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www.epa.gov/iris
f/EPA
DEVELOPMENT OF A RELATIVE
POTENCY FACTOR (RPF) APPROACH
FOR POLYCYCLIC AROMATIC
HYDROCARBON (PAH) MIXTURES
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
February 2010
NOTICE
This document is an External Review draft. This information is distributed solely for the
purpose of pre-dissemination peer review under applicable information quality guidelines. It has
not been formally disseminated by EPA. It does not represent and should not be construed to
represent any Agency determination or policy. It is being circulated for review of its technical
accuracy and science policy implications.
U.S. Environmental Protection Agency
Washington, DC
-------
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 or recommendation for use.
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EXECUTIVE SUMMARY
The U.S. Environmental Protection Agency's (U.S. EPA's) Integrated Risk Information
System (IRIS) Program is releasing for scientific review a relative potency factor (RPF)
approach for polycyclic aromatic hydrocarbon (PAH) mixtures as one approach for assessing
cancer risk from exposure to PAH mixtures. The RPF analysis under review is not a
reassessment of individual PAH carcinogenicity, but rather provides a cancer risk estimate for
PAH mixtures by summing doses of component PAHs after scaling the doses (with RPFs)
relative to the potency of an index PAH (i.e., benzo[a]pyrene). The cancer risk is then estimated
using the dose-response curve for the index PAH. RPFs for seven individual PAHs were
developed in the U.S. EPA (1993) Provisional Guidance for Quantitative Risk Assessment of
PAHs (Provisional Guidance} and are utilized extensively within U.S. EPA program offices and
other regulatory agencies. The RPF analysis provided in the current report includes more recent
data and an analysis of both tumorigenicity and genotoxicity data for PAHs.
The Supplemental Guidance for Conducting Health Risk Assessment of Chemical
Mixtures (U.S. EPA, 2000) indicates that approaches based on whole mixtures are preferred to
component approaches, such as the RPF approach. Risk assessment approaches based on
toxicity evaluations of whole mixtures inherently address specific interactions among PAHs and
account for the toxicity of unidentified components of PAH mixtures. They also do not require
assumptions regarding the toxicity of individual components (e.g., dose additivity or response
additivity). While whole mixture assessment is preferred, there are challenges associated with
using these approaches. There are very few toxicity data available for whole PAH mixtures and,
in most cases, chemical analyses of the composition of mixtures are limited. In addition, PAH-
containing mixtures tend to be very complex; the composition of these mixtures appears to vary
across sources releasing these mixtures to the environment and in various environmental media
in which they occur. For these reasons, a whole mixtures approach may not always be
practicable for risk assessment purposes. This report provides recommendations for
development of the RPF approach for PAH mixtures health risk assessment and includes:
(1) A rationale for recommending an RPF approach (Chapter 2);
(2) A summary of previous approaches for developing the RPF approach for PAHs
(Chapter 3);
(3) An evaluation of the carcinogenicity of individual PAHs (Chapter 4);
(4) Methods for dose-response assessment and individual study RPF calculation (Chapter 5);
(5) Selection of PAHs for inclusion in the RPF approach (Chapter 6);
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(6) Derivation of RPFs for selected PAHs (Chapter 7); and
(7) Characterization of strengths, weaknesses, and uncertainties associated with the RPF
approach to PAH cancer risk assessment (Chapter 8).
The RPF approach involves two key assumptions related to the application of a dose-
additivity model: (1) a imilar toxicological action of PAH components in the mixture; and
(2) interactions among PAH mixture components do not occur at low levels of exposure typically
encountered in the environment. Mechanistic studies indicate that the mutagenic and tumor-
initiating activity of carcinogenic PAHs requires metabolic activation to reactive intermediates
(e.g., dihydrodiol epoxides, quinones, radical cations), which covalently modify
deoxyribonucleic acid (DNA) targets resulting in mutation, and that tumor promotion and
progression phases may involve parent compound binding to the Ah receptor (AhR) and
subsequent alterations of gene expression or a cell proliferation response to metabolite
cytotoxicity (see Section 2.4, Similarities in Mode of Carcinogenic Action for PAHs, and
Figure 2-3, Overview of the proposed key events in the mode of action for PAH
carcinogenicity). As such, there is evidence that an assumption of a similar toxicological action
is reasonable; however, the carcinogenic process for individual PAHs is likely related to some
unique combination of multiple molecular events resulting from the formation of several reactive
species. The second assumption of no interactions at low levels of exposure is also reasonable,
but cannot be conclusively demonstrated in experimental systems (see Section 2.8, Dose
Additivity of PAHs in Combined Exposures). Use of the RPF approach assumes that doses of
component chemicals that act in a similar manner can be added together, after scaling the
potencies relative to the index chemical. The assumptions of toxicological similarity and no
interaction effects at low environmental exposure levels that are inherent in the dose-additivity
model are generally supported by the experimental data for PAHs (see Sections 2.4 and 2.7).
Several approaches have been used previously for the determination of RPFs for PAHs
(see Chapter 3). In the published literature, RPF values were proposed in at least one analysis
for a total of 27 PAHs (see Table 3-1). Because these approaches generally relied on similar
bioassay data and modeling methods, the resulting RPF values are considered comparable for
most PAHs across analyses.
There is a large PAH database on carcinogenicity in animal bioassays, genotoxicity in
various test systems, and bioactivation to tumorigenic and/or genotoxic metabolic intermediates.
The RPF analysis presented here includes only unsubstituted PAHs with three or more fused
aromatic rings containing only carbon and hydrogen atoms, because these are the most widely
studied members of the PAH chemical class. The study types that were considered most useful
for RPF derivation were rodent carcinogenicity bioassays (all routes) in which one or more PAH
was tested at the same time as benzo[a]pyrene. In addition, in vivo and in vitro data for cancer-
related endpoints in which one or more PAH and benzo[a]pyrene was tested simultaneously were
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obtained, including studies on the formation of DNA adducts, mutagenicity, chromosomal
aberrations, sister chromatid exchange frequency, aneuploidy, DNA damage/repair/
recombination, unscheduled DNA synthesis, and cell transformation. Although it would be
possible to calculate RPFs from studies where a PAH and benzo[a]pyrene were tested by the
same laboratory using the same test system but at different times, this approach was not
considered because it could introduce differences in the dose-response information that are
unrelated to the chemical (e.g., variability associated with laboratory environment conditions,
animal handling, food supply, etc.). Thus, studies in which benzo[a]pyrene was not tested
simultaneously with another PAH were not considered in the RPF calculations.
Studies of AhR binding/activation were not considered for use in deriving RPFs because
there does not appear to be a clear relationship between affinity for the AhR and carcinogenic
potency. For example, highly mutagenic fjord-region PAHs are potent carcinogens despite
exhibiting lower AhR affinity (reviewed by Bostrom et al., 2002). Likewise, some PAHs that
strongly activate the AhR, such as benzo[k]fluoranthene (Machala et al., 2001), are only weakly
carcinogenic. In addition, some studies have demonstrated the formation of DNA adducts in the
liver of AhR knock-out mice following intraperitoneal or oral exposure to benzo[a]pyrene
(Sagredo et al., 2006; Uno et al., 2006; Kondraganti et al., 2003), indicating that Ah
responsiveness is not strictly required for metabolic activation and genotoxicity. These findings
suggest that there may be alternative (i.e., non-AhR-mediated) mechanisms of benzo[a]pyrene
activation in the mouse liver, and that AhR affinity would not be a good predictor of
carcinogenic potency. Also, several studies indicate that AhR-mediated CYP1 Al induction
potency does not correlate well with carcinogenic potency. These studies compared CYP1 Al
induction potency for several PAHs using assays to measure ethoxyresorufm O-deethylase
(EROD) activity, CYP1 Al protein, and messenger ribonucleic acid (mRNA) levels, or chemical-
activated luciferase reporter gene expression (Bosveld et al., 2002; Machala et al., 2001; Bols et
al., 1999; Till et al., 1999; Willett et al., 1997).
Several study types were excluded from the database because they did not provide
carcinogenicity or cancer-related endpoint information for individual PAHs. These include
biomarker studies measuring DNA adducts in humans, studies of PAH metabolism, and studies
of PAH mixtures. Although these studies contain important information on human exposure to
PAH mixtures and the mode of action for PAH toxicity, they generally do not contain dose-
response information that would be useful for calculation of RPF estimates.
A database of primary literature relevant to the RPF approach for PAHs was developed by
performing a comprehensive review of the scientific literature dating from the 1950s through
2009 on the carcinogenicity and genotoxicity of PAHs. The search identified over 900 individual
publications for a target list of 74 PAHs (see Table 2-1) that have been identified in
environmental media or for which toxicological data are available. Review of these publications
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resulted in the identification of more than 600 papers that included carcinogenicity or cancer-
related endpoint data on at least one PAH and benzo[a]pyrene tested at the same time.
References in the PAH database were sorted into the following major categories: cancer
bioassays, in vivo studies of cancer-related endpoints, and in vitro studies of cancer-related
endpoints. These categories were further sorted by route (for bioassays) or by endpoint (for
cancer-related endpoints). Each study was reviewed, and critical study details were extracted
into tables for each individual endpoint (see Chapter 4). The tables also include an initial
determination of whether the data from each study meet selection criteria for use in the RPF
analysis. Studies with data on selected PAHs and benzo[a]pyrene were considered for RPF
determination, even if a particular PAH has not been classified by U.S. EPA or International
Agency for Research on Cancer (IARC) as a carcinogen. Studies were included in the analysis if
the following selection criteria were met:
• Benzo[a]pyrene was tested simultaneously with another PAH;
• A statistically increased incidence of tumors was observed with benzo[a]pyrene
administration, compared with control incidence;
• Benzo[a]pyrene produced a statistically significant change in a cancer-related
endpoint finding;
• Quantitative results were presented;
• The carcinogenic response observed in either the benzo[a]pyrene- or other PAH-
treated animals at the lowest dose level was not saturated (i.e., tumor incidence at the
lowest dose was <90%), with the exception of tumor multiplicity findings; and
• There were no study quality concerns or potential confounding factors that precluded
use (e.g., no concurrent control, different vehicles, strains, etc. were used for the
tested PAH and benzo[a]pyrene; use of cocarcinogenic vehicle; PAHs of questionable
purity; unexplained mortality in treated or control animals).
If the above criteria were met, studies were selected for use in the analysis regardless of
whether positive or nonpositive results were reported. Studies with positive findings were used
for calculation of RPFs. Studies with nonpositive findings were used in a weight of evidence
evaluation to select PAHs for inclusion in the RPF approach (see Section 6.1).
Dose-response data were extracted from studies with positive findings that met selection
criteria. For studies that reported results graphically, individual data points were extracted using
digitizing software. In all, over 300 data sets were extracted, reflecting dose-response data from
at least one study for 51 of the 74 PAHs included in the analysis. All of the extracted data are
presented in Appendix C of this report.
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While tumor multiplicity data from tumor bioassays are not generally used to estimate
cancer potency., these data were included in the dose-response assessment in order to determine
whether they could serve as a reliable measure of relative cancer potency. Several bioassays
reported data on both tumor incidence and tumor number, providing information that was later
used to compare relative potencies estimated from these two endpoints. Statistical analyses were
performed on tumor bioassay data to determine whether the tumor incidence or multiplicity
observed at a particular dose represented a statistically significant increase over controls. If
statistical analyses were not described in the original report, incidence data were analyzed using
Fisher's exact test and the Cochran-Armitage trend test. Positive findings were indicated by a
significant (p < 0.05) difference for at least one dose group by comparison to control (in Fisher's
exact or an equivalent test) or a significant dose-response trend (Cochran-Armitage or
equivalent) for multidose studies. For tumor bioassay data reported as tumor count, a t-test was
conducted (when variance data were available) to determine whether the count was significantly
different from control (p < 0.05). The results of the statistical analyses are shown with the dose-
response data in Appendix C. Statistical analyses of the cancer-related endpoint data were not
conducted; the study author's conclusions as to response (positive or nonpositive) was used.
Chapter 5 describes the methods used for both the dose-response assessment and the RPF
calculation in detail. The general equation for estimating an RPF was the ratio of the slope of the
dose-response curve for the subject PAH to the slope of the dose-response curve for
benzo[a]pyrene. For bioassay data, tumor incidences were modeled using the multistage model
within the U.S. EPA Benchmark Dose (BMD) Software (Version 1.3.2). For cancer-related
endpoint data in quantal form, this model was also used; for continuous data (either tumor
multiplicity or cancer-related endpoint data), the simplest continuous model (linear) within the
software was applied. Whenever the data allowed, benchmark response (BMR) values of 10%
for quantal data and 1 standard deviation (SD) from the control value for continuous data were
used to calculate the slope by linear extrapolation to the origin for consistency across data sets.
Alternative BMR values were used in select instances, as described in Section 5.3. For data sets
that included only a single dose, or those for which no model fit was achieved with the selected
models, a point estimate RPF1 was calculated. As Table G-2 indicates, final RPFs for five
compounds (benz[a]anthracene, benz[b,c]aceanthrylene, benz[j]aceanthrylene,
dibenzo[a,h]pyrene, and naphtho[2,3-e]pyrene) are based exclusively on point estimates; the
remaining 19 PAHs had at least one dataset that could be modeled (see Appendix G).
The RPFs calculated from individual studies for each PAH were used in a weight of
evidence evaluation to select PAHs for inclusion in the RPF approach (see Chapter 6) and in the
derivation of a final RPF for each compound (Chapter 7). The selection of PAHs to be included
JFor the purpose of this report, the term "point estimate RPF" is used to describe an RPF calculated from a single
point on the dose-response curve for both the PAH of interest and benzo[a]pyrene. This term distinguishes the RPF
from one calculated using a BMD modeling result from multidose data.
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in the RPF approach began with an evaluation of whether the available data were adequate to
assess the carcinogenicity of each compound. At least one RPF value was calculated for each of
51 PAHs. For 16 of these compounds, only a single RPF value derived from an in vitro cancer-
related endpoint (primarily mutagenicity assays) was available (see Table 6-1). Due to the
limited data available for these 16 compounds, no further evaluation of these PAHs was
conducted, and they were not selected for inclusion in the RPF approach.
For the remaining 35 PAHs, a weight of evidence evaluation (see Figure 6-1) was
conducted to assess the evidence that each PAH could induce a carcinogenic response. For the
purposes of this analysis, PAHs were assumed to be carcinogenic due to toxicological similarity
to the indicator compound, benzo[a]pyrene. The weight of evidence approach was developed to
determine whether the available information for each PAH was adequate for inclusion in the RPF
approach. If the data were not considered adequate, then the PAH was excluded. In vivo tumor
bioassays that included benzo[a]pyrene were given the greatest weight in assessing the
carcinogenicity of a given PAH; data from other bioassays and cancer-related endpoint studies
were used to supplement the weight of evidence when the bioassay data that included
benzo[a]pyrene were conflicting or nonpositive. Structural alerts for PAH carcinogenicity or
mutagenicity (as defined in Section 2.5 as the presence of a classic bay or fjord region in a PAH
containing at least four benzene rings) were noted in the evaluation for each PAH, but were not
used explicitly in the weight of evidence evaluation.
The weight of evidence evaluation (Chapter 6) indicated that the available data were
adequate to determine that 24 of the 35 PAHs were carcinogenic, that 3 PAHs (anthracene,
phenanthrene, and pyrene) were not carcinogenic, and that data were inadequate to evaluate the
carcinogenicity for 8 PAHs. The eight PAHs with inadequate data were excluded from the RPF
approach. For the three PAHs for which there were sufficient data to conclude that they were not
carcinogenic (i.e., robust nonpositive tumor bioassay data and cancer-related endpoint data), a
final RPF of zero was recommended. While there is little quantitative difference between
selecting a final RPF of zero for a given PAH and excluding that PAH from the RPF approach,
this is an important distinction for uncertainty analysis. There is substantial uncertainty in the
risk associated with PAHs that are excluded from the RPF approach due to inadequate data;
these compounds could be of low or high potency. However, for PAHs with an RPF of zero,
there is evidence to suggest that these compounds are not carcinogenic, and the uncertainty
associated with the cancer risk for these compounds is markedly reduced.
For each of the remaining 24 compounds, a final nonzero RPF was derived. A number of
options were considered for deriving an RPF from among the numerous values calculated for
each individual PAH. These options included: prioritizing bioassay RPFs from different
exposure routes based on environmentally relevant routes; prioritizing bioassay RPFs based on
target organs considered relevant to human susceptibility to PAH carcinogenesis; prioritizing
RPFs based on quality of the underlying study; prioritizing cancer-related endpoints by their
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correlation with bioassay potency (i.e., ability to predict bioassay potency); and aggregating
RPFs across all bioassays, across all cancer-related endpoints, or across all endpoints. In the
end, it was concluded that the available data did not provide a clear scientific basis for
prioritizing RPFs except for a preference for bioassay data over cancer-related endpoints. As a
consequence, final RPFs were derived from bioassay data for any PAH that had at least one RPF
based on a bioassay.
For each carcinogenic PAH with bioassay data, the average RPF was calculated from
bioassays with positive results. For those PAHs that did not have an estimated RPF based on a
bioassay, but for which the weight of evidence evaluation indicated a carcinogenic response
(e.g., dibenz[a,c]anthracene), the final RPF was calculated from all cancer-related endpoint
studies with positive results. In both cases, nonpositive results were not included in the
calculation. The final RPF for each PAH was reported to one significant figure. The range of
RPF values was also reported. Presenting the RPFs in this manner provides an average and
maximum estimate for each PAH that has data from multiple studies.
Several options were considered for the determination of final RPFs (e.g., arithmetic
mean, geometric mean, weighted average, maximum, or order of magnitude estimates). The
arithmetic mean and range were chosen as a simple approach to describing the calculated RPF
values available for each PAH. Other estimates were not considered appropriate due to the
limited number of RPF values calculated for most PAHs and the variability in the RPF estimates.
Most PAHs (18/24, 73%) had <3 calculated RPF values and the range of RPF values was greater
than an order of magnitude for several compounds (7/24 PAHs). The variability in RPF
estimates is likely due to differences in study design parameters (e.g., route, species/strain,
exposure duration, exposure during sensitive time periods, initiation versus promotion and
complete carcinogenesis protocols, tumor incidence versus multiplicity reporting) and dose-
response methods (modeled versus point estimates). Calculation of a weighted average was not
possible because there is no clear scientific rationale for choosing among study types or tumor
data outcomes. Providing order of magnitude estimates, as has been previously done for
estimating RPFs for PAHs, was not considered to be superior to calculating simple means.
Including the range in the estimated RPFs was considered to be informative to the user for
characterizing uncertainty.
Once a final RPF was derived for a given PAH, the resulting value was assigned a
relative confidence rating of high, medium, or low confidence. The relative confidence rating
characterized the nature of the database upon which the final RPF was based. Confidence
rankings were based on the robustness of the database. For final RPFs based on tumor bioassay
data, confidence ratings considered both the available tumor bioassays and the availability of
supporting data for cancer-related endpoints. The most important factors that were considered
included the availability of in vivo data and whether multiple exposure routes were represented.
Other database characteristics that were considered included the availability of more than one in
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vivo study, and whether effects were evident in more than one sex or species. Very low relative
confidence was reserved for final RPFs based on cancer-related endpoint data only (e.g.,
dibenz[a,c]anthracene). An RPF of zero was only applied if the data implied high or medium
relative confidence.
Table 1 shows the average RPFs based on tumor bioassay data with their associated range
and relative confidence ratings, and an overview of the tumor bioassay database (total number of
studies, exposure routes tested, species tested, and sexes tested) for each PAH. Table 2 shows
the average RPF for dibenz[a,c]anthracene, the only RPF based on cancer-related endpoint data,
with its associated range, relative confidence rating, and an overview of the database for this
compound.
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Table 1. PAHs with final RTFs based on tumor bioassay data
PAH
Anthanthrene
Anthracene
B enz [a] anthracene
Benz[b,c]aceanthrylene, 11H-
Benzo [bjfluoranthene
Benzo [c]fluorene
Benz [e] aceanthry lene
Benzo [g,h,i]pery lene
Benz [j ] aceanthry lene
Benzo [j]fluoranthene
Benzo [kjfluoranthene
Benz [1] aceanthry lene
Chrysene
Cyclopenta[c,d]pyrene
Cyclopenta[d,e,f]chrysene, 4H-
Dibenzo [a,e]fluoranthene
Dibenzo [a,e]py rene
Dibenz [a,h] anthracene
Dibenzo [a,h]pyrene
Dibenzo [a,i]py rene
Dibenzo [a,l]py rene
Fluoranthene
Indeno [ 1,2,3 -c,d]pyrene
Naphtho[2,3-e]pyrene
Phenanthrene
Pyrene
Average
RPF
0.4
0
0.2
0.05
0.8
20
0.8
0.009
60
0.3
0.03
5
0.1
0.4
0.3
0.9
0.4
10
0.9
0.6
30
0.08
0.07
0.3
0
0
Range of
RPFs
0.2-0.5
0
0.02-0.4
0.05
0.1-2
1-50
0.6-0.9
0.009
60
0.01-1
0.03-0.03
4-7
0.04-0.2
0.07-1
0.2-0.5
0.7-1
0.3-0.4
l^K)
0.9
0.5-0.7
10-40
0.009-0.2
0.07
0.3
0
0
Relative
confidence
Medium
Medium3
Medium
Low
High
Medium
Low
Low
Low
High
Medium
Low
High
Medium
Low
Low
Low
High
Low
Low
Medium
Low
Low
Low
High
Medium
Number of
datasets
2
1 (nonpositive)
3
1
5
2
2
1
1
5
2
2
7
5
2
2
2
3
1
2
3
5
1
1
3 (nonpositive)
7 (nonpositive)
Exposure routes tested
Dermal, lung implantation
Dermal
Dermal, intraperitoneal
Dermal
Dermal, intraperitoneal, lung implantation
Oral, intraperitoneal
Dermal
Lung implantation
Intraperitoneal
Dermal, intraperitoneal, lung implantation
Dermal, lung implantation
Dermal
Dermal, intraperitoneal, lung implantation
Dermal, intraperitoneal
Dermal
Dermal
Dermal
Dermal, intraperitoneal, lung implantation
Dermal
Dermal
Dermal, intraperitoneal
Intraperitoneal
Lung implantation
Dermal
Dermal, intraperitoneal, lung implantation
Dermal, intraperitoneal
Species
tested
Mouse, rat
Mouse
Mouse
Mouse
Mouse, rat
Mouse
Mouse
Rat
Mouse
Mouse, rat
Mouse, rat
Mouse
Mouse, rat
Mouse
Mouse
Mouse
Mouse
Mouse, rat
Mouse
Mouse
Mouse
Mouse
Rat
Mouse
Mouse, rat
Mouse
Sexes tested
Female
Female
Female, male
Female
Female, male
Female
Female, male
Female
Male
Female, male
Female
Female, male
Female, male
Female, male
Female
Female
Female
Female, male
Female
Female
Female, male
Female, male
Female
Female
Female, male
Female, male
""Reflects availability of data from anthracene exposure via another exposure route in a study that did not include benzo[a]pyrene.
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Table 2. PAHs with final RTFs based on cancer-related endpoint data
(no tumor bioassay data available)
PAH
Dibenz[a,c]anthracene
Average
RPF
4
Range of
RPFs
0.04-50
Relative
confidence
Very low
Types of studies
Total =14 studies
One in vivo DNA adduct
Six in vitro bacterial
mutagenicity
One in vitro mammalian
mutagenicity
One in vitro morphological/
malignant transformation
Three in vitro DNA damage
Two in vitro DNA adducts
Multiple dose studies
Total = 6 studies
Four in vitro bacterial
mutagenicity
One in vitro DNA
damage
One in vitro DNA
adduct
The cancer risk for a PAH mixture of concern is determined by multiplying the
benzo[a]pyrene equivalent dose or concentration by the benzo[a]pyrene cancer toxicity value
(e.g., oral slope factor). Benzo[a]pyrene equivalents are calculated by multiplying the
concentration (or dose) of a particular PAH component in the mixture by its RPF. The proposed
RPF approach considers each of the bioassay types used for RPF derivation to be equivalent for
the purpose of determining relative potency to benzo[a]pyrene.
According to the Supplemental Guidance for Assessing Susceptibility from Early-Life
Exposure to Carcinogens (U.S. EPA, 2005b), benzo[a]pyrene is carcinogenic by a mutagenic
mode of action. A common mutagenic mode of action for other carcinogenic PAHs is
hypothesized based on information available for the indicator chemical, benzo[a]pyrene (U.S.
EPA, 2005b). When assessing PAH cancer risks for lifestages under 16 years of age, or for
lifetime exposures that include early-life exposures, the RPF values should be applied with
specific exposure information to the benzo[a]pyrene cancer risk estimates including adjustment
for early-life susceptibility, through the application of age-dependent adjustment factors
(ADAFs).
A description of uncertainties and limitations is crucial to interpretation of the RPF
approach for PAH mixtures risk assessment (see Chapter 8). Many of the general uncertainties
related to chemical-specific risk assessment are also applicable to the proposed RPF approach for
PAHs (e.g., appropriateness of animal models, low-dose and interspecies extrapolation,
variability within the human population). Use of a component-based approach for mixtures risk
assessment leads to additional uncertainties related to adequate characterization of the mixture
and the potential interactions that may occur between individual components within the mixture
(i.e., PAHs and other chemicals). The RPF approach is limited by the small number of PAHs for
which there are analytical chemistry and toxicology data, and thus may result in underestimation
of actual cancer risks from complex PAH mixtures. There are uncertainties and limitations
related to the size and nature of the PAH database, the human relevance of animal data,
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assumptions regarding mode of action and dose additivity, and cross-route extrapolation.
Specific uncertainties that are related to dose-response assessment (i.e., calculation of RPFs) and
the selection of single RPF values for each PAH are also discussed in Chapter 8.
In summary, the current analysis represents a significant improvement upon the previous
component-based approaches for PAH mixtures risk assessment. One of the most important
improvements is the consideration of data from a comprehensive review of the scientific
literature dating from the 1950s through 2008 on the carcinogenicity and genotoxicity of PAHs.
The search identified over 900 individual publications for a target list of 74 PAHs that have been
identified in environmental media and for which toxicological data are available. Review of
these publications resulted in the identification of more than 600 papers that included
carcinogenicity or cancer-related endpoint data on at least one PAH and benzo[a]pyrene tested at
the same time. Dose-response data were extracted, and RPFs from individual studies were
calculated from over 300 data sets representing 51 individual PAHs. For 35 compounds, a
weight of evidence evaluation was conducted to select PAHs for inclusion in the RPF approach;
data were inadequate to conduct such an evaluation for the remaining 16 compounds. A final
RPF was derived for each PAH based on tumor bioassay data (if available) or cancer-related
endpoint data (if no tumor bioassay RPFs were available). Final RPFs were derived for
27 PAHs, significantly increasing the number of PAHs that can be addressed through this
approach. Each RPF was assigned a relative confidence rating reflecting the nature of the tumor
bioassay or cancer-related endpoint database that was used to derive the final RPF for that PAH.
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CONTENTS
LIST OF TABLES xvii
LIST OF FIGURES xxi
LIST OF ABBREVIATIONS AND ACRONYMS xxiv
AUTHORS, CONTRIBUTORS, AND REVIEWERS xxvi
1. BACKGROUND FOR THE DEVELOPMENT OF RELATIVE POTENCY FACTOR
APPROACH FOR PAH MIXTURES HEALTH ASSESSMENT 1
2. RATIONALE FOR RECOMMENDING AN RPF APPROACH 2
2.1. PAHs AS A CHEMICAL CLASS 4
2.2. THE TOXICOLOGICAL DATABASE FOR PAHs 20
2.3. BENZO[A]PYRENE AS AN INDEX CHEMICAL 21
2.4. SIMILARITIES IN MODE OF CARCINOGENIC ACTION FOR PAHs 23
2.5. STRUCTURAL ALERTS FORPAH CARCINOGENESIS 34
2.6. SIMILARITIES IN RELATIVE POTENCY ACROSS ENDPOINTS 35
2.7. SIMILARITIES IN RELATIVE POTENCY ESTIMATES ACROSS SPECIES
AND EXPOSURE ROUTES 37
2.8. DOSE ADDITIVITY OF PAHs IN COMBINED EXPOSURES 38
3. DISCUS SIGN OF PREVIOUSLY PUBLISHED RPF APPRO ACHES 44
3.1. PREVIOUS EFFORTS TO VALIDATE THE RPF APPROACH 52
4. EVALUATION OF THE CARCINOGENICITY OF INDIVIDUAL PAHs 55
4.1. DATABASE OF STUDIES ON PAH CARCINOGENICITY AND CANCER-
RELATED ENDPOINTS 55
4.2. STUDIES IN HUMANS 57
4.3. STUDIES IN ANIMALS 57
4.3.1. In Vivo Cancer Bioassays in Animals 86
4.3.1.1. Dermal Exposure 86
4.3.1.2. Intraperitoneal Exposure 89
4.3.1.3. Subcutaneous Injection Exposure 90
4.3.1.4. Oral Exposure 91
4.3.1.5. Other Routes 92
4.3.2. In Vivo Studies of Cancer-Related Endpoints 93
4.3.2.1. DNAAdducts 93
4.3.2.2. Clastogenicity or Sister Chromatid Exchange Frequency 95
4.3.2.3. In Vivo Mutagenicity 96
4.3.3. In Vitro Studies of Cancer-Related Endpoints 97
4.3.3.1. Bacterial Mutageni city 97
4.3.3.2. Mammalian Mutageni city 98
4.3.3.3. Morphological/Malignant Cell Transformation 99
4.3.3.4. DNAAdducts 100
4.3.3.5. DNADamage/Repair 101
4.3.3.6. Clastogenicity or Sister Chromatid Exchange Frequency 102
4.4. SUMMARY OF INFORMATION AVAILABLE TO DEVELOP RPFs FOR
INDIVIDUAL PAHs 103
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5. METHODS FOR DOSE-RESPONSE ASSESSMENT AND RPF CALCULATION 104
5.1. CHOICE OF DOSE-RESPONSE DATA 104
5.1.1. Dose-Response Data for Tumor Bioassays 104
5.1.2. Dose-Response Data for Cancer-Related Endpoint Studies 105
5.2. OVERALL FORM OF RPF ESTIMATE 106
5.3. RPF CALCULATION FOR MULTIDOSE DATASETS 106
5.4. RPF CALCULATION FOR SINGLE DOSE DATASETS 108
5.5. DOSE CONVERSION FOR RPF CALCULATION 109
5.6. SPECIAL CONSIDERATIONS FOR RPF CALCULATION USING TUMOR
BIOASSAYDATA 110
5.7. SPECIAL CONSIDERATIONS FOR RPF CALCULATION USING CANCER-
RELATED ENDPOINT DATA Ill
6. SELECTION OF PAHs FOR INCLUSION IN RELATIVE POTENCY APPROACH 113
6.1. METHOD FOR SELECTING PAHs FOR INCLUSION IN RELATIVE POTENCY
APPROACH 114
6.2. WEIGHT OF EVIDENCE EVALUATION FOR 35 INDIVIDUAL PAHs 117
7. DERIVATION OF FINAL RPFs FOR SELECTED PAHs 190
7.1. METHODS FOR DERIVING FINAL RPFs 190
7.2. CONFIDENCE RATINGS FOR FINAL RPFs 194
7.3. APPLICATION OF RPFs FOR ASSESSING CANCER RISKS FROM EXPOSURE
TO PAH MIXTURES 196
7.4. SUSCEPTIBILITY FROM EARLY LIFE EXPOSURE TO CARCINOGENS 196
8. UNCERTAINTIES AND LIMITATIONS ASSOCIATED WITH THE RPF APPROACH
198
8.1. DOSE-RESPONSE ASSESSMENT FOR INDIVIDUAL PAHs 199
8.2. SELECTION OF PAHs FOR INCLUSION IN RPF APPROACH 201
8.3. DERIVATION OF A FINAL RPF FOR EACH PAH 203
8.4. USE OF ANIMAL DATA TO PREDICT HUMAN CANCER RISK FOR PAHs 208
8.5. ASSUMPTIONS OF A COMMON MODE OF ACTION AND DOSE ADDITIVITY 210
8.6. EXTRAPOLATION OF RPFs ACROSS EXPOSURE ROUTES 211
9. REFERENCES 218
APPENDIX A. SECONDARY SOURCES REVIEWED FOR IDENTIFICATION OF
PRIMARY LITERATURE A-l
APPENDIX B. BIBLIOGRAPHY OF STUDIES WITHOUT BENZO[A]PYRENE AS
A REFERENCE COMPOUND B-l
B.I. BIBLIOGRAPHY OF BIOASSAYS WITHOUT BENZO[A]PYRENE B-4
B.2. BIBLIOGRAPHY OF STUDIES ON CANCER-RELATED ENDPOINTS
WITHOUT BENZO[A]PYRENE B-10
APPENDIX C. DOSE-RESPONSE DATA FOR POTENCY CALCULATIONS C-l
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APPENDIX D. BENCHMARK DOSE MODELING OUTPUTS D-l
D.I. DERMAL BIO AS SAYS D-l
D.2. INTRAPERITONEAL BIOASSAYS D-43
D.3. LUNG IMPLANTATION BIO AS SAYS D-82
D.5. BACTERIAL MUTAGENICITY D-l 17
D.6. MAMMALIAN MUTAGENICITY D-126
D.7. MALIGNANT TRANSFORMATION D-154
D.8. IN VITRO DNA DAMAGE D-186
APPENDIX E. CALCULATION OF RPFs E-l
APPENDIX F. EXAMPLE CALCULATION OF RPF DETECTION LIMIT F-l
APPENDIX G: EVALUATION OF ALTERNATIVES FOR RANKING RPFs G-1
G.I. OPTIONS FOR RANKING TUMOR BIO AS SAY RPFs G-l
G.2. RANKING NONBIO AS SAY DAT A G-7
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LIST OF TABLES
1. PAHs with final RPFs based on tumor bioassay data xi
2. PAHs with final RPFs based on cancer-related endpoint data (no tumor bioassay data
available) xii
2-1. PAHs evaluated in the RPF analysis 5
2-2. Studies of binary mixtures of PAHs and tumorigenicity 40
3-1. Comparison among various relative potency estimates for PAHs from the published
literature and regulatory agencies (1984-2004) 45
4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAH 59
4-2. Study summaries: intraperitoneal bioassays of benzo[a]pyrene and at least one other
PAH 63
4-3. Study summaries: subcutaneous bioassays of benzo[a]pyrene and at least one other
PAH 65
4-4. Study summaries: oral bioassays of benzo[a]pyrene and at least one other PAH 66
4-5. Study summaries: other route bioassays of benzo[a]pyrene and at least one other
PAH 67
4-6. Study summaries: in vivo DNA adducts with benzo[a]pyrene and at least one other
PAH 68
4-7. Study summaries: in vivo clastogenicity or sister chromatid exchange with
benzo[a]pyrene and at least one other PAH 70
4-8. Study summaries: in vivo mutagenicity with benzo[a]pyrene and at least one other
PAH 72
4-9. Study summaries: in vitro bacterial mutagenicity with benzo[a]pyrene and at least one
other PAH 73
4-10. Study summaries: in vitro mammalian mutagenicity assays with benzo[a]pyrene
and at least one other PAH 76
4-11. Study summaries: in vitro morphological/malignant cell transformation with
benzo[a]pyrene and at least one other PAH 79
4-12. Study summaries: in vitro DNA adducts with benzo[a]pyrene and at least one
other PAH 81
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4-13. Study summaries: in vitro DNA damage, repair, or synthesis with benzo[a]pyrene
and at least one other PAH 82
4-14. Study summaries: in vitro clastogenicity or sister chromatid exchange with
benzo[a]pyrene and at least one other PAH 84
5-1. Comparison between molar and mass-based RPF 110
6-1. PAHs with only one RPF from a single in vitro cancer-related endpoint study and
excluded from RPF approach 113
6-2. Results of weight of evidence evaluation for 27 PAHs selected for inclusion in the
RPF approach 118
7-1. Final RPFs based on tumor bioassay data 193
7-2. Final RPFs based on cancer-related endpoint data (no tumor bioassay data available) 194
7-3. Relative confidence ratings for RPFs 195
7-4. Sample calculation of estimated cancer risk for benz[a]anthracene with the
application of ADAFs 197
8-1. Results of simple linear regression of log-transformed average tumor bioassay
RPF versus log average genotoxicity RPF 206
8-2. PAHs with RPFs of varying relative confidence 207
8-3. Comparisons among average tumor bioassay RPF values by exposure route and
target organ 213
B-l. Bioassays with and without benzo[a]pyrene by PAH B-2
C-l. Dermal bioassays: dose-response information for incidence data C-2
C-2. Dermal bioassays: dose-response information for tumor multiplicity C-8
C-3. Intraperitoneal bioassays: dose-response information for incidence data C-l3
C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicity C-23
C-5. Lung implantation bioassays: dose-response information for incidence data C-27
C-6. Oral bioassays: dose-response information for incidence data C-31
C-7. Oral bioassays: dose-response information for tumor multiplicity C-31
C-8. In vitro bacterial mutagenicity: data use C-32
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C-9. In vitro bacterial mutagenicity: dose-response data C-35
C-10. In vitro mammalian mutagenicity: data use C-53
C-ll. In vitro mammalian mutagenicity: dose-response data C-56
C-12. In vitro malignant/morphological cell transformation: data use C-64
C-13. In vitro malignant/morphological cell transformation: dose-response data C-66
C-14. In vitro DNA adducts: data use C-74
C-15. In vitro DNA adducts: dose-response data C-75
C-16. In vitro DNA damage: data use C-78
C-ll. In vitro DNA damage: dose-response data C-79
C-18. In vitro clastogenicity: data use C-84
C-19. In vitro clastogenicity: dose-response data C-85
C-20. In vivo DNA adducts: data use C-86
C-21. In vivo DNA adducts: dose-response data C-87
C-22. In vivo clastogenicity: data use C-95
C-23. In vivo clastogenicity: dose-response data C-96
E-l. Dermal bioassays: RPF calculations for incidence data E-2
E-2. Dermal bioassays: RPF calculations for multiplicity data E-5
E-3. Intraperitoneal bioassays: RPF calculations for incidence data E-6
E-4. Intraperitoneal bioassays: RPF calculations for multiplicity data E-8
E-5. Lung implantation bioassays: RPF calculations (incidence data) E-9
E-6. Oral bioassays: RPF calculations (incidence and multiplicity data) E-10
E-7. In vivo DNA adducts: RPF calculations E-ll
E-8. In vivo clastogenicity or sister chromatid exchange: RPF calculation E-14
E-9. In vitro bacterial mutagenicity: RPF calculations E-16
E-10. In vitro mammalian mutagenicity: RPF calculations E-22
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E-ll. In vitro morphological/malignant transformation: RPF calculation E-25
E-12. In vitro DNAadducts: RPF calculations E-27
E-13. In vitro DNA damage: RPF calculations E-29
E-14. In vitro clastogenicity or sister chromatid exchange: RPF calculations E-31
F-l. Example data for calculation of RPF detection limit F-l
G-l. Comparisons among average nonzero tumor bioassay-based RPF values by
exposure route G-3
G-2. Comparisons among average nonzero tumor bioassay-based RPF values by
calculation method G-6
G-3. Results of simple linear regression of log-transformed average genotoxicity RPF
versus log average tumor bioassay RPF G-8
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1 LIST OF FIGURES
2
3
4 2-1. Structural features of PAHs 20
5
6 2-2. Metabolic pathways for benzo[a]pyrene 24
7
8 2-3. Overview of the proposed key events in the mode of action for PAH carcinogenicity 26
9
10 2-4. Structures of the four stereoisomeric adduct moieties, anti-[EaP]-N2-dG., derived from
11 the trans- or cis- covalent binding of (+)-a«ft'-BaP diol epoxide or (-)-awft'-BaP diol
12 epoxide to dG residues in DNA 27
13
14 2-5. Depurinating adducts of benzo[a]pyrene formed by one-electron oxidation 28
15
16 2-6. Spectrum of DNA adducts anticipated with PAH o-quinones 29
17
18 2-7. Interaction of PAHs with the AhR - regulation of genes related to induction of
19 metabolism and cell differentiation and proliferation 31
20
21 6-1. Weight of evidence analysis of for selection of PAHs to be included in the RPF
22 approach 115
23
24 6-2. 2,3-Acepyrene (ACEP) RPFs 120
25
26 6-3. Anthanthrene (AA) RPFs 122
27
28 6-4. Anthracene (AC) RPFs 124
29
30 6-5. Benz[a]anthracene (BaA) RPFs 126
31
32 6-6. 1 lH-Benz[b,c]aceanthrylene (BbcAC) RPFs 128
33
34 6-7. Benzo[b]fluoranthene (BbF) RPFs 130
35
36 6-8. 1 lH-Benzo[b]fluorene (BbFE) RPFs 132
37
38 6-9. Benzo[c]fluorene (BcFE) RPFs 134
39
40 6-10. Benz[e]aceanthrylene (BeAC) RPFs 136
41
42 6-11. Benzo[e]pyrene (BeP) RPFs 138
43
44 6-12. Benzo[g,h,i]fluoranthene (BghiF) RPFs 140
45
46 6-13. Benzo[g,h,i]perylene (BghiP) RPFs 142
47
48 6-14. Benz[j]aceanthrylene(BjAC)RPFs 144
49
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1 6-15. Benzo[j]fluoranthene(BjF)RPFs 146
2
3 6-16. Benzo[k]fluoranthene (BkF) RPFs 148
4
5 6-17. Benz[l]aceanthrylene (B1AC) RPFs 150
6
7 6-18. Chrysene (CH) RPFs 152
8
9 6-19. Coronene (CO) RPFs 154
10
11 6-20. Cyclopenta[c,d]pyrene (CPcdP) RPFs 156
12
13 6-21. Cyclopenta[d,e,f]chrysene (CPdefC) RPFs 158
14
15 6-22. Dibenz[a,c]anthracene (DBacA) RPFs 160
16
17 6-23. Dibenzo[a,e]fluoranthene (DBaeF) RPFs 162
18
19 6-24. Dibenzo[a,e]pyrene (DBaeP) RPFs 164
20
21 6-25. Dibenz[a,h]anthracene (DBahA) RPFs 166
22
23 6-26. Dibenzo[a,h]pyrene (DBahP) RPFs 168
24
25 6-27. Dibenzo[a,i]pyrene (DbaiP) RPFs 170
26
27 6-28. Dibenzo[a,l]pyrene (DBalP) RPFs 173
28
29 6-29. Fluoranthene (FA) RPFs 175
30
31 6-30. Fluorene (FE) RPFs 177
32
33 6-31. Indeno[l,2,3-c,d]pyrene(IP)RPFs 179
34
35 6-32. Naphtho[2,3-e]pyrene (N23eP) RPFs 181
36
37 6-33. Perylene (Pery) RPFs 183
38
39 6-34. Phenanthrene (PH) RPFs 185
40
41 6-35. Pyrene (Pyr) RPFs 187
42
43 6-36. Triphenylene (Tphen) RPFs 189
44
45 8-1. Correlation between incidence and multiplicity RPFs 204
46
47 G-l. Average bioassay RPF versus average in vivo DNA adduct RPF G-9
48
49 G-2. Average bioassay RPF versus average in vivo nonbioassay RPF G-10
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1
2 G-3. Average bioassay RPF versus average nonbioassay RPF G-ll
o
J
4 G-4. Average bioassay RPF versus average in vitro nonbioassay RPF G-12
5
6
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LIST OF ABBREVIATIONS AND ACRONYMS
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
ADAF
AEL
Ah
AhR
ATSDR
AUC
BMD
BMR
CASRN
CCRIS
CHO
CYP
dG
DMSO
DNA
DSSTOX
EOPP
EROD
HPRT
IARC
IRIS
MGP
MN-PCE
mRNA
MVK
NTP
OEHHA
PAC
PAH
PCB
PCR
PEF
QSAR
RNA
RPF
RTD
SD
TK
TIDAL
TEF
TK
TPA
TSCATS
age-dependent adjustment factor
acceptable exposure level
aryl hydrocarbon
Ah receptor
Agency for Toxic Substances and Disease Registry
area under the curve
benchmark dose
benchmark response
Chemical Abstract Service Registry Number
Chemical Carcinogenesis Research Information System
Chinese hamster ovary
cytochrome P450
deoxyguanosine
dimethyl sulfoxide
deoxyribonucleic acid
Distributed Structure-Searchable Toxicity
estimated order of potential potency
ethoxyresorufin O-deethylase
hypoxanthine-guanine phosphoribosyl transferase gene
International Agency for Research on Cancer
Integrated Risk Information System
manufactured gas plant
micronuleated polychromatic erythrocyte
messenger ribonucleic acid
Moolgavkar-Venson-Knudsen two-stage model
National Toxicology Program
Office of Environmental Health Hazard Assessment, California EPA
polycyclic aromatic compound
polycyclic aromatic hydrocarbon
polychlorinated biphenyl
polymerase chain reaction
potency equivalency factor
quantitative structure activity relationship
ribonucleic acid
relative potency factor
relative tumor dose
standard deviation
thymidine kinase locus
time-integrated DNA adduct level
toxicity equivalency factor
thymidine kinase
12-O-tetra-decanoylphorbol-13-acetate
Toxic Substances Control Act Test Submissions
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1 U.S. EPA U.S. Environmental Protection Agency
2 WHO World Health Organization
o
J
4 Abbreviations for PAH chemical names are provided in Table 2-1.
5
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1 AUTHORS, CONTRIBUTORS, AND REVIEWERS
2
3 PROJECT CO-MANAGERS
4
5 Lynn Flowers, Ph.D., DABT
6 National Center for Environmental Assessment
7 Office of Research and Development
8 U.S. Environmental Protection Agency
9 Washington, DC
10
11 Martin Gehlhaus, III
12 National Center for Environmental Assessment, IRIS Program
13 Office of Research and Development
14 U.S. Environmental Protection Agency
15 Washington, DC
16
17 AUTHORS
18
19 Lynn Flowers, Ph.D., DABT
20 National Center for Environmental Assessment
21 Office of Research and Development
22 U.S. Environmental Protection Agency
23 Washington, DC
24
25 Martin Gehlhaus, III
26 National Center for Environmental Assessment, IRIS Program
27 Office of Research and Development
28 U.S. Environmental Protection Agency
29 Washington, DC
30
31 Karen Hogan
32 National Center for Environmental Assessment, IRIS Program
33 Office of Research and Development
34 U.S. Environmental Protection Agency
35 Washington, DC
36
37 Channa Keshava, Ph.D.
38 National Center for Environmental Assessment, IRIS Program
39 Office of Research and Development
40 U.S. Environmental Protection Agency
41 Washington, DC
42
43 Glenn Rice, Ph.D.
44 National Center for Environmental Assessment
45 Office of Research and Development
46 U.S. Environmental Protection Agency
47 Cincinnati, OH
48
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1 Jamie Strong, Ph.D.
2 National Center for Environmental Assessment, IRIS Program
3 Office of Research and Development
4 U.S. Environmental Protection Agency
5 Washington, DC
6
7 Linda Teuschler, Ph.D.
8 National Center for Environmental Assessment
9 Office of Research and Development
10 U.S. Environmental Protection Agency
11 Cincinnati, OH
12
13 Stephen Nesnow, Ph.D.
14 Environmental Carcinogenesis Division
15 National Health and Environmental Effects Research Laboratory
16 Office of Research and Development
17 Research Triangle Park, NC
18
19 Chao Chen, Ph.D.
20 National Center for Environmental Assessment
21 Office of Research and Development
22 Washington, DC
23
24 Heather Carlson-Lynch, S.M.
25 Syracuse Research Corporation, Inc.
26 Syracuse, NY
27
28 Julie Stickney, Ph.D., DABT
29 Syracuse Research Corporation, Inc.
30 Syracuse, NY
31
32 Peter R. McClure, Ph.D., DABT
33 Syracuse Research Corporation, Inc.
34 Syracuse, NY
35
36 Amber B acorn
37 Syracuse Research Corporation, Inc.
38 Syracuse, NY
39
40
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1 1. BACKGROUND FOR THE DEVELOPMENT OF A RELATIVE POTENCY
2 FACTOR APPROACH FOR PAH MIXTURES HEALTH ASSESSMENT
o
4 This analysis focuses on the relative potency factor (RPF) approach that is based on
5 component PAHs in PAH mixtures. U.S. EPA held a peer consultation workshop to outline some
6 of the important issues related to approaches for PAH mixtures risk assessment. These issues are
7 discussed in Peer Consultation Workshop on Approaches to Poly cyclic Aromatic Hydrocarbon
8 (PAH) Health Assessment (U.S. EPA, 2002) and the accompanying discussion document. Health
9 assessments for 15 unsubstituted, nonheterocyclic poly cyclic aromatic hydrocarbons (PAHs)
10 with three or more rings are currently entered on EPA's IRIS database. Benzo[a]pyrene is the
11 only PAH for which there are robust animal dose-response data for the oral, dermal, and
12 inhalation routes.
13 In 1993, U.S. EPA published the Provisional Guidance for Quantitative Risk Assessment
14 of PAHs (Provisional Guidance}. The Provisional Guidance recommended estimated orders of
15 potential potency (EOPP) for individual PAHs that could be used in a component-based
16 approach to PAH mixtures risk assessment. The Provisional Guidance recommended EOPPs for
17 seven PAHs categorized as Group B2 (probable human carcinogens) under the 1986 U.S. EPA
18 Cancer Guidelines: benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluor-
19 anthene, chrysene, dibenz[a,h]anthracene, and indeno[l,2,3-c,d]pyrene (U.S. EPA, 1993). The
20 current analysis extends the 1993 Provisional Guidance and provides recommendations for
21 further development of this approach to PAH mixtures risk assessment. The assessment includes
22 the following:
23
24 (1) A rationale for recommending an order of potency, or RPF, approach;
25
26 (2) A summary of previous approaches for developing the RPF approach for PAHs;
27
28 (3) Identification of individual carcinogenic PAHs that could be included in the RPF
29 approach;
30
31 (4) Identification of potential index chemicals;
32
33 (5) Presentation of the available literature for in vivo carcinogenicity and both in vivo and in
34 vitro cancer-related endpoint assays for individual PAHs;
35
36 (6) Development of a recommendation for the RPF approach for PAH mixtures; and
37
38 (7) Characterization of strengths, weaknesses, and uncertainties associated with the
39 recommended approaches.
40
41
42
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1 2. RATIONALE FOR RECOMMENDING AN RTF APPROACH
2
3
4 PAHs are a concern as human health hazards, because many PAHs are demonstrated
5 tumorigenic agents in animal bioassays and are active in in vivo or in vitro tests for genotoxicity
6 or deoxyribonucleic acid (DNA) damage. PAHs do not occur in the environment as isolated
7 entities; they primarily occur in complex mixtures generated from the combustion or pyrolysis of
8 substances containing carbon and hydrogen. Several complex mixtures of PAHs have been
9 classified as possibly carcinogenic, probably carcinogenic, or carcinogenic to humans (Straif et
10 al., 2005; U.S. EPA, 2002; Bostrom et al., 2002; WHO, 1998; ATSDR, 1995; IARC, 1985,
11 1984a, b, 1983).
12 In accordance with U.S. EPA (2000, 1986) guidance for health risk assessment of
13 chemical mixtures, assessment of the cancer risk from long-term human exposure to a particular
14 PAH mixture would best be conducted with quantitative information on the dose-response
15 relationship for cancer from chronic exposure to the mixture of concern. When data for the
16 mixture of concern are not available, U.S. EPA (2000, 1986) guidance recommends using
17 toxicity data on a "sufficiently similar" mixture. However, quantitative cancer dose-response
18 information exists only for a few complex mixtures generated from the combustion or pyrolysis
19 of organic matter; for example, tobacco smoke, coke oven emissions, and emissions from roofing
20 tar pots (see Bostrom et al., 2002; Albert et al., 1983). U.S. EPA's IRIS database currently
21 includes assessments for only three PAH-containing mixtures: coke oven emissions, creosote,
22 and diesel emissions. The availability of oral carcinogenicity bioassays of manufactured gas
23 plant (MGP) residue (Weyand et al., 1995) and coal tar preparations (Gulp et al., 1998; Gaylor et
24 al., 1998) has expanded the PAH mixture cancer database.
25 Component-based approaches, involving an analysis of the toxicity of components of the
26 mixture, are recommended when appropriate toxicity data on a complex mixture of concern, or
27 on a "sufficiently similar" mixture, are unavailable (U.S. EPA, 2000, 1986). Component-based
28 approaches involving dose addition (such as the RPF approach) are recommended when
29 components in the mixture are judged to act in a lexicologically similar manner. In the RPF
30 approach, doses of component chemicals that act in a lexicologically similar manner are added
31 together, after scaling the doses relative to the potency of an index chemical (U.S. EPA, 2000,
32 1986). Then, using the dose-response curve of the index chemical, the response to the total
33 equivalent dose in the mixture is estimated. The index compound is typically the best-studied
34 member of the class with the largest body of available data describing exposure and health
35 effects. The index chemical should have a quantitative dose-response assessment of acceptable
36 scientific quality and must have (or be expected to have) similar toxic effects to the rest of the
37 members of the class.
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1 For exposure situations in which dose-response data for the PAH mixture or a sufficiently
2 similar mixture are not available (e.g., the source of the PAH contamination may be mixed or
3 unknown), there are at least three practical advantages of an RPF approach that uses
4 benzo[a]pyrene as the index PAH:
5
6 (1) Benzo[a]pyrene is routinely assayed and detected in environmental media contaminated
7 with PAH mixtures;
8
9 (2) Benzo[a]pyrene is the only PAH for which robust cancer dose-response data involving
10 chronic exposures are available; and
11
12 (3) There is a large database of studies in which the potency of benzo[a]pyrene is compared
13 with the potency of other PAHs in various assays.
14
15 The database includes animal tumorigenicity2 assays involving dermal or parenteral
16 administration, and in vivo and in vitro assays of cancer-related endpoints (e.g., various
17 genotoxic endpoints). Thus, RPFs for a number of PAHs can be derived.
18 The RPF approach involves two key assumptions related to the application of a dose-
19 additivity model: (1) the assumption of similar toxicological action; and (2) the assumption that
20 interactions among PAH mixture components do not occur at low levels of exposure typically
21 encountered in the environment.
22 Mechanistic studies indicate that the mutagenic and tumor-initiating activity of most
23 carcinogenic PAHs requires metabolic activation to reactive intermediates (e.g., stereospecific
24 dihydrodiol epoxides). For several PAHs (e.g., benzo[a]pyrene, dibenz[a,h]anthracene,
25 dibenzo[a,l]pyrene), there is evidence that DNA damage associated with metabolism can lead to
26 mutations in cancer-related genes. Tumor promotion and progression by PAHs may involve
27 parent compound binding to the aryl hydrocarbon (Ah) receptor and subsequent alterations of
28 gene expression, as well as by cell proliferation in response to cytotoxic effects from metabolites
29 (see Section 2.4, Similarities in Mode of Carcinogenic Action for PAHs). As such, there is
30 evidence that an assumption of similar toxicological action is reasonable; however, the
31 carcinogenic process for individual PAHs is likely to be related to some unique combination of
32 multiple molecular events resulting from the formation of several reactive species. The second
33 assumption of no interactions at low levels of exposure is also reasonable, but has not been
34 conclusively demonstrated in experimental systems (see Section 2.8, Dose Additivity of PAHs in
35 Combined Exposures).
36 Key limitations to the RPF approach, relative to whole mixture approaches, are:
37 (1) RPFs have been derived for a limited number of PAHs; and (2) cancer risks from non-PAH
38 components, unidentified PAHs, and heterocyclic and substituted PAHs in PAH mixtures are not
throughout this report, the term "tumorigenicity" is used to describe the production of either benign or malignant
tumors.
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1 estimated. The first of these limitations is being addressed, to the degree allowable by available
2 data, by the derivation of RPFs for numerous PAHs as discussed in Chapters 4 through 7 of this
3 report. If non-PAH carcinogenic components are identified and quantified in the complex
4 mixture of concern and appropriate dose-response data are available, the second limitation can be
5 addressed by adding the cancer risk from PAH components estimated by the RPF approach to
6 cancer risks estimated for the non-PAH carcinogenic components of the mixture. Previous
7 efforts to validate the RPF approach using data for PAH mixtures are discussed in Section 3.1.
