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&EPA
EPA/635/R-10/006C
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
June 2012
NOTICE
This document is an Interagency Science Consultation draft. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable
information quality guidelines. It has not been formally disseminated by EPA. It does not
represent and should not be construed to represent any Agency determination or policy. It
is being circulated for review of its technical accuracy and science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review ofbenzo[a]pyrene
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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Toxicological Review ofbenzo[a]pyrene
CONTENTS
APPENDIX A. OTHER AGENCY AND INTERNATIONAL ASSESSMENTS A-l
APPENDIX B. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-
REPONSE ANALYSIS B-3
TOXICOKINETICS B-3
HUMAN STUDIES B-16
ANIMAL BIOASSAYS B-36
OTHER PERTINENTTOXICITY INFORMATION B-88
APPENDIX C. DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE
VALUES FOR EFFECTS OTHER THAN CANCER AND THE DERIVATION OF
CANCER RISK ESTIMATES C-l
DOSE-RESPONSE MODELING FOR DERVIATION OF RFD C-l
INHALATION DOSIMETRY MODELING FOR RFC DERIVATION C-23
DOSE-RESPONSE MODELING FOR CANCER RISK VALUES C-26
DOSE-RESPONSE MODELING FOR THE INHALATION UNIT RISK C-58
DOSE-RESPONSE MODELING FOR THE DERMAL SLOPE FACTOR C-65
ALTERNATIVE APPROACHES FOR CROSS-SPECIES SCALING OF THE
DERMAL SLOPE FACTOR C-97
APPENDIX D. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND
EPA'S DISPOSITION D-l
REFERENCES FOR APPENDICES 1
This document is a draft for review purposes only and does not constitute Agency policy.
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TABLES AND FIGURES
Table A-l. Health assessments and regulatory limits by other national and international agencies A-
1
Figure B-l. Metabolic pathways for benzo[a]pyrene B-8
Figure B-2. The stereospecific activation of benzo[a]pyrene B-9
Table B-l. Exposure to benzo[a]pyrene and mortality from cardiovascular diseases in a European
cohort of asphalt paving workers B-17
Table B-2. Exposure to benzo[a]pyrene and mortality from cardiovascular diseases in a Canadian
cohort of male aluminum smelter workers B-19
Table B-3. Exposure-related effects in Chinese coke oven workers or warehouse controls exposed
to benzo[a]pyrene in the workplace B-25
Table B-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 B-26
Table B-5. Background information on Chinese coke oven workers or warehouse controls exposed
to benzo[a]pyrene in the workplace B-27
Table B-6. Exposure-related effects in male Wistar rats exposed to benzo[a]pyrene by gavage 5
days/week for 5 weeks B-36
Table B-7. Exposure-related effects in Wistar rats exposed to benzo[a]pyrene by gavage 5
days/week for 5 weeks B-40
Table B-8. Means ± SDa for liver and thymus weights in Wistar rats exposed to benzo[a]pyrene by
gavage 5 days/week for 90 days B-42
Table B-9. Incidences of exposure-related neoplasms in Wistar rats treated by gavage with
benzo[a]pyrene, 5 days/week, for 104 weeks B-44
Table B-10. Incidences of alimentary tract tumors in Sprague-Dawley rats chronically exposed to
benzo[a]pyrene in the dietor by gavage in caffeine solution B-47
Table B-ll. Incidence of nonneoplastic and neoplastic lesions in female B6C3Fi mice fed
benzo[a]pyrene in the diet for up to 2 years B-49
Table B-12. Other oral exposure cancer bioassays in mice B-51
Table B-13. Incidence of respiratory and upper digestive tract tumors in male hamsters treated for
life with benzo[a]pyrene by inhalation B-56
Table B-l4. Number of animals with pharynx and larynx tumors in male hamsters exposed by
inhalation to benzo[a]pyrene for life B-57
Table B-15. Skin tumor incidence and time of appearance in male C57L mice dermally exposed to
benzo[a]pyrene for up to 103 weeks B-59
Table B-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 B-60
Table B-17. Tumor incidence in female Swiss mice dermally exposed to benzo[a]pyrene for up to
93 weeks B-61
Table B-l8. Skin tumor incidence in female NMRI and Swiss mice dermally exposed to
benzo[a]pyrene B-62
Table B-19. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene.... B-62
Table B-20. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene.... B-63
Table B-21. Skin tumor incidence and time of appearance in female CFLP mice dermally exposed to
benzo[a]pyrene for 104 weeks B-64
Table B-22. Skin tumor incidence in female NMRI mice dermally exposed to benzo[a]pyrene for life
B-65
Table B-23. Skin tumor incidence in male C3H/HeJ mice dermally exposed to benzo[a]pyrene for
This document is a draft for review purposes only and does not constitute Agency policy.
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24 months B-66
Table B-24. Mortality and cervical histopathology incidences in female ICR mice exposed to
benzo[a]pyrene via gavage for 14 weeks B-70
Table B-25. Means ± SD for ovary weight in female Sprague-Dawley rats B-72
Table B-26. Reproductive effects in male and female CD-I Fl mice exposed in utero to
benzo[a]pyrene B-74
Table B-27. Effect of prenatal exposure to benzo[a]pyrene on indices of reproductive performance
in Fl female NMRI mice B-76
Table B-28. Exposure-related effects in Long Evans Hooded rats exposed to benzo[a]pyrene by
gavage daily in utero from GD14-GD17 B-80
Table B-29. Exposure-related effects in Swiss Albino OF1 mice exposed as pups to benzo[a]pyrene
in breast milk from dams treated by gavage daily from PND1 - PND14 B-82
Table B-30. Pregnancy outcomes in female F344 rats treated with benzo[a]pyrene on CDs 11-21
by inhalation B-84
Table B-31. In vitro genotoxicity studies of benzo[a]pyrene in non-mammalian cells B-88
Table B-32. In vitro genotoxicity studies of benzo[a]pyrene in mammalian cells B-89
Table B-33. In vivo genotoxicity studies ofbenzo[a]pyrene B-l
Table C-l. Means ± SDa for thymus weight in male Wistar rats exposed to benzo[a]pyrene by
gavage 5 days/week for 90 days C-2
Table C-2. Model predictions for decreased thymus weight in male Wistar rats—90 days C-2
Figure C-l. Fit of linear model (nonconstant variance) to data on decreased thymus weight in male
Wistar rats—90 days C-3
Table C-3. Means ± SDa for thymus weight in female Wistar rats exposed to benzo[a]pyrene by
gavage 5 days/week for 90 days C-6
Table C-4. Model predictions for decreased thymus weight in female Wistar rats—90 days C-6
Figure C-2. Fit of linear model (constant variance) to data on decreased thymus weight in female
Wistar rats—90 days C-7
Table C-5. Means ± SDs for ovary weight in female Sprague-Dawley rats C-10
Table C-6. Model predictions for decreased ovary weight in female Sprague-Dawley rats C-10
Figure C-3. Fit of linear/polynomial (1°) model to data on decreased ovary weight C-ll
Table C-7. Means ± SDs for Escape Latency and Time Spent in Target Quadrant C-14
Table C-8. Model predictions for increase in Morris water maze test for escape latency, male and
female rats C-14
Figure C-4. Fit of Hill model to data on Morris water maze test escape latency C-15
Table C-9. Model predictions for decrease in Morris water maze test for time spent in target
quadrant, male and female rats C-18
Figure C-5. Fit of Exponential 4 model to data on Morris water maze time spent in target quadrant.
C-18
Table C-10. Incidence of cervical epithelial hyperplasia C-21
Table C-ll. Model predictions for increased incidence of epithelial hyperplasia in female ICR miceC-
21
Figure C-6. Human fractional deposition C-23
Figure C-7. Rat fractional deposition C-24
Table C-12. Tumor incidence data, with time to death with tumor; male rats exposed by gavage to
benzo[a]pyrene—Kroese etal. (2001) C-29
Table C-13. Tumor incidence data, with time to death with tumor; female rats exposed by gavage to
benzo[a]pyrene—Kroese etal. (2001) C-31
Table C-14. Tumor incidence, with time to death with tumor; female mice exposed to
benzo[a]pyrene via diet—Beland and Gulp (1998) C-33
Table C-15. Derivation of HEDs to use for BMD modeling of Wistar rat tumor incidence data from
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Kroeseetal. (2001) C-34
Table C-16. Derivation of HEDs for dose-response modeling of B6C3Fi female mouse tumor
incidence data from Beland and Gulp (1998) C-35
Table C-17. Summary of model selection and modeling results for best-fitting multistage-Weibull
models, using time-to-tumor data for rats from Kroese et al. (1981) C-36
Table C-18. Summary of human equivalent overall oral slope factors, based on male and female rat
tumor incidence C-55
Table C-19. Summary of model selection among multistage-We ibull models fit to alimentary tract
tumor data for female mice C-55
Table C-20. Individual pathology and tumor occurrence data for male Syrian hamsters exposed to
benzo[a]pyrene via inhalation for lifetime—Thyssenetal. (1981) C-58
Table C-21. Summary of model selection among multistage-Weibull models fit to tumor data for
male hamsters C-60
Table C-22. Skin tumor incidence, benign or malignant in female Swiss or NMRI mice dermally
exposed to benzo[a]pyrene C-68
Table C-23. Skin tumor incidence, benign or malignant, in C57L male mice dermally exposed to
benzo[a]pyrene C-69
Table C-24. Skin tumor incidence, benign or malignant, in female CFLP mice dermally exposed to
benzo[a]pyrene C-69
Table C-25. Skin tumor incidence, benign or malignant, in male C3H/HeJ mice dermally exposed to
benzo[a]pyrene C-70
Table C-26. Summary of 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....C-
71
Figure C-8. Fit of multistage model to skin tumors in C57L mice exposed dermally to
benzo[a]pyrene (Poel, 1959); graph and model output C-72
Figure C-9. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to
benzo[a]pyrene (Roe et al., 1970); graph and model output C-75
Figure C-10. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Schmidt et al., 1973); graph and model output C-77
Figure C-ll. Fit of multistage model to skin tumors in female Swiss mice exposed dermally to
benzo[a]pyrene (Schmidt et al., 1973); graph and model output C-79
Figure C-12. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Schmahl etal., 1977); graph and model output C-81
Figure C-13. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Habs etal., 1980); graph and model output C-83
Figure C-14. Fit of multistage model to skin tumors in female NMRI mice exposed dermally to
benzo[a]pyrene (Habs etal., 1984); graph and model output C-85
Figure C-15. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1983); graph and model output C-87
Figure C-16. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984); graph and model output C-89
Figure C-17. Fit of log-logistic model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984); graph and model output C-91
Figure C-18. Fit of multistage model to skin tumors in female CFLP mice exposed dermally to
benzo[a]pyrene (Grimmer et al., 1984), highest dose dropped; graph and model output C-93
Figure C-19. Fit of multistage model to skin tumors in male CeH/HeJ mice exposed dermally to
benzo[a]pyrene (Sivaketal., 1997); graph and model output C-95
Table C-27. Alternative approaches to cross-species scaling C-100
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ABBREVIATIONS
Toxicological Review ofbenzo[a]pyrene
3-MC 3-methylcholanthrene
8-OHdG 8-hydroxydeoxyguanosine
ADAF age-dependent adjustment factor
Ah aryl hydrocarbon
AHH aryl hydrocarbon hydroxylase
AhR Ah receptor
AIC Akaike's Information Criterion
AKR aldo-keto reductase
ALT alanine aminotransferase
ANOVA analysis of variance
ATSDR Agency for Toxic Substances and
Disease Registry
AUC area under the curve
BMD benchmark dose
BMDL benchmark dose, 95% lower bound
BMDS Benchmark Dose Software
BMR benchmark response
BPDE benzo[a]pyrene-7,8-diol-9,10-epoxide
BPQ benzo[a]pyrene-7,8-quinone
BrdU bromodeoxyuridine
BSM benzene-soluble matter
BUN blood urea nitrogen
CA chromosomal aberration
CASRN Chemical Abstracts Service Registry
Number
CHO Chinese hamster ovary
CI confidence interval
CNS central nervous system
CONSAAM Conversational SAAM
COX cyclooxygenase
CYP cytochrome
CYP450 cytochrome P450
dG-N2-BPDE 10p-(deoxyguanosin-N2-yl>
7p,8
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Toxicological Review ofbenzo[a]pyrene
RfD reference dose
RN reaction network
RNA ribonucleic acid
ROS reactive oxygen species
RR relative risk
s.c. subcutaneous
SAAM Simulation, Analysis and Modeling
SAM S-adenosylmethionine
SCC squamous cell carcinoma
SCE sister chromatid exchange
SD standard deviation
SE standard error
SEM standard error of the mean
SIR standardized incidence ratio
SNP single nucleotide polymorphisms
SPF specific pathogen-free
SRBC sheep red blood cell
SSB single strand break
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
TPA 12-0-tetradecanoylphorbol-13-acetate
TWA time-weighted average
UCL upper confidence limit
UDP uridine diphosphate
UF uncertainty factor
WBC white blood cells
WT wild type
WTC World Trade Center
XP xeroderma pigmentosum
XPA xeroderma pigmentosum group A
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APPENDIX A. OTHER AGENCY AND
INTERNATIONAL ASSESSMENTS
Table A-l. Health assessments and regulatory limits by other national
and international agencies
Organization
Toxicity value or determination
Non-cancer: oral value
CalEPA(2010)
The concentration of 4 u.g/L (ADD = 1.7 x 10"3 mg/kg-day) for benzo[a]pyrene in
water for noncarcinogenic effects was derived from a LOAEL of 5 mg/kg-day for
renal toxicity from Knuckles et al. (2001), a UF of 3,000.
Non-cancer: inhalation value
WHO
(1996, 2003)
Health Canada
(1986, 2005)
The guideline value for benzo[a]pyrene in drinking water of 0.7 u.g/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 (MAC) for benzo[a]pyrene in drinking
water of 0.01 u.g/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 jug//. benzo[a]pyrene correspond to
lifetime excess cancer risks oflO'4, 10~5, and 10~6.
Cancer: Oral value
CalEPA(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, 2010)
CalEPA(1994)
EU (2005)
Does not recommend specific guideline values for 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 IUR from coke oven emissions. The concentrations 0.0012, 1.2 x 10'4, and 1.2
x 10'5 HQ/m3 benzo[a]pyrene correspond to lifetime excess cancer risks oflO'4, 10~5,
andlO'6.
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 one calendar year) as a
marker of PAH carcinogenic risk. Does not include information for how target value
was derived.
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Cancer characterization
IARC(2010)
NTP (2011)
CalEPA(2000)
Health Canada
(1986, 1988)
Carcinogenic to humans (Group 1) (based on mechanistic data)
"reasonably anticipated to be a human carcinogen"
"Sufficient reason for concern regarding the carcinogenic potential of this toxicant in
humans."
Probably carcinogenic to man
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i APPENDIX B. INFORMATION IN SUPPORT OF
2 HAZARD IDENTIFICATION AND DOSE-REPONSE
3 ANALYSIS
4 TOXICOKINETICS
5 Overview
6 Benzo[a]pyrene is absorbed following exposure by inhalation, oral, 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 conjungates 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 Absorption
20 The absorption of benzo[a]pyrene has been studied in humans and laboratory animals for
21 inhalation, ingestion and dermal exposure. Studies of workers occupationally exposed to
22 benzo[a]pyrene have qualitatively demonstrated absorption via inhalation by correlating
23 concentrations of benzo [a]pyrene in the air and benzo [a]pyrene metabolites in the exposed
24 worker's urine. Occuational exposures to benzo[a]pyrene measured with personal air samplers
25 were correlated to urine concentrations of benzo[a]pyrene-9,10-dihydrodiol, a specific metabolite
26 of benzo[a]pyrene, in 24 hour aggregate urine samples by Grimmer etal., 1994. Theamountof
27 benzo[ajpyrene extracted- from personal air monitoring devices (a surrogate for ambient PAHs) of
28 coke oven workers were correlated with r-7,t-8,9,c 10 tetrahydroxy-7,8,9,10-
29 tetrahydrobenzo[a]pyrene (trans-anti-benzo[a]pyrene-tetrol, a specific metabolite of
30 benzo[a]pyrene) in the worker's urine by Wu etal. (2002). In both of these studies only a very
31 small fraction (< 1%) of the inhaled benzo[a]pyrene was recovered from urine, consistent with
32 studies in animals that find urine is not a major route of elimination for benzo[a]pyrene (as
33 described in the excretion section below). These occupational studies cannot be used to quantify
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1 absorption through inhalation-only exposure in humans because the persistence of
2 benzo[a]pyrene-contaminated particulate matter on surfaces and food may lead to exposures via
3 additional routes (Bostrum et al., 2002). Nevertheless, the observation of benzo[a]pyrene
4 metabolites in excreta of exposed humans provides qualitative evidence for benzo[a]pyrene
5 absorption, at least some of which is likely to occur via inhalation.
6 Results from studies of animals following intratracheal instillation of benzo[a]pyrene
7 provide supporting, quantitative evidence that absorption by the respiratory tract is rapid (Bevan
8 and Ulman, 1991; Gerde et al. 1993 b; Weyand and Bevan, 1986; 1987). Following intratracheal
9 instillation of 1 |ig 3H-labeled benzo[a]pyrene/kg dissolved in triethylene glycol to Sprague-Dawley
10 rats, radioactivity rapidly appeared in the liver (reaching a maximum of about 21% of the
11 administered dose within 10 minutes). Elimination of radioactivity from the lung was biphasic,
12 with elimination half-times of 5 and 116 minutes (Weyand and Bevan, 1986). In bile-cannulated
13 rats, bile collected for 6 hours after instillation accounted for 74% of the administered radioactivity
14 (Weyand and Bevan, 1986). The results are consistent with rapid and extensive absorption by the
15 respiratory tract and rapid entry into hepatobiliary circulation following intratracheal instillation.
16 The respiratory tract absorption may also be affected by the vehicle, since higher amounts of
17 benzo[a]pyrene were excreted in bile when administered with hydrophilic triethylene glycol than
18 with lipophilic solvents ethyl laurate or tricaprylin (Bevan and Ulman, 1991). Particle-bound
19 benzo [a]pyrene deposited in the respiratory tract is absorbed and cleared more slowly than the
20 neat compound (Gerde etal., 2001).
21 Studies conducted to assess levels of benzo[a]pyrene metabolites or benzo[a]pyrene-DNA
22 adduct levels in humans exposed to benzo[a]pyreneby the oral route are not adequate to develop
23 quantitative estimates of oral bioavailability. The concentration of benzo[a]pyrene was below
24 detection limits (<0.1 |ig/person) in the feces of eight volunteers who had ingested broiled meat
25 containing approximately 8.6 |ig of benzo[a]pyrene (Hecht et al., 1979). However, studies in
26 laboratory animals demonstrate benzo[a]pyrene is absorbed via ingestion. Studies of rats and pigs
27 measured the oral bioavailability of benzo[a]pyrene in the range from 10 to 40% (Ramesh et al.,
28 2001b; Fothetal., 1988; Cavretetal., 2003; Hecht etal., 1979). The absorption of benzo [a] pyrene
29 may depend on the vehicle. Intestinal absorption of benzo[a]pyrene was enhanced in rats when the
30 compound was solubilized in lipophilic compounds such as triolein, soybean oil, and high-fat diets,
31 as compared with fiber- or protein-rich diets (O'Neill et al., 1991; Kawamura et al., 1988). Aqueous
32 vehicles, quercetin, chlorogenic acid, or carbon particles reduced biliary excretion of
33 benzo[a]pyrene, while lipid media such as corn oil increased it (Stavric and Klassen, 1994). The
34 addition of wheat bran to the benzo[a]pyrene containing diets increased fecal excretion of
35 benzo[a]pyrene (Mirvish etal., 1981).
36 Studies of benzo[a]pyrene metabolites or DNA adducts measured in humans exposed
37 dermally to benzo[a]pyrene-containing mixtures demonstrate that benzo[ajpyrene is absorbed
38 dermally. One study of dermal absorption in human volunteers found absorption rate constants
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Toxicological Review ofbenzo[a]pyrene
1 ranging from 0.036 to 0.135/hour over a 45 minute exposure, suggesting 20-56% of the dose
2 would be absorbed within 6 hours (Van Rooij et al., 1993). Dermal absorption rates varied 69%
3 between different anatomical sites (forehead, shoulder, volar forearm, palmar side of the hand,
4 groin, and ankle) and only 7% between different individual volunteers (Van Rooij et al., 1993). The
5 overall absorbed amount of benzo[a]pyrene in explanted viable skin samples from tissue donors
6 (maintained in short-term organ cultures) exposed for 24 hours ranged from 0.09 to 2.6% of the
7 dose (Kao etal., 1985; Wester etal., 1990). Similar amounts of penetration were measured in skin
8 samples from other species including marmosets, rats, and rabbits (Kao et al., 1985). Skin from
9 mice allowed more of the dose to penetrate (more than 10%), while that of guinea pig let only a
10 negligible percentage of the dose penetrate (Kao etal., 1985). The vehicle for benzo[a]pyrene
11 exposure is an important factor in skin penetration. Exposure of female Sprague-Dawley rats and
12 female rhesus monkeys topically to benzo[a]pyrene in crude oil or acetone caused approximately 4-
13 fold more extensive absorption than benzo[a]pyrene in soil (Wester et al., 1990; Yang etal., 1989).
14 The viscosity of oil product used as a vehicle also changed skin penetration with increased uptake
15 of benzo[a]pyrene for oils with decreased viscosity (Potter et al., 1999). Metabolism is also an
16 important determinant of permeation, with very low rates observed in nonviable skin (Kao et al.,
17 1985).
18 Distribution
19 No adequate quantitative studies of benzo[a]pyrene tissue distribution in exposed humans
20 were identified. Obana et al. (1981) observed low levels of benzo[a]pyrene in liver and fat tissues
21 from autopsy samples. However, prior exposure histories were not available for the donors.
22 Nevertheless, the identification of benzo[a]pyrene metabolites or DNA adducts in tissues and
23 excreta of PAH-exposed populations suggest that benzo[a]pyrene is widely distributed.
24 Distribution of benzo[a]pyrene has been studied in laboratory animals for multiple routes
25 of exposure, including inhalation, ingestion, dermal and intravenous. Exposure to benzo[a]pyrene
26 in various species (Sprague-Dawley rats, Gunn rats, guinea pigs, and hamsters) results in wide
27 distribution throughout the body and rapid uptake into well-perfused tissues (i.e. lung, kidney, and
28 liver) (Weyand and Bevan, 1987; Weyand and Bevan, 1986). Route of administration of
29 benzo[a]pyrene has little influence on the tissue distribution with similar results from studies of
30 inhalation (or intratracheal instillation), oral, i.v. and dermal exposures (Weyand and Bevan, 1987;
31 Weyand and Bevan, 1986; Morse and Carlson, 1985; Saunders et al., 2002; Neubert and Tapken
32 1988; Moir et al., 1998). Intratracheal instillation of radiolabeled benzo[a]pyrene in mice resulted
33 in increased radioactivity in lung-associated lymph nodes, suggesting distribution of
34 benzo[a]pyrene or its metabolites via the lymph (Schnizlein et al. 1987). Rats with biliary cannulas
35 had high excretion of benzo[a]pyrene and benzo[a]pyrene metabolites in bile. The benzo[a]pyrene
36 thioether and glucuronic acid-conjugated metabolites in intestines indicated enterohepatic
37 recirculation of benzo[a]pyrene and benzo[a]pyrene metabolites (Weyand and Bevan, 1986). The
38 vehicle for delivery of inhalated benzo[a]pyrene impacts the distribution with aerosolized
This document is a draft for review purposes only and does not constitute Agency policy.
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1 benzo[a]pyrene more readily absorbed directly in the respiratory tract than particle-adsorbed
2 benzo[a]pyrene (which is cleared by the mucociliary and then ingested) (Sun et al., 1982).
3 Exposure of pregnant rats and mice to benzo[a]pyrene via inhalation and ingestion showed a wide
4 tissue distribution of benzo[a]pyrene, consistent with other studies and demonstrated placental
5 transfer of benzo[a]pyrene and its metabolites (Withey et al., 1993; Neubert and Tapken 1988;
6 Shendrikova and Aleksandrov, 1974). The reactive metabolites of benzo[a]pyrene are also
7 transported in the blood and may be distributed to tissues incapable of benzo[a]pyrene
8 metabolism, such. Serum of benzo[a]pyrene-treated mice incubated with splenocytes or salmon
9 sperm DNA resulted in adduct formation, suggesting that reactive benzo[a]pyrene metabolites
10 were systemically distributed and available for interaction with target tissues (Ginsberg and
11 Atherholt, 1989).
12 Metabolism
13 The metabolic pathways of benzo[a]pyrene (Figure B-l) and variation in species, strains,
14 organ system, age and sex have been studied extensively with in vitro and in vivo experiments. In
15 addition, there have been numerous studies of exposed humans or animals with subsequent
16 detection of benzo[a]pyrene metabolites in tissues or excreta. For example, elevated frequency of a
17 detected urinary metabolite (7,8,9,10-tetrol) was observed in patients treated with coal tar
18 medication (Bowman et al., 1997), demonstrating extensive metabolism of benzo[a]pyrene in
19 humans.
20 Phase I metabolism results in a number of reactive metabolites such as epoxide
21 intermediates, dihydrodiols, phenols, quinones, and their various combinations that are likely to
22 contribute to the toxic effects of benzo[a]pyrene (e.g. dihydrodiol epoxides and quinones). The
23 Phase II metabolism of benzo[a]pyrene metabolites protects cellular macromolecules from binding
24 with reactive benzo[a]pyrene diolepoxides and radical cations. These metabolic process include
25 glutathione conjugation of diol epoxides, sulfation and glucuronidation of phenols, and reduction of
26 quinones by NADPH:quinone oxidoreductase (NQO). Numerous reviews on the metabolism of
27 benzo[a]pyrene are available (Miller and Ramos, 2001; WHO, 1998; ATSDR, 1995; Conney et al.,
28 1994; Grover, 1986; Levin et al., 1982; Gelboin, 1980). Key concepts have been adapted largely
29 from these reviews and supplemented with recent findings.
30 Phase I metabolism
31 Phase I reactions of benzo[a]pyrene are catalyzed primarily by CYP450 and produce
32 metabolites including epoxides, dihydrodiols, phenols and quinones (Figure B-2). The first step of
33 Phase I metabolism is reaction of benzo[a]pyrene into epoxides, the four major forms of which are
34 the 2,3-, 4,5-, 7,8-, and 9,10-isomers (Gelboin, 1980). Once formed, these epoxides may undergo
35 three different routes of metabolism: (1) spontaneous rearrangement to phenols, (2) hydration to
36 trans-dihydrodiols catalyzed by microsomal epoxide hydrolase, or (3) the Phase II detoxification of
37 binding with glutathione (either spontaneously or catalyzed by cytosolic glutathione-S-transferases
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1 (IARC 1983)). The metabolism of benzo[a]pyrene to phenols occurs for 5 phenol isomers (1-, 3-, 6-,
2 7, and 9-OH benzo[a]pyrene) (Pelkonen etal. 1982). The hydration of benzo[a]pyrene epoxides to
3 trans-dihydrodiols occurs for all four major epoxide isomers (2,3-, 4,5-, 7,8-, and 9,10-). The
4 7,8-oxide is the focus of much of the study of benzo[a]pyrene metabolism, since it is a precursor to
5 the potent DNA-binding metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). BPDE is formed
6 from benzo[a]pyrene 7,8-transdiol by multiple mechanisms including catalysis by CYPs (Deutsch
7 1979; Grover 1986), myeloperoxidase (MPO) (Mallet 1991), or prostaglandin h synthase (PHS, also
8 known as cyclooxygenase COX) (Marnett 1990), and lipid peroxidation (Byczkowski 1990). The
9 diolepoxides can react further by spontaneously hydrolyzing to tetrols (Hall and Grover 1988).
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OH
BaP7,8-dio|.9.10.epmide
0'
6-oxo-BaP radical
OH
BaP 1,6-hydroquinone
Q- 0
BaP 1,6-semiquinone BaP 1,6-quinone
F BaP 3.6 I
Lsemiquinone J
BaP6,12-quinone
OH
BaP 6.12-hydroquinone
OH
BaP3.6-hydroquinone
BaP3,6-quinone
1
2 Source: Miller and Ramos (2001).
3 Figure B-l. Metabolic pathways for benzo[a]pyrene.
4 The metabolism of benzo[a]pyrene, proceeds with a high degree of stereoselectivity. Liver
5 microsomes from rats stereospecifically oxidize the 7,8-bond of benzo[a]pyrene to yield almost
6 exclusively the (+)-benzo[a]pyrene-(7,8)-oxide (see Figure B-2). Each enantiomer of the 7,8-oxide
7 is stereospecifically converted by epoxide hydrolase (EH) to a different dihydrodiol and further
8 metabolism of the (-)-benzo[a]pyrene-7,8-dihydrodiol enantiomer by rat GYP enzymes
9 preferentially yields (+)-benzo[a]pyrene-7R,8S-diol-9S,10R-epoxide [(+)-anti- benzo[a]pyrene-7,8-
10 diol-9,10-epoxide (BPDE)], which is believed to be the most potent carcinogen among the four
11 stereoisomers (Figure B-2). Formation of these stereoisomers does not occur at equimolar ratios,
12 and the ratios differ between biological systems. For example a study in rabbit livers demonstrated
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1 that purified microsomes oxidized the (-)-benzo[a]pyrene-7,8-dihydrodiol to isomeric diol epoxides
2 in a ratio ranging from 1.8:1 to 11:1 in favor of the (+)-anti-BPDE isomer (Deutsch etal., 1979).
HO
t
OH
10
12 1
ml2 Mixed Function
j 3 Oxidase System
(+)-BP-7R,8S-diol-9S, 10R-epoxide
Mixed Function (+) anti BPDE
Oxidase System o s
(-)-BP-7R,8S-diol-9R, 10S-epoxide
(-) syn BPDE
765
HO ^
OH
(+)-BP-7S,8R-diol-9S, 10R-epoxide
(+) syn BPDE
O
(-)-BP-7,8-oxide
HO ~m
OH
(-)-BP-7S,8R-diol-9R, 10S-epoxide
A (-) anti BPDE
5 Source: Grover (1986).
6 Figure B-2. The stereospecific activation of benzo[a]pyrene.
7 Several studies have attempted to determine which GYP isozyme is predominantly
8 responsible for the metabolism of benzo[a]pyrene. Dermal administration of [3H]-benzo[a]pyrene
9 to mice that have an Ah receptor (AhR) knock-out (AhR-/-) had significantly decreased formation
10 of (+)-anti-BPDE-DNA adducts compared to WT and IB I-/- mice (Kleiner etal. 2004). Gavage
11 administration of benzo[a]pyrene in AhR knock-out mice found the AhR-/- mice (with lower levels
12 of CYP1A1) had higher levels of protein adducts and unmetabolized benzo[a]pyrene than the
13 AhR+/+ or +/- mice (Sagredo et al., 2006). Similarly, CYP1A1 (-/-) knock-out mice administered
14 benzo[a]pyrene in feed for 18 days had higher steady-state blood levels of benzo[a]pyrene and
15 benzo[a]pyrene-DNA adducts (Uno etal. 2006). DNA post-labeling studies of mice administered by
16 gavage demonstrated higher benzo[a]pyrene-DNAadduct levels in CYP1A1(-/-) than CYP1A1(+/+)
17 mice in liver, small intestines, spleen and bone marrow (Uno et al., 2004). These findings establish
18 important roles inbenzo[a]pyrene metabolism for CYP1A1, but the relationship is not clear
19 between the GYP enzymes and biological activation or detoxification.
20 Another important factor in evaluating variability in the metabolic activation of
21 benzo[a]pyrene by GYP P450s is the effect of functional polymorphisms, which has been the subject
22 of numerous reviews (e.g., Wormhoudt et al., 1999). Recombinant CYP1A1 allelic variants
23 produced BPDE with generally lower catalytic activity and Km values than the WT allele (Schwarz
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1 et al., 2001). However, the formation of diol epoxides is stereospecific, with the allelic variants
2 producing about three times the amount of (±)-anti-BPDE isomers as compared to the
3 stereoisomers, (±)-syn-BPDE (Schwarz et al., 2001). In a study of occupational exposures to
4 benzo[a]pyrene, no relationship was observed between benzo[a]pyrene metabolite formation and
5 the CYP1A1 Mspl polymorphism (Wu et al., 2002).
6 Another metabolic pathway of benzo[a]pyrene metabolism is the conversion of
7 transdihydrodiol-benzo[a]pyrene or 6-OH benzo[a]pyrene into quinones, primarily the 1,6-, 3,6-,
8 7,8- and 6,12- isomers. Transdihydrodiol-benzo[a]pyrene such as (+/-)-anti-BPDE can be
9 converted in a redox cycling reaction into benzo[a]pyrene-7,8-quinone (BPQ) catalyzed by
10 dihydrodiol dehydrogenase (DD). This reaction pathway produces peroxide anion radicals,
11 benzo[a]pyrene semiquinone radicals, hydroxyl radicals, and H202 which in turn can causes
12 extensive DNA fragmentation (Penning 1999; Flowers etal., 1996; 1997).
13 6-Hydroxybenzo[a]pyrene can be oxidized into 6-oxo-benzo[a]pyrene semi-quinone radical and
14 further metabolized into 1,6-, 3,6-, or 6,12-quinones spontaneously, or catalytically by
15 prostaglandin endoperoxide synthetase (Eling, etal 1983).
16 Phase II metabolism
17 The reactive products of phase I metabolism are subject to the action of several phase II
18 conjugation and detoxification enzyme systems that display preferential activity for specific
19 oxidation products of benzo[a]pyrene. These phase II reactions play a critical role in protecting
20 cellular macromolecules from binding with reactive benzo[a]pyrene diolepoxides, radical cations,
21 or ROS. Therefore, the balance between Phase I activation of benzo[a]pyrene and its metabolites
22 and detoxification by Phase II processes is an important determinant of toxicity.
23 The diol epoxides formed from benzo[a]pyrene metabolism by Phase I reactions are not
24 usually found as urinary metabolites. Rather, they are detected as adducts of nucleic acids or
25 proteins o further metabolized by glutathione (GSH) conjugation, glucuronidation, and sulfation.
26 These metabolites make up a significant portions of total metabolites in excreta or tissues For
27 example, the identified metabolites in bile 6 hours after a 2 [J.g/kg benzo[a]pyrene dose by
28 intratracheal instillation to male Sprague-Dawley rats were 49% glucuronides (quinol
29 diglucuronides or monglucuronides), 30.4% thioether conjugates, 6.2% sulfate conjugates, and
30 14.4% unconjugated metabolites (Bevan and Sadler, 1992).
31 Conjugation of benzo[a]pyrene with GSH is catalyzed by GSTs. Numerous studies using
32 human GSTs expressed in mammalian cell lines have demonstrated the ability of GST to metabolize
33 benzo[a]pyrene diol epoxides. Isolated human GST have significant catalytic activity toward
34 benzo[a]pyrene-derived diol epoxides and (±)anti-BPDE with variation in activity across GST
35 isoforms (Dreij etal. 2002; Robertson etal. 1986; Rojas etal. 1998). Benzo[a]pyrene quinones can
36 also be conjugated with glutathione (Agarwal et al. 1991; IARC 1983). This compelling evidence for
37 a role of GSTs in the metabolism of reactive benzo[a]pyrene metabolites has triggered several
3 8 molecular epidemiology studies. However, recent studies on the impact of polymorphism on
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1 adduct levels in PAH-exposed human populations did not show a clear relationship between the
2 Phase I (CYP1A1, EH), or Phase II (GST) enzyme polymorphisms and formation of DNA adducts
3 (Hemminki et al., 1997) or blood protein adducts (Pastorelli et al., 1998).
4 Conjugation with UDP-glucuronide catalyzed by UGT enzymes is another important
5 detoxification mechanism for oxidative benzo[a]pyrene metabolites. UGT isoforms, as well as their
6 allelic variants, are expressed and have glucuronidation activity toward benzo[a]pyrene-derived
7 phenols and diols in the aerodigestive tract (tongue, tonsil, floor of the mouth, larynx, esophagus),
8 but not lung or liver (Zheng et al., 2002; Fang and Lazarus 2004). UGT activity also shows
9 significant interindividual variability. Incubation of lymphocytes with benzo [a]pyrene resulted in
10 covalent binding to protein with a 143-fold interindividual variability and a statistically significant
11 inverse correlation between glucuronidation and protein binding (Hu and Wells, 2004).
12 Sulfotransferases can catalyze the formation of sulfates of benzo[a]pyrene metabolites. In
13 rat or mouse liver, cytosolic sulfotransferase (in the presence of 3'-phosphoadenosine 5'-
14 phosphosulfate) catalyzes formation of sulfates of three benzo[a]pyrene metabolites:
15 benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol, benzo[a]pyrene-7,8-dihydrodiol, and benzo [a]pyrene-
16 7,8,9,10-tetrol. The benzo[a]pyrene-7,8,9,10-tetrahydro-7-ol-sulfate is able to form potentially
17 damaging DNA adducts (Surh and Tannenbaum, 1995). In human lung tissue 3-
18 hydroxybenzo[a]pyrene conjugation to sulfate produces benzo[a]pyrene-3-yl-hydrogen sulfate, a
19 very lipid soluble compound that would not be readily excreted in the urine (Cohen et al. 1976).
20 Although not specific for benzo[a]pyrene, there is now considerable evidence that genetic
21 polymorphisms of the GST, UGT, and EH genes impart an added risk to humans for developing
22 cancer. Of some significance to the assessment of benzo [a]pyrene maybe that smoking, in
23 combination with genetic polymorphism at several gene loci, increases the risk for bladder cancer
24 (Moore etal., 2004; Choietal., 2003; Parketal., 2003) and lung cancer (Alexandrie etal., 2004; Lin
25 et al., 2003). Coke oven workers (who are exposed to PAHs, including benzo[a]pyrene)
26 homozygous at the P187S site of the NQ01 gene (an inhibitor of benzo[a]pyrene-quinone adducts
27 with DNA), or carrying the null variant of the GSTM1 gene, had a significantly increased risk of
28 chromosomal damage in peripheral blood lymphocytes. Meanwhile, the risk was much lower than
29 controls in subjects with a variant allele at the HllSYsite of the EH gene (Lengetal., 2004).
30 Tissue-specific Metabolism
31 Benzo[ajpyrene metabolism has been demonstrated in vivo in laboratory animals for
32 various tissues via multiple routes including inhalation, ingestion and dermal absorption. Nasal
33 instillation or inhalation of benzo[a]pyrene in monkeys, dogs, rats and hamsters resulted in the
34 formation of dihydrodiols, phenols, quinones, and tetrols in the nasal mucus and lung (Petridou-
35 Fischer etal. 1988; Weyand and Bevan 1986,1987a, 1988; Dahl etal. 1985; Wolff etal. 1989b). In
36 rats, the fractions of metabolites in the lung at 6 hours after instillation were: 20% unmetabolised
37 benzo[a]pyrene, 16% conjugates or polyhydroxylated compounds, 10.7% 4,5-, 7,8-, and 9,10-
38 dihydrodiols, 9.3% 1,6-, 3,6-, 6,12- quinone, and 6.9% 3- and 9-hydroxybenzo[a]pyrene (Weyand
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1 and Bevan 1986). In hamsters, approximately 50% of the benzo[a]pyrene instilled was
2 metabolized in the nose (nasal tissues had the highest metabolic acitivity per-gram of the
3 respiratory tract tissues), and the metabolites produced were similar to other species (Dahl et al.
4 1985).
5 In vitro studies of human and laboratory cells and cell lines provide further quantitative and
6 mechanistic details of the metabolism of benzo[a]pyrene in the cells of the respiratory tract, skin,
7 liver and other tissues. Tracheobronchial tissues in culture of several species (including humans,
8 mice, rats, hamsters, and bovines) were all found to metabolize benzo[a]pyrene extensively to
9 phenols, diols, tetrols, quinones, and their conjugates (Autrup et al., 1980). The results show a high
10 degree of interindividual variability (a 33-fold difference in human bronchus, a 5-fold variation in
11 human trachea, and a 3-fold difference in bovine bronchus), but minimal variation among
12 individuals of the laboratory animal species (Autrup et al., 1980). Human bronchial epithelial and
13 lung tissue conjugated benzo [ajpyrene metabolites to glutathione and sulfates, but not with
14 glucuronide (Autrup et al. 1978; Cohen et al. 1976; Kiefer et al. 1988). The binding of
15 benzo [a]pyrene metabolites with DNA in primary human hepatocytes was associated with the
16 amount of unconjugated 7,8-dihydrodiol (Monteith et al. 1987).
17 Human and animal skin is able to metabolize benzo[a]pyrene. Human skin samples
18 maintained in short term organ culture (i.e., human epithelial tissue, samples from human hair
19 follicles, and melanocytes isolated from adult human skin) can metabolize benzo[a]pyrene into
20 dihydrodiols, phenolas, quinones and glucuronide and sulfate conjugates (Hall & Griver, 1988; Merk
21 etal., 1987; Alexandrov et al., 1990; Agarwal etal., 1991). The permeation of benzo[a]pyrene in
22 skin is linked to benzo[a]pyrene metabolism. Nonviable skin is unable to metabolize
23 benzo[a]pyrene (the permeation into nonviable skin is lower than viable skin) as measured in a
24 range of species including humans, rat, mouse, rabbit and marmoset (Kao etal., 1985). Viable
25 human skin samples treated with 2 [ig/cm2 [14C]-benzo[a]pyrene in acetone and incubated for
26 24 hours produced the following proportions of benzo[a]pyrene metabolites; 52% water-soluble
27 compounds, 8% polar compounds, 17% diols, 1% phenols, 2.5% quinones and 18% unmetabolized
28 benzo[ajpyrene (Kao etal., 1985).
29 Benzo [ajpyrene is also metabolized by multiple reproductive tissues including prostate,
30 endometrium, cervical epithelial and styromal, and testes (Williams et al., 2000; Bao et al., 2002;
31 Melikian etal., 1999; Ramesh etal., 2003). Exposure of fetal tissues to reactive benzo[a]pyrene
32 metabolites in utero is a concern. Transport of benzo[a]pyrene and benzo[a]pyrene metabolites to
33 fetal tissues including plasma, liver, hippocampus and cerebral cortex has been demonstrated in
34 multiple studies (McCabe and Flynn, 1990; Neubert and Tapken, 1988; Shendrikova and
35 Aleksandrov, 1974), and benzo[a]pyrene is metabolized by human fetal esophageal cell culture
36 (Chakradeo et al. 1993).
