavagefor 14 weeks D-70
Table D-25. Means ± SD for ovary weight in female Sprague-Dawley rats D-73
Table D-26. Reproductive effects in male and female CD-I Fl mice exposed in utero to
benzo[a]pyrene D-75
Table D-27. Effect of prenatal exposure to benzo[a]pyrene on indices of reproductive
performance in Fl female NMRI mice D-76
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-81
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
toPND14 D-82
Table D-30. Pregnancy outcomes in female F344 rats treated with benzo[a]pyrene on GDs 11-
21 by inhalation D-85
Table D-31. In vitro genotoxicity studies of benzo[a]pyrene in non-mammalian cells D-88
Table D-32. In vitro genotoxicity studies of benzo[a]pyrene in mammalian cells D-90
Table D-33. In vivo genotoxicity studies of benzo[a]pyrene D-95
Table D-34. Search terms and the number of studies retrieved from the gene expression
omnibus and array express microarray repositories D-115
Table D-35. Mapping of group numbers to time/dose groups D-118
Table E-l. Non-cancer 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 (Chenetal., 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., 2011b); 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 Gulp, 1998) E-39
Table E-12. Derivation of HEDs to use for BMD modeling of Wistar rat tumor incidence data
from Kroese et al. (2001) E-42
Table E-13. Derivation of HEDs for dose-response modeling of B6C3F! female mouse tumor
incidence data from Beland and Gulp (1998) E-42
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-43
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-66
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 Gulp, 1998) E-66
Table E-17. Individual pathology and tumor occurrence data for male Syrian golden hamsters
exposed to benzo[a]pyrene via inhalation for lifetime—Thyssen et al. (1981)a E-70
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-72
Table E-19. 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-20. Skin tumor incidence, benign or malignant, in C57L male mice dermally exposed to
benzo[a]pyrene; data from Poel (1959) E-80
Table E-21. 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-22. Skin tumor incidence, benign or malignant, in male C3H/HeJ mice dermally exposed
to benzo[a]pyrene; data from Sivak et al. (1997) E-81
Table E-23. Summary of BMD model selection and modeling results for best-fitting multistage
models, for multiple data sets of skin tumors in mice following dermal
benzo[a]pyrene exposure E-82
Table E-24. Alternative approaches to cross-species scaling E-110
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-112
Figure D-4. Aryl hydrocarbon receptor pathway D-119
Figure D-5. DNA Damage pathway D-120
Figure D-6. Nrf2 pathway D-122
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 etalv 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 etal., 2012) E-18
Figure E-6. Fit of exponential model (4) to data on elevated plus maze open arm maze entries
(Chen etal., 2012) E-22
Figure E-7. Fit of log-logistic model to data on cervical epithelial hyperplasia (Gao et al., 2011b) 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-45
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-55
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-57
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
etal., 2001) E-60
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-63
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-65
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-68
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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-74
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-76
Figure E-22. Fit of multistage model to skin tumors in C57L mice exposed dermally to
benzo[a]pyrene (Poel, 1959) E-83
Figure E-23. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to
benzo[a]pyrene (Roe et al., 1970) E-85
Figure E-24. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Schmidt et al., 1973) E-87
Figure E-25. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to
benzo[a]pyrene (Schmidt et al., 1973) E-89
Figure E-26. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Schmahl et al., 1977) E-91
Figure E-27. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Habs et al., 1980) E-93
Figure E-28. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Habs et al., 1984) E-95
Figure E-29. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1983) E-97
Figure E-30. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984) E-99
Figure E-31. Fit of log-logistic model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984) E-101
Figure E-32. 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-103
Figure E-33. Fit of multistage model to skin tumors in male CeH/HeJ mice exposed dermally to
benzo[a]pyrene (Sivak et al., 1997) E-105
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ABBREVIATIONS
Supplem en tal Inform ation —Benzo[a]pyren e
1-OH-Py 1-hydroxypyrene HR
AchE acetylcholine esterase Hsp90
Ah aryl hydrocarbon Ig
AHH aryl hydrocarbon hydroxylase IHD
AhR Ah receptor i.p.
AIC Akaike's Information Criterion IRIS
AMI acute myocardial infarction i.v.
ANOVA analysis of variance KEGG
ARNT Ah receptor nuclear translocator
AST aspartate transaminase LDH
BMD benchmark dose LH
BMDL benchmark dose, 95% lower bound LOAEL
BMDS Benchmark Dose Software MAP
BMR benchmark response MLE
BPDE benzo[a]pyrene-7,8-diol-9,10-epoxide MMAD
BrdU bromodeoxyuridine MN
BSM benzene-soluble matter mRNA
BUN blood urea nitrogen MS
CA chromosomal aberration NCE
CASRN Chemical Abstracts Service Registry NK
Number NMDA
CHO Chinese hamster ovary nNOS
CI confidence interval NOAEL
GYP cytochrome NQO
CYP450 cytochrome P450 NRC
dbcAMP dibutyl cyclic adenosine OR
monophosphate PAH
DMSO dimethyl sulfoxide PBMC
DNA deoxyribonucleic acid PBPK
EC European Commission PCE
EH epoxide hydrolase PCR
ELISA enzyme-linked immunosorbent assay PND
eNOS endothelial nitric oxide synthase POD
EROD 7-ethoxyresorufin-O-deethylase RBC
ETS environmental tobacco smoke RfC
Fe203 ferrous oxide RfD
GABA gamma-aminobutyric acid RNA
GD gestational day ROS
GI gastrointestinal RR
GJIC gap junctional intercellular SCC
communication SCE
GSH reduced glutathione SD
GST glutathione-S-transferase SE
GSTM1 glutathione-S-transferase Ml SEM
hCG human chorionic gonadotropin SHE
HED human equivalent dose SIR
HFC high-frequency cells SMR
HPLC high-performance liquid SOD
chromatography SSB
hprt hypoxanthine guanine phosphoribosyl TCDD
transferase TK
hazard ratio
heat shock protein 90
immunoglobulin
ischemic heart disease
intraperitoneal
Integrated Risk Information System
intravenous
Kyoto Encyclopedia of Genes and
Genomes
lactate dehydrogenase
luteinizing hormone
lowest-observed-adverse-effect level
mitogen-activated protein
maximum likelihood estimate
mass median aerodynamic diameter
micronucleus
messenger ribonucleic acid
mass spectrometry
normochromatic erythrocyte
natural-killer
N-methyl-D-aspartate
neuronal nitric oxide system
no-observed-adverse-effect level
NADPH:quinone oxidoreductase
National Research Council
odds ratio
polycyclic aromatic hydrocarbon
peripheral blood mononuclear cell
physiologically based pharmacokinetic
polychromatic erythrocyte
polymerase chain reaction
postnatal day
point of departure
red blood cell
reference concentration
reference dose
ribonucleic acid
reactive oxygen species
relative risk
squamous cell carcinoma
sister chromatid exchange
standard deviation
standard error
standard error of the mean
Syrian hamster embryo
standardized incidence ratio
standardized mortality ratio
superoxide dismutase
single strand break
2,3,7,8-tetrachlorodibenzo-p-dioxin
thymidine kinase
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TPA 12-0-tetradecanoylphorbol-13-acetate
TUNEL terminal deoxynucleotidyl transferase
dUTP nick end labeling
TWA time-weighted average
UCL upper confidence limit
WESPOC water escape pole climbing
WT wild type
WTC World Trade Center
XPA xeroderma pigmentosum group A
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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 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 (HSDB. 2012). The structural formula is presented
11 in Figure A-l. The physical and chemical properties of benzo[a]pyrene are shown in Table A-l.
12
13
Benzo[a]pyrene
Figure A-l. Structural formula of benzo[a]pyrene.
14
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Table A-l. Chemical and physical properties of benzo[a]pyrene
CASRN 50-32-8
Synonyms
Melting point
Boiling point
Vapor pressure, at 20°C
Density
Flashpoint (open cup)
Water solubility at 25°C
LogKow
Odor threshold
Molecular weight
Conversion factors3
Empirical formula
Benzo[d,e,f]chrysene;
3,4-benzopyrene,
3,4-benzpyrene; benz[a]pyrene; BP; BaP
179-179.3°C
310-312°CatlOmm Hg
5 x 10"7 mm Hg
1.351 g/cm3
No data
1.6-2.3 x 10"3 mg/L
6.04
No data
252.32
1 ppm = 10.32 mg/m3
C2oHl2
ChemlDplus(2012)
O'Neiletal. (2001)
O'Neiletal. (2001)
Verschueren (2001)
IARC (1973)
Howard and Meylan (1997); ATSDR
(1995)
Verschueren (2001)
O'Neiletal. (2001)
Verschueren (2001)
ChemlDplus(2012)
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
Calculated based on the ideal gas law, PV = nRJ at 25°C: ppm = mg/m3 x 24.45 4- molecular weight.
No reference to any commercial use for purified benzo[a]pyrene, other than for research
purposes, was found. The earliest research reference for benzo[a]pyrene was related to the
identification of coal tar constituents associated with human skin tumors [Phillips. 1983: Cook et
al.. 1933]. It is found ubiquitously in the environment, primarily as a result of incomplete
combustion emissions (Bostrometal.. 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, cigarettes, and various industrial
combustion processes (ATSDR. 1995). Benzo[a]pyrene is also found in soot and coal tars. Mahler
etal. (2005) 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. 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).
Inhalation. The Agency for Toxic Substances and Disease Registry (ATSDR. 1995) reported
average indoor concentrations of benzo[a]pyrene of 0.37-1.7 ng/m3 for smokers and 0.27-
0.58 ng/m3 for non-smokers. Naumovaetal. (2002) measured PAHs in 55 non-smoking 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
This document is a draft for review purposes only and does not constitute Agency policy.
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1 observed an average indoor/outdoor ratio of approximately 0.7 [Naumova etal., 2002]. Mitra and
2 Wilson [1992] measured benzo[a]pyrene air levels in Columbus, Ohio, and found elevated indoor
3 levels in homes with smokers. The measured average concentration was 1.38 ng/m3 for outdoor
4 air; indoor concentrations were 0.07 ng/m3 for homes with electrical utilities, 0.91 ng/m3 for
5 homes with gas utilities, 0.80 ng/m3 for homes with gas utilities and a fireplace, 2.75 ng/m3 for
6 homes with gas utilities and smokers, and 1.82 ng/m3 for homes with gas utilities, smokers, and a
7 fireplace [Mitra and Wilson. 1992]. Mitra and Ray [1995] evaluated data on benzo[a]pyrene air
8 levels in Columbus, Ohio, and reported average concentrations of 0.77 ng/m3 inside homes and
9 0.23 ng/m3 outdoors. Park etal. [2001] measured an average ambient level of benzo[a]pyrene in
10 Seabrook, Texas during 1995-1996 of 0.05 ng/m3 (vapor plus particulate]. Park etal. [2001] also
11 reported average ambient air levels from earlier studies as 1.0 ng/m3 for Chicago, 0.19 ng/m3 for
12 Lake Michigan, 0.01 ng/m3 for Chesapeake Bay, and 0.02 ng/m3 for Corpus Christie, Texas. Petry et
13 al. [1996] conducted personal air sampling during 1992 at five workplaces in Switzerland: carbon
14 anode production, graphite production, silicon carbide production, bitumen paving work, and metal
15 recycling. Table A-2 summarizes the benzo[a]pyrene air concentration data from the previous
16 studies.
17
This document is a draft for review purposes only and does not constitute Agency policy.
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Table A-2. Benzo[a]pyrene concentrations in air
Setting
Outdoor, urban
Los Angeles, California
Houston, Texas
Elizabeth, New Jersey
Seabrook, Texas
Columbus, Ohio
Indoor, residential
Los Angeles, California
Houston, Texas
Elizabeth, New Jersey
Columbus, Ohio
Columbus, Ohio
Homes with smokers
Homes without smokers
Occupational
Aluminum production
Coke production
Carbon anode production, Switzerland
Graphite production, Switzerland
Silicon carbide production, Switzerland
Metal recovery, Switzerland
Bitumen paving, Switzerland
Year
1999-2000
1999-2000
1999-2000
1995-1996
1986-1987
1999-2000
1999-2000
1999-2000
1986-1987
1992
1992
1992
1992
1992
n
19
21
15
NA
8
19
21
15
8
10
30
16
14
5
9
Concentration
(ng/m3)
0.065
0.025
0.14
0.05
0.23
0.078
0.020
0.055
0.77
0.07-2.75
0.37-1.7
0.27-0.58
30-530
150-6,720
8,000
1,100
83
36
14
10
Reference
Naumova et al. (2002)
Naumova et al. (2002)
Naumova et al. (2002)
Park et al. (2001)
Mitraand Ray (1995)
Naumova et al. (2002)
Naumova et al. (2002)
Naumova et al. (2002)
Mitra and Ray (1995)
Mitra and Wilson (1992)
ATSDR(1995)
ATSDR(1995)
ATSDR(1995)
Retry et al. (1996); ATSDR
(1995)
Retry etal. (1996)
Retry etal. (1996)
Retry etal. (1996)
Retry etal. (1996)
Retry etal. (1996)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
NA = not available.
Santodonato etal. [1981] estimated adult daily intake from inhalation as 9-43 ng/day. The
European Commission [EC. 2002] reported benzo[a]pyrene air levels in Europe during the 1990s as
0.1-1 ng/m3 in rural areas and 0.5-3 ng/m3 in urban areas. The mean intake via inhalation for an
adult non-smoker was estimated as 20 ng/day. Naumova etal. [2002] focused on non-smoking
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. The processing and cooking of foods is viewed as the dominant pathway of PAH
contamination in foods [Bostrom et al.. 2002]. 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 [Li etal.. 2009].
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Kazerouni et al. [2001] measured benzo[a]pyrene in a variety of commonly consumed foods
2 collected from grocery stores and restaurants in Maryland (analyzed as a composite from 4-
3 6 samples of each food type). The foods were tested after various methods of cooking; the results
4 are reported in Table A-3. The concentrations were combined with food consumption data to
5 estimate intake. The intakes of the 228 subjects ranged from approximately 10 to 160 ng/day, with
6 about 30% in the 40-60 ng/day range. The largest contributions to total intake were reported as
7 bread, cereal, and grain (29%) and grilled/barbecued meats (21%).
Table A-3. Benzo[a]pyrene levels in food
Food
Meat
Fried or broiled beef
Grilled beef
Fried or broiled chicken
Grilled chicken
Fish
Smoked fish
Bread
Breakfast cereals
Vegetable oil
Eggs
Cheese
Butter
Milk
Fruit
Concentration (ng/g)
0.01-0.02
0.09-4.9
0.08-0.48
0.39-4.57
0.01-0.24
0.1
0.1
0.02-0.3
0.02
0.03
<0.005
<0.005
0.02
0.01-0.17
9
10
11
12
13
14
15
16
17
18
19
20
21
Source: Kazerouni et al. (2001).
Kishikawa et al. (2003) 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 European Union countries, the mean or national-
averaged dietary intake of benzo[a]pyrene for an adultperson was estimated in the range of 0.05-
0.29 |ig/day (EC. 2002). In the United Kingdom, average intakes on a ng kg-1 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. 2002). The contribution from grilled foods
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1 appeared less important in Europe than in the United States because grilled foods are consumed
2 less often [EC. 20021.
3 Dermal. The general population can be exposed dermally to benzo[a]pyrene when
4 contacting soils or materials that contain benzo[a]pyrene, such as soot or tar. Exposure can also
5 occur via the use of dermally applied pharmaceutical products that contain coal tars, including
6 formulations used to treat conditions such as eczema and psoriasis [IARC. 2010).
7 PAHs are commonly found in all types of soils. ATSDR [1995] reported benzo[a]pyrene
8 levels in soil of 2-1,300 [ig/kg in rural areas, 4.6-900 [ig/kg in agricultural areas, 165-220 [ig/kg in
9 urban areas, and 14,000-159,000 [ig/kg at contaminated sites (before remediation). The soil levels
10 for all land uses appear highly variable. The levels are affected by proximity to roads/combustion
11 sources, use of sewage-sludge-derived amendments on agricultural lands, particle size, and organic
12 carbon content. Weinberg et al. [1989] reported that PAH levels in soils generally increased during
13 the 1900s and that sediment studies suggest that some declines may have occurred since the 1970s.
14 An illustration of benzo[a]pyrene levels in soil is presented in Table A-4.
15
This document is a draft for review purposes only and does not constitute Agency policy.
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Table A-4. Levels of benzo[a]pyrene in soil
Reference
Butler etal. (1984)
Vogt etal. (1987)
Yang etal. (1991)
Trapido (1999)
Nam et al. (2008)
Mielke etal. (2001)
Nadal etal. (2004)
Maliszewska-Kordvbach et al. (2009)
Wilcke (2000)
Location
United Kingdom
Norway
Norway
Australia
Poland
Estonia
Estonia
Estonia
Estonia
Estonia
Estonia
Estonia
Estonia
United Kingdom
Norway
New Orleans
Spain
Spain
Spain
Spain
Poland
Various temperate
Various temperate
Various temperate
Various temperate
Bangkok
Brazil
Land Type
Urban
Industrial
Rural
Residential
Agricultural
Urban
Urban
Urban
Urban
Rural
Rural
Rural
Rural
Rural
Rural
Urban
Industrial-chemical
Industrial-petrochemical
Residential
Rural
Agricultural
Arable
Grassland
Forest
Urban
Urban-tropical
Forest-tropical
Concentration
Mean (|ig/kg)
1,165
321
14
363
22
106
398
1,113
1,224
6.8
15
27
31
46
5.3
276
100
18
56
22
30
18
19
39
350
5.5
0.3
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1
2
3
APPENDIX B. ASSESSMENTS BY OTHER NATIONAL
AND INTERNATIONAL HEALTH AGENCIES
4
5
Table B-l. Health assessments and regulatory limits by other national and
international agencies
Organization
Toxicity Value or Determination
Non-cancer: oral value
Cal/EPA(2010)
The concentration of 4 ng/L (Acceptable Daily Dose = 1.7 x 10"B mg/kg-day) for benzo[a]pyrene
in water for noncarcinogenic effects was derived from a lowest-observed-adverse-effect level
(LOAEL) of 5 mg/kg-day for renal toxicity from Knuckles et al. (2001). an uncertainty factor of
3,000.
Non-cancer: inhalation value
WHO (2003, 1996)
Health Canada
(2010, 1998)
The guideline value for benzo[a]pyrene in drinking water of 0.7 ng/L was based on a cancer
slope factor of 0.46 (mg/kg-day)"1 derived from Neal and Rigdon (1967) and a lifetime excess
cancer risk of 10 5.
The Maximum Acceptable Concentration for benzo[a]pyrene in drinking water of 0.01 ug/L was
derived from Neal and Rigdon (1967) using a drinking water consumption rate of 1.5 L/day,
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.)
Cancer: oral value
Cal/EPA(2010)
Cancer slope factor of 2.9 (mg/kg-day)"1 derived from Gulp et al. (1998). This includes an age
sensitivity factor of 1.7.
Cancer: inhalation value
WHO (2000, 1997)
Cal/EPA(1994)
EU (2005)
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.
The inhalation unit risk of 1.1 (mg/m3)"1 was derived based on Thyssen et al. (1981).
Target value of 1 ng/m3 benzo[a]pyrene (averaged over 1 calendar year) as a marker of PAH
carcinogenic risk. Does not include information for how target value was derived.
Cancer characterization
IARC (2010)
NTP (2011)
Cal/EPA(2010)
Health Canada
(1998)
Carcinogenic to humans (Group 1) (based on mechanistic data).
"Reasonably anticipated to be a human carcinogen." (First classified in 1981.)
"Sufficient reason for concern regarding the carcinogenic potential of this toxicant in humans."
Probably carcinogenic to man.
6
7
8
CalEPA = California Environmental Protection Agency; EU = European Union; IARC = International Agency for
Research on Cancer; NTP = National Toxicology Program; WHO = World Health Organization.
This document is a draft for review purposes only and does not constitute Agency policy.
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i APPENDIX C LITERATURE SEARCH STRATEGY
2 KEYWORDS
3
4
Table C-l. Literature search strategy keywords for benzo[a]pyrene
Database
Set ft
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 "l,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"[maj'r]) OR
("benzo a pyrene/metabolism"[MeSH Terms] AND (humans[MeSH Terms] OR
animals[MeSH Terms]))))) OR (("Benzo a pyrene"[tw] OR "Benzo d, e, f
chrysene"[tw] OR "Benzo def chrysene"[tw] OR "3,4-Benzopyrene"[tw] OR "1,2-
Benzpyrene"[tw] OR "3,4-BP"[tw] OR "Benz(a)pyrene"[tw] OR "3,4-
Benzpyren"[tw] OR "3,4-Benzpyrene"[tw] OR "4,5-Benzpyrene"[tw] OR "6,7-
Benzopyrene"[tw] OR Benzopirene[tw] OR "benzo[alpha]pyrene"[tw] OR
(("B(a)P"[tw] OR BaP[tw]) AND (pyrene*[tw] OR benzopyrene*[tw] OR pah[tw] OR
pahs[tw] OR polycyclic aromatic hydrocarbon[tw] OR polycyclic aromatic
hydrocarbons[tw])))AND ("Benzopyrenes/adverse effects"[MeSH Terms] OR
"Benzopyrenes/antagonistsand inhibitors"[MeSH Terms] OR
5,184
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"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:
I960's-2/14/2012
Search date:
2/14/2012
IB
(((50-32-8 [rn] OR "benzo a pyrene" OR "benzo d e f 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 "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") 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 lymphocyte OR lymphocytes OR lymphocytosis OR lymphokines
OR monocyte OR monocytes) OR (abnormal OR abnormalities OR abnormality OR
abort OR aborted OR abortion OR aborts OR cleft OR clefts OR development OR
developmental OR embryo OR embryologic OR embryology OR embryonic OR
embryos OR fertile OR fertilities OR fertility OR fetal OR fetus OR fetuses OR foetal
OR foetus OR foetuses OR gestation OR gestational OR infertile OR infertility OR
malform OR malformation OR malformations OR malformed OR malforms OR
neonatal OR neonatally OR neonate OR neonates OR newborn OR newborns OR
ova OR ovaries OR ovary OR ovum OR perinatal OR perinatally OR placenta OR
placental OR placentas OR postnatal OR postnatally OR pregnancies OR pregnancy
OR pregnant OR prenatal OR prenatally OR reproduction OR reproductive OR
sperm OR spermatid OR spermatids OR spermatocidal OR spermatocyte OR
spermatocytes OR spermatogenesis OR spermatogonia OR spermatozoa OR sterile
OR sterility OR teratogen OR teratogenesis OR teratogenic OR teratogenicities OR
teratogenicity OR teratogens OR weaned OR weaning OR weanling OR weanlings
OR zygote OR zygotes) OR (ames OR aneuploid OR aneuploidy OR chromosomal
OR chromosome OR chromosomes OR clastogen OR clastogenesis OR clastogenic
OR clastogenicities OR clastogenicity OR clastogens OR cytogenesis OR
cytogenetic OR cytogenetics OR dna OR dominant lethal OR gene OR genes OR
genetic OR genotoxic OR genotoxicities OR genotoxicity OR genotoxin OR
genotoxins OR hyperploid OR hyperploidy OR micronuclei OR micronucleus OR
mitotic OR mutagen OR mutagenesis OR mutagenicities OR mutagenicity OR
25,621
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TSCATS, TSCATS2,
TSCA recent
notices
Date range:
TSCATS2 2000-
2/14/2012;
TSCATS, TSCA
notices no limit
Search date:
2/14/2012
Toxcenter
Date range: 2000-
2/14/2012
Search date:
2/14/2012
1C
1D1
mutagens OR mutate OR mutated OR mutating OR mutation OR mutations OR
recessive lethal OR sister chromatid) OR (cancer OR cancerous OR cancers OR
carcinogen OR carcinogenesis OR carcinogenic OR carcinogenicities OR
carcinogenicity OR carcinogens OR carcinoma OR carcinomas OR cocarcinogen OR
cocarcinogenesis OR cocarcinogenic OR cocarcinogens OR lymphoma OR
lymphomas OR neoplasm OR neoplasms OR neoplastic OR oncogene OR
oncogenes OR oncogenic OR precancerous OR tumor OR tumorigenesis OR
tumorigenic OR tumorigenicities OR tumorigenicity OR tumors OR tumour OR
tumourigenesis OR tumourigenic OR tumourigenicity OR tumours))
50-32-8
((50-32-8 NOT (patent/dt OR tscats/fs)) AND (py>2007 OR ed>20080930) AND
(chronic OR immunotox? OR neurotox? OR toxicokin? OR biomarker? OR
neurolog? OR pharmacokin? OR subchronic OR pbpk OR epidemiology/st,ct, it)
OR acute OR subacute OR Id50# OR Ic50# OR (toxicity OR adverse OR
poisoning)/st,ct,it OR inhal? OR pulmon? OR nasal? OR lung? OR respir? OR
occupation? OR workplace? OR worker? OR oral OR orally OR ingest? OR gavage?
OR diet OR diets OR dietary OR drinking(w)water OR (maximum and
concentration? and (allowable OR permissible)) OR (abort? OR abnormalit? OR
embryo? OR cleft? OR fetus? OR foetus? OR fetal? OR foetal? OR fertil? OR
malform? OR ovum OR ova OR ovary OR placenta? OR pregnan? OR prenatal OR
perinatal? OR postnatal? OR reproduc? OR steril? OR teratogen? OR sperm OR
spermac? OR spermag? OR spermati? OR spermas? OR spermatob? OR
spermatoc? OR spermatog? OR spermatoi? OR spermatol? OR spermator? OR
spermatox? OR spermatoz? OR spermatu? OR spermi? OR spermo? OR neonat?
OR newborn OR development OR developmental? OR zygote? OR child OR
children OR adolescen? OR infant OR wean? OR offspring OR age(w)factor? OR
dermal? OR dermis OR skin OR epiderm? OR cutaneous? OR carcinog? OR
cocarcinog? OR cancer? OR precancer? OR neoplas? OR tumor? OR tumour? OR
oncogen? OR lymphoma? OR carcinom? OR genetox? OR genotox? OR mutagen?
OR genetic(w)toxic? OR nephrotox? OR hepatotox? OR endocrin? OR estrogen?
OR androgen? OR hormon? OR rat OR rats OR mouse OR mice OR muridae OR dog
OR dogs OR rabbit? OR hamster? OR pig OR pigs OR swine OR porcine OR goat OR
goats OR sheep OR monkey? OR macaque? OR marmoset? OR primate? OR
mammal? OR ferret? OR gerbil? OR rodent? OR lagomorpha OR baboon? OR
bovine OR canine OR cat OR cats OR feline OR pigeon? OR occupation? OR
worker? OR epidem?) AND (biosis/fs OR (caplus/fs AND (rat OR rats OR mouse OR
mice OR guinea pig OR muridae OR dog OR dogs OR rabbit? OR hamster? OR pig
OR pigs OR swine OR porcine OR goat OR goats OR sheep OR monkey? OR
macaque? OR marmoset? OR primate? OR mammal? OR ferret? OR gerbil? OR
rodent? OR lagomorpha OR baboon? OR bovine OR canine OR cat OR cats OR
feline OR pigeon? OR occupation? OR worker? OR epidem? OR human? OR
hominidae OR mammal? OR subject? OR patient? OR genotox? OR mutat? OR
mutag?)))) OR (((50-32-8 NOT (patent/dt OR tscats/fs)) NOT (py>2007 OR
ed>20080930)) AND py>1999 AND (((caplus/fs OR biosis/fs) AND (cancer? OR
carcinog? OR carcinom? OR cocarcinog? OR lymphoma? OR neoplas? OR
oncogen? OR precancer? OR tumor? OR tumour?)/ti,ct,st,it OR (ames assay OR
ames test OR aneuploid? OR chromosom? OR clastogen? OR cytogen? OR dna OR
dominant lethal OR genetic OR gene? OR genotox? OR hyperploid? OR
micronucle? OR mitotic OR mutagen? OR mutat? OR recessive lethal OR sister
62 TSCATS
(health effects)
0 TSCATS2
1 recent notices
4,344
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chromatid)/ti,ct,st,it OR (brain OR cerebral OR cognition OR cognitive OR
encephal? OR nerve? OR nervous OR neural OR neurolog? OR neuron? OR
neurop? OR neurotox? OR spinal cord)/ti,ct,st,it OR (antibod? OR antigen? OR
autoimmun? OR cytokine? OR granulocyte? OR immun? OR inflamm? OR
interferon? OR interleukin? OR leukocyte? OR lymph? OR lymphocyt? OR
monocyt?)/ti,ct,st,it OR (abnormal? OR abort? OR cleft? OR development OR
developmental OR embryo? OR endocrine OR fertil? OR fetal? OR fetus? OR
foetal? OR foetus? OR gestation? OR infertil? OR malform? OR neonat? OR
newborn? OR ova OR ovaries OR ovary OR ovum)/ti,ct,st,it OR (perinatal? OR
placenta? OR postnatal? OR pregnan? OR prenatal? OR reproduc? OR sperm? OR
steril? OR teratogen? OR wean? OR zygote?)/ti,ct,st,it) OR ((chronic OR
immunotox? OR neurotox? OR toxicokin? OR biomarker? OR neurolog? OR
pharmacokin? OR subchronic OR pbpk OR epidemiology/st,ct, it) OR acute OR
subacute OR Id50# OR Ic50# OR (toxicity OR adverse OR poisoning)/st,ct,it OR
inhal? OR pulmon? OR nasal? OR lung? OR respir? OR occupation? OR workplace?
OR worker? OR oral OR orally OR ingest? OR gavage? OR diet OR diets OR dietary
OR drinking(w)water OR (maximum and concentration? and (allowable OR
permissible)) OR (abort? OR abnormalit? OR embryo? OR cleft? OR fetus? OR
foetus? OR fetal? OR foetal? OR fertil? OR malform? OR ovum OR ova OR ovary
OR placenta? OR pregnan? OR prenatal OR perinatal? OR postnatal? OR reproduc?
OR steril? OR teratogen? OR sperm OR spermac? OR spermag? OR spermati? OR
spermas? OR spermatob? OR spermatoc? OR spermatog? OR spermatoi? OR
spermatol? OR spermator? OR spermatox? OR spermatoz? OR spermatu? OR
spermi? OR spermo? OR neonat? OR newborn OR development OR
developmental? OR zygote? OR child OR children OR adolescen? OR infant OR
wean? OR offspring OR age(w)factor? OR dermal? OR dermis OR skin OR epiderm?
OR cutaneous? OR carcinog? OR cocarcinog? OR cancer? OR precancer? OR
neoplas? OR tumor? OR tumour? OR oncogen? OR lymphoma? OR carcinom? OR
genetox? OR genotox? OR mutagen? OR genetic(w)toxic? OR nephrotox? OR
hepatotox? OR endocrin? OR estrogen? OR androgen? OR hormon? OR rat OR rats
OR mouse OR mice OR muridae OR dog OR dogs OR rabbit? OR hamster? OR pig
OR pigs OR swine OR porcine OR goat OR goats OR sheep OR monkey? OR
macaque? OR marmoset? OR primate? OR mammal? OR ferret? OR gerbil? OR
rodent? OR lagomorpha OR baboon? OR bovine OR canine OR cat OR cats OR
feline OR pigeon? OR occupation? OR worker? OR epidem?) AND (cancer? OR
carcinog? OR carcinom? OR cocarcinog? OR lymphoma? OR neoplas? OR
oncogen? OR precancer? OR tumor? OR tumour?) OR (ames assay OR ames test
OR aneuploid? OR chromosom? OR clastogen? OR cytogen? OR dna OR dominant
lethal OR genetic OR gene? OR genotox? OR hyperploid? OR micronucle? OR
mitotic OR mutagen? OR mutat? OR recessive lethal OR sister chromatid) OR
(brain OR cerebral OR cognition OR cognitive OR encephal? OR nerve? OR nervous
OR neural OR neurolog? OR neuron? OR neurop? OR neurotox? OR spinal cord)
OR (antibod? OR antigen? OR autoimmun? OR cytokine? OR granulocyte? OR
immun? OR inflamm? OR interferon? OR interleukin? OR leukocyte? OR lymph?
OR lymphocyt? OR monocyt?) OR (abnormal? OR abort? OR cleft? OR
development OR developmental OR embryo? OR endocrine OR fertil? OR fetal?
OR fetus? OR foetal? OR foetus? OR gestation? OR infertil? OR malform? OR
neonat? OR newborn? OR ova OR ovaries OR ovary OR ovum) OR (perinatal? OR
placenta? OR postnatal? OR pregnan? OR prenatal? OR reproduc? OR sperm? OR
steril? OR teratogen? OR wean? OR zygote?) AND (medline/fs OR biosis/fs OR
(caplus/fs AND (rat OR rats OR mouse OR mice OR guinea pig OR muridae OR dog
OR dogs OR rabbit? OR hamster? OR pig OR pigs OR swine OR porcine OR goat OR
goats OR sheep OR monkey? OR macaque? OR marmoset? OR primate? OR
mammal? OR ferret? OR gerbil? OR rodent? OR lagomorpha OR baboon? OR
bovine OR canine OR cat OR cats OR feline OR occupation? OR worker? OR
epidem? OR human? OR hominidae OR mammal? OR subject? OR patient? OR
genotox? OR mutat? OR mutag?))))))
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Combined
Reference Set
1
(duplicates eliminated through electronic screen)
20,700
Secondary Refinement
Combined
Reference Set with
Additional Terms
Applied
2
forestomach* OR tongue* OR (auditory AND canal*) OR (ear* AND canal*) OR
esophagus* OR esophageal* OR larynx* OR laryngeal* OR pharynx* OR
pharyngeal* OR ((lung* OR pulmonary OR skin*) AND (neoplasm* OR tumor* OR
tumour* OR papilloma* OR carcinoma*)) OR leukemia* OR leukaemia* OR
sperm* OR testic* OR fertilit*OR infertilit* OR testosterone OR ((testis OR testes)
AND (weight* OR mass*)) OR epididymis* OR epididymal* OR seminiferous OR
((cervical* OR cervix*) AND hyperplasia*) OR ovary OR ovaries OR ovarian OR
primordial OR corpora lutea OR corpus luteum OR estrous* OR estrus* OR
thymus* OR spleen* OR spleno* OR immunoglobulin* OR immunoglobin* OR
((immune OR immun*) AND (suppress* OR immunosuppress*)) OR (functional
AND observational AND battery) OR neurobehavioral*OR neurobehavioural* OR
rotarod* OR nerve* AND conduction* OR locomotor* OR neuromuscular* OR
weight* OR neurodevelopment* OR ((neuro* OR brain*) AND (development* OR
developing)) OR intelligence* OR cognition* OR cognitive* OR learn* OR memory
OR righting*
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1
2
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i APPENDIX D. INFORMATION IN SUPPORT OF
2 HAZARD IDENTIFICATION AND DOSE-RESPONSE
3 ANALYSIS
4 D.I. TOXICOKINETICS
5 D.I.I. Overview
6 Benzo[a]pyrene is absorbed following exposure by oral, inhalation, and dermal routes. The
7 rate and extent of absorption are dependent upon the exposure medium. The presence of
8 benzo[a]pyrene in body fat, blood, liver, and kidney and the presence of benzo[a]pyrene
9 metabolites in serum and excreta demonstrate wide systemic tissue distribution. Benzo[a]pyrene
10 metabolism occurs in essentially all tissues, with high metabolic capacity in the liver and significant
11 metabolism in tissues at the portal of entry (lung, skin, and gastrointestinal [GI] tract) and in
12 reproductive tissues. Stable metabolic products identified in body tissues and excreta are very
13 diverse and include phenols, quinones, and dihydrodiols. These classes of metabolites are typically
14 isolated as glucuronide or sulfate ester conjugates in the excreta, but can also include glutathione
15 conjugates formed from quinones or intermediary epoxides. The primary route of metabolite
16 elimination is in the feces via biliary excretion, particularly following exposure by the inhalation
17 route. To a lesser degree, benzo[a]pyrene metabolites are eliminated via urine. Overall,
18 benzo[a]pyrene is eliminated quickly with a biological half-life of several hours.
19 D.1.2. Absorption
20 The absorption of benzo[a]pyrene has been studied in humans and laboratory animals for
21 inhalation, ingestion, and dermal exposure. In the environment, human exposure to
22 benzo[a]pyrene predominantly occurs via contact with insoluble carbonaceous particles (e.g., soot,
23 diesel particles) to which organic compounds, such as PAHs, are adsorbed.
24 Studies of workers occupationally exposed to benzo[a]pyrene have qualitatively
25 demonstrated absorption via inhalation by correlating concentrations of benzo[a]pyrene in the air
26 and benzo[a]pyrene metabolites in the exposed workers' urine. Occupational exposures to
27 benzo[a]pyrene measured with personal air samplers were correlated to urine concentrations of
28 benzo[a]pyrene-9,10-dihydrodiol, a specific metabolite of benzo[a]pyrene, in 24-hour aggregate
29 urine samples by Grimmer et al. (1994). The amount of benzo[a]pyrene extracted from personal
30 air monitoring devices (a surrogate for ambient PAHs) of coke oven workers were correlated with
31 r-7,t-8,9,c 10 tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-benzo[a]pyrene-tetrol, a
32 specific metabolite of benzo[a]pyrene) in the workers' urine by Wuetal. (2002). In both of these
33 studies, only a very small fraction (<1%) of the inhaled benzo[a]pyrene was recovered from urine,
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1 consistent with studies in animals that find urine is not a major route of elimination for
2 benzo[a]pyrene (as described in the excretion section below). These occupational studies cannot
3 be used to quantify absorption through inhalation-only exposure in humans because the
4 persistence of benzo[a]pyrene-contaminated particulate matter on surfaces and food may lead to
5 exposures via additional routes (Bostrom et al.. 2002). Nevertheless, the observation of
6 benzo[a]pyrene metabolites in excreta of exposed humans provides qualitative evidence for
7 benzo[a]pyrene absorption, at least some of which is likely to occur via inhalation. This conclusion
8 is supported by studies in experimental animals, which indicate that benzo[a]pyrene is readily
9 absorbed from carbonaceous particles following inhalation exposure [Gerde etal.. 2001: Hood et
10 al.. 20001
11 Results from studies of animals following intratracheal instillation of benzo[a]pyrene
12 provide supporting, quantitative evidence that absorption by the respiratory tract is rapid [Gerde et
13 al.. 1993: Bevan and Ulman. 1991: Weyand and Bevan. 1987.1986]. Following intratracheal
14 instillation of 1 ug tritiated benzo[a]pyrene/kg dissolved in triethylene glycol to Sprague-Dawley
15 rats, radioactivity rapidly appeared in the liver (reaching a maximum of about 21% of the
16 administered dose within 10 minutes). Elimination of radioactivity from the lung was biphasic,
17 with elimination half-times of 5 and 116 minutes (Weyand and Bevan. 1986). In bile-cannulated
18 rats, bile collected for 6 hours after instillation accounted for 74% of the administered radioactivity
19 (Weyand and Bevan. 1986). The results are consistent with rapid and extensive absorption by the
20 respiratory tract and rapid entry into hepatobiliary circulation following intratracheal instillation.
21 The respiratory tract absorption may also be affected by the vehicle, since higher amounts of
22 benzo[a]pyrene were excreted in bile when administered with hydrophilic triethylene glycol than
23 with lipophilic solvents ethyl laurate or tricaprylin (Bevan and Ulman. 1991]. Particle-bound
24 benzo[a]pyrene deposited in the respiratory tract is absorbed and cleared more slowly than the
25 neat compound (Gerde etal., 2001].
26 Studies conducted to assess levels of benzo[a]pyrene metabolites or benzo[a]pyrene-
27 deoxyribonucleic acid (DNA] adduct levels in humans exposed to benzo[a]pyrene by the oral route
28 are not adequate to develop quantitative estimates of oral bioavailability. The concentration of
29 benzo[a]pyrene was below detection limits (<0.1 ug/person] in the feces of eight volunteers who
30 had ingested broiled meat containing approximately 8.6 ug of benzo[a]pyrene (Hechtetal.. 1979].
31 However, studies in laboratory animals demonstrate that benzo[a]pyrene is absorbed via ingestion.
32 Studies of rats and pigs measured the oral bioavailability of benzo[a]pyrene in the range of 10-40%
33 fCavretetal.. 2003: Ramesh etal.. 2001b: Fothetal.. 1988: Hechtetal.. 19791 The absorption of
34 benzo[a]pyrene may depend on the vehicle. Intestinal absorption of benzo[a]pyrene was enhanced
35 in rats when the compound was solubilized in lipophilic compounds such as triolein, soybean oil,
36 and high-fat diets, as compared with fiber- or protein-rich diets (O'Neill etal.. 1991: Kawamura et
37 al.. 1988]. Aqueous vehicles, quercetin, chlorogenic acid, or carbon particles reduced biliary
38 excretion of benzo[a]pyrene, while lipid media such as corn oil increased it (Stavric and Klassen.
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1 1994]. The addition of wheat bran to the benzo[a]pyrene-containing diets increased fecal excretion
2 ofbenzo[a]pyrene [Mirvishetal.. 1981].
3 Studies of benzo[a]pyrene metabolites or DNA adducts measured in humans exposed
4 dermally to benzo[a]pyrene-containing PAH mixtures demonstrate that benzo[a]pyrene is
5 absorbed dermally. One study of dermal absorption in volunteers found absorption rate constants
6 ranging from 0.036 to 0.135/hour over a 45-minute exposure, suggesting that 20-56% of the dose
7 would be absorbed within 6 hours [VanRooij etal.. 1993]. Dermal absorption rates varied 69%
8 between different anatomical sites (forehead, shoulder, volar forearm, palmar side of the hand,
9 groin, and ankle] and only 7% between different individual volunteers [VanRooij etal.. 1993].
10 Metabolism is also an important determinant of permeation, with very low rates observed in
11 nonviable skin [Kao etal., 1985]. The overall absorbed amount of benzo[a]pyrene in explanted
12 viable skin samples from tissue donors (maintained in short-term organ cultures] exposed for
13 24 hours ranged from 0.09 to 2.6% of the dose (Wester etal.. 1990: Kao etal.. 1985]. Similar
14 amounts of penetration were measured in skin samples from other species including marmosets,
15 rats, and rabbits (Kao etal.. 1985]. Skin from mice allowed more of the dose to penetrate (>10%],
16 while that of guinea pig let only a negligible percentage of the dose penetrate (Kao etal.. 1985].
17 The vehicle for benzo[a]pyrene exposure is an important factor in skin penetration.
18 Exposure of female Sprague-Dawley rats and female rhesus monkeys topically to benzo[a]pyrene in
19 crude oil or acetone caused approximately fourfold more extensive absorption than
20 benzo[a]pyrene in soil (Wester et al.. 1990: Yang et al.. 1989]. The viscosity of oil product used as a
21 vehicle also changed skin penetration with increased uptake of benzo[a]pyrene for oils with
22 decreased viscosity (Potter etal.. 1999]. Soil properties also greatly impact dermal absorption.
23 Reduced absorption of benzo[a]pyrene occurs with increasing organic carbon content of the soil
24 and increased soil aging (i.e., contact time between soil and chemical] (Turkalletal.. 2008: Roy and
25 Singh. 2001: Yang etal.. 1989].
26 D.1.3. Distribution
27 No adequate quantitative studies of benzo[a]pyrene tissue distribution in exposed humans
28 were identified. Obanaetal. (1981] observed low levels of benzo[a]pyrene in liver and fat tissues
29 from autopsy samples. However, prior exposure histories were not available for the donors.
30 Nevertheless, the identification of benzo[a]pyrene metabolites or DNA adducts in tissues and
31 excreta of PAH-exposed populations suggest that benzo[a]pyrene is widely distributed.
32 Distribution of benzo[a]pyrene has been studied in laboratory animals for multiple routes
33 of exposure, including inhalation, ingestion, dermal, and intravenous (i.v.]. Exposure to
34 benzo[a]pyrene in various species (Sprague-Dawley rats, Gunn rats, guinea pigs, and hamsters]
35 results in wide distribution throughout the body and rapid uptake into well-perfused tissues (i.e.,
36 lung, kidney, and liver] (Weyand and Bevan, 1987,1986]. Benzo[a]pyrene and its metabolites are
37 distributed systemically after administration via many routes of administration including
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1 inhalation (or intratracheal instillation), oral, i.v., and dermal exposures [Saunders etal., 2002: Moir
2 etal.. 1998: NeubertandTapken. 1988: WeyandandBevan. 1987.1986: Morse and Carlson. 19851.
3 Intratracheal instillation of radiolabeled benzo[a]pyrene in mice resulted in increased radioactivity
4 in lung-associated lymph nodes, suggesting distribution of benzo[a]pyrene or its metabolites via
5 the lymph [Schnizlein et al.. 1987). Rats with biliary cannulas had high excretion of benzo[a]pyrene
6 and benzo[a]pyrene metabolites in bile. The benzo[a]pyrene thioether and glucuronic acid-
7 conjugated metabolites in intestines indicated enterohepatic recirculation of benzo[a]pyrene and
8 benzo[a]pyrene metabolites [Weyand and Bevan. 1986]. The vehicle for delivery of inhalated
9 benzo[a]pyrene impacts the distribution, with aerosolized benzo[a]pyrene more readily absorbed
10 directly in the respiratory tract than particle-adsorbed benzo[a]pyrene (which is cleared by the
11 mucociliary and then ingested) (Sun etal., 1982]. Exposure of pregnant rats and mice to
12 benzo[a]pyrene via inhalation and ingestion showed a wide tissue distribution of benzo[a]pyrene,
13 consistent with other studies, and demonstrated placental transfer of benzo[a]pyrene and its
14 metabolites (Withey etal.. 1993: Neubert and Tapken. 1988: Shendrikova and Aleksandrov. 1974].
15 The reactive metabolites of benzo[a]pyrene are also transported in the blood and may be
16 distributed to tissues incapable of benzo[a]pyrene metabolism. Serum of benzo[a]pyrene-treated
17 mice incubated with splenocytes or salmon sperm DNA resulted in adduct formation, suggesting
18 that reactive benzo[a]pyrene metabolites were systemically distributed and available for
19 interaction with target tissues (Ginsberg and Atherholt. 1989].
20 D.1.4. Metabolism
21 The metabolic pathways of benzo[a]pyrene (Figure D-l] and variation in species, strains,
22 organ system, age, and sex have been studied extensively with in vitro and in vivo experiments. In
23 addition, there have been numerous studies of exposed humans or animals with subsequent
24 detection of benzo[a]pyrene metabolites in tissues or excreta. For example, elevated frequency of a
25 detected urinary metabolite (7,8,9,10-tetrol] was observed in patients treated with coal tar
26 medication (Bowman etal.. 1997]. demonstrating extensive metabolism of benzo[a]pyrene in
27 humans.
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1
2
3
BaP7,8-transdio
l-OH BaP
9-OH BaP
t
BaPl,2-oxide BaP 2,3-oxide
tl
3-OH BaP
BaP 9,10-transdiol BaP 9,10-oxide
Benzo[a]pyrene
V
3-OH BaP
BaP 7,8-oxitle
OH 7-OHBaP
BaP 7,8-diol-9,10-epoxide
^ \.
f BaP 6,12 1
[semiquinonej
\, BaP 1,6-hydroquinone BaP 1,6 semiquinone
6-oxo-BaP radical-41 _ -. BaP 1,6 quinone
|_semlquinonej
*^
BaP6,12-quinone BaPG.lZ-hydraqulnone BaP 3,6-hydroquinone
BaP 3,6-quinone
Source: Miller and Ramos (2001).
Figure D-l. Metabolic pathways for benzo[a]pyrene.
4 Phase I metabolism results in a number of reactive metabolites such as epoxides,
5 dihydrodiols, phenols, quinones, and their various combinations that are likely to contribute to the
6 toxic effects of benzo[a]pyrene (e.g., phenols, dihydrodiols, epoxides and quinones). The Phase II
7 metabolism of benzo[a]pyrene metabolites protects the cells and tissues from the toxic effects of
8 benzo[a]pyrene phenols, dihydrodiols and epoxides by converting them to water soluble products
9 that are eliminated. Also, the Phase II metabolism of some benzo[a]pyrene dihydrodiols prevents
10 them from further bioactivation to reactive forms that bind to cellular macromolecules.These
11 metabolic process include glutathione conjugation of diol epoxides, sulfation and glucuronidation of
12 phenols, and reduction of quinones by NADPH:quinone oxidoreductase (NQO). Numerous reviews
13 on the metabolism of benzo[a]pyrene are available [Miller and Ramos. 2001: IPCS. 1998: ATSDR.
14 1995: ConneyetaL 1994: Grover. 1986: Levin etal.. 1982: Gelboin. 1980). Key concepts have been
15 adapted largely from these reviews and supplemented with recent findings.
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1 Phase I Metabolism
2 Phase I reactions of benzo[a]pyrene are catalyzed primarily by cytochrome P450 (CYP450)
3 and produce metabolites including epoxides, dihydrodiols, phenols, and quinones (Figure D-2). The
4 first step of Phase I metabolism is the oxidation of benzo[a]pyrene that forms a series of epoxides,
5 the four major forms of which are the 2,3-, 4,5-, 7,8-, and 9,10-isomers [Gelboin. 1980). Once
6 formed, these epoxides may undergo three different routes of metabolism: (1] spontaneous
7 rearrangement to phenols, (2) hydration to trans-dihydrodiols catalyzed by microsomal epoxide
8 hydrolase, or (3) the Phase II detoxification of binding with glutathione (either spontaneously or
9 catalyzed by cytosolic glutathione-S-transferases (GSTs) flARC. 198311 The metabolism of
10 benzo[a]pyrene to phenols results in five phenol isomers (1-, 3-, 6-, 7, and 9-OH benzo[a]pyrene)
11 (Pelkonen and Nebert. 1982]. Four benzo[a]pyrene epoxides (2,3-, 4,5-, 7,8-, and 9,10-) are
12 hydrated to trans-dihydrodiols. Benzo[a]pyrene-7,8-diol (formed from benzo[a]pyrene-7,8-oxide)
13 has been the focus of much of the study of benzo[a]pyrene metabolism. Benzo[a]pyrene-7,8-diol is
14 the metabolic precursor to the potent DNA-binding metabolite, benzo[a]pyrene-7,8-diol-9,10-
15 epoxide (BPDE). BPDE is formed from trans-benzo[a]pyrene 7,8-diol by multiple mechanisms
16 including catalysis by cytochromes (CYPs) (Grover. 1986: Deutschetal.. 1979]. myeloperoxidase
17 (Mallet etal.. 1991). or prostaglandin h synthase (also known as cyclooxygenase) (Marnett. 1990).
18 and lipid peroxidation (Byczkowski and Kulkarni, 1990]. The diolepoxides can react further by
19 spontaneously hydrolyzing to tetrols (Hall and Grover. 1988].
20 The metabolism of benzo[a]pyrene proceeds with a high degree of stereoselectivity. Liver
21 microsomes from rats stereospecifically oxidize the 7,8-bond of benzo[a]pyrene to yield almost
22 exclusively the (+]-benzo[a]pyrene-(7,8]-oxide (see Figure D-2]. Each enantiomer of
23 benzo[a]pyrene-7,8-oxide is stereospecifically converted by epoxide hydrolase (EH] to a different
24 stereoisomeric trans dihydrodiol. The (+]-benzo[a]pyrene-7,8-oxide is preferentially hydrated to
25 the (-]-trans-benzo[a]pyrene-7,8-dihydrodiol enantiomer by rat GYP enzymes and the (-]-trans-
26 benzo[a]pyrene-7,8-dihydrodiol is preferentially oxidized by GYP enzymes to (+]-benzo[a]pyrene-
27 7R,8S-diol-9S,10R-epoxide [(+]-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE]], which is the
28 most potent carcinogen among the four stereoisomers (Figure D-2]. Formation of these
29 stereoisomers does not occur at equimolar ratios, and the ratios differ between biological systems.
30 For example, a study in rabbit livers demonstrated that purified microsomes oxidized the
31 (-]-benzo[a]pyrene-7,8-dihydrodiol to isomeric diol epoxides in a ratio ranging from 1.8:1 to 11:1
32 in favor of the (+]-anti-BPDE isomer (Deutsch etal.. 1979].
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1
2
3
'+)-BP-7R,8S-diol-9S,10R-epoxide
Function (+)antiBPDE
ase System
(-)-BP-7R,8S-diol-9R,10S-epoxide
(-) syn BPDE
+)-BP-7S,8R-diol-9S,10R-epoxide
(+) syn BPDE
Epoxide Hydrolase H0^^^ Oxidase System
OH
(-)-BP-7,8-oxide (+)-BP-7]B-diol
(-)-BP-7S,8R-diol-9R,10S-epoxide
(-) anti BPDE
Source: Grover (1986).
Figure D-2. The stereospecific activation of benzo[a]pyrene.
4 Several studies have attempted to determine which GYP isozyme is predominantly
5 responsible for the metabolism of benzo[a]pyrene. Dermal administration of tritiated
6 benzo[a]pyrene to mice that have an aryl hydrocarbon (Ah) receptor (AhR) knock-out (AhR-/-)
7 had significantly decreased formation of (+)-anti-BPDE-DNA adducts compared to wild type (WT)
8 and 1B1-/- mice [Kleiner et al.. 2004]. Gavage administration of benzo[a]pyrene in AhR knock-out
9 mice found that the AhR-/- mice (with lower levels of CYP1A1) had higher levels of protein
10 adducts and unmetabolized benzo[a]pyrene than the AhR+/+ or +/- mice (Sagredo etal.. 2006).
11 Similarly, CYP1A1 (-/-) knock-out mice administered benzo[a]pyrene in feed for 18 days had
12 higher steady-state blood levels of benzo[a]pyrene and benzo[a]pyrene-DNA adducts (Uno etal..
13 2006]. These findings establish important roles inbenzo[a]pyrene metabolism for CYP1A1, but the
14 relationship is not clear between the GYP enzymes and biological activation or detoxification.
15 Another important factor in evaluating variability in the metabolic activation of
16 benzo[a]pyrene by CYP450s is the effect of functional polymorphisms, which has been the subject
17 of numerous reviews (e.g.. Wormhoudtetal.. 1999). RecombinantCYPlAl allelic variants
18 produced BPDE with generally lower catalytic activity and Km values than the WT allele (Schwarz
19 etal.. 2001]. However, the formation of diol epoxides is stereospecific, with the allelic variants
20 producing about 3 times the amount of (±)-anti-BPDE isomers as compared to the stereoisomer,
21 (±)-syn-BPDE (Schwarz etal., 2001]. In a study of occupational exposures to benzo[a]pyrene, no
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1 relationship was observed between benzo[a]pyrene metabolite formation and the CYP1A1 Mspl
2 polymorphism [Wu etal.. 2002].
3 Another pathway of benzo[a]pyrene metabolism is the conversion of benzo[a]pyrene to
4 6-OH benzo[a]pyrene which can be further oxidized into quinones, primarily the 1,6-, 3,6-, and
5 6,12- isomers. Trans-benzo[a]pyrene-7,8-dihydrodiol can be converted by aldo-keto reductases
6 (AKR) to 7,8-dihydroxybenzo[a]pyrene (benzo[a]pyrene-7,8-catechol) which auto-oxidizes to
7 benzo[a]pyrene-7,8-quinone (BPQ). BPQ can undergo redox cycling in the presence of cellular
8 reducing equivalents. This reaction pathway produces reactive oxygen species (ROS) including
9 peroxide anion radicals, benzo[a]pyrene semiquinone radicals, hydroxyl radicals, and HzOz, which
10 in turn can causes extensive DNA fragmentation [Penning etal.. 1999: Flowers etal.. 1997: Flowers
11 etal., 1996]. 6-Hydroxybenzo[a]pyrene can be oxidized into 6-oxo-benzo[a]pyrene semi-quinone
12 radical and further metabolized into 1,6-, 3,6-, or 6,12-quinones spontaneously, or catalytically by
13 prostaglandin endoperoxide synthetase [Elingetal.. 1986]. The GYP and AKR enzymes both can
14 metabolize trans-benzo[a]pyrene-7,8-dihydrodiol to different metabolites, BPDE and BPQ.
15 Reconstituted in vitro systems of human lung cells show GYP enzymes have faster steady state
16 reaction rate constants than AKR and basal expression of AKR is higher than GYP in lung cells,
17 suggesting that AKR and GYP enzymes compete for metabolism of trans-benzo[a]pyrene-7,8-
18 dihydrodiol [Quinn and Penning. 2008].
19 Phase II Metabolism
20 The reactive products of Phase I metabolism are subject to the action of several phase II
21 conjugation and detoxification enzyme systems that display preferential activity for specific
22 oxidation products of benzo[a]pyrene. These Phase II reactions play a critical role in protecting
23 cellular macromolecules from binding with reactive benzo[a]pyrene diolepoxides, radical cations,
24 or reactive oxygen species (ROS]. Therefore, the balance between Phase I activation of
25 benzo[a]pyrene and its metabolites and detoxification by Phase II processes is an important
26 determinant of toxicity.
27 The diol epoxides formed from benzo[a]pyrene metabolism by Phase I reactions are not
28 usually found as urinary metabolites. Rather, they are detected as adducts of nucleic acids or
29 proteins or further metabolized by glutathione (GSH] conjugation, glucuronidation, and sulfation.
30 These metabolites make up a significant portion of total metabolites in excreta or tissues. For
31 example, the identified metabolites in bile 6 hours after a 2 [J.g/kg benzo[a]pyrene dose by
32 intratracheal instillation to male Sprague-Dawley rats were 49% glucuronides (quinol
33 diglucuronides or monglucuronides], 30.4% thioether conjugates, 6.2% sulfate conjugates, and
34 14.4% unconjugated metabolites [Bevan and Sadler. 1992].
35 Conjugation of benzo[a]pyrene with GSH is catalyzed by GSTs. Numerous studies using
36 human GSTs expressed in mammalian cell lines have demonstrated the ability of GST to metabolize
37 benzo[a]pyrene diol epoxides. Isolated human GSTs have significant catalytic activity toward
38 benzo[a]pyrene-derived diol epoxides and (±]anti-BPDE with variation in activity across GST
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1 isoforms [Dreij etal., 2002: Rojas etal., 1998: Robertson etal., 1986]. Benzo[a]pyrene quinones
2 can also be conjugated with glutathione [Agarwal etal., 1991: IARC, 1983]. This compelling
3 evidence for a role of GSTs in the metabolism of reactive benzo[a]pyrene metabolites has triggered
4 several molecular epidemiology studies. However, recent studies on the impact of polymorphism
5 on adduct levels in PAH-exposed human populations did not show a clear relationship between the
6 Phase I (CYP1A1, EH], or Phase II (GST] enzyme polymorphisms and formation of DNA adducts
7 [Hemminki et al.. 1997] or blood protein adducts [Pastorelli etal.. 1998].