8 These validation efforts compared the cancer risk of a PAH mixture measured experimentally
9 with the cancer risk that was predicted using the RPF method but were limited by the small
10 number of compounds for which RPFs and analytical data were available (Muller et al., 1997;
11 McClure, 1996; Goldstein et al., 1994; Clement Associates, 1990, 1988; Krewski et al., 1989).
12 Validation of the updated approach presented here would be of value, either using previous data
13 on PAH mixtures (human and animal) or using new data collected with the main purpose of
14 evaluating the validity of the approach.
15
16 2.1. PAHs AS A CHEMICAL CLASS
17 The PAH chemical class has been variously defined to include organic compounds
18 containing either two or more, or three or more, fused rings made up of carbon and hydrogen
19 atoms (i.e., unsubstituted parent PAHs and their alkyl-substituted derivatives) (WHO, 1998).
20 Most PAHs are high-melting, high-boiling point, lipophilic compounds, predominately generated
21 from the incomplete combustion or pyrolysis of organic matter. The PAH chemical class
22 includes alkylated PAHs (e.g., 1,4-dimethylphenanthrene and 5-methylchrysene), but not
23 heterocyclic compounds containing N, S, or O or PAHs substituted with N-, S-, or O-containing
24 groups; these are included in a larger chemical class, often referred to as polycyclic aromatic
25 compounds (PACs) (WHO, 1998). The number of chemicals that comprise the PAHs class is
26 unknown; however, there are thought to be hundreds of individual PAHs present as components
27 of complex mixtures (WHO, 1998). The analysis presented here is limited in focus to include
28 only unsubstituted PAHs with three or more fused aromatic rings containing only carbon and
29 hydrogen atoms, because these are the most widely studied members of the PAH chemical class.
30 Naphthalene is a widely studied two-ring PAH compound; however, a separate toxicological
31 review and carcinogenicity assessment is being developed by the IRIS Program for this
32 compound and it is not included in this RPF approach. The list of PAH compounds that were
33 considered for inclusion in this analysis is presented in Table 2-1 along with the Chemical
34 Abstracts Service Registry Numbers (CASRNs) and the abbreviations that are utilized in tables
35 throughout the report.
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Benzo[a]pyrene
BaP
252.31
Aceanthrylene
202-03-09
ACEA
202.26
Acenaphthene
83-32-9
AN
154.21
Acenaphthylene
208-96-8
ANL
152.20
Acephenanthrylene
201-06-9
APA
202.26
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Acepyrene, 2,3-
25732-74-5
ACEP
228.29
Anthanthrene
191-26-4
AA
276.34
Anthracene
120-12-7
AC
178.23
Benzacenaphthylene
76774-50-0
BAN
202.26
B enz [a] anthracene
56-55-3
BaA
228.29
Benzo [a]fluoranthene
203-33-8
BaF
252.32
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Benzo[a]fluorene
238-84-6
BaFE
216.28
Benzo[a]perylene
191-85-5
BaPery
302.38
Benz[b,c]aceanthrylene, 11H-
202-94-8
BbcAC
240.30
Benz [b] anthracene
(naphthacene)
92-24-0
BbA
228.29
Benzo[b]chrysene
214-17-5
BbC
278.35
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Benzo [b]fluoranthene
205-99-2
BbF
252.32
Benzo [b]fluorene, 11H
243-17-4
BbFE
216.28
Benzo [b]perylene
197-70-6
BbPeiy
302.38
Benzo[c]chrysene
194-69-4
BcC
278.35
Benzo [c]fluorene
205-12-9
BcFE
216.28
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Benzo [c]phenanthrene
195-19-7
BcPH
228.29
Benz [e] aceanthry lene
199-54-2
BeAC
252.32
Benzo [e]pyrene
192-97-2
BeP
252.32
Benzo [g,h,i]fluoranthene
203-12-3
BghiF
226.28
Benzo [g,h,i]pery lene
191-24-2
BghiP
276.34
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Benzo[g]chrysene
196-78-1
BgC
278.35
Benz |] ] aceanthry lene
202-33-5
BjAC
252.32
Benzo[j]fluoranthene
205-82-3
BjF
252.32
Benzo fkjfluoranthene
207-08-9
BkF
252.32
Benz [1] aceanthry lene
211-91-6
B1AC
252.32
Benzophenanthrene
65777-08-4
BPH
228.29
10
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Chrysene
218-01-9
CH
228.29
Coronene
191-07-1
CO
300.36
Cyclopent[h,i]aceanthrylene
131581-33-4
CPhiACEA
226.28
Cyclopenta[c,d]pyrene
27208-37-3
CPcdP
226.28
Cyclopenta[d,e,f]chrysene, 4H-
202-98-2
CPdefC
240.30
Cyclopenta[d,e,f]phenanthrene
203-64-5
CPdefPH
190.24
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Cyclopenta[h,i]acephenanthrylene
114959-37-4
CPhiAPA
226.28
Cyclopentaphenanthrene
219-08-9
CPPH
216.28
Cyclopenteno-l,2-benzanthracene, 5,6-
7099-43-6
CPBA
268.36
Dibenz[a,c]anthracene
(benzotriphenylene)
215-58-7
DBacA
278.35
Dibenzo[a,c]fluorene, 13H-
201-65-0
DBacFE
266.34
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Dibenzo [a,e]fluoranthene
5385-75-1
DBaeF
302.38
Dibenzo [a,e]py rene
192-65-4
DBaeP
302.38
Dibenzo [a,f]fluoranthene
(indeno [1,2,3 -fg] naphthacene)
203-11-2
DBafF
302.38
Dibenzo [a,g]fluorene, 13H-
207-83-0
DBagFE
266.34
Dibenz[a,h]anthracene
53-70-3
DBahA
278.35
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Dibenzo [a,h]pyrene
189-64-0
DBahP
302.38
Dibenzo [a,i]py rene
189-55-9
DBaiP
302.38
Dibenzo [a,l]py rene
191-30-0
DBalP
302.38
Dibenzo [b,e]fluoranthene
2997-45-7
DBbeF
302.38
Dibenzo [e,l]pyrene
(dibenzo[fg,op]naphthacene)
192-51-8
DBelP
302.38
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Dibenzo [h,rst]pentaphene
192-47-2
DBhrstPent
352.43
Dibenz \j ,mno] acephenanthry lene
153043-82-4
DBjmnoAPH
276.34
Dibenz [k,mno]acephenanthrylene
153043-81-3
DBkmnoAPH
276.34
Dihydroaceanthrylene, 1,2-
641-48-5
DACEA
204.27
Fluoranthene
206-44-0
FA
202.26
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Fluorene
86-73-7
FE
166.22
Indeno [ 1,2,3 -c,d]fluoranthene
193-43-1
IF
276.34
Indeno [ 1,2,3 -c,d]pyrene
193-39-5
IP
276.34
Naphth[l ,2,3 -mno]acephenanthrylene
113779-16-1
N123mnoAPH
276.34
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Naphtho[ 1,2-b]fluoranthene
111189-32-3
N12bF
302.38
Naphtho[2, l-a]fluoranthene
203-20-3
N21aF
302.38
Naphtho[2,3-a]pyrene
(naphtho [2,1,8-qra]naphthacene)
196-42-9
N23aP
302.38
Naphtho[2,3-e]pyrene
(dibenzo [de,qr] naphthacene)
193-09-9
N23eP
302.38
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Table 2-1. PAHs evaluated in the RTF analysis
PAH
(common synonyms)
CASRN
Abbreviation
Structure
Molecular
weight
(g/mol)
Pentacene
135-48-8
PCE
278.35
Pentaphene
(dibenzphenanthrene, 2,3:6,7-)
222-93-5
Pent
278.35
Perylene
198-55-0
Pery
252.32
Phenanthrene
85-01-8
PH
178.23
Picene
213-46-7
Pic
278.35
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Table 2-1. PAHs evaluated in the RTF analysis
1
PAH
(common synonyms)
Pyrene
Tribenzofluoranthene 3,4-10,11-12,13-
Triphenylene
CASRN
129-00-0
13579-05-0
217-59-4
Abbreviation
Pyr
TBF
Tphen
Structure
^
CC
[
l""
%X^
/
Vv
^
^
\
=<
x^^t/^^
~^^^
!3
)
Molecular
weight
(g/mol)
202.26
352.43
228.29
2 Unsubstituted PAHs have been further classified into alternant and nonalternant
3 compounds. Alternant PAHs are those compounds composed solely of fused benzene rings,
4 while nonalternant PAHs contain both benzene and five carbon rings. Among alternant PAHs,
5 important structural features related to enhanced mutagenicity and carcinogenicity include the
6 presence of at least four rings (Bostrom et al., 2002). Common structural features of PAH
7 compounds are illustrated in Figure 2-1 .
19
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Benzo[a]pyrene Pyrene
Examples of Alternant PAHs
Fluorene
Fluoranthene
Examples of Nonalternant PAHs
Fjord-region
Chrysene
Benzo[c]phenanthrene
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Bay-region and Fjord-regions of PAHs
Figure 2-1. Structural features of PAHs.
2.2. THE TOXICOLOGICAL DATABASE FOR PAHs
Over the last 30- to 50-years, a large PAH database has been generated including studies
of carcinogenicity in animal bioassays, genotoxicity in various test systems, and metabolism
(bioactivation) to tumorigenic and/or genotoxic intermediates. Carcinogenicity and genotoxicity
data are sufficient to classify a number of individual PAHs as possibly carcinogenic to humans
(WHO, 1998; U.S. EPA, 1993; IARC, 1989, 1986, 1985, 1984a, b, 1983). Other PAHs have
been tested for tumorigenicity and/or genotoxicity, but either nonpositive or equivocal results
were obtained; for many PAHs, positive results were only observed in genotoxicity assays (e.g.,
pyrene). Many studies have been performed to provide further understanding about the
carcinogenic mode of action of PAHs (see Bostrom et al., 2002; WHO, 1998; ATSDR, 1995).
Therefore, the PAH database contains studies that evaluate:
20
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1 • Metabolism to reactive intermediates;
2 • Characterization of PAH-DNA adducts;
3 • Mutagenicity of PAHs in bacterial and mammalian cells;
4 • Mutation spectra in identified oncogene and tumor suppressor genes;
5 • Clastogenic effects;
6 • Cell transformation; and
7 • Initiation and promotion of carcinogenicity (complete carcinogenesis).
8
9 A limitation to the database is the lack of data from long-term oral or inhalation cancer
10 studies for most individual PAH compounds. The only PAH for which there are robust animal
11 dose-response data is benzo[a]pyrene (Kroese et al., 2001; Gulp et al., 1998, 1996a, b; Thyssen
12 etal., 1981, 1980; Rigdon et al., 1969; Rigdon and Neal, 1969, 1966; Neal and Rigdon, 1967).
13 Furthermore, most of the toxicological data available for PAHs relate to cancer or genotoxicity.
14 Available information on the systemic, noncarcinogenic effects of PAHs is limited, although
15 immunological, neurotoxic, and developmental effects have been noted in animal studies and
16 some human studies (for earlier reviews, see WHO, 1998; ATSDR, 1995). As a result, the
17 relative potency methodology described here is applied only to cancer risk assessment for PAHs.
18
19 2.3. BENZO[A]PYRENE AS AN INDEX CHEMICAL
20 Because long-term animal studies are not available for many individual PAHs, it is
21 necessary to choose an appropriate index chemical for comparison of relative carcinogenic
22 potency. The index compound is typically the best-studied member of the class, with the largest
23 body of available data describing exposure and health effects. The index chemical should have a
24 quantitative dose-response assessment of acceptable scientific quality and must have (or be
25 expected to have) similar toxic effects to the rest of the members of the class.
26 Although the PAH composition of complex mixtures varies, benzo[a]pyrene is
27 considered to be present in significant amounts in certain occupational environments and urban
28 settings (WHO, 1998; Petry et al., 1996; ATSDR, 1995). Benzo[a]pyrene is one of the most
29 potent of the carcinogenic PAHs and has, therefore, been proposed to contribute significantly to
30 the carcinogenicity of a PAH mixture, even when present in low concentrations (Petry et al.,
31 1996). Benzo[a]pyrene is also the best-studied PAH compound, with carcinogenicity bioassay
32 data available for several routes of exposure and a considerable number of studies on
33 carcinogenic mode of action. Benzo[a]pyrene has been characterized as reasonably anticipated
34 to be a human carcinogen (NTP, 2005) or carcinogenic to humans (Straif, 2005).
35 The laboratory animal database for benzo[a]pyrene is robust. Benzo[a]pyrene has been
36 shown to induce tumors at the site of administration and at distal sites in numerous studies.
37 Dose-response data for tumors are available for the oral, inhalation, and dermal routes of
38 administration in multiple species. There are methodological limitiations associated with the
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1 inhalation data (Thyssen et al., 1981), although positive findings in intratracheal instillation
2 studies support the observed positive response. Dermal exposure studies with several strains of
3 mice also provide data on dose-related tumor incidences (Albert et al., 1991; Warshawsky and
4 Barkley, 1987; Habs et al., 1984, 1980; Nesnow et al., 1983; Wynder et al., 1957).
5 The animal carcinogenicity database for benzo[a]pyrene includes several well-conducted
6 oral cancer bioassays. Kroese et al. (2001) conducted a well-designed gavage study of
7 benzo[a]pyrene carcinogenicity and found that benzo[a]pyrene induced tumors at multiple sites
8 in rats of both sexes, specifically in the liver, forestomach, auditory canal, and oral cavity. In
9 another well-conducted study, using Ah-responsive B6C3Fi female mice exposed to
10 benzo[a]pyrene in the diet (Beland and Gulp, 1998; Gulp et al., 1998), only portal-of-entry
11 tumors were found, including papillomas and/or carcinomas of the forestomach, esophagus,
12 tongue, and larynx. Earlier, a number of related studies were conducted to evaluate the
13 carcinogenicity of benzo[a]pyrene in feed in Ah-responsive white Swiss mice (Rigdon and Neal,
14 1969, 1966; Neal and Rigdon, 1967). These studies were not conducted using standard, modern
15 toxicological methods and have several limitations, including inconsistent dosing protocols;
16 varying ages of the animals; use of benzene as a solvent; small numbers of animals; and
17 evaluation of only a limited number of tissues. These studies do, however, provide useful dose-
18 response information on benzo[a]pyrene carcinogenicity. Following oral administration via
19 feeding of benzo[a]pyrene, site-of-contact tumors (both papillomas and carcinomas) were
20 induced in the forestomach, esophagus, and larynx of mice (Gulp et al., 1998; Neal and Rigdon,
21 1967) and rats (Brune et al., 1981). The results following inhalation, dermal, or oral exposure
22 are further supported by numerous mechanistic studies or assays using infant mice, susceptible
23 transgenic strains, or Ah-receptor knockout mice.
24 Benzo[a]pyrene is a complete carcinogen and likely acts by initiating tumors through
25 direct DNA damage as well as by promoting tumor growth. Benzo[a]pyrene has been shown to
26 be mutagenic in multiple assay systems. Several modes of carcinogenic action are possible.
27 These include:
28
29 (1) Alteration of pathways regulating cell proliferation and survival (Tannheimer et al.,
30 1998);
31
32 (2) Inhibition of intracellular communication (Sharovskaia et al., 2003; Blaha et al.,
33 2002; Rummel et al., 1999);
34
35 (3) Altered intracellular Ca2+ signaling (Tannheimer et al., 1998);
36
37 (4) Modulation of cell survival, cell proliferation, and altered growth via generation of
38 oxidative stress and activation of oxidant stress signaling (Burdick et al., 2003; Miller
39 and Ramos, 2001);
40
41 (5) Altered apoptosis processes (Chen et al., 2003);
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1
2 (6) Dysregulation of normal circulating hormone levels or activity affecting
3 tumorigenesis in reproductive tissues (Safe and Wormke, 2003; Archibong et al.,
4 2002) or the central nervous system (Dasgupta and Lahiri, 1992);
5
6 (7) Disruption of cell cycle kinetics in breast cancer cells (Jeffy et al., 2002, 2000); and
7
8 (8) Disruption of DNA repair through alteration of ribonucleic acid (RNA) polymerase
9 activity (Shah and Bhattacharya, 1989).
10
11 Oral (dietary) carcinogenicity bioassays are available that compare MGP residue
12 (Weyand et al., 1995) or coal tar preparations (Culp et al., 1998; Gaylor et al., 1998) with
13 benzo[a]pyrene. In both cases, there were significant differences in the target organ distribution
14 of tumors between benzo[a]pyrene and complex mixtures of PAHs. Following dietary
15 administration, benzo[a]pyrene-induced tumors were observed primarily at the point of contact
16 (i.e., the forestomach), while MGP residue and coal tar produced tumors in the lung, liver,
17 forestomach, skin, and other organs. Tissue-specific differences in metabolic activation and
18 DNA binding of PAHs may contribute to the observed differences in target organ sensitivity
19 (Weyand and Wu, 1995; Culp and Beland, 1994). However, a dietary study in A/J mice
20 (Weyand et al., 2004) showed that benzo[a]pyrene could induce significant increases in the
21 incidences of lung adenomas and forestomach carcinomas. Further, a gavage study in rats
22 (Kroese et al., 2001) demonstrated that oral exposure to benzo[a]pyrene could induce tumors in
23 the liver and auditory canal; no lung tumors were observed. The latter two studies indicate that,
24 contrary to the conclusions of earlier studies, benzo[a]pyrene can induce tumors at distal sites.
25 In summary, benzo[a]pyrene is the most appropriate compound to use as an index
26 chemical for carcinogenic PAHs. It is well-studied, with a robust database of both bioassay data
27 and mode of action information. Benzo[a]pyrene is a complete carcinogen with both initiating
28 and promoting properties, is among the most potent PAH carcinogens, and is prevalent in many
29 complex environmental mixtures. No alternative index chemical was identified from the list of
30 target PAHs.
31
32 2.4. SIMILARITIES IN MODE OF CARCINOGENIC ACTION FOR PAHs
33 Toxicological similarity of chemicals is the basis for the assumption of dose additivity
34 that underlies the RPF approach (U.S. EPA, 1990). The carcinogenic mode of action for PAHs
35 has been extensively reviewed (Ramesh, 2004; CCME, 2003; Bostrom et al., 2002; Larsen and
36 Larsen, 1998; WHO, 1998; Muller et al., 1997; Sjogren et al., 1996; ATSDR, 1995; Malcolm
37 and Dobson, 1994; U.S. EPA, 1990). Key events that have been associated with PAH
38 carcinogenicity include:
39
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Oxidative metabolism to reactive intermediates that covalently bind to DNA, RNA,
and proteins (benzo[a]pyrene metabolism is illustrated in Figure 2-2);
Formation of DNA adducts;
Tumor initiation due to mutations in cancer-related genes (e.g., tumor suppressor
genes or oncogenes); and
Tumor promotion related to cytotoxicity and formation of reactive oxygen species,
and/or Ah receptor (AhR) affinity and upregulation of genes related to
biotransformation, growth, and differentiation.
BaP 1,6-hydroquinont BaP 1,6-semtquinone BaP 1.6-quinone
Reprinted from Impact of cellular metabolism on the biological effects of benzo[a]pyrene
and related hydrocarbons, 2001 by Miller, KP; Ramos, KS; with permission of Taylor &
Francis.
Source: Miller and Ramos (2001).
Figure 2-2. Metabolic pathways for benzo[a]pyrene.
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1 Formation of reactive intermediates andDNA adducts. Each of the key events identified
2 above is affected by the chemical structure of the individual PAH. At least three distinct
3 molecular mechanisms have been proposed to explain the tumor initiation process of PAHs (Xu
4 et al., 2009; Jiang et al., 2007, 2005; Xue and Warshawsky, 2005; Bolton et al., 2000; Penning et
5 al., 1999; Harvey, 1996; Cavalieri and Rogan, 1995). These modes of action include the
6 formation of diol epoxides, radical cations, and o-quinones (Figure 2-3). Diol epoxide formation
7 leads to stable and unstable DNA adducts, mainly at guanine and adenine, which can lead to
8 mutations in proto-oncogenes and tumor-suppressor genes. Radical cation formation may lead to
9 the generation of unstable adducts at guanine and adenine, leading to apurinic sites and mutation
10 in HRAS. o-Quinone formation could lead to stable and unstable DNA adducts and generation of
11 reactive oxygen species, inducing mutations in RP53. The evidence supporting the role of these
12 reactive metabolites in tumor initiation includes a characterization of the specific DNA adducts
13 arising from PAH metabolism and observations of mutagenesis resulting from direct exposure.
14 Figure 2-3 illustrates the proposed key steps in the mode of action for PAH carcinogenesis.
15 These include the interaction of reactive metabolites with DNA to form adducts, induction of
16 depurination, transversion mutations (e.g., GC^-TA or AT^-TA), and oxidative damage to
17 DNA, and tumor promotion mediated by AhR-mediated effects on gene regulation.
18
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Key Events in the Mode of Action for PAH Carcinogenicity
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Progression
Upregulation of
les related to
biotransformation,
growth, and
differentiation
Figure 2-3. Overview of the proposed key events in the mode of action for
PAH carcinogenicity.
The formation of diol epoxides is a proposed key step in the most established mode of
action for PAH-induced carcinogenicity. Extensive studies of the metabolism of carcinogenic
PAHs suggest that bay- and fjord-region diol epoxides are some of the ultimate reactive
metabolites of PAHs (Jerina et al., 1978; Jerina and Lehr, 1977). These metabolites are
generally formed through cytochrome P450 (CYP) oxidation to form epoxides and epoxide
hydrolase cleavage resulting in diol formation. CYP1 Al appears to be the primary isozyme
involved in diol epoxide formation; however, other isozymes may also contribute to PAH
metabolism (i.e., CYPIA2, CYP1B1, CYP3A4) (Bostrom et al., 2002; ATSDR, 1995). Non-
alternant PAHs, composed of fused benzenoid and five-membered rings, may be metabolized
through other pathways resulting in the formation of reactive intermediates that bind to DNA.
Classic bay- and fjord-region diol epoxides may be formed from these compounds; however,
epoxide formation at cyclopenta-ring structures has also been demonstrated to result in DNA
adduct formation (Bostrom et al., 2002).
Many studies have been performed to evaluate the formation of DNA adducts following
in vivo or in vitro exposure to PAHs. Diol epoxide metabolites interact preferentially with the
exocyclic amino groups of deoxyguanine and deoxyadenine (Geacintov et al., 1997; Jerina et al.,
26
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1 1991). Adducts may give rise to mutations, unless these adducts are removed by DNA repair
2 processes prior to replication. The stereochemical nature of the diol epoxide metabolite (i.e.,
3 anti- versus syn-diol epoxides) affects the number and type of adducts and mutation that occurs.
4 Figure 2-4 presents the structures of four stereoisomeric adducts arising from the interaction of
5 benzo[a]pyrene diol epoxide metabolites with the deoxyguanosine (dG) residues in DNA
6 (Geacintov et al., 1997). Transversion mutations (e.g., GC—>TA or AT—>TA) are the most
7 common type of mutation found in mammalian cells following diol epoxide exposure (Bostrom
8 et al., 2002).
R HO,,,
HO
10S (+)-trans-anti-[BaP]-N2-dG
R
HO
OH
(-)-trans-anti-[BaP]-N2-dG
10
11
12
13
14
15
16
17
18
19
R
HO
OH
10R (+)-c/s-an//-[BaP]-A/2-dG
Source: Geacintov et al. (1997).
OH
10S (-)-c/s-a/7f/-[BaP]-AP-dG
Figure 2-4. Structures of the four stereoisomeric adduct moieties,
anti-\Ba¥]-N2-AG, derived from the trans- or cis- covalent binding of
(+)-a«ft'-BaP diol epoxide or (-)-a«ft'-BaP diol epoxide to dG residues in DNA.
Radical cation formation involves a one-electron oxidation that produces electrophilic
radical cation intermediates (Cavalieri and Rogan, 1995, 1992). Oxidation of this type can occur
by CYP or peroxidase enzymes (i.e., horseradish peroxidase, prostaglandin H synthetase).
Radical cations can be further metabolized to phenols and quinones (Cavalieri et al., 1988a), or
27
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1
2
o
3
4
5
6
7
10
11
12
13
14
15
16
17
18
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23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
they can form unstable adducts with DNA that ultimately result in depurination (Cavalieri et al.,
2005, 1993; Rogan et al., 1993). Radical cations have been shown to play a major role in
formation of DNA adducts for several carcinogenic PAHs (e.g., 7,12-dimethylbenzanthracene,
benzo[a]pyrene, dibenzo[a,l]pyrene). The predominant depurinating adducts occur at the
N-3 and N-7 positions of adenine and the C-8 and N-7 positions of guanine (Cavalieri and
Rogan, 1995; Li et al., 1995). Figure 2-5 illustrates three depurinating adducts of
benzo[a]pyrene formed by one-electron oxidation. Abasic sites resulting from base depurination
undergo error-prone excision repair to induce mutations. In the case of dibenzo[a,l]pyrene-
treated mouse skin, repair error from abasic sites resulted in H-ras oncogene mutations that
underwent rapid clonal expansion and regression (Chakravarti et al., 2000). H-ras mutations in
mouse skin papillomas also corresponded to adenine and guanine depurinating adducts resulting
from exposure to dibenzo[a,l]pyrene, 7,12-dimethyl-benz[a]anthracene, benzo[a]pyrene, and
benzo[a]pyrene-7,8-dihydrodiol (Chakravarti et al., 2008).
o
BaP-6-C8-guanine
BaP-6-N7-guanine
BaP-6-N7-adenine
Reprinted from Central role of radical cations in metabolic activation of poly cyclic
aromatic hydrocarbons, 1995 by Cavalieri, EL; Rogan, EG; with permission of Taylor &
Francis.
Source: Cavalieri and Rogan (1995).
Figure 2-5. Depurinating adducts of benzo[a]pyrene formed by one-electron
oxidation.
o-Quinone metabolites of PAHs are formed by enzymatic dehydrogenation of
dihydrodiols (Bolton et al., 2000; Penning et al., 1999; Harvey, 1996; ATSDR, 1995).
Dihydrodiol dehydrogenase enzymes are members of the a-keto reductase gene superfamily.
o-Quinone metabolites are potent cytotoxins, are weakly mutagenic, and are capable of
producing a broad spectrum of DNA damage. These metabolites can interact directly with DNA
and can also result in production of reactive oxygen species (i.e., hydroxyl and superoxide
radicals) that may produce further cytotoxicity and DNA damage. The DNA damage caused by
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1
2
o
3
4
5
6
7
9
10
11
12
13
14
15
16
17
o-quinones may include the formation of stable adducts (Balu et al., 2006), N-7 depurinating
adducts (McCoull et al., 1999), oxidative base damage (i.e., 8-oxo-2'-dG or 8-oxo-dG) (Park et
al., 2006, 2005), and strand scission (Flowers-Geary et al., 1997). The reactive oxygen species
generated by the o-quinone of benzo[a]pyrene and other PAH o-quinones have been shown to
induce mutation in the p53 tumor suppressor gene (Park et al., 2008; Shen et al., 2006; Yu et al.,
2002). Figure 2-6 illustrates the spectrum of DNA adducts associated with PAH o-quinones.
o-Quinone adducts
ROS modifications
stable adducts
translesional synthesis
G to T transversions
depurinating adducts
apurinic sites
G to T transversions
8'-oxo-dG
base pair mismatch
G to T transversions
base propenals
Source: Bolton et al. (2000).
Figure 2-6. Spectrum of DNA adducts anticipated with PAH o-quinones.
The cytotoxicity of o-quinone metabolites may also contribute to tumor promotion via
inflammatory responses leading to cell proliferation (Burdick et al., 2003).
Genotoxicity andmutagenicity. The genotoxicity and mutagenicity of PAHs have been
demonstrated in various bacterial and mammalian assays (see Section 4.3.2 below) (reviewed in
WHO, 1998; ATSDR, 1995). Mutagenesis of PAHs in the Ames assay (Salmonella
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1 typhimuriuni) as well as other bacterial assays requires the presence of a mammalian metabolic
2 enzyme system. In most cases, this is supplied by postmitochondrial supernatant (S9) from the
3 liver of rodents treated with an enzyme inducer. Mammalian cell mutagenesis in Chinese
4 hamster V79 cells and mouse lymphoma L5178Y cells also requires metabolic activation in the
5 form of a rodent S9 mix or co-cultivation with metabolically active rodent cells (i.e., cell-
6 mediated assay). Several studies have noted a correlation between mutagenic potency and tumor
7 initiation potency in the two-stage dermal carcinogenicity assay for multiple PAH compounds
8 (LaVoie et al., 1985, 1979; Raveh et al., 1982).
9 Tumor promotion and the AhR. The ability of certain PAHs to act as tumor promoters as
10 well as initiators may increase their carcinogenic potency (Andrews et al., 1978). The
11 promotional effects of PAHs appear to be related to AhR affinity and the upregulation of genes
12 related to growth and differentiation (Bostrom et al., 2002). Figure 2-7 illustrates the function of
13 the AhR and depicts the genes regulated by this receptor as belonging to two major functional
14 groups (i.e., induction of metabolism or regulation cell differentiation and proliferation). PAHs
15 bind to the cytosolic AhR in complex with heat shock protein 90. The ligand-bound receptor is
16 then transported to nucleus in complex with the AhR nuclear translocator protein. The AhR
17 complex interacts with AhR elements of DNA to increase the transcription of proteins associated
18 with induction of metabolism and regulation of cell differentiation and proliferation.
19
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
*
PAH
-»tAHR|Hsp90
[Hsp90
AHR
Hsp90
Hsp90
Hsp90
Hsp90
ARNT
Enhanced
specific
mRNA
production
m
St..
|AHR
ARNT
AHREDNA
Increased
synthesis of
PAH metabolizing
enzymes
Increased
synthesis of
proteins that
regulate cell
differentiation and
proliferation
Reprinted from Molecular biology of the aromatic hydrocarbon (dioxin) receptor, 1994
by Okey, AB; et al. with permission of Elsevier.
Source: Okey etal. (1994).
Figure 2-7. Interaction of PAHs with the AhR - regulation of genes related
to induction of metabolism and cell differentiation and proliferation.
Tumor promotion and cytotoxicity. PAHs are metabolized to o-quinones, which are
cytotoxic and can generate reactive oxygen species (Bolton et al., 2000; Penning, 1999). PAH
o-quinones reduce the viability and survival of rat and human hepatoma cells (Flowers-Geary et
al., 1996, 1993). Inflammatory responses to cytotoxicity may contribute to the tumor promotion
process. For example, benzo[a]pyrene quinones (1,6-, 3,6-, and 6,12-benzo[a]pyrene-quinone)
generated reactive oxygen species and increased cell proliferation by enhancing the epidermal
growth factor receptor pathway in cultured breast epithelial cells (Burdick et al., 2003). Dermal
exposure of mice to dibenzo[a,l]pyrene and dimethyl-benz[a]anthracene resulted in an
inflammatory response that was correlated with epidermal hyperplasia and skin tumor promotion
(Casale et al., 2000, 1997). The extent of epidermal hyperplasia was correlated with the cytokine
mRNA response in lymph nodes and skin of treated mice (Casale et al., 2000).
Genetic targets and tumor formation. DNA adducts and oncogenes/tumor suppressor
gene mutations have been demonstrated in tumor tissue from humans and laboratory animals.
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1 DeMarini et al. (2001) demonstrated mutations in the p53 tumor suppressor gene and the K-ras
2 oncogene in the lung tumors of nonsmokers, whose tumors were associated with exposure to
3 smoky coal. Lung tumors were obtained from 24 nonsmoking women from China (age 30-
4 63 years, mean age 48.5 ± 8.8 years) who used smoky coal in their homes without chimneys.
5 Bronchioloalveolar adenocarcinoma and acinar adenocarcinoma were observed in 54 and 46% of
6 the women studied, respectively. The observed mutations in lung tumors were primarily G—>T
7 transversions at either K-ras or p53. The mutation hotspots in the lung tumors that were
8 examined corresponded with hot spots for PAH adducts (codon 154), cigarette smoke associated
9 mutations (codon 249), and both of these events together (codon 273). The mutation spectrum
10 was described as unique and consistent with exposure to PAHs in the absence of cigarette smoke.
11 Mutations in the K-ras, H-ras, and p53 genes were assessed in forestomach tumors
12 (n = 31) of mice fed benzo[a]pyrene in the diet (0, 5, 25, or 100 ppm) for 2 years (Gulp et al.,
13 2000). Sixty-eight percent of 31 forestomach tumors analyzed had K-ras mutations, which were
14 G—>T or C transversions in codon 12 or 13. H-ras (codon 13) and p53 mutations characterized
15 as G—>T or C transversions were also each found in 10% of forestomach tumors.
16 [32P]-postlabeling of forestomach DNA of benzo[a]pyrene-treated mice revealed one maj or
17 adduct characterized as dG N2 BPDE. In mice exposed to benzo[a]pyrene at several
18 concentrations in the diet for 4 weeks (5, 25, and 100 ppm), there was an approximate linear
19 relationship between the daily dose of benzo[a]pyrene (in units of jig/day) and the concentration
20 of dG-N2-BPDE-DNA adducts in the forestomach (Gulp et al., 2000, 1996a). In contrast, the
21 tumor dose-response data in mice exposed for 2 years showed a sharp increase in incidence
22 between the 5-ppm group (6% of mice had forestomach tumors) and the 25-ppm group (78% had
23 forestomach tumors) (Gulp et al., 1996a). The appearance of increased levels of BPDE-DNA
24 adducts in the target tissue at 28 days is temporally consistent with the contribution of these
25 adducts to the initiation of forestomach tumors at 25 and 100 ppm benzo[a]pyrene in the diet.
26 However, the absence of a sharp increase in the BPDE-DNA relationship between 5 and 25 ppm
27 benzo[a]pyrene is consistent with the possible contributions of mutagenic modes of action other
28 than the diol epoxide pathway (i.e., formation of depurinated DNA adducts from the radical
29 cation or aldo-keto-reductase pathways and reactive oxygen species DNA damage from the aldo-
30 keto-reductase pathway).
31 A series of experiments designed to evaluate the mechanistic relationship between PAH
32 DNA adducts, oncogene mutations, and lung tumorigenesis were performed in the A/J mouse
33 lung model (Nesnow et al., 1998a, 1996, 1995; Mass et al., 1993). Tumorigenic potency in the
34 lung of A/J mice varied over 2 orders of magnitude following a single intraperitoneal injection of
35 seven PAHs of varying structure (benzo[a]pyrene, benzo[b]fluoranthene, benz[j]aceanthrylene,
36 dibenz[a,h]anthracene, dibenzo[a,l]pyrene, cyclopenta[c,d]pyrene, and 5-methylchrysene).
37 When considering the non-alkylated PAHs, the number of lung adenomas per mouse was highest
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1 for benz[j]aceanthrylene and cyclopenta[c,d]pyrene, each of which contain a pentacyclic ring
2 feature. The major DNA adducts identified in the mouse lung included:
o
4 (1) Bay region diol epoxide adducts for benzo[a]pyrene, dibenz[a,h]anthracene, and
5 5-methylcholanthrene;
6
7 (2) Phenolic diol epoxide adducts for benzo[b]fluoranthene;
8
9 (3) Cyclopenta-ring adducts for cyclopenta[c,d]pyrene and benz[j]aceanthrylene;
10
11 (4) Bisdihydrodiol epoxide adducts for dibenz[a,h]anthracene; and
12
13 (5) Fjord-region diol epoxide adducts for dibenzo[a,l]pyrene (Nesnow et al., 1998a,
14 1996, 1995; Mass et al., 1993).
15
16 Guanine adducts were most common for all PAHs; however, adenine adducts were also
17 demonstrated for dibenzo[a,l]pyrene and benz[j]aceanthrylene. Quantitative analysis of DNA
18 adducts by [32P]-postlabeling illustrates the importance of measuring DNA adduct levels over
19 time. A time-integrated DNA adduct level (TIDAL) was linearly related to the dose of a
20 particular PAH. The relationship of TIDAL level to tumor formation was similar for PAHs that
21 produce different types of adducts and different mutations in the Ki-ras oncogene. This suggests
22 that the probability of tumor formation for these PAHs may be related to the extent of overall
23 DNA damage and repair rather than the formation of a specific adduct at specific sites.
24 The DNA sequence analysis of Ki-ras mutations in lung adenomas at codons 12 and 61
25 was generally consistent with the DNA adduct data in that PAHs that produced guanine adducts
26 also produced Ki-ras guanine mutations (Nesnow et al., 1998a, 1996, 1995; Mass et al., 1993).
27 Cyclopenta[c,d]pyrene, benz[j]aceanthrylene, and 5-methylchrysene produced large numbers of
28 adenomas per mouse (>90) and also produced a large proportion of tumors with CGT mutations
29 at Ki-ras codon 12. Cyclopenta-ring adduct formation by cyclopenta[c,d]pyrene and
30 benz[j]aceanthrylene was correlated with the formation of GGT—>CGT mutations at Ki-ras
31 codon 12. The primary mutation type for benzo[a]pyrene, benzo[b]fluoranthene, and
32 dibenzo[a,l]pyrene was the GGT^TGT mutation, which is associated with the formation of diol
33 epoxide guanine adducts. Dibenz[a,h]anthracene did not induce mutations in Ki-ras codons 12
34 or 61; however, diol epoxide guanine adducts and lung adenomas in A/J mice were observed.
35 This suggests that a different genetic target may be involved in carcinogenicity of this
36 compound.
37 H-ras mutations were studied in skin papillomas of SENCAR mice resulting from dermal
38 initiation by benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol (400 nmol) followed by
39 12-O-tetra-decanoylphorbol-13-acetate (TPA) promotion (Chakravarti et al., 2008). Polymerase
40 chain reaction (PCR) amplification of the H-ras gene and sequencing revealed that codon 13
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1 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the
2 relative levels of depurinating adducts of guanine and adenine, despite the formation of
3 significant amounts of stable DNA adducts.
4 Other studies also suggest that multiple genetic targets may be involved in PAH
5 mutagenicity and carcinogenicity (Conney et al., 2001; Smith et al., 2000). Smith et al. (2000)
6 indicated that diol epoxide adducts and mutations were observed in the p53 tumor suppressor
7 gene following in vitro exposure of cultured human bronchial epithelial cells to metabolites of
8 benzo[a]pyrene, chrysene, benzo[c]phenanthrene, and benzo[g]chrysene. PAH adducts and
9 corresponding mutations preferentially formed at lung mutational hot spots (codons 154, 157,
10 158, 245, 248, and 273), suggesting that PAHs may contribute to the mutation spectrum
11 observed in human lung cancer. Conney et al. (2001) provided evidence that dose-dependent
12 differences may exist for the mutation spectra seen in PAH-induced tumors. Skin papillomas
13 induced by benzo[a]pyrene in female mice were examined for mutations in the c-Ha-ras proto-
14 oncogene. The major difference between high- and low-dose groups was mutations at exon 2 of
15 the c-Ha-ras gene, with the proportion of AT base pair mutations higher in the low-dose group.
16 Dose-dependent changes in the mutation profile were also evident in Chinese hamster V79 cells
17 exposed to the diol epoxides of benzo[a]pyrene and benzo[c]phenanthrene (i.e., the proportion of
18 AT mutations decreased with increasing concentration).
19 In conclusion, there is evidence that an assumption of a similar toxicological action is
20 reasonable for PAHs; however, the carcinogenic process for individual PAHs is likely to be
21 related to some unique combination of multiple molecular events resulting from formation of
22 several reactive species. For these reasons, the use of an RPF approach to estimate cancer risk
23 associated with PAH exposure is considered appropriate. A common mutagenic mode of action
24 for carcinogenic PAHs is hypothesized based on information available for the indicator
25 chemical, benzo[a]pyrene (U.S. EPA, 2005b). The uncertainties and limitations related to the
26 mode of action assumption for PAH-induced cancer are further discussed in Section 8.5.
27
28 2.5. STRUCTURAL ALERTS FOR PAH CARCINOGENESIS
29 The carcinogenic activity of PAH compounds is influenced by specific structural
30 features. For example, alternant PAHs having four or more benzene rings exhibit greater
31 carcinogenic potency than PAHs with two or three benzene rings (Bostrom et al., 2002). The
32 carcinogenic activity of PAHs is also related to the specific arrangement of the benzene rings.
33 As described in Section 2.4, PAHs that form bay- and fjord-region diol or dihydrodiol epoxides
34 are more potent carcinogens compared with linear PAHs that lack this structural feature
35 (Bostrum et al., 2002). These metabolites are resistant to detoxification due to stereochemical
36 effects and, consequently, are more likely to be mutagenic and cause cancer (Buterin et al., 2000;
37 Chang et al., 1981; Buening et al., 1979; MacLeod et al., 1979; Flesher et al., 1976).
38 Dihydrodiol epoxides formed at other positions on the PAH molecule (i.e., not the bay- or fjord-
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1 regions) are more accessible to glutathione transferase detoxification and are less potent
2 mutagens and carcinogens (MacLeod et al., 1979; Flesher et al., 1976). Nonalternant PAHs
3 containing fused benzenoid and five-membered rings, can also be metabolized to bay- and fjord-
4 region diol epoxides (Bostrum et al., 2002); however, epoxide formation at the cyclopenta- ring
5 structure may also contribute to carcinogenicity (Bostrum et al., 2002; Nyholm et al., 1996).
6 PAHs with at least four rings and a classic bay- or fjord-region (formed entirely by
7 benzene rings; see Figure 2-1) may be characterized as containing structural alerts for
8 carcinogenesis. However, this structural characterization is likely to be overly simplistic and
9 other features may be important to carcinogenesis. Recent studies have applied quantitative
10 structure activity relationship (QSAR) methods to evaluate the relationship between specific
11 PAH structural features and mechanistic events related to carcinogenesis (Bruce et al., 2008;
12 Vijayalakshmi et al., 2008).
13
14 2.6. SIMILARITIES IN RELATIVE POTENCY ACROSS ENDPOINTS
15 Studies that have evaluated the association between cancer-related endpoints and
16 tumorigencity of PAHs are briefly summarized below.
17 Several studies have been performed that compare the bacterial or mammalian cell
18 mutagenicity of various PAHs with tumor initiating activity or complete carcinogenesis
19 (Blackburn et al., 1996; LaVoie et al., 1985, 1981, 1979; Raveh et al., 1982; Andrews et al.,
20 1978). In general, mutagenicity appears to correlate best with tumor initiation. Complete
21 carcinogenicity is not well-predicted by positive findings in short-term mutagenicity assays.
22 Andrews et al. (1978) tested 24 PAHs for bacterial mutagenicity in the Ames test and compared
23 these findings to evidence of carcinogenicity (parent and metabolites) from previously published
24 studies. Positive findings of both mutagenicity and carcinogenicity were only reported for 14 of
25 the 24 PAHs evaluated. Eight of the 10 remaining PAHs were found to be mutagenic in the
26 Ames assay, but were not carcinogenic in animal studies. LaVoie et al. (1979) compared the
27 mutagenicity, tumor-initiating activity, and complete carcinogenicity of several series of
28 structurally related PAHs. Tumor-initiating activity was found to correspond with complete
29 carcinogenicity. Quantitation of mutagenicity in the Ames assay for structurally related PAHs
30 failed to provide a reliable indication of tumor-initiating activity or complete carcinogenicity. In
31 addition, mutagenicity results could not be used to predict which PAHs would be
32 noncarcinogenic. Many PAHs were active mutagens, but were not shown to be carcinogenic.
33 Studies using methylated derivatives of anthracene demonstrated a correlation between
34 mutagenicity of specific metabolites and tumor initiating activity in mouse skin (LaVoie et al.,
35 1985). Raveh et al. (1982) reported that the mutagenic response to PAHs in Chinese hamster
36 V79 cells was similar to the skin tumor initiating activity observed in SENCAR mice.
37 Benzo[a]pyrene was demonstrated to be a more potent mutagen and skin tumor initiator than
38 cyclopenta[c,d]pyrene.
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1 Blackburn et al. (1996) compared the predictive power of a mutagenicity test (the
2 Modified Ames Test, which uses enhanced extraction techniques and greater levels of S9 to
3 improve performance when oils are tested) and DNA adduct formation (measured by
4 P32-postlabelling) to predict the dermal carcinogenicity of 120 PAH-containing oils. The
5 Modified Ames Test provided greater accuracy in predicting carcinogenicity (96%). In addition,
6 the mutagenicity index estimated from this test correlated strongly (r2 > 0.83) with PAH content
7 of the oils. The DNA adduct assay predicted carcinogenicity correctly with about 73% accuracy;
8 however, the study authors indicated that the lower predictability may have resulted from the use
9 of adduct data that were collected while the assay was still undergoing development.
10 Sjogren et al. (1996) performed a multivariate analysis of data for 29 PAHs to evaluate
11 the relevance of different biological assays to the carcinogenic properties of PAHs. This analysis
12 considered carcinogenicity (International Agency for Research on Cancer [IARC] weight of
13 evidence and QSAR predictions), bacterial mutagenicity, inhibition or enhancement of bacterial
14 mutagenicity, AhR affinity, and enzyme induction. Bacterial mutagenicity data were poorly
15 correlated with observed and predicted cancer data, while AhR affinity variables were
16 statistically relevant to describe these data.
17 Other studies suggest that the relationship between affinity for the AhR and carcinogenic
18 potency is unclear. For example, highly mutagenic fjord-region PAHs are potent carcinogens
19 despite exhibiting lower AhR affinity (reviewed by Bostrom et al., 2002). Likewise, some PAHs
20 that strongly activate the AhR, such as benzo[k]fluoranthene (Machala et al., 2001), are only
21 weakly carcinogenic. In addition, some studies have demonstrated the formation of DNA
22 adducts in the liver of AhR knock-out mice following intraperitoneal or oral exposure to
23 benzo[a]pyrene (Sagredo et al., 2006; Uno et al., 2006; Kondraganti et al., 2003), indicating that
24 Ah responsiveness is not strictly required for metabolic activation and genotoxicity. These
25 findings suggest that there may be alternative (i.e., non-AhR mediated) mechanisms of
26 benzo[a]pyrene activation in the mouse liver, and that AhR affinity would not be a good
27 predictor of carcinogenic potency.
28 AhR-mediated CYP1 Al induction by PAHs is considered to contribute to tumorigenesis
29 by increasing the production of DNA-reactive metabolites (Ayrton et al., 1990). However,
30 CYP1 Al induction potency alone does not appear to correlate well with carcinogenic potency of
31 PAHs. Ethoxyresorufm O-deethylase (EROD) activity was evaluated as a measure of CYP1 Al
32 induction in rat hepatocytes (Bosveld et al., 2002; Till et al., 1999; Willett et al., 1997) and trout
33 liver cells (Bols et al., 1999). Till et al. (1999) additionally measured levels of CYP1A1 protein
34 and mRNA. Machala et al. (2001) measured PAH activation of the AhR using a chemical -
35 activated luciferase reporter gene assay. Comparable results were observed across studies, and
36 benzo[k]fluoranthene was consistently demonstrated to be the most potent inducer of CYP1 Al.
37 Chrysene, benzo[b]fluoranthene, dibenz[a,h]anthracene, and indeno[l,2,3-c,d]pyrene were also
38 demonstrated to be more potent inducers of CYP1 Al than benzo[a]pyrene. However, most of
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1 these PAH compounds (except dibenz[a,h]anthracene) are considerably less potent as
2 carcinogens in animal bioassays.
3 Ross et al. (1995) evaluated the relationship between TIDAL values for DNA adduct
4 formation and lung adenoma formation in A/J mice. The TIDAL value versus tumor relationship
5 was similar for five different PAHs, suggesting a correlation between adduct levels and tumor
6 formation (regression analysis was not performed). As described above, the relationship of
7 TIDAL level to tumor formation was similar for PAHs that produce different types of adducts
8 and different mutations in the Ki-ras oncogene, suggesting that the probability of tumor
9 formation may be related to the extent of overall DNA damage and repair (Nesnow et al., 1998a,
10 1996, 1995; Mass et al., 1993).
11 To summarize, various cancer-related endpoints have been associated with PAH
12 carcinogenicity. Tumor initiation ability was shown to correspond well with complete
13 carcinogenicity, while some studies suggested that bacterial mutagenesis assays of individual
14 PAHs were not highly correlated with tumor formation (Sjogren et al., 1996; Lavoie et al., 1979).
15 DNA adduct formation corresponded with lung adenoma formation in A/J mice for several
16 PAHs (Sjogren et al., 1996; Ross et al., 1995; LaVoie et al., 1979). The development of RPFs in
17 this analysis considered both tumorigenicity and cancer-related endpoints (e.g., mutagenicity,
18 clastogenicity, morphological transformation). Studies of AhR binding/activation were not
19 considered for use in deriving RPFs because there does not appear to be a clear relationship
20 between affinity for the AhR and carcinogenic potency of PAHs.
21
22 2.7. SIMILARITIES IN RELATIVE POTENCY ESTIMATES ACROSS SPECIES AND
23 EXPOSURE ROUTES
24 Available studies suggest that the potency of individual PAHs is generally consistent
25 across species and study protocols. The consistency of potency estimates based on in vivo
26 tumorigenicity studies conducted using different study protocols and exposure routes in varying
27 species/strains of test animals is summarized below.
28 Nisbet and LaGoy (1992) and Clement Associates (1988) reported that RPFs for PAHs
29 are reasonably consistent across different study protocols using varying species/strains of
30 laboratory animals. RPF estimates were calculated in multiple test systems including mouse skin
31 complete carcinogenesis studies, mouse skin tumor initiation studies, studies in rat lung
32 (implantation), other rat studies (intrapulmonary injection, subcutaneous injection), and newborn
33 mouse studies (intraperitoneal injection). The RPF estimates for specific PAHs calculated from
34 different assay systems varied by less than an order of magnitude. The relative potency of
35 individual PAHs to benzo[a]pyrene was also shown to be very similar when based on data in
36 different strains of mice using different mouse tumor initiation models (Slaga and Fisher, 1983).
37 Muller et al. (1997) compared the relative potency of benzo[a]pyrene and 3-methylcholanthrene
38 from data generated in three species (rat, mouse, and hamster). Similar RPF values (i.e., within a
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1 factor of 2) were derived for oral exposures in mice, rats, and hamsters. In their comparison
2 across different exposure routes (oral, respiratory, and dermal), Muller et al. (1997) reported
3 similar relative potencies for benzo[a]pyrene and 3-methylcholanthrene (within a factor of 2) for
4 data from rats exposed via oral and respiratory routes, and for mice exposed via oral and dermal
5 routes. The relative potency for respiratory exposure in mice was an order of magnitude lower
6 than relative potencies for the other two exposure routes.
7 Schneider et al. (2002) performed a more recent evaluation of the impact of exposure
8 route on the determination of RPFs. Potency ratios were calculated for several carcinogenicity
9 bioassays by dividing the carcinogenic potency of a PAH mixture by the carcinogenic potency of
10 benzo[a]pyrene as a single substance. The potency ratios were observed to vary by exposure
11 route and target organ. For example, potency ratios associated with forestomach tumors from
12 oral exposure ranged from 0.7 to 1.2 (i.e., the potencies of the PAH mixtures and benzo[a]pyrene
13 to induce forestomach tumors were approximately equal). This suggested that these tumors may
14 be attributable to the benzo[a]pyrene content of the mixture. Potency ratios for skin tumor
15 production from dermal exposure ranged from 2 to 11, whereas RPFs calculated for lung tumors
16 from oral exposure, pulmonary implantation, or inhalation were greater than 20. These results
17 suggested that the benzo[a]pyrene content of PAH mixtures may be only slightly responsible for
18 lung and dermal carcinogenicity. Schneider et al. (2002) suggested that RPF estimates should be
19 derived separately for oral, dermal, and inhalation exposure using studies with the relevant
20 exposure pathway.
21 To summarize, there is some consistency within the in vivo carcinogenicity database for
22 relative potency estimates derived from different species and strains exposed by various routes,
23 although this is an area for which further research is needed. However, Schneider et al. (2002)
24 have cautioned that potency ratios appear to cluster by exposure route and target organ and have
25 suggested that route-specific RPFs be developed. There is also some concern regarding the use
26 of benzo[a]pyrene as an index chemical to estimate lung cancer from PAH mixtures, considering
27 that the lung is relatively insensitive to benzo[a]pyrene-induced tumorigenicity following oral
28 exposure (Gaylor et al., 1998). Section 8.6 provides a comparison of RPF values calculated in
29 this report, using bioassay data from different exposure routes and study designs. RPF values
30 were comparable across most exposure routes, with the exception of the newborn mouse
31 intraperitoneal injection studies.
32
33 2.8. DOSE ADDITIVITY OF PAHs IN COMBINED EXPOSURES
34 Use of the RPF approach assumes that doses of component chemicals that act in a similar
35 manner can be added together, after scaling the potencies relative to the index chemical, and that
36 interaction effects do not occur at low environmental exposure levels (U.S. EPA, 2000, 1986).
37 The level of confidence in the RPF approach is increased if dose additivity can be demonstrated
38 experimentally, even with simple mixtures. For PAHs, the assumption of dose additivity at low
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1 exposures cannot be confirmed or refuted based on the available experimental data. It appears
2 that interactions may occur at higher doses of complex PAH mixtures (see below).
3 The complexity of potential interactions for tumorigenesis of binary mixtures of PAHs is
4 illustrated in Table 2-2. The nature of the interaction varies with the PAHs evaluated and the
5 study conditions (e.g., vehicle used, dose selection, study method). Many studies were designed
6 to evaluate the combined administration of a known carcinogen with either a weak carcinogen or
7 a noncarcinogenic PAH. The true nature of the interaction (i.e., additive, synergistic, or
8 antagonistic) can be difficult to determine in studies wherein the tumorigenic response is not
9 measured for both PAHs given alone and in combination. These studies can distinguish between
10 an enhanced or cocarcinogenic response and an inhibitory response, but a further classification
11 cannot be made. The interactions described as cocarcinogenic in Table 2-2 may be either
12 additive or synergistic in nature.
13
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Table 2-2. Studies of binary mixtures of PAHs and tumorigenicity
Reference
Cavalieri et al., 1983
DiGiovanni et al., 1982
Falketal., 1964
Laviketal., 1942
Pfeiffer, 1973
Slagaetal., 1979
Steiner, 1955; Steiner
and Falk, 1951
Van Duuren and
Goldschmidt, 1976;
Goldschmidtetal.,
1973
Van Duuren etal.,
1973
Warshawsky etal.,
1993
Endpoint
Mouse skin
carcinogenicity
Skin tumor initiation in
mice
Sarcoma induction in
mice by subcutaneous
injection
Mouse skin tumors
Sarcoma induction in
mice by subcutaneous
injection
Skin tumor initiation in
mice
Sarcoma induction in
mice by subcutaneous
injection
Mouse skin
carcinogenicity
Mouse skin
carcinogenicity
Mouse skin
carcinogenicity
Findings
BaP and CPcdP given together resulted in a
synergistic effect at low and intermediate doses;
three- to sevenfold increase in relative risk at
intermediate dose of both BaP and CPcdP as
compared to the sum of the relative risk for the
same dose of each PAH given alone.
BeP increased BaP tumor initiation (30%f),
inhibited tumor initiation by DMBA (84% J.) and
DBahA (48%J.) and produced no change in
combination with 3-MC; DBacA inhibited tumor
initiation by DMBA (92%|), DBahA (39%|), and
3-MC (61%J.) and produced no change in
combination with BaP.
PH inhibited tumor response of DBahA in ethyl
laurate vehicle (approximately 30%J,, estimated
from graph); tumor response was enhanced in
triethylene glycol vehicle (approximately 50%| to
100% tumor-bearing animals, estimated from
graph).
3-MC and BaP, DBahA, or BaA essentially
additive.
BaP and DBahA less than additive; tumor response
for combined treatment was within 10% of DBahA
response.
BeP, Pyr, or FA increased skin tumor initiation by
BaP (30, 35, and 23%|, respectively); BeP, Pyr, or
FA decreased skin tumor initiation by DMBA (84,
50, and 34%|, respectively).