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1 Elimination
2 Benzo[a]pyrene metabolites have been detected in the urine of exposed humans, but the
3 fecal excretion has not been investigated in any detail. Studies of benzo[a]pyrene elimination in
4 animals following exposure via inhalation, ingestion and dermal routes have shown benzo[a]pyrene
5 is excreted preferentially in the feces in multiple species of laboratory animals including rat, mice,
6 hamsters, guinea pigs, monkeys and dogs (Petridou-Fischer et al., 1988; Wolff et al., 1989; Sun et al.,
7 1982; Wang et al., 2003; Weyand and Bevan, 1987; Yang et al., 1989; Hecht et al., 1979; Likhachev
8 et al., 1992). The metabolites in bile are primarily benzo[a]pyrene conjugates, predominately
9 thioether conjugates of varying extent in different species (Weyand and Bevan, 1987). Six hours
10 after a single intratracheal instillation of benzo[a]pyrene (2 |J.g/kg) to male Sprague-Dawley rats,
11 relative metabolite levels were 31.2% diglucuronides, 30.4% thioether conjugates, 17.8%
12 monoglucuronides, 6.2% sulfate conjugates, and 14.4% unconjugated metabolites (Bevan and
13 Sadler, 1992). Rats administered benzo[a]pyrene via i.v. excrete a larger fraction in urine than via
14 inhalation or oral exposure, suggesting an important role for enterohepatic circulation of
15 benzo[a]pyrene metabolite conjugates (Moir etal., 1998; Weyand and Bevan, 1986; Hirom etal.,
16 1983). The vehicle impacts the amount of benzo[a]pyrene excreted and may in part be due to the
17 elimination rate or to other factors such as the absorption rate. For [3H]-benzo[a]pyrene
18 administered to Sprague-Dawley rats in hydrophilic triethylene glycol, 70.5% of the dose was
19 excreted into bile within 6 hours. If lipophilic solvents ethyl laurate and tricaprylin were used as
20 vehicles, 58.4 and 56.2% of the dose were excreted (Bevan and Ulman, 1991). In addition to
21 benzo[a]pyrene and its metabolites, adducts of benzo[a]pyrene with nucleotides have also been
22 identified as a small fraction of the administered dose in feces and urine of animals. The level of
23 BPDE adducts with guanine detected in urine of male Wistar rats was dose-dependent. 48 hours
24 after dosing with 100 [J.g/kg tritiated benzo[a]pyrene, 0.15% of the administered benzo[a]pyrene
25 dose was excreted in the urine as an adduct with guanine (Autrup and Seremet, 1986). Overall, the
26 data in humans and laboratory animals are sufficient to describe benzo[a]pyrene elimination
27 qualitatively but to limited to estimate quantitative rates of elimination.
28 Physiologically based pharmacokinetic models
29 Several toxicokinetic or pharmacokinetic models of benzo[a]pyrene have been developed
30 for rodents (rat and hamster). However, human models have only been developed via allometric
31 scaling, and metabolic parameters in humans have not been calibrated against in vivo toxicokinetic
32 data or in vitro experiments.
33 Bevan and Weyand (1988) performed compartmental pharmacokinetic analysis of
34 distribution of radioactivity in male Sprague-Dawley rats, following the intratracheal instillation of
35 benzo[a]pyrene to normal and bile duct-cannulated animals (Weyand and Bevan, 1987,1986).
36 However, implicit simulation approaches were used, as opposed to physiologically-based
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1 approaches. The model calculated linear rate constants among compartments, and assumed the
2 kinetics of benzo[a]pyrene and its metabolites were the same
3 Roth and Vinegar (1990) reviewed the capacity of the lung to impact the disposition of
4 chemicals and used benzo[a]pyrene as a case study. A PBPK model was presented based on data
5 from Wiersma and Roth (1983a, b) and was evaluated against tissue concentration data from
6 Schlede et al. (1970). The model was structured with compartments for arterial blood, venous
7 blood, lung, liver, fat, and slowly as well as rapidly perfused tissues. Metabolism in liver and lung
8 was estimated using kinetic data from control rats and rats pretreated with 3-MC to induce
9 benzo[a]pyrene metabolism. The results of PBPK simulations showed that induction of
10 metabolizing enzymes increased the amount of benzo[a]pyrene cleared by the lungs relative to the
11 liver. An adequate fit was obtained for some compartments; however tissue-level data for
12 calibration and validation of this model were limited.
13 Moir et al. (1998) conducted a pharmacokinetic study on benzo[a]pyrene to obtain data for
14 model development Rats were injected with varying doses of [14C]-benzo[a]pyrene to 15 mg/kg
15 and blood, liver, fat, and richly perfused tissue were sampled varying time points after dosing. Moir
16 (1999) then described a model for lung, liver, fat, richly and slowly perfused tissues, and venous
17 blood, with saturable metabolism occurring in the liver. The fat and richly perfused tissues were
18 modeled as diffusion-limited, while the other tissues were flow-limited. The model predicted the
19 blood benzo[a]pyrene concentrations well, although it overestimated the 6 mg/kg results at longer
20 times (>100 minutes). The model also produced a poor fit to the liver data. The model simulations
21 were also compared to data of Schlede etal. (1970), who had injected rats with 0.056 mg/kg body
22 weight of benzo[a]pyrene. The model predicted blood and fatbenzo[a]pyrene concentrations well,
23 but still poorly predicted liver benzo[a]pyrene concentrations. The model included only one
24 saturable metabolic pathway, and only parent chemical concentrations were used to establish the
25 model. No metabolites were included in the model. This model was re-calibrated by Crowell etal.
26 (2011) by optimizing against additional rodent data and altering partition coefficient derivation.
27 However, it still did not incorporate metabolites, and some tissues continued to exhibit poor model
28 fits.
29 An attempt to scale the Moir et al. (1998) rodent PBPK model to humans, relevant to risk
30 assessment of oral exposures to benzo[a]pyrene, was presented by Zeilmaker et al. (1999a, b). The
31 PBPK model for benzo[a]pyrene was derived from an earlier model for TCDD in rats (Zeilmaker and
32 van Eijkeren, 1997). Most compartments were perfusion-limited, and tissues modeled included
33 blood, adipose (with diffusion limitation), slowly and richly perfused tissues, and the liver.
34 However, there was no separate compartment for the lung. The liver compartment featured the
35 AhR-dependent CYP450 induction mechanism and DNA adduct formation as a marker for
36 formation of genotoxic benzo[a]pyrene metabolites. It was assumed that DNA adduct formation
37 and the bulk benzo[a]pyrene metabolism were mediated by two different metabolic pathways. The
38 model was experimentally calibrated in rats with the data for EROD and formation of DNA adducts
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1 in the liver after i.v. administration of a single dose and per os (p.o.) administration of a single or
2 repeated doses of benzo[a]pyrene (Zeilmaker et al., 1999a).
3 Zeilmaker et al. (1999b) assumed identical values for several parameters in rats and
4 humans (i.e. benzo[a]pyrene tissue partition coefficients, AhR concentration in liver, rate constant
5 for the decay of the benzo[a]pyrene-CYP450 complex, half-life of the CYP450 protein, fraction and
6 rate of GI absorption of benzo[a]pyrene, and rates of formation and repair of DNAadducts in liver).
7 The basal GYP45 0 activity in humans was assumed to be lower than that in rat liver. The
8 mechanism of AhR-dependent induction of CYP450 dominated the simulated benzo[a]pyrene-DNA
9 adduct formation in the liver. The results of PBPK model simulations indicated that the same dose
10 of benzo[a]pyrene administered to rats or humans might produce one order of magnitude higher
11 accumulation of DNA adducts in human liver when compared with the rat (Zeilmaker et al., 1999b).
12 Even though the model of Zeilmaker et al. (1999b) represents a major improvement in
13 predictive modeling of benzo[a]pyrene toxicokinetics, the interspecies extrapolation introduce
14 significant uncertainties. As emphasized by the authors, the conversion of benzo[a]pyrene to its
15 mutagenic and carcinogenic metabolites could not be explicitly modeled in human liver because no
16 suitable experimental data were available. According to the authors, improvement of the model
17 would require direct measurements of basal activities of CYP1A1 and CYP1A2 and formation of
18 benzo[a]pyrene-DNA adducts in human liver. Metabolic clearance of benzo[a]pyrene in the lungs
19 was also not addressed. Additionally, the toxicokinetic modeling by Zeilmaker et al. (1999b)
20 addressed only one pathway of benzo[a]pyrene metabolic activation, a single target organ (the
21 liver), and one route of administration (oral). In order to model health outcomes of exposures to
22 benzo[a]pyrene, the PBPK model needs to simulate rate of accumulation of benzo[a]pyrene-DNA
23 adducts and/or the distribution and fate of benzo[a]pyrene metabolites (e.g., BPDE) that bind to
24 DNA and other macromolecules. Alternatively, stable toxic metabolites (e.g., trans-anti-tetrol-
25 benzo[a]pyrene) may be used as an internal dose surrogate. While the metabolic pattern of
26 benzo[a]pyrene has been relatively well characterized qualitatively in animals, the quantitative
27 kinetic relationships between the more complex metabolic reactions in potential target organs are
28 not yet well defined.
29 Recommendations for the use of PBPK models in toxicity value derivation
30 PBPK models for benzo[a]pyrene were evaluated to determine the capability to extrapolate
31 from rats to humans, or between oral and inhalation exposure routes. Due to significant
32 uncertainties with respect to the inter-species scaling of the metabolic parameters between rats
33 and humans, these models were not used for cross-species extrapolation. Furthermore, no
34 complete mechanistic PBPK model for the inhalation route was identified, nor was there a model
35 for humans that simulates the typical inhalation exposure to benzo[a]pyrene on poorly soluble
36 carbonaceous particles. This precluded the model's use for cross-route extrapolation to the
37 inhalation pathway.
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1 HUMAN STUDIES
2 Non-Cancer Endpoints
3 Cardiovascular Endpoints
4 Burystn et al. (2005) reported the association of death from cardiovascular disease with
5 B[a]P exposure in a cohort of 12,367 male European asphalt workers (Table B-l). These workers
6 were first employed in asphalt paving between 1913 and 1999, and worked at least one season.
7 Average duration of follow-up was 17 ± 9 years (mean ± SD), encompassing 193,889 person-years
8 of observation. Worker exposure to coal tar was estimated using industrial process and hygiene
9 information and modeling (presented in a previous report), and coal tar exposure was found to be
10 the strongest determinant of exposure to B[a]P. Benzo[a]pyrene exposure was assessed
11 quantitatively using measurement-driven mixed effects exposure models, using data collected from
12 other asphalt industry workers, and this model was constructed and validated previously. Due to
13 limited data availability, only information regarding the primary cause of death was collected, and
14 this analysis was limited to diseases of the circulatory system (ICD codes 390 - 459), specifically
15 ischemic heart disease (IHD: ICD codes 410 - 414). Diesel exhaust exposure was also assessed in
16 this cohort, but varied little among the asphalt pavers, and was not associated with risk of death
17 from cardiovascular disease. 0.25% of the cohort was lost to follow-up, and 0.38% emigrated
18 during the course of observation. Relative risks and associated 95% confidence intervals were
19 estimated using Poisson regression, and all models included exposure index for agent of interest
20 (coal tar or B[a]P), age, calendar period of exit from cohort, total duration of employment and
21 country, using the category of lowest exposure as the reference. Confounding by tobacco smoke
22 exposure was considered in relation to the strength of its association with cardiovascular disease
23 and the smoking prevalence in the population. The RR attributed to cigarette smoking in former
24 and current smokers was assumed to be 1.2 and 2, respectively, based upon literature reports.
25 From analysis of smoking incidence in a sub-cohort, the following smoking distribution was
26 proposed: in the lowest exposure group, 40% never smokers, 30% former smokers and 30%
27 current smokers; among the highest exposed, the proportion shifted to 20/30/50%, respectively.
28 Exposed subjects were stratified into quintiles based upon IHD mortality, with 83 - 86
29 deaths per exposure category, composing approximately 2/3 of the 660 cardiovascular disease-
30 related deaths. Both cumulative and average exposure indices for B[a]P were positively associated
31 with IHD mortality, with a RR of approximately 1.6 in the highest exposure quintile from both
32 metrics, independent of total employment duration. Similar monotonic trends were observed for
33 all cardiovascular diseases (combined), although a dose-response relationship was evident only for
34 IHD and not hypertension or other individual heart disease categories. Similar trends were also
35 observed for coal tar exposure and IHD. Adjusting the RR to account for possible confounding by
36 smoking yields a RR of 1.39 under the assumptions mentioned above, and is still elevated (1.21) if
37 the contribution of smoking to cardiovascular disease etiology was greater than the original
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1 assumptions. Furthermore, the RR for the high vs. low exposure quintile is 1.24 even if the
2 distribution of non-smokers/former smokers/current smokers shifts to 0/30/70%, using the
3 original assumptions of cigarette smoke casual potency.
4
5
Table B-l. Exposure to benzo[a]pyrene and mortality from
cardiovascular diseases in a European cohort of asphalt paving workers
Effect measured
Cumulative exposure (ng/m3 -years)
0 - 189a
189 - 501
502-931
932 - 2012
>2013
Pfor
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
Ischemic heart disease
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
Pfor
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
Ischemic heart disease
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
6
7
8
9
10
11
12
13
14
15
16
17
a Reference category
Source: Burstyn et al. (2005).
Friesen et al. (2010) examined the association between B [a]P exposure and deaths from
chronic non-malignant disease in a cohort of 6.423 male and 603 female Canadian aluminum
smelter workers (Table B-2). Inclusion criteria required at least 3 years of continuous employment
in either the smelter facility or power-generating station from 1954 - 1997, with worker history
collected up through 1999. This cohort was probabilistically linked to the Canadian national
mortality database for external comparison to the British Columbia population and calculation of
standardized mortality ratios, which were adjusted for age, sex and time period. Ninety-five %
confidence intervals were calculated for the SMRs assuming a Poisson distribution. Internal
comparisons were also made during the analysis of IHD mortality in male workers, calculating
hazard ratios (HR) for IHD with or without acute myocardial infarction (AMI) after 1969, as AMI
could not be differentiated from other IHD on death certificates issued previously. HRs were
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1 calculated using Cox regression models, with age as a metamarker of time, also including smoking
2 status, time since 1st employed and work location status. Smoking information for 77% of this
3 updated cohort was collected by questionnaire, and workers categorized as 75% ever-smokers and
4 25% never-smokers. Quantitative exposure to coal tar pitch volatiles were estimated by B[a]P
5 measurements, calculated by a job classification and time-based exposure matrix, as described in a
6 previous report; annual arithmetic mean values were calculated for exposures from 1977 - 2000,
7 while pre-1977 levels were backwards-extrapolated from 1977 values, incorporating major
8 technological changes in time periods as appropriate.
9 Cumulative exposure metrics were highly skewed. Cumulative B[a]P with a 5-year lag (past
10 B[a]P exposure) and cumulative B[a]P in the most recent 5 years (recent B [a]P exposure) were only
11 slightly positively correlated (r = 0.10, P < 0.001). Current B[a]P exposure was highly correlated
12 with cumulative exposure for the most recent 5 years of exposure (r = 0.86, P < 0.001), but not with
13 5-year lagged cumulative exposure (r = 0.03, P < 0.001). Lagged cumulative exposure metrics (0 -
14 10 years) were all highly correlated with each other (r = 0.96, all P's < 0.001); lagged metrics for
15 cumulative exposure were used to distinguish between effects of current versus long-term
16 exposure.
17 When exposed workers were pooled and compared externally to non-exposed referents, the
18 IHD and AMI standardized mortality ratios were all < 1.00 for males, and the only significant
19 association in females was an SMR of 1.27 for AMI. For internal comparisons, exposed males were
20 stratified into quintiles based upon IHD mortality, with approximately 56 deaths per exposure
21 category. 5-year lagged cumulative B[a]P exposure was significantly associated with elevated risk
22 of IHD mortality, HR = 1.62 (95% CI: 1.06, 2.46) in the highest exposure quintile, while no
23 association was observed between most recent (5 years) exposure and mortality. Restricting IHD
24 events to only AMI (1969 onward) resulted in similar monotonic trends, albeit of lower statistical
25 significance. No association was observed between B [a] P exposure and non-AMI IHD. While there
26 was little difference in the exposure-response association among 0, 2 and 5-year lagged data, 10-
27 year lagged data resulted in a weaker association. All risk estimates were strengthened by the
28 incorporation of work status and time-since-hire to account for the healthy worker effect, as
29 evidenced by the SMR of 0.87 (95% CI: 0.82, 0.92) for all chronic non-malignant diseases combined
30 in male exposed workers versus external referents. Using a continuous variable, the authors
31 calculated thatthe risk of death from IHD to be 1.002 (95% CI: 1.000,1.005) per [ig/m3 from
32 cumulative B[a]P exposure; however, visual inspection of the categorical relationships indicated
33 that the association is nonlinear, suggesting that this value may be an underestimate. Restricting
34 the cohort to only members who died within 30 days of active employment at the worksite,
35 cumulative B[a]P exposure was not significantly associated with IHC or AMI, although the HR for
36 the highest exposure group was 2.39 (95% CI: 0.95, 6.05). Exposure-response relationships were
37 similarly examined in male smelter workers for chronic obstructive pulmonary disease (COPD) and
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1 cerebrovascular disease, but neither was significantly associated with cumulative B[a]P exposure in
2 either internal or external comparisons.
4
5
Table B-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-year lag (u.g/ms -year)
0
0 - 7.79
7.79 - 24.3
24.3 - 66.7
>66.7
Pfor
trend3
Continuous13
All ischemic heart disease (1957 onward)
Deaths
P-YC of follow-
up
HR
95% Cl
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
P-YC of follow-
up
HR
95% Cl
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 A
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
6
7
8
9
10
11
12
13
14
15
16
17
18
19
a Two-sided test for trend using the person-year-weighted mean value for each category as a linear,
continuous variable.
b Exposure variable was entered as a continuous, linear variable in the model
c P-Y, person-years
Source: Friesen et al. (2010).
Reproductive and Developmental Endpoints
Wu etal. (2010) conducted a study of benzo[a]pyrene-DNAadduct levels in relation to risk
of fetal death in Tianjin, China. This case-control study included women who experienced a missed
abortion before 14 weeks gestational age (i.e., a fetal death that remained in utero and therefore
required surgical intervention). Cases were matched by age and gravidity to controls (women
undergoing induced abortion due to an unplanned or unwanted pregnancy). The study excluded
women who smoked, women with chronic disease and pregnancy complications, and women with
occupational exposures to PAHs. Residency within Tianjin for at least 1 year was also an eligibility
criterion. The participation rate was high: 81/84 eligible cases participated and 81/89 eligible
controls participated. Data pertaining to demographic characteristics, reproductive history, and
factors relating to potential PAH exposure were collected using a structured interview, and samples
from the aborted tissue were obtained. In two of the four hospitals used in the study, blood
samples from the women (n = 51 cases and 51 controls) were also collected. The presence of
benzo[a]pyrene-BPDE adducts was assessed in the blood and tissue samples using HPLC. There
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1 was no correlation between blood and aborted tissue levels of benzo[a]pyrene adducts (r = -0.12
2 for the 102 blood-tissue pairs, r = -0.02 for the 51 case pairs and r = -0.21 for the 51 control pairs).
3 (The authors noted that there was little difference between women with and without blood
4 samples in terms of the interview-based measures collected or in terms of the DNA-adduct levels in
5 aborted tissue.) Benzo[a]pyrene-adduct levels were similar but slightly lower in the aborted tissue
6 of cases compared with controls (mean ± SD 4.8 ± 6.0 incases and 6.0 ± 7.4 in controls, p = 0.29). In
7 the blood samples, however, benzo[a]pyrene-adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ±
8 2.2 in cases and controls, respectively, p < 0.001). In logistic regression analyses using a continuous
9 adduct measure, the OR was 1.35 (95% CI 1.11-1.64) per adduct/108 nucleotide. These results
10 were adjusted for education and household income, but were very similar to the unadjusted results.
11 Categorizing exposure at the median value resulted in an adjusted OR of 4.27 (95% CI 1.41-12.99)
12 in the high compared with low benzo[a]pyrene-adduct group. There was no relation between
13 benzo[a]pyrene-adduct levels in the aborted tissue and missed abortion in the logistic regression
14 analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93-1.02) or dichotomous
15 exposure measure (adjusted OR 0.76, 95% CI 0.37-1.54). Associations between missed abortion
16 and several interview-based measures of potential PAH exposure were also seen: adjusted OR 3.07
17 (95% CI 1.31-7.16) for traffic congestion near residence, 3.52 (95% CI 1.44-8.57) for commuting
18 by walking, 3.78 (95% CI 1.11-12.87) for routinely cooked during pregnancy, and 3.21 (95% CI
19 0.98-10.48) for industrial site or stack near residence, but there was no association with other
20 types of commuting (e.g., by bike, car, or bus).
21 Perera etal. (2005a) studied 329 nonsmoking pregnant women (30 ± 5 years old) possibly
22 exposed to PAHs from fires during the 4 weeks after 09/11/2001. Maternal and umbilical cord
23 blood levels of benzo[a]pyrene (BPDE)-DNA adducts were highest in study participants who lived
24 within 1 mile of the WTC, with an inverse correlation between cord blood levels and distance from
25 the WTC. Neither cord blood adduct level nor ETS alone was positively correlated with adverse
26 birth outcomes. However, the interaction between ETS exposure and cord blood adducts was
27 significantly associated with reduced birth weight and head circumference. Among babies exposed
28 to ETS in utero, a doubling of cord blood benzo[a]pyrene-DNA adducts was associated with an 8%
29 decrease in birth weight (p = 0.03) and a 3% decrease in head circumference (p = 0.04).
30 Perera et al. (2005b) compared various exposures—ETS, nutrition, pesticides, material
31 hardship—with birth outcomes (length, head circumference, cognitive development). ETS
32 exposure and intake of PAH-rich foods by pregnant women were determined by questionnaire.
33 Levels of benzo[a]pyrene diol epoxide (BPDE)-DNA adducts were determined in umbilical cord
34 blood collected at delivery. The study population consisted of Dominican or African-American
35 nonsmoking pregnant women (n = 529;24±5 years old) free of diabetes, hypertension, HIV, and
36 drug or alcohol abuse. Benzo[a]pyrene adducts, ETS, and dietary PAHs were not significantly
37 correlated with each other. However, the interaction between benzo[a]pyrene-DNA adducts and
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1 ETS exposure was significantly associated with reduced birth weights (-6.8%; p = 0.03) and
2 reduced head circumference (-2.9%; p = 0.04).
3 Tang et al. (2006) measured benzo[a]pyrene diol epoxide (BPDE)-DNA adducts in maternal
4 and umbilical cord blood obtained at delivery from a cohort of 150 nonsmoking women and their
5 newborns in China. Exposure assessment was related to the seasonal operation of a local, coal-fired
6 power plant; however, airborne PAH concentrations were not measured. Dietary PAH intake was
7 not included as a covariate because it did not significantly contribute to the final models, but ETS,
8 sex, and maternal height and weight were considered as covariates. DNA adduct levels were
9 compared to several birth outcomes and physical development parameters, such as gestational age
10 at birth; infant sex, birth weight, length, head circumference, and malformations; maternal height
11 and pregnancy weight total weight gain; complications of pregnancy and delivery; and medications
12 used during pregnancy.
13 High cord blood adduct levels were significantly associated with reduced infant/child
14 weight at 18 months (P = -0.048, p = 0.03), 24 months (P = -0.041, p = 0.027), and 30 months of age
15 (P = -0.040, p = 0.049); decreased birth head circumference was marginally associated with DNA
16 adductlevels (P = -0.011, p = 0.057). Maternal adduct levels were correlated neither with cord
17 blood adduct levels nor with fetal and child growth. Among female infants, cord blood adduct levels
18 were significantly associated with smaller birth head circumference (p = 0.022) and with lower
19 weight at 18 months (p = 0.014), 24 months (p = 0.012), and 30 months of age (p = 0.033), and with
20 decreased body length at 18 months of age (p = 0.033). Among male infants, the corresponding
21 associations were also inverse but were not statistically significant
22 Considerable evidence of a deleterious effect of smoking on male and female fertility has
23 accumulated from epidemiological studies of time to pregnancy, ovulatory disorders, semen
24 quality, and spontaneous abortion (reviewed in Waylen et al., 2009; Cooper and Moley, 2008;
25 Scares and Melo, 2008). In addition, the effect of smoking, particularly during the time of the
26 perimenopausal transition, on acceleration of ovarian senescence (menopause) has also been
27 established (Midgette and Baron, 1990). More limited data are available pertaining specifically to
28 measures of benzo[a]pyrene and reproductive outcomes.
29 Neal etal. (2008, 2007) examined levels of benzo[a]pyrene and other PAHs infollicular
30 fluid and serum sample from 36 women undergoing in vitro fertilization at a clinic in Toronto, and
31 compared the successful conception rate in relation to benzo[a]pyrene levels. The women were
32 classified by smoking status, with 19 current cigarette smokers, 7 with passive or sidestream
33 smoke exposure (i.e., nonsmoker with a partner who smoked), and 10 nonsmokers exposed. An
34 early follicular phase blood sample and follicular fluid sample from the follicle at the time of ovum
35 retrieval were collected and analyzed for the presence of benzo[a]pyrene, acenapthelene,
36 phenanthrene, pyrene, and chrysene using gas chromatography/MS (detection limit 5 pg/mL). The
37 frequency of nondectable levels of serum benzo[a]pyrene was highest in the nonsmoking group
38 (60.0,14.3, and 21.0% below detection limit in nonsmoking, sidestream smoke, and active smoking
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1 groups, respectively). A similar pattern was seen with follicular fluid benzo[a]pyrene (30.0,14.3,
2 and 10.5% below detection limit in nonsmoking, sidestream smoke, and active smoking groups,
3 respectively). In the analyses comparing mean values across groups, an assigned value of 0 was
4 used for nondetectable samples. Follicular fluid benzo[a]pyrene levels were higher in the active
5 smoking group (mean ± SE, 1.32 ± 0.68 ng/mL) than in the sidestream (0.05 ± 0.01 ng/mL) or
6 nonsmoking (0.03 ± 0.01 ng/mL) groups (p = 0.04). The between-group differences in serum
7 benzo[a]pyrene levels were not statistically significant (0.22 ± 0.15, 0.98 ± 0.56, and 0.40 ±
8 0.13 ng/mL in nonsmoking, sidestream smoke, and active smoking groups, respectively), and there
9 were no differences in relation to smoking status. Among active smokers, the number of cigarettes
10 smoked per day was strongly correlated with follicular fluid benzo[a]pyrene levels (r = 0.7, p <
11 0.01). Follicular fluid benzo[a]pyrene levels were significantly higher among the women who did
12 not conceive (1.79 ng/mL ± 0.86) compared with women who did get pregnant (mean
13 approximately 0.10 ng/mL, as estimated from graph) (p < 0.001), but serum levels of
14 benzo[a]pyrene were not associated with successful conception.
15 A small case-control study conducted between August 2005 and February 2006 in Lucknow
16 city (Uttar Pradesh), India examined PAH concentrations in placental tissues (Singh et al., 2008) in
17 relation to risk of preterm birth. The study included 29 cases (delivery between 28 and <36 weeks
18 of gestation) and 31 term delivery controls. Demographic data smoking history, reproductive
19 history, and other information were collected by interview, and a 10 g sample of placental tissue
20 was collected from all participants. Concentration of specific PAHs in placental tissue was
21 determined using HPLC. In addition to benzo[a]pyrene, the PAHs assayed were naphthalene,
22 acenapththylene, phenanthrene, fluorene, anthracene, benzo(a)anthracene, fluoranthene, pyrene,
23 benzo(k)fluoranthene, benzo(b)fluoranthene, benzo(g,h,i)perylene, and dibenzo(a,h)anthracene.
24 PAH exposure in this population was from environmental sources and from cooking. The age of
25 study participants ranged from 20 to 35 years. There was little difference in birth weight between
26 cases and controls (mean 2.77 kg and 2.75 kg in the case and control groups, respectively).
27 Placental benzo[a]pyrene levels were lower than the levels of the other PAHs detected (mean 8.83
28 ppb in controls for benzo[a]pyrene compared with 25-30 ppb for anthracene,
29 benzo(k)fluoranthene, benzo(b)fluoranthene, and dibenzo(a,h)anthracene, 59 ppb for
30 acenaphthylene, and 200-380 ppm for naphthalene, phenanthrene, fluoranthene, and pyrene;
31 nondetectable levels of fluorine, benzo(a)anthracene, and benzo(g,h,i)perylene were found). There
32 was little difference in benzo[a]pyrene levels between cases (mean ± SE 13.85 ± 7.06 ppb) and
33 controls (8.83 ± 5.84 ppb), but elevated levels of fluoranthene (325.91 ± 45.14 and 208.6 ± 21.93
34 ppb in cases and controls, respectively, p < 0.05) and benzo(b)fluoranthene (61.91 ± 12.43 and
35 23.84 ± 7.01 ppb in cases and controls, respectively, p < 0.05) were seen.
36 Wuetal. (2010) conducted a study of benzo[a]pyrene-DNAadduct levels in relation to risk
37 of fetal death in Tianjin, China. This case-control study included women who experienced a missed
38 abortion 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 HPLC. There
11 was no correlation between blood and aborted tissue levels of benzo[a]pyrene adducts (r = -0.12
12 for the 102 blood-tissue pairs, r = -0.02 for the 51 case pairs and r = -0.21 for the 51 control pairs).
13 (The authors noted that there was little difference between women with and without blood
14 samples in terms of the interview-based measures collected or in terms of the DNA-adduct levels in
15 aborted tissue.) Benzo[a]pyrene-adduct levels were similar but slightly lower in the aborted tissue
16 of cases compared with controls (mean ± SD 4.8 ± 6.0 in cases and 6.0 ± 7.4 in controls, p = 0.29). In
17 the blood samples, however, benzo[a]pyrene-adduct levels were higher in cases (6.0 ± 4.7 and 2.7 ±
18 2.2 in cases and controls, respectively, p < 0.001). In logistic regression analyses using a continuous
19 adduct measure, the OR was 1.35 (95% CI 1.11-1.64) per adduct/108 nucleotide. These results
20 were adjusted for education and household income, but were very similar to the unadjusted results.
21 Categorizing exposure at the median value resulted in an adjusted OR of 4.27 (95% CI 1.41-12.99)
22 in the high compared with low benzo[a]pyrene-adduct group. There was no relation between
23 benzo[a]pyrene-adduct levels in the aborted tissue and missed abortion in the logistic regression
24 analyses using either the continuous (adjusted OR 0.97, 95% CI 0.93-1.02) or dichotomous
25 exposure measure (adjusted OR 0.76, 95% CI 0.37-1.54). Associations between missed abortion
26 and several interview-based measures of potential PAH exposure were also seen: adjusted OR 3.07
27 (95% CI 1.31-7.16) for traffic congestion near residence, 3.52 (95% CI 1.44-8.57) for commuting
28 by walking, 3.78 (95% CI 1.11-12.87) for routinely cooked during pregnancy, and 3.21 (95% CI
29 0.98-10.48) for industrial site or stack near residence, but there was no association with other
30 types of commuting (e.g., by bike, car, or bus).
31 Neurotoxicity
32 Niu et al. (2010) studied 176 Chinese coke-oven workers with elevated B[a]P exposure and
33 compared them against 48 referents (workers in a supply warehouse), matched by socioeconomic
34 status, lifestyle and health. Blood levels of monoamine, amino acid and chloine neurotransmitters
35 were measured, and the WHO Neurobehavioral Core Test Battery (NCTB) was administered to
36 assess emotional state, learning, memory and hand-eye coordination. The authors self-designed a
37 study questionnaire to gather information on worker education, vocational history, smoking and
38 drinking habits, personal habits, personal and family medical history, as well as any current
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1 symptoms and medications used in the pervious several weeks. Workers were excluded from the
2 study for any of the following criteria: reported feeling depressed at any point during the previous
3 6 months; had taken medicine in the previous 2 weeks which could affect nervous system function;
4 or if they reported undertaking vigorous exercise less than 48 hrs previously. "Smoking" was
5 defined as > 10 cigarettes/day during the past year. Similarly, "drinking" was defined as
6 wine/beer/spirits consumed > 3 times/week for the past 6 months. Workplace environmental
7 sampling stations were established at each of the physical work locations, including the referent's
8 warehouse, and dual automatic air sampling pumps collected samples at personal breathing zone
9 height for 6 hours/day, over 3 consecutive days. B[a]P content was determined by HPLC, and
10 relative exposure was compared to post-shift urine levels of aB[a]P metabolite, 1-hydroxypyrene
11 (1-OH-Py). Blood was collected in the morning before breakfast; monoamine (norepinephrine and
12 dopamine) and amino acid (Glu, Asp, Gly, and GABA) neurotransmitter levels were determined by
13 HPLC, acetylcholine (Ach) levels determined by hydroxyamine chromometry, and Ach esterase
14 (AchE) levels measured in lysed RBCs using activity kits.
15 B[a]P mean concentrations were 19.56 ± 13.2,185.96 ± 38.6 and 1623.56 ± 435.8 ng/m3 at
16 the bottom, side and top of the coke oven, respectively, all of which were higher than the mean at
17 the referents' warehouse (10.26 ± 7.6 ng/m3). The authors did not report stratified analysis by
18 different levels of B[a]P exposure, and reported only comparisons between the referents and all
19 exposed workers combined (Table B-3), or between workers grouped by urinary B[a]P metabolite
20 1-OH-Py levels (Table B-4). There were no significant differences in age, education, smoking or
21 alcohol use between the coke oven and warehouse workers. Urinary 1-OH-Py levels were 32%
22 higher in coke oven workers compared to the referent group, corresponding to the higher levels of
23 B[a]P detected in all coke oven workstation compared to the supply warehouse. Performance in
24 two neurobehavioral function tests, digit span and forward digit span, were significantly decreased
25 in the exposed oven workers versus control group; when stratified by urinary metabolite level,
26 scores significantly decreased with increasing 1-OH-Py levels. Of the neurotransmitters assessed,
27 norepinephrine, dopamine, Asp and GABA were significantly decreased in exposed versus control
28 workers; norepinephrine and Asp were also significantly and inversely related with 1-OH-Py levels.
29 Dopamine levels appeared to decrease with increased urinary metabolite levels, although the
30 relationship was not statistically significant GABA levels were highly variable, and appeared to
31 increase with increasing 1-OH-Py levels, although this relationship was statistically significant.
32 Acetylcholine levels were 4-fold higher in coke oven workers compared to referents, and AchE
33 actiivty 30% lower; both Ach and AchE were significantly associated with urinary B[a]P metabolite
34 levels, although Ach increased and AchE activity decreased with increasing 1-OH-Py. The authors
3 5 reported results of correlation analysis, indicating that digit span scores correlated negatively with
36 Ach and positively with AchE (coefficients of-0.230, -0.276 and 0.120, 0.170, respectively),
37 although no indication of statistical significance was given. No other associations were reported.
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Table B-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 %)
Age (yr)
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 B[a]P metabolite (u.mol/mol Cr; 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)
Asp (u.g/ml)
Glu (u.g/ml)
GABA (ng/ml)
Ach (u.g/ml)
AchE activity (U/mg protein)
62.54 ±58.07
1566.28 ±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
1425.85 ±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
Source: Niu et al. (2010).
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Table B-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 categoried by 1-OH-Py level
0 - 3.09
u,mol/mol Cr
33
3.09-3.90
u,mol/mol Cr
72
3.90-5.53
u,mol/mol Cr
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)
Asp (u.g/ml)
Glu (u.g/ml)
GABA (ng/ml)
Ach (u.g/ml)
AchE activity (U/mg
protein)
67.31 ±67.45
1614.45 ± 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
1482.30 ±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
1405.06 ±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
Source: Niu et al. (2010).
Immuno toxicity
Zhang et al. (2012) studied 129 Chinese coke-oven workers with elevated B[a]P exposure
and compared them against 37 referents (workers in a supply warehouse), matched by
socioeconomic status, lifestyle and health. Area B[a]P levels were quantified in the various work
areas, and the primary endpoint was the level of early and late apoptosis in PBMCs isolated from
each worker sub-group 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 pervious 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 B[a]P exposure: 34
oven bottom workers, 48 oven side workers, and 47 oven top workers. Workplace environmental
sampling stations were established at each of the physical work locations, including the referent's
warehouse, and dual automatic air sampling pumps collected samples at personal breathing zone
height for 6 hours/day, over 3 consecutive days. B[a]P content was determined by HPLC, and
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relative exposure was compared to post-shift urine levels of a B[a]P metabolite, 1-hydroxypyrene
(1-OH-Py). Collected and purified PBMCs were incubated with Annexin-V and PI prior to analysis
by flow cytometry; early apoptotic cells were considered to be Annexin V+/PI-, while late apoptotic
cells were considered Annexin V+/PI+.
All apoptosis data was displayed graphically, and in all groupings early:late apoptotic
PBMCs occurred at an approximate 2:1 frequency. PBMC apoptosis was similar in each of the three
coke oven worker groups, which were all statistically significantly higher than referents
(approximately 2-fold) for both early and late apoptosis. While self-reported smoking incidence
varied significantly among the 4 worker groups, stratification by smoking years or smoking index
did not reveal any significant association with PBMC apoptosis. Multiple linear stepwise regression
analysis suggested that urine 1-OH-Py levels and years of coke oven operation were positively
associated with increased early and late PMBC apoptosis (Table B-5), and that years of ethanol
consumption was negatively associated with only early apoptosis. These associations were tested
by stratifying workers into three groups by urinary 1-OH-Py levels or coke oven operation years,
and in both cases, the groups with the highest urinary metabolite levels or longest oven operating
experience had statistically significantly higher levels of both early and late apoptotic PBMCs, vs.
the lowest or shortest duration groups, respectively. Likewise, when sorted into groups based
upon years of ethanol consumption, the highest ethanol "years of consumption" group had
statistically significantly lower early apoptosis rates when compared to the lowest ethanol
consuming group.
Table B-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
1623.5 ± 435.8
47
P value
Background information (mean ± SD or %)
Age (yr)
Working years (yr)
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 B[a]P metabolite (u.mol/mol Cr; mean ± SD)
1-OH-Py
2.78 ±1.04
3. 22 ±0.81*
3.51 ±0.55*
3. 66 ±0.58*
0.000
* p < 0.05 significantly different from control mean
Source: Zhang et al. (2012).
23
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1 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 end points include
5 chromosomal aberration (CA), sister chromatid exchange (SCE), micronucleus (MN) formation,
6 hypoxanthine guanine phosphoribosyl transferase (hprt) mutation frequency, and glycophorin A
7 mutation frequency (Gyorffy et al., 2008). These biomarkers are often incorporated in multi-
8 endpoint studies with other biomarkers of exposure. Because they indicate related but different
9 endpoints, there is often a lack of correlation between the different categories of biomarkers.
10 Merlo et al. (1997) evaluated DNA adduct formation (measured by [32P]-postlabelling) and
11 MN in WBCs of 94 traffic policemen versus 52 residents from the metropolitan area of Genoa, Italy.
12 All study subjects wore personal air samplers for 5 hours of one work shift, and levels of
13 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), butMN 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., 1994,1993). In the first study, involving 48 workers, several biomarkers were analyzed for
24 dose-response and interindividual variability (Perera et al., 1993). PAH-DNA adducts were
25 determined in WBCs using an immunoassay as described in Section 4.1.2.2.1 and enzyme-linked
26 immunosorbent assay with fluorescence detection. Mutations at the hprt locus were also measured
27 in WBC DNA. The latter assay is based on the fact that each cell contains only one copy of the hprt
28 gene, which is located on the X-chromosome. While male cells have only one X-chromosome,
29 female cells inactivate one of the two X-chromosomes at random. The gene is highly sensitive to
30 mutations such that in the event of a crucial mutation in the gene, enzyme activity disappears
31 completely from the cell. In addition, mutations at the glycophorin A gene locus were measured in
32 red blood cells (RBCs). The glycophorin A mutation frequency was not correlated with either
33 benzo[a]pyrene exposure or PAH-DNA adduct formation. However, both PAH-DNA adductlevels
34 and hprt mutation frequency increased with increasing benzo[a]pyrene exposure. In addition,
35 there was a highly significant correlation between incidence of hprt mutations and PAH-DNA
36 adduct levels (p = 0.004).
37 In a second study, Perera et al. (1994) surveyed 64 iron foundry workers with assessments
38 conducted in 2 successive years; 24 of the workers provided blood samples in both years. Exposure
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1 to benzo[a]pyrene, collected by personal and area sampling in the first year of the study, ranged
2 from <5 to 60 ng/m3 and was estimated to have decreased by 40% in the second year. The levels of
3 PAH-DNA adducts were roughly 50% lower in the 2nd year, presumably reflecting decreased
4 exposure. The longer-lived hprt mutations were not as strongly influenced by the decreasing
5 exposure to benzo[a]pyrene. Study subjects who did not have detectable levels of DNA adducts
6 were excluded from the study. As in the previous study, a strong correlation between DNA adduct
7 levels and incidence of hprt mutations was observed (Perera etal., 1993).
8 Kalina et al. (1998) studied several cytogenetic markers in 64 coke oven workers and
9 34 controls employed at other locations within the same plant. Airborne benzo[a]pyrene and seven
10 other carcinogenic PAHs were collected by personal air samplers, which showed ambient
11 benzo[a]pyrene concentrations ranging widely from 0.002 to 50 ug/m3 in coke oven workers and
12 from 0.002 to 0.063 ug/m3 in controls. CAs, SCEs, high-frequency cells (HFCs), and SCE
13 heterogeneity index were all significantly increased with benzo[a]pyrene exposure. Except for
14 increases in HFCs, no effect of smoking was observed. Consistent with studies of PAH-DNA adduct
15 formation, reduced cytogenetic response at high exposure levels produced a nonlinear dose-
16 response relationship. The authors also evaluated the potential influence of polymorphisms in
17 enzymes involved in the metabolism of benzo[a]pyrene. Glutathione S-transferase Ml (GSTM-1)
18 and N-acetyl transferase-2 polymorphisms were studied and no evidence of the two gene
19 polymorphisms having any influence on the incidence of cytogenetic damage was found.
20 Motykiewicz et al. (1998) conducted a similar study of genotoxicity associated with
21 benzo[a]pyrene exposure in 67 female residents of a highly polluted industrial urban area of Upper
22 Silesia, Poland, and compared the results to those obtained from 72 female residents of another
23 urban but less polluted area in the same province of Poland. Urinary mutagenicity and 1-
24 hydroxypyrene levels, PAH-DNA adducts in oral mucosa cells (detected by immunoperoxidase
25 staining), SCEs, HFCs, CAs, bleomycin sensitivity, and GSTM-1 and CYP1A1 polymorphisms in blood
26 lymphocytes were investigated. High volume air samplers and gas chromatography were used to
27 quantify ambient benzo[a]pyrene levels, which were 3.7 ng/m3 in the polluted area and 0.6 ng/m3
28 in the control area during the summer. During winter, levels rose to 43.4 and 7.2 ng/m3 in the two
29 areas, respectively. The cytogenetic biomarkers (CA and SCE/HFC), urinary mutagenicity, and
30 urinary 1-hydroxypyrene excretion were significantly increased in females from the polluted area,
31 and differences appeared to be more pronounced during winter time. PAH-DNA adduct levels were
32 significantly increased in the study population, when compared to the controls, only in the winter
33 season. No difference in sensitivity to bleomycin-induced lymphocyte chromatid breaks was seen
34 between the two populations. As with the study by Kalina et al. (1998), genetic polymorphisms
3 5 assumed to affect the metabolic transformation of benzo [a]pyrene were not associated with any
36 difference in the incidence of DNA damage.
37 In a study of Thai school boys in urban (Bangkok) and rural areas, bulky (including but not
38 limited to BPDE-type) DNA adduct levels were measured in lymphocytes along with DNA SSBs,
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1 using the comet assay, and DNA repair capacity (Tuntawiroon et al., 2007). Ambient air and
2 personal breathing zone measurements indicated that Bangkok school children experienced
3 significantly higher exposures to benzo[a]pyrene and total PAHs. A significantly higher level of
4 SSBs (tail length 1.93 ± 0.09 versus 1.28 ± 0.12 ^m, +51%; p < 0.001) was observed in Bangkok
5 school children when compared with rural children, and this parameter was significantly
6 associated with DNA adduct levels. A significantly reduced DNA repair capacity (0.45 ±0.01 versus
7 0.26 ± 0.01 y-radiation-induced deletions per metaphase, -42%; p < 0.001) was also observed in the
8 city school children, again significantly associated with DNA adduct levels. It was not evident why
9 higher environmental PAH exposure would be associated with lowered DNA repair capacity.