8 Conjugation with uridine diphosphate-glucuronide catalyzed by UDP-
9 glucuronosyltransferase (UGT] enzymes is another important detoxification mechanism for
10 oxidative benzo[a]pyrene metabolites. UGT isoforms, as well as their allelic variants, are expressed,
11 and have glucuronidation activity toward, benzo[a]pyrene-derived phenols and diols in the
12 aerodigestive tract (tongue, tonsil, floor of the mouth, larynx, esophagus], but not in the lung or
13 liver [Fang and Lazarus. 2004: Zheng etal.. 2002]. UGT activity also shows significant
14 interindividual variability. Incubation of lymphocytes with benzo[a]pyrene resulted in covalent
15 binding to protein with a 143-fold interindividual variability and a statistically significant inverse
16 correlation between glucuronidation and protein binding [Hu and Wells. 2004].
17 Sulfotransferases can catalyze the formation of sulfates of benzo[a]pyrene metabolites. In
18 rat or mouse liver, cytosolic sulfotransferase (in the presence of 3'-phosphoadenosine 5'-phospho-
19 sulfate] catalyzes formation of sulfates of three benzo[a]pyrene metabolites: benzo[a]pyrene-
20 7,8,9,10-tetrahydro-7-ol, benzo[a]pyrene-7,8-dihydrodiol, andbenzo[a]pyrene-7,8,9,10-tetrol. The
21 benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol-sulfate is able to form potentially damaging DNA adducts
22 (Surh and Tannenbaum. 1995]. In human lung tissue 3-hydroxybenzo[a]pyrene conjugation to
23 sulfate produces benzo[a]pyrene-3-yl-hydrogen sulfate, a very lipid soluble compound that would
24 not be readily excreted in the urine (Cohen et al.. 1976].
25 Although not specific for benzo[a]pyrene, there is now considerable evidence that genetic
26 polymorphisms of the GST, UGT, and EH genes impart an added risk to humans for developing
27 cancer. Of some significance to the assessment of benzo[a]pyrene maybe that smoking, in
28 combination with genetic polymorphism at several gene loci, increases the risk for bladder cancer
29 (Moore etal.. 2004: Choi etal.. 2003: Park etal.. 2003] and lung cancer (Alexandrie etal.. 2004: Lin
30 etal.. 2003]. Coke oven workers (who are exposed to PAHs, including benzo[a]pyrene]
31 homozygous atthe P187S site of the NQ01 gene (an inhibitor of benzo[a]pyrene-quinone adducts
32 with DNA], or carrying the null variant of the glutathione-S-transferase Ml (GSTM1] gene, had a
33 significantly increased risk of chromosomal damage in peripheral blood lymphocytes. Meanwhile,
34 the risk was much lower than controls in subjects with a variant allele atthe HllSYsite of the EH
35 gene (Lengetal.. 2004].
36 Tissue-specific Metabolism
37 Benzo[a]pyrene metabolism has been demonstrated in vivo in laboratory animals for
38 various tissues via multiple routes including inhalation, ingestion, and dermal absorption.
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1 Metabolism of benzo[a]pyrene at the site of administration such as in the respiratory tract, the GI
2 tract or the skin impact the amount of benzo[a]pyrene and its form asbenzo[a]pyrene or one of the
3 metabolites that reach systemic circulation. Nasal instillation or inhalation of benzo[a]pyrene in
4 monkeys, dogs, rats, and hamsters resulted in the formation of dihydrodiols, phenols, quinones, and
5 tetrols in the nasal mucus and lung [Wolff etal.. 1989: Petridou-Fischer etal.. 1988: Weyand and
6 Lavoie. 1988: Weyand and Bevan. 1987.1986: DahletaL 19851 In rats, the fractions of
7 metabolites in the lung at 6 hours after instillation were: 20% unmetabolized benzo[a]pyrene, 16%
8 conjugates or polyhydroxylated compounds, 10.7% 4,5-, 7,8-, and 9,10-dihydrodiols, 9.3% 1,6-, 3,6-,
9 and 6,12-quinone, and 6.9% 3- and 9-hydroxybenzo[a]pyrene [Weyand and Bevan. 1986]. In
10 hamsters, approximately 50% of the benzo[a]pyrene instilled was metabolized in the nose (nasal
11 tissues had the highest metabolic activity per-gram of the respiratory tract tissues), and the
12 metabolites produced were similar to other species [Dahl etal.. 1985).
13 In vitro studies of human and laboratory cells and cell lines provide further quantitative and
14 mechanistic details of the metabolism of benzo[a]pyrene in the cells of the respiratory tract, skin,
15 liver, and other tissues. Tracheobronchial tissues in culture of several species (including humans,
16 mice, rats, hamsters, and bovines) were all found to metabolize benzo[a]pyrene extensively to
17 phenols, diols, tetrols, quinones, and their conjugates (Autrup etal.. 1980). The results show a high
18 degree of interindividual variability (a 33-fold difference in human bronchus, a 5-fold variation in
19 human trachea, and a 3-fold difference in bovine bronchus), but minimal variation among
20 individuals of the laboratory animal species (Autrup etal.. 1980). Human bronchial epithelial and
21 lung tissue conjugated benzo[a]pyrene metabolites to glutathione and sulfates, but not with
22 glucuronide (Kiefer etal.. 1988: Autrup etal.. 1978: Cohen etal.. 1976). Lung tissue slices exposed
23 to benzo[a]pyrene induced expression of CYP1A1 and CYP1B1 at levels 10-20 times higher than in
24 the liver (Harriganetal.. 2006) and total levels of benzo[a]pyrene-DNA adducts were
25 approximately 2-6 times greater in the lung slices than liver (Harrigan et al., 2004).
26 Benzo[a]pyrene undergoes extensive metabolism in the GI tract and liver after oral
27 administration. In rats after administration of an oral dose, the majority of benzo[a]pyrene
28 detected in organs are metabolites (Ramesh et al.. 2004: Ramesh et al.. 2001b: Yamazaki and
29 Kakiuchi. 1989). In rats administered a 100 nmol dose, greater than 90% was recovered in portal
30 blood as metabolites (Bock etal.. 1979). Orally administered benzo[a]pyrene produced strong
31 induction of CYP1A1 in the intestine of mice (Brooks etal.. 1999). DNA post-labeling studies of
32 mice administered benzo[a]pyrene by gavage demonstrated higher benzo[a]pyrene-DNA adduct
33 levels in CYP1A1(-/-) than CYP1A1(+/+) mice in small intestines fUno etal.. 20041 To compare
34 the relative roles of the liver and intestine in benzo[a]pyrene metabolism and absorption a
35 multicompartment perfusion system was developed and found that benzo[a]pyrene is extensively
36 metabolized by the intestinal Caco-2 cells and benzo[a]pyrene and its metabolites are transported
37 to the apical side of the Caco-2 cells away from the liver HepG2 cells (Choi etal.. 2004).
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1 Dermal exposure in humans and animals resulted in benzo[a]pyrene metabolism and the
2 permeation of benzo[a]pyrene in skin is linked to benzo[a]pyrene metabolism. Human skin
3 samples maintained in short-term organ culture (i.e., human epithelial tissue, samples from human
4 hair follicles, and melanocytes isolated from adult human skin) can metabolize benzo[a]pyrene into
5 dihydrodiols, phenolas, quinones, and glucuronide and sulfate conjugates [Agarwal etal.. 1991:
6 Alexandrovetal.. 1990: Hall and Grover. 1988: MerketaL 19871 Nonviable skin is unable to
7 metabolize benzo[a]pyrene (the permeation into nonviable skin is lower than viable skin) as
8 measured in a range of species including humans, rat, mouse, rabbit and marmoset (Kao etal.,
9 1985). Viable human skin samples treated with 2 [ig/cm2 [14C]-benzo[a]pyrene in acetone and
10 incubated for 24 hours produced the following percentages of benzo[a]pyrene metabolites; 52%
11 water-soluble compounds, 8% polar compounds, 17% diols, 1% phenols, 2.5% quinones, and 18%
12 unmetabolizedbenzo[a]pyrene (Kao etal.. 1985).
13 Benzo[a]pyrene that reaches systemic circulation is also metabolized by multiple tissues
14 that are targets of benzo[a]pyrene toxicity, including reproductive tissues such as prostate,
15 endometrium, cervical epithelial andstromal, and testes (Rameshetal.. 2003: Bao etal.. 2002:
16 Williams etal.. 2OOP: Melikianetal.. 1999).
17 Age-speciflc Metabolism
18 Metabolism of benzo[a]pyrene occurs in the developing fetus and in children, as indicated
19 by DNA or protein adducts or urinary metabolites (Naufal et al.. 2010: Ruchirawat et al.. 2 010: Suter
20 etal.. 2010: Mielzynska etal.. 2006: Pereraetal.. 2005a: Tang etal.. 1999: Whyatt etal.. 1998).
21 Transport of benzo[a]pyrene and benzo[a]pyrene metabolites to fetal tissues including plasma,
22 liver, hippocampus, and cerebral cortex has been demonstrated in multiple studies (McCabe and
23 Flynn, 1990: Neubert and Tapken, 1988: Shendrikova and Aleksandrov, 1974), andbenzo[a]pyrene
24 is metabolized by human fetal esophageal cell culture (Chakradeo etal.. 1993). While expression of
25 GYP enzymes are lower in fetuses and infants, the liver to body mass ratio and increased blood flow
26 to liver in fetuses and infants may compensate for the decreased expression of GYP enzymes
27 (Ginsberg etal.. 2004). Prenatal exposure to benzo[a]pyrene upregulates CYP1A1 and may
28 increase the formation of benzo[a]pyrene-DNA adducts (Wu etal.. 2003a). Activity of Phase II
29 detoxifying enzymes in neonates and children is adequate for sulfation but decreased for
30 glucuronidation and glutathione conjugation (Ginsbergetal., 2004). The conjugation of
31 benzo[a]pyrene-4,5-oxide with glutathione was approximately one-third less in human fetal than
32 adult liver cytosol (Pacifici etal.. 1988). The differential Phase I and II enzyme expression and
33 activity in the developing fetus and in children are consistent with an expectation that these
34 lifestages can be more susceptible to benzo[a]pyrene toxicity.
35 D.I.5. Elimination
36 Benzo[a]pyrene metabolites have been detected in the urine of exposed humans, but fecal
37 excretion has not been investigated in any detail. Studies of benzo[a]pyrene elimination in animals
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1 following exposure via inhalation, ingestion, and dermal routes have shown that benzo[a]pyrene is
2 excreted preferentially in the feces in multiple species of laboratory animals including rat, mice,
3 hamsters, guinea pigs, monkeys, and dogs [Wangetal.. 2003: Likhachevetal.. 1992: Wolff etal..
4 1989: Yang etal.. 1989: Petridou-Fischer etal.. 1988: Weyand and Bevan. 1987: Sun etal.. 1982:
5 Hechtetal.. 1979}. The metabolites in bile are primarily benzo[a]pyrene conjugates,
6 predominantly thioether conjugates of varying extent in different species [Weyand and Bevan.
7 1987]. Six hours after a single intratracheal instillation of benzo[a]pyrene (2 [J.g/kg] to male
8 Sprague-Dawley rats, relative metabolite levels were 31.2% diglucuronides, 30.4% thioether
9 conjugates, 17.8% monoglucuronides, 6.2% sulfate conjugates, and 14.4% unconjugated
10 metabolites [Bevan and Sadler. 1992}. Rats administered benzo[a]pyrene via i.v. excrete a larger
11 fraction in urine than via inhalation or oral exposure, suggesting an important role for
12 enterohepatic circulation of benzo[a]pyrene metabolite conjugates [Moir etal.. 1998: Weyand and
13 Bevan. 1986: Hirom etal.. 1983]. The vehicle impacts the amount of benzo [ajpyrene excreted and
14 may, in part, be due to the elimination rate or to other factors such as the absorption rate. For
15 tritiated benzo[a]pyrene administered to Sprague-Dawley rats in hydrophilic triethylene glycol,
16 70.5% of the dose was excreted into bile within 6 hours. If lipophilic solvents, ethyl laurate and
17 tricaprylin, were used as vehicles, 58.4 and 56.2% of the dose was excreted, respectively [Bevan
18 andUlman, 1991]. In addition to benzo[a]pyrene and its metabolites, adducts of benzo [a]pyrene
19 with nucleotides have also been identified as a small fraction of the administered dose in feces and
20 urine of animals. The level of BPDE adducts with guanine detected in urine of male Wistar rats was
21 dose-dependent Forty-eight hours after dosing with 100 [J.g/kg tritiated benzo[a]pyrene, 0.15% of
22 the administered benzo[a]pyrene dose was excreted in the urine as an adduct with guanine [Autrup
23 and Seremet. 1986]. Overall, the data in humans and laboratory animals are sufficient to describe
24 benzo[ajpyrene elimination qualitatively, but are limited in estimating quantitative rates of
25 elimination.
26 D.2. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS
27 Several toxicokinetic or pharmacokinetic models of benzo [a]pyrene have been developed
28 for rodents (rat and hamster]. However, human models have only been developed via allometric
29 scaling, and metabolic parameters in humans have not been calibrated against in vivo toxicokinetic
30 data or in vitro experiments.
31 Bevan and Weyand [1988] performed compartmental pharmacokinetic analysis of
32 distribution of radioactivity in male Sprague-Dawley rats, following the intratracheal instillation of
33 benzo[a]pyrene to normal and bile duct-cannulated animals [Weyand and Bevan. 1987.1986].
34 However, implicit simulation approaches were used, as opposed to physiologically-based
35 approaches. The model calculated linear rate constants among compartments, and assumed that
36 the kinetics of benzo [ajpyrene and its metabolites were the same.
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1 Roth and Vinegar [1990] reviewed the capacity of the lung to impact the disposition of
2 chemicals and used benzo[a]pyrene as a case study. A PBPK model was presented based on data
3 from Wiersma and Roth [1983a. b] and was evaluated against tissue concentration data from
4 Schlede etal. [1970]. The model was structured with compartments for arterial blood, venous
5 blood, lung, liver, fat, and slowly and rapidly perfused tissues and an adequate fit was obtained for
6 some compartments; however, tissue-level data for calibration and validation of this model were
7 limited. Metabolism in liver and lung was estimated using kinetic data from control rats and rats
8 pretreated with 3-methylcholanthrene [3MC] to induce benzo[a]pyrene metabolism. In
9 microsomal preparations from control and SMC induced rat livers and lungs benzo[a]pyrene
10 hydroxylase activity was 1,000-fold greater in liver. In isolated rat lungs the clearance of
11 benzo[a]pyrene was about one-sixth of the clearance in isolated rat livers and in 3MC-pretreated
12 rats the clearance in lungs and livers were of similar magnitude. The PBPK simulations model
13 based on this data showed that for a bolus intravascular injection of benzo[a]pyrene in rats the
14 majority of benzo[a]pyrene metabolism usually occurs in the liver. Except for cases when rats are
15 pretreated with enzyme inducing agents or where the exposure occurs via inhalation the metabolic
16 clearance in the lung is minor.
17 Moir etal. [1998] conducted a pharmacokinetic study on benzo[a]pyrene to obtain data for
18 model development Rats were injected with varying doses of [14C]-benzo[a]pyrene to 15 mg/kg,
19 and blood, liver, fat, and richly perfused tissue were sampled varying time points after dosing. Moir
20 [1999] then described a model for lung, liver, fat, richly and slowly perfused tissues, and venous
21 blood, with saturable metabolism occurring in the liver. The fat and richly perfused tissues were
22 modeled as diffusion-limited, while the other tissues were flow-limited. The model predicted the
23 blood benzo[a]pyrene concentrations well, although it overestimated the 6 mg/kg results at longer
24 times (>100 minutes]. The model also produced a poor fit to the liver data. The model simulations
25 were also compared to data of Schlede etal. [1970], who injected rats with 0.056 mg/kg body
26 weight of benzo[a]pyrene. The model predicted blood and fat benzo[a]pyrene concentrations well,
27 but still poorly predicted liver benzo[a]pyrene concentrations. The model included only one
28 saturable metabolic pathway, and only parent chemical concentrations were used to establish the
29 model. No metabolites were included in the model. This model was re-calibrated by Crowell et al.
30 [2011] by optimizing against additional rodent data and altering partition coefficient derivation.
31 However, it still did not incorporate metabolites, and some tissues continued to exhibit poor model
32 fits.
33 An attempt to scale the Moir etal. [1998] rodent PBPK model to humans, relevant to risk
34 assessment of oral exposures to benzo[a]pyrene, was presented by Zeilmaker et al. [1999a] and
35 Zeilmaker et al. [1999b]. The PBPK model for benzo[a]pyrene was derived from an earlier model
36 for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats [Zeilmaker and van Eijkeren. 1997]. Most
37 compartments were perfusion-limited, and tissues modeled included blood, adipose (with diffusion
38 limitation], slowly and richly perfused tissues, and liver. However, there was no separate
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1 compartment for the lung. The liver compartment featured the AhR-dependent CYP450 induction
2 mechanism and DNA adduct formation as a marker for formation of genotoxic benzo[a]pyrene
3 metabolites. It was assumed that DNA adduct formation and the bulk benzo[a]pyrene metabolism
4 were mediated by two different metabolic pathways. The model was experimentally calibrated in
5 rats with the data for 7-ethoxyresorufin-O-deethylase (EROD) and formation of DNA adducts in the
6 liver after i.v. administration of a single dose and per os administration of a single or repeated doses
7 ofbenzo[a]pyrene [Zeilmaker etal., 1999a].
8 Zeilmaker et al. [1999b] assumed identical values for several parameters in rats and
9 humans (i.e., benzo[a]pyrene tissue partition coefficients, AhR concentration in liver, rate constant
10 for the decay of the benzo[a]pyrene-CYP450 complex, half-life of the CYP450 protein, fraction and
11 rate of GI absorption of benzo[a]pyrene, and rates of formation and repair of DNA adducts in liver).
12 The basal CYP450 activity in humans was assumed to be lower than that in rat liver. The
13 mechanism of AhR-dependent induction of CYP450 dominated the simulated benzo[a]pyrene-DNA
14 adduct formation in the liver. The results of PBPK model simulations indicated that the same dose
15 ofbenzo[a]pyrene administered to rats or humans might produce one order of magnitude higher
16 accumulation of DNA adducts in human liver when compared with the rat [Zeilmaker etal., 1999b].
17 Even though the model of Zeilmaker etal. (1999b) represents a major improvement in
18 predictive modeling of benzo[a]pyrene toxicokinetics, the interspecies extrapolation introduces
19 significant uncertainties. As emphasized by the authors, the conversion of benzo[a]pyrene to its
20 mutagenic and carcinogenic metabolites could not be explicitly modeled in human liver because no
21 suitable experimental data were available. According to the authors, improvement of the model
22 would require direct measurements of basal activities of CYP1A1 and CYP1A2 and formation of
23 benzo[a]pyrene-DNA adducts in human liver. Metabolic clearance of benzo[a]pyrene in the lungs
24 was also not addressed. Additionally, the toxicokinetic modeling by Zeilmaker et al. (1999b)
25 addressed only one pathway of benzo[a]pyrene metabolic activation, a single target organ (the
26 liver), and one route of administration (oral). In order to model health outcomes of exposures to
27 benzo[a]pyrene, the PBPK model needs to simulate rate of accumulation of benzo[a]pyrene-DNA
28 adducts and/or the distribution and fate of benzo[a]pyrene metabolites (e.g., BPDE) that bind to
29 DNA and other macromolecules. Alternatively, stable toxic metabolites (e.g., trans-anti-tetrol-
30 benzo[a]pyrene) may be used as an internal dose surrogate. While the metabolic pattern of
31 benzo[a]pyrene has been relatively well characterized qualitatively in animals, the quantitative
32 kinetic relationships between the more complex metabolic reactions in potential target organs are
33 not yet well defined.
34 D.2.1. Recommendations for the Use of PBPK Models in Toxicity Value Derivation
35 PBPK models for benzo[a]pyrene were evaluated to determine the capability to extrapolate
36 from rats to humans, or between oral and inhalation exposure routes. Due to significant
37 uncertainties with respect to the inter-species scaling of the metabolic parameters between rats
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1 and humans, these models were not used for cross-species extrapolation. Furthermore, no
2 complete mechanistic PBPK model for the inhalation route was identified, nor was there a model
3 for humans that simulates the typical inhalation exposure to benzo[a]pyrene on poorly soluble
4 carbonaceous particles. This precluded the model's use for cross-route extrapolation to the
5 inhalation pathway.
6 D.3. HUMAN STUDIES
7 D.3.1. Non-Cancer Endpoints
8 Cardiovascular Endpoints
9 Burstynetal. [2005] reported the association of death from cardiovascular disease with
10 benzo[a]pyrene exposure in a cohort of 12,367 male European asphalt workers (Table D-l). These
11 workers were first employed in asphalt paving between 1913 and 1999, and worked at least one
12 season. Average duration of follow-up was 17 ± 9 years (mean ± standard deviation [SD]),
13 encompassing 193,889 person-years of observation. Worker exposure to coal tar was estimated
14 using industrial process and hygiene information and modeling (presented in a previous report),
15 and coal tar exposure was found to be the strongest determinant of exposure to benzo[a]pyrene.
16 Benzo[a]pyrene exposure was assessed quantitatively using measurement-driven mixed effects
17 exposure models, using data collected from other asphalt industry workers, and this model was
18 constructed and validated previously. Due to limited data availability, only information regarding
19 the primary cause of death was collected, and this analysis was limited to diseases of the circulatory
20 system (ICD codes 390-459), specifically ischemic heart disease (IHD: ICD codes 410-414). Diesel
21 exhaust exposure was also assessed in this cohort, but varied little among the asphalt pavers, and
22 was not associated with risk of death from cardiovascular disease. Of the initial cohort, 0.25% was
23 lost to follow-up and 0.38% emigrated during the course of observation. Relative risks and
24 associated 95% confidence intervals (CIs) were estimated using Poisson regression, and all models
25 included exposure index for agent of interest (coal tar or benzo[a]pyrene), age, calendar period of
26 exit from cohort, total duration of employment, and country, using the category of lowest exposure
27 as the reference. Confounding by tobacco smoke exposure was considered in relation to the
28 strength of its association with cardiovascular disease and the smoking prevalence in the
29 population. The relative risk (RR) attributed to cigarette smoking in former and current smokers
30 was assumed to be 1.2 and 2, respectively, based upon literature reports. From analysis of smoking
31 incidence in a subcohort, the following smoking distribution was proposed: in the lowest exposure
32 group, 40% never-smokers, 30% former smokers, and 30% current smokers; among the highest
33 exposed, the proportion shifted to 20/30/50%, respectively.
34 Exposed subjects were stratified into quintiles based upon IHD mortality, with 83-
35 86 deaths per exposure category, composing approximately 2/3 of the 660 cardiovascular disease-
36 related deaths. Both cumulative and average exposure indices for benzo[a]pyrene were positively
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1 associated with IHD mortality, with a RR of approximately 1.6 in the highest exposure quintile from
2 both metrics, independent of total employment duration. Similar mono tonic trends were observed
3 for all cardiovascular diseases (combined), although a dose-response relationship was evident only
4 for IHD and not hypertension or other individual heart disease categories. Similar trends were also
5 observed for coal tar exposure and IHD. Adjusting the RR to account for possible confounding by
6 smoking yields a RR of 1.39 under the assumptions mentioned above, and is still elevated (1.21) if
7 the contribution of smoking to cardiovascular disease etiology was greater than the original
8 assumptions. Furthermore, the RR for the high versus low exposure quintile is 1.24 even if the
9 distribution of non-smokers/former smokers/current smokers shifts to 0/30/70%, using the
10 original assumptions of cigarette smoke casual potency.
11
12
Table D-l. Exposure to benzo[a]pyrene and mortality from cardiovascular
diseases in a European cohort of asphalt paving workers
Effect Measured
Cumulative Exposure (ng/ms-yrs)
0-189a
189-501
502-931
932-2,012
>2,013
p- value
for Trend
Diseases of the circulatory system
Deaths
RR
95% Cl
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% Cl
Effect Measured
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
Average Exposure (ng/m3)
0-68a
68-105
106-146
147-272
>273
0.06
p-value
for Trend
Diseases of the circulatory system
Deaths
RR
95% Cl
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% Cl
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
15
16
17
18
Reference category.
Source: Burstvn et al. (2005).
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Friesenetal. [2010] examined the association between benzo[a]pyrene exposure and
2 deaths from chronic non-malignant disease in a cohort of 6,423 male and 603 female Canadian
3 aluminum smelter workers (Table D-2). Inclusion criteria required at least 3 years of continuous
4 employment in either the smelter facility or power-generating station from 1954 to 1997, with
5 worker history collected up through 1999. This cohort was probabilistically linked to the Canadian
6 national mortality database for external comparison to the British Columbia population and
7 calculation of standardized mortality ratios (SMRs), which were adjusted for age, sex, and time
8 period. Ninety-five percent CIs were calculated for the SMRs assuming a Poisson distribution.
9 Internal comparisons were also made during the analysis of IHD mortality in male workers,
10 calculating hazard ratios (HRs) for IHD with or without acute myocardial infarction (AMI) after
11 1969, as AMI could not be differentiated from other IHD on death certificates issued previously.
12 HRs were calculated using Cox regression models, with age as a metamarker of time, also including
13 smoking status, time since first employed and work location status. Smoking information for 77%
14 of this updated cohort was collected by questionnaire, and workers were categorized as 75% ever-
15 smokers and 25% never-smokers. Quantitative exposure to coal tar pitch volatiles were estimated
16 by benzo[a]pyrene measurements, calculated by a job classification and time-based exposure
17 matrix, as described in a previous report; annual arithmetic mean values were calculated for
18 exposures from 1977 to 2000, while pre-1977 levels were backwards-extrapolated from 1977
19 values, incorporating major technological changes in time periods as appropriate.
20 Cumulative exposure metrics were highly skewed. Cumulative benzo[a]pyrene with a
21 5-year lag (past benzo[a]pyrene exposure) and cumulative benzo[a]pyrene in the most recent
22 5 years (recent benzo[a]pyrene exposure) were only slightly positively correlated (r = 0.10,
23 p < 0.001). Current benzo[a]pyrene exposure was highly correlated with cumulative exposure for
24 the most recent 5 years of exposure (r = 0.86, p < 0.001), but not with 5-year lagged cumulative
25 exposure (r = 0.03, p < 0.001). Lagged cumulative exposure metrics (0-10 years) were all highly
26 correlated with each other (r = 0.96, all p-values < 0.001); lagged metrics for cumulative exposure
27 were used to distinguish between effects of current versus long-term exposure.
28 When exposed workers were pooled and compared externally to non-exposed referents, the
29 IHD and AMI SMRs were all <1.00 for males, and the only significant association in females was an
30 SMR of 1.27 for AMI. For internal comparisons, exposed males were stratified into quintiles based
31 upon IHD mortality, with approximately 56 deaths per exposure category. Five-year lagged
32 cumulative benzo[a]pyrene exposure was significantly associated with elevated risk of IHD
33 mortality, HR = 1.62 (95% CI 1.06-2.46) in the highest exposure quintile, while no association was
34 observed between most recent (5 years) exposure and mortality. Restricting IHD events to only
35 AMI (1969 onward) resulted in similar monotonic trends, albeit of lower statistical significance. No
36 association was observed between benzo[a]pyrene exposure and non-AMI IHD. While there was
37 little difference in the exposure-response association among 0-, 2-, and 5-year lagged data, 10-year
38 lagged data resulted in a weaker association. All risk estimates were strengthened by the
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1 incorporation of work status and time-since-hire to account for the healthy worker effect, as
2 evidenced by the SMR of 0.87 (95% CI 0.82-0.92) for all chronic non-malignant diseases combined
3 in male exposed workers versus external referents. Using a continuous variable, the authors
4 calculated that the risk of death from IHD to be 1.002 (95% CI 1.000-1.005) per [ig/m3 from
5 cumulative benzo[a]pyrene exposure; however, visual inspection of the categorical relationships
6 indicated that the association is nonlinear, suggesting that this value may be an underestimate.
7 Restricting the cohort to only members who died within 3 0 days of active employment at the
8 worksite, cumulative benzo[a]pyrene exposure was not significantly associated with IHC or AMI,
9 although the HR for the highest exposure group was 2.39 (95% CI 0.95-6.05). Exposure-response
10 relationships were similarly examined in male smelter workers for chronic obstructive pulmonary
11 disease and cerebrovascular disease, but neither was significantly associated with cumulative
12 benzo[a]pyrene exposure in either internal or external comparisons.
13
14
Table D-2. Exposure to benzo[a]pyrene and mortality from cardiovascular
diseases in a Canadian cohort of male aluminum smelter workers
Effect Measured
Categorical Cumulative Exposure with a 5-yr Lag (ug/ms-yr)
0
0-7.79
7.79-24.3
24.3-66.7
>66.7
p-value
for
Trend3
Continuous13
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
/Acute myocardial infarction (1969 onward)
Deaths
Person-years of
follow-up
HR
95% CI
0
35
25,071
1
referent
0-7.51
37
30,454
1.14
0.71-1.82
7.51-27.7
37
34,621
1.21
0.75-1.96
27.7-67.4
38
24,081
1.36
0.84-2.45
>67.4
37
13,261
1.46
0.87-2.45
0.19
184
127,488
1.001
0.997-1.005
15
16
17
18
19
20
21
22
23
24
25
aTwo-sided test for trend using the person-year-weighted mean value for each category as a linear, continuous
variable.
bExposure variable was entered as a continuous, linear variable in the model.
Source: Friesen et al. (2010).
Reproductive and Developmental Endpoints
Wuetal. (2010) conducted a study of benzo[a]pyrene-DNA adduct levels in relation to risk
of fetal death in Tianjin, China. This case-control study included women who experienced a delayed
miscarriage before 14 weeks gestational age (i.e., a fetal death that remained in utero and therefore
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1 required surgical intervention). Cases were matched by age and gravidity to controls (women
2 undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded
3 women who smoked, women with chronic disease and pregnancy complications, and women with
4 occupational exposures to PAHs. Residency within Tianjin for at least 1 year was also an eligibility
5 criterion. The participation rate was high: 81/84 eligible cases participated and 81/89 eligible
6 controls participated. Data pertaining to demographic characteristics, reproductive history, and
7 factors relating to potential PAH exposure were collected using a structured interview, and samples
8 from the aborted tissue were obtained. In two of the four hospitals used in the study, blood
9 samples from the women (n = 51 cases and 51 controls) were also collected. The presence of
10 benzo[a]pyrene-BPDE adducts was assessed in the blood and tissue samples using high-
11 performance liquid chromatography (HPLC). There was no correlation between blood and aborted
12 tissue levels of benzo[a]pyrene adducts (r = -0.12 for the 102 blood-tissue pairs, r = -0.02 for the
13 51 case pairs and r = -0.21 for the 51 control pairs). (The authors noted that there was little
14 difference between women with and without blood samples in terms of the interview-based
15 measures collected or in terms of the DNA-adduct levels in aborted tissue.) Benzo[a]pyrene-adduct
16 levels were similar but slightly lower in the aborted tissue of cases compared with controls
17 (mean ± SD 4.8 ± 6.0 in cases and 6.0 ± 7.4 in controls, p = 0.29). In the blood samples, however,
18 benzo[a]pyrene-adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ± 2.2 in cases and controls,
19 respectively, p < 0.001). In logistic regression analyses using a continuous adduct measure, the
20 odds ratio (OR) was 1.35 (95% CI 1.11-1.64) per adduct/108 nucleotide. These results were
21 adjusted for education, household income, and gestational age, but were very similar to the
22 unadjusted results. Categorizing exposure at the median value resulted in an adjusted OR of 4.27
23 (95% CI 1.41-12.99) in the high compared with low benzo[a]pyrene-adduct group. There was no
24 relation between benzo[a]pyrene-adduct levels in the aborted tissue and miscarriage in the logistic
25 regression analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93-1.02) or
26 dichotomous exposure measure (adjusted OR 0.76, 95% CI 0.37-1.54). Associations between
27 miscarriage and several interview-based measures of potential PAH exposure were also seen:
28 adjusted ORs of 3.07 (95% CI 1.31-7.16) for traffic congestion near residence, 3.52 (95% CI 1.44-
29 8.57) for commuting by walking, 3.78 (95% CI 1.11-12.87) for routinely cooked during pregnancy,
30 and 3.21 (95% CI 0.98-10.48) for industrial site or stack near residence, but there was no
31 association with other types of commuting (e.g., by bike, car, or bus).
32 Pereraetal. (2005a) studied 329 non-smoking pregnant women (30 ± 5 years old) possibly
33 exposed to PAHs from fires at the World Trade Center (WTC) during the 4 weeks after 09/11/2001.
34 Maternal and umbilical cord blood levels of benzo [a]pyrene (BPDE)-DNA adducts were highest in
35 study participants who lived within 1 mile of the WTC, with an inverse correlation between cord
36 blood levels and distance from the WTC. Neither cord blood adduct level nor environmental
37 tobacco smoke (ETS) alone was positively correlated with adverse birth outcomes. However, the
38 interaction between ETS exposure and cord blood adducts was significantly associated with
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1 reduced birth weight and head circumference. Among babies exposed to ETS in utero, a doubling of
2 cord blood benzo[a]pyrene-DNA adducts was associated with an 8% decrease in birth weight
3 (p = 0.03) and a 3% decrease in head circumference (p = 0.04).
4 Pereraetal. [2005b), a reanalysis of Pereraetal. [2004),, compared various exposures—
5 ETS, nutrition, pesticides, material hardship—with birth outcomes (length, head circumference,
6 cognitive development). ETS exposure and intake of PAH-rich foods by pregnant women were
7 determined by questionnaire. Levels of BPDE-DNA adducts were determined in umbilical cord
8 blood collected at delivery. The study population consisted of Dominican or African-American non-
9 smoking pregnant women (n = 214;24±5 years old) free of diabetes, hypertension, HIV, and drug
10 or alcohol abuse. Benzo[a]pyrene adducts, ETS, and dietary PAHs were not significantly correlated
11 with each other. However, the interaction between benzo[a]pyrene-DNA adducts and ETS
12 exposure was significantly associated with reduced birth weights (-6.8%; p = 0.03) and reduced
13 head circumference (-2.9%; p = 0.04).
14 Tangetal. (2006) measured BPDE-DNA adducts in maternal and umbilical cord blood
15 obtained at delivery from a cohort of 150 non-smoking women and their newborns in China.
16 Exposure assessment was related to the seasonal operation of a local, coal-fired power plant;
17 however, airborne PAH concentrations were not measured. Dietary PAH intake was not included as
18 a covariate because it did not significantly contribute to the final models, but ETS, sex, and maternal
19 height and weight were considered as covariates. DNA adduct levels were compared to several
20 birth outcomes and physical development parameters, such as gestational age at birth; infant sex,
21 birth weight, length, head circumference, and malformations; maternal height and pregnancy
22 weight total weight gain; complications of pregnancy and delivery; and medications used during
23 pregnancy.
24 High cord blood adduct levels were significantly associated with reduced infant/child
25 weight at 18 months (P = -0.048, p = 0.03), 24 months (P = -0.041, p = 0.027), and 30 months of age
26 (P = -0.040, p = 0.049); decreased birth head circumference was marginally associated with DNA
27 adduct levels (P = -0.011, p = 0.057). Maternal adductlevels were correlated neither with cord
28 blood adduct levels nor with fetal and child growth. Among female infants, cord blood adduct levels
29 were significantly associated with smaller birth head circumference (p = 0.022) and with lower
30 weight at 18 months (p = 0.014), 24 months (p = 0.012), and 30 months of age (p = 0.033), and with
31 decreased body length at 18 months of age (p = 0.033). Among male infants, the corresponding
32 associations were also inverse but were not statistically significant
33 Considerable evidence of a deleterious effect of smoking on male and female fertility has
34 accumulated from epidemiological studies of time to pregnancy, ovulatory disorders, semen
35 quality, and spontaneous abortion (reviewed in Waylenetal., 2009: Cooper and Moley, 2008:
36 Spares and Melo, 2008). In addition, the effect of smoking, particularly during the time of the
37 perimenopausal transition, on acceleration of ovarian senescence (menopause) has also been
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1 established [Midgette and Baron, 1990]. More limited data are available pertaining specifically to
2 measures of benzo[a]pyrene and reproductive outcomes.
3 Nealetal. [2008] examined levels of benzo[a]pyrene and other PAHs in follicular fluid and
4 serum sample from 36 women undergoing in vitro fertilization at a clinic in Toronto, and compared
5 the successful conception rate in relation to benzo[a]pyrene levels. The women were classified by
6 smoking status, with 19 current cigarette smokers, 7 with passive or sidestream smoke exposure
7 (i.e., non-smoker with a partner who smoked], and 10 non-smokers exposed. An early follicular
8 phase blood sample and follicular fluid sample from the follicle at the time of ovum retrieval were
9 collected and analyzed for the presence of benzo[a]pyrene, acenapthelene, phenanthrene, pyrene,
10 and chrysene using gas chromatography/mass spectrometry (MS] (detection limit 5 pg/mL]. The
11 frequency of nondectable levels of serum benzo[a]pyrene was highest in the non-smoking group
12 (60.0,14.3, and 21.0% below detection limit in non-smoking, sidestream smoke, and active
13 smoking groups, respectively]. A similar pattern was seen with follicular fluid benzo[a]pyrene
14 (30.0,14.3, and 10.5% below detection limit in non-smoking, sidestream smoke, and active
15 smoking groups, respectively]. In the analyses comparing mean values across groups, an assigned
16 value of 0 was used for nondetectable samples. Follicular fluid benzo[a]pyrene levels were higher
17 in the active smoking group (mean ± standard error [SE], 1.32 ± 0.68 ng/mL] than in the sidestream
18 (0.05 ± 0.01 ng/mL] or non-smoking (0.03 ± 0.01 ng/mL] groups (p = 0.04]. The between-group
19 differences in serum benzo[a]pyrene levels were not statistically significant (0.22 ± 0.15, 0.98 ±
20 0.56, and 0.40 ±0.13 ng/mL in non-smoking, sidestream smoke, and active smoking groups,
21 respectively], and there were no differences in relation to smoking status. Among active smokers,
22 the number of cigarettes smoked per day was strongly correlated with follicular fluid
23 benzo[a]pyrene levels (r = 0.7, p < 0.01]. Follicular fluid benzo[a]pyrene levels were significantly
24 higher among the women who did not conceive (1.79 ± 0.86 ng/mL] compared with women who
25 did getpregnant (mean approximately 0.10 ng/mL, as estimated from graph] (p < 0.001], but
26 serum levels of benzo [a]pyrene were not associated with successful conception.
27 A small case-control study conducted between August 2005 and February 2006 in Lucknow
28 city (Uttar Pradesh], India examined PAH concentrations in placental tissues (Singh etal., 2008] in
29 relation to risk of preterm birth. The study included 29 cases (delivery between 28 and <36 weeks
30 of gestation] and 31 term delivery controls. Demographic data smoking history, reproductive
31 history, and other information were collected by interview, and a 10 g sample of placental tissue
32 was collected from all participants. Concentration of specific PAHs in placental tissue was
33 determined using HPLC. In addition to benzo[a]pyrene, the PAHs assayed were naphthalene,
34 acenapththylene, phenanthrene, fluorene, anthracene, benzo [a] anthracene, fluoranthene, pyrene,
35 benzo[k]fluoranthene, benzo[b]fluoranthene, benzo [g,h,i]perylene, and dibenzo[a,h]anthracene.
36 PAH exposure in this population was from environmental sources and from cooking. The age of
37 study participants ranged from 20 to 35 years. There was little difference in birth weight between
38 cases and controls (mean 2.77 and 2.75 kg in the case and control groups, respectively]. Placental
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1 benzo[a]pyrene levels were lower than the levels of the other PAHs detected (mean 8.83 ppb in
2 controls for benzo[a]pyrene compared with 25-30 ppb for anthracene, benzo[k]fluoranthene,
3 benzo[b]fluoranthene, and dibenzo[a,h]anthracene, 59 ppb for acenaphthylene, and 200-380 ppm
4 for naphthalene, phenanthrene, fluoranthene, and pyrene; nondetectable levels of fluorine,
5 benzo[a]anthracene, and benzo[g,h,i]perylene were found). There was little difference in
6 benzo[a]pyrene levels between cases (mean ± SE 13.85 ± 7.06 ppb) and controls (8.83 ± 5.84 ppb),
7 but elevated levels of fluoranthene (325.91 ± 45.14 and 208.6 ± 21.93 ppb in cases and controls,
8 respectively, p < 0.05) and benzo[b]fluoranthene (61.91 ± 12.43 and 23.84 ± 7.01 ppb in cases and
9 controls, respectively, p < 0.05) were seen.
10 Neurotoxicity
11 Niuetal. (2010) studied 176 Chinese coke-oven workers with elevated benzo[a]pyrene
12 exposure and compared them against 48 referents (workers in a supply warehouse), matched by
13 socioeconomic status, lifestyle and health. Blood levels of monoamine, amino acid and chloine
14 neurotransmitters were measured, and the World Health Organization Neurobehavioral Core Test
15 Battery was administered to assess emotional state, learning, memory, and hand-eye coordination.
16 The authors self-designed a study questionnaire to gather information on worker education,
17 vocational history, smoking and drinking habits, personal habits, personal and family medical
18 history, as well as any current symptoms and medications used in the previous several weeks.
19 Workers were excluded from the study for any of the following criteria: if they reported feeling
20 depressed at any point during the previous 6 months; if they had taken medicine in the previous
21 2 weeks that could affect nervous system function; or if they reported undertaking vigorous
22 exercise less than 48 hours previously. "Smoking" was defined as >10 cigarettes/day during the
23 past year. Similarly, "drinking" was defined as wine/beer/spirits consumed >3 times/week for the
24 past 6 months. Workplace environmental sampling stations were established at each of the
25 physical work locations, including the referent's warehouse, and dual automatic air sampling
26 pumps collected samples at personal breathing zone height for 6 hours/day, over 3 consecutive
27 days. Benzo [a]pyrene content was determined by HPLC, and relative exposure was compared to
28 post-shift urine levels of a benzo [a]pyrene metabolite, 1-hydroxypyrene (1-OH-Py). Blood was
29 collected in the morning before breakfast; monoamine (norepinephrine and dopamine) and amino
30 acid (glutamate, aspartate, glycine, and gamma-aminobutyric acid [GABA]) neurotransmitter levels
31 were determined by HPLC, acetylcholine levels determined by hydroxyamine chromometry, and
32 acetylcholine esterase (AchE) levels measured in lysed red blood cells (RBCs) using activity kits.
33 Benzo[a]pyrene mean concentrations were 19.56 ± 13.2,185.96 ± 38.6, and 1,623.56 ±
34 435.8 ng/m3 atthe bottom, side, and top of the coke oven, respectively, all of which were higher
35 than the mean at the referents' warehouse (10.26 ± 7.6 ng/m3). The authors did not report
36 stratified analysis by different levels of benzo [ajpyrene exposure, and reported only comparisons
37 between the referents and all exposed workers combined (Table D-3), or between workers grouped
38 by urinary benzo[a]pyrene metabolite 1-OH-Py levels (Table D-4). There were no significant
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1 differences in age, education, or smoking or alcohol use between the coke oven and warehouse
2 workers. Urinary 1-OH-Py levels were 32% higher in coke oven workers compared to the referent
3 group, corresponding to the higher levels of benzo[a]pyrene detected in all coke oven workstation
4 compared to the supply warehouse. Performance in two neurobehavioral function tests, digit span
5 and forward digit span, were significantly decreased in the exposed oven workers versus control
6 group; when stratified by urinary metabolite level, scores significantly decreased with increasing
7 1-OH-Py levels. Of the neurotransmitters assessed, norepinephrine, dopamine, aspartate and GABA
8 were significantly decreased in exposed versus control workers; norepinephrine and aspartate
9 were also significantly and inversely related with 1-OH-Py levels. Dopamine levels appeared to
10 decrease with increased urinary metabolite levels, although the relationship was not statistically
11 significant GABA levels were highly variable, and appeared to increase with increasing 1-OH-Py
12 levels, although this relationship was statistically significant Acetylcholine levels were fourfold
13 higher in coke oven workers compared to referents, and AchE activity was 30% lower; both
14 acetylcholine and AchE were significantly associated with urinary benzo[a]pyrene metabolite
15 levels, although acetylcholine increased and AchE activity decreased with increasing 1-OH-Py. The
16 authors reported the results of correlation analysis, indicating that digit span scores correlated
17 negatively with acetylcholine and positively with AchE (coefficients of-0.230, -0.276 and 0.120,
18 0.170, respectively), although no indication of statistical significance was given. No other
19 associations were reported.
20
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1
2
Table D-3. Exposure-related effects in Chinese coke oven workers or
warehouse controls exposed to benzo[a]pyrene in the workplace
Effect Measured
Exposure Group
Controls (n = 48)
Exposed Workers (n = 176)
p-value
Background information (mean ± SD, incidence or percent)
Age (yrs)
Education (junior/senior)
Smoking
Drinking
39.71 ±7.51
23/25
77%
27%
37.86 ±6.51
110/66
64%
39%
0.098
0.068
0.093
0.140
Urine benzo[a]pyrene metabolite (u.mol/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
Digit span
Forward digit span
413.88 ± 95.40
17.31 ±4.54
10.65 ± 2.42
437.39 ± 88.44
15.47 ± 4.08
9.25 ±2.64
0.109
0.006
0.001
Neurotransmitter concentrations (mean ± SD)
Norepinephrine (ng/mL)
Dopamine (ng/mL)
Aspartate (ug/mL)
Glutamate (u.g/mL)
GABA(u.g/mL)
Acetylcholine (u.g/mL
AchE activity (U/mg protein)
62.54 ± 58.07
1,566.28 ± 317.64
2.13 ±1.66
11.21 ±5.28
2.52 ±5.16
172.60 ±67. 19
71.31 ±46.18
40.62 ± 29.78
1,425.85 ± 422.66
1.58 ±0.99
9.68 ±5.72
1.01 ±2.21
704.00 ± 393.86
50.27 ± 34.02
0.000
0.029
0.004
0.074
0.004
0.000
0.012
3
4
5
6
Source: Niu etal. (2010).
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1
2
3
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
Effect Measured
Number of Subjects
Exposure Group Categorized by 1-OH-Py Level
0-3.09 umol/mol
Creatinine
33
3.09-3.90 umol/mol
Creatinine
72
3.90-5.53 umol/mol
Creatinine
36
p-value
Neurobehavioral function tests (mean ± SD)
Digit span
Forward digit span
Backward digit span
Right dotting
18.24 ±4.58
10.85 ±2. 12
7.20 ± 3.07
152. 15 ±35.43
16.04 ± 4.24
9.80 ± 2.86
6.38 ±2.55
153.80 ± 31.55
15.78 ±3.71
9.58 ±2.33
6.20 ±2.15
167.22 ±59.21
0.003
0.019
0.089
0.094
Neurotransmitter concentrations (mean ± SD)
Norepinephrine (ng/mL)
Dopamine (ng/mL)
Aspartate (ng/mL)
Glutamate (ug/mL)
GABA(ug/mL)
Acetylcholine (ug/mL)
AchE activity (U/mg protein)
67.31 ±67.45
1,614.45 ± 683.57
2.29 ±2.13
11.56 ±8.92
1.40 ±3.59
334.66 ± 83.75
68.17 ±9.28
36.97 ±23.58
1,482.30 ± 323.66
1.61 ±0.71
9.93 ±4.14
1.42 ± 3.44
483.71 ±57.87
54.98 ±4.23
46.75 ±35.88
1,405.06 ± 332.23
1.47 ±0.58
9.06 ± 3.30
1.56 ±3.24
665.85 ± 94.34
52.64 ±4.60
0.002
0.134
0.001
0.070
0.964
0.030
0.043
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Source: Niu et al. (2010).
Immunotoxicity
Zhang etal. [2012] studied 129 Chinese coke-oven workers with elevated benzo[a]pyrene
exposure and compared them against 37 referents (workers in a supply warehouse), matched by
socioeconomic status, lifestyle, and health. Area benzo[a]pyrene levels were quantified in the
various work areas, and the primary endpoint was the level of early and late apoptosis in
peripheral blood mononuclear cells (PBMCs) isolated from each worker subgroup the morning
following an overnight fast. The authors self-designed a study questionnaire to gather information
on worker education, vocational history, smoking and drinking habits, personal habits, personal
and family medical history, as well as any current symptoms and medications used in the previous
several weeks. "Smoking" was defined as >10 cigarettes/day during the past year, with "smoking
index" defined as cigarettes/day x years smoking. Similarly, "drinking" was defined as wine/beer/
spirits consumed >3 times/week for the past 6 months, and "drinking index" defined as grams of
alcohol consumed/day x years drinking. Exposed workers were categorized by physical worksite
location and expected differences in benzo[a]pyrene exposure: 34 oven bottom workers, 48 oven
side workers, and 47 oven top workers. Workplace environmental sampling stations were
established at each of the physical work locations, including the referent's warehouse, and dual
automatic air sampling pumps collected samples at personal breathing zone height for 6 hours/day,
This document is a draft for review purposes only and does not constitute Agency policy.
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1 over 3 consecutive days. Benzo[a]pyrene content was determined by HPLC, and relative exposure
2 was compared to post-shift urine levels of a benzo[a]pyrene metabolite, 1-OH-Py. Collected and
3 purified PBMCs were incubated with Annexin-V and PI prior to analysis by flow cytometry; early
4 apoptotic cells were considered to be Annexin V+/PI-, while late apoptotic cells were considered
5 Annexin V+/PI+.
6 All apoptosis data were displayed graphically, and in all groupings, early:late apoptotic
7 PBMCs occurred at an approximate 2:1 frequency. PBMC apoptosis was similar in each of the three
8 coke oven worker groups, which were all statistically significantly higher than referents
9 (approximately twofold) for both early and late apoptosis. While self-reported smoking incidence
10 varied significantly among the worker groups, stratification by smoking years or smoking index did
11 not reveal any significant association with PBMC apoptosis. Multiple linear step wise regression
12 analysis suggested that urine 1-OH-Py levels and years of coke oven operation were positively
13 associated with increased early and late PMBC apoptosis (Table D-5), and thatyears of ethanol
14 consumption was negatively associated with only early apoptosis. These associations were tested
15 by stratifying workers into three groups by urinary 1-OH-Py levels or coke oven operation years,
16 and in both cases, the groups with the highest urinary metabolite levels or longest oven operating
17 experience had statistically significantly higher levels of both early and late apoptotic PBMCs versus
18 the lowest or shortest duration groups, respectively. Likewise, when sorted into groups based
19 upon years of ethanol consumption, the highest ethanol "years of consumption" group had
20 statistically significantly lower early apoptosis rates when compared to the lowest ethanol
21 consuming group.
22
23
Table D-5. Background information on Chinese coke oven workers or
warehouse controls exposed to benzo[a]pyrene in the workplace
Effect Measured
Number of Subjects
Exposure Group (ng/m3; mean ± SD)
10.2 ± 7.6
37
19.5 ± 13.2
34
185.9 ± 38.6
48
1,623.5 ± 435.8
47
p-value
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
24
25
26
27
28
*p < 0.05 significantly different from control mean.
Source: Zhang etal. (2012).
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1 D.3.2. Cancer-related Endpoints
2 Benzo[a]pyrene-Induced Cytogenetic Damage
3 Many studies measure cytogenetic damage as biomarkers of early biological effects which
4 also reflect exposure to genotoxic chemicals. Standard cytogenetic endpoints include chromosomal
5 aberration (CA), sister chromatid exchange (SCE), micronucleus (MN) formation, hypoxanthine
6 guanine phosphoribosyl transferase (hprt) mutation frequency, and glycophorin A mutation
7 frequency [Gyorffy etal., 2008]. These biomarkers are often incorporated in multi-endpoint
8 studies with other biomarkers of exposure. Because they indicate related but different endpoints,
9 there is often a lack of correlation between the different categories of biomarkers.
10 Merlo etal. [1997] evaluated DNAadductformation (measuredby [32P]-postlabelling] and
11 MN in white blood cells (WBCs] of 94 traffic policemen versus 52 residents from the metropolitan
12 area of Genoa, Italy. All study subjects wore personal air samplers for 5 hours of one work shift,
13 and levels of benzo[a]pyrene and other PAHs were measured. Policemen were exposed to 4.55 ng
14 benzo[a]pyrene/m3 air, compared with urban residents who were exposed to 0.15 ng/m3. DNA
15 adduct levels in policemen were 35% higher than in urban residents (p = 0.007], but MN in urban
16 residents were 20% higher than in policemen (p = 0.02]. Linear regressions of DNA adducts and
17 MN incidence, respectively, versus benzo[a]pyrene exposure levels did not reveal significant
18 correlations.
19 Perera and coworkers assessed DNA damage in Finnish iron foundry workers in two
20 separate studies and using three methodologies. Based on results from personal sampling and
21 stationary monitoring in both studies, three levels of benzo[a]pyrene air concentrations were
22 defined: low (<5 ng/m3 benzo[a]pyrene], medium (5-12 ng/m3], and high (>12 ng/m3] (Perera et
23 al., 1994: Perera etal., 1993]. In the first study, involving 48 workers, several biomarkers were
24 analyzed for dose-response and interindividual variability (Perera etal.. 1993]. PAH-DNA adducts
25 were determined in WBCs using an immunoassay and enzyme-linked immunosorbent assay with
26 fluorescence detection. Mutations at the hprt locus were also measured in WBC DNA. The latter
27 assay is based on the fact that each cell contains only one copy of the hprt gene, which is located on
28 the X-chromosome. While male cells have only one X-chromosome, female cells inactivate one of
29 the two X-chromosomes at random. The gene is highly sensitive to mutations such that in the event
30 of a crucial mutation in the gene, enzyme activity disappears completely from the cell. In addition,
31 mutations at the glycophorin A gene locus were measured in RBCs. The glycophorin A mutation
32 frequency was not correlated with either benzo[a]pyrene exposure or PAH-DNA adduct formation.
33 However, both PAH-DNA adduct levels and hprt mutation frequency increased with increasing
34 benzo[a]pyrene exposure. In addition, there was a highly significant correlation between incidence
35 of hprt mutations and PAH-DNA adduct levels (p = 0.004].
36 In a second study, Perera etal. (1994] surveyed 64 iron foundry workers with assessments
37 conducted in 2 successive years; 24 of the workers provided blood samples in both years. Exposure
38 to benzo[a]pyrene, collected by personal and area sampling in the firstyear of the study, ranged
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1 from <5 to 60 ng/m3 and was estimated to have decreased by 40% in the second year. The levels of
2 PAH-DNA adducts were roughly 50% lower in the 2nd year, presumably reflecting decreased
3 exposure. The longer-lived hprt mutations were not as strongly influenced by the decreasing
4 exposure to benzo[a]pyrene. Study subjects who did not have detectable levels of DNA adducts
5 were excluded from the study. As in the previous study, a strong correlation between DNA adduct
6 levels and incidence of hprt mutations was observed [Pereraetal.. 1993).
7 Kalina et al. [1998] studied several cytogenetic markers in 64 coke oven workers and
8 34 controls employed at other locations within the same plant. Airborne benzo[a]pyrene and seven
9 other carcinogenic PAHs were collected by personal air samplers, which showed ambient
10 benzo[a]pyrene concentrations ranging widely from 0.002 to 50 [ig/m3 in coke oven workers and
11 from 0.002 to 0.063 [ig/m3 in controls. CAs, SCEs, high-frequency cells (HFCs), and SCE
12 heterogeneity index were all significantly increased with benzo[a]pyrene exposure. Except for
13 increases in HFCs, no effect of smoking was observed. Consistent with studies of PAH-DNA adduct
14 formation, reduced cytogenetic response at high exposure levels produced a nonlinear dose-
15 response relationship. The authors also evaluated the potential influence of polymorphisms in
16 enzymes involved in the metabolism of benzo[a]pyrene. GSTM1 and N-acetyl transferase-2
17 polymorphisms were studied and no evidence of the two gene polymorphisms having any influence
18 on the incidence of cytogenetic damage was found.
19 Motykiewicz etal. [1998] conducted a similar study of genotoxicity associated with
20 benzo[a]pyrene exposure in 67 female residents of a highly polluted industrial urban area of Upper
21 Silesia, Poland, and compared the results to those obtained from 72 female residents of another
22 urban but less polluted area in the same province of Poland. Urinary mutagenicity and 1-OH-Py
23 levels, PAH-DNA adducts in oral mucosa cells (detected by immunoperoxidase staining], SCEs,
24 HFCs, CAs, bleomycin sensitivity, and GSTM1 and CYP1A1 polymorphisms in blood lymphocytes
25 were investigated. High volume air samplers and gas chromatography were used to quantify
26 ambient benzo[a]pyrene levels, which were 3.7 ng/m3 in the polluted area and 0.6 ng/m3 in the
27 control area during the summer. During winter, levels rose to 43.4 and 7.2 ng/m3 in the two areas,
28 respectively. The cytogenetic biomarkers (CA and SCE/HFC], urinary mutagenicity, and urinary
29 1-OH-Py excretion were significantly increased in females from the polluted area, and differences
30 appeared to be more pronounced during winter time. PAH-DNA adduct levels were significantly
31 increased in the study population, when compared to the controls, only in the winter season. No
32 difference in sensitivity to bleomycin-induced lymphocyte chromatid breaks was seen between the
33 two populations. As with the study by Kalina etal. [1998]. genetic polymorphisms assumed to
34 affect the metabolic transformation of benzo [ajpyrene were not associated with any difference in
35 the incidence of DNA damage.
36 In a study of Thai school boys in urban (Bangkok] and rural areas, bulky (including but not
37 limited to BPDE-type] DNA adduct levels were measured in lymphocytes along with DNA single
38 strand breaks (SSBs], using the comet assay, and DNA repair capacity [Tuntawiroon et al.. 2007].