DBahA and 3-MC in combination roughly additive;
BaA and CH in combination resulted in a
synergistic effect (9%| above additive response);
BaA and DBahA in combination resulted in
inhibition (48%J, below additive response).
BeP, BghiP, Pyr, or FA and BaP increased tumors
over BaP alone (>50% increase in incidence, also
t multiplicity); no tumors were observed for PAHs
without BaP.
BaP and BghiP had cocarcinogenic effect (23%|
over BaP response alone).
Nontumorigenic dose of BaP increased tumor
incidence produced by CH (16%|), AC (8%|), and
FA (8%t).
Net effect
S
Co, I
Co, I
A
I
Co, I
A, S, and I
S
Co
S
1
2
o
5
4
5
3-MC = 3-methylchloanthrene; A = additive; Co = cocarcinogenic (enhanced tumorigenicity, study design does not
allow for determination of A or S); DMBA = 7,12-dimethyl-benz[a]anthracene; I = inhibitory; S = synergistic
Slooff et al. (1989) reviewed the available data addressing the carcinogenicity of
individual PAHs and in combination. It was concluded that a generally additive effect was
observed following administration of more than two different PAHs in weight ratios similar to
those occurring in ambient air or in various emissions. Combinations of only two PAHs
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1 produced either additive, synergistic, or inhibitory effects. The complexity of the interaction
2 among single PAH compounds is thought to be related to effects on metabolic enzyme systems
3 including induction processes and competitive inhibition. The generally additive response noted
4 for a more complex mixture may reflect the balance between inhibitory and synergistic
5 processes.
6 Additivity has been observed in carcinogenicity studies of complex mixtures of PAHs.
7 Schmahl et al. (1977) evaluated the production of skin tumors following combined dermal
8 treatment with 11 PAHs found as constituents of automobile exhaust. Tumor findings were
9 presented separately for two groups of PAHs. High potency carcinogens (Group 1) included
10 benzo[a]pyrene, dibenz[a,h]anthracene, benz[a]anthracene, and benzo[b]fluoranthene. Lower
11 potency PAHs (Group 2) included anthracene, benzo[e]pyrene, benzo[g,h,i]perylene, chrysene,
12 fluoranthene, phenanthrene, and pyrene. Chronic dermal exposure to PAHs in both groups
13 resulted in an additive response when compared to the tumor response for each group alone.
14 Nesnow et al. (1998b) evaluated lung tumor formation in A/J mice following combined
15 administration of five carcinogenic PAH compounds (benzo[a]pyrene, benzo[b]fluoranthene,
16 dibenz[a,h]anthracene, 5-methylchrysene, and cyclopenta[c,d]pyrene). High and low doses were
17 selected for each PAH in this study based on toxicity, survival, range of response, and predicted
18 tumor yield. The ratio of PAH doses was designed to simulate PAH ratios found in
19 environmental air and emissions samples. PAHs were administered to mice in a 25 factorial
20 study design yielding 32 dose groups (combination of five PAHs at high and low doses). The
21 formation of lung adenomas was evaluated 8 months following intraperitoneal injection of PAH
22 mixtures. A response surface model was used to evaluate specific interactions among PAHs.
23 The results of the study indicated that greater-than-additive effects were seen at low doses, while
24 less-than-additive effects were observed at high doses. However, the magnitude of the
25 interactions was relatively small (twofold), suggesting that potential interactions are limited in
26 extent.
27 Dermal application of binary mixtures of PAHs has also been shown to produce additive,
28 synergistic, and inhibitory effects on DNA binding in mouse skin (Hughes and Phillips, 1993,
29 1990). Hermann (1981) demonstrated that many PAHs could both enhance and inhibit the
30 bacterial mutagenicity of benzo[a]pyrene depending on the relative concentrations in the binary
31 mixture. Binary mixtures of benzo[a]pyrene and benzo[e]pyrene produced a synergistic
32 response in the TA98 strain of S. typhimurium (which detects frameshift mutations) and
33 antagonistic and additive effects in strain TA100 (which detects a broad spectrum of mutations)
34 depending on the concentration (Hass et al., 1981). Binary mixtures of PAHs have also been
35 shown to produce antagonistic or less-than-additive effects in the Ames assay of bacterial
36 mutagenicity (Barrai et al., 1992; Salamone et al., 1979a). Vaca et al. (1992) demonstrated an
37 additive effect for sister chromatid exchange induction by combined administration of
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1 benzo[a]pyrene and fluoranthene in human peripheral lymphocytes cocultured with
2 polychlorinated biphenyl-induced rodent liver cells.
3 The effects of binary PAH mixtures on gene expression, DNA adduct formation,
4 apoptosis, and cell cycle are additive compared to the effects of the individual compounds in
5 human hepatoma cells (HepG2) (Staal et al., 2007). Equimolar and equitoxic mixtures of
6 benzo[a]pyrene with either dibenzo[a,l]pyrene, dibenz[a,h]anthracene, benzo[b]fluoranthene,
7 fluoranthene, or 1-methylphenanthrene were studied. PAH mixtures showed an additive effect
8 on apoptosis and on cell cycle blockage. The effects of binary mixtures of PAHs on gene
9 expression were generally additive or slightly antagonistic.
10 Additivity has also been observed for the mutagenicity of PAHs administered as a
11 complex mixture (Bostrom et al., 1998; Kaden et al., 1979). Kaden et al. (1979) evaluated the
12 bacterial mutagenicity of the PAH fraction of kerosene soot using resistance to 8-azaguanine as a
13 genetic marker for forward mutation in S. typhimurium. Approximately half of the PAHs tested
14 (34 of 70) produced a significant increase in the mutant fraction in this assay system. The
15 mutagenicity of the complex soot mixture was demonstrated to be approximately equal to the
16 additive mutagenicity of the individual components. Bostrom et al. (1998) reported additivity in
17 the Ames test of bacterial mutagenesis (i.e., reversion to histidine independence) for a mixture of
18 four PAHs (benzo[a]pyrene, benz[a]anthracene, fluorene, and pyrene) using four different strains
19 of S. typhimurium.
20 Mechanistic studies have suggested that the outcome of the interaction between two
21 PAHs in a binary mixture is dependent on changes in metabolism. PAHs can act as both
22 inducers and competitive inhibitors of the CYP enzymes that are responsible for generation of
23 reactive metabolites. Benzo[e]pyrene has been shown to alter the oxidative metabolism of
24 benzo[a]pyrene, which may be related to the cocarcinogenic effect seen in skin tumor initiation
25 studies (Baird et al., 1984). Alterations in the types and amounts of benzo[a]pyrene metabolites
26 suggest that benzo[e]pyrene-induced changes may be isozyme specific (Smolarek and Baird,
27 1984). An increase in the formation of benzo[a]pyrene DNA adducts has also been
28 demonstrated for coadministration of benzo[e]pyrene in SENCAR mouse skin (Smolarek et al.,
29 1987). Fluoranthene and pyrene have been shown to increase the formation of benzo[a]pyrene-
30 DNA adducts in mouse skin following a combined treatment (Rice et al., 1988, 1984).
31 Enhancement of the metabolism of benzo[a]pyrene to diol epoxide metabolites and subsequent
32 DNA binding may explain the increased carcinogenic effect in this case. Phenanthrene did not
33 increase the formation of benzo[a]pyrene-DNA adducts and was not shown to be cocarcinogenic
34 following combined administration with benzo[a]pyrene in this study. Cherng et al. (2001)
35 demonstrated that benzo[g,h,i]perylene increased the formation of benzo[a]pyrene adducts in
36 hepatoma cells (HepG2) by enhancing benzo[a]pyrene induction of CYP1A1. Benzo[g,h,i]-
37 perylene increased the nuclear accumulation of the AhR and/or the activation of the AhR to a
38 DNA-binding form (Cherng et al., 2001). Benzo[k]fluoranthene altered the metabolic profile of
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1 benz[a]anthracene by increasing the activity of CYP1A1 (Schmoldt et al., 1981). The bacterial
2 mutagenicity of benz[a]anthracene was enhanced by use of a rodent liver S9 that was obtained
3 from animals previously exposed to other PAHs (Norpoth et al., 1984). Coadministration of
4 benzo[a]pyrene and benz[a]anthracene to hamster embryo cell cultures resulted in decreases in
5 the metabolism of benzo[a]pyrene, the level of DNA binding, and the mutation frequency in
6 hamster V79 cells (Smolarek et al., 1986).
7 In summary, combined administration of binary mixtures of PAHs can result in several
8 types of joint action (i.e., additive, synergistic, or antagonistic). The nature of the joint action
9 appears to be dependent on the characteristics of the individual PAHs, related changes in
10 metabolism and possibly the test species/strain. PAHs can act as both inducers and competitive
11 inhibitors of the CYP enzymes that are responsible for generation of reactive metabolites.
12 Additivity has been observed for some complex mixtures of PAHs, suggesting a balance in the
13 relative metabolism of individual PAHs. For the purposes of this analysis, an assumption is
14 made that the combination of individual PAHs results in additive effects. Additional research is
15 needed to characterize the validity of this assumption.
16
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1 3. DISCUSSION OF PREVIOUSLY PUBLISHED RPF APPROACHES
2
3
4 There are multiple analyses available for the derivation of relative potency estimates for
5 individual PAHs. All of these analyses utilize benzo[a]pyrene as the index chemical. Table 3-1
6 compares relative cancer potency values for PAHs presented by several authors. A review of the
7 derivation of these relative potency values follows.
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Table 3-1. Comparison among various relative potency estimates for PAHs from the published literature and
regulatory agencies (1984-2004)
PAH
Acenaphthene
Acenaphthylene
Anthanthrene
Anthracene
Benzo[a]pyrene
Benz[a]anthracene
Benzo [b] fluoranthene
Benzo[c]phenanthrene
Benzo[e]pyrene
Benzo[g,h,i]perylene
Benzo [j ] fluoranthene
Benzo [k] fluoranthene
Chrysene
Coronene
Cyclopenta[c,d]
pyrene
Dibenz[a,h]
anthracene
Dibenz[a,c]anthracene
Dibenzo[a,e]pyrene
Dibenzo[a,h]pyrene
Dibenzo[a,i]pyrene
Dibenzo[a,l]pyrene
Fluoranthene
Fluorene
Indenof 1,2,3-
c,d]pyrene
Perylene
Abbr
AN
ANL
AA
AC
BaP
BaA
BbF
BcPH
BeP
BghiP
BjF
BkF
CH
CO
CPcdP
DBahA
DBacA
DBaeP
DBahP
DBaiP
DBalP
FA
FE
IP
Pery
U.S.
EPA
(1993)
1
0.1
0.1
0.01
0.001
1
0.1
Chu
and
Chen
(1984)
1
0.013
0.08
0.004
0.001
0.69
0.017
Clement
(1988)
0.32
1
0.145
0.14
0.004
0.022
0.061
0.066
0.0044
0.023
1.11
0.232
Clement
(1990)
0.316
1
0.1228
0.007
0.0212
0.0523
0.0523
0.278
Rugen et
al. (1989)
1
0.004-
0.006
0.0235
0.0763
0.599
0.00599
Slooffet
al. (1989)
0
1
0-0.04
0.01-0.03
0.03-0.09
0.05-0.89
0-0.06
0-0.08
Kroese
etal.
(2001)
0
1
0.1
0.03
<0.1
0.1-0.03
0.01
0.1
Nisbet
and
LaGoy
(1992)
0.001
0.001
0.01
1
0.1
0.1
0.01
0.1
0.01
5
0.001
0.001
0.1
Malcolm
and
Dobson
(1994)
0.001
0.001
0.01
1
0.1
0.1
0.01
0.01
0.1
0.1
0.01
0.001
0.1
1
0.1
0.001
0.001
0.1
0.001
Meek
etal.
(1994)
1
0.06
0.05
0.04
0.12
Muller
etal.
(1997)
0.28
1
0.014
0.11
0.023
0
0.012
0.045
0.037
0.026
0.012
0.89
1.2
1.1
0.067
Larsen and
Larsen
(1998)
0.3
0.0005
1
0.005
0.1
0.023
0.002
0.02
0.05
0.05
0.03
0.02
1.1
0.2
1
0.1
1
0.05
0.1
Collins
etal.
(1998)
1
0.1
0.1
0.1
0.1
0.01
1
10
10
10
0.1
Cali-
fornia
EPA
(2004)
0.62
0.52
0.17
11
12
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Table 3-1. Comparison among various relative potency estimates for PAHs from the published literature and
regulatory agencies (1984-2004)
PAH
Phenanthrene
Pyrene
Abbr
PH
Pyr
U.S.
EPA
(1993)
Chu
and
Chen
(1984)
Clement
(1988)
0.081
Clement
(1990)
Rugen et
al. (1989)
Slooffet
al. (1989)
0.01
Kroese
etal.
(2001)
<0.01
Nisbet
and
LaGoy
(1992)
0.001
0.001
Malcolm
and
Dobson
(1994)
0.001
0.001
Meek
etal.
(1994)
Muller
etal.
(1997)
0.00064
0
Larsen and
Larsen
(1998)
0.0005
0.001
Collins
etal.
(1998)
Cali-
fornia
EPA
(2004)
Abbr = abbreviation
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1 U.S. EPA (1993) presented RPFs (termed EOPPs) for seven PAHs (benzo[a]pyrene,
2 benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene,
3 dibenz[a,h]anthracene, indeno[l,2,3-c,d]pyrene) as Provisional Guidance for the risk evaluation
4 of PAHs. On the IRIS database, the current entries for all seven of these compounds contain a
5 cancer weight of evidence classification of Group B2 (probable human carcinogen, based on
6 sufficient evidence of carcinogenicity in animals) (www.epa.gov/iris). U.S. EPA (1993)
7 indicated that the data for PAHs did not meet the criteria for the development of toxicity
8 equivalency factors (TEFs). In particular, the existing database was limited primarily to studies
9 of metabolism, genotoxicity, and cancer, and the assumptions of the dose-additivity model (i.e.,
10 toxicological similarity and no interactions at low concentrations) were not proven or refuted.
11 The EOPP terminology was used because this approach was limited to skin painting data and
12 was based on benzo[a]pyrene exposure from a single (oral) pathway (for the derivation of the
13 slope factor). This analysis considered only a small subset of PAHs routinely measured in PAH
14 mixtures at hazardous waste sites. The EOPP values were based on previous evaluations
15 conducted by Chu and Chen (1984) and Clement Associates (1988) and were calculated for
16 various test systems (i.e., mouse skin carcinogenesis, subcutaneous injection in mice,
17 intrapulmonary administration to rats, tumor initiation on mouse skin, and intraperitoneal
18 injection in newborn mice) (Clement Associates, 1988). Various statistical methods for
19 combining data sets were considered; however, final EOPP values were based on a single test
20 system (skin painting) and were rounded to the closest order of magnitude. The EOPPs were
21 recommended for the oral exposure route only, because the quantitative dose-response
22 assessment for benzo[a]pyrene was from an oral carcinogenicity bioassay (i.e., an oral cancer
23 slope factor). This recommendation was, however, complicated by the fact that the EOPPs were
24 derived from comparisons based on dermal exposure.
25 Chu and Chen (1984) presented RPF values for the seven PAH compounds described in
26 the Provisional Guidance described above (U.S. EPA, 1993) (see Table 3-1). These values were
27 calculated using mouse skin painting data only. Tumor incidence data were modeled using the
28 linearized multistage model and the resulting EDio and ql* (upper confidence limit of the linear
29 slope) were presented for target PAHs and benzo[a]pyrene. The RPFs listed in Table 3-1
30 represent the ratio of the ql * value for a PAH compound to the ql * value for benzo[a]pyrene
31 (i.e., ql*PAH-ql*Bap).
32 Clement Associates (1988) identified 11 published studies that concurrently compared
33 the carcinogenicity of benzo[a]pyrene with one or more other PAHs, and used the data to derive
34 relative cancer potencies for 13 PAHs, including benzo[a]pyrene. Test protocols used in this
35 analysis included mouse skin complete carcinogenesis, initiation-promotion on mouse skin,
36 subcutaneous injection into mice, lung implantation in rats, and intraperitoneal injection into
37 newborn mice. Tumor incidence data were fit to a simplified version of the Moolgavkar-
38 Venson-Knudsen (MVK) two-stage model and to the linearized multistage model to obtain low-
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1 dose cancer potency values (transition rates and low-dose slope factors, respectively). Most of
2 the estimates were derived using data for multiple exposure levels and controls, but some were
3 based on a single exposure level and a control. RPFs were calculated as the ratio of the
4 estimated transition rate or slope factor for a particular PAH to the corresponding values for
5 benzo[a]pyrene from the same study. Clement Associates (1988) selected representative RPFs
6 for each of the studied PAHs based on evaluations of the quality of the studies from which the
7 estimates were obtained.
8 Clement Associates (1990) also derived relative cancer potencies for eight PAHs based
9 on tumor incidence data from rat lung implantation data only (Deutsch-Wenzel, 1983). The data
10 were restricted to a single group of studies using a defined experimental protocol in order to
11 address issues of questionable data quality associated with other studies. Data quality concerns
12 cited for other studies include variation in survival, saturation of the carcinogenic effect,
13 outmoded pathological classification, and inadequate controls. The RPF values based on rat lung
14 implantation data were comparable to those originally derived by Clement Associates (1988)
15 (see Table 3-1).
16 Rugen et al. (1989) proposed a relative potency approach to establish acceptable
17 exposure levels (AELs) for six carcinogenic PAHs in drinking water (listed in Table 3-1). These
18 authors reviewed mouse skin painting studies in which the cancer potency of benzo[a]pyrene
19 was compared with those of other PAHs (Bingham and Falk, 1969; Wynder and Hoffmann,
20 1961, 1959a, b). The following relationship was used to calculate conversion factors to derive
21 AELs for these PAHs from the AEL for benzo[a]pyrene: relative tumor dose (RTD) =
22 (di/ni)/(d2/n2); where di and ni represented a dosage level and associated tumor incidence after a
23 given exposure duration to a certain PAH, PAHi, and d2 and n2 represented similar quantities for
24 exposure to the index PAH, benzo[a]pyrene, for the same exposure duration. The AEL for a
25 particular PAH was then derived with the following relationship: AEL(PAHi) = AEL(benzo[a]Pyrene) x
26 RTD(pAHi)- In this approach, RTDs for PAHs more potent than benzo[a]pyrene were less
27 than 1 and RTDs for PAHs less potent than benzo[a]pyrene were greater than 1. The reciprocal
28 of the RTDs derived by Rugen et al. (1989) were comparable to the RPFs presented by other
29 authors and are presented as such in Table 3-1.
30 The Netherlands (RIVM) proposed RPF values for 10 PAHs (naphthalene, anthracene,
31 phenanthrene, fluoranthene, chrysene, benz[a]anthracene, benzo[k]fluoranthene, benzo[a]pyrene,
32 benzo[g,h,i]perylene, and indeno[l,2,3-c,d]pyrene) (Slooff et al., 1989). RPFs were calculated
33 as a ratio of ED50 values that were calculated using a simple linear model. For dermal studies in
34 which the latency period was determined, the tumor incidence was divided by latency and
35 concentration, and the values were averaged for the different concentrations. Kroese et al.
36 (2001) provided an update of the RPF values calculated by Slooff et al. (1989) by incorporating
37 more recent evaluations conducted by other authors (Larsen and Larsen, 1998; Nesnow et al.,
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1 1998b; Muller, 1997; Nisbet and LaGoy, 1992). The RPF values for chrysene and fluoranthene
2 were decreased, while other values remained similar to those originally proposed (see Table 3-1).
3 Nisbet and LaGoy (1992) proposed toxicity equivalence factors for 17 PAHs commonly
4 found at hazardous waste sites. These authors reviewed published studies in which the
5 tumorigenic potencies of one or more PAHs were compared with benzo[a]pyrene (essentially the
6 same as those reviewed by Clement Associates, 1988) and rounded, to an order of magnitude, the
7 estimates presented by Clement Associates (1988) for seven carcinogenic PAHs (dibenz[a,h]-
8 anthracene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, indeno[l,2,3-c,d]-
9 pyrene, benzo[g,h,i]perylene, and chrysene) (see Table 3-1). Nisbet and LaGoy (1992) argued
10 that the rounded estimates more accurately reflected the uncertainty in the estimates than the
11 values presented by Clement Associates (1988). Nisbet and LaGoy (1992) stated that Clement
12 Associates (1988) proposed a TEF of 0.32 for anthracene (CASRN 120-12-7), but examination
13 of the original report shows that Clement Associates (1988) proposed this value for anthanthrene
14 (CASRN 191-26-4) and did not propose a value for anthracene. Nisbet and LaGoy (1992)
15 assigned a value of 0.01 to anthracene. In addition, Nisbet and LaGoy (1992) arbitrarily
16 assigned TEFs of 0.001 to eight other PAHs for which adequate evidence of carcinogenicity in
17 animals was not available (acenaphthene, acenaphthylene, fluoranthene, fluorene, 2-methyl-
18 naphthalene, naphthalene, phenanthrene, and pyrene). In defense of this assignment, the
19 argument was made that some of these PAHs have been shown to have some, albeit limited,
20 evidence for carcinogenic or genotoxic activity in some studies (e.g., phenanthrene and
21 naphthalene3). The RPF value proposed for dibenz[a,h]anthracene was substantially higher than
22 that proposed by Clement Associates (1988). Nisbet and LaGoy (1992) indicate that their
23 analysis of the dose-response data suggests that an RPF value of 5 is more appropriate for
24 environmental exposures where the chemically-related tumor incidence rate would be
25 approximately <25%.
26 Malcolm and Dobson (1994) used RPFs for 23 PAHs to calculate environmental
27 assessment levels for atmospheric PAHs (sponsored by the Great Britain Department of the
28 Environment). The RPFs were derived from previously reported review papers (Nisbet and
29 LaGoy, 1992; Rugen et al., 1989; Clement Associates, 1988; Chu and Chen, 1984), as well as the
30 primary literature describing pulmonary implant, skin painting, subcutaneous injection, and
31 mouse skin DNA binding studies. No information was provided regarding the methodology used
32 to derive RPFs from specific experimental studies. The proposed RPF values for individual
33 PAHs were the highest values reported in the literature. Many of the RPF values are similar to
34 those reported by Nisbet and LaGoy (1992). RPFs were additionally reported for
35 benzo[e]pyrene, coronene, cyclopenta[c,d]pyrene, dibenz[a,c]anthracene, and perylene. The
36 benzo[e]pyrene and cyclopenta[c,d]pyrene RPFs were apparently calculated directly from mouse
3It should be noted that a recent bioassay for naphthalene has shown increased incidence of nasal tumors in exposed
rats (NTP, 2000).
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1 skin painting studies (Habs et al., 1980; Hoffmann and Wynder, 1966; Wynder and Hoffmann,
2 1959a, b). Coronene and perylene were arbitrarily assigned RPF values of 0.001 given the IARC
3 and U.S. EPA designation as "not classifiable as to human carcinogen!city" (similar approach to
4 Nisbet and LaGoy, 1992). Dibenz[a,c]anthracene was assigned an RPF value of 0.1 based on the
5 IARC designation of "possibly carcinogenic to humans."
6 Health Canada (Meek et al., 1994) proposed RPFs for five PAHs (benzo[a]pyrene,
7 benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[j]fluoranthene, and indeno[l,2,3-cd]pyrene)
8 based on the results of multistage modeling of incidence data in Osborne-Mendel rats treated by
9 lung implantation (Deutsch-Wenzel et al., 1983). Values were based on a comparison of the
10 doses that caused a 5% increase in tumor incidence (ED0s). RPFs were calculated as the ratio of
11 the ED05 for benzo[a]pyrene to the ED0s for a specific PAH compound.
12 The Ontario Ministry of Environment and Energy (Muller et al., 1997) proposed RPF
13 values for 209 PAHs using data from dermal studies in mouse skin or rat lung bioassays. Most
14 of these PAHs were alkylated PAHs, PAH metabolites, or heterocyclic PAH compounds. The
15 17 unsubstituted PAHs that were evaluated in this analysis are listed in Table 3-1. Muller et al.
16 (1997) derived a standard time of observation in order to account for varying study duration
17 across experiments. Several dose-response models were considered for the evaluation of tumor
18 incidence and multiplicity, and linear regression was selected as the preferable method.
19 Tumorigenic potency (i.e., the slope of incidence/mg) was determined separately for each data
20 set based on the following order of preference regarding study type: tumor initiation in
21 CD-I mice, tumor initiation in SENCAR mice, rat lung implantation, and complete
22 carcinogenicity in C57BL mice. RPFs were determined as the ratio of PAH potency to the
23 potency of benzo[a]pyrene. RPF values derived by Muller et al. (1997) were comparable to
24 values estimated by other authors.
25 Larsen and Larsen (1998) estimated RPFs for 23 PAHs based on a compilation of
26 available carcinogenicity data in animals using oral, pulmonary, and skin application of PAHs.
27 The authors indicated that these values represent an entirely subjective estimate of relative
28 potency; however, further detail regarding the derivation of RPF estimates was not provided.
29 Collins et al. (1998) developed RPFs (termed potency equivalency factors [PEFs]) for
30 21 PAHs; 10 of these were either methyl- or nitro-substituted or heterocyclic PAHs. A hierarchy
31 of data types was utilized to provide an order of preference for data utilization in calculating
32 RPFs. Because the analysis focused on PAHs as air contaminants, tumor data from inhalation
33 studies were preferred (although none were found), followed by intratracheal or intrapulmonary
34 instillation, oral administration, skin-painting, and subcutaneous or intraperitoneal injection.
35 Genotoxicity and structure activity data were considered the least-preferred data type for
36 calculation of RPFs. Collins et al. (1998) noted that a wide range of PEFs were observed for
37 individual chemicals using different types of data (e.g., mutagenicity versus tumor data). The
38 basis for the derivation of individual RPF values was presented in a California EPA (2002)
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1 technical support document. RPF values for benz[a]anthracene, benzo[b]fluoranthene,
2 benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[l,2,3-c,d]pyrene, and chrysene were similar
3 to those described by Clement Associates (1988). Additional RPFs for dibenzo[a,e]pyrene,
4 dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and dibenzo[a,l]pyrene were calculated using mouse
5 skin and rat mammary gland data (Cavalieri et al., 1991, 1989). A cancer slope factor was
6 directly calculated for dibenz[a,h]anthracene using the tumor incidence data from a drinking
7 water study (Snell and Stewart, 1962). The relative potency of dibenz[a,h]anthracene was
8 estimated to be 0.1, when compared to the oral potency for benzo[a]pyrene.
9 Revised California EPA RPFs were recently developed for benzo[b]fluoranthene,
10 benzo[j]fluoranthene, chrysene, dibenzo[a,h]pyrene, and dibenzo[a,i]pyrene (California EPA,
11 2004). Cancer potency estimates were derived from lung adenoma data in newborn mice treated
12 by intraperitoneal injection. Potency estimates represented the upper 95% confidence limit on
13 the linear term of the multistage model fit for the newborn mouse dose-response data. Because
14 benzo[a]pyrene was demonstrated to be 75 times more toxic in newborn mouse intraperitoneal
15 assays than in adult oral studies, oral equivalent potencies for individual PAHs were derived by
16 adjusting the cancer potency downward by a factor of 75. The RPFs listed in Table 3-1 were
17 calculated as the ratio of the oral equivalent potency for a PAH to the oral potency estimate for
18 benzo[a]pyrene. This methodology resulted in a significant increase in RPF values for
19 benzo[b]fluoranthene, benzo[j]fluoranthene, and chrysene when compared with other
20 approaches.
21 In summary, several approaches are available for the determination of RPFs for PAHs.
22 RPF values are proposed in at least one study for a total of 27 PAHs (see Table 3-1). Because
23 these approaches generally rely on similar bioassay data and modeling methods, the resulting
24 RPF values are fairly comparable for most PAHs across studies. Reports by Larsen and Larsen
25 (1998) and Malcolm and Dobbs (1994) did not provide sufficient information on the
26 methodology used to calculate RPF estimates and are therefore more uncertain. Variable RPF
27 estimates were reported for benz[a]anthracene, chrysene, and indeno[l,2,3-c,d]pyrene. RPF
28 values were also highly variable for dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene,
29 and dibenzo[a,l]pyrene; however, these were only presented in a few recent studies. As
30 indicated above, the recent California EPA (2004) approach to estimating RPFs provides
31 considerably higher RPF values for benzo[b]fluoranthene, benzo[j]fluoranthene, and chrysene,
32 compared with other approaches.
33 U.S. EPA is reevaluating the RPF approach for PAHs in this analysis due to the evolution
34 of the state of the science and increased understanding of PAH toxicology. A great deal of
35 scientific research on PAHs has been conducted since the 1993 Provisional Guidance was
36 developed. Toxicological data are available for a larger number of PAHs and cancer-related
37 endpoints. However, the database for PAHs still does not meet the criteria for the derivation of
38 TEFs. U.S. EPA (2000) defines TEFs as special types of RPFs that are derived when there are
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1 abundant data supporting a specific mode of action that is pertinent to all health endpoints. RPFs
2 may be derived when the mode of action is less certain or is known for only a subset of all health
3 endpoints. The major differences in the use of TEFs and RPFs is that TEFs are applied to all
4 health endpoints, exposure routes, and exposure durations (U.S. EPA, 2000), while RPFs may be
5 limited to specific endpoints, routes, or durations. In the case of PAHs, there are inadequate data
6 to identify a specific mode of action that is applicable across all health endpoints. Most of the
7 available toxicological data are limited to cancer endpoints and there are few data on the
8 potential mode(s) of action for other effects. As a result, the more generalized RPF approach is
9 considered appropriate for PAHs.
10
11 3.1. PREVIOUS EFFORTS TO VALIDATE THE RPF APPROACH
12 Several studies have attempted to validate the RPF approach by comparing the cancer
13 risk of a PAH mixture measured experimentally with the cancer risk that was predicted using the
14 RPF method (Muller et al., 1997; McClure, 1996; Goldstein et al., 1994; Clement Associates,
15 1990, 1988; Krewski et al., 1989). These studies provide semi-quantitative information on the
16 overall uncertainty in using a component-based approach. Consistent findings were not reported
17 across these studies. Some studies suggested that the RPF approach would closely predict the
18 cancer risks associated with PAH mixtures, while others indicated that cancer risks might be
19 over- or underestimated.
20 Clement Associates (1988) evaluated the usefulness of selected RPFs to predict the tumor
21 incidence observed in a mouse skin painting assay. Schmahl et al. (1977) exposed groups of
22 mice to multiple doses of benzo[a]pyrene alone or to one of two defined mixtures of PAHs. The
23 first of these mixtures was comprised of benzo[a]pyrene, dibenz[a,h]anthracene,
24 benz[a]anthracene, and benzo[b]fluoranthene. The second mixture contained seven PAHs:
25 phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[e]pyrene, and
26 benzo[g,h,i]perylene. The predicted tumor incidences for the animals treated with the mixtures
27 were calculated from benzo[a]pyrene equivalents of the mixture and dose-response modeling of
28 the Schmahl et al. (1977) data for benzo[a]pyrene alone. Predicted tumor incidences for the first
29 mixture were comparable to observed tumor incidences, while predicted values were greater than
30 the observed values for the second mixture.
31 Clement Associates (1990) examined the utility of a relative potency approach, in which
32 relative cancer potency estimates of eight PAHs were used, to predict the cancer potencies of
33 each of four complex mixtures containing many PAHs and other substances: gasoline engine
34 exhaust condensate, flue-gas condensate from coal-fired residential furnaces, diesel engine
35 exhaust condensate, and sidestream smoke condensate of cigarettes. Relative cancer potencies
36 (compared to benzo[a]pyrene) for each of the four complex mixtures were calculated using a
37 simplified version of the MVK two-stage model and tumor incidence data from a series of
38 published rat lung implantation studies that examined the carcinogenicity of each complex
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1 mixture, various subfractions of the mixtures, and benzo[a]pyrene (Grimmer et al., 1988,
2 1987a, b, 1984). Lung implantation data (Deutsch-Wenzel, 1983) were used to calculate RPFs
3 for benzo[b]fluoranthene, benzo[e]pyrene, benzo[j]fluoranthene, benzo[k]fluoranthene,
4 indeno[l,2,3-c,d]pyrene, anthanthrene, benzo[g,h,i]perylene, and benzo[a]pyrene. The sum of
5 the benzo[a]pyrene exposure equivalents for the eight PAHs (i.e., the sum of the products of the
6 relative cancer potencies of the eight PAHs multiplied by their concentrations in the respective
7 complex mixtures) accounted for only minor fractions of the total carcinogenicity of each of the
8 four complex mixtures. When the assumption was made that each of the eight PAHs was as
9 potent as benzo[a]pyrene, the sum of the benzo[a]pyrene equivalents still accounted for only
10 minor fractions of the carcinogenicity of each mixture. Clement Associates (1990) concluded
11 that the cancer risk associated with a complex PAH mixture could not be estimated reliably from
12 measurements of a few indicator components, and further speculated that the underestimation
13 occurred because complex mixtures that occur in the environment contain many PAHs that have
14 not been studied in cancer tests, but may be carcinogenic. In addition, complex PAH mixtures
15 found in the environment contain other potential carcinogens including substituted and
16 heterocyclic PAHs and non-PAH components.
17 Krewski et al. (1989) compared the observed tumor response rate for two PAH mixtures
18 in mice with the tumor response predicted using the RPFs for 13 individual PAHs; chemical
19 characterization of the mixture was not provided. With the exception of the highest dose, the
20 predicted tumor response for mixture 1 was similar to the observed response. For mixture 2, the
21 predicted tumor response value was higher than the observed response.
22 Goldstein et al. (1994) compared the experimental carcinogenicity of a MGP residue to
23 the predicted cancer risk using the Nisbet and LaGoy (1992) RPF scheme. The RPF method
24 underestimated the carcinogenicity of the mixture. The lack of correspondence was suggested to
25 be related to the presence of unidentified carcinogens in the mixture or possible synergistic
26 interactions between PAHs.
27 McClure et al. (1996) compared the tumor response predicted using U.S. EPA's 1993
28 provisional values (i.e., EOPPs) to the observed response reported in studies of mice exposed to
29 synthetic and complex mixtures of PAHs. The results of this analysis were mixed. EOPP values
30 closely predicted the mouse tumor response to subcutaneous or dermal application of synthetic
31 mixtures containing relatively potent carcinogens, while overestimating the response to synthetic
32 mixtures containing only relatively weak carcinogens (similar to findings of Clement Associates,
33 1988). Mouse skin tumor initiation with several coal liquids was closely predicted by the EOPP
34 approach; however, this method underestimated the tumor response from lung implantation of
35 coal furnace emission condensate and its PAH-containing neutral fraction.
36 The validation analyses that were performed by Muller et al. (1997) consisted of
37 component versus whole mixture risk comparisons using data for smoky coal and coke oven
38 emissions. The human lung cancer risks that were estimated using the RPF approach were
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1 compared to the whole mixture cancer risk derived from epidemiology studies. The relative
2 content of PAHs (compared to benzo[a]pyrene) in the mixture was determined analytically (for
3 smoky coal and coke oven emissions) or was estimated as a standard mixture assumed to
4 represent an average PAH profile. The RPF method produced PAH cancer risk estimates that
5 were significantly lower than the risk estimates derived from epidemiology studies.
6
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1 4. EVALUATION OF THE CARCINOGENICITY OF INDIVIDUAL PAHs
2
3
4 4.1. DATABASE OF STUDIES ON PAH CARCINOGENICITY AND CANCER-
5 RELATED ENDPOINTS
6 A database of primary literature relevant to the RPF approach for PAHs was developed.
7 This was accomplished through the following means:
8
9 • Definition of the study types that were considered relevant to relative potency
10 development;
11
12 • Review of reference lists from review articles and other secondary sources;
13
14 • Identification of selected PAHs to be included in search of open literature;
15
16 • Performance of targeted searches of open literature on selected PAHs; and
17
18 • Population of the database with references and meaningful keywords.
19
20 The study types that were considered most useful for RPF derivation were rodent
21 carcinogenicity bioassays (all routes) in which one or more PAH was tested at the same time as
22 benzo[a]pyrene. In addition, in vivo and in vitro data for cancer-related endpoints (in which one
23 or more PAH and benzo[a]pyrene was tested simultaneously) were obtained, including studies
24 on the formation of DNA adducts, mutagenicity, chromosomal aberrations, aneuploidy, DNA
25 damage/repair/recombination, unscheduled DNA synthesis, and cell transformation. Although it
26 would be possible to calculate RPFs from studies where a PAH and benzo[a]pyrene were tested
27 by the same laboratory using the same test system but at different times, this approach was not
28 considered because it could introduce differences in the dose-response information that are
29 unrelated to the chemical (e.g., variability associated with laboratory environment conditions,
30 animal handling, food supply). Thus, studies in which benzo[a]pyrene was not tested
31 simultaneously with another PAH were not considered for use in calculating RPFs. Studies that
32 did not include benzo[a]pyrene were, however, considered useful for evaluating the weight of
33 evidence for selecting PAHs to be included in the RPF approach.
34 Several study types were initially excluded from the database because they did not
35 provide carcinogenicity or cancer-related endpoint information for individual PAHs. These
36 include biomarker studies measuring DNA adducts in humans, studies of PAH metabolism, and
37 studies of PAH mixtures. Although these studies contain important information on human
38 exposure to PAH mixtures and the mode of action for PAH toxicity, they generally do not
39 contain dose-response information that would be useful for calculation of RPF estimates. In
40 addition to the primary bioassay and cancer-related endpoint studies described above, the RPF
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1 database also includes information on PAH mode of carcinogenic action, interactions among
2 PAHs in mixtures, and the influence of exposure route on carcinogenic action of PAHs.
3 Primary studies were identified through the review of available secondary sources and
4 review articles, supplemented by a targeted literature search. A complete list of the secondary
5 sources that were reviewed is contained in Appendix A. A literature search strategy was
6 developed by first constructing a list of the individual PAHs to be included. The list of PAHs
7 was restricted to unsubstituted PAHs with three or more fused aromatic rings containing only
8 carbon and hydrogen atoms, because these are the most widely studied members of the PAH
9 chemical class. Heterocyclic PACs or PAHs with substituted groups (e.g., alkyl, hydroxyl,
10 sulfhydryl, amino, or nitro groups) were not included. An initial search yielded a list of PAHs
11 for which toxicological data are available. Individual PAHs were then chosen for the literature
12 search because they were known to have toxicological information relevant to cancer, and in
13 most cases, their presence in environmental sources of PAH exposure was known. Using these
14 criteria and excluding benzo[a]pyrene, 74 PAHs were identified from primary and secondary
15 sources (see Table 2-1 in Chapter 2).
16 A search of the open literature was conducted in the MEDLINE (PubMed) database for
17 the 74 PAHs that were identified. This database encompasses many of the studies that would
18 also be found in TOXLINE and CANCERLIT (the latter is no longer available as a separate
19 database). MEDLINE was searched by CASRN in conjunction with cancer and cancer-related
20 endpoint keywords. The search was not limited by publication date to ensure that all relevant
21 studies were identified. A few compounds did not show any result when searched by CASRN.
22 For these PAHs, an additional search by name was conducted. Search results, including
23 MEDLINE keywords, were downloaded directly into the working RPF database.
24 In addition to MEDLINE, computer searches of the following databases and websites
25 were conducted: IARC, World Health Organization (WHO), Agency for Toxic Substances and
26 Disease Registry (ATSDR), Health Canada, the National Toxicology Program (NTP), California
27 EPA's Office of Environmental Health Hazard Assessment (OEHHA), the Substance Registry
28 System, the Chemical Carcinogenesis Research Information System (CCRIS), the Toxic
29 Substance Control Act Test Submission (TSCATS) database, and the Distributed Structure-
30 Searchable Toxicity (DSSTOX) database.
31 Primary and secondary studies were entered in the RPF database and relevant keywords
32 (identifying study type, whether benzo[a]pyrene was included, route of administration, target
33 organ, etc.) were identified for each study. The list of keywords was developed in order to
34 facilitate database searching for references on a specific topic. Quality assurance procedures
35 were employed to ensure that database references were properly keyword-coded for retrieval.
36
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1 4.2. STUDIES IN HUMANS
2 Numerous studies have evaluated cancer outcomes in PAH-exposed individuals
3 (reviewed in Bostrom et al., 2002; WHO, 1998; ATSDR, 1995; IARC, 1987, 1983, 1973).
4 However, since these exposures were to complex mixtures containing multiple PAH
5 carcinogens, they did not provide adequate data to evaluate the human carcinogenicity of
6 individual PAH compounds. Epidemiology studies have focused on occupational exposure to
7 PAH mixtures. Emissions from coke production, coal gasification, aluminum production, iron
8 and steel founding, coal tars, coal tar pitches, and soot have produced lung cancer in humans
9 (Bostrom et al., 2002). Skin and scrotal cancers have resulted from exposure to coal tar, coal tar
10 pitches, nonrefmed mineral oils, shale oils, and soot (Larsen and Larsen, 1998; WHO, 1998;
11 ATSDR, 1995). Occupational studies clearly demonstrate exposure-response relationships for
12 PAH mixtures; however, quantitative estimates of risk are limited primarily to lung cancer in
13 coke oven workers (Bostrom et al., 2002; Larsen and Larsen, 1998; ATSDR, 1995).
14 Biomonitoring of exposure to PAHs includes measurement of DNA and protein adducts
15 and measurement of urinary metabolites of PAHs, studies on genetic polymorphisms of CYP450
16 and other enzymes, and changes in PAH metabolism (Bostrom et al., 2002; Larsen and Larsen,
17 1998; ATSDR, 1995). While these studies demonstrate the degree of exposure to PAHs from
18 various settings, quantitative dose-response data for humans exposed to individual PAHs are not
19 available. Cancer-related endpoint studies that were performed using human cell lines are
20 presented with similar assays in other mammalian species in Section 4.3.
21
22 4.3. STUDIES IN ANIMALS
23 The database of studies investigating cancer or cancer-related endpoints in animals
24 exposed to PAHs is extensive. For the purpose of developing relative potency estimates, only
25 those studies that included at least one selected PAH and benzo[a]pyrene as a reference
26 compound were reviewed. Studies were excluded if PAH potency comparisons were not
27 conducted in the same laboratory in concurrent experiments. Studies without benzo[a]pyrene are
28 listed in two separate bibliographies in Appendix B. Table B-l shows PAHs that were assayed
29 with or without benzo[a]pyrene. Table B-l shows that 32 of the 74 PAHs were assayed with
30 benzo[a]pyrene; an additional 14 PAHs were not tested in the same study as benzo[a]pyrene.
31 The remaining 28 PAHs either have only cancer-related endpoint data, or have neither bioassays
32 nor cancer-related endpoint data. Bioassays without benzo[a]pyrene were considered in the
33 weight of evidence evaluation for individual PAHs (Section 6.1). Studies that provided only
34 information on PAH mixtures or PAH metabolites were not reviewed or summarized for this
35 analysis.
36 References in the database were sorted by keyword into the following major categories:
37 cancer bioassays, in vivo studies of cancer-related endpoints, and in vitro studies of cancer-
38 related endpoints. These categories were further divided by route (for bioassays) or by endpoint
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1 (for cancer-related endpoints). Each study was reviewed, and critical study details were
2 extracted into tables (Tables 4-1 through 4-14) for each individual endpoint. Studies with data
3 on selected PAHs and benzo[a]pyrene were used, even if a particular PAH has not been
4 evaluated by U.S. EPA or IARC for carcinogenicity. Studies were included in the analysis if the
5 following selection criteria were met:
6
7 • Benzo[a]pyrene was tested simultaneously with another PAH;
8
9 • A statistically increased incidence of tumors was observed with benzo[a]pyrene
10 administration;
11
12 • Benzo[a]pyrene produced a statistically significant change in a cancer-related
13 endpoint finding;
14
15 • Quantitative results were presented;
16
17 • The carcinogenic response observed in either the benzo[a]pyrene- or other PAH-
18 treated animals at the lowest dose level was not saturated (i.e., tumor incidence at the
19 lowest dose was <90%); and
20
21 • There were no study quality concerns or potential confounding factors that precluded
22 use (e.g., no concurrent control, different vehicles, strains, etc. were used for the
23 tested PAH and benzo[a]pyrene; use of cocarcinogenic vehicle; PAHs of questionable
24 purity; unexplained mortality in treated or control animals).
25
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Table 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAH
Record
number
Reference
Mouse"
strain
Exposure
Follow up
Vehicle
Promoter
Tumor type
Positive
result
Nonpositive
result
Meets selection
criteria?
Comments
Complete carcinogenicity studies
480
600
22390
19320
22400
13640
13650
620
17660
610
19760
18570
Bingham and Falk,
1969
Habsetal., 1980
Wynder and
Hoffmann, 1959a
LaVoie et al., 1979
Wynder and
Hoffmann, 1959b
Cavalieri et al., 1983
Cavalieri et al.,
1981b
Hoffmann and
Wynder, 1966
Cavalieri et al., 1977
Higginbotham et al.,
1993
Masuda and
Kagawa, 1972
Hechtetal., 1974
CH3/He
NMRI
Swiss
HA/ICR
Swiss
albino
Swiss
Swiss
Swiss
Ha/ICR/
Mil Swiss
Swiss
Swiss
Ha/ICR/
Mil Swiss
Ha/ICR/
Mil Swiss
3 times/wk
2 times/wk
(4 times for
CO) for life
3 times/wk
3 times/wk
3 times/wk
2 times/wk
for 48 wk
2 times/wk
for 30 wk
3 times/wk
for 12 mo
2 times/wk
for 30 wk
2 times/wk
3 times/wk
for
60 applica-
tions
3 times/wk
for 17 mo
50 wk
Until
moribund or
dead
6-14 mo
Unspecified
10-22 mo
Until 2 cm
tumor or
61 wk
Until 2 cm
tumor,
moribund, or
57 wk
Up to 1 5 mo
Until
moribund,
dead, or
after 70 wk
40 wk
7 mo
72 wk
Toluene
or n-do-
decane
Acetone
(DMSO
for CO)
Cyclo-
hexane
Acetone
Acetone
Acetone
Acetone
Dioxane
Acetone
Acetone
Dioxane
Acetone
None
None
None
None
None
None
None
None
None
None
None
None
Malignant
and benign
Papilloma,
carcinoma,
sarcoma
Papilloma,
carcinoma
Unspecified
Papilloma,
carcinoma
Papilloma,
adenoma,
carcinoma
Primarily
squamous
cell
carcinoma
Papillomas
Papilloma,
kerato-
acanthoma,
carcinoma
Papilloma,
carcinoma
Unspecified
Unspecified
BaA
BbF
BbF, BjF
CH, BbF,
BjF,
DBaeP,
DBahP,
DBaiP
CH,
DBahA,
DBaiP
CPcdP
CPcdP
DBaeP,
DBahP,
DBaiP,
DBaeF
DBahP,
AA
DBaiP
DBaiP
CH
BkF, BjF, CPcdP,
CO, IP
BghiF, BkF
AC, Pyr, BghiF,
BkF, AA, BeP,
DBelP, IP,
BghiP, N23eP
AC, BeP, Pyr, FA
ACEP
BaA
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
No
No
BaP administered in different vehicle.
n-Dodecane cocarcinogenic with BaA.
No concurrent untreated, toluene, or
n-dodecane control.
Deaths prior to first tumor appearance.
No concurrent control.
Reiterates data published elsewhere.
Deaths prior to first tumor appearance.
Not clear if BaP administered
simultaneously. No concurrent
control.
Reports both incidence and
multiplicity.
Tumor incidence not useable because
BaP tumor incidence was 100%.
Tumor multiplicity data available for
dose-response assessment.
Paper in German. Paper reports
compound as DBaiP; LaCassagne et
al. (1968) state that it is actually
DBaeF. DBahP incidence >90% at
lowest dose.
DBahP incidence >90% at lowest
dose.
No tumors with BaP.
No concurrent untreated or vehicle
control; lowest dose DBaiP gave
100% incidence.
BaP dose not reported.
59
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Table 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAH
Record
number
21310
23310
23840
Reference
Shubiketal., 1960
Pfeiffer and Allen,
1948
Barry et al., 1935
Mouse"
strain
Syrian
golden
hamster
Rhesus
monkey
Un-
specified
Exposure
2 times/wk
for 10 wk
various
2 times/wk
Follow up
75 wk
Various
1-2+ yr
Vehicle
Mineral
oil
Sesame
oil
Benzene
Promoter
None
None
None
Tumor type
None
Various
Epithelioma,
papilloma
Positive
result
Multiple
Multiple
Nonpositive
result
DBahA, BaA
Meets selection
criteria?
No
No
No
Comments
Small number of animals (5/sex/dose).
Sequential exposure to multiple
compounds; no concurrent untreated
control.
Test compounds from various sources
gave differing results; purity may be
suspect; use of benzene vehicle
confounds tumorigenicity results; no
benzene or untreated control.
Initiation studies
24800
21410
630
16310
10200
18570
22500
Nesnow et al., 1984
Slaga et al., 1978
LaVoie et al., 1982
Weyand et al., 1992
El-Bayoumy et al.,
1982
Hechtetal., 1974
Van Duuren et al.,
1966
SENCAR
CD-I
CrliCD-
1[ICR]
BR
Crl:CD-l
CrliCD-
1[ICR]
BR
Ha/ICR/
Mil Swiss
ICR/HA
Single
Single
10 subdoses
every other d
5 or
10 applica-
tions given
every other d
10 subdoses
every other d
10 subdoses
every other d
Single
31 wk
27 wk
Unspecified
Until
promotion
complete
Unspecified
Until
promotion
complete
63 wk
Acetone
Acetone
Acetone
Acetone
Acetone
Acetone
Acetone
TPA 2 ng
2 times/wk
for 30 wk
TPA 10 ug
2 times/wk
for 26 wk
TPA 2.5 ug
3 times/wk
for 20 wk
TPA 2.5 ug
3 times/wk
for 20 wk
TPA 2.5 ug
3 times/wk
for 25 wk
TPA 2.5 ug
3 times/wk
for 20 wk
Croton resin,
25 Ug
3 times/wk
Papilloma
Papilloma
Primarily
squamous
cell
papilloma
Unspecified
Primarily
squamous
cell
papilloma
Unspecified
Papilloma,
carcinoma
BeAC,
B1AC
BaA
BbF, BjF,
BkF
BjF
CH
CH
CH, BbF
Pery, Pyr
BghiF
Yes
Yes
Yes
Yes
Yes
Yes
No
Reports both incidence and
multiplicity.
Tumor incidence data not useable
because BaP gave 93% tumor
incidence. Tumor multiplicity data
available for dose-response
assessment.
Reports both incidence and
multiplicity.
Tumor incidence data not useable
because BaP gave 100% tumor
incidence. Tumor multiplicity data
available for dose-response
assessment. DNA adducts,
mutagenicity also evaluated.
Tumor incidence data not useable
because single dose CH gave 100%
tumor incidence; BaP gave 90% tumor
incidence. Tumor multiplicity data
available for dose-response
assessment.
Reports both incidence and
multiplicity.
BaP gave 100% tumor incidence.
Corollary data with acetone only as
promotion agent not included.
60
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Table 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAH
Record
number
24300
19320
21420
15640
620
610
13660
19360
13650
20830
Reference
Rice et al., 1985
LaVoie et al., 1979
Slaga, et al., 1980
Raveh et al., 1982
Hoffmann and
Wynder, 1966
Higginbotham et al.,
1993
Cavalieri et al., 1991
LaVoie et al., 1985
Cavalieri et al.,
1981b
Roe, 1962
Mouse"
strain
CD-I
HA/ICR
Swiss
albino
SENCAR
SENCAR
Ha/ICR/
Mil Swiss
SENCAR
SENCAR
Crl:CD/l
(ICR)BR
CD-I
Albino
Exposure
10 subdoses
every other d
10 subdoses
every other d
Single
Single
Single
Single
Single
10 subdoses
every other d
10 subdoses
every other d
Single
Follow up
Until
promotion
complete
Until
promotion
complete
15 wk
25 wk
6 mo
27 wk
16 wk and
27 wk (two
experiments)
Unspecified
57 wk
Until
promotion
complete
Vehicle
Acetone
Acetone
or
dioxane
Acetone
Un-
specified
Dioxane
Acetone
Acetone
Acetone
Acetone
Acetone
Promoter
TPA
0.0025%
3 times/wk
for 20 wk
TPA 2.5 ug
3 times/wk
for 20 wk or
croton oil
2.5%
3 times/wk
TPA 2 ug
2 times/wk
TPA 2 ng
2 times/wk
for 25 wk
Croton oil
TPA
2.6 nmol,
2 times/wk
TPA
3. 24 nmol
2 times/wk
for 1 1 wk
TPA 2.5 ug
3 times/wk
for 20 wk
TPA
0.017umol
2 times/wk
for 40 wk
Croton oil
once/wk for
20 wk
Tumor type
Unspecified
Unspecified
Papilloma
Papilloma
Papillomas
Papillomas,
few
carcinomas
Primarily
papilloma
Unspecified
Papilloma
Papilloma
Positive
result
CH,
CPdefC
CH,
DBaeP,
DBahP,
DBaiP,
N23eP
CH,
DBahA,
CPcdP
DBaeF,
DBaeP,
DBahP,
DBaiP,
N23eP
DBaiP
DBaiP
CPcdP
Nonpositive
result
FA, AA, DBelP,
BghiP, IP
BeP, DBacA
IP, AA, BghiP,
DBelP
AC
ACEP
PH
Meets selection
criteria?
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Comments
Tumor incidence data not useable
because all compounds gave >90%
tumor incidence. Tumor multiplicity
data available for dose-response
assessment.
Reiterates data published elsewhere.
Not clear if BaP done simultaneously
but protocol, vehicle, and follow-up
are the same. Reports both incidence
and multiplicity.
Reports both incidence and
multiplicity.
Paper reports compound as DBaiP;
LaCassagne et al. (1968) state that it is
actually DBaeF.
No tumors with BaP.
Tumor incidence data not useable
because lowest dose DBaiP gave
>90% tumor incidence. Tumor
multiplicity data from both
experiments available for dose-
response assessment.
Reports both incidence and
multiplicity.
BaP not simultaneous.
61
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Table 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAH
Record
number
16440
17450
18680
19420
13660
15700
Reference
Woodetal., 1980
Bruneetal., 1978
Hoffmann et al.,
1972
LaVoieetal., 1981
Cavalieri et al., 1991
Rice et al., 1988
Mouse"
strain
CD-I
NMRI
Ha/ICR/
Mil Swiss
HA/ICR
Swiss
albino
SENCAR
CD-I
Exposure
Single
Unspecified
10 subdoses
every other d
10 subdoses
every other d
Single
10 subdoses
every other d
Follow up
27 wk
Unspecified
Until
promotion
complete
Unspecified
27 wk
24 wk
Vehicle
Acetone
Un-
specified
Acetone
Acetone
Acetone
Acetone
Promoter
TPA 16 nmol
2 times/wk
for 26 weeks
TPA
Croton oil
2.5% for
20 wk
TPA 2.5 ug
3 times/wk
for 20 wk
None
TPA 2.5 ug
3 times/wk
for 20 wk
Tumor type
Unspecified
Unspecified
Unspecified
Unspecified
Primarily
papilloma
Unspecified
Positive
result
DBalP
CH,
BbcAC,
CPdefC
Nonpositive
result
Pyr, CPcdP
AC
FA
PH
Meets selection
criteria?
Yes
No
Yes
Yes
Yes
Yes
Comments
Study design not reported. Results
reported qualitatively.
Initiating dose only; no promoter.
Tumor incidence data not useable
because lowest dose DBalP gave
>90% tumor incidence. Tumor
multiplicity data available for dose-
response assessment.
Not clear if BaP done simultaneously
for all PAHs.
Cocarcinogenicity studies
18700
21430
21840
21850
21920
Horton and
Christian, 1974
Slaga et al., 1979
Van Duuren and
Goldschmidt, 1976
Van Duuren et al.,
1973
Warshawsky et al.,
1993
C3H
CD-I
ICR/Ha
Swiss
ICR/HA
C3H/HEJ
2 times/wk
for 80 wk
Single
3 times/wk
3 times/wk
for 52 wk
2 times/wk
82 wk
30 wk
368 or 440 d
52 wk
Until lesion
developed or
104 wk
n-Do-
decane/
decalin
mixture
Acetone
Acetone
Acetone
Toluene
or n-do-
decane
None
TPA 10 ug
2 times/wk
for 30 wk
None
None
None
Carcinoma,
papilloma
Papilloma
Papilloma
None
Unspecified
DBacA,
Pyr
BeP
CH, FA, Tphen,
Pery,
Pyr, BghiP, BeP,
FA
Pyr, BghiP, BeP
AC, CH, Pyr, FA,
PH
No
No
Yes
No
No
Not clear if BaP done simultaneously.