10 However, because the personal breathing zone PAH levels and DNA adduct levels were not
11 associated with each other, it is conceivable that the city school children had a priori lower DNA
12 repair capacities that contributed significantly to the high adduct levels. The authors considered
13 genetic differences between the two study populations as a possible reason for this observation.
14 Epidemiologic Findings in Humans
15 The association between human cancer and contact with PAH-containing substances, such
16 as soot, coal tar, and pitch, has been widely recognized since the early 1900s (Bostrom et al., 2002).
17 Although numerous epidemiology studies establish an unequivocal association between PAH
18 exposure and human cancer, defining the causative role for benzo[a]pyrene and other specific PAHs
19 remains a challenge. In essentially all reported studies, either the benzo[a]pyrene exposure and/or
20 internal dose are not known, or the benzo[a]pyrene carcinogenic effect cannot be distinguished
21 from the effects of other PAH and non-PAH carcinogens. Nevertheless, three types of investigations
22 provide support for the involvement of benzo[a]pyrene in some human cancers: molecular
23 epidemiology studies; population- and hospital-based case-control studies; and occupational cohort
24 studies. In some cohort studies, benzo[a]pyrene exposure concentrations were measured and thus
25 provide a means to link exposure intensity with observed cancer rates. In case-control studies, by
26 their nature, benzo[a]pyrene and total PAH doses can only be estimated.
27 Molecular Epidemiology and Case-Control Cancer Studies
28 Defective DNA repair capacity leading to genomic instability and, ultimately, increased
29 cancer risk is well documented (Wu et al., 2007, 2005). Moreover, sensitivity to mutagen-induced
30 DNA damage is highly heritable and thus represents an important factor that determines individual
31 cancer susceptibility. Based on studies comparing monozygotic and dizygotic twins, the genetic
32 contribution to BPDE mutagenic sensitivity was estimated to be 48.0% (Wu et al., 2007). BPDE has
33 been used as an etiologically relevant mutagen in case-control studies to examine the association
34 between elevated lung and bladder cancer risk and individual sensitivity to BPDE-induced DNA
35 damage. Mutagen sensitivity is determined by quantifying chromatid breaks or DNA adducts in
36 phytohemagglutinin-stimulated peripheral blood lymphocytes as an indirect measure of DNA
37 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 (Li et al., 2001). Lung cancer cases showed consistent statistically significant
4 elevations in induced BPDE-DNA adducts in lymphoctes, 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 (odds ratios [ORs] 1.11,1.62,
8 and 3.23; trend test, p < 0.001). In a related hospital-based, case-control study involving 155 lung
9 cancer patients and 153 healthy controls, stimulated peripheral blood lymphocytes were exposed
10 to BPDE in vitro (Wu et al., 2005). DNA damage/repair was evaluated in lymphocytes using the
11 comet assay, and impacts on cell cycle checkpoints were measured using a fluorescence-activated
12 cell-sorting method. The lung cancer cases exhibited significantly higher levels of BPDE-induced
13 DNA damage than the controls (p < 0.001), with lung cancer risk positively associated with
14 increasing levels of lymphocyte DNA damage when grouped in quartiles (trend test, p < 0.001). In
15 addition, lung cancer patients demonstrated significantly shorter cell cycle delays in response to
16 BPDE exposure to 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 (Gu et al., 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% confidence interval [CI] 3.26-8.59). Categorization of patients into tertiles for BPDE
25 sensitivity relative to controls demonstrated a dose-related association between BPDE-induced
26 9p21 damage and bladder cancer risk. Collectively, the results of molecular epidemiology studies
27 with lung and bladder cancer patients indicate that individuals with a defective ability to repair
28 BPDE-DNA adducts are at increased risk for cancer and, moreover, that specific genes linked to
29 tumorigenesis 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 et al., 2005), another involving 626 pancreatic cancer
34 cases and 530 controls (Li etal., 2007), and a third involving 146 colorectal adenoma cases and 228
35 controls (Sinha et al., 2005), dietary intake of benzo[a]pyrene was estimated using food frequency
36 questionnaires. In all studies, the primary focus was on estimated intake of benzo[a]pyrene (and
37 other carcinogens) derived from cooked meat Overall, cases when compared with controls had
3 8 higher intakes of benzo [ajpyrene and other food carcinogens, leading to the conclusion that
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1 benzo[a]pyrene plays a role in the etiology of these tumors in humans. In a supportive follow-up
2 case-control study of colorectal adenomas, levels of leukocyte PAH-DNA adducts were significantly
3 higher in cases when compared with controls (p = 0.02), using a method that recognizes BPDE and
4 several other PAHs bound to DNA (Gunter et al., 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 (Bosetti etal., 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 Bosetti et al., 2007; Armstrong et al., 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
18 Cancer incidence in aluminum and electrode production plants
19 Exposure to benzo[a]pyrene and BSM in aluminum smelter workers is strongly associated
20 with bladder cancer and weakly associated with lung cancer (Boffetta et al., 1997; Tremblay et al.,
21 1995; Armstrong et al., 1994; Gibbs, 1985; Theriault et al., 1984). In an analysis of pooled data from
22 nine cohorts of aluminum production workers, 688 respiratory tract cancer cases were observed
23 versus 674.1 expected (pooled RR 1.03; CI 0.96-1.11) (Bosetti et al., 2007). A total of 196 bladder
24 cancer cases were observed in eight of the cohorts, compared with 155.7 expected (pooled relative
25 risk [RR] 1.29; CI 1.12-1.49). Based on estimated airborne benzo[a]pyrene exposures from a meta-
26 analysis of eight cohort studies, the predicted lung cancer RR per 100 ug/m3-years of cumulative
27 benzo[a]pyrene exposure was 1.16 (95% CI 1.05-1.28) (Armstrong etal., 2004).
28 Spinelli et al. (2006) reported a 14-year update to a previously published historical cohort
29 study (Spinelli etal., 1991) of Canadian aluminum reduction plant workers. The results confirmed
30 and extended the findings from the earlier epidemiology study. The study surveyed a total of 6,423
31 workers with >3 years of employment at an aluminum reduction plant in British Columbia, Canada,
32 between the years 1954 and 1997, and evaluated all types of cancers. The focus was on cumulative
33 exposure to coal tar pitch volatiles, measured as BSM and as benzo[a]pyrene. Benzo[a]pyrene
34 exposure categories were determined from the range of predicted exposures over time from
35 statistical exposure models. There were 662 cancer cases, of which approximately 98% had
36 confirmed diagnoses. The overall cancer mortality rate (standardized mortality ratio 0.97; CI 0.87-
37 1.08) and cancer incidence rate (standardized incidence ratio [SIR] 1.00; CI 0.92-1.08) were not
3 8 different from that of the British Columbia general population. However, this study identified
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1 significantly increased incidence rates for cancers of the bladder (SIR 1.80; CI 1.45-2.21) and the
2 stomach (SIR 1.46; CI 1.01-2.04). The lung cancer incidence rate was only slightly higher than
3 expected (SIR 1.10; CI 0.93-1.30). Significant dose-response associations with cumulative
4 benzo[a]pyrene exposure were seen for bladder cancer (p trend < 0.001), stomach cancer (p trend
5 < 0.05), lung cancer (p trend < 0.001), non-Hodgkin lymphoma (p trend < 0.001), and kidney cancer
6 (p trend < 0.01), although the overall incidence rates for the latter three cancer types were not
7 significantly elevated versus the general population. Similar cancer risk results were obtained
8 using BSM as the exposure measure; the cumulative benzo[a]pyrene and BSM exposures were
9 highly correlated (r = 0.94).
10 In several occupational cohort studies of workers in Norwegian aluminum production
11 plants, personal and stationary airborne PAH measurements were performed.
12 In a study covering 11,103 workers and 272,554 person x years of PAH exposure, cancer
13 incidence was evaluated in six Norwegian aluminum smelters (Romundstad et al., 2000a, b).
14 Reported estimates of PAH exposure concentrations reached a maximum of 3,400 [J.g/m3 PAH
15 (680 [ig/m3 benzo[a]pyrene). The overall number of cancers observed in this study did not differ
16 significantly from control values (SIR 1.03; CI 1.0-1.1). The data from this study showed
17 significantly increased incidences for cancer of the bladder (SIR 1.3; CI 1.1-1.5) and elevated, but
18 not significant, SIRs for larynx (SIR 1.3; CI 0.8-1.9), thyroid (SIR 1.4; CI 0.7-2.5), and multiple
19 myeloma (SIR 1.4; CI 0.9-1.9). Incidence rates for bladder, lung, pancreas, and kidney cancer (the
20 latter three with SIRs close to unity) were subjected to a cumulative exposure-response analysis.
21 The incidence rate for bladder cancer showed a trend with increasing cumulative exposure and
22 with increasing lag times (up to 3 0 years) at the highest exposure level. The incidence of both lung
23 and bladder cancers was greatly increased in smokers. The authors reported that using local
24 county rates rather than national cancer incidence rates as controls increased the SIR for lung
25 cancer (SIR 1.4; CI 1.2-1.6) to a statistically significant level.
26
27 Cancer incidence in coke oven, coal gasification, and iron and steel foundry workers
28 An increased risk of death from lung and bladder cancer is reported in some studies
29 involving coke oven, coal gasification, and iron and steel foundry workers (Bostrom et al., 2002;
30 Boffetta et al., 1997). An especially consistent risk of lung cancer across occupations is noted when
31 cumulative exposure is taken into consideration (e.g., RR of 1.16 per 100 unity-years for aluminum
32 smelter workers, 1.17 for coke oven workers, and 1.15 for coal gasification workers). In an analysis
33 of pooled data from 10 cohorts of coke production workers, 762 lung cancer cases were observed
34 versus 512.1 expected (pooled RR 1.58; CI 1.47-1.69) (Bosetti et al., 2007). Significant variations in
35 risk estimates among the studies were reported, particularly in the large cohorts (RRs of 1.1,1.2,
36 2.0, and 2.6). There was no evidence for increased bladder cancer risk in the coke production
37 workers. Based on estimated airborne benzo[a]pyrene exposures from a meta-analysis of 10
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1 cohort studies, the predicted lung cancer RR per 100 ug/m3-years of cumulative benzo[a]pyrene
2 exposure was 1.17 (95% CI 1.12-1.22) (Armstrong et al., 2004).
3 A meta-analysis of data from five cohorts of gasification workers reported 251 deaths from
4 respiratory tract cancer, compared with 104.7 expected (pooled RR 2.58; 95% CI 2.28-2.92)
5 (Bosetti et al., 2007). Pooled data from three of the cohorts indicated 18 deaths from urinary tract
6 cancers, versus 6.0 expected (pooled RR 3.27; 95% CI 2.06-5.19). Based on estimated airborne
7 benzo[a]pyrene exposures from a meta-analysis of four gas worker cohort studies, the predicted
8 lung cancer RRper 100 ug/m3-years of cumulative benzo[a]pyrene exposure was 1.15 (95% CI
9 1.11-1.20) (Armstrong et al., 2004).
10 Increased risks were reported in iron and steel foundry workers for cancers of the
11 respiratory tract, bladder, and kidney. In an analysis of pooled data from 10 cohorts,
12 1,004 respiratory tract cancer cases were observed versus 726.0 expected (pooled RR 1.40;
13 CI 1.31-1.49) (Bosetti etal., 2007). A total of 99 bladder cancer cases were observed in seven of the
14 cohorts, compared with 83.0 expected (pooled RR 1.29; CI 1.06-1.57). For kidney cancer, 40 cases
15 were observed compared with 31.0 expected based on four studies (pooled RR 1.30; 95% CI 0.95-
16 1.77).
17 Xu et al. (1996) conducted a nested case-control study, surveying the cancer incidence
18 among 196,993 active or retired workers from the Anshan Chinese iron and steel production
19 complex. A large number of historical benzo[a]pyrene measurements (1956-1995) were available.
20 The study included 610 cases of lung cancer and 292 cases of stomach cancer, with 959 age- and
21 gender-matched controls from the workforce. After adjusting for nonoccupational risk factors such
22 as smoking and diet, significantly elevated risks for lung cancer and stomach cancer were identified
23 for subjects employed for >15 years, with ORs varying among job categories. For either type of
24 cancer, highest risks were seen among coke oven workers: lung cancer, OR = 3.4 (CI 1.4-8.5);
25 stomach cancer, OR = 5.4 (CI 1.8-16.0).
26 There were significant trends for long-term, cumulative benzo[a]pyrene exposure versus
27 lung cancer (p = 0.004) or stomach cancer (p = 0.016) incidence. For cumulative total
28 benzo[a]pyrene exposures of <0.84, 0.85-1.96,1.97-3.2, and >3.2, respectively, the ORs for lung
29 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
30 cumulative total benzo[a]pyrene exposures of <0.84, 0.85-1.96,1.97-3.2, and >3.2, the ORs for
31 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),
32 respectively. However, the investigators noted that additional workplace air contaminants were
33 measured, which might have influenced the outcome. Of these, asbestos, silica, quartz, and iron
34 oxide-containing dusts may have been confounders. For lung cancers, cumulative exposures to
35 total dust and silica dust both showed significant dose-response trends (p = 0.001 and 0.007,
36 respectively), while for stomach cancer, only cumulative total dust exposure showed a marginally
37 significant trend (p = 0.061). For cumulative total dust exposures of <69, 69-279, 280-882, and
38 >883 mg/m3, the ORs for lung cancer were 1.4 (CI 1.2-1.9), 1.2 (CI 1.0-2.19), 1.4 (CI 1.0-2.0), and
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Toxicological Review ofbenzo[a]pyrene
1 1.9 (CI 1.3-2.5), respectively. For cumulative silica dust exposures of <3.7, 3.7-10.39,10.4-27.71,
2 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),
3 and 1.8 (CI 1.2-2.5), respectively. For cumulative total dust exposures of <69, 69-279, 280-882,
4 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
5 1.6 (CI 1.1-2.5), respectively.
6 Exposure-response data from studies of coke oven workers in the United States have often
7 been used to derive quantitative risk estimates for PAH mixtures, and for benzo[a]pyrene as an
8 indicator substance (Bostrom et al., 2002). However, there are numerous studies of coke oven
9 worker cohorts that do notprovide estimates of benzo[a]pyrene exposure. An overview of the
10 results of these and other studies can be obtained from the review of Boffetta et al. (1997).
11
12 Cancer incidence in asphalt workers and roofers
13 These groups encompass different types of work (asphalt paving versus roofing) and also
14 different types of historical exposure that have changed from using PAH-rich coal tar pitch to the
15 use of bitumen or asphalt, both of which are rather low in PAHs due to their source (crude oil
16 refinery) and a special purification process. Increased risks for lung cancer were reported in large
17 cohorts of asphalt workers and roofers; evidence for increased bladder cancer risk is weak
18 (Burstynetal., 2007; Partanen and Boffetta, 1994; Chiazze et al., 1991; Hansen, 1991,1989;
19 Hammond etal., 1976). In an analysis of pooled data from two cohorts of asphalt workers, 822 lung
20 cancer cases were observed versus 730.7 expected (pooled RR 1.14; 95% CI 1.07-1.22) (Bosetti et
21 al., 2007). In two cohorts of roofers, analysis of pooled data indicated that 138 lung cancer cases
22 were observed, compared with 91.9 expected (pooled RR 1.51; 95% CI 1.28-1.78) (Bosetti etal.,
23 2007).
24
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1 ANIMAL BIOASSAYS
2 Oral Bioassays
3 Subchronic Studies
4 De Jong et al. (1999) treated male Wistar rats (eight/dose group) with benzo[a]pyrene
5 (98.6% purity) dissolved in soybean oil by gavage 5 days/week for 35 days at doses of 0, 3,10, 30,
6 or 90 mg/kg-day (adjusted doses: 0, 2.14, 7.14, 21.4, and 64.3 mg/kg-day). At the end of the
7 exposure period, rats were necropsied, organ weights were determined, and major organs and
8 tissues were prepared for histological examination (adrenals, brain, bone marrow, colon, caecum,
9 jejunum, heart, kidney, liver, lung, lymph nodes, esophagus, pituitary, spleen, stomach, testis, and
10 thymus). Blood was collected for examination of hematological endpoints, but there was no
11 indication that serum biochemical parameters were analyzed. Immune parameters included
12 determinations of serum immunoglobulin (Ig) levels (IgG, IgM, IgE, and IgA), relative spleen cell
13 distribution, and spontaneous cytotoxicity of spleen cell populations determined in a natural-killer
14 (NK) cell assay.
15 Body weight gain was decreased beginning at week 2 at the high dose of 90 mg/kg-day;
16 there was no effect at lower doses (De Jong et al., 1999). Hematology revealed a dose-related
17 decrease in RBC count, hemoglobin, and hematocrit at >10 mg/kg-day (Table B-6). A minimal but
18 significant increase in mean cell volume and a decrease in mean cell hemoglobin concentration
19 were noted at 90 mg/kg-day, and may indicate dose-related toxicity for the RBCs and/or RBC
20 precursors in the bone marrow. A decrease in WBCs, attributed to a decrease in the number of
21 lymphocytes (approximately 50%) and eosinophils (approximately 90%), was observed at
22 90 mg/kg-day; however, there was no effect on the number of neutrophils or monocytes. A
23 decrease in the cell number in the bone marrow observed in the 90 mg/kg-day dose group was
24 consistent with the observed decrease in the RBC and WBC counts at this dose level. In the
25 90 mg/kg-day dose group, brain, heart, kidney, and lymph node weights were decreased and liver
26 weight was increased (Table B-6). Decreases in heart weight at 3 mg/kg-day and in kidney weight
27 at 3 and 30 mg/kg-day were also observed, but these changes did not show dose-dependent
28 responses. Dose-related decreases in thymus weight were statistically significant at >10 mg/kg-
29 day (Table B-6).
30 Table B-6. Exposure-related effects in male Wistar rats exposed to
31 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)
Dose (mg/kg-d)
0
14.96 ± 1.9
8.7 ±0.2
3
13. 84 ±3.0
8.6 ±0.2
10
13.69 ±1.8a
8.3 ±0.2
30
13.58 ±2.9a
7.8 ±0.4
90
8.53±l.la
7.1±0.4a
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Effect
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 of thymus;
mean ± SD; n = 6-8)
Dose (mg/kg-d)
0
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
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
282a
1,864
934a
l,761a
9,567
472 ± 90
590
123
148
18
74.4 ±2. 2
10
9.8±0.2a
0.47±0.01a
86 ±31
104 ± 28
78 ±67
228 ±351
59 ±13
31 ±8
34±3a
40 ±9
24 ±5
24 ±7
300
1,859
1,000
1,899
11,250
438 ± 64a
538
160
130a
19
79. 2 ±5. 9
30
9.5 ± 0.4a
0.46 ± 0.02a
67 ± 16a
106 ± 19
72 ±22
145 ± 176
63 ±10
27 ±8
32±4a
36 ±2
22 ±4
19 ±2
293
1,784
967
l,790a
11,118
388 ± 71a
596
141
158
17
75.8 ±4.0
90
8.6±0.6a
0.43±0.02a
81 ±26
99 ±29
39 ± 19a
75 ±55
41 ± 10a
19±4a
23±4a
44 ±6
26 ±4
27 ±5
250a
l,743a
863a
l,626a
12,107a
198 ± 65a
505
89a
107a
10a
68.9 ± 5. 2a
1
2
3
4
5
6
7
Significantly (p < 0.05) different from control mean. For body weight and organ weight means, SDs
were only reported for thymus weights.
Source: De Jong et al. (1999).
Statistically significant reductions were also observed in the relative cortex surface area of
the thymus and thymic medullar weight at 90 mg/kg-day, but there was no difference in cell
proliferation between treated and control animals using the proliferating cell nuclear antigen
(PCNA) technique. Changes in the following immune parameters were noted: dose-related and
statistically significant decrease in the relative number of B cells in the spleen at 10 (13%),
30 (18%), and 90 mg/kg-day (41%); significant decreases in absolute number of cells harvested in
the spleen (31%), in the number of B cells in the spleen (61%), and NK cell activity in the spleen
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1 (E:T ratio was 40.9 ± 28.4% that of the controls) at 90 mg/kg-day; and a decrease in serum IgM
2 (33%) and IgA (61%) in rats treated with 30 and 90 mg/kg-day, respectively. The decrease in the
3 spleen cell count was attributed by the study authors to the decreased B cells and suggested a
4 possible selective toxicity of benzo[a]pyrene to B cell precursors in the bone marrow. The study
5 authors considered the decrease in IgA and IgM to be due to impaired production of antibodies,
6 suggesting a role of thymus toxicity in the decreased (T-cell dependent) antibody production. In
7 addition to the effects on the thymus and spleen, histopathologic examination revealed treatment-
8 related lesions only in the liver and forestomach at the two highest dose levels, but the incidence
9 data for these lesions were not reported by De Jong et al. (1999). Increased incidence for
10 forestomach basal cell hyperplasia (p < 0.05 by Fisher's exact test) was reported at 30 and
11 90 mg/kg-day, and increased incidence for oval cell hyperplasia in the liver was reported at
12 90 mg/kg-day (p < 0.01, Fisher's exact test). The results indicate that 3 mg/kg-day was a no-
13 observed-adverse-effect level (NOAEL) for effects on hematological parameters (decreased RBC
14 count, hemoglobin, and hematocrit) and immune parameters (decreased thymus weight and
15 percent of B cells in the spleen) noted in Wistar rats at 10 mg/kg-day (the lowest-observed-
16 adverse-effect level [LOAEL]) and above. Lesions of the liver (oval cell hyperplasia) and
17 forestomach (basal cell hyperplasia) occurred at doses >30 mg/kg-day.
18 Knuckles etal. (2001) exposed male and female F344 rats (20/sex/dose group) to
19 benzo[a]pyrene (98% purity) at doses of 0, 5, 50, or 100 mg/kg-day in the diet for 90 days. Food
20 consumption and body weight were monitored, and the concentration of benzo[a]pyrene in the
21 food was adjusted every 3-4 days to maintain the target dose. The authors indicated that the actual
22 intake of benzo[a]pyrene by the rats was within 10% of the calculated intake, and the nominal
23 doses were not corrected to actual doses. Hematology and serum chemistry parameters were
24 evaluated. Urinalysis was also performed. Animals were examined for gross pathology, and
25 histopathology was performed on selected organs (stomach, liver, kidney, testes, and ovaries).
26 Statistically significant decreases in RBC counts and hematocrit level (decreases as much as 10 and
27 12%, respectively) were observed in males at doses >50 mg/kg-day and in females at 100 mg/kg-
28 day. A maximum 12% decrease (statistically significant) in hemoglobin level was noted in both
29 sexes at 100 mg/kg-day. Blood chemistry analysis showed a significant increase in blood urea
30 nitrogen (BUN) only in high-dose (100 mg/kg-day) males. Histopathology examination revealed an
31 apparent increase in the incidence of abnormal tubular casts in the kidney in males at 5 mg/kg-day
32 (40%), 50 mg/kg-day (80%), and 100 mg/kg-day (100%), compared to 10% in the controls. Only
33 10% of the females showed significant kidney tubular changes at the two high-dose levels
34 compared to zero animals in the female control group. The casts were described as molds of distal
3 5 nephron lumen and were considered by the study authors to be indicative of renal dysfunction.
36 From this study, male F344 rats appeared to be affected more severely by benzo[a]pyrene
37 treatment than the female rats. However, the statistical significance of the kidney lesions are
38 unclear. Several reporting gaps and inconsistencies regarding the reporting of kidney
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1 abnormalities in Knuckles et al. (2001) make interpretation of the results difficult Results of
2 histopathological kidney abnormalities (characterized primarily as kidney casts) were presented
3 graphically and the data were not presented numerically in this report. No indication was given in
4 the graph that any groups were statistically different than controls, although visual examination of
5 the magnitude of response and error bars appears to indicate a fourfold increase in kidney casts in
6 males compared to the control group (40 compared to 10%). The figure legend reported the data
7 as "percentage incidence of abnormal kidney tissues" and reported values as mean ± SD. However,
8 the text under the materials and methods section stated that Fisher's exact test was used for
9 histopathological data, which would involve the pairwise comparison of incidence and not means.
10 There are additional internal inconsistencies in the data presented. The data appeared to indicate
11 that incidences for males were as follows: control, 10%; 5 mg/kg-day, 40%; 50 mg/kg-day, 80%;
12 and 100 mg/kg-day, 100%; however, these incidences are inconsistent with the size of the study
13 groups, which were reported as 6-8 animals per group. The study authors were contacted, but did
14 not respond to EPA's request for clarification of study design and/or results. Due to issues of data
15 reporting, aLOAEL could not be established for the increased incidence of kidney lesions. Based on
16 the statistically significant hematological effects including decreases in RBC counts, hematocrit, and
17 BUN, the NOAEL in males was 5 mg/kg-day and the LOAEL was 50 mg/kg-day, based on in F344
18 rats. No exposure-related histological lesions were identified in the stomach, liver, testes, or
19 ovaries in this study.
20 In a range-finding study, Wistar (specific pathogen-free [SPF] Riv:TOX) rats (10/sex/dose
21 group) were administered benzo[a]pyrene (97.7% purity) dissolved in soybean oil by gavage at
22 dose levels of 0,1.5, 5,15, or 50 mg/kg body weight-day, 5 days/week for 5 weeks (Kroese et al.,
23 2001). Behavior, clinical symptoms, body weight, and food and water consumption were
24 monitored. None of the animals died during the treatment period. Animals were sacrificed
25 24 hours after the last dose. Urine and blood were collected for standard urinalysis and
26 hematology and clinical chemistry evaluation. Liver enzyme induction was monitored based on
27 EROD activity in plasma. Animals were subjected to macroscopic examination, and organ weights
28 were recorded. The esophagus, stomach, duodenum, liver, kidneys, spleen, thymus, lung, and
29 mammary gland (females only) from the highest-dose and control animals were evaluated for
30 histopathology. Intermediate-dose groups were examined if abnormalities were observed in the
31 higher-dose groups.
32 A significant, but not dose-dependent, increase in food consumption in males at >1.5 mg/kg-
33 day and a decrease in food consumption in females at >5 mg/kg-day was observed (Kroese et al.,
34 2001). Water consumption was statistically significantly altered in males only: a decrease at 1.5, 5,
35 and 15 mg/kg-day and an increase at 50 mg/kg-day. Organ weights of lung, spleen, kidneys,
36 adrenals, and ovaries were not affected by treatment. There was a dose-related, statistically
37 significant decrease in thymus weight in males at 15 and 20 mg/kg-day (decreased by 28 and 33%,
38 respectively) and a significant decrease in thymus weight in females at 50 mg/kg-day (decreased by
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Toxicological Review ofbenzo[a]pyrene
1 17%) (Table B-7). In both sexes, liver weight was statistically significantly increased only at
2 50 mg/kg-day by about 18% (Table B-7).
3 Table B-7. Exposure-related effects in Wistar rats exposed to benzo[a]-
4 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 ± 20a
317 ±30
3/10
3/10a
50
7.20±0.18a
5.03±0.15a
317±21a
271 ± 16a
7/10
7/10a
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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, 50 mg/kg-day, so the livers from intermediate-dose groups were,
therefore, not examined. An increased incidence of brown pigmentation of red pulp (hemosiderin)
in the thymus was observed in treated animals of both sexes. However, this tissue was not
examined in intermediate-dose groups. This range-finding, 5-week study identified a NOAEL of
5 mg/kg-day and a LOAEL of 15 mg/kg-day, based on decreased thymus weight and forestomach
hyperplasia in Wistar rats.
Kroese etal. (2001) exposed Wistar (Riv:TOX) rats (10/sex/dose group) to benzo[a]pyrene
(98.6% purity, dissolved in soybean oil) by gavage at 0, 3, 10, or 30 mg/kg body weight-day,
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1 5 days/week for 90 days. The rats were examined daily for behavior and clinical symptoms and by
2 palpation. Food and water consumption, body weights, morbidity, and mortality were monitored.
3 At the end of the exposure period, rats were subjected to macroscopic examination and organ
4 weights were recorded. Blood was collected for hematology and serum chemistry evaluation, and
5 urine was collected for urinalysis. All gross abnormalities, particularly masses and lesions
6 suspected of being tumors, were evaluated. The liver, stomach, esophagus, thymus, lung, spleen,
7 and mesenteric lymph node were examined histopathologically. In addition, cell proliferation in
8 forestomach epithelium was measured as the prevalence of S-phase epithelial cells displaying
9 bromodeoxyuridine (BrdU) incorporation.
10 There were no obvious effects on behavior of the animals, and no difference was observed
11 in survival or food consumption between exposed animals and controls (Kroese et al., 2001).
12 Higher water consumption and slightly lower body weights than the controls were observed in
13 males but not females at the high dose of 30 mg/kg-day. Hematological investigations showed only
14 nonsignificant, small dose-related decreases in RBC count and hemoglobin level in both sexes.
15 Clinical chemistry evaluation did not show any treatment-related group differences or dose-
16 response relationships for alanine aminotransferase (ALT), serum aspartate transaminase (AST),
17 lactate dehydrogenase (LDH), or creatinine, but a small dose-related decrease in y-glutamyl
18 transferase (GGT) activity was observed in males only. Urinalysis revealed an increase in urine
19 volume in males at 30 mg/kg-day, which was not dose related. At the highest dose, both sexes
20 showed increased levels of urinary creatinine and a dose-related increase in urinary protein.
21 However, no further investigation was conducted to determine the underlying mechanisms for
22 these changes. At necropsy, reddish to brown/gray discoloration of the mandibular lymph nodes
23 was consistently noted in most rats; occasional discoloration was also observed in other regional
24 lymph nodes (axillary). Statistically significant increases in liver weight were observed at 10 and
25 30 mg/kg-day in males (15 and 29%) and at 30 mg/kg-day in females (17%). A decrease in thymus
26 weight was seen in both sexes at 30 mg/kg-day (17 and 33% decrease in females and males,
27 respectively, compared with controls) (Table B-8). At 10 mg/kg-day, thymus weight in males was
28 decreased by 15%, but the decrease did not reach statistical significance.
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1
2
Table B-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 ± 1.30b
5.76 ±0.71
330 ± 60
300 ± 40
30
9.67±1.17b
6.48±0.78b
270 ± 40b
230±30b
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
aReported as SE, but judged to be SD (and confirmed by study authors).
Significantly (p < 0.05) different from control mean; student t-test (unpaired, two-tailed); n =
10/sex/group.
Source: Kroese et al. (2001).
Histopathologic examination revealed what was characterized by Kroese et al. (2001) as
basal cell disturbance in the epithelium of the forestomach in males (p < 0.05) and females
(p < 0.01) at 30 mg/kg-day. The basal cell disturbance was characterized by increased number of
basal cells, mitotic figures, and remnants of necrotic cells; occasional early nodule development;
infiltration by inflammatory cells (mainly histiocytes); and capillary hyperemia, often in
combination with the previous changes (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
resultof 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 Bioassavs
5 Kroese etal. (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 et al., 2001). The organs and tissues collected and prepared for microscopic
15 examination included: brain, pituitary, heart, thyroid, salivary glands, lungs, stomach, oesophagus,
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 ~60
27 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 4-5; 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 B-9).
38 The first liver tumors were detected in week 35 in high-dose male rats. Liver tumors were
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Toxicological Review ofbenzo[a]pyrene
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; Table
3 B-9). Forestomach tumors were associated with the basal cell proliferation observed (without
4 diffuse hyperplasia) in the forestomach of rats in the preliminary range-finding and 90-day
5 exposure studies described previously in Section 4.2.1. At the highest dose level, 59% of rats with
6 forestomach tumors had malignant carcinomas (60/102; Table B-9). Other tissue sites with
7 distinctly elevated incidences of tumors in the 30 mg/kg-day dose group included the oral cavity
8 (papilloma and squamous cell carcinoma [SCC]) in both sexes, and the jejunum (adenocarcinoma),
9 kidney (cortical adenoma), and skin (basal cell adenoma and carcinoma) in male rats (Table B-9).
10 In addition, auditory canal tumors (carcinoma or squamous cell papilloma originating from pilo-
11 sebaceous units including the Zymbal's gland) were also detected in both sexes at 30 mg/kg-day,
12 but auditory canal tissue was not histologically examined in the lower dose groups and the controls
13 (Table B-9). Gross examination revealed auditory canal tumors only in the high-dose group.
14 Table B-9. Incidences of exposure-related neoplasms in Wistar rats
15 treated by gavage with benzo[a]pyrene, 5 days/week, for 104 weeks
Site
Oral cavity
Papilloma
SCC
Basal cell adenoma
Sebaceous cell carcinoma
Oesophagus
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'1
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
20/5 lc
10/5 lc
7/52c
32/52c
1/52
1/52
0/0
0/0
0/0
9/3 lc
9/3 lc
4/31
1/31
0/52
0/52
0/52
25/52c
25/52c
1/52
50/52C
0/52
0/52
1/20
1/20
13/20C
Males"
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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
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
3
0/24
0/24
0/24
0/24
7/5 2C
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
10
2/37
5/37
0/37
0/37
18/5 2C
25/52c
1/51
15/52C
23/52c
0/52
7/52c
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
30a
10/38C
11/38C
2/38
2/38
17/52C
35/52c
8/49c
4/52
45/52c
1/52
8/5 2C
0/52
3/52
0/33
4/33
19/33C
1/33
10/5 lc
4/51
5/51
4/51
8/5 lc
0/51
1/52
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.
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.
Source: Kroese et al. (2001).
1
2 Kroese et al. (2001) did not systematically investigate nonneoplastic lesions detected in rats
3 sacrificed during the 2-year study, because the focus was to identify and quantitate tumor
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1 occurrence. However, incidences were reported for nonneoplastic lesions in tissues or organs in
2 which tumors were detected (i.e., oral cavity, oesophagus, forestomach, jejunum, liver, kidney, skin,
3 mammary, and auditory canal). The reported nonneoplastic lesions associated with exposure were
4 the forestomach basal cell hyperplasia and clear cell foci of cellular alteration in the liver.
5 Incidences for forestomach basal cell hyperplasia in the control through high-dose groups were
6 1/52, 8/51,13/51, and 2/52 for females and 2/50, 8/52, 8/52, and 0/52 in males. Incidences for
7 hepatic clear cell foci of cellular alteration were 22/52, 33/52, 4/52, and 2/52 for females and
8 8/52, 22/52,1/52, and 1/52 for males. These results indicate that the lowest dose group, 3 mg/kg-
9 day, was a LOAEL for increased incidence of forestomach hyperplasia and hepatic histological
10 changes in male and female Wistar rats exposed by gavage to benzo[a]pyrene for up to 104 weeks
11 (see Table 4-5). The lack of an increase in incidence of these nonneoplastic lesions in the
12 forestomach and liver at the intermediate and high doses (compared with controls) were
13 associated with increased incidences of forestomach and liver tumors at these dose levels. The
14 authors of this study note that non-neoplastic effects were not quantified in organs with tumors.
15 As an adjunct study to the 2-year gavage study with Wistar rats, Kroese et al. (2001)
16 sacrificed additional rats (6/sex/group) after 4 and 5 months of exposure (0,1, 3,10, or 30 mg/kg-
17 day) for analysis of DNA adduct formation in WBCs and major organs and tissues. Additional rats
18 (6/sex/time period) were exposed to 0.1 mg/kg-day benzo[a]pyrene for 4 and 5 months for
19 analysis of DNA adduct formation. Usingthe [32P]-postlabeling technique, five benzo[a]pyrene-DNA
20 adducts were identified in all of the examined tissues at 4 months (WBCs, liver, kidney, heart, lung,
21 skin, forestomach, glandular stomach, brain). Only one of these adducts (adduct 2) was identified
22 based on co-chromatography with a standard. This adduct, identified as 10p-(deoxyguanosin-N2-
23 yl)-7p,8a,9a-trihydroxy-7,8,9,10 tetrahydro-benzo[a]pyrene (dG-N2-BPDE), was the predominant
24 adduct in all organs of female rats exposed to 10 mg/kg-day, except the liver and kidney, in which
25 another adduct (unidentified adduct 4) was predominant. Levels of total adducts (number of
26 benzo[a]pyrene-DNA adducts per 1010 nucleotides) in examined tissues (from the single 10 mg/kg-
27 day female rat) showed the following order: liver > heart > kidney > lung > skin > forestomach «
28 WBCs > brain. Mean values for female levels of total benzo[a]pyrene-DNA adducts (number per
29 1010 nucleotides) in four organs showed the same order, regardless of exposure group: liver > lung
30 > forestomach « WBCs; comparable data for males were not reported). Mean total benzo[a]pyrene-
31 DNA adduct levels in livers increased in both sexes from about 100 adducts per 1010 nucleotides at
32 0.1 mg/kg-day to about 70,000 adducts per 1010 nucleotides at 30 mg/kg-day. In summary, these
33 results suggest that total benzo[a]pyrene-DNA adduct levels in tissues at 4 months were not
34 independently associated with the carcinogenic responses noted after 2 years of exposure to
35 benzo[a]pyrene. The liver showed the highest total DNA adduct levels and a carcinogenic response,
36 but total DNA adduct levels in heart, kidney, and lung (in which no carcinogenic responses were
37 detected) were higher than levels in forestomach and skin (in which carcinogenic responses were
38 detected).
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Toxicological Review ofbenzo[a]pyrene
1 Groups of Sprague-Dawley rats (32/sex/dose) were fed diets delivering a daily dose of
2 0.15 mg benzo[a]pyrene/kg body weight every ninth day or 5 times/week (Brune et al., 1981).
3 Other groups (32/sex/dose) were given gavage doses of 0.15 mgbenzo[a]pyrene (in aqueous 1.5%
4 caffeine solution)/kg every ninth day, every third day, or 5 times/week. The study included an
5 untreated control group (to compare with the dietary exposed groups) and a gavage vehicle control
6 group (each with 32 rats/sex). Rats were treated until moribundity or death occurred, with
7 average annual doses are reported in Table 4-6 (mg/kg-year, calculated by Brune et al. [1981]).
8 The following tissues were prepared for histopathological examination: tongue, larynx, lung, heart,
9 trachea, esophagus, stomach, small intestine, colon, rectum, spleen, liver, urinary bladder, kidney,
10 adrenal gland, and any tissues showing tumors or other gross changes. Survival was similar among
11 the groups, with the exception that the highest gavage-exposure group showed a decreased median
12 time of survival (Table B-10). Increased incidences of portal-of-entry tumors (forestomach,
13 esophagus, and larynx) were observed in all of the gavage-exposed groups and in the highest
14 dietary exposure group (Table B-10). Following dietary administration, all observed tumors were
15 papillomas. Following gavage administration, two malignant forestomach tumors were found (one
16 each in the mid- and high-dose groups) and the remaining tumors were benign. The data in Table
17 4-6 show that the carcinogenic response to benzo[a]pyrene was stronger with the gavage protocol
18 compared with dietary exposure, and that no distinct difference in response was apparent between
19 the sexes. Tumors at distant sites (mammary gland, kidney, pancreas, lung, urinary bladder, testes,
20 hematopoietic, and soft tissue) were not considered treatment-related as they were also observed
21 at similar rates in the control group (data not provided). The study report did not address
22 noncancer systemic effects.
23 Table B-10. Incidences of alimentary tract tumors in Sprague-Dawley
24 rats chronically exposed to benzo[a]pyrene in the diet or by gavage in
25 caffeine solution
Average annual
dose (mg/kg-yr)
Estimated average
daily dose3
(mg/kg-d)
Forestomach tumors'5
Total alimentary tract
tumors0 (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%)d
26/64 (40.1%)e
14/64 (21.9%)e
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%)d
3/64 (4.7%)
3/64 (4.7%)
10/64 (15.6%)
129
128
131
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Toxicological Review ofbenzo[a]pyrene
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): Male: 6/32, 7/32, 15/32, 8/32
Female: 0/32, 6/32, 11/32, 6/32
Diet (control, high dose): Male: 3/32, 3/32, 8/32
Female: 0/32, 0/32, 2/32
dSignificantly (p < 0.1) different from control using a modified %2test that accounted for group
differences in survival time.
Significantly (p < 0.05) different from control using a modified %2 test that accounted for group
differences in survival time.
Source: Brune et al. (1981).
1
2 In the other modern cancer bioassay with benzo[a]pyrene, female B6C3Fi mice (48/dose
3 group) were administered benzo[a]pyrene (98.5% purity) at concentrations of 0 (acetone vehicle),
4 5, 25, or 100 ppm in the diet for 2 years (Beland and Gulp, 1998; Gulp et al., 1998). This study was
5 designed to compare the carcinogenicity of coal tar mixtures with that of benzo[a]pyrene and
6 included groups of mice fed diets containing one of several concentrations of two coal tar mixtures.
7 Benzo[a]pyrene was dissolved in acetone before mixing with the feed. Control mice received only
8 acetone-treated feed. Female mice were chosen because they have a lower background incidence of
9 lung tumors than male B6C3Fi mice. Gulp et al. (1998) reported that the average daily intakes of
10 benzo[a]pyrene in the 25- and 100-ppm groups were 104 and 430 ug/day, but did not report
11 intakes for the 5-ppm group. Based on the assumption that daily benzo[a]pyrene intake at 5 ppm
12 was one-fifth of the 25-ppm intake (about 21 ug/day), average daily doses for the three
13 benzo[a]pyrene groups are estimated at 0.7, 3.3, and 16.5 mg/kg-day. Estimated doses were
14 calculated using time-weighted average (TWA) body weights of 0.032 kg for the control, 5- and 25-
15 ppm groups and 0.026 kg for the 100-ppm group (estimated from graphically presented data).
16 Food consumption, body weights, morbidity, and mortality were monitored at intervals, and lung,
17 kidneys, and liver were weighed at sacrifice. Necropsy was performed on all mice that died during
18 the experiment or survived to the end of the study period. Limited histopathologic examinations
19 (liver, lung, small intestine, stomach, tongue, esophagus) were performed on all control and high-
20 dose mice and on all mice that died during the experimental period, regardless of treatment group.
21 In addition, all gross lesions found in mice of the low- and mid-dose groups were examined
22 histopathologically.
23 None of the mice administered 100 ppm benzo[a]pyrene survived to the end of the study,
24 and morbidity/mortality was 100% by week 78. Decreased survival was also observed at 25 ppm
25 with only 27% survival at 104 weeks, compared with 56 and 60%, in the 5-ppm and control groups,
26 respectively. In the mid- and high-dose group, 60% of mice were alive at about 90 and 60 weeks,
27 respectively. Early deaths in exposed mice were attributed to tumor formation rather than other
28 causes of systemic toxicity. Food consumption was not statistically different in
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Toxicological Review ofbenzo[a]pyrene
1 benzo[a]pyrene-exposed and control mice. Body weights of mice fed 100 ppm were similar to
2 those of the other treated and control groups up to week 46, and after approximately 52 weeks,
3 body weights were reduced in 100-ppm mice compared with controls. Body weights for the 5- and
4 25-ppm groups were similar to controls throughout the treatment period. Compared with the
5 control group, no differences in liver, kidney, or lung weights were evident in any of the treated
6 groups (other organ weights were not measured).