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1 Ambient air and personal breathing zone measurements indicated that Bangkok school children
2 experienced significantly higher exposures to benzo[a]pyrene and total PAHs. A significantly
3 higher level of SSBs (tail length 1.93 ± 0.09 versus 1.28 ± 0.12 ^m, +51%; p < 0.001) was observed
4 in Bangkok school children when compared with rural children, and this parameter was
5 significantly associated with DNA adduct levels. A significantly reduced DNA repair capacity (0.45 ±
6 0.01 versus 0.26 ± 0.01 y-radiation-induced deletions per metaphase, -42%; p < 0.001) was also
7 observed in the city school children, again significantly associated with DNA adduct levels. It was
8 not evident why higher environmental PAH exposure would be associated with lowered DNA repair
9 capacity. However, because the personal breathing zone PAH levels and DNA adduct levels were
10 not associated with each other, it is conceivable that the city school children had a priori lower DNA
11 repair capacities that contributed significantly to the high adduct levels. The authors considered
12 genetic differences between the two study populations as a possible reason for this observation.
13 D.3.3. Epidemiologic Findings in Humans
14 The association between human cancer and contact with PAH-containing substances, such
15 as soot, coal tar, and pitch, has been widely recognized since the early 1900s (Bostrometal., 2002).
16 Although numerous epidemiology studies establish an unequivocal association between PAH
17 exposure and human cancer, defining the causative role for benzo[a]pyrene and other specific PAHs
18 remains a challenge. In essentially all reported studies, either the benzo[a]pyrene exposure and/or
19 internal dose are not known, or the benzo[a]pyrene carcinogenic effect cannot be distinguished
20 from the effects of other PAH and non-PAH carcinogens. Nevertheless, three types of investigations
21 provide support for the involvement of benzo[a]pyrene in some human cancers: molecular
22 epidemiology studies; population- and hospital-based case-control studies; and occupational cohort
23 studies. In some cohort studies, benzo[a]pyrene exposure concentrations were measured and thus
24 provide a means to link exposure intensity with observed cancer rates. In case-control studies, by
25 their nature, benzo[a]pyrene and total PAH doses can only be estimated.
26 Molecular Epidemiology and Case-Control Cancer Studies
27 Defective DNA repair capacity leading to genomic instability and, ultimately, increased
28 cancer risk is well documented (Wuetal., 2007: Wuetal., 2005). Moreover, sensitivity to mutagen-
29 induced DNA damage is highly heritable and thus represents an important factor that determines
30 individual cancer susceptibility. Based on studies comparing monozygotic and dizygotic twins, the
31 genetic contribution to BPDE mutagenic sensitivity was estimated to be 48.0% (Wuetal.. 2007).
32 BPDE has been used as an etiologically relevant mutagen in case-control studies to examine the
33 association between elevated lung and bladder cancer risk and individual sensitivity to BPDE-
34 induced DNA damage. Mutagen sensitivity is determined by quantifying chromatid breaks or DNA
35 adducts in phytohemagglutinin-stimulated peripheral blood lymphocytes as an indirect measure of
36 DNA repair capacity.
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1 In a hospital-based, case-control study involving 221 lung cancer cases and 229 healthy
2 controls, DNA adducts were measured in stimulated peripheral blood lymphocytes after incubation
3 with BPDE in vitro [Lietal.. 2001). Lung cancer cases showed consistent statistically significant
4 elevations in induced BPDE-DNA adducts in lymphocytes, compared with controls, regardless of
5 subgroup by age, sex, ethnicity, smoking history, weight loss, or family history of cancer. The
6 lymphocyte BPDE-induced DNA adduct levels, when grouped by quartile using the levels in controls
7 as cutoff points, were significantly dose-related with lung cancer risk (ORs 1.11,1.62, and 3.23;
8 trend test, p < 0.001). In a related hospital-based, case-control study involving 155 lung cancer
9 patients and 153 healthy controls, stimulated peripheral blood lymphocytes were exposed to BPDE
10 in vitro [Wu etal.. 2005). DNA damage/repair was evaluated in lymphocytes using the comet assay,
11 and impacts on cell cycle checkpoints were measured using a fluorescence-activated cell-sorting
12 method. The lung cancer cases exhibited significantly higher levels of BPDE-induced DNA damage
13 than the controls (p < 0.001), with lung cancer risk positively associated with increasing levels of
14 lymphocyte DNA damage when grouped in quartiles (trend test, p < 0.001). In addition, lung cancer
15 patients demonstrated significantly shorter cell cycle delays in response to BPDE exposure to
16 lymphocytes, which correlated with increased DNA damage.
17 Sensitivity to BPDE-induced DNA damage in bladder cancer patients supports the results
18 observed in lung cancer cases. In a hospital-based, case-control study involving 203 bladder cancer
19 patients and 198 healthy controls, BPDE-induced DNA damage was specifically evaluated at the
20 chromosome 9p21 locus in stimulated peripheral blood lymphocytes [Guetal., 2008). Deletions of
21 9p21, which includes critical components of cell cycle control pathways, are associated with a
22 variety of cancers. After adjusting for age, sex, ethnicity, and smoking status, individuals with high
23 BPDE-induced damage at 9p21 were significantly associated with increased bladder cancer risk
24 (OR 5.28; 95% CI 3.26-8.59). Categorization of patients into tertiles for BPDE sensitivity relative to
25 controls demonstrated a dose-related association between BPDE-induced 9p21 damage and
26 bladder cancer risk. Collectively, the results of molecular epidemiology studies with lung and
27 bladder cancer patients indicate that individuals with a defective ability to repair BPDE-DNA
28 adducts are at increased risk for cancer and, moreover, that specific genes linked to tumorigenesis
29 pathways may be molecular targets for benzo[a]pyrene and other carcinogens.
30 Due to the importance of the diet as a benzo[a]pyrene exposure source, several population-
31 and hospital-based, case-control studies have investigated the implied association between dietary
32 intake of benzo[a]pyrene and risk for several tumor types. In a study involving 193 pancreatic
33 cancer cases and 674 controls (Anderson etal.. 2005). another involving 626 pancreatic cancer
34 cases and 530 controls (Lietal., 2007), and a third involving 146 colorectal adenoma cases and
35 228 controls (Sinha etal., 2005), dietary intake of benzo[a]pyrene was estimated using food
36 frequency questionnaires. In all studies, the primary focus was on estimated intake of
37 benzo[a]pyrene (and other carcinogens) derived from cooked meat. Overall, cases when compared
38 with controls, had higher intakes of benzo[a]pyrene and other food carcinogens, leading to the
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1 conclusion thatbenzo[a]pyrene plays a role in the etiology of these tumors in humans. In a
2 supportive follow-up case-control study of colorectal adenomas, levels of leukocyte PAH-DNA
3 adducts were significantly higher in cases when compared with controls (p = 0.02), using a method
4 that recognizes BPDE and several other PAHs bound to DNA (Gunter etal.. 2007].
5 Cohort Cancer Studies
6 Epidemiologic studies of workers in PAH-related occupations indicate increased human
7 cancer risks associated with iron and steel production, roofing, carbon black production, and
8 exposure to diesel exhaust [Bosettietal., 2007]. Exposure to benzo[a]pyrene is only one of
9 numerous contributors to the cancer risk from complex PAH-containing mixtures that occur in the
10 workplace. Although some occupational cohort studies report measured or estimated inhalation
11 exposure concentrations for benzo[a]pyrene, none report biomarkers of internal benzo[a]pyrene
12 dose in study subjects (reviewed in Bosettietal., 2007: Armstrongetal., 2004]. Several of these
13 cohort studies (summarized below] demonstrate a positive exposure-response relationship with
14 cumulative PAH exposure using benzo[a]pyrene—or a proxy such as benzene-soluble matter (BSM]
15 that can be converted to benzo[a]pyrene—as an indicator substance. These studies provide insight
16 and support for the causative role of benzo[a]pyrene in human cancer.
17 Cancer incidence in aluminum and electrode production plants
18 Exposure to benzo[a]pyrene and BSM in aluminum smelter workers is strongly associated
19 with bladder cancer and weakly associated with lung cancer (Boffetta et al., 1997: Tremblay etal.,
20 1995: Armstrongetal.. 1994: Gibbs. 1985: Theriaultetal.. 1984]. In an analysis of pooled data from
21 nine cohorts of aluminum production workers, 688 respiratory tract cancer cases were observed
22 versus 674.1 expected (pooled RR 1.03; CI 0.96-1.11] (Bosettietal.. 2007]. A total of 196 bladder
23 cancer cases were observed in eight of the cohorts, compared with 155.7 expected (pooled RR 1.29;
24 CI 1.12-1.49]. Based on estimated airborne benzo[a]pyrene exposures from a meta-analysis of
25 eight cohort studies, the predicted lung cancer RR per 100 [ig/m3-years of cumulative
26 benzo[a]pyrene exposure was 1.16 (95% CI 1.05-1.28] (Armstrong etal.. 2004].
27 Spinelli et al. (2006] reported a 14-year update to a previously published historical cohort
28 study (Spinelli etal., 1991] of Canadian aluminum reduction plant workers. The results confirmed
29 and extended the findings from the earlier epidemiology study. The study surveyed a total of
30 6,423 workers with >3 years of employment at an aluminum reduction plant in British Columbia,
31 Canada, between the years 1954 and 1997, and evaluated all types of cancers. The focus was on
32 cumulative exposure to coal tar pitch volatiles, measured as BSM and as benzo[a]pyrene.
33 Benzo[a]pyrene exposure categories were determined from the range of predicted exposures over
34 time from statistical exposure models. There were 662 cancer cases, of which approximately 98%
35 had confirmed diagnoses. The overall cancer mortality rate (SMR 0.97; CI 0.87-1.08] and cancer
36 incidence rate (standardized incidence ratio [SIR] 1.00; CI 0.92-1.08] were not different from that
37 of the British Columbia general population. However, this study identified significantly increased
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1 incidence rates for cancers of the bladder (SIR 1.80; CI 1.45-2.21) and stomach (SIR 1.46; CI 1.01-
2 2.04). The lung cancer incidence rate was only slightly higher than expected (SIR 1.10; CI 0.93-
3 1.30). Significant dose-response associations with cumulative benzo[a]pyrene exposure were seen
4 for bladder cancer (p < 0.001), stomach cancer (p < 0.05), lung cancer (p < 0.001), non-Hodgkin
5 lymphoma (p < 0.001), and kidney cancer (p < 0.01), although the overall incidence rates for the
6 latter three cancer types were not significantly elevated versus the general population. Similar
7 cancer risk results were obtained using BSM as the exposure measure; the cumulative
8 benzo[a]pyrene and BSM exposures were highly correlated (r = 0.94).
9 In several occupational cohort studies of workers in Norwegian aluminum production
10 plants, personal and stationary airborne PAH measurements were performed.
11 In a study covering 11,103 workers and 272,554 person x years of PAH exposure, cancer
12 incidence was evaluated in six Norwegian aluminum smelters (Romundstad etal.. 2000a) and
13 (Romundstad et al., 2000b). Reported estimates of PAH exposure concentrations reached a
14 maximum of 3,400 [ig/m3 PAH (680 [ig/m3 benzo[a]pyrene). The overall number of cancers
15 observed in this study did not differ significantly from control values (SIR 1.03; CI 1.0-1.1). The
16 data from this study showed significantly increased incidences for cancer of the bladder (SIR 1.3;
17 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;
18 CI 0.7-2.5), and multiple myeloma (SIR 1.4; CI 0.9-1.9). Incidence rates for bladder, lung, pancreas,
19 and kidney cancer (the latter three with SIRs close to unity) were subjected to a cumulative
20 exposure-response analysis. The incidence rate for bladder cancer showed a trend with increasing
21 cumulative exposure and with increasing lag times (up to 30 years) at the highest exposure level.
22 The incidence of both lung and bladder cancers was greatly increased in smokers. The authors
23 reported that using local county rates rather than national cancer incidence rates as controls
24 increased the SIR for lung cancer (SIR 1.4; CI 1.2-1.6) to a statistically significant level.
25 Cancer incidence in coke oven, coal gasification, and iron and steel foundry workers
26 An increased risk of death from lung and bladder cancer is reported in some studies
27 involving coke oven, coal gasification, and iron and steel foundry workers (Bostrom etal., 2002:
28 Boffettaetal., 1997). An especially consistent risk of lung cancer across occupations is noted when
29 cumulative exposure is taken into consideration (e.g., RR of 1.16 per 100 unity-years for aluminum
30 smelter workers, 1.17 for coke oven workers, and 1.15 for coal gasification workers). In an analysis
31 of pooled data from 10 cohorts of coke production workers, 762 lung cancer cases were observed
32 versus 512.1 expected (pooled RR 1.58; CI 1.47-1.69) (Bosetti etal.. 2007). Significant variations in
33 risk estimates among the studies were reported, particularly in the large cohorts (RRs of 1.1,1.2,
34 2.0, and 2.6). There was no evidence for increased bladder cancer risk in the coke production
35 workers. Based on estimated airborne benzo[a]pyrene exposures from a meta-analysis of
36 10 cohort studies, the predicted lung cancer RR per 100 [ig/m3-years of cumulative benzo[a]pyrene
37 exposure was 1.17 (95% CI 1.12-1.22) (Armstrongetal.. 2004).
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1 A meta-analysis of data from five cohorts of gasification workers reported 251 deaths from
2 respiratory tract cancer, compared with 104.7 expected (pooled RR 2.58; 95% CI 2.28-2.92)
3 [Bosettietal.. 2007). Pooled data from three of the cohorts indicated 18 deaths from urinary tract
4 cancers, versus 6.0 expected (pooled RR 3.27; 95% CI 2.06-5.19). Based on estimated airborne
5 benzo[a]pyrene exposures from a meta-analysis of four gas worker cohort studies, the predicted
6 lung cancer RRper 100 [J.g/m3-years of cumulative benzo[a]pyrene exposure was 1.15 (95% CI
7 1.11-1.20) (Armstrong etal.. 2004).
8 Increased risks were reported in iron and steel foundry workers for cancers of the
9 respiratory tract, bladder, and kidney. In an analysis of pooled data from 10 cohorts,
10 1,004 respiratory tract cancer cases were observed versus 726.0 expected (pooled RR 1.40;
11 CI 1.31-1.49) (Bosetti et al., 2007). Atotal of 99 bladder cancer cases were observed in seven of the
12 cohorts, compared with 83.0 expected (pooled RR 1.29; CI 1.06-1.57). For kidney cancer, 40 cases
13 were observed compared with 31.0 expected based on four studies (pooled RR 1.30; 95% CI 0.95-
14 1.77).
15 Xu etal. (1996) conducted a nested case-control study, surveying the cancer incidence
16 among 196,993 active or retired workers from the Anshan Chinese iron and steel production
17 complex. A large number of historical benzo[a]pyrene measurements (1956-1995) were available.
18 The study included 610 cases of lung cancer and 292 cases of stomach cancer, with 959 age- and
19 gender-matched controls from the workforce. After adjusting for nonoccupational risk factors such
20 as smoking and diet, significantly elevated risks for lung cancer and stomach cancer were identified
21 for subjects employed for >15 years, with ORs varying among job categories. For either type of
22 cancer, highest risks were seen among coke oven workers: lung cancer, OR = 3.4 (CI 1.4-8.5) and
23 stomach cancer, OR = 5.4 (CI 1.8-16.0).
24 There were significant trends for long-term, cumulative benzo[a]pyrene exposure versus
25 lung cancer (p = 0.004) or stomach cancer (p = 0.016) incidence. For cumulative total
26 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
27 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
28 cumulative total benzo[a]pyrene exposures of <0.84, 0.85-1.96,1.97-3.2, and >3.2 |ig/m3-year, the
29 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),
30 respectively. However, the investigators noted that additional workplace air contaminants were
31 measured, which might have influenced the outcome. Of these, asbestos, silica, quartz, and iron
32 oxide-containing dusts may have been confounders. For lung cancers, cumulative exposures to
33 total dust and silica dust both showed significant dose-response trends (p = 0.001 and 0.007,
34 respectively), while for stomach cancer, only cumulative total dust exposure showed a marginally
35 significant trend (p = 0.061). For cumulative total dust exposures of <69, 69-279, 280-882, and
36 >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
37 1.9 (CI 1.3-2.5), respectively. For cumulative silica dust exposures of <3.7, 3.7-10.39,10.4-27.71,
38 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),
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1 and 1.8 (CI 1.2-2.5), respectively. For cumulative total dust exposures of <69, 69-279, 280-882,
2 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
3 1.6 (CI 1.1-2.5), respectively.
4 Exposure-response data from studies of coke oven workers in the United States have often
5 been used to derive quantitative risk estimates for PAH mixtures, and for benzo[a]pyrene as an
6 indicator substance (Bostrom etal.. 2002). However, there are numerous studies of coke oven
7 worker cohorts thatdo notprovide estimates of benzo[a]pyrene exposure. An overview of the
8 results of these and other studies can be obtained from the review of Boffettaetal. (1997).
9 Cancer incidence in asphalt workers and roofers
10 These groups encompass different types of work (asphalt paving versus roofing) and also
11 different types of historical exposure that have changed from using PAH-rich coal tar pitch to the
12 use of bitumen or asphalt, both of which are rather low in PAHs due to their source (crude oil
13 refinery) and a special purification process. Increased risks for lung cancer were reported in large
14 cohorts of asphalt workers and roofers; evidence for increased bladder cancer risk is weak
15 (Burstyn etal.. 2007: Partanen andBoffetta. 1994: Chiazze etal.. 1991: Hansen. 1991.1989:
16 Hammond et al., 1976). In an analysis of pooled data from two cohorts of asphalt workers, 822 lung
17 cancer cases were observed versus 730.7 expected (pooled RR 1.14; 95% CI 1.07-1.22) (Bosetti et
18 al.. 2007). In two cohorts of roofers, analysis of pooled data indicated that 138 lung cancer cases
19 were observed, compared with 91.9 expected (pooled RR 1.51; 95% CI 1.28-1.78) (Bosetti etal..
20 2007).
21 D.4. ANIMAL STUDIES
22 D.4.1. Oral Bioassays
23 Subchronic Studies
24 De Jong etal. (1999) treated male Wistar rats (eight/dose group) with benzo[a]pyrene
25 (98.6% purity) dissolved in soybean oil by gavage 5 days/week for 35 days at doses of 0, 3,10, 30,
26 or 90 mg/kg-day (adjusted doses: 0, 2.14, 7.14, 21.4, and 64.3 mg/kg-day). Atthe end of the
27 exposure period, rats were necropsied, organ weights were determined, and major organs and
28 tissues were prepared for histological examination (adrenals, brain, bone marrow, colon, caecum,
29 jejunum, heart, kidney, liver, lung, lymph nodes, esophagus, pituitary, spleen, stomach, testis, and
30 thymus). Blood was collected for examination of hematological endpoints, but there was no
31 indication that serum biochemical parameters were analyzed. Immune parameters included
32 determinations of serum immunoglobulin (Ig) levels (IgG, IgM, IgE, and IgA), relative spleen cell
33 distribution, and spontaneous cytotoxicity of spleen cell populations determined in a natural-killer
34 (NK) cell assay.
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Supplem en tal Inform ation —Benzo[a]pyren e
1 Body weight gain was decreased beginning at week 2 at the high dose of 90 mg/kg-day;
2 there was no effect at lower doses [De Jongetal.. 1999]. Hematology revealed a dose-related
3 decrease in RBC count, hemoglobin, and hematocrit at >10 mg/kg-day (Table D-6). A minimal but
4 significant increase in mean cell volume and a decrease in mean cell hemoglobin concentration
5 were noted at 90 mg/kg-day, and may indicate dose-related toxicity for the RBCs and/or RBC
6 precursors in the bone marrow. A decrease in WBCs, attributed to a decrease in the number of
7 lymphocytes (approximately 50%) and eosinophils (approximately 90%), was observed at
8 90 mg/kg-day; however, there was no effect on the number of neutrophils or monocytes. A
9 decrease in the cell number in the bone marrow observed in the 90 mg/kg-day dose group was
10 consistent with the observed decrease in the RBC and WBC counts at this dose level. In the
11 90 mg/kg-day dose group, brain, heart, kidney, and lymph node weights were decreased and liver
12 weight was increased (Table D-6). Decreases in heart weight at 3 mg/kg-day and in kidney weight
13 at 3 and 30 mg/kg-day were also observed, but these changes did not show dose-dependent
14 responses. Dose-related decreases in thymus weight were statistically significant at >10 mg/kg-
15 day (Table D-6).
16
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1
2
Table D-6. Exposure-related effects in male Wistar rats exposed to
benzo[a]pyrene by gavage 5 days/week for 5 weeks
Effect
Hematologic effects
(mean ± SD; n = 7-8)
WBCs (109/L)
RBCs (109/L)
Hemoglobin (mmol/L)
Hematocrit (L/L)
Serum Ig levels
(mean ± SD; n = 7-8)
IgM
IgG
IgA
IgE
Cellularity (mean ± SD; n = 7-8)
Spleen (cell number x 107)
Bone marrow (G/L)
Spleen cell distribution (%)
B cells
T cells
Th cells
Ts cells
Body (g) and organ (mg) weights
(means; n = 7-8)
Body weight
Brain
Heart
Kidney
Liver
Thymus
Spleen
Mandibular lymph nodes
Mesenteric lymph nodes
Popliteal lymph nodes
Thymus cortex surface area
(% of total surface area ofthymus;
mean ± SD; n = 6-8)
Dose (mg/kg-d)
0
14.96 ± 1.9
8.7 ±0.2
10.5 ± 0.2
0.5 ±0.01
100 ± 13
100 ± 40
100 ± 28
100 ± 65
59 ±15
31 ±7
39±4
40 ±9
23 ±7
24 ±5
305
1,858
1,030
1,986
10,565
517 ± 47
551
152
165
19
77.9 ±3.8
3
13.84 ± 3.0
8.6 ±0.2
10.4 ± 0.3
0.5 ±0.01
87 ±16
141 ± 106
73 ±29
50 ±20
71 ±14
36 ±5
36 ±2
48 ±12
26 ±7
26 ±6
282*
1,864
934*
1,761*
9,567
472 ± 90
590
123
148
18
74.4 ± 2.2
10
13.69 ± 1.8
8.3 ±0.2*
9.8 ±0.2*
0.47 ±0.01*
86 ±31
104 ± 28
78 ±67
228 ±351
59 ±13
31 ±8
34 ±3*
40 ±9
24 ±5
24 ±7
300
1,859
1,000
1,899
11,250
438 ± 64*
538
160
130*
19
79.2 ±5.9
30
13.58 ±2.9
7.8 ±0.4*
9.5 ±0.4*
0.46 ± 0.02*
67 ± 16*
106 ± 19
72 ±22
145 ± 176
63 ±10
27 ±8
32 ±4*
36 ±2
22 ±4
19 ±2
293
1,784
967
1,790*
11,118
388 ± 71*
596
141
158
17
75.8 ±4.0
90
8.53 ±1.1*
7.1 ±0.4*
8.6 ±0.6*
0.43 ±0.02*
81 ±26
99 ±29
39 ± 19*
75 ±55
41 ± 10*
19 + 4*
23 ±4*
44 ±6
26 ±4
27 ±5
250*
1,743*
863*
1,626*
12,107*
198 ±65*
505
89*
107*
10*
68.9 ±5.2*
3
4
5
6
7
8
9
10
11
12
13
*Significantly (p < 0.05) different from control mean. For body weight and organ weight means, SDs were only
reported for thymus weights.
Source: DeJongetal. (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
technique. Changes in the following immune parameters were noted: dose-related and statistically
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1 significant decrease in the relative number of B cells in the spleen at 10 (13%), 30 (18%), and
2 90 mg/kg-day (41%); significant decreases in absolute number of cells harvested in the spleen
3 (31%), in the number of B cells in the spleen (61%), and NK cell activity in the spleen (E:T ratio was
4 40.9 ± 28.4% that of the controls) at 90 mg/kg-day; and a decrease in serum IgM (33%) and IgA
5 (61%) in rats treated with 30 and 90 mg/kg-day, respectively. The decrease in the spleen cell count
6 was attributed by the study authors to the decreased B cells and suggested a possible selective
7 toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The study authors considered
8 the decrease in IgA and IgM to be due to impaired production of antibodies, suggesting a role of
9 thymus toxicity in the decreased (T-cell dependent) antibody production. In addition to the effects
10 on the thymus and spleen, histopathologic examination revealed treatment-related lesions only in
11 the liver and forestomach at the two highest dose levels, but the incidence data for these lesions
12 were not reported by De long et al. (1999). Increased incidence for forestomach basal cell
13 hyperplasia (p < 0.05 by Fisher's exact test) was reported at 30 and 90 mg/kg-day, and increased
14 incidence for oval cell hyperplasia in the liver was reported at 90 mg/kg-day (p < 0.01, Fisher's
15 exact test). The results indicate that 3 mg/kg-day was a no-observed-adverse-effect level (NOAEL)
16 for effects on hematological parameters (decreased RBC count, hemoglobin, and hematocrit) and
17 immune parameters (decreased thymus weight and percent of B cells in the spleen) noted in Wistar
18 rats at 10 mg/kg-day (the lowest-observed-adverse-effectlevel [LOAEL]) and above. Lesions of the
19 liver (oval cell hyperplasia) and forestomach (basal cell hyperplasia) occurred at doses >30 mg/kg-
20 day.
21 Knuckles etal. (2001) exposed male and female F344 rats (20/sex/dose group) to
22 benzo[a]pyrene (98% purity) at doses of 0, 5, 50, or 100 mg/kg-day in the diet for 90 days. Food
23 consumption and body weight were monitored, and the concentration of benzo[a]pyrene in the
24 food was adjusted every 3-4 days to maintain the target dose. The authors indicated that the actual
25 intake of benzo[a]pyrene by the rats was within 10% of the calculated intake, and the nominal
26 doses were not corrected to actual doses. Hematology and serum chemistry parameters were
27 evaluated. Urinalysis was also performed. Animals were examined for gross pathology, and
28 histopathology was performed on selected organs (stomach, liver, kidney, testes, and ovaries).
29 Statistically significant decreases in RBC counts and hematocrit level (decreases as much as 10 and
30 12%, respectively) were observed in males at doses >50 mg/kg-day and in females at 100 mg/kg-
31 day. A maximum 12% decrease (statistically significant) in hemoglobin level was noted in both
32 sexes at 100 mg/kg-day. Blood chemistry analysis showed a significant increase in blood urea
33 nitrogen (BUN) only in high-dose (100 mg/kg-day) males. Histopathology examination revealed an
34 apparent increase in the incidence of abnormal tubular casts in the kidney in males at 5 mg/kg-day
35 (40%), 50 mg/kg-day (80%), and 100 mg/kg-day (100%), compared to 10% in the controls. Only
36 10% of the females showed significant kidney tubular changes at the two high-dose levels
37 compared to zero animals in the female control group. The casts were described as molds of distal
38 nephron lumen and were considered by the study authors to be indicative of renal dysfunction.
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1 From this study, male F344 rats appeared to be affected more severely by benzo[a]pyrene
2 treatment than the female rats. However, the statistical significance of the kidney lesions is unclear.
3 Several reporting gaps and inconsistencies regarding the reporting of kidney abnormalities in
4 Knuckles etal. [2001] make interpretation of the results difficult. Results of histopathological
5 kidney abnormalities (characterized primarily as kidney casts) were presented graphically and the
6 data were not presented numerically in this report. No indication was given in the graph that any
7 groups were statistically different than controls, although visual examination of the magnitude of
8 response and error bars appears to indicate a fourfold increase in kidney casts in males compared
9 to the control group (40 compared to 10%). The figure legend reported the data as "percentage
10 incidence of abnormal kidney tissues" and reported values as mean ± SD. However, the text under
11 the materials and methods section stated that Fisher's exact test was used for histopathological
12 data, which would involve the pairwise comparison of incidence and not means. There are
13 additional internal inconsistencies in the data presented. The data appeared to indicate that
14 incidences for males were as follows: control, 10%; 5 mg/kg-day, 40%; 50 mg/kg-day, 80%; and
15 100 mg/kg-day, 100%; however, these incidences are inconsistent with the size of the study
16 groups, which were reported as 6-8 animals per group. The study authors were contacted, but did
17 not respond to EPA's request for clarification of study design and/or results. Due to issues of data
18 reporting, a LOAEL could not be established for the increased incidence of kidney lesions. Based on
19 the statistically significant hematological effects including decreases in RBC counts, hematocrit, and
20 BUN, the NOAEL in males was 5 mg/kg-day and the LOAEL was 50 mg/kg-day, based on in F344
21 rats. No exposure-related histological lesions were identified in the stomach, liver, testes, or
22 ovaries in this study.
23 In a range-finding study, Wistar (specific pathogen-free Riv:TOX) rats (10/sex/dose group)
24 were administered benzo[a]pyrene (97.7% purity) dissolved in soybean oil by gavage at dose levels
25 of 0,1.5, 5,15, or 50 mg/kg body weight-day, 5 days/week for 5 weeks (Kroese etal., 2001).
26 Behavior, clinical symptoms, body weight, and food and water consumption were monitored. None
27 of the animals died during the treatment period. Animals were sacrificed 24 hours after the last
28 dose. Urine and blood were collected for standard urinalysis and hematology and clinical chemistry
29 evaluation. Liver enzyme induction was monitored based on EROD activity in plasma. Animals
30 were subjected to macroscopic examination, and organ weights were recorded. The esophagus,
31 stomach, duodenum, liver, kidneys, spleen, thymus, lung, and mammary gland (females only) from
32 the highest-dose and control animals were evaluated for histopathology. Intermediate-dose groups
33 were examined if abnormalities were observed in the higher-dose groups.
34 A significant, but not dose-dependent, increase in food consumption in males at >1.5 mg/kg-
35 day and a decrease in food consumption in females at >5 mg/kg-day was observed (Kroese etal.,
36 2001). Water consumption was statistically significantly altered in males only: a decrease at 1.5, 5,
37 and 15 mg/kg-day and an increase at 50 mg/kg-day. Organ weights of lung, spleen, kidneys,
38 adrenals, and ovaries were not affected by treatment. There was a dose-related, statistically
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1 significant decrease in thymus weight in males at 15 and 50 mg/kg-day (decreased by 28 and 33%,
2 respectively) and a significant decrease in thymus weight in females at 50 mg/kg-day (decreased by
3 17%) (Table D-7). In both sexes, liver weight was statistically significantly increased only at
4 50 mg/kg-day by about 18% (Table D-7).
5
6
Table D-7. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene
by gavage 5 days/week for 5 weeks
Organ
Liver weight (g; mean ± SD)
Males
Females
Thymus weight (mg; mean ± SD)
Males
Females
Basal cell hyperplasia of the
forestomach (incidence with slight
severity)
Males
Females
Dose (mg/kg-d)
0
6.10 ±0.26
4.28 ±0.11
471 ± 19
326 ± 12
1/10
0/10
1.5
6. 19 ±0.19
4.40 ± 0.73
434 ± 20
367 ± 23
1/10
1/10
5
6.13 ±0.10
4.37 ±0.11
418 ± 26
351 ±25
4/10
1/10
15
6.30 ±0.14
4.67 ±0.17
342 ± 20*
317 ± 30
3/10
3/10*
50
7.20 ±0.18*
5.03 ±0.15*
317 ±21*
271 ±16*
7/10
7/10*
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
*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 (Kroese etal.. 2001). The
induction of liver microsomal EROD was not accompanied by any adverse histopathologic findings
in the liver at the highest dose, 5 0 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.
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Supplem en tal Inform ation —Benzo[a]pyren e
1 Kroese etal. [2001] exposed Wistar (Riv:TOX) rats (10/sex/dose group) to benzo[a]pyrene
2 (98.6% purity, dissolved in soybean oil) by gavage at 0, 3,10, or 30 mg/kg body weight-day,
3 5 days/week for 90 days. The rats were examined daily for behavior and clinical symptoms and by
4 palpation. Food and water consumption, body weights, morbidity, and mortality were monitored.
5 At the end of the exposure period, rats were subjected to macroscopic examination and organ
6 weights were recorded. Blood was collected for hematology and serum chemistry evaluation, and
7 urine was collected for urinalysis. All gross abnormalities, particularly masses and lesions
8 suspected of being tumors, were evaluated. The liver, stomach, esophagus, thymus, lung, spleen,
9 and mesenteric lymph node were examined histopathologically. In addition, cell proliferation in
10 forestomach epithelium was measured as the prevalence of S-phase epithelial cells displaying
11 bromodeoxyuridine (BrdU) incorporation.
12 There were no obvious effects on behavior of the animals, and no difference was observed
13 in survival or food consumption between exposed animals and controls [Kroese etal., 2001).
14 Higher water consumption and slightly lower body weights than the controls were observed in
15 males but not females at the high dose of 30 mg/kg-day. Hematological investigations showed only
16 nonsignificant, small dose-related decreases in RBC count and hemoglobin level in both sexes.
17 Clinical chemistry evaluation did not show any treatment-related group differences or dose-
18 response relationships for alanine aminotransferase, serum aspartate transaminase (AST), lactate
19 dehydrogenase (LDH), or creatinine, but a small dose-related decrease in y-glutamyl transferase
20 activity was observed in males only. Urinalysis revealed an increase in urine volume in males at
21 30 mg/kg-day, which was not dose related. At the highest dose, both sexes showed increased levels
22 of urinary creatinine and a dose-related increase in urinary protein. However, no further
23 investigation was conducted to determine the underlying mechanisms for these changes. At
24 necropsy, reddish to brown/gray discoloration of the mandibular lymph nodes was consistently
25 noted in most rats; occasional discoloration was also observed in other regional lymph nodes
26 (axillary). Statistically significant increases in liver weight were observed at 10 and 30 mg/kg-day
27 in males (15 and 29%) and at 30 mg/kg-day in females (17%). A decrease in thymus weight was
28 seen in both sexes at 30 mg/kg-day (17 and 33% decrease in females and males, respectively,
29 compared with controls) (Table D-8). At 10 mg/kg-day, thymus weight in males was decreased by
30 15%, but the decrease did not reach statistical significance.
31
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1
2
Table D-8. Means ± SDa for liver and thymus weights in Wistar rats exposed to
benzo[a]pyrene by gavage 5 days/week for 90 days
Organ
Liver weight (g)
Males
Females
Thymus weight (mg)
Males
Females
Dose (mg/kg-d)
0
7.49 ±0.97
5.54 ±0.70
380 ± 60
320 ± 60
3
8.00 ± 0.85
5.42 ±0.76
380 ± 110
310 ± 50
10
8.62 ± 1.30*
5.76 ±0.71
330 ± 60
300 ± 40
30
9.67 ±1.17*
6.48 ±0.78*
270 ± 40*
230 ± 30*
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
29
30
31
*Significantly (p < 0.05) different from control mean; student t-test (unpaired, two-tailed); n = 10/sex/group.
Reported as SE, but judged to be SD (and confirmed by study authors).
Source: Kroese et al. (2001).
Histopathologic examination revealed what was characterized by Kroese etal. [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 [Kroese etal., 2001). Incidences for these lesions (also
described as "slight basal cell hyperplasia") in the 0, 3,10, and 30-mg/kg-day groups were 0/10,
2/10, 3/10, and 7/10, respectively, in female rats and 2/10, 0/10, 6/10, and 7/10, respectively, in
male rats. Nodular hyperplasia was noted in one animal of each sex at 30 mg/kg-day. A significant
(p < 0.05) increase in proliferation of forestomach epithelial cells was detected at doses >10 mg/kg-
day by morphometric of analysis of nuclei with BrdU incorporation. The mean numbers of BrdU-
staining nuclei per unit surface area of the underlying lamina muscularis mucosa were increased by
about 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.
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1 The target organs of benzo[a]pyrene toxicity in this 90-day dietary study of Wistar rats
2 were the forestomach, thymus, and liver. The LOAEL for forestomach hyperplasia, decreased
3 thymus weight, and thymus atrophy was 30 mg/kg-day and the NOAEL was 10 mg/kg-day.
4 Chronic Studies and Cancer Bioassays
5 Kroese et al. [2001] exposed Wistar (Riv:TOX) rats (52/sex/dose group) to benzo[a]pyrene
6 (98.6% purity) in soybean oil by gavage at nominal doses of 0, 3,10, or 30 mg/kg-day, 5 days/week,
7 for 104 weeks. Mean achieved dose levels were 0, 2.9, 9.6, and 29 mg/kg-day. Additional rats
8 (6/sex/group) were sacrificed after 4 and 5 months of exposure for analysis of DNA adduct
9 formation in blood and major organs and tissues. The rats were 6 weeks old at the start of
10 exposure. The rats were examined daily for behavior and clinical symptoms and by palpation.
11 Food and water consumption, body weights, morbidity, and mortality were monitored during the
12 study. Complete necropsy was performed on all animals that died during the course of the study,
13 were found moribund, or at terminal sacrifice [organ weight measurement was not mentioned in
14 the report by Kroese etal., 2001). The organs and tissues collected and prepared for microscopic
15 examination included: brain, pituitary, heart, thyroid, salivary glands, lungs, stomach, esophagus,
16 duodenum, jejunum, ileum, caecum, colon, rectum, thymus, kidneys, urinary bladder, spleen, lymph
17 nodes, liver pancreas, adrenals, sciatic nerve, nasal cavity, femur, skin including mammary tissue,
18 ovaries/uterus, and testis/accessory sex glands. Some of these tissues were examined only when
19 gross abnormalities were detected. All gross abnormalities, particularly masses and lesions that
20 appeared to be tumors, were also examined.
21 At 104 weeks, survival in the control group was 65% (males) and 50% (females), whereas
22 mortality in the 30 mg/kg-day dose group was 100% after about week 70. At 80 weeks, survival
23 percentages were about 90, 85, and 75% in female rats in the 0, 3, and 10 mg/kg-day groups,
24 respectively; in males, respective survival percentages were ~95, 90, and 85% at 80 weeks.
25 Survival of 50% of animals occurred at 104,104, ~90, and 60 weeks for control through high-dose
26 females; for males, the respective times associated with 65% survival were 104,104,104, and
27 ~60 weeks. The high mortality rate in high-dose rats was attributed to liver or forestomach tumor
28 development, not to noncancer systemic effects. After 2 0 weeks, body weight was decreased
29 (compared with controls by >10%) in 30-mg/kg-day males, but not in females. This decrease was
30 accompanied by a decrease in food consumption. Body weights and food consumption were not
31 adversely affected in the other dose groups compared to controls. In males, there was a dose-
32 dependent increase in water consumption starting at week 13, but benzo[a]pyrene treatment had
33 no significant effects on water consumption in females.
34 Tumors were detected at significantly elevated incidences at several tissue sites in female
35 and male rats at doses >10 and >3 mg/kg-day, respectively (Table D-9) (Kroese etal., 2001). The
36 tissue sites with the highest incidences of tumors were the liver (hepatocellular adenoma and
37 carcinoma) and forestomach (squamous cell papilloma and carcinoma) in both sexes (Table D-9).
38 The first liver tumors were detected in week 35 in high-dose male rats. Liver tumors were
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1 described as complex, with a considerable proportion (59/150 tumors) metastasizing to the lungs.
2 At the highest dose level, 95% of rats with liver tumors had malignant carcinomas (95/100;
3 Table D-9). Forestomach tumors were associated with the basal cell proliferation observed
4 (without diffuse hyperplasia) in the forestomach of rats in the preliminary range-finding and
5 90-day exposure studies. At the highest dose level, 59% of rats with forestomach tumors had
6 malignant carcinomas (60/102; Table D-9). Other tissue sites with significantly elevated incidences
7 of tumors in the 30 mg/kg-day dose group included the oral cavity (papilloma and squamous cell
8 carcinoma [SCC]) in both sexes, and the jejunum (adenocarcinoma), kidney (cortical adenoma), and
9 skin (basal cell adenoma and carcinoma) in male rats (Table D-9). In addition, auditory canal
10 tumors (carcinoma or squamous cell papilloma originating from pilo-sebaceous units including the
11 Zymbal's gland) were also detected in both sexes at 30 mg/kg-day, but auditory canal tissue was
12 not histologically examined in the lower dose groups and the controls (Table D-9). Gross
13 examination revealed auditory canal tumors only in the high-dose group.
14
15
Table D-9. Incidences of exposure-related neoplasms in Wistar rats treated by
gavage with benzo[a]pyrene, 5 days/week, for 104 weeks
Site
Oral cavity
Papilloma
SCC
Basal cell adenoma
Sebaceous cell carcinoma
Esophagus
Sarcoma undifferentiated
Rhabdomyosarcoma
Fibrosarcoma
Forestomach
Squamous cell papilloma
SCC
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Cholangiocarcinoma
Anaplastic carcinoma
Auditory canal
Benign tumor
Squamous cell papilloma
Carcinoma
Dose (mg/kg-d)
0
3
10
30a
Females'3
0/19
1/19
0/19
0/19
0/52
0/52
0/52
1/52
0/52
0/52
0/52
0/52
0/52
0/0
0/0
0/0
0/21
0/21
0/21
0/21
0/52
1/52
0/52
3/51
3/51
2/52
0/52
0/52
0/52
0/0
0/1
0/1
0/9
0/9
1/9
0/9
2/52
4/52
3/52
20/51*
10/51*
7/52*
32/52*
1/52
1/52
0/0
0/0
0/0
9/31*
9/31*
4/31
1/31
0/52
0/52
0/52
25/52*
25/52*
1/52
50/52*
0/52
0/52
1/20
1/20
13/20*
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Site
Oral cavity
Papilloma
sec
Basal cell adenoma
Sebaceous cell carcinoma
Forestomach
Squamous cell papilloma
sec
Jejunum
Adenocarcinoma
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Cholangiocarcinoma
Kidney
Cortical adenoma
Adenocarcinoma
Urothelial carcinoma
Auditory canal
Benign
Squamous cell papilloma
Carcinoma
Sebaceous cell adenoma
Skin and mammary
Basal cell adenoma
Basal cell carcinoma
sec
Keratoacanthoma
Trichoepithelioma
Fibrosarcoma
Fibrous histiocytoma (malignant)
Dose (mg/kg-d)
0
3
10
30a
Males"
0/24
1/24
0/24
0/24
0/52
0/52
0/51
0/52
0/52
0/52
0/52
0/52
0/52
0/1
0/1
0/1
0/1
2/52
1/52
0/52
1/52
0/52
0/52
0/52
0/24
0/24
0/24
0/24
7/52*
1/52
0/50
3/52
1/52
0/52
0/52
0/52
0/52
0/0
0/0
0/0
0/0
0/52
1/52
1/52
0/52
1/52
3/52
0/52
2/37
5/37
0/37
0/37
18/52*
25/52*
1/51
15/52*
23/52*
0/52
7/52*
2/52
0/52
1/7
0/7
2/7
0/7
1/52
0/52
1/52
1/52
2/52
5/52
1/52
10/38*
11/38*
2/38
2/38
17/52*
35/52*
8/49*
4/52
45/52*
1/52
8/52*
0/52
3/52
0/33
4/33
19/33*
1/33
10/51*
4/51
5/51
4/51
8/51*
0/51
1/52
1
2
3
4
5
6
7
8
9
10
11
12
*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.
blncidences 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).
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1 Kroese etal. [2001] did not systematically investigate nonneoplastic lesions detected in rats
2 sacrificed during the 2-year study, because the focus was to identify and quantitate tumor
3 occurrence. However, incidences were reported for nonneoplastic lesions in tissues or organs in
4 which tumors were detected (i.e., oral cavity, esophagus, forestomach, jejunum, liver, kidney, skin,
5 mammary, and auditory canal). The reported nonneoplastic lesions associated with exposure were
6 the forestomach basal cell hyperplasia and clear cell foci of cellular alteration in the liver.
7 Incidences for forestomach basal cell hyperplasia in the control through high-dose groups were
8 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
9 hepatic clear cell foci of cellular alteration were 22/52, 33/52, 4/52, and 2/52 for females and
10 8/52, 22/52,1/52, and 1/52 for males. These results indicate thatthe lowest dose group, 3 mg/kg-
11 day, was a LOAEL for increased incidence of forestomach hyperplasia and hepatic histological
12 changes in male and female Wistar rats exposed by gavage to benzo[a]pyrene for up to 104 weeks
13 (see Table D-9). The lack of an increase in incidence of these nonneoplastic lesions in the
14 forestomach and liver at the intermediate and high doses (compared with controls) were
15 associated with increased incidences of forestomach and liver tumors at these dose levels. The
16 authors of this study note that nonneoplastic effects were not quantified in organs with tumors.
17 As an adjunct study to the 2-year gavage study with Wistar rats, Kroese etal. (2001)
18 sacrificed additional rats (6/sex/group) after 4 and 5 months of exposure (0,1, 3,10, or 30 mg/kg-
19 day) for analysis of DNA adduct formation in WBCs and major organs and tissues. Additional rats
20 (6/sex/time period) were exposed to 0.1 mg/kg-day benzo[a]pyrene for 4 and 5 months for
21 analysis of DNA adduct formation. Using the [32P]-postlabeling technique, five benzo[a]pyrene-DNA
22 adducts were identified in all of the examined tissues at 4 months (WBCs, liver, kidney, heart, lung,
23 skin, forestomach, glandular stomach, brain). Only one of these adducts (adduct 2) was identified
24 based on co-chromatography with a standard. This adduct, identified as 10p-(deoxyguanosin-
25 N2-yl)-7p,8a,9a-trihydroxy-7,8,9,10 tetrahydro-benzo[a]pyrene, was the predominant adduct in all
26 organs of female rats exposed to 10 mg/kg-day, except the liver and kidney, in which another
27 adduct (unidentified adduct 4) was predominant Levels of total adducts (number of
28 benzo[a]pyrene-DNA adducts per 1010 nucleotides) in examined tissues (from the single 10 mg/kg-
29 day female rat) showed the following order: liver > heart > kidney > lung > skin > forestomach «
30 WBCs > brain. Mean values for female levels of total benzo[a]pyrene-DNA adducts (number per
31 1010 nucleotides) in four organs showed the same order, regardless of exposure group: liver > lung
32 > forestomach « WBCs; comparable data for males were not reported. Mean total benzo[a]pyrene-
33 DNA adduct levels in livers increased in both sexes from about 100 adducts per 1010 nucleotides at
34 0.1 mg/kg-day to about 70,000 adducts per 1010 nucleotides at 30 mg/kg-day. In summary, these
35 results suggest that total benzo[a]pyrene-DNA adduct levels in tissues at 4 months were not
36 independently associated with the carcinogenic responses noted after 2 years of exposure to
37 benzo[a]pyrene. The liver showed the highest total DNA adduct levels and a carcinogenic response,
38 but total DNA adduct levels in heart, kidney, and lung (in which no carcinogenic responses were
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1 detected) were higher than levels in forestomach and skin (in which carcinogenic responses were
2 detected).
3 Groups of Sprague-Dawley rats (32/sex/dose) were fed diets delivering a daily dose of
4 0.15 mg benzo[a]pyrene/kg body weight every ninth day or 5 times/week [Brune etal., 1981).
5 Other groups (32/ sex/dose) were given gavage doses of 0.15 mgbenzo[a]pyrene (in aqueous 1.5%
6 caffeine solution)/kg every ninth day, every third day, or 5 times/week. The study included an
7 untreated control group (to compare with the dietary exposed groups) and a gavage vehicle control
8 group (each with 32 rats/sex). Rats were treated until moribundity or death occurred, with
9 average annual doses reported in Table D-10 [mg/kg-year, calculated by Brune etal. (1981)]. The
10 following tissues were prepared for histopathological examination: tongue, larynx, lung, heart,
11 trachea, esophagus, stomach, small intestine, colon, rectum, spleen, liver, urinary bladder, kidney,
12 adrenal gland, and any tissues showing tumors or other gross changes. Survival was similar among
13 the groups, with the exception that the highest gavage-exposure group showed a decreased median
14 time of survival (Table D-10). Significantly increased incidences of portal-of-entry tumors
15 (forestomach, esophagus, and larynx) were observed in all of the gavage-exposed groups and in the
16 highest dietary exposure group (Table D-10). Following dietary administration, all observed
17 tumors were papillomas. Following gavage administration, two malignant forestomach tumors
18 were found (one each in the mid- and high-dose groups) and the remaining tumors were benign.
19 The data in Table D-10 show that the carcinogenic response to benzo[a]pyrene was stronger with
20 the gavage protocol compared with dietary exposure, and that no distinct difference in response
21 was apparent between the sexes. Tumors at distant sites (mammary gland, kidney, pancreas, lung,
22 urinary bladder, testes, hematopoietic, and soft tissue) were not considered treatment-related as
23 they were also observed at similar rates in the control group (data not provided). The study report
24 did not address noncancer systemic effects.
25
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1
2
3
Table D-10. 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 Dose3
(mg/kg-d)
Forestomach Tumors
Total Alimentary Tract
Tumorsc (Larynx,
Esophagus, Forestomach)
Median
Survival Time
(wks)
Benzo[a]pyrene by gavage in 1.5% caffeine solution
0
6
18
39
0
0.016
0.049
0.107
3/64 (4.7%)
12/64 (18.8%)*
26/64(40.1%)**
14/64 (21.9%)**
6/64 (9.4%)
13/64 (20.3%)
26/64 (40.6%)
14/64 (21.9%)
102
112
113
87
Benzo[a]pyrene in diet
0
6
39
0
0.016
0.107
2/64(3.1%)
1/64 (1.6%)
9/64 (14.1%)*
3/64 (4.7%)
3/64 (4.7%)
10/64 (15.6%)
129
128
131
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
29
30
31
32
33
*Significantly (p < 0.1) different from control using a modified % test that accounted for group differences in
survival time.
**Significantly (p < 0.05) different from control using a modified %2 test that accounted for group differences in
survival time.
aAverage annual dose divided by 365 days.
bNo sex-specific forestomach tumor incidence data were reported by Brune et al. (1981).
cSex-specific incidences for total alimentary tract tumors were reported as follows:
Gavage (control, high dose):
Diet (control, high dose):
Source: Brune et al. (1981).
Male: 6/32, 7/32, 15/32, 8/32
Female: 0/32, 6/32,11/32, 6/32
Male: 3/32, 3/32, 8/32
Female: 0/32, 0/32, 2/32
In the other modern cancer bioassay with benzo[a]pyrene, female B6C3Fi mice (48/dose
group) were administered benzo[a]pyrene (98.5% purity) at concentrations of 0 (acetone vehicle),
5, 25, or 100 ppm in the diet for 2 years (Beland and Gulp. 1998: Gulp etal.. 1998). This study was
designed to compare the carcinogenicity of coal tar mixtures with that of benzo[a]pyrene and
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. Gulp etal. (1998) reported that the average daily intakes of
benzo[a]pyrene in the 25- and 100-ppm groups were 104 and 430 |ig/day, but did not report
intakes for the 5-ppm group. Based on the assumption that daily benzo[a]pyrene intake at 5 ppm
was one-fifth of the 2 5-ppm intake (about 21 |ig/day), average daily doses for the three
benzo[a]pyrene groups are estimated as 0.7, 3.3, and 16.5 mg/kg-day. Estimated doses were
calculated using time-weighted average (TWA) body weights of 0.032 kg for the control, 5- and
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1 25-ppm groups and 0.026 kg for the 100-ppm group (estimated from graphically presented data).
2 Food consumption, body weights, morbidity, and mortality were monitored at intervals, and lung,
3 kidneys, and liver were weighed at sacrifice. Necropsy was performed on all mice that died during
4 the experiment or survived to the end of the study period. Limited histopathologic examinations
5 (liver, lung, small intestine, stomach, tongue, esophagus) were performed on all control and high-
6 dose mice and on all mice that died during the experimental period, regardless of treatment group.
7 In addition, all gross lesions found in mice of the low- and mid-dose groups were examined
8 histopathologically.
9 None of the mice administered 100 ppm benzo[a]pyrene survived to the end of the study,
10 and morbidity/mortality was 100% by week 78. Decreased survival was also observed at 25 ppm
11 with only 27% survival at 104 weeks, compared with 56 and 60%, in the 5-ppm and control groups,
12 respectively. In the mid- and high-dose group, 60% of mice were alive at about 90 and 60 weeks,
13 respectively. Early deaths in exposed mice were attributed to tumor formation rather than other
14 causes of systemic toxicity. Food consumption was not statistically different in benzo[a]pyrene-
15 exposed and control mice. Body weights of mice fed 100 ppm were similar to those of the other
16 treated and control groups up to week 46, and after approximately 52 weeks, body weights were
17 reduced in 100-ppm mice compared with controls. Body weights for the 5- and 25-ppm groups
18 were similar to controls throughout the treatment period. Compared with the control group, no
19 differences in liver, kidney, or lung weights were evident in any of the treated groups (other organ
20 weights were not measured).
21 Papillomas and/or carcinomas of the forestomach, esophagus, tongue, and larynx at
22 elevated incidences occurred in groups of mice exposed to 25 or 100 ppm, butno exposure-related
23 tumors occurred in the liver or lung (Beland and Gulp, 1998: Gulp et al., 1998). The forestomach
24 was the most sensitive tissue, demonstrated the highest tumor incidence among the examined
25 tissues, and was the only tissue with an elevated incidence of tumors at 25 ppm (Table D-ll). In
26 addition, most of the forestomach tumors in the exposed groups were carcinomas, as 1, 31, and 45
27 mice had forestomach carcinomas in the 5-, 25-, and 100-ppm groups respectively. Nonneoplastic
28 lesions were also found in the forestomach at significantly (p < 0.05) elevated incidences:
29 hyperplasia at >25 ppm and hyperkeratosis at >25 ppm (Table D-ll). The esophagus was the only
30 other examined tissue showing elevated incidence of a nonneoplastic lesion (basal cell hyperplasia,
31 see Table D-ll). Tumors (papillomas and carcinomas) were also significantly elevated in the
32 esophagus and tongue at 100 ppm (Table D-ll). Esophogeal carcinomas were detected in 1 mouse
33 at 25 ppm and 11 mice at 100 ppm. Tongue carcinomas were detected in seven 100-ppm mice; the
34 remaining tongue tumors were papillomas. Although incidences of tumors of the larynx were not
35 significantly elevated in any of the exposed groups, a significant dose-related trend was apparent
36 (Table D-ll).
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1
2
Table D-ll. Incidence of nonneoplastic and neoplastic lesions in female
B6C3Fi mice fed benzo[a]pyrene in the diet for up to 2 years
Tissue and Lesion
Liver (hepatocellular adenoma)
Lung (alveolar/bronchiolar adenoma and/or carcinoma)
Forestomach (papilloma and/or carcinoma)
Forestomach (hyperplasia)
Forestomach (hyperkeratosis)
Esophagus (papilloma and/or carcinoma)
Esophagus (basal cell hyperplasia)
Tongue (papilloma and/or carcinoma)
Larynx (papilloma and/or carcinoma)
Incidence (%)
Benzo[a]pyrene Concentration (ppm) in Diet
0
5
25
100
Average Daily Doses (mg/kg-d)
0
2/48
(2)
5/48
(10)
l/48a
(2)
13/48a
(27)
13/48a
(27)
0/48a
(0)
l/48a
(2)
0/49a
(0)
0/35a
(0)
0.7
7/48
(15)
0/48
(0)
3/47
(6)
23/47
(49)
22/47
(47)
0/48
(0)
0/48
(0)
0/48
(0)
0/35
(0)
3.3
5/47
(11)
4/45
(9)
36/46*
(78)
33/46*
(72)
33/46*
(72)
2/45
(0)
5/45
(11)
2/46
(4)
3/34
(9)
16.5
0/45
(0)
0/48
(0)
46/47*
(98)
37/47*
(79)
38/47*
(81)
27/46*
(59)
30/46*
(65)
23/48*
(48)
5/38
(13)
3
4
5
6
7
8
9
10
11
12
13
14
15
*Significantly different from control incidence (p < 0.05); using a modified Bonferonni procedure for multiple
comparisons to the same control.
Significant (p < 0.05) dose-related trend calculated for incidences of these lesions.
Sources: Beland and Gulp (1998): Gulp et al. (1998).
Neal and Rigdon [1967] fedbenzo[a]pyrene (purity not reported) at concentrations of 0,1,
10, 20, 30, 40, 45, 50,100, and 250 ppm to male and female CFW-Swiss mice in the diet.
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
44.4 mg/kg-day. The age of the mice ranged from 17 to 180 days old and the treatment time was
iCalculation: 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 from 1 to 197 days; the size of the treated groups ranged from 9 to 73. There were 289 mice
2 (number of mice/sex not stated) in the control group. No forestomach tumors were reported at 0,
3 0.2, or 1.8 mg/kg-day. The incidence of forestomach tumors at 20, 30, 40, 45, 50,100, and 250 ppm
4 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, 23/34,
5 19/23, and 66/73, respectively.
6 Other Oral Exposure Cancer Bioassays in Mice
7 Numerous other oral exposure cancer bioassays in mice have limitations that restrict their
8 usefulness for characterizing dose-response relationships between chronic-duration oral exposure
9 to benzo[a]pyrene and noncancer effects or cancer, but collectively, they provide strong evidence
10 that oral exposure to benzo[a]pyrene can cause portal-of-entry site tumors (see Table D-12 for
11 references).
12
Table D-12. Other oral exposure cancer bioassays in mice
Species/Strain
Rat/Sprague-
Dawley
Mouse/Ha ICR
Exposure
Groups of rats (32/sex/dose)
were fed diets delivering a
daily dose of 0.15 mg
benzo[a]pyrene/kg body
weight every 9th day or
5 times/week (Brune et al..
1981). Other groups (32/
sex/dose) were given gavage
doses of 0.15 mg
benzo[a]pyrene (in aqueous
1.5% caffeine solution)/kg
every 9th day, every 3rd day,
or 5 times/week.
Groups of 12-20 mice (10
wks old) were fed
benzo[a]pyrene in the diet
(0.1, 0.3, or 1.0 mg/g diet) for
12-20 wks. Estimated doses
were 14.3, 42.0, or
192 mg/kg-d.
Results
Larynx, esophagus, and
forestomach tumors
Dose
(gavage)
0 6/64
0.016 13/64
0.049 26/64
0.107 14/64
Dose
/ j; 4.\
(diet)
0 3/64
0.016 3/64
0.107 10/64
Incidence with
forestomach tumors:
Low, 11/20 (18 wks)
Mid, 13/19 (20 wks)
High, 12/12 (12 wks)
Comments
Doses are annual
averages.
Nonstandard
treatment
protocol involved
animals being
treated for <5
d/wk; relatively
high control
incidence
compared to
other gavage
studies.
Less-than-
lifetime exposure
duration; only
stomachs were
examined for
tumors; tumors
found only in
forestomach.
Reference
Brune et al.