Experiments with decalin
(noncarcinogen) and 50/50 decalin/
dodecane mix (cocarcinogenic). No
data for BaP in 50/50 mix. No vehicle
control in decalin.
No concurrent control. Study aimed at
exploring interactions; not clear if BaP
done simultaneously.
Qualitative results reported.
No tumors with BaP.
"Except where noted, all studies were conducted in mice.
DMSO = dimethyl sulfoxide
62
DRAFT - DO NOT CITE OR QUOTE
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Table 4-2. Study summaries: intraperitoneal bioassays of benzo[a]pyrene and at least one other PAH
Record
number
Reference
Mouse strain"
Exposure
Follow
up
Vehicle
Target
organ(s)
Tumor type(s)
Positive
result
Non-
positive
result
Meets selection
criteria?
Comments
Newborn mouse studies
13610
17560
640
7510
22040
22510
Busby et al.,
1984
Busby et al.,
1989
LaVoie et al.,
1987
LaVoie et al.,
1994
Weyand and
LaVoie, 1988
Wislocki et al.,
1986
Swiss-
Webster
BLU:Ha
(ICR)
Swiss-
Webster
BLU:Ha
(ICR)
CD-I
CD-I
CD-I
CD-I
1st, 8th, 15thd
1st, 8th, 15thd
1st, 8th, 15thd
1st, 8th, 15thd
1st, 8th, 15thd
1st, 8th, 15thd
26 wk
26 wk
52 wk
12 mo
Not
reported
12 mo
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Lung
Lung
Lung,
liver
Lung,
liver
Lung,
liver
Lung,
liver,
lymphatic
system
Adenoma,
adenocarcinoma
Adenoma,
adenocarcinoma
Adenoma,
hepatoma
Foci, adenoma,
carcinoma
Unspecified
Adenoma,
carcinoma,
lymphoma
FA
FA
BbF, BjF
FA
Not
reported
CH, BaA
Pyr, CH
BkF, IP
Pyr
Yes
Yes
Yes
Yes
No
Yes
Tumor incidence data not useable
because lowest dose BaP gave >90%
tumor incidence. Tumor multiplicity
data available for dose-response
assessment.
Reports both incidence and multiplicity.
Reports both incidence and multiplicity.
Abstract only; dose-response information
not included.
Reports both incidence and multiplicity.
Studies in A/J mice
11190
23960 and
23450
22670
24590
Mass et al., 1993
Nesnow et al.,
1998a, 1995
Nesnow et al.,
1996
Nesnow et al.,
1998b
A/J
A/J
A/J
A/J
Single
Single
Single
Single
8 mo
8 mo
8 mo
8 mo
Tri-
caprylin
Tri-
caprylin
Tri-
caprylin
Tri-
caprylin
Lung
Lung
Lung
Lung
Adenoma,
carcinoma
Adenoma
Adenoma
Adenoma
BjAC
BbF,
DBahA,
CPcdP
BbF,
DBahA,
CPcdP
CPcdP,
BbF,
DBahA,
BjAC,
DBalP
No
No
No
Yes
Reiterates data reported elsewhere
(Record 24590).
Reiterates data reported elsewhere
(Record 24590).
(Reiterates data reported elsewhere
(Record 24590).)
Raw data (both incidence and
multiplicity) obtained courtesy of S.
Nesnow.
63
DRAFT - DO NOT CITE OR QUOTE
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Table 4-2. Study summaries: intraperitoneal bioassays of benzo[a]pyrene and at least one other PAH
Record
number
20920
24801
Reference
Rossetal., 1995
Weyand et al.,
2004
Mouse strain"
A/J
A/J
Exposure
Single
Single
Follow
up
240 d
260 d
Vehicle
Tri-
caprylin
Tri-
caprylin
Target
organ(s)
Lung
Lung
Tumor type(s)
Adenoma
Adenoma
Positive
result
BbF,
DBahA,
CPcdP
BcFE
Non-
positive
result
Pyr
Meets selection
criteria?
No
Yes
Comments
Reiterates data reported elsewhere
(Record 24590).
"All studies were conducted in mice.
64
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-3. Study summaries: subcutaneous bioassays of benzo[a]pyrene and at least one other PAH
Record
number
23840
220
18350
23200
660
23310
24290
24310
21560
Reference
Barry et al., 1935
Bryan and
Shimkin, 1943
Grant and Roe,
1963
Homburger et al.,
1972
Pfeiffer, 1977
Pfeiffer and Allen,
1948
Rask-Nielson,
1950
Roe and Waters,
1967
Steiner, 1955
Species
Mouse
Mouse
Mouse
Hamster
Mouse
Monkey
Mouse
Mouse
Mouse
Strain
Unspeci-
fied
C3H
Albino
Various
NMRI
Rhesus
Street
Swiss
albino
C57BL
Exposure
site
Unspecified
Right axilla
Neck
Groin
Neck
Various
Thymus,
lung,
mammary
area
Not
specified
Interscapular
Exposure
Single
Single
1st d after
birth
Single
Single
Various
Single
Istd after
birth
Single
Follow
up
1-2+ yr
until
20mm
tumor
52-62 wk
52 wk
114wk
variable
30 mo
50-60 wk
22-28 mo
Vehicle
Lard
Tricaprylin
Aqueous
gelatin
Tricaprylin
Tricaprylin
Sesame oil
Paraffin
Aqueous
gelatin
Tricaprylin
Target
organ(s)
Injection
site
Injection
site
Lung
Injection
site; lung
Injection
site
Various
Various
Liver
Injection
site
Tumor
type(s)
Sarcoma
Unspecified
Adenoma
Various
Sarcoma
Various
Various
Hepatoma
Sarcoma
Positive
result
Multiple
DBahA
BaA
DBahA
Multiple
DBahA
PH
DBahA,
BaA, CH
Nonpositive
result
PH
AC, PH
Meets selection
criteria?
No
No
Yes
No
No
No
No
No
No
Comments
Test compounds from
various sources gave
differing results; purity
may be suspect; no
untreated control.
No concurrent untreated
control.
Study aimed at
evaluating strain
specificity of
tumorigenicity. BaA
results equivocal. Not
clear if BaP treatment
simultaneous. "Aged"
mice used as controls;
aged mice allowed to
live 16 weeks longer.
Less than 10% of
100 control mice alive
at 1 14 wk; control data
not provided.
Sequential exposure to
multiple compounds; no
concurrent untreated
control.
Number of control and
exposed varies by
tumor type reported;
BaP nontumorigenic;
DBahA results
equivocal; results
unclear.
Study methodology and
results not detailed; PH
results equivocal.
No concurrent untreated
control; study aimed at
evaluating interactions.
65
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Table 4-4. Study summaries: oral bioassays of benzo[a]pyrene and at least one other PAH
Record
number
17280
23880
24801
Reference
Biancifiori and Caschera,
1962
Huggins and Yang, 1962
Weyand et al., 2004
Species
Mouse
Rat
Mouse
Strain
BALB/c
Sprague-
Dawley
A/J
Exposure
route
Gavage
Gavage
Diet
Exposure
2 times/wk,
15 wk
Single
Daily,
260 d
Follow up
Variable;
50-60 wk
Not
reported
260 d
Target
organ(s)
Mammary
gland
Mammary
gland
Lung
Tumor
type(s)
Carcinomas
and
sarcomas
Unspecified
Adenoma
Positive
result
DBahA
BcFE
Non-
positive
result
BaA, PH
Meets selection
criteria?
No
No
Yes
Comments
Tumors observed after DBahA only
in pseudopregnant mice, not virgin
mice.
Untreated control information not
included.
66
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-------
Table 4-5. Study summaries: other route bioassays of benzo[a]pyrene and at least one other PAH
Record
number
21750
17620
13660
21620
20280
17940
22000
21500
23910
Reference
Topping et al., 1981
Cavalieri et al.,
1988b
Cavalieri et al., 1991
Sugiyama, 1973
Pataki and Huggins,
1969
Deutsch-Wenzel et
al., 1983
Wenzel-Hartung et
al., 1990
Soltetal., 1987
Nikonova, 1977
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Hamster
Mouse
Strain
F344
Sprague-
Dawley
Sprague-
Dawley
Long-
Evans
Sprague-
Dawley
Osborne-
Mendel
Osborne-
Mendel
Syrian
golden
A
Exposure route
Implantation in
transplanted
tracheas
Intramammillary
Intramammillary
Intramuscular
Intravenous
Lung implantation
Lung implantation
Painting buccal
pouch
Subcutaneous (FO)
and transplacental
(Fl)
Exposure
Release
from pellet
Single
Single
Single
3 doses 3 d
apart
Release
from pellet
Release
from pellet
2 times/wk
for 20 wk
GD 18 or
19
Follow up
28 mo
20 wk
Until 2 cm
tumor or
24 wk
9 mo
98 d
Until
moribund
or dead
Until
moribund
or dead
Up to
44 wk
lyr
Vehicle
Beeswax
pellet
None
Trioctanoin
Sesame oil
Lipid
emulsion
Beeswax/
trioctanoin
Beeswax/
trioctanoin
Paraffin oil
Sunflower
oil
Target
organ(s)
Tracheal
epithelium
Mammary
Mammary,
other
Injection
site
Mammary
Lung
Lung
Buccal
pouch
Lung,
mammary,
liver,
injection
site
Tumor type(s)
Carcinoma,
sarcoma
Adeno-
carcinoma,
adenofibroma,
fibrosarcoma
Adeno-
carcinoma,
adenofibroma,
fibrosarcoma,
squamous cell
carcinoma
Sarcoma
Unspecified
Carcinoma,
sarcoma
Carcinoma
Carcinoma
Adenoma
Positive
result
DBalP
BbF,
BjF,
BkF,
IP, AA,
BghiP
CH,
DBahA
Non-
positive
result
BeP
DBahA,
BaA
BaA
BaA
BeP
PH
BaA
Pyr
Meets
selection
criteria?
No
No
No
No
No
Yes
Yes
No
No
Comments
Interaction
information
included.
Control data
from untreated
mammary
glands of same
rats.
DBalP
produced
tumors in all
animals at the
lowest dose.
BaP gave 100%
tumor
incidence.
No control
group.
Fewer than
20 animals per
group; negative
result.
Transplacental
exposure not
quantified.
67
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Table 4-6. Study summaries: in vivo DNA adducts with benzo[a]pyrene and at least one other PAH
Record
number
6210
17420
17630
18810
18790
10900
13200
11190
8010
22670
23960
24590
22810
20650
20920
16310
22040
Reference
Arifetal., 1997
Brookes and
Lawley, 1964
Cavalieri et al.,
1981a
Hughes and
Phillips, 1990
Hughes and
Phillips, 1991
Koganti et al.,
2000
Li et al., 2002
Mass et al.,
1993
Nesnow et al.,
1993b
Nesnow et al.,
1996
Nesnow et al.,
1995
Nesnow et al.,
1998a
Phillips et al
1979
Reddy etal.,
1984
Ross et al.,
1995
Weyand etal.,
1992
Weyand and
LaVoie, 1988
Route of
administration
Intramammillary
Dermal
Dermal
Dermal
Dermal
Oral-diet
Gavage or oral-
diet
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Dermal
Dermal
Intraperitoneal
Dermal
Intraperitoneal
Exposure
frequency
Single dose
Single dose
Single dose
Single dose
Single dose
14 d
1 time/d for 1—
4 d; diet 14 d
Single dose
Single dose
Single dose
Single dose
Single dose
Single dose
4 doses (0, 6,
30, 54 hr)
Single dose
Single dose
Postnatal d 1,
8, 15
Hours between
dosing and
sacrifice
48
various to —12 d
4,24
0.5, 1,2,4,7,21,
84 d
24
not stated
24, 48, 72
1, 3, 7, 14, 28, 56 d
7d
7d
various to 2 1 d
19 24 48 72 96
120, 144
24
0, 1, 3, 5, 7, 14, 21 d
24
24
Tissue analyzed
Mammary
epithelium, lung
Skin
Skin
Skin, lung
Skin
Lung
Mammary gland
and liver; lung
Lung
Lung, liver,
peripheral blood
lymphocytes
Lung
Lung
Lung
Skin
Skin
Lung
Skin
Lung, liver
Method of
analysis
[32P] postlabeling
[3H] prelabeling
[3H] or [14C]
prelabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[3H]-Prelabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
PAHs evaluated"
DBalP
DBacA, DBahA
CPcdP, ACEP
DBalP, DBaeP,
DBahP, DBaiP
DBaeP
BcFE, BaFE, BbFE
BcFE
BjAC
BbF
BbF, DBahA,
CPcdP
BbF, DBahA,
CPcdP
BbF, CPcdP,
DBahA, DBalP
BaA DBacA
DBahA
AC, BaA, BghiP,
BeP, CH, DBacA,
DBahA, Pery, Pyr
BbF, CPcdP,
DBahA
BjF
BbF, BjF, BkF
Meets selection
criteria?
Yes
No
Yes
Yes
No
No
No
Yes
Yes
No
No
Yes
Yes
No
No
No
No
Comments
Data on individual compounds not
reported.
24-hr experiment with DBaeP and
DBalP; 84-d experiment with all.
No quantitative information; abstract
only.
Not quantified.
Not quantified; BaP administered by
gavage, BcFE admin in diet.
Peaks differ temporally; study also
correlates number of adducts in
organs.
Not quantified.
Not quantified.
Used data from Ross et al., 1995 (ref
20920) to calculate slope.
Semiquantitative data only.
Reiterates data published elsewhere
(Record 24590).
Not quantified.
No quantitative data; abstract only.
68
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Table 4-6. Study summaries: in vivo DNA adducts with benzo[a]pyrene and at least one other PAH
Record
number
24801
24790
Reference
Weyand et al.,
2004
Kligerman et
al., 2002
Route of
administration
Oral-diet or
intraperitoneal
Intraperitoneal
and oral
Exposure
frequency
14 d diet;
single dose
intraperitoneal
Single dose
Hours between
dosing and
sacrifice
24
7d
Tissue analyzed
Lung,
forestomach
Peripheral blood
lymphocytes
Method of
analysis
[32P] postlabeling
[32P] postlabeling
PAHs evaluated"
BcFE
BaA, BbF, CH
Meets selection
criteria?
Yes
Yes
Comments
Data in both rats and mice.
"Positive findings were reported for all PAHs evaluated.
69
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Table 4-7. Study summaries: in vivo clastogenicity or sister chromatid exchange with benzo[a]pyrene and at
least one other PAH
Record
number
24740
14270
17190
19030
24720
24790
20200
20230
20950
21050
21770
Reference
Allen et al., 1999
He and Baker,
1991
Bayer, 1978
Katzetal., 1981
Kligerman et al.,
1986
Kligerman et al.,
2002
Oshiro et al.,
1992
Paikaetal., 1981
Roszinsky-
Kocher et al.,
1979
Salamone et al.,
1981
Tsuchimoto and
Matter, 1981
Species
Mice
Mice
Hamsters
Mice
Mice
Mice and
rats
Mice
Mice
Hamsters
Mice
Mice
Strain
A/Jor
p53 +/+,
+/-, and
HRA/Skh
hairless
Chinese
B6C3Fi/
BR
C57BL6
CD-I
Swiss
mice; CD
rats
CD-I
CBA/J
Chinese
B6C3Fi
CD-I
Route of
administration
Intraperitoneal
Dermal
Intraperitoneal
Intraperitoneal
Gavage
Oral and
intraperitoneal
Peroral
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Vehicle
Tricaprylin
Acetone
Tricaprylin
DMSO
Corn oil
Sunflower
seed oil
Polyethylene
glycol
DMSO
Tricapryline
Not
specified
DMSO
Exposure
Single
Single
Single
At 0 and
24 hr
Single
Single
1 time/d,
4d
single
2 doses
24 hr
apart
2 doses
24 hr
apart
2 doses
24 hr
apart
Hours between
dosing and
sacrifice
48 or 72 hr
24 hr
24 hr for
aberrations; 30 hr
for micronuclei
various; 24, 30,
48, 72 hr after last
dose
23.5-25 hr
7d
24 hr after 2nd
and 4th treatment
16-20 hr
24 hr after 2nd
treatment
24, 48, 72 hr after
2nd treatment
6 hr after 2nd
treatment
Tissue
analyzed
Bone
marrow or
peripheral
blood
Keratino-
cytes
Bone
marrow
Bone
marrow
Peripheral
blood
Whole
blood or
mono-
nuclear
leukocytes
Peripheral
blood
Bone
marrow
Bone
marrow
Bone
marrow
Bone
marrow
Clasto-
genic
end point
Micro-
nuclei
Micro-
nuclei
Gaps,
breaks,
micro-
nuclei,
sister
chromatid
exchanges
micro-
nuclei
Sister
chromatid
exchanges
Sister
chromatid
exchange,
micro-
nuclei
Micro-
nuclei
Sister
chromatid
exchanges
Sister
chromatid
exchanges,
aberrations
Micro-
nuclei
Micro-
nuclei
Positive
results
DBalP
CH
PH (high
dose
only)
B1AC
BaA,
BbF, CH
PH, CH,
DBahA,
BaA,
BbF, BeP
Non-
positive
results
Pyr
DBaiP,
AC,
BghiP, Pyr
Pyr, AC
Pyr
AC
AC, Pyr
Pyr
Meets
selection
criteria?
Yes
Yes
Yes
No
Yes
Yes
No
No
Yes
Yes
Yes
Comments
No quantitative data.
All positive for sister
chromatid exchange
via intraperitoneal
administration;
mixed results for oral
administration.
No quantitative data;
published as abstract.
No quantitative data.
Positive results for
sister chromatid
exchanges, not
aberrations.
70
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Table 4-7. Study summaries: in vivo clastogenicity or sister chromatid exchange with benzo[a]pyrene and at
least one other PAH
Record
number
21390
21620
Reference
Sirianni and
Huang, 1978
Sugiyama, 1973
Species
Mice
Rats
Strain
C3H/SI
Long-
Evans
Route of
administration
V79 cells in dif-
fusion chamber
implanted in
peritoneal
cavity of mice
Intravenous
Vehicle
Lipid
emulsion
Exposure
Single
Hours between
dosing and
sacrifice
12, 24 hr
Tissue
analyzed
Chinese
hamster
V79 cells
Bone
marrow
Clasto-
genic
end point
Sister
chromatid
exchanges
Gaps,
breaks
Positive
results
Non-
positive
results
AC, Pyr,
Pery
BaA
Meets
selection
criteria?
Yes
Yes
Comments
71
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Table 4-8. Study summaries: in vivo mutagenicity with benzo[a]pyrene and at least one other PAH
Record
number
18130
13980
11190
23960
22670
24590
21370
21830
22450
Reference
Fahmy and Fahmy,
1980
Frolich and Wurgler,
1990
Mass et al., 1993
Nesnowetal., 1995
Nesnow et al., 1996
Nesnowetal., 1998b
Simmon et al., 1979
Valencia and
Houtchens, 1981
Zijlstra and Vogel,
1984
Species/strain
Drosophila
melanogaster
D. melanogaster
A/3 mice
A/J mice
A/J mice
A/J mice
Swiss Webster
mice
D. melanogaster
D. melanogaster
Route of
administration
Suspension in
media
Suspension in
media
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
PAHs
intramuscular or
peroral;
microorganisms
intraperitoneal
Filter feeding
Abdominal
injection
Exposure
frequency/follow up
48-72 hr
48-72 hr
3 d/8 mo
Single injection/
8 mo
Single injection/
8 mo
Single injection/
8 mo
Single injection/4 hr
48-72 hr
Not applicable
Mutagenic end point
Somatic mutation; eye color
mosaicism
Somatic mutation and
recombination test; wing
spots
Mutations in codon 12 of
the Ki-ras oncogene; PCR
and DNA sequencing of
lung tumor DNA
Mutations in codon 12 of
the Ki-ras oncogene; PCR
and DNA sequencing of
lung tumor DNA
Mutations in codon 12 of
the Ki-ras oncogene; PCR
and DNA sequencing of
lung tumor DNA
Mutations in codons 12 and
61 of the Ki-ras oncogene;
PCR and DNA sequencing
of lung tumor DNA
Intraperitoneal host
mediated assay;
mutagenicity in S.
typhimurium and
Saccharomyes cerevisiae of
recovered microorganisms
Sex-linked recessive lethal
test
Sex-linked recessive lethal
test; 2-3 translocation and
ring-X loss
Positive
result
BjAC
BbF,
DBahA,
CPcdP
BbF,
DBahA,
CPcdP
BbF,
DBahA,
CPcdP,
BjAC,
DBalP
Non-
positive
result
BaA
BaA
AC, BaA,
BeP, CH,
PH
Pyr
BaA
Meets
selection
criteria?
Yes
No
No
No
No
No
No
No
No
Comments
Inconsistent results for BaA; significant
effects only seen with cross-breeding of
strains selected for enhanced metabolic
activity (not standard strains).
Quantitative dose-response data were
not available. Different mutation
sequences observed; GGT— >TGT for
BaP and GG1WCGT for BjAC;
mutation sequence for BjAC may
correlate with cyclopenta-adduct
formation.
Quantitative dose-response data were
not available. GGT— >TGT mutations
for BaP and BbF; GG1WCGT for
CPcdP; no mutations seen for DBahA.
Quantitative dose-response data were
not available. GGT— >TGT mutations
for BaP and BbF; GGT^CGT for
CPcdP; no mutations seen for DBahA.
Quantitative dose-response data were
not available. Mutations in codon 12,
GGT^TGT for BaP, BbF, and DBalP;
GGT^CGT for CPcdP and BjAC; no
mutations seen for DBahA; GTT
mutations seen for all other PAHs. Only
DBalP caused mutations in codon 61.
Assay was not considered sensitive
enough for detecting carcinogens.
Results were negative for BaP.
Results were negative for BaP.
72
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Table 4-9. Study summaries: in vitro bacterial mutagenicity with benzo[a]pyrene and at least one other PAH
Record
number
17030
23830
23660
17380
9560
17590
17630
9620
24030
13860
18030
18050
18180
24080
14080
14170
14260
Reference
Andrews et al.,
1978
Baker et al., 1980
Bartsch et al., 1980
Bosetal., 1988
Carver et al., 1985
Carver et al., 1986
Cavalieri et al.,
1981a
Chang etal., 2002
De Flora et al., 1984
Devanesan etal.,
1990
Dunkeletal., 1984
Eisenstadt and
Gold, 1978
Florin et al., 1980
Gibson et al., 1978
Gold and
Eisenstadt, 1980
Outline et al., 1982
Hassetal., 1981
Salmonella strain(s)
TA100, TA1527,
TA1538
TA100
TA100, TA1535,
TA98
TA98, TA100
TA98, TA100
TA100
TM677
TA100
TA1535, TA1537,
TA1538, TA98,
TA100
TA100, TA98
TA1535, TA1537,
TA1538, TA98,
TA100
TA1537, TA100
TA98, TA100
TA1535, TA1537,
TA1538, TA98
TA100
TA98, TA100
TA98, TA100
Activation system
Ar S9 and others
Guinea pig MC S9
and others
Rat MC S9
Rat Ar S9
S9
Ar rat and Ar hamster
S9
ArS9
Rat Ar S9
Rat AR S9
Rat Ar S9
Rat, mouse, hamster
ArS9
Rat Ar S9
Rat Ar and MC S9
Nonenzymatic
(gamma radiation)
Rat MC S9
Rat Ar S9 compare to
PGS from ram
seminal vesicles
Rat Ar S9
Positive result
AA, DBahA, DBajA, DBacA, BghiP,
BeP
DBaiP, BaA, DBacA, DBahA
BaA
PH, Pyr
Pery
BaA, BghiF, Pery
CPcdP, ACEP, Pyr
BghiF, BcPH
BaA, Pery, BeP
DBaeP, DBalP
BaA, BeP, PH, Pyr
CPcdP
BaA, CH, Pery, CO
BaA, BghiP, CH, FE, Pyr
CPcdP
BaA, CH
Nonpositive
result
AC
AC
DBahA, AC,
Pic, Tphen
BeP
Meets
selection
criteria?
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
Yes
Comments
TA100 results include BaP.
Qualitative data for other PAHs (no BaP);
quantitative data with BaP comparison for
PHandPyrinTAlOO.
The response varied at different
concentrations of S9; BaP was more potent
at low S9 while Pery was more potent at
high S9.
Qualitative data also presented for other
PAHs. S9 concentration varied;
400 uL/plate optimal.
BaP data from previous publication used.
Dose-response data not provided for Pyr.
No concurrent control.
Dose-response data not provided.
AN, PH also tested; toxicity interfered with
mutagenicity testing.
BaP and CPcdP maximal responses
occurred at different S9 levels.
BaP tested in TA98, BaA and CH tested in
TA100.
73
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Table 4-9. Study summaries: in vitro bacterial mutagenicity with benzo[a]pyrene and at least one other PAH
Record
number
18650
10670
19000
24680
19320
19360
23650
15170
20220
20530
20450
20490
20560
20880
21000
21040
Reference
Hermann, 1981
Johnsen et al., 1997
Kadenetal., 1979
Lafleuretal., 1993
LaVoie et al., 1979
LaVoie et al., 1985
McCann et al., 1975
Norpothetal., 1984
Pahlman and
Pelkonen, 1987
Penman et al., 1980
Phillipson and
loannides, 1989
Poncelet et al., 1978
Probst etal., 1981
Rosenkranz and
Poirier, 1979
Sakaietal., 1985
Salamone et al.,
1979a
Salmonella strain(s)
TA98
TA98
TM677
TM677
TA98, TA100
TA98, TA100
TA1535, TA1537,
TA98, TA100
TA100
TA100
TM677
TA100
TA1530, TA1535,
TA1537, TA1538,
TA98, TA100
TA1530, TA1535,
TA1537, TA1538,
TA98, TA100
TA1530, TA1535
TA97, TA98, TA100
TA1535, TA1537,
TA1538, TA98,
TA100
Activation system
Rat Ar S9
Rat control or PB S9
Rat Ar or PB S9
ArPMS
Rat Ar S9
Rat Ar S9
Rat Ar S9
Rat and mouse S9;
induction by Clophen
ASOand 18PAHs
S9 from control, MC,
or TCDD treated rats
and mice
Rat Ar or PB S9
S9 isolated from
mouse, hamster, rat,
pig, and human
S9 (origin unknown)
Rat Ar S9
Uninduced rat S9
Rat Ar S9
Rat Ar S9
Positive result
BbA, BaA, CH, FA, Tphen, BeP,
DBacA, DBahA, BbF, Pery, DBalP,
DBaiP, AA, CO
BjAC, B1AC
AN, ANL, Pyr, BbFE, CPcdP, BaA, CH,
Tphen, FA, BeP, Pery, BghiP, AA,
DBacA, DBahA, DBbeF
CPcdP, APA, ACEA, CPhiAPA,
CPhiACEA
BeP, Pery
DBaiP, BeP, DBacA, DBahA, CH, BaA
BaA
BaA, CH, Tphen, DBacA, DBahA
Pery, CPcdP, DBacA
BaA, DBaiP, DBahA
CO, Tphen, FA, BghiP
BbA, DBacA
FE (equiv.), AC, PH, FA, CH, Pyr, BeP,
Pery, BghiP, CO
BaA, BeP (equiv.), BghiP, DBaiP, BPH,
CH, CO, DBacA, PCE
Nonpositive
result
AC, PH, FE,
Pyr, BbFE
FE, AC, PH, Pic,
CO
AC
Pyr, AC, PH, FE
AN, AC, PH,
FE, Pyr, BeP,
Pery, PCE
BbF
AC, DBahA,
PH, Pyr, DBaiP
AC, BaA, BeP,
CH, PH
AC, BaFE,
BbFE, FA, Pery,
Pyr
Meets
selection
criteria?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
No
No
Yes
Yes
No
Comments
Mutagenic activity relative to BaP reported.
Several other PAHs were evaluated, but not
concurrent with BaP.
S9 composition was different for BaA and
BaP; result cannot be compared.
No concurrent control values were reported.
Qualitative data reported in published
abstract.
Data reported as minimum mutagenic
concentration (nmol/mL).
Increase in spontaneous mutation rate was
indicated, but dose data were not provided.
74
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Table 4-9. Study summaries: in vitro bacterial mutagenicity with benzo[a]pyrene and at least one other PAH
Record
number
13260
11860
21360
21640
16180
16440
Reference
Salamone et al.,
1979b
Sangaiah et al.,
1983
Simmon, 1979a
Teranishi et al.,
1975
Utesch et al., 1987
Wood et al., 1980
Salmonella strain(s)
TA98, TA100
TA1535, TA1537,
TA1538, TA98,
TA100
TA1535, TA1536,
TA1537, TA1538,
TA98, TA100
TA1535, TA1536,
TA1537, TA1538
TA100
TA98, TA100
Activation system
Rat Ar S9
Rat Ar S9
Rat Ar S9
S9 from rats treated
with PB and MC or
DBahA
Intact or
homogenized
hepatocytes from Ar
treated rats
Rat Ar S9 and
purified MFO
enzymes system
Positive result
DBaiP
BjAC
BaA, BeP
DBaiP, DBaeP
BaA
CPcdP
Nonpositive
result
AC, CH, PH
DBahA, BaA,
BeP
Meets
selection
criteria?
No
Yes
Yes
Yes
Yes
Yes
Comments
Dose-response data were not completely
reported; maximal response information
(dose and number of revertants) was
presented in text; BaP max response at
different S9 than DBaiP.
Dose-response data for BaP was presented
for TA98 only.
Ar = Arochlor 1254-treated; MC = 3-methylcholanthrene-treated; PB = phenobarbital-treated; PMS = postmitochondrial supernatant
75
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Table 4-10. Study summaries: in vitro mammalian mutagenicity assays with benzo[a]pyrene and at least one
other PAH
Record
number
16900
16920
16930
16940
16910
13440
17140
24670
18260
14250
18750
18740
24120
18990
24720
Reference
Allen-Hoffmann and
Rheinwald, 1984
Amacher and Paillet,
1982
Amacher and Paillet,
1983
Amacher and Turner,
1980
Amacher et al., 1980
Bairdetal., 1984
Barfknecht et al., 1982
Durant et al., 1999
Gehlyetal., 1982
Hassetal., 1982
Huberman, 1975
Huberman and Sachs,
1976
Huberman and Sachs,
1974
Jotz and Mitchell,
1981
Kligerman et al., 1986
Cell type
Human epidermal
keratinocyte
Mouse lymphoma cells
(L5178Y)
Mouse lymphoma cells
(L5178Y)
Mouse lymphoma cells
(L5178Y)
Mouse lymphoma cells
(L5178Y)
V79 Chinese hamster cells
TK6 human lymphoblast cells
HlAlv2 human
lymphoblastoid cells
C3H/10T1/2 clone 8 mouse
fibroblast cells
V79 Chinese hamster cells
V79 Chinese hamster cells
V79 Chinese hamster cells
V79 Chinese hamster cells
Mouse lymphoma cells
(L5178Y)
Mouse lymphoma cells
(L5178Y)
Metabolic activation
None
Syrian golden hamster S9 mix or
cocultivated hamster hepatocytes
Cocultivated rat hepatocytes
S9 from eight rodent species or
strain; one rat strain induced by Ar
Rat Ar and noninduced S9
Hamster embryo cells
Rat Ar S9
Transfected with cyplal cDNA
None
Hamster embryo cells
Hamster cells
Hamster embryo cells
Hamster embryo cells
Rat Ar S9
Rat Ar S9
Mutagenesis assay
6-Thioguanine resistance (HPRT)
Trifluorothymidine resistance
(thymidine kinase locus [TK])
Trifluorothymidine resistance (TK)
Trifluorothymidine resistance (TK)
Trifluorothymidine resistance (TK)
6-Thioguanine resistance (HPRT)
Trifluorothymidine resistance (TK)
Trifluorothymidine resistance (TK)
Ouabain resistance (HPRT)
Ouabain and 6-thioguanine
resistance (HPRT)
8-Azaguanine resistance (HPRT)
Ouabain and 8-azaguanine
resistance (HPRT)
8-Azaguanine resistance (HPRT)
Trifluorothymidine resistance (TK)
Trifluorothymidine resistance (TK)
Positive
result
BaA
AC, BaA
BaA
FA, BaA,
CH, Tphen,
CPcdP
BaPery,
BbPery,
DBaeF,
DBafF,
DBahP,
DBaiP,
DBelP,
N23aP,
N23eP
DBaiP,
DBahP
DBacA,
DBahA
(both weak)
Pyr
B1AC
Non-
positive
result
BaA
BaA
AC, Pyr
BeP
PH, AC,
ACEP
DBjlF,
N12bF
BeP
BaA, Pyr
Pyr, PH,
CH, BaA
BaA
Meets selection
criteria?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Comments
AC data not
useable; BaP
not
simultaneous.
76
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Table 4-10. Study summaries: in vitro mammalian mutagenicity assays with benzo[a]pyrene and at least one
other PAH
Record
number
19180
24680
24170
7550
19870
20040
11450
15630
15640
21410
21720
21730
Reference
Krahn and
Heidelberger, 1977
Lafleur et al., 1993
Langenbach et al.,
1983
Li and Lin, 1996
Mishraetal., 1978
Myhr and Caspary,
1988
Nesnow et al., 1984
Raveh and Huberman,
1983
Raveh et al., 1982
Slagaetal., 1978
Tong et al., 1983
Tongetal., 1981b
Cell type
V79 Chinese hamster cells
MCL-3 human
lymphoblastoid cells
V79 Chinese hamster cells
HS1 HeLa cells (human
epithelial cells)
Fischer rat embryo cells
infected with Rauscher
leukemia virus
Mouse lymphoma cells
(L5178Y)
V79 Chinese hamster cells
V79 Chinese hamster cells
V79 Chinese hamster cells
V79 Chinese hamster cells
Rat liver epithelial cells
(ARL-18)
Rat liver epithelial cells
(ARL-18)
Metabolic activation
Rat MC S9
Transfected with cypla2 and
cyp2a6 cDNA
Cocultivation with primary rodent
cells from liver, lung, kidney, and
bladder
None
Rat Ar S9
Rat Ar and noninduced S9
Rat Ar S9
Hamster embryo fibroblasts
Hamster embryo fibroblasts
Hamster embryo cells
None
Mutagenesis assay
6-Thioguanine resistance (HPRT)
Trifluorothymidine resistance (TK)
Ouabain resistance (HPRT)
6-Thioguanine resistance (HPRT)
Ouabain resistance (HPRT)
Trifluorothymidine resistance (TK)
6-Thioguanine resistance (HPRT)
6-Thioguanine resistance (HPRT);
phorbol myristate acetate used to
enhance recovery
Ouabain and 6-thioguanine
resistance (HPRT)
Ouabain resistance (HPRT)
6-Thioguanine resistance (HPRT)
6-Thioguanine resistance (HPRT)
Positive
result
BaA,
DBacA,
DBahA
CPcdP,
ACEA,
CPhiACEA
BaA
AC, BaA,
BeP
B1AC,
BeAC,
BjAC
CPcdP
CPcdP
BaA (weak)
Non-
positive
result
APA,
CPhiAPA,
BghiF
AC
AC, PH,
Pyr, BeP
BaA
BaA, BeP,
Pyr
BeP, Pyr,
BaA
Meets selection
criteria?
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Comments
DBacA and
DBahA data
not useable;
treatment
different than
BaP.
Results
reported as
ranges.
Mutagenicity
correlated with
skin tumor
initiation.
Repeats data
from
Record 21730
Tong et al.,
1981b
77
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Table 4-10. Study summaries: in vitro mammalian mutagenicity assays with benzo[a]pyrene and at least one
other PAH
Record
number
16190
21900
Reference
Vacaetal., 1992
Wangenheim and
Bolcsfoldi, 1988
Cell type
UV-sensitive Chinese hamster
ovary (CHO) cells
Mouse lymphoma cells
(L5178Y)
Metabolic activation
Rat Ar S9
Rat Ar S9
Mutagenesis assay
6-Thioguanine resistance (HPRT)
Trifluorothymidine resistance (TK)
Positive
result
FA
Pyr, FE
Non-
positive
result
Meets selection
criteria?
Yes
Yes
Comments
HPRT = hypoxanthine-guanine phosphoribosyl transferase mutagenicity assay (resistance to 6-thioguanine, 8-azaguanine, or ouabain); TK = thymidine kinase mutagenicity assay (resistance to
trifluorothymidine)
78
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Table 4-11. Study summaries: in vitro morphological/malignant cell transformation with benzo[a]pyrene and
at least one other PAH
Record
number
13390
17610
17730
24750
17970
17990
23630
18020
18080
23640
18260
14130
23890
14640
14700
14850
19870
24710
24700
7980
7990
8000
20120
Reference
Atchison et al., 1985
Casto, 1979
Chen and
Heidelberger, 1969
Davis, 1999
DiPaolo et al., 1969
DiPaolo et al., 1972
DiPaolo et al., 1973
Dunkeletal., 1981
Emuraetal., 1980
Evans and DiPaolo,
1975
Gehlyetal., 1982
Greb et al., 1980
Kakunaga, 1973
Krolewskietal., 1986
Laaksonen et al., 1983
Lubetetal., 1983
Mishraetal., 1978
Mohapatra et al., 1987
Nesnow et al., 1990
Nesnow et al., 1997
Nesnow et al., 1994
Nesnow et al., 1993a
Nesnow et al., 1991
Cell type
BALB/3T3 mouse embryo fibroblasts
Syrian golden hamster embryo cells
Adult C3H mouse ventral prostate cells
C3H10T1/2 cells
Syrian golden hamster embryo cells
BALB/3T3
Syrian golden hamster embryo cells
Balb/3T3, Syrian golden hamster embryo,
and Rauscher murine leukemia virus-
infected F344 rat embryo cells
Syrian golden hamster fetal lung cells
Strain 2 guinea pig fetal cells
C3H10T1/2CL8 mouse embryo fibroblasts
BHK 21/CL 13
BALB/3T3 subclone A3 1-714
C3H10T1/2CL8 mouse embryo fibroblasts
Newborn NMRI nu/nu nude mouse skin
fibroblasts
C3H10T1/2CL8 mouse embryo fibroblasts
Rauscher leukemia virus-infected Fischer
rat embryo
C3H10T1/2CL8 mouse embryo fibroblasts
Human neonatal foreskin fibroblasts
C3H10T1/2CL8 mouse embryo fibroblasts
C3H10T1/2CL8 mouse embryo fibroblasts
C3H10T1/2CL8 mouse embryo fibroblasts
C3H10T1/2CL8 mouse embryo fibroblasts
Metabolic activation system
None
None
Cocultivated irradiated C3H
mouse embryonic fibroblasts
None
Cocultivated irradiated
Sprague-Dawley rat fetal
cells
None
In vivo (transplacental)
exposure
None
None
None
None
Rat Ar S9
None
None
None
None
None
None
None
None
None
None
None
Positive result
DBahA
DBahA
DBalP, DBaeP,
BcC, BgC, BcPH
DBahA, BaA,
BeP, DBacA
BaA
BbF, BaA, IP
CH, BaA, BbF,
DBahA, BeP
CPcdP
BaA
BeP
Be AC, BjAC,
B1AC
B1AC
DBalP
DBahA
DBkmnoAPH
Nonpositive
result
FA, Pyr
Pyr
DBacA, Pyr
Pyr, PH
AC, Pyr
AC, PH, Pyr
BeP, PH, AC
BkF, BeP
AC, Pyr, PH
BeP
PH, AC
PH, Pyr
AC
AC, DBahA, PH
AC, PH, Pyr,
BeP
BkAC
DBjmnoAPH,
N123mnoAPH
ACEA
Meets selection
criteria?
Yes
Yes
No
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Comments
Control data not provided.
Control data not provided.
No quantitative information.
Qualitative data only for R-MuLV-
RE cells. BaA positive in SHEM,
equivocal in Balb/3T3.
No quantitative information.
Not clear if BaP administered
simultaneously.
No quantitative information.
79
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Table 4-11. Study summaries: in vitro morphological/malignant cell transformation with benzo[a]pyrene and
at least one other PAH
Record
number
23720
8490
Reference
Pienta et al., 1977
Sheuetal., 1994
Cell type
Syrian golden hamster embryo
BALB/3T3 A31-1-1
Metabolic activation system
Cocultivated X-irradiated
cells of same type
None
Positive result
BaA, DBahA
Nonpositive
result
CH, BeP, Pyr,
AC, DBacA, PH
Pyr, BaA, CH
Meets selection
criteria?
Yes
Yes
Comments
80
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Table 4-12. Study summaries: in vitro DNA adducts with benzo[a]pyrene and at least one other PAH
Record
number
16890
6300
9510
6570
13780
22800
10660
10670
13200
7870
7990
20120
21200
24810
Reference
Allen and Coombs,
1980
Binkova et al., 2000
Bryla and Weyand,
1992
Cherngetal., 2001
Cooper et al., 1982
Grover and Sims, 1968
Johnsen et al., 1998
Johnsen et al., 1997
Li et al., 2002
Melendez-Colon et al.,
2000
Nesnow et al., 1994
Nesnowetal., 1991
Segerback and
Vodicka, 1993
Baird et al., 2002
Cell type or DNA source
Mouse embryo cells from TO mice
Human diploid lung fibroblast
cells
Calf thymus DNA
Human hepatoma HepG2 cells
Fibroblasts and epithelial cells
from Wistar rat mammary tissue
Salmon testes DNA
Human lymphocytes and human
promyelocytic HL-60 cells
Rat lung Clara cells, Type 2 cells,
and macrophages
MCF-7 cells or rat lung DNA
Human mammary carcinoma
MCF-7 cells and leukemia HL-60
cells
C3H10T1/2CL8 fibroblasts
C3H10T1/2 cells
Calf thymus DNA
MCF-7 cells
Incubation
time
24 hr
Various up to
24 hr
Ihr
24 hr
24 hr
Not specified
24 hr
2hr
7-24 hr
4 or 24 hr
24 hr
24 hr
3hr
24 hr
Activation system
None
None
None
None
None
Rat liver microsomes
None
PCB pretreatment of
whole animals
Human mammary
microsomes with rat
lung DNA
None
None
None
Rat Ar S9
Morpholinos
inhibition (antisense
oligomer that blocks
protein synthesis of
CYPIA1)
Method of analysis
[3H] prelabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[3H] prelabeling
[3H] prelabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling
[32P] postlabeling,
3H-binding
[32P] postlabeling
PAHs
evaluated"
BaA
DBalP
BaA, DBacA,
PH
BghiP
BaA
DBahA, DBacA,
BaA, Pyr, PH
BjAC, B1AC
BjAC, B1AC
DBalP, BcPH,
DBahA
DBalP
DBahA
ACEA
CH, BaA, BbF,
DBahA, FA,
BghiP, Pyr
DBalP
Meets selection
criteria?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
No
Comments
PH did not form measurable
DNA adducts. Adduct
formation enhanced when
reacted under white light.
BghiP did not form measurable
DNA adducts.
BaA formed little or no
measurable DNA adducts.
No quantitative results.
No adducts formed in HL-60
cells that lack significant P450
activity.
No quantitative results.
Measures repair of adducts only,
not synthesis.
Confounded by CYP1A1
inhibition by morpholinos.
"Except where noted, positive findings were reported for all PAHs evaluated.
81
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Table 4-13. Study summaries: in vitro DNA damage, repair, or synthesis with benzo[a]pyrene and at least one
other PAH
Record
number
16840
17610
24030
18030
23790
10670
10660
19270
19680
19690
19730
19740
23800
Reference
Agrelo and Amos,
1981
Casto, 1979
De Flora etal., 1984
Dunkel et al., 1984
Ichinotsubo etal.,
1977
Johnsen et al., 1997
Johnsenetal., 1998
Lake etal., 1978
Mamber et al., 1983
Mane et al., 1990
Martin and
McDermid, 1981
Martin etal., 1978
McCarroll et al.,
1981
Cell type
Human fibroblasts
Syrian golden hamster
embryo
Escherichia coli WP2,
WP67, and CM871
E. coli WP-2 uvrA
E. coli Rec BC
Rat lung Clara cells, Type 2
cells, and macrophages
Human lymphocytes and
human promyelocytic HL-
60 cells
Human foreskin epithelial
cells
E. coli WP2 and WP100
Human and rat mammary
epithelial cells
HeLa S3 cells
HeLa S3 cells
E. coli WP2, WP2 uvrA,
WP67, CM611, WP100,
W3110polA+, and
p3478pola-
Metabolic activation
Rat Ar S9
Intrinsic
Rat Ar S9
Rat, mouse, hamster
ArS9
S9 (origin unknown)
PCB pretreatment of
whole animals
Rat or human liver
microsomes
None
Rat Ar S9
None
PB-induced rat liver
postmitochondrial
supernatant
3-MC induced rat
liver
postmitochondrial
supernatant
Rat Ar S9
1 ml point
Unscheduled DNA
synthesis
Unscheduled DNA
synthesis
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
Unscheduled DNA
synthesis
DNA damage
Inhibition of DNA
synthesis
Unscheduled DNA
synthesis
Unscheduled DNA
synthesis
DNA damage
Assay
[3H] Thymidine
uptake
[3H] Thymidine
uptake
Differential
killing repair-
deficient strains
Differential
killing repair-
deficient strains
Alkaline elution
Alkaline elution
[3H] Thymidine
uptake
Growth
inhibition of
repair deficient
strains
[3H] Thymidine
uptake
[3H] Thymidine
uptake
[3H] Thymidine
uptake
Differential
killing repair-
deficient strains
Positive result
Pyr
AC, BaA
BaA, BeP, PH, Pyr
DBaiP, DBahA
BjAC, B1AC
DBahA
BaA (in human MEC
only)
Pyr (authors:
"dubious" result)
BeP, BaA, DBacA,
DBahA
Nonpositive
result
DBahA, Pyr,
PH
Pery, BeP
AC
BjAC, B1AC
AC, BeP, PH,
Pyr
AC, FE, Pyr
BeP
AC
Pyr, AC
AC, PH
Meets selection
criteria?
Yes
Yes
No
No
Yes
No
Yes
No
Yes
No
No
Yes
Yes
Comments
Semiquantitative
data.
Dose-response
data not
provided.
No untreated
control.
Doses reported
as ranges.
Positive response
for BaA not
observed
consistently.
No quantitative
information.
82
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Table 4-13. Study summaries: in vitro DNA damage, repair, or synthesis with benzo[a]pyrene and at least one
other PAH
Record
number
19830
19850
20050
20560
20810
23900
20880
20940
21380
21720
21730
21790
16190
22260
Reference
Mersch-
Sundermann et al.,
1992
Miloetal., 1978
Nagabhushan et al.,
1990
Probst etal., 1981
Robinson and
Mitchell, 1981
Rosenkranz and
Leifer, 1980
Rosenkranz and
Poirier, 1979
Rossman et al.,
1991
Simmon, 1979b
Tongetal., 1983
Tongetal., 1981b
Tweats, 1981
Vacaetal., 1992
Williams et al.,
1982
Cell type
E. coli PQ37
Human skin fibroblast NF
and Detroit 550 cells
Hamster buccal pouch
epithelial cells and tissue
fragments
Rat hepatocyte primary
culture
Human fibroblasts WI-38
cells
E. coli pol Al-
E. coli pol Al-
E. coli WP2s(X)
S. cerevisiae D3
Rat hepatocyte primary
culture
Rat hepatocyte primary
culture
E. coli WP2, WP67(uvrA
polA), CM871 (uvrAlexA
recA)
CHO cells
Rat hepatocyte primary
culture
Metabolic activation
Rat Ar S9
None
Not specified
None
Rat Ar S9
Rat liver S9
Uninduced rat S9
Rat liver S9
Rat Ar S9
None
None
Rat Ar S9
Rat Ar S9
None
1 ml point
Induction of SOS
system
DNA damage
Inhibition of DNA
synthesis
Unscheduled DNA
synthesis
Unscheduled DNA
synthesis
DNA damage
DNA damage
DNA damage
induced
recombination
Unscheduled DNA
synthesis
Unscheduled DNA
synthesis
DNA damage
DNA damage
Unscheduled DNA
synthesis
Assay
SOS chromotest
Alkaline elution
[3H] Thymidine
uptake
[3H] Thymidine
uptake
[3H] Thymidine
uptake
Differential
killing repair-
deficient strains
Differential
killing repair-
deficient strains
A prophage
induction
Colony
pigmentation on
adenine medium
[3H] Thymidine
uptake
[3H] Thymidine
uptake
Differential
killing repair-
deficient strains
Alkaline elution
[3H] Thymidine
uptake
Positive result
AA, BaA, BbF, BghiF,
BjF, BbFE, BghiP,
BeP, CH, DBacA,
DBahA, DBalP,
DBahP, DBaiP, FA,
IP, PH, Tphen
BbA, DBacA
Pyr (with activation)
AC, DBacA, DBahA,
PH
BaA
BaA
FA
Nonpositive
result
AC, BaFE,
CO, FE, Pery,
Pyr
AC, Pyr, PH,
BeP
BaA
AC, DBahA,
PH, Pyr,
DBaiP, FE,
BeP
AC, BaA, BeP,
CH, PH
AC, BaA, BeP,
CH, PH
BeP, FA, Pyr
AC, BaA, BeP,
CH, PH
BeP, AC, CH,
Pyr
BeP, AC, CH,
Pyr
Pyr, AC
Pyr, BeP
Meets selection
criteria?
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
No
Comments
Abstract only.
BaA inhibited
synthesis 4%.
Artifact of
counting method
resulted in
control responses
reported as
negative values.
Repeats data
from21730Tong
etal., 1981b.
No quantitative
information.
No untreated or
vehicle control.
No quantitative
information.
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Table 4-14. Study summaries: in vitro clastogenicity or sister chromatid exchange with benzo[a]pyrene and at
least one other PAH
Record
number
16740
17890
17930
18120
23640
18260
14620
14640
19690
19770
20020
20340
Reference
Abe and Sasaki, 1977
Dean, 1981
DeSalvia et al., 1988
Evans and Mitchell,
1981
Evans, and DiPaolo,
1975
Gehlyetal., 1982
Kochhar, 1982
Krolewski et al., 1986
Mane et al., 1990
Matsuokaetal., 1979
Murison, 1988
Perry and Thomson,
1981
Cell type
Pseudodiploid Chinese
hamster D-6
Near-diploid epithelial-
type rat liver RLi
Male Chinese hamster
liver epithelial cells
CHO
Diploid strain 2 guinea
pig fetal cells
CH3/10T1/2 clone 8
mouse fibroblasts
Chinese hamster V79
CH3/10T1/2 clone 8
mouse embryo cells
Chinese hamster V79
cells
Male Chinese hamster
lung
P3 clonal isolate from
human epithelial
teratocarcinoma
CHO cells
Metabolic activation
None
None
None
Rat Ar S9
None
None
None
None
With and without rat mam-
mary epithelial cell coculture
Rat Ar S9
BJ-0 1 5 human breast
epithelial cell coculture
Rat Ar S9
Clastogenic
endpoint(s)
Aberrations and sister
chromatid exchanges
Various aberrations
Sister chromatid
exchanges
Sister chromatid
exchanges
Aneuploidy
Sister chromatid
exchanges
Aberrations including
gaps, rings, breaks,
fragments, exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Aberrations and sister
chromatid exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Positive
results
Pyr (with
activation)
BaA
CPcdP
BaA
CPcdP
Pyr
Non-
positive
results
AC, Pyr
AC, Pyr
Pyr, FA
AC
BeP
BeP
PH
BeP
AC
Meets selection
criteria?
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
No
No
No
Comments
Semiquantitative results.
No untreated or vehicle control.
No quantitative data. Pyr, PH also
evaluated using different protocol without
BaP reference.
Dose-dependent increase in the percentage
cells with aberrations.
CPcdP appears to increase sister chromatid
exchanges in dose-dependent fashion (two
doses).
Not clear if BaP administered simultane-
ously. No untreated control.
Not clear if BaP administered
simultaneously; no concurrent control.
No untreated control.
84
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Table 4-14. Study summaries: in vitro clastogenicity or sister chromatid exchange with benzo[a]pyrene and at
least one other PAH
Record
number
20500
21710
21720
8780
8850
21980
Reference
Popescu et al., 1977
Tongetal., 1981a
Tongetal., 1983
Vienneau et al., 1995
Warshawsky et al.,
1995
Weinstein et al., 1977
Cell type
Chinese hamster
V79-4 cells
Adult rat liver
epithelial (ARL 18)
cells
Adult rat liver
epithelial (ARL 18)
cells
UDP-Glucuronosyl-
transferases-deficient
rat (RHA-J/J) skin
fibroblasts
Human lymphocytes
Human diploid
fibroblasts (WI-38)
Metabolic activation
With or without irradiated
Syrian golden hamster
secondary embryo feeder cells
None
None
None
None
With or without rat Ar s9
Clastogenic
endpoint(s)
Aberrations and sister
chromatid exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Micronuclei
Micronuclei and sister
chromatid exchanges
Chromosomal
damage, mitotic
index, abnormal
metaphases
Positive
results
Pery, Pyr
BaA
BaA
Non-
positive
results
PH
BeP, Pyr,
AC
BeP, Pyr,
AC
BeP
BaA
Pyr
Meets selection
criteria?
No
Yes
No
Yes
Yes
Yes
Comments
BaP increased sister chromatid exchanges
but Pyr and Pery increased aberrations.
Pery increased aberrations w/o activation.
60% of Pyr treated cells (activated)
polyploid. Increased aberrations in
polyploid cells.
Repeats data from Record 21710 Tong et
al., 1981a.
85
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1 If the above criteria were met, studies were selected for use in the analysis regardless of
2 whether positive or nonpositive results were reported. Studies with positive findings were used
3 for calculation of RPFs. Studies with nonpositive findings were used in a weight of evidence
4 evaluation for selecting PAHs for inclusion in the RPF approach (discussed later in Section 6.1).
5 To be considered adequate for use in the analysis, nonpositive bioassays were selected only if
6 two additional conditions were met: (1) at least 20 animals were used per dose group, and
7 (2) animals were observed for at least 6 months. More strict criteria were applied to nonpositive
8 studies due to the difficulty in demonstrating the absence of an effect. For example, if a positive
9 tumor response (i.e., statistically significant increase in incidence) was observed after 3 months
10 of treatment with a given PAH, the positive finding is clear; however, if no response (or a
11 nonsignificant response) was observed after 3 months, the absence of response might reflect a
12 lack of carcinogenic action, but might also have resulted from inadequate follow-up time. The
13 use of these additional criteria for nonpositive studies served to ensure that PAHs would not be
14 treated as noncarcinogenic based on inadequate nonpositive bioassays.
15 Study design details, findings, limitations, and a determination of whether the study met
16 selection criteria are presented in Tables 4-1 through 4-14 for each study reviewed in each
17 category. Except where noted, positive and nonpositive findings reported in the table are based
18 on the author's determination. When statistical analysis of tumor bioassay data was not included
19 in the pertinent publication, statistical analysis was conducted to determine whether the response
20 differed from control. In the sections that follow, overviews of the data available in each
21 category are presented. The overviews address the nature of the studies available, concise
22 information on general study methods, general findings for the tested compounds, and key
23 strengths and limitations of the available data for relative potency development.
24
25 4.3.1. In Vivo Cancer Bioassays in Animals
26 The PAH database contained a large number of cancer bioassay studies in which one or
27 more PAHs was evaluated along with benzo[a]pyrene. The vast majority of the tumor bioassay
28 studies were mouse skin painting studies (n = 43). In addition, there were 12 intraperitoneal
29 studies, 9 subcutaneous exposure studies, 3 oral studies, and 9 studies using miscellaneous
30 exposure routes.