7 Papillomas and/or carcinomas of the forestomach, esophagus, tongue, and larynx at
8 elevated incidences occurred in groups of mice exposed to 25 or 100 ppm, butno exposure-related
9 tumors occurred in the liver or lung (Table B-ll; Beland and Gulp, 1998; Gulp et al., 1998). The
10 forestomach was the most sensitive tissue, and demonstrated the highest tumor incidence among
11 the examined tissues and was the only tissue with an elevated incidence of tumors at 25 ppm
12 (Table B-ll). In addition, most of the forestomach tumors in the exposed groups were carcinomas,
13 as 1, 31, and 45 mice had forestomach carcinomas in the 5-, 25-, and 100-ppm groups respectively.
14 Nonneoplastic lesions were also found in the forestomach at significantly (p < 0.05) elevated
15 incidences: hyperplasia at >5 ppm and hyperkeratosis at >25 ppm (Table B-ll). The esophagus
16 was the only other examined tissue showing elevated incidence of a nonneoplastic lesion (basal cell
17 hyperplasia, see Table B-ll). Tumors (papillomas and carcinomas) were also significantly elevated
18 in the esophagus and tongue at 100 ppm (Table B-ll). Esophogeal carcinomas were detected in 1
19 mouse at 25 ppm and in 11 mice at 100 ppm. Tongue carcinomas were detected in seven 100-ppm
20 mice; the remaining tongue tumors were papillomas. Although incidences of tumors of the larynx
21 were not significantly elevated in any of the exposed groups, a significant dose-related trend was
22 apparent (Table B-ll).
23 Table B-ll. Incidence of nonneoplastic and neoplastic lesions in female
24 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)
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/48b
(2)
13/48b
(27)
13/48b
0.7
7/48
(15)
0/48
(0)
3/47
(6)
23/47
(49)
22/47
3.3
5/47
(11)
4/45
(9)
36/46a
(78)
33/46a
(72)
33/46a
16.5
0/45
(0)
0/48
(0)
46/47a
(98)
37/47a
(79)
38/47a
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Esophagus (papilloma and/or carcinoma)
Esophagus (basal cell hyperplasia)
Tongue (papilloma and/or carcinoma)
Larynx (papilloma and/or carcinoma)
(27)
0/48b
(0)
l/48b
(2)
0/49b
(0)
0/35b
(0)
(47)
0/48
(0)
0/48
(0)
0/48
(0)
0/35
(0)
(72)
2/45
(0)
5/45
(11)
2/46
(4)
3/34
(9)
(81)
27/46a
(59)
30/46a
(65)
23/48a
(48)
5/38
(13)
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).
1
2 Neal and Rigdon (1967) fed benzo[a]pyrene (purity not reported) at concentrations of 0,1,
3 10, 20, 30, 40, 45, 50,100, and 250 ppm to male and female CFW-Swiss mice in the diet
4 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
5 44.4 mg/kg-day. The age of the mice ranged from 17 to 180 days old and the treatment time was
6 from 1 to 197 days; the size of the treated groups ranged from 9 to 73. There were 289 mice
7 (number of mice/sex not stated) in the control group. No forestomach tumors were reported at 0,
8 0.2, or 1.8 mg/kg-day. The incidence of forestomach tumors at 20, 30, 40, 45, 50,100, and 250 ppm
9 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,
10 19/23, and 66/73, respectively.
11 Other Oral Exposure Cancer Bioassavs in Mice
12 Numerous other oral exposure cancer bioassays in mice have limitations that restrict their
13 usefulness for characterizing dose-response relationships between chronic-duration oral exposure
14 to benzo[a]pyrene and noncancer effects or cancer, but collectively, they provide strong evidence
15 that oral exposure to benzo[a]pyrene can cause portal-of-entry site tumors (see Table B-12 for
16 references).
Calculation: mg/kg-day = (ppm in feed x kg food/day)/kg body weight. Reference food
consumption rates of 0.0062 kg/day (males) and 0.0056 kg/day (females) and reference body
weights of 0.0356 kg (males) and 0.0305 kg (females) were used (U.S. EPA, 1988) and resulting
doses were averaged between males and females.
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Table B-12. Other oral exposure cancer bioassays in mice
Species/strain
Rat/Sprague-
Dawley
Mouse/HalCR
Mouse/HalCR
Mouse/HalCR
Exposure
Groups of Sprague-
Dawley rats
(32/sex/dose) were fed
diets delivering a daily
dose of 0.15 mg
benzo[a]pyrene/kg body
weight every 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.
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.
Results
Dose larynx, esophagus,
and forestomach
(gavage) tumors
0 6/64
0.016 13/64
0.049 26/64
0.107 14/64
(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)
Incidence with
forestomach tumors:
Control 0/9
Low 6/9
High 9/9
8/20 exposed mice had
forestomach tumors.
Comments
Doses are annual
averages.
Nonstandard
treatment
protocol involved
animals being
treated for <5
days/week;
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.
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.
Reference
Brune et al.,
1981
Wattenberg
,1972
Triolo et al.,
1977
Wattenberg
,1974
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Toxicological Review ofbenzo[a]pyrene
Species/strain
Mouse/CD-I
Mouse/BALB
Mouse/C3H
Mouse/albino
Mouse/albino
Exposure
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.
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.
Results
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.
Incidence of mice (that
survived at least to 60 d)
with forestomach
papillomas:
Dose (u.g) Incidence
(Experiment 1)
(Experiment 2)
Control 0/17
0/18
12.5 3/17
2/18
50 0/17
1/17
200 8/17
NE
Gastric tumors were
observed at the following
incidence:
Control 0/158
8 mg benzo[a]pyrene
total 13/160
Comments
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.
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.
Reference
EI-Bayoumy,
1985
Biancifiori
etal., 1967
Berenblum
and Haran,
1955
Field and
Roe, 1965
Chouroulink
ovet al.,
1967
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Toxicological Review ofbenzo[a]pyrene
Species/strain
Mouse/CFW
Mouse/Swiss
albino
Mouse/ICR
Exposure
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
bygavage. Surviving mice
were killed at 18 mo of
age and examined for
macroscopic tumors.
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.
Results
ppm Exposure
Forestomach tumor
(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:
Dose (u.g) - Carcinoma
Papilloma
0 0/65
2/65
50 1/61
20/61
Incidence of mice with
forestomach neoplasms
Experiment 1 23/24
Experiment 2 19/20
Comments
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 (see also
Rigdon and Neal,
1969, 1967,
1966).
Less-than-lifetime
duration of
exposure;
exposure-related
tumors only
found in
forestomach.
Less-than-lifetime
duration of
exposure; only
stomachs were
examined for
tumors; tumors
found only in
forestomach;
nonexposed
controls were not
mentioned.
Reference
Neal and
Rigdon,
1967
Roeetal.,
1970
Benjamin et
al., 1988
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Toxicological Review ofbenzo[a]pyrene
Species/strain
Mouse/white
Mouse/A/HeJ
Mouse/A/J
Mouse/A/J
Exposure
Groups of 16-30 mice
were given
benzo[a]pyrene in
triethylene glycol (0.001-
10 mg) wkly 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 u.g/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).
Groups 40 female mice (8
wks old) were given 0 or
0.25 mg benzo[a]pyrene
(in 2%emulphor) by
gavage 3 times/wk for
8 wks. Mice were killed
at 9 mo of age and
examined for lung or
forestomach tumors.
Results
Tumors in stomach
antrum
Dose (mg) - Carcinoma
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
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
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.
Less-than-lifetime
duration of
exposure; only
lungs and Gl tract
were examined
for tumors.
Reference
Fedorenko
and
Yansheva,
1967; as
cited in U.S.
EPA, 199 la
Wattenberg
,1974
Weyand et
al., 1995
Robinson et
al., 1987
NE = not evaluated
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Toxicological Review ofbenzo[a]pyrene
1 Inhalation Studies
2 Short-term and Subchronic Studies
3 Wolff et al. (1989) exposed groups of 40 male and 40 female F344/Crl rats, via nose only, to
4 7.5 mg benzo[a]pyrene/m3 for 2 hours/day, 5 days/week for 4 weeks (corresponding to a TWA of
5 0.45 mg/m3). Rats were 10-11 weeks old atthe beginning of the experiment Benzo[a]pyrene
6 (>98% pure) aerosols were formed by heating and then condensing the vaporized benzo[a]pyrene.
7 The particle MMAD was 0.21 |im. Subgroups of these animals (six/sex/dose) were exposed for
8 4 days or 6 months after the end of the 4-week exposure to radiolabeled aluminosilicate particles.
9 Lung injury was assessed by analyzing clearance of radiolabeled aluminosilicate particles and via
10 histopathologic evaluations. Body and lung weights, measured in subgroups from 1 day to 12
11 months after the exposure did not differ between controls and treated animals. Radiolabeled
12 particle clearance did not differ between the control and treated groups, and there were no
13 significant lung lesions. This study identified a NOAEL for lung effects of 0.45 mg/m3 for a short-
14 term exposure.
15 Chronic Studies and Cancer Bioassavs
16 Thyssen et al. (1981) conducted an inhalation study in which male Syrian golden hamsters
17 were exposed to benzo[a]pyrene for their natural lifetime. Groups of 20-30 animals (8 weeks old)
18 were exposed by nose-only inhalation to NaCl aerosols (controls; 240 [ig NaCl/m3) or
19 benzo[a]pyrene condensed onto NaCl aerosols atthree nominal concentrations of 2,10, or 50 mg
20 benzo[a]pyrene/m3 for 3-4.5 hours/day, 5 days/week for 1-41 weeks, followed by 3 hours/day,
21 7 days/week for the remainder of study (until hamsters died or became moribund). Thyssen et al.
22 (1981) reported average measured benzo[a]pyrene concentrations to be 0, 2.2, 9.5, or 46.5 mg/m3.
23 More than 99% of the particles were between 0.2 and 0.5 |im in diameter, and over 80% had
24 diameters between 0.2 and 0.3 |im. The particle analysis of the aerosols was not reported to
25 modern standards (MMAD and geometric SD were not reported). Each group initially consisted of
26 24 hamsters; final group sizes were larger as animals dying during the first 12 months of the study
27 were replaced.
28 Survival was similar in the control, low-dose, and mid-dose groups, but was significantly
29 decreased in the high-dose group. Average survival times in the control, low-, mid-, and high-dose
30 groups were 96.4 ± 27.6, 95.2 ± 29.1, 96.4 ± 27.8, and 59.5 ± 15.2 weeks, respectively. After the 60*
31 week, body weights decreased and mortality increased steeply in the highest dose group.
32 Histologic examination of organs (a complete list of organs examined histologically was not
33 reported by Thyssen et al. [1981]) revealed a dose-related increase in tumors in the upper
34 respiratory tract, including the nasal cavity, pharynx, larynx, and trachea, and in the digestive tract
35 in the mid- and high-dose groups (Table B-13). A statistical analysis was not included in the
36 Thyssen et al. (1981) report. No lung tumors were observed. Squamous cell tumors in the
37 esophagus and forestomach were also observed in the high-dose group, presumably as a
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Toxicological Review ofbenzo[a]pyrene
1 consequence of mucociliary particle clearance. Tumors were detected in other sites, but none of
2 these appeared to be related to exposure. The results indicated that the pharynx and larynx,
3 including the epiglottis, were the main cancer targets (Table B-13).
4
5
Table B-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(107.1115.5)
1/26 (115)
0
6/26 (97.2 ± 16.9)
0
1/26 (119)
1/25 (79)
13/25(67.6112.1)
3/25(63.3133.3)
0
14/25 (67.5 ± 12.2)
2/25 (70, 79)
1/25 (72)
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Effective number of animals in control group: n = 27.
bEffective number of animals in 2 mg/m3 dose group: n = 27.
cMean±SD.
Source: Thyssen et al. (1981).
Under contract to the U.S. EPA, Clement Associates (1990) obtained the individual animal
data (including individual animal pathology reports, time-to-death data, and exposure chamber
monitoring data) collected by Thyssen et al. (1981). Re-analysis of the original data revealed
several errors and omissions in the published report 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 In addition, actual exposure concentrations
varied widely from week to week. 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 deal with this problem, Clement Associates (1990) 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.
For this assessment, the individual animal pathology reports prepared by Thyssen et al.
(1981) and obtained by Clement Associates (1990) were examined to independently assess the
numbers of hamsters with tumors in the larynx, pharynx, and nose in each group. Table B-14
presents the number of animals with tumors in the larynx and pharynx and the numbers of animals
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Toxicological Review ofbenzo[a]pyrene
1
2
3
4
5
6
in each exposure group. Numbers of animals with either laryngeal or pharyngeal tumors are also
noted in Table B-14, since these two types of tumors arise in close anatomical proximity from
similar cell types. Examination of the individual animal pathology reports also showed that all of
the nasal, forestomach, esophageal, and tracheal tumors occurred in animals that also had either
laryngeal or pharyngeal tumors, 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.
Table B-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
group15
27
27
26
34
Larynxb
Malignant
0
0
8
9
All
0
0
11
12
Pharynx15
Malignant
0
0
7
17
All
0
0
9
18
Larynx or pharynx,
combined0
Malignant
0
0
11
17
All
0
0
16
18
9
10
11
12
13
14
15
16
17
18
19
20
21
22
aAs calculated by Clement Associates (1990) from air monitoring data collected by Thyssen and
colleagues.
bAs counted from information in Table E-l in Appendix E, which was obtained from examination of
individual animal pathology reports prepared by Thyssen and colleagues and obtained by Clement
Associates.
cAs counted from information in Table E-l 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 thatbenzo[a]pyrene is
tumorigenic, but do not provide data useful for characterizing dose-response relationships because
of their design (Kobayashi, 1975; Reznik-Schuller and Mohr, 1974; Henry etal., 1973; Mohr, 1971;
Saffiotti etal., 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)
were found at higher dosage levels (0.25-2 mgbenzo[a]pyrene once per week for 30-52 weeks) in
four multiple-dose studies (Feron and Kruysse, 1978; Ketkar et al., 1978; Feron et al., 1973; Saffiotti
et al., 1972). These studies identify the respiratory tract as a cancer target with exposure to
benzo[a]pyrene by intratracheal instillation and provide supporting evidence for the
carcinogenicity of benzo[a]pyrene atportal-of-entry sites.
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Toxicological Review ofbenzo[a]pyrene
1 Dermal studies
2 Skin-Tumor Initiation-Promotion Assays
3 Results from numerous studies indicate that acute dermal exposure to benzo[a]pyrene
4 induces skin tumors in mice when followed by repeated exposure to a potent tumor promoter
5 (Weyand et al., 1992; Cavalieri et al., 1991,1981; Rice et al., 1985; El-Bayoumy et al., 1982; LaVoie
6 et al., 1982; Raveh et al., 1982; Slaga et al., 1980,1978; Wood et al., 1980; Hoffmann et al., 1972).
7 The typical exposure protocol in these studies involved the application of a single dose of
8 benzo[a]pyrene (typically >20 nmol per mouse) to dorsal skin of mice followed by repeated
9 exposure to a potent tumor promoter, such as 12-0-tetradecanoylphorbol-13-acetate (TPA).
10 Carcinogenicity Bioassays
11 Repeated application of BaP to skin (in the absence of exogenous promoters) has been
12 variously demonstrated to induce skin tumors in mice, rats, rabbits, and guinea pigs (IARC, 2010,
13 1983,1973; WHO, 1998; ATSDR, 1995). Mice have been most extensively studied, presumably
14 because of early evidence that they may be more sensitive than other animal species, but
15 comprehensive comparison of species differences in sensitivity to lifetime dermal exposure are not
16 available. Early studies of complete dermal carcinogenicity in other species (rats, hamsters, guinea
17 pigs, and rabbits) have several limitations which make them not useful for dose-response analysis
18 (see IARC, 1973 for descriptions of studies by Nakano et al., 1937, Shubik et al., 1960; Oberling et
19 al., 1937; Schiirch and Winterstein, 1935; Wynder et al., 1957). The limitations in these studies
20 include inadequate reporting of the amount of BaP applied, use of the carcinogen benzene as a
21 vehicle, and less than lifetime exposure duration.
22 This section discusses complete carcinogenicity bioassays in mice that provide the best
23 available dose-response data for skin tumors caused by repeated dermal exposure to BaP (Sivak et
24 al., 1997; Higginbotham et al., 1993; Albert et al., 1991; Habs et al., 1984,1980; Grimmer et al.,
25 1984, 1983; Schmahl et al., 1977; Schmidt et al., 1973; Roe et al., 1970; Poel, 1960, 1959). Early
26 studies of BaP complete carcinogenicity in mouse skin (Wynder and Hoffman 1959; Wynder et al,
27 1957) are notfurther described herein, because the investigators applied solutions of BaP at
28 varying concentrations on the skin, but did not report volumes applied. As such, applied doses in
29 these studies cannot be determined. Other complete carcinogenicity mouse skin tumor bioassays
30 with BaP are available, but these are not described further in this review, because: (1) they only
31 included one BaP dose level (e.g., Emmett et al., 1981) or only dose levels inducing 90-100%
32 incidence of mice with tumors (e.g., Wilson and Holland, 1988; Warshawsky and Barkley, 1987) and
33 thus provide no information about the shape of the dose-response relationship; (2) they used a 1-
34 time/week (e.g., Nesnow et al., 1983) or 1-time every 2 weeks (e.g., Levin et al., 1977) exposure
35 protocol, which is less useful for extrapolating to daily human exposure; or (3) they used a vehicle
36 demonstrated to interact with or enhance benzo[a]pyrene carcinogenicity (Bingham and Falk,
37 1969).
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Toxicological Review ofbenzo[a]pyrene
1
2
3
4
5
6
7
9
10
Poel (1959) applied benzo[a]pyrene in toluene to shaved interscapular skin of groups of
13-56 male C57L mice at doses of 0, 0.15, 0.38, 0.75, 3.8,19, 94,188, 376, or 752 ug, 3 times/week
for up to 103 weeks or until the appearance of a tumor by gross examination (3 times weekly).
Some organs (not further specified) and interscapular skin in sacrificed mice were examined
histologically. With increasing dose level, the incidence of mice with skin tumors increased and the
time of tumor appearance decreased (see Table B-15). Doses >3.8 ugwere associated with 100%
mortality after increasingly shorter exposure periods, none greater than 44 weeks. Poel (1959) did
not mention the appearance of exposure-related tumors in tissues other than interscapular skin.
Table B-15. Skin tumor incidence and time of appearance in male C57L
mice dermally exposed to benzo[a]pyrene for up to 103 weeks
Dose (ug)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)
-
42-44c
24
36
21-25
11-21
8-19
9-18
4-15
5-13
Incidence of mice
with epidermoid
carcinoma11
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
25-44c
22-43
20-35
19-35
19-30
11
12
13
14
15
16
17
18
19
20
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.
cRanges reflect differing information in Tables 4 and 6 of Poel (1959).
Source: Poel (1959).
Poel (1960) applied benzo[a]pyrene in a toluene vehicle to shaved interscapular skin of
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,
19.0, 94.0, or 470 ug benzo[a]pyrene per application, until mice died or a skin tumor was observed.
Time ranges for tumor observations were provided, but not times of death for mice without tumors,
so it was not possible to evaluate differential mortality among all dose groups or the length of
exposure for mice without tumors. With increasing dose level, the incidence of mice with skin
tumors increased and the time of tumor appearance decreased (Table B-16). The lowest dose level
did not induce an increased incidence of mice with skin tumors in any strain, but strain differences
in susceptibility were evident at higher dose levels. SWR and CSHeB mice showed skin tumors at
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1 doses >0.38 ugbenzo[a]pyrene, whereas AH/e mice showed tumors at doses >19 ug
2 benzo[a]pyrene (Table B-16). Except for metastases of the skin tumors to lymph nodes and lung,
3 Poel (1960) did not mention the appearance of exposure-related tumors in tissues other than
4 interscapular skin.
5 Table B-16. Skin tumor incidence and time of appearance in male SWR,
6 C3HeB, and A/He mice dermally exposed to benzo [ajpyrene for life or
7 until a skin tumor was detected
Dose (ug)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 first
tumor
appearance
(wks)
-
55-55
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 first
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 fist
tumor
appearance
(wks)
-
-
-
-
-
21-40
14-31
4-21
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).
9 Roe et al. (1970) treated groups of 50 female Swiss mice with 0 (acetone vehicle), 0.1, 0.3,1,
10 3, or 9 ug benzo[a]pyrene applied to the shaved dorsal skin 3 times/week for up to 93 weeks; all
11 surviving mice were killed and examined for tumors during the following 3 weeks. The dorsal skin
12 of an additional control group was shaved periodically but was not treated with the vehicle. Mice
13 were examined every 2 weeks for the development of skin tumors at the site of application.
14 Histologic examinations included: (1) all skin tumors thought to be possibly malignant; (2) lesions
15 of other tissues thought to be neoplastic; and (3) limited nonneoplastic lesions in other tissues. As
16 shown in Table B-17, markedly elevated incidences of mice with skin tumors were only found in the
17 two highest dose groups (3 or 9 ug), compared with no skin tumors in the control groups.
18 Malignant skin tumors (defined as tumors with invasion or penetration of the panniculus carnosus
19 muscle) were detected in 4/41 and 31/40 mice in the 3- and 9-ug groups, respectively, surviving to
20 atleastSOO days. Malignant lymphomas were detected in all groups, but the numbers of cases were
21 not elevated compared with expected numbers after adjustment for survival differences. Lung
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1 tumors were likewise detected in control and exposed groups at incidences that were not
2 statistically different
3 Table B-17. Tumor incidence in female Swiss mice dermally exposed to
4 benzo[a]pyrenefor up to 93 weeks
Dose (ug)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
incidence0
19/44 (43%)
12/47 (26%)
11/43 (26%)
10/43 (23%)
16/44 (36%)
23/42 (55%)
9/40 (23%)
Lung tumor
incidence0
12/41 (29%)
10/46 (22%)
10/40 (25%)
13/43 (30%)
15/43 (35%)
12/40 (30%)
5/40 (13%)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
aDoses were applied 3 times/week for up to 93 weeks to shaved dorsal skin.
bNumerator: 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: Roeetal. (1970).
Schmidt et al. (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
twice weekly with doses of 0, 0.05, 0.2, 0.8, or 2 [ig until spontaneous death occurred or until an
advanced carcinoma was observed. Skin carcinomas were identified by the presence of crater-
shaped ulcerations, infiltrative growth, and the beginning of physical wasting (i.e., cachexia).
Necropsy was performed for all animals, and histopathological examination of the dermal site of
application and any other tissues with gross abnormalities was conducted. Skin tumors were
observed at the two highest doses in both strains of female mice (see Table B-18), with induction
periods of 53.0 and 75.8 weeks for the 0.8 and 2.0 ug NMRI mice and 57.8 and 60.7 weeks for the
Swiss mice, respectively. The authors indicated that the latency period for tumor formation was
highly variable and significant differences among exposure groups could not be identified, but no
further timing information was available, including overall survival. Carcinoma was the primary
tumor type seen after lifetime application of benzo[a]pyrene to mouse skin.
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1
2
Table B-18. Skin tumor incidence in female NMRI and Swiss mice
dermally exposed to benzo[a]pyrene
Dose (ug)a'b
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%)
3
4
5
6
7
8
9
10
11
12
13
14
15
16
aMice were exposed until natural death or until
blndicated doses were applied 2 times/week to
Source: Schmidt et al. (1973).
they developed a carcinoma at the site of application.
shaved skin of the back.
Schmahl et al. (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 u,g in 20 uL 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,
which the authors attributed to autolyis; no information was provided concerning when tumors
appeared in the relevant groups, how long treatment lasted in each group, or any times of death.
Necropsy was performed on all mice and the skin of the back, as well as any organs that exhibited
macroscopic changes, were examined histopathologically. The incidence of all types of skin tumors
was increased in a dose-related manner compared to controls (see Table B-19). Carcinoma was the
primary tumor type observed following chronic dermal exposure to benzo[a]pyrene, and skin
papillomas occurred infrequently. Dermal sarcoma was not observed.
Table B-19. Skin tumor incidence in female NMRI mice dermally
exposed to benzo[a]pyrene
Dose (ug)a'b
0
1
Skin tumor incidence
(all types)
1/81 (1%)
11/77 (14%)
Incidence of papilloma
0/81 (0%)
1/77 (1%)
Incidence of carcinoma
0/81 (0%)
10/77 (13%)
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1.7
3
25/88 (28%)
45/81 (56%)
0/88 (0%)
2/81 (3%)
25/88 (28%)
43/81 (53%)
1
2
3
4
5
6
7
8
9
10
11
12
aMice were exposed until natural death or until they developed a carcinoma at the site of application.
blndicated doses were applied 2 times/week to shaved skin of the back.
Source: Schmahl et al. (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 ugin 20 uL 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 B-20).
Table B-20. Skin tumor incidence in female NMRI mice dermally
exposed to benzo[a]pyrene
Dose (ug)a'b
0 (acetone)
1.7
2.8
4.6
Skin tumor incidence
0/35 (0%)
8/34 (24%)
24/35 (68%)
22/36 (61%)
Age-standardized tumor incidence0
0%
24.8%
89.3%
91.7%
13
14
15
16
17
18
19
20
21
22
aMice were exposed until natural death or until they developed a carcinoma at the site of application.
blndicated doses were applied 2 times/week to shaved skin of the back.
cMortality data of the total study population were used to derive the age-standardized tumor incidence.
Source: Habs et al. (1980).
Grimmer etal. (1984,1983) 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 u,g 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 B-21).
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1 Carcinoma was the primary tumor type observed and a dose-response relationship was evident for
2 carcinoma formation and incidence of all types of skin tumors.
3 Table B-21. Skin tumor incidence and time of appearance in female
4 CFLP mice dermally exposed to benzo[a]pyrene for 104 weeks
Dose (ug)a
Skin tumor incidence
(all types)
Incidence of
papilloma
Incidence of
carcinoma
Tumor appearance
in weeks
Grimmer etal. (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%)
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
Grimmer etal. (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%)
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)
5
6
7
8
9
10
11
12
13
14
15
16
17
Indicated doses were applied twice/week to shaved skin of the back.
bMean±SD.
'Median with 95% Cl.
Sources: Grimmer et al. (1984,1983)
Habs etal. (1984) appliedbenzo[a]pyrene (in 0.01 mL acetone) to the shaved interscapular
skin of female NMRI mice at doses of 0, 2, or 4 ug, 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 ug/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 B-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.
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1
2
Table B-22. Skin tumor incidence in female NMRI mice dermally
exposed to benzo[a]pyrene for life
Dose (u,g)a'b
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)
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
aMice were exposed until natural death or until they developed an invasive tumor at the site of
application.
blndicated 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 ug/treatment) benzo[a]pyrene (>99% pure) in
acetone 2 times weekly for 40 weeks (Higginbotham et al., 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,
liver, kidneys, spleen, urinary bladder, uterus, and ovaries, as well as any other grossly abnormal
tissue. Lung adenomas occurred in each group (1/27, 2/24,1/23,1/23), and other tumors were
noted in isolated mice (i.e., malignant lymphoma [spleen] in one low-dose and one mid-dose mouse;
malignant lymphoma with middle organ involvement in one high-dose mouse; and hemangioma
[liver] in one mid-dose mouse) and were not considered dose related. In addition, benzo[a]pyrene
showed no skin tumors under the conditions of this bioassay.
Sivak et al. (1997) designed a study to compare the carcinogenicity of condensed asphalt
fumes (including benzo[a]pyrene and other PAHs) with several doses of benzo[a]pyrene alone. For
the purposes of this assessment, the exposure groups exposed to PAH mixtures are not discussed.
Groups of 30 male C3H/HeJ mice were treated dermally twice/week to 0, 0.0001, 0.001, or 0.01%
(0, 0.05, 0.5, or 5 |ig) benzo[a]pyrene in a 50 uL volume of cyclohexanone/acetone (1:1) for 104
weeks beginning at 8 weeks of age. Mice dying during the exposure period or sacrificed at the 24
month termination were necropsied; mice with skin tumors that persisted for 4 consecutive weeks
with diameters > 3 cm were sacrificed before the study termination and also necropsied. Skin
samples and any grossly observed lesions were subjected to histopathological examination.
Carcinomas and sarcomas were referred to as carcinomas, whereas papillomas, keratoacanthomas,
and fibromas were referred to as papillomas. The incidences of mice with skin tumors and mean
survival times for each group are shown in Table B-23. All high-dose mice died before the final
sacrifice, and 80% showed scabs and sores at the site of application. The time of first tumor
appearance was not reported for the tumor-inducing groups, but from a plot of the tumor incidence
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Toxicological Review ofbenzo[a]pyrene
2
3
5
6
in the high-dose group versus treatment days, an estimate of ~320 days (~43 weeks) is obtained
for this group. The extent of deaths prior to 1 year in each group was not provided, so that the
reported incidence may underestimate the tumor rate of animals exposed long enough to develop
tumors. However, the crude skin tumor rates show an increasing trend in incidence.
Table B-23. Skin tumor incidence in male C3H/HeJ mice dermally
exposed to benzo [ajpyrene for 24 months
Dose (u,g)a
0 cyclohexanone/acetone
(1:1)
0.05
0.5
5.0
Skin tumor incidence
(all types)"
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
7
8
9
10
1 1
12
13
14
15
16
1 7
18
19
20
21
22
23
24
25
Indicated doses were applied twice/week to shaved dorsal skin.
b Number 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: Sivak et al. (1997).
To examine dose-response relationships and the time course of benzo [a] pyre ne- induced
skin damage, DNA adduct formation, and tumor formation, groups of 43-85 female Harlan mice
were treated dermally with 0, 16, 32, or 64 u,g of benzo [ajpyrene in 50 uL of acetone once per week
for 29 weeks (Albert et al., 1991). Interscapular skin of each mouse was clipped 3 days before the
first application and every 2 weeks thereafter. Additional groups of mice were treated for 9 weeks
with 0, 8, 16, 32, or 64 u,g radiolabeled benzo [ajpyrene to determine BPDE-DNA adduct formation
in the epidermis at several time points (1, 2, 4, and 9 weeks). Tumor formation was monitored only
in the skin.
No tumors were present in vehicle-treated or untreated control mice. In exposed groups,
incidences of mice with skin tumors were not reported, but time-course data for cumulative
number of tumors per mouse, corrected for deaths from nontumor causes, were reported. Tumors
began appearing after 12-14 weeks of exposure for the mid- and high-dose groups and at 18 weeks
for the low-dose group. At study termination (35 weeks after start of exposure), the mean number
of tumors per mouse was approximately one per mouse in the low- and mid-dose groups and eight
per mouse in the high-dose group; indicating that most, if not all, mice in each exposure group
developed skin tumors and that the tumorigenic response was greatest in the highest dose group.
The majority of tumors were initially benign, with an average time of 8 weeks for progression from
benign papillomas to malignant carcinomas. Epidermal damage occurred in a dose-related manner
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Toxicological Review ofbenzo[a]pyrene
1 (more severe in the high-dose group than in the low- and mid-dose groups) and included
2 statistically significant increases (compared with controls) in: [3H]-thymidine labeling and mitotic
3 indices; incidence of pyknotic and dark cells (signs of apoptosis); and epidermal thickness. Only a
4 minor expansion of the epidermal cell population was observed. In the high-dose group, indices of
5 epidermal damage increased to a plateau by 2 weeks of exposure. The early time course of
6 epidermal damage indices was not described in the low- or mid-dose groups, since data for these
7 endpoints were only collected at 20, 24, and 30 weeks of exposure. An increased level of BPDE-
8 DNA adducts, compared with controls, was apparent in all exposed groups after 4 weeks of
9 exposure in the following order: 64>32>16>8 [ig/week. The time-course data indicate that
10 benzo[a]pyrene-induced increases in epidermal damage indices and BPDE-DNA adducts preceded
11 the appearance of skin tumors.
12 Reproductive and Developmental Toxicity Studies
13 Oral
14 In a study evaluating the combined effects of DBF and benzo[a]pyrene on the male
15 reproductive tract, Chen et al. (2011) administered benzo[a]pyrene alone in corn oil via daily
16 gavage at 5 mg/kg-day to 30 male Sprague-Dawley rats (28-30 days old); a group of 30 rats
17 received only vehicle. Body weight was measured weekly. Groups of 10 rats per group were
18 sacrificed after 4, 8, and 12 weeks of exposure. At sacrifice, blood was collected for analysis of
19 serum testosterone levels by radioimmunoassay. The testes and epidiymides were weighed, and
20 the right testis and epididymis were examined microscopically. The left epididymis was used for
21 evaluation of sperm parameters (sperm count and morphology). Oxidative stress, as measured by
22 superoxide dismutase, glutathione peroxidase, and catalase activity and malondioaldehyde levels,
23 was evaluated in the left testis of each rat Exposure to benzo [a]pyrene did not affect body weight,
24 and no signs of toxicity were seen. Testes and epididymides weights of exposed rats were similar
25 to controls at all time points. Sperm counts and percent abnormal sperm were also similar to
26 controls at 4 and 8 weeks of exposure, but were significantly (p<0.05) different from controls after
27 12 weeks of exposure to benzo[a]pyrene (29% decrease in sperm count and 54% increase in
28 percent abnormal sperm). Serum testosterone levels were significantly increased relative to
29 controls after 4 weeks (>two-fold higher) and 8 weeks (~1.5-fold higher) of benzo [ajpyrene
30 exposure, but were comparable to controls after 12 weeks. Histopathology evaluation of the testes
31 revealed irregular and disordered arrangement of germ cells in the seminiferous tubules of treated
32 rats; the authors did not report incidence or severity of these changes. Among measures of
33 testicular oxidative stress, only catalase activity was significantly affected by benzo[a]pyrene
34 exposure, showing an increase of ~50% after 12 weeks of exposure. These data suggest a LOAEL of
35 5 mg/kg-day (the only dose tested) for decreased sperm count, increased percentage of abnormal
36 sperm, altered testosterone levels, and histopathology changes in the testes following 13 weeks of
37 exposure.
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1 Chung etal. (2011) evaluated the effects of low-dose benzo[a]pyrene exposure on
2 spermatogenesis, and the role of altered steroidogenesis on the sperm effects. Groups of 20-25
3 male Sprague-Dawley rats (8 wks old) were given daily gavage doses of 0, 0.001, 0.01, or 0.1
4 mg/kg-day benzo[a]pyrene in DMSO for 90 consecutive days. At the end of exposure, the animals
5 were sacrificed for removal of the pituitary, testes, and epididymides, and collection of serum and
6 testicular interstitial fluid. Subgroups of each exposure group were used for various analyses.
7 Serum levels of testosterone and LH were measured, as was testosterone concentration in the
8 interstitial fluid (ELISA assays). Body and testes weights were recorded. Sections of the testis
9 were analyzed for apoptotic germ cells using TUNEL assay. Evaluation of the epididymis included
10 histopathology as well as measurement of caput and caudal epididymal tubule diameters. In
11 addition, sperm were isolated from the cauda epididymis for analysis of sperm number and
12 motility, acrosomal integrity, and immunocytochemistry for ADAMS (a disintegrin and
13 metallopeptidse domain 3; a sperm surface protein associated with fertilization).
14 Leydig cells were isolated from the right testis of animals from each dose group and
15 cultured with or without human chorionic gondatotropin (hCG) or dibutyl cyclic adenosine
16 monophosphate (dbcAMP) to evaluate testosterone production (Chung et al., 2011). Cultured
17 Leydig cells were also subjected to western blot and immunocytochemistry analyses to evaluate
18 changes in the expression of genes involved in steroidogenesis (StAR[steroidogenic acute
19 regulatory protein], p450scc [p450 side-chain cleavage], and 3p-HSD[3p-hydroxysteroid
20 dehydrogenase isomerase]). Finally, pituitary gland extracts were evaluated for LH protein
21 content using immunohistochemistry. Data were reported graphically and analyzed by ANOVA
22 followed by Duncan's post hoc test, using a p-value cutoff of 0.05 for significant difference.
23 At termination of exposure, body weights of treated animals were similar to controls, as
24 were absolute testes weights (Chung et al., 2011). Testosterone concentrations in both serum and
25 testicular interstitial fluid were significantly reduced at the high dose of benzo[a]pyrene (0.1
26 mg/kg-day); based on visual inspection of the data, the mean serum concentration in this group
27 was ~20% of the control and the mean intersitital fluid concentration was ~60% of the control
28 (n=9 animals/dose for these evaluations). In addition, baseline production of testosterone by
29 cultured Leydig cells was significantly decreased (~50% based on data shown graphically) at 0.1
30 mg/kg-day. Both hCG- and dbcAMP-stimulated testosterone production measurements were lower
31 (~60% lower than controls) in Leydig cells from rats exposed to either 0.01 or 0.1 mg/kg-day.
32 Serum LH was significantly increased at both 0.01 and 0.1 mg/kg-day (~65-75% higher than
33 controls based on visual inspection of graphs); concordant increases in the intensity of LH
34 immunoreactivity were evident in pituitary extracts from exposed rats.
35 Dose-related increases in the number of apoptotic germ cells, primarily spermatogonia,
36 were demonstrated both via TUNEL assay and caspase-3 staining; the number per tubule was
37 significantly increased over control at all doses (Chung etal., 2011). Numbers of sperm were lower
38 in the treatment groups, but did not differ significantly from the control group. However, sperm
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1 motility was significantly reduced in exposed groups compare with control. The authors did not
2 report sperm motility for all dose groups, but showed only the significant decrease in the 0.01
3 mg/kg-day mid-dose group (~30% lower than controls based on visual inspection of graph).
4 Acrosomal integrity (measured by LysoTracker staining) was diminished in sperm heads from
5 exposed rats; likewise, the expression of ADAMS protein was downregulated by exposure to
6 benzo[a]pyrene; the authors reported a significant decrease in the 0.01 mg/kg-day group but did
7 not provide details of the analysis of other exposure groups. Histopathology examination of the
8 caput and cauda epididymides revealed dose-related decreases in both cauda and caput tubule
9 diameters that were statistically significantly lower than controls at all doses (~10-30% smaller
10 mean diameter than control based on measurements of 175 tubules collected from 5 samples in
11 each group; data reported graphically).
12 Statistically significant effects observed at the lowest dose (0.001 mg/kg-day) of
13 benzo[a]pyrene in this study included decreased caput and cauda epididymal tubule diameters
14 (~10-15% lower than controls) and increased numbers of apoptotic germ cells (~twofold higher
15 than controls) by TUNEL assay (Chung etal., 2011). The authors reported that "sperm motility was
16 significantly reduced in the benzo[a]pyrene-exposed groups in comparison to that of the control"
17 but provided quantitative data only for the middle dose group, which exhibited a ~30% decrease in
18 percent motile sperm. No statistically significant decrease in sperm count was reported at any
19 dose. The middle dose (0.01 mg/kg-day) is considered to be a LOAEL, based on reduced sperm
20 motility.
21 Gao etal. (2011) examined effects of benzo[a]pyrene exposure via on cervical cell
22 morphology. Female ICRmice (18-22 g) were exposed to doses of 0, 2.5, 5, or 10 mg/kg twice per
23 week for 14 weeks, either by oral gavage or by intraperitoneal injection (for this review, only oral
24 results are reported). After adjustment for equivalent continuous dosing (2/7 days/week), the
25 equivalent daily doses are estimated to be 0.7,1.4, 2.9 mg/kg-day. Both vehicle (sesame oil) and
26 untreated control groups were maintained. Body weights were determined weekly. Groups of 26
27 mice per dose per exposure route were sacrificed at the end of exposure for evaluation of cervical
28 weight and histopathology. Additional groups of 10 mice were exposed for 14 weeks and used for
29 determination of lipid peroxidation (malondialdehyde and glutathione-S-transferase levels) and
30 CYP1A1 activity (EROD) in both liver and cervix, as well as creatine kinase activity, AST activity, and
31 IL-6 levels in cervix and serum.
32 Mortality was observed in all exposure groups with the exception of the low dose oral
33 exposure group; the authors did not indicate the timing or causes of death (Gao etal., 2011). There
34 were no control deaths. Mortality incidences in the oral exposure groups (low to high dose) were
35 0/26 (untreated control), 0/26 (vehicle control), 0/26,1/36, and 2/26. Benzo[a]pyrene treatment
36 resulted in dose-dependent decreases in body weight gain. In the high dose group of both
37 treatments, body weight began to decline after ~7 weeks of exposure. Based on visual examination
38 of data presented graphically, mean terminal body weights in the low, mid-, and high-dose oral
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1
2
3
4
5
6
7
10
11
exposure groups were ~10,15, and 30% lower (respectively) than the vehicle control mean. The
untreated control mean body weight for the oral exposure group was similar to the vehicle control
mean body weight. Cervical weight as a function of body weight was not affected by oral
benzo[a]pyrene exposure. Microscopic examination of the cervix revealed increased incidences of
epithelial hyperplasia and inflammatory cells in the cervix of all groups of exposed mice, and
atypical hyperplasia of the cervix in mice exposed to 1.4 or 2.9 mg/kg benzo[a]pyrene. Statistical
analysis of the findings was conducted, but was poorly reported in the publication. Table B-24
shows the incidences in the oral exposure groups, along with the results of Fisher's exact tests
performed for this review.
Table B-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
Atypical hyperplasia of cervix
Inflammatory cells in cervix
Dose (mg/kg-d)
Untreated
control
0/26
0/26
0/26
2/26
Vehicle
control
0/26
0/26
0/26
3/26
0.7
0/26
4/26
0/26
10/26a
1.4
1/26
6/25a
2/25
12/25a
2.9
2/26
7/24a
4/24a
18/24a
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Significantly different from vehicle control by Fisher's exact test performed for this review (one-sided p
<0.05).
Source: Gao et al. (2011).
Levels of malondialdehyde in both the cervix and liver were significantly higher than
controls in all dose groups of animals treated by either oral (1.5 to 2-fold higher in the cervix and
~3-fold to 7-fold higher in the liver after oral exposure p<0.05) or intraperitoneal exposure.
Concomitant decreases in GST activity (~15% to 50% lower than controls in the cervix and ~30%
to 60% lower in the liver after oral exposure; p<0.05) were also observed at all doses and in both
organs and both treatments. EROD activity was increased in the cervix (~4- to ~12-fold) and liver
(~12- to ~35-fold) of all exposure groups. Measurement of CK and AST activity in the cervix and
serum also showed significant increases at all doses and after both exposures (~1.5- to 2-fold in the
cervix, and ~20% to 50% higher than controls in the liver after oral exposure). Finally, levels of the
inflammatory cytokine IL-6 were significantly (p<0.05) increased in the cervix of all treated mice,
and were markedly increased (from more than two-fold higher than untreated or vehicle controls
at the low dose, to ~six-fold higher at the high dose) in the serum of treated mice.
Based on the observations of decreased body weight and increased cervical epithelial
inflammation and hyperplasia, a LOAEL of 0.7 mg/kg-day (the lowest dose tested) is identified for
this study.
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1 Mohamed etal. (2010) investigated multi-generational effects in male mice following
2 exposure of 6-week old-C57BL/6 mice (10/group) to 0 (corn oil), 1, or 10 mg/kg-day
3 benzo[a]pyrene for 6 weeks by gavage. Following final treatment, male mice were allowed to
4 stabilize for 1 week prior to being mated with two untreated female mice to produce an
5 Fl generation. Male mice were sacrificed 1 week after mating. Fl males were also mated with
6 untreated female mice as were F2 males. The mice of the Fl, F2, and F3 generations were not
7 exposed to benzo[a]pyrene. The FO, Fl, F2, and F3 mice were all sacrificed at the same age
8 (14 weeks) and endpoints including testis histology, sperm count, sperm motility, and in vitro
9 sperm penetration (of hamster oocytes) were evaluated. These endpoints were analyzed
10 statistically using analysis of variance (ANOVA) and Tukey's honest significance test and results
11 were reported graphically as means ± SD.