(1981)
Wattenberg
(1972)
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Species/Strain
Mouse/Ha ICR
Mouse/Ha ICR
Mouse/CD-I
Mouse/BALB
Mouse/C3H
Exposure
Groups of nine mice (9 wks
old) were fed benzo[a]pyrene
in the diet (0, 0.2, or 0.3 mg/g
diet) for 12 wks and
sacrificed. Estimated doses
were 0, 27.3, or 41 mg/kg-d.
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.
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.
25 mice (8 wks old) were
given 0.5 mg benzo[a]pyrene
2 times/wk for 15 wks.
19 mice (about 3 mo old)
were given 0.3 mLof 0.5%
benzo[a]pyrene in
polyethylene glycol-400 by
gavage, once/d for 3 d.
Results
Incidence with
forestomach tumors:
Control, 0/9
Low, 6/9
High, 9/9
8/20 exposed mice had
forestomach tumors
Incidence with
forestomach tumors:
Exposed, 17/20 (85%)
Controls, 0/24
5/25 mice had squamous
carcinomas of the
forestomach; tumors were
detected 28-65 wks after
treatment
By 30 wks, 7/10 mice had
papillomas; no carcinomas
were evident
Comments
Less-than-
lifetime exposure
duration;
glandular
stomach, lung,
and livers from
control and
exposed mice
showed no
tumors.
Less-than-
lifetime exposure
duration; only
stomachs were
examined for
tumors; tumors
found only in
forestomach; no
nonexposed
controls were
mentioned.
Less-than-
lifetime exposure
duration; only
stomach were
examined for
tumors; tumors
found only in
forestomach.
Less-than-
lifetime exposure
duration; the
following details
were not
reported:
inclusion of
controls,
methods for
detecting
tumors, and body
weight data.
Less-than-
lifetime exposure
duration.
Reference
Triolo et al.
(1977)
Wattenberg
(1974)
EI-Bayoumy
(1985)
Biancifiori et
al. (1967)
Berenblum
and Haran
(1955)
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Species/Strain
Mouse/albino
Mouse/albino
Mouse/CFW
Mouse/Swiss
albino
Exposure
Groups of 17-18 mice were
given single doses of
benzo[a]pyrene and allowed
to survive until terminal
sacrifice at 569 d.
Groups of about 160 female
mice (70 d of age; strain
unknown) were given 0 or
8 mg benzo[a]pyrene mixed
in the diet over a period of
14 mo.
Groups of mice (mixed sex)
were fed benzo[a]pyrene in
the diet (dissolved in benzene
and mixed with diet) at 0, 1,
10, 20, 30, 40, 45, 50, 100, or
250 ppm in the diet.
Groups of mice (9-14 wks
old) were given single doses
of 0 or 0.05 mg
benzo[a]pyrene in
polyethylene glycol-400 by
gavage. Surviving mice were
killed at 18 mo of age and
examined for macroscopic
tumors.
Results
Incidence of mice (that
survived at least to 60 d)
with forestomach
papillomas:
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
Gastric tumors were
observed at the following
incidence:
Control, 0/158
8 mg benzo[a]pyrene total,
13/160
Fore-
stomach
Exposure tumor
ppm (d) incidence
1 110 0/25
10 110 0/24
20 110 1/23
30 110 0/37
40 110 1/40
45 110 4/40
50 152 24/34
100 110 19/23
250 118 66/73
Forestomach tumor
incidence:
Carcinoma
Dose (ng) Papilloma
0 0/65
2/65
50 1/61
20/61
Comments
Less-than-
lifetime exposure
duration; Gl tract
examined for
tumors with
hand lens; body
weight data not
reported.
Close to lifetime
exposure
duration; daily
dose levels and
methods of
detecting tumors
were not clearly
reported.
Less-than-
lifetime exposure
duration; no
vehicle control
group; animals
ranged from
3 wks to 6 mo old
at the start of
dosing; only
alimentary tract
was examined for
tumors.
Less-than-
lifetime duration
of exposure;
exposure-related
tumors only
found in
forestomach.
Reference
Field and Roe
(1965)
Chouroulinko
vetal. (1967)
Neal and
Rigdon (1967)
Roe et al.
(1970)
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Species/Strain
Mouse/ICR
Mouse/white
Mouse/A/HeJ
Mouse/A/J
Exposure
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.
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.
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.
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).
Results
Incidence of mice with
forestomach neoplasms
Experiment 1, 23/24
Experiment 2, 19/20
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
12/12 exposed mice had
lung tumors
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
Comments
Less-than-
lifetime duration
of exposure; only
stomachs were
examined for
tumors; tumors
found only in
forestomach;
nonexposed
controls were not
mentioned.
Less-than-
lifetime exposure
duration.
Less-than-
lifetime exposure
duration; only
lungs examined
for tumors; no
nonexposed
controls were
mentioned.
Close to lifetime
exposure
duration; A/J
strain of mice
particularly
sensitive to
chemically
induced cancer;
only lungs and
stomachs were
examined for
tumors.
Reference
Benjamin et
al. (1988)
Fedorenko
and Yansheva
(1967); as
cited in U.S.
EPA(1991a)
Wattenberg
(1974)
Weyand et al.
(1995)
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Species/Strain
Mouse/A/J
Exposure
Groups 40 female mice (8
wks old) were given 0 or
0.25 mg benzo[a]pyrene (in
2% emulphor) by gavage
3 times/wkfor 8 wks. Mice
were killed at 9 mo of age
and examined for lung or
forestomach tumors.
Results
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
Comments
Less-than-
lifetime duration
of exposure; only
lungs and Gl tract
were examined
for tumors.
Reference
Robinson et
al. (1987)
2 D.4.2. Inhalation Studies
3 Short-term and Subchronic Studies
4 Wolff etal. [1989] exposed groups of 40 male and 40 female F344/Crl rats, via nose only, to
5 7.5 mg benzo[a]pyrene/m3 for 2 hours/day, 5 days/week for 4 weeks (corresponding to a TWA of
6 0.45 mg/m3). Rats were 10-11 weeks old at the beginning of the experiment Benzo[a]pyrene
7 (>98% pure) aerosols were formed by heating and then condensing the vaporized benzo[a]pyrene.
8 The particle mass median aerodynamic diameter (MMAD) was 0.21 |im. Subgroups of these
9 animals (six/sex/dose) were exposed for 4 days or 6 months after the end of the 4-week exposure
10 to radiolabeled aluminosilicate particles. Lung injury was assessed by analyzing clearance of
11 radiolabeled aluminosilicate particles and via histopathologic evaluations. Body and lung weights,
12 measured in subgroups from 1 day to 12 months after the exposure did not differ between controls
13 and treated animals. Radiolabeled particle clearance did not differ between the control and treated
14 groups, and there were no significant lung lesions. This study identified a NOAEL for lung effects of
15 0.45 mg/m3 for a short-term exposure.
16 Chronic Studies and Cancer Bioassays
17 Thyssenetal. [1981] conducted an inhalation study in which male Syrian golden hamsters
18 were exposed to benzo[a]pyrene for their natural lifetime. Groups of 20-30 animals (8 weeks old]
19 were exposed by nose-only inhalation to NaCl aerosols (controls; 240 |ig NaCl/m3] or
20 benzo[a]pyrene condensed onto NaCl aerosols at three target concentrations of 2,10, or 50 mg
21 benzo[a]pyrene/m3 for 3-4.5 hours/day, 5 days/week for 1-41 weeks, followed by 3 hours/day,
22 7 days/week for the remainder of study (until hamsters died or became moribund]. Thyssen et al.
23 [1981] reported average measured benzo[a]pyrene concentrations to be 0, 2.2, 9.5, or 46.5 mg/m3.
24 More than 99% of the particles were between 0.2 and 0.5 [im in diameter, and over 80% had
25 diameters between 0.2 and 0.3 [im. The particle analysis of the aerosols was not reported to
26 modern standards (MMAD and geometric SD were not reported]. Each group initially consisted of
27 24 hamsters; final group sizes were larger as animals dying during the first 12 months of the study
28 were replaced.
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1 Survival was similar in the control, low-dose, and mid-dose groups, but was significantly
2 decreased in the high-dose group. Average survival times in the control, low-, mid-, and high-dose
3 groups were 96.4 ± 27.6, 95.2 ± 29.1, 96.4 ± 27.8, and 59.5 ± 15.2 weeks, respectively. After the 60th
4 week, body weights decreased and mortality increased steeply in the highest dose group.
5 Histologic examination of organs [a complete list of organs examined histologically was not
6 reported by Thyssenetal. [1981]] revealed a dose-related increase in tumors in the upper
7 respiratory tract, including the nasal cavity, pharynx, larynx, and trachea, and in the digestive tract
8 in the mid- and high-dose groups (Table D-13). A statistical analysis was not included in the
9 Thyssenetal. [1981] report No lung tumors were observed. Squamous cell tumors in the
10 esophagus and forestomach were also observed in the high-dose group, presumably as a
11 consequence of mucociliary particle clearance. Tumors were detected in other sites, but none of
12 these appeared to be related to exposure. The results indicated that the pharynx and larynx,
13 including the epiglottis, were the main cancer targets (Table D-13].
14
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1
2
Table D-13. Incidence of respiratory and upper digestive tract tumors in male
hamsters treated for life with benzo[a]pyrene by inhalation
Tumor site
Nasal cavity
Larynx
Trachea
Lung
Pharynx
Esophagus
Forestomach
Reported benzo[a]pyrene concentration (mg/m3)
Oa
2b
10
50
Tumor incidence (latency in wksc)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3/26 (116 ± 1.5)
8/26 (107.1 ±15.5)
1/26 (115)
0
6/26 (97.2 ± 16.9)
0
1/26 (119)
1/25 (79)
13/25 (67.6 ± 12.1)
3/25 (63.3 ± 33.3)
0
14/25 (67.5 ± 12.2)
2/25 (70, 79)
1/25 (72)
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
29
Effective number of animals in control group: n = 27.
bEffective number of animals in 2 mg/m3 dose group: n = 27.
cMean±SD.
Source: Thvssen et al. (1981).
Under contract to the U.S. EPA, Clement Associates obtained the individual animal data
(including individual animal pathology reports, time-to-death data, and exposure chamber
monitoring data) collected by Thyssen et al. [U.S. EPA, 1990a]. Review of the original data revealed
several discrepancies in the reported exposure protocol. The actual exposure protocol was as
follows: 4.5 hours/day, 5 days/week on weeks 1-12; 3 hours/day, 5 days/week on weeks 13-29;
3.7 hours/day, 5 days/week on week 30; 3 hours/day, 5 days/week on weeks 31-41; and
3 hours/day, 7 days/week for the reminder of the experiment
Analytical chamber monitoring data were generally recorded about once or twice per week,
with some exceptions ranging from no measurements for a three week period to as many as five
measurements in one week. Individual measurements (in mg/m3) ranged from 0.2 to 4.52,1.16 to
19.2, and 0.96 to 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%. Because different animals were started at different times, each individual animal had an
exposure history somewhat different than others in the same exposure group. In order to address
this variability, U.S. EPA (1990a) used the original individual animal data to calculate average
continuous lifetime exposures for each individual hamster. Group averages of individual average
continuous lifetime exposure concentrations were 0, 0.25,1.01, and 4.29 mg/m3 for the control
through high-exposure groups. These averages were approximately 10% lower than the averages
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1 across the entire duration of the study, with 95% confidence limits for the average exposure level
2 among animals in each group varying within 2%.
3 The individual animal pathology reports prepared by Thyssenetal. [1981] were examined
4 to assess the joint incidence of tumors in the larynx and pharynx in each group and the relative
5 incidences of malignant tumors [U.S. EPA (1990a). Table D-14 presents the number of animals with
6 tumors in the larynx and pharynx and the numbers of animals in each exposure group. Numbers of
7 animals with either laryngeal or pharyngeal tumors are also noted in Table D-14, since these two
8 types of tumors arise in close anatomical proximity from similar cell types. Examination of the
9 individual animal pathology reports also showed that all of the nasal, forestomach, esophageal, and
10 tracheal tumors occurred in animals that also had either laryngeal or pharyngeal tumors, except for
11 two animals in the mid-dose group that displayed nasal tumors (one malignant and one benign)
12 without displaying tumors in the pharynx or larynx.
13
14
Table D-14. Number of animals with pharynx and larynx tumors in male
hamsters exposed by inhalation to benzo[a]pyrene for life
Average
Continuous
Benzo[a]pyrene
Concentration3
(mg/m3)
Control
0.25
1.01
4.29
Number of
Hamsters in
Groupb
27
27
26
34
Larynxb
Malignant
0
0
8
9
All
0
0
11
12
Pharynxb
Malignant
0
0
7
17
All
0
0
9
18
Larynx or Pharynx,
Combinedc
Malignant
0
0
11
17
All
0
0
16
18
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
aAs calculated by Clement Associates (U.S. EPA, 1990a) from air monitoring data measured by Thyssen et al.
(1981).
bAs counted from information in Table E-16 in Appendix E which was obtained from examination of individual
animal pathology reports prepared by Thyssen and colleagues and obtained by Clement Associates.
°As counted from information in Table E-16 in Appendix E. Nasal, forestomach, esophageal, and tracheal tumors
occurred in hamsters that also had tumors in the larynx or pharynx, except for two animals in the mid-dose group
that displayed nasal tumors (one malignant and one benign) without displaying tumors in the pharynx or larynx.
Several studies have investigated the carcinogenicity of benzo[a]pyrene in hamsters
exposed by intratracheal instillation. Single-dose studies verified that benzo[a]pyrene is
tumorigenic, but do not provide data useful for characterizing dose-response relationships because
of their design fKobayashi. 1975: Renzik-Schiiller and Mohr. 1974: Henry etal.. 1973: Mohr. 1971:
Saffiotti et al., 1968: Gross etal., 1965: Herrold and Dunham, 1962]. 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 (Kunstler, 1983]. Tumorigenic responses (mostly in the respiratory tract]
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1 were found at higher dosage levels (0.25-2 mgbenzo[a]pyrene once per week for 30-52 weeks) in
2 four multiple-dose studies (Feron and Kruysse. 1978: Ketkar etal.. 1978: Feronetal.. 1973: Saffiotti
3 etal.. 1972). These studies identify the respiratory tract as a cancer target with exposure to
4 benzo[a]pyrene by intratracheal instillation and provide supporting evidence for the
5 carcinogenicity of benzo[a]pyrene atportal-of-entry sites.
6 D.4.3. Dermal studies
7 Skin- Tumor Initiation-Promotion Assays
8 Results from numerous studies indicate that acute dermal exposure to benzo[a]pyrene
9 induces skin tumors in mice when followed by repeated exposure to a potent tumor promoter
10 fWeyand etal.. 1992: Cavalieri etal.. 1991: Rice etal.. 1985: El-Bayoumy etal.. 1982: Lavoieetal..
11 1982: RavehetaL 1982: Cavalieri etal.. 1981: Slagaetal.. 1980: Wood etal.. 1980: Slagaetal..
12 1978: Hoffmann etal., 1972]. The typical exposure protocol in these studies involved the
13 application of a single dose of benzo[a]pyrene (typically >20 nmol per mouse) to dorsal skin of mice
14 followed by repeated exposure to a potent tumor promoter, such as 12-0-tetradecanoylphorbol-
15 13-acetate (TPA).
16 Carcinogenicity Bioassays
17 Repeated application of benzo[a]pyrene to skin (in the absence of exogenous promoters)
18 has been variously demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs
19 (IARC. 2010: IPCS. 1998: ATSDR. 1995: IARC. 1983.1973). Mice have been most extensively
20 studied, presumably because of early evidence that they may be more sensitive than other animal
21 species, but comprehensive comparison of species differences in sensitivity to lifetime dermal
22 exposure are not available. Early studies of complete dermal carcinogenicity in other species (rats,
23 hamsters, guinea pigs, and rabbits) have several limitations that make them not useful for dose-
24 response analysis [see IARC (1973) for descriptions of studies]. The limitations in these studies
25 include inadequate reporting of the amount of benzo[a]pyrene applied, use of the carcinogen
26 benzene as a vehicle, and less-than-lifetime exposure duration.
27 This section discusses complete carcinogenicity bioassays in mice that provide the best
28 available dose-response data for skin tumors caused by repeated dermal exposure to
29 benzo[a]pyrene (Sivaketal.. 1997: Higginbothametal.. 1993: Albert etal.. 1991: Grimmer etal..
30 1984: Habs etal.. 1984: Grimmer etal.. 1983: Habs etal.. 1980: Schmahl etal.. 1977: Schmidt etal..
31 1973: Roe etal., 1970: Poel, 1960,1959). Early studies of benzo[a]pyrene complete carcinogenicity
32 in mouse skin (Wynder and Hoffmann, 1959: Wynder etal., 1957] are not further described herein,
33 because the investigators applied solutions of benzo[a]pyrene at varying concentrations on the
34 skin, but did not report volumes applied. As such, applied doses in these studies cannot be
35 determined. Other complete carcinogenicity mouse skin tumor bioassays with benzo[a]pyrene are
36 available, but these are not described further in this review, because: (1) they only included one
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1 benzo[a]pyrene dose level [e.g., Emmett et al., 1981] or only dose levels inducing 90-100%
2 incidence of mice with tumors (e.g., Wilson and Holland, 1988: Warshawsky and Barkley, 1987] and
3 thus provide no information about the shape of the dose-response relationship; (2] they used a
4 1-time/week [e.g., Nesnow et al., 1983] or 1-time every 2 weeks [e.g., Levin etal., 1977] exposure
5 protocol, which is less useful for extrapolating to daily human exposure; or (3] they used a vehicle
6 demonstrated to interact with or enhance benzo[a]pyrene carcinogenicity [Bingham and Falk.
7 1969].
8 Poel [1959] applied benzo[a]pyrene in toluene to shaved interscapular skin of groups of
9 13-56 male C57L mice at doses of 0, 0.15, 0.38, 0.75, 3.8,19, 94,188, 376, or 752 u.g, 3 times/week
10 for up to 103 weeks or until the appearance of a tumor by gross examination (3 times weekly].
11 Some organs (not further specified] and interscapular skin in sacrificed mice were examined
12 histologically. With increasing dose level, the incidence of mice with skin tumors increased and the
13 time of tumor appearance decreased (see Table D-15]. Doses >3.8 [igwere associated with 100%
14 mortality after increasingly shorter exposure periods, none greater than 44 weeks. Poel (1959] did
15 not mention the appearance of exposure-related tumors in tissues other than interscapular skin.
16
17
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
Dose (u,g)a
0 (toluene)
0.15
0.38
0.75
3.8
19
94
188
376
752
Incidence of Mice with
Gross Skin Tumors
0/33 (0%)
5/55 (9%)
11/55 (20%)
7/56 (13%)
41/49 (84%)
38/38 (100%)
35/35 (100%)
12/14 (86%)
14/14 (100%)
13/13 (100%)
Time of First Tumor
Appearance (wks)
-
42-44°
24
36
21-25
11-21
8-19
9-18
4-15
5-13
Incidence of Mice
with Epidermoid
Carcinoma13
0/33 (0%)
0/55 (0%)
2/55 (4%)
4/56 (7%)
32/49 (65%)
37/38 (97%)
35/35 (100%)
10/14 (71%)
12/14 (86%)
13/13 (100%)
Length of Exposure
Period (wks)
92
98
103
94
82
25-44°
22-43
20-35
19-35
19-30
18
19
20
21
22
23
24
25
Indicated doses were applied to interscapular skin 3 times/week for up to 103 weeks or until time of appearance
of a grossly detected skin tumor.
bCarcinomas were histologically confirmed.
°Ranges reflect differing information in Tables 4 and 6 of Poel (1959).
Source: Poel (1959).
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1
2 Poel [1960] applied benzo[a]pyrene in a toluene vehicle to shaved interscapular skin of
3 groups of 14-25 male SWR, CSHeB, or A/He mice 3 times/week at doses of 0, 0.15, 0.38, 0.75, 3.8,
4 19.0, 94.0, or 470 |igbenzo[a]pyrene per application, until mice died or a skin tumor was observed.
5 Time ranges for tumor observations were provided, but not times of death for mice without tumors,
6 so it was not possible to evaluate differential mortality among all dose groups or the length of
7 exposure for mice without tumors. With increasing dose level, the incidence of mice with skin
8 tumors increased and the time of tumor appearance decreased (Table D-16). The lowest dose level
9 did not induce an increased incidence of mice with skin tumors in any strain, but strain differences
10 in susceptibility were evident at higher dose levels. SWR and CSHeB mice showed skin tumors at
11 doses >0.38 [igbenzo[a]pyrene, whereas AH/e mice showed tumors at doses >19 [ig
12 benzo[a]pyrene (Table D-16). Except for metastases of the skin tumors to lymph nodes and lung,
13 Poel (I960) did not mention the appearance of exposure-related tumors in tissues other than
14 interscapular skin.
15
16
17
Table D-16. Skin tumor incidence and time of appearance in male SWR,
CSHeB, and A/He mice dermally exposed to benzo[a]pyrene for life or until a
skin tumor was detected
Dose (u,g)a
0 (toluene)
0.15
0.38
0.75
3.8
19.0
94.0
470.0
SWR Mice
Tumor
Incidence13
0/20 (0%)
0/25 (0%)
2/22 (9%)
15/18 (83%)
12/17 (70%)
16/16 (100%)
16/17 (94%)
14/14 (100%)
Time of
Tumor
Appearance
(wks)
-
55
25-72
25-51
12-28
9-17
5-11
CSHeB Mice
Tumor
Incidence13
0/17 (0%)
0/19 (0%)
3/17 (18%)
4/17 (24%)
11/18 (61%)
17/17 (100%)
18/18 (100%)
17/17 (100%)
Time of
Tumor
Appearance
(wks)
-
-
81-93
51-93
35-73
13-32
10-22
4-19
A/He Mice
Tumor
Incidence13
0/17 (0%)
0/18 (0%)
0/19 (0%)
0/17 (0%)
0/17 (0%)
21/23 (91%)
11/16 (69%)
17/17 (100%)
Time of
Tumor
Appearance
(wks)
-
-
-
-
-
21-40
14-31
4-21
18
19
20
21
22
23
24
25
Indicated doses were applied 3 times/week for life or until a skin tumor was detected. Mice were 10-14 weeks
old at initial exposure.
blncidence of mice exposed >10 weeks with a skin tumor.
Source: Poel (1960).
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1 Roe etal. [1970] treated groups of 50 female Swiss mice with 0 (acetone vehicle), 0.1, 0.3,1,
2 3, or 9 [ig benzo[a]pyrene applied to the shaved dorsal skin 3 times/week for up to 93 weeks; all
3 surviving mice were killed and examined for tumors during the following 3 weeks. The dorsal skin
4 of an additional control group was shaved periodically but was not treated with the vehicle. Mice
5 were examined every 2 weeks for the development of skin tumors at the site of application.
6 Histologic examinations included: (1) all skin tumors thought to be possibly malignant; (2) lesions
7 of other tissues thought to be neoplastic; and (3) limited nonneoplastic lesions in other tissues. As
8 shown in Table D-17, markedly elevated incidences of mice with skin tumors were only found in
9 the two highest dose groups (3 and 9 [ig), compared with no skin tumors in the control groups.
10 Malignant skin tumors (defined as tumors with invasion or penetration of the panniculus carnosus
11 muscle) were detected in 4/41 and 31/40 mice in the 3- and 9-u.g groups, respectively, surviving to
12 at least 300 days. Malignant lymphomas were detected in all groups, but the numbers of cases were
13 not elevated compared with expected numbers after adjustment for survival differences. Lung
14 tumors were likewise detected in control and exposed groups at incidences that were not
15 statistically different
16
17
Table D-17. Tumor incidence in female Swiss mice dermally exposed to
benzo[a]pyrene for up to 93 weeks
Dose (u,g)a
No treatment
Acetone
0.1
0.3
1
3
9
Cumulative Number of Mice with Skin
Tumor/Survivors
200 d
0/48
0/49
0/45
0/46
0/48
0/47
0/46
300 d
0/43
0/47
1/42
0/42
0/43
0/41
4/40
400 d
0/40
0/45
1/35
0/37
0/37
1/37
21/32
500 d
0/31
0/37
1/31
0/30
1/30
7/35
28/21
600 d
0/21
0/23
1/22
0/19
1/18
8/24
33/8
700 d
0/0
0/0
1/0
0/0
1/0
8/0
34/0
Skin Tumor
Incidence13
0/43 (0%)
0/47 (0%)
1/42 (2%)
0/42 (0%)
1/43 (2%)
8/41 (20%)
34/46 (74%)
Malignant
Lymphoma
lncidencec
19/44 (43%)
12/47 (26%)
11/43 (26%)
10/43 (23%)
16/44 (36%)
23/42 (55%)
9/40 (23%)
Lung Tumor
lncidencec
12/41 (29%)
10/46 (22%)
10/40 (25%)
13/43 (30%)
15/43 (35%)
12/40 (30%)
5/40 (13%)
18
19
20
21
22
23
24
25
26
27
28
29
30
31
aDoses were applied 3 times/week for up to 93 weeks to shaved dorsal skin.
bl\lumerator: number of mice detected with a skin tumor. Denominator: number of mice surviving to 300 days for
all groups except the highest dose group. For the highest dose group (in which skin tumors were first detected
between 200 and 300 days), the number of mice surviving to 200 days was used as the denominator.
Numerator: number of mice detected with specified tumor. Denominator: number of mice surviving to 300 days
unless a tumor was detected earlier, in which case, the number dying before 300 days without a tumor was
subtracted from the number of animals reported to have been examined.
Source: Roe etal. (1970).
Schmidt etal. [1973] dermally administered benzo[a]pyrene in acetone to female NMRI
mice (100/group) and female Swiss mice. Benzo[a]pyrene was applied to the shaved dorsal skin
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1 twice weekly at doses of 0, 0.05, 0.2, 0.8, or 2 [ig until spontaneous death occurred or until an
2 advanced carcinoma was observed. Skin carcinomas were identified by the presence of crater-
3 shaped ulcerations, infiltrative growth, and the beginning of physical wasting (i.e., cachexia).
4 Necropsy was performed for all animals, and histopathological examination of the dermal site of
5 application and any other tissues with gross abnormalities was conducted. Skin tumors were
6 observed at the two highest doses in both strains of female mice (see Table D-18), with induction
7 periods of 53.0 and 75.8 weeks for the 0.8 and 2.0 ^g NMRI mice and 57.8 and 60.7 weeks for the
8 Swiss mice, respectively. The authors indicated that the latency period for tumor formation was
9 highly variable, and significant differences among exposure groups could not be identified, but no
10 further timing information was available, including overall survival. Carcinoma was the primary
11 tumor type seen after lifetime application of benzo[a]pyrene to mouse skin.
12
13
Table D-18. Skin tumor incidence in female NMRI and Swiss mice dermally
exposed to benzo[a]pyrene
Dose (u,g)a
Skin Tumor Incidence (All Types)
Incidence of Papilloma
Incidence of Carcinoma
Female NMRI mice
0 (acetone)
0.05
0.2
0.8
2
0/100 (0%)
0/100 (0%)
0/100 (0%)
2/100 (2%)
30/100 (30%)
0/100 (0%)
0/100 (0%)
0/100 (0%)
0/100 (0%)
2/100 (2%)
0/100 (0%)
0/100 (0%)
0/100 (0%)
2/100 (2%)
28/100 (28%)
Female Swiss mice
0 (acetone)
0.05
0.2
0.8
2
0/80 (0%)
0/80 (0%)
0/80 (0%)
5/80 (6%)
45/80 (56%)
0/80 (0%)
0/80 (0%)
0/80 (0%)
0/80 (0%)
3/80 (4%)
0/80 (0%)
0/80 (0%)
0/80 (0%)
5/80 (6%)
42/80 (52%)
14
15
16
17
18
19
20
21
22
23
24
aMice were exposed until natural death or until they developed a carcinoma at the site of application; indicated
doses were applied 2 times/week to shaved skin of the back.
Source: Schmidt etal. (1973).
Schmahl etal. (1977] applied benzo[a]pyrene 2 times/week to the shaved dorsal skin of
female NMRI mice (100/group) at doses of 0,1,1.7, or 3 [igin 20 [J.L acetone. The authors reported
that animals were observed until natural death or until they developed a carcinoma at the site of
application. The effective numbers of animals at risk was about 80% of the nominal group sizes,
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1 which the authors attributed to autolysis; no information was provided concerning when tumors
2 appeared in the relevant groups, how long treatment lasted in each group, or any times of death.
3 Necropsy was performed on all mice and the skin of the back, as well as any organs that exhibited
4 macroscopic changes, were examined histopathologically. The incidence of all types of skin tumors
5 was increased in a dose-related manner compared to controls (see Table D-19). Carcinoma was the
6 primary tumor type observed following chronic dermal exposure to benzo[a]pyrene, and skin
7 papillomas occurred infrequently. Dermal sarcoma was not observed.
8
9
Table D-19. Skin tumor incidence in female NMRI mice dermally exposed to
benzo[a]pyrene
Dose (u,g)a
0
1
1.7
3
Skin Tumor Incidence (All Types)
1/81 (l%)b
11/77 (14%)
25/88 (28%)
45/81 (56%)
Incidence of Papilloma
0/81 (0%)
1/77 (1%)
0/88 (0%)
2/81 (3%)
Incidence of Carcinoma
0/81 (0%)
10/77 (13%)
25/88 (28%)
43/81 (53%)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
aMice were exposed until natural death or until they developed a carcinoma at the site of application; indicated
doses were applied 2 times/week to shaved skin of the back.
bSarcoma.
Source: Schmahletal. (1977).
Habs etal. [1980] applied benzo[a]pyrene to the shaved interscapular skin of female NMRI
mice (40/group) at doses of 0,1.7, 2.8, or 4.6 |ig in 20 \\L acetone twice weekly, from 10 weeks of
age until natural death or gross observation of infiltrative tumor growth. Latency of tumors, either
as time of first appearance or as average time of appearance of tumors, was not reported. Necropsy
was performed on all animals, and the dorsal skin, as well as any organs showing gross alterations
at autopsy, was prepared for histopathological examination. Age-standardized mortality rates,
using the total population of the experiment as the standard population, were used to adjust tumor
incidence findings in the study. Benzo[a]pyrene application was associated with a statistically
significant increase in the incidence of skin tumors at each dose level (see Table D-20).
27
28
Table D-20. Skin tumor incidence in female NMRI mice dermally exposed to
benzo[a]pyrene
Dose (u,g)a
0 (acetone)
1.7
Skin Tumor Incidence
0/35 (0%)
8/34 (24%)
Age-Standardized Tumor Incidence13
0%
24.8%
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2.8
4.6
24/35 (68%)
22/36 (61%)
89.3%
91.7%
aMice were exposed until natural death or until they developed a carcinoma at the site of application; indicated
doses were applied 2 times/week to shaved skin of the back.
bMortality data of the total study population were used to derive the age-standardized tumor incidence.
Source: Habs et al. (1980).
Grimmer et al. [1984] and Grimmer et al. [1983] applied benzo[a]pyrene (in 0.1 ml of a
1:3 solution of acetone:dimethyl sulfoxide [DMSO]] to the interscapular skin of female CFLP mice
(65-80/group) 2 times/week for 104 weeks. Doses were 0, 3.9, 7.7, and 15.4 u,g in the 1983
experiment, and 0, 3.4, 6.7, and 13.5 [ig in the 1984 experiment. Mice were observed until
spontaneous death, unless an advanced tumor was observed or if animals were found moribund.
Survival information was not provided; incidences reflect the number of animals placed on study.
Necropsy was performed on all mice. Histopathological examination of the skin and any other
organ showing gross abnormalities was performed. Chronic dermal exposure to benzo[a]pyrene
produced a dose-related increase in skin tumor incidence and a decrease in tumor latency (see
Table D-21). Carcinoma was the primary tumor type observed and a dose-response relationship
was evident for carcinoma formation and incidence of all types of skin tumors.
20
21
Table D-21. Skin tumor incidence and time of appearance in female CFLP mice
dermally exposed to benzo[a]pyrene for 104 weeks
Dose (ug)a
Skin Tumor Incidence
(All Types)
Grimmer et al. (1983)
0 (1:3 Solution of
acetone:DMSO)
3.9
7.7
15.4
0/80 (0%)
22/65 (34%)
39/64 (61%)
56/64 (88%)
Grimmer et al. (1984)
0 (1:3 Solution of
acetone:DMSO)
3.4
6.7
13.5
0/65 (0%)
43/64 (67%)
53/65 (82%)
57/65 (88%)
Incidence of
Papilloma
Incidence of
Carcinoma
Tumor Appearance in
Weeks
0/80 (0%)
7/65 (11%)
5/64 (8%)
2/64 (3%)
0/80 (0%)
15/65 (23%)
34/64 (53%)
54/64 (84%)
-
74.6 ± 16.78b
60.9 ± 13.90
44.1 ±7.66
0/65 (0%)
6/64 (9%)
8/65 (12%)
4/65 (6%)
0/65 (0%)
37/64 (58%)
45/65 (69%)
53/65 (82%)
-
61 (53-65)c
47 (43-50)
35 (32-36)
22
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Indicated doses were applied twice/week to shaved skin of the back.
bMean±SD.
'Median with 95% Cl.
Sources: Grimmer et al. (1984) and Grimmer et al. (1983).
Habs etal. [1984] applied benzo[a]pyrene (in 0.01 mL acetone) to the shaved interscapular
skin of female NMRI mice at doses of 0, 2, or 4 [ig, 2 times/week for life. Animals were observed
twice daily until spontaneous death, unless an invasive tumor was observed. All animals were
necropsied and histopathological examination was performed on the dorsal skin and any other
organ with gross abnormalities. Chronic dermal exposure to benzo[a]pyrene did not affect body
weight gain, but appeared to reduce survival at the highest dose with mean survival times of 691,
648, and 528 days for the 0, 2, and 4 [J.g/day groups, respectively. The total length of exposure for
each group was not reported, but can be inferred from the survival data. Latency also was not
reported. Benzo[a]pyrene application resulted in a dose-related increase the incidence of total skin
tumors and skin carcinomas (see Table D-22). Hematopoietic tumors (at 6/20, 3/20, and 3/20)
and lung adenomas (at 2/20,1/20, and 0/20) were observed in the controls and in the
benzo[a]pyrene treatment groups, but did not appear to be treatment related according to the
study authors.
21
22
Table D-22. Skin tumor incidence in female NMRI mice dermally exposed to
benzo[a]pyrene for life
Dose (ng)a
0 (Acetone)
2
4
Skin Tumor Incidence
(All Types)
0/20 (0%)
9/20 (45%)
17/20 (85%)
Incidence of
Papilloma
0/20 (0%)
2/20 (10%)
0/20 (0%)
Incidence of
Carcinoma
0/20 (0%)
7/20 (35%)
17/20 (85%)
Mean Survival Time,
Days (95% Cl)
691 (600-763)
648 (440-729)
528 (480-555)
23
24
25
26
27
28
29
30
31
32
33
34
35
aMice were exposed until natural death or until they developed an invasive tumor at the site of application;
indicated doses were applied 2 times/week to shaved interscapular skin.
Source: Habs et al. (1984).
Groups of 23-27 female Ah-receptor-responsive Swiss mice were treated on a shaved area
of dorsal skin with 0,1, 4, or 8 nmol (0, 0.25,1, or 2 [ig/treatment) benzo[a]pyrene (>99% pure) in
acetone 2 times weekly for 40 weeks (Higginbotham etal.. 1993). Surviving animals were
sacrificed 8 weeks later. Complete necropsies were performed, and tissues from the treated area,
lung, liver, kidney, spleen, urinary bladder, ovary, and uterus were harvested for histopathologic
examination. Histopathologic examination was performed on tissues from the treated area, lungs,
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1 liver, kidneys, spleen, urinary bladder, uterus, and ovaries, as well as any other grossly abnormal
2 tissue. Lung adenomas occurred in each group (1/27, 2/24,1/23,1/23), and other tumors were
3 noted in isolated mice (i.e., malignant lymphoma [spleen] in one low-dose and one mid-dose mouse;
4 malignant lymphoma with middle organ involvement in one high-dose mouse; and hemangioma
5 [liver] in one mid-dose mouse) and were not considered dose related. In addition, benzo[a]pyrene
6 showed no skin tumors under the conditions of this bioassay.
7 Sivaketal. (1997) designed a study to compare the carcinogenicity of condensed asphalt
8 fumes (including benzo[a]pyrene and other PAHs) with several doses of benzo[a]pyrene alone. For
9 the purposes of this assessment, the exposure groups exposed to PAH mixtures are not discussed.
10 Groups of 30 male C3H/HeJ mice were treated dermally twice/week to 0, 0.0001, 0.001, or 0.01%
11 (0, 0.05, 0.5, or 5 |ig) benzo[a]pyrene in a 50 [J.L volume of cyclohexanone/acetone (1:1) for
12 104 weeks beginning at 8 weeks of age. Mice dying during the exposure period or sacrificed at the
13 24 month termination were necropsied; mice with skin tumors that persisted for 4 consecutive
14 weeks with diameters >3 cm were sacrificed before the study termination and also necropsied.
15 Skin samples and any grossly observed lesions were subjected to histopathological examination.
16 Carcinomas and sarcomas were referred to as carcinomas, whereas papillomas, keratoacanthomas,
17 and fibromas were referred to as papillomas. The incidences of mice with skin tumors and mean
18 survival times for each group are shown in Table D-23. All high-dose mice died before the final
19 sacrifice, and 80% showed scabs and sores at the site of application. The time of first tumor
20 appearance was not reported for the tumor-inducing groups, but from a plot of the tumor incidence
21 in the high-dose group versus treatment days, an estimate of ~320 days (~43 weeks) is obtained
22 for this group. The extent of deaths prior to 1 year in each group was not provided, so the reported
23 incidence may underestimate the tumor rate of animals exposed long enough to develop tumors.
24 However, the crude skin tumor rates show an increasing trend in incidence.
25
26
Table D-23. Skin tumor incidence in male C3H/HeJ mice dermally exposed to
benzo[a]pyrene for 24 months
Dose (u,g)a
0 cyclohexanone/acetone (1:1)
0.05
0.5
5.0
Skin Tumor Incidence
(All Types)b
0/30 (0%)
0/30 (0%)
5/30 (20%)
27/30 (90%)
Number of Mice that Died
Before Final Sacrifice
19
15
15
30
Mean Survival Time
(days)
607
630
666
449
27
28
29
30
31
32
33
Indicated doses were applied twice/week to shaved dorsal skin.
bNumber of skin tumor-bearing mice. In the high-dose group, 1 papilloma and 28 carcinomas were detected; in
the 0.5 u.g group, 2 papillomas and 3 carcinomas were detected.
Source: Sivaketal. (1997).
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1
2 To examine dose-response relationships and the time course of benzo[a]pyrene-induced
3 skin damage, DNA adduct formation, and tumor formation, groups of 43-85 female Harlan mice
4 were treated dermally with 0,16, 32, or 64 |ig of benzo[a]pyrene in 50 [J.L of acetone once per week
5 for 29 weeks [Albert etal.. 1991). Interscapular skin of each mouse was clipped 3 days before the
6 first application and every 2 weeks thereafter. Additional groups of mice were treated for 9 weeks
7 with 0, 8,16, 32, or 64 |ig radiolabeled benzo[a]pyrene to determine BPDE-DNA adduct formation
8 in the epidermis at several time points (1, 2, 4, and 9 weeks). Tumor formation was monitored only
9 in the skin.
10 No tumors were present in vehicle-treated or untreated control mice. In exposed groups,
11 incidences of mice with skin tumors were not reported, but time-course data for cumulative
12 number of tumors per mouse, corrected for deaths from nontumor causes, were reported. Tumors
13 began appearing after 12-14 weeks of exposure for the mid- and high-dose groups and at 18 weeks
14 for the low-dose group. At study termination (3 5 weeks after start of exposure), the mean number
15 of tumors per mouse was approximately one per mouse in the low- and mid-dose groups and eight
16 per mouse in the high-dose group, indicating that most, if not all, mice in each exposure group
17 developed skin tumors and that the tumorigenic response was greatest in the highest dose group.
18 The majority of tumors were initially benign, with an average time of 8 weeks for progression from
19 benign papillomas to malignant carcinomas. Epidermal damage occurred in a dose-related manner
20 (more severe in the high-dose group than in the low- and mid-dose groups) and included
21 statistically significant increases (compared with controls) in: [3H]-thymidine labeling and mitotic
22 indices; incidence of pyknotic and dark cells (signs of apoptosis); and epidermal thickness. Only a
23 minor expansion of the epidermal cell population was observed. In the high-dose group, indices of
24 epidermal damage increased to a plateau by 2 weeks of exposure. The early time course of
25 epidermal damage indices was not described in the low- or mid-dose groups, since data for these
26 endpoints were only collected at 20, 24, and 30 weeks of exposure. An increased level of BPDE-
27 DNA adducts, compared with controls, was apparent in all exposed groups after 4 weeks of
28 exposure in the following order: 64>32>16>8 |ig/week. The time-course data indicate that
29 benzo[a]pyrene-induced increases in epidermal damage indices and BPDE-DNA adducts preceded
30 the appearance of skin tumors.
31 D.4.4. Reproductive and Developmental Toxicity Studies
32 Oral
33 In a study evaluating the combined effects of DBF and benzo[a]pyrene on the male
34 reproductive tract, Chen etal. (2011) administered benzo[a]pyrene alone in corn oil via daily
35 gavage at 5 mg/kg-day to 30 male Sprague-Dawley rats (28-30 days old); a group of 30 rats
36 received only vehicle. Body weight was measured weekly. Groups of 10 rats per group were
37 sacrificed after 4, 8, and 12 weeks of exposure. At sacrifice, blood was collected for analysis of
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1 serum testosterone levels by radioimmunoassay. The testes and epididymides were weighed, and
2 the right testis and epididymis were examined microscopically. The left epididymis was used for
3 evaluation of sperm parameters (sperm count and morphology). Oxidative stress, as measured by
4 superoxide dismutase (SOD), glutathione peroxidase, and catalase activity and malondialdehyde
5 levels, was evaluated in the left testis of each rat Exposure to benzo[a]pyrene did not affect body
6 weight, and no signs of toxicity were seen. Testes and epididymides weights of exposed rats were
7 similar to controls at all time points. Sperm counts and percent abnormal sperm were also similar
8 to controls at 4 and 8 weeks of exposure, but were significantly (p < 0.05) different from controls
9 after 12 weeks of exposure to benzo[a]pyrene (29% decrease in sperm count and 54% increase in
10 percent abnormal sperm). Serum testosterone levels were significantly increased relative to
11 controls after 4 weeks (>2-fold higher) and 8 weeks (~1.5-fold higher) of benzo[a]pyrene exposure,
12 but were comparable to controls after 12 weeks. Histopathology evaluation of the testes revealed
13 irregular and disordered arrangement of germ cells in the seminiferous tubules of treated rats; the
14 authors did not report incidence or severity of these changes. Among measures of testicular
15 oxidative stress, only catalase activity was significantly affected by benzo[a]pyrene exposure,
16 showing an increase of ~50% after 12 weeks of exposure. These data suggest a LOAEL of 5 mg/kg-
17 day (the only dose tested) for decreased sperm count, increased percentage of abnormal sperm,
18 altered testosterone levels, and histopathology changes in the testes following 13 weeks of
19 exposure.
20 Chung etal. (2011) evaluated the effects of low-dose benzo[a]pyrene exposure on
21 spermatogenesis and the role of altered steroidogenesis on the sperm effects. Groups of 20-
22 25 male Sprague-Dawley rats (8 weeks old) were given daily gavage doses of 0, 0.001, 0.01, or
23 0.1 mg/kg-day benzo[a]pyrene inDMSO for 90 consecutive days. At the end of exposure, the
24 animals were sacrificed for removal of the pituitary, testes, and epididymides, and collection of
25 serum and testicular interstitial fluid. Subgroups of each exposure group were used for various
26 analyses. Serum levels of testosterone and luteinizing hormone (LH) were measured, as was
27 testosterone concentration in the interstitial fluid (enzyme-linked immunosorbent assays [ELISA]).
28 Body and testes weights were recorded. Sections of the testis were analyzed for apoptotic germ
29 cells using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.
30 Evaluation of the epididymis included histopathology as well as measurement of caput and caudal
31 epididymal tubule diameters. In addition, sperm were isolated from the cauda epididymis for
32 analysis of sperm number and motility, acrosomal integrity, and immunocytochemistry for ADAMS
33 (a disintegrin and metallopeptidase domain 3; a sperm surface protein associated with
34 fertilization).
35 Leydig cells were isolated from the right testis of animals from each dose group and
36 cultured with or without human chorionic gonadotropin (hCG) or dibutyl cyclic adenosine
37 monophosphate (dbcAMP) to evaluate testosterone production (Chung etal.. 2011). Cultured
38 Leydig cells were also subjected to western blot and immunocytochemistry analyses to evaluate
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1 changes in the expression of genes involved in steroidogenesis (steroidogenic acute regulatory
2 protein, p450 side-chain cleavage, and 3(3-hydroxysteroid dehydrogenase isomerase). Finally,
3 pituitary gland extracts were evaluated for LH protein content using immunohistochemistry. Data
4 were reported graphically and analyzed by analysis of variance (ANOVA) followed by Duncan's post
5 hoc test, using a p-value cutoff of 0.05 for significant difference.
6 At termination of exposure, body weights of treated animals were similar to controls, as
7 were absolute testes weights [Chung etal., 2011]. Testosterone concentrations in both serum and
8 testicular interstitial fluid were significantly reduced at the high dose of benzo [ajpyrene
9 (0.1 mg/kg-day); based on visual inspection of the data, the mean serum concentration in this
10 group was ~20% of the control and the mean interstitial fluid concentration was ~60% of the
11 control (n = 9 animals/dose for these evaluations). In addition, baseline production of testosterone
12 by cultured Leydig cells was significantly decreased (~50% based on data shown graphically) at
13 0.1 mg/kg-day. Both hCG- and dbcAMP-stimulated testosterone production measurements were
14 lower (~60% lower than controls) in Leydig cells from rats exposed to either 0.01 or 0.1 mg/kg-
15 day. Serum LH was significantly increased at both 0.01 and 0.1 mg/kg-day (~65-75% higher than
16 controls based on visual inspection of graphs); concordant increases in the intensity of LH
17 immunoreactivity were evident in pituitary extracts from exposed rats.
18 Dose-related increases in the number of apoptotic germ cells, primarily spermatogonia,
19 were demonstrated both via TUNEL assay and caspase-3 staining; the number per tubule was
20 significantly increased over control at all doses [Chung etal., 2011). Numbers of sperm were lower
21 in the treatment groups, but did not differ significantly from the control group. However, sperm
22 motility was significantly reduced in exposed groups compared with controls. The authors did not
23 report sperm motility for all dose groups, but showed only the significant decrease in the
24 0.01 mg/kg-day mid-dose group (~30% lower than controls based on visual inspection of graph).
25 Acrosomal integrity (measured by LysoTracker staining) was diminished in sperm heads from
26 exposed rats; likewise, the expression of ADAMS protein was downregulated by exposure to
27 benzo[a]pyrene; the authors reported a significant decrease in the 0.01 mg/kg-day group, but did
28 not provide details of the analysis of other exposure groups. Histopathology examination of the
29 caput and cauda epididymides revealed dose-related decreases in both cauda and caput tubule
30 diameters that were statistically significantly lower than controls at all doses (~10-30% smaller
31 mean diameter than control based on measurements of 175 tubules collected from five samples in
32 each group; data reported graphically).
33 Statistically significant effects observed at the lowest dose (0.001 mg/kg-day) of
34 benzo[a]pyrene in this study included decreased caput and cauda epididymal tubule diameters
35 (~10-15% lower than controls) and increased numbers of apoptotic germ cells (~twofold higher
36 than controls) by TUNEL assay (Chung etal., 2011). The authors reported that "sperm motility was
37 significantly reduced in the benzo[a]pyrene-exposed groups in comparison to that of the control"
38 but provided quantitative data only for the middle dose group, which exhibited a ~30% decrease in
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1 percent motile sperm. No statistically significant decrease in sperm count was reported at any
2 dose. The middle dose (0.01 mg/kg-day) is considered to be a LOAEL based on reduced sperm
3 motility.
4 Gao etal. [201 Ib] examined effects of benzo[a]pyrene exposure via on cervical cell
5 morphology. Female ICRmice (18-22 g) were exposed to doses of 0, 2.5, 5, or 10 mg/kg twice per
6 week for 14 weeks, either by gavage or by intraperitoneal (i.p.) injection (for this review, only oral
7 results are reported). After adjustment for equivalent continuous dosing (2/7 days/week), the
8 equivalent daily doses are estimated to be 0.7,1.4, and 2.9 mg/kg-day. Both vehicle (sesame oil)
9 and untreated control groups were maintained. Body weights were determined weekly. Groups of
10 26 mice per dose per exposure route were sacrificed at the end of exposure for evaluation of
11 cervical weight and histopathology. Additional groups of 10 mice were exposed for 14 weeks and
12 used for determination of lipid peroxidation (malondialdehyde and glutathione-S-transferase
13 levels) and CYP1A1 activity (EROD) in both liver and cervix, as well as creatine kinase activity, AST
14 activity, and IL-6 levels in cervix and serum.
15 Mortality was observed in all exposure groups with the exception of the low-dose oral
16 exposure group; the authors did not indicate the timing or causes of death (Gao etal., 2011b).
17 There were no control deaths. Mortality incidences in the oral exposure groups (low to high dose)
18 were 0/26 (untreated control), 0/26 (vehicle control), 0/26,1/36, and 2/26. Benzo[a]pyrene
19 treatment resulted in dose-dependent decreases in body weight gain. In the high-dose group of
20 both treatments, body weight began to decline after ~7 weeks of exposure. Based on visual
21 examination of data presented graphically, mean terminal body weights in the low-, mid-, and high-
22 dose oral exposure groups were ~10,15, and 30% lower (respectively) than the vehicle control
23 mean. The untreated control mean body weight for the oral exposure group was similar to the
24 vehicle control mean body weight. Cervical weight as a function of body weight was not affected by
25 oral benzo[a]pyrene exposure. Microscopic examination of the cervix revealed increased
26 incidences of epithelial hyperplasia and inflammatory cells in the cervix of all groups of exposed
27 mice, and atypical hyperplasia of the cervix in mice exposed to 1.4 or 2.9 mg/kg benzo[a]pyrene.
28 Statistical analysis of the findings was conducted, but was poorly reported in the publication. Table
29 D-24 shows the incidences in the oral exposure groups, along with the results of Fisher's exact tests
30 performed for this review.
31
32
Table D-24. Mortality and cervical histopathology incidences in female ICR
mice exposed to benzo[a]pyrene via gavage for 14 weeks
Endpoint
Mortality
Cervical epithelial hyperplasia
Dose (mg/kg-d)
Untreated
Control
0/26
0/26
Vehicle
Control
0/26
0/26
0.7
0/26
4/26
1.4
1/26
6/25*
2.9
2/26
7/24*
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Atypical hyperplasia of cervix
Inflammatory cells in cervix
0/26
2/26
0/26
3/26
0/26
10/26*
2/25
12/25*
4/24*
18/24*
1
2 *Significantly different from vehicle control by Fisher's exact test performed for this review (one-sided p < 0.05).
3
4 Source: Gao etal. (2011b).
5 Levels of malondialdehyde in both the cervix and liver were significantly higher than
6 controls in all dose groups of animals treated by either oral (1.5-2-fold higher in the cervix and
7 ~3-7-fold higher in the liver after oral exposure, p < 0.05) or i.p. exposure. Concomitant decreases
8 in GST activity (~15-50% lower than controls in the cervix and ~30-60% lower in the liver after
9 oral exposure, p < 0.05) were also observed at all doses and in both organs and both treatments.
10 EROD activity was increased in the cervix (~4—12-fold) and liver (~12—35-fold) of all exposure
11 groups. Measurement of creatine kinase and AST activity in the cervix and serum also showed
12 significant increases at all doses and after both exposures (~1.5-2-fold in the cervix, and ~20-50%
13 higher than controls in the liver after oral exposure). Finally, levels of the inflammatory cytokine
14 IL-6 were significantly (p < 0.05) increased in the cervix of all treated mice, and were markedly
15 increased (from more than twofold higher than untreated or vehicle controls at the low dose, to
16 ~sixfold higher at the high dose) in the serum of treated mice.
17 Based on the observations of decreased body weight and increased cervical epithelial
18 inflammation and hyperplasia, a LOAEL of 0.7 mg/kg-day (the lowest dose tested) is identified for
19 this study.
20 Mohamedetal. (2010) investigated multi-generational effects in male mice following
21 exposure of 6-week old-C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day
22 benzo[a]pyrene for 6 weeks by gavage. Following final treatment, male mice were allowed to
23 stabilize for 1 week prior to being mated with two untreated female mice to produce an
24 Fl generation. Male mice were sacrificed 1 week after mating. Fl males were also mated with
25 untreated female mice, as were F2 males. The mice of the Fl, F2, and F3 generations were not
26 exposed to benzo[a]pyrene. The FO, Fl, F2, and F3 mice were all sacrificed at the same age
27 (14 weeks) and endpoints including testis histology, sperm count, sperm motility, and in vitro
28 sperm penetration (of hamster oocytes) were evaluated. These endpoints were analyzed
29 statistically using AN OVA and Tukey's honest significance test and results were reported
30 graphically as means ± SD.
31 Testicular atrophy was observed in the benzo[a]pyrene treatment groups, but was not
32 statistically different than controls. Statistically significant reductions were observed in epididymal
33 sperm counts of FO and Fl generations treated with the high or low dose of benzo[a]pyrene. For FO
34 and Fl generations, epididymal sperm counts were reduced approximately 50 and 70%,
35 respectively, in the low- and high-dose groups. Additionally, sperm motility was statistically
36 significantly decreased at the high dose in the FO and Fl generations. Sperm parameters of the F3
37 generation were not statistically different from controls. An in vitro sperm penetration assay
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1 revealed statistically significantly reduced fertilization in FO and Fl generations of the low- and
2 high-dose groups. However, the value of this in vitro test is limited as it bypasses essential
3 components of the intact animal system [U.S. EPA. 1996}. Based on decreased epididymal sperm
4 counts of FO and Fl generations, a LOAEL of 1 mg/kg-day was established from this study (no
5 NOAEL was identified).
6 Arafaetal. [2009] exposed groups of 12 male Swiss albino rats to benzo[a]pyrene in olive
7 oil (0 or 50 mg/kg-day via gavage) for 10 consecutive days, either alone or after similar treatment
8 with 200 mg/kg-day of the flavonoid hesperidin, which has been shown to exert anti-inflammatory,
9 antioxidant, and anticarcinogenic activity. One day after the final dose, the animals were sacrificed
10 for removal of the cauda epididymides and testes. Epididymal sperm count and motility were
11 assessed, as was daily sperm production in the testes. The study authors also investigated the
12 testicular activity of LDH, SOD, and GST, as well as GSH, malondialdehyde, and protein content The
13 testes were examined under light microscope.
14 Relative testes weights (normalized to body weight) of benzo[a]pyrene exposed-animals
15 were significantly decreased compared with controls (35% lower, p < 0.05) (Arafa et al., 2009). In
16 addition, exposure to benzo[a]pyrene alone resulted in significantly decreased sperm count,
17 numbers of motile sperm, and daily sperm production (~40% decrease from control in each
18 parameter, p < 0.05). Effects on sperm count and production were abolished by hesperidin
19 pretreatment, but the number of motile sperm remained significantly depressed (compared with
20 the control group) in the group exposed to both benzo[a]pyrene and hesperidin. Measures of
21 antioxidant enzymes and lipid peroxidation showed statistically significant induction of oxidative
22 stress in the testes of benzo[a]pyrene-exposed rats. With the exception of the decrease in testicular
23 GSH content (which was partially mitigated), pretreatment with hesperidin eliminated the effects of
24 benzo[a]pyrene on lipid peroxidation and antioxidant enzymes.
25 Xu etal. (2010) treated female Sprague-Dawley rats (6/group) to 0 (corn oil only), 5, or
26 10 mg/kg-day benzo[a]pyrene by gavage every other day for a duration of 60 days. This resulted in
27 TWA doses of 0, 2.5, and 5 mg/kg-day over the study period of 60 days. Endpoints examined
28 included ovary weight, estrous cycle, 17B-estradiol blood level, and ovarian follicle populations
29 (including primordial, primary, secondary, atretic, and corpora leutea). Animals were observed
30 daily for any clinical signs of toxicity and following sacrifice, gross pathological examinations were
31 made and any findings were recorded. All animals survived to necropsy. A difference in clinical
32 signs was not observed for the treated groups and body weights were not statistically different in
33 treated animals (although they appear to be depressed 6% at the high dose). Absolute ovary
34 weight was statistically significantly reduced in both the low- and high-dose groups (11 and 15%,
35 respectively) (see Table D-25). Animals treated with the high dose were noted to have a
36 statistically significantly prolonged duration of the estrous cycle and nonestrus phase compared to
37 controls. Animals in the high-dose group also had statistically significantly depressed levels of
38 estradiol (by approximately 25%) and decreased numbers of primordial follicles (by approximately
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1 20%). This study also indicated a strong apoptotic response of ovarian granulosa cells as visualized
2 through TUNEL labeling; however, the strongest response was seen at the low dose; decreased
3 apoptosis was also observed at the high dose. Based on decreased ovary weight following a 60-day
4 oral exposure to benzo[a]pyrene, a LOAEL of 2.5 mg/kg-day was established from this study (no
5 NOAEL was identified).
6 Table D-25. Means ± SD for ovary weight in female Sprague-Dawley rats
Ovary weight (g)
Body weight (g)
Dose (mg/kg-d)a
0
0.160 ±0.0146
261.67 ± 12.0
2.5
0.143 ±0.0098*
249.17 ± 11.2
5
0.136 ±0.0098*
247.25 ± 11.2
7
8 *Statistically different from controls (p < 0.05) using one-way ANOVA.
9
10 aTWA doses over the 60-day study period.