31
32 4.3.1.1. Dermal Exposure
33 A summary of the 43 dermal bioassays is provided in Table 4-1. These studies were all
34 conducted in mice. Fifteen studies tested the complete carcinogenicity of PAHs, while
35 23 studies tested PAHs as initiators in initiation-promotion protocols. In some cases, both
36 complete and initiation-promotion studies were reported in the same reference. For these
37 references, two entries are included in the table.
86 DRAFT - DO NOT CITE OR QUOTE
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1 Complete carcinogenicity studies were conducted in mice using either dropper or
2 paintbrush application. Swiss mice were typically preferred for these studies. PAHs, usually in
3 acetone, were applied to the shaved interscapular skin 2 or 3 times/week. The duration of
4 exposure varied from 10 weeks up to about 70 weeks; most studies continued exposure for at
5 least 30 weeks. Skin tumor counts were recorded on a weekly basis, and animals were sacrificed
6 when tumors reached a minimum size (e.g., 2 cm) or when the animals were moribund. These
7 studies generally focused exclusively on skin papillomas and carcinomas. Skin tumor data were
8 reported as incidence (i.e., number of animals with tumors) and/or tumor count (mean number of
9 tumors per animal) (indicated in Table 4-1).
10 Several PAHs consistently (in two or more studies) proved to be complete carcinogens in
11 mouse skin painting assays, including benzo[b]fluoranthene, benzo[j]fluoranthene,
12 cyclopenta[c,d]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and
13 dibenzo[a,l]pyrene. Chrysene gave positive results in two complete carcinogenicity studies
14 (LaVoie et al., 1979; Wynder and Hoffmann, 1959) and equivocal results in a third (Hecht et al.,
15 1974). Anthanthrene, dibenzo[a,e]fluoranthene, and dibenz[a,h]anthracene each gave positive
16 turnorigenicity results in a single assay (Cavalieri et al., 1977; Hoffmann and Wynder, 1966; and
17 Wynder and Hoffmann, 1959; respectively). Nonpositive or equivocal results were reported for
18 benzo[k]fluoranthene, benzo[g,h,i]fluoranthene, dibenzo[e,l]pyrene, indeno[l,2,3-c,d]pyrene,
19 benzo[g,h,i]perylene, naphtho[2,3-e]pyrene, anthracene, pyrene, fluoranthene, 2,3-acepyrene,
20 benz[a]anthracene, coronene, and benzo[e]pyrene (see Table 4-1).
21 According to LaCassagne et al. (1968), in studies conducted prior to 1966, the compound
22 reported as dibenzo[a,l]pyrene was actually dibenzo[a,e]fluoranthene. In the text and tables of
23 this report, data from Hoffmann and Wynder (1966) are reported as dibenzo[a,e]fluoranthene in
24 Table 4-1.
25 The initiation studies in Table 4-1 were performed under a generally consistent protocol,
26 as follows. During the early part of the second telogen phase of the hair cycle (at about 7-
27 8 weeks of age), PAHs in acetone were applied to the shaved interscapular skin of mice. In
28 general, female Swiss, CD-I, or SENCAR mice were used. Some studies used dropper
29 administration, but the majority employed a painting method using a camel's hair brush. About
30 half of the initiation studies used a single initiation dose, while the other half administered the
31 initiating compound in 10 subdoses given every other day. One to 2 weeks after the final
32 initiating dose, promotion was begun with twice or thrice weekly applications of a promoting
33 agent, usually TPA or croton oil. The dose of the promoting agent varied by study. Promotion
34 usually continued for about 20 weeks (with a range across studies from 11 to 26 weeks). The
35 incidence of skin papillomas was recorded on a weekly basis until the promotion period was
36 ended. Papillomas were removed at random for histological verification. Some studies reported
37 the number of tumors per animal; some reported only the incidence.
87 DRAFT - DO NOT CITE OR QUOTE
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1 The initiation studies in Table 4-1 consistently showed positive tumorigenicity across two
2 or more studies for the following compounds: benzo[j]fluoranthene, benzo[b]fluoranthene,
3 chrysene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, dibenzo[a,l]pyrene, and
4 cyclopenta[d,e,f]chrysene. In at least one study, benzo[k]fluoranthene, benz[l]aceanthrylene,
5 benz[e]aceanthrylene, naphtho[2,3-e]pyrene, dibenz[a,h]anthracene, dibenz[a,c]anthracene, and
6 benz[b,c]aceanthrylene showed positive initiating activity. Nonpositive results were reported for
7 pyrene, perylene, benzo[g,h,i]fluoranthene, fluoranthene, anthanthrene, dibenzo[e,l]pyrene,
8 benzo[g,h,i]perylene, indeno[l,2,3-c,d]pyrene, benzo[e]pyrene, anthracene, 2,3-acepyrene, and
9 phenanthrene. Cyclopenta[c,d]pyrene gave nonpositive results in one study (Wood et al., 1980)
10 and positive results in two studies (Raveh et al., 1982; Cavalieri et al., 1981b) (see Table 4-1).
11 The vast majority of the initiation and complete carcinogenicity studies were conducted
12 in female mice; thus, data on gender differences in skin tumor susceptibility are not available.
13 A few studies using dermal application (Warshawsky et al., 1993; Slaga et al., 1979; Van
14 Duuren and Goldschmidt, 1976; Horton and Christian, 1974; Van Duuren et al., 1973) were
15 designed to evaluate the cocarcinogenicity of two or more PAHs, or of a single PAH with
16 dodecane as a vehicle. These were primarily complete carcinogenicity studies, wherein PAHs
17 were administered together over a chronic time period, although Slaga et al. (1979) used an
18 initiation-promotion design. Study design was similar to other complete carcinogenicity
19 experiments. In these studies, the carcinogenicity of single PAHs was evaluated for comparison
20 with the results obtained when the PAHs were administered with a cocarcinogen. Data on single
21 PAHs (without a cocarcinogen) were generally limited to single dose levels. In the
22 cocarcinogenesis studies, only dibenz[a,c]anthracene, benzo[e]pyrene, and pyrene gave positive
23 results when administered without a cocarcinogen; results for pyrene were judged to be
24 equivocal in the absence of statistical confirmation. The PAHs chosen for cocarcinogenesis
25 studies were often those traditionally understood to be nontumorigenic or weakly tumorigenic
26 when administered alone (e.g., perylene, pyrene, benzo[e]pyrene, benzo[g,h,i]perylene,
27 phenanthrene, fluoranthene).
28 Several issues relating to the potential use of the dermal bioassay data for relative
29 potency development were identified during study review. Several studies did not include a
30 concurrent untreated or vehicle-treated control group (Masuda and Kagawa, 1972; Bingham and
31 Falk, 1969; Wynder and Hoffmann, 1959a, b). In a number of reports, it appears that bioassays
32 were done in batches and reported in a single publication. In these cases, it appears that
33 benzo[a]pyrene treatment may not have been undertaken concurrently with all of the compounds
34 in the report. For some of these studies (Horton and Christian, 1974; Bingham and Falk, 1969),
35 there are differences in the choice of vehicle or promoter, or other issues that argue against using
36 the benzo[a]pyrene data for direct comparison. In several other studies, however (Rice et al.,
37 1988; Slaga et al., 1980; Van Duuren and Goldschmidt, 1976; Wynder and Hoffmann, 1959), the
38 protocols (including vehicle and promoting agent) appear to have been the same.
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1 Among the dermal tumor bioassay studies in Table 4-1, 24 studies met the selection
2 criteria for use in this analysis.
o
J
4 4.3.1.2. Intraperitoneal Exposure
5 Twelve cancer bioassay s in the literature used intraperitoneal injection. Six of these
6 studies were carried out in newborn mice, while the other six used adult A/J mice. The studies
7 were focused on lung and liver tumorigenicity after PAH exposure; one study also examined
8 forestomach lesions. Study summaries for all of these references are reported in Table 4-2.
9 Tumor data were reported as incidence (i.e., number of animals with tumors) and/or tumor count
10 (mean number of tumors per animal) (indicated in Table 4-2).
11 Newborn mouse studies. Six cancer bioassays in newborn mice were identified (LaVoie
12 et al., 1994, 1987; Busby et al., 1989, 1984; Weyand and LaVoie, 1988; Wislocki et al., 1986).
13 In general, PAHs were administered intraperitoneally to newborn mice (usually of the Swiss or
14 CD-I strains). The dosing schedule called for l/7th, 2/7ths, and 4/7ths of the total dose to be
15 administered on the 1st, 8th, and 15th days of life. Typically, the mice were sacrificed at either
16 6 months or 1 year, and lung and/or liver tumors were identified and classified.
17 The studies in newborn mice showed a distinct gender difference in liver tumorigenicity.
18 Male mice appear to be substantially more susceptible to liver tumor induction than females. In
19 contrast, both male and female mice developed lung tumors after exposure. Three studies
20 (LaVoie et al., 1994; Busby et al., 1989, 1984) reported that fluoranthene induced lung tumors in
21 both male and female mice, while one study reported that fluoranthene induced liver tumors in
22 male mice only (LaVoie et al., 1994). LaVoie et al. (1987) reported that benzo[b]fluoranthene
23 and benzo[j]fluoranthene induced lung adenomas in both male and female mice, but induced
24 liver tumors only in males. Wislocki et al. (1986) reported that treatment with benz[a]anthracene
25 resulted in a significant increase in liver tumors in male mice. In this study, benz[a]anthracene
26 treatment resulted in an increased incidence of lung tumors in both males and females, although
27 the tumor incidence was significantly increased only for females. The same authors (Wislocki et
28 al., 1986) reported a significant increase in liver tumors in male mice treated with chrysene, but
29 no increase in lung tumorigenicity. The lack of lung tumorigenicity in mice treated with
30 chrysene was also reported by Busby et al. (1989).
31 Nonpositive tumorigenicity results in newborn mouse assays were reported for pyrene,
32 chrysene, benzo[k]fluoranthene, and indeno[l,2,3-c,d]pyrene (Busby et al., 1989; LaVoie et al.,
33 1987).
34 Most of the data from the newborn mouse assays met the criteria for relative potency
35 development, although Weyand and LaVoie (1988) is an abstract and does not provide dose-
36 response information. LaVoie et al. (1994) noted that liver tumorigenicity in newborn mice
37 exposed to weak tumorigenic agents may not be fully realized for 12 months; thus, the failure to
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1 observe liver tumors in studies of shorter duration (Busby et al., 1989, 1984) may result from the
2 longer latency and should be taken into consideration in using these data.
3 Lung adenoma A/J mouse studies. Six studies (Nesnow et al., 1998a, b, 1996, 1995; Ross
4 et al., 1995; Mass et al., 1993) were carried out in 6- to 8-week-old A/J mice by the same
5 laboratory using a standard protocol (Table 4-2). Mice were given a single intraperitoneal
6 injection of PAH in tricaprylin and followed for 8 months. Upon sacrifice, the lungs were
7 removed and adenomas were counted. Tumor multiplicity was reported, while tumor incidence
8 was not. Several of these studies include estimates of relative potency based on statistical
9 analysis of the tumor multiplicity data. These studies report positive tumor findings (reported as
10 an increase in the number of tumors per animal) for all of the PAHs tested (benz[j]aceanthrylene,
11 benzo[b]fluoranthene, dibenz[a,h]anthracene, cyclopenta[c,d]pyrene, and dibenzo[a,l]pyrene).
12 One additional study by a different group (Weyand et al., 2004) used the same study design to
13 assess effects of benzo[c]fluorene. In this study, both lung adenomas and forestomach lesions
14 were evaluated after 8 months. Both benzo[c]fluorene and benzo[a]pyrene were associated with
15 increased incidences of lung adenomas but not with increased forestomach lesions.
16 Among the intraperitoneal tumor bioassay studies in Table 4-2, nine studies met the
17 selection criteria for use in this analysis.
18
19 4.3.1.3. Subcutaneous Injection Exposure
20 Nine studies employing a subcutaneous exposure design were identified. All of the
21 subcutaneous exposure studies are more than 25 years old; the most recent is Pfeiffer (1977).
22 Study descriptions are presented in Table 4-3.
23 Two studies utilized newborn mice (Roe and Waters, 1967; Grant and Roe, 1963). In
24 these studies, phenanthrene was administered subcutaneously to newborn albino mice on the first
25 day of life. Ten mice of each group were sacrificed after 52 weeks, and the remaining animals
26 were sacrificed at 62 weeks. Grant and Roe (1963) evaluated lung tumorigenicity and observed
27 no increase with phenanthrene, while Roe and Waters (1967) reported liver tumors in the same
28 group of mice. Roe and Waters (1967) reported an elevated incidence of liver tumors in male
29 mice exposed subcutaneously to phenanthrene; however, it is not clear whether the difference
30 was significant. Roe and Waters (1967) is a brief communication with limited details of the
31 study design and results.
32 In most of the remaining studies, single subcutaneous doses of one or more PAH and
33 benzo[a]pyrene were administered to mice, followed 1-2.5 years later by an evaluation of
34 injection site and other tumors. Tumors at the injection site were most commonly reported;
35 however, in some studies, investigators also examined other organs for tumors (Homburger et
36 al., 1972; Roe and Waters, 1967; Grant and Roe, 1963; Rask-Nielsen, 1950; Pfeiffer and Allen,
37 1948).
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1 Most of the subcutaneous bioassays suffer from critical shortcomings in design or
2 reporting. One study used "aged" mice for controls, allowing these animals to live 16 weeks
3 longer than the treated group (Homburger et al., 1972). Three studies gave apparently positive
4 results for dibenz[a,h]anthracene (i.e., substantial tumor induction) (Pfeiffer, 1977; Steiner, 1955;
5 Bryan and Shimkin, 1943). However, neither Bryan and Shimkin (1943) nor Steiner (1955)
6 included untreated control groups. Pfeiffer (1977) included an untreated control group in which
7 there was 90% mortality prior to sacrifice of the treated animals; data on tumor incidence in
8 controls were not reported. Several other studies (Pfeiffer and Allen, 1948; Barry et al., 1935)
9 also did not include a concurrent untreated or vehicle-treated control group. These studies were
10 not used for dose-response assessment due to the lack of appropriate controls.
11 Fundamental flaws were observed in two older studies. Pfeiffer and Allen (1948)
12 examined the effects of PAHs in Rhesus monkeys. Individual animals were exposed
13 sequentially to several PAHs via multiple exposure routes; thus, the effect of any individual PAH
14 or benzo[a]pyrene cannot be discerned. Barry et al. (1935) treated mice with PAHs from varying
15 sources and of varying purity. Given the age of the study and the attendant issues with
16 nomenclature, purity, and analysis of the treatment compounds, data from this study are excluded
17 from use in relative potency development.
18 Among the subcutaneous tumor bioassay studies in Table 4-3, only a single study met
19 selection criteria for use in this analysis.
20
21 4.3.1.4. Oral Exposure
22 The literature search identified three oral bioassays that included benzo[a]pyrene and at
23 least one other PAH. Critical aspects of the study design for these studies are reported in
24 Table 4-4.
25 Biancifiori and Caschera (1962) compared the induction of mammary tumors in virgin
26 and pseudopregnant mice (female mice mated with vasectomized males) after gavage exposure
27 to dibenz[a,h]anthracene or benzo[a]pyrene. Tumor incidence was increased in pseudopregnant
28 mice given 1 mg/week of either compound for 15 weeks, but not in virgin mice given the same
29 dose. The relevance of the positive findings in pseudopregnant mice is uncertain given that an
30 increased incidence of tumors was not observed in virgin mice treated at the same dose. One
31 possible explanation for the disparate findings is that circulating hormones in pseudopregnant
32 mice differed from those in virgin mice and interacted with the PAH to enhance tumor
33 formation. Huggins and Yang (1962) also evaluated mammary tumor incidence after a single
34 oral PAH exposure. Sprague-Dawley rats were given gavage doses of benzo[a]pyrene,
35 benz[a]anthracene, or phenanthrene. This study did not include an untreated or vehicle-treated
36 control group. No tumors were observed in the rats treated with either benz[a]anthracene or
37 phenanthrene, while mammary tumors were observed in eight of the nine benzo[a]pyrene-treated
38 animals.
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1 Weyand et al. (2004) conducted an oral bioassay in which female A/J mice were fed diets
2 containing benzo[c]fluorene or benzo[a]pyrene throughout the study. At sacrifice after 260 days,
3 lung adenomas were counted and forestomach lesions were characterized. Exposure to
4 benzo[c]fluorene and benzo[a]pyrene resulted in significantly increased incidences of lung
5 adenomas, but only benzo[a]pyrene exposure resulted in forestomach neoplasms. This was the
6 only oral study that met the selection criteria for use in this analysis.
7
8 4.3.1.5. Other Routes
9 Nine bioassays were available that did not fit into other exposure route categories (i.e.,
10 dermal, intraperitoneal, subcutaneous, or oral) (see Table 4-5). Among these were studies using
11 intramammillary, intramuscular, and intravenous injection as well as lung implantation, tracheal
12 implantation, and transplacental exposure after subcutaneous injection. Seven studies were in
13 rats, with one each in mice and hamsters.
14 Deutsch-Wenzel et al. (1983) and Wenzel-Hartung et al. (1990) implanted
15 PAH-containing pellets (consisting of beeswax and trioctanoin) into the lungs of inbred female
16 Osborne-Mendel rats. Lung tumor incidence was reported for a total of 10 PAHs and
17 benzo[a]pyrene. The authors reported relative potency estimates based on the lung tumor data.
18 Lung tumors were induced by benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluor-
19 anthene, benzo[g,h,i]perylene, indeno[l,2,3-c,d]pyrene, anthanthrene, chrysene, and
20 dibenz[a,h]anthracene. Nonpositive findings were reported for benzo[e]pyrene and
21 phenanthrene.
22 Cavalieri et al. (1991) treated Sprague-Dawley rats with single intramammillary
23 injections of dibenzo[a,l]pyrene into the left mammary glands and followed them for up to
24 24 weeks. Tumors of the mammary gland, mesenchymal tissue, or skin were recorded.
25 Dibenzo[a,l]pyrene produced tumors in all animals at both doses.
26 In six studies, tumors were not induced after exposure to any target PAH.
27 Intramammillary injection of dibenz[a,h]anthracene and benz[a]anthracene did not induce
28 mammary tumors in rats (Cavalieri et al., 1988b). Pregnant mice receiving subcutaneous
29 injection of pyrene did not develop tumors, nor did their offspring (Nikonova, 1977). Rats
30 treated either intravenously or intramuscularly with benz[a]anthracene did not develop either
31 mammary or injection site tumors (Pataki and Huggins, 1969). Similarly, benz[a]anthracene was
32 not tumorigenic after intramuscular injection in rats (Sugiyama, 1973) or buccal pouch painting
33 in hamsters (Solt et al., 1987). Finally, benzo[e]pyrene was not tumorigenic when it was
34 implanted into tracheas transplanted subcutaneously into isogenic rats (Topping et al., 1981).
35 Among the tumor bioassays that used alternative exposure routes in Table 4-5, four
36 studies met the selection criteria for use in this analysis.
37
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1 4.3.2. In Vivo Studies of Cancer-Related Endpoints
2 The database of cancer-related endpoints measured after in vivo exposure to PAHs is
3 much smaller than the in vitro database. Endpoints examined after in vivo exposure include
4 mutagenicity, DNA adducts, and clastogenicity or sister chromatid exchange. As with the in
5 vitro database, only studies of selected PAHs that included benzo[a]pyrene as a reference
6 compound were reviewed. Each study that was reviewed for consideration in relative potency
7 development is presented in tabular format in subsequent sections. The tables summarize study-
8 specific information and indicate whether a particular study is considered useful for dose-
9 response assessment. The text provides an overall description of the available studies, including
10 a general description of the methodology used for each study type, the results, and the
11 weaknesses or problems associated with specific studies or study types.
12
13 4.3.2.1. DNA Adducts
14 Nineteen studies evaluating DNA adduct formation for PAHs and benzo[a]pyrene were
15 identified in the database (Table 4-6). Nine studies presented quantitative data for DNA adduct
16 formation and are discussed below. Among studies with data potentially useful for RPF
17 derivation, the route of exposure was intramammillary injection in one study (Arif et al., 1997),
18 intraperitoneal injection in seven studies (Weyand et al., 2004; Kligerman et al., 2002; Nesnow
19 et al., 1998a, 1996, 1995; Ross et al., 1995; Mass et al., 1993), dermal in three studies (Hughes
20 and Phillips, 1990; Cavalieri et al., 1981b; Phillips et al., 1979), and oral in two studies (Weyand
21 et al., 2004; Kligerman et al., 2002). Adducts were identified by [32P]-postlabeling in all of the
22 studies except for two by Phillips et al. (1979) and Cavalieri et al. (1981b), which utilized
23 [3H]- or [14C]-radiolabeled PAHs. Three papers described experiments with a single time
24 point(s) at 24 or 48 hours or 14 days (Weyand et al., 2004; Arif et al., 1997; Hughes and Phillips,
25 1990), whereas the rest had multiple time points. The duration of exposure was as short as
26 4 hours (Cavalieri et al., 1981b), although 24 hours was usually the first time point(s) in time-
27 course studies. The longest duration for a time-course study was 84 days (Hughes and Phillips,
28 1990), but most were <3 weeks. The tissues evaluated included mammary epithelium (Arif et
29 al., 1997), skin (Hughes and Phillips, 1990; Cavalieri et al., 1981b; Phillips et al., 1979), liver
30 and peripheral blood lymphocytes (Kligerman et al., 2002; Nesnow et al., 1993b), lung (Weyand
31 et al., 2004; Nesnow et al., 1998a, 1993b; Arif et al., 1997; Ross et al., 1995; Mass et al., 1993;
32 Hughes and Phillips, 1990), and forestomach (Weyand et al., 2004).
33 Dermal exposure studies typically involved application of the chemical in solution to the
34 shaved dorsal skin of mice (Hughes and Phillips, 1990; Cavalieri et al., 1981b; Phillips et al.,
35 1979). After the scheduled sacrifice, the treated skin was excised and frozen; a scalpel was used
36 to scrape away the dermis from the epidermis that was subsequently powdered in liquid nitrogen.
37 In one study, the lung was also excised and frozen in liquid nitrogen (Hughes and Phillips,
38 1990). DNA was isolated from the frozen epidermis or lung. Liquid scintillation counting was
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1 used to quantify DNA adducts to PAH labeled with [3H] or [14C] (Cavalieri et al., 1981b; Phillips
2 etal., 1979). For [32P]-postlabeling, DNA was treated to selectively dephosphorylated
3 nonadducted nucleotides; after postlabeling, adducts were resolved by sequential anion-exchange
4 thin layer chromatography on polyethyleneimine-cellulose plates in several directions using three
5 solvents (Hughes and Phillips, 1990). Adduct spots on chromatograms were located by
6 autoradiography, after which the spots were excised and radioactivity levels were determined by
7 Cerenkov counting.
8 Most studies reported the mean number of adducts formed within a tissue per unit of
9 DNA, with time-course data displayed graphically. Peak values were sometimes called out
10 specifically in the text or tables. As the shapes of dose-response curves differ among different
11 PAHs, the peak value is an imprecise measure for comparing the relative adduct-forming
12 potency of the different compounds. The TIDAL has also been used for reporting results for a
13 time-course study (Ross et al., 1995). The TIDAL value is the area under the curve (AUC) for
14 adduct persistence (based on the rate of adduct formation and repair) for the duration of the
15 study. The TIDAL value expresses the total DNA adduct burden experienced by the tissue from
16 the time of treatment to the end of the study. The TIDAL versus administered dose curve
17 provides a convenient way to compare adduct-forming potency for different PAHs in time-
18 course experiments. An important limitation of the TIDAL approach is the inherent assumption
19 that the ratios of specific adducts are relatively constant across dose and time course. Ross et al.
20 (1995) demonstrated that this assumption was valid for several different PAHs; however, it was
21 also noted that two adducts of benzo[a]pyrene in rat liver did not conform to this general pattern.
22 Ross et al. (1995) presented data for lung adenoma incidence (measured at 8 months) in
23 several ways: as a function of administered dose, as a function of adduct levels per dose
24 measured 24 hours after dosing (results for 3 days postdosing were mentioned but not shown), as
25 a function of TIDAL values measured over 21 days (during which period, adduct levels were
26 specifically quantified), and as a function of TIDAL values extrapolated to 8 months. The
27 relative tumor induction potencies of the studied PAHs were similar for each assay for a single
28 PAH when described as functions of administered dose, the adduct levels per dose at 3 days, the
29 TIDAL values over 21 days, or the TIDAL values extrapolated to 8 months. The relative
30 potencies for tumor incidence as a function of adduct levels at 24 hours were not similar to those
31 associated with the other measures of exposure. Ross et al. (1995) suggested that
32 pharmacokinetic differences in adduct formation among the PAHs were responsible for the
33 discrepancy, but suggested that peak levels could be used to compare the potencies of different
34 PAHs if adduct formation for those PAHs followed similar kinetics.
35 DNA adduct experiments were carried out in replicate and were usually analyzed
36 statistically. It should be noted that, based on the work of Ross et al. (1995), relative potencies
37 determined from studies that administered a single dose level and measured adducts at a single
38 time point will be less reliable unless the shapes of the adduct formation curves are similar.
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1 However, the single dose and single measurement studies were also used for dose-response
2 assessment.
3 Among the in vivo DNA adduct studies shown in Table 4-6, nine studies met the
4 selection criteria for use in this analysis.
5
6 4.3.2.2. Clastogenicity or Sister Chromatid Exchange Frequency
7 The database included 13 studies in which clastogenic effects or frequency of sister
8 chromatid exchanges of benzo[a]pyrene and at least one other PAH were tested in whole animal
9 systems. Table 4-7 lists the studies along with important study design details. The clastogenic
10 endpoints measured in these studies were micronuclei, chromosome gaps and breaks, and
11 nonspecific aberrations; sister chromatid exchanges were also measured. These studies were all
12 conducted in rodents, including mice, rats, and hamsters.
13 Eight of the studies evaluated micronuclei, sister chromatid exchanges, or chromosome
14 gaps or breaks in bone marrow from treated mice or hamsters (Allen et al., 1999; Katz et al.,
15 1981; Paika et al., 1981; Salamone et al., 1981; Tsuchimoto and Matter, 1981; Roszinsky-Kocher
16 et al., 1979; Bayer, 1978; Sugiyama, 1973). In these studies, one or two doses of PAH were
17 injected intraperitoneally into the animals, and sacrifice occurred at various time points thereafter
18 (typically 24 hours after). Bone marrow smears were examined microscopically and scored for
19 micronuclei, sister chromatid exchanges, gaps, or breaks.
20 He and Baker (1991) applied multiple dose levels of chrysene or phenanthrene to the skin
21 of hairless mice and harvested keratinocytes upon sacrifice 24 hours later. The keratinocytes
22 were incubated for 2 days and treated with cytochalasin B to identify binucleated cells. After
23 4 days in vitro, cells were mounted on slides and examined microscopically for micronuclei.
24 Results were reported as the percent of binucleated cells with one or more micronuclei among
25 the total number of binucleated cells scored. Chrysene treatment resulted in a dose-related
26 increase in micronuclei, while pyrene did not.
27 Kligerman et al. (2002, 1986) measured sister chromatid exchanges and/or micronuclei in
28 the blood of mice or rats given a single dose of PAH either orally or intraperitoneally. The study
29 by Oshiro et al. (1992) involved two or four oral doses of pyrene or anthracene in mice. Blood
30 obtained from the tail 24 hours after the last treatment was examined microscopically and
31 micronuclei were scored in polychromatic erythrocytes. In an unusual study design, Sirianni and
32 Huang (1978) measured sister chromatid exchanges in V79 cells placed in a diffusion chamber
33 implanted in the peritoneal cavity of mice.
34 Thirteen individual PAHs were evaluated in these studies. Only chrysene gave positive
35 results for more than one endpoint (for sister chromatid exchange and micronucleus frequency;
36 He and Baker, 1991; Roszinsky-Kocher et al., 1979). Five other PAHs (phenanthrene,
37 dibenz[a,h]anthracene, benz[a]anthracene, benzo[b]fluoranthene, and benzo[e]pyrene) increased
38 the frequency of sister chromatid exchange in hamster bone marrow after intraperitoneal
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1 administration (Roszinsky-Kocher et al., 1979). Bayer (1978) also reported an increase in sister
2 chromatid exchange frequency in hamster bone marrow after phenanthrene administration (high
3 dose only). Anthracene and pyrene consistently gave nonpositive results in several studies
4 (Oshiro et al., 1992; He and Baker, 1991; Katz et al., 1981; Paika et al., 1981; Salamone et al.,
5 1981; Tsuchimoto and Matter, 1981; Roszinsky-Kocher et al., 1979; Sirianni and Huang, 1978).
6 Dibenzo[a,i]pyrene and benzo[g,h,i]perylene each gave nonpositive results in an assay for bone
7 marrow micronuclei (Katz et al., 1981).
8 Among studies with positive results, only He and Baker (1991), Kligerman et al. (1986),
9 and Bayer (1978) administered PAHs at multiple dose levels. Bayer (1978) observed a positive
10 response only with the highest dose of phenanthrene. Of the single dose studies, only
11 Roszinsky-Kocher et al. (1979) reported responses clearly differing from controls.
12 Among the in vivo clastogenicity or sister chromatid exchange studies shown in
13 Table 4-7, 10 studies met the selection criteria for use in this analysis.
14
15 4.3.2.3. In Vivo Mutagenicity
16 The PAH database contains several studies that evaluate specific mutagenic endpoints
17 following in vivo exposure to PAHs (see Table 4-8). These studies include mutagenicity
18 experiments in Drosophila melanogaster, an intraperitoneal host-mediated assay using
19 Salmonella strains or yeast, and DNA sequence analysis of specific codons in the Ki-ras
20 oncogene in mouse lung tumors.
21 Most Drosophila studies administered PAH compounds to either the suspension media or
22 to the diet for 48-72 hours prior to cross-mating and analysis of mutations (Frolich and Wurgler,
23 1990; Valencia and Houtchens, 1981; Fahmy and Fahmy, 1980). One study used abdominal
24 injection as an exposure pathway (Zijlstra and Vogel, 1984). The mutagenic endpoints evaluated
25 included somatic mutations (i.e., eye color mosaicism, wing spots) (Frolich and Wurgler, 1990;
26 Fahmy and Fahmy, 1980) or sex-linked recessive lethal mutations (Zijlstra and Vogel, 1984;
27 Valencia and Houtchens, 1981). Only two PAHs were evaluated in the Drosophila studies in
28 addition to benzo[a]pyrene (benz[a]anthracene and pyrene), and the results were either
29 nonpositive or inconsistent in all studies (Frolich and Wurgler, 1990; Zijlstra and Vogel, 1984;
30 Valencia and Houtchens, 1981; Fahmy and Fahmy, 1980). A significant effect was seen for
31 benz [a] anthracene only with cross-breeding of strains selected for enhanced metabolic activity
32 (Frolich and Wurgler, 1990). No effect was observed using the standard strains.
33 An intraperitoneal host-mediated assay was described by Simmon et al. (1979). Five
34 PAHs (anthracene, benz [a] anthracene, benzo[e]pyrene, chrysene, and phenanthrene) were
35 administered to Swiss Webster mice by gavage or intramuscular injection (single dose only).
36 Microorganisms (S. typhimurium and Saccharomyces cerevisiae) were injected intraperitoneally
37 into exposed mice and were recovered 4 hours later for mutation analysis. Nonpositive results
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1 were observed and the host-mediated assay system was considered insensitive for detecting
2 carcinogenic PAHs.
3 A series of studies have investigated the mutation sequence in codons 12 and 61 of the
4 Ki-ras oncogene from PAH-induced lung adenomas in A/J mice (Nesnow et al., 1998a, 1996,
5 1995; Mass et al., 1993). As discussed in Section 2.4 (Similarities in Mode of Carcinogenic
6 Action for PAHs), the purpose of these studies was to correlate the tumorigenic potency of
7 specific PAHs with the formation of DNA adducts and the mutation of specific codons in the
8 Ki-ras oncogene. Six non-alkylated PAHs were utilized in these studies (benzo[a]pyrene,
9 benz[j]aceanthrylene, benzo[b]fluoranthene, dibenz[a,h]anthracene, cyclopenta[c,d]pyrene, and
10 dibenzo[a,l]pyrene). Mutation analysis of the Ki-ras oncogene at codons 12 and 61 was carried
11 out in PAH-induced lung adenomas using PCR amplification and dideoxy nucleotide sequencing
12 methods. The primary mutation type for benzo[a]pyrene, benzo[b]fluoranthene, and
13 dibenzo[a,l]pyrene was the GGT^TGT mutation. This guanine mutation was correlated with
14 the formation of diol epoxide guanine adducts. The GGT—>CGT mutation was the primary
15 mutation type for benz[j]aceanthrylene and cyclopenta[c,d]pyrene. The CGT mutation was
16 associated with the formation of cyclopenta-guanine adducts and increased tumorigenic potency
17 (i.e., >90 adenomas per mouse) in A/J mice. Dibenz[a,h]anthracene was the only PAH evaluated
18 that did not induce mutations in Ki-ras codons 12 or 61. This compound produced diol epoxide
19 guanine adducts and lung adenomas in A/J mice, suggesting a possible interaction at a different
20 genetic target. The Ki-ras mutation analysis data were presented as percent of tumors with a
21 specific mutation at either codon 12 or 61. No dose-response data were provided.
22 Among the in vivo mutagenicity studies shown in Table 4-8, only one study met the
23 selection criteria for use in this analysis.
24
25 4.3.3. In Vitro Studies of Cancer-Related Endpoints
26 Many in vitro studies of cancer-related endpoints are present in the PAH database. As
27 previously discussed, only those studies that included at least one selected PAH and
28 benzo[a]pyrene as a reference compound were reviewed. Each study that was reviewed for the
29 purpose of RPF development is included in Tables 4-9 through 4-14. The tables summarize
30 study-specific information and indicate whether a particular study is considered useful for dose-
31 response assessment. The text provides an overall description of the available studies, including
32 a general description of the methodology used for each study type, the results, and the
33 weaknesses or problems associated with specific studies or study types.
34
35 4.3.3.1. Bacterial Mutagenicity
36 The bacterial mutagenicity of many PAHs has been extensively studied (39 studies with
37 benzo[a]pyrene; see Table 4-9). All of the studies used the Ames assay in S. typhimurium. A
38 total of 38 PAHs have been evaluated for their ability to induce mutations in bacterial systems.
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1 The Ames Salmonella assay is a bacterial reverse mutation assay, which measures the
2 frequency at which histidine-independent bacteria arise from histidine-requiring bacterial strains
3 in the presence of a chemical mutagen. The results are generally expressed as either the number
4 of revertant colonies per plate or the number of revertants/nmol of the test compound (calculated
5 from the linear portion of the dose-response curve). Several strains of S. typhimurium have been
6 used to evaluate specific PAH mutation types; for example, TA98, TA1537, and TA1538 detect
7 various frameshift mutations, TA1535 responds to base-pair substitution, and TA100 responds to
8 a broad spectrum of mutations. Metabolism to reactive intermediates is required for PAH
9 mutagenicity in Salmonella and many metabolic activation systems have been employed. Rat
10 liver postmitochondrial supernatant (known as S9) from Aroclor-induced rats is most often used,
11 although other rodent species and enzyme inducers are sometimes employed. Isolated rat
12 hepatocytes or purified mixed-function oxidase enzymes were occasionally utilized for metabolic
13 activation of PAHs.
14 Of the PAHs tested for bacterial mutagenicity, most were considered positive in at least
15 one study under optimal study conditions. Compounds that produced nonpositive results in
16 multiple studies include anthracene, fluorene, phenanthrene, and pyrene. The primary weakness
17 of the bacterial mutagenicity database for PAHs is the limited amount of multiple-dose data for
18 many PAHs. Many studies report findings at a single dose level for several PAHs.
19 Among the in vitro bacterial mutagenicity studies shown in Table 4-9, 29 studies met the
20 selection criteria for use in this analysis.
21
22 4.3.3.2. Mammalian Mutagenicity
23 Studies that evaluate the mutagenicity of target PAHs in mammalian cells are described
24 in Table 4-10 (29 studies). The most common cell types used in these studies were the
25 V79 Chinese hamster cells and the L5178Y mouse lymphoma cells. Other cell types include
26 human epidermal keratinocytes, TK6 human lymphoblasts, human epithelial cells (HS1 HeLa),
27 human foreskin fibroblasts (D-550), mouse fibroblasts, rat embryo cells, rat liver epithelial cells
28 (ARL-18), and Chinese hamster ovary (CHO) cells. A total of 14 PAHs have been evaluated for
29 their ability to induce mutations in mammalian cell systems.
30 Each of the mammalian cell assays detects forward mutations that confer resistance to a
31 toxic chemical. Mutations in the hypoxanthine-guanine phosphoribosyl transferase gene (HPRT)
32 result in resistance to purine analogs such as 6-thioguanine, 8-azaguanine, and ouabain. HPRT
33 mutations induced by PAHs were most often measured in V79 Chinese hamster cells, but have
34 also been detected in human, rat, and mouse cell lines. Forward mutation at the thymidine
35 kinase (TK) locus is measured as colony growth in the presence of thymidine analogs (e.g.,
36 trifluorothymidine or 5-bromo-2'-deoxyuridine). PAH-induced TK mutations were measured in
37 mouse lymphoma cells (L5178Y) and human lymphoblasts. Forward mutation assays are
38 considered to respond to a variety of mutation types (including frameshift, base-pair substitution,
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1 deletions, and rearrangements or complex mutations). Exogenous metabolic activation is
2 required for PAH mutagenicity in most mammalian cell assays. This was accomplished using a
3 rat liver S9 mix or cocultivation with other rodent cells able to metabolize PAHs to reactive
4 intermediates (i.e., hamster embryo cells, fibroblasts, or hepatocytes; rat hepatocytes). The
5 results of forward mutation assays in mammalian cell lines are generally expressed as mutant
6 frequency/ 10X survivors.
7 Of the 26 PAHs tested for mammalian cell mutagenicity, all were considered positive in
8 at least one study under optimal study conditions. Compounds that produced nonpositive results
9 in some studies include anthracene, benzo[e]pyrene, phenanthrene, and pyrene. Benzo[a]-
10 anthracene produced positive findings in seven studies and nonpositive findings in four studies.
11 The mammalian mutagenicity studies generally provide more multidose data than the bacterial
12 mutagenicity studies.
13 Among the in vitro mammalian mutagenicity studies shown in Table 4-10, 27 studies met
14 the selection criteria for use in this analysis.
15
16 4.3.3.3. Morphological/Malignant Cell Transformation
17 Twenty-five studies examined the capacity of benzo[a]pyrene and other PAHs to
18 transform cells in culture (Table 4-11). All of these studies were conducted using mammalian
19 cells, most commonly mouse or hamster embryo cells. A few studies added feeder cells or rat
20 liver homogenate to enhance metabolic activation in the test system; however, the majority relied
21 on the intrinsic metabolic capacity of the cells. The general test protocol involved seeding the
22 cultured cells in Petri dishes followed by exposure to a solution of the test compound, usually for
23 a period of 24 hours. The cells were then cultured for about 6 weeks before being fixed and
24 stained. Transformed colonies (foci) were scored based on characteristics such as cell piling,
25 criss-crossing, basophilic staining, and/or invasion of surrounding (nontransformed) cell
26 monolayer. In studies conducted by some laboratories, foci were classified as Type II or
27 Type III; the latter category included those with invasion of the surrounding monolayer, highly
28 criss-crossed arrays, and deep staining. Data were generally reported as the number of foci
29 (colony of transformed cells) per dish or per surviving cells and/or the percent of dishes with
30 foci.
31 In a few cases (e.g., Greb et al., 1980), transformation was assessed by growth of treated
32 cells in soft agar. Transformed cell colonies growing in semi-solid agar are capable of
33 anchorage-independent growth.
34 Three studies (Evans and DiPaolo, 1975; Kakunaga, 1973; DiPaolo et al., 1972)
35 confirmed the identification of malignant cells by injecting the transformed cells into rodents and
36 following tumor induction in the animals. In all three cases, cells identified as transformed gave
37 rise to tumors, while the cells without these characteristics did not.
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1 Cell transformation assays were identified that included 22 individual PAHs other than
2 benzo[a]pyrene. Dibenz[a,h]anthracene consistently gave rise to transformed cells in all but one
3 of the seven studies in which it was tested. Cyclopenta[c,d]pyrene, indeno[l,2,3-c,d]pyrene,
4 benzo[j]aceanthralene, benz[e]aceanthrylene, and dibenz[k,mno]acephenanthrylene were each
5 tested in a single study and gave positive results. Benz[a]anthracene, pyrene, phenanthrene,
6 benzo[e]pyrene, and anthracene each gave nonpositive results in a number of studies, while
7 fluoranthene, benzo[k]fluoranthene, dibenz[j,mno]acephenanthrylene, naphth[ 1,2,3-mno]ace-
8 phenanthrylene, and aceanthrylene were each tested in a single study and gave nonpositive
9 results. Only a single dose of the target PAH was applied in 8 of the 26 studies of in vitro
10 morphological/malignant cell transformation.
11 Among the in vitro morphological/malignant transformation studies shown in Table 4-11,
12 19 studies met the selection criteria for use in this analysis.
13
14 4.3.3.4. DNAAdducts
15 Several studies (14) were identified in which DNA adducts were measured after either
16 whole cells or extracted DNA were incubated with benzo[a]pyrene and at least one other PAH.
17 Table 4-12 shows general study details for these studies. Most of the studies involved
18 measurement of DNA adducts in whole mammalian cells, while some measured adducts formed
19 when PAHs were incubated with extracted DNA. Whole cells were usually incubated with
20 PAHs for about 24 hours, while extracted DNA was exposed to PAH solutions for a shorter time
21 period (1-3 hours). Some of the studies added metabolic activation (usually rat liver
22 microsomes) to the incubation solution. Melendez-Colon et al. (2000) evaluated DNA adduct
23 formation after dibenzo[a,l]pyrene exposure in two cell types: one having significant CYP450
24 activity (MCF-7 cells) and one lacking significant CYP450 activity (HL-60). The authors
25 reported that adducts were formed in the cells having CYP450 activity, but no adducts were
26 formed in the cells lacking such activity.
27 Identification and quantification of adducts was generally done using a [32P]-postlabeling
28 assay as follows. After exposure, DNA was isolated and digested to mononucleotides.
29 Mononucleotides were radiolabeled with [32P]-ATP, separated with thin layer chromatography,
30 and visualized by autoradiography. Relative adduct labeling was measured using a scintillation
31 counter. A few early studies used [3H]-labeled PAHs to identify and quantify adducts. In some
32 cases, adducts were identified by high-performance liquid chromatography and gas
33 chromatography-mass spectrometry.
34 The 14 studies reviewed examined 15 PAHs other than benzo[a]pyrene. Apart from
35 phenanthrene, which did not result in measurable DNA adducts when incubated with calf thymus
36 DNA under various conditions (Bryla and Weyand, 1992), each of the PAHs produced
37 measurable DNA adducts in at least one study.
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1 Major limitations associated with some of the in vitro DNA adduct data for relative
2 potency development include the lack of data at multiple PAH exposure levels, the use of
3 extracted DNA rather than whole cell assays, and the inconsistent use of extrinsic metabolic
4 activation sources. Only three studies with positive adduct findings reported adduct
5 measurements at multiple doses (concentrations) of PAH (Binkova et al., 2000; Melendez-Colon,
6 2000; Bryla and Weyand, 1992). Three studies used extracted DNA rather than whole cells to
7 measure DNA binding (Segerback and Vodicka, 1993; Bryla and Weyand, 1992; Grover and
8 Sims, 1968). Finally, the available studies on DNA adduct formation use cell types with varying
9 degrees of PAH metabolic capacity, with and without added metabolic activation sources. Both
10 the types and the quantities of DNA adducts formed are likely to depend on the level of
11 metabolic activation for most PAHs.
12 Among the in vitro DNA adduct studies shown in Table 4-12, 10 studies met the
13 selection criteria for use in this analysis.
14
15 4.3.3.5. DNA Damage/Repair
16 Twenty-four reports in the database evaluated the effects of one or more PAHs on DNA
17 damage, repair, or synthesis. Table 4-13 summarizes the study design information and results of
18 these studies. Studies included measures of unscheduled DNA synthesis and DNA damage.
19 Unscheduled DNA synthesis was generally measured by increased radiolabeled (3H) thymidine
20 uptake in treated cells versus untreated cells. DNA damage was measured either using the
21 alkaline elution assay for DNA strand breakage in mammalian cells, or using the differential
22 killing of DNA repair-deficient bacterial strains. Metabolic activation of PAHs was most often
23 accomplished using a rat liver S9 mix.
24 Twenty-eight different PAHs have been tested for effects on DNA in one or more assays.
25 In general, pyrene, anthracene, phenanthrene, perylene, fluorene, and benzo[e]pyrene gave
26 nonpositive results in multiple studies. Chrysene gave nonpositive results in four assays and
27 positive results in one assay (Mersch-Sundermann et al., 1992). More positive than nonpositive
28 results were reported for benz[a]anthracene, dibenz[a,h]anthracene, and dibenz[a,c]anthracene.
29 Other PAHs were tested only once, or gave roughly an equal frequency of positive and
30 nonpositive responses in these assays.
31 Although a large number of PAHs have been tested for DNA damage/repair, the database
32 includes both bacterial and mammalian cells and several different genotoxic endpoints. In
33 addition, the use of external metabolic activation, or cell types with intrinsic metabolic capacity,
34 was inconsistent across these studies. These limitations make it difficult to compare studies
35 using the same target PAHs.
36 Among the in vitro DNA damage/repair studies shown in Table 4-13, 15 studies met the
37 selection criteria for use in this analysis.
38
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1 4.3.3.6. Clastogenicity or Sister Chromatid Exchange Frequency
1 The database contains 18 studies in which clastogenicity or sister chromatid exchange
3 frequency was measured in cultured cells after exposure to benzo[a]pyrene and at least one other
4 PAH (Table 4-14). A wide variety of cell types was used in these assays, including hamster
5 liver, lung, CHO, and V79 cells; rat liver epithelial cells; human teratocarcinoma epithelial cells;
6 rat and human mammary epithelial cells; mouse, rat, and human fibroblasts; human
7 lymphocytes; and guinea pig fetal cells. A number of the studies used a metabolic activation
8 system, typically either rat liver S9 or coculture with a cell type able to metabolize PAHs. While
9 laboratory methods varied widely, the general approach involved treating the cultured cells with
10 a solution of the test compound, either with or without metabolic activation. Usually,
11 bromodeoxyuridine was added to the growth medium to provide a means of staining metaphase
12 chromosomes, and colcemid was used to arrest mitotic cells. Chromosomes were examined
13 microscopically and aberrations or exchanges were scored visually. In most cases, the endpoint
14 examined was frequency of sister chromatid exchanges. Other endpoints included frequency of
15 micronuclei and scoring of chromosomal aberrations such as breaks, gaps, deletions, etc.
16 Only eight PAHs (anthracene, benz[a]anthracene, benzo[e]pyrene, cyclopenta-
17 [c,d]pyrene, fluoranthene, perylene, phenanthrene, and pyrene) have been tested for clastogenic
18 effects in vitro. In many cases, the available studies were aimed at evaluating the validity of a
19 given test system to predict carcinogenicity. In these studies, a range of compounds of known or
20 believed carcinogenicity were used. Often, benzo[a]pyrene was included as a known carcinogen,
21 and other PAHs were chosen because they were known or believed to be noncarcinogenic or
22 weakly carcinogenic.
23 Among the tested compounds, four gave positive results in at least one study. With few
24 exceptions, PAHs administered without metabolic activation gave nonpositive responses in these
25 assays. Cyclopenta[c,d]pyrene was reported to increase the frequency of sister chromatid
26 exchanges in two assays, one with and one without metabolic activation (Murison, 1988;
27 Krolewski et al., 1986). Benz[a]anthracene gave positive results in three studies of sister
28 chromatid exchange induction (Mane et al., 1990; Tong et al., 1983, 1981a) and nonpositive
29 results in a fourth (Warshawsky et al., 1995). Kochhar (1982) reported a dose-dependent
30 increase in chromosomal aberrations in V79 cells treated with benz[a]anthracene in the absence
31 of metabolic activation. Perylene increased aberrations in one system (Popescu et al., 1977), but
32 did not increase sister chromatid exchanges in another (Sirianni and Huang, 1978). Likewise,
33 pyrene gave positive results in a number of studies that included metabolic activation (Evans and
34 Mitchell, 1981; Perry and Thomson, 1981; Popescu et al., 1977) and nonpositive results in
35 several that did not include activation (DeSalvia et al., 1988; Tong et al., 1983, 1981a; Dean,
36 1981; Abe and Sasaki, 1977).
37 The clastogenicity and sister chromatid exchange data for PAHs are variable with respect
38 to cell type and use of extrinsic metabolic activation. Some cells have intrinsic metabolic
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1 activity, while others require activation from an external source. The degree to which metabolic
2 activation is required for PAHs to exert a clastogenic effect in cell cultures is not well
3 established. Another limitation of these data stems from the fact that a small number of PAHs,
4 many traditionally believed to be noncarcinogenic or weakly carcinogenic, have been tested for
5 clastogenic effects in vitro.
6 Among the in vitro clastogenicity/sister chromatid exchange studies shown in Table 4-14,
7 10 studies met the selection criteria for use in this analysis.
8
9 4.4. SUMMARY OF INFORMATION AVAILABLE TO DEVELOP RPFs FOR
10 INDIVIDUAL PAHs
11 The PAH database contains several different types of data that may be used to estimate
12 relative potencies of individual PAHs. The data were summarized in Section 4.3 and include in
13 vivo tumor bioassays using various routes of exposure and data for cancer-related endpoints
14 from both in vivo and in vitro studies. As discussed above, the concurrent testing of
15 benzo[a]pyrene as a reference compound was considered essential to allow for RPF calculation.
16 The introduction to Section 4.3 lists criteria for selecting studies or data sets for use in the
17 analysis. Studies that met these criteria were used in the development of the RPF approach.
18 Chapter 5 discusses methods used for dose-response assessment and RPF calculation from each
19 study or dataset, and Chapter 6 discusses the selection of PAHs to be included in the RPF
20 approach using a weight of evidence evaluation of the available data. Chapter 7 describes the
21 derivation of final RPFs for each PAH included in the analysis.
22
23
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1 5. METHODS FOR DOSE-RESPONSE ASSESSMENT AND RTF CALCULATION
2
3
4 A discussion of the available data on PAH carcinogenicity and cancer-related endpoints
5 and criteria for selection of studies was presented in Chapter 4. This section describes the
6 selection of dose-response data and methods for dose-response assessment and RPF calculation
7 from the selected datasets. The dose-response data extracted from each study with positive
8 results and the results of the statistical analyses are shown in Appendix C. Appendix C also
9 contains information regarding the source of the dose-response data (i.e., the figure or table
10 number from the study and the particular data points that were used in the dose-response
11 assessment) and additional comments on the use of the data for dose-response assessment and
12 RPF calculation. The results of the RPF calculations are shown in tables in Appendix E. These
13 tables provide summary information for each study, including the PAHs that were tested, the
14 data used to estimate the slopes (point estimate4 or BMD model result), the calculated RPF
15 value, and any specific comments related to the data analysis.
16
17 5.1. CHOICE OF DOSE-RESPONSE DATA
18 For each of the endpoints evaluated in Chapter 4 (dermal, intraperitoneal, subcutaneous,
19 oral, and other route bioassays; in vivo DNA adducts; in vivo clastogenicity or sister chromatid
20 exchange frequency; in vitro bacterial and mammalian mutagenicity; in vitro morphological/
21 malignant transformation; in vitro clastogenicity or sister chromatid exchange frequency; and
22 other in vitro endpoints [DNA adducts, unscheduled DNA synthesis, DNA damage, etc.]), there
23 was at least one study that met selection criteria. For those studies with positive findings, dose-
24 response data were extracted for dose-response assessment and calculation of RPFs.
25
26 5.1.1. Dose-Response Data for Tumor Bioassays
27 Data on both benign and malignant tumors were included in the dose-response
28 assessment. In cases where the combined incidence of benign and malignant tumors was
29 reported, these data were selected; however, in some cases, only benign or only malignant tumor
30 incidence was reported. These data were also considered appropriate for derivation of RPFs.
31 There is evidence for progression from benign to malignant tumors (e.g., dermal papillomas
32 progressing to carcinomas) in studies of benzo[a]pyrene (for example, see Albert et al., 1991),
33 and other PAHs are assumed to be lexicologically similar to benzo[a]pyrene. Thus, even when a
34 study reported only the incidence of benign tumors, these data were used in the dose-response
35 assessment.
4For the purpose of this report, the term "point estimate RPF" is used to describe an RPF calculated from a single
point on the dose-response curve for both the PAH of interest and benzo[a]pyrene. This term distinguishes the RPF
from one calculating using a BMD modeling result from multidose data.
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1 While tumor multiplicity data from tumor bioassays are not generally used to estimate
2 cancer potency., these data were included in the dose-response assessment in order to determine
3 whether they could serve as a reliable measure of relative cancer potency. Several bioassays
4 reported data on both tumor incidence and tumor number, providing information that could later
5 be used to compare relative potencies estimated from these two endpoints.
6 As discussed in Section 4.3, statistics were used for tumor bioassay data to determine
7 whether the tumor incidence or multiplicity observed at a particular dose represented a
8 statistically significant increase over controls. If statistical analyses were not described in the
9 original report, incidence data were analyzed using Fisher's exact test and the Cochran-Armitage
10 trend test. Positive findings were indicated by a significant (p < 0.05) difference for at least one
11 dose group by comparison to control (in Fisher's exact or an equivalent test) or a significant
12 dose-response trend (Cochran-Armitage or equivalent) for multidose studies. For tumor bioassay
13 data reported as tumor count, a t-test was conducted (when variance data were available) to
14 determine whether the count was significantly different from control (p < 0.05). The results of
15 the statistical analyses are shown with the dose-response data in Appendix C.
16 The tumor bioassays that reported both incidence and tumor count were unique in
17 offering two different datasets for the same study. For each dose of each PAH in the tumor
18 bioassays, the decision to calculate an RPF, and in some instances, the selection of the point of
19 departure, was based on whether the tumor incidence or count was statistically significantly
20 increased over the control; if there was a significant increase, an RPF was calculated. There was
21 a single instance where the tumor count was statistically significantly increased, but the
22 incidence of tumors was not. In female mice exposed at the high dose of fluoranthene in the
23 study by Busby et al. (1984), the lung tumor count was significantly increased (albeit borderline,
24 p = 0.0343) while the incidence was not, and neither was statistically significantly increased at
25 the lower dose. As there were no higher doses in this study, it is possible that the two measures
26 might have produced consistent findings at higher doses. For the purpose of this analysis, the
27 multiplicity data from this study were treated as an independent measure of carcinogenic
28 potency, and an RPF was calculated for the statistically increased tumor count irrespective of the
29 analysis of incidence. It should be noted that average tumor count can be skewed by an unusual
30 response in a single animal, and no information was available to determine whether such
31 response represented an anomaly unrelated to exposure or an unusual susceptibility to the
32 exposure. Thus, reliance on statistical analysis of mean tumor count alone as a measure of
33 carcinogenic response may be subject to additional uncertainty.
34
35 5.1.2. Dose-Response Data for Cancer-Related Endpoint Studies
36 For cancer-related endpoint data, each study authors' conclusions regarding a positive or
37 nonpositive response for each PAH were accepted, and RPFs were calculated when positive
38 results were reported. Data that were reported in graphical format in published studies of cancer-
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1 related endpoints were digitized (Grab It!™ Graph Digitizer, Datatrend Software) to identify the
2 dose-response data points. In a few cases, the only cancer-related endpoint data in a given
3 publication were reported as relative potency (relative to benzo[a]pyrene). For these
4 publications, which included only in vitro cancer-related endpoint data (primarily mutagenicity),
5 the relative potency estimates calculated by the authors were used without modification (except
6 for dose adjustment where appropriate; see Section 5.5).