12 Testicular atrophy was observed in the benzo[a]pyrene treatment groups, but was not
13 statistically different than controls. Statistically significant reductions were observed in epididymal
14 sperm counts of FO and Fl generations treated with the high or low dose of benzo[a]pyrene. For FO
15 and Fl generations, epididymal sperm counts were reduced approximately 50 and 70%,
16 respectively, in the low- and high-dose groups. Additionally, sperm motility was statistically
17 significantly decreased at the high dose in the FO and Fl generations. Sperm parameters of the F3
18 generation were not statistically different from controls. An in vitro sperm penetration assay
19 revealed statistically significantly reduced fertilization in FO and Fl generations of the low- and
20 high-dose groups. However, the value of this in vitro test is limited as it bypasses essential
21 components of the intact animal system (U.S. EPA, 1996). Based on decreased epididymal sperm
22 counts of FO and Fl generations, a LOAEL of 1 mg/kg-day was established from this study (no
23 NOAEL was identified).
24 Arafa et al. (2009) exposed groups of 12 male Swiss albino rats to benzo[a]pyrene in olive
25 oil (0 or 50 mg/kg-day via gavage) for 10 consecutive days, either alone or after similar treatment
26 with 200 mg/kg-day of the flavonoid hesperidin, which has been shown to exert anti-inflammatory,
27 antioxidant, and anticarcinogenic activity. One day after the final dose, the animals were sacrificed
28 for removal of the cauda epididymides and testes. Epididymal sperm count and motility were
29 assessed, as was daily sperm production in the testes. The study authors also investigated the
30 testicular activity of LDH, SOD, and GST, as well as GSH, malondialdehyde, and protein content. The
31 testes were examined under light microscope.
32 Relative testes weights (normalized to body weight) of benzo[a]pyrene exposed-animals
33 were significantly decreased compared with controls (35% lower, p < 0.05) (Arafa et al., 2009). In
34 addition, exposure to benzo[a]pyrene alone resulted in significantly decreased sperm count,
35 numbers of motile sperm, and daily sperm production (~40% decrease from control in each
36 parameter, p < 0.05). Effects on sperm count and production were abolished by hesperidin
37 pretreatment, but the number of motile sperm remained significantly depressed (compared with
38 the control group) in the group exposed to both benzo[a]pyrene and hesperidin. Measures of
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1 antioxidant enzymes and lipid peroxidation showed statistically significant induction of oxidative
2 stress in the testes of benzo[a]pyrene-exposed rats. With the exception of the decrease in testicular
3 GSH content (which was partially mitigated), pretreatment with hesperidin eliminated the effects of
4 benzo[a]pyrene on lipid peroxidation and antioxidant enzymes.
5 Xu et al. (2010) treated female Sprague-Dawley rats (6/group) to 0 (corn oil only), 5, or 10
6 mg/kg-day benzo[a]pyrene by gavage every other day for a duration of 60 days. This resulted in
7 TWA doses of 0, 2.5, and 5 mg/kg-day over the study period of 60 days. Endpoints examined
8 included ovary weight, estrous cycle, 17B-estradiol blood level, and ovarian follicle populations
9 (including primordial, primary, secondary, atretic, and corpora leutea). Animals were observed
10 daily for any clinical signs of toxicity and following sacrifice, gross pathological examinations were
11 made and any findings were recorded. All animals survived to necropsy. A difference in clinical
12 signs was not observed for the treated groups and body weights were not statistically different in
13 treated animals (although they appear to be depressed 6% at the high dose). Absolute ovary
14 weight was statistically significantly reduced in both the low- and high-dose groups (11 and 15%,
15 respectively) (see Table B-25). Animals treated with the high dose were noted to have a
16 statistically significantly prolonged duration of the estrous cycle and nonestrus phase compared to
17 controls. Animals in the high-dose group also had statistically significantly depressed levels of
18 estradiol (by approximately 25%) and decreased numbers of primordial follicles (by approximately
19 20%). This study also indicated a strong apoptotic response of ovarian granulosa cells as visualized
20 through TUNEL labeling; however, the strongest response was seen at the low dose; decreased
21 apoptosis was also observed at the high dose. Based on decreased ovary weight following a 60-day
22 oral exposure to benzo[a]pyrene, a LOAEL of 2.5 mg/kg-day was established from this study (no
23 NOAEL was identified).
24 Table B-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 ±0.0098b
249. 17 ±11. 2
5
0.136 ±0.0098b
247.25 ± 11.2
aTWA doses over the 60-day study period.
Statistically different from controls (p < 0.05) using one-way ANOVA.
Source: Xuetal. (2010).
25
26 Zheng et al. (2010) treated male Sprague-Dawley rats to 0 (corn oil only), 1, or 5 mg/kg-day
27 benzo[a]pyrene by daily gavage for a duration of 30 (8/group) or 90 days (8/group). At necropsy,
28 the left testis of each animal was collected and weighed. Testes testosterone concentrations were
29 determined by radioimmunassay and results were expressed as ng/g testis and reported
30 graphically. Testicular testosterone was statistically significantly decreased in the high-dose group
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1 approximately 15% following 90 days of exposure. The low-dose group also appeared to have a
2 similar average depression of testosterone levels; however, the change did not reach statistical
3 significance. Testosterone levels measured in animals sacrificed following 30 days of
4 benzo[a]pyrene exposure were not statistically different than controls. Based on decreased
5 testicular testosterone levels following a 90-day oral exposure to benzo[a]pyrene, a LOAEL of 5
6 mg/kg-day and a NOAEL of 1 mg/kg-day were identified.
7 McCallister et al. (2008) administered 0 or 300 [J.g/kg benzo[a]pyrene by gavage in peanut
8 oil to pregnant Long Evans rats (n = 5 or 6) on CDs 14-17. At this exposure level, no significant
9 changes were see in number of pups per litter, pup growth, or liver to body weight ratios in control
10 compared to benzo[a]pyrene exposed offspring. Treatment-related differences in brain to body
11 weight ratios were observed only on PNDs 15 and 30. Decreases in cerebrocortical mRNA
12 expression of the glutamatergic N-methyl-D-aspartate (NMDA) receptor subunitwas significantly
13 reduced (50%) in treated offspring compared to controls. In addition, in utero exposed offspring
14 exhibited decreased evoked cortical neuronal activity in the barrel field cortex when tested at PNDs
15 90-120.
16 Rigdon and Neal (1965) administered diets containing 1,000 ppm benzo[a]pyrene to
17 pregnant mice (nine/group) on CDs 10-21 or 5-21. The pups were reported as appearing
18 generally normal at birth, but cannibalism was elevated in the exposed groups. These results are in
19 contrast with an earlier study (Rigdon and Rennels, 1964) in which rats (strain not specified) were
20 fed diets containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and
21 during gestation. In the earlier study, five of eight treated females mated with untreated males
22 became pregnant, but only one delivered live young. The treated dam that delivered had two live
23 and two stillborn pups; one dead pup was grossly malformed. In the remaining treated females,
24 vaginal bleeding was observed on CDs 23 or 24. In the inverse experimental design, three of six
25 controls mated to benzo[a]pyrene-treated males became pregnant and delivered live young.
26 Visceral and skeletal examinations of the pups were not conducted. These studies were limited by
27 the small numbers of animals, minimal evaluation of the pups, lack of details on days of treatment
28 (food consumption, weight gain), and the occurrence of cannibalism.
29 Reproductive effects of in utero exposure via oral route
30 MacKenzie and Angevine (1981) conducted a two-generation reproductive and
31 developmental toxicity study for benzo[a]pyrene in CD-I mice. Benzo[a]pyrene was administered
32 by gavage in 0.2 mL of corn oil to groups of 30 or 60 pregnant (the FO generation) mice at doses of
33 0,10, 40, or 160 mg/kg-day on CDs 7-16 only. Therefore, unlike the standard two-generation
34 study, Fl animals were exposed only in utero. Fl offspring were evaluated for postnatal
35 development and reproductive function as follows. Fl pups (four/sex when possible) were allowed
36 to remain with their mothers until weaning on PND 20. Crossover mating studies were then
37 conducted. Beginning at 7 weeks of age, each Fl male mouse (n = 20-45/group) was allowed to
38 mate with two untreated virgin females for 5-day periods for 25 days (for a total exposure of 10
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1 untreated females/Fl male), after which time the males were separated from the females.
2 Fourteen days after separation from the males (i.e., on days 14-19 of gestation), the females were
3 sacrificed and the numbers of implants, fetuses, and resorptions were recorded. The F2 fetuses
4 were then examined for gross abnormalities. Similarly, each Fl female mouse (n = 20-55/group),
5 beginning at 6 weeks of age, was paired with an untreated male for a period of 6 months. Males
6 were replaced if the females failed to produce a litter during the first 30-day period. All F2 young
7 were examined for gross abnormalities on day 1 of life and their weights were recorded on day 4 of
8 age. This F2 group was sacrificed on day 20 postpartum, while the Fl female was left with a male
9 until the conclusion of the study. At 6 weeks of age, gonads of groups of 10 male and 10 female Fl
10 mice exposed to 0,10, or 40 mg/kg-day benzo[a]pyrene in utero were subjected to gross pathology
11 and histologic examinations.
12 No maternal toxicity was observed. The number of FO females with viable litters at
13 parturition at the highest dose was statistically significantly reduced by about 35% (Table B-2 6),
14 but progeny were normal by gross observation. Parturition rates of the low- and mid-dose groups
15 were unaffected by treatment, and litter sizes of all treated groups were similar to the control group
16 throughout lactation. However, body weights of the Fl pups in the mid- and high-dose groups were
17 statistically significantly decreased on PND 20, by 7 and 13%, respectively, and in all treated pups
18 on PND 42, 6, 6, and 10% for the low, mid, and high dose, respectively (Table B-26). The number of
19 Fl pups surviving to PNDs 20 and 42 was significantly reduced at the high dose (p < 0.01), by 8 and
20 16%, respectively. When Fl males were bred to untreated females and Fl females were mated
21 with untreated males, a marked dose-related decrease in fertility of >3 0% was observed in both
22 sexes, starting at the lowest exposure. There were no treatment-associated gross abnormalities or
23 differences in body weights in the F2 offspring.
24 Table B-26. Reproductive effects in male and female CD-I Fl mice
25 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 indexc
Fl female fertility indexd
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±0.3b
52.0b
65.7b
40
44/60 (73%)
10.4±0.1b
28.0±0.2b
4.7b
0.0b
160
13/30 (43%)b
9.7±0.2b
26.8 ± 0.4b
0.0b
0.0b
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aPregnant FO mice were administered daily doses of benzo[a]pyrene in corn oil on GDs 7-16.
Significantly (p < 0.05) different from control by unspecified tests.
Beginning 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.
dBeginning 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).
1
2 Exposure to benzo[a]pyrene caused a marked dose-related decrease in the size of the
3 gonads. In Fl males, testes weights were statistically significantly reduced. Testes from animals
4 exposed in utero to 10 and 40 mg/kg-day weighed approximately 60 and 18%, respectively, of the
5 weight of testes from the control animals (no F2 offspring were produced in the high-dose group).
6 This was confirmed by histopathologic observation of atrophic seminiferous tubules in the
7 40 mg/kg-day group that were smaller than those of controls and were empty except for a basal
8 layer of cells. The number of interstitial cells in the testes was also increased in this group. Males
9 from the 10 mg/kg-day group showed limited testicular damage; although all exhibited evidence of
10 tubular injury, each animal had some seminiferous tubules that displayed active spermatogenesis.
11 Ovarian tissue was absent or reduced in Fl females such that organ weights were not possible to
12 obtain. Examination of available tissue in these females revealed hypoplastic ovaries with few
13 follicles and corpora lutea (10 mg/kg-day) or with no evidence of folliculogenesis (40 mg/kg-day).
14 Ovarian tissue was not examined in highest-dose females.
15 The LOAEL in this study was 10 mg/kg-day, based on decreases in mean pup weight (<5%)
16 atPND 42 of Fl offspring of dams treated with 10, 40, or 160 mg/kg-daybenzo[a]pyrene, marked
17 decreases in the reproductive capacity (as measured by fertility index) of both male and female Fl
18 offspring exposed at all three treatment levels of benzo[a]pyrene (by approximately 30% in males
19 and females), decreased litter size (by about 20%) in offspring of Fl dams, and the dramatic
20 decrease in size and alteration in anatomy of the gonads of both male and female Fl mice exposed
21 to 10 and 40 mg/kg-day benzo[a]pyrene in utero. A NOAEL was not identified.
22 In another reproductive and developmental toxicity study, benzo[a]pyrene was
23 administered by gavage in corn oil to nine female NMRI mice at a dose of 10 mg/kg-day on GDs 7-
24 16; a group of nine controls received corn oil (Kristensen et al., 1995). Body weights were
25 monitored. FO females were kept with their offspring until after weaning (21 days after delivery).
26 At 6 weeks of age, one Fl female from each litter (n = 9) was caged with an untreated male. The
27 F2 offspring were inspected for gross deformities at birth, weight and sex were recorded 2 days
28 after birth, and the pups were sacrificed. The Fl females were sacrificed after 6 months of
29 continuous breeding. The effects of benzo[a]pyrene treatment on fertility, ovary weights, follicles,
30 and corpora lutea were evaluated. FO females showed no signs of general toxicity, and there was no
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1
2
3
4
5
6
7
effect on their fertility. Fl females had statistically significantly lower median numbers of offspring,
number of litters, and litter sizes and a statistically significantly greater median number of days
between litters as compared with the controls (Table B-27). At necropsy, the Fl females from
treated FO females had statistically significantly reduced ovary weights; histologic examination of
the ovaries revealed decreased numbers of small, medium, or large follicles and corpora lutea
(Table B-27). Only one dose group was used in this study, with decreased Fl female fertility
observed following in utero exposure at the LOAEL of 10 mg/kg-day; no NOAEL was identified.
Table B-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
exposeda(10 mg/kg-d)
22b (0-86)
3b (0-8)
8b (3-11)
21b (20-23)
9b (7-13)
Ob (0-68)
Ob (0-57)
Ob (0-19)
Ob (0-14)
10
11
12
13
14
15
16
17
18
19
20
21
22
aGroups of nine female NMRI FO mice were administered 0 or 10 mg benzo[a]pyrene/kg-day by gavage
in corn oil on GDs 7-16. One Fl female from each litter was continuously bred with an untreated male
for 6 months.
Significantly (p < 0.05) different from control group by Wilcoxon rank sum test or Kruskall-Wallis two-
tailed test.
Source: Kristensen et al. (1995).
Chen et al., (2012) treated male and female neonatal Sprague-Dawley rats (10/sex/group)
with benzo[a]pyrene (unspecified purity) dissolved in peanut oil by gavage daily from post-natal
day (PND) 5 - 11, at doses of 0.02, 0.2 or 2 mg/kg in 3 mL vehicle/kg b.w., determined individually
based upon daily measurements. This time period was described as representing the brain growth
spurt in rodents, analogous to brain developmental occurring from the third trimester to 2 years of
age in human infants. Breeding was performed by pairs of nine week old rats, with delivery
designated as PNDO. Litters were culled to 8 pups/dam (4/ea male and female, when possible) and
randomly redistributed at PND1 among the nursing dams; dams themselves were rotated every 2-3
days to control for caretaking differences, and cage-side observations of maternal behavior were
made daily. One male and female from each litter were assigned per treatment group, and the
following physical maturation landmarks were assessed daily in all treatment groups until weaning
atPND21: incisor eruption, eye opening, development of fur, testis decent and vaginal opening.
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1 Neonatal sensory and motor developmental tests were administered to pups during the
2 preweaning period at PNDs 12,14,16 and 18, and were behavioral tests administered to rats as
3 adolescents (PND 35, 36) or as adults (PND 70, 71): each rat was only tested during one
4 developmental period. All dosing was performed from 1300 - 1600 hrs, and behavioral testing was
5 during the "dark" period from 1900 - 2300 hrs, although tests were performed in a lighted
6 environment. Pups were observed individually and weighed daily, the order of testing litters was
7 randomized each day, and all observations were recored by investigators blinded to group
8 treatment.
9 Sensory and motor developmental tests including the surface righting reflex test, negative
10 geotaxis test, and cliff aversion test were performed only once, while the forelimb grip strength test
11 was assessed during three 60 second trials on PND12. Rat movements during the open-field test
12 were recorded by camera, and two blinded investigators scored movement and rearing separately
13 during a 5 min. evaluation period. Blinded investigators directly observed video monitoring of rat
14 movements during the elevated plus maze, and after a 5 min. free exploration period, recored
15 number of entries into the closed and open arms, the time spent in the open arms, and latency to
16 the first arm entry. Assessment of the Morris water maze was slightly different, in that the rats
17 were habituated to the testing pool by a 60 second swim without a platform on the day prior to
18 testing. The rats were then tested during a 60 second swim with a hidden platform present at a
19 constant position each day for four days; on the fifth day, the rats were evaluated during a 60
20 second probe swim without a platform. The number of times each animal crossed the original
21 platform location and the duration of time spent in the platform quadrant were recorded during
22 this final evaluation. One pup/sex/litter were assigned for behavioral testing to each of four tracks:
23 Track 1, surface righting reflex test, cliff aversion test, and open-field test (PND 12-18); Track 2,
24 negative geotaxis test, forelimb grip strength test, and open-field test (PND 12 - 20); Track 3,
25 elevated plus maze, Morris water maze, and open-field test (PND 34 - 36); Track 4, elevated plus
26 maze, Morris water maze, and open-field test (PND 69 - 71). All results were presented in
27 graphical form only.
28 No significant effects on pup body weight were observed during the 7-day treatment period
29 (PND 5 - 11). Three-way ANOVA (time xB[a]P treatment x sex) indicated that effects of B[a]P were
30 not sex-dependent throughout the 71 day experiment, so both sexes were pooled together. From
31 this pooled analysis, pups in the 2 mg/kg treatment group gained significantly less weight at both
32 PND36 and PND71. There were no differences among treatment groups in incisor eruption, eye
33 opening, development of fur, testis decent or vaginal opening.
34 For all measurements of neonatal sensory and motor development, results from both sexes
35 were analyzed together since B[a]P was reported to have no significant interaction with sex by 3-
36 way ANOVA. No significant differences were observed in either the cliff aversion or forelimb grip
37 strength tests. In the surface righting reflex test, latency was increased in the 0.2 mg/kg group at
38 PND12, in the 0.02 and 2 mg/kg groups at PND14, in only the high dose 2 mg/kg group at PND16,
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1 and was not significantly different in any group at PND18. At PND12 there was a dose-related
2 increase in negative geotaxis latency associated with 0.02, 2 and 2 mg/kg B[a]P, which was also
3 present in the 2 mg/kg group at PND14, but returned to control levels at PND16 and PND18. In the
4 open field test, there were no significant differences in either locomotion or rearing activity at
5 PND18 or 20. At PND34, the 2 mg/kg group exhibited significantly increased movement, but
6 increases in rearing were not significant. At PND69, increased locomotion was observed in both the
7 0.2 and 2 mg/kg groups, while rearing was significantly increased in only the 2 mg/kg treatment
8 group.
9 The elevated plus maze performance was only evaluated in adolescent and adult rats.
10 Unlike the previous tests, 3-way ANOVA revealed a statistically significant interaction between
11 neonatal B[a]P treatment and sex, so male and female performance was analyzed independently.
12 No significant differences in PND35 males were observed, and the only significant observation in
13 PND35 females was increased time spent in the open maze arms by 2 mg/kg treatment group.
14 Significantly decreased latency time to first open arm entry was observed in PND70 males and
15 females in both 0.2 and 2 mg/kg treatment groups; these groups also spent significantly more time
16 in open maze arms, along with the 0.02 mg/kg female group. PND70 2 mg/kg males, along with 0.2
17 and 2 mg/kg females, entered more frequently into open arms and less frequently into closed arms
18 than vehicle controls. In the Morris water maze, escape latency (time to reach the platform during
19 each of the four testing days) was consistently increased in the 2 mg/kg treatment group of both
20 sexes, in both adolescent and adult animals. These increases were statistically significant in both
21 males and females treated with 2 mg/kg B[a]P at both PND39 and PND74, and were also
22 significantly elevated in 0.2 mg/kg animals of both sexes at PND74. Likewise, performance during
23 the fifth test day, in the absence of the escape platform, was significantly adversely affected by both
24 metrics (decreased time spent in the target quadrant and decreased number of attempts to cross
25 the platform location) in 2 mg/kg rats of both sexes at both PND40 and PND75. PND75 females
26 treated with 0.2 mg/kg B[a]P also showed significant decreases in both performance metrics, while
27 PND75 0.2 mg/kg males only demonstrated significant differences in "time spent in target
28 quadrant". Swim speed was also assessed, but there were no differences among any treatment
29 group at either age evaluated.
30 Jules et al., (2009) treated pregnant Long Evans Hooded (LEH) rats with benzo[a]pyrene
31 (unspecified purity) dissolved in 0.875 mL peanut oil by gavage daily from GD14 - GD17, at doses of
32 150, 300, 600 and 1,200 |ig B[a]P /kgb.w., with animals weighed daily. Cage-side observations
33 were performed until pup weaning, and litter size evaluated for each treatment group. Pups from 4
34 - 5 individual litters were analyzed for each endpoint, which was independently repeated for a total
35 of 3 replicates. Delivery was designated PNDO, and pups were harvested from PNDO - 15 for B[a]P
36 metabolite identification, or for other endpoints as young adults at PND53. Systolic/diastolic blood
37 pressure and heart rate was recorded by a volume pressure recording sensor and occlusion tail-cuff
38 applied to conscious, non-anesthetized animals. Animals were preconditioned to the restraint
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1 device and tail-cuff by daily acclimatization sessions during PND46 - 50, to minimize stress effects
2 during data collection. Cardiac function values were averaged from 15 readings each collected over
3 a 1 minute interval every other minute for 30 minutes on PND53. Whole blood was collected from
4 the heart and aorta prior to surgical resection and tissue processing. Plasma and heart tissue B[a]P
5 metabolite content was quantified by reverse-phase HPLC with UV and fluorescence detection,
6 while heart and aortic tissue was subjected to SDS-PAGE for qualitative protein analysis, and RNA
7 extraction. Quantitative RT-PCR was performed for levels of angiotensin II (Angll), neuronal NOS
8 (nNOS), endothelial NOS (eNOS) and 7,8-Dihydrobiopterin oxidoreductase (BH4/BH2
9 oxidoreductase). Total RNA was also used to probe a cDNA microarray, and targets with > 2-fold
10 changes in expression were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and
11 Gene Ontology (GO) biological process pathway analysis.
12 No significant differences in litter size or pup weight gain from PNDO - 15 were reported in
13 any treatment group, and no convulsions, tremors or abnormal movements were reproducibly
14 observed. Most analytical data was reported graphically, as mean ± SEM of three replicates of 3 - 5
15 offspring measured/group. Plasma and heart tissue total B[a]P metabolite levels were maximal at
16 PNDO (the first time point sampled) and progressively decreased from PNDO - 13. Compared to the
17 low-dose group (150 |J.g/kg), plasma metabolite levels were significantly elevated in the 600 and
18 1,200 |ig/kgB[a]P groups through PND13, while heart metabolite levels were significantly
19 increased through PND11. Metabolites in mid-dose group, 300 [J.g/kg, trended between the 150
20 and 600 [ig/kg group levels from PNDO - 7, while not achieving statistically significant differences
21 in pair-wise comparisons. Three principle groups of B [a]P metabolites were identified. More than
22 70% of the total heart metabolite burden was composed of diol metabolites through PND13, while
23 the more reactive hydroxyl metabolites increased in relative composition from PND9 - 13, and the
24 dione population remained constant at < 5%.
25 Cardiovascular function was evaluated in pups exposed in utero to 600 or 1,200 [ig/kg B[a]P
26 vs. controls. A dose-related and statistically significant increase in both systolic (20, 50%) and
27 diastolic pressure (30, 80%) was observed in mid and high-dose pups, respectively. Heart rate was
28 also significantly altered; a 10% increased heart rate was reported in the 600 [ig/kg B[a]P group,
29 while the average heart rate of the 1,200 [ig/kg B[a]P groups decreased 8%. Cardiac tissue eNOS
30 protein levels fluctuated as a result of B [a] P treatment; in both the 600 and 1,200 [J.g/kg groups,
31 eNOS expression by semi-quantitative SDS-PAGE was significantly decreased at PNDO and PND5,
32 while it was significantly elevated above controls atPNDIO and PND15. While eNOS expression in
33 the 600 [ig/kg B[a]P group had returned to control levels by PND53, eNOS expression was
34 significantly higher (approximately 2-fold) in the 1,200 [ig/kg group. Compared to vehicle-treated
35 controls, cardiac message levels of nNOS and eNOS were not significantly affected by B[a]P
36 treatment at PNDO, and while nNOS mRNA levels were 2-fold higher at PND53 in the 600 [ig/kg
37 group, and eNOS mRNA was 3-fold higher in the 1,200 [J.g/kg group, consistent with the increased
38 eNOS protein levels detected at PND53. Message levels of BH4/BH2 oxidoreductase were
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1 suppressed in both B[a]P treatment groups at PNDO, and while mRNA expression remained
2 suppressed at PND53 in the 600 ug/kg group, BH4/BH2 message returned to control levels in the
3 1,200 ug/kg group. Angiotensin II mRNA levels were 1.8-fold higher in both B[a]P groups at PNDO,
4 and while expression increased to 5-fold more than controls at PND53 in the 600 ug/kg group,
5 Angll expression remained closer to 1.5-fold greater in the high-dose group. The following
6 pathways were identified as being enriched by 1,200 ug/kg B[a]P treatment in utero using KEGG
7 analysis, and correcting for multiple comparisons using the false-discovery rate method: PPARy,
8 renin-angiotensin system (Angll, adiponectin C1Q and collagen domain, adrenergic(33R, tachykinin
9 Rl), hematopoietic cell lineage, CYP450 metabolism (CYP2a2, CYP7al and CYP2bl2), retinol
10 metabolism, cell adhesion molecules-CAMs, primary bile acid biosynthesis and tight junctions.
11 Table B-28. Exposure-related effects in Long Evans Hooded rats
12 exposed to benzo[a]pyrene by gavage daily in utero from GD14 - GDI?
Effect measured
Heart rate (bmp; 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*
*Significantly (p < 0.05) different from control mean; n = 4-5/replicate, 3 replicates performed.
Source: Jules et al. (2012).
13
14 Bouayed et al., (2009) treated nursing female Swiss Albino OF1 mice (5/dose group) with
15 benzo[a]pyrene (unspecified purity) dissolved in avocado oil by gavage daily while nursing pups
16 from PND1 - 14 at 0, 2 or 20mg/kg-day in 10 mL/kg b.w., individually determined each day. Prior
17 to benzo[a]pyrene treatment, Swiss Albino litters were culled to 10 pups (5/sex when possible),
18 and nurturing females assigned to litters that were stratified randomly to achieve equivalent mean
19 pup litter body weights across the designated treatment groups. As the effects of B[a]P on maternal
20 nurturing behavior was unknown, dam behavior was visually monitored daily until weaning.
21 Furthermore, maternal nurturing performance from PNDO - 21 was assessed by two methods: a
22 nest-building test administered q.2.d., where nest quality/complexity was scored 15 minutes after
23 cotton material was supplied; and pup retrieval, in which latency to return the displaced pup to the
24 nest was measured twice and averaged, was evaluated q.d. At the indicated times 2 mice/sex/litter
25 were randomly selected, weighed, and brains resected for later mRNA expression analysis (n =
26 20/group).
27 Pup neuromotor maturation and behavior was assessed during pre-weaning by four
28 standard methods (administered between 1000 - 1300 on testing days, and in temporal order as
29 indicated): 1) righting reflex test, maximum duration 120 seconds, administered on PNDs 3, 5, 7 and
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1 9; 2) negativegeotaxis test, maximum duration 120 seconds, administered on PNDs 5, 7, 9 and 11;
2 3) forelimb grip test, duration until failure, administered on PNDs 9 and 11; and 4) open field test, 6
3 minute evaluation of locomotor activity and rearing following a 1 minute habituation period,
4 administered on PND15. Adolescent function was evaluated by three methods: water escape pole
5 climbing (WESPOC) test, administered at PND20, in which the time to find the pole, time to climb the
6 pole, and the time to reach the safety platform were reported; elevated plus maze, administered at
7 PND32 for 5 minutes, in which the latency time to first open arm entry, number of entries into open
8 arms, total number of entries, percent of time spent in open arms, and percent of entries into open
9 arms was determined; and Y-maze spontaneous alternation test, administered at PND40 for 5
10 minutes, in which the % spontaneous alternation was calculated by: [(the number of successful
11 overlapping triplets)/(total number of arm entries - 2) x 100%].
12 Benzo[a]pyrene treatment did not significantly affect the body weight of nursing mothers
13 during the 2 week treatment period. Since three-way AN OVA indicated that changes in pup weight
14 as a result of B[a]P treatment were not sex-dependent, data from male and female pups were
15 combined. B[a]P treatment of nursing mothers was associated with a 8-9% weight gain in pups
16 nursing from the 2 mg/kg group, and a 10-12% weight gain in pups from the 20 mg/kg group at
17 PND12 - 20. While not significantly different from PND26 - 40, pup weight in the 20 mg/kg group
18 was continuously higher than either the 2 mg/kg group or vehicle-treated controls. There were no
19 significant differences in pup brain weight or eye opening observed. Likewise, B[a]P treatment of
20 nursing mothers did not affect nest-building interest or quality, and while not significantly
21 impacting pup retrieval time, the retrieval latency period was observed to increase with increasing
22 treatment duration in both B[a]P groups vs. controls.
23 Behavioral test data was reported graphically, as mean ± SEM of n = 20/group. For the pre-
24 weaning neuromotor developmental tests, B[a]P treatment was found to not depend on sex, and so
25 data from male and female pups was combined. Pups nursing from mothers administered 2 or 20
26 mg/kg-day B [a]P had significantly elevated righting reflex times at PNDS - 5, which decreased to
27 control times at PND7 - 9. Only pups from the 20 mg/kg treatment group demonstrated
28 significantly increased negative geotaxis latency, which was 2-fold greater than controls at PNDs 5,
29 7 and 9, but returned to control levels at PND11. Interestingly, mice in the 20 mg/kg group had
30 increased forelimb grip strength, which was significantly greater than control mice at PND9 and 11,
31 corresponding to increased body weight in the B[a]P-treated mice vs. controls. Mice in the 2 mg/kg
32 group also performed better than controls at PND9, but were equivalent at PND11. No treatment or
33 sex-related effects were reported on locomotion or rearing activity during the open field test Sex-
34 dependency on test performance became evident during the analysis of the WESPOC test data:
3 5 female pups were not significantly affected using any metric, while males in the 2 0 mg/kg group
36 demonstrated a statistically significantly longer pole-grasping latency (3-fold), and took 13-times
37 longer to escape the pole and board the safety platform, vs. vehicle controls. While performance of
38 male pups from the 2 mg/kg group was not statistically significantly worse than vehicle controls by
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
pair-wise comparison, latency for both pole-grasping and escape in this treatment group
contributed to a significant trend for treatment dose and these effects. In the evaluation of the
elevated plus maze, treatment effects did not appear to depend upon sex, so both male and female
performance was analyzed together. Mice in both B[a]P treatment groups demonstrated
significantly decreased latency time to first entering an open arm (30 - 50%), as well as entered
open arms 2-times more frequently and spent twice as much time there vs. vehicle controls. While
mice in the 2 mg/kg treatment group entered into closed arms 20% less frequently than controls,
mice in the 20 mg/kg group were not significantly different. Likewise, mice nursing from mothers
treated with 2 mg/kg B[a]P performed 15% more spontaneous alternations in the Y-maze
spontaneous alternation test compared to controls, while mice in the high-dose group were not
significantly different The brains of pups nursing from the 20 mg/kg group expressed
approximately 50% lower levels of 5-hydroxytryptamine (serotonin) 1A (5HT1A), and mu 1-opioid
(MORI) mRNA, and a trend was observed in the low-dose group as well. No significant changes in
alpha-ID adrenergic (ADRAID) or gamma-aminobutyric acid A (GABAA) mRNA levels were
detected.
Table B-29. Exposure-related effects in Swiss Albino OF1 mice exposed
as pups to benzo[a]pyrene in breast milk from dams treated by gavage
daily from PND1 - PND14
Effect measured
Pup body weight (g; mean ± SEM, n = 20)
PNDO
PND4
PND8
PND12
PND20
PND26
PND32
PND40
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
19
20
21
22
23
24
25
** p < 0.01, *** p < 0.001 significantly different from control mean
Source: Bouayed et al. (2009).
Reproductive effects in adults and repeated oral exposure
Rigdon and Neal (1965) conducted a series of experiments to assess the reproductive
effects of orally administered benzo[a]pyrene to Ah-responsive white Swiss mice. Female animals
(number not stated) were administered benzo[a]pyrene at 250, 500, or 1,000 ppm in the feed
before or during a 5-day mating period. Based on the initial body weight, the doses can be
estimated as 32, 56, and 122 mg/kg-day, respectively. No effect on fertility was observed at any
treatment dose, even when animals were fed 1,000 ppm benzo[a]pyrene for 20 days prior to
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1 mating, but interpretation of this finding was marred by large variability in numbers of pregnant
2 females and litter sizes for both treated and control mice. In separate experiments, the fertility of
3 five male mice/group was not affected by exposure to 1,000 ppm in food for up to 30 days prior to
4 mating with untreated females. Histologic examinations showed that male mice fed 500 ppm
5 benzo[a]pyrene for 30 days had spermatozoa present in their testes; further details were not
6 provided. The only treatment-related effect was a lack of weight gain related to feed unpalatability.
7 While this study suggests that premating exposure of male or female mice to doses up to
8 122 mg/kg-day for 20 days may not affect fertility, the sample sizes were too small and study
9 designs were too inconsistent to provide reliable NOAELs and LOAELs for
10 reproductive/developmental toxicity.
11 In an earlier study (Rigdon and Rennels, 1964), rats (strain not specified) were fed diets
12 containing benzo[a]pyrene at 1,000 ppm for approximately 28 days prior to mating and during
13 gestation. In this study, five of eight treated females mated with untreated males became pregnant,
14 but only one delivered live young. The treated dam that delivered had two live and two stillborn
15 pups; one dead pup was grossly malformed. In the remaining treated females, vaginal bleeding was
16 observed on CDs 23 or 24. In the inverse experimental design, three of six controls mated to
17 benzo[a]pyrene-treated males became pregnant and delivered live young. Visceral and skeletal
18 examinations of the pups were not conducted. These studies are insufficiently reported and of
19 insufficient design (e.g., inadequate numbers of animals for statistical analysis) to provide reliable
20 NOAELs or LOAELs for reproductive effects from repeated oral exposure to benzo[a]pyrene.
21 Inhalation
22 Reproductive toxicity and in utero exposure via inhalation
23 Archibong et al. (2002) evaluated the effect of exposure to inhaled benzo[a]pyrene on fetal
24 survival and luteal maintenance in timed-pregnant F344 rats. Prior to exposure on GD 8,
25 laparotomy was performed to determine the number of implantation sites, and confirmed pregnant
26 rats were divided into three groups, consisting of rats that had four to six, seven to nine, or more
27 than nine conceptuses in utero. Rats in these groups were then assigned randomly to the treatment
28 groups or control groups to ensure a similar distribution of litter sizes. Animals (10/group) were
29 exposed to benzo[a]pyrene:carbon black aerosols at concentrations of 25, 75, or 100 [ig/m3 via
30 nose-only inhalation, 4 hours/day on CDs 11-20. Control animals were either sham-exposed to
31 carbon black or remained entirely unexposed. Results of particle size analysis of generated
32 aerosols were reported by several other reports from this laboratory (Inyang et al., 2003; Ramesh
33 etal., 2001a; Hoodetal., 2000). Aerosols showed a trimodal distribution with averages of 95%
34 cumulative mass with diameters <15.85 |im; 89% <10 |im; 55% <2.5 |im; and 38% <1 |im (Inyang
35 etal., 2003). Ramesh etal. (2001a) reported that the (MMAD ± geometric SD) for the 55% mass
36 fraction with diameters <2.5 [im was 1.7 ± 0.085. Progesterone, estradiol-17p, and prolactin
37 concentrations were determined in plasma collected on CDs 15 and 17. Fetal survival was
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
calculated as the total number of pups divided by the number of all implantation sites determined
on GD 8. Individual pup weights and crown-rump length per litter per treatment were determined
on PND 4 (PND 0 = day of parturition).
Archibong et al. (2002) reported that exposure of rats to benzo[a]pyrene caused
biologically and statistically significant (p < 0.05) reductions in fetal survival compared with the
two control groups; fetal survival rates were 78.3, 38.0, and 33.8% per litter at 25, 75, and
100 ug/m3, respectively, and 96.7% with carbon black or 98.8% per litter in untreated controls (see
Table 4-24). Consequently, the number of pups per litter was also decreased in a concentration-
dependent manner. The decrease was ~50% at 75 ug/m3 and ~65% at 100 ug/m3, compared with
sham-exposed and unexposed control groups. No effects on hormone levels were observed on
CDs 15 or 17 at the low-dose. Biologically significant decreases in mean pup weights (expressed as
g per litter) of >5% were observed at doses >75 ug/m3 (14 and 16% decreases at 75 and 100
ug/m3, respectively, p < 0.05). Exposure to benzo[a]pyrene did not affect crown-rump length (see
Table B-30).
Table B-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/m3)
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±0.5b
78.3±4.1b
10.5 ±0.2
28.0 ±0.6
75
9.0 ±0.2
4.2±0.1b
38.0 ± 2. lb
9.1±0.2b
27.3 ±0.7
100
8.8 ±0.1
3.0±0.2b
33.8±1.3b
8.9±0.1b
27.9 ±0.7
17
18
19
20
21
22
23
24
25
aValues presented as means ± SEM.
br-
'Significantly different from controls at p < 0.05 by one-tailed post-hoc t-testing following ANOVA.
Source: Archibong et al. (2002).
Benzo[a]pyrene exposure at 75 ug/m3 caused a statistically significant decrease in plasma
progesterone, estradiol, and prolactin on GD 17; these levels were not determined in the rats
exposed to 100 ug/m3 (Archibong et al., 2002). Plasma prolactin is an indirect measure of the
activity of decidual luteotropin, a prolactin-like hormone whose activity is necessary for luteal
maintenance during pregnancy in rats. Control levels of prolactin increased from GD 15 to 17, but
this increase did not occur in the rats exposed to 75 ug/m3. Although the progesterone
concentration at 75 ug/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
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1 loss of fetuses was thought to be caused by the lower prolactin levels rather than progesterone
2 deficiency. The reduced circulating levels of progesterone and estradiol-17(3 among
3 benzo[a]pyrene-treated rats were thought to be a result of limited decidual luteotropic support for
4 the corpora lutea. The authors proposed the following mechanism for the effects of benzo[a]pyrene
5 on fertility: benzo[a]pyrene or its metabolites decreased prolactin and decidual luteotropin levels,
6 compromising the luteotropic support for the corpora lutea and thereby decreasing the plasma
7 levels of progesterone and estradiol-17p. The low estradiol-17p may decrease uterine levels of
8 progesterone receptors, thereby resulting in fetal mortality. Based on biologically and statistically
9 significant decreases in pups/litter and percent fetal survival/per litter, the LOAEL was 25 ug/m3;
10 no NOAEL was identified.
11 Neurotoxicity and in utero exposure via inhalation
12 To evaluate the effects of benzo[a]pyrene on the developing CNS, Wormley et al. (2004)
13 exposed timed-pregnant F344 rats (10/group) to benzo[a]pyrene:carbon black aerosols by nose-
14 only inhalation on CDs 11-21 for 4 hours/day at a concentration of 100 ug/m3. Results of particle
15 size analysis of genenerated aerosols were reported by other reports from this laboratory (Ramesh
16 etal., 2001a; Hoodetal., 2000). Particle size analysis of a 100-ug/m3 aerosol showed a trimodal
17 distribution with averages of 95% cumulative mass with diameters <15.85 um; 90% <10 um;
18 67.5% <2.5 um; and 66.2% <1 um; the MMAD ± geometric SD for the latter fraction was 0.4 ± 0.02
19 um (Hoodetal., 2000). Dams were maintained to term and pups were weaned on PND 30.
20 Benzo[a]pyrene reduced the number of live pups to one-third of control values without affecting
21 the number of implantation sites. During PNDs 60-70, electrical stimulation and evoked field
22 potential responses were recorded via electrodes implanted into the brains of the animals. Direct
23 stimulation of perforant paths in the entorhinal region revealed a diminution in long-term
24 potentiation of population spikes across the perforant path-granular cell synapses in the dentate
25 gyrus of the hippocampus of Fl generation benzo[a]pyrene-exposed animals; responses in exposed
26 offspring were about 25% weaker than in control offspring. Additionally, NMDA receptor subunit 1
27 protein (important for synaptic functioning) was down-regulated in the hippocampus of
28 benzo[a]pyrene exposed Fl pups. The authors interpreted their results as suggesting that
29 gestational exposure to benzo[a]pyrene inhalation attenuates the capacity for long-term
30 potentiation (a cellular correlate of learning and memory) in the Fl generation.
31 In another study by this same group of investigators, Wu et al. (2003) evaluated the
32 generation of benzo[a]pyrene metabolites in Fl generation pups, as well as the developmental
33 profile for AhR and mRNA. In this study, confirmed pregnant F344 rats were exposed to
34 benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 ug/m3 via nose-only inhalation,
35 4 hours/day, for 10 days (CDs 11-21). Control animals were exposed to carbon black (sham) to
36 control for inert carrier effects or they remained untreated. Each benzo[a]pyrene concentration
37 had its own set of controls (carbon black and untreated). Two randomly selected pups were
38 sacrificed on each of PND 0, 3, 5,10,15, 20, and 30. Body, brain, and liver weights were recorded.
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1 Benzo[a]pyrene metabolites were analyzed in the cerebral cortex, hippocampus, liver, and plasma.
2 A dose-related increase in plasma and cortex benzo[a]pyrene metabolite concentrations in pups
3 was observed. Dihydrodiols (4,5-; 7,8-; 9,10-) dominated the metabolite distribution profile up to
4 PND 15 and the hydroxy (3-OH-benzo[a]pyrene; 9-OH-benzo[a]pyrene) metabolites after PND 15
5 at 100 [ig/m3 (the only exposure concentration reported). Results indicated a dose-related
6 decrease in the ratio of the total number of pups born per litter to the total number of implantation
7 sites per litter. The number of resorptions at 75 andlOO [ig/m3, butnotat25 [J.g/m3,was
8 statistically significantly increased compared with controls.