11
12 Source: Xu et al. (2010).
13
14
15 Zheng etal. [2010) treated male Sprague-Dawley rats to 0 (corn oil only), 1, or 5 mg/kg-day
16 benzo[a]pyrene by daily gavage for a duration of 30 (8/group) or 90 days (8/group). At necropsy,
17 the left testis of each animal was collected and weighed. Testes testosterone concentrations were
18 determined by radioimmunassay and results were expressed as ng/g testis and reported
19 graphically. Testicular testosterone was statistically significantly decreased in the high-dose group
20 approximately 15% following 90 days of exposure. The low-dose group also appeared to have a
21 similar average depression of testosterone levels; however, the change did not reach statistical
22 significance. Testosterone levels measured in animals sacrificed following 30 days of
23 benzo[a]pyrene exposure were not statistically different than controls. Based on decreased
24 testicular testosterone levels following a 90-day oral exposure to benzo[a]pyrene, a LOAEL of
25 5 mg/kg-day and a NOAEL of 1 mg/kg-day were identified.
26 McCallister et al. (2008) administered 0 or 300 |J.g/kg benzo[a]pyrene by gavage in peanut
27 oil to pregnant Long-Evans rats (n = 5 or 6) on gestational days (CDs) 14-17. At this exposure level,
28 no significant changes were see in number of pups per litter, pup growth, or liver to body weight
29 ratios in control compared to benzo[a]pyrene exposed offspring. Treatment-related differences in
30 brain to body weight ratios were observed only on postnatal days (PNDs) 15 and 30. Decreases in
31 cerebrocortical messenger ribonucleic acid (mRNA) expression of the glutamatergic N-methyl-
32 D-aspartate (NMDA) receptor subunit was significantly reduced (50%) in treated offspring
33 compared to controls. In addition, in utero exposed offspring exhibited decreased evoked cortical
34 neuronal activity in the barrel field cortex when tested at PNDs 90-120.
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1 Rigdon and Neal [1965] administered diets containing 1,000 ppm benzo[a]pyrene to
2 pregnant mice (nine/group) on CDs 10-21 or 5-21. The pups were reported as appearing
3 generally normal at birth, but cannibalism was elevated in the exposed groups. These results are in
4 contrast with an earlier study [Rigdon and Rennels, 1964] in which rats (strain not specified] were
5 fed diets containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and
6 during gestation. In the earlier study, five of eight treated females mated with untreated males
7 became pregnant, but only one delivered live young. The treated dam that delivered had two live
8 and two stillborn pups; one dead pup was grossly malformed. In the remaining treated females,
9 vaginal bleeding was observed on CDs 23 or 24. In the inverse experimental design, three of six
10 controls mated to benzo[a]pyrene-treated males became pregnant and delivered live young.
11 Visceral and skeletal examinations of the pups were not conducted. These studies were limited by
12 the small numbers of animals, minimal evaluation of the pups, lack of details on days of treatment
13 (food consumption, weight gain], and occurrence of cannibalism.
14 Reproductive Effects of In Utero Exposure Via Oral Route
15 Mackenzie and Angevine (1981] conducted a two-generation reproductive and
16 developmental toxicity study for benzo[a]pyrene in CD-I mice. Benzo[a]pyrene was administered
17 by gavage in 0.2 mL of corn oil to groups of 30 or 60 pregnant (the FO generation] mice atdoses of
18 0,10, 40, or 160 mg/kg-day on CDs 7-16 only. Therefore, unlike the standard two-generation
19 study, Fl animals were exposed only in utero. Fl offspring were evaluated for postnatal
20 development and reproductive function as follows. Fl pups (four/sex when possible] were allowed
21 to remain with their mothers until weaning on PND 20. Crossover mating studies were then
22 conducted. Beginning at 7 weeks of age, each Fl male mouse (n = 20-45/group] was allowed to
23 mate with two untreated virgin females for 5-day periods for 25 days (for a total exposure of
24 10 untreated females/Fl male], after which time the males were separated from the females.
25 Fourteen days after separation from the males (i.e., on days 14-19 of gestation], the females were
26 sacrificed and the numbers of implants, fetuses, and resorptions were recorded. The F2 fetuses
27 were then examined for gross abnormalities. Similarly, each Fl female mouse (n = 20-55/group],
28 beginning at 6 weeks of age, was paired with an untreated male for a period of 6 months. Males
29 were replaced if the females failed to produce a litter during the first 30-day period. All F2 young
30 were examined for gross abnormalities on day 1 of life and their weights were recorded on day 4.
31 This F2 group was sacrificed on day 20 postpartum, while the Fl female was left with a male until
32 the conclusion of the study. At 6 weeks of age, gonads of groups of 10 male and 10 female Fl mice
33 exposed to 0,10, or 40 mg/kg-day benzo[a]pyrene in utero were subjected to gross pathology and
34 histologic examinations.
35 No maternal toxicity was observed. The number of FO females with viable litters at
36 parturition at the highest dose was statistically significantly reduced by about 35% (Table D-26],
37 but progeny were normal by gross observation. Parturition rates of the low- and mid-dose groups
38 were unaffected by treatment, and litter sizes of all treated groups were similar to the control group
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1 throughout lactation. However, body weights of the Fl pups in the mid- and high-dose groups were
2 statistically significantly decreased on PND 20, by 7 and 13%, respectively, and in all treated pups
3 on PND 42, 6, 6, and 10% for the low, mid, and high dose, respectively (Table D-26). The number of
4 Fl pups surviving to PNDs 20 and 42 was significantly reduced at the high dose (p < 0.01), by 8 and
5 16%, respectively. When Fl males were bred to untreated females and Fl females were mated
6 with untreated males, a marked dose-related decrease in fertility of >30% was observed in both
7 sexes, starting at the lowest exposure. There were no treatment-associated gross abnormalities or
8 differences in body weights in the F2 offspring.
9
10
Table D-26. Reproductive effects in male and female CD-I Fl mice exposed in
utero to benzo[a]pyrene
Effect
FO mice with viable litters at parturition
Mean ± SEM pup weight (g) at PND 20
Mean ± SEM pup weight (g) at PND 42
Fl male fertility indexb
Fl female fertility index0
Dose (mg/kg-d)a
0
46/60 (77%)
11.2 ±0.1
29.9 ±0.2
80.4
100.0
10
21/30 (70%)
11.6 ±0.1
28.2 ±0.3*
52.0*
65.7*
40
44/60 (73%)
10.4 ±0.1*
28.0 ±0.2*
4.7*
0.0*
160
13/30 (43%)*
9.7 ±0.2*
26.8 ±0.4*
0.0*
0.0*
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
*Significantly (p < 0.05) different from control by unspecified tests.
aPregnant FO mice were administered daily doses of benzo[a]pyrene in corn oil on GDs 7-16.
bBeginning at 7 weeks of age, each Fl male mouse (20-45/group) was exposed to 10 untreated females over a
period of 25 days. Index = (females pregnant/females exposed to males) x 100.
Beginning at 6 weeks of age, each Fl female mouse (20-55/group) was cohabitated with an untreated male for a
period of 6 months.
SEM = standard error of the mean.
Source: Mackenzie and Angevine (1981).
Exposure to benzo[a]pyrene caused a marked dose-related decrease in the size of the
gonads. In Fl males, testes weights were statistically significantly reduced. Testes from animals
exposed in utero to 10 and 40 mg/kg-day weighed approximately 42 and 82%, respectively, of the
weight of testes from the control animals (no F2 offspring were produced in the high-dose group).
This was confirmed by histopathologic observation of atrophic seminiferous tubules in the
40 mg/kg-day group that were smaller than those of controls and were empty except for a basal
layer of cells. The number of interstitial cells in the testes was also increased in this group. Males
from the 10 mg/kg-day group showed limited testicular damage; although all exhibited evidence of
tubular injury, each animal had some seminiferous tubules that displayed active spermatogenesis.
Ovarian tissue was absent or reduced in Fl females such that organ weights were not possible to
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1 obtain. Examination of available tissue in these females revealed hypoplastic ovaries with few
2 follicles and corpora lutea (10 mg/kg-day) or with no evidence of folliculogenesis (40 mg/kg-day).
3 Ovarian tissue was not examined in highest-dose females.
4 The LOAEL in this study was 10 mg/kg-day based on decreases in mean pup weight (<5%)
5 at PND 42 of Fl offspring of dams treated with 10, 40, or 160 mg/kg-day benzo[a]pyrene, marked
6 decreases in the reproductive capacity (as measured by fertility index) of both male and female Fl
7 offspring exposed at all three treatment levels of benzo[a]pyrene (by approximately 30% in males
8 and females), decreased litter size (by about 20%) in offspring of Fl dams, and the dramatic
9 decrease in size and alteration in anatomy of the gonads of both male and female Fl mice exposed
10 to 10 and 40 mg/kg-day benzo[a]pyrene in utero. A NOAEL was not identified.
11 In another reproductive and developmental toxicity study, benzo[a]pyrene was
12 administered by gavage in corn oil to nine female NMRI mice at a dose of 10 mg/kg-day on CDs 7-
13 16: a group of nine controls received corn oil (Kristensenetal., 1995). Body weights were
14 monitored. FO females were kept with their offspring until after weaning (21 days after delivery).
15 At 6 weeks of age, one Fl female from each litter (n = 9) was caged with an untreated male. The
16 F2 offspring were inspected for gross deformities at birth, weight and sex were recorded 2 days
17 after birth, and the pups were sacrificed. The Fl females were sacrificed after 6 months of
18 continuous breeding. The effects of benzo[a]pyrene treatment on fertility, ovary weights, follicles,
19 and corpora lutea were evaluated. FO females showed no signs of general toxicity, and there was no
20 effect on fertility. Fl females had statistically significantly lower median numbers of offspring,
21 number of litters, and litter sizes and a statistically significantly greater median number of days
22 between litters as compared with the controls (Table D-27). At necropsy, the Fl females from
23 treated FO females had statistically significantly reduced ovary weights; histologic examination of
24 the ovaries revealed decreased numbers of small, medium, or large follicles and corpora lutea
25 (Table D-27). Only one dose group was used in this study, with decreased Fl female fertility
26 observed following in utero exposure at the LOAEL of 10 mg/kg-day; no NOAEL was identified.
27
28
Table D-27. Effect of prenatal exposure to benzo[a]pyrene on indices of
reproductive performance in Fl female NMRI mice
Endpoint (Median with Range in Parentheses)
Number of F2 offspring
Number of F2 litters
F2 litter size (number of pups per litter)
Number of d between F2 litters
Fl ovary weight (mg)
Number of small follicles
Number of medium follicles
Number of large follicles
Number of corpora lutea
Control3
92 (26-121)
8 (3-8)
11.5 (6-15)
20.5 (20-21)
13 (13-20)
44 (1-137)
9 (5-25)
14 (6-23)
16 (6-35)
Benzo[a]pyrene Exposed3
(10 mg/kg-d)
22* (0-86)
3* (0-8)
8* (3-11)
21* (20-23)
9* (7-13)
0* (0-68)
0* (0-57)
0* (0-19)
0* (0-14)
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1
2 *Significantly (p < 0.05) different from control group by Wilcoxon rank sum test or Kruskall-Wallis two-tailed test.
3
4 aGroups of nine female NMRI FO mice were administered 0 or 10 mg benzo[a]pyrene/kg-day by gavage in corn oil
5 on GDs 7-16. One Fl female from each litter was continuously bred with an untreated male for 6 months.
6
7 Source: Kristensen etal. (1995).
8
9 Chen etal. [2012] treated male and female neonatal Sprague-Dawley rats (10/sex/group)
10 with benzo[a]pyrene (unspecified purity) dissolved in peanut oil by gavage daily on PNDs 5-11, at
11 doses of 0.02, 0.2, or 2 mg/kg in 3 mL vehicle/kg body weight, determined individually based upon
12 daily measurements. This time period was described as representing the brain growth spurt in
13 rodents, analogous to brain developmental occurring from the third trimester to 2 years of age in
14 human infants. Breeding was performed by pairs of 9-week-old rats, with delivery designated as
15 PND 0. Litters were culled to eight pups/dam (four males and four females, when possible) and
16 randomly redistributed at PND 1 among the nursing dams; dams themselves were rotated every 2-
17 3 days to control for caretaking differences, and cage-side observations of maternal behavior were
18 made daily. One male and female from each litter were assigned per treatment group, and the
19 following physical maturation landmarks were assessed daily in all treatment groups until weaning
20 at PND 21: incisor eruption, eye opening, development of fur, testis decent, and vaginal opening.
21 Neonatal sensory and motor developmental tests were administered to pups during the
22 preweaning period at PNDs 12,14,16, and 18, and were behavioral tests administered to rats as
23 adolescents (PNDs 35 and 36) or as adults (PNDs 70 and 71): each rat was only tested during one
24 developmental period. All dosing was performed from 1300 to 1600 hours, and behavioral testing
25 was during the "dark" period from 1900 to 2300 hours, although tests were performed in a lighted
26 environment Pups were observed individually and weighed daily, the order of testing litters was
27 randomized each day, and all observations were recorded by investigators blinded to group
28 treatment
29 Sensory and motor developmental tests, including the surface righting reflex test, negative
30 geotaxis test, and cliff aversion test, were performed only once, while the forelimb grip strength test
31 was assessed during three 60-second trials on PND 12. Rat movements during the open-field test
32 were recorded by camera, and two blinded investigators scored movement and rearing separately
33 during a 5-minute evaluation period. Blinded investigators directly observed video monitoring of
34 rat movements during the elevated plus maze, and after a 5-minute free exploration period,
35 recorded number of entries into the closed and open arms, time spent in the open arms, and latency
36 to the first arm entry. Assessment of the Morris water maze was slightly different, in that the rats
37 were habituated to the testing pool by a 60-second swim without a platform on the day prior to
38 testing. The rats were then tested during a 60-second swim with a hidden platform present at a
39 constant position each day for 4 days; on the 5th day, the rats were evaluated during a 60-second
40 probe swim without a platform. The number of times each animal crossed the original platform
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1 location and the duration of time spent in the platform quadrant were recorded during this final
2 evaluation. One pup/sex/litter were assigned for behavioral testing to each of four tracks: Track 1,
3 surface righting reflex test, cliff aversion test, and open-field test (PNDs 12-18); Track 2, negative
4 geotaxis test, forelimb grip strength test, and open-field test (PNDs 12-20); Track 3, elevated plus
5 maze, Morris water maze, and open-field test (PNDs 34-36); and Track 4, elevated plus maze,
6 Morris water maze, and open-field test (PNDs 69-71). All results were presented in graphic form
7 only.
8 No significant effects on pup body weight were observed during the 7-day treatment period
9 (PNDs 5-11). Three-way AN OVA (time x benzo[a]pyrene treatment x sex) indicated that effects of
10 benzo[a]pyrene were not sex-dependent throughout the 71-day experiment, so both sexes were
11 pooled together. From this pooled analysis, pups in the 2 mg/kg treatment group gained
12 significantly less weight at both PND 36 and 71. There were no differences among treatment
13 groups in incisor eruption, eye opening, development of fur, testis decent, or vaginal opening.
14 For all measurements of neonatal sensory and motor development, results from both sexes
15 were analyzed together since benzo[a]pyrene was reported to have no significant interaction with
16 sex by 3-way AN OVA. No significant differences were observed in either the cliff aversion or
17 forelimb grip strength tests. In the surface righting reflex test, latency was increased in the
18 0.2 mg/kg group at PND 12, in the 0.02 and 2 mg/kg groups at PND 14, in only the high-dose group
19 at PND 16, and was not significantly different in any group at PND 18. At PND 12, there was a dose-
20 related increase in negative geotaxis latency associated with 0.02, 2, and 2 mg/kg benzo[a]pyrene,
21 which was also present in the 2 mg/kg group at PND 14, but returned to control levels at PND 16
22 and 18. In the open field test, there were no significant differences in either locomotion or rearing
23 activity at PND 18 or 20. At PND 34, the 2 mg/kg group exhibited significantly increased
24 movement, but increases in rearing were not significant. At PND 69, increased locomotion was
25 observed in both the 0.2 and 2 mg/kg groups, while rearing was significantly increased in only the
26 2 mg/kg treatment group.
27 The elevated plus maze performance was only evaluated in adolescent and adult rats.
28 Unlike the previous tests, 3-way AN OVA revealed a statistically significant interaction between
29 neonatal benzo[a]pyrene treatment and sex, so male and female performance was analyzed
30 independently. No significant differences in PND 35 males were observed, and the only significant
31 observation in PND 35 females was increased time spent in the open maze arms by the 2 mg/kg
32 treatment group. Significantly decreased latency time to first open arm entry was observed in
33 PND 70 males and females in both 0.2 and 2 mg/kg treatment groups; these groups also spent
34 significantly more time in open maze arms, along with the 0.02 mg/kg female group. At PND 70, the
35 2 mg/kg males, along with the 0.2 and 2 mg/kg females, entered more frequently into open arms
36 and less frequently into closed arms than the vehicle controls. In the Morris water maze, escape
37 latency (time to reach the platform during each of the four testing days) was consistently increased
38 in the 2 mg/kg treatment group of both sexes, in both adolescent and adult animals. These
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1 increases were statistically significant in both males and females treated with 2 mg/kg
2 benzo[a]pyrene at both PNDs 39 and 74, and were also significantly elevated in 0.2 mg/kg animals
3 of both sexes at PND 74. Likewise, performance during the 5th test day, in the absence of the escape
4 platform, was significantly adversely affected by both metrics (decreased time spent in the target
5 quadrant and decreased number of attempts to cross the platform location) in 2 mg/kg rats of both
6 sexes at both PNDs 40 and 75. PND 75 females treated with 0.2 mg/kg benzo[a]pyrene also
7 showed significant decreases in both performance metrics, while PND 75 0.2 mg/kg males only
8 demonstrated significant differences in "time spent in target quadrant". Swim speed was also
9 assessed, but there were no differences among any treatment group at either age evaluated.
10 Tules etal. [2012] treated pregnant Long-Evans Hooded rats with benzo[a]pyrene
11 (unspecified purity) dissolved in 0.875 mL peanut oil by gavage daily on CDs 14-17, at doses of
12 150, 300, 600, and 1,200 [igbenzo[a]pyrene/kg body weight, with animals weighed daily. Cage-
13 side observations were performed until pup weaning, and litter size evaluated for each treatment
14 group. Pups from 4 to 5 individual litters were analyzed for each endpoint, which was
15 independently repeated for a total of 3 replicates. Delivery was designated PND 0, and pups were
16 harvested on PNDs 0-15 for benzo[a]pyrene metabolite identification, or for other endpoints as
17 young adults at PND 53. Systolic/diastolic blood pressure and heart rate was recorded by a volume
18 pressure recording sensor and occlusion tail-cuff applied to conscious, non-anesthetized animals.
19 Animals were preconditioned to the restraint device and tail-cuff by daily acclimatization sessions
20 during PNDs 46-50, to minimize stress effects during data collection. Cardiac function values were
21 averaged from 15 readings each collected over a 1-minute interval every other minute for
22 30 minutes on PND 53.
23 No significant differences in litter size or pup weight gain from PND 0 to 15 were reported
24 in any treatment group, and no convulsions, tremors, or abnormal movements were reproducibly
25 observed. Most analytical data were reported graphically, as mean ± standard error of the mean
26 (SEM) of three replicates of 3-5 offspring measured/group. Plasma and heart tissue total
27 benzo[a]pyrene metabolite levels were maximal at PND 0 (the first time point sampled) and
28 progressively decreased from PNDs 0 to 13. Compared to the low-dose group (150 |J.g/kg), plasma
29 metabolite levels were significantly elevated in the 600 and 1,200 [J.g/kg benzo[a]pyrene groups
30 through PND 13, while heart metabolite levels were significantly increased through PND 11.
31 Metabolites in mid-dose group, 300 [J.g/kg, trended between the 150 and 600 [ig/kg group levels
32 from PND 0 to 7, while not achieving statistically significant differences in pair-wise comparisons.
33 Three principal groups of benzo[a]pyrene metabolites were identified. More than 70% of the total
34 heart metabolite burden was composed of diol metabolites through PND 13, while the more
35 reactive hydroxyl metabolites increased in relative composition from PND 9 to 13, and the dione
36 population remained constant at <5%.
37 Cardiovascular function was evaluated in pups exposed in utero to 600 or 1,200 [ig/kg
38 benzo[a]pyrene versus controls (see Table D-28). A dose-related and statistically significant
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1 increase in both systolic (20, 50%) and diastolic pressure (30, 80%) was observed in mid- and
2 high-dose pups, respectively. Heart rate was also significantly altered; a 10% increased heart rate
3 was reported in the 600 [ig/kg benzo[a]pyrene group, while the average heart rate of the 1,200
4 Mg/kg benzo[a]pyrene groups decreased 8%.
5
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1
2
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
Heart rate (bpm; mean ± SEM)
Dose (mg/kg-d)
0
504.6 ± 15.7
0.600
554.6 ±26.2*
1.20
466.3 ± 16.9*
Blood pressure measured by tail cuff (mmHg; mean ± SEM)
Systolic pressure
Diastolic pressure
131.6 ± 1.2
85.0 ±4.2
151.6 ±45*
113.0 ±3.3*
200.4 ± 2.4*
155.6 ±3.2*
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
29
30
31
32
33
*Significantly (p < 0.05) different from control mean; n = 4-5/replicate, 3 replicates performed.
Source: Jules etal. (2012).
Bouayedetal. (2009a) treated nursing female Swiss Albino OF1 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 group and a 10-12% weight gain in pups from the 20 mg/kg
8 group at PNDs 12-20 (see Table D-29). While not significantly different from PND 26 to 40, pup
9 weight in the 20 mg/kg group was continuously higher than either the 2 mg/kg group or vehicle-
10 treated controls. There were no significant differences in pup brain weight or eye opening
11 observed. Likewise, benzo[a]pyrene treatment of nursing mothers did not affect nest-building
12 interest or quality, and while not significantly impacting pup retrieval time, the retrieval latency
13 period was observed to increase with increasing treatment duration in both benzo[a]pyrene groups
14 versus controls.
15
16
17
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 PND 14
Pup Body Weight (g; mean ± SEM, n = 20)
PNDO
PND4
PNDS
PND 12
PND 20
PND 26
PND 32
PND 40
Dose (mg/kg-d)
0
1.70 ± 0.02
3.01 ±0.08
5.08 ±0.1
6.57 ±0.12
12.51 ±0.24
17.71 ±0.49
24.47 ± 0.55
30.55 ±0.94
2
1.73 ± 0.02
3.08 ± 0.06
5.26 ± 0.09
7. 16 ±0.06*
13.55 ±0.25**
18.60 ±0.36
25.59 ±0.57
30.90 ±0.93
20
1.74 ±0.02
3. 16 ±0.04
5.30 ±0.08
7.39 ±0.05*
13.79 ±0.14*
18.35 ±0.34
25.38 ±0.54
31.78 ±0.97
18
19
20
21
22
23
24
25
26
27
28
29
30
31
*p < 0.001 significantly different from control mean.
**p<0.01.
Source: Bouaved et al. (2009a).
Behavioral test data was reported graphically, as mean ± SEM of n = 20/group. For the pre-
weaning neuromotor developmental tests, benzo[a]pyrene treatment was found to not depend on
sex; therefore, data from male and female pups were combined. Pups nursing from mothers
administered 2 or 20 mg/kg-day benzo[a]pyrene had significantly elevated righting reflex times at
PNDs 3-5, which decreased to control times at PNDs 7-9. Only pups from the 20 mg/kg 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
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1 20 mg/kg group had increased forelimb grip strength, which was significantly greater than control
2 mice at PNDs 9 and 11, corresponding to increased body weight in the benzo[a]pyrene-treated mice
3 versus controls. Mice in the 2 mg/kg group also performed better than controls at PND 9, but were
4 equivalent at PND 11. No treatment or sex-related effects were reported on locomotion or rearing
5 activity during the open field test. Sex-dependency on test performance became evident during the
6 analysis of the WESPOC test data: female pups were not significantly affected using any metric,
7 while males in the 20 mg/kg group demonstrated a statistically significantly longer pole-grasping
8 latency (threefold), and took 13 times longer to escape the pole and board the safety platform
9 versus vehicle controls. While performance of male pups from the 2 mg/kg group was not
10 statistically significantly worse than vehicle controls by pair-wise comparison, latency for both
11 pole-grasping and escape in this treatment group contributed to a significant trend for treatment
12 dose and these effects. In the evaluation of the elevated plus maze, treatment effects did not appear
13 to depend upon sex, so both male and female performance was analyzed together. Mice in both
14 benzo[a]pyrene treatment groups demonstrated decreased latency time to first entering an open
15 arm (30-50%), as well as entered open arms 2-times more frequently and spent twice as much
16 time there versus vehicle controls. While mice in the 2 mg/kg treatment group entered into closed
17 arms 20% less frequently than controls, mice in the 20 mg/kg group were not significantly
18 different. Likewise, mice nursing from mothers treated with 2 mg/kg benzo[a]pyrene performed
19 15% more spontaneous alternations in the Y-maze spontaneous alternation test compared to
20 controls, while mice in the high-dose group were not significantly different. The brains of pups
21 nursing from the 20 mg/kg group expressed approximately 50% lower levels of
22 5-hydroxytryptamine (serotonin) 1A (5HT1A), and mu 1-opioid (MORI) mRNA, and a trend was
23 observed in the low-dose group as well. No significant changes in alpha-ID adrenergic or GABA-A
24 mRNA levels were detected.
25 Reproductive Effects in Adults and Repeated Oral Exposure
26 Rigdon and Neal (1965) conducted a series of experiments to assess the reproductive
27 effects of orally administered benzo[a]pyrene to Ah-responsive white Swiss mice. Female animals
28 (number not stated) were administered benzo[a]pyrene at 250, 500, or 1,000 ppminthe feed
29 before or during a 5-day mating period. Based on the initial body weight, the doses can be
30 estimated as 32, 56, and 122 mg/kg-day, respectively. No effect on fertility was observed at any
31 treatment dose, even when animals were fed 1,000 ppm benzo[a]pyrene for 20 days prior to
32 mating, but interpretation of this finding was marred by large variability in numbers of pregnant
33 females and litter sizes for both treated and control mice. In separate experiments, the fertility of
34 five male mice/group was not affected by exposure to 1,000 ppm in food for up to 30 days prior to
35 mating with untreated females. Histologic examinations showed that male mice fed 500 ppm
36 benzo[a]pyrene for 30 days had spermatozoa present in their testes; further details were not
37 provided. The only treatment-related effect was a lack of weight gain related to feed unpalatability.
38 While this study suggests that premating exposure of male or female mice to doses up to
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1 122 mg/kg-day for 20 days may not affect fertility, the sample sizes were too small and the study
2 designs were too inconsistent to provide reliable NOAELs and LOAELs for reproductive/
3 developmental toxicity.
4 In an earlier study [Rigdon and Rennels, 1964], rats (strain not specified) were fed diets
5 containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and during
6 gestation. In this study, five of eight treated females mated with untreated males became pregnant,
7 but only one delivered live young. The treated dam that delivered had two live and two stillborn
8 pups; one dead pup was grossly malformed. In the remaining treated females, vaginal bleeding was
9 observed on CDs 23 or 24. In the inverse experimental design, three of six controls mated to
10 benzo[a]pyrene-treated males became pregnant and delivered live young. Visceral and skeletal
11 examinations of the pups were not conducted. These studies are insufficiently reported and of
12 insufficient design (e.g., inadequate numbers of animals for statistical analysis) to provide reliable
13 NOAELs or LOAELs for reproductive effects from repeated oral exposure to benzo[a]pyrene.
14 D.4.5. Inhalation
15 Reproductive Toxicity and In Utero Exposure Via Inhalation
16 Archibong et al. (2002) evaluated the effect of exposure to inhaled benzo[a]pyrene on fetal
17 survival and luteal maintenance in timed-pregnant F344 rats. Prior to exposure on GD 8,
18 laparotomy was performed to determine the number of implantation sites, and confirmed pregnant
19 rats were divided into three groups, consisting of rats that had four to six, seven to nine, or more
20 than nine conceptuses in utero. Rats in these groups were then assigned randomly to the treatment
21 groups or control groups to ensure a similar distribution of litter sizes. Animals (10/group) were
22 exposed to benzo[a]pyrene:carbon black aerosols at concentrations of 25, 75, or 100 [ig/m3 via
23 nose-only inhalation, 4 hours/day on CDs 11-20. Control animals were either sham-exposed to
24 carbon black or remained entirely unexposed. Results of particle size analysis of generated
25 aerosols were reported by several other reports from this laboratory (Inyangetal., 2003: Ramesh
26 etal., 2001a: Hoodetal., 2000). Aerosols showed a trimodal distribution (average of cumulative
27 mass, diameter) <95%, 15.85 ^m; 89%, <10 urn; 55%, <2.5 ^m; and 38%, <1 ^m (Inyangetal..
28 2003). Ramesh etal. (2001a) reported that the MMAD (± geometric SD) for the 55% mass fraction
29 with diameters <2.5 |im was 1.7 ± 0.085. Progesterone, estradiol-17(3, and prolactin concentrations
30 were determined in plasma collected on CDs 15 and 17. Fetal survival was calculated as the total
31 number of pups divided by the number of all implantation sites determined on GD 8. Individual
32 pup weights and crown-rump length per litter per treatment were determined on PND 4
33 (PND 0 = day of parturition).
34 Archibong et al. (2001) reported that exposure of rats to benzo[a]pyrene caused
35 biologically and statistically significant (p < 0.05) reductions in fetal survival compared with the
36 two control groups; fetal survival rates were 78.3, 38.0, and 33.8% per litter at 25, 75, and
37 100 [J.g/m3, respectively, and 96.7% with carbon black or 98.8% per litter in untreated controls (see
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1 Table D-30). Consequently, the number of pups per litter was also decreased in a concentration-
2 dependent manner. The decrease was ~50% at 75 [ig/m3 and ~65% at 100 [ig/m3, compared with
3 sham-exposed and unexposed control groups. No effects on hormone levels were observed on
4 CDs 15 or 17 atthe low dose. Biologically significant decreases in mean pup weights (expressed as
5 g per litter) of >5% were observed at doses >75 [J.g/m3(14 and 16% decreases at 75 and
6 100 [J.g/m3, respectively, p < 0.05). Exposure to benzo[a]pyrene did not affect crown-rump length
7 (see Table D-30).
8
9
Table D-30. Pregnancy outcomes in female F344 rats treated with
benzo[a]pyrene on CDs 11-21 by inhalation
Parameter3
Implantation sites
Pups per litter
Survival (litter %)
Pup weight (g/litter)
Crown-rump length
(mm/litter)
Administered Concentration of Benzo[a]pyrene (u.g/ms)
0 (Unexposed
Control)
8.6 ±0.2
8.5 ±0.2
98.9 ±1.1
10.6 ±0.1
29.4 ±0.6
0
(Carbon Black)
8.8 ±0.1
8.7 ±0.2
96.7 ±1.7
8.8 ±0.1
29.3 ±0.5
25
8.8 ±0.5
7.4 ±0.5*
78.3 ±4.1*
10.5 ± 0.2
28.0 ±0.6
75
9.0 ±0.2
4.2 ±0.1*
38.0 ±2.1*
9.1 ±0.2*
27.3 ±0.7
100
8.8 ±0.1
3.0 ±0.2*
33.8 ±1.3*
8.9 ±0.1*
27.9 ±0.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
*Significantly different from controls at p < 0.05 by one-tailed post-hoc t-testing following ANOVA.
aValues presented as means ± SEM.
Source: Archibong et al. (2002).
Benzo[a]pyrene exposure at 75 [ig/rn3 caused a statistically significant decrease in plasma
progesterone, estradiol, and prolactin on GD 17; these levels were not determined in the rats
exposed to 100 [ig/rn3 (Archibong etal.. 2002). Plasma prolactin is an indirect measure of the
activity of decidual luteotropin, a prolactin-like hormone whose activity is necessary for luteal
maintenance during pregnancy in rats. Control levels of prolactin increased from GD 15 to 17, but
this increase did not occur in the rats exposed to 75 [ig/m3. Although the progesterone
concentration at 75 [ig/m3 was significantly lower than in controls on GD 17, the authors thought
that the circulating levels should have been sufficient to maintain pregnancy; thus, the increased
loss of fetuses was thought to be caused by the lower prolactin levels rather than progesterone
deficiency. The reduced circulating levels of progesterone and estradiol-17p among
benzo[a]pyrene-treated rats were thought to be a result of limited decidual luteotropic support for
the corpora lutea. The authors proposed the following mechanism for the effects of benzo[a]pyrene
on fertility: benzo[a]pyrene or its metabolites decreased prolactin and decidual luteotropin levels,
compromising the luteotropic support for the corpora lutea and thereby decreasing the plasma
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1 levels of progesterone and estradiol-17p. The low estradiol-17p may decrease uterine levels of
2 progesterone receptors, thereby resulting in fetal mortality. Based on biologically and statistically
3 significant decreases in pups/litter and percent fetal survival/per litter, the LOAEL was 25 [ig/m3;
4 no NOAEL was identified.
5 Neurotoxicity and In Utero Exposure Via Inhalation
6 To evaluate the effects of benzo[a]pyrene on the developing central nervous system,
7 Wormleyetal. [2004] exposed timed-pregnant F344 rats (10/group) to benzo[a]pyrene:carbon
8 black aerosols by nose-only inhalation on CDs 11-21 for 4 hours/day at a concentration of
9 100 [J.g/m3. Results of particle size analysis of generated aerosols were reported by other reports
10 from this laboratory [Rameshetal.. 2001a: Hood etal.. 2000). Particle size analysis of a 100-^g/m3
11 aerosol showed a trimodal distribution (average of cumulative mass, diameter): <95%, 15.85 [im;
12 90%, <10 urn; 67.5%, <2.5 ^m; and 66.2%, <1 [im; the MMAD ± geometric SD for the latter fraction
13 was 0.4 ± 0.02 |im [Hoodetal., 2000]. Dams were maintained to term and pups were weaned on
14 PND 30. Benzo[a]pyrene reduced the number of live pups to one-third of control values without
15 affecting the number of implantation sites. During PNDs 60-70, electrical stimulation and evoked
16 field potential responses were recorded via electrodes implanted into the brains of the animals.
17 Direct stimulation of perforant paths in the entorhinal region revealed a diminution in long-term
18 potentiation of population spikes across the perforant path-granular cell synapses in the dentate
19 gyrus of the hippocampus of Fl generation benzo[a]pyrene-exposed animals; responses in exposed
20 offspring were about 25% weaker than in control offspring. Additionally, NMDA receptor subunit 1
21 protein (important for synaptic functioning) was down-regulated in the hippocampus of
22 benzo[a]pyrene-exposed Fl pups. The authors interpreted their results as suggesting that
23 gestational exposure to benzo[a]pyrene inhalation attenuates the capacity for long-term
24 potentiation (a cellular correlate of learning and memory) in the Fl generation.
25 In another study by this same group of investigators, Wu etal. (2003a) evaluated the
26 generation of benzo[a]pyrene metabolites in Fl generation pups, as well as the developmental
27 profile for AhR and mRNA. In this study, confirmed-pregnant F344 rats were exposed to
28 benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 [ig/m3 via nose-only inhalation,
29 4 hours/day, for 10 days (CDs 11-21). Control animals either were exposed to carbon black (sham)
30 to control for inert carrier effects or remained untreated. Each benzo[a]pyrene concentration had
31 its own set of controls (carbon black and untreated). Two randomly selected pups were sacrificed
32 on each of PNDs 0, 3, 5,10,15, 20, and 30. Body, brain, and liver weights were recorded.
33 Benzo[a]pyrene metabolites were analyzed in the cerebral cortex, hippocampus, liver, and plasma.
34 A dose-related increase in plasma and cortex benzo[a]pyrene metabolite concentrations in pups
35 was observed. Dihydrodiols (4,5-; 7,8-; 9,10-) dominated the metabolite distribution profile up to
36 PND 15 and the hydroxy (3-OH-benzo[a]pyrene; 9-OH-benzo[a]pyrene) metabolites after PND 15
37 at 100 [ig/m3 (the only exposure concentration reported). Results indicated a dose-related
38 decrease in the ratio of the total number of pups born per litter to the total number of implantation
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1 sites per litter. The number of resorptions at 75 andlOO [ig/m3, butnotat25 [J.g/m3,was
2 statistically significantly increased compared with controls.
3 Adult Male Reproductive Effects and Repeated Inhalation Exposure
4 Inyangetal. [2003] evaluated the effect of subacute exposure to inhaled benzo[a]pyrene on
5 testicular steroidogenesis and epididymal function in rats. Male F344 rats (10/group), 13 weeks of
6 age, were exposed to benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 [ig/m3 via nose-only
7 inhalation, 4 hours/day for 10 days. Control animals either were exposed to carbon black (sham) to
8 control for exposure to the inert carrier or remained untreated. Each benzo[a]pyrene
9 concentration had its own set of controls (carbon black and untreated). Aerosols showed a
10 trimodal distribution (average of cumulative mass, diameter): 95%, <15.85 [im; 89%, <10 [im; 55%,
11 <2.5 [im; and 38%, <1 [im (Inyangetal.. 2003): an earlier report from this laboratory indicated that
12 the 55% mass fraction had a MMAD ± geometric SD of 1.7 ± 0.085 (Rameshetal.. 2001a). Blood
13 samples were collected at 0, 24, 48, and 72 hours after cessation of exposure to assess the effect of
14 benzo[a]pyrene on systemic concentrations of testosterone and LH, hormones that regulate
15 testosterone synthesis. Reproductive endpoints such as testis weight and motility and density of
16 stored (epididymal) sperm were evaluated.
17 Regardless of the exposure concentration, inhaled benzo[a]pyrene did not affect testis
18 weight or the density of stored sperm compared with controls. However, inhaled benzo[a]pyrene
19 caused a concentration-dependent reduction in the progressive motility of stored sperm.
20 Progressive motility was similar at 75 and 100 [J.g/m3, but these values were significantly lower
21 (p < 0.05) than in any other group. The reduction in sperm motility postcessation of exposure was
22 thoughtto be the result of benzo[a]pyrene limiting epididymal function. Benzo[a]pyrene exposure
23 to 75 [J.g/m3 caused a decrease in circulating concentrations of testosterone compared with controls
24 from the time of cessation of exposure (time 0) to 48 hours posttermination of exposure (p < 0.05).
25 However, the decrease was followed by a compensatory increase in testosterone concentration at
26 72 hours postcessation of exposure. Exposure to 75 [ig/m3 caused a nonsignificant increase in
27 plasma LH concentrations at the end of exposure compared with controls, which increased further
28 and turned significant (p < 0.05) for the remaining time of the study period. The decreased plasma
29 concentration of testosterone, accompanied by an increased plasma LH level, was thought to
30 indicate that benzo[a]pyrene did not have a direct effect on LH. The authors also noted that the
31 decreased circulating testosterone may have been secondary to induction of liver CYP450 enzymes
32 by benzo[a]pyrene. The authors concluded that subacute exposure to benzo[a]pyrene contributed
33 to impaired testicular endocrine function that ultimately impaired epididymal function. For this
34 study, the NOAEL was 25 [J.g/m3 and the LOAEL was 75 [ig/m3, based on a statistically significant
35 reduction in the progressive motility of stored sperm and impairment of testicular function with
36 10 days of exposure at 75 [ig/m3.
37 In a follow-up study with longer exposure duration, adult male F344 rats (10 per group)
38 were exposed to benzo[a]pyrene:carbon black aerosols at 75 [ig/m3 via nose-only inhalation,
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1 4 hours/day for 60 days [Archibongetal., 2008: Rameshetal., 2008]. Rats in the control group
2 were subjected to the nose-only restraint, but were not exposed to the carbon black carrier. Blood
3 samples were collected at 0, 24, 48, and 72 hours after exposure terminated, and the animals were
4 sacrificed for tissue analyses following the last blood sampling. Data were analyzed statistically for
5 benzo[a]pyrene effects on weekly body weights, total plasma testosterone and LH concentrations,
6 testis weights, density of stored spermatozoa, sperm morphological forms and motility,
7 benzo[a]pyrene metabolite concentrations and aryl hydrocarbon hydroxylase (AHH) activity, and
8 morphometric assessments of testicular histologies. Relative to controls, the results indicated 34%
9 reduced testis weight (p < 0.025), reduced daily sperm production (p < 0.025), and reduced
10 intratesticular testosterone concentrations (p < 0.025). Plasma testosterone concentrations were
11 reduced to about one-third of the level in controls on the last day of exposure (day 60) and at 24,
12 48, and 72 hours later (p < 0.05). However, plasma LH concentrations in benzo[a]pyrene-exposed
13 rats were elevated throughout the blood sampling time periods compared with controls (p < 0.05).
14 In testis, lung, and liver, AHH activity and benzo[a]pyrene-7,8-dihydrodiol (precursor to the
15 DNA-reactive BPDE) and benzo[a]pyrene-3,6-dione metabolites were significantly (p < 0.05)
16 elevated relative to controls. Progressive motility and mean density of stored spermatozoa were
17 significantly reduced (p < 0.05). Weekly body weight gains were not affected by benzo[a]pyrene
18 exposure. These results indicate that a 60-day exposure of adult male rats to benzo[a]pyrene:
19 carbon black aerosols at 75 [ig/m3 produced decreased testis weight; decreased intratesticular and
20 plasma testosterone concentrations; and decreased sperm production, motility, and density.
21 D.5. OTHER PERTINENT TOXICITY INFORMATION
22
23 D.5.1. Genotoxicity Information
24
25 Information regarding the genotoxicity of benzo[a]pyrene in in vitro and in vivo systems is
26 presented in Tables D-31, D-32, and D-33.
27
28
Table D-31. In vitro genotoxicity studies of benzo[a]pyrene in non-
mammalian cells
Result
+S9
-S9
Reference
Endpoint/test system: prokaryotic cells
Forward mutation
Salmonella typhimurium TM677
S. typhimurium TM677
+
+
-
ND
Rastetter et al. (1982)
Babsonetal. (1986)
Reverse mutation
This document is a draft for review purposes only and does not constitute Agency policy.
D-88 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
S. typhimurium TA98, TA1538
S. typhimurium TA98, TA100, TA1538
S. typhimurium TA1538, TA98
S. typhimurium TA98, TA100, TA1537
S. typhimurium TA98, TA100
S. typhimurium TA98
S. typhimurium TA98, TA100
S. typhimurium TA98, TA100
S. typhimurium TA98
S. typhimurium TA98, TA100
S. typhimurium TA98, TA100, TA1538
S. typhimurium TA97, TA98, TA100
S. typhimurium TA97, TA98, TA100, TA1537
S. typhimurium TA97, TA98, TA100
S. typhimurium TA98
S. typhimurium TA98, TA100
S. typhimurium TA98
S. typhimurium TA98, TA100
S. typhimurium TA98
S. typhimurium TA98
S. typhimurium TA98
S. typhimurium TA98
S. typhimurium TA100
S. typhimurium TA100
S. typhimurium TA100
S. typhimurium TA100
S. typhimurium TA100
S. typhimurium TA100
S. typhimurium TA100
S. typhimurium TA1537, TA1538
S. typhimurium TA1537, TA1538
S. typhimurium TA1537
S. typhimurium TA1538
S. typhimurium TA1538
S. typhimurium TA1535
S. typhimurium TA 1535
S. typhimurium TA 1535
S. typhimurium TA1535
Result
+S9
+
+
+
+
+
+
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
-
-
-
-
-S9
ND
ND
-
-
-
-
ND
-
ND
ND
-
-
-
ND
-
-
ND
ND
ND
ND
ND
ND
ND
-
ND
ND
ND
ND
ND
-
-
ND
ND
-
-
-
ND
-
Reference
Ames et al. (1975)
McCannetal. (1975)
Wood etal. (1976)
Epleretal. (1977)
Obermeier and Frohberg (1977)
Pitts etal. (1978)
LaVoie etal. (1979)
Simmon (1979a)
Hermann (1981)
Alfheim and Ramdahl (1984)
Glatt etal. (1985)
Sakai etal. (1985)
Glatt etal. (1987)
Marino (1987)
Alzieu etal. (1987)
Prasanna etal. (1987)
Ampvetal. (1988)
Bos etal. (1988)
Lee and Lin (1988)
Antignacetal. (1990)
Gao etal. (1991)
Balansky etal. (1994)
Norpothetal. (1984)
Carver etal. (1986)
Pahlman and Pelkonen (1987)
Tang and Friedman (1977)
Bruce and Meddle (1979)
Phillipson and loannides (1989)
Balanskv etal. (1994)
Ames etal. (1973)
Glatt etal. (1975)
Oesch etal. (1976)
Egert and Greim (1976)
Rosenkranz and Poirier (1979)
Ames etal. (1973)
Glatt etal. (1975)
McCannetal. (1975)
Epleretal. (1977)
DNA damage
This document is a draft for review purposes only and does not constitute Agency policy.
D-89 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
Eschehchia coli/pol A
E. coli/differential killing test
E. co// WP2-WP100/rec-assay
E. coli/SOS chromotest Pq37
Result
+S9
+
+
+
+
-S9
-
-
ND
-
Reference
Rosenkranz and Poirier (1979)
Tweats (1981)
Mamberetal. (1983)
Mersch-Sundermann et al. (1992)
Endpoint/test system: nonmammalian eukaryotes
Mitotic recombination
Saccharomyces cerevisiae D4-RDII
S. cerevisiae D3
ND
-
-
-
Siebertetal. (1981)
Simmon (1979b)
1
2
+ = positive; - = negative; ND = not determined.
3
4
Table D-32. In vitro genotoxicity studies of benzo[a]pyrene in mammalian
cells
Assay/Test System
Result
+S9
-S9
Reference
Forward mutation
Human AHH-1 lymphoblastoid cells
Human lymphoblast (AHH-1) cells (hprt)
Human lymphoblastoid (AHH-1) cell line
Human fibroblast (MRC5CV1) cell line (hprt)
Human lymphoblast (TK) cells
Human lymphoblast (TK6) cells
Human embryonic epithelial (EUE) cells
Human HSC172 lung fibroblasts
Human Q3-wp normal lung keratinocytes
Human SCC-13Y lung keratinocytes
Mouse /ocZtransgenic Muta™Mouse primary
hepatocytes
Mouse L5178Y/HGPRT
Mouse lymphoma (L5178Y/TK+/-) cells
Mouse lymphoma (L5178Y/TK+/-) cells
Mouse lymphoma (L5178Y/TK+/-) cells
Mouse lymphoma (L5178Y/TK+/-) cells
Chinese hamster ovary (CHO) cells (aprt)
CHOcells(5 marker loci)
Chinese hamster V79 cells (co-cultured with
irradiated HepG2 cells)
ND
ND
ND
-
ND
+
ND
+
+
ND
ND
+
+
+
+
+
+
+
+
+
+
+
ND
+
ND
+
-
ND
+
+
-
-
ND
-
ND
ND
+
ND
Danheiser et al. (1989)
Crespietal. (1985)
Chen etal. (1996)
Haneltetal. (1997)
Barfknecht etal. (1982)
Crespietal. (1985)
Rocchi etal. (1980)
Gupta and Goldstein (1981)
Allen-Hoffmann and Rheinwald
(1984)
Allen-Hoffmann and Rheinwald
(1984)
Chen etal. (2010)
Clive etal. (1979)
Clive etal. (1979)
Amacher et al. (1980); Amacher
and Turner (1980)
Amacher and Paillet (1983)
Arce et al. (1987)
Yang etal. (1999)
Gupta and Singh (1982)
Diamond etal. (1980)
This document is a draft for review purposes only and does not constitute Agency policy.
D-90 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
Assay/Test System
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
Rat/Fischer, embryo cells/Qua R
Result
+S9
+
+
+
ND
-S9
ND
ND
ND
+
Reference
Hubermanetal. (1976)
Arce et al. (1987)
O'Donovan (1990)
Mishraetal. (1978)
DNA damage
DNA adducts
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Human fibroblast (MRC5CV1) cell line
Human hepatoma (HepG2) cell line
Hamster tracheal cells
Chinese hamster V79 lung epithelial cells
Virus transformed SHE and mouse C3H10T1/2
cells
Mouse lymphoma (L5178Y/TK+/-) cells
Rat tracheal cells
ND
ND
ND
ND
+
ND
ND
+
ND
+
ND
+
+
+
+
ND
+
+
ND
+
ND
+
Wienckeetal. (1990)
Li et al. (2001)
Wu et al. (2005)
Gu et al. (2008)
Haneltetal. (1997)
Tarantini etal. (2009)
Roggeband etal. (1994)
Arce et al. (1987)
Arce etal. (1987)
Arce et al. (1987)
Roggeband etal. (1994)
Unscheduled DNA synthesis
HeLa cells
Human fibroblasts
Human fibroblasts
Human HepG2
Hamster primary embryo cells
Hamster tracheal cells
Rat hepatocytes
Rat tracheal cells
+
+
+
ND
ND
ND
ND
ND
ND
ND
-
+
+
+
+
-
Martin etal. (1978)
Agrelo and Amos (1981)
Robinson and Mitchell (1981)
Valentin-Severin et al. (2004)
Casto etal. (1976)
Roggeband etal. (1994)
Michalopoulos et al. (1978)
Roggeband etal. (1994)
DNA repair
Human mammary epithelial cells
Human skin fibroblasts
Baby hamster kidney (BHK21/cl3) cells
secondary mouse embryo fibroblasts (C57BL/6)
and human lymphocytes
Rat/F344 hepatocytes
ND
ND
ND
ND
ND
+
+
+
+
+
Leadonetal. (1988)
Milo etal. (1978)
Feldman etal. (1978)
Shinohara and Cerutti (1977)
Williams etal. (1982)
Cytogenetic damage
CAs
Human blood cells
Human WI38 fibroblasts
Chinese hamster lung cells
Chinese hamster V79-4 lung epithelial cells
ND
+
+
-
+
-
-
-
Salamaetal. (2001)
Weinstein et al. (1977)
Matsuokaetal. (1979)
Popescu etal. (1977)
This document is a draft for review purposes only and does not constitute Agency policy.
D-91 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
Assay/Test System
Mouse lymphoma (L5178Y/TK+/-) cells
Rat Liver RL1 cells
Result
+S9
+
+
-S9
ND
ND
Reference
Arce et al. (1987)
Dean (1981)
MN
Human AHH-1 lymphoblastoid cells
Human HepG2 liver cells
Human lymphoblastoid (TK) cells
Human MCL-5 lymphoblastoid cells
Human peripheral blood lymphocytes
Chinese hamster V79 cells
Chinese hamster V79-MZ cells
ND
ND
ND
ND
+
ND
ND
+
+
+
+
ND
+
+
Crofton-Sleigh et al. (1993)
Wuetal. (2003a)
Fowler et al. (2010)
Crofton-Sleigh et al. (1993)
Lo Jacono et al. (1992)
Whitwell et al. (2010)
Matsuokaetal. (1999)
DMA strand breaks
Human sperm
Human peripheral blood lymphocytes
Human fibroblast (MRC5CV1) cell line
Human hepatoma (HepG2) cell line
Human prostrate carcinoma (DU145) cell line
Mouse embryo fibroblast (C3H/10T1/2 CL 8)
cells
Rat CIS trachea epithelial cells
Rat lymphocytes
+
+
+
ND
ND
ND
ND
ND
+
+
ND
+
+
+
+
+
Sipinen etal. (2010)
Rodriguez-Romero et al. (2012)
Hanelt etal. (1997)
Tarantini etal. (2009)
Nwagbaraetal. (2007)
Lubet etal. (1983)
Cosma and Marchok (1988);
Cosmaetal. (1988)
(Gao etal., 1991)
SCEs
Human C-HC-4 and C-HC-20 hepatoma cells
Human diploid fibroblast (TIG-II) cell line
Human fibroblasts
Human blood cells
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Human peripheral blood lymphocytes
Chinese hamster Don-6 cells
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
CHO cells
ND
+
ND
ND
ND
ND
ND
ND
+
+
ND
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
-
ND
+
-
ND
ND
ND
+
-
Abe etal. (1983a, b)
Huh etal. (1982)
Juhl etal. (1978)
Salamaetal. (2001)
Rudiger etal. (1976)
Craig-Holmes and Shaw (1977)
Schonwald etal. (1977)
Wiencke etal. (1990)
Tohda etal. (1980)
Lo Jacono etal. (1992)
Abe etal. (1983a, b)
Popescu etal. (1977)
Mane etal. (1990)
Woiciechowski et al. (1981)
Arce et al. (1987)
Kulkaetal. (1993a)
deRaat(1979)
This document is a draft for review purposes only and does not constitute Agency policy.
D-92 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
Assay/Test System
CHO cells
CHO cells
CHO cells
Chinese hamster lung cells
Rabbit peripheral blood lymphocytes
Rat ascites hepatoma AH66-B
Rat esophageal tumor Rl
Rat hepatocyte (immortalized) cell lines (NRL
cl-B, NRLcl-C, andARL)
Rat hepatoma (Reuber H4-II-E) cells
Rat liver cell line ARL18
Rat pleural mesothelial cells
Result
+S9
+
ND
ND
ND
ND
ND
ND
+
ND
ND
ND
-S9
-
+
+
+
+
+
+
ND
+
+
+
Reference
Husgafvel-Pursiainen et al.
(1986)
Wolff and Takehisa (1977)
Pal etal. (1978)
Shimizuetal. (1984)
Takehisa and Wolff (1978)
Abeetal. (1983a, b)
Abeetal. (1983a, b)
Kulkaetal. (1993b)
Dean etal. (1983)
Tong etal. (1981)
Achard etal. (1987)
Aneuploidy
Chinese hamster V79-MZ cells
ND
+
Matsuokaetal. (1998)
Cell transformation
Human BEAS-2B lung cells
Human breast epithelial (MCF-10F, MCF-7, T24)
cell lines
Baby hamster kidney (BHK21/cl3) cells
Golden hamster embryo cells
Syrian hamster embryo (SHE) cells
SHE cells
SHE cells
SHE cells/focus assay
Fetal Syrian hamster lung (FSHL) cells
Virus infected rat embryo RLV/RE and RAT cells;
mouse embryo AKR/Me cells; Syrian hamster
embryo cells
Virus transformed SHE and mouse C3H10T1/2
cells
Mouse C3H/10T1/2 embryo fibroblasts
Mouse embryo fibroblast (C3H/10T1/2 CL 8)
cells
Mouse embryo fibroblast (C3H/10T1/2 CL 8)
cells
Mouse SHE cells; BALB/c-3t3 cells; C3H/10T1/2
cells; prostate cells
Mouse BALB/C-3T3 cells
ND
ND
+
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
+
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
van Agen etal. (1997)
Calaf and Russo (1993)
Greb etal. (1980)
Mager etal. (1977)
Dipaolo et al. (1971); Dipaolo et
al. (1969)
Dunkel etal. (1981)
Leboeuf etal. (1990)
Casto etal. (1977)
Emura et al. (1987); Emura et
al. (1980)
Heidelbergeretal. (1983)
Arce et al. (1987)
Nesnow et al. (2002); Nesnow
etal. (1997)
Peterson et al. (1981)
Lubet etal. (1983)
Heidelbergeretal. (1983)
Dunkel etal. (1981)
This document is a draft for review purposes only and does not constitute Agency policy.
D-93 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
Assay/Test System
Mouse BALB/C-3T3 cells
Mouse BALB/C-3T3 clone A31-1-1
Rat/Fischer, embryo cells (leukemia virus
transformed)
Rat/Fischer, embryo cells/OuaR
Result
+S9
ND
ND
ND
ND
-S9
+
+
+
+
Reference
Matthews (1993)
Little and Vetrovs (1988)
Dunkeletal. (1981)
Mishraetal. (1978)
1
2
3
4
+ = positive; - = negative; CHO = Chinese hamster ovary; ND = not determined; SHE = Syrian hamster embryo;
TK = thymidine kinase.
This document is a draft for review purposes only and does not constitute Agency policy.
D-94 DRAFT—DO NOT CITE OR QUOTE
-------
Table D-33. In vivo genotoxicity studies of benzo[a]pyrene
r>
C
§
Q
I
5
c
0 §
s|
1
Q.
o
§
n 9.
Endpoint
Mutation
Mutation,
germline
Test system
Human, blood
T lymphocytes (smokers
and non-smokers); hprt
locus mutation assay
Mouse, T-stock, (SEC x
C57BL)F1, (C3H x 101)F1,
or(C3HxC57BL)Flfor
females; (101 xC3H)Fl or
(CSHxlOl)Fl for males;
dominant-lethal mutation
assay
Test conditions
T-cells of lung cancer patients (smokers
and non-smokers from lung cancer
patients and population controls with
known smoking status) analyzed for hprt
locus mutations.
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 from 9 to 11 hrs after mating
and first-cleavage metaphase
chromosomes prepared 20 hrs after
mating.
Results
+
+
Dose
Smokers and
non-smokers
500 mg/kg
Comment
Splicing mutations, base-pair
substitutions, frameshift, and
deletion mutations observed.
Smokers and non-smokers had
GC->TAtransversions (13 and 6%,
respectively) and GC->AT
transitions (24 and 35%,
respectively) in hprt gene
consistent with in vitro
mutagenicity of benzo[a]pyrene.
The percent of dominant lethal
mutations were in the order of
T-stock = (C3H x 101)F1 > (SEC x
C57BL)F1 >(C3HxC57BL)Fl.
Reference
Hackman
etal.
(2000)
Generoso
etal.
(1979)
c
1
I
a
B
.
o
a
I
Co
o
73
P
O
1
-------
r>
C
§
Q
I
i
Q.
§
Endpoint
Mutation,
germline
Mutation,
germline
Mutations
and BPDE-
DNA
adducts,
germline
Mutations
and BPDE-
DNA
adducts,
germline
Test system
Mouse, male stocks: (101
xC3H)Fl; female stocks
(A): (101xC3H)Fl, (B):
(C3H x 101)F1, (C): (C3H
xC57BL)Fl, (D):(SECx
C57BL)F1, (E):T-stock
females; dominant lethal
mutations
Mouse, male stocks:
(101 xC3H)Fl; female
stocks (A): (101xC3H)Fl,
(B): (C3H x 101)F1,
(C): (C3H x C57BL)F1,
(D):(SECxC57BL)Fl,
(E):T-stock females;
heritable translocations
Mouse, C57BL/6, ell
transgenic (Big Blue®)
Mouse, C57BL/6 males,
WT and Xpc"7" with
pUR288/ocZ reporter
gene
Test conditions
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.
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.
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.
Benzo[a]pyrene given via gavage in
sunflower oil 3 times/wk for 1, 4, or 6 wks
(Xpc"7") or 6 wks (WT). Spleen, testis, and
sperm cells analyzed for lacZ mutation
frequency, and DNA adducts analyzed in
testis by [32P]-postlabeling.
Results
+
+
+
Dose
500 mg/kg
500 mg/kg
50 mg/kg
13 mg/kg
Comment
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).