7
8 5.2. OVERALL FORM OF RTF ESTIMATE
9 The overall goal of the dose-response analysis was to calculate ratios representing the
10 relative potency of a given PAH compared with benzo[a]pyrene (i.e., RPFs). For all datasets, the
11 RPF was defined as the ratio (PAH;:BaP) of the slopes of the dose-response curves in the low-
12 dose region, following Equation 5-1 below:
13
14 RPF = slope PAH; + slope BaP (5-1)
15
16 Data available for calculation of RPFs consisted of both quantal and continuous
17 endpoints. Quantal endpoints included tumor incidence or incidence of cancer-related endpoints
18 (including frequency of mutations). Continuous endpoint datasets included tumor counts
19 (number of tumors per animal) or cancer-related endpoints of a continuous-variable nature (e.g.,
20 number of sister chromatid exchanges, number of morphologically transformed colonies). Dose-
21 response assessment methods were specific to each type of endpoint (quantal or continuous) and
22 differed depending on whether there were multiple dose groups or a single dose group in the
23 dataset. Methods for multidose and single dose quantal and continuous data are described below.
24
25 5.3. RPF CALCULATION FOR MULTIDOSE DATASETS
26 Dose-response modeling using U.S. EPA's Benchmark Dose Software (Version 2.1.1 or
27 1.3.2) was conducted on multiple-dose data sets to estimate potency for both the target PAHs and
28 benzo[a]pyrene. Modeled estimates consider information about the shape of the dose-response
29 curve and are thus preferred over using a single dose group as the point of departure.
30 Dose-response modeling. For multidose quantal data, the multistage model was used and
31 the degree of the polynomial was assumed to equal the number of dose groups minus 2. The
32 multistage model was selected because it is the preferred model for cancer risk assessment of
33 animal bioassay data, and it provided a consistent model form for all of the datasets. For tumor
34 bioassay data, the multistage-cancer model was selected, while other quantal data were modeled
35 using the multistage model (both have the same model form and yield the same result). For
36 multidose continuous data, the linear model was selected for all datasets, as it is the simplest
37 model form for continuous data. For both quantal and continuous datasets, the goodness-of-fit
38 criteria were used to evaluate model fit. If the model did not provide adequate fit to the data,
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1 high-dose groups were sequentially eliminated in an effort to achieve adequate fit, except when
2 truncating the data would result in the loss of datapoints at response levels in the range of the
3 benzo[a]pyrene response. The focus of the modeling effort is on the low dose and response
4 region, so doses and responses much higher than the benchmark response (BMR) are not as
5 informative and can be eliminated to improve model fit. If dose-group elimination did not
6 improve the model fit, a point-estimate ratio approach was used (see Section 5.4). The BMD
7 modeling outputs for all datasets that were successfully modeled are shown in Appendix D.
8 Selection of BMR: Multidose data for both PAH and benzo[a]pyrene. For tumor
9 incidence data, the BMR used in estimating the point of departure was a 10% increase in tumor
10 incidence over controls (extra risk form). For cancer-related endpoints such as frequency of
11 mutations, endpoint-specific points of departure were selected based on the background/control
12 frequency of the endpoint and the detection limit of the assay. For example, a 1% frequency was
13 selected for a control mutation frequency of 1/10,000 and a detection limit of two- to threefold
14 above background.
15 For multidose continuous data, the BMR used in estimating the point of departure was a
16 change of 1 standard deviation (1 SD) from the control mean. In the event that multiple-dose
17 continuous data were reported in the absence of SD values, a point estimate ratio approach was
18 employed to calculate the slope (see Section 5.4).
19 Selection of BMR: Multidose data for PAH, single dose benzo [a]pyrene. Some studies
20 included only one dose of benzo[a]pyrene as a positive control, while providing multiple-dose
21 data for a selected PAH. In these cases, dose-response modeling was performed for the selected
22 PAH and the BMR used for modeling was the observed response for benzo[a]pyrene adjusted for
23 background response. For tumor incidence data, for example, if the benzo [a] pyrene dose was
24 associated with a 60% extra risk for tumors, the BMR chosen for modeling the data for the PAH
25 was 60% extra risk. RPFs were then calculated using a ratio of the slope factors calculated with
26 equivalent points of departure (e.g., BMD6o). The goal of this approach was to compare PAH
27 potencies at similar response locations on the dose-response curve. There is uncertainty
28 associated with relative potency estimates calculated at the high end of the dose-response curves
29 and using the resultant RPF for low-exposure scenarios, because the relative potency relationship
30 between any two PAHs may be different at the low end, compared with the high end, of the
31 dose-response curves. The uncertainties and limitations associated with the use of high-dose
32 data to estimate relative potency are further discussed in Chapter 7. Data sets for which tumor
33 incidence was >90% in the lowest dose group were not used to calculate potency estimates and
34 RPFs, because the response is near plateau and such data provide insufficient information on the
35 slope of the dose-response relationship.
36 For continuous data, when a point estimate was used to estimate the slope for
37 benzo[a]pyrene and modeling was used to estimate the slope for a given PAH, the BMR used for
38 BMD modeling was a point value set at the response (e.g., mean number of tumors per animal
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1 for tumor multiplicity data) observed in the benzo[a]pyrene group, adjusted for response in the
2 control group. This approach is consistent with the BMR used for quantal data when only a
3 single benzo[a]pyrene dose group was available. Provided that a linear model is fit to continuous
4 data, the choice of a higher BMR would not appreciably change the RPF.
5 Selection of point of departure. The point of departure selected for slope estimation was
6 the BMD estimate rather than the lower confidence limit on the BMD. The BMD, as the central
7 or "best" estimate of the dose associated with the selected BMR, was considered a more stable
8 basis for comparison between the potency of the selected PAH and benzo[a]pyrene, and thus for
9 calculation of relative potency, than the lower confidence limit.
10 Extrapolation from point of departure. The slopes of the dose-response curves in the
11 low-dose regions were calculated by linear extrapolation to the origin from the model-predicted
12 points of departure. Equation 5-2 below shows the calculation of slope from multidose quantal
13 data.
14
15 Slope = [0.1/BMDi<,] (5-2)
16
17 Equation 5-3 below shows the calculation of slope from multidose continuous data.
18
19 Slope = [lSDchange]/[BMD1SD] (5-3)
20
21 5.4. RPF CALCULATION FOR SINGLE DOSE DATASETS
22 A number of studies reported data for only single doses of benzo[a]pyrene and other
23 PAHs; for these studies, a point estimate approach was used to calculate the RPF. A point
24 estimate approach was also used to calculate RPFs for multidose datasets when model fit was not
25 achieved, when variance data were not available for continuous data, or when problems with
26 model implementation were encountered.
27 Selection of point of departure. When only one dose of each compound was used, there
28 was only one choice for the point of departure. However, when multidose data were available,
29 but a point estimate approach was used, the point of departure was chosen as follows. For tumor
30 bioassay data, the lowest dose associated with a statistically significant increase in tumor
31 incidence or multiplicity over control values was selected as the point of departure. Variance
32 was not reported for tumor multiplicity data in any of the dermal studies and for some of the
33 intraperitoneal studies, so the corresponding incidence data were used to determine the dose at
34 which a significant difference from control was observed.
35 The benzo[a]pyrene dose chosen in most instances was the lowest dose associated with a
36 significant increase in tumor count or incidence. For tumor multiplicity data, the PAH dose
37 chosen for the point estimate RPF calculation was the lowest dose associated with a tumor count
38 similar to that observed at the selected benzo[a]pyrene dose (similar to selecting a BMR similar
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1 to the benzo[a]pyrene incidence). In the case of two dermal initiation studies conducted by
2 Cavalieri et al. (1991), however, the tumor count at the lowest dose of dibenzo[a,l]pyrene was
3 much higher than the tumor count at the lowest benzo[a]pyrene dose associated with statistical
4 significance. In order to compare the doses associated with similar tumor counts (i.e., at a
5 similar place on the dose-response curve), a higher benzo[a]pyrene dose was chosen for the RPF
6 calculation. A comparison of the RPFs calculated using this approach with RPFs calculated
7 using the lowest dose associated with a statistically significant increase over controls for both
8 dibenzo[a,l]pyrene and benzo[a]pyrene showed only small differences in the RPF values
9 (9 versus 10 in the 16-week study and 39 versus 42 in the 27-week study). A similar approach
10 was used to calculate the RPF for Bj AC using the intraperitoneal multiplicity data from Mass et
11 al. (1993).
12 For cancer-related endpoint data, statistical analysis was not always available for each
13 dose group. For these data, the lowest dose that produced a near maximal change in the assay of
14 concern was selected as the point of departure. That is, the highest dose in the linear portion of
15 the dose-response curve (identified by visual display of the data) was selected in these cases.
16 Extrapolation from point of departure. As with multiple dose slope estimations, point
17 estimate slope calculations also used the extra risk form. Thus, for single dose quantal data, the
18 slope was calculated by linear extrapolation to the origin after an extra risk adjustment of the
19 observed response (Equation 5-4):
20
21 Slope = [(response at dose - control response) ^ (1 - control response)] + dose (5-4)
22
23 For single dose continuous data, the slope was calculated by linear extrapolation to the
24 origin after adjustment of the observed response in the PAH-treated animals for the control
25 response (Equation 5-5).
26
27 Slope = [(value of variable at dose) - (value of variable)COntroi] ^ dose (5-5)
28
29 5.5. DOSE CONVERSION FOR RPF CALCULATION
30 Some of the studies used to calculate RPFs reported doses or test concentrations on a
31 molar basis (e.g., (imol per mouse, |imol/L), rather than a mass basis (mg or jig). The molar
32 ratio differs from the mass ratio for any PAH with a molecular weight that differs from that of
33 benzo[a]pyrene; thus, for these compounds, an RPF expressed on a mass basis will differ from
34 that expressed on a molar basis. Table 5-1 shows a hypothetical example for fluoranthene, a
35 PAH with a molecular weight that differs from benzo[a]pyrene by 20%. As the table shows, the
36 RPF differs depending on which dose units are used.
37
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Table 5-1. Comparison between molar and mass-based RPF
FA
BaP
Response
0.1
0.1
Dose in mol
5
1
Molecular
weight
(g/mol)
202.26
252.32
Dose in g
1,011
252
Molar RPF
0.20
1
Mass RPF
0.25
1
1
2 In order to ensure that comparisons across endpoints used consistent units, the doses used
3 to calculate RPFs were converted to mass-based units using the molecular weight of the relevant
4 PAH prior to estimating the RPF. While the RPF ratio is nominally unitless, it should be
5 interpreted as the ratio of the dose of PAH to the dose of benzo[a]pyrene. Since RPFs will be
6 used in conjunction with a PAH dose and benzo[a]pyrene cancer potency in mass units (oral
7 slope factors and inhalation unit risks reported in units of [mg/kg-day]"1 and [jig/m3]"1,
8 respectively); it is important to use mass-based RPFs. Alternatively, if a molar RPF ratio were to
9 be used, it would be applied with PAH doses and benzo[a]pyrene cancer potency values
10 estimated on a molar basis; this would require a significant shift in the way PAH risks are
11 calculated compared to other carcinogens. Therefore, the mass-based RPF was selected to be
12 consistent with dose metrics used to calculate cancer risk.
13
14 5.6. SPECIAL CONSIDERATIONS FOR RPF CALCULATION USING TUMOR
15 BIOASSAY DATA
16 Several dermal bioassays reported significant mortality prior to the appearance of the first
17 skin tumor. For these data sets, an assumption was made that the number of animals at risk for
18 tumor development was equal to the total number of animals alive at the time of the appearance
19 of the first tumor. Benign and malignant tumor types within the same target organ were
20 combined for calculation of the RPF. The total incidence of animals with either a benign or
21 malignant lesion was directly reported in each study (i.e., the number of animals with adenoma
22 or carcinoma).
23 Tumor incidence data reported for different target organs within the same group of
24 animals were analyzed separately unless the joint incidence (incidence of either tumor type in
25 each dose group) was reported in the publication. Liver and lung tumors were reported in
26 newborn mice exposed to PAHs by intraperitoneal injection (LaVoie et al., 1994, 1987; Busby et
27 al., 1989, 1984; Weyand and LaVoie, 1988; Wislocki et al., 1986). In most studies, tumor
28 incidence was reported separately for the different target organs and could not be combined as
29 the joint incidence was unknown. A gender difference was observed in the newborn mouse
30 studies, with liver tumors observed in male mice only, and lung tumors reported for both male
31 and female mice. The tumor incidence data were, therefore, evaluated separately for male and
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1 female mice. RPF values were calculated separately for male and female mice and for lung
2 tumor incidence and liver tumor incidence in these studies.
o
3
4 5.7. SPECIAL CONSIDERATIONS FOR RPF CALCULATION USING CANCER-
5 RELATED ENDPOINT DATA
6 The in vitro studies of cancer-related endpoints included measurements of bacterial
7 mutagenicity, mammalian mutagenicity, morphological/malignant cell transformation, DNA
8 adduct formation, DNA damage or repair, and clastogenicity or sister chromatid exchange
9 frequency. Many of the studies describing in vitro cancer-related endpoints provide dose-
10 response data under varying study conditions. For example, bacterial mutagenesis studies used
11 multiple strains, different metabolic activation processes, and/or varying assay systems. In order
12 to limit the number of datasets used for dose-response analysis of in vitro mutagenicity studies,
13 and to provide a consistent basis for comparing RPFs for different PAHs, data associated with
14 the conditions that maximized the benzo[a]pyrene response within a particular study were used
15 for the dose-response assessment of PAHs. It should be noted that in several studies, test
16 conditions that were optimal for benzo[a]pyrene were not necessarily optimal for the selected
17 PAH (see Appendix C for specific studies). The uncertainties and limitations associated with
18 this approach are discussed further in Chapter 8.
19 For time-course studies of DNA adducts, results were reported as either AUC or peak
20 formation of adducts. AUC was considered preferable for dose-response assessment, because
21 this measure considers both adduct formation and repair. Adducts measured in more than one
22 organ were summed to derive a total measure of adduct formation (standardized per unit amount
23 of DNA).
24 The data for bacterial and mammalian cell mutagenicity and malignant cell
25 transformation were sometimes expressed as a mutation or transformation frequency (i.e.,
26 mutants/total cell count or transformed cells/total cells). For multiple-dose studies, these quantal
27 variables were evaluated using the multistage model as described above. Problems were
28 sometimes encountered when using the multistage model for incidence data of this type. In some
29 cases, modifying the initial parameters in the multistage algorithm facilitated convergence. In a
30 select few cases, the quantal linear model was used when the multistage model would not
31 converge. If neither the multistage nor quantal linear models provided adequate fit, a point
32 estimate approach was used. If possible, the point estimates for both benzo[a]pyrene and the
33 target PAH were chosen at a comparable response level (e.g., the doses of benzo[a]pyrene and
34 the target PAH that both gave two mutants in 105 cells). However, in many cases, a comparable
35 response rate was not available. In these instances, the RPF was derived from slopes calculated
36 by linear extrapolation from the peak response.
37 As noted earlier, for studies that included only one dose of benzo[a]pyrene and multiple
38 dose data for a selected PAH, the BMR selected for dose-response modeling for the selected
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1 PAH was the benzo[a]pyrene response with the background or control response subtracted. In
2 some instances, when the benzo[a]pyrene response level greatly exceeded the response at the
3 highest dose of the selected PAH, the software would fail to calculate the BMD at the
4 benzo[a]pyrene response level. In these instances, a point estimate approach using the peak
5 response for the selected PAH was used.
6 The individual study RPFs calculated for each PAH were used in a weight of evidence
7 evaluation to select PAHs for inclusion in the RPF approach (see Chapter 6) and in the derivation
8 of a final RPF for each compound (Chapter 7).
9
10
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6. SELECTION OF PAHs FOR INCLUSION IN RELATIVE POTENCY APPROACH
2
3
4
5
6
7
8
9
10
11
The selection of PAHs to be included in the RPF approach began with an evaluation of
whether the available data were adequate to assess the carcinogen!city of each compound. At
least one RPF value was calculated for each of 51 PAHs. For 16 of these compounds, only a
single RPF value derived from an in vitro cancer-related endpoint (primarily mutagenicity
assays) was available. These PAHs are shown in Table 6-1. Due to the limited data available for
these 16 compounds, no further evaluation of these PAHs was conducted, and they were not
selected for inclusion in the RPF approach.
Table 6-1. PAHs with only one RPF from a single in vitro cancer-related
endpoint study and excluded from RPF approach
PAH
Aceanthrylene
Acenaphthene
Acenaphthylene
Acephenanthrylene
Benzo[a]perylene
Benz [b] anthracene
Benzo[b]perylene
Benzo [c]phenanthrene
Cyclopent[h,i]aceanthrylene
Cyclopent[h,i]acephenanthrylene
Dibenzo [a,f]fluoranthene
Dibenz[aj ] anthracene
Dibenzo [b,e]fluoranthene
Dibenzo [e,l]pyrene
Dibenz[k,mno]acephenanthrylene
Naphtho[2,3-a]pyrene
CASRN
202-03-09
83-32-9
208-96-8
201-06-9
191-85-5
92-24-9
197-70-6
195-19-7
131581-33-4
114959-37-4
203-11-2
224-41-9
2997-45-7
192-51-8
153043-81-3
196-42-9
Abbreviation
ACEA
AN
ANL
APA
BaPery
BbA
BbPery
BcPH
CPhiACEA
CPhiAPA
DBafF
DBajA
DBbeF
DBelP
DBkmnoAPH
N23aP
12
13
14
15
16
17
18
19
20
21
22
The remaining 35 PAHs had RPF values calculated from at least one in vivo dataset or at
least two in vitro cancer-related endpoint datasets. For these compounds, a weight of evidence
approach was used to determine whether the available data (including the calculated RPFs as
well as nonpositive studies that met selection criteria) were adequate to include each compound
in the RPF approach. Using the calculated RPFs in the weight of evidence evaluation allowed
consideration of the magnitude of calculated RPFs in assessing carcinogenicity. When data were
not considered adequate, the PAH was excluded from the RPF approach. When data were
considered adequate for a given PAH, it was selected for inclusion.
A PAH with adequate evidence to suggest no carcinogenicity was selected for inclusion
in the RPF approach and assigned an RPF of zero. While there is little quantitative difference
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1 between selecting a final RPF of zero for a given PAH and excluding that PAH from the RPF
2 approach, this is an important distinction for uncertainty analysis. There is substantial
3 uncertainty in the risk associated with a PAH that is excluded from the RPF approach due to
4 inadequate data; this compound could be of low or high potency. However, for a PAH with an
5 RPF of zero, there is evidence to suggest that this compound is not carcinogenic, and the
6 uncertainty associated with the cancer risk is markedly reduced. For anthracene, phenanthrene,
7 and pyrene, it has been determined that the available data support a practical RPF of zero. The
8 weight of evidence analysis is outlined in Section 6.1 and the results are described in narratives
9 for each of the 35 individual PAHs (Section 6.2). Chapter 7 describes how the RPFs from
10 multiple datasets were used to derive final RPFs for those PAHs selected for inclusion in the
11 approach, and reports the final RPF information for each PAH.
12
13 6.1. METHOD FOR SELECTING PAHs FOR INCLUSION IN RELATIVE POTENCY
14 APPROACH
15 For each of the 35 PAHs, a weight of evidence evaluation was conducted to assess the
16 evidence that each PAH could induce a carcinogenic response. For the purposes of this analysis,
17 PAHs were assumed to be carcinogenic by inferring toxicological similarity to the indicator
18 compound, benzo[a]pyrene. The weight of evidence approach was developed to determine
19 whether the available information for each PAH was adequate for inclusion of the PAH in the
20 RPF approach. Figure 6-1 shows the decision tree that was used to evaluate the data for each
21 PAH and to determine whether it should be included in the RPF approach. The weight of
22 evidence evaluation concluded with one of two possible outcomes:
23
24 (1) The data reviewed are adequate to evaluate carcinogen!city and the PAH should be
25 included in the RPF analysis, or
26
27 (2) The data reviewed are inadequate to assess carcinogenicity and the PAH should be
28 excluded from the RPF analysis.
29
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Has PAH been tested in
tumor bioassay with
BaPa?
Yes
No
Did PAH give positive
result in any tumor
bioassays with BaP?
Yes
No
Do other tumor bioassaysb
and/or cancer-related
endpoint data0 provide
adequate data to assess
carcinogenicity?
1
2
3
4
5
6
7
8
9
10
11
12
13
14
aBioassays with benzo[a]pyrene that met study quality criteria (includes studies with
nonpositive results).
bOther bioassays include those that did not test benzo[a]pyrene and/or those that were not
suitable for RPF derivation (e.g., incidence at lowest dose exceeded 90%).
cCancer-related endpoint data examined in this process included studies of DNA adducts,
clastogenicity or sister chromatid exchange, mutagenicity, morphological transformation,
DNA damage, unscheduled DNA synthesis, etc. that included the selected PAH and
benzo[a]pyrene.
Figure 6-1. Weight of evidence analysis of for selection of PAHs to be
included in the RPF approach.
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1 In vivo tumor bioassays that included benzo[a]pyrene were given the greatest weight in
2 assessing the carcinogenicity of a given PAH; data from other bioassays and cancer-related
3 endpoint studies were used to supplement the weight of evidence when the bioassay data that
4 included benzo[a]pyrene were conflicting or nonpositive. Structural alerts for PAH
5 carcinogenicity or mutagenicity (specifically, at least four aromatic rings, or the presence of a
6 classic bay or fjord region formed entirely by aromatic rings) were noted in the evaluation for
7 each PAH, but were not used explicitly in the weight of evidence evaluation.
8 When there were bioassays including benzo[a]pyrene with positive findings, and none
9 with nonpositive findings for a given PAH, that compound was selected for inclusion in the RPF
10 approach, and no further evaluation of cancer-related endpoint data was conducted. However,
11 the cancer-related endpoint findings for these compounds were noted in the individual PAH
12 narratives (Section 6.2). Among the PAHs included in this analysis, there were none with
13 positive bioassay data and robust nonpositive cancer-related endpoint data. Were this instance to
14 arise, it would require special consideration, as it might imply a different mode of carcinogenic
15 action than the PAHs addressed herein.
16 Bioassays that met selection criteria (see Section 4.3) were included in the weight of
17 evidence analysis, regardless of whether positive or nonpositive results were found. However,
18 the weight of evidence evaluation assumed that a given compound may be active in one system
19 (e.g., newborn mouse) and inactive or weakly active in another (e.g., dermal initiation). Thus,
20 when conflicting results were observed in different test systems, different species, or different
21 genders, the PAH was assumed to be carcinogenic based on the positive findings and was
22 included in the RPF approach.
23 In order to evaluate the results of bioassays with positive and nonpositive results in the
24 same test system, an "RPF detection limit" was conceptualized as a means of approximating the
25 minimum RPF that could be determined with respect to the design of the study. The "RPF
26 detection limit" was defined as the RPF determined by the lowest response that would have been
27 statistically significant for the subject PAH and the actual benzo[a]pyrene response. The lowest
28 statistically significant response was calculated using the incidence of tumors in the control
29 group, number of animals in the group treated with the subject PAH, and Fisher's exact test5
30 (employing a one-sided^-value < 0.05). Appendix F provides an example calculation of an
31 "RPF detection limit." The utility of this concept is in weighing positive and nonpositive
32 bioassay results. If all of the nonpositive studies for a subject PAH had "RPF detection limits" in
33 excess of or in the range of what is observed in the positive studies, then it is plausible that the
34 nonpositive studies may not have been sufficiently sensitive to estimate the RPF appropriate to
35 the subject PAH. In this event, the PAH was considered carcinogenic and was included in the
36 RPF approach.
5This calculation was implemented using trial and error within the Fisher's exact test in the online statistical
calculator, GraphPad6.
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1 If there were no bioassays with benzo[a]pyrene for a given compound, all of the selected
2 bioassays gave nonpositive results, or inconsistent results could not be explained by test system
3 or "RPF detection limit", then the results of other bioassays (those without benzo[a]pyrene, or
4 those rejected from dose-response assessment exclusively because of concerns associated with
5 benzo[a]pyrene) and cancer-related endpoint data were evaluated. The weight of evidence
6 analysis then considered all of the following information: bioassays with benzo[a]pyrene, other
7 bioassays, and cancer-related endpoint data. If these data were determined to be inadequate to
8 assess the carcinogenicity for a given PAH, then that compound was excluded from the RPF
9 approach. If the data were considered adequate to assess the carcinogenicity, the compound was
10 retained and a final RPF was derived. Section 6.2 below describes the weight of evidence
11 evaluation for each of the 35 PAHs. Section 7.1 describes how final RPFs were derived for the
12 27 PAHs selected for inclusion in the RPF approach.
13
14 6.2. WEIGHT OF EVIDENCE EVALUATION FOR 35 INDIVIDUAL PAHs
15 For each PAH, the structure is shown along with a brief reference to any structural alerts
16 for carcinogenicity (specifically, more than three aromatic rings and/or bay or fjord region in
17 alternant PAH). Next, a brief narrative describing the weight of evidence evaluation is given,
18 with a graphical representation of the data that were available for RPF calculation (Figures 6-2 to
19 6-35). The graph for each compound provides a visual representation of the database of studies
20 that included both the subject PAH and benzo[a]pyrene. The solid bars show the values of the
21 RPFs calculated from all studies with positive findings. The x-axis label shows the reference for
22 the pertinent study. The RPFs are color-coded to distinguish among in vivo tumor bioassays
23 based on incidence data, in vivo tumor bioassays based on multiplicity data, in vivo cancer-
24 related endpoint studies, and in vitro cancer-related endpoint studies. Within these categories,
25 the RPFs are ordered (left to right in the graph) from highest to lowest, with positive results
26 shown before nonpositive results.
27 For each nonpositive bioassay, an empty, dotted bar shows what is termed the "RPF
28 detection limit" (see Section 6.1 for description). Missing bars designate cancer-related studies
29 that resulted in nonpositive findings. An RPF detection limit for nonpositive cancer-related
30 studies was not included, because comparisons between nonpositive and positive studies were
31 complicated by the wide variety of study conditions (e.g., test species and strains, metabolic
32 activation sources, assay systems).
33 Each narrative concludes with a statement as to whether the subject PAH was selected for
34 inclusion in the PAH RPF approach. The weight of evidence evaluation for the 35 PAHs with at
35 least one in vivo RPF or at least two in vitro cancer-related endpoint RPFs resulted in the
36 selection of 27 PAHs for inclusion in the RPF approach (see Table 6-2) and the exclusion of
37 8 PAHs from the approach.
38
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Table 6-2. Results of weight of evidence evaluation for 27 PAHs selected for
inclusion in the RPF approach
Adequate data: selected for inclusion in RPF approach
PAH
Benzo[a]pyrene
Anthanthrene
Anthracene
B enz [a] anthracene
Benz[b,c]aceanthrylene,
11H-
Benzo [bjfluoranthene
Benzo[c]fluorene
Benz [e] aceanthry lene
Benzo [g,h,i]pery lene
Benz |j ] aceanthry lene
BenzoO]fluoranthene
Benzo [kjfluoranthene
Benz [1] aceanthry lene
Chrysene
CASRN
50-32-8
191-26-4
120-12-7
56-55-3
202-94-8
205-99-2
205-12-9
199-54-2
191-24-2
202-33-5
205-82-3
207-08-9
211-91-6
218-01-9
Abbreviation
BaP
AA
AC
BaA
BbcAC
BbF
BcFE
BeAC
BghiP
BjAC
BjF
BkF
B1AC
CH
PAH
Cyclopenta[c,d]pyrene
Cyclopenta[d,e,f]chrysene,
4H-
Dibenz [a,c] anthracene
Dibenzo[a,e]fluoranthene
Dibenzo[a,e]pyrene
Dibenz [a,h] anthracene
Dibenzo[a,h]pyrene
Dibenzo[a,i]pyrene
Dibenzo[a,l]pyrene
Fluoranthene
Indeno [ 1 ,2,3 -c,d]pyrene
Naphtho [2, 3 -e]py rene
Phenanthrene
Pyrene
CASRN
27208-37-3
202-98-2
215-58-7
5385-75-1
192-65-4
53-70-3
189-64-0
189-55-9
191-30-0
206-44-0
193-39-5
193-09-9
85-01-8
129-00-0
Abbreviation
CPcdP
CPdefC
DBacA
DBaeF
DBaeP
DBahA
DBahP
DBaiP
DBalP
FA
IP
N23eP
PH
Pyr
Inadequate data
PAH
Acepyrene, 2,3-
Benzo[b]fluorene, 11H-
Benzo[e]pyrene
Benzo [g,h,i]fluoranthene
CASRN
25732-74-5
243-17-4
192-97-2
203-12-3
Abbreviation
ACEP
BbFE
BeP
BghiF
PAH
Coronene
Fluorene
Perylene
Triphenylene
CASRN
191-07-1
86-73-7
198-55-0
217-59-4
Abbreviation
CO
FE
Pery
Tphen
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2,3-Acepyrene (ACEP)
1
3
4 2,3-Acepyrene (CASRN 25732-74-5) is a nonalternant PAH comprised of four aromatic
5 rings and one five-membered ring. 2,3-Acepyrene does not contain a classic bay or fjord region
6 in its structure.
7 Five datasets for 2,3-acepyrene met selection criteria and included benzo[a]pyrene
8 (shown in Figure 6-2). Dermal initiation and complete carcinogenicity bioassays in mice
9 resulted in nonpositive findings (both published by Cavalieri et al., 1981b). RPF detection limits
10 for these studies were 0.09 and 0.02, respectively. The limited cancer-related data are mixed,
11 with one positive dataset for in vivo DNA adduct formation, one positive bacterial mutagenicity
12 dataset (both published by Cavalieri et al., 1981a), and one nonpositive mammalian mutagenicity
13 dataset (Barfknecht et al., 1982). There are no bioassays of 2,3-acepyrene without
14 benzo[a]pyrene. Overall, the database for 2,3-acepyrene is both limited and inconsistent. The
15 database for 2,3-acepyrene does not provide adequate information with which to assess
16 carcinogenicity; this PAH was not selected for inclusion in the RPF approach.
17
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100
10 -
1 -
o
-------
Anthanthrene (AA)
2
3
4 Anthanthrene (CASRN 191-26-4) is an alternant PAH comprised of six fused aromatic
5 rings. Anthanthrene does not have a bay or fjord region in its structure.
6 There are seven datasets for anthanthrene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-3). The database includes three in vivo tumor bioassays, three
8 bacterial mutagenicity datasets, and one in vitro DNA damage dataset. Statistically increased
9 tumor incidences were reported in both a rat lung implantation bioassay (Deutsch-Wenzel et al.,
10 1983) and a dermal complete carcinogenicity bioassay in mice (Cavalieri et al., 1977). No
11 increase over control tumor incidence was reported in a dermal initiation study (Hoffmann and
12 Wynder, 1966), but the RPF detection limit for this study was 0.3. All of the cancer-related
13 endpoint studies gave positive results. Because conflicting bioassay data can be explained by
14 differences in study design (initiation versus complete dermal carcinogenicity), anthanthrene was
15 considered carcinogenic and selected for inclusion in the RPF approach.
16
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100
10 --
1 -•
s
I
0.1 --
0.01 --
0.001
RPF detection limit
fornonpositive
bioassay
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Cavalieriet al., 1977 Deutsch-Wenzeletal., Hoffman and Wynder, Andrews etal., 1978 Hermann,1981 Mersch-Sundermann et Kadenetal., 1979
1983 1966 al., 1992
Reference
Figure 6-3. Anthanthrene (AA) RTFs.
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Anthracene (AC)
2
3
4 Anthracene (CASRN 120-12-7) is an alternant PAH comprised of three fused aromatic
5 rings. Anthracene does not have a bay or fjord region in its structure, and contains less than four
6 aromatic rings.
7 Thirty-seven datasets for anthracene met selection criteria and included benzo[a]pyrene,
8 including 1 dermal initiation tumor bioassay, 3 in vivo clastogenicity or sister chromatid
9 exchange datasets, 10 bacterial mutagenicity datasets, 4 mammalian mutagenicity datasets,
10 6 morphological/malignant cell transformation datasets, and 13 in vitro DNA adduct, DNA
11 damage, or clastogenicity datasets (Figure 6-4). The single dermal initiation bioassay gave a
12 nonpositive result, with an RPF detection limit of 0.2 (LaVoie et al., 1985). Only two datasets
13 gave positive results: an in vitro bacterial mutagenicity assay and an in vitro study of DNA
14 damage. The remaining 35 datasets reported nonpositive findings. To confirm the nonpositive
15 findings in the one tumor bioassay that included benzo[a]pyrene, other bioassays and cancer-
16 related endpoint data for anthracene were considered in the weight of evidence evaluation. In
17 bioassays without benzo[a]pyrene, anthracene did not induce a statistically significant increase in
18 tumor incidence in two dermal initiation studies (LaVoie et al., 1983; Salaman and Roe, 1956)
19 and a lung implantation bioassay (Stanton, 1972). Scribner (1973) reported a weak tumorigenic
20 response in a dermal initiation study in mice (4/28 mice developed papillomas by week 35 after
21 dermal treatment with 10 (imol anthracene in benzene followed by twice weekly treatment with
22 TPA, as compared with 0/30 control mice, p = 0.048).
23 In vitro assays of mutagenicity (both bacterial and mammalian) are nearly all nonpositive
24 for anthracene (13/14 studies). Studies of morphological/malignant cell transformation were all
25 nonpositive. Finally, in numerous in vitro studies of DNA damage or clastogenicity, anthracene
26 has given nonpositive results (12/13). Sakai et al. (1985) reported a mutagenic response in
27 bacteria treated with anthracene, and Rossman et al. (1991) observed evidence of unscheduled
28 DNA synthesis in Escherichia coli treated with anthracene. Overall, the weight of evidence
29 suggests that anthracene is not carcinogenic. In addition, anthracene lacks all three known
30 structural alerts (at least four rings, bay or fjord region) for PAH carcinogen!city and/or
31 mutagenicity. Because the weight of evidence evaluation suggests that the data are adequate to
32 assess the carcinogenicity of anthracene, this compound was selected for inclusion in the RPF
33 approach and assigned an RPF of zero.
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£ 0.1 -
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Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
RPF detection limits
fornonpositive
bioassays
f <** *"
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-------
Benzfajanthracene (BaA)
2
3
4 Benz[a]anthracene (CASRN 56-55-3) is an alternant PAH comprised of four fused
5 aromatic rings. Benz[a]anthracene contains a bay region but no fjord region in its structure.
6 There are 65 datasets for benz[a]anthracene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-5). Included in the database are tumor bioassays (5), in vivo DNA
8 adduct studies (4), in vivo clastogenicity studies (4), an in vivo mutagenicity study (1), bacterial
9 mutagenicity (15), mammalian mutagenicity (14), morphological/malignant cell transformation
10 assays (6), and in vitro studies of DNA damage, adducts, or clastogenicity (16). There are five
11 tumor bioassay datasets of benz[a]anthracene that included benzo[a]pyrene; four gave positive
12 results and one gave a nonpositive result. The positive findings were in different genders tested in
13 a newborn mouse study using intraperitoneal injection (Wislocki et al., 1986); the datasets
14 included both tumor incidence and multiplicity data for both sexes. Positive results were also
15 reported in a dermal initiation study (Slaga et al., 1978). The one nonpositive bioassay (Cavalieri
16 et al., 1977) was a dermal complete carcinogenicity study with an RPF detection limit of 0.2.
17 Benz[a]anthracene was shown to form DNA adducts when administered in vivo in both rats and
18 mice via injection and gavage (Kligerman et al., 2002). Mutagenicity and morphological/
19 malignant cell transformation assays of benz[a]anthracene were predominantly positive, as were
20 studies of other cancer-related endpoints.
21 Given that the differing bioassay results can be attributed to different test systems and
22 study design, benz[a]anthracene was considered carcinogenic and was selected for inclusion in
23 the RPF approach.
24
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1 •-
s
I
0.1 -
0.01 •
RPF detection limit
fornonpositive
bioassay
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
JL
0.001
^?<^^^
B^ K'fr I
S" & e
4V5^ 'c$^«^
* Missing bar indicates nonpositive cancer-related endpoint study
Figure 6-5. Benz[a]anthracene (BaA) RTFs*.
Reference
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HH-Benz[b,c]aceanthrylene (BbcAC)
2
3
4 1 lH-Benz[b,c]aceanthrylene (CASRN 202-94-8) is a nonalternant PAH comprised of
5 four aromatic rings and one five-membered ring. 1 lH-Benz[b,c]aceanthrylene does not contain
6 a classic bay or fjord region in its structure.
7 There was only one dataset for benz[b,c]aceanthrylene that met selection criteria and
8 included benzo[a]pyrene (Figure 6-6). This multidose dermal initiation study resulted in an RPF
9 estimate of 0.05 (Rice et al., 1988). Benz[b,c]aceanthrylene has not been tested in any bioassay
10 without benzo[a]pyrene. There are no cancer-related endpoint data for benz[b,c]aceanthrylene.
11 As the only available bioassay of this PAH was positive, benz[b,c]aceanthrylene was considered
12 carcinogenic and was selected for inclusion in the RPF approach.
13
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Benzo[b]fluoranthene (BbF)
2
3
4 Benzo[b]fluoranthene (CASRN 205-99-2) is a nonalternant PAH comprised of four
5 aromatic rings and one five-membered ring. Benzo[b]fluoranthene contains one classic bay
6 region but no fjord region in its structure.
7 There were 22 datasets of benzo[b]fluoranthene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-7). Included in the database are in vivo tumor bioassay datasets (8), in
9 vivo DNA adduct datasets (7), in vivo clastogenicity datasets (3), mutagenicity and
10 morphological/malignant cell transformation datasets (3), and an in vitro DNA damage dataset
11 (1). Statistically significant increases in tumor incidence and/or multiplicity were reported in
12 male mice tested in two newborn mouse bioassays using intraperitoneal injection (Nesnow et al.,
13 1998b; LaVoie et al., 1987), in dermal initiation (LaVoie et al., 1982) and dermal complete
14 carcinogenicity (Habs et al., 1980) bioassays, and in a rat lung implantation bioassay (Deutsch-
15 Wenzel et al., 1983). The one nonpositive result was in female mice tested in the newborn
16 mouse bioassay; the RPF detection limit was 0.8 (LaVoie et al., 1987). A number of studies
17 showed that benzo[b]fluoranthene forms DNA adducts when administered in vivo to rats or mice
18 via injection or gavage (Kligerman et al., 2002; Nesnow et al., 1998b, 1993b). One mutagenicity
19 assay and two morphological/malignant cell transformation assays of benzo[b]fluoranthene were
20 positive, as were studies of other cancer-related endpoints; there were no nonpositive studies of
21 cancer-related endpoints. Given that the differing bioassay results can be attributed to different
22 genders, benz[a]anthracene was considered carcinogenic and was selected for inclusion in the
23 RPF approach.
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10 -•
1 -•
o
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s
0.1 -•
o.oi --
0.001 -I
RPF detection limit
fornonpositive
bioassay
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
1
4°
S?
Figure 6-7. Benzo[b]fluoranthene (BbF) RTFs.
**• y **•
Reference
?'
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HH-Benzo[b]fluorene (BbFE)
4 1 lH-Benzo[b]fluorene (CASRN 243-17-4) is a nonalternant PAH comprised of three
5 aromatic rings and one five-membered ring. 1 lH-Benzo[b]fluorene does not contain a classic
6 bay or fjord region in its structure.
7 There were three datasets for 1 lH-benzo[b]fluorene that met selection criteria and
8 included benzo[a]pyrene (Figure 6-8): two mutagenicity datasets and an in vitro DNA damage
9 dataset. There are no bioassays of 1 lH-benzo[b]fluorene that included benzo[a]pyrene, so
10 bioassays without benzo[a]pyrene and cancer-related endpoint data were considered. LaVoie et
11 al. (1981) conducted a study of skin tumor initiation in mice treated with 1 mg 1 lH-benzo[b]-
12 fluorene followed by 20 weeks of treatment with TPA. The incidence of tumor-bearing animals
13 (4/20) was not significantly increased over controls (1/20) (LaVoie et al., 1981). The limited
14 cancer-related endpoint data were mixed, with one positive mutagenicity study (Kaden et al.,
15 1979), one nonpositive mutagenicity study (Hermann, 1981), and one positive in vitro study of
16 DNA damage (Mersch-Sundermann et al., 1992). Overall, the database for 1 lH-benzo[b]-
17 fluorene is both limited and inconsistent. Because the database for 1 lH-benzo[b]fluorene does
18 not provide adequate information with which to assess carcinogenicity, this PAH was not
19 selected for inclusion in the RPF approach.
20
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0.1 -
0.01 -
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Kadenetal., 1979
* Missingbar indicatesnonpositive cancer-related endpoint study
Figure 6-8. llH-Benzo[b]fluorene (BbFE) RTFs*.
Mersch-Sundermann etal., 1992
Reference
Hermann, 1981
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Benzo[c]fluorene (BcFE).
2
3
4
5
6
7
8
9
10
11
12
13
14
Benzo[c]fluorene (CASRN 205-12-9) is a nonalternant PAH comprised of three aromatic
rings and one five-membered ring. Benzo[c]fluorene does not contain a classic bay or fjord
region in its structure.
There were six datasets for benzo[c]fluorene that met selection criteria and included
benzo[a]pyrene (Figure 6-9); all gave positive results. The database includes oral and
intraperitoneal in vivo tumor bioassays (each reporting both incidence and multiplicity) and in
vivo DNA adduct data. Significantly increased lung tumor incidence and tumor multiplicity
were reported after both oral and intraperitoneal exposure (Weyand et al., 2004). As the
available bioassays that included benzo[a]pyrene were positive, benzo[c]fluorene was considered
carcinogenic and was selected for inclusion in the RPF approach.
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10 -•
1 -•
o
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j3
13
0.1 -•
0.01 -•
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-re la ted endpoint
In vitro cancer-related endpoint
Weyandetal.,2004 Weyandetal., 2004 Weyandetal., 2004 Weyandetal., 2004 Weyandetal., 2004 Weyandetal., 2004
Reference
Figure 6-9. Benzo[c]fluorene (BcFE) RTFs.
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Benzfejaceanthrylene (BeAC).
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Benz[e]aceanthrylene (CASRN 199-54-2) is a nonalternant PAH comprised of four
aromatic rings and one five-membered ring. Benzfejaceanthrylene contains a classic bay region
but no fjord region in its structure.
There were six datasets for benz[e]aceanthrylene that met selection criteria and included
benzo[a]pyrene (Figure 6-10); all gave positive results. The database includes an in vivo tumor
bioassay in two sexes (each reporting both incidence and multiplicity), a mammalian
mutagenicity study, and a morphological/malignant cell transformation study. Significantly
increased tumor incidence and tumor multiplicity were reported for both male and female mice
in a dermal initiation bioassay in mice (Nesnow et al., 1984). As the available bioassay that
included benzo[a]pyrene was positive, benz[e]aceanthrylene was considered carcinogenic and
was selected for inclusion in the RPF approach.
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10 -•
1 -•
o
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s
0.1 -•
0.01 -•
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Nesnowet al., 1984 Nesnowet al., 1984 Nesnowet al., 1984 Nesnowet al., 1984 Nesnowet al., 1984b Mohapatraetal., 1987
Reference
Figure 6-10. Benz[e]aceanthrylene (BeAC) RTFs.
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Benzofejpyrene (BeP)
4 Benzo[e]pyrene (192-97-2) is an alternant PAH comprised of five fused aromatic rings.
5 Benzo[e]pyrene contains two bay regions but no fjord region in its structure.
6 Thirty-seven datasets for benzo[e]pyrene met selection criteria and included
7 benzo[a]pyrene: 2 tumor bioassays, 1 in vivo clastogenicity dataset, 12 bacterial mutagenicity
8 datasets, 4 mammalian mutagenicity datasets, 7 morphological/malignant cell transformation
9 datasets, and 11 in vitro DNA damage or clastogenicity datasets (Figure 6-11). No increase in
10 tumor incidence was observed when benzo[e]pyrene was tested alone as part of a dermal
11 cocarcinogenicity bioassay (Van Duuren and Goldschmidt, 1976). When tested in a lung
12 implantation bioassay in rats, benzo[e]pyrene exposure did not result in a significant increase in
13 tumor incidence (Deutsch-Wenzel et al., 1983). The RPF detection limits of these studies were
14 approximately 0.01 and 0.1. To confirm the nonpositive findings in the available tumor
15 bioassays that included benzo[a]pyrene, other bioassays and cancer-related endpoint data were
16 considered. In bioassays without benzo[a]pyrene, benzo[e]pyrene gave nonpositive results in a
17 dermal initiation bioassay (1 mg/mouse; Van Duuren et al., 1968) and a newborn mouse bioassay
18 (0.7 (imol; Chang et al., 1981). A significant increase in tumor incidence was reported in a
19 single-concentration dermal initiation study in mice; 11/13 surviving mice (20 were treated) had
20 papillomas by week 35 after dermal treatment with 10 |imol benzo[e]pyrene in benzene
21 (p < 0.0001), followed by twice weekly treatment with TPA; no control mice had papillomas
22 (Scribner, 1973).
23 In vitro assays of mutagenicity (both bacterial and mammalian) and morphological/
24 malignant cell transformation give inconsistent results for benzo[e]pyrene; 11/23 studies were
25 positive and the rest were nonpositive. Positive studies include a mix of bacterial mutagenicity
26 and morphological/malignant cell transformation assays; four mammalian mutagenicity assays
27 were nonpositive. One study of in vivo clastogenicity and two studies of in vitro DNA damage
28 were positive, while nine studies of in vitro DNA damage or clastogenicity were nonpositive.
29 While the database for benzo[e]pyrene is quite large, the results are inconsistent; as a
30 result, no conclusion can be drawn as to carcinogenicity. This PAH was not selected for
31 inclusion in the RPF approach.
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0.
1UU
10 -
1 -
0.1 -
0.01 -
nm -
| | Positive bioassay (incidence)
| | Positive bioassay (multiplicity)
I In vivo cancer-related endpoint
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limits for
nonpositive
bioassays
1\
r~\
i \
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i
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-
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-
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-------
Benzo[g,h,i]fluoranthene (BghiF)
2
o
5
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Benzo[g,h,i]fluoranthene (CASRN 203-12-3) is a nonalternant PAH comprised of four
aromatic rings and one five-membered ring. Benzo[g,h,i]fluoranthene does not contain a classic
bay or fjord region in its structure.
There were six datasets for benzo[g,h,i]fluoranthene that met selection criteria and
included benzo[a]pyrene (Figure 6-12). A dermal initiation bioassay in mice (Van Duuren et al.,
1966) did not result in a statistically significant increase in tumor incidence; the RPF detection
limit was 0.06. There were no other bioassays that met selection criteria. There were three
positive bacterial mutagenicity studies (Chang et al., 2002; Lafleur et al., 1993; Carver et al.,
1986), one positive study of in vitro DNA damage (Mersch-Sundermann et al., 1992), and a
mammalian mutagenicity study with nonpositive results (Lafleur et al., 1993). The RPF values
for the positive cancer-related endpoint datasets ranged from 0.6 to 1. Overall, the database for
benzo[g,h,i]fluroanthene is both limited and inconsistent. Because the database for
benzo[g,h,i]fluoranthene does not provide adequate information with which to assess
carcinogenicity, this PAH was not selected for inclusion in the RPF approach.
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I
0.1 -
0.01 -
0.001
RPF detection limits
fornonpositive
bioassays
Positive bio assay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Van Duurenetal., 1966 LaFleuretal., 1993 Carver etal., 1986 Chang etal., 2002
Reference
* Missing bar indicates nonpositive cancer-related endpoint study
Figure 6-12. Benzo[g,h,i]fluoranthene (BghiF) RTFs*.
Mersch-Sundermannetal., LaFleuretal., 1993
1992
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Benzo[g,h,i]perylene (BghiP)
2
3
4 Benzo[g,h,i]perylene (CASRN 191-24-2) is an alternant PAH comprised of six fused
5 aromatic rings. Benzo[g,h,i]perylene contains a bay region but no fjord region in its structure.
6 There were 10 datasets for benzo[g,h,i]perylene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-13). The database includes three in vivo tumor bioassays, four
8 bacterial mutagenicity datasets, an in vitro DNA damage dataset, and two in vitro DNA adduct
9 datasets. Of the three bioassays, positive findings were only reported in one: a rat lung
10 implantation bioassay (Deutsch-Wenzel et al., 1983) that resulted in an RPF estimate of 0.009.
11 In a dermal initiation bioassay (Hoffmann and Wynder, 1966) and a dermal cocarcinogenicity
12 bioassay (Van Duuren and Goldschmidt, 1976), there was no statistically significant increase in
13 tumor incidence, but these studies had relatively insensitive RPF detection limits (around 0.1)
14 compared with the positive study. There were four positive mutagenicity studies; all were
15 conducted in bacterial systems. Studies of in vitro DNA adducts and DNA damage were
16 positive. Because the inconsistent bioassay results can be attributed to different test systems
17 (different species and route), benzo[g,h,i]perylene was considered carcinogenic and was selected
18 for inclusion in the RPF approach.
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0.01 --
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
RPF detection limits for
nonpositive bioassays
Deutsch-Wenzel Hoffmannand VanDuurenand Andrews etal., Sakaietal., 1985 Gibson etal., Kadenetal.,
etal.,1983 Wynder, 1966 Goldschmidt, 1978 1978 1979
1976
Mersch- Segerbackand Cherngetal.
Sundermannet Vodicka, 1993 2001
al.,1992
* Missing bar indicates nonpositive cancer-related endpoint study
Figure 6-13. Benzo[g,h,i]perylene (BghiP) RTFs*.
Reference
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Benz[j]aceanthrylene (BjAC)
2
3
4 Benz[j]aceanthrylene (CASRN 202-33-5) is a nonalternant PAH comprised of four
5 aromatic rings and one five-membered ring. Benz[j]aceanthrylene contains a classic bay region
6 but no fjord region in its structure.
7 There were 12 datasets for benz[j]aceanthrylene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-14); all of the studies gave positive results. The database includes one
9 in vivo tumor bioassay dataset, one in vivo DNA adduct dataset, four mutagenicity or
10 morphological/malignant cell transformation datasets, and six in vitro DNA damage or DNA
11 adduct datasets. In a bioassay of benz[j]aceanthrylene that used intraperitoneal injection in an
12 A/J mouse system (Mass et al., 1993), all mice treated with benz[j]aceanthrylene developed
13 tumors (incidence of 100% at doses of 20-100 mg/kg; incidence for benzo[a]pyrene was 63-
14 100% across the same dose range), precluding the derivation of an RPF using incidence data.
15 However, tumor multiplicity (average number of tumors per animal) data were available for
16 dose-response modeling and resulted in an RPF estimate of 60. Benz[j]aceanthrylene treatment
17 resulted in a pronounced increase in the average number of tumors per animal (59.45 tumors per
18 animal at 20 mg/kg), much higher than benzo[a]pyrene treatment (5.05 tumors per animal at
19 100 mg/kg), indicating that this compound is very potent in this test system. In a dermal
20 initiation bioassay that did not include benzo[a]pyrene, benz[j]aceanthrylene induced papillomas
21 in 90% of mice treated with an initiating dose of 40 jig (compared with 5% incidence in
22 controls). As the available bioassay that included benzo[a]pyrene was positive and suggested
23 that this compound is very potent, benz[j]aceanthrylene was considered carcinogenic and was
24 selected for inclusion in the RPF approach.
25
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-
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^ Positive bioassay (incidence)
I | Positive bioassay (multiplicity)
I | In vivo cancer-related endpoint
| | In vitro cancer-related endpoint
Mass etal., Mass etal., Johnsenetal., Johnsenetal., Johnsenetal., Sangaiah et Nesnowetal., Mohapatraet Johnsenetal., Johnsenetal., Johnsenetal., Johnsenetal.
1993 1993 1997 1997 1997 al., 1983 1984 al., 1987 1998 1998 1998 1998
Reference
Figure 6-14. Benz[j]aceanthrylene (BjAC) RTFs.
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Benzofjjfluoranthene (BjF)
2
3
4 Benzo[j]fluoranthene (CASRN 205-82-3) is a nonalternant PAH comprised of four
5 aromatic rings and one five-membered ring. Benzo[j]fluoranthene does not contain a classic bay
6 or fjord region in its structure.
7 There were eight datasets for benzo[j]fluoranthene that met selection criteria and
8 included benzo[a]pyrene (Figure 6-15): seven in vivo tumor bioassay datasets and one in vitro
9 study of DNA damage. Of the seven bioassay datasets, significant increases in tumor incidence
10 or count were observed in all but one. Significant increases in tumor incidence were reported in
11 both male and female mice tested in a newborn mouse bioassay using intraperitoneal injection of
12 single doses (LaVoie et al., 1987), a mouse dermal initiation study (LaVoie et al., 1982), and a
13 rat lung implantation bioassay (Deutsch-Wenzel et al., 1983). Significant increases in tumor
14 multiplicity were reported in two mouse dermal initiation studies (Weyand et al., 1992; LaVoie
15 et al., 1982). The one nonpositive bioassay was a mouse dermal complete carcinogenicity
16 bioassay with an RPF detection limit of 0.1 (Habs et al., 1980). The in vitro study of DNA
17 damage gave positive results (Mersch-Sundermann et al., 1992). Because the inconsistent
18 bioassay results can be attributed to different test systems or study design, benzo[j]fluroanthene
19 was considered carcinogenic and was selected for inclusion in the RPF approach.
20
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PH
rt
!*.
o
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0.1 --
0.01 --
0.001
B
Positive bio assay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
n
RPF detection
limit for
nonpositive
bioassay
JL
-H
LaVoieet al., 1987 LaVoieet al., 1987 LaVoieet al., 1982 LaVoieet al., 1982 Deutsch-Wenzel et Weyandet al., 1992 Habsetal., 1980 Mersch-
al., 1983 Sundermannetal..
1992
Reference
Figure 6-15. Benzo[j]fluoranthene (BjF) RTFs.
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Benzo[k]fluoranthene (BkF)
1
3
4 Benzo[k]fluoranthene (CASRN 207-08-9) is a nonalternant PAH comprised of four
5 aromatic rings and one five-membered ring. Benzo[j]fluoranthene does not contain a classic bay
6 or fjord region in its structure.
7 There were five datasets for benzo[k]fluoranthene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-16). The database includes four in vivo tumor bioassay datasets and
9 one morphological/malignant cell transformation dataset. Statistically significant increases in
10 tumor incidence and tumor count were reported in a mouse dermal initiation study (LaVoie et al.,
11 1982) and increased tumor incidence was reported in a rat lung implantation bioassay (Deutsch-
12 Wenzel et al., 1983). No significant increase in tumor incidence was observed in a dermal
13 complete carcinogenicity study with an RPF detection limit of 0.1 (Habs et al., 1980). The
14 morphological/malignant cell transformation study (Emura et al., 1980) was nonpositive.
15 Because the inconsistent bioassay results can be attributed to different test systems or study
16 design (dermal initiation versus dermal complete carcinogenicity), benzo[k]fluroanthene was
17 considered carcinogenic and was selected for inclusion in the RPF approach.
18
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0.01 --
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
RPF detection limits for
nonpositive bioassays
Deutsch-Wenzel et al..
1983
LaVoieetal., 1982
* Missingbar indicatesnonpositive cancer-related endpoint study
LaVoieetal., 1982
Reference
HabsetaL 1980
EmuraetaL 1980
Figure 6-16. Benzo[k]fluoranthene (BkF) RTFs*.
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Benzfljaceanthrylene (BIAC)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Benzfljaceanthrylene (CASRN 211-91-6) is a nonalternant PAH comprised of four
aromatic rings and one five-membered ring. Benz[l]aceanthrylene does not contain a classic bay
or fjord region in its structure.
There were 16 datasets for benz[l]aceanthrylene that met selection criteria and included
benzo[a]pyrene (Figure 6-17); all of the studies gave positive results. The database includes four
in vivo tumor bioassay datasets, five mutagenicity or morphological/malignant cell
transformation datasets, one in vivo clastogenicity dataset, and six in vitro DNA adduct or DNA
damage datasets. Significant increases in tumor count and multiplicity were reported in both
male and female mice in a dermal initiation bioassay (Nesnow et al., 1984). All of the cancer-
related endpoint studies were positive as well. Relative potency estimates for most of the
available datasets were >1.0, suggesting equivalent or greater potency than benzo[a]pyrene. As
the available bioassays that included benzo[a]pyrene were positive, benz[l]aceanthrylene was
considered carcinogenic and was selected for inclusion in the RPF approach.