9 Adult male reproductive effects and repeated inhalation exposure
10 Inyangetal. (2003) evaluated the effect of subacute exposure to inhaled benzo[a]pyrene on
11 testicular steroidogenesis and epididymal function in rats. Male F344 rats (10/group), 13 weeks of
12 age, were exposed to benzo[a]pyrene:carbon black aerosols at 25, 75, or 100 [ig/m3 via nose-only
13 inhalation, 4 hours/day for 10 days. Control animals were either exposed to carbon black (sham) to
14 control for exposure to the inert carrier, or they remained untreated. Each benzo[a]pyrene
15 concentration had its own set of controls (carbon black and untreated). Aerosols showed a
16 trimodal distribution with averages of 95% cumulative mass <15.85 |im; 89% <10 |im; 55% <2.5
17 |im; and38% <1 |im (Inyangetal., 2003); an earlier report from this laboratory indicated that the
18 55% mass fraction had a MMAD ± geometric SD of 1.7 ± 0.085 (Rameshetal., 2001a). Blood
19 samples were collected at 0, 24, 48, and 72 hours after cessation of exposure to assess the effect of
20 benzo[a]pyrene on systemic concentrations of testosterone and luteinizing hormone (LH),
21 hormones that regulate testosterone synthesis. Reproductive endpoints such as testis weight and
22 motility and density of stored (epididymal) sperm were evaluated.
23 Regardless of the exposure concentration, inhaled benzo[a]pyrene did not affect testis
24 weight or the density of stored sperm compared with controls. However, inhaled benzo[a]pyrene
25 caused a concentration-dependent reduction in the progressive motility of stored sperm.
26 Progressive motility was similar at 75 and 100 [ig/m3, but these values were significantly lower (p <
27 0.05) than in any other group. The reduction in sperm motility postcessation of exposure was
28 thought to be the result of benzo[a]pyrene limiting epididymal function. Benzo[a]pyrene exposure
29 to 75 [J.g/m3 caused a decrease in circulating concentrations of testosterone compared with controls
30 from the time of cessation of exposure (time 0) to 48 hours posttermination of exposure (p < 0.05).
31 However, the decrease was followed by a compensatory increase in testosterone concentration at
32 72 hours postcessation of exposure. Exposure to 75 [ig/m3 caused a nonsignificant increase in
33 plasma LH concentrations at the end of exposure compared with controls, which increased further
34 and turned significant (p < 0.05) for the remaining time of the study period. The decreased plasma
3 5 concentration of testosterone, accompanied by an increased plasma LH level, was thought to
36 indicate thatbenzo[a]pyrene did not have a direct effect on LH. The authors also noted thatthe
37 decreased circulating testosterone may have been secondary to induction of liver CYP450 enzymes
38 by benzo[a]pyrene. The authors concluded that subacute exposure to benzo[a]pyrene contributed
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Toxicological Review ofbenzo[a]pyrene
1 to impaired testicular endocrine function that ultimately impaired epididymal function. Based on
2 this study, the NOAEL was 25 [J.g/m3 and the LOAEL was 75 [ig/m3, based on a statistically
3 significant reduction in the progressive motility of stored sperm and impairment of testicular
4 function with 10 days of exposure at 75 [ig/m3.
5 In a follow-up study with longer exposure duration, adult male F344 rats (10 per group)
6 were exposed to benzo[a]pyrene:carbon black aerosols at 75 [ig/m3 via nose-only inhalation,
7 4 hours/day for 60 days (Archibong et al., 2008; Ramesh et al., 2008). Rats in the control group
8 were subjected to the nose-only restraint, but were not exposed to the carbon black carrier. Blood
9 samples were collected at 0, 24, 48, and 72 hours after exposure terminated, and the animals
10 sacrificed for tissue analyses following the last blood sampling. Data were analyzed statistically for
11 benzo[a]pyrene effects on weekly body weights, total plasma testosterone and LH concentrations,
12 testis weights, density of stored spermatozoa, sperm morphological forms and motility,
13 benzo[a]pyrene metabolite concentrations and AHH activity, and morphometric assessments of
14 testicular histologies. Relative to controls, the results indicated 34% reduced testis weight (p <
15 0.025), reduced daily sperm production (p < 0.025) and reduced intratesticular testosterone
16 concentrations (p < 0.025). Plasma testosterone concentrations were reduced to about one-third of
17 the level in controls on the last day of exposure (day 60) and at 24, 48, and 72 hours later (p < 0.05).
18 However, plasma LH concentrations in benzo[a]pyrene exposed rats were elevated throughout the
19 blood sampling time periods compared with controls (p < 0.05). In testis, lung, and liver, AHH
20 activity, and benzo[a]pyrene-7,8-dihydrodiol (precursor to the DNA-reactive BPDE) and
21 benzo[a]pyrene-3,6-dione metabolites were significantly (p < 0.05) elevated relative to controls.
22 Progressive motility and mean density of stored spermatozoa were significantly reduced (p < 0.05).
23 Weekly body weight gains were not affected by benzo[a]pyrene exposure. These results indicate
24 that 60-day exposure of adult male rats to benzo[a]pyrene:carbon black aerosols at 75 [J.g/m3
25 produced decreased testis weight; decreased intratesticular and plasma testosterone
26 concentrations; and decreased sperm production, motility, and density.
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Toxicological Review ofbenzo[a]pyrene
1 OTHER PERTINENT TOXICITY INFORMATION
2 Table B-31. In vitro genotoxicity studies of benzo[a]pyrene in non-
3 mammalian cells
Result
S9
S9
Reference
Endpoint/test system: prokaryotic cells
Forward mutation
Salmonella typhimurium TM677
S. typhimurium TM677
+
+
-
ND
Rastetter et al., 1982
Babson et al., 1986
Reverse mutation
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
+
+
+
+
+
+
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
-
+
+
+
+
ND
ND
-
-
-
ND
-
ND
ND
-
-
-
ND
-
-
ND
ND
ND
ND
ND
ND
ND
-
ND
Ames et al., 1975
McCann et al., 1975
Wood et al., 1976
Epleretal., 1977
Obermeier and Frohberg,
1977
Pitts et al., 1978
LaVoie et al., 1979
Simmon, 1979a
Hermann, 1981
Alfheim and Randahl, 1984
Glatt et al., 1985
Sakaietal., 1985
Glatt et al., 1987
Marino, 1987
Alzieu et al., 1987
Prasanna et al., 1987
Ampyetal., 1988
Bos et al., 1988
Lee and Lin, 1988
Antignac et al., 1990
Gaoetal., 1991
Balanskyetal., 1994
Norpoth et al., 1984
Carver et al., 1986
Pahlman and Pelkonen, 1987
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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
+
+
+
-
+
+
+
+
+
-
-
-
-
S9
ND
ND
ND
ND
-
-
ND
ND
-
-
-
ND
-
Reference
Tang and Friedman, 1977
Bruce and Meddle, 1979
Phillipson and loannides, 1989
Balanskyetal., 1994
Ames et al., 1973
Glatt et al., 1975
Oesch et al., 1976
Egert and Greim, 1976
Rosenkranz and Poirier, 1979
Ames et al., 1973
Glatt et al., 1975
McCann et al., 1975
Epleretal., 1977
DIMA damage
E. co///pol A
E. co///differential killing test
E. co// WP2-WP100/rec-assay
E. CO///SOS chromotest Pq37
+
+
+
+
-
-
ND
Rosenkranz and Poirier, 1979
Tweats, 1981
Mamberetal., 1983
Mersch-Sundermann et al.,
1992
Endpoint/test system: nonmammalian eukaryotes
Mitotic recombination
S. cerevisiae D4-RDII
S. cerevisiae D3
ND
-
-
-
Siebert et al., 1981
Simmon, 1979b
1
2
+ = positive; - = negative; ND = not determined
Table B-32. In vitro genotoxicity studies of benzo[a]pyrene in
mammalian cells
dssay/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
ND
ND
ND
+
+
+
Danheiseretal., 1989
Crespi et al., 1985
Chen et al., 1996
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Assay /test system
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)
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
Chinese hamster V79 lung epithelial cells
Rat/Fischer, embryo cells/OuaR
Result
+S9
—
ND
+
ND
+
+
ND
ND
+
+
+
+
+
+
+
+
+
+
+
ND
-S9
ND
+
ND
+
-
ND
+
+
-
-
ND
-
ND
ND
+
ND
ND
ND
ND
+
Reference
Haneltetal., 1997
Barfknechtetal., 1982
Crespi et al., 1985
Rocchi et al., 1980
Gupta and Goldstein, 1981
Allen-Hofmann and
Rheinwald, 1984
Allen-Hofmann and
Rheinwald, 1984
Chen etal., 2010
Clive et al., 1979
Clive et al., 1979
Amacher and Turner, 1980;
Amacheretal., 1980
Amacher and Paillet, 1983
Arce et al., 1987
Yang et al., 1999
Gupta and Singh, 1982
Diamond etal., 1980
Huberman, 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
ND
ND
ND
ND
+
ND
ND
+
+
+
+
+
ND
+
+
ND
Wiencke et al., 1990
Li et al., 2001
Wu et al., 2005
Gu et al., 2008
Haneltetal., 1997
Tarantini et al., 2009
Roggeband et al., 1994
Arce et al., 1987
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Toxicological Review ofbenzo[a]pyrene
Assay /test system
Virus transformed SHE and mouse
C3H10T1/2 cells
Mouse lymphoma (L5178Y/TK+/-) cells
Rat tracheal cells
Result
+S9
ND
+
ND
-S9
+
ND
+
Reference
Arce et al., 1987
Arce et al., 1987
Roggeband et al., 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 et al., 1978
Agrelo and Amos, 1981
Robinson and Mitchell,
1981
Valentin-Severin et al., 2004
Casto et al., 1976
Roggeband et al., 1994
Michalopoulos et al., 1978
Roggeband et al., 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
Miloetal., 1978
Feldmanetal., 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
Mouse lymphoma (L5178Y/TK+/-) cells
Rat Liver RL1 cells
ND
+
+
—
+
+
+
-
-
—
ND
ND
Salamaetal., 2001
Weinstein et al., 1977
Matsuoka et al., 1979
Popescu et al., 1977
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
ND
ND
ND
ND
+
ND
+
+
+
+
ND
+
Crofton-Sleigh et al., 1993
Wu et al., 2003
Fowler etal., 2010
Crofton-Sleigh et al., 1993
LoJaconoetal., 1992
Whitwell etal., 2010
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Assay /test system
Chinese hamster V79-MZ cells
Result
+S9
ND
-S9
+
Reference
Matsuoka et al., 1999
DNA 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
+
+
+
+
+
Sipinenetal., 2010
Rodriguez-Romero et al.,
2012
Haneltetal., 1997
Tarantini et al., 2009
Nwagbara, 2007
Lubet et al., 1983
Cosma and Marchok, 1988;
Cosma et al., 1988
Gaoetal., 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
CHO cells
CHO cells
ND
+
ND
ND
ND
ND
ND
ND
+
+
ND
+
+
+
+
ND
+
+
ND
+
+
+
+
+
+
+
+
-
ND
+
-
ND
ND
ND
+
-
—
+
Abe et al., 1983a, b
Huh etal., 1982
Juhletal., 1978
Salamaetal., 2001
Rudigeretal., 1976
Craig-Holmes and Shaw,
1977
Schonwald et al., 1977
Wiencke et al., 1990
Tohda et al., 1980
LoJaconoetal., 1992
Abe et al., 1983a, b
Popescu et al., 1977
Mane etal., 1990
Wojciechowski et al., 1981
Arce et al., 1987
Kulka et al., 1993a
de Raat, 1979
Husgafvel-Pursiainen et al.,
1986
Wolff and Takehisa, 1977
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Toxicological Review ofbenzo[a]pyrene
Assay /test system
CHO cells
Chinese hamster lung cells
Rabbit peripheral blood lymphocytes
Rat ascites hepatoma AH66-B
Rat esophageal tumor Rl
Rat hepatocyte (immortalized) cell lines
(NRLcl-B, NRLcl-C, and ARL)
Rat hepatoma (Reuber H4-II-E) cells
Rat liver cell lineARLlS
Rat pleural mesothelial cells
Result
+S9
ND
ND
ND
ND
ND
+
ND
ND
ND
-S9
+
+
+
+
+
ND
+
+
+
Reference
Pal et al., 1978
Shimizu etalv 1984
Takehisa and Wolff, 1978
Abe et al., 1983a, b
Abe et al., 1983a, b
Kulkaetal., 1993b
Dean et al., 1983
long et al., 1981
Achard et al., 1987
Aneuploidy
Chinese hamster V79-MZ cells
ND
+
Matsuoka et al., 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-313 cells
Mouse BALB/c-313 cells
Mouse BALB/c-313 clone A31-1-1
ND
ND
+
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
+
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
van Agen et al., 1997
Calaf and Russo, 1993
Greb et al., 1980
Mager et al., 1977
DiPaoloetal., 1971,1969
Dunkeletal., 1981
LeBoeuf etal., 1990
Casto et al., 1977
Emura et al., 1987, 1980
Heidelberger et al., 1983
Arce et al., 1987
Nesnow et al., 2002, 1997
Peterson et al., 1981
Lubet et al., 1983
Heidelberger et al., 1983
Dunkeletal., 1981
Matthews, 1993
Little and Vetroys, 1988
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Assay /test system
Rat embryo cells/SA7 virus transformation
Rat/Fischer, embryo cells (leukemia virus
transformed)
Rat/Fischer, embryo cells/OuaR
Result
+S9
ND
ND
ND
-S9
+
+
+
Reference
DiPaolo and Casto, 1976
Dunkeletal., 1981
Mishra et al., 1978
+ = positive; - = negative; ND = not determined; SHE = Syrian hamster embryo; TK = thymidine kinase
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1
2
Table B-33. In vivo genotoxicity studies of benzo[a]pyrene
Endpoint
Mutation
Mutation,
germline
Test system
Human, blood
T lymphocytes
(smokers and
nonsmokers); hprt
locus mutation assay
Mouse, T-stock, (SEC x
C57BL)F1, (C3H x
101)F1, or(C3Hx
C57BL)F1 for females;
(101xC3H)Flor(C3H
x 101)F1 for males;
dominant-lethal
mutation assay
Test conditions
T-cells of lung cancer patients
(smokers and nonsmokers from lung
cancer patients and population
controls with known smoking status)
analyzed for hprt locus mutations.
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
nonsmokers
500 mg/kg
Comment
Splicing mutations, base-pair
substitutions, frameshift, and
deletion mutations observed.
Smokers and nonsmokers 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 = (C3Hxl01)Fl>
(SECxC57BL)Fl >
(C3HxC57BL)Fl
Reference
Hackman
et al., 2000
Generoso
et al., 1979
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Toxicological Review ofbenzo[a]pyrene
Endpoint
Mutation,
germline
Mutation,
germline
Mutations
and BPDE-
DNA
adducts,
germline
Test system
Mouse, male stocks:
(101 xC3H)Fl; female
stocks (A):
(101xC3H)Fl, (B):
(C3Hxl01)Fl, (C):
(C3HxC57BL)Fl,
(D):(SECxC57BL)Fl,
(E):T-stock females;
dominant lethal
mutations
Mouse, male stocks:
(101 xC3H)Fl; female
stocks (A): (101 x
C3H)F1, (B):(C3Hx
101)F1, (C): (C3H x
C57BL)F1, (D):(SECx
C57BL)F1, (E): T-stock
females; heritable
translocations
Mouse, C57BL/6, ell
transgenic (Big Blue®)
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 LC-
MS/MS SRM with 15N-deoxyguanosine
labeling.
Results
+
-
+
Dose
500 mg/kg
500 mg/kg
50 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
post-treatment).
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
(GOTA transversions).
Reference
Generoso
et al., 1982
Generoso
et al., 1982
Olsen et
al., 2010
This document is a draft for review purposes only and does not constitute Agency policy.
B-2 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
Mutations
and BPDE-
DNA
adducts,
germline
Mutations
and BPDE-
DNA
adducts
Mutation
Test system
Mouse, C57BL/6 males,
wild type and Xpc-/~
with pUR288/ocZ
reporter gene
Mouse, C57BL/6 lacZ
transgenic
Mouse, C57BL female x
T-strain male; somatic
mutation assay
Test conditions
Benzo[a]pyrene given via gavage in
sunflower oil 3 times/wk for 1, 4, or 6
wks (Xpc7") or 6 wks (Wt). Spleen,
testis, and sperm cells analyzed for
lacZ mutation frequency, and DNA
adducts analyzed in testis by 32P-
postlabeling.
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.
Results
+
+
+
Dose
13 mg/kg
50 mg/kg
100 or
500 mg/kg
Comment
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.
BPDE-dG adduct levels peaked
between 5 and 7 days
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 days 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.
Reference
Verhofstad
etal., 2011
Boerrigter,
1999
Russell,
1977
This document is a draft for review purposes only and does not constitute Agency policy.
B-3 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
Mutation
Mutation
Mutation
Mutation
Mutation
Test system
Mouse, /ocZtransgenic
(Muta™Mouse)
Mouse, /ocZtransgenic
(Muta™Mouse)
Mouse, C57BL/6J Dlb-1
congenic; Dlb-1 locus
assay
Mouse, C57BL/6 (lacZ
negative and XPA+/+
and XPA~'~); hprt
mutations in T
lymphocytes
Mouse, Cockayne
syndrome-deficient
(Csfo^V; heterozygous
(Csb+/~) and WT
controls (Csb+/+); hprt
mutation frequency
assay
Test conditions
Benzo[a]pyrene given via gavage in
olive oil daily for 28 consecutive d;
sacrificed 3 d after last dosing; 4
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.
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 p.o. alone
or 96 hrs following a single i.p. dosing
with 10 u.g/kgTCDD.
Gavage in corn oil 3 times/wk for 0, 1,
5, 9, or 13 wks; sacrificed 7 wks after
last treatment.
Csb-/yiacZ+/- and Csb+/yiacZ+/- 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 collected.
Results
+
+
+
+
+
Dose
25, 50, and 75
mg/kg-day
125 mg/kg-day
40 mg/kg
13 mg/kg
13 mg/kg
Comment
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
Benzo[a]pyrene caused a dose-
dependent increase in mutant
frequency; i.p. route showed
higher mutant frequency than
p.o. 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
Csb~f~ mice; hprt mutations
significantly higher in Csb~f~
mice than control mice. BPDE-
dGuo adducts in hprt gene are
preferentially removed in WT
mice than Csb~f~ mice.
Reference
Lemieux et
al., 2011
Hakura et
al., 1998
Brooks et
al., 1999
Bol et al.,
1998
Wijnhoven
et al., 2000
This document is a draft for review purposes only and does not constitute Agency policy.
B-4 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
Mutation
Mutation
Mutation
Mutation
Mutation
Mutation
Test system
Mouse, B6C3F1;
forestomach H-ros,
K-ros, and p53
mutations
Mouse, lacZ/galE
(Muta™ Mouse); skin
painting study
Mouse, T-strain
Mouse, 129/Ola (WT);
hprt mutations in
splenic T lymphocytes
Mouse, A/J, male
Mouse, CD-I; skin
papillomas (Ha-ros
mutations)
Test conditions
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; DNAfrom skin, liver,
and lung analyzed for mutations.
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 nmol TPA for 14
wks. One month after stopping TPA
application, papillomas were collected
and DNAfrom 10 individual papillomas
were analyzed for Ha-ros mutations by
PCR and direct sequencing.
Results
+
+skor
Li,Lu
+
+
+
+
Dose
5, 25, or
100 ppm
1.25 or
2.5 mg/kg
(25 or
50 u.g/mouse)
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
Comment
68% K-ros (codons 12,13), 10%
H-ros (codon 13), 10% p53
mutations; all G->T
transversions
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.
Dose-dependent increase in
hprt mutation frequency.
Dose-dependent increase in
lung tissue K-ros codon 12
G->T mutation frequency.
About 90% of papillomas
contained Ha-ros mutations, all
of them being transversions at
codons 12 (20% GGA->GTA),
13 (50% GGC->GTC), and 61
(20% CAA->CTA).
Reference
Gulp et al.,
2000
Dean et al.,
1998
Davidson
and
Dawson,
1976
Bol et al.,
1998
Meng et
al., 2010
Colapietro
et al., 1993
This document is a draft for review purposes only and does not constitute Agency policy.
B-5 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
Mutation
BPDE-
DNA
adducts
BPDE-
DNA
adducts
Test system
Rat, Wistar
Human, WBCs
Human, WBCs
Test conditions
Single dose by gavage; urine and feces
collected 0-24, 24-48, and 48-72 hrs
posttreatment; urine and extracts of
feces tested in S. typhimurium TA100
strain with or without S9 mix and (3-
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.
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.
Results
+
+
+
Dose
0, 1, 5, 10, or
100 mg/kg
Comment
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 (3-glucuronidase ±
S9 mix.
Percentages of subjects with
adduct levels > 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.
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 NER
capacity.
Reference
Willems et
al., 1991
Pavanello
et al., 1999
Pavanello
et al., 2005
This document is a draft for review purposes only and does not constitute Agency policy.
B-6 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
BPDE-
DNA
adducts
BPDE-
DNA
adducts
Test system
Human, peripheral
lymphocytes
Human, maternal and
umbilical cord blood
Test conditions
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 nonsmoking pregnant
women exposed to emissions from
fires during the 4 weeks following the
collapse of the World Trade Center
(WTC) building in New York City on
09/11/2001.
Results
+
+
Dose
Comment
Forty-two percent of the
participants had elevated anti-
BPDE-DNAadduct 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/year), and longtime
periods spent outdoors (>4
versus <4 hours/day) 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
et al., 2006
Perera et
al., 2005a
This document is a draft for review purposes only and does not constitute Agency policy.
B-7 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
Test system
Human, WBCs
Human, WBCs
Mouse, /ocZtransgenic
(Muta™Mouse)
Mouse, (Ahr+/+, Ahr+/~,
Ahr'~)
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; 4
organs analyzed for DNA adducts using
32P-postlabeling with nuclease PI
digestion enrichment.
Gavage; sacrificed 24 hrs
posttreatment.
Results
+
+
+
+
Dose
<0. 15, 0.15-4,
or >4 u.g/m3 of
benzo[a]pyren
e
0.14 u.g/m3
25, 50, and 75
mg/kg-day
100 mg/kg
Comment
PAH exposure, CYP1A1 status
and smoking significantly
affected DNA adduct levels,
i.e., CYPlAl(*l/*2 or *2A/*2a)
> CYP1A1*1/*1; occupational >
environmental exposure;
smokers > nonsmokers;
adducts increased with dose
and duration of smoking.
Median detectable BPDE-DNA
adducts in workers vs. controls
not significant due to low
number of subjects (9 workers,
26 controls); 4/9 workers had
adducts substantially higher
than all controls. No significant
difference between smokers
and nonsmokers; no
correlation with air
benzo[a]pyrene levels and
adduct levels.
Highest adduct levels observed
in liver, followed by glandular
stomach, small intestine, and
bone marrow
No induction of CYP in Ahr'7',
but all alleles positive for
adduct formation.
Reference
Rojas et
al., 2000
Mensing et
al., 2005
Lemieux et
al., 2011
Sagredo et
al., 2006
This document is a draft for review purposes only and does not constitute Agency policy.
B-8 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
Test system
Mouse, C57BL/6J
Cyplal(+/-) and
Cyplal (-/-)
Mouse, B6C3Fi
Mouse, BALB/c
Mouse, BALB/cAnN
(BALB), CBA/JN (CBA);
[32P]-postlabeling assay
Mouse, BALB/c, skin
Test conditions
Single i.p. injection; sacrificed 24 hrs
posttreatment; liver DNA analyzed by
[32P]-postlabeling assay.
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.
Results
+
+
+
+
+
Dose
500 mg/kg
5 ppm (32
wks) and 100
ppm (4 wks)
140 u.Ci/100 g
body weight
50 and
200 mg/kg
4x 1.2 u.mol/
animal
Comment
BPDE-DNAadduct levels
fourfold higher in Cyplal(-f-)
mice than Cyplal(+/-) mice.
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.
Reference
Uno et al.,
2001
Gulp et al.,
2000
Gangar et
al., 2006
Wu etal.,
2008
Reddy et
al., 1984
This document is a draft for review purposes only and does not constitute Agency policy.
B-9 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
BPDE-
DNA
adducts
Test system
Mouse, Swiss,
epidermal and dermal
skin
Rat, CD, peripheral
blood lymphocytes,
lungs, and liver
Rat, Sprague-Dawley,
liver
Rat, Lewis, lung and
liver
Rat, F344;
[32P]-postlabeling assay
Test conditions
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 Pl-endhanced [32P]-
postlabeling assay.
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-oxodG 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.
Results
Dose
250 nmol in
150 ul
acetone
2.5 mg/animal
100 mg/kg
10 mg/kg
0, 5, 50, or
100 mg/kg
Comment
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.
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-
oxodG levels in urine and
decreased levels in liver and
lung.
Adduct levels linear at low and
intermediate doses, nonlinear
at high dose.
Reference
Oueslati et
al., 1992
Ross et al.,
1991
Reddy et
al., 1984
Briede et
al., 2004
Ramesh
and
Knuckels,
2006
This document is a draft for review purposes only and does not constitute Agency policy.
B-10 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
BPDE-
DNA
adducts
CAs
CAs
CAs
Test system
Rat, Wistar; liver and
peripheral blood
lymphocyte adducts
Mouse, C57 (high AHH
inducible) and DBA
(lowAHHinducible)
strains; 11-d-old
embryos; adult bone
marrows
Mouse, 1C3F1 hybrid
(101/ElxC31xEl)Fl;
CAs in bone marrow
Rat, Wistar; peripheral
blood lymphocytes
Test conditions
Single dose by gavage; sacrificed 24 hrs
post-dosing; peripheral blood
lymphocytes and liver DNA analyzed
by [32P]-postlabelingfor BP-DNA
adducts.
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
of post-dosing; 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.
Results
+
+
+
-
Dose
0, 10, or
100 mg/kg
150 mg/kg
63 mg/kg
0, 10, 100, or
200 mg/kg
Comment
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.
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 BP-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.
Reference
Willems et
al., 1991
Adler et
al., 1989
Adler and
Ingwersen,
1989
Willems et
al., 1991
This document is a draft for review purposes only and does not constitute Agency policy.
B-ll DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
CAs
MN
MN
MN
MN
MN
MN
Test system
Hamster; bone
marrow
Mouse, /ocZtransgenic
(Muta™Mouse)
Mouse, B6C3Fi
(hybrid)
Mouse, CD-I and
BDF1; bone marrow
Mouse, CD-I and
BDF1, peripheral blood
reticulocytes
Mouse, ICR[Hsd:
(ICR)Br]
Mouse, Swiss albino;
bone marrow
Test conditions
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; % of PCEs and NCEs
reported.
i.p. injection; several doses given to
calculate LD50.
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.
Results
+
+
+
+
+
+
+
Dose
25, 50, or
100 mg/kg
25, 50, and 75
mg/kg-day
232 mg/kg
(LD50/7);
259 mg/kg
(LD50/4)
250, 500,
1,000, or
2,000 mg/kg
62.5, 125, 250,
or 500 mg/kg
150 mg/kg
75 mg/kg
Comment
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 % PCEs
and NCEs at all doses.
Study conducted to determine
the toxicity of benzo[a]pyrene
(LD50).
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.
Reference
Bayer,
1978
Lemieux et
al., 2011
Salamone
etal., 1981
Shimada et
al., 1990
Shimada et
al., 1992
Harper et
al., 1989
Koratkar et
al., 1993
This document is a draft for review purposes only and does not constitute Agency policy.
B-12 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
Endpoint
MN
MN
MN
MN
MN
MN
MN
MN
Test system
Mouse, Swiss; bone
marrow polychromatic
erythrocytes
Mouse, CD-I and
MS/Ae strains
Mouse, BDF1, bone
marrow
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
Test conditions
Given by gavage and sacrificed 36 hrs
posttreatment.
i.p. and p.o. 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
polychromatic erythrocytes.
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
3d.
Results
+
+
+
+
+
+
+
+
Dose
75 mg/kg
62.5, 125, 250,
or 500 mg/kg
0, 25, 50, or
60 mg/kg
0.5, 5, 50, 100,
or
500 mg/mous
e
0.4, 1, 2, or
4mg
62.5, 125, 250,
500, or
1,000 mg/kg
25 mg/kg
Comment
Good dose response by both
routes, strains; i.p. better than
P.O.; 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.
Exposure to sunlight simulator
to evaluate photogenotoxicity
and chemical exposure.
Maximum response seen at 72
hrs posttreatment.
Reference
Rao and
Nandan,
1990
Awogi and
Sato, 1989
Balansky et
al., 1994
He and
Baker,
1991
Nishikawa
et al., 2005
Hara et al.,
2007
Shimada et
al., 1992
De Flora et
al., 1991
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Endpoint
MN
MN
MN
DNA
strand
breaks
DNA
strand
breaks
DNA
strand
breaks
Test system
Rat, Sprague-Dawley,
bone marrow cells
Hamster; bone marrow
Fish (carp, rainbow
trout, clams); blood
and hemolymph
Rat, Sprague-Dawley;
comet assay
Aquatic organisms:
carp (Cyprinus carpio),
rainbow trout
(Oncorhynchus my kiss),
and clams (Spisula
sachalinensis); Comet
assay
Rat, Brown Norway
Test conditions
Intratracheal instillation, once/day for
3d.
Single, i.p. injection of benzo[a]pyrene
dissolved in tricaprylin; animals
sacrificed 30 hours post-exposure.
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, and
lymphocytes, 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.
Results
-
-
+
+
+
-
Dose
25 mg/kg
100, 300, or
500 mg/kg
0.05, 0.25, 0.5,
orl ppm
Experiment
#1: 3mgof
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
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.
Reference
De Flora et
al., 1991
Bayer,
1978
Kim and
Hyun, 2006
Garry et
al., 2003a,
b
Kim and
Hyun, 2006
Mullaart et
al., 1989
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Endpoint
UDS
UDS
UDS
UDS
UDS
SCEs
Test system
Rat, F344
Mouse, HOS:HR-1
hairless; skin
Rat, Brown Norway;
liver
Mouse, (C3Hfxl01)Fl
hybrid, germ cells
Mouse, early
spermatid
Hamster; SCEs in bone
marrow
Test conditions
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.
Single topical application on two spots
on the backs after stripping stratum
corneum with adhesive tape to
enhance penetration; sacrificed 24 hr
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.
Results
+
—
—
+
Dose
100 mg/kg
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
Comment
Benzo[a]pyrene was negative
at both time points.
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.
Reference
Mirsalis et
al., 1982
Mori et al.,
1999
Mullaart et
al., 1989
Sega, 1979
Sega, 1982
Roszinsky-
Kocher et
al., 1979
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Endpoint
SCEs
SCEs
SCEs
SCEs
SCEs
SCEs
Test system
Hamster
Hamster; fetal liver
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
Test conditions
Animals implanted s.c. with BrdU
tablet; 2 hrs later given phorone (125
or 250 mg/kg) i.p.; another 2 hrs later
dosed i.p. with benzo[a]pyrene; 24 hrs
post-BrdU dosing, animals injected
with colchicine 10 mg/kg body weight,
sacrificed 2 hrs later; bone marrow
from femur prepared for SCE assay.
i.p. injection to pregnant animals on
GDs 11, 13, or 15; fetal liver SCEs were
analyzed.
NA
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
48 hrs with 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.
Results
+
+
+
+
+
+
Dose
50 or
100 mg/kg
50 and
125 mg/kg
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
Comment
SCEs increased with low dose
of phorone significantly.
Produced doubling of SCE
frequency.
Frequency of SCEs increased
>40 mg/kg body weight
SCEs and BP-DNA adducts in
the order of C57BI/6(AHH-
inducible) < DBA/2 (AHH-
noninducible).
SCEs and BP-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.
Reference
Bayer et
al., 1981
Pereira et
al., 1982
Bayer,
1978
Wielgosz
etal., 1991
Wielgosz
etal., 1991
Willems et
al., 1991
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Endpoint
Mutation
Mutation
Mutation
Mutation
Mutation
Mutation
Test system
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
D. melanogaster,
Canton-S (WT) males,
FM6 (homozygousfor
an X chromosome)
females; sex-linked
recessive lethal test
D. melanogaster;
somatic mutation, eye
color mosaicism
Test conditions
Base males exposed to benzo[a]pyrene
were mated with virgin females of
Berlin K or me/-9L1strains.
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.
Adult male flies were fed on filters
soaked in benzo[a]pyrene for 48 or 72
hrs; treated and control males mated
with FM6 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
then discarded and the eggs allowed
to hatch; larvae fed on benzo[a]pyrene
deposited on food surface and the
emerging adult males scored for
mosaic eye sectors.
Results
+
+
-
+
-
+
Dose
10 mM
5 or 7.5 mM
2 or 5 mM
0.1-4 mM
250 or 500
ppm
1, 2, or 3 mM
Comment
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.
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
Vogel et
al., 1983
Vogel et
al., 1983
Zijlstra and
Vogel,
1984
Vogel et
al., 1983
Valencia
and
Houtchens,
1981
Fahmy and
Fahmy,
1980
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Endpoint
Cell trans-
formation
Test system
Hamster, LVG:LAK
strain (virus free);
transplacental host-
mediated assay
Test conditions
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
3 mg/100 g
body weight
Comment
Reference
Quarles et
al., 1979
1
2
CSB = Cockayne syndrome; FM6 = 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; Li = liver; Lu = lung; Sk = skin; DOS = unscheduled DNA synthesis; XPA = xeroderma
pigmentosum group A
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 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 (Bolton et al., 2000; Penning,
6 1999). Benzo[a]pyrene o-quinones reduce the viability and survival of rat and human hepatoma
7 cells (Flowers-Geary et al., 1996,1993). Cytotoxicity was also induced by benzo[a]pyrene and
8 BPDE in a human prostate carcinoma cell line (Nwagbara et al., 2007). Inflammatory responses to
9 cytotoxicity may contribute to the tumor promotion process. For example, benzo[a]pyrene
10 quinones (1,6-, 3,6-, and 6,12-benzo[a]pyrene-quinone) generated ROS and increased cell
11 proliferation by enhancing the epidermal growth factor receptor pathway in cultured breast
12 epithelial cells (Burdick et al., 2 0 0 3).
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, b) exposed Sprague-Dawley rats by inhalation to benzo[a]pyrene with or without ferrous
16 oxide (Fe20s) particles. They found that benzo[a]pyrene alone or in combination with Fe20s
17 particles elicited mRNA and protein synthesis of the inflammatory cytokine, IL-1. Tamaki et al.
18 (2004) also demonstrated a benzo[a]pyrene-induced increase in IL-1 expression in a human
19 fibroblast-like synoviocyte cell line (MH7A). Benzo[a]pyrene increases the expression of the mRNA
20 for CCL1, an inflammatory chemokine, in human macrophages (N'Diaye et al., 2006). The
21 benzo[a]pyrene-induced increase in CCL1 mRNA was inhibited by the potent AhR antagonist,
22 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 (Bostrom et al., 2002). Figure B-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 translocater. 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
*
PAH
Hsp90
Hsp90
ARNT
Enhanced
specific
mRNA
production
•^
IB
^r
AHR
ARNT
AHREDNA
Increased
synthesis of
PAH metabolizing
enzymes
Increased
synthesis of
proteins that
regulate cell
differentiation and
proliferation
AHREoNA = Ah-responsive elements of DNA; ARNT = Ah receptor nuclear translocater; Hsp90 = heat
shock protein 90
Source: Okey etal. (1994).
Figure B-3. Interaction of PAHs with the AhR.
Binding to the AhR induces enzymes that increase the formation of reactive metabolites,
resulting in DNA binding and, eventually, tumor initiation. In addition, with persistent exposure,
the ligand-activated AhR triggers epithelial hyperplasia, which provides the second step leading
from tumor initiation to promotion and progression (Nebertetal., 1993). Ma and Lu (2007)
reviewed several studies of benzo[a]pyrene toxicity and tumorigenicity in mouse strains with high
and low affinity AhRs. Disparities were observed in the tumor pattern and toxicity of
Ah-responsive (+/+ and +/-) and Ah-nonresponsive (-/-) mice. Ah-responsive mice were more
susceptible to toxicity and tumorigenicity in proximal target tissues such as the liver, lung, and skin.
For example, Shimizu etal. (2000) reported that AhR knock-out mice (-/-), treated with
benzo[a]pyrene by s.c. injection or dermal painting, did not develop skin cancers at the treatment
site, while AhR-responsive (+/+) or heterozygous (+/-) mice developed tumors within 18-25 weeks
after treatment Benzo[a]pyrene treatment increased CYP1A1 expression in the skin and liver of
AhR-positive mice (+/- or +/+), but CYP1A1 expression was not altered by benzo[a]pyrene
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Toxicological Review ofbenzo[a]pyrene
1 treatment in AhR knock-out mice (-/-)• Talaskaetal. (2006) also showed that benzo[a]pyrene
2 adduct levels in skin were reduced by 50% in CYP1A2 knock-out mice and by 90% in AhRknock-
3 out mice compared with WT C57B16/J mice following a single dermal application of 33 mg/kg
4 benzo[a]pyrene for 24 hours. Ma and Lu (2007) further noted that Ah-nonresponsive mice were at
5 greater risk of toxicity and tumorigenicity in remote organs, distant from the site of exposure (i.e.,
6 bone marrow). As an example, Uno et al. (2006) showed thatbenzo[a]pyrene (125 mg/kg-day, p.o.
7 for 18 days) caused marked wasting, immunosuppression, and bone marrow hypocellularity in
8 CYP1A1 knock-out mice, but not in WT mice.
9 Some studies have demonstrated the formation of DNA adducts in the liver of AhR knock-
10 out mice following i.p. or oral exposure to benzo[ajpyrene (Sagredo etal., 2006; Uno etal., 2006;
11 Kondraganti et al., 2003). These findings suggest that there may be alternative (i.e., non-AhR
12 mediated) mechanisms of benzo [a] pyrene activation in the mouse liver. Sagredo etal. (2006)
13 studied the relationship between the AhR genotype and GYP metabolism in different organs of the
14 mouse. AhR+/+, +/~, and -/- mice were treated once with 100 mg/kg benzo[a]pyrene by gavage.
15 CYP1A1, CYP1B1, and AhR expression was evaluated in the lung, liver, spleen, kidney, heart, and
16 blood, via real-time or reverse transcriptase polymerase chain reaction, 24 hours after treatment.
17 CYP1A1 RNA was increased in the lung and liver and CYP1B1 RNA was increased in the lung
18 following benzo[a]pyrene treatment in AhR+/+ and+/- mice (generally higher in heterozygotes).
19 Benzo [a]pyrene treatment did not induce CYP1A1 or CYP1B1 enzymes in AhR-/- mice. The
20 expression of CYP1A1 RNA, as standardized to (3-actin expression, was generally about 40 times
21 that of CYP1B1. The concentration of benzo[a]pyrene metabolites and the levels of DNA and
22 protein adducts were increased in mice lacking the AhR, suggesting that there may be an
23 AhR-independent pathway for benzo[a]pyrene metabolism and activation. The high levels of
24 benzo [ajpyrene DNA adducts in organs other than the liver of AhR-/- mice may be the result of slow
25 detoxification of benzo[a]pyrene in the liver, allowing high concentrations of the parent compound
26 to reach distant tissues.
27 Uno et al. (2006) also demonstrated a paradoxical increase in liver DNA adducts in AhR
28 knock-out mice following oral exposure to benzo[ajpyrene. WT C57BL/6 mice and several knock-
29 out mouse strains (CYP1A2-/- and CYP1B1-/- single knock-out, CYP1A1/1B1-/- and CYP1A2/1B1-/-
30 double knock-out) were studied. Benzo[ajpyrene was administered in the feed at 1.25,12.5, or 125
31 mg/kg for 18 days (this dose is well tolerated by WT C57BL/6 mice for 1 year, but lethal within 30
32 days to the CYP1A1-/- mice). Steady-state blood levels of benzo[a]pyrene, reached within 5 days of
33 treatment, were -25 times higher in CYP1A1-/- and -75 times higher in CYP1A1/1B1-/- than in WT
34 mice, while clearance was similar to WT mice in the other knock-out mouse strains. DNA adduct
35 levels, measured by [32P]-postlabeling in liver, spleen, and bone marrow, were highest in the
36 CYP1A1-/- mice at the two higher doses, and in the CYP1A1/1B1-/- mice at the mid dose only.
37 Adduct patterns, as revealed by 2-dimensional chromatography, differed substantially between
3 8 organs in the various knock-out types.
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Toxicological Review ofbenzo[a]pyrene
1 Dertinger etal. (2001, 2000) demonstrated that AhR signaling may play a role in
2 cytogenetic damage caused by benzo[a]pyrene. The in vivo formation of MN in peripheral blood
3 reticulocytes of C57B1/6J mice induced by a single i.p. injection of benzo[a]pyrene (150 mg/kg) was
4 eliminated by prior treatment with the potent AhR antagonist 3'-methoxy-4'-nitroflavone. This
5 antagonist also protected AhR null allele mice from benzo[a]pyrene-induced increases in MN
6 formation, suggesting that 3'-methoxy-4'-nitroflavone may also act through a mechanism
7 independent of the AhR (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 et al., 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 et al. (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 4-1), 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 (GJIC)
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 (Sharovskaya et al., 2006;
35 Yamasaki, 1990).
36 Blaha et al. (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
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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).
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Toxicological Review ofbenzo[a]pyrene
i APPENDIX C. 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. Except where other software is
7 noted, all endpoints were modeled using the U.S. EPA's Benchmark Dose Software (BMDS; U.S. EPA,
8 2012; version 2.0 or later). The preambles for the cancer and non-cancer parts below describe the
9 common practices used in evaluating the model fit and selecting the appropriate model for
10 determining the POD, as outlined in the draft Benchmark Dose Technical Guidance Document (U.S.
11 EPA, 2000).
12 DOSE-RESPONSE MODELING FOR DERVIATION OF RFD
13 Evaluation of Model Fit
14 For each dichotomous endpoint, BMDS dichotomous models were fitted to the data using
15 the maximum likelihood method. Each model was tested for goodness-of-fit using a chi-square
16 goodness-of-fit test (x2 p-value < 0.10 indicates lack of fit). Other factors were also used to assess
17 model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the
18 vicinity of the BMR.
19 For each continuous endpoint, BMDS continuous models were fitted to the data using the
20 maximum likelihood method. Model fit was assessed by a series of tests as follows. For each model,
21 first the homogeneity of the variances was tested using a likelihood ratio test (BMDS Test 2). If Test
22 2 was not rejected (x2 p-value > 0.10), the model was fitted to the data assuming constant variance.
23 If Test 2 was rejected (x2 p-value < 0.10), the variance was modeled as a power function of the
24 mean, and the variance model was tested for adequacy of fit using a likelihood ratio test (BMDS
25 Test 3). For fitting models using either constant variance or modeled variance, models for the mean
26 response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 4, with x2 p-value <
27 0.10 indicating inadequate fit). Other factors were also used to assess the model fit, such as scaled
28 residuals, visual fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.
29 Model Selection
30 For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as
31 estimated by the profile likelihood method) and AIC value were used to select a best-fit model from
32 among the models exhibiting adequate fit. If the BMDL estimates were "sufficiently close," that is,
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Toxicological Review ofbenzo[a]pyrene
1 differed by at most threefold, the model selected was the one that yielded the lowest AIC value. If
2 the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the POD.
3 Decreased thvmus weight, males fKroese et al, 20011
4
5
Table C-l. Means ± SDa for thymus weight in male Wistar rats exposed
to benzo[a]pyrene by gavage 5 days/week for 90 days
Organ
Thymus weight
(mg), males
Dose (mg/kg-d)
0
380 ± 60
3
380 ± 110
10
330 ± 60
30
270 ± 40b
6
7
aReported as SE, but judged to be SD (and confirmed by study authors).
Significantly (p < 0.05) different from control mean; student t-test (unpaired, two-tailed); n
10/sex/group.