No significant differences were
observed between treated and
control progeny.
Exposed spermatocytes acquired
persistent BPDE-DNA adducts;
exposed spermatogonia gave rise
to spermatocytes with mutations
consistent with a benzo[a]pyrene
spectrum (GC>TAtransversions).
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.
Reference
Generoso
etal.
(1982)
Generoso
etal.
(1982)
Olsen et al.
(2010)
Verhofstad
etal.
(2011)
-------
r>
C
§
Q
I
-
§
Endpoint
Mutations
and BPDE-
DNA
adducts
Mutation
Mutation
Mutation
Test system
Mouse, C57BL/6 lacZ
transgenic
Mouse, C57BL female x
T-strain male; somatic
mutation assay
Mouse, lacZ transgenic
(Muta™Mouse)
Mouse, lacZ transgenic
(Muta™Mouse)
Test conditions
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.
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.
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.
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.
Results
Dose
50 mg/kg
100 or
500 mg/kg
25, 50, and 75
mg/kg-day
125 mg/kg-day
Comment
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.
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.
Highest lacZ mutation frequency
observed in small intestine,
followed by bone marrow,
glandular stomach, and liver.
Highest mutation frequency
observed in colon followed by
ileum > forestomach > bone
marrow = spleen > glandular
stomach > liver = lung >
kidney = heart.
Reference
Boerrigter
(1999)
Russell
(1977)
Lemieux et
al. (2011)
Hakura et
al. (1998)
o
-------
r>
C
§
Q
I
i
Q.
§
n 9.
H 5'
m ^
O
P
P
Endpoint
Mutation
Mutation
Mutation
Mutation
Mutation
Test system
Mouse, C57BL/6J Dlb-1
congenic; Dlb-1 locus
assay
Mouse, C57BL/6 (lacZ
negative and XPA and
XPA1'}; hprt mutations in
T lymphocytes
Mouse, Cockayne
syndrome-deficient
(Csb'''); heterozygous
(Csb+/~) and WT controls
(Csb+/+); hprt mutation
frequency assay
Mouse, B6C3Fi,
forestomach H-ras, K-ras,
and p53 mutations
Mouse, lacZ/galE (Muta™
Mouse); skin painting
study
Test conditions
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
10u.g/kgTCDD.
Gavage in corn oil 3 times/wk for 0, 1, 5, 9,
or 13 wks; sacrificed 7 wks after last
treatment.
Csb'^/lacZ^' and Csb+/~/lacZ+/~ mice were
dosed i.p. with benzo[a]pyrene 3 times/wk
for 5, 9, or 13 wks; for hprt mutation
frequency analysis mice were sacrificed
3 wks after last treatment; splenocytes
collected; for lacZ mutation frequency
analysis, mice were sacrificed 3 d after last
treatment and liver, lung, and spleen were
collected.
Benzo[a]pyrene given in feed in a 2-yr
chronic feeding 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;
DNA from skin, liver, and lung analyzed for
mutations.
Results
+
+
+
+
+skor
Li.Lu
Dose
40 mg/kg
13 mg/kg
13 mg/kg
5, 25, or
100 ppm
1.25 or
2.5 mg/kg
(25 or
50 |jg/mouse)
Comment
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.
Mutation sensitivity:
XPA''' > XPA*'\
lacZ mutation frequency detected
in all tissues but no differences
between WT and Csfa"7" mice; hprt
mutations significantly higher in
Csb~'~ mice than control mice.
BPDE-dGuo adducts in hprt gene
are preferentially removed in WT
mice than Csfa"7" mice.
68% K-ras (codons 12, 13), 10%
H-ras (codon 13), 10% p53
mutations; all G->Ttransversions.
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.
Reference
Brooks et
al. (1999)
Boletal.
(1998)
Wiinhoven
etal.
(2000)
Gulp etal.
(2000)
Dean et al.
(1998)
1
-------
r>
C
§
Q
I
i
Q.
§
Endpoint
Mutation
Mutation
Mutation
Mutation
Mutation
BPDE-DNA
adducts
Test system
Mouse, T-strain
Mouse, 129/Ola (WT);
hprt mutations in splenic
T lymphocytes
Mouse, A/J, male
Mouse, CD-I; skin
papillomas (Ha-ras
mutations)
Rat, Wistar
Human, WBCs
Test conditions
Benzo[a]pyrene given to pregnant mice by
gavage in 0.5 mL corn oil on GDs 5-10.
Single i.p. injection followed by sacrifice
7 wks posttreatment.
Single i.p. injection followed by sacrifice
28 days posttreatment.
Female mice were initiated topically with
a single dose of benzo[a]pyrene and 1 wk
after initiation promoted twice weekly
with 5 nmolTPAfor 14 wks. One month
after stopping TPA application, papillomas
were collected and DNAfrom 10 individual
papillomas was analyzed for Ha-ras
mutations by PCR and direct sequencing.
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.
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.
Results
+
+
+
+
+
+
Dose
10 mg/mouse
(5x2 mg)
0, 50, 100, 200,
or 400 mg/kg
0, 0.05, 0.5, 5,
or 50 mg/kg
600
nmol/mouse
0, 1, 5, 10, or
100 mg/kg
Comment
Dose-dependent increase in hprt
mutation frequency.
Dose-dependent increase in lung
tissue K-ras codon 12 G->T
mutation frequency.
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).
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.
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.
Reference
Davidson
and
Dawson
(1976)
Bol et al.
(1998)
Meng et al.
(2010)
Colapietro
etal.
(1993)
Willemset
al. (1991)
Pavanello
etal.
(1999)
-------
3
In'
r>
C
§
Q
I
O O
o 2.
•^
i
Q.
§
n a
Endpoint
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
Test system
Human, WBCs
Human, peripheral
lymphocytes
Human, maternal and
umbilical cord blood
Test conditions
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.
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.
Maternal and umbilical cord blood
obtained following normal delivery from
329 non-smoking pregnant women
exposed to emissions from fires during the
4 weeks following the collapse of the WTC
building in New York City on 09/11/2001.
Results
+
+
+
Dose
Comment
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 NER capacity.
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.
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.
Reference
Pavanello
etal.
(2005)
Pavanello
etal.
(2006)
Perera et
al. (2005b)
O
73
P
O
1
-------
r>
C
§
Q
I
O O
I-1
i
Q.
I
§
Endpoint
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
Test system
Human, WBCs
Human, WBCs
Mouse, /ocZtransgenic
(Muta™Mouse)
Mouse, (Ahfh, Ahr*'~,
Ahr1')
Mouse, C57BL/6J
Cypla !(+/-) and Cypla 1
Test conditions
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.
Coke oven workers were exposed to PAHs
and benzo[a]pyrene-WBC DNA analyzed
by HPLC-fluorescence detection for BPDE-
DNA adducts.
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.
Gavage; sacrificed 24 hrs posttreatment.
Single i.p. injection; sacrificed 24 hrs
posttreatment; liver DNA analyzed by
[32P]-postlabeling assay.
Results
+
+
+
Dose
<0. 15, 0.15-4,
or >4 u.g/m3 of
benzo[a]pyren
e
0.14u.g/m3
25, 50, and
75 mg/kg-day
100 mg/kg
500 mg/kg
Comment
PAH exposure, CYP1A1 status and
smoking significantly affected
DNA adduct levels, i.e.,
CYPlAl(*l/*2 or *2/4/*2a) >
CYP1A1*1/*1; occupational >
environmental exposure;
smokers > non-smokers; adducts
increased with dose and duration
of smoking.
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
non-smokers; no correlation with
air benzo[a]pyrene levels and
adduct levels.
Highest adduct levels observed in
liver, followed by glandular
stomach, small intestine, and
bone marrow.
No induction of CYP in Nnf'', but
all alleles positive for adduct
formation.
BPDE-DNA adduct levels fourfold
higher in Cyplal(-/-) mice than
Cypla '!(+/-) mice.
Reference
Roias et al.
(2000)
Mensing et
al. (2005)
Lemieuxet
al. (2011)
Sagredo et
al. (2006)
Uno et al.
(2001)
o
-------
r>
C
§
Q
I
O O
NJ 2.
•^
i
Q.
I
§
Endpoint
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
Test system
Mouse, B6C3F!
Mouse, BALB/c
Mouse, BALB/cAnN
(BALB), CBA/JN (CBA);
[32P]-postlabeling assay
Mouse, BALB/c, skin
Mouse, Swiss, epidermal
and dermal skin
Rat, CD, peripheral blood
lymphocytes, lungs, and
liver
Test conditions
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.
Single i.p. injection; sacrificed 12 hrs
postinjection; liver and forestomach
collected; DNA binding of [3H]-benzo[a]-
pyrene analyzed by scintillation counting.
Animals dosed i.p. with or without 24 hr
pretreatment with TCDD.
Four doses of benzo[a]pyrene topically
applied to the shaved backs of animals at
0, 6, 30, and 54 hrs; sacrificed 1 day after
last treatment; DNA analyzed by
[32P]-postlabeling assay.
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.
Single i.p. injection; sacrificed 3 d
posttreatment; DNA analyzed by Nuclease
Pi-enhanced [32P]-postlabeling assay.
Results
+
+
+
+
+
+
Dose
5 ppm (32 wks)
and 100 ppm
(4 wks)
140 u.Ci/100 g
body weight
50 and
200 mg/kg
4 x 1.2 u.mol/
animal
250 nmol in
150 u.L acetone
2.5 mg/animal
Comment
Linear dose-response in 4-wk
study; the 5 ppm groups showed
a plateau after 4 wks of feeding.
Liver DNA had threefold higher
binding of benzo[a]pyrene than
that of forestomach.
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.
Five adducts spots detected.
Both cells positive for
benzo[a]pyrene adducts;
epidermis > dermis; adducts
persisted up to 7 d with a gradual
decline in levels.
BPDE-dG as major adducts and
several minor adducts detected
in all tissues.
Reference
Gulp et al.
(2000)
Gangar et
al. (2006)
Wu et al.
(2008)
Reddy et al.
(1984)
Oueslati et
al. (1992)
Ross et al.
(1991)
-------
r>
C
§
Q
I
1
S|
1
Q.
Endpoint
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
BPDE-DNA
adducts
Test system
Rat, Sprague-Dawley, liver
Rat, Lewis, lung and liver
Rat, F344;
[32P]-postlabeling assay
Rat, Wistar; liver and
peripheral blood
lymphocyte adducts
Test conditions
Single i.p. injection followed by sacrifice at
4 hrs posttreatment; liver DNA isolated
and analyzed by [32P]-postlabeling assay.
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.
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.
Single dose by gavage; sacrificed 24 hrs
postdosing; peripheral blood lymphocytes
and liver DNA analyzed by
[32P]-postlabeling for BPDE-DNA adducts.
Results
+
+
+
+
Dose
100 mg/kg
10 mg/kg
0, 5, 50, or
100 mg/kg
0, 10, or
100 mg/kg
Comment
Two adduct spots detected.
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.
Adduct levels linear at low and
intermediate doses, nonlinear at
high dose.
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.
Reference
Reddyetal.
(1984)
Briede et
al. (2004)
Ramesh
and
Knuckles
(2006)
Willems et
al. (1991)
c
1
I
a
B
> 'A
I
§
n 9.
.
o
a
I
Co
o
73
P
O
1
-------
r>
C
§
Q
I
O O
*»
i
Q.
§
Endpoint
CAs
CAs
CAs
CAs
MN
Test system
Mouse, C57 (high AHH
inducible) and DBA (low
AHH inducible) strains;
11-d-old embryos; adult
bone marrows
Mouse, 1C3F1 hybrid
(101/ElxC31xEl)Fl;
CAs in bone marrow
Rat, Wistar; peripheral
blood lymphocytes
Hamster; bone marrow
Mouse, /ocZtransgenic
(Muta™Mouse)
Test conditions
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.
Single dose by gavage; sacrificed 30 hrs
postdosing; bone marrow from femur
isolated and analyzed for CAs.
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.
Single, i.p. injection of benzo[a]pyrene
dissolved in tricapryline; animals sacrificed
24 hrs post-exposure.
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.
Results
+
+
+
+
Dose
150 mg/kg
63 mg/kg
0, 10, 100, or
200 mg/kg
25, 50, or
100 mg/kg
25, 50, and
75 mg/kg-d
Comment
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
BaP-induced CAs and AHH
inducibility. No differences in
bone marrow mitotic index of
males of different strains
between control and treatment
groups.
Significant increase in CAs in
benzo[a]pyrene-treated animals
compared to controls.
No difference between control
and treatment groups at any dose
or at any sampling time observed.
Benzo[a]pyrene induced CAs at
50 mg/kg body weight only, with
negative responses at the low
and high dose.
Statistically significant, dose-
dependent increases in percent
of PCEs and NCEs at all doses.
Reference
Adler et al.
(1989)
Adler and
Ingwersen
(1989)
Willems et
al. (1991)
Bayer
(1978)
Lemieuxet
al. (2011)
O
1
-------
r>
C
§
Q
I
O O
on
i
Q.
I
§
Endpoint
MN
MN
MN
MN
MN
MN
MN
Test system
Mouse, CD-land BDF1;
bone marrow
Mouse, CD-land BDF1,
peripheral blood
reticulocytes
Mouse, ICR [Hsd: (ICR)Br]
Mouse, Swiss albino;
bone marrow
Mouse, Swiss; bone
marrow polychromatic
erythrocytes
Mouse, CD-I and MS/Ae
strains
Mouse, BDF1, bone
marrow
Test conditions
Dosed orally once, twice, or thrice at 24-hr
intervals; sacrificed 24 hrs after last
treatment.
Given single i.p injection; tail blood
collected at 24-hr intervals from 0 to
72 hrs.
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.
Given orally in corn oil; sacrificed 24 hrs
post-exposure.
Given by gavage and sacrificed 36 hrs
posttreatment.
i.p. and oral administration.
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.
Results
+
+
+
+
+
+
+
Dose
250, 500,
1,000, or
2,000 mg/kg
62.5, 125, 250,
or 500 mg/kg
150 mg/kg
75 mg/kg
75 mg/kg
62.5, 125, 250,
or 500 mg/kg
0, 25, 50, or
60 mg/kg
Comment
Significant increase at all doses;
no dose-response; double dosing
at 500 mg/kg dose gave best
response.
Maximum response seen at 48
hrs posttreatment.
All groups significantly higher
than controls for MN; fetal liver
more sensitive than any other
group.
Good dose-response by both
routes, strains; i.p. better than
oral; MS/Ae strain more sensitive
than CD-I strain.
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.
Reference
Shimada et
al. (1990)
Shimada et
al. (1992)
Harper et
al. (1989)
Koratkar et
al. (1993)
Rao and
Nandan
(1990)
Awogi and
Sato (1989)
Balansky et
al. (1994)
O
1
-------
r>
C
§
Q
I
1
o o
en 2.
1
Q.
51
^ i?
§
n 9.
Endpoint
MN
MN
MN
MN
MN
MN
MN
MN
Test system
Mouse, HRA/Skh hairless,
keratinocytes
Mouse, HOS:HR-1,
hairless; skin micronuclei
Mouse, HR-1 hairless, skin
(benzo[a]pyrene with
slight radiation)
Rat, Sprague-Dawley,
peripheral blood
reticulocytes
Rat, Sprague-Dawley,
pulmonary alveolar
macrophages
Rat, Sprague-Dawley,
bone marrow cells
Hamster; bone marrow
Fish (carp, rainbow trout,
clams); blood and
hemolymph
Test conditions
Single topical application.
Topical application once daily for 3 d;
sacrificed 24 hrs after last treatment.
Given single i.p injection; tail blood
collected at 24-hr intervals from 0 to
96 hrs.
Intratracheal instillation, once/day for 3 d.
Intratracheal instillation, once/day for 3 d.
Single, i.p. injection of benzo[a]pyrene
dissolved in tricaprylin; animals sacrificed
30 hours post-exposure.
Results
+
+
+
+
+
-
+
Dose
0.5, 5, 50, 100,
or
500 mg/mouse
0.4, 1, 2, or
4 mg
62.5, 125, 250,
500, or
1,000 mg/kg
25 mg/kg
25 mg/kg
100, 300, or
500 mg/kg
0.05, 0.25, 0.5,
orl ppm
Comment
Exposure to sunlight simulator to
evaluate photogenotoxicity and
chemical exposure.
Maximum response seen at 72
hrs posttreatment.
Reference
He and
Baker
(1991)
Nishikawa
etal.
(2005)
Hara et al.
(2007)
Shimada et
al. (1992)
De Flora et
al. (1991)
De Flora et
al. (1991)
Bayer
(1978)
Kim and
Hyun
(2006)
C
1
|
a
B
.
o
a
I
Co
o
73
P
O
1
-------
r>
C
§
Q
I
HI'
O O
-J
i
Q.
O
§
n 9.
Endpoint
DNA
strand
breaks
DNA
strand
breaks
DNA
strand
breaks
UDS
Test system
Rat, Sprague-Dawley;
comet assay
Aquatic organisms: carp
(Cyphnus carpio), rainbow
trout (Oncorhynchus
mykiss), and clams
(Spisula sachalinensis);
Comet assay
Rat, Brown Norway
Rat, F344
Test conditions
Instilled intratracheally 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.
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.
UDS determined after 5 and 18 hrs of a
single intragastric dosing.
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.
Results
+
+
_
_
Dose
Experiment #1:
3 mg of
benzo[a]pyren
e; Experiment
#2: dose-
response study
with 0.75, 1.5,
or 3 mg
benzo[a]pyren
e
0.05, 0.25, 0.5,
and 1 ppm
62.5 mg/kg
100 mg/kg
Comment
All time points showed significant
increase in SSBs (Experiment #1);
a dose-response in SSBs was
observed (Experiment #2).
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.
Negative at both time points.
Benzo[a]pyrene was negative at
both time points.
Reference
Garry et al.
12003a, b)
Kim and
Hyun
(2006)
Mullaart et
al. (1989)
Mirsalis et
al. (1982)
c
1
I
a
B
.
o
a
I
Co
o
73
P
O
1
-------
r>
C
§
Q
I
o o
00 3
I
§
Endpoint
UDS
UDS
UDS
UDS
SCEs
SCEs
SCEs
Test system
Mouse, HOS:HR-1
hairless; skin
Rat, Brown Norway; liver
Mouse, (CSHfxlOl)Fl
hybrid, germ cells
Mouse, early spermatid
Hamster; SCEs in bone
marrow
Hamster
Hamster; fetal liver
Test conditions
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.
Single intragastric injection; sacrificed at
5 and 18 hrs post-injection.
i.p. injection of benzo[a]pyrene;
[3H]-thymidine injection later.
i.p. injection.
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.
Animals implanted subcutaneously 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.
i.p. injection to pregnant animals on
GDs 11, 13, or 15; fetal liver SCEs were
analyzed.
Results
+
-
-
+
+
+
Dose
0, 0.25, 0.5,
and 1% (w/v) in
acetone
62.5 mg/kg
0.3 mL
250-500 mg/kg
450 mg/kg
50 or
100 mg/kg
50 and
125 mg/kg
Comment
UDS index showed a dose-
dependent increase up to 0.5%
benzo[a]pyrene dose and then
plateaued.
Benzo[a]pyrene was negative at
both time points.
Concentration not specified.
Reviewed by Sotomayor and Sega
(2000).
Significant increase in metaphase
SCEs in benzo[a]pyrene-treated
animals compared to vehicle-
treated controls.
SCEs increased with low dose of
phorone significantly.
Produced doubling of SCE
frequency.
Reference
Mori et al.
(1999)
Mullaart et
al. (1989)
Sega (1979)
Sega (1982)
Roszinsky-
Kocher et
al. (1979)
Bayer et al.
(1981)
Pereira et
al. (1982)
O
1
-------
r>
C
§
Q
I
o o
ID 2.
•^
i
Q.
51
^ i?
§
n 9.
H 5'
m ^
O
P
P
Endpoint
SCEs
SCEs
SCEs
SCEs
Mutation
Mutation
Mutation
Mutation
Test system
Hamster; bone marrow
Mouse, DBA/2 and
C57BL/6, bone marrow
cells
Mouse, DBA/2 and
C57BL/6, splenic
lymphocytes
Rat, Wistar; peripheral
blood lymphocytes
Drosophila melanogaster,
sex-linked recessive lethal
test
D. melanogaster, sex-
linked recessive lethal
test
D. melanogaster, Berlin-K
and Oregon-K strains; sex-
linked recessive lethal
test
D. melanogaster, sex-
linked recessive lethal
test
Test conditions
Not available
Two intragastric injections given; mice
implanted with BrdU tablets, sacrificed on
d 5, SCEs estimated.
Two intragastric injections given; mice
killed on 5th day and cells cultured for
48hrswith BrdU.
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.
Base males exposed to benzo[a]pyrene
were mated with virgin females of Berlin K
or mei-9L1 strains.
Adult Berlin males treated orally with
benzo[a]pyrene.
Benzo[a]pyrene dissolved in special fat
and injected into the abdomen of flies.
Male Berlin K larvae treated with
benzo[a]pyrene for 9-11 d.
Results
+
+
+
+
±
+
+
Dose
2.5, 25, 40, 50,
75, or
100 mg/kg
10 or
100 mg/kg
10 or
100 mg/kg
0, 10, 100, or
200 mg/kg
10 mM
5 or 7.5 mM
2 or 5 mM
0.1-4 mM
Comment
Frequency of SCEs increased
>40 mg/kg body weight.
SCEs and BaP-DNA adducts in the
order of C57BI/6 (AHH-inducible)
< DBA/2 (AHH-noninducible).
SCEs and BaP-DNA adducts in the
order of C57BI/6 (AHH-inducible)
< DBA/2 (AHH-noninducible).
Linear dose-response at any
sampling time; however,
significant at the highest dose
only; no interaction between
dose and sampling time.
Data inconclusive due to low
fertility rates of mei-9L1 females.
Low mutagenic activity.
Negative at both doses.
Threefold enhancement in lethals
in treated versus controls.
Reference
Bayer
(1978)
Wielgosz et
al. (1991)
Wielgosz et
al. (1991)
Willemset
al. (1991)
Vogel et al.
(1983)
Vogel et al.
(1983)
Ziilstra and
Vogel
(1984)
Vogel et al.
(1983)
1
-------
r>
C
§
Q
I
HI'
I-* O
O 2.
•^
i
Q.
O
§
n 9.
Endpoint
Mutation
Mutation
Cell trans-
formation
Test system
D. melanogaster, Canton-
S (WT) males, FM6
(homozygous for an X-
chromosome) females;
sex-linked recessive lethal
test
D. melanogaster; somatic
mutation, eye color
mosaicism
Hamster, LVG:LAK strain
(virus free);
transplacental host-
mediated assay
Test conditions
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.
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.
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.
Results
Dose
250 or 500
ppm
1, 2, or 3 mM
3 mg/100 g
body weight
Comment
Authors report incomplete
dissolution of benzo[a]pyrene in
DMSO as a possible cause of
negative result.
Benzo[a]pyrene was effective as a
mutagen; no dose-response
observed.
Reference
Valencia
and
Houtchens
(1981)
Fahmy and
Fahmy
(1980)
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.
c
1
I
a
B
.
o
a
I
Co
o
73
P
O
1
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1 D.5.2. Tumor Promotion and Progression
2 Cytotoxicity and Inflammatory Response
3 The cytotoxicity of benzo[a]pyrene metabolites may contribute to tumor promotion via
4 inflammatory responses leading to cell proliferation [Burdick et al.. 2003). Benzo[a]pyrene is
5 metabolized to o-quinones, which are cytotoxic, and can generate ROS [Boltonetal., 2000: Penning
6 etal., 1999]. Benzo[a]pyrene o-quinones reduce the viability and survival of rat and human
7 hepatoma cells [Flowers-Geary etal., 1996: Flowers-Geary etal., 1993]. Cytotoxicity was also
8 induced by benzo[a]pyrene and BPDE in a human prostate carcinoma cell line [Nwagbaraetal..
9 2007]. Inflammatory responses to cytotoxicity may contribute to the tumor promotion process.
10 For example, benzo[a]pyrene quinones (1,6-, 3,6-, and 6,12-benzo[a]pyrene-quinone] generated
11 ROS and increased cell proliferation by enhancing the epidermal growth factor receptor pathway in
12 cultured breast epithelial cells [Burdick etal.. 2003].
13 Several studies have demonstrated that exposure to benzo[a]pyrene increases the
14 production of inflammatory cytokines, which may contribute to cancer progression. Garcon et al.
15 [2001a] and Garcon etal. [2001b] exposed Sprague-Dawley rats by inhalation to benzo[a]pyrene
16 with or without ferrous oxide (Fe20s] particles. They found that benzo[a]pyrene alone or in
17 combination with Fe20s particles elicited mRNA and protein synthesis of the inflammatory
18 cytokine, IL-1. Tamaki etal. [2004] also demonstrated abenzo[a]pyrene-induced increase in IL-1
19 expression in a human fibroblast-like synoviocyte cell line [MH7A]. Benzo[a]pyrene increases the
20 expression of the mRNA for CCL1, an inflammatory chemokine, in human macrophages [N'Diaye et
21 al., 2006]. The benzo[a]pyrene-induced increase in CCL1 mRNA was inhibited by the potent AhR
22 antagonist, 3'-methoxy-4'-nitroflavone.
23 AhR-mediated Effects
24 The promotional effects of benzo[a]pyrene may also be related to AhR affinity and the
25 upregulation of genes related to biotransformation (i.e., induction of CYP1A1], growth, and
26 differentiation [Bostrometal., 2002]. Figure D-3 illustrates the function of the AhR and depicts the
27 genes regulated by this receptor as belonging to two major functional groups (i.e., induction of
28 metabolism or regulation cell differentiation and proliferation]. PAHs bind to the cytosolic AhR in
29 complex with heat shock protein 90 [Hsp90]. The ligand-bound receptor is then transported to
30 nucleus in complex with the Ah receptor nuclear translocator. The AhR complex interacts with the
31 Ah responsive elements of the DNA to increase the transcription of proteins associated with
32 induction of metabolism and regulation of cell differentiation and proliferation.
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2
3
4
5
PAH
4 AUD I HsnQCll
AHR
Hsp90
Hsp90
Hsp90
Hsp90
ARNT
Enhanced
specific
mRNA
production
-••
X.
|AHR
ARNT
AHREDNA
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; Hsp90 = heat shock
protein 90
Source: Okeyetal. (1994).
Figure D-3. Interaction of PAHs with the AhR.
7 Binding to the AhR induces enzymes that increase the formation of reactive metabolites,
8 resulting in DNA binding and, eventually, tumor initiation. In addition, with persistent exposure,
9 the ligand-activated AhR triggers epithelial hyperplasia, which provides the second step leading
10 from tumor initiation to promotion and progression [Nebertetal.. 1993). Ma and Lu [2007]
11 reviewed several studies of benzo[a]pyrene toxicity and tumorigenicity in mouse strains with high
12 and low affinity AhRs. Disparities were observed in the tumor pattern and toxicity of
13 Ah-responsive (+/+ and +/-) and Ah-nonresponsive (-/-) mice. Ah-responsive mice were more
14 susceptible to toxicity and tumorigenicity in proximal target tissues such as the liver, lung, and skin.
15 For example, Shimizuetal. [2000] reported that AhR knock-out mice (-/-], treated with
16 benzo[a]pyrene by subcutaneous injection or dermal painting, did not develop skin cancers at the
17 treatment site, while AhR-responsive (+/+] or heterozygous (+/-] mice developed tumors within
18 18-25 weeks after treatment. Benzo[a]pyrene treatment increased CYP1A1 expression in the skin
19 and liver of AhR-positive mice (+/- or +/+], but CYP1A1 expression was not altered by
20 benzo[a]pyrene treatment in AhR knock-out mice (-/-]. Talaskaetal. [2006] also showed that
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1 benzo[a]pyrene adduct levels in skin were reduced by 50% in CYP1A2 knock-out mice and by 90%
2 in AhR knock-out mice compared with WT C57B16/J mice following a single dermal application of
3 33 mg/kg benzo[a]pyrene for 24 hours. MaandLu [2007] further noted that Ah-nonresponsive
4 mice were at greater risk of toxicity and tumorigenicity in remote organs, distant from the site of
5 exposure (i.e., bone marrow). As an example, Uno etal. [2006] showed thatbenzo[a]pyrene
6 (125 mg/kg-day, orally for 18 days] caused marked wasting, immunosuppression, and bone
7 marrow hypocellularity in CYP1A1 knock-out mice, but not in WT mice.
8 Some studies have demonstrated the formation of DNA adducts in the liver of AhR knock-
9 out mice following i.p. or oral exposure to benzo[a]pyrene [Sagredo etal., 2006: Uno etal., 2006:
10 Kondraganti etal.. 2003]. These findings suggest that the re may be alternative (i.e., no n-AhR
11 mediated] mechanisms of benzo[a]pyrene activation in the mouse liver. Sagredo etal. [2006]
12 studied the relationship between the AhR genotype and GYP metabolism in different organs of the
13 mouse. AhR+/+, +/-, and -/- mice were treated once with 100 mg/kg benzo[a]pyrene by gavage.
14 CYP1A1, CYP1B1, and AhR expression was evaluated in the lung, liver, spleen, kidney, heart, and
15 blood, via real-time or reverse transcriptase PCR, 24 hours after treatment CYP1A1 RNA was
16 increased in the lung and liver and CYP1B1 RNA was increased in the lung following
17 benzo[a]pyrene treatment in AhR+/+ and +/- mice (generally higher in heterozygotes].
18 Benzo[a]pyrene treatment did not induce CYP1A1 or CYP1B1 enzymes in AhR-/- mice. The
19 expression of CYP1A1 RNA, as standardized to (3-actin expression, was generally about 40 times
20 that of CYP1B1. The concentration of benzo[a]pyrene metabolites and the levels of DNA and
21 protein adducts were increased in mice lacking the AhR, suggesting that there may be an
22 AhR-independent pathway for benzo[a]pyrene metabolism and activation. The high levels of
23 benzo[a]pyrene DNA adducts in organs other than the liver of AhR-/- mice may be the result of slow
24 detoxification of benzo[a]pyrene in the liver, allowing high concentrations of the parent compound
25 to reach distant tissues.
26 Uno etal. [2006] also demonstrated a paradoxical increase in liver DNA adducts in AhR
27 knock-out mice following oral exposure to benzo[a]pyrene. WT C57BL/6 mice and several knock-
28 out mouse strains (CYP1A2-/- and GYP IB I-/- single knock-out, CYP1A1/1B1-/- and CYP1A2/1B1-/-
29 double knock-out] were studied. Benzo[a]pyrene was administered in the feed at 1.25,12.5, or
30 125 mg/kg for 18 days (this dose is well-tolerated by WT C57BL/6 mice for 1 year, but lethal within
31 30 days to the CYP1A1-/- mice]. Steady-state blood levels of benzo[a]pyrene, reached within 5 days
32 of treatment, were -25 times higher in CYP1A1-/- and -75 times higher in CYP1A1/1B1-/- than in
33 WT mice, while clearance was similar to WT mice in the other knock-out mouse strains. DNA
34 adduct levels, measured by [32P]-postlabeling in liver, spleen, and bone marrow, were highest in the
35 CYP1A1-/- mice at the two higher doses, and in the CYP1A1/1B1-/- mice at the mid dose only.
36 Adduct patterns, as revealed by 2-dimensional chromatography, differed substantially between
37 organs in the various knock-out types.
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1 AhR signaling may play a role in cytogenetic damage caused by benzo[a]pyrene [Dertinger
2 etal., 2001: Dertinger etal., 2000]. The in vivo formation of MN in peripheral blood reticulocytes of
3 C57B1/6J mice induced by a single i.p. injection of benzo[a]pyrene (150 mg/kg) was eliminated by
4 prior treatment with the potent AhR antagonist 3'-methoxy-4'-nitroflavone. This antagonist also
5 protected AhR-null allele mice from benzo[a]pyrene-induced increases in MN formation, suggesting
6 that 3'-methoxy-4'-nitroflavone may also act through a mechanism independent of the AhR
7 [Dertinger etal.. 2000].
8 Several in vitro studies have suggested that the AhR plays a role in the disruption of cell
9 cycle control, possibly leading to cell proliferation and tumor promotion following exposure to
10 benzo[a]pyrene [Andrysik etal.. 2007: Chung etal.. 2007: Chen etal.. 2003]. Chung etal. [2007]
11 showed that benzo[a]pyrene-induced cytotoxicity and apoptosis in mouse hepatoma [Hepalclc7]
12 cells occurred through a p53 and caspase-dependent process requiring the AhR. An accumulation
13 of cells in the S-phase of the cell cycle (i.e., DNA synthesis and replication] was also observed,
14 suggesting that this process may be related to cell proliferation. Chen etal. [2003] also
15 demonstrated the importance of the AhR in benzo[a]pyrene-7,8-dihydrodiol- and BPDE-induced
16 apoptosis in human HepG2 cells. Both the dihydrodiol and BPDE affected Bcl2 (a member of a
17 family of apoptosis suppressors] and activated caspase and p3 8 mitogen-activated protein (MAP]
18 kinases, both enzymes that promote apoptosis. When the experiments were conducted in a cell line
19 that does not contain Ah receptor nuclear translocator (see Figure D-3], the dihydrodiol was not
20 able to initiate apoptotic event sequences, indicating that activation to BPDE by CYP1A1 was
21 required. BPDE did not induce apoptosis-related events in a p38-defective cell line, illustrating the
22 importance of MAP kinases in this process. In rat liver epithelial cells (WB-F344 cells], in vitro
23 exposure to benzo[a]pyrene resulted in apoptosis, a decrease in cell number, an increase in the
24 percentage of cells in S-phase (comparable to a proliferating population of WB-F334 cells], and
25 increased expression of cell cycle proteins (e.g., cyclin A] (Andrysik et al., 2007]. Benzo[a]pyrene-
26 induced apoptosis was attenuated in cells transfected with a dominant-negative mutation of the
27 AhR.
28 Inhibition of gap junctional intercellular communication (GTIC]
29 Gap junctions are channels between cells that allow substances of a molecular weight up to
30 roughly 1 kDa to pass from one cell to the other. This process of metabolic cooperation is crucial
31 for differentiation, proliferation, apoptosis, and cell death and consequently for the two epigenetic
32 steps of tumor formation, promotion, and progression. Chronic exposure to many toxicants results
33 in down-regulation of gap junctions. For tumor promoters, such as TPA or TCDD, inhibition of
34 intercellular communication is correlated with their promoting potency (Sharovskayaetal., 2006:
35 Yamasaki. 1990].
36 Blahaetal. (2002] surveyed the potency of 35 PAHs, including benzo[a]pyrene, to inhibit
37 GJIC. The scrape loading/dye transfer assay was employed using a rat liver epithelial cell line that
38 was incubated in vitro for 15, 30, or 60 minutes with 50 [M benzo[a]pyrene. After incubation, cells
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1 were washed, and then a line was scraped through the cells with a surgical blade. Cells were
2 exposed to the fluorescent dye lucifer yellow for 4 minutes and then fixed with formalin. Spread of
3 the dye from the scrape line into cells remote from the scrape was estimated under a fluorescence
4 microscope. Benzo[a]pyrene reduced spread of the dye after 30 minutes of exposure
5 (approximately 50% of control). Recovery of GJIC was observed 60 minutes after exposure.
6 Sharovskaya et al. [2006] studied the effects of carcinogenic and noncarcinogenic PAHs on
7 GJIC in HepG2 cells. Individual carcinogenic PAHs inhibited GJIC in a temporary fashion (70-100%
8 within 24 hours), but removal of the PAH from culture reversed the effect Noncarcinogenic PAHs
9 had very little effect on GJIC. Benzo[a]pyrene at 20 [M inhibited GJIC completely within 24 hours,
10 while its noncarcinogenic homolog, benzo[e]pyrene, produced <20% inhibition. The effect was not
11 AhR-dependent, because benzo[a]pyrene inhibited GJIC in HepG2 cells to the same extent as in
12 hepatoma G27 cells, which express neither CYP1A1 nor AhR. The authors concluded that the
13 effects of benzo[a]pyrene and benzo[e]pyrene on GJIC were direct (i.e., not caused by metabolites).
14
15 D.5.3. Benzo[a]pyrene Transcriptomic Microarray Analysis
16 The objective of this analysis was to use transcriptomic microarray analysis to help inform
17 the cancer mode of action for benzo[a]pyrene. A systematic review and meta-analysis approach
18 was used to (1) identify studies, (2) analyze the raw data, (3) assess data quality, and (4) combine
19 evidence from multiple studies to identify genes that were reproducibly active across all of the
20 studies.
21 The Gene Expression Omnibus and Array Express microarray repositories were searched
22 for studies thatusedbenzo[a]pyrene as a test chemical and raw data were available. The search
23 terms used and the number of studies retrieved are listed in Table D-34. Many of the search terms
24 included terms for specific polycyclic aromatic hydrocarbon mixtures (PAH), as benzo[a]pyrene is
25 commonly used as a reference chemical in PAH mixture studies, to ensure the available and usable
26 benzo[a]pyrene microarray data were identified.
27 Table D-34. Search terms and the number of studies retrieved from the gene
28 expression omnibus and array express microarray repositories
Search Term
Coal tar
Polycyclic aromatic hydrocarbons
B[a]P
Diesel
Smoke NOT cigarette
Benzo[a]pyrene
Fuel oil
Cigarette smoke
Tobacco smoke
Number of Microarray Studies Retrieved
2
13
52
11
16
53
1
63
16
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Supplem en tal Inform ation —Benzo[a]pyren e
1 Forty responsive gene expression datasets were identified, representing 26 peer-reviewed
2 publications. These datasets were further culled for analysis by focusing on publicly available
3 results and species and organs represented by more than one available dataset on the same
4 microarray platform. Crossing microarray platforms and species boundaries adds significant
5 uncertainty to the interpretation with respect to comparisons of the probes being measured, how
6 those different probes align to the genome and are mapped to specific genes, and creates an open
7 question regarding the discovery and mapping of orthologous genes across species. Thus, the
8 analysis included two studies that focused on mouse in vivo transcriptomic studies of the liver
9 (Gene Expression Omnibus accessions: GSE24907 and GSE18789).
10 The first study [Malik etal.. 2012). GSE24907, exposed five male Muta mice (a LacZ
11 transgenic mouse line) per group to 25, 50, 75 mg/kg benzo[a]pyrene or olive oil vehicle for 28
12 days by oral gavage. The second study[Yauk et al.. 2 011). GSE18789, exposed 27-30 day old male
13 B6C3Fi mice to 150 mg/kg benzo[a]pyrene by oral gavage for 3 days and sacrificed 4 hours or 24
14 hours after the final dose. Both studies were subjected to study quality evaluation by the
15 Systematic Omics Analysis Review (SOAR) tool.
16 SOAR was developed to assist in the quick and transparent identification of studies that are
17 suitable for hazard assessment development. SOAR consists of a series of objective questions that
18 examine the overall study quality of a transcriptomic microarray study. SOAR combines questions
19 from the Toxicological Reliability Assessment (ToxR) Tool, the Minimum Information About a
20 Microarray Experiment (MIAME) standard, and the Checklist for Exchange and Interpretation of
21 Data from a Toxicology Study. Both studies were determined to be relevant and suitable for hazard
22 assessment development using SOAR.
23 Data Analysis Overview
24 Raw data for both studies were obtained from the Gene Expression Omnibus
25 (http://www.ncbi.nlm.nih.gov/geo/) using the GEOquery package (Davis and Meltzer. 2007) in
26 Bioconductor (a bioinformatics software repository for packages that may be used in the R
27 statistical environment). Each study was pre-processed, normalized, subjected to quality control
28 analysis (see below) and analyzed independently to determine the number of active genes using a
29 fold-change cut-off, and then a subsequent p-value cut-off.
30 Pre-processing involves the acquisition of data, background subtraction (not performed
31 here), and synthesis of gene expression data across multiple probesets (only for Affymetrix data,
32 and only if analysis is performed on a probeset basis). Normalization is the mathematical
33 adjustment of data to correct. Data were normalized using fastlo within-groups to control for
34 technical variance (Eckel etal., 2005).
35 The raw microarray data from both studies were analyzed for quality using Principal
36 Components Analysis (PCA) and boxplot analysis. Principal Components Analysis is commonly
37 used for cluster analysis based on the variance within the dataset The PCA algorithm (in this case
38 we used singular value decomposition) can be thought of projecting the data into a
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1 multidimensional space, and drawing an axis through the data cloud to explain the largest amount
2 of variance. The next axis is drawn through the cloud to explain the next largest amount of variance
3 while also being orthogonal to the first axis (e.g., the Y-axis is orthogonal to the X-axis in a Cartesian
4 plane). The idea is that samples will naturally cluster in a way that is easily visualized in a simple 2-
5 dimensional plot, where the axis representing the largest variance is the X-axis. For quality control
6 purposes, observation of samples from the same biological grouping (e.g., all of the controls, or all
7 of the samples treated the same way for the same duration) clustered in the X-Y plane is preferable.
8 The samples in GSE24907 separated mostly by group when the normalized data were visualized by
9 PCA. The boxplots exhibited a somewhat compressed interquartile range. Overall, the data were
10 deemed to be of high enough quality to continue analysis, although the compressed interquartile
11 range could manifest data compression issues which may decrease the overall statistical power
12 The normalized samples in GSE18709 also separated mostly by group; however, one
13 benzo[a]pyrene treated 24 hour sample and one 4 hour control sample clustered distantly from the
14 rest of their groups. This raises concerns that there remains a significant amount of variance in the
15 data that the normalization could not overcome. This variance may decrease the overall statistical
16 power of the meta-analysis. The boxplots of normalized data for this study were more compressed
17 than that for GSE24907.
18 Data were analyzed using limma and an empirical Bayes moderated t-test (Smyth, 2004).
19 Following analysis, active genes were identified. A gene was considered active if it exhibited a 1.5
20 fold-change andap-value < 0.1 in at least one condition or group (e.g., time-point or dose).
21 A data mining/pathway analysis approach was undertaken using the GeneGo Metacore
22 software and using the active gene lists. This approach compares the pathways identified from
23 bioinformatics analyses of the active gene lists from both studies. The active gene lists from both
24 studies were analyzed using the GeneGo Metacore software. The data were mined to identify
25 GeneGo Metacore pathways that represent a large number of genes from both datasets. Gene
26 expression data were overlaid only for those conditions where the gene was at least 1.5 fold up- or
27 down-regulated. The GeneGo pathways were analyzed for relevance to the hypothesized mode of
28 action for benzo [a]pyrene, and for pathways that may illustrate new modes of action. This analysis
29 is strictly an exploratory pathway analysis to help inform the interpretation of the transcriptomics
30 data.
31 The pathway analysis is a powerful method for comparing study results and identifying
32 consistency than a direct comparison of the active gene list For instance, differentially expressed
33 gene (DEC) lists reported in the peer-reviewed literature are not reproducible across similar
34 studies (Shi etal.. 2008: Chuangetal.. 2007: Ein-Dor etal.. 2005: Lossos etal.. 2004: Fortuneletal..
35 2003). In one example, three different studies aimed at identifying genes that confer "sternness"
36 (i.e., genes which are responsible for conferring stem-cell like capabilities) each yielded 230, 283,
37 and 385 active genes, yet the overlap between them was only one gene (Fortuneletal.. 2003). This
38 demonstrates that the use of simple Venn diagrams to show the overlap of genes across studies are
This document is a draft for review purposes only and does not constitute Agency policy.
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1 not as informative as pathway analysis, and are less likely to provide support to potential MOA
2 hypotheses.
3
4
5
6
7
8
9
10
11
12
13
14
Three candidate pathways were identified. These are:
• AhR signaling
• DNA damage regulation of the Gl/S phase transition
• Nrf2 regulation of oxidative stress
Gene differential expression is represented on the pathway map as a "thermometer" next to
the protein symbol. Upregulation is symbolized by an upward pointing thermometer, where the
length of the red bar represents a relative Iog2 fold-change. Downregulation is symbolized by a
downward pointing thermometer, where the length of the blue bar represents a relative Iog2 fold-
change. A red line connecting proteins represents inhibition. A green line connecting proteins
represents activation. A symbol legend accompanies this report
Table D-35. Mapping of group numbers to time/dose groups
Number Under Thermometer
In Figures D-4-D-6
2
3
4
5
6
Dose
150 mg/kg
150 mg/kg
75 mg/kg
50 mg/kg
25 mg/kg
Time Point
3 d exposure (sacrificed 4 hr after
final dose)
3 d exposure (sacrificed 24 hr after
final dose)
28 d exposure
28 d exposure
28 d exposure
Reference
Yauketal. (2011)
Yauketal. (2011)
Malik etal. (2012)
Malik etal. (2012)
Malik etal. (2012)
15
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2
3
4
5
Polychjorinated Polycyor{nated PolyctJIotjnated
Dibehzo^fans B!pi)e;»yls DibeVorfoxins Blfcs^jgfnents
BAFF
(TNFSF13B)
TGM3 CathepsinD hypoxia induced
HIF1 activation
Figure D-4. Aryl hydrocarbon receptor 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-35). For
instance, NRF2 is upregulated in the 25 mg/kg.
6 Aryl Hydrocarbon receptor signaling
7 The AhR regulates the transcription of several genes, including xenobiotic metabolism
8 genes (Figure D-4). It appears that benzo[a]pyrene is activating the AhR in these studies based on
9 the expression of many of its transcriptional targets. Relevant to further analysis and investigating
10 the mode of action, the c-Myc gene is upregulated at 4 and 24 hours in the time-course and at the 50
11 mg/kg dose in the dose-response, while Nrf2 is upregulated at the 4 hour time-point and at the 25
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1
2
3
4
5
7
8
9
10
11
12
13
14
15
16
17
mg/kg and 75 mg/kg doses. c-Myc has been shown to be upregulated following exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin, and a putative dioxin response element has been detected in
the c-Myc promoter [Dere etal.. 2011: Kimetal.. 2000). The AhR has been demonstrated to bind
and regulate the Nrf2 promoter fDere etal.. 2011: Lo etal.. 2011: Nair etal.. 20081.
DK^ l*DK4 Cvelirffl)
Regulation o(G1/S
•ansrtion {parti)
Sister chroma1
I
I Brcal as a
transcription
regulator
Transition and termination of DNA replicalion
&
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 thatpSS is activated.
DNA Damage Signaling
The strong upregulation of p21 andMDM2 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 hour and 75 mg/kg, further suggesting that that p53 may initially be
upregulated at times prior to 4 hour 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
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1 with the upregulation of Cyclin D and PCNA at 75 mg/kg (among other conditions), this suggests a
2 pro-mitotic shift may be occurring which could lead to cellular proliferation in the liver in the mice
3 exposed to 75 mg/kg per day.
4 Nr/2 Signaling
5 Nrf2 transcription may be upregulated by benzo[a]pyrene through activation of the AhR
6 (Figure D-4). The Nrf2 protein heterodimerizes with the MafF protein (Surhetal., 2008: Marini et
7 al., 2002: Kimetal., 2000] to regulate the transcription of Phase II metabolism and anti-oxidative
8 enzymes (Figure D-6). Activated p53 competes with Nrf2 anti-oxidant signaling, perhaps to ensure
9 a large oxidative stress response is present in the cell to promote the induction of apoptosis
10 (Faraonio etal.. 2006). Upregulation of Cul3 at 4 hours and the 75 mg/kg dose in concert with the
11 upregulation of ubiquitin at the same time and dose suggests that repression of Nrf2 activity may
12 occur. This would support the p53-mediated pro-oxidant hypothesis, which is further substaniated
13 by the lack of upregulation of anti-oxidant genes at 75 mg/kg, with the exception of GCL cat.
14
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DJ-1 stabilizes NRF2 protein
by promoting NRF2 dissociation
from KEAP1 and thus escaping
proteasomal degradation during
oxidative stress
PI3K cat class IA 2.7.1.153
Ptdlns(3,4,5)P3
Fyn and GSK3 beta kinases-mediated
phosphorylation regulates NRF2 interaction
with CRM1 and its nuclear export
UGT1A1 GSTM3 GSTP1 GSTA3 GST
SOD1 PRDX1 TXNR
Anti-oxidative enzymes
Thioredoxin GCL can GCL reg
Phase II detoxifying enzymes
Scavenging of Reactive
oxygen species
Protection of the cell from reactive oxygen
species and the products of peroxidation
1
2 Figure D-6. Nrf2 pathway. Nrf2 is upregulated 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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1 The transcriptomics data support a potential mutagenic and cellular proliferation mode of
2 action. The transcriptomics data support the hypothesis that DNA damage is occurring at 4 hours
3 following three daily doses of 150 mg/kg-day of benzo[a]pyrene and 75 mg/kg-day for 28 days.
4 This is supported by the transcriptional activation of p53 target genes, including p21 and MDM2.
5 The transcriptional data further suggest that p53 signaling may be waning under these conditions,
6 as ubiquitin and MDM2 are both upregulated, and work together to degrade p53. Furthermore, the
7 transcriptional upregulation of Cyclin D in the 75 mg/kg-day exposure may result in enough Cyclin
8 D protein to overcome the p21 inhibitory competition for CDK4, allowing for Gl/S phase transition
9 to occur. In addition, the upregulation of PCNA in the 75 mg/kg-day exposure group together with
10 upregulation of ubiquitin further supports the argument that cells are moving towards a more Gl/S
11 phase transition friendly environment Translesion synthesis (i.e., a DNA repair/bypass
12 mechanism, whereby DNA adducts are allowed to remain in newly synthesized DNA, so as to allow
13 the cell to continue with DNA synthesis and complete the cell cycle) by ubiquitinated PCNA may
14 favor mutagenesis if the Gl/S phase transition occurs by allowing DNA adducts to persist in
15 daughter cells.
16 There are a number of areas of uncertainty within the transcriptomics data that require
17 additional research. For instance, transcriptomics data only measure changes in gene expression;
18 these studies did not monitor changes in protein or metabolite expression, which would be more
19 indicative of an actual cellular state change. Inferences of protein activation and changes in protein
20 activity and cellular signaling are made based on the transcriptomics data. Further research is
21 required at the molecular level to demonstrate that the cellular signaling events being inferred are
22 actually taking place, and that these events result in phenotypic changes, consistent with the overall
23 mode of action. The studies also have inherent uncertainty with respect to extrapolation from short
24 term, high dose studies to low dose exposures across a lifetime. In addition, this work uses a
25 hypothesized MOA in the liver to support an overall MOA.
26
27
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i APPENDIX E. DOSE-RESPONSE MODELING FOR
2 THE DERIVATION OF REFERENCE VALUES FOR
3 EFFECTS OTHER THAN CANCER AND THE
4 DERIVATION OF CANCER RISK ESTIMATES
5 This appendix provides technical detail on dose-response evaluation and determination of
6 points of departure (POD) for relevant toxicological endpoints, organized by risk value (reference
7 value or cancer risk value). Except where other software is noted, all endpoints were modeled
8 using the U.S. EPA's Benchmark Dose Software [BMDS; (U.S. EPA. 2012a): version 2.0 or later]. The
9 preambles for the cancer and non-cancer parts below describe the practices used in evaluating the
10 model fit and selecting the appropriate model for determining the POD, as outlined in the
11 Benchmark Dose Technical Guidance (U.S. EPA. 2012b).
12 E.I. NON-CANCER ENDPOINTS
13 E.I.I. Reference Dose (RfD)
14 Evaluation of Model Fit
15 For each dichotomous endpoint, BMDS dichotomous models were fitted to the data using
16 the maximum likelihood method. For the log-logistic and dichotomous Hill models, slope
17 parameters were restricted to be >1; for the gamma and Weibull models, power parameters were
18 restricted to be >1; and for the multistage models, betas were restricted to be non-negative (bi >0).
19 Each model was tested for goodness-of-fit using a chi-square goodness-of-fit test (x2 p-value < 0.10
20 indicates lack of fit). Other factors were also used to assess model fit, such as scaled residuals,
21 visual fit, and adequacy of fit in the low-dose region and in the vicinity of the benchmark response
22 (BMR).
23 For each continuous endpoint, BMDS continuous models were fitted to the data using the
24 maximum likelihood method. For the polynomial models, betas were restricted to be non-negative (in
25 the case of increasing response) or non-positive (in the case of decreasing response data); and for
26 the Hill, power, and exponential models, power parameters were restricted to be >1. Model fit was
27 assessed by a series of tests as follows. For each model, first the homogeneity of the variances was
28 tested using a likelihood ratio test (BMDS Test 2). If Test 2 was not rejected (x2 p-value > 0.10), the
29 model was fitted to the data assuming constant variance. If Test 2 was rejected (x2 p-value < 0.10),
30 the variance was modeled as a power function of the mean, and the variance model was tested for
31 adequacy of fit using a likelihood ratio test (BMDS Test 3). For fitting models using either constant
32 variance or modeled variance, models for the mean response were tested for adequacy of fit using a
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1 likelihood ratio test (BMDS Test 4, with x2 p-value < 0.10 indicating inadequate fit). Other factors
2 were also used to assess the model fit, such as scaled residuals, visual fit, and adequacy of fit in the
3 low-dose region and in the vicinity of the BMR.
4
5
6
7
8
9
10
11
12
Model Selection
For each endpoint selected for modeling (see Table E-l), the BMDL estimate (95% lower
confidence limit on the benchmark dose [BMD], as estimated by the profile likelihood method) and
Akaike's Information Criterion (AIC) value were used to select a best-fit model from among the
models exhibiting adequate fit. If the BMDL estimates were "sufficiently close," that is, differed by
at most threefold, the model selected was the one that yielded the lowest AIC value. If the BMDL
estimates were not sufficiently close, the lowest BMDL was selected as the POD.
Table E-l. Non-cancer endpoints selected for dose-response modeling for
benzo[a]pyrene: RfD
Study
Kroese et
al. (2001)
Kroese et
al. (2001)
Xu et al.
(2010)
Chen et
al. (2012)
Gao et al.
(2011b)
Endpoint
Thymus
weight (mg)
Thymus
weight (mg)
Ovary
weight (mg)
Morris
water maze
Elevated
plus maze
Cervical
epithelial
hyperplasia
Species / Sex
Rat (Wistar) /
Male
Rat (Wistar) /
Female
Sprague-
Dawley /
Female
Sprague-
Dawley /
Male and
Female
Sprague-
Dawley /
Female
ICR/ Female
Doses and Effect Data
Dose (mg/kg-d)
Mean±SDa
Dose (mg/kg-d)
Mean±SDa
Dose (mg/kg-d)b
Mean ±SD
Dose (mg/kg-d)
Escape latency
(sec); mean ±SD
Time spent in
target quadrant
(sec); mean ± SD
Number of open
arm entries
Dose (mg/kg-d)c
Incidence
0
380 ± 60
0
320 ± 60
0
0.160±
0.0146
0
9.89 ±5.76
33.6 ±8.92
10.24 ±
1.905
0
0/26
3
380 ± 110
3
310 ± 50
2.5
0.143 ±
0.0098**
0.02
12.5 ±5. 10
31.9 ±8.63
10.36 ±
3.048
0.71
4/26
10
330 ± 60
10
300 ± 40
5
0.136 ±
0.0098**
0.2
19.1 ±5.85
16.6 ±5.74
12.89 ±
2.667
1.4
6/25
30
270 ± 40*
30
230 ± 30*
2.0
33.5 ±
9.93
11.1 ±
5.12
16.39 ±
3.048
2.9
7/24
13
14
15
16
17
18
*Significantly (p < 0.05) different from control mean; student t-test (unpaired, two-tailed); n = 10/sex/group.
**Statistically different (p < 0.05) from controls using one-way ANOVA.
aReported as SE, but judged to be SD (and confirmed by study authors).
bTWA doses over the 60-day study period.
cDoses converted to mg/kg-d after adjustment for equivalent continuous dosing (2/7 d/wk).
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1 Modeling Results
2 Below are tables and figures summarizing the modeling results for the non-cancer
3 endpoints modeled (see Tables E-2 through E-8 and Figures E-l through E-7).
4
5
6
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
Model
Variance
p-valuea
Goodness of Fit
p-value
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
Constant variance
Linear
0.01
0.74
384.84
12.97
8.97
Nonconstant variance
Hillb
Linear, Polynomial (2-degree), Powerc
Insufficient degrees of freedom
0.30
0.23
380.71
16.40
11.30
7
8
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
Linear Model with 0.95 Confidence Level
o
Q.
c
(0
OJ
450
400
350
300
250
0 5 10 15 20
dose
9 15:3310/152009
10 BMDs and BMDLs indicated are associated with a change of 1 SD from the control, and are in units of mg/kg-day.
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
Figure E-l. Fit of linear model (nonconstant variance) to data on decreased
thymus weight in male Wistar rats—90 days (Kroese etal.. 2001).
3
4
5
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7
8
9
10
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Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\benzoIalpyrene\RfD\Kroese2001\90day\thymusweight\male\durationadjusted\2Linkrolin.(
d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzoUlpyrene\RfD\Kroese2001\90day\thymusweight\male\durationadjusted\2Linkrolin.p
It
Dependent variable = mean
Independent variable = dose
The polynomial coefficients are restricted to be negative
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial 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_l
lalpha 1 -1 0.048 -0.061
rho -1 1 -0.048 0.061
beta_0 0.048 -0.048 1 -0.84
beta 1 -0.061 0.061 -0.84 1
Parameter Estimates
Variable
lalpha
rho
beta_0
beta 1
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-37.9473
1.38062
346.558
-7.11189
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
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1
J.
2
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0 10 380 379 60 84.3 0.
2.1 10 380 368 110 78.8 0
7.1 10 330 342 60 66.6 -0
21. 4 10 270 269 40 37 . 9 0.
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var$$e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var$$e(ij)} = exp(lalpha + rho*ln (Mu (i) ) )
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e (i)
Var$$e(i)} = SigmaA2
Likelihoods of Interest
Model Log (likelihood) # Param's AIC
Al -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)
Test 2: Are Variances Homogeneous? (Al vs A2)
Test 3: Are variances adequately modeled? (A2 vs. A3)
Test 4: 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.)
Tests of Interest
Test -2*log (Likelihood Ratio) Test df p-value
Test 1 25.3602 6 0.0002928
Test 2 10.8874 3 0.01235
Test 3 2.42069 2 0.2981
Test 4 2.93983 2 0.2299
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
0392
.475
.591
0908
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1
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11
BMDL =
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1
2
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
etal.. 2001); BMR = 1 SD change from the control mean
Model (constant variance)
Hillb
Linearc
Polynomial (2-degree)c'd
Powerb
Goodness of Fit
Variance p-
valuea
Mean p-valuea
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
NA
0.17
0.17
0.81
0.77
349.12
350.80
10.52
13.29
7.64
7.77
NA
4
5
6
7
8
9
10
11
12
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.