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^T ^ ^ ^ ^ ^ ^ ^ ^ ^
^ ^ ^ ** ^ & f >n ^
Reference
Figure 6-17. Benz[l]aceanthrylene (B1AC) RTFs.
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Chrysene (CH)
1
3
4 Chrysene (CASRN 218-01-9) is an alternant PAH comprised of four fused aromatic
5 rings. Chrysene contains two bay regions but no fjord region in its structure.
6 There were 40 datasets for chrysene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-18). Included in the database are 13 in vivo tumor bioassay datasets,
8 4 in vivo DNA adduct datasets, 3 in vivo clastogenicity datasets, 11 mutagenicity datasets,
9 3 morphological/malignant cell transformation datasets, and 6 in vitro studies of DNA damage,
10 adducts, or clastogenicity. Among the bioassays that included benzo[a]pyrene, 11 reported
11 significant increases in tumor incidence or tumor multiplicity, and 3 did not. Significant
12 increases in tumor incidence and/or multiplicity were reported in three dermal initiation studies
13 in mice (Rice et al., 1988; Slaga et al., 1980; Hecht et al., 1974), a newborn mouse study in
14 males (Wislocki et al., 1986), and a rat lung implantation bioassay (Wenzel-Hartung et al.,
15 1990). Female mice tested in the newborn mouse assay published by Wislocki et al. (1986) did
16 not have a significant increase in tumor incidence, resulting in one of the three nonpositive
17 studies. The other two nonpositive findings were in males and females tested in another
18 newborn mouse bioassay (Busby et al., 1989). The bioassays with nonpositive findings had RPF
19 detection limits between 0.06 and 0.2. Conflicting results in male mice were reported in the two
20 newborn mouse bioassays (Busby et al., 1989; Wislocki et al., 1986). The major difference
21 between the two studies is the duration of follow-up; Busby et al. (1989) sacrificed the mice at
22 26 weeks, while Wislocki et al. (1986) followed the mice for a full year. LaVoie et al. (1994)
23 observed that liver tumor induction in the newborn mouse bioassay is not fully realized until the
24 mice have reached 1 year of age, and the positive findings by Wislocki et al. (1986) indeed
25 reflect liver tumors in the male mice. Chrysene was shown to form DNA adducts when
26 administered in vivo in both rats and mice via injection and gavage (Kligerman et al., 2002).
27 Bacterial and mammalian mutagenicity and morphological/malignant cell transformation assays
28 of chrysene were all positive, as were studies of clastogenicity tested in vivo. In contrast, results
29 from in vitro studies of DNA adducts, DNA damage, and clastogenicity were not consistent.
30 Because the inconsistent bioassay results can be attributed to different study designs
31 (gender, follow-up time), chrysene was considered carcinogenic and was selected for inclusion in
32 the RPF approach.
33
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nonpositive bioassays
- - I^M 11 I M ^ i I M F 11 I M M^I! -i • '\' -\> M I I M M I I M M I I M M I M I M I M I I M M I I M M I I M M I I M M—i—i—i—i—i—i—i—h
>^^X^
^^^^^^^^^^^^^^^%^*'
A& ^ (^ NN ^^^ 6,^^ AJ^ ^ GT^
^ yf ^ 4> ^° ^°
* Mis sing bar indicates nonpositive cancer-related endpoint study
Figure 6-18. Chrysene (CH) RTFs*.
Reference
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Coronene (CO)
2
3
4 Coronene (CASRN 191-07-1) is an alternant PAH comprised of seven fused aromatic
5 rings. Coronene contains no bay or fjord regions in its structure.
6 There were six datasets for coronene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-19). A dermal complete carcinogenicity bioassay in mice did not
8 result in a statistically significant increase in tumor incidence (Habs et al., 1980); the RPF
9 detection limit was 0.06. To confirm the nonpositive findings in the one tumor bioassay that
10 included benzo[a]pyrene, other bioassays and cancer-related endpoint data were considered.
11 There was one bioassay of coronene that did not include benzo[a]pyrene. Van Duuren et al.
12 (1968) conducted a dermal initiation bioassay of coronene using groups of 20 mice (0.5 mg
13 coronene in 0.5 mL benzene, followed by croton resin treatment until death). Although the
14 authors characterized coronene as a weak tumor initiator, the incidence of tumors was not
15 significantly increased over concurrent controls. The limited cancer-related endpoint data were
16 mixed, with three positive bacterial mutagenicity studies (with RPFs ranging from 0.01 to 0.5),
17 one nonpositive bacterial mutagenicity study, and a nonpositive in vitro DNA damage study.
18 Overall, the database for coronene is both limited and inconsistent. Because the database
19 for coronene does not provide adequate information with which to assess carcinogenicity, this
20 PAH was not selected for inclusion in the RPF approach.
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In vitro cancer-related endpoint
RPF detection limits for
nonpositive bioassays
J
-+-
-+-
-+-
-+-
•+•
Habsetal., 1980 Hermann,1981 Sakaietal., 1985 Florin etal., 1980 Kadenetal., 1979 Mersch-Sundermannet
al., 1992
* Missingbarindicatesnonpositive cancer-related endpoint study
Reference
Figure 6-19. Coronene (CO) RTFs*.
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Cyclopenta[c,d]pyrene (CPcdP)
\
2
3
4 Cyclopenta[c,d]pyrene (CASRN 27208-37-3) is a nonalternant PAH comprised of four
5 aromatic rings and one five-membered ring. Cyclopenta[c,d]pyrene does not contain a classic
6 bay or fjord region in its structure.
7 There were 25 datasets for cyclopenta[c,d]pyrene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-20). The database includes 11 in vivo tumor bioassay datasets, 2 in
9 vivo DNA adduct datasets, 11 studies of mutagenicity or morphological/malignant cell
10 transformation, and a single study of in vitro clastogenicity. Nine of the 11 tumor bioassay
11 datasets and all of the cancer-related endpoint studies gave positive results. Statistically
12 significant increases in tumor incidence and/or multiplicity were reported in two dermal
13 complete carcinogenicity bioassay (Cavalieri et al., 1983, 1981b), two dermal initiation
14 bioassays (Raveh et al., 1982; Cavalieri et al., 1981b), and an intraperitoneal study using adult
15 A/J mice (Nesnow et al., 1998b). Bioassays in which no significant increase in tumorigenicity
16 was observed included a dermal initiation (Wood et al., 1980) and complete carcinogenicity
17 study (Habs et al., 1980); these studies had RPF detection limits of 0.1 and 0.03, respectively.
18 After obtaining nonpositive results for low initiating doses of cyclopenta[c,d]pyrene, Wood et al.
19 (1980) repeated their experiment with higher doses and observed statistically significant
20 increases in tumor incidence. In the latter experiment, benzo[a]pyrene was not included, so an
21 RPF could not be calculated from these data. The study design of the nonpositive complete
22 carcinogenicity bioassay was quite similar to that of the two positive studies of this type, with the
23 exception of the mouse strain used; Habs et al. (1980) used NMRI mice, while Cavalieri et al.
24 (1983, 198Ib) used Swiss mice. Although the differing results in dermal complete
25 carcinogenicity studies may be explained by slight differences in strain susceptibility, these two
26 strains are of common origin, which argues against this explanation.
27 The available cancer-related endpoint data indicate that cyclopenta[c,d]pyrene is
28 mutagenic and capable of morphological/malignant cell transformation in vitro; a single study of
29 in vitro clastogenicity was also positive. Overall, the data supporting a finding of
30 carcinogenicity for cyclopenta[c,d]pyrene are very consistent, and this compound was selected
31 for inclusion in the RPF approach.
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fornonpositive
bioassays
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
I ' ' I ' ' I ' ' I ' ' I ' ' I '—' I ' ' I ' ' I ' ' I ' ' I
c?
0° _**
Reference
Figure 6-20. Cyclopenta[c,d]pyrene (CPcdP) RTFs.
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4H-Cyclopenta[d, e,fjchrysene (CPdefC)
1
3
4 4H-Cyclopenta[d,e,f]chrysene (CASRN 202-98-2) is a nonalternant PAH comprised of
5 four aromatic rings and one five-membered ring. 4H-Cyclopenta[d,e,f]chrysene contains a
6 classic bay region but no fjord region in its structure.
7 There were two datasets for 4H-cyclopenta[d,e,f]chrysene that met selection criteria and
8 included benzo[a]pyrene (Figure 6-21); both were multidose dermal initiation datasets (Rice et
9 al., 1988, 1985). Rice et al. (1988) reported a statistically significant increase in tumor incidence
10 in a multidose dermal initiation study. In the second study, the incidence of tumors after
11 treatment with cyclopenta[d,e,f]chrysene exceeded 90%, precluding RPF derivation from
12 incidence data, but tumor multiplicity data were available for RPF calculation (Rice et al., 1985).
13 Cyclopenta[d,e,f]chrysene has not been tested in a bioassay without benzo[a]pyrene; however,
14 sterically hindered diol epoxides of this compound have given positive results in a newborn
15 mouse assay (Amin et al., 1995). Because the bioassay of cyclopenta[d,e,f]chrysene was
16 positive, this PAH was considered carcinogenic and was selected for inclusion in the RPF
17 approach.
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o.i --
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0.001
RiceetaL 1988
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-relatedendpoint
In vitro cancer-related endpoint
RiceetaL, 1985
Reference
Figure 6-21. Cyclopenta[d,e,f]chrysene (CPdefC) RTFs.
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Dibenz[a,c]anthracene (DBacA)
2
3
4 Dibenz[a,c]anthracene (CASRN 215-58-7) is an alternant PAH comprised of five fused
5 aromatic rings. Dibenz[a,c]anthracene contains three bay regions but no fiord region in its
6 structure.
7 There were 15 datasets for dibenz[a,c]anthracene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-22). The database includes a single in vivo study of DNA adducts,
9 nine mutagenicity or morphological/malignant cell transformation studies, and five studies of in
10 vitro DNA damage or adducts. One morphological/malignant cell transformation assay gave
11 nonpositive results, while the remaining studies were positive. In the absence of positive
12 bioassays with benzo[a]pyrene, other bioassays and cancer-related data were considered to
13 evaluate the carcinogenicity of dibenz[a,c]anthracene.
14 Conflicting results were reported in three dermal initiation bioassays of
15 dibenz[a,c]anthracene in which benzo[a]pyrene was not included. Van Duuren et al. (1970)
16 observed a tumor incidence of 95% (19/20, compared to 1/20 controls) when mice were treated
17 with an initiating dose of 1 mg dibenz [a, c] anthracene in benzene followed by thrice weekly
18 treatment with phorbol myristate acetate. In contrast, there was no significant increase in tumor
19 formation when the same initiating dose was followed by thrice weekly application of croton
20 resin (Van Duuren et al., 1968); however, the latency to first tumor was substantially reduced
21 (65 versus 150 days in controls). Latency was also substantially reduced in the study by Van
22 Duuren et al. (1970), in which the first tumor appeared after 74 days, compared with 338 days in
23 controls.
24 Cancer-related endpoint data for dibenz[a,c]anthracene are predominantly positive
25 (8/9 mutagenicity or morphological/malignant cell transformation studies and 5/5 studies of in
26 vitro DNA adducts or DNA damage). Although the conflicting bioassay data are not easily
27 explained, the high incidence of tumors (19/20) in the study by Van Duuren et al. (1970) and the
28 reduced latency to tumor formation in both studies, coupled with predominantly positive cancer-
29 related endpoint data, suggest that dibenz [a, c] anthracene is carcinogenic. Contributing to this
30 conclusion is the observation that dibenz[a,c]anthracene is an alternant PAH with known
31 structural alerts for carcinogenicity (more than three rings, and three bay regions). Thus,
32 dibenz [a, c]anthracene was selected for inclusion in the RPF approach.
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Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-relatedendpoint
In vitro cancer-related endpoint
/ / /
/
/
jf Jr ^
* Missing bar in dicatesnonpositive cancer-relatedendpoint study
Reference
Figure 6-22. Dibenz[a,c]anthracene (DBacA) RTFs*.
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Dibenzo[a,e]fluoranthene (DBaeF)
2
3
4 Dibenzo[a,e]fluoranthene (CASRN 5385-75-1) is a nonalternant PAH comprised of five
5 aromatic rings and one five-membered ring. Dibenzo[a,e]fluoranthene contains a classic bay
6 region but no fiord region in its structure.
7 There were three datasets for dibenzo[a,e]fluoranthene that met selection criteria and
8 included benzo[a]pyrene (Figure 6-23); all gave positive results. The database includes two in
9 vivo tumor bioassays and one mammalian mutagenicity study. Statistically significant increases
10 in tumor incidence were reported in dermal initiation and complete carcinogenicity bioassays in
11 mice (both reported by Hoffmann and Wynder, 1966). As the available bioassays for
12 dibenzo[a,e]fluoranthene were positive, this compound was considered carcinogenic and was
13 selected for inclusion in the RPF approach.
14
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Hoffmann and Wynder 1966
Hoffmann and Wynder 1966
Reference
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Durantetal., 1999
Figure 6-23. Dibenzo[a,e]fluoranthene (DBaeF) RTFs.
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Dibenzo[a,e]pyrene (DBaeP)
2
3
4 Dibenzo[a,e]pyrene (CASRN 192-65-4) is an alternant PAH comprised of six fused
5 aromatic rings. Dibenzo[a,e]pyrene contains three bay regions but no fjord region in its
6 structure.
7 There were three datasets for dibenzo[a,e]pyrene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-24). The database includes two in vivo tumor bioassay datasets and
9 one in vitro bacterial mutagenicity dataset, all of which gave positive results. Statistically
10 significant increases in tumor incidence were reported in dermal initiation and complete
11 carcinogeni city bioassays in mice (Hoffmann and Wynder, 1966). The complete carcinogenicity
12 bioassay was confounded by significant toxicity-related mortality unrelated to tumors (Hoffmann
13 and Wynder, 1966). The one bacterial mutagenicity study reported positive results. Because the
14 available bioassays with benzo[a]pyrene were both positive, dibenzo[a,e]pyrene was considered
15 carcinogenic and was selected for inclusion in the RPF approach.
16
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Hoffmann and Wynder 1966
Hoffmann and Wynder 1966
Reference
Figure 6-24. Dibenzo[a,e]pyrene (DBaeP) RTFs.
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-re la ted endpoint
Teranishiet al., 1975
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Dibenz[a,h]anthracene (DBahA)
2
3
4 Dibenz[a,h]anthracene (CASRN 53-70-3) is an alternant PAH comprised of five fused
5 aromatic rings. Dibenz [a, h] anthracene contains two bay regions but no fiord region in its
6 structure.
7 There were 31 datasets for dibenz[a,h]anthracene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-25). Included in the database are in vivo tumor bioassay datasets (5),
9 in vivo DNA adduct datasets (2), an in vivo clastogenicity dataset, mutagenicity datasets (10),
10 morphological/malignant cell transformation datasets (6), and in vitro DNA damage, adducts, or
11 clastogenicity datasets (7). There were three tumor bioassays for dibenz[a,h]anthracene that
12 included benzo[a]pyrene, and all resulted in statistically significant increases in tumor incidence
13 and/or multiplicity. The bioassays were in three different test systems: a rat lung implantation
14 study (Wenzel-Hartung et al., 1990), a mouse dermal initiation study reporting both incidence
15 and multiplicity (Slaga et al., 1980), and an intraperitoneal study in A/J mice (Nesnow et al.,
16 1998b). Dibenz[a,h]anthracene was shown to form DNA adducts when administered in vivo to
17 mice via intraperitoneal injection (Nesnow et al., 1998b) and dermal application (Phillips et al.,
18 1979). Mutagenicity and morphological/malignant cell transformation assays of
19 dibenz[a,h]anthracene were predominantly positive (13/16), as were studies of other cancer-
20 related endpoints. Because the available bioassays with benzo[a]pyrene were positive,
21 dibenz [a,h]anthracene was considered carcinogenic and was selected for inclusion in the RPF
22 approach.
23
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n
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Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
0 001 I™!1 M™!™!1 M1 M1 M1 M11!1 M1 M1 M1 M1 M1 M11!1 M1 M1 l|l l|l l|l l|ll|l l|l l|l M I 1 I I
" *"*" *" *" *" *" *" *
*" *"" **"
* Mis sing bar indicates nonpositive cancer-related endpoint study
Figure 6-25. Dibenz[a,h]anthracene (DBahA) RTFs*.
Rcf crcn CG
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Dibenzo[a,h]pyrene (DBahP)
2
3
4 Dibenzo[a,h]pyrene (CASRN 189-64-0) is an alternant PAH comprised of six fused
5 aromatic rings. Dibenzo[a,h]pyrene contains two bay regions but no fjord region in its structure.
6 There were five datasets for dibenzo[a,h]pyrene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-26); all gave positive results. The database includes one in vivo
8 bioassay dataset, one in vivo DNA adduct dataset, two in vitro mammalian mutagenicity
9 datasets, and one in vitro DNA damage dataset. A statistically significant increase in tumor
10 incidence was reported in a dermal initiation bioassay in mice (Hoffmann and Wynder, 1966).
11 In addition, two dermal studies of complete carcinogenicity that included benzo[a]pyrene gave
12 positive results, but no RPF could be calculated because the incidence of tumors in the mice
13 exposed to dibenzo[a,h]pyrene was >90% at the lowest dose tested (Cavalieri et al., 1977;
14 Hoffmann and Wynder, 1966) and tumor multiplicity was not reported. As all of the available
15 bioassays that included benzo[a]pyrene showed exposure-related tumorigenic responses,
16 dibenzo[a,h]pyrene was considered carcinogenic and was selected for inclusion in the RPF
17 approach.
18
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Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-relatedendpoint
In vitro cancer-related endpoint
Hoffmann and Wynder 1966 Hughes and P hillips 1990
Durantet al., 1999
Reference
Hassetal., 1982
Mersch-Sundermann et al..
1992
Figure 6-26. Dibenzo[a,h]pyrene (DBahP) RPFs.
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Dibenzo[a,i]pyrene (DBaiP)
2
3
4 Dibenzo[a,i]pyrene (CASRN 189-55-9) is an alternant PAH comprised of six fused
5 aromatic rings. Dibenzo[a,i]pyrene contains two bay regions but no fjord region in its structure.
6 There were 12 datasets for dibenzo[a,i]pyrene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-27); all gave positive results. The database includes two in vivo
8 bioassay datasets, one in vivo DNA adduct dataset, seven in vitro mutagenicity datasets, and two
9 in vitro DNA damage datasets. Statistically significant increases in tumor incidence were
10 reported in dermal initiation and complete carcinogenicity bioassays in mice, both published by
11 Hoffmann and Wynder (1966). The cancer-related endpoint studies were all positive. As the
12 available bioassays that included benzo[a]pyrene were both positive, dibenzo[a,i]pyrene was
13 considered carcinogenic and was selected for inclusion in the RPF approach.
14
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ta
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Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
1
n
-+-
-+-
-+-
•+•
•+•
•+•
•+•
-H
Hoffmann an dHoffmann and Hughes and Durantetal., Hassetal., Teranishiet Hermann, Mersch- Phillipsonand Baker etal., IchinotsuboetMcCann etal.
Wynder 1966 Wynder 1966 Phillips 1990 1999 1982 al., 1975 1981 Sundermannetloannides 1989 1980 al., 1977 1975
al., 1992)
* Missingbar indicatesnonpositive genotoxicity study
Figure 6-27. Dibenzo[a,i]pyrene (DbaiP) RTFs*.
Reference
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Dibenzo[a,l]pyrene (DBalP).
2
3
4 Dibenzo[a,l]pyrene (CASRN 191-30-0) is an alternant PAH comprised of six fused
5 aromatic rings. Dibenzo[a,l]pyrene contains both a bay region and a fjord region in its structure.
6 There were 16 datasets for dibenzo[a,l]pyrene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-28); all of the studies gave positive results. The database includes four
8 in vivo tumor bioassay datasets, three in vivo DNA adduct datasets, one bacterial mutagenicity
9 dataset, one morphological/malignant cell transformation dataset, four in vivo clastogenicity
10 datasets, and three in vitro DNA adduct or DNA damage datasets.
11 In three bioassays of dibenzo[a,l]pyrene included benzo[a]pyrene, RPFs could not be
12 calculated using incidence data, because the incidence of tumors associated with the lowest dose
13 of dibenzo[a,l]pyrene exceeded 90% (two dermal initiation experiments in mice and an
14 intramammilary injection study in rats, both reported by Cavalieri et al., 1991); however, tumor
15 multiplicity data were reported for the dermal initiation experiments and were used to calculate
16 RPFs of 10 and 40. Nesnow et al. (1998b) provided tumor multiplicity and incidence data6 in
17 A/J mice exposed intraperitoneally; both endpoints indicated an RPF of-30. Because the
18 available studies indicated that dibenzo[a,l]pyrene may be much more potent benzo[a]pyrene,
19 other studies were also examined to confirm the potency of this compound.
20 Dibenzo[a,l]pyrene treatment resulted in significant increases in tumor incidence in seven
21 bioassays that did not include benzo[a]pyrene, including two dermal initiation studies (Gill et al.,
22 1994; Cavalieri et al., 1989), a dermal complete carcinogenicity study (Nakatsuru et al., 2004),
23 an intramammilary injection study in rats (Cavalieri et al., 1989), a newborn mouse bioassay
24 (Platt et al., 2004), an intraperitoneal bioassay using A/J mice (Prahalad et al., 1997), and a
25 gavage bioassay comparing the responses of cyplBl wild-type and null mice (Buters et al.,
26 2002). In several of these studies, there was significant toxicity associated with dibenzo[a,l]-
27 pyrene treatment. Tumor incidences were very high in most of the studies, including the gavage
28 study (Buters et al., 2002), which reported an overall tumor incidence of 100% in cyplBl wild-
29 type mice treated with a single dose of dibenzo[a,l]pyrene. A recent study examining in utero
30 and/or lactational exposure to dibenzo[a,l]pyrene showed that mouse pups exposed during late
31 gestation develop T-cell lymphomas between 3 and 6 months of age, as well multiple lung and
32 liver tumors (Castro et al., 2008). All of the cancer-related data for dibenzo[a,l]pyrene were
33 positive and resulted in high RPF estimates, including in vivo and in vitro studies of DNA
6Data were obtained courtesy of S. Nesnow.
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1 adducts, in vivo clastogenicity studies, morphological/malignant cell transformation studies,
2 bacterial mutagenicity studies, and in vitro DNA damage or DNA adduct studies.
3 The weight of evidence supporting a finding of carcinogenicity for dibenzo[a,l]pyrene is
4 strong and suggests that this compound is very potent; thus, it was selected for inclusion in the
5 RPF approach.
6
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I Positive bioassay (incidence)
I Positive bioassay (multiplicity)
] In vivo cancer-related endpoint
| In vitro cancer-related endpoint
c/ c/ v v
^
v
Reference
Figure 6-28. Dibenzo[a,l]pyrene (DBalP) RTFs.
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Fluoranthene (FA)
2
3
4 Fluoranthene (CASRN 206-44-0) is a nonalternant PAH comprised of three aromatic
5 rings and one five-membered ring. Fluoranthene does not contain a classic bay or fjord region in
6 its structure.
7 There were 21 datasets for fluoranthene that met selection criteria and included
8 benzo[a]pyrene (Figure 6-29). Included in the database are in vivo tumor bioassay datasets (11),
9 bacterial and mammalian mutagenicity datasets (5), a morphological/malignant cell
10 transformation assay, and in vitro studies of DNA damage, DNA adducts, or clastogenicity (4).
11 Of the bioassay datasets that included benzo[a]pyrene, nine gave positive results and two gave
12 nonpositive results. Statistically significant increases in tumor incidence and tumor multiplicity
13 were reported in newborn mouse bioassays (in male and female mice [LaVoie et al., 1994] and in
14 female mice [Busby et al., 1989]). The tumor incidence was not significantly increased by
15 fluoranthene in a mouse dermal initiation study with an RPF detection limit of 0.01 (Hoffman et
16 al., 1972) and when fluoranthene was tested alone in a dermal cocarcinogenicity bioassay with
17 an RPF detection limit of 0.1 (Van Duuren and Goldschmidt, 1976). In another newborn mouse
18 bioassay (Busby et al., 1984) that reported both incidence and multiplicity, the lowest dose of
19 benzo[a]pyrene resulted in a tumor incidence of >90%, precluding RPF calculation from the
20 incidence data; however, multiplicity data were available. Statistical analysis of the data for
21 fluoranthene demonstrated positive findings for both incidence and multiplicity in male mice, but
22 the results for the two endpoints were inconsistent in females. In female mice exposed at the
23 high dose of fluoranthene in a newborn mouse bioassay reported by Busby et al. (1984), the lung
24 tumor count was significantly increased (albeit borderline, p = 0.0343) while the incidence was
25 not (p > 0.05), and neither was statistically significantly increased at the lower dose. For the
26 purpose of this analysis, the multiplicity data were treated as an independent measure of
27 carcinogenic potency, and an RPF was calculated for the statistically increased tumor count in
28 female mice.
29 The mutagenicity studies of fluoranthene were all positive, but in vitro studies of DNA
30 damage, DNA adducts, and clastogenicity gave inconsistent results. Because the inconsistent
31 bioassay results can be attributed to different test systems (different exposure route and/or
32 gender) or study design, fluoranthene was considered carcinogenic and was selected for
33 inclusion in the RPF approach.
34
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100
10 ••
ta
PH
1 ••
0.01 --
0.001
RPF detection limits for
nonpositive bioassays
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
&
Mis sing bar indicates nonpositive cancer- related en dpoint study
Rpfpl'PIlCP
Figure 6-29. Fluoranthene (FA) RTFs*.
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Fluorene (FE)
2
o
J
4 Fluorene (CASRN 86-73-7) is a nonalternant PAH comprised of two aromatic rings and
5 one five-membered ring. Fluorene does not contain a classic bay or fjord region in its structure.
6 There were nine datasets for fluorene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-30). There were no tumor bioassays of fluorene that included
8 benzo[a]pyrene, so other bioassays and cancer-related endpoint data were considered. LaVoie et
9 al. (1980) conducted a study of skin tumor initiation in mice treated with 1 mg fluorene followed
10 by 20 weeks of treatment with TPA; the study did not include benzo[a]pyrene. The incidence of
11 tumor-bearing animals (5%) was not significantly increased over controls (0%) (LaVoie et al.,
12 1980). The limited cancer-related endpoint data were mixed, with three positive and four
13 nonpositive mutagenicity datasets, and two nonpositive in vitro DNA damage datasets. Overall,
14 the database for fluorene is both limited and inconsistent. Because the database for fluorene does
15 not provide adequate information with which to assess carcinogenicity, this PAH was not
16 selected for inclusion in the RPF approach.
17
18
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o
OJ
J3
13
100
10 -
1 -
0.1 -
0.01 -
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Sakaietal., 1985 Gibson etal., 1978 Wangenheim and Hermann, 1981 Kaden etal., 1979 McCann etal., Pahlman and Mamber etal., Mersch-
Bolcsfoldi 1988 1975 Pelkonen, 1987 1983 SundermannetaL
1992
: Missing bar indicates nonpositive cancer-related endpoint study
Reference
Figure 6-30. Fluorene (FE) RTFs*.
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Indeno[l,2,3-c,d]pyrene (IP)
1
3
4 Indeno[l,2,3-c,d]pyrene (CASRN 193-39-5) is a nonalternant PAH comprised of five
5 aromatic rings and one five-membered ring. Indeno[l,2,3-c,d]pyrene does not contain a classic
6 bay or fiord region in its structure.
7 There were five datasets for indeno[l,2,3-c,d]pyrene that met selection criteria and
8 included benzo[a]pyrene (Figure 6-31). There are three tumor bioassays, one in vitro study of
9 morphological/malignant cell transformation (Emura et al., 1980), and one in vitro study of DNA
10 damage (Mersch-Sundermann et al., 1992). Of the three tumor bioassays, only one, a rat lung
11 implantation study (Deutsch-Wenzel et al., 1983), reported a statistically significant increase in
12 tumor incidence or multiplicity; the RPF was 0.07. Nonpositive findings were reported in mouse
13 dermal initiation (Hoffmann and Wyner, 1966) and complete carcinogenicity (Habs et al., 1980)
14 studies with RPF detection limits in the range of 0.1-0.3. Because the inconsistent bioassay
15 results can be attributed to different test systems (different species and route), and the
16 nonpositive studies may not have been sufficiently sensitive to detect an effect, indeno-
17 [l,2,3-c,d]pyrene was considered carcinogenic and was selected for inclusion in the RPF
18 approach.
19
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100
10 --
1 --
PH
«
!*.
o
0.1 --
0.01 --
0.001
RPF detection limits for
nonpositive bioassays
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Deutsch-Wenzel et al., 1983 Hoffmannand Wynder 1966 Habs etal., 1980 Mersch-Sundermann et al., 1992 Emura et al., 1980
Reference
Figure 6-31. Indeno[l,2,3-c,d]pyrene (IP) RTFs.
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Naphtho[2,3-e]pyrene (N23eP)
2
3
4
5
6
7
8
9
10
11
12
Naphtho[2,3-e]pyrene (CASRN 193-09-9) is an alternant PAH comprised of six fused
aromatic rings. Naphtho[2,3-e]contains two bay regions but no fjord region in its structure.
There were two datasets for naphtho[2,3-e]pyrene that met selection criteria and included
benzo[a]pyrene (Figure 6-32): a tumor bioassay dataset and an in vitro mammalian mutagenicity
dataset (both were positive). The tumor bioassay was a single dose dermal initiation bioassay
(Hoffmann and Wynder, 1966). As the available bioassay reported a statistically significant
increase in tumor incidence, naphtho[2,3-e]pyrene was considered carcinogenic, and was
selected for inclusion in the RPF approach.
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100 T
10 -•
1 -•
o
u
s
0.1 --
0.01 --
0.001
Hoffmann and Wynder 1966
Reference
Figure 6-32. Naphtho[2,3-e]pyrene (N23eP) RTFs.
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Durantet al., 1999
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Perylene (Pery)
4 Perylene (CASRN 198-55-0) is an alternant PAH comprised of five fused aromatic rings.
5 Perylene contains two bay regions but no fjord region in its structure.
6 There were 11 datasets for perylene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-33). The database includes an in vivo tumor bioassay dataset, an in
8 vivo clastogenicity dataset, eight bacterial mutagenicity datasets, and an in vitro DNA damage
9 dataset. The single tumor bioassay, a dermal initiation study, gave nonpositive results for
10 perylene (El-Bayoumy et al., 1982); the RPF detection limit was 0.01. To confirm the
11 nonpositive bioassay findings, other bioassays and cancer-related endpoint data were considered.
12 In a study that did not include benzo[a]pyrene, Van Duuren et al. (1970) did not observe an
13 increase in tumor incidence over controls when mice were treated by dermal application with an
14 initiating dose of 0.8 mg perylene in benzene followed by thrice weekly treatment with phorbol
15 myristate acetate for 58 weeks. However, seven of the eight bacterial mutagenicity studies gave
16 positive results, while perylene tested nonpositive in one bacterial mutagenicity study, the
17 clastogenicity study, and the DNA damage study. Overall, the database for perylene is both
18 limited and inconsistent. Because the database for perylene does not provide adequate
19 information with which to assess carcinogenicity, this PAH was not selected for inclusion in the
20 RPF approach.
21
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10 -
1 -
fan
PH
£ 0.1
0.01 -
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
RPF detection limits for
nonpositive bioassays
•+•
•+•
-+-
-+-
-+-
-+-
•+•
•+•
•+•
-H
El-Bayoumyet Sirianniand Kadenetal., Carveretal., Sakaietal., 1985 Hermann, 1981 LaVoieetal., DeFloraetal., Florinetal., Pahlman and Mersch-
al., 1982 Huang, 1978 1979 1986 1979 1984 1980 Pelkonen, 1987 Sundermannet
al., 1992
* Missingbar indicatesnonpositive cancer-related endpoint study
Figure 6-33. Perylene (Pery) RTFs*.
Reference
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Phenanthrene (PH)
2
3
4 Phenanthrene (CASRN 85-01-8) is an alternant PAH comprised of three fused aromatic
5 rings. Phenanthrene contains a bay region in its structure, but has less than four aromatic rings.
6 There were 34 datasets for phenanthrene that met selection criteria and included
7 benzo[a]pyrene, including 3 in vivo tumor bioassay datasets, 2 in vivo clastogenicity datasets,
8 11 mutagenicity datasets, 6 morphological/malignant cell transformation datasets, and 12 in vitro
9 studies of DNA adducts, DNA damage, or clastogenicity (Figure 6-34). Only 7 studies reported
10 positive results; the remaining 27 studies reported nonpositive findings, including all 3 bioassays.
11 Nonpositive findings were reported in the three bioassays that included benzo[a]pyrene,
12 including a lung implantation study in rats (Wenzel-Hartung et al., 1990), a dermal initiation
13 study in mice (LaVoie et al., 1981), and a subcutaneous study in mice (Grant and Roe, 1963).
14 To confirm the nonpositive findings, other bioassays and cancer-related endpoint data were
15 considered. In bioassays without benzo[a]pyrene, phenanthrene did not induce significant
16 increases in tumors in a newborn mouse assay using a total dose of 1.4 (imol (Buening et al.,
17 1979) or in two dermal initiation assays (Wood et al., 1979; Salaman and Roe, 1956) using doses
18 of 10 (imol and 540 mg, respectively. However, 12/30 mice developed papillomas by week 35
19 after dermal treatment with 10 |imol phenanthrene (in benzene) followed by twice weekly
20 treatment with TPA; no control mice had papillomas (Scribner, 1973). The response was
21 statistically significantly increased over controls (p < 0.01).
22 In vitro assays of mutagenicity and morphological/malignant cell transformation were
23 predominantly nonpositive for phenanthrene. One of the two positive studies (Sakai et al., 1988)
24 reported a poor dose-response relationship for phenanthrene. Two studies found evidence of
25 clastogenicity after in vivo administration of phenanthrene (Roszinsky-Kocher et al., 1979;
26 Bayer, 1978). However, in the study by Bayer (1978), only the high dose gave a significant
27 response, and there was not a significant dose-response trend. When phenathrene was tested in
28 in vitro studies of DNA adducts, DNA damage, and clastogenicity, the results were
29 predominantly nonpositive (9/12 studies). Overall, the database for phenanthrene is substantial,
30 and the weight of evidence suggests that this PAH is not carcinogenic. Based on the large
31 number of nonpositive bioassays and the abundant evidence that phenanthrene lacks genotoxic
32 action, this compound was selected for inclusion in the RPF approach and assigned an RPF of
33 zero.
34
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100
10 -
1 -
o
3j
s
0.1 -
0.01 -
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
RPF detection limits for
nonpositive bioassays
r~\
1 Missingbar indicates nonpositive cancer-related endpoint study
Figure 6-34. Phenanthrene (PH) RPFs*.
Reference
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1 Pyrene (Pyr)
2
4
5 Pyrene (CASRN 129-00-0) is an alternant PAH comprised of four fused aromatic rings.
6 Pyrene does not contain a bay or fjord region in its structure.
7 There were 49 datasets for pyrene that met study quality criteria and included
8 benzo[a]pyrene (Figure 6-35). Included in the database are in vivo tumor bioassay datasets (7),
9 in vivo clastogenicity datasets (5), bacterial and mammalian mutagenicity datasets (14),
10 morphological/malignant cell transformation datasets (7), and in vitro DNA damage, DNA
11 adducts, or clastogenicity datasets (16). There were seven bioassay s of pyrene that included
12 benzo[a]pyrene; all gave nonpositive results. Nonpositive results were reported in two newborn
13 mouse bioassays in which both males and females were tested (Busby et al., 1989; Wislocki et
14 al., 1986), two studies of dermal initiation (El-Bayoumy et al., 1982; Wood et al., 1980), and a
15 dermal cocarcinogenesis bioassay (Van Duuren and Goldschmidt, 1976). RPF detection limits in
16 these studies ranged from about 0.01 to 0.1 (see Figure 6-35). In an intraperitoneal bioassay
17 using A/J mice that included benzo[a]pyrene, the authors reported that pyrene treatment did not
18 induce lung adenomas (Ross et al., 1995); data were not reported, so an RPF detection limit
19 could not be estimated. In bioassays without benzo[a]pyrene, pyrene did not induce a significant
20 increase in tumors in a dermal initiation bioassay (Salaman and Roe, 1956). Scribner (1973)
21 reported a weak tumorigenic response in a dermal initiation study in mice (5/29 mice developed
22 papillomas 35 weeks after dermal treatment with 10 |imol pyrene in benzene followed by twice
23 weekly treatment with TPA as compared with 0/30 control mice, p = 0.02).
24 In vitro assays of bacterial and mammalian mutagenicity and morphological/malignant
25 cell transformation were predominantly nonpositive for pyrene. In five studies of clastogenicity
26 in animals exposed in vivo to pyrene, no evidence of clastogenic effects was reported. Further,
27 in vitro studies of DNA adducts, DNA damage, and clastogenicity using pyrene also largely
28 reported nonpositive results. Overall, the database for pyrene is substantial, and the weight of
29 evidence suggests that this PAH is not carcinogenic. Based on the large number of nonpositive
30 bioassays and the abundant evidence that pyrene lacks genotoxic action, this compound was
31 selected for inclusion in the RPF approach and assigned an RPF of zero.
32
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RPF detection limits for
nonpositive bioassays
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r
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n n
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-------
Triphenylene (TPhen)
4 Triphenylene (CASRN 217-59-4) is an alternant PAH comprised of four fused aromatic
5 rings. Triphenylene contains several bay regions but no fjord region in its structure.
6 There were six datasets for triphenylene that met selection criteria and included
7 benzo[a]pyrene (Figure 6-36); all but one of the studies gave positive results. The database
8 includes five mutagenicity studies (four positive and one nonpositive) and a study of in vitro
9 DNA damage. There were no bioassays of triphenylene that met selection criteria, and no
10 bioassays without benzo[a]pyrene. Although all of the available cancer-related endpoint studies
11 for triphenylene gave positive results, the database is very limited, consisting of only a few in
12 vitro mutagenicity and DNA damage studies. The RPFs for cancer-related endpoints ranged
13 from 0.02 to 0.4. Because the database for triphenylene does not provide adequate information
14 with which to assess carcinogenicity, this PAH was not selected for inclusion in the RPF
15 approach.
16
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100
10 •
PH
«
it-
o
0.1
0.01
0.001
Positive bioassay (incidence)
Positive bioassay (multiplicity)
In vivo cancer-related endpoint
In vitro cancer-related endpoint
Mersch-Sundermann et Hermann, 1981
al., 1992
Kadenet al., 1979 Barfknecht et al., 1982 Pahlman and Pelkonen Gibsonetal., 1978
1987
* Missingbar indicatesnonpositive cancer-related endpoint study
Figure 6-36. Triphenylene (Tphen) RTFs*.
Reference
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1 7. DERIVATION OF FINAL RTFs FOR SELECTED PAHs
2
3
4 The weight of evidence evaluation (Chapter 6) indicates that the available data are
5 adequate to suggest that 24 of the 27 PAHs are carcinogenic, 3 PAHs (anthracene, phenanthrene,
6 and pyrene) exhibited no carcinogenicity, and data are inadequate to evaluate the carcinogenicity
7 of eight PAHs. The 8 PAHs with inadequate data are excluded from the RPF analysis.
8 For the three PAHs for which there were sufficient data to conclude that they were not
9 carcinogenic (i.e., robust nonpositive tumor bioassay data and cancer-related endpoint data), a
10 final RPF of zero was recommended. While there is little quantitative difference between
11 selecting a final RPF of zero for a given PAH and excluding that PAH from the RPF approach,
12 this is an important distinction for uncertainty analysis. There is substantial uncertainty in the
13 risk associated with PAHs that are excluded from the RPF analysis due to inadequate data, as
14 these compounds could be of low or high potency. However, for PAHs with an RPF of zero,
15 there is evidence to suggest that these compounds are not carcinogenic, and the uncertainty
16 associated with the cancer risk for these compounds is markedly reduced.
17 For each of the remaining 24 compounds, a final nonzero RPF was derived. A number of
18 options were considered for deriving a final RPF from among the numerous values calculated for
19 each individual PAH. These options included: prioritizing bioassay RPFs from different
20 exposure routes based on environmentally relevant routes; prioritizing bioassay RPFs based on
21 target organs considered relevant to human susceptibility to PAH carcinogenesis; prioritizing
22 RPFs based on quality of the underlying study; prioritizing cancer-related endpoints by their
23 correlation with bioassay potency (i.e., ability to predict bioassay potency); and combining (i.e.,
24 averaging) RPFs across all bioassays, across all cancer-related endpoints, or across all endpoints.
25 Appendix G details analyses that were undertaken to assess various options for ranking or
26 prioritizing RPFs. It was concluded that the available data did not provide a basis for prioritizing
27 RPFs except for a preference for bioassay data over cancer-related endpoints. As a consequence,
28 final RPFs were derived from bioassay data for any PAH that had at least one RPF based on a
29 bioassay. For carcinogenic PAHs without bioassay data, final RPFs were calculated from all
30 cancer-related endpoint datasets with positive results (see next section).
31
32 7.1. METHODS FOR DERIVING FINAL RPFs
33 For each carcinogenic PAH with bioassay data, the average RPF was calculated from
34 bioassay datasets with positive results (nonpositive bioassay results were not included in the
35 calculation). For those PAHs that did not have any RPF based on a bioassay, but for which the
36 weight of evidence evaluation indicated a carcinogenic response (e.g., dibenz[a,c]anthracene),
37 the average RPF was calculated from all cancer-related endpoint datasets with positive results
38 (again, nonpositive results were not included in the calculation). The range of RPF values was
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1 also reported. Presenting the average and the range provides an average and maximum estimate
2 for each PAH that has data from multiple studies.
3 Several options were considered for the estimation of a final RPF, including arithmetic
4 mean, geometric mean, weighted average, maximum, or order of magnitude estimates. The
5 arithmetic mean and range were chosen as a simple approach to describing the calculated RPF
6 values available for each PAH. Other estimates were not considered due to the limited number
7 of individual RPF values calculated for most PAHs and the variability in the RPF estimates.
8 There were usually not enough data (3 or fewer RPFs for 17/23 PAHs with nonzero RPFs) to
9 assess the shape of the RPF distribution for any given PAH; thus, a geometric mean was not
10 considered. Further, the range of RPF values from tumor bioassays was greater than an order of
11 magnitude for several compounds (6/23 PAHs). The variability in RPF estimates is likely due to
12 differences in study design parameters (e.g., route, species/strain, exposure duration, exposure
13 during sensitive time periods, initiation versus complete carcinogenesis protocol, tumor
14 incidence versus tumor multiplicity reporting) and dose-response methods (modeled versus point
15 estimates). Calculation of a weighted average was considered, but without a rationale for
16 assigning weights among study types or among tumor data outcomes, using a weighting
17 approach might increase uncertainty.
18 Several previous approaches for generating RPF values for PAHs have used order-of-
19 magnitude estimates (Collins et al., 1998; Malcolm and Dobson, 1994; U.S. EPA, 1993; Nisbet
20 and LaGoy, 1992, see Chapter 3). The presentation of the arithmetic mean (and range) of RPFs
21 for each PAH reflects the available data better than an order-of-magnitude approach.
22 The range was reported as a measure of variability instead of a confidence interval on the
23 average RPF. The input data for each average RPF (bioassay RPFs of different route, species,
24 sex, and target organ, or cancer-related endpoint data across a wide variety of assays and test
25 conditions) reflect such heterogeneity in study design that confidence limits would not provide
26 the statistical precision that they typically convey. All tumor bioassay RPFs (across all exposure
27 routes, species, and sexes, and including both tumor incidence and tumor multiplicity RPFs)
28 were combined to estimate the mean and range for each PAH, except as follows. Only nonzero
29 RPFs were included in the calculation of the final RPF and range for each PAH
30 While tumor multiplicity data from tumor bioassays are not generally used to estimate
31 cancer potency, these data were included in the dose-response assessment in order to determine
32 whether they could serve as a reliable measure of relative cancer potency. Several bioassays
33 reported data on both tumor incidence and tumor number, providing information that was used to
34 compare relative potencies estimated from these two endpoints. The comparison between RPFs
35 calculated from incidence and tumor multiplicity data from the same experiment showed these
36 values to be highly correlated (r2 = 0.76; see further discussion in Chapter 8), indicating that
37 multiplicity RPFs are reasonably predictive of incidence RPFs. When both incidence and
38 multiplicity RPFs were calculated for the same group of animals, the results for each endpoint
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1 could not be considered independent, so the higher of the two values was included in the average
2 and the lower value was excluded. As discussed further in Chapter 8, in 70% of the cases where
3 data for both incidence and multiplicity were used to calculate RPFs, the RPF associated with
4 incidence was the higher of the two (or the two values were equal) and was therefore included in
5 the average, omitting the corresponding multiplicity RPF.
6 When separate RPFs were calculated for different target organs in the same group of
7 animals, the higher value of the two RPFs was included in the average and range, and the lower
8 value was dropped from the combined data. Different RPFs were calculated for liver and lung
9 tumors in male mice (females did not develop liver tumors) in newborn mouse studies. This
10 occurrence applied only to benz[a]anthracene, chrysene, and fluoranthene tested in studies
11 reported by LaVoie et al. (1994) and Wislocki et al. (1986).
12 When separate RPFs were calculated for male and female animals in the same study
13 (generally, these were also newborn mouse studies), both sex-specific RPFs were included in the
14 aggregation, as these were two separate groups of animals. In the one dermal study that included
15 both sexes (Nesnow et al., 1984), the male and female RPFs differed by only -50% for both
16 benz[c]aceanthrylene and benz[l]aceanthrylene. In the newborn mouse studies that resulted in
17 nonzero RPFs for both males and females (LaVoie et al., 1994, 1987; Wislocki et al., 1986), the
18 male RPF was typically three- to fivefold higher than the female RPF. Final RPFs that included
19 both male and female values from the same study were calculated for three PAHs:
20 benzo[j]fluoranthene, benz[a]anthracene, and fluoranthene.
21 Table 7-1 shows the average RPFs based on tumor bioassay data with their associated
22 range, and an overview of the tumor bioassay database (total number of studies, exposure routes
23 tested, species tested, and sexes tested) for each PAH. Table 7-2 shows the average RPF for
24 dibenz[a,c]anthracene, the only RPF based on cancer-related endpoint data, with its associated
25 range, and an overview of the database for this compound.
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Table 7-1. Final RTFs based on tumor bioassay data
PAH
Anthanthrene
Anthracene
B enz [a] anthracene
Benz[b,c]aceanthrylene, 11H-
Benzo [bjfluoranthene
Benzo[c]fluorene
Benz [e] aceanthry lene
Benzo [g,h,i]pery lene
Benz |] ] aceanthry lene
Benzo tj]fluoranthene
Benzo [kjfluoranthene
Benz [1] aceanthry lene
Chrysene
Cyclopenta[c,d]pyrene
Cyclopenta[d,e,f]chrysene, 4H-
Dibenzo [a,e]fluoranthene
Dibenzo [a,e]py rene
Dibenz[a,h]anthracene
Dibenzo [a,h]pyrene
Dibenzo [a,i]py rene
Dibenzo [a,l]py rene
Fluoranthene
Indeno [ 1,2,3 -c,d]pyrene
Naphtho[2,3-e]pyrene
Phenanthrene
Pyrene
Average RPF
0.4
0
0.2
0.05
0.8
20
0.8
0.009
60
0.3
0.03
5
0.1
0.4
0.3
0.9
0.4
10
0.9
0.6
30
0.08
0.07
0.3
0
0
Range of RPFs
0.2-0.5
0
0.02-0.4
0.05
0.1-2
1-50
0.6-0.9
0.009
60
0.01-1
0.03-0.03
4-7
0.04-0.2
0.07-1
0.2-0.5
0.7-1
0.3-0.4
1-40
0.9
0.5-0.7
10-40
0.009-0.2
0.07
0.3
0
0
Number of datasets
2
1 (nonpositive)
3
1
5
2
2
1
1
5
2
2
7
5
2
2
2
3
1
2
3
5
1
1
3 (nonpositive)
7 (nonpositive)
Exposure routes tested
Dermal, lung implantation
Dermal
Dermal, intraperitoneal
Dermal
Dermal, intraperitoneal, lung
implantation
Oral, intraperitoneal
Dermal
Lung implantation
Intraperitoneal
Dermal, intraperitoneal, lung
implantation
Dermal, lung implantation
Dermal
Dermal, intraperitoneal, lung
implantation
Dermal, intraperitoneal
Dermal
Dermal
Dermal
Dermal, intraperitoneal, lung
implantation
Dermal
Dermal
Dermal, intraperitoneal
Intraperitoneal
Lung implantation
Dermal
Dermal, intraperitoneal, lung
implantation
Dermal, intraperitoneal
Species tested
Mouse, rat
Mouse
Mouse
Mouse
Mouse, rat
Mouse
Mouse
Rat
Mouse
Mouse, rat
Mouse, rat
Mouse
Mouse, rat
Mouse
Mouse
Mouse
Mouse
Mouse, rat
Mouse
Mouse
Mouse
Mouse
Rat
Mouse
Mouse, rat
Mouse
Sexes tested
Female
Female
Female, male
Female
Female, male
Female
Female, male
Female
Male
Female, male
Female
Female, male
Female, male
Female, male
Female
Female
Female
Female, male
Female
Female
Female, male
Female, male
Female
Female
Female, male
Female, male
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Table 7-2. Final RPFs based on cancer-related endpoint data (no tumor
bioassay data available)
PAH
Dibenz[a,c]anthracene
Average RPF
4
Range of RPFs
0.04-50
Types of studies
Total =14 studies
One in vivo DNA adduct
Six in vitro bacterial
mutagenicity
One in vitro mammalian
mutagenicity
One in vitro morphological/
malignant transformation
Three in vitro DNA damage
Two in vitro DNA adducts
Multiple dose studies
Total = 6 studies
Four in vitro bacterial
mutagenicity
One in vitro DNA damage
One in vitro DNA adduct
1
2 7.2. CONFIDENCE RATINGS FOR FINAL RPFs
3 Once a final RPF was derived for a given PAH, the resulting value was assigned a
4 relative confidence rating of high, medium, low, or very low. The relative confidence rating
5 characterized the nature of the database upon which the final RPF was based. Confidence
6 rankings were based on the robustness of the database. For final RPFs based on tumor bioassay
7 data, confidence ratings considered both the available tumor bioassays and the availability of
8 supporting data for cancer-related endpoints. The most important factors that were considered
9 included the availability of in vivo data and whether multiple exposure routes were represented.
10 Other database characteristics that were considered included the availability of more than one in
11 vivo study, and whether effects were evident in more than one sex or species. The database
12 characteristics of exposure route, species, and gender are somewhat related (i.e., not independent
13 variables). For example, intraperitoneal injection studies were generally performed in both male
14 and female mice while lung implantation studies were conducted in rats only. An increase in the
15 number of exposure routes tested also results in generation of data for multiple species and
16 genders. The factors that were considered in the relative confidence rating for each RPF are
17 illustrated in Table 7-3.
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Table 7-3. Relative confidence ratings for RTFs
PAH
Benzo [b]fluoranthene
Benzo tj]fluoranthene
Chrysene
Dibenz[a,h]anthracene
Phenanthrene
Anthanthrene
Anthracene
B enz [a] anthracene
Benzo [c]fluorene
Benzo [kjfluoranthene
Cyclopenta[c,d]pyrene
Dibenzo [a,l]pyrene
Pyrene
Benz[b,c]aceanthrylene, 11H-
Benz [e] aceanthry lene
Benzo [g,h,i]pery lene
Benz \j ] aceanthry lene
Benz [1] aceanthry lene
Cyclopenta[d,e,f]chrysene, 4H-
Dibenzo [a,e]fluoranthene
Dibenzo [a,e]py rene
Dibenzo [a,h]pyrene
Dibenzo [a,i]py rene
Fluoranthene
Indeno [ 1,2,3 -c,d]pyrene
Naphtho[2,3-e]pyrene
Dibenz[a,c]anthracene
Relative
confidence
High
High
High
High
High
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Very low
Tumor bioassay data
In vivo data
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
>1 Exposure
route
•/
•/
•/
•/
•/
•/
S*
•/
•/
•/
•/
•/
•/
>2 Exposure
routes
•/
S
S
s
s
>1 Species
•/
•/
•/
•/
•/
•/
S*
•/
>1 Gender
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
•/
Supporting data
for cancer-related
endpoints
•/
S
S
s
s
s
•/
•/
•/
•/
•/
s
s
s
s
s
•/
•/
•/
•/
•/
s
s
s
aBioassays of anthracene without benzo[a]pyrene included dermal studies in mice and a lung implantation study in rats.
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1 Very low relative confidence was used to describe final RPFs based on cancer-related
2 endpoint data only (e.g., dibenz[a,c]anthracene).
3 For RPFs of zero, the confidence rating considered both the available tumor bioassays
4 (with and without benzo[a]pyrene) and the size and consistency of the cancer-related endpoint
5 database. An RPF of zero was only applied if the data implied high or medium relative
6 confidence. For anthracene, phenanthrene, and pyrene, the available data support a practical
7 RPF of zero.
8
9 7.3. APPLICATION OF RPFs FOR ASSESSING CANCER RISKS FROM EXPOSURE
10 TO PAH MIXTURES
11 In the proposed RPF approach, the cancer risk associated with exposure to a particular
12 mixture of PAHs is assumed to equal the sum of the risks associated with exposure to individual
13 carcinogenic components. Because quantitative cancer risk values are available only for
14 benzo[a]pyrene, exposure units (either concentrations or doses, in units of mass) for other PAHs
15 found in the mixture are expressed in terms of benzo[a]pyrene equivalents. These are summed
16 with benzo[a]pyrene to obtain an estimate of the total benzo[a]pyrene equivalents (in
17 concentration or dose) presented by the mixture. Benzo[a]pyrene equivalents for PAH
18 components in a particular mixture are calculated by multiplying the concentration (or dose) of a
19 particular PAH component in the mixture by its RPF. The total benzo[a]pyrene equivalents for a
20 particular mixture of PAHs is calculated as follows:
21
22 E = ZRPFjCj + X
23
24 where:
25 E = the benzo[a]pyrene equivalent exposure presented by the mixture
26 RPFj = relative potency factor of the jth PAH detected in the mixture
27 Cj = dose or concentration of the jth PAH detected in the mixture
28 X = dose or concentration of benzo[a]pyrene in the mixture.
29
30 The cancer risk for the PAH mixture is determined by multiplying the benzo[a]pyrene
31 equivalent dose or concentration by the benzo[a]pyrene cancer toxicity value (e.g., oral slope
32 factor). The proposed RPF approach considers each of the bioassay types used for RPF
33 derivation to be equivalent for the purpose of determining relative potency to benzo[a]pyrene.
34 The uncertainty associated with using a single RPF to derive benzo[a]pyrene equivalents for
35 multiple exposure routes is discussed in Section 8.6.
36
37 7.4. SUSCEPTIBILITY FROM EARLY LIFE EXPOSURE TO CARCINOGENS
38 According to the Supplemental Guidance for Assessing Susceptibility from Early Life
39 Exposure to Carcinogens (U.S. EPA, 2005b), benzo[a]pyrene is carcinogenic by a mutagenic
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1
2
o
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
mode of action. For example, an acute dosing study using benzo[a]pyrene suggests that early-
lifestage exposure would lead to an increased incidence of tumors compared with adult
exposures of a similar dose and duration (EPA, 2005b). Mice that were treated with
benzo[a]pyrene (75 or 150 ug/g body weight intraperitoneal) within 24 hours of birth or at
15 days of age developed hepatomas at a higher incidence than similarly treated animals at
42 days of age (Vesselinovitch et al., 1975, as cited in EPA 2005b).
The Supplemental Guidance establishes age-dependent adjustment factors (ADAFs) for
three specific age groups. The ADAFs and their age groupings are 10 for <2 years, 3 for 2-<16
years, and 1 for >16 years (U.S. EPA, 2005b). The 10- and 3-fold adjustments in slope factor are
to be combined with age-specific exposure estimates when estimating cancer risks from early life
(<16 years age) exposure to PAHs.