Table C-2. Model predictions for decreased thymus weight in male
Wistar rats—90 days
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
Hill"
Linear, Polynomial (2-
degree), Power"
Insufficient degrees of freedom
0.30
0.23
380.71
16.40
11.30
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Toxicological Review ofbenzo[a]pyrene
Linear Model with 0.95 Confidence Level
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450
400
350
300
250
Linear
BMDL
BMD
10
dose
15
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Figure C-l. Fit of linear model (nonconstant variance) to data on
decreased thymus weight in male Wistar rats—90 days.
BMDs and BMDLs indicated are associated with a change of 1 SD from the
control, and are in units of mg/kg-day.
d)
BMDS Model Run
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
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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 0.288754
1.38062 7.94967
346.558 411.351
-7.11189 -3.17249
Model Descriptions for likelihoods calculated
Likelihoods of Interest
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Model Log (likelihood) #
Al -189.116991
A2 -183.673279
A3 -184.883626
fitted -186.353541
R -196.353362
Explanation of Tests
Param's AIC
5 388.233982
8 383.346558
6 381.767253
4 380.707081
2 396.706723
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (Al v
s 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 25.3602 6
Test 2 10.8874 3
Test 3 2.42069 2
Test 4 2.93983 2
The p-value for Test 1 is less than .05.
difference between response and/or varianc
It seems appropriate to model the data
The p-value for Test 2 is less than .1. A
model appears to be appropriate
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 = 16.4008
BMDL = 11.2965
(A3 vs. fitted)
and Test 2 will be the same.)
df p-value
0.0002928
0. 01235
0.2981
0.2299
There appears to be a
es among the dose levels
non-homogeneous variance
The modeled variance appears
The model chosen seems
deviations from the control mean
53
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 Decreased thvmus weight, females CKroese et al. 20011
2
3
Table C-3. Means ± SDa for thymus weight in female Wistar rats
exposed to benzo[a]pyrene by gavage 5 days/week for 90 days
Organ
Thymus weight
(mg) - Females
Dose (mg/kg-d)
0
320 ± 60
3
310 ±50
10
300 ± 40
30
230±30b
4
5
aReported as SE, but judged to be SD (and confirmed by study authors).
Significantly (p < 0.05) different from control mean; student t-test (unpaired, two-tailed); n =
10/sex/group.
Table C-4. Model predictions for decreased thymus weight in female
Wistar rats—90 days
Model (constant variance)
Hillb
Linear0
Polynomial (2-degree)c'd
Powerb
Variance
p-valuea
Mean
p- value3
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
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bPower restricted to >1.
Coefficients restricted to be negative.
dLowest degree polynomial with an adequate fit is reported.
BMD/BMC = maximum likelihood estimate (MLE) of the dose/concentration associated with the
selected BMR; NA = not applicable; model failed to generate
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Linear Model with 0.95 Confidence Level
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340
320
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BMDL
BMD
0
10
dose
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BMDs and BMDLs indicated are associated with a change of 1 SD from the
control, and are in units of mg/kg-day.
Figure C-2. Fit of linear model (constant variance) to data on decreased
thymus weight in female Wistar rats—90 days.
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
^:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\9Oday\thymusweight\female\durationadjusted\2Linkrolin
. (d)
Gnuplot Plotting File:
^:\USEPA\IRIS\benzo[a]pyrene\RfD\Kroese2001\9Oday\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
Parameter Estimates
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
1098.16 2811.69
303.558 340.73
-5.84334 -2.56026
44.
44.
44.
44.
Model Descriptions for likelihoods calculated
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Likelihoods of Int
Model Log (likelihood)
Al -171.357252
A2 -168.857234
A3 -171.357252
fitted -171.562118
R -181.324151
Explanation of Tests
erest
# Param's AIC
5 352.714504
8 353.714467
5 352.714504
3 349.124237
2 366.648303
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 24.9338 6
Test 2 5. 00004 3
Test 3 5.00004 3
Test 4 0.409733 2
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 = 10.5228
BMDL = 7.64037
? (A3 vs. fitted)
and Test 2 will be the same.)
df p-value
0.0003512
0.1718
0.1718
0. 8148
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
58
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 Decreased ovary weight—female rats. 60 days (Xu et al, 2010)
2 Table C-5. Means ± SDs for ovary weight in female Sprague-Dawley rats
Organ
Ovary weight (mg)
Dose (mg/kg-d)a
0
0.160 ±0.0146
2.5
0.143 ±0.0098b
5
0.136 ±0.0098b
aTWA doses over the 60-day study period.
Statistically different (p < 0.05) from controls using one-way ANOVA.
Table C-6. Model predictions for decreased ovary weight in female
Sprague-Dawley rats
Model
Power
Linear, Polynomial (1°)
Goodness-of-fit
p-value
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
NA
0.39
-138.67
2.27
1.49
NA = not applicable; model failed to generate
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Linear Model with 0.95 Confidence Level
0.1
0.1
0.13
Linear
16:0312/142010
BMDL
BMD
2 3
dose
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Figure C-3. Fit of linear/polynomial (1°) model to data on decreased
ovary weight.
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
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
This document is a draft for review purposes only and does not constitute Agency policy.
C-ll DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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Default Initial Parameter Values
alpha = 0.000136
rho = 0 Specified
beta_0 = 0.158333
beta 1 = -0.0048
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
4.12162e-005 0.000196562
0.150369 0.166298
-0.00726768 -0.00233232
Model Descriptions for likelihoods calculated
Model
Al
A2
A3
fitted
This document is a draft for review purposes only and does not constitute Agency policy.
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R 67.008505
Explanation of Tests
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 varianc
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
es among the dose levels
A homogeneous variance
The modeled variance appears
The model chosen seems
deviations from the control mean
49
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Toxicological Review ofbenzo[a]pyrene
Morris water maze results—male and female Sprague-Dawley rats. Chen et al. (2012)
Data from Morris water maze was presented graphically in Chen et al., 2012, but dose group
means and standard deviations 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 ANOVA, and allowing for interactions),
and because there was no rationale or information available suggesting there would be sex-
mediated differences for these neurologic tests.
Table C-7. Means ± SDs for Escape Latency and Time Spent in Target
Quadrant
Test
Escape latency
(sec)
Time spent in
target quadrant
(sec)
Dose (mg/kg-d)
0
9.89 ±5. 76
33.6 ±8.92
0.02
12.5 ±5. 10
31.9 ±8.63
0.2
19.1 ±5. 85
16.6 ±5.74
2.0
33. 5 ±9.93
11.1 ±5. 12
11
12
Table C-8. Model predictions for increase in Morris water maze test for
escape latency, male and female rats
Model3
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
a Includes modeling of heterogeneous variances (BMDS Test 3, p = 0.313).
b Power parameter n was estimated to be 1 (boundary of parameter space).
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Hill Model with 0.95 Confidence Level
Figure C-4. Fit of Hill model to data on Morris water maze test escape
latency.
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Hill Model. (Version: 2.16; Date: 04/06/2011)
Input Data File: C:\Documents and Settings\jfox\My Documents\_CURRENTWORK\_CAST
plus\BaP\BMDS\hil_Chen.FM.latency_Hil-ModelVariance-BMRlStd-Restrict.(d)
Gnuplot Plotting File: C:\Documents and Settings\jfox\My Documents\_CURRENTWORK\_CAST
plus\BaP\BMDS\hil_Chen.FM.latency_Hil-ModelVariance-BMRlStd-Restrict.plt
Tue Apr 24 14:41:26 2012
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
This document is a draft for review purposes only and does not constitute Agency policy.
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95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-1.0229
0.33391
8.86283
20.9783
Variable
lalpha
rho
intercept
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
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
Model
Al
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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Q
o
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A2 -186.795503
A3 -187.957975
fitted -188.169983
R -234.549118
Explanation of Tests
8 389.591006
6 387.915949
5 386.339965
2 473.098237
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 95.5072 6
Test 2 12.008 3
Test 3 2.32494 2
Test 4 0.424016 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 less than .1.
model appears to be appropriate
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 = 0. 106284
BMDL = 0.0609511
? (A3 vs. fitted)
and Test 2 will be the same.)
df p-value
<.0001
0. 007356
0.3127
0.5149
There appears to be a
ces among the dose levels
A non-homogeneous variance
The modeled variance appears
The model chosen seems
deviations from the control mean
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Table C-9. Model predictions for decrease in Morris water maze test for
time spent in target quadrant, male and female rats
Model3
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
BMDisD
(mg/kg-d)
0.065
0.084
0.071
1.23
BMDL1SD
(mg/kg-d)
0.043
0.044
0.038
0.98
' Includes modeling of heterogenous variances (BMDS Test 3, p = 0.919).
5 NA: insufficient degrees of freedom to evaluate chi-square.
Exponential Model 4 with 0.95 Confidence Level
40
35
30
25
20
15
10
Exponential
BMDL BMD
0.5
1
dose
1.5
14:3504/242012
5
6
I?
;2
3
:4
6
:s
9
20
21
22
23
Figure C-5. Fit of Exponential 4 model to data on Morris water maze
time spent in target quadrant.
Exponential Model. (Version: 1.7; Date: 12/10/2009)
Input Data File: C:\Documents and Settings\...\exp_Chen.FM.target_Exp-ModelVariance-
BMRlStd-Down.(d) ~ ~
BMDS Model Run
Note: Y[dose] is the median response for exposure = dose;
sign = +1 for increasing trend in data;
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 sign = -1 for decreasing trend.
2
3 Model 2 is nested within Models 3 and 4.
4 Model 3 is nested within Model 5.
5 Model 4 is nested within Model 5.
8 Dependent variable = Mean
9 Independent variable = Dose
0 Data are assumed to be distributed: normally
1 Variance Model: exp(lnalpha +rho *ln(Y[dose]))
2 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i))
4 Total number of dose groups = 4
5 Total number of records with missing values = 0
6 Maximum number of iterations = 250
7 Relative Function Convergence has been set to: le-008
8 Parameter Convergence has been set to: le-008
20 MLE solution provided: Exact
21
24
25
26
27
28
29
30
31
32
33
34
35
36 Parameter Estimates
37
38 Variable Model 4
7
L
49
50 Table of Stats From Input Data
51
52 Dose N Obs Mean Obs Std Dev
54 -- -
55
56
57
58
59
60 Estimated Values of Interest
61
62 Dose Est Mean Est Std Scaled Residual
63
64
65
66
67
68
71
72
73 Model Al: Yij = Mu(i) + e(ij)
74 Var{e(ij)} = SigmaA2
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Toxicological Review ofbenzo[a]pyrene
2
3
4 Model A3: Yij = Mu(i) + e(ij)
5 Var{e(ij)} = exp(lalpha + log(mean(i)
9
0
1 Likelihoods of Interest
2
3 Model Log(likelihood) DF AIC
4
5 Al
6 A2
7 A3
8 R
_9 4
20
21
22 Additive constant for all log-likelihoods = -73.52. This constant added to the
23 above values gives the log-likelihood including the term that does not
24 depend on the model parameters.
25
26
27 Explanation of Tests
28
29
30
31
32
33
34
35
36 Tests of Interest
37
38 Test -2*log (Likelihood Ratio) D. F.
7
8
9
50
51
52
54
55
56
57
58
59
60 Benchmark Dose Computations:
61
62 Specified Effect = 1.000000
63
64 Risk Type = Estimated standard deviations from control
65
66 Confidence Level = 0.950000
67
68 BMD = 0.0650194
9
0 BMDL = 0.0432761
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1
2
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
Cervical epithelial hyperplasia -female ICR mice (Gao et al. 2011)
Table C-10. Incidence of cervical epithelial hyperplasia
Observation
Cervical epithelial hyperplasia
Dose (mg/kg-day)a
0
0/26
0.71
4/26
1.4
6/25
2.9
7/24
a doses converted to mg/kg-day after adjustment for equivalent continuous dosing (2/7
days/week)
Table C-ll. Model predictions for increased incidence of epithelial
hyperplasia in female ICR mice
Model3
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
Logistic Model. (Version: 2.13; Date: 10/28/2009)
Input Data File: C:\Users\hclynch\Documents\_Active Projects\_FA498 IRIS\xBaP\IASC Aug
2011\bmd modeling\lnl_gao 2011 inflamm cells_0pt.(d)
Gnuplot Plotting File: C:\Users\hclynch\Documents\_Active Projects\_FA498
IRIS\xBaP\IASC Aug 2011\bmd modeling\lnl_gao 2011 inflamm cells Opt.pit
BMDS Model Run
User has chosen the log transformed model
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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
—* ^x
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
Asymptotic Correlation Matrix of Parameter Estimates
( ***
int
intercept
Variable
background
intercept
slope
The model parameter (s) -background -slope
have been estimated at a boundary point, or have been specified by the user
and do not appear in the correlation matrix )
ercept
1
Parameter Estimates
95.0% Wald Confidence Interval
Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
0 * * *
-1.6502 * * *
1 * * *
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Dose Est
0.0000 0.
0 . 7100 0 .
1.4000 0.
2 . 9000 0 .
ChiA2 = 0.86
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-39.4267 4
-39.8502 1 0.847034 3 0.8382
-45.7739 1 12.6945 3 0.005346
81.7004
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 26 0.000
1200 3.119 4.000 26 0.532
2119 5.297 6.000 25 0.344
3577 8.584 7.000 24 -0.675
d.f. = 3 P-value = 0.8360
Computation
0.1
= Extra risk
0. 95
0.578668
0.368701
59
60
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
INHALATION DOSIMETRY MODELING FOR RFC DERIVATION
Wed, 03/17/2010. 02:07:20 PM EOT
Region: Entire Lung
0.750
0.600
« 0.450
LJ_
E
O
S 0.300
Q
0.150
0.0
0.621
0.449
Species & Model Info:
Species/Geometry: Human Limited
FRC Volume: 3300.00 ml
Head Volume: 50.00 ml
Breathing Route: nasal
Breathing Parameters:
Tidal Volume: 860.00 nil
Breathing Frequency: 16.00 1/min
Inspiratory Fraction: 0.50
Pause Fraction: 0.00
Particle Properties:
Diameter: MVIAD: 1.70 urn
OSD: 1.00
Concentration: 4.20 ug/m'S
4 Figure C-6. Human fractional deposition.
5 Species = humanlimited
6 FRC = 3300.0
7 Head volume = 50.0
8 Density =1.0
9 Number of particles calculated = single
10 Diameter = 1.7000000000000002 urn MMAD
11 Inhalability = yes
12 GSD =1.0
13 Breathing interval: One single breath
14 Concentration =4.2
15 Breathing Frequency = 16.0
16 Tidal Volume = 860.0
17 Inspiratory Fraction = 0.5
18 Pause Fraction = 0.0
19 Breathing Route = nasal
20
21 Region: Entire Lung
22 Region: Entire Lung
23 Region Deposition Fraction
24 —
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 Head 0.449
2 TB 0.045
3 P 0.127
4 Total 0.621
Wed, 03/17/2010, 02:15:27 PM EOT
Region: Entire Lung
0.250 i-
0.200
0.150
sz
o
£ 0.100
0.050
0.0
0.181
Head TB
Total
Region
Species 6 Model Info:
Species/Geometry: Rat
FRC Vblume: 4.00 ml
Head Vblume: 0.42 ml
Breathing Route: nasal
Breathing Parameters:
Tidal Vblume: 1.80 ml
Breathing Frequency: 102.00 1/rnin
Inspiratory Fraction: 0.50
Pause Fraction: 0.00
Particle Properties:
Diameter: MMAD: 1.70 urn
GSD: 1.00
Concentration: 4.20 ugAri"3
8 Figure C-7. Rat fractional deposition.
9 Species = rat
10 FRC =4.0
11 Head volume =0.42
12 Density =1.0
13 Number of particles calculated = single
14 Diameter = 1.7000000000000002 urn MMAD
15 Inhalability = yes
16 GSD =1.0
17 Breathing interval: One single breath
18 Concentration = 4.2
19 Breathing Frequency = 102.0
20 Tidal Volume =1.8
21 Inspiratory Fraction = 0.5
22 Pause Fraction = 0.0
23 Breathing Route = nasal
24
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1
2
3
4
5
6
7
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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Toxicological Review ofbenzo[a]pyrene
1 DOSE-RESPONSE MODELING FOR CANCER RISK VALUES
2 The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend thatthe
3 method used to characterize and quantify cancer risk from a chemical is determined by what is
4 known about the mode of action of the carcinogen and the shape of the cancer dose-response curve.
5 No biologically based models for BaP carcinogenicity following oral, inhalation, or dermal exposure
6 were identified.
7 Methods for the Oral Slope Factor and Inhalation Unit Risk
8 Due to the occurrence of multiple tumor types, earlier occurrence with increasing exposure,
9 and early termination of the high-dose group in each of the oral and inhalation carcinogenicity
10 studies (see Appendix B for study details), methods that can reflect the influence of competing risks
11 and intercurrent mortality on site-specific tumor incidence rates are preferred. EPA has generally
12 used a model that incorporates the time at which death-with-tumor occurred as well as the dose;
13 the multistage-Weibull model is multistage in dose and Weibull in time, and has the form:
14
15 P(d, t) = l- exp[-(q0 + qid + q2tf +... + q*d*) x(t± t0)c],
16
17 where P(d, t) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent
18 exposure in this case) and age t (in bioassay weeks); parameters qt > 0, for i = 0,1,..., k; t is the time
19 at which the tumor was observed; and c is a parameter which characterizes the change in response
20 with age. The parameter to represents the time between when a potentially fatal tumor becomes
21 observable and when it causes death, and is generally set to 0 either when all tumors are
22 considered incidental or because of a lack of data to estimate the time reliably. The dose-response
23 analyses were conducted using the computer software program MultiStage-Weibull (U.S. EPA,
24 2010), which is based on Weibull models drawn from Krewskietal. (1983). Parameters were
25 estimated using the method of maximum likelihood. From specific model fits using stages up to n-1,
26 where n is the number of dose groups, the model fit with the lowest AIC was selected.
27 Two general characteristics of the observed tumor types were considered prior to
28 modeling; allowance for different, although unidentified modes of action, and allowance for relative
29 severity of tumor types. First, etiologically different tumor types were not combined across sites
30 prior to modeling (that is, overall counts of tumor-bearing animals were not tabulated) in order to
31 allow for the possibility that different tumor types could have different dose-response relationships
32 due to different underlying mechanisms or factors, such as latency. Consequently, all of the tumor
33 types were also modeled separately.
34 Additionally, the multistage-Weibull model can address relative severity of tumor types by
3 5 distinguishing between tumors as being either fatal or incidental to the death of an animal in order
36 to adjust partially for competing risks. In contrast to fatal tumors, incidental tumors are those
37 tumors thought not to have caused the death of an animal. Cause-of-death information for most
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 early animal deaths was provided by the investigators of both of the bioassays. In the rat study,
2 tumors of the forestomach or liver were the principal cause of death for most animals dying or
3 sacrificed (due to moribundity) before the end of the study, while tumors of the forestomach were
4 the most common cause of early deaths in the mouse study.
5 PODs for estimating low-dose risk were identified at doses at the lower end of the observed
6 data, generally corresponding to 10% extra risk, where extra risk is defined as [P(d) - P(0)]/[l -
7 P(0)]. The lifetime oral cancer slope factor for humans is defined as the slope of the line from the
8 lower 95% bound on the exposure at the POD to the control response (slope factor = 0.1/BMDLio).
9 This slope, a 95% upper confidence limit (UCL) represents a plausible upper bound on the true risk.
10 Although the time-to-tumor modeling helps account for competing risks associated with
11 decreased survival times and other tumors, considering the tumor sites individually still does not
12 convey the total amount of risk potentially arising from the sensitivity of multiple sites—that is, the
13 risk of developing any combination of the increased tumor types, not just the risk of developing all
14 simultaneously. One approach suggested in the Guidelines for Carcinogen Risk Assessment (U.S. EPA,
15 2005a) would be to estimate cancer risk from tumor-bearing animals. EPA traditionally used this
16 approach until the National Resource Council (NRC) document Science and Judgment (NRC, 1994)
17 made a case that this approach would tend to underestimate overall risk when tumor types occur in
18 a statistically independent manner. In addition, application of one model to a composite data set
19 does not accommodate biologically relevant information that may vary across sites or may only be
20 available for a subset of sites. For instance, the time courses of the multiple tumor types evaluated
21 varied, as is suggested by the variation in estimates of c, from 1.5 (e.g., male rat skin or mammary
22 gland basal cell tumors), indicating relatively little effect of age on tumor incidence, to 3.7 (e.g., male
23 mouse alimentary tract tumors), indicating a more rapidly increasing response with increasing age
24 (in addition to exposure level). The result of fitting a model with parameters that can reflect
25 underlying mechanisms, such as z in the multistage-Weibull model, would be difficult to interpret
26 with composite data (i.e., counts of tumor-bearing animals). A simpler model, such as the
27 multistage model, could be used for the composite data but relevant biological information would
28 then be ignored.
29 Following the recommendations of the NRC (1994) regarding combining risk estimates,
30 statistical methods that can accommodate the underlying distribution of slope factors are optimal,
31 such as through maximum likelihood estimation or through bootstrapping or Bayesian analysis.
32 However, these methods have not yet been extended to models such as the multistage-Weibull
33 model. A method involving the assumption that the variability in the slope factors could be
34 characterized by a normal distribution is detailed below (U.S. EPA, 2010). Using the results in
35 female rats to illustrate, the overall risk estimate involved the following steps:
36
37 (1) It was assumed that the tumor groupings modeled above were statistically independent—
3 8 that is, that the occurrence of a liver tumor was not dependent upon whether there was a
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 forestomach tumor. This assumption cannot currently be verified, and if not correct, could
2 lead to an overestimate of risk from summing across tumor sites. However, NRC (1994)
3 argued that a general assumption of statistical independence of tumor-type occurrences
4 within animals was not likely to introduce substantial error in assessing carcinogenic
5 potency from rodent bioassay data.
6
7 (2) The models previously fitted to estimate the BMDs and BMDLs were used to extrapolate to a
8 lower level of risk (R), in order to reach the region of each estimated dose-response
9 function where the slope was reasonably constant and upper bound estimation was still
10 numerically stable. For these data, a 10-3 risk was generally the lowest risk necessary. The
11 oral slope factor for each site was then estimated by R/BMDL.R, as for the estimates for each
12 tumor site above.
13
14 (3) The maximum likelihood estimates (MLE) ofunit potency (thatis, risk per unit of exposure)
15 estimated by R/BMDR, were summed across the alimentary tract, liver, and
16 jejunum/duodenum in female rats.
17
18 (4) An estimate of the 95% (one-sided) upper bound on the summed oral slope factor was
19 calculated by assuming a normal distribution for the individual risk estimates, and deriving
20 the variance of the risk estimate for each tumor site from its 95% UCL according to the
21 formula:
22
23 95% UCL = MLE + 1.645 x SD,
24 rearranged to:
25 s.d. = (UCL - MLE) / 1.645,
26
27 where 1.645 is the t-statistic corresponding to a one-sided 95% CI and >120 degrees of freedom,
28 and the SD is the square root of the variance of the MLE. The variances (variance = SD2) for each
29 site-specific estimate were summed across tumor sites to obtain the variance of the sum of the
30 MLEs. The 95% UCL on the sum of MLEs was calculated from the expression above for the UCL,
31 using the variance of the sum of the MLE to obtain the relevant SD (SD = variance1/2).
32 The results of this analysis are provided in Table C-17.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1
2
Table C-12. Tumor incidence data, with time to death with tumor; male
rats exposed by gavage to benzo[a]pyrene—Kroese et al. (2001)
Dose
(mg/kg-d)
0
3
Week of
death
44
80
82
84
89
90
91
92
93
94
95
96
97
98
100
104
105
108
109
29
40
74
76
79
82
92
93
94
95
98
107
108
109
Total
examined
1
1
1
1
1
3
1
1
1
1
2
2
1
1
3
1
1
7
22
1
1
1
1
1
1
2
1
1
2
1
10
15
14
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
0
1
0
0
0
0
0
0
0
0
0
4
2
1
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
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
0
0
0
0
0
0
0
0
1
3
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
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
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
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
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Dose
(mg/kg-d)
10
30
Week of
death
39
47
63
68
69
77
80
81
84
86
90
95
97
100
102
103
104
107
108
109
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
2
1
2
1
1
1
1
1
1
1
3
1
1
1
1
3
12
11
6
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
0
0
1
2
1
0
0
1
1
0
1
3
1
1
1
1
3
12
11
5
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
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
1
0
1
0
0
1
1
0
0
0
1
0
2
0
0
0
1
0
Liver tumors
Incidental
0
0
0
0
0
1
1
0
0
1
0
2
0
1
1
1
3
11
11
3
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
1
0
0
0
1
0
0
0
0
0
0
0
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
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
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
0
0
0
0
0
0
0
0
1
0
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
0
0
0
0
0
0
0
1
0
0
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
0
0
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
"Incidental" denotes presence of tumors not
tumors reported by the study investigators to
known to have caused death of particular animals.
have caused death of particular animals.
'Fatal" denotes incidence of
This document is a draft for review purposes only and does not constitute Agency policy.
C-30 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
1
2
Table C-13. Tumor incidence data, with time to death with tumor;
female rats exposed by gavage to benzo[a]pyrene—Kroese et al. (2001)
Dose
(mg/kg-d)
0
3
10
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
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
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
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
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
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
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
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
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
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
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
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
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.
C-31 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
Dose
(mg/kg-d)
30
Week of
death
104
105
106
107
108
109
26
44
47
48
54
55
56
57
58
59
60
61
62
63
64
66
67
68
69
71
72
Total
examined
1
2
1
5
7
4
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
1
1
1
5
7
2
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
0
0
0
0
0
0
1
1
1
0
0
0
0
0
1
0
1
1
1
Liver tumors
Incidental
1
1
0
5
7
2
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
0
0
0
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
0
0
0
0
0
0
3
0
0
0
1
0
0
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.
C-32 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
1
2
Table C-14. Tumor incidence, with time to death with tumor; female
mice exposed to benzo[a]pyrene via diet—Beland and Culp (1998)
Dose group
(ppm in diet)
0
5
25
Week of death
31
74
89
91
93
94
97
98
99
100
101
104
105
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
2
1
2
2
2
2
1
2
2
1
29
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
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
1
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.
C-33 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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
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.
1
2
Table C-15. Derivation of HEDs to use for BMD modeling of Wistar rat
tumor incidence data from Kroese et al. (2001)
Benzo[a]pyrene dose (mg/kg-d)
TWA body weight (kg)
Interspecies scaling
factor3
HEDb (mg/kg-d)
Male
3
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
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.
bHED = administered dose x scaling factor.
This document is a draft for review purposes only and does not constitute Agency policy.
C-34 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
1
2
Table C-16. Derivation of HEDs for dose-response modeling of B6C3Fi
female mouse tumor incidence data from Beland and Culp (1998)
Benzo[a]pyrene
dose in diet
(ppm)
5
25
100
Intake (ng/d)
21
104
430
TWA body weight
average (kg)
0.032
0.032
0.027
Administered
dosea (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
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.
C-35 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
1
2
Table C-17. Summary of model selection and modeling results for best-
fitting multistage-Weibull models, using time-to-tumor data for rats
from Kroese etal. (1981)
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: squamouscell
tumors
Oral cavity and
forestomach:
squamous cell 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
Model selection rationale
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
Lowest AIC, 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.
C-36 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1 Male Rat (Kroese et al, 2001): Squamous Cell Papilloma or Carcinoma in Oral Cavity or Forestomach
2
O
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: OralForstKroeseM3.(d)
^
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
22
23
24
25
26
27
28 Default Initial Parameter Values
2.y c = 3.6
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 t o
47
48 beta 1
49
50 beta 3
51
52
53 Parameter Estimates
54 95.0% Wald Confidence Interval
55 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
56 c 3.74559 0.447309 2.86888 4.6223
57 tO 41.4581 2.14975 37.2447 45.6716
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.
C-37 DRAFT—DO NOT CITE OR QUOTE
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1
2
O
4
5
6
8
9
10
11
12
13
14
15
16
17
18
Toxicological Review ofbenzo[a]pyrene
U Total Expected Response
0
Minimum observation time for F tumor context =
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified
effect =
BMD =
BMDL =
BMDU =
44
Incidental Risk: OralForstKroeseM3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00 Dose = 0.54
19
20
OD
CD
OD
CD
CD ~~
CN
CD
CD
I I I I I I
0 20 40 60 80 100
S 1
OD
CD
-------
Toxicological Review ofbenzo[a]pyrene
1 Male Rat (Kroese et al, 2001): Hepatocellular Adenoma or Carcinoma
2
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: LiverKroeseMS.(d)
7
8
9
10
11
12
13
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
23
24
25
26
27
28
29
30 Default Initial Parameter Values
31 c = 3.6
32 t_0 = 34.6667
33 beta_0 = 0
34 beta_l = 2.73535e-009
35 beta_2 = 8.116e-028
36 beta 3 = 1.43532e-008
37
38
39 Asymptotic Correlation Matrix of Parameter Estimates
40 ( *** The model parameter(s) -beta_0 -beta_2
41 have been estimated at a boundary point, or have been specified by the user,
42 and do not appear in the correlation matrix )
43
44
45
46
47
48 t o
49
50 beta 1
51
52 beta 3
53
54
55 Parameter Estimates
56
57 Variable
58
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 -138.544 6
71
72
73
This document is a draft for review purposes only and does not constitute Agency policy.
C-39 DRAFT—DO NOT CITE OR QUOTE
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1
2
O
4
5
6
8
9
10
11
12
13
14
15
16
17
18
Toxicological Review ofbenzo[a]pyrene
Total Expected Response
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified
effect =
BMD =
BMDL =
BMDU =
19
20
Incidental Risk: Hepatocellular_Kroese_M3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00
Dose = 0.54
Probability
oo
o
0 ~
o
o
\
1 1 1 1 1 1
0 20 40 60 80 100
Probability
oo
o
0 ~
o
«-> i i " T i r "i
0 20 40 60 80 100
Time
Time
Dose= 1.81
Dose= 5.17
oo
\ \ I I I T
0 20 40 60 80 100
2
CL
p
O
\ \
0 20 40 60 80 100
Time
Time
This document is a draft for review purposes only and does not constitute Agency policy.
C-40 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1 Male Rat (Kroese et al, 2001): Duodenum or Jejunum Adenocarcinoma
2
o
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: DuoJejKroeseM3.(d)
^
9
10
11
12
13
14
15 Dependent variable = CONTEXT
16 Independent variables = DOSE, TIME
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 = 1
22 Degree of polynomial = 3
24
25
26 User specifies the following parameters:
27 t 0 0
28
29
30
31
32
33
34 Default Initial Parameter Values
35 c 1.63636
36
37
38
39
40
41
42
43 Asymptotic Correlation Matrix of Parameter Estimates
44 ( *** The model parameter(s) -t_0 -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 c beta 3
49
50 c 1-1
51
52 beta 3-11
53
54
55 Parameter Estimates
56 95.0% Wald Confidence Interval
57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
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 -28.4387 5
70
71
72 Data Summary
73 CONTEXT
74 C F I U Total Expected Response
This document is a draft for review purposes only and does not constitute Agency policy.
C-41 DRAFT—DO NOT CITE OR QUOTE
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1
2
O
4
5
6
8
9
10
11
12
13
14
15
16
51
43
Specified effect =
BMD =
BMDL =
BMDU =
Toxicological Review ofbenzo[a]pyrene
Incidental Risk: DuoJej_Kroese_M3
17
18
19
Dose= 0.00
Dose= 0.54
t
la
ro
-Q
o
Dl
if)
CD
0
0
t
la
ro
-Q
o
Dl
CD 1 1 1 1 ~~l 1 '
0 20 40 60 80 100
—
T —
CD
0
0
0
I
0
I I
20 40
I I I
60 80 100
Time
Time
Dose= 1.81
Dose= 5.17
\ \ \ \ \
20 40 60 80 100
\
0
\ \ \ \ \
20 40 60 80 100
Time
Time
This document is a draft for review purposes only and does not constitute Agency policy.
C-42 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
2 Male Rat (Kroese et al. 2001): Skin or Mammary Gland Basal Cell Tumors
O
4 =======================================================================
5 Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009)
6 Solutions are obtained using donlp2-intv, (c) by P. Spellucci
7 Input Data File: SKinMamBasalKroeseM3.(d)
^
10
11
12
13
14
15
16 Dependent variable = CONTEXT
17 Independent variables = DOSE, TIME
18
19 Total number of observations = 208
20 Total number of records with missing values = 0
21 Total number of parameters in model = 6
22 Total number of specified parameters = 1
23 Degree of polynomial = 3
24
25
26
27 User specifies the following parameters:
28 t o = o
29
30
31
32
33
34
35 Default Initial Parameter Values
36 c = 1.38462
37 t_0 = 0 Specified
38 beta_0 = 3.84298e-005
39 beta_l = 1.06194e-028
40 beta_2 = 0
41 beta 3 = 6.84718e-006
42
43
44 Asymptotic Correlation Matrix of Parameter Estimates
45 ( *** The model parameter(s) -t_0 -beta_l -beta_2
46 have been estimated at a boundary point, or have been specified by the user,
47 and do not appear in the correlation matrix )
48
49 c beta 0 beta 3
50 -
51 c 1-1 -i
52
53
54
55
56
57
58
59
60 Variable
61
62
63
64
65
66
67
68
69
70
71 Log (likelihood) # Param AIC
This document is a draft for review purposes only and does not constitute Agency policy.
C-43 DRAFT—DO NOT CITE OR QUOTE
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1
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O
4
5
6
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Specified effect =
BMD =
BMDL =
BMDU =
Toxicological Review ofbenzo[a]pyrene
Total Expected Response
Incidental Risk: Skin Mam Basal Kroese M3
Dose= 0.54
Dose= 1.81
J^
—
!5
CD
.a
a.
oo
0
•
^^
0
—
0
- - mmm ^m t
^
±^
!5
CD
r2
2
CL
O 1 ill 1 1
oo
CD
•
^^
0
—
0
o ~S 1 1 - r -i - 1 — •
0 20 40 60 80
0 20 40 60 80
Time
Time
23
Dose= 5.17
.Q
CD
.Q
O
oq
C3
q
c>
Time
0 20 40 60 80
This document is a draft for review purposes only and does not constitute Agency policy.
C-44 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1 Male Rat (Kroese et al, 2001): Skin or Mammary Gland Squamous Cell Tumors
2
o
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: SKinMamSCCKroeseMS.(d)
^
9
10
11
12
13
14
15 Dependent variable = CONTEXT
16 Independent variables = DOSE, TIME
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 = 1
22 Degree of polynomial = 3
24
25
26 User specifies the following parameters:
27 t 0 0
28
29
30
31
32
33
34 Default Initial Parameter Values
35 0=3
36
37
38
39
40
41
42
43 Asymptotic Correlation Matrix of Parameter Estimates
44 ( *** The model parameter(s) -t_0 -beta_0 -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
51
52
53
54
55
56 Parameter Estimates
57 95.0% Wald Confidence Interval
58 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
59
60
61
62
63
64
65
66
67
68
69 Log (likelihood) # Param
70 Fitted Model -27.652 5
71
72
73 Data Summary
74 CONTEXT
This document is a draft for review purposes only and does not constitute Agency policy.
C-45 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1
2
O
4
5
6
8
9
10
11
12
13
14
15
U Total Expected Response
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
16
Specified
effect =
BMD =
BMDL =
Incidental Risk: OralForstKroeseM3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose= 0.00 Dose= 0.54
17
OD
CD
UD
CD
rsj
CD
O
CD
^ I I I I
20 40 60 80 100
OD
CD
GD
CD
OJ
CD
CD
CD
I I I I I
20 40 60 80 100
Time
Time
Dose= 1.81
Dose= 5.17
OD
CD
o
CL
CD
CD
O 'If O-I-
I I I I I
20 40 60 80 100
:=-!
,±±
LE
CTJ
0
CL
• — i
OD
CD
tfl
CD
CD ~~
CM
CD _
i — i
0» 0«^^«»4
/
/
/ +*
i
f f
J
1 J
._--/ .»^rf^
20 40 60 80 100
Time
Time
This document is a draft for review purposes only and does not constitute Agency policy.
C-46 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1 Male Rat (Kroese et al, 2001): Kidney Urothelial Carcinomas
2
o
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: KidneyUrothelialCarKroeseM3.(d)
^
9
10
11
12
13
14
15 Dependent variable = CONTEXT
16 Independent variables = DOSE, TIME
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 = 1
22 Degree of polynomial = 3
24
25
26 User specifies the following parameters:
27 t 0 0
28
29
30
31
32
33
34 Default Initial Parameter Values
35 c 1.63636
36
37
38
39
40
41
42
43 Asymptotic Correlation Matrix of Parameter Estimates
44 ( *** The model parameter(s) -t_0 -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 c beta 3
49
50 c 1-1
51
52 beta 3-11
53
54
55 Parameter Estimates
56 95.0% Wald Confidence Interval
57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
58 c 1.74897 3.79403 -5.68719 9.18512
59 beta_0 0 NA
60 beta_l 0 NA
61 beta 2 0 NA
62
63
64
65
66
67
68
69 Log (likelihood) # Param
70 Fitted Model -11.3978 5
71
72
73 Data Summary
74 CONTEXT
This document is a draft for review purposes only and does not constitute Agency policy.
C-47 DRAFT—DO NOT CITE OR QUOTE
-------
1
2
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4
5
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13
14
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16
Toxicological Review ofbenzo[a]pyrene
U Total Expected Response
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified
effect =
BMD =
BMDL =
Incidental Risk: Kidney_Kroese_M3
Dose = 0.00
Dose= 0.54
^
o
(TJ
O
2
n
in
o
o
o
in
o _
o
o
o
^
.a
(TJ
_Q
2
CL
"-1 1 1 1 1 1 1
0 20 40 60 80 100
in
o
o
o
in
o _
o
o
o
"-1 1 1 1 1 1 1
0 20 40 60 80 100
17
18
Time
Time
Dose= 1.81
Dose= 5.17
in
o
= o
.a
§
o
8
o
TTi
I
20
I
40
I
60
I
80
in
o
= o
.a
2.
CL
s J
o
8 J
o
100
20
I
40
I
60
80 100
Time
Time
This document is a draft for review purposes only and does not constitute Agency policy.
C-48 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1 Female Rat (Kroese et al, 2001): Oral Cavity or Forestomach, Squamous Cell Papilloma or Carcinoma
2
3 =======================================================================
4
5
6
7
8
9
10
11
12
13
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
23
24
25
26
27
28
29
30 Default Initial Parameter Values
31 c = 3.6
32 t_0 = 45.1111
33 beta_0 = 1.11645e-009
34 beta_l = 4.85388e-009
35 beta_2 = 0
36 beta 3 = 1.95655e-008
37
38 Asymptotic Correlation Matrix of Parameter Estimates
39 ( *** The model parameter(s) -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
57 95.0% Wald Confidence Interval
58 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
59 c 3.52871 0.701117 2.15454
60 t_0 46.553 5.93306 34.9244
61 beta_0 1.53589e-009 5.40523e-009 -9.05817e-009
62 beta~l 7.57004e-009 2.9647e-008 -5.05369e-008
63
64
65
66
67
68
69
70 Log (likelihood) # Param
71 Fitted Model -94.5119 6
72
73
This document is a draft for review purposes only and does not constitute Agency policy.
C-49 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1
2
O
4
5
6
8
9
10
11
12
13
14
15
16
17
U Total Expected Response
Minimum observation time for F tumor context =
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect =
BMD =
BMDL =
BMDU =
Incidental Risk: OralForstKroeseFS
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00 Dose = 0.49
18
±i
'o
CD
O
p
Q.
00
0
_
1
^
o
0
0 ~"
\
1 1 1 1 1 1
-1—*
&
CD
O
Q.
00
0
_
1
^
0
0
0 ~"
O IB
1 1 1 1 1 1
0 20 40 60 80 100
0 20 40 60 80 100
Time
Time
Dose = 1 .62
Dose = 4.58
-i— •
o
CD
.a
o
00
o
_
«.
/•
fr
:~-\ ^C'
>,
S3.
CD
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o
or'?
,
•
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e *
I I I I I I
0 20 40 60 80 100
1 1 1 1 1 1
0 20 40 60 80 100
Time
Time
This document is a draft for review purposes only and does not constitute Agency policy.
C-50 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1 Female Rat [Kroese etal., 2001]: Hepatocellular Adenoma or Carcinoma
2
O
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: LiverKroeseF3.(d)
7 Fri Apr 16 09:08:03 2010
8
9
10 Timer to Tumor Model, Liver Hepatocellular Tumors, Kroese et al, Female
12
13
14
15
16
17
18
19
20 Dependent variable = CONTEXT
21 Independent variables = DOSE, TIME
22
23 Total number of observations = 208
24 Total number of records with missing values = 0
25 Total number of parameters in model = 6
26 Total number of specified parameters = 0
27 Degree of polynomial = 3
28
29
30
31
32
33
34
35 Default Initial Parameter Values
36 c 3.6
37 t_0 = 31.7778
38 beta_0 = 0
39 beta_l = 4.9104e-031
40 beta_2 = 5.45766e-030
41 beta 3 = 3.44704e-008
42
43
44 Asymptotic Correlation Matrix of Parameter Estimates
45 ( *** The model parameter(s) -beta_0 -beta_l -beta_2
46 have been estimated at a boundary point, or have been specified by the user,
47 and do not appear in the correlation matrix )
48
49 c t o
50
51
52
53 t o
54
55 beta 3
56
57
58 Parameter Estimates
59
60 Variable
61
62
63
64
65
66
67
68
69
70
71
72 Log (likelihood) # Param
73 Fitted Model -228.17 6
This document is a draft for review purposes only and does not constitute Agency policy.
C-51 DRAFT—DO NOT CITE OR QUOTE
-------
1
2
O
4
5
6
8
9
10
11
12
13
14
15
16
17
18
19
20
Toxicological Review ofbenzo[a]pyrene
Total Expected Response
44
Specified effect =
BMD =
BMDL =
BMDU =
21
Incidental Risk: Hepatocellular_Kroese_F3
points show nonparam. est. for Incidental (unfilled) and Fatal (filled)
Dose = 0.00
Dose = 0.49
-1— '
CD
2
CL
oo
o
,-f.
0 ~
_
0
-i— '
CD
2
Q.
^ 1 1 1 1 1 1
0 20 40 60 80 100
oo
O
^.
0
0
o
1 1 1 1
0 20 40 60
=«CMt4» — •
1 1
80 100
Time
Time
Dose = 1 .62
Dose = 4.58
\ \ \ \ \ \
0 20 40 60 80 100
>s °°.
= °
!Q
s -*
2 °
Q_
C3
O
n i i i i i
0 20 40 60 80 100
Time
Time
22
This document is a draft for review purposes only and does not constitute Agency policy.
C-52 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1 Female Rat [Kroese et al.. 2001): Duodenum or lejunum Adenocarcinoma
2
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: DuoJejKroeseFS.(d)
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
22
23
24
25
26
27
28
29
30
31
32 Default Initial Parameter Values
33
34
35
36
37
38
39
40
41 Asymptotic Correlation Matrix of Parameter Estimates
42 ( *** The model parameter(s) -t_0 -beta_0 -beta_l -beta_2
43 have been estimated at a boundary point, or have been specified by the user,
44 and do not appear in the correlation matrix )
45
46 c beta 3
47
48 c 1-1
49
50 beta 3-11
51
52
53
54
55 Variable
56
57
58
59
60
61
62
63
64
65
66 Log (likelihood) # Param
67 Fitted Model -13.8784 5
68
69
70
71
72
73
This document is a draft for review purposes only and does not constitute Agency policy.