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Linear Model with 0.95 Confidence Level
o
Q.
o:
c
(0
OJ
360
340
320
300
280
260
240
220
200
Linear
BMDL
BMD
0 5 10 15 20
dose
1 16:2710/152009
2 BMDs and BMDLs indicated are associated with a change of 1SD from the control, and are in units of mg/kg-day.
3 Figure E-2. Fit of linear model (constant variance) to data on decreased
4 thymus weight in female Wistar rats—90 days (Kroese et al.. 2001).
5
6
7
8
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Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\benzoUlpyrene\RfD\Kroese2001\90day\thymusweight\female\durationadjusted\2Linkrolin
. (d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzoUlpyrene\RfD\Kroese2001\90day\thymusweight\female\durationadjusted\2Linkrolin
.pit
Thu Oct 15 16:27:44 2009
This document is a draft for review purposes only and does not constitute Agency policy.
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A constant variance model is fit
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 1
rho = 0 Specified
beta_0 = 322.144
beta 1 = -4.2018
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
alpha
beta_0
beta 1
Parameter Estimates
Variable
alpha
beta_0
beta 1
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
1098.16 2811.69
303.558 340.73
-5.84334 -2.56026
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
This document is a draft for review purposes only and does not constitute Agency policy.
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Likelihoods of Interest
# Param's
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (Al vs A2)
Test 3: Are variances adequately modeled? (A2 vs. A3)
Test 4: 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.)
Tests of Interest
-2*log(Likelihood Ratio) Test df
p-value
Test
Test 1
Test 2
Test 3
Test 4
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 3 is greater than .1. The modeled variance appears
to be appropriate here
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 10.5228
BMDL =
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1
2
3
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
(Xuetal.. 2010); BMR = 1 SD change from the control mean
Model
Power
Linear, Polynomial (1°)
Goodness of Fit
p-value
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
NAa
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)
en
£=
O
Q.
a:
CO
0)
0.18
0.17
0.16
0.15
0.14
0.13
Linear
BMDL
BMD
16:0312/142010
dose
7
8
Figure E-3. Fit of linear/polynomial (1°) model to data on decreased ovary
weight fXuetal.. 2010).
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11
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13
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15
16
Polynomial Model. (Version: 2.16; Date: 05/26/2010)
Input Data File
C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_lSD.(d)
Gnuplot Plotting File
C:/USEPA/BMDS212/Data/benzo[a]pyrene/Bap_AbsOvaryWeight/Xu2010_AbsOvaryWeight_Linear_lSD.plt
Tue Dec 14 13:51:32 2010
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7
8 Dependent variable = Mean
9 Independent variable = Dose
10 rho is set to 0
11 Signs of the polynomial coefficients are not restricted
12 A constant variance model is fit
13
14 Total number of dose groups = 3
15 Total number of records with missing values = 0
16 Maximum number of iterations = 250
17 Relative Function Convergence has been set to: le-008
18 Parameter Convergence has been set to: le-008
19
20
21
22 Default Initial Parameter Values
23 alpha = 0.000136
24 rho = 0 Specified
25 beta_0 = 0.158333
26 beta 1 = -0.0048
27
28
29 Asymptotic Correlation Matrix of Parameter Estimates
30
31 ( *** The model parameter (s) -rho
32 have been estimated at a boundary point, or have been specified by the user,
33 and do not appear in the correlation matrix )
34
35 alpha beta 0 beta 1
36
37 alpha 1 4e-010 -4.5e-010
38
39 beta 0 4e-010 1 -0.77
40
41 beta 1 -4.5e-010 -0.77 1
42
43
44
45 Parameter Estimates
46
47 95.0% Wald Confidence Interval
48 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
49 alpha 0.000118889 3.96296e-005 4.12162e-005 0.000196562
50 beta_0 0.158333 0.00406354 0.150369 0.166298
51 beta 1 -0.0048 0.00125904 -0.00726768 -0.00233232
52
53
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55
56
57
58
59
60 o e
61 2.5 6
62 56
63
64
65
66 Model Descriptions for likelihoods calculated
67
68
69
70
71
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Model A2: Yij = Mu(i) + e(ij)
Var$$e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var$$e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e (i)
Var$$e (i) } = SigmaA2
Likelihoods of Int
Model Log (likelihood)
Al 72.766595
A2 73.468565
A3 72.766595
fitted 72.335891
R 67.008505
Explanation of Tests
erest
# Param's AIC
4 -137.533190
6 -134.937129
4 -137.533190
3 -138.671782
2 -130.017010
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (Al
vs A2)
Test 3: Are variances adequately modeled? (A2 vs. A3)
Test 4 : Does the Model for the Mean Fit
(Note: When rho=0 the results of Test 3
Tests of Interest
Test -2*log (Likelihood Ratio) Test
Test 1 12.9201 4
Test 2 1.40394 2
Test 3 1.40394 2
Test 4 0.861408 1
The p-value for Test 1 is less than .05.
difference between response and/or varian
It seems appropriate to model the data
The p-value for Test 2 is greater than .1
model appears to be appropriate here
The p-value for Test 3 is greater than .1
to be appropriate here
The p-value for Test 4 is greater than .1
to adequately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard
Confidence level = 0.95
BMD = 2.27159
BMDL = 1.49968
? (A3 vs. fitted)
and Test 2 will be the same.)
df p-value
0.01167
0.4956
0.4956
0 . 3533
There appears to be a
ces among the dose levels
A homogeneous variance
The modeled variance appears
The model chosen seems
deviations from the control mean
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4
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 etal.. 2012); BMR = 1 SD change from the control
mean
Model"
Hillb
Exponential 4, 5
Polynomial (2°)
Linear, Power
Exponential 2, 3
Goodness of Fit
p-value
0.515
0.466
0.423
0.002
<0.001
AIC
386.3
386.4
386.6
396.7
400.3
BMD1SD
(mg/kg-d)
0.106
0.115
0.123
0.543
0.815
BMDL1SD
(mg/kg-d)
0.061
0.071
0.083
0.421
0.687
5
6
7
8
9
10
11
12
13
14
15
16
Includes modeling of heterogeneous variances (BMDS Test 3, p = 0.313).
bPower parameter n was estimated to be 1 (boundary of parameter space).
Data from Morris water maze was presented graphically in Chen etal. [2012], but dose
group means and SDs were provided upon request by the study authors, which enabled modeling of
this endpoint In addition, the data for male and female rats were combined for dose-response
analysis because there was no substantive difference between males and females for each dose
group (supported by statistical testing using two-way AN OVA, and allowing for interactions), and
because there was no rationale or information available suggesting there would be sex-mediated
differences for these neurobehavioral tests.
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Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -n
have been estimated at a boundary point, or have been specified by the user,
lalpha
rho
intercept
Variable
lalpha
rho
intercept
v
n
k
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev
Model Descriptions for likelihoods calculated
Model A3: Yij = Mu(i) + e(ij)
Var$$e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Likelihoods of Interest
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1
2 Model Log(likelihood) # Param's AIC
3 Al
4 A2
5 A3
6 fitted
7 R
8
9
10 Explanation of Tests
11
12 Test 1: Do responses and/or variances differ among Dose levels?
13 (A2 vs. R)
14 Test 2: Are Variances Homogeneous? (Al vs A2)
15 Test 3: Are variances adequately modeled? (A2 vs. A3)
16 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
17 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.'
18
19 Tests of Interest
20
21 Test -2*log(Likelihood Ratio) Test df
22
23 Test 1
24 Test 2
25 Test 3
26 Test 4
27
28
29
30
31
32 The p-value for Test 2 is less than .1. A non-homogeneous variance
33 model appears to be appropriate
34
35
36
37
38 The p-value for Test 4 is greater than .1. The model chosen seems
39 to adequately describe the data
40
41
42 Benchmark Dose Computation
43
44 Specified effect = 1
45
46 Risk Type = Estimated standard deviations from the control mean
47
48 Confidence level = 0.95
49
50 BMD = 0.106284
51
52 BMDL = 0.0609511
53
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1
2
3
4
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
Model"
Exponential 4
Exponential 5
Hill
Linear, Power, Polynomial (1°, 2°, 3°)
Goodness of Fit
p-value
0.576
NAb
NAb
<0.001
AIC
395.4
397.1
397.1
433.1
BMD1SD
(mg/kg-d)
0.065
0.084
0.071
1.23
BMDL1SD
(mg/kg-d)
0.043
0.044
0.038
0.98
5
6
7
Includes modeling of heterogenous variances (BMDS Test 3, p = 0.919).
bNA: insufficient degrees of freedom to evaluate x2.
8
9
10
cc
40
35
30
25
20
15
10
iMDL
Exponential Model 4 with 0.95 Confidence Level
Exponential
BMD
0
14:3504/242012
0.5
1
dose
1.5
Figure E-5. Fit of Exponential 4 model to data on Morris water maze time
spent in target quadrant (Chen etal.. 2012).
11
12
13
14
15
16
17
18
19
20
21
22
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13 Dependent variable = Mean
14 Independent variable = Dose
15 Data are assumed to be distributed: normally
16 Variance Model: exp(lnalpha +rho *ln(Y[dose]))
17 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
18
19 Total number of dose groups = 4
20 Total number of records with missing values = 0
21 Maximum number of iterations = 250
22 Relative Function Convergence has been set to: le-008
23 Parameter Convergence has been set to: le-008
24
25 MLE solution provided: Exact
26
27
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30 Variable Model 4
31
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41 Parameter Estimates
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43 Variable Model 4
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This document is a draft for review purposes only and does not constitute Agency policy.
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1 2 11.18 4.824 -0.03277
2
3
4
5 Other models for which likelihoods are calculated:
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 Likelihoods of Interest
21
22 Model Log (likelihood) DF
23
24 Al -197.0118 5
25 A2 -192.448 8
26 A3 -192.5331 6
27 R -238.8696 2
28 4 -192.6894 5
29
30
31 Additive constant for all log-likelihoods = -73.52. This constant added to the
32 above values gives the log-likelihood including the term that does not
33 depend on the model parameters.
34
35
36 Explanation of Tests
37
38
39
40
41
42 Test 6a: Does Model 4 fit the data? (A3 vs 4)
43
44
45 Tests of Interest
46
47 Test -2*log(Likelihood Ratio) D. F. p-value
48
49 Test 1
50 Test 2
51 Test 3 0.1701 2
52 Test 6a 0.3126 1
53
54
55 The p-value for Test 1 is less than .05. There appears to be a
56 difference between response and/or variances among the dose
57 levels, it seems appropriate to model the data.
58
59
60
61
62
63
64
65 The p-value for Test 6a is greater than .1. Model 4 seems
66 to adequately describe the data.
67
68
69
70
71
This document is a draft for review purposes only and does not constitute Agency policy.
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1
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8
Risk Type = Estimated standard deviations from control
Confidence Level = 0.950000
BMD = 0.0650194
BMDL = 0.0432761
9
10
11
Table E-7. Summary of BMD modeling results for elevated plus maze: open
arm entries for females at PND 70 (Chen etal.. 2012); BMR = 1 SD change from
the control mean
Model"
Exponential (M2)
Exponential (M3)
Exponential (M4)
Exponential (M5)
Hill
Polynomial 1°
Polynomial 2°
Polynomial 3°
Power
Goodness of fit
p-valueb
0.107
0.840
NA
NA
0.129
AIC
125.93
123.51
125.47
125.47
125.57
BMD1SD
(mg/kg-d)
1.086
0.184
0.194
0.193
0.964
BMDL1SD
(mg/kg-d)
0.845
0.086
0.087
0.066
0.713
12
13
14
15
16
17
18
19
20
21
a Constant variance models are presented (BMDS Test 2 p-value = 0.46), with the selected model in bold. Scaled
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,
respectively.
For exponential model M3, parameter d = 1, reducing it to M2.
For the power model, the power parameter estimate was 1 (boundary of parameter space). For the polynomial
2° and 3° models, the b2 and b3 coefficient estimates were 0 (boundary of parameter space). Consequently,
these three models all reduced to the polynomial 1° model.
bExponential M5 and Hill model required four parameters and there are four dose groups, leaving no d.f. for the
goodness-of-fit test. Therefore these were not considered for model selection
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18
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12
10
Exponential Model 4 with 0.95 Confidence Level
12:35 08/02 2012
2
3
Figure E-6. Fit of exponential model (4) to data on elevated plus maze open
arm maze entries (Chenetal.. 2012).
4
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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_ChenF070_Exp-ConstantVariance-BMRlStd-Up.(d)
Gnuplot Plotting File: C:\Documents and Settings\jfox\My Documents\_CURRENTWORK\_CAST
plus\BaP\BMDS\exp_ChenF070_Exp-ConstantVariance-BMRlStd-Up.pit
Thu Aug 02 12:35:33 2012
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 * dose)Ad}
Model 4: Y[dose] = a * [c-(c-l) * exp{-b * dose}]
Model 5: Y[dose] = a * [c-(c-l) * exp{-(b * dose)AdK
Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
rho is set to 0.
A constant variance model is fit.
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1
2
3
4
5 MLE solution provided: Exact
6
7
8
9
10 Variable
11
12
13
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15
16
17
18
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20
21
22
23 Parameter Estimates
24
25 Variable Model 4
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41
42
43
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45 Estimated Values of Interest
46
47 Dose Est Mean Est Std Scaled Residual
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71 Likelihoods of Interest
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1
2
3
*J
4
5
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78
^U
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Additive cons
above values
depend on the
Test 1: Does
Test 2: Are
Test 3: Are
Test 6a: Does
Test
Test 1
Test 2
Test 3
Test 6a
The p-value
difference
levels, it
The p-value
variance mo
The p-value
Model Log (likelihood) DF AIC
Al -57.73371 5 125.4674
A2 -56.43655 8 128.8731
A3 -57.73371 5 125.4674
R -71.03323 2 146.0665
4 -57.75397 4 123.5079
tant for all log-likelihoods = -36.76. This constant added to the
gives the log-likelihood including the term that does not
model parameters.
Explanation of Tests
response and/or variances differ among Dose levels? (A2 vs. R)
Variances Homogeneous? (A2 vs. Al)
variances adequately modeled? (A2 vs. A3)
Model 4 fit the data? (A3 vs 4)
Tests of Interest
-2*log (Likelihood Ratio) D. F. p-value
29.19 6 < 0. 0001
2.594 3 0.4585
2.594 3 0.4585
0 . 04053 1 0.8404
for Test 1 is less than .05. There appears to be a
between response and/or variances among the dose
seems appropriate to model the data.
for Test 2 is greater than .1. A homogeneous
del appears to be appropriate here.
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 adequately describe the data.
Benchmark Dos
e Computations:
Specified Effect = 1.000000
Risk
Confidence
BMDL =
Type = Estimated standard deviations from control
Level = 0 . 950000
BMD = 0.184087
0. 0864691
60
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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 etal.. ZOllb); BMR = 1 SD change from the control
mean
Model
Gamma
Logistic
Log-logistic
Probit
Log-Probit
Multistage
Goodness of Fit
p-value
0.6874
0.1422
0.8360
0.1544
0.0775
0.6874
AIC
82.2821
88.4607
81.7004
88.1151
88.2004
82.2821
BMD1SD
(mg/kg-d)
0.659
1.422
0.578
1.326
1.012
0.659
BMDL1SD
(mg/kg-d)
0.452
1.052
0.369
0.979
0.686
0.452
6
7
•
I
C
o
13
ro
0.5
0.4
0.3
0.2
0.1
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMDL BMD
0.5
1.5
dose
2.5
19:01 08/262011
Figure E-7. Fit of log-logistic model to data on cervical epithelial hyperplasia
(Gao etal.. ZOllb)
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Logistic Model. (Version: 2.13; Date: 10/28/2009)
2 Input Data File: C:\Users\hclynch\Documents\_Active Projects\_FA498 IRIS\xBaP\IASC Aug
3 2011\bmd modeling\lnl_gao 2011 inflamm cells_0pt.(d)
4 Gnuplot Plotting File: C:\Users\hclynch\Documents\_Active Projects\_FA498
5 IRIS\xBaP\IASC Aug 2011\bmd modeling\lnl_gao 2011 inflamm cells_0pt.pit
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20 Total number of observations = 4
21 Total number of records with missing values = 0
22 Maximum number of iterations = 250
23 Relative Function Convergence has been set to: le-008
24 Parameter Convergence has been set to: le-008
25
26
27
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30
31
32
33
34
35
36
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38
39
40
41 and do not appear in the correlation matrix )
42
43 intercept
44
45 intercept 1
46
47
48
49
50
51 95.0% Wald Confidence Interval
52 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
53
54
55
56
57 * - Indicates that this value is not calculated.
58
59
60
61
62
63 Model Log(likelihood) # Param's Deviance Test d.f. P-value
64 Full model
65 Fitted model
66 Reduced model
67
68 AIC:
69
70
71
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2 Dose Est. Prob.
3 -
4
5
6
7
8
9 ChiA2 =0.86 d.f. = 3
10
11
12 Benchmark Dose Computation
13
14 Specified effect = 0.1
15
16 Risk Type = Extra risk
17
18 Confidence level = 0.95
19
20 BMD = 0.578668
21
22 BMDL = 0.368701
23
24
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Supplem en tal Inform ation —Benzo[a]pyren e
1 Reference Concentration (RfC)
2 Candidate studies for the development of the RfC were not amenable to BMD modeling.
3 DosimetryModeling for Estimation of Human Equivalent Concentrations
4 As discussed in Section 2.2.2, the human equivalent concentration (HEC) was calculated
5 from the PODADj by multiplying by a DAF, which, in this case, was the regional deposited dose ratio
6 (RDDRER) for extrarespiratory (i.e., systemic) effects. The observed developmental effects are
7 considered systemic in nature (i.e., extrarespiratory) and the normalizing factor for
8 extrarespiratory effects of particles is body weight. The RDDRER was calculated as follows:
9
10 BWA (VE)H (FTOT)H
11 where:
12 BW = body weight (kg)
13 VE = ventilation rate (L/minute)
14 FTOT = total fractional deposition
15
16 The total fractional deposition includes particle deposition in the nasal-pharyngeal,
17 tracheobronchial, and pulmonary regions. FTOT for both animals and humans was calculated using
18 the Multi-Path Particle Dosimetry model, a computational model used for estimating human and rat
19 airway particle deposition and clearance (Multi-Path Particle Dosimetry [MPPD]; Version 2.0 ©
20 2006, publicly available through the Hamner Institute). See model output below.
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Supplem en tal Inform ation —Benzo[a]pyren e
Wed, 03/17/2010. 02:07:20 PM EOT
Region: Entire Lung
1
2
0.750 i-
0.600
0.450
S 0.300
Q.
ID
Q
0.150
0.0
0.621
0.449
Head
Total
Region
Species & Model Info:
Species/Geometry: Human Limited
FRC \6lume: 3300.00 ml
Head Volume: 50.00 ml
Breathing Route: nasal
Breathing Parameters:
Tidal Vblurne: 860.00 ml
Breathing Frequency: 16.00 1/min
Inspiratory Fraction: 0.50
Pause Fraction: 0.00
Particle Properties:
Diameter: MvlAD: 1.70 urn
GSD: 1.00
Concentration: 4.20 ug/m"3
Figure E-8. Human fractional deposition.
3 Species = humanlimited
4 FRC = 3300.0
5 Head volume = 50.0
6 Density =1.0
7 Number of particles calculated = single
8 Diameter = 1.7000000000000002 urn MMAD
9 Inhalability = yes
10 GSD =1.0
11 Breathing interval: One single breath
12 Concentration = 4.2
13 Breathing Frequency = 16.0
14 Tidal Volume = 860.0
15 Inspiratory Fraction = 0.5
16 Pause Fraction = 0.0
17 Breathing Route = nasal
18
19 Region: Entire Lung
20 Region: Entire Lung
21 Region Deposition Fraction
22
23 Head 0.449
24 TB 0.045
25 P 0.127
26 Total 0.621
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Wed, 03/17/2010. 02:15:27 PM EOT
Region: Entire Lung
1
2
0.250 i-
0.200
1 0.150
O
ID
Q
0.100
0.050
0.0
0.181
0.072
Head TB
Total
Region
Species & Model Info:
Species/Geometry: Rat
FRC Vblume: 4.DD ml
Head Vblume: 0.42 ml
Breathing Route: nasal
Breathing Parameters:
Tidal Vblume: 1.80 nil
Breathing Frequency: 102.00 1Anin
Inspiratory Fraction: 0.50
Pause Fraction: 0.00
Particle Properties:
Diameter: MvlAD: 1.70 urn
GSD: 1.00
Concentration: 4.20 pg/hi"3
Figure E-9. Rat fractional deposition.
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
29
Species = rat
FRC =4.0
Head volume =0.42
Density =1.0
Number of particles calculated = single
Diameter = 1.7000000000000002 urn MMAD
Inhalability = yes
GSD =1.0
Breathing interval: One single breath
Concentration = 4.2
Breathing Frequency = 102.0
Tidal Volume =1.8
Inspiratory Fraction = 0.5
Pause Fraction = 0.0
Breathing Route = nasal
Region: Entire Lung
Region: Entire Lung
Region Deposition Fraction
Head 0.072
TB 0.041
P 0.068
Total 0.181
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[a]pyren e
1 E.2. Cancer Endpoints
2 E.2.1. Dose-Response Modeling for the Oral Slope Factor
3 Dose-Response Models
4 Due to the occurrence of multiple tumor types, earlier occurrence with increasing exposure,
5 and early termination of the high-dose group in the oral carcinogenicity studies (see Appendix D for
6 study details), methods that can reflect the influence of competing risks and intercurrent mortality
7 on site-specific tumor incidence rates are preferred. EPA has generally used a model that
8 incorporates the time at which death-with-tumor occurred as well as the dose; the multistage-
9 Weibull model is multistage in dose and Weibull in time, and has the form:
10 P(d, t) = l- exp[-(q0 + qid + q2d* + ... + qkdk] x (t ± t0)c],
11 where P(d, t) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent
12 exposure in this case) and age t (in bioassay weeks); parameters qi > 0, for / = 0,1,..., k; t is the time
13 at which the tumor was observed; and c is a parameter which characterizes the change in response
14 with age. The parameter to represents the time between when a potentially fatal tumor becomes
15 observable and when it causes death, and is generally set to 0 either when all tumors are
16 considered incidental or because of a lack of data to estimate the time reliably. The dose-response
17 analyses were conducted using the computer software program MultiStage-Weibull (U.S. EPA,
18 2010), which is based on Weibull models drawn from Krewskietal. (1983). Parameters were
19 estimated using the method of maximum likelihood. From specific model fits using stages up to n -
20 1, where n is the number of dose groups, the model fit with the lowest AIC was selected.
21 E.2.2. Data Adjustments Prior to Modeling
22 Two general characteristics of the observed tumor types were considered prior to
23 modeling; allowance for different, although unidentified modes of action, and allowance for relative
24 severity of tumor types. First, etiologically different tumor types were not combined across sites
25 prior to modeling (that is, overall counts of tumor-bearing animals were not tabulated) in order to
26 allow for the possibility that different tumor types could have different dose-response relationships
27 due to different underlying mechanisms or factors, such as latency. Consequently, all of the tumor
28 types were also modeled separately.
29 Additionally, the multistage-Weibull model can address relative severity of tumor types by
30 distinguishing between tumors as being either fatal or incidental to the death of an animal in order
31 to adjust partially for competing risks. In contrast to fatal tumors, incidental tumors are those
32 tumors thought not to have caused the death of an animal. Cause-of-death information for most
33 early animal deaths was provided by the investigators of both bioassays. In the rat study of Kroese
This document is a draft for review purposes only and does not constitute Agency policy.
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1 etal. [2001], tumors of the forestomach or liver were the principal cause of death for most animals
2 dying or sacrificed (due to moribundity] before the end of the study, while tumors of the
3 forestomach were the most common cause of early deaths in the mouse study of Belandand Gulp
4 [1998]. The incidence data modeled are listed in Tables E-9 (male rats], E-10 (female rats], and
5 E-11 (female mice].
6 Human-equivalent dose estimates used for dose-response modeling were based on scaling
7 by body weight3/4, as there were no pharmacokinetic models or data to inform another approach.
8 The dose estimates are provided in Table E-12 [Kroese etal., 2001] and E-13 [Beland and Gulp,
9 1998].
10 Evaluation of model flt and model selection:
11 Each model was examined for adequacy of fit in the low-dose region and in the vicinity of
12 the benchmark response [BMR] of 10% extra risk. In general, the model fit with the lowest AIC was
13 selected, except when model fit near the BMR and in the low-dose region was improved by
14 including an additional stage (parameter] in the model.
15 PODs for estimating low-dose risk were identified at doses at the lower end of the observed
16 data, generally corresponding to 10% extra risk, where extra risk is defined as [P(d] - P(0]]/[l -
17 P(0]]. The lifetime oral cancer slope factor for humans is defined as the slope of the line from the
18 lower 95% bound on the exposure atthe POD to the control response (slope factor = 0.1/BMDLio].
19 This slope, a 95% upper confidence limit [UCL], represents a plausible upper bound on the true
20 risk.
21 Overall risk
22 Although the time-to-tumor modeling helps account for competing risks associated with
23 decreased survival times and other tumors, considering the tumor sites individually still does not
24 convey the total amount of risk potentially arising from the sensitivity of multiple sites (i.e., the risk
25 of developing any combination of the increased tumor types, not just the risk of developing all
26 simultaneously]. One approach suggested in the Guidelines for Carcinogen Risk Assessment [U.S.
27 EPA, 2005] would be to estimate cancer risk from tumor-bearing animals. EPA traditionally used
28 this approach until the National Research Council (NRC] document Science and Judgment in Risk
29 Assessment [NRC. 1994] made a case that this approach would tend to underestimate overall risk
30 when tumor types occur in a statistically independent manner. In addition, application of one
31 model to a composite data set does not accommodate biologically relevant information that may
32 vary across sites or may only be available for a subset of sites. For instance, the time courses of the
33 multiple tumor types evaluated varied, as is suggested by the variation in estimates of c, from
34 1.5 (e.g., male rat skin or mammary gland basal cell tumors], indicating relatively little effect of age
35 on tumor incidence, to 3.7 (e.g., male mouse alimentary tract tumors], indicating a more rapidly
36 increasing response with increasing age (in addition to exposure level]. The result of fitting a
37 model with parameters that can reflect underlying mechanisms, such as z in the multistage-Weibull
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1 model, would be difficult to interpret with composite data (i.e., counts of tumor-bearing animals). A
2 simpler model, such as the multistage model, could be used for the composite data but relevant
3 biological information would then be ignored.
4 Following the recommendations of the NRG [1994] regarding combining risk estimates,
5 statistical methods that can accommodate the underlying distribution of slope factors are optimal,
6 such as through maximum likelihood estimation or through bootstrapping or Bayesian analysis.
7 However, these methods have not yet been extended to models such as the multistage-Weibull
8 model. A method involving the assumption that the variability in the slope factors could be
9 characterized by a normal distribution is detailed below [U.S. EPA, 2010]. Using the results in
10 female rats to illustrate, the overall risk estimate involved the following steps:
11 1] It was assumed that the tumor groupings modeled above were statistically independent
12 (i.e., that the occurrence of a liver tumor was not dependent upon whether there was a
13 forestomach tumor]. This assumption cannot currently be verified, and if not correct, could
14 lead to an overestimate of risk from summing across tumor sites. However, NRG (1994]
15 argued that a general assumption of statistical independence of tumor-type occurrences
16 within animals was not likely to introduce substantial error in assessing carcinogenic
17 potency from rodent bioassay data.
18 2] The models previously fitted to estimate the BMDs and BMDLs were used to extrapolate to a
19 lower level of risk (R], in order to reach the region of each estimated dose-response
20 function where the slope was reasonably constant and upper bound estimation was still
21 numerically stable. For these data, a 10~3 risk was generally the lowest risk necessary. The
22 oral slope factor for each site was then estimated by R/BMDL.R, as for the estimates for each
23 tumor site above.
24 3] The maximum likelihood estimates (MLE] of unit potency (i.e., risk per unit of exposure]
25 estimated by R/BMDn, were summed across the alimentary tract, liver, and
26 jejunum/duodenum in female rats.
27 4] An estimate of the 95% (one-sided] upper bound on the summed oral slope factor was
28 calculated by assuming a normal distribution for the individual risk estimates, and deriving
29 the variance of the risk estimate for each tumor site from its 95% UCL according to the
30 formula:
31
32 95% UCL = MLE + 1.645 x SD,
33 rearranged to:
34 SD = (UCL-MLE]/1.645,
35
36 where 1.645 is the t-statistic corresponding to a one-sided 95% CI and >120 degrees of
37 freedom, and the SD is the square root of the variance of the MLE. The variances (variance =
38 SD2] for each site-specific estimate were summed across tumor sites to obtain the variance
39 of the sum of the MLEs. The 95% UCL on the sum of MLEs was calculated from the
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
expression above for the UCL, using the variance of the sum of the MLE to obtain the
relevant SD (SD = variance1/2).
4 Dose-Response Modeling for the Oral Slope Factor
5
6
7
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 etal.
20011
Dose
(mg/kg-d)
0
Week of
Death
44
80
82
84
89
90
91
92
93
94
95
96
97
98
100
104
105
108
109
Total
Examined
1
1
1
1
1
3
1
1
1
1
2
2
1
1
3
1
1
7
22
Numbers of Animals with:
Oral Cavity or
Forestomach
Tumors
Incidental3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Fatal3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Liver Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Fatal
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Duodenum
or Jejunum
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Skin or Mammary
Gland
Basal Cell
Tumors
Incidental
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
Squamous
Cell
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Kidney
Urothelial
Carcinoma
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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[a]pyren e
Dose
(mg/kg-d)
3
10
Week of
Death
29
40
74
76
79
82
92
93
94
95
98
107
108
109
39
47
63
68
69
77
80
81
84
86
90
95
97
100
102
103
104
107
108
109
Total
Examined
1
1
1
1
1
1
2
1
1
2
1
10
15
14
1
2
1
2
1
1
1
1
1
1
1
3
1
1
1
1
3
12
11
6
Numbers of Animals with:
Oral Cavity or
Forestomach
Tumors
Incidental3
0
1
0
0
0
0
0
0
0
0
0
4
2
1
0
0
1
2
1
0
0
1
1
0
1
3
1
1
1
1
3
12
11
5
Fatal3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Liver Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
1
3
0
0
0
0
0
0
1
1
0
0
1
0
2
0
1
1
1
3
11
11
3
Fatal
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
Duodenum
or Jejunum
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
Skin or Mammary
Gland
Basal Cell
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
Squamous
Cell
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
Kidney
Urothelial
Carcinoma
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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[a]pyren e
Dose
(mg/kg-d)
30
Week of
Death
32
35
37
44
45
47
48
49
50
51
52
53
56
58
59
60
61
62
63
64
65
66
67
68
70
71
73
76
Total
Examined
1
1
1
1
2
1
1
1
1
1
4
1
2
2
2
2
3
5
5
2
3
1
3
1
2
1
1
1
Numbers of Animals with:
Oral Cavity or
Forestomach
Tumors
Incidental3
1
1
1
0
2
1
1
1
1
1
3
1
1
2
2
1
2
5
5
2
2
1
1
1
2
1
0
1
Fatal3
0
0
0
1
0
0
0
0
0
0
1
0
1
0
0
1
1
0
0
0
1
0
2
0
0
0
1
0
Liver Tumors
Incidental
0
1
0
1
2
1
1
1
1
1
3
1
1
2
2
1
1
0
4
1
1
0
2
1
1
1
1
0
Fatal
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
1
2
4
1
1
2
1
1
0
1
0
0
1
Duodenum
or Jejunum
Tumors
Incidental
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
3
1
0
0
0
1
0
1
0
0
0
Skin or Mammary
Gland
Basal Cell
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
1
1
0
1
0
0
0
0
2
0
3
0
1
0
1
1
1
1
Squamous
Cell
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
2
0
1
0
0
1
0
0
Kidney
Urothelial
Carcinoma
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
0
0
0
0
0
0
0
0
1
2
3
a"lncidental" denotes presence of tumors not known to have caused death of particular animals. "Fatal" denotes
incidence of tumors reported by the study investigators to have caused death of particular animals.
This document is a draft for review purposes only and does not constitute Agency policy.
E-36 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
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 etal..
2001)
Dose
(mg/kg-d)
0
3
Week of
Death
64
69
75
104
106
107
108
109
8
47
52
60
65
76
77
83
85
86
88
93
94
97
107
108
109
Total
Examined
1
1
1
1
4
7
7
30
1
1
1
1
1
1
1
2
1
1
1
2
1
1
6
9
21
Numbers of Animals with:
Oral Cavity or Forestomach
Tumors
Incidental3
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
1
Fatal3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Liver Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
Fatal
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Duodenum or
Jejunum
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
This document is a draft for review purposes only and does not constitute Agency policy.
E-37 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Dose
(mg/kg-d)
10
Week of
Death
42
43
44
45
48
55
59
75
76
77
80
81
82
83
85
86
87
88
89
91
95
96
98
99
102
104
105
106
107
108
109
Total
Examined
1
1
1
1
1
1
1
1
2
2
1
1
1
1
2
1
1
2
1
1
1
1
2
3
1
1
2
1
5
7
4
Numbers of Animals with:
Oral Cavity or Forestomach
Tumors
Incidental3
0
0
0
0
0
0
0
0
0
0
1
1
1
0
1
1
0
1
1
0
0
0
2
3
1
1
1
1
5
7
2
Fatal3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Liver Tumors
Incidental
0
0
0
0
0
1
0
1
1
0
1
0
1
1
1
0
1
1
0
0
0
0
1
1
0
1
1
0
5
7
2
Fatal
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
1
1
1
0
0
1
2
1
0
1
1
0
0
0
Duodenum or
Jejunum
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
This document is a draft for review purposes only and does not constitute Agency policy.
E-38 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Dose
(mg/kg-d)
30
Week of
Death
26
44
47
48
54
55
56
57
58
59
60
61
62
63
64
66
67
68
69
71
72
Total
Examined
1
4
3
1
1
3
2
2
4
2
1
2
2
3
5
3
2
1
4
4
2
Numbers of Animals with:
Oral Cavity or Forestomach
Tumors
Incidental3
0
4
3
1
0
3
2
2
3
1
0
2
2
3
5
3
1
1
3
3
1
Fatal3
0
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
1
0
1
1
1
Liver Tumors
Incidental
0
3
2
0
1
1
0
2
0
0
1
0
1
0
0
0
0
0
1
1
0
Fatal
0
1
1
1
0
2
2
0
4
2
0
2
1
3
5
3
2
1
3
3
2
Duodenum or
Jejunum
Tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
1
0
0
1
2
3
a"lncidental" denotes presence of tumors not known to have caused death of particular animals. "Fatal" denotes
incidence of tumors indicated by the study investigators to have caused death of particular animals.
4
5
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 Gulp. 1998)
Dose Group
(ppm in Diet)
0
Week of Death
31
74
89
91
93
94
97
98
99
100
101
104
105
Total Examined
1
1
2
1
2
2
2
2
1
2
2
1
29
Number of Animals with Alimentary Tract
Squamous Cell Tumors
Fatal3
0
0
0
0
0
0
0
0
0
0
0
0
0
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
1
This document is a draft for review purposes only and does not constitute Agency policy.
E-39 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Dose Group
(ppm in Diet)
5
25
Week of Death
25
55
83
86
87
88
90
94
95
96
97
98
101
102
105
44
47
64
70
77
80
81
84
85
86
88
89
90
93
94
96
97
98
99
100
101
102
104
105
Total Examined
1
1
1
1
2
2
1
1
2
1
2
2
2
2
27
1
1
1
1
1
1
1
2
1
1
1
1
4
3
2
3
1
1
2
1
1
2
1
13
Number of Animals with Alimentary Tract
Squamous Cell Tumors
Fatal3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
1
1
1
1
1
0
4
2
2
0
1
1
1
1
0
2
1
0
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
2
0
0
1
0
0
0
0
10
This document is a draft for review purposes only and does not constitute Agency policy.
E-40 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Dose Group
(ppm in Diet)
100
Week of Death
39
40
42
47
49
50
53
55
56
57
58
59
60
61
62
63
64
65
66
68
69
70
71
72
73
74
79
Total Examined
1
1
1
2
1
1
1
3
1
1
1
3
1
3
5
4
3
2
3
1
2
2
1
1
1
1
1
Number of Animals with Alimentary Tract
Squamous Cell Tumors
Fatal3
1
1
1
2
0
1
0
3
1
1
1
3
1
3
5
4
3
2
3
1
2
2
1
1
1
1
1
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
a"lncidental" denotes presence of tumors not known to have caused death of particular animals. "Fatal" denotes
incidence of tumors indicated by the study investigators to have caused death of particular animals.
This document is a draft for review purposes only and does not constitute Agency policy.
E-41 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
Table E-12. Derivation of HEDs to use for BMD modeling of Wistar rat tumor
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
10
30
0.349
0.349
0.288
0.27
0.27
0.25
0.54
1.81
5.17
Female
3
10
30
0.222
0.222
0.222
0.24
0.24
0.24
0.49
1.62
4.85
3
4
5
6
aScaling factors were calculated using U.S. EPA (1988) reference body weights for humans (70 kg), and the TWA
body weights for each dose group: rat-to-human = (TWA body weight/70)0'25 = scaling factor.
bHuman equivalent dose (HED) = administered dose x scaling factor.
7
8
Table E-13. Derivation of HEDs for dose-response modeling of B6C3Fi female
mouse tumor incidence data from Beland and Gulp (1998)
Benzo[a]pyrene
Dose in Diet
(ppm)
5
25
100
Intake (ug/d)
21
104
430
TWA Body
Weight Average
(kg)
0.032
0.032
0.027
Administered
Dose3 (mg/kg-d)
0.7
3.3
16.5
Scaling Factorb
0.15
0.15
0.14
HEDC (mg/kg-d)
0.10
0.48
2.32
9
10
11
12
13
14
15
Administered doses in mg/kg-day were calculated from dietary concentrations of benzo[a]pyrene using the TWA
body weight and reported food intakes for mice.
bScaling factors were calculated using U.S. EPA (1988) reference body weights for humans (70 kg), and the TWA
body weights for each dose group: mouse-to-human = (TWA body weight/70)
CHED = administered dose x scaling factor.
1 = scaling factor.
This document is a draft for review purposes only and does not constitute Agency policy.
E-42 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1 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 BaP. The model outputs and graphs following each of these
4 tables (Figures E-10 through E-19) provide more details for the best-fitting models in each case.
5
6
7
8
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
Male
rats
Female
rats
Endpoints
Oral cavity and
forestomach:
squamouscell
tumors
Hepatocellular
tumors
Duodenum and
jejunum tumors
Kidney: uroethelial
carcinoma
Skin and mammary
gland: basal cell
tumors
Skin and mammary
gland: squamous
cell tumors
Oral cavity and
forestomach:
squamouscell
tumors
Hepatocellular
tumors
Duodenum and
jejunum tumors
Model
Stages
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
AIC
577.8
407.6
229.0
367.3
301.5
289.1
69.6
65.9
66.9
31.9
31.7
32.8
110.6
105.1
104.7
63.5
64.3
65.3
277.1
211.6
201.0
595.5
774.9
468.3
37.9
37.0
37.8
BMD10
0.104
0.678
0.453
0.181
0.472
0.651
2.64
3.04
3.03
9.16
5.71
4.65
1.88
2.58
2.86
3.36
2.75
2.64
0.245
0.428
0.539
0.146
0.370
0.575
6.00
4.33
3.43
BMDL10 -
BMDU10
0.281-0.612
0.449-0.772
2.38-3.87
2.50-9.01
2.35-3.62
1.77-4.42
0.328-0.717
0.507-0.630
1.95-5.70
Basis for Model Selection
Lowest AIC, best fit to low dose data
Lowest AIC, best fit to low dose data
Best fit to data
Best fit to data
Lowest AIC, best fit to low dose data
Best fit to low dose data
Lowest AIC, best fit to low dose data
Lowest AIC, best fit to low dose data
Best fit to low dose data
This document is a draft for review purposes only and does not constitute Agency policy.
E-43 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1 Male rat (Kroese etal.. 20011: Squamous cell papilloma or carcinoma in oral cavity or
2 forestomach
3
4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
6 Input Data File: OralForstKroeseMS.(d)
8
9
10
11
12
13
14 Dependent variable = CONTEXT
15 Independent variables = DOSE, TIME
16
17 Total number of observations = 208
18 Total number of records with missing values = 0
19 Total number of parameters in model = 6
20 Total number of specified parameters = 0
21 Degree of polynomial = 3
23
24
25
26
27
28
29
30 t_0 = 39.1111
31 beta_0 = 0
32 beta_l = 8.8911e-009
33 beta_2 = 1.60475e-031
34 beta 3 = 1.95818e-008
35
36
37 Asymptotic Correlation Matrix of Parameter Estimates
38 ( *** The model parameter(s) -beta_0 -beta_2
39 have been estimated at a boundary point, or have been specified by the user,
40 and do not appear in the correlation matrix )
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55 Variable
56
57
58
59
60
61
62
63 NA - Indicates that this parameter has hit a bound implied by some inequality constraint
64 and thus has no standard error.
65
66
67 Log (likelihood) # Param
68 Fitted Model -108.512 6
69
70
71
72
This document is a draft for review purposes only and does not constitute Agency policy.
E-44 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
U Total Expected Response
0
Minimum observation time for F tumor context =
Computation
Incidental
Extra
0 . 9
104
Specified effect =
BMD =
BMDL =
BMDU =
44
Incidental Risk: OralForstKroeseM3
points show nonpararn. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00 Dose = 0.54
oq
CD
LD
CD
CD
CD
I
20
I
40
>-.
la
60
Time
80 100
CO
CD
LD
CD
CD ~~
CM
CD
CD
CD ~~l
0 «JOO .— /•ffi'
I I I I ! I
0 20 40 60 80 100
Time
Dose= 1.81
Dose = 5.17
LTD
CD
:=-.
= LD
15 CD
CD
CD
I I I I I
20 40 60 80 100
Time
LTD
CD
-s T
CD
CD
\
I
I
I
I
0 20 40 60 80 100
Time
17
18
19
20
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)
This document is a draft for review purposes only and does not constitute Agency policy.
E-45 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1 Male rat (Kroese etal. 2001): Hepatocellular adenoma or carcinoma
2
3 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
5 Input Data File: LiverKroeseM3.(d)
6
7
8
9
10
11
12
13
14 Dependent variable = CONTEXT
15 Independent variables = DOSE, TIME
16
17 Total number of observations = 208
18 Total number of records with missing values = 0
19 Total number of parameters in model = 6
20 Total number of specified parameters = 0
21 Degree of polynomial = 3
23
24
25
26
27
28
29 Default Initial Parameter Values
O\J c = 3.6
31 t_0 = 34.6667
32 beta_0 = 0
33 beta_l = 2.73535e-009
34 beta_2 = 8.116e-028
35 beta 3 = 1.43532e-008
36
37
38 Asymptotic Correlation Matrix of Parameter Estimates
39 ( *** The model parameter(s) -beta_0 -beta_2
40 have been estimated at a boundary point, or have been specified by the user,
41 and do not appear in the correlation matrix )
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56 Variable
57
58
59
60
61
62
63
64 NA - Indicates that this parameter has hit a bound implied by some inequality constraint
65 and thus has no standard error.
66
67
68 Log (likelihood) # Param
69 Fitted Model -138.544 6
70
71
72
73
74
This document is a draft for review purposes only and does not constitute Agency policy.
E-46 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
This document is a draft for review purposes only and does not constitute Agency policy.
E-47 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Dose= 0.00
Incidental Risk: Hepatocellular_Kroese_M3
points show nonparam. est. for Incidental (unfilled
Dose = 0.54
.a
ro
.£2
P
CL
oo
o _
-
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1 Male rat (Kroese et al. 2001): Duodenum or jejunum adenocarcinoma
2
3 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
5 Input Data File: DuoJejKroeseM3.(d)
6
7
8
9
10
11
12
13
14 Dependent variable = CONTEXT
15 Independent variables = DOSE, TIME
16
17 Total number of observations = 208
18 Total number of records with missing values = 0
19 Total number of parameters in model = 6
20 Total number of specified parameters = 1
21 Degree of polynomial = 3
23
24
25 User specifies the following parameters:
26 t 0 0
27
28
29
30
31
32
33 Default Initial Parameter Values
34 c 1.63636
35 t_0 = 0 Specified
36 beta_0 = 4.31119e-027
37 beta_l = 2.96347e-025
38 beta_2 = 0
39 beta 3 = 1.76198e-006
40
41
42 Asymptotic Correlation Matrix of Parameter Estimates
43 ( *** The model parameter(s) -t_0 -beta_0 -beta_l -beta_2
44 have been estimated at a boundary point, or have been specified by the user,
45 and do not appear in the correlation matrix )
46
47
48
49 c 1-1
50
51 beta 3-11
52
53
54
55
56 Variable
57
58
59
60
61
62
63 NA - Indicates that this parameter has hit a bound implied by some inequality constraint
64 and thus has no standard error.
65
66
67 Log (likelihood) # Param
68 Fitted Model -28.4387 5
69
70
71
72
73
74
This document is a draft for review purposes only and does not constitute Agency policy.
E-49 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
51
43
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Specified effect = 0.1
Confidence level = 0.9
Time = 104
Specified effect =
BMD =
BMDL =
BMDU =
Incidental Risk: DuoJej_Kroese_M3
Dose= 0.00
Dose= 0.54
(0
-Q
O
O .
0 -
CD _
(0
-Q
O
O _,
CD -
CD _
\ \ \ i i r
0 20 40 60 80 100
\\ \ \ i r
0 20 40 60 80 100
Time
Time
Dose= 1.81
Dose= 5.17
(0
O
CL
If)
CD
O
CD -
CD _
\
r
(0
o
it
o _
20 40 60 80 100
\ \ \ \ i r
0 20 40 60 80 100
Time
Time
15
16
17
18
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.. 20011
19
This document is a draft for review purposes only and does not constitute Agency policy.
E-50 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1 Male rat (Kroese etal. 2001): Skin or mammary gland basal cell tumors
2
3 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
5 Input Data File: SKinMamBasalKroeseM3.(d)
6
7
8
9
10
11
12
13
14 Dependent variable = CONTEXT
15 Independent variables = DOSE, TIME
16
17 Total number of observations = 208
18 Total number of records with missing values = 0
19 Total number of parameters in model = 6
20 Total number of specified parameters = 1
21 Degree of polynomial = 3
23
24
25 User specifies the following parameters:
26 t 0 0
27
28
29
30
31
32
33 Default Initial Parameter Values
34 c 1.38462
35 t_0 = 0 Specified
36 beta_0 = 3.84298e-005
37 beta_l = 1.06194e-028
38 beta_2 = 0
39 beta 3 = 6.84718e-006
40
41
42 Asymptotic Correlation Matrix of Parameter Estimates
43 ( *** The model parameter(s) -t_0 -beta_l -beta_2
44 have been estimated at a boundary point, or have been specified by the user,
45 and do not appear in the correlation matrix )
46
47
48
49
50
51
52
53
54
55
56 Parameter Estimates
57
58 Variable
59
60
61
62
63
64
65 NA - Indicates that this parameter has hit a bound implied by some inequality constraint
66 and thus has no standard error.
67
68
69 Log (likelihood) # Param
70 Fitted Model -47.3623 5
71
72
73
74
This document is a draft for review purposes only and does not constitute Agency policy.
E-51 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
U Total Expected Response
Benchmark Dose C
Risk Response =
Risk Type
Confidence level =
Time
1
1
13
Computation
Incidental
Extra
0. 9
104
Specified effect =
BMD =
BMDL =
BMDU =
Incidental Risk: Skin Mam Basal Kroese M3
Dose = 0.54
Dose= 1.81
.a
CD
.a
o
cq —
d
p —
d _
\ \ \ \ \
20 40 60 80
.a
CD
.a
o
o —
T
0 20 40 60 80
Time
Time
Dose= 5.17
.a
CD
.a
o
oq
d
p
d
\ \
0 20 40 60 80
Time
15
16
17
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)
18
This document is a draft for review purposes only and does not constitute Agency policy.
E-52 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1 Male rat (Kroese etal. 2001): Skin or mammary gland squamous cell tumors
2
3 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
5 Input Data File: SKinMamSCCKroeseMS.(d)
6
7
8
9
10
11
12
13
14 Dependent variable = CONTEXT
15 Independent variables = DOSE, TIME
16
17 Total number of observations = 208
18 Total number of records with missing values = 0
19 Total number of parameters in model = 6
20 Total number of specified parameters = 1
21 Degree of polynomial = 3
23
24
25 User specifies the following parameters:
26 t 0 0
27
28
29
30
31
32
33 Default Initial Parameter Values
34 3
35 t_0 = 0 Specified
36 beta_0 = 0
37 beta_l = 1.25256e-008
38 beta_2 = 1.25627e-030
39 beta 3 = 3.34696e-009
40
41
42 Asymptotic Correlation Matrix of Parameter Estimates
43 ( *** The model parameter(s) -t_0 -beta_0 -beta_2
44 have been estimated at a boundary point, or have been specified by the user,
45 and do not appear in the correlation matrix )
46
47 c beta 1 beta 3
48
49 c 1 -0.99 -1
50
51 beta_l -0.99 1
52 beta 3 -1 0.99
53
54
55
56
57 Variable
58
59
60
61
62
63
64
65
66
67
68 Log (likelihood) # Param
69 Fitted Model -27.652 5
70
71
72
73
74
This document is a draft for review purposes only and does not constitute Agency policy.
E-53 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
5
6
7
8
9
10
11
12
13
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect = 0.1
BMD = 2.6414
BMDL = 1.76931
BMDU = 4.42145
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 Inform ation —Benzo[a]pyren e
Incidental Risk: OralForstKroeseM3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose= 0.00 Dose= 0.54
D_
oq
czi
CN
czi
CD
CD
\
20
40 60
Time
I
80
100
oq
czi
>^
:t± CD
15 CD
CT5
-§ ^
CN
czi
CZI
CD
I
20
\
60
Time
\
80
100
Dose= 1.81
Dose= 5.17
CO
CD
!=^
= ^
15 CD
O
CD
I I I I I
20 40 60 80 100
Time
CO
CD
2
CN
CD
O
czi
I I I I I
20 40 60 80 100
Time
2
3
4
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
etal.. 20011
This document is a draft for review purposes only and does not constitute Agency policy.
E-55 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1 Male rat (Kroese et al. 2001): Kidney urothetial carcinomas
2
3 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
5 Input Data File: KidneyUrothelialCarKroeseM3.(d)
6
7
8
9
10
11
12
13
14 Dependent variable = CONTEXT
15 Independent variables = DOSE, TIME
16
17 Total number of observations = 208
18 Total number of records with missing values = 0
19 Total number of parameters in model = 6
20 Total number of specified parameters = 1
21 Degree of polynomial = 3
23
24
25 User specifies the following parameters:
26 t 0 0
27
28
29
30
31
32
33 Default Initial Parameter Values
34 c 1.63636
35 t_0 = 0 Specified
36 beta_0 = 3.78734e-027
37 beta_l = 1.59278e-027
38 beta_2 = 2.718e-024
39 beta 3=4.96063e-007
40
41
42 Asymptotic Correlation Matrix of Parameter Estimates
43 ( *** The model parameter(s) -t_0 -beta_0 -beta_l -beta_2
44 have been estimated at a boundary point, or have been specified by the user,
45 and do not appear in the correlation matrix )
46
47
48
49 c 1-1
50
51 beta 3-11
52
53
54
55
56 Variable
57
58
59
60
61
62
63 NA - Indicates that this parameter has hit a
64 bound implied by some inequality constraint
65 and thus has no standard error.
66
67
68 Log (likelihood) # Param
69 Fitted Model -11.3978 5
70
71
72
73
74
This document is a draft for review purposes only and does not constitute Agency policy.
E-56 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Dose = 0.00
£
2
ro
_Q
2
CL
in
o _
o
^—
0 _
8
0 _
8
0
1 1 1 1 1 1
0 20 40 60 80 100
Incidental Risk: Kidney_Kroese_M3
Dose = 0.54
in
o _
2.
CL
8
o _
8
o _
Time
\ I I I
20 40 60 80
Time
100
in
o _
8
o _
8
o _
Dose = 1.81
••••• ••••
in
o _
1= O
2
CL
\IIIIr^
0 20 40 60 80 100
Time
o _
8
o _
8
o _
Dose= 5.17
20
n I
40 60
Time
80 100
15
16
17
Figure E-15. Fit of multistage Weibull model to kidney urothelial tumors of
male rats exposed orally to benzo[a]pyrene (Kroese etal.. 2001)
18
This document is a draft for review purposes only and does not constitute Agency policy.
E-57 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1 Female rat (Kroese etal. 2001): Oral cavity or forestomach, squamous cell papilloma or
2 carcinoma
3
4 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
5 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
6 Input Data File: OralForstKroeseF3.(d)
8
9
10
11
12
13 The parameter betas are restricted to be positive
14
15 Dependent variable = CONTEXT
16 Independent variables = DOSE, TIME
17
18 Total number of observations = 208
19 Total number of records with missing values = 0
20 Total number of parameters in model = 6
21 Total number of specified parameters = 0
22 Degree of polynomial = 3
24
25
26
27
28
29 Default Initial Parameter Values
O\J c = 3.6
31 t_0 = 45.1111
32 beta_0 = 1.11645e-009
33 beta_l = 4.85388e-009
34 beta_2 = 0
35 beta 3 = 1.95655e-008
36
37 Asymptotic Correlation Matrix of Parameter Estimates
38 ( *** The model parameter(s) -beta_2
39 have been estimated at a boundary point, or have been specified by the user,
40 and do not appear in the correlation matrix )
41
42
43
44
45
46 t o
47
48 beta 0
49
50 beta 1
51
52 beta 3
53
54
55
56 95.0% Wald Confidence Interval
57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
58 c 3.52871 0.701117 2.15454
59 t_0 46.553 5.93306 34.9244
60 beta_0 1.53589e-009 5.40523e-009 -9.05817e-009
61 beta 1 7.57004e-009 2.9647e-008 -5.05369e-008
62
63
64
65 NA - Indicates that this parameter has hit a bound implied by some inequality constraint
66 and thus has no standard error.
67
68
69 Log (likelihood) # Param
70 Fitted Model -94.5119 6
71
72
73
This document is a draft for review purposes only and does not constitute Agency policy.
E-58 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2 C F I U Total Expected Response
3 DOSE
4 0 51 0 1 0
5 0.49 46 0 6 0
6 1.6 22 0 30 0
7 4.6 2 743 0
8
9 Minimum observation time for F tumor context =
10
11 Benchmark Dose Computation
12 Risk Response = Incidental
13 Risk Type = Extra
14 Confidence level = 0.9
15 Time = 104
16
Specified effect =
BMD =
BMDL = 0.328135
BMDU = 0.717127
This document is a draft for review purposes only and does not constitute Agency policy.
E-59 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Dose = 0.00
Incidental Risk: OralForstKroeseFS
points show nonparam. est. for Incidental (unfilled
Dose = 0.49
^2
£
p
Q.
00
O _
•sf —
O _
o -
o
..-, ,-^
.a
E
Q
Q_
00
o _
•st —
o _
o -
o
O CD
._, -•• ••»•-•«-•»'•• m
\ \ i i r
0 20 40 60 80 100
Time
\\ \ i i r
0 20 40 60 80 100
Time
Dose = 1.62
Dose = 4.58
•Q
CD
O
P
Q.
00
0 _
•^1" ~
o _
o -
0
ODCOD
f
•mtarn
^x
,.'•' s
•Q
CD
2
Q.
00
0 _
•^1" ~~
O _
o -
0
tjHMmt
09* o
/ •
/
/
/ •
--- '' • it ^^
\ \ i i r
0 20 40 60 80 100
Time
\ \ \ I I T
0 20 40 60 80 100
Time
2
3
4
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.
E-60 DRAFT—DO NOT CITE OR QUOTE
-------
Supplem en tal Inform ation —Benzo[a]pyren e
1 Female rat (Kroese etal. 2001]: Hepatocellular adenoma or carcinoma
2
3 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
4 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
5 Input Data File: LiverKroeseF3.(d)
6 Fri Apr 16 09:08:03 2010
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22 Total number of observations = 208
23 Total number of records with missing values = 0
24 Total number of parameters in model = 6
25 Total number of specified parameters = 0
26 Degree of polynomial = 3
27
28
29
30
31
32
33
34 Default Initial Parameter Values
35 c 3.6
36 t_0 = 31.7778
37 beta_0 = 0
38 beta_l = 4.9104e-031
39 beta_2 = 5.45766e-030
40 beta 3 = 3.44704e-008
41
42
43
44 ( *** The model parameter(s) -beta_0 -beta_l -beta_2
45 have been estimated at a boundary point, or have been specified by the user,
46 and do not appear in the correlation matrix )
47
48
49
50 c
51
52 t o
53
54 beta 3
55
56
57
58 95.0% Wald Confidence Interval
59 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
60 c 3.11076 0.549208 2.03434 4.18719
61 tO 38.6965 5.21028 28.4846 48.9085
62
63
64
65
66
67 NA - Indicates that this parameter has hit a bound implied by some inequality constraint
68 and thus has no standard error.
69
70
71 Log (likelihood) # Param
72 Fitted Model -228.17 6
73
74
This document is a draft for review purposes only and does not constitute Agency policy.
E-61 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3 C F I U Total Expected Response
4
5
6
7
8
9
10 Minimum observation time for F tumor context = 44
11
12
13
14
15
16
17
18
772/s document is a draft for review purposes only and does not constitute Agency policy.
E-62 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Dose = 0.00
Incidental Risk: Hepatocellular_Kroese_F3
points show nonparam. est. for Incidental (unfilled
Dose = 0.49
CD
0
Q.
oo -
o _
0 _
o -
0
.Q
CD
0
Q.
\ i i i i r
0 20 40 60 80 100
oo —
o _
0 _
o —
0
1 1 1 1 1 1
0 20 40 60 80 100
Time
Time
Dose = 1.62
Dose = 4.58
I I I I T
0 20 40 60 80 100
CD
2
Q.
00
O
CD
O
\\ \ i i
0 20 40 60 80 100
Time
Time
2
3
4
Figure E-17. Fit of multistage Weibull model to hepatocellular adenomas or
carcinomas in female rats exposed orally to benzo[a]pyrene (Kroese etal..
2001)
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Female rat (Kroese etal.. 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: DuoJejKroeseF3.(d)
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
This document is a draft for review purposes only and does not constitute Agency policy.
E-63 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
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
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
User specifies the following parameters:
t 0 0
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 )
1
-1
beta_3
-1
1
Parameter Estimates
Variable
- Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -13.8784 5
Total Expected Response
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
This document is a draft for review purposes only and does not constitute Agency policy.
E-64 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Incidental Risk: DuoJej_Kroese_F3
Dose= 0.00
Dose = 0.49
>s
~
JD
s
O
CL
LO
d _
o
^~
d _
o
d _
o
o
d
( • ( !•
>s
~
JD
s
O
CL
1 1 1 1 1 1
0 20 40 60 80 100
Time
LO
T—
d _
o
^~
d _
o
d _
o
o
d _
I I I I I I
0 20 40 60 80 100
Time
Dose= 1.62
Dose= 4.58
>s T-
-t-*
:= O
I O
o •<-
D- O
cp
ci _
o
cp
ci _
1 I I I I
20 40 60 80 100
Time
>s T-
-t-«
:= O
I O
O ^~
D- O
cp
ci _
o
cp
ci _
\ I I I I
20 40 60 80 100
Time
2
3
4
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..2Q01)
This document is a draft for review purposes only and does not constitute Agency policy.
E-65 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
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 etal.. 2001)
Data set
Males
Females
Tumor Site
Oral cavity/
forestomach
Liver
Duodenum/
jejunum
Skin/mammary
gland: basal cell
Skin/mammary
gland: squam. cell
Kidney
BMDooi
6.37 x 10"3
2.00 x 10"2
6.42 x 10"1
6.06 x 10"1
7.06 x 10"2
9.84 x 10"1
BMDLooi
2.86 x 10"3
5.30 x 10"3
4.21 x 10"2
4.24 x 10"2
2.11xlO"2
7.48 x 10"2
Sum, risk values at BMD0oi:
Risk Value3 at
BMDooi
1.57 x 10"1
5.00 x 10"2
1.56 x 10"3
1.65 x 10"3
1.42 x 10"2
1.02 x 10"3
2.25 x 10"1
BMDLooi
3.50 x 10"1
1.89 x 10"1
2.38 x 10"2
2.36 x 10"2
4.75 x 10"2
1.34 x 10"2
SD
1.17 x 10"1
8.42 x 10"2
1.35 x 10"2
1.33 x 10"2
2.03 x 10"2
7.51 x 10"3
Sum, SD2:
Overall SDb:
Upper bound on sum of risk estimates0:
Oral cavity/
forestomach
Liver
Duodenum/
jejunum
3.45 x 10"3
1.53 x 10"2
5.85 x 10"2
1.01 x 10"2
1.22 x 10"1
7.27 x 10"1
Sum, risk values at BMD0oi:
2.90 x 10"1
6.54 x 10"2
1.71 x 10"2
1.09 x 10"1
SD2
1.38 x 10"2
7.09 x 10"3
1.82 x 10"4
1.78 x 10"4
4.10xlO"4
5.64 x 10"5
2.17 x 10"2
1.47 x 10"1
Proportion
of Total
Variance
0.64
0.33
0.01
0.01
0.02
0.00
4.68 x 10"1
9.92 x 10"2
8.21 x 10"3
1.38 x 10"3
1.16 x 10"1
3.48 x 10"2
9.56 x 10"3
Sum, SD2:
Overall SD:
Upper bound on sum of risk estimates0:
1.35 x 10"2
1.21 x 10"3
9.13 x 10"5
1.48 x 10"2
1.22 x 10"1
0.91
0.08
0.01
3.09 x 10"1
4
5
6
7
"Risk value = 0.001/BMDL001.
"Overall SD = (sum, SD2)0'5.
°Upper bound on the overall risk estimate = sum of BMD001 risk values + 1.645 x overall SD.
8
9
10
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 Gulp. 1998)
Model
Stages
1
2
3
AIC
688.5
629.2
624.5
BMD10
0.104
0.102
0.127
BMDL10-BMDU10
0.071 - 0.179
Basis for Model Selection
Lowest AIC, best fit to low dose data
11
12
This document is a draft for review purposes only and does not constitute Agency policy.
E-66 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1 Female mice (Beland and Gulp. 19981: Alimentary tractsquamous cell tumors
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
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
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:\mswlO-09\benzo[a]pyrene_FemaleSquamF3i.(d)
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
c
t_0
beta_0
beta_l
beta_2
beta 3
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
9.54705
23.677
3.14019e-015
2.55825e-014
This document is a draft for review purposes only and does not constitute Agency policy.
E-67 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
BMD =
BMDL =
BMDU =
Incidental Risk: BaP_FenialeSqiiamF3i
points show nonpararn. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00
Dose= 0.10
CO
a
LO
o
-=T
CD
r-i
0
o
CD ~~l
^^0
1 1 1 1 1 1
0 20 40 60 80 100
CO
a
~ LO
15 o
fa
-0 TT
o . —
£ °
CN
0
o
CD
1 1 1 1 1 1
0 20 40 60 80 100
Time Time
Dose = 0.48
Dose= 2.32
CO
CD
>^
:= ^
15 CD
05
I 3
CN
CD
CD
CD
I I I I I
20 40 60 80 100
Time
CO
CD
i= 'P
15 CD
! s
CN
CD
CD
CD
I I I I I
20 40 60 80 100
Time
21
22
23
24
25
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..2Q01)
This document is a draft for review purposes only and does not constitute Agency policy.
E-68 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
1 E.2.3. Dose-Response Modeling for the Inhalation Unit Risk
2 Modeling Methods
3 As with the tumor data used for the oral slope factor (see Section E.2.1, Dose Response-
4 modeling for the Oral Slope Factor), there was earlier occurrence of tumors with increasing
5 exposure, and early termination of the high-dose group [Thyssen et al., 1981: see Appendix D for
6 study details]. The computer software program Multistage Weibull [U.S. EPA, 2010] was used as
7 described in the analysis of the oral carcinogenicity data. See Section E.2.1 for details of the
8 modeling methods.
9 Data adjustments prior to modeling
10 Thyssen etal. [1981] did not determine cause of death for any of the animals. Since the
11 investigators for the oral bioassays considered the same tumors to be fatal at least some of the time,
12 bounding estimates for the Thyssen etal. [1981] data were developed by treating the tumors
13 alternately as either all incidental or all fatal. In either case, therefore, an estimate of to (the time
14 between a tumor first becoming observable and causing death] could not be estimated. The data
15 analyzed are summarized in Table E-17. Group average TWA continuous exposures, based on
16 chamber air monitoring data and individual hamsters' time on study, of 0, 0.25,1.01, and 4.29
17 mg/m3 corresponded to the 0, 2,10, and 50 mg/m3 nominal study concentrations, respectively (U.S.
18 EPA. 1990a].
19
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
Table E-17. Individual pathology and tumor occurrence data for male Syrian
golden hamsters exposed to benzo[a]pyrene via inhalation for lifetime—
Thyssen etal. (1981)a
Nominal
Exposure
Concentration
(mg/m3)
0
2
Time on Study
17
39
45
79
83
85
86
88
89
90
101
102
103
106
108
109
112
115
116
122
123
124
125
127
132
14
35
53
59
71
78
80
85
87
88
93
98
99
102
103
108
111
113
114
115
116
Number
Examined
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Papillomas, Polyps, Papillary Polyps, Squamous Cell Carcinomas
Larynx
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ob
0
0
0
ob
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Pharynx
ob
0
0
0
0
ob
0
0
0
0
0
0
0
0
0
0
0
0
ob
0
0
0
0
ob
0
ob
0
0
0
0
0
0
0
0
0
0
ob
0
0
0
0
0
0
0
0
0
Trachea
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Esophagus
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Forestoma
ch
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Nasal
Cavity
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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Supplem en tal Inform ation —Benzo[a]pyren e
Nominal
Exposure
Concentration
(mg/m3)
10
50
Time on Study
117
120
122
133
31
32
52
67
73
76
80
85
94
100
102
105
111
113
114
115
116
117
118
122
124
125
20
21
25
29
30
34
36
37
40
41
43
47
48
51
56
57
60
63
64
66
68
70
71
72
Number
Examined
1
1
2
2
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
4
1
1
1
1
1
2
1
1
1
2
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Papillomas, Polyps, Papillary Polyps, Squamous Cell Carcinomas
Larynx
0
0
ob
0
0
0
0
0
0
0
1
0
1
0
0
1
0
0
1
1
0
1
3
1
1
0
ob
ob
ob
ob
ob
ob
ob
ob
lb
0
0
1
0
0
1
0
0
0
0
1
0
1
1
1
Pharynx
0
0
ob
0
0
0
0
0
0
2
0
0
0
0
1
1
1
1
1
ob
0
0
lc
0
1
0
ob
ob
ob
ob
ob
ob
ob
ob
lb
0
0
1
1
ob
1
1
1
0
1
1
1
1
1
1
Trachea
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
ob
ob
ob
ob
ob
ob
ob
ob
lb
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
Esophagus
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
Forestoma
ch
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
Nasal
Cavity
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
od
0
0
1
1
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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Supplem en tal Inform ation —Benzo[a]pyren e
Nominal
Exposure
Concentration
(mg/m3)
Time on Study
73
79
Number
Examined
2
4
Papillomas, Polyps, Papillary Polyps, Squamous Cell Carcinomas
Larynx
2
3
Pharynx
2
4
Trachea
0
1
Esophagus
0
1
Forestoma
ch
0
0
Nasal
Cavity
0
1
1
2
3
4
5
6
7
8
9
10
11
12
Histopathology incidence from U.S. EPA(1990a)
bTissue was not examined for one animal of total examined.
"Tissue was not examined for two animals of total examined.
dAn adenocarcinoma was observed in this tissue, but not included in the dose-response analysis because it was of
a different cell type than the other tumors listed. It was judged to be an isolated finding not clearly associated
with exposure.
Modeling Results
Table E-18 summarizes the modeling results supporting the derivation of an inhalation unit
risk value for BaP. The model outputs and graphs (Figures E-20 and E-21) following Table E-18
provide more details for the best-fitting models.
13
14
15
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 etal.. 1981)
Tumor Context
All tumors considered
incidental to cause of death
All tumors considered to be
cause of death
Model
Stages
1
2
1
2
3
AIC
58.0
47.9
327.3
302.9
299.0
BMD10
0.090
0.285
0.136
0.421
0.648
BMDL10
0.064
0.198
0.104
0.343
0.461
Basis for Model Selection
Lowest AIC, best fit to data (BMDU10 = 0.350)
Lowest AIC; best fit to data (BMDU10 = 0.719)
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Output for squamous cell neoplasia following inhalation exposure to benzo[a]pyrene: all
tumors considered incidental to cause of death
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Supplem en tal Inform ation —Benzo[a]pyren e
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
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
Total number of specified parameters = 1
Degree of polynomial = 2
c = 3.6
t 0 0
beta_0 = 1.18657e-031
beta_l = 1.49e-030
beta 2 = 6.10362e-008
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -t_0 -beta_0 -beta_l
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
1
-1
-1
1
Parameter Estimates
Variable
Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -19.967 4
Data Summary
Class
F I
Total Expected Response
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Specified effect = 0.1
Confidence level = 0.9
104
BMD
BMDL
BMDU
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Supplem en tal Inform ation —Benzo[a]pyren e
Incidental Risk: BaP-Thyssen_inc2st
Dose = 0.00
Dose = 0.25
.a
ro
.a
2.
a.
cq -
0 _
O _
q -
c> _
0 20
1 I I T
60 100
.a
co
.a
2
CL
cq -
0 _
0 _
60
100
Time
Time
Dose = 1.00
Dose = 4.29
s
oo —
o _
q -
o _
\ \ \ \ I I T
0 20 60 100
\\ I I I I T
0 20 60 100
Time
Time
2
3
4
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.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Output for respiratory tract tumors: all tumors considered to be cause of death
Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
Solutions are obtained using donlp2-intv, (c) by P. Spellucci
Input Data File: C:\msw\benzo[a]pyrene-Thyssen_allfatal_noU_3st.(d)
Total number of observations = 96
Total number of records with missing values = 0
Total number of parameters in model = 6
Total number of specified parameters = 1
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Supplem en tal Inform ation —Benzo[a]pyren e
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
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
User specifies the following parameters:
t 0 0
Default Initial Parameter Values
c = 4.5
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 )
1
-1
-1
1
Parameter Estimates
Variable
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Log(likelihood) # Param
Fitted Model -144.522 5
U Total
Minimum observation time for F tumor context =
=P
Confidence level =
Time
BMD
BMDL
BMDU
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Supplem en tal Inform ation —Benzo[a]pyren e
Fatal Risk: BaP-Thyssen_allfatal_iiQU_3st
Dose= 0.00
Dose= 0.25
^
EE
OJ
O
n
00
O
^
O
—
0
••••I •
.^
EE
O5
O
Q_
<-" 1 1 1 1 1 1 1
0 20 60 100
00
O
^
O
—
0
<-" I I I I I I I
0 20 60 100
Time
Time
Dose= 1.00
Dose= 4.29
>-,
15
oi
O
CL
oo
CD
CD ~
O
•
*
*,
•7
LJ 1 1 1 1 1 1 1
0 20 60 100
Probability
oo
CD
CD ~
O
J
<-» I I I I I I I
0 20 60 100
Time
Time
2
3
4
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.
5 E.2.4. Dose-Response Modeling for the Dermal Slope Factor
6 Modeling methods
7 For each endpoint, multistage models [BMDS; fU.S. EPA. 2012a): v 2.1] were fitted to the
8 data using the maximum likelihood method. Each model was tested for goodness-of-fit using a chi-
9 square goodness-of-fit test (x2 p-value < 0.05 indicates lack of fit). Other factors were used to
10 assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in
11 the vicinity of the BMR. The BMDL estimate (95% lower confidence limit on the BMD, as estimated
12 by the profile likelihood method) and AIC value were used to select a best-fit model from among the
13 models exhibiting adequate fit. The data modeled are summarized in Tables E-19 through E-22.
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1 Data adjustments prior to modeling
2 Roe etal. [1970] applied benzo[a]pyrene dermally for 93 weeks or until natural death; with
3 the exception of the highest dose group, each group still had approximately 20 animals at 86 weeks
4 (see Table E-19). The tumors were first observed in the lowest and highest dose groups during the
5 interval of weeks 29-43. Mice that died before week 29 were likely not at risk of tumor
6 development However, because tumor incidence and mortality were reported in 100-day
7 intervals, mice that had not been on study long enough to develop tumors were not easily
8 identifiable. Incidence denominators reflect the number of animals alive at week 29, and may thus
9 tend to lead to underestimates of tumor risk if the number of animals at risk has been
10 overestimated.
11 Schmidt etal. [1973] did not report survival information; instead, the authors provided
12 incidences based on the numbers of mice initially included in each dose group at the start of the
13 study. Overall latency was reported for the two high-dose groups in each series, but these data only
14 describe the survival of mice with tumors (animals were removed from study when a tumor
15 appeared]. It is not clear how long exposures lasted overall in each dose group, or whether some
16 mice may have died on study from other causes before tumors appeared. While it is possible that
17 no mice died during the study, all of the other studies considered here demonstrate mortality.
18 However, the data were modeled as reported, recognizing the possibility of underestimating risk
19 associated with incidences reported and lack of duration of exposure (see Table E-19].
20 Schmahl etal. [1977] reported that reduced numbers of animals at risk (77-88 mice per
21 dose group compared with the initial group sizes of 100] resulted from varying rates of autolysis.
22 No other survival or latency information was provided, so all exposures were assumed to have
23 lasted for 104 weeks and were modeled as reported. Given the results of the other studies, it seems
24 possible that the numbers at risk in each group may be overestimated, which could lead to an
25 underestimate of lifetime risk (see Table E-19].
26 Habs etal. [1980] reported age-standardized skin tumor incidence rates, indicating earlier
27 mortality in the two highest dose groups (2.8 and 4.6 [ig/application]. These rates were used to
28 estimate the number at risk in the dose-response modeling, by dividing the number of mice with
29 tumors by the age-standardized rates. Exposure lasted longer than 104 weeks in the two lower
30 exposure groups, at about 120 and 112 weeks, and until about 88 weeks in the highest exposure
31 group. Incidence in the two lower exposure groups may be higher than if the exposure had lasted
32 just 104 weeks. There was mortality in the first 52 weeks of exposure, about 10-15% in the three
33 exposure groups, but because there was no information concerning when tumors first appeared, it
34 is not possible to determine how much the early mortality may have impacted the number of mice
35 at risk in each group (see Table E-19].
36 Habs etal. [1984] reported mean survival times (with 95% CIs] for each dose group. The
37 CIs supported the judgment that the control and lower dose groups were treated for 104 weeks.
38 The higher dose group (4 |ig/application] was probably treated for <104 weeks, because the upper
39 95% confidence limit for the mean survival was approximately 79 weeks. However, since it was
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Supplem en tal Inform ation —Benzo[a]pyren e
1 not possible to estimate a more realistic duration for this group, an estimate of 104 weeks was used
2 (see Table E-19).
3 The studies by Poel [1960.1959] were conducted in male mice and used toluene as the
4 vehicle. In addition to a control group, the 1959 study included nine dose groups of one mouse
5 strain (C57L) and the 1960 study included seven dose groups of three other mouse strains. Both
6 studies demonstrated high mortality and tumor incidence at higher exposure levels. All C57L mice
7 in dose groups with >3.8 |ig/application died by week 44 of the study [Poel, 1959]. Therefore,
8 these five dose groups were omitted prior to dose-response modeling because of the relatively
9 large uncertainty in extrapolating cancer risk as a result of lifetime exposure. Four dose groups in
10 addition to control remained. Among these groups, mice survived and were exposed until
11 weeks 83-103. According to the lifespan ranges provided, at least one mouse in each dose group
12 died before the first appearance of tumor, but insufficient information was available to determine
13 how many; consequently, the incidence denominators were not adjusted. The dose-response data
14 are summarized in Table E-20.
15 For the Poel [1960] studies, all tumors in the highest three dose groups for each of the three
16 mouse strains had occurred by week 40. While these observations support concern for cancer risk,
17 as noted above such results are relatively uncertain for estimating lifetime cancer risk. In addition,
18 there was no information indicating duration of exposure for the mice without tumors; although
19 exposure was for lifetime, it might have been as short as for the mice with tumors. Overall, these
20 datasets did not provide sufficient information to estimate the extent of exposure associated with
21 the observed tumor incidence. Consequently, the experiments reported by Poel [1960] were not
22 used for dose-response modeling.
23 Grimmer etal. [1984]: Grimmer etal. [1983], studied female CFLP mice, using
24 acetone:DMSO (1:3) as the vehicle. Mean or median latency times were reported (as well as
25 measures of variability), but no information concerning overall length of exposure or survival was
26 included in the results. The total of tumor-bearing mice and the reported percentages of mice with
27 any skin tumors was reported and varied, at most, one animal from the number of animals initially
28 placed on study. The decreasing latency and variability and increasing tumor incidence with
29 increasing benzo[a]pyrene exposure suggests that exposure probably did not last for 104 weeks in
30 at least the high-dose group, but the available information did not provide duration of exposure.
31 The data reported were modeled under the assumption that at least some animals in each group
32 were treated and survived until week 104 (see Table E-21).
33 Sivaketal. [1997] exposed male C3H/HeJ mice dermally to benzo[a]pyrene in
34 cyclohexanone/acetone (1:1) for 24 months, and reported mean survival times for each group. All
35 high-dose mice died before the final sacrifice. From the information provided, it is apparent that
36 the animals in the control and lower two dose groups survived until study termination at week 104.
37 The study authors did not report how long treatment in the highest dose group lasted, but
38 estimation of the figure from the publication suggest that exposure duration was 74 weeks (see
39 Table E-22).
40
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Supplem en tal Inform ation —Benzo[a]pyren e
1
2
3
4
Table E-19. 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). Habs etal. (1980). Habs etal.
f!9841
Study
Roe etal.
(1970)a'b
Schmidt et
al. (1973)°
Schmahl et
al. (1977)°
Habs etal.
(1980)c'f
Habs etal.
(1984)c
Mouse
Strain
Swiss
NMRI
Swiss
NMRI
NMRI
NMRI
Dose (u,g)
0 (acetone)
0.1
0.3
1
3
9
0 (acetone)
0.05
0.2
0.8
2
0 (acetone)
0.05
0.2
0.8
2
0 (acetone)
1
1.7
3
0 (acetone)
1.7
2.6
4.6
0 (acetone)
2
4
Average
Daily
Dose
(Hg/d)
0
0.04
0.13
0.43
1.29
3.86
0
0.01
0.06
0.23
0.57
0
0.01
0.06
0.23
0.57
0
0.29
0.49
0.86
0
0.49
0.74
1.31
0
0.57
1.14
First
Appearance
of Tumor
(wks)
29-43
57-71
43-57
29-43
53e
76e
58e
61e
NR
NR
NR
NR
NR
NR
NR
NR
Length of
Exposure
(wks)
93
93
93
93
93
93
104d
104
104
104
104
104
104
104
104
104
104
104
104
104
128
120
112
88
104
104
104
Lifetime
Average
Daily Dose
(ug/d)
0.00
0.03
0.09
0.31
0.92
2.76
0
0.01
0.06
0.23
0.57
0
0.01
0.06
0.23
0.57
0
0.29
0.49
0.86
0
0.49
0.74
0.80
0
0.57
1.14
Skin Tumor
Incidence (All Types)
0/49 (0%)
1/45 (2%)
0/46 (0%)
1/48 (2%)
8/47 (20%)
34/46 (74%)
0/100 (0%)
0/100 (0%)
0/100 (0%)
2/100 (2%)
30/100 (30%)
0/80 (0%)
0/80 (0%)
0/80 (0%)
5/80 (6%)
45/80 (56%)
1/81 (1%)
11/77 (14%)
25/88 (28%)
45/81 (56%)
0/35 (0%)
8/34 (24.8%)
24/27 (89.3%)
22/24 91.7%)
0/20 (0%)
9/20 (45%)
17/20 (85%)
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6
7
8
9
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11
12
13
14
15
16
17
18
19
aDoses were applied 3 times/wk 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/wk to shaved skin of the back. Mice were exposed until natural death or until they
developed a carcinoma at the site of application.
Exposure periods not reported were assumed to be 104 weeks; indicated in italics.
eCentral 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table E-20. Skin tumor incidence, benign or malignant, in C57L male mice
dermally exposed to benzo[a]pyrene; data from Poel (1959)
Study
Poel (1959)
Mouse
Strain
C57L
Dose (u.g)a
0 (toluene)
0.15
0.38
0.75
3.8
Average
Daily Dose
(ug/d)
0
0.06
0.16
0.32
1.63
First
Appearance of
Tumor (wks)
_
42
24
36
21-25
Length of
Exposure
(wks)
92
98
103
94
82
Lifetime
Average Daily
Doseb
0.00
0.05
0.16
0.24
0.80
Skin Tumor
Incidence (All
Types)c
0/33 (0%)
5/55 (9%)
11/55 (20%)
7/56 (13%)
41/49 (84%)
3
4
5
6
7
8
aDoses were applied to interscapular skin 3 times/wk for up to 103 wks or until time of appearance of a grossly
detected skin tumor. See Table E-15 for data of five highest dose groups (19-752 ug) in which all mice died by
wk 44. These groups were not considered for dose-response modeling.
bSee Section 2.5.2 of Toxicological Review for discussion of extrapolation to lifetime average daily doses.
cTumors were histologically confirmed as epidermoid carcinomas.
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10
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Table E-21. 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)
Study
Grimmer et
al. (1983)
Grimmer et
al. (1984)
Dose (ug)a
0(1:3
acetone:DMSO)
3.9
7.7
15.4
0(1:3
acetone:DMSO)
3.4
6.7
13.5
Average
Daily Dose
(ug/d)
0
1.1
2.2
4.4
0
0.97
1.9
3.9
Mean or
Median Time
of Tumor
Appearance
(wks)
74.6 ± 16.8d
60.9 ± 13.9
44.1 ±7.7
61 (53-65)e
47 (43-50)
35 (32-36)
Length of
Exposure
(wks)b
104
104
104
104
104
104
104
104
Lifetime
Average Daily
Dose
(ug/d)
0
1.1
2.2
4.4
0
0.97
1.9
3.9
Skin Tumor
Incidence (All
Types)c
0/80 (0%)
22/65 (34%)
39/64 (61%)
56/64 (88%)
0/80 (0%)
43/64 (67%)
53/65 (82%)
57/65 (88%)
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13
14
15
16
17
18
19
a Indicated doses were applied twice/week to shaved skin of the back for up to 104 weeks.
b Assumed exposure period is indicated in italics.
c Incidence denominators were calculated from reported tumor-bearing animals and reported percentages.
dMean±SD.
e Median and 95% confidence limit.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table E-22. Skin tumor incidence, benign or malignant, in male C3H/HeJ mice
dermally exposed to benzo[a]pyrene; data from Sivaketal. (1997)
Dose (ug)a
0 (1:1 cyclohexanone/acetone)
0.05
0.5
5.0
Average
Daily Dose
(ug/d)
0
0.01
0.14
1.4
First
Appearance
of Tumor
(wks)
-
-
NR
~43
Length of
Exposure
(wks)b
104
104
104
74
Lifetime
Average Daily
Dose
(ug/d)
0.0
0.01
0.14
0.51
Skin Tumor
Incidence (All
Types)b
0/30 (0%)
0/30 (0%)
5/30 (17%)
27/30 (90%)
3
4
5
6
7
8
9
10
11
12
Indicated doses were applied twice/week to shaved dorsal skin.
bNumber of skin tumor-bearing mice.
NR = not reported.
Modeling Results
The modeling results are summarized in Table E-23. The modeling details are provided
with Figures E-22 through E-33.
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Table E-23. Summary of BMD model selection and modeling results for best-
fitting multistage models, for multiple data sets of skin tumors in mice
following dermal benzo[a]pyrene exposure
Data set
Poel (1959)
MaleC57L
Roe etal. (1970)
Female Swiss
Schmidt et al.
(1973)
Female NMRI
Schmidt et al.
(1973)
Female Swiss
Schmahl etal.
(1977)
Female NMRI
Habs etal. (1980)
Female NMRI
Habs etal. (1984)
Female NMRI
Grimmer et al.
(1983)
Female CFLP
Grimmer et al.
(1984)b
Female CFLP
Sivaket al.
(1997)
Male CeH/HeJ
Model
Multistage 1°
Multistage 2°
Multistage 3°
Multistage 4°
Multistage 1°
Multistage 2°
Multistage 3°
Multistage 1°
Multistage 2°
Multistage 3°
Multistage 1°
Multistage 2°
Multistage 3°
Multistage 4°
Multistage 1°
Multistage 2°
Multistage 3°
Multistage 1°
Multistage 2°
Multistage 3°
Multistage 1°
Multistage 2°
Multistage 1°
Multistage 2°
Multistage 3°
Multistage 1°
LogLogistic
Dichotomous-
Hill
LogProbit
Gamma,
Weibull
Logistic
Probit
Multistage 1°,
high dose
dropped
Multistage 1°
Multistage 2°
Multistage 3°
Goodness-of-
Fit
P-
value
0.011
0.027
0.053
0.068
0.110
0.485
0.485
0.008
0.609
0.999
<0.01
0.514
0.983
0.983
0.136
0.939
0.939
0.0
0.009
0.207
0.577
1.000
0.850
0.972
0.972
0.003
0.919
1.000
0.047
0.003
0.0
0.0
0.499
0.059
0.998
0.998
AIC
191.5
188.6
186.9
186.2
131.1
123.6
123.6
162.7
147.4
143.9
178.0
153.3
151.3
151.3
298.4
296.3
296.3
96.5
84.4
76.7
48.4
47.6
219.9
221.1
221.1
205.3
195.8
197.7
200.2
205.3
250.5
255.4
57.8
48.6
48.6
BMD10
(Hg/d)
0.070
0.134
0.127
0.123
0.318
0.748
0.748
0.256
0.329
0.381
0.116
0.216
0.282
0.282
0.140
0.233
0.233
0.063
0.198
0.294
0.078
0.171
0.245
0.292
0.292
0.132
1.07
0.902
1.33
0.132
2.03
2.29
1.21
0.036
0.109
0.109
BMDL10
(Hg/d)
0.057
0.078
0.078
0.077
0.249
0.480
0.480
0.194
0.287
0.326
0.093
0.192
0.223
0.223
0.117
0.149
0.143
0.050
0.143
0.215
0.056
0.060
0.208
0.213
0.213
0.113
0.479
0.533
1.11
0.113
1.76
2.03
1.01
0.026
0.058
0.052
Basis for Model Selection3
No significant improvement in model
fit with higher stage
No significant improvement in model
fit with higher stages
No significant improvement in model
fit with higher stages
No significant improvement in model
fit with higher stage
No significant improvement in model
fit with higher stage
Only model with adequate fit
No significant improvement in model
fit with higher stage
No significant improvement in model
fit with higher stages
(Higher stages did not provide better fit)
Lowest AIC among adequately fitting
models.
(Same as Multistage 1°)
No significant improvement in model
fit with higher stage
Figure
Number
E-22
E-23
E-24
E-25
E-26
E-27
E-28
E-29
E-30
E-31
E-32
E-33
4
5
6
7
8
Adequate 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).
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
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
0.7
0.8
2
3
Figure E-22. Fit of multistage model to skin tumors in C57L mice exposed
dermally to benzo[a]pyrene (Poel. 1959).
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Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_1959_MultiCanc3_0.1.(d)
Gnuplot Plotting File:
\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_1959_MultiCanc3_0.l.plt
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
Default Initial Parameter Values
Background = 0.0449589
Beta(l) = 0.490451
Beta (2) = 0
Beta(3) = 2.68146
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Asymptotic C
orrelation Matrix of Parameter Estimates
( *** The model parameter (s) -Beta (2)
have b
and do
Background
Background 1
Beta(l) -0.87
Beta(3) 0.74
Variable
Background
Beta (1)
Beta (2)
Beta (3)
* - Indicates that this
een estimated at a boundary point, or have been specified by the user
not appear in the correlation matrix )
Beta(l) Beta(3)
-0.87 0.74
1 -0.92
-0.92 1
Parameter Estimates
95.0% Wald Confidence Interval
Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
0 . 0176699 * * *
0.79766 * * *
0 * * *
2.17146 * * *
value is not calculated.
Analysis of Deviance Table
Model Log (likelihood) # Param's Deviance Test d.f. P-value
Full model
Fitted model
Reduced model
AIC:
Dose Est. Prob
0 . 0000 0 . 0177
0 0500 0 0563
0.1600 0.1430
0.2400 0.2128
0.8000 0.8293
ChiA2 = 5.88 d.f.
-87.1835 5
-90.4265 3 6.48606 2 0.03905
-141.614 1 108.86 4 <.0001
186.853
Goodness of Fit
Scaled
Expected Observed Size Residual
0 . 583 0 . 000 33 -0.770
3.098 5. 000 55 1. 112
7.866 11.000 55 1.207
11.917 7.000 56 -1.605
40. 635 41. 000 49 0. 139
= 2 P-value = 0.0528
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
Taken together, (0.0777
interval for the BMD
Multistage Cancer Slope
0.1
Extra risk
0. 95
0. 126567
0. 0777875
0.272961
875, 0.272961) is a 90 % two-sided confidence
Factor = 1.28555
72
This document is a draft for review purposes only and does not constitute Agency policy.
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-a
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Supplem en tal Inform ation —Benzo[a]pyren e
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o
Variable Estimate Std. Err.
Background
Beta(1)
Beta(2)
Beta (3)
Beta (4)
Beta (5)
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Goodness of Fit
Prob.
d.f. =3
BMD =
BMDL =
BMDU =
% two-sided confidence
This document is a draft for review purposes only and does not constitute Agency policy.
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o
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o
=5
ro
0.4
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
BMDL
BMD
0.1
0.2
0.3
dose
0.4
0.5
3
4
Figure E-24. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Schmidt et al.. 1973).
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Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data
::\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidtl973femaleNMRI\2MulSchMS_.(d)
Gnuplot Plotting
::\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidtl973femaleNMRI\2MulSchMS_.pit
File
File
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
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(l) = 0
Beta(2) = 1.11271
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 )
Variable
Background
Beta(1)
Beta(2)
Estimate
0
n
Model
Full model
Fitted model
Reduced model
Goodness of Fit
Est. Prob.
d.f. = 4
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0. 95
0.329464
0.286624
0.384046
% two-sided confidence
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
I
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o
'•8
ro
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0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
0.1
0.2
0.3
dose
0.4
0.5
3
4
Figure E-25. Fit of multistage model to skin tumors in female Swiss mice
exposed dermally to benzo[a]pyrene (Schmidt et al.. 1973).
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Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidtl973swissmice\3MulSchMS_.(d)
Gnuplot Plotting
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidtl973swissmice\3MulSchMS_.pit
File
File
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
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(l) = 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)
1 -0.99
-0.99 1
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Model
Full model
Fitted model
Reduced model
1
AIC: 151.326
Goodness of Fit
Est._Prob. Expected Observed
ChiA2 = 0.16 d.f. = 3
Benchmark Dose Computation
0.1
Extra risk
Confidence level = 0.95
BMD =
BMDL =
BMDU =
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
I
c
o
••§
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0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
0.1
0.2
0.3
0.4
dose
0.5
0.6
0.7
0.8
0.9
2
3
Figure E-26. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Schmahl et al.. 1977).
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File
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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
This document is a draft for review purposes only and does not constitute Agency policy.
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**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l) Beta(2)
Background 1 -0.67 0.47
Beta(l) -0.67 1 -0.94
Beta(2) 0.47 -0.94 1
Parameter Estimates
Variable Estimate Std. Err.
Background
Beta(1)
Beta(2)
- Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
P-value
Prob.
Goodness of Fit
Expected Observed Size
81
88
81
d.f. = 1
BMD =
BMDL =
BMDU =
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[a]pyren e
Multistage Cancer Model with 0.95 Confidence Level
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
0.7
0.8
2
3
Figure E-27. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Habs etal.. 1980).
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Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: M:\_BMDS\msc_BAP_HABS1980_MultiCanc3_0.1.(d)
Gnuplot Plotting File: M:\_BMDS\msc_BAP_HABS1980_MultiCanc3_0.1.plt
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
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 4.23649
Beta (3) = 0
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
have been estimated
(s) -Background -Beta(l) -Beta (2)
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
Variable Estimate
Background 0
Beta(l) 0
Beta (2) 0
Beta(3) 4.1289
* - Indicates that this value is not
Analysis of
Model Log (likelihood) #
Full model -34.8527
Fitted model -37.3373
Reduced model -82.5767
AIC: 76.6745
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
•^ ^ ^
Tt- Tt- Tt-
* ^ *
* * *
calculated.
Deviance Table
Param's Deviance Test d.f. P-value
4
1 4.96903 3 0.1741
1 95.4478 3 <.0001
Goodness of Fit
Dose Est. Prob. Expected
0 . 0000 0 . 0000 0 . 000
0.4900 0.3848 13.082
0.7400 0.8123 21.933
0.8000 0.8792 21.102
ChiA2 = 4.56 d.f. =3 P
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)
interval for the BMD
Multistage Cancer Slope Factor =
Scaled
Observed Size Residual
0 . 000 35 0 . 000
8.000 34 -1.791
24.000 27 1.019
22.000 24 0.563
-value = 0.2067
is a 90 % two-sided confidence
0.46479
67
This document is a draft for review purposes only and does not constitute Agency policy.
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0.8
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c
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
BMDL BMD
0
0.2
0.4
0.6
dose
0.8
2
3
Figure E-28. Fit of multistage model to skin tumors in female NMRI mice
exposed dermally to benzo[a]pyrene (Habs etal.. 1984).
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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
The form of the probability function is:
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
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(l) = 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(l) 1
Parameter Estimates
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
AIC:
d.f. =2
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0. 95
0.0778926
0.0558466
0.111853
% two-sided confidence
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[a]pyren e
Multistage Cancer Model with 0.95 Confidence Level
0.8
0.6
!
I °-4
0.2
Multistage Cancer
Linear extrapolation
4.5
2
3
Figure E-29. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al.. 1983).
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Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmerl983CFLPmice\lMulGriMS_.(d)
Gnuplot Plotting
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmerl983CFLPmice\lMulGriMS_.pit
File
File
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
This document is a draft for review purposes only and does not constitute Agency policy.
E-97 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
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**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
Default Initial Parameter Values
Background = 0
Beta(l) = 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)
Variable
Background
Beta(1)
- Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
P-value
Prob.
d.f. =3
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0. 95
0.244816
0.208269
0.289606
% two-sided confidence
This document is a draft for review purposes only and does not constitute Agency policy.
E-98 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Multistage Cancer Model with 0.95 Confidence Level
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
EM PL
3.5
2
3
Figure E-30. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al.. 1984).
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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(l) = 0.502556
This document is a draft for review purposes only and does not constitute Agency policy.
E-99 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
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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(l)
Beta(l) 1
Variable
Background
Beta(l)
Parameter Estimates
Estimate Std. Err.
0 *
0.796546 *
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
AIC:
Log(likelihood)
-95.8385
-101.643
-175.237
205.287
# Param's Deviance Test d.f.
4
1 11.61 3
1 158.797 3
P-value
0.008846
<.0001
Dose
0.0000
0.9700
1.9100
3.9000
Est. Prob.
0.0000
0.5382
0.7816
0.9552
Gooc
Expected
0.000
34.446
50.804
62.091
Iness of Fi1
Observed
0.000
43.000
53.000
57.000
Size
65
64
65
65
Scaled
Residual
0.000
2.145
0.659
-3.054
ChiA2 = 14.36
d.f. = 3
P-value = 0.0025
Benchmark Dose Computation
Specified effect =
Risk Type =
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
0.132272
0.113427
0.154848
Taken together, (0.113427, 0.154848) is a 90
interval for the BMD
% two-sided confidence
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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Supplem en tal Inform ation —Benzo[a]pyren e
Log-Logistic Model with 0.95 Confidence Level
0.6
0.4
0.2
Log-Logistic
BMDL
BMD
0.5
1.5
2
dose
2.5
3.5
2
3
Figure E-31. Fit of log-logistic model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al.. 1984).
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Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data
::\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmerl984_Grimmerl984_0.70u.(d)
Gnuplot Plotting
::\Usepa\BMDS21\Data\lnl_benzo[a]pyrene_Grimmerl984_Grimmerl984_0.70u.plt
File
File
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
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 b
and do
intercept
intercept
slope
Variable
background
intercept
slope
1
-0. 68
* - Indicates that this
Model
Full model
Fitted model
Reduced model
AIC:
Dose Est
0.0000 0.
0 . 9700 0 .
1.9100 0.
3 . 9000 0 .
ChiA2 = 0.17
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
een estimated at a boundary point, or have been specified by the use
not appear in the correlation matrix )
slope
-0. 68
1
Parameter Estimates
95.0% Wald Confidence Interval
Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
0 * * *
0.783559 * * *
0 . 922655 * * *
value is not calculated.
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
. Prob
0000
6804
7991
8849
d.f.
-95.8385 4
-95.9236 2 0.17031 2 0.9184
-175.237 1 158.797 3 <.0001
195. 847
Goodness of Fit
Scaled
Expected Observed Size Residual
0.000 0.000 65 0.000
43.543 43.000 64 -0.146
51.941 53.000 65 0.328
57.516 57.000 65 -0.200
= 2 P-value = 0. 9190
Computation
=
=
=
=
=
0 . 7
Extra risk
0. 95
1.07152
0.478669
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[a]pyren e
0.8
0.6
0.4
0.2
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
1.5
dose
2
3
4
Figure E-32. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al.. 1984). highest dose
dropped.
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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
This document is a draft for review purposes only and does not constitute Agency policy.
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Asympt
( **
*
otic Correlati
The mod
on Matrix
el param
eter
have been estimated
and do
(s)
at a
of Parameter Estimates
-Background
not appear in the
boundary point, or have been specified by the user
correlation matrix )
Beta (1)
Beta (1)
1
Parameter
Variable
Estimat
Background
Beta (1)
* - Indicates that this
Mod
Full
Fitted
Reduced
el
model
model
model
AIC:
0. 99711
value i
Analysi
Estimates
95.0% Wald Confidence Interval
e Std. Err. Lower Conf. Limit Upper Conf. Limit
0
7
s
g
not
of
Log (likelihood) #
_
_
71.5928
72 . 2756
-134.46
146.551
Tt- Tt- Tt-
* * *
calculated.
Deviance Table
Param
3
1
1
Goodnes
Chi
Dose
0.0000
0 9700
1. 9100
A2 = 1
Est
.39
0.
0 .
0.
Benchmark Dose
Specified
Risk
Type
Confidence
. Prob.
0000
6199
8511
d.f.
Expected
0
39
55
000
671
322
s
's Deviance Test d.f. P-value
1.36568 2 0.5052
125.735 2 <.0001
of Fit
Scaled
Observed Size Residual
0
43
53
= 2 P-value
000 65 0.000
000 64 0 . 857
000 65 -0.809
= 0.4992
Computation
effect =
0
= Extra ri
leve
1
=
BMD =
BMDL =
BMDU =
Taken together.
inte
rval f
Multistage
or th
Cane
(1. 00734
0
er
BMD
Slope
0.
9
7
sk
5
1.20745
1.007
1.457
, 1.457
Factor
34
89
8
=
9) i
s a 9
0
0
% two-sided confidence
6949
63
This document is a draft for review purposes only and does not constitute Agency policy.
E-104 DRAFT—DO NOT CITE OR QUOTE
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Supplem en tal Inform ation —Benzo[a]pyren e
Multistage Cancer Model with 0.95 Confidence Level
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
0.5
2
3
Figure E-33. Fit of multistage model to skin tumors in male CeH/HeJ mice
exposed dermally to benzo[a]pyrene (Sivak et al.. 1997).
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Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivak!993_MultiCanc2_0.1.(d)
Gnuplot Plotting File
::\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Sivakl993_MultiCanc2_0.l.plt
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Parameter Estimates
Std. Err.
Variable
Background
Beta(1)
Beta(2)
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
P-value
Est. Prob.
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0. 95
0.108575
0.058484
0.129641
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Alternative Approaches for Cross-Species Scaling of the Dermal Slope Factor
2 Several publications that develop a dermal slope factor for benzo[a]pyrene are available in
3 the peer-reviewed literature [Knaflaetal.. 2011: Knaflaetal.. 2006: Hussainetal.. 1998: LaGoy and
4 Quirk. 1994: Sullivan etal.. 19911 With the exception of the Knaflaetal. (2011). none of these
5 approaches applied quantitative adjustments to account for interspecies differences, though the
6 proposed slope factors were developed to account for human risk. Knaflaetal. (2011) qualitatively
7 discuss processes that could affect the extrapolation between mice and humans, including skin
8 metabolic activity adduct formation, stratum corneum thickness, epidermal thickness, etc.
9 Ultimately, the authors apply an adjustment based on the increased epidermal thickness of human
10 skin on the arms and hands compared to mouse interscapular epidermal thickness. They
11 hypothesize thatthe carcinogenic potential of benzo[a]pyrene may be related to the thickness of
12 the epidermal layer.
13 Because there is no established methodology for cross-species extrapolation of dermal
14 toxicity, several alternative approaches were evaluated. Each approach begins with the POD of
15 0.066 [ig/day that was based on a 10% extra risk for skin tumors in male mice. Based on the
16 assumptions of each approach, a dermal slope factor for humans is calculated. The discussion of
17 these approaches uses the following abbreviations:
18
19 DSF = dermal slope factor
20 PODM = point of departure (for 10% extra risk) from mouse bioassay, in [ig/day
21 BWM= mouse body weight = 0.035 kg (assumed)
22 BWn = human body weight = 70 kg (assumed)
23 SAn = total human surface area = 19,000 cm2 (assumed)
24 SAM = total mouse surface area = 100 cm2 (assumed)
25 Approach 1. No interspecies adjustment to daily applied dose (POD) in mouse model
26 Under this approach, a given mass of benzo[a]pyrene, applied daily, would pose the same risk in an
27 animal or in humans, regardless of whether it is applied to a small surface area or to a larger
28 surface area at a proportionately lower concentration.
29
30
31 DSF = 0.1/PODM
32 DSF = 0.1/0.068 ^g/day = 1.5 (ng/day)-1
33
34
35 Assumptions: The same mass of benzo[a]pyrene, applied daily, would have same potency in mice as
36 in human skin regardless of treatment area.
37 Approach 2. Cross-species adjustment based on whole body surface-area scaling
38 Under this approach, animals and humans are assumed to have equal lifetime cancer risk with
39 equal average whole body exposures in loading units (|ig/cm2-day). As long as doses are low
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1 enough that risk is proportional to the mass of applied compound, the daily dermal dose of
2 benzo[a]pyrene can be normalized over the total surface area.
3
4
5 POD Og/cm2-day) = PODM/sA Og/cm2-day) = PODM Og/day) / SAM (cm*)
6 POD = (0.068 ng/day) / 100 cm2 = 0.00068 [ig/cm2-day
7 DSF = 0.1/(0.00068 |ig/cm2-day) * 147 (ng/cm2-day) *
8
9
10 Assumptions: Mouse and human slope factors are equipotent if total dermal dose is averaged over
11 equal fractions of the entire surface area. Tumor potency of benzo[a]pyrene is assumed to be
12 related to overall dose and not dose per unit area. For example, a human exposed to 0.01 [J.g/day
13 on 10 cm2 would be assumed to have the same potential to form a skin tumor as someone treated
14 with 0.01 [ig/day over 19,000 cm2 (assumed human surface area).
15 Approach 3. Cross-species adjustment based on body weight
16 Under this approach, a given mass of benzo[a]pyrene is normalized relative to the body weight of
17 the animal or human.
18
19
20 PODM/ BWM= 0.068 ng/0.035 kg-day = 1.9 ng/kg-day
21 DSF = 0.1/1.9 ng/kg-day = 0.051 (ng/kg-day) *
22
23
24 Assumptions: The potency of point of contact skin tumors is related to bodyweight and humans and
25 mice would have an equal likelihood of developing skin tumors based on a dermal dose per kg
26 basis.
27
28 Issues: Skin cancer following benzo[a]pyrene exposure is a local effect and not likely dependent on
29 body weight
30
31 Approach 4. Cross-species adjustment based on allometric scaling using body weight to the
32 3/4 power
33 Under this approach, rodents and humans exposed to the same daily dose of a carcinogen, adjusted
34 for B W3/4, would be expected to have equal lifetime risks of cancer. That is, a lifetime dose
35 expressed as |ig/kg3/4-day would lead to an equal risk in rodents and humans. This scaling reflects
36 the empirically observed phenomena of more rapid distribution, metabolism, and clearance in
37 smaller animals. The metabolism of benzo[a]pyrene to reactive intermediates is a critical step in
38 the carcinogenicity of benzo[a]pyrene, and this metabolism occurs in the skin.
39
40
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1 POD (jig/day) = PODM (^g/day) x (BWH / BWM)3/4
2 POD (tig/day) = 0.068 ng/day x (70 kg / 0.035 kg)3/4 = 20.3 ng/day
3 DSF = 0.1/(20.3 Lig/day) * 0.0049 (ng/day) *
4
5
6 Assumptions: Risk at low doses of benzo[a]pyrene is dependent on absolute dermal dose and not
7 dose per unit of skin, meaning that a higher exposure concentration of benzo[a]pyrene contacting a
8 smaller area of exposed skin could carry the same risk of skin tumors as a lower exposure
9 concentration of benzo[a]pyrene that contacts a larger area of skin.
10
11 Issues: It is unclear if scaling of doses based on bodyweight ratios will correspond to differences in
12 metabolic processes in the skin of mice and humans.
13 Synthesis of the alternative approaches to cross-species scaling
14 A comparison of the above approaches is provided in Table E-24. The lifetime risk from a nominal
15 human dermal exposure to benzo[a]pyrene over a 5% area of exposed skin (approximately 950
16 cm2), estimated at 1 x 1Q-4 [ig/day, is calculated for each of the approaches in order to judge
17 whether the method yields risk estimates that are unrealistically high.
18 Other potential interspecies adjustments
19 The above discussion presents several mathematical approaches that result from varying
20 assumptions about what is the relevant dose metric for determining equivalence across species.
21 Biological information (that is not presently comprehensive or detailed enough to develop robust
22 models) that could be used in future biologically based models for cross-species extrapolation
23 include:
24 a. Quantitative information on interspecies differences in partitioning from exposure medium
25 to the skin and absorption through the skin
26 b. Thickness of the stratum corneum between anatomical sites and between species
27 c. Thickness of epidermal layer
28 d. Skin permeability
29 e. Metabolic activity of skin
30 f. Formation of DNA adducts in skin
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Table E-24. Alternative approaches to cross-species scaling
Approach
1. Mass-per-
day scaling
2. Surface-
area scaling
3. Body-
weight scaling
4. Allometric
scaling (BW3/4)
Assumptions
Equal mass per day (u.g/d), if applied to equal areas of skin (cm ), will affect similar
numbers of cells across species. Cancer risk is proportional to the area (cm2)
exposed if the loading rate (u.g/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 (u.g/cm2-day) and skin area exposed (cm2) that have the
same product when multiplied, will result in the same risk.
Equal mass per day (u.R/d), if applied to equal 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 (u.g/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.
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 (u.g/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 (u.g/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 (u.g/day) and body weight
(kg) that have the same result when divided will result in the same risk.
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.
Dose metric
M-g/day
u.g/cm2-day
u.g/kg-day
Mg/day
Dermal Slope
Factor
1.5 per u.g/d
147 per
u.g/cm2-d
0.051 per
u.g/kg-d
0.0049 per
Hg/d
Risk at nominal
exposure
(0.0004 ug/day)a
6 x 10"4
3 x 10"6
3 x 10"7
2 x 10"6
r>
C
§
Q
I
i
Q.
I
§
O
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).
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Supplem en tal Inform ation —Benzo[a]pyren e
i APPENDIX F. DOCUMENTATION OF
2 IMPLEMENTATION OF THE 2011 NATIONAL
3 RESEARCH COUNCIL RECOMMENDATIONS
4 Background: On December 23, 2011, The Consolidated Appropriations Act, 2012, was
5 signed into law2. The report language included direction to EPA for the IRIS Program related to
6 recommendations provided by the National Research Council (NRC) in their review of EPA's draft
7 IRIS assessment of formaldehyde3. The report language included the following:
8
9 The Agency shall incorporate, as appropriate, based on chemical-specific datasets
10 and biological effects, the recommendations of Chapter 7 of the National Research
11 Council's Review of the Environmental Protection Agency's Draft IRIS Assessment of
12 Formaldehyde into the IRIS process... For draft assessments released in fiscal year
13 2012, the Agency shall include documentation describing how the Chapter 7
14 recommendations of the National Academy of Sciences (NAS) have been
15 implemented or addressed, including an explanation for why certain
16 recommendations were not incorporated.
17
18 The NRC's recommendations, provided in Chapter 7 of their review report, offered
19 suggestions to EPA for improving the development of IRIS assessments. Consistent with the
20 direction provided by Congress, documentation of how the recommendations from Chapter 7 of the
21 NRC report have been implemented in this assessment is provided in the table below. Where
22 necessary, the documentation includes an explanation for why certain recommendations were not
23 incorporated.
24 The IRIS Program's implementation of the NRC recommendations is following a phased
25 approach that is consistent with the NRC's "Roadmap for Revision" as described in Chapter 7 of the
26 formaldehyde review report. The NRC stated that "the committee recognizes that the changes
27 suggested would involve a multi-year process and extensive effort by the staff at the National
28 Center for Environmental Assessment and input and review by the EPA Science Advisory Board and
29 others."
30 Phase 1 of implementation has focused on a subset of the short-term recommendations,
31 such as editing and streamlining documents, increasing transparency and clarity, and using more
32 tables, figures, and appendices to present information and data in assessments. Phase 1 also
33 focused on assessments near the end of the development process and close to final posting. The
34 IRIS benzo[a]pyrene assessment is in Phase 2 and represents a significant advancement in
2Pub. L. No. 112-74, Consolidated Appropriations Act, 2012.
3National Research Council, 2011. 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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1 implementing the NRC recommendations shown in Table F-l below. The Program is implementing
2 all of these recommendations but recognizes that achieving full and robust implementation of
3 certain recommendations will be an evolving process with input and feedback from the public,
4 stakeholders, and external peer review committees. Phase 3 of implementation will incorporate
5 the longer-term recommendations made by the NRC as outlined below in Table F-2, including the
6 development of a standardized approach to describe the strength of evidence for noncancer effects.
7 On May 16, 2012, EPA announced4 that as a part of a review of the IRIS Program's assessment
8 development process, the NRC will also review current methods for weight-of-evidence analyses
9 and recommend approaches for weighing scientific evidence for chemical hazard identification.
10 This effort is included in Phase 3 of EPA's implementation plan.
11
12
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
eliminate any redundancies or inconsistencies.
4EPA 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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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
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 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
Implemented. The Dose-Response Analysis section of
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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
are advanced for consideration in calculating the RfCs
and unit risks need to be expanded. All candidate
RfCs should be evaluated together with the aid of
graphic displays that incorporate selected
information on attributes relevant to the database.
the new document structure provides a clear explanation
of the rationale used to select and advance studies that
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 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 RfD's. 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 (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.
This document is a draft for review purposes only and does not constitute Agency policy.
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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.
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.
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.
11. Establish standard protocols for evidence
identification.
12. Develop a template for description of the search
approach.
13. Use a database, such as the Health and
Environmental Research Online (HERO) database, to
capture study information and relevant quantitative
data.
Evidence Evaluation: Hazard Identification and
Implemented. EPA has created Chemical Assessment
Support Teams to formalize an internal process to
provide additional overall quality control for the
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 is 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.
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.epa.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.
Implemented. Standardized tables have been developed
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National Research Council recommendations
that EPA is implementing in the short term
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-
evidence, and utility as a basis for deriving reference
values and unit risks.
15. Develop templates for evidence tables, forest
plots, or other displays.
16. Establish protocols for review of major types of
studies, such as epidemiologic and bioassay.
Selection of Studies for Derivation of Reference
Values and Unit Risks (p. 165)
17. Establish clear guidelines for study selection.
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 points of
departure (such as benchmark dose, no-observed-
adverse-effect level, and lowest observed-adverse-
effect level), and assessment of the analyses that
underlie the points of departure.
19. Provide explanation of the risk-estimation
modeling processes (for example, a statistical or
Implementation status
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
addition, exposure-response arrays are utilized in the
assessment to provide a graphical representation of
points of departure 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.
Implemented. Templates for evidence tables and
exposure-response arrays have been developed and are
utilized in Section 1.1.
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.
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.
Implemented. The rationale for the selection of the
points of departure for the organ/system specific oral
reference values is provided in Section 2.1. The rationale
for the selection of the point of departure and the
inhalation dosimetry modeling (for the approximation of
a human equivalent concentration) for the derivation of
the inhalation reference value is transparently described
in Section 2.2. The benchmark dose modeling for
candidate reference values is transparently described in
the Supplemental Information (Appendix E).
Implemented. The risk-estimation modeling processes
used to develop cancer risk estimates for benzo[a]pyrene
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National Research Council recommendations
that EPA is implementing in the short term
Implementation status
biologic model fit to the data) that are used to
develop a unit risk estimate.
are described in Section 2 of the lexicological 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
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.
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 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.
1
2
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
announced5 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.
5EPA 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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National Research Council recommendations
that EPA is generally implementing in the long-
term (p. # in NRC report)
Implementation status
Calculation of Reference Values and Unit Risks (pp.
165-166)
7. Assess the sensitivity of derived estimates to model
assumptions and end points 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 end points.
This document is a draft for review purposes only and does not constitute Agency policy.
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i APPENDIX G. SUMMARY OF EXTERNAL PEER
2 REVIEW AND PUBLIC COMMENTS AND EPA'S
3 DISPOSITION
4
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1
2 REFERENCES FOR APPENDICES
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1 Blaha, L; Kapplova, P; Vondracek, J; Upham, B; Machala, M. (2002). Inhibition of gap-junctional
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26 Bostrom, CE; Gerde, P; Hanberg, A; Jernstrom, B; Johansson, C; Kyrklund, T; Rannug, A;
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28 guidelines for polycyclic aromatic hydrocarbons in the ambient air [Review]. Environ
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38 Bowman, ED; Rothman, N; Hackl, C; Santella, RM; Weston, A. (1997). Interindividual variation in
39 the levels of certain urinary polycyclic aromatic hydrocarbon metabolites following
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1 medicinal exposure to coal tar ointment. Biomarkers 2: 321-327.
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1 Cal/EPA (California Environmental Protection Agency). (2010). Public health goals for chemicals
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25 Cavret, S; Laurent, C; Feidt, C; Laurent, F; Rychen, G. (2003). Intestinal absorption of 14C from
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37 impairments that emerge postnatally and continue into adolescence and adulthood.
38 Toxicol Sci 125: 248-261. http://dx.doi.org/10.1093/toxsci/kfr265
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12 Chen, X; An, H; Ao, L; Sun, L; Liu, W; Zhou, Z; Wang, Y; Cao, J. (2011). The combined toxicity of
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34 http://dx.doi.0rg/10.1016/i.mrgentox.2010.02.012
35 Zeilmaker, MJ; van Eijkeren, JCH. (1997). Modeling of Ah-receptor dependent P450 induction I.
36 Cellular model definition and its corporation in a PBPK model of 2,3,7,8-TCDD.
37 (604138.001). Bilthoven, The Netherlands: National Institute of Public Health and the
38 Environment (RIVM). http://www.rivm.nl/bibliotheek/rapporten/604138001.html
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[a]pyren e
1 Zeilmaker, MJ; van Eijkeren, JCH; Kroese, ED. (1999a). PBPK simulated DNA adduct formation:
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4 http://www.rivm.nl/bibliotheek/rapporten/658603009.html
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9 Zhang, HM; Nie, JS; Li, X; Niu, Q. (2012). Characteristic analysis of peripheral blood mononuclear
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13 benzo(a)pyrene alters IL-13 secretion and subset expression of testicular macrophages,
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21 http://dx.doi.org/10.1016/0027-5107(84)90074-5
22
23
This document is a draft for review purposes only and does not constitute Agency policy.
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