C-53 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1
2
O
4
5
6
8
9
10
11
12
48
Benchmark Dose Computation
Risk Response = Incidental
Risk Type = Extra
Confidence level = 0.9
Time = 104
Specified effect =
BMD =
BMDL =
BMDU =
13
Incidental Risk: DuoJej_Kroese_F3
Dose = 0.00
Dose = 0.49
s
o
CL
LO
d
LT>
O
d
O
O
I
20
40
60
S
o
CL
80 100
d
o
d
in
o _
d
o
o
d
I I I I I I
0 20 40 60 80 100
Time
Time
Dose= 1.62
Dose = 4.58
s
o
D.
LT>
o _
o
o _
\
20
40
60
JD
s
o
CL
o
o
d
p _
d
o
o _
80 100
\
20
40
I
60
\
80
100
Time
Time
14
15
This document is a draft for review purposes only and does not constitute Agency policy.
C-54 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofbenzo[a]pyrene
1
2
Table C-18. Summary of human equivalent overall oral slope factors,
based on male and female rat tumor incidence
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
2.11 x 10"2
7.48 x 10"2
Sum, risk values at BMD001:
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 BMD001:
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
4.10 x 10"4
5.64 x 10"5
2.17 x 10"2
1.47 x 10"1
Properties
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
3.09E-01
3
4
"Risk value = 0.001/BMDL001.
bOverall SD = (sum, SD2)0 5.
"Upper bound on the overall risk estimate = sum of BMD0oi risk values + 1.645 x overall SD.
Source of data: Kroese et al. (2001).
Table C-19. Summary of model selection among multistage-Weibull
models fit to alimentary tract tumor data for female mice
5
6
7
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
Model selection rationale
Lowest AIC, best fit to low dose data
Source of data: Beland and Gulp (1998)
This document is a draft for review purposes only and does not constitute Agency policy.
C-55 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
1 Female Mice fBeland and Culv, 19981: Alimentary Tract Sauamous Cell Tumors
4
5
6
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
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55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
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)
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
User Inputs Initial Parameter Values
c 2
t_0 = 15
beta_0 = 1.6e-014
beta_l = 0
beta_2 = 5.5e-012
beta 3 = 4.4e-012
Asymptotic Correlation Matrix of Parameter Estimates
c tO beta 0 beta 1
Variable
c
t_0
beta_0
beta_l
beta_2
beta 3
Upper Conf. Limit
9.54705
23.677
3.14019e-015
2.55825e-014
7.93813e-013
1.19919e-012
This document is a draft for review purposes only and does not constitute Agency policy.
C-56 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
1
2
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6
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11
12
13
14
15
16
17
18
19
20
Minimum observation time for F tumor context =
dp
Confidence level =
Time
BMD =
BMDL =
BMDU =
21
22
Incidental Risk: BaP_FemaleSquamF3i
points show nonpararm. est for Incidental (unfilled) and Fatal (filled)
Dose= 0.00
Dose= 0.10
-Jr
•^
cc
o
CL
i — i
OD
CD
UD
CD
^t
CD
fN
CD
CD
,,r--ii
i — i
OD
CD
;t± UD
15 CD
C13
O ^ —
d °
r-i
CD
CD
o
« * !•>••-»«
° 1 1 1 1 1 1 ° 1 1 1 1 1 1
0 20 40 60 80 100 0 20 40 60 80 100
Time
Time
Dose= 0.48
Dose= 2.32
OD
CD
r-i
CD
p
CD
I I I I I
20 40 60 80 100
Time
OD
CD
I 3
p
CD
I
20
40
I I I
60 80 100
Time
This document is a draft for review purposes only and does not constitute Agency policy.
C-57 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
1 DOSE-RESPONSE MODELING FOR THE INHALATION UNIT RISK
2 As with the tumor data used for the oral slope factor (see Dose Response-modeling for the
3 Oral Slope Factor Section), there was earlier occurrence of tumors with increasing exposure, and
4 early termination of the high-dose group (Thyssen et al., 1981; see Appendix B for study details).
5 The computer software program MSW (U.S. EPA, 2010) was used as described in the analysis of the
6 oral carcinogenicity data.
7 Thyssen et al. (1981) did not determine cause of death for any of the animals. Bounding
8 estimates for the Thyssen et al. (1981) data were developed by treating the tumors alternately as
9 either all incidental or all fatal. In either case, therefore, an estimate of to (the time between a
10 tumor first becoming observable and causing death) could not be estimated. The data analyzed are
11 summarized in Table C-20, the results are summarized in Table C-21, and the modeling details
12 follow.
13 Table C-20. Individual pathology and tumor occurrence data for male
14 Syrian hamsters exposed to benzo [ajpyrene via inhalation for lifetime-
15 Thyssen etal. (1981).
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
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
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
oa
0
0
0
oa
0
0
0
0
0
0
Pharynx
oa
0
0
0
0
oa
0
0
0
0
0
0
0
0
0
0
0
0
oa
0
0
0
0
oa
0
oa
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
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
Forestomach
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
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Toxicological Review ofbenzo[a]pyrene
Nominal
exposure
concentration
(mg/m3)
10
50
Time on
study
85
87
88
93
98
99
102
103
108
111
113
114
115
116
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
Number
examined
1
1
1
1
1
1
1
1
1
1
1
1
1
1
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
Papillomas, Polyps, Papillary polyps, Squamous cell carcinomas
Larynx
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
oa
0
0
0
0
0
0
0
1
0
1
0
0
1
0
0
1
1
0
1
3
1
1
0
oa
oa
oa
oa
oa
oa
oa
oa
la
0
0
1
0
0
1
0
Pharynx
0
0
0
0
oa
0
0
0
0
0
0
0
0
0
0
0
oa
0
0
0
0
0
0
2
0
0
0
0
1
1
1
1
1
oa
0
0
lb
0
1
0
oa
oa
oa
oa
oa
oa
oa
oa
la
0
0
1
1
oa
1
1
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
1
1
0
0
0
0
0
oa
oa
oa
oa
oa
oa
oa
oa
la
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
0
0
0
0
0
0
0
0
0
0
Forestomach
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
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
oc
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
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Toxicological Review ofbenzo[a]pyrene
Nominal
exposure
concentration
(mg/m3)
Time on
study
60
63
64
66
68
70
71
72
73
79
Number
examined
1
1
1
1
1
1
1
1
2
4
Papillomas, Polyps, Papillary polyps, Squamous cell carcinomas
Larynx
0
0
0
1
0
1
1
1
2
3
Pharynx
1
0
1
1
1
1
1
1
2
4
Trachea
0
0
0
0
0
0
1
0
0
1
Esophagus
0
0
0
0
0
1
0
0
0
1
Forestomach
0
0
1
0
0
0
0
0
0
0
Nasal cavity
0
0
0
0
0
0
0
0
0
1
aTissue was not examined for one animal of total examined.
bTissue was not examined for two animals of total examined.
cAn 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.
1
2
Table C-21. Summary of model selection among multistage-Weibull
models fit to tumor data for male hamsters
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
Model selection rationale
Lowest AIC, best fit to data
(BMDU10 = 0.350)
Lowest AIC; best fit to data
(BMDU10 = 0.719)
3 Data source: Thyssen etal. (1981)
4 Output for squamous cell neoplasia following inhalation exposure to BaP: all tumors considered
5 incidental to cause of death
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
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_inc2st.(d)
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)Ac *
(beta 0+beta l*doseAl+beta 2*doseA2)}
Dependent variable = Class
Independent variables = Cone, Time
Total number of observations = 96
Total number of records with missing values = 0
Total number of parameters in model = 5
Total number of specified parameters = 1
Degree of polynomial = 2
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Toxicological Review ofbenzo[a]pyrene
4
5
6
8
9
10
11
12
13
14
15
16
17
18
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
c 3.6
t_o = o
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
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 -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
Time
BMD
BMDL
BMDU
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Toxicological Review ofbenzo[a]pyrene
Incidental Risk: BaP-Thyssen_inc2st
Dose = 0.00
Dose = 0.25
Probability
oo
o
o ~"
o
«-» i i r i T~ r™r*
0 20 60 100
Probability
oo
o
o ~"
o
o | | '| H "j~"|"— y '
0 20 60 100
Time
Time
Dose = 1.00
Dose = 4.29
p
o
n i r
o 20
\ \
60
Time
i r
100
100
Time
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Toxicological Review ofbenzo[a]pyrene
1 Output for respiratory tract tumors: all tumors considered to be cause of death
2
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: C:\msw\benzo[a]pyrene-Thyssen allfatal noU 3st.(d)
7
8
9
10
11
12
13
14
15
16
17
18 Total number of observations = 96
19 Total number of records with missing values = 0
20 Total number of parameters in model = 6
21 Total number of specified parameters = 1
22 Degree of polynomial = 3
23
24
25
26 User specifies the following parameters:
27 t 0 = 0
28
29
30
31
32
33
34 Default Initial Parameter Values
35 c = 4.5
36 t_0 = 0 Specified
37 beta_0 = 0
38 beta_l = 1.37501e-010
39 beta_2 = 2.84027e-010
40 beta 3 = 1.44668e-037
41
42
43 Asymptotic Correlation Matrix of Parameter Estimates
44 ( *** The model parameter(s) -t_0 -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 c beta 3
49
50 c 1-1
51
52 beta 3-11
53
54
55 Parameter Estimates
56
57 Variable
58
59
60
61
62
63
64 NA - Indicates that this parameter has hit a
65 bound implied by some inequality constraint
66 and thus has no standard error.
67
68
69 Log (likelihood) # Param
70 Fitted Model -144.522 5
71
72
73
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23
24
Toxicological Review ofbenzo[a]pyrene
1
2
O
4
5
6
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Class
F I
0 0
0 0
18 0
18 0
U Total
BMD
BMDL
BMDU
Fatal Risk: BaP-Thv$sen_allfatal_noU_3st
Dose= 0.00
Dose= 0.25
^
=
~s
o
n
CO
o
,-j.
o
—
o
.^
=
OJ
o
D_
<-> \ \ I I I 1 1
0 20 60 100
CO
O
^
o
—
o
<-> I I I I I I I
0 20 60 100
Time
Time
Dose= 1.00
Dose= 4.29
^
.~t±
-Q
OJ
-Q
O
Q.
CO
o
—
CD ~
0
*
*
J
f
/
^
."t±
-D
(C
-Q
O
a.
'-> I I I I I I I
0 20 60 100
CO
o
—
CD ~
0
*
/
I
/
/
XV
L-1 I I I I I I I
0 20 60 100
Time
Time
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Toxicological Review ofbenzo[a]pyrene
1 DOSE-RESPONSE MODELING FOR THE DERMAL SLOPE FACTOR
2 Modeling methods:
3 For each endpoint, multistage models (BMDS; U.S. EPA, 2012;v2.1) were fitted to the data
4 using the maximum likelihood method. Each model was tested for goodness-of-fit using a chi-
5 square goodness-of-fit test (x2 p-value < 0.05 indicates lack of fit). Other factors were used to
6 assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in
7 the vicinity of the BMR. The BMDL estimate (95% lower confidence limit on the BMD, as estimated
8 by the profile likelihood method) and AIC value were used to select a best-fit model from among the
9 models exhibiting adequate fit. The data modeled are summarized in Tables C-22 through C-25.
10 The modeling results are summarized in Table C-26. The modeling details are provided with
11 Figures C- 8 through C-19.
12 Data adjustments prior to modeling:
13 Roe et al. (1970) applied benzo[a]pyrene dermally for 93 weeks or until natural death; with
14 the exception of the highest dose group, each group still had approximately 20 animals at 86 weeks
15 (see Table C-22). The tumors were first observed in the lowest and highest dose groups during the
16 interval of weeks 29-43. Mice that died before week 29 were likely not at risk of tumor
17 development However, because tumor incidence and mortality were reported in 100-day
18 intervals, mice that had not been on study long enough to develop tumors were not easily
19 identifiable. Incidence denominators re fleet the number of animals alive at week 29, and may thus
20 tend to lead to underestimates of tumor risk if the number of animals at risk has been
21 overestimated.
22 Schmidt et al. (1973) did not report survival information; instead, the authors provided
23 incidences based on the numbers of mice initially included in each dose group at the start of the
24 study. Overall latency was reported for the two high-dose groups in each series, but these data only
25 describe the survival of mice with tumors (animals were removed from study when a tumor
26 appeared). It is not clear how long exposures lasted overall in each dose group, or whether some
27 mice may have died on study from other causes before tumors appeared. While it is possible that
28 no mice died during the study, all of the other studies considered here demonstrate mortality.
29 However, the data were modeled as reported, recognizing the possibility of underestimating risk
30 associated with incidences reported and lack of duration of exposure. (See Table C-22.)
31 Schmahl et al. (1977) reported that reduced numbers of animals at risk (77-88 mice per
32 dose group compared with the initial group sizes of 100) resulted from varying rates of autolysis.
33 No other survival or latency information was provided, so all exposures were assumed to have
34 lasted for 104 weeks and were modeled as reported. Given the results of the other studies, it seems
35 possible that the numbers at risk in each group may be overestimated, which could lead to an
36 underestimate of lifetime risk. (See Table C-22.)
37 Habs et al. (1980) reported age-standardized skin tumor incidence rates, indicating earlier
38 mortality in the two highest dose groups (2.8 and 4.6 |ig/application). These rates were used to
39 estimate the number at risk in the dose-response modeling, by dividing the number of mice with
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Toxicological Review ofbenzo[a]pyrene
1 tumors by the age-standardized rates. Exposure lasted longer than 104 weeks in the two lower
2 exposure groups, at about 120 and 112 weeks, and until about 88 weeks in the highest exposure
3 group. Incidence in the two lower exposure groups may be higher than if the exposure had lasted
4 just 104 weeks. There was mortality in the first 52 weeks of exposure, about 10-15% in the three
5 exposure groups, but because there was no information concerning when tumors first appeared, it
6 is not possible to determine how much the early mortality may have impacted the number of mice
7 at risk in each group. (See Table C-22.)
8 Habs et al. (1984) reported mean survival times (with 95% CIs) for each dose group. The
9 CIs supported the judgment that the control and lower dose groups were treated for 104 weeks.
10 The higher dose group (4 [ig/application) was probably treated for <104 weeks, because the upper
11 95% confidence limit for the mean survival was approximately 79 weeks. However, since it was
12 not possible to estimate a more realistic duration for this group, an estimate of 104 weeks was
13 used. (See Table C-22.)
14 The studies by Poel (1960,1959) were conducted in male mice and used toluene as the
15 vehicle. In addition to a control group, the 1959 study included nine dose groups of one mouse
16 strain (C57L) and the 1960 study included seven dose groups of three other mouse strains. Both
17 studies demonstrated high mortality and tumor incidence at higher exposure levels. All C57L mice
18 in dose groups with >3.8 |ig/application died by week 44 of the study (Poel, 1959). Therefore,
19 these five dose groups were omitted prior to dose-response modeling because of the relatively
20 large uncertainty in extrapolating cancer risk as a result of lifetime exposure. Four dose groups in
21 addition to control remained. Among these groups, mice survived and were exposed until weeks
22 83-103. According to the lifespan ranges provided, at least one mouse in each dose group died
23 before the first appearance of tumor, but insufficient information was available to determine how
24 many; consequently, the incidence denominators were not adjusted. The dose-response data are
25 summarized in Table C-23.
26 For the Poel (1960) studies, all tumors in the highest three dose groups for each of the three
27 mouse strains had occurred by week 40. While these observations support concern for cancer risk,
28 as noted above such results are relatively uncertain for estimating lifetime cancer risk. In addition,
29 there was no information indicating duration of exposure for the mice without tumors; although
30 exposure was for lifetime, it might have been as short as for the mice with tumors. Overall, these
31 datasets did not provide sufficient information to estimate the extent of exposure associated with
32 the observed tumor incidence. Consequently, the experiments reported by Poel (1960) were not
33 used for dose-response modeling.
34 Grimmer et al. (1984,1983), studied female CFLP mice, using acetone:DMSO (1:3) as the
35 vehicle. Mean or median latency times were reported (as well as measures of variability), but no
36 information concerning overall length of exposure or survival was included in the results. The total
37 of tumor-bearing mice and the reported percentages of mice with any skin tumors was reported
3 8 and varied, at most, one animal from the number of animals initially placed on study. The
39 decreasing latency and variability and increasing tumor incidence with increasing benzo[a]pyrene
40 exposure suggests that exposure probably did not last for 104 weeks in at least the high-dose
41 group, but the available information did not provide duration of exposure. The data reported were
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Toxicological Review ofbenzo[a]pyrene
1 modeled under the assumption that at least some animals in each group were treated and survived
2 until week 104. (See Table C-24.)
3 Sivak et al. (1997), exposed male C3H/HeJ mice dermally to benzo[a]pyrene in
4 cyclohexanone/acetone (1:1) for 24 months, and reported mean survival times for each group. All
5 high-dose mice died before the final sacrifice. From the information provided, it is apparent that
6 the animals in the control and lower two dose groups survived until study termination at week 104.
7 The study authors did not report how long treatment in the highest dose group lasted, but
8 estimation of the figure from the publication suggest that exposure duration was 74 weeks. (See
9 Table C-25).
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2
3
Toxicological Review ofbenzo[a]pyrene
Table C-22. Skin tumor incidence, benign or malignant in female Swiss
or NMRI mice dermally exposed to benzo[a]pyrene
Study
Roeetal.,
1970a,b
Schmidt et
al., 1973°
Schmahl et
al., 1977°
Habsetal.,
1980c'f
Habsetal.,
1984°
Mouse
strain
Swiss
NMRI
Swiss
NMRI
NMRI
NMRI
Dose (ug)
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
(ug/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%)
aDoses were applied 3 times/week for up to 93 weeks to shaved dorsal skin.
bNumerator: number of mice detected with a skin tumor. Tumors were thought to be malignant based on
invasion or penetration of the panniculus carnosus muscle. Denominator: number of mice surviving to 29 weeks
(200 days).
cDoses were applied 2 times/week to shaved skin of the back. Mice were exposed until natural death or until they
developed a carcinoma at the site of application. Schmidt et al. (1973): At 0.23 u.g/d, all tumors were malignant in
both strains; at 0.57 u.g/d, tumors were predominately malignant: 28/30 for NMRI and 42/45 for Swiss. Schmahl
et al., (1977): malignant/total tumors were 10/11, 25/25, and 43/45 for the 1-, 1.7-, and 3-u.g/d groups. Habs et al.
(1984): malignant/total tumors were 7/9 and 17/11 for the 2- and 4-u.g/d groups.
Exposure periods not reported were assumed to be 104 weeks; indicated in italics.
eCentral tendency estimates; range or other variability measure not reported.
The 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 C-23. Skin tumor incidence, benign or malignant, in C57L male
mice dermally exposed to benzo[a]pyrene
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
aDoses were applied to interscapular skin 3 times/week for up to 103 weeks or until time of appearance of a
grossly detected skin tumor. See Table B-15 for data of five highest dose groups (19-752 u.g) in which all mice
died by week 44. These groups were not considered for dose-response modeling.
bSee Section 2.5.2. of Toxicological Reivew for discussion of extrapolation to lifetime average daily doses.
cTumors were histologically confirmed as epidermoid carcinomas.
Table C-24. Skin tumor incidence, benign or malignant, in female CFLP
mice dermally exposed to benzo[a]pyrene
Study
Grimmer et al.,
1983
Grimmer et al.,
1984
Dose (u,g)a
0(l:3acetone:DMSO)
3.9
7.7
15.4
0(l:3acetone: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 ± 16.8b
60.9 ±13.9
44.1 ±7.7
—
61 (53-65)c
47 (43-50)
35 (32-36)
Length of
exposure
(wks)d
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)0
0/80 (0%)
22/65 (34%)
39/64 (61%)
56/64 (88%)
0/80 (0%)
43/64 (67%)
53/65 (82%)
57/65 (88%)
5
6
Indicated doses were applied twice/week to shaved skin of the back for up to 104 weeks.
bMean±SD.
cMedian and 95% confidence limit.
dAssumed exposure period is indicated in italics.
Incidence denominators were calculated from reported tumor-bearing animals and reported percentages.
Grimmer et al. (1983): malignant/total tumors were 15/22, 34/39, and 54/56 for the low- through high-dose groups.
Grimmer et al. (1984): malignant /total tumors were 37/43, 45/53, and 53/57 for the low- through high-dose
groups.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table C-25. Skin tumor incidence, benign or malignant, in male
C3H/HeJ mice dermally exposed to benzo[a]pyrene
Dose (u,g)a
0 (1:1 cyclohexanone/acetone)
0.05
0.5
5.0
Average
daily dose
(Hg/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
typesjc
0/30 (0%)
0/30 (0%)
5/30 (17%)
27/30 (90%)
Indicated doses were applied twice/week to shaved dorsal skin.
bAssumed exposure period is indicated in italics.
°Number 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.
NR = not reported
Source: Sivaketal. (1997).
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Table C-26. Summary of 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
Roeetal., 1970
female Swiss
Schmidt etal., 1973
female NMRI
Schmidt etal., 1973
female Swiss
Schmahletal., 1977
female NMRI
Habsetal., 1980
female NMRI
Habsetal., 1984
female NMRI
Grimmer etal., 1983
female CFLP
Grimmer etal., 1984b
female CFLP
Sivak etal., 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°
Log Logistic
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
(ug/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
(ug/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
C-9
C-10
C-ll
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
3 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 for Grimmer 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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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
0.1
4
5
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1
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19
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21
22
23
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26
27
28
29
30
31
32
33
34
35
36
37
38
Figure C-8. Fit of multistage model to skin tumors in C57L mice exposed
dermally to benzo[a]pyrene (Poel, 1959); graph and model output.
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:
C:\Usepa\BMDS21\Data\msc_benzo[a]pyrene_Poel_1959_MultiCanc3_0.1.pit
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/xl-beta2*dose/x2-betaS*doseA3)
The parameter betas are restricted to be positive
Dependent variable = NumAff
Independent variable = LADD
Total number of observations = 5
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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Background =
Beta(l) =
Beta(2) =
Beta(3) =
0.0449589
0.490451
0
2.68146
the user,
Background
Beta(l)
Beta (3)
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(2)
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Background Beta(l) Beta(3)
1 -0.87 0.74
-0.87 1 -0.92
0.74 -0.92 1
Interval
Variable
Limit
Parameter Estimates
Estimate Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Background
Beta(l)
Beta(2)
Beta(3)
0.0176699
0.79766
0
2.17146
* - Indicates that this value is not
Model
Full model
Fitted model
Reduced model
AIC:
Dose Est
0.0000 0.
0.0500 0.
0.1600 0.
0.2400 0.
0.8000 0.
Analysis of
Log (likelihood) #
-87.1835
-90.4265
-141.614
186.853
*
*
*
*
calculated.
Deviance Table
Param' s Deviance Test
5
3 6.48606
1 108.86
Goodness of Fit
. Prob. Expected Observed Size
0177 0.583
0563 3.098
1430 7.866
2128 11.917
8293 40.635
0.000 33
5.000 55
11.000 55
7.000 56
41.000 49
*
*
*
*
d.f. P-value
2 0.03905
4 <.0001
Scaled
Residual
-0.770
1.112
1.207
-1.605
0.139
2 = 5.88 d.f. = 2 P-value = 0.0528
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
1 Confidence level = 0.95
2
3 BMD = 0.126567
4
5 BMDL = 0.0777875
6
7 BMDU = 0.272961
8
9 Taken together, (0.0777875, 0.272961) is a 90 % two-sided confidence
10 interval for the BMD
11
12 Multistage Cancer Slope Factor = 1.28555
13
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.5
1.5
2.5
dose
2
3
4
6
1
8
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18
19
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21
22
23
24
25
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30
31
32
33
34
35
36
37
Figure C-9. Fit of multistage model to skin tumors in female Swiss mice
exposed dermally to benzo[a]pyrene (Roe etal., 1970); graph and
model output.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta (3) -Beta (4) -Beta(5)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
- Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Est. Prob.
# Param's
Test d.f.
P-value
d.f. =3
BMD =
BMDL =
BMDU =
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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Multistage Cancer Model with 0.95 Confidence Level
0.4
Multistage Cancer
Li near extrapolation
BMDL
BMD
0.1
0.2
0.3
dose
0.4
0.5
4
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21
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24
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30
31
32
33
34
35
36
Figure C-10. Fit of multistage model to skin tumors in female NMRI
mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph
and model output.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\benzoUlpyrene\dermalslopefactor\Schmidtl973femaleNMRI\2MulSchMS_. (d)
Gnuplot Plotting File:
C:\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidtl973femaleNMRI\2MulSchMS_.pit
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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Asymptotic Correlation Matrix of Parameter Estimates
and do not appear in the correlation matrix )
Beta (2)
1
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
Model
Full model
Fitted model
Reduced model
AIC:
P-value
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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Toxicological Review ofbenzo[a]pyrene
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
BMDL BMP
0.1
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0.3
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0.4
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Figure C-ll. Fit of multistage model to skin tumors in female Swiss
mice exposed dermally to benzo[a]pyrene (Schmidt et al., 1973); graph
and model output.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidtl973swissmice\3MulSchMS_. (d)
Gnuplot Plotting File:
\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmidtl973swissmice\3MulSchMS_.pit
This document is a draft for review purposes only and does not constitute Agency policy.
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**** incorporate
Asympt
( ***
user,
B
Beta (2)
Beta (3)
Variable
Limit
Background
Beta (1)
Beta (2)
Beta (3)
these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0.338951
Beta(3) = 3.8728
otic Correlation Matrix of Parameter Estimates
The model parameter (s) -Background -Beta(l)
have been estimated at a boundary point, or have been spe
and do not appear in the correlation matrix )
eta(2) Beta(3)
1 -0.99
-0.99 1
Parameter Estimates
95.0% Wald Confid'
Estimate Std. Err. Lower Conf. Limit
0 * *
0 * *
0.108125 * *
4.31441 * *
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Dose Est
0.0000 0.
0 . 0100 0 .
0.0600 0.
0 . 2300 0 .
0.5700 0.
ChiA2 = 0.16
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-valu
-73.5285 5
-73.6628 2 0.268637 3 0.
-150.708 1 154.359 4 <.0
151.326
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 80 0.000
0000 0.001 0.000 80 -0.035
0013 0.106 0.000 80 -0.325
0566 4.524 5.000 80 0.230
5657 45.260 45.000 80 -0.059
d.f. =3 P-value = 0. 9833
Computation
0.1
= Extra risk
0. 95
0.282007
0.223401
0.309888
Taken together, (0.223401, 0.309888) is a 90 % two-sided confidence
interval for the
Multistage Cancer
BMD
Slope Factor = 0.447626
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
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BMD
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Figure C-12. Fit of multistage model to skin tumors in female NMRI
mice exposed dermally to benzo[a]pyrene (Schmahl etal., 1977); graph
and model output.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahll977femaleNMRI\2MulschMS_. (d)
Gnuplot Plotting File:
\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Schmahll977femaleNMRI\2MulschMS_.plt
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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Limit
**** 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.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
P-value
Est. Prob.
81
88
81
d.f. = 1
Confidence level =
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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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
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Linear extrapolation
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Figure C-13. Fit of multistage model to skin tumors in female NMRI
mice exposed dermally to benzo[a]pyrene (Habs etal., 1980); graph
and model output.
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
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Asymptotic C
( ***
The mo
orrelation Matrix of Parameter Estimates
del parameter
have been estimated
user,
Beta (3)
and do
Beta (3)
1
(s) -Background -Beta(l) -Beta (2)
at a boundary point, or have been specified by the
not appear in the correlation matrix )
Parameter Estimates
Variable
Limit
Background
Beta (1)
Beta (2)
Beta (3)
* - Indicates that this
Model
Full model
Fitted model
Reduced model
AIC:
Estimate
0
0
0
4 . 1289
value is not
Analysis of
Log (likelihood) #
-34.8527
-37.3373
-82.5767
76. 6745
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf.
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 Es
0.0000 0
0 . 4900 0
0.7400 0
0 . 8000 0
ChiA2 = 4.56
Benchmark Dos
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together,
interval for the
Multistage Cance
t. Prob
.0000
.3848
.8123
.8792
d.f.
Expected
0.000
13. 082
21. 933
21. 102
= 3 P
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
e Computation
=
=
=
=
=
=
0.1
Extra risk
0. 95
0.294407
0.215151
0.320955
(0.215151, 0.320955)
BMD
r Slope
Factor =
is a 90 % two-sided confidence
0.46479
70
This document is a draft for review purposes only and does not constitute Agency policy.
C-84 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
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Linear extrapolation
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Figure C-14. Fit of multistage model to skin tumors in female NMRI
mice exposed dermally to benzo[a]pyrene (Habs etal., 1984); graph
and model output.
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
This document is a draft for review purposes only and does not constitute Agency policy.
C-85 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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Default Initial Parameter Values
Background = 0
Beta(l) = 1.66414
Beta (1)
and do not appear in the correlation matrix )
Beta(1)
1
Variable
Background
Beta(1)
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Model
Full model
Fitted model
Reduced model
Goodness of Fit
Dose Est._Prob. Expected Observed Size
d.f. =2
Benchmark Dose Computation
0.1
Extra risk
Confidence level = 0.95
BMD =
BMDL =
BMDU =
Multistage Cancer Slope Factor =
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
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Linear extrapolation
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Figure C-15. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer etal., 1983); graph and
model output.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
::\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmerl983CFLPmice\lMulGriMS_.(d)
Gnuplot Plotting File:
::\USEPA\IRIS\benzo[a]pyrene\dermalslopefactor\Grimmerl983CFLPmice\lMulGriMS_.pit
This document is a draft for review purposes only and does not constitute Agency policy.
C-87 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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Beta(1)
Beta(1)
Variable
Background
Beta(1)
Parameter Estimates
Std. Err.
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
P-value
Est. 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.
C-88 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
0.6
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EMDL, 3MD
Multistage Cancer
Linear extrapolation
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Figure C-16. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer et al., 1984); graph and
model output.
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\Usepa\BMDS21\Data\msc_benzoUlpyrene_Grimmerl984_MultiCancl_0.1. (d)
Gnuplot Plotting File:
C:\Usepa\BMDS21\Data\msc_benzoUlpyrene_Grimmerl984_MultiCancl_0.l.plt
Wed Apr 27 17:11:28 2011
Total number of observations = 4
Total number of records with missing value
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.
C-89 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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Beta (1)
*** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by the
and do not appear in the correlation matrix )
Beta(1)
1
Limit
Variable
Background
Beta(1)
Estimate
Std. Err.
Model
Full model
Fitted model
Reduced model
AIC:
# Param's Deviance Test d.f.
4
1 11.61 3
1 158.797 3
P-value
Prob.
d.f. =3
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
This document is a draft for review purposes only and does not constitute Agency policy.
C-90 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
Log-Logistic Model with 0.95 Confidence Level
0.6
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BMDL
BMD
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Figure C-17. Fit of log-logistic model to skin tumors in female CFLP
mice exposed dermally to benzo[a]pyrene (Grimmer etal., 1984); graph
and model output.
This document is a draft for review purposes only and does not constitute Agency policy.
C-91 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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Asymptotic Correlation Matrix of Parameter Estimates
and do not appear in the correlation matrix )
intercept slope
intercept 1 -0.68
slope -0.68 1
Parameter Estimates
Interval
Variable
Conf. Limit
background
intercept
slope
Estimate
Std. Err.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
Goodness of Fit
Est. Prob.
Benchmark Dose Computation
Specified effect = 0.7
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.07152
BMDL = 0.478669
This document is a draft for review purposes only and does not constitute Agency policy.
C-92 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
0.8
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Multistage Cancer
Li near extrapolation
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Figure C-18. Fit of multistage model to skin tumors in female CFLP mice
exposed dermally to benzo[a]pyrene (Grimmer etal., 1984), highest
dose dropped; graph and model output.
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File: C: /Usepa/_BaP/msc_BaP_Griminerl984_drophidose_MultiCancl_0 . 7 . (d)
Gnuplot Plotting File:
C: /Usepa/_BaP/msc_BaP_Griminerl984_drophidose_MultiCancl_0 . 7 .pit
[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 = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0806622
Beta(l) = 0.88595
Asymptotic Correlation Matrix of Parameter Estimates
This document is a draft for review purposes only and does not constitute Agency policy.
C-93 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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( ***
The model parameter (s) -Background
have been estimated
user,
and do
not appear in
at a boundary point, or have been specified by the
the correlation matrix )
Beta(l)
Beta(l)
1
Parameter Estimates
Variable
Limit
Background
Beta(l)
* - Indicates that this
Estimate
0
0.997117
value is not
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf.
* * *
* * *
calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log (likelihood) #
-71.5928
-72.2756
-134.46
146.551
Param's Deviance Test d.f. P-value
3
1 1.36568 2 0.5052
1 125.735 2 <.0001
Goodness of Fit
Dose Est. Prob
0.0000 0
0.9700 0
1.9100 0
ChiA2 =1.39
.0000
.6199
.8511
d.f .
Expected
0.000
39.671
55.322
Scaled
Observed Size Residual
0.000 65 0.000
43.000 64 0.857
53.000 65 -0.809
= 2 P-value = 0.4992
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together,
interval for the
=
=
_
=
=
=
0.7
Extra risk
0.95
1.20745
1.00734
1.45789
(1.00734, 1.45789) is
BMD
Multistage Cancer Slope
Factor =
a 90 % two-sided confidence
0.6949
64
This document is a draft for review purposes only and does not constitute Agency policy.
C-94 DRAFT—DO NOT CITE OR QUOTE
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0.6
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Toxicological Review ofbenzo[a]pyrene
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.1
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Figure C-19. Fit of multistage model to skin tumors in male CeH/HeJ
mice exposed dermally to benzo[a]pyrene (Sivak et al., 1997); graph
and model output.
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:
C:\Usepa\BMDS21\Data\msc_benzoUlpyrene_Sivakl993_MultiCanc2_0.l.plt
Total number of observations = 4
Total number of records with missing value
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.
C-95 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofbenzo[a]pyrene
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Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Variable Estimate Std. Err.
Background
Beta(1)
Beta(2)
- Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
P-value
Prob.
d.f. =3
Specified effect =
Risk Type
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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Toxicological Review ofbenzo[a]pyrene
1 ALTERNATIVE APPROACHES FOR CROSS-SPECIES SCALING OF THE DERMAL SLOPE
2 FACTOR
3 Several publications which develop a dermal slope factor for benzo[a]pyrene are available
4 in the peer reviewed literature (Knafla et al., 2010; 2006; Hussain et al., 1998; LaGoy and Quirk
5 1994; Sullivan et al., 1991). With the exception of the 2010 Knafla etal. publication, none of these
6 approaches applied quantitative adjustments to account for interspecies differences, though the
7 proposed slope factors were developed to account for human risk. Knafla et al. (2010) qualitatively
8 discuss processes which could affect the extrapolation between mice and humans including skin
9 metabolic activity adduct formation, stratum corneum thickness, epidermal thickness, etc.
10 Ultimately, the authors apply an adjustment based on the increased epidermal thickness of human
11 skin on the arms and hands compared to mouse interscapular epidermal thickness. They
12 hypothesize that the carcinogenic potential of benzo[a]pyrene may be related to the thickness of
13 the epidermal layer.
14 Because there is no established methodology for cross-species extrapolation of dermal
15 toxicity, several alternative approaches were evaluated. Each approach begins with the POD of
16 0.066 [J.g/day that was based on a 10% extra risk for skin tumors in male mice. Based on the
17 assumptions of each approach, a dermal slope factor for humans is calculated. The discussion of
18 these approaches uses the following abbreviations:
19
20 DSF = dermal slope factor
21 PODM = point of departure (for 10% extra risk) from mouse bioassay, in [ig/day
22 BWM= mouse body weight = 0.035 kg (assumed)
23 BWn = human body weight = 70 kg (assumed)
24 SAn = total human surface area = 19,000 cm2 (assumed)
25 SAM = total mouse surface area = 100 cm2 (assumed)
26
27 Approach 1. No interspecies adjustment to daily applied dose (POD) in mouse model
28 Under this approach, a given mass of benzo[a]pyrene, applied daily, would pose the same
29 risk in an animal or in humans, regardless of whether it is applied to a small surface area or to a
30 larger surface area at a proportionately lower concentration.
31
32 DSF=0.1/PODM
33
34 DSF= 0.1/0.068 ^g/day = 1.5 (ug/day)1
35
36 Assumptions: The same mass of benzo[a]pyrene, applied daily, would have same potency in
37 mice as in human skin regardless of treatment area.
38
39 Approach 2. Cross-species adjustment based on whole body surface-area scaling
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Toxicological Review ofbenzo[a]pyrene
1 Under this approach, animals and humans are assumed to have equal lifetime cancer risk
2 with equal average whole body exposures in loading units ([ig/cm2-day). As long as doses are low
3 enough that risk is proportional to the mass of applied compound, the daily dermal dose of
4 benzo[a]pyrene can be normalized over the total surface area.
5
6 POD Og/cm2-day) = PODM/sA Og/cm2-day) = PODM Og/day) / SAM (cm*)
7
8 POD = (0.068 ng/day) / 100 cm2
9 = 0.00068 [ig/cm2-day
10
11 DSF = 0.1/(0.00068 [ig/cm2-day) * 147 (ng/cm2-day) *
12
13 Assumptions: Mouse and human slope factors are equipotent if total dermal dose is
14 averaged over equal fractions of the entire surface area. Tumor potency of benzo[a]pyrene is
15 assumed to be related to overall dose and not dose per unit area. For example, a human exposed to
16 0.01 [J.g/day on 10 cm2 would be assumed to have the same potential to form a skin tumor as
17 someone treated with 0.01 [ig/day over 19,000 cm2 (assumed human surface area).
18
19 Approach 3. Cross-species adjustment based on body weight
20 Under this approach, a given mass of benzo[a]pyrene is normalized relative to the body
21 weight of the animal or human. This approach has been used for oral doses for noncancer effects.
22
23 PODM/ BWM= 0.068 ng/0.035 kg-day = 1.9 ng/kg-day
24
25 DSF = 0.1/1.9 ng/kg-day = 0.051 (ug/kg-day) 1
26
27 Assumptions: The potency of point of contact skin tumors is related to bodyweight and
28 humans and mice would have an equal likelihood of developing skin tumors based on a dermal dose
29 per kg basis.
30
31 Issues: Skin cancer following benzo[a]pyrene exposure is a local effect and not likely
32 dependent on body weight.
33
34 Approach 4. Cross-species adjustment based on allometric scaling using body weight to the
35 3/4 power
36 Under this approach, rodents and humans exposed to the same daily dose of a carcinogen,
37 adjusted for BW3/4, would be expected to have equal lifetime risks of cancer. That is, a lifetime dose
38 expressed as |ig/kg3/4-day would lead to an equal risk in rodents and humans. This scaling reflects
39 the empirically observed phenomena of more rapid distribution, metabolism, and clearance in
40 smaller animals. The metabolism of benzo[a]pyrene to reactive intermediates is a critical step in
41 the carcinogenicity of benzo[a]pyrene, and this metabolism occurs in the skin.
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Toxicological Review ofbenzo[a]pyrene
1
2 POD (ug/day) = PODM (^g/day) x (BWH / BWM)3/4
3
4 POD (tig/day) = 0.068 ng/day x (70 kg / 0.035 kg)3/4
5 = 20.3[ig/day
6
7 DSF = 0.1/(20.3 Lig/day) * 0.0049 (ng/day)-1
8
9 Assumptions: Risk at low doses of benzo[a]pyrene is dependent on absolute dermal dose
10 and not dose per unit of skin, meaning a higher exposure concentration of benzo[a]pyrene
11 contacting a smaller area of exposed skin could carry the same risk of skin tumors as a lower
12 exposure concentration of benzo [a] pyrene that contacts a larger area of skin.
13
14 Issues: It is unclear if scaling of doses based on bodyweight ratios will correspond to
15 differences in metabolic processes in the skin of mice and humans.
16
17 Synthesis of the alternative approaches to cross-species scaling
18 A comparison of the above approaches is provided in Table C-27 below. The lifetime risk
19 from a nominal human dermal exposure to benzo [ajpyrene over a 5% area of exposed skin
20 (approximately 950 cm2), estimated at 1 x 10 ~4 [ig/day*, is calculated for each of the approaches in
21 order to judge whether the method yields risk estimates that are unrealistically high.
22
23 Other potential interspecies adjustments
24 The above discussion presents several mathematical approaches that result from varying
25 assumptions about what is the relevant dose metric for determining equivalence across species.
26 Biological information (that is not presently comprehensive or detailed enough to develop robust
27 models) that could be used in future biologically based models for cross-species extrapolation
28 include:
29 a. Quantitative information on interspecies differences in partitioning from exposure medium
30 to the skin and absorption through the skin
31 b. Thickness of the stratum corneum between anatomical sites and between species
32 c. Thickness of epidermal layer
33 d. Skin permeability
34 e. Metabolic activity of skin
35 f. Formation of DNA adducts in skin
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Table C-27. 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)
\1_» V V /
Assumptions
Equal mass per day (u.g /d), if applied to equal areas of skin (cm2), will affect similar
numbers of cells across species. Cancer risk is proportional to the area (cm2) exposed if
the loading rate (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
ug/day
DSF
1.3 per
u.g/day
128 per
u.g/cm2-day
f\ f\JI C n mr
0.045 per
us/ks-dav
r*o/ lxo « M y
0.0043 per u.g
/day
Risk at nominal
exposure
(0.0001 ng/day)*
1 x 10"4
7 x 10"7
6 x 10"8
4 x 10"7
2 * Nominal exposure calculated as a geometric mean of average daily doses (u.g/day) calculated from a range of benzo[a]pyrene soil concentrations (1-1000
3 ppb) reported from non-contaminated rural/agricultural soils (ATSDR, 1995) and a range of standard exposure assumptions.
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i APPENDIX D. SUMMARY OF EXTERNAL PEER
2 REVIEW AND PUBLIC COMMENTS AND EPA'S
3 DISPOSITION
4
5
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1
2 REFERENCES FOR APPENDICES
3 Multiple references published in the same year by the same author(s) have been assigned a letter
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47
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31 Cavalieri, EL; Higginbotham, S; RamaKrishna, NV; et al. (1991) Comparative dose-response tumorigenicity
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8 Chung, JY; Kim, JY; Kim, WR; et al. (2007) Abundance of aryl hydrocarbon receptor potentiates
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17 Colapietro, AM; Goodell, AL; Smart, RC. (1993) Characterization of benzo[a]pyrene-initiated mouse skin
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19
20 Cohen, GM; Haws, SM; Moore, BP; et al. (1976) Benzo(a)pyrene-3-yl hydrogen sulphate, a major ethyl acetate-
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24 Conney, AH; Chang, RL; Jerina, DM; et al. (1994) Studies on the metabolism of benzo [ajpyrene and dose-
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28 Cooper, AR; Moley, KH. (2008) Maternal tobacco use and its preimplantation effects on fertility: more reasons
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31 Cosma, GN; Marchok, AC. (1988) Benzo [ajpyrene- and formaldehyde-induced DNA damage and repair in rat
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33
34 Cosma, GN; Jamasbi, R; Marchok, AC. (1988) Growth inhibition and DNA damage induced by benzo [ajpyrene
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36
37 Craig-Holmes, AP; Shaw, MW. (1977) Effects of six carcinogens on SCE frequency and cell kinetics in cultured
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39
40 Crespi, CL; Altman, JD; Marietta, MA. (1985) Xenobiotic metabolism and mutation in a human lymphoblastoid
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