Because a mutagenic mode of action for benzo[a]pyrene carcinogenicity is sufficiently
supported in laboratory animals and relevant to humans, and in the absence of chemical-specific
data to evaluate differences in susceptibility, increased early-life susceptibility is assumed and
the ADAFs should be applied, as appropriate. A common mutagenic mode of action for
carcinogenic PAHs is hypothesized based on information available for the indicator chemical,
benzo[a]pyrene (U.S. EPA, 2005b). In the absence of chemical-specific data to evaluate
differences in susceptibility, increased early-life susceptibility to the 24 PAHs (for which RPFs
were derived) in this analysis is assumed and the ADAFs should be applied, along with exposure
information, as appropriate (see Table 7-4 for example).
Some of the studies used to derive RPFs for the PAHs were conducted in newborn mice.
The RPFs calculated from the newborn mouse studies reflect only the potency of the tested PAH
relative to that ofbenzofajpyrene, and do not take into account the potency of the PAH
administered in newborn or young animals relative to the potency of the same PAH administered
to adult animals. The ADAF should be applid to account for the latter difference.
Table 7-4. Sample calculation of estimated cancer risk for
benz [a] anthracene with the application of ADAFs
Age group
0-<2
2-<16
>16
ADAF
10
3
1
Benzo[a]pyrene oral slope
factor (per mg/kg-d)
7.3
7.3
7.3
Adjusted
benzo[a]pyrene
cancer risk estimate
73
24
7.3
RPF
0.2
0.2
0.2
Benz[a]anthracene estimated
cancer risk (per mg/kg-d)
15
4.8
1.5
27
28
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1 8. UNCERTAINTIES AND LIMITATIONS ASSOCIATED WITH THE RTF
2 APPROACH
o
J
4
5 A description of uncertainties and limitations is an important component of the RPF
6 approach for PAH mixtures risk assessment. Many of the general uncertainties related to
7 chemical-specific risk assessment are also applicable to the proposed RPF approach for PAHs.
8 These include issues related to selection of an appropriate animal model, low-dose and
9 interspecies extrapolation, and variability within the human population. Use of a component-
10 based approach to mixtures risk assessment leads to additional uncertainties, e.g., the lack of
11 experimental data on potential interactions among individual components within the mixture
12 (i.e., among PAHs and with other chemicals).
13 The feasibility of conducting a robust component-based approach for PAH mixtures
14 (RPF approach) was evaluated by a PAH mixtures peer consultation workshop (U.S. EPA,
15 2002). Included in the discussion was a general evaluation of U.S. EPA's Provisional Guidance
16 (U.S. EPA, 1993). Workshop participants highlighted the following limitations of the 1993
17 guidance:
18
19 (1) The approach only considered a small subset of PAHs (i.e., unsubstituted PAHs only,
20 no heterocyclic compounds or nitro- or alkyl- substituted PAHs);
21
22 (2) There are no human toxicity data for any individual PAH;
23
24 (3) The assumption of additivity may not be valid, and there may be interactions among
25 PAHs or between PAHs and other components of a mixture (e.g., metals);
26
27 (4) PAHs may generally have a common mode of action (i.e., mutagenicity), but multiple
28 modes of action for carcinogenesis are possible; and
29
30 (5) The EOPP approach was limited to the oral exposure route (i.e., a recommendation
31 was made not to apply the factors to dermal and inhalation exposures).
32
33 The current analysis represents a significant improvement upon the previous component-
34 based approach for PAH mixtures risk assessment. One of the most important improvements is a
35 comprehensive review of the scientific literature dating from the 1950s through 2009 on the
36 carcinogenicity and genotoxicity of PAHs. The search identified over 900 individual
37 publications for a target list of 74 PAHs that had been identified in environmental media or for
38 which toxicological data were available. Review of these publications resulted in the
39 identification of more than 600 papers that included carcinogenicity or cancer-related endpoint
40 data on at least one PAH and benzo[a]pyrene tested at the same time. Dose-response data were
41 extracted, and individual RPFs were calculated from over 300 data sets representing
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1 51 individual PAHs. For 35 PAHs, a weight of evidence evaluation was conducted to select
2 compounds for inclusion in the RPF approach; data were inadequate to conduct such an
3 evaluation for the remaining 16 compounds. A final RPF was derived for each PAH based on
4 tumor bioassay data (if available) or cancer-related endpoint data if no tumor bioassay RPFs
5 were available. Final RPFs were derived for 27 PAHs (see Table 7-2), significantly increasing
6 the number of PAHs that can be addressed through this approach. Each RPF was assigned a
7 relative confidence rating reflecting the size and diversity of the tumor bioassay or cancer-related
8 endpoint database that was used to derive the final RPF for that PAH.
9 Despite these improvements, many of the uncertainties highlighted during the 2002 peer
10 consultation workshop (U.S. EPA, 2002) also apply to the current analysis. The following
11 sections describe some specific uncertainties and limitations associated with the development
12 and use of RPFs for PAHs. The uncertainties that are specific to the approach presented herein
13 are discussed below in Sections 8.1 and 8.2. Sections 8.3-8.6 discuss the general uncertainties
14 associated with a component-based approach to PAH mixtures risk assessment. These include
15 the number of PAHs included in the approach, human relevance of animal data, assumptions
16 regarding mode of action and dose additivity, and cross-route extrapolation.
17
18 8.1. DOSE-RESPONSE ASSESSMENT FOR INDIVIDUAL PAHs
19 Several uncertainties and limitations are specifically associated with the selection of data
20 and dose-response assessment methodology used in this analysis to derive RPFs for PAHs.
21 Uncertainties are associated with the following decisions:
22
23 • Inclusion of data from studies reporting the occurrence of benign tumors in derivation
24 of RPFs;
25
26 • Use of a single dose-response model for quantal or continuous data;
27
28 • Use of varying BMR levels;
29
30 • Use of tumor incidence data at the upper end of the dose-response curve (e.g., >75%
31 incidence) to calculate some RPFs;
32
33 • Use of tumor multiplicity data to calculate some RPFs;
34
35 • Use of single-dose point estimates7 to calculate some RPFs;
36
37 • Reliance on data from cancer-related endpoint studies in the absence of bioassays;
38 and
39
7In this report, the term "point estimate RPF" is used to describe an RPF calculated from a single point on the dose-
response curve for both the PAH of interest and benzo[a]pyrene. This term distinguishes the RPF from one
calculating using a BMD modeling result from multidose data.
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1 • Use of cancer-related data from assay conditions that maximize the benzo[a]pyrene
2 response, even though these conditions were not necessarily optimal for other PAHs.
3
4 The decision was made to employ a single dose-response model for either quantal or
5 continuous data due to the large number of data sets that needed be analyzed from the PAH
6 database. The multistage model for incidence data and the linear model for continuous data were
7 considered to be broadly applicable to different types of data as simple curve-fitting models. In
8 some cases, the goodness-of-fit criteria indicated that the selected model did not fit the data. In
9 these cases, high-dose groups were sequentially eliminated until an adequate fit was achieved,
10 but other model structures (e.g., gamma, probit, logistic, etc.) were not considered.
11 Tumor bioassay data were modeled at a BMR of 10% in order to target the low end of the
12 dose-response curve as the point of departure for slope estimation. When this was not feasible,
13 usually because only a single dose was used for benzo[a]pyrene, an attempt was made to match
14 individual target PAH response levels to the benzo[a]pyrene response chosen for the point
15 estimate. This assumes that the shape of the dose-response curve is similar for the target PAH
16 and benzo[a]pyrene (also a necessary assumption of dose additivity) and that the slope is
17 constant across the dose-response curve. These assumptions may not hold, especially in studies
18 of tumor incidence where the point estimate benzo[a]pyrene response was very high or near
19 maximal. In many cases, the dose of benzo[a]pyrene selected as the positive control produced
20 near maximal tumor incidence in exposed animals (i.e., >75%). There is uncertainty associated
21 with comparing potency estimates at the high end of the dose-response curves and using the
22 resultant RPF to estimate risks associated with low environmental exposures. The relative
23 potency relationship between any two PAHs may be different at the low end, compared with the
24 high end, of the dose-response curves.
25 It is not clear whether relative potency values estimated at the high end of the dose-
26 response curve are reasonably predictive of relative potency at low environmental exposure
27 levels. For this reason, additional uncertainty is involved in using RPFs that are not based on a
28 BMR of 10% (especially those RPFs that are based on responses exceeding 75%) to estimate
29 risks associated with low exposures.
30 If model fit was not achieved, then a point-estimate ratio approach was used. Point
31 estimate ratios were also used for several other reasons:
32
33 (1) Only a single dose group was tested;
34
35 (2) When the standard deviation or number of replicates were not reported for continuous
36 data sets; or
37
38 (3) High-dose groups from multiple dose data sets were not usable due to a saturated
39 tumor response (>90% incidence in the lowest exposure group).
40
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1 The point estimate approach is most reliable when the chosen point is in the linear
2 portion of the dose-response curve. In many cases, however, especially for single-dose data, it
3 was not possible to determine whether the chosen point was in a linear or nonlinear portion of
4 the dose-response curve. The dose-response relationship observed in many studies of cancer-
5 related endpoints was nonlinear at high doses. Whenever possible, the point estimate was chosen
6 from the linear portion of the dose-response curve (i.e., before the response plateau that occurs at
7 high doses). Of 50 individual RPFs calculated from tumor incidence data, 21 were calculated
8 using a point of departure incidence <25%, 19 were calculated using a point of departure
9 incidence between 25 and 75%, and the remaining 10 were calculated using a point of departure
10 incidence between 75 and 90%. Thus, only 20% of the individual RPFs for tumor incidence data
11 were calculated from a point high (>75 and <90% incidence) on the dose-response curve.
12 For a few PAHs tested in older dermal bioassays, the authors reported mortality prior to
13 the appearance of the first tumor. For these data sets, an assumption was made that the number
14 of animals at risk for tumor development was equal to the total number of animals alive at the
15 time of the appearance of the first tumor. This approach ensures that the incidence is not
16 underestimated by including animals that did not survive long enough to develop tumors. As this
17 assumption applied to a small number of RPFs (specifically, individual RPFs for chrysene,
18 dibenzo[a,e]pyrene, dibenzo[a,e]fluoranthene, and dibenzo[a,h]pyrene calculated from data
19 reported by Hecht et al. [1974] and Hoffmann and Wynder [1966]), it had little impact on the
20 overall analysis.
21 RPFs were also calculated for many cancer-related endpoints. Many of the studies
22 describing in vitro cancer-related endpoints provided dose-response data under varying study
23 conditions. For example, bacterial mutagenesis studies utilized multiple strains, different
24 metabolic activation processes, and varying assay systems. In order to minimize the amount of
25 data used for dose-response analysis of in vitro mutagenicity studies, and to provide a consistent
26 basis for comparing RPFs for different PAHs, the data from conditions that maximize the
27 benzo[a]pyrene response within a particular study were used for the dose-response assessment.
28 In several studies, the conditions that were optimal for benzo[a]pyrene were not necessarily
29 optimal for the target PAH. For example, the concentration of S9 mix that produced the highest
30 mutation rate for benzo[a]pyrene did not produce a maximal response for perylene or
31 cyclopenta[c,d]pyrene (Carver et al., 1986; Eisenstadt and Gold, 1978). In vitro data were only
32 used in the derivation of a single final RPF (for dibenz[a,c]anthracene; see Table 7-2); thus, the
33 uncertainties associated with the use of cancer-related endpoint data are important for
34 dibenz[a,c]anthracene, but have minimal impact on the proposed RPFs for the other 26 PAHs.
35
36 8.2. SELECTION OF PAHs FOR INCLUSION IN RPF APPROACH
37 One of the uncertainties highlighted by the peer consultation workshop (U.S. EPA, 2002)
38 stemmed from the fact that U.S. EPA's 1993 provisional EOPP approach only considered a small
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1 subset of PAHs (i.e., unsubstituted PAHs only, no heterocyclic compounds or nitro- or alkyl-
2 substituted PAHs), and EOPPs were available for only seven PAHs. Although the present report
3 considered a larger number of PAHs than previous analyses (the toxicological literature was
4 searched for data on 74 individual PAHs identified in environmental media or for which there
5 were toxicological data), the focus of this analysis remains limited to unsubstituted PAHs with
6 three or more fused aromatic rings containing only carbon and hydrogen atoms. Thus, the RPF
7 analysis presented here does not account for the possible carcinogenicity of substituted or
8 heterocyclic PAHs that may be present in complex mixtures. This may result in an
9 underestimation of PAH mixture cancer risk.
10 Of the 74 unsubstituted PAHs with three or more aromatic rings, there were studies
11 including benzo[a]pyrene that were suitable for RPF calculation for 51 compounds. The
12 methodology for selecting PAHs for inclusion in the RPF approach from among these 51 PAHs
13 is described in Chapter 6. At the outset, 16 PAHs were excluded because only one or two in
14 vitro cancer-related endpoint RPFs were available. The remaining 35 PAHs were evaluated
15 using a weight of evidence approach. The primary uncertainties associated with the selection
16 process relate to:
17
18 (1) The use of a weight of evidence approach that focused on tumor bioassays including
19 benzo[a]pyrene as opposed to a comprehensive cancer assessment to select PAHs for
20 inclusion in the approach; and
21
22 (2) The exclusion of PAHs with limited or inconclusive data.
23
24 The weight of evidence approach was used due to the large number of compounds that
25 were under consideration. The approach was structured as a decision tree that focused primarily
26 on cancer bioassays that included benzo[a]pyrene, and only considered other data (e.g., bioassays
27 that did not include benzo[a]pyrene, or cancer-related data) when cancer bioassays with
28 benzo[a]pyrene were unavailable, nonpositive, or inconsistent (see Figure 6-1). The data
29 collection for this analysis was centered on studies that included benzo[a]pyrene, as these studies
30 would be most useful for RPF calculation. Consequently, information from bioassays that
31 included benzo[a]pyrene were readily available for use in the weight of evidence determinations.
32 Bioassays that did not include benzo[a]pyrene and cancer-related endpoint data were considered
33 only when there were conflicting or nonpositive results in the studies that did include
34 benzo[a]pyrene. There is uncertainty in drawing conclusions as to carcinogenicity based on a
35 narrow subset of the available database. Other elements of a more comprehensive weight of
36 evidence determination that were not considered include: cancer-related endpoint data from
37 studies that did not include benzo[a]pyrene; information on tumorigenicity of metabolites;
38 information on formation of reactive metabolites; other mechanistic data (e.g., AhR reactivity,
39 inhibition of gap junction intercellular communication, etc.); and QSAR assessment.
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1 A number of PAHs (24 of 51 PAHs that had at least one RPF value) were excluded from
2 the relative potency approach because the available data were inadequate to draw a conclusion as
3 to carcinogenicity (see Tables 6-1 and 6-2). All of these PAHs had at least one RPF, indicating
4 that the compounds were active in at least one cancer-related endpoint assay. Excluding these
5 PAHs from the approach increases the uncertainty in assessing risks from a mixture that includes
6 them, particularly if the excluded PAHs constitute a large fraction of the mixture.
7 In summary, RPFs were proposed for only 27 of the 74 PAHs initially considered,
8 because the remaining 47 compounds did not have adequate data. Thus, even among the subset
9 of PAHs upon which this analysis was focused, RPFs were only recommended for only about
10 one-third of the compounds. Because only a fraction of any given PAH mixture can be
11 evaluated using the RPF approach, it is important to note as part of the uncertainty evaluation of
12 a risk assessment using these RPFs that there is some proportion of the total mixture (i.e., mass
13 fraction) that is comprised of compounds that are not considered in the component-based
14 approach.
15
16 8.3. DERIVATION OF A FINAL RPF FOR EACH PAH
17 The methodology for deriving a final RPF value and assigning a relative confidence
18 rating is described in Sections 7.1 and 7.2. The primary uncertainties associated with RPF
19 derivati on rel ate to:
20
21 (1) Combining RPFs across multiple exposure routes, species, sexes, tumor types, and
22 studies;
23
24 (2) Inclusion of RPFs based on tumor multiplicity data in the combined data;
25
26 (3) Inclusion of RPFs from female newborn mice when male RPF values were
27 demonstrably higher;
28
29 (4) Use of an arithmetic mean to derive final RPFs; and
30
31 (5) Use of cancer-related endpoint data to derive final RPFs for compounds without
32 tumor bioassay RPFs.
33
34 A variety of options were considered for prioritizing and/or combining RPFs.
35 Appendix G describes analyses that were undertaken to assess options for prioritizing RPFs. As
36 the appendix indicates, the current state of knowledge does not suggest a clear biological basis
37 for prioritizing RPFs. As a result, RPFs were combined across exposure routes, species, sexes,
38 tumor types, dose-response methods, and studies.
39 In addition to tumor incidence data, tumor multiplicity data were used to calculate RPFs.
40 The relationship between tumor incidence RPFs and tumor multiplicity RPFs is not known;
41 however, this analysis resulted in the calculation of both incidence and multiplicity RPFs for
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1 24 individual datasets. These data were plotted, and a linear regression analysis was performed
2 to assess the correlation between these two relative potency estimates. Figure 8-1 shows the
3 results.
10
1:1 Correspondence Line
Incidence RPF
100
5 Figure 8-1. Correlation between incidence and multiplicity RTFs.
6
7 As shown in Figure 8-1, there is a high degree of correspondence between incidence and
8 multiplicity RPFs calculated from results in the same animals, with one exception (see circled
9 data point). The regression analysis indicated an r2 of 0.76 for the correlation when the outlier
10 was excluded, or only 0.28 when it was included. The outlier datapoint reflects the incidence
11 and multiplicity RPFs for benzo[c]fluorene calculated for the one oral study (Weyand et al.,
12 2004). All of the other datapoints reflect incidence and multiplicity RPFs for dermal or
13 intraperitoneal exposure studies; thus, one possible explanation for the outlier is that the
14 relationship between incidence and multiplicity after oral exposure differs from the relationship
15 after exposure via other routes. However, there was good correspondence between incidence
16 and multiplicity in dermal and intraperitoneal studies, despite the marked differences in
17 absorption, distribution, and metabolism of PAHs administered by these two exposure routes.
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1 Compound-specific differences in the association between incidence and multiplicity RPFs also
2 seem unlikely; the dataset shown in Figure 8-1 also includes a comparison between incidence
3 and multiplicity RPFs for benzo[c]fluorene in an intraperitoneal exposure study, and there is
4 good correspondence between the two (RPF = 1 for incidence and RPF = 0.6 for multiplicity).
5 The most plausible explanation for the outlier is that the basis for the multiplicity RPF in the oral
6 study of benzo[c]fluorene (RPF = 50) was estimated using a point high on the dose-response
7 curve (incidence was 100%), at which a large mean number of tumors per animal (46 ± 2.8) was
8 recorded, while the incidence RPF (RPF = 5) for the same study was estimated using BMD
9 modeling at a response point lower on the curve (BMR of 0.7). All of the other comparisons
10 between incidence and multiplicity RPFs from the same set of animals were based on
11 multiplicity responses <10 tumors per animal. Although there is little information with which to
12 explore this hypothesis, it is possible that RPFs for multiplicity that are calculated using
13 unusually high tumor number are not reliable measures of relative incidence potency. This could
14 result from changes in the slope of the tumor number versus dose curve at high tumor number, or
15 from methodology limitations that hamper accurate measurement of high tumor numbers.
16 Notwithstanding the one outlier, as the remaining incidence and multiplicity RPFs from
17 the same study were highly correlated, only one of the two metrics (the higher of the incidence or
18 multiplicity RPF from the same study) was included in the average and range. Figure 8-1 shows
19 that multiplicity RPFs exhibit a slight tendency to underestimate the RPF from incidence data
20 (more points are to the right of the 1:1 correspondence line); thus, the higher value was usually
21 calculated from incidence data. Specifically, 15/24 incidence RPFs were higher than the
22 corresponding multiplicity RPF from the same study, and 2/24 of the incidence and multiplicity
23 RPFs were identical. Thus, only 7/24 multiplicity RPFs were higher than their corresponding
24 incidence RPFs.
25 As discussed in Section 7.1, in newborn mouse studies that resulted in nonzero RPFs for
26 both males and females (LaVoie et al., 1994, 1987; Wislocki et al., 1986), the male RPF was
27 typically three- to fivefold higher than the female RPF, but both were included in the final RPF
28 calculation. Final RPFs that included both male and female values from the same study were
29 calculated for three PAHs: benzo[a]anthracene, benzo[j]fluoranthene, and fluoranthene. An
30 alternative approach would be to select the RPF associated with the most sensitive sex (i.e.,
31 males) and to omit the female RPF from the final calculation. The net effect of including female
32 RPFs for these three compounds is to reduce the average RPF and, in some cases, to reduce the
33 lower limit of the range of RPFs. For benzo[a]anthracene and benzo[j]fluoranthene, the final
34 RPF is unchanged whether or not the female RPF is included. For fluoranthene, inclusion of the
35 female RPFs yields a final RPF of 0.08, while excluding the female RPFs would result in a final
36 RPF of 0.1.
37 Final RPFs were calculated as the arithmetic mean and range of RPFs from tumor
38 bioassay data when such data were available. Presenting the average and the range provides both
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1
2
o
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
an average and a maximum estimate for each PAH that has data from multiple studies. Other
options for deriving a central tendency RPF include geometric mean, median, weighted average,
and order of magnitude estimates. The arithmetic mean represents a simple approach to
describing the calculated RPF values available for each PAH. There were usually not enough
data (<3 RPFs for 18/24 PAHs with nonzero RPFs) to assess the shape of the RPF distribution
for any given PAH, so a geometric mean was not considered. Calculation of a weighted average
was considered, but without a clear biological rationale for assigning weights among study types
or tumor data outcomes, using a weighting approach might increase uncertainty. Finally, the use
of simple means and ranges of estimated RPFs rather than order of magnitude estimates, as has
been previously done for estimating RPFs for PAHs, was considered to better reflect the
available data and provide a clearer characterization of uncertainty.
Cancer-related endpoint data were relied upon for the derivation of an RPF for only one
PAH (dibenz[a,c]anthracene). For this compound, there were no tumor bioassay data suitable for
the determination of an RPF. However, cancer-related endpoint data provided qualitative
support for the finding of carcinogenicity for this compound (see individual narrative for this
compound in Section 6.2). Although the mutagenic mode of action for benzo[a]pyrene (U.S.
EPA, 2005b) suggests that, in general, these endpoints may be relevant to PAH carcinogenicity,
the predictive value of a positive response in these tests has not been conclusively demonstrated.
Thus, there is considerable uncertainty in an RPF based on cancer-related endpoint data.
Appendix G includes analysis of the correlation between average RPFs calculated from cancer-
related endpoint data and tumor bioassay data. As shown in Table 8-1, and further discussed in
Appendix G, cancer-related endpoint RPFs are reasonably predictive of tumor bioassay RPFs;
however, the relationship between these RPFs and the relative potency of a given PAH in
humans exposed via environmentally relevant routes is unknown.
Table 8-1. Results of simple linear regression of log-transformed average
tumor bioassay RPF versus log average genotoxicity RPF
Genotoxicity endpoint
All in vivo DNA adducts
All in vivo nonbioassays
All nonbioassay endpoints (in vitro and in vivo)
All in vitro nonbioassays
All in vivo micronuclei and sister chromatid exchanges
All in vitro mutagenicity
r2
0.64
0.55
0.40
0.39
0.39
0.032
Slope
1.22
1.16
1.10
0.91
0.81
0.33
/7-Value
O.01
0.01
0.01
0.01
>0.05 (nonsignificant)
>0.05 (nonsignificant)
n
10
11
20
19
6
17
26
27
28
29
30
For three PAHs (anthracene, phenanthrene, and pyrene), a final RPF of zero was
recommended. As noted earlier in Chapter 6, there is little quantitative difference between
selecting a final RPF of zero for a given PAH and excluding that PAH from the RPF approach.
However, excluding PAHs from the RPF approach implies substantial uncertainty (these
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2
o
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
compounds could be of low or high potency), while assigning an RPF of zero suggests lower
uncertainty because there is evidence to suggest that these compounds are not carcinogenic.
Nevertheless, there remains uncertainty in the RPFs for these three compounds, as all of them
included one or more studies suggesting activity in cancer-related endpoint assays. In addition, it
is possible that available bioassay studies for these compounds may not provide sufficient
sensitivity to allow for a potency comparison with benzo[a]pyrene; thus, the RPF of zero should
not be considered a characterization of the inherent carcinogen!city of anthracene, phenanthrene,
or pyrene.
In the present analysis, RPFs for individual PAHs were based on data of varying quality
and reproducibility, so there is additional uncertainty in risks estimated for mixtures containing
differing concentrations of individual PAHs. Confidence ratings were assigned to each RPF to
qualitatively characterize the uncertainty in each individual RPF. Table 8-2 shows the
distribution of PAHs with RPFs of each confidence rating. As the table indicates, there are
5 PAHs with RPFs of high confidence, 8 PAHs with RPFs of medium confidence, 13 PAHs with
RPFs of low confidence, and 1 PAH with an RPF of very low confidence. The confidence
ratings assigned to the RPFs may be used to qualitatively assess the uncertainty in a mixtures risk
assessment that utilizes the RPFs. For example, if a high proportion of the total cancer risk
predicted for a given mixture is attributable to benzo[a]pyrene and other PAHs with RPFs of
high or medium confidence, then the confidence in the overall cancer risk assessment will be
relatively high. If, in contrast, benzo[a]pyrene contributes a relatively small fraction of the
overall risk, and/or the mixture consists primarily of PAHs with RPFs of low confidence, then
the confidence in the overall cancer risk assessment will be correspondingly lower. Thus, it will
be important to consider the relative contribution of benzo[a]pyrene to the total risk, as well as
the relative confidence ratings of the RPF values for component PAHs, in the uncertainty
evaluation for cancer risk assessments that employ these RPFs.
Table 8-2. PAHs with RPFs of varying relative confidence
High confidence RPF
Medium confidence RPF
Low confidence RPF
Very low confidence RPF
Benzo [b]fluoranthene
Benzo tj]fluoranthene
Chrysene
Dibenz[a,h]anthracene
Phenanthrene
Anthanthrene
Anthracene
B enz [a] anthracene
Benzo [c]fluorene
Benzo fkjfluoranthene
Cyclopenta[c,d]pyrene
Dibenzo [a,l]py rene
Pyrene
Benz[b,c]aceanthrylene, 11H-
Benz [e] aceanthry lene
Benzo[g,h,i]perylene
Benz |] ] aceanthry lene
Benz [1] aceanthry lene
Cyclopenta[d,e,f]chrysene, 4H-
Dibenzo [a,e]fluoranthene
Dibenzo [a,e]pyrene
Dibenzo [a,h]pyrene
Dibenzo [a,i]pyrene
Fluoranthene
Indeno[l,2,3-c,d]pyrene
Naphtho [2,3 -e]pyrene
Dibenz [a,c] anthracene
27
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1 8.4. USE OF ANIMAL DATA TO PREDICT HUMAN CANCER RISK FOR PAHs
2 Section 4.2 briefly summarizes the epidemiology and human biomarker data related to
3 exposure to PAH mixtures and carcinogenicity. Exposure to certain PAH mixtures is clearly
4 associated with cancer in humans. Epidemiology studies evaluating emissions from coke
5 production, coal gasification, aluminum production, iron and steel founding, coal tars, coal tar
6 pitches, and soot have demonstrated associations between exposure and increased risk of lung
7 cancer in humans (see review of Bostrom et al., 2002). Skin and scrotal cancers have been
8 associated with exposure to coal tar, coal tar pitches, nonrefmed mineral oils, shale oils, and soot
9 (Larsen and Larsen, 1998; WHO, 1998; ATSDR, 1995). While human epidemiology data may
10 be sufficient for the purpose of quantifying the cancer risks associated with exposure to a few
11 PAH mixtures, there are no data for many mixtures; hence the need for other approaches
12 including surrogate-mixture and component-based approaches. As noted by the peer
13 consultation workshop (U.S. EPA, 2002), there are no human data on cancer response to
14 individual PAHs that could be used as the basis for, or as a supplement to, a component-based
15 approach. As a result, the RPF approach relies on animal bioassay data to predict human cancer
16 risk associated with individual PAHs.
17 The use of animal bioassays in predicting relative carcinogenic potency in humans
18 represents a source of uncertainty in this approach. As there are no human data on cancer
19 response to individual PAHs, including benzo[a]pyrene, there can be no quantitative evaluation
20 of uncertainty in extrapolating from RPFs based on animal bioassay data to relative potency in
21 humans. Possible species differences in toxicokinetics, toxicodynamics, and mode of action
22 contribute to the uncertainty. Cancer-related endpoint data are available using human cells (e.g.,
23 epidermal keratinocytes, lymphoblasts, human epithelial cells) for the evaluation of
24 mutagenicity, DNA adducts, unscheduled DNA synthesis, DNA damage, and clastogenicity or
25 sister chromatid exchange frequency (see Section 4.3). Findings in human cells were generally
26 consistent with those in other mammalian cells; however, whether this finding of consistency
27 extends to effects in vivo, and specifically to formation of tumors, is not known.
28 In addition, animal bioassays use various routes of administration (e.g., intraperitoneal
29 and subcutaneous injection), which may not be directly relevant to expected routes of exposure
30 for humans. It is difficult to determine whether the relative potency based on animal bioassays
31 using injection routes of exposure is predictive of relative potency that would be observed in
32 humans exposed through environmentally relevant exposure routes (see further discussion of
33 exposure-route uncertainties in Section 8.6). An additional source of uncertainty in the use of
34 animal bioassay data stems from differences in the doses used in animal bioassays as compared
35 with low doses received by humans exposed in the environment. Mechanistic data, primarily
36 obtained using benzo[a]pyrene, provide support for the human relevance of PAH tumorigenicity
37 in animals. There is evidence linking three pathways activating benzo[a]pyrene to DNA-reactive
38 agents [(+)-anti-BPDE, radical cations, benzo[a]pyrene-7,8-dione, and reactive oxygen species]
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1 with key mutational events in genes (p53 tumor suppressor gene and H-ras or K-ras oncogenes)
2 that can lead to tumor initiation. Results in support of mutagenic modes of action via the diol
3 epoxide and radical cation pathways include in vivo results in animals. All of these activation
4 pathways occur in human tissues, and associations have been made between spectra of mutations
5 in the p53 tumor suppressor gene or ras oncogenes induced by benzo[a]pyrene metabolites with
6 spectra of mutations in these genes in tumor tissue from benzo[a]pyrene-exposed animals or
7 tumor tissue in humans.
8 Support for the association between the diol epoxide pathway and tumor initiation
9 includes observation that: (+)-anti-BPDE activated the H-ras-1 proto-oncogene to transform
10 NIH/3T3 cells via G^T point mutations in the 12th codon (Marshall et al., 1984); (+)-anti-
11 BPDE reacts with the p53 tumor suppressor gene at several hotspots mutated in lung cancer
12 patients (Denissenko et al., 1996; Puisieux et al., 1991); the spectra of p53 and K-ras mutations
13 in lung tumors of nonsmoking patients, chronically exposed to smoky coal emissions, was
14 consistent with (+)-anti-BPDE mutations in these genes (DeMarini et al., 2001); elevated BPDE-
15 DNA adducts have been observed in coke oven workers and chimney sweepers (Pavanello et al.,
16 1999); and the spectra of mutation in the K-ras, H-ras, and p53 genes in forestomach tumors of
17 mice fed benzo[a]pyrene in the diet for 2 years were consistent with (+)-anti-BPDE DNA
18 reactions (Culp et al., 2000).
19 Support for the radical cation pathway includes observations that depurinated adducts,
20 (expected products from reactions of benzo[a]pyrene radical cations with DNA) accounted for
21 74% of identified DNA adducts in mouse skin exposed to benzo[a]pyrene (Rogan et al., 1993)
22 and 9/13 examined tumors from mice exposed to dermal applications of benzo[a]pyrene had
23 H-ras oncogene mutations attributed to depurinated DNA adducts from benzo[a]pyrene radical
24 cations (Chakravarti et al., 1995).
25 Support for the aldo-keto reductase pathway includes in vitro demonstration that several
26 types of DNA damage can occur from o-quinones and reactive oxygen species (Park et al., 2006;
27 Balu et al., 2004; McCoull et al., 1999; Flowers-Geary et al., 1997, 1996), benzo[a]pyrene-
28 7,8-dione can induce mutations in the p53 tumor suppressor gene using an in vitro yeast reporter
29 gene assay (Park et al., 2008; Shen et al., 2006; Yu et al., 2002), and dominant p53 mutations
30 induced by benzo[a]pyrene,7,8-dione in this system corresponded with p53 mutation hotspots
31 observed in human lung cancer tissue (Park, 2008).
32 All three activation pathways are expected to occur in human tissues (Jiang et al., 2007),
33 and associations have been made between spectra of mutations in the p53 tumor suppressor gene
34 or ras oncogenes induced by benzo[a]pyrene metabolites with spectra of mutations in these genes
35 in tumor tissue from benzo[a]pyrene-exposed animals or humans. In particular, DeMarini et al.
36 (2001) demonstrated mutations in the p53 tumor suppressor gene and the K-ras oncogene in the
37 lung tumors of nonsmokers, whose tumors were associated with exposure to smoky coal.
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1 The available information supporting these actions for benzo[a]pyrene is consistent with
2 what is known about the mode of action for other PAHs demonstrated to induce cancer in
3 animals, including cyclopenta[cd]pyrene, dibenz[a,h]anthracene, and dibenzo[a,l]pyrene
4 (Cogliano et al., 2008; Straif et al., 2005). All PAHs that have been studied require metabolic
5 activation to produce carcinogenic responses in animals, and there is evidence for activation to
6 DNA reactive intermediates via several pathways (Straif et al., 2005; Xue and Warshawsky,
7 2005; WHO, 1998; Cavalieri and Rogan, 1995). For example, incubation of rat liver
8 microsomes with dibenzo[a,l]pyrene, a PAH that is more tumorigenically potent than
9 benzo[a]pyrene in mouse skin and rat mammary tissue, formed depurinated DNA adducts from
10 the radical cation pathway, as well as DNA adducts from the diol epoxide pathway (Cavalieri
11 and Rogan, 1995).
12 In summary, the relevance of animal bioassay data to the prediction of human
13 carcinogenic potency remains a significant area of uncertainty in the use of this and other
14 approaches to PAH cancer risk assessment. However, mechanistic data on benzo[a]pyrene and
15 other PAHs provide evidence that the molecular events leading to PAH-induced tumor formation
16 in animals are relevant to humans.
17
18 8.5. ASSUMPTIONS OF A COMMON MODE OF ACTION AND DOSE ADDITIVITY
19 A discussion of the potential modes of action for PAH carcinogenicity is presented in
20 Section 2.4. Individual carcinogenic PAHs are linked by a common effect (i.e., tumorigenicity),
21 which may occur through multiple mechanisms. Reactive metabolites produced during
22 metabolic transformations of PAHs include diol epoxides, reactive oxygen species, radical
23 cations, and o-quinones. The formation of these metabolites is not mutually exclusive, and the
24 carcinogenic process for PAHs is likely to be related to some combination of molecular events
25 resulting from formation of several reactive species. Reactive metabolites of PAHs interact with
26 DNA to form adducts and produce DNA damage resulting in mutations in cancer-related genes
27 such as tumor suppressor genes or oncogenes. These events appear to reflect the initiation
28 potency of an individual PAH (e.g., strong mutagens are generally potent initiators) (Sjogren et
29 al., 1996). Certain PAHs exhibit promotional effects that may be related to cytotoxicity and the
30 formation of reactive oxygen species, AHR affinity, and the upregulation of genes related to
31 biotransformation (i.e., induction of CYP1A1), growth, and differentiation (Bostrom et al.,
32 2002). The inhibition of gap junctional intracellular communication is also related to tumor
33 promotion by PAHs (Bostrom et al., 2002). The ability of certain PAHs to act as tumor
34 promoters as well as initiators may increase their carcinogenic potency in animal bioassays
35 conducted at high doses. Initiation potency may be more relevant to low-level environmental
36 exposure in humans (Bostrom et al., 2002; Sjogren et al., 1996); however, the proposed RPF
37 approach is not unduly affected by this as it relies largely on high-dose animal bioassay data for
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1 selecting RPF values. This represents an uncertainty in the use of the RPF approach in
2 estimating human cancer risks from PAHs.
3 Conceptually, the uncertainty related to relative potency for initiation versus promotion
4 could be reduced by using separate RPF schemes for each part of the carcinogenic process. This
5 would require selection of indicator compounds that best represent the initiation and promotion
6 processes, and use of mechanistic data to determine relative potency for each process (i.e.,
7 mutagenicity for initiation, AhR binding, or enzyme induction for promotion). There are several
8 problems with this approach, including the lack of data to support the selection of indicator
9 compounds and the complete carcinogenic nature of many PAHs (i.e., they act as both initiators
10 and promoters). The initiation and promotion potency of an individual PAH is determined by its
11 chemical structure. Some PAHs are strong mutagens, but have low affinity for the AhR (e.g.,
12 fjord-region PAHs) (Bostrum et al., 2002; Sjogren et al., 1996). Other PAHs are complete
13 carcinogens, with initiating properties (i.e., mutagenesis) and AhR affinity leading to tumor
14 promotion (e.g., benzo[a]pyrene, dibenz[a,h]anthracene) (Bostrum et al., 2002; Sjogren et al.,
15 1996). Benzo[a]pyrene is considered a good indicator compound for similar PAHs with
16 complete carcinogenic activity. However, the relative potency of other PAHs, especially those
17 that act primarily via either initiation or promotion, may be over- or underestimated.
18 There is evidence that an assumption of similar toxicological action is reasonable for
19 PAHs; however, the carcinogenic process for individual PAHs is likely to be related to some
20 unique combination of multiple molecular events resulting from formation of several reactive
21 species. The absence of a clearly-defined common mode of action increases the level of
22 uncertainty associated with the use of an RPF approach. It is not possible to determine whether
23 cancer risks would be under- or overestimated by using a PAH RPF approach that assumes a
24 common mode of action. The assumption that interactions among PAH mixture components do
25 not occur at low levels of exposure cannot be conclusively demonstrated using experimental
26 approaches. The experimental data relating to dose additivity for PAH carcinogenicity are
27 discussed in Section 2.8. It appears that interactions may occur at higher doses of PAH mixtures
28 given in combination. This remains a significant uncertainty in the proposed RPF approach.
29
30 8.6. EXTRAPOLATION OF RPFs ACROSS EXPOSURE ROUTES
31 The peer consultation workshop (U.S. EPA, 2002) also identified uncertainty in
32 extrapolation of RPFs across exposure routes. As with the 1993 Provisional Guidance., RPFs
33 proposed in this analysis are also based on in vivo bioassay data collected using various routes of
34 administration (e.g., dermal, intraperitoneal, subcutaneous, intramammillary, intramuscular, or
35 intravenous injection, as well as lung implantation, tracheal implantation, and transplacental
36 exposure after subcutaneous injection). The RPF approach considers each bioassay type
37 equivalent for the purpose of determining relative potency to benzo[a]pyrene.
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1 Table 8-3 compares the average RPFs (calculated from raw numbers and rounded to one
2 significant digit) based on tumor bioassay data for each PAH across exposure routes. Dermal
3 studies are shown collectively as well as separated by study type (complete or initiation).
4 Likewise, intraperitoneal studies are shown grouped as well as separated by target organ (lung
5 and liver).
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Table 8-3. Comparisons among average tumor bioassay RPF values by exposure route and target organ
PAH
AA
AC
BaA
BbcAC
(1,12-MBA)
BbF
BcFE
BeAC
BghiP
BjAC
BjF
BkF
B1AC
CH
CPcdP
CPdefC
DBacA
DBaeF
DBaeP
DBahA
DBahP
DBaiP
DBalP
FA
IP
N23eP
Dermal
n
1
-
1
1
2
-
2
-
-
2
1
2
5
4
2
-
2
2
1
1
2
2
-
-
1
Average
0.5
-
0.02
0.05
0.4
-
0.8
-
-
0.03
0.03
5
0.1
0.3
0.3
-
0.9
0.4
1
0.9
0.6
30
-
-
0.3
Dermal
complete
n
1
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
1
1
-
-
1
-
-
-
-
Average
0.5
-
-
-
0.3
-
-
-
-
-
-
-
-
0.4
-
-
1
0.3
-
-
0.7
-
-
-
-
Dermal initiation
n
-
-
1
1
1
-
2
-
-
2
1
2
5
2
2
-
1
1
1
1
1
2
-
-
1
Average
-
-
0.02
0.05
0.4
-
0.8
-
-
0.03
0.03
5
0.1
0.2
0.3
-
0.7
0.4
1
0.9
0.5
30
-
-
0.3
Intraperitoneal
n
-
-
2
-
2b
1
-
-
1
2b
-
-
1
1
-
-
-
-
1
-
-
1
5
-
-
Average
-
-
0.2a
-
lc
ld
-
-
60d
OT
-
-
0.2a
ld
-
-
-
-
40d
-
-
30d
0.08a
-
-
Intraperitoneal,
target organ =
lung
n
-
-
1
-
1
1
-
-
1
1
-
-
-
1
-
-
-
-
1
-
-
1
4
-
-
Average
-
-
0.08
-
1
1
-
-
60
0.4
-
-
-
1
-
-
-
-
40
-
-
30
0.05
-
-
Intraperitoneal,
target organ =
liver
n
-
-
2
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
1
-
-
Average
-
-
0.4
-
-
-
-
-
-
1
-
-
0.2
-
-
-
-
-
-
-
-
-
0.2
-
-
Lung
implantation
n
1
-
-
-
1
-
-
1
-
1
1
-
1
-
-
-
-
-
1
-
-
-
-
1
-
Average
0.2
-
-
-
0.1
-
-
0.009
-
0.03
0.03
-
0.04
-
-
-
-
-
2
-
-
-
-
0.07
-
Oral
n
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Average
-
-
-
-
-
50
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
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Table 8-3. Comparisons among average tumor bioassay RPF values by exposure route and target organ
PAH
PH
Pyr
Dermal
n
-
-
Average
-
-
Dermal
complete
n
-
-
Average
-
-
Dermal initiation
n
-
-
Average
-
-
Intraperitoneal
n
-
-
Average
-
-
Intraperitoneal,
target organ =
lung
n
-
-
Average
-
-
Intraperitoneal,
target organ =
liver
n
-
-
Average
-
-
Lung
implantation
n
-
-
Average
-
-
Oral
n
-
-
Average
-
-
"Newborn mouse model.
bNumber of intraperitoneal RPFs includes those calculated for combined lung and liver incidence; these are not included in numbers of RPFs with lung or liver tumors.
Includes both newborn mouse and adult A/J mouse models.
dAdult A/J mouse model.
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1
2 The table shows a marked difference between the oral and intraperitoneal RPFs for
3 benzo[c]fluorene (BcFE) (RPF = 50 for oral multiplicity and RPF = 1 for intraperitoneal
4 incidence). However, as discussed earlier, this difference may result more from the use of a high
5 tumor number to calculate the oral multiplicity RPF for this compound than route differences; if
6 the oral incidence RPF is used for comparison, the two routes are more similar (RPF = 1 for
7 intraperitoneal incidence versus RPF = 5 for oral incidence). Based on the latter comparison,
8 which represents the only data with which to compare oral RPFs with those calculated from
9 other routes, there appears to be fairly good correspondence between intraperitoneal and oral
10 RPFs; however, this is based on only one PAH.
11 Based on the comparisons in the table, RPFs based on initiation and complete dermal
12 carcinogenicity studies are similar (within a factor of 2). However, there are few PAHs with
13 both types of dermal studies.
14 With respect to other route comparisons, the table generally shows that RPFs calculated
15 from lung implantation and dermal studies are of the same order of magnitude, while RPFs
16 calculated from intraperitoneal studies are higher for most compounds. The intraperitoneal RPF
17 for dibenzo[a,l]pyrene is similar to its dermal RPF. At first glance, one might attribute the
18 higher intraperitoneal RPFs calculated from newborn mouse assays (footnoted "a" in the table)
19 to greater sensitivity of the newborn mouse, compared with an adolescent or adult mouse, to the
20 carcinogenic action of PAHs. However, since the RPFs reflect potency of the PAH relative to
21 benzo[a]pyrene, and not potency of the newborn mouse relative to other systems, the higher RPF
22 cannot reflect a greater sensitivity of the animal model, since both the PAH of interest and
23 benzo[a]pyrene have been tested in the same model. There is little information to evaluate
24 whether RPFs from newborn mouse studies tend to be higher or lower than the adult A/J mouse
25 model when both are exposed via intraperitoneal injection. Only one compound,
26 benzo[b]fluoranthene (BbF), had RPFs calculated from both newborn mouse and adult A/J
27 mouse models, and the values were similar; the newborn mouse RPF was 2, while the A/J mouse
28 RPF was 1. In summary, it is not clear whether the intraperitoneal RPFs are higher than dermal
29 or lung implantation RPFs due to route-specific differences or animal model differences (for
30 example, differential metabolism in various animal systems).
31 Cross-route extrapolation of relative potency estimates is a necessary, though uncertain,
32 aspect of the RPF approach. It is difficult to determine which of the available study types (e.g.,
33 dermal, intraperitoneal, intratracheal) is most predictive of potential risks from oral and
34 inhalation exposure in humans. In order to prioritize bioassays by exposure route, robust data
35 are needed on relative potencies for oral and inhalation exposures for comparison with relative
36 potencies based on other exposure routes.
37 The inhalation RPF scheme used by the California EPA (2004) employed a hierarchy of
38 bioassay data based on exposure route (inhalation studies were preferred, followed by
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1 intratracheal or intrapulmonary instillation, oral administration, skin-painting, and subcutaneous
2 or intraperitoneal injection). Apart from the obvious preference for exposure routes that targeted
3 the respiratory tract (inhalation, intratracheal, intrapulmonary), the basis for prioritizing the other
4 exposure routes is not evident. Pufulete et al. (2004), who were also focused on PAHs as air
5 contaminants, suggested that the clearance of PAHs after intratracheal instillation may be similar
6 to clearance after inhalation exposure. The authors acknowledged that the high concentrations of
7 PAHs used in intratracheal and intrapulmonary instillation studies may lead to major differences
8 in pharmacokinetics, compared with inhalation exposure (Pufulete et al., 2004). Nevertheless,
9 the authors suggested that intratracheal instillation of low doses of PAHs might be an appropriate
10 surrogate exposure model for assessing relative potency of inhalation exposure. It is important
11 to note that no intratracheal instillation studies were identified in the search for studies from
12 which to calculate RPFs; thus, the information provided by Pufulete et al. (2004) is not directly
13 useful for suggesting route-specific RPFs. Pufulete et al. (2004) did not provide any specific
14 information on the relevance of intrapulmonary administration (a route used in several of the
15 bioassays used to calculate RPFs) to inhalation exposure.
16 As noted by U.S. EPA (2004), cross-route extrapolation would be contraindicated if there
17 were convincing toxicokinetic evidence that absorption of PAHs does not occur by one or more
18 exposure routes. Available data on the absorption of PAHs indicate that, in general, PAHs are
19 readily absorbed via ingestion, inhalation, and dermal exposure routes; however, the rate of
20 uptake varies with route and other factors (e.g., matrix, intake of fats and oils) (ATSDR, 1995).
21 Evidence for absorption of PAHs through these routes includes measurement of PAH-DNA
22 adducts at sites distal from the route of entry, measurement of urinary metabolites, and
23 radiotracer studies in animals (ATSDR, 1995). U.S. EPA (2004) indicated that demonstration of
24 any degree of uptake for each of the routes of interest is sufficient to allow the qualitative
25 judgment to apply the route-to-route extrapolation; thus, cross-route extrapolation is supported
26 by current data on the bioavailability of PAHs across several exposure routes.
27 U.S. EPA (2004, 1994) also noted that point-of-entry toxicity may be considered contrary
28 evidence for cross-route extrapolation. With respect to PAHs, available information on this issue
29 is mixed. The one inhalation bioassay of benzo[a]pyrene (Thyssen et al., 1981) identified the
30 upper respiratory tract as the site of tumor formation, suggesting a point-of-entry effect;
31 however, the authors did not specify the organs that were examined histologically in the study.
32 Dermal bioassays of benzo[a]pyrene have generally evaluated only skin tumors, precluding their
33 use in determining whether distal tumors are induced. A number of early oral cancer bioassays
34 of benzo[a]pyrene suggested that tumor formation was limited to the forestomach (Rigdon and
35 Neal, 1969, 1966; Neal and Rigdon, 1967). In oral carcinogenicity bioassays of MGP residue
36 (Weyand et al., 1995) and coal tar preparations (Culp et al., 1998; Gaylor et al., 1998) that
37 included separate groups exposed to benzo[a]pyrene, there were significant differences in target
38 organ distribution of tumors between benzo[a]pyrene and the complex mixtures.
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1 Benzo[a]pyrene-induced tumors were observed primarily at the point of contact (i.e., the
2 forestomach), while MGP residue and coal tar produced tumors in the lung, liver, forestomach,
3 skin, and other organs. Other PAHs (e.g., benzo[c]fluorene) were proposed as the primary
4 compounds responsible for tumors at distal sites such as the lung (Koganti et al., 2000; Gulp et
5 al., 1998). However, a gavage study in rats (Kroese et al., 2001) and a dietary study in A/J mice
6 (Weyland et al., 2004) each demonstrated that oral exposure to benzo[a]pyrene could induce
7 tumors at distal sites, including the lung, liver, and auditory canal. Tissue-specific differences in
8 metabolic activation and DNA binding of PAHs may contribute to the observed differences in
9 target organ sensitivity (Weyand and Wu, 1995; Gulp and Beland, 1994).
10 In summary, available information provides some support for cross-route extrapolation.
11 Absorption of PAHs across oral, inhalation, and dermal routes is evident and, while many of the
12 cancer bioassays of benzo[a]pyrene suggested tumor formation limited to the point-of-entry, at
13 least one recent study (Kroese et al., 2001) suggests that tumors may also be induced at distal
14 sites. Furthermore, there is evidence that other PAHs (e.g., benzo[c]fluorene) may induce
15 tumors at distal sites after oral exposure (Weyand et al., 2004; Koganti et al., 2000; Gulp et al.,
16 1998). However, cross-route extrapolation of RPFs is a significant source of uncertainty in this
17 approach.
18 Another approach to the issue of route-to-route extrapolation would be to prefer RPFs
19 derived from particular target tissues deemed relevant to the exposure route of interest. For
20 example, RPFs based on lung tumor data might be preferred for use in inhalation risk
21 assessment. To examine whether lung tumor RPFs were consistent across routes, RPFs
22 calculated from lung tumor potency in intraperitoneal studies (both newborn mouse and adult
23 A/J mouse models) were compared with RPFs from lung implantation studies in Table 8-3.
24 RPFs for both intraperitoneal-lung and lung implantation studies were available for only four
25 compounds (benzo[b]fluoranthene, benzo[j]fluoranthene, chrysene, and dibenz[a,h]anthracene);
26 for each of these, the intraperitoneal lung tumor RPF exceeded the lung implantation RPF. No
27 information assessing the concordance between lung tumor potency after intraperitoneal
28 administration and inhalation cancer potency was identified in the literature. The use of the final
29 RPFs derived in this analysis across all routes of exposure is recommended given the information
30 outlined above and in the absence of data to indicate otherwise.
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APPENDIX A. SECONDARY SOURCES REVIEWED FOR IDENTIFICATION OF
PRIMARY LITERATURE
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hydrocarbons (PAHs). Atlanta, GA.
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Proposition 65 carcinogens benzo[b]fluoranthene, benzo[j]fluoranthene, chrysene, dibenzo[a,h]pyrene,
dibenzo[a,i]pyrene, and 5-methyl chrysene by the oral route. Office of Environmental Health Hazard Assessment,
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prepared for the U.S. EPA. September 30, 1990.
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hydrocarbons and polycyclic aromatic hydrocarbon derivatives. Regul Toxicol Pharmacol 28:45-54.
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chemicals to humans. Lyon, France, pp. 65-159.
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oil and major petroleum fuels. In: IARC monographs on the evaluation of carcinogenic risk of chemicals to
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assessment for exposure to PAH. In: Fifteenth international symposium on polycyclic aromatic compounds:
chemistry, biology and environmental impact. Belgirate, Italy, 19-22 September 1995. Ispra, Joint Research Centre
European Commission, pp. 161.
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environmental exposures in Canada. Environ Carcinog Ecotoxicol Rev C 12(2):443-452.
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hydrocarbons (PAH). Part I. Hazard identification and dose-response assessment. Ontario: Ontario Ministry of
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Regul Toxicol Pharmacol 16:290-300.
Petry, T; Schmid, P; Schlatter, C. (1996) The use of toxic equivalency factors in assessing occupational and
environmental health risk associated with exposure to airborne mixtures of polycyclic aromatic hydrocarbons
(PAHs). Chemosphere 32(4):639-648.
Rugen, PJ; Stern, CD; Lamm, SH. (1989) Comparative carcinogenicity of the PAHs as abasis for acceptable
exposure levels (AELs) in drinking water. Regul Toxicol Pharmacol 9(3):273-283.
Schneider, K; Roller, M; Kalberlah, F; et al. (2002) Cancer risk assessment for oral exposure to PAH mixtures. J
Appl Toxicol 22(l):73-83.
Sjogren, M; Ehrenberg, L; Rannug, U. (1996) Relevance of different biological assays in assessing initiating and
promoting properties of polycyclic aromatic hydrocarbons with respect to carcinogenic potency. Mutat Res
358(1):97-112.
Slooff, W; Janus, JA; Matthijsen, AJCM; et al. (1989) Integrated criteria document PAHs (PDF includes addendum
by Montizaan). Bilthoven, The Netherlands. National Institute of Public Health and the Environment (RIVM).
SRC (Syracuse Research Corporation). (1993) Estimating cancer risk from exposure to PAHs: a relative potency
approach. SRC TR-93-045. Draft report prepared for U.S. EPA, Environmental Criteria and Assessment Office,
Cincinnati, OH.
U.S. EPA (Environmental Protection Agency). (1990) Drinking water criteria document for polycyclic aromatic
hydrocarbons. Cincinnati, OH: Office of Health and Environmental Assessment, Environmental Criteria and
Assessment Office.
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U.S. EPA (Environmental Protection Agency). (1993) Provisional guidance for quantitative risk assessment of
polycyclic aromatic hydrocarbons. Cincinnati, OH: Office of Health and Environmental Assessment,
Environmental Criteria and Assessment Office.
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1 APPENDIX B. BIBLIOGRAPHY OF STUDIES WITHOUT BENZO[A]PYRENE AS A
2 REFERENCE COMPOUND
3
4
5
6
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Table B-l. Bioassays with and without benzo[a]pyrene by PAH
PAH"
Aceanthrylene
Acenaphthene
Acenaphthylene
Acephenanthrylene
Acepyrene, 2,3-
Anthanthrene
Anthracene
Benz[a]anthracene
Benz[b]anthracene
Benz[b,c]aceanthrylene, 11H-
Benz[e]aceanthrylene
Benz[j]aceanthrylene
Benz[l]aceanthrylene
Benzacenaphthylene
Benzo [a] fluoranthene
Benzo [a] fluorene
Benzo[a]perylene
Benzo [b] chrysene
Benzo[b]fluoranthene
HH-Benzo[b]fluorene
Benzo[b]perylene
Benzo[c]chrysene
Benzo [c] fluorene
Benzo [c] phenanthrene
Benzo[e]pyrene
Benzo[g]chrysene
Benzo[g,h,i]fluoranthene
Benzo[g,h,i]perylene
Benzo[j]fluoranthene
Benzo[k]fluoranthene
Benzophenanthrene
Chrysene
Coronene
Cyclopenta[c,d]pyrene
Cyclopenta[d,e,f]chrysene, 4H-
Cyclopenta[d,e,f]phenanthrene, 4H-
Cyclopenta[h,i]acephenanthrylene
CASRN
202-03-09
83-32-9
208-96-8
201-06-9
25732-74-5
191-26-4
120-12-7
56-55-3
92-24-9
202-94-8
199-54-2
202-33-5
211-91-6
76774-50-0
203-33-8
238-84-6 or
30777-18-5
191-85-5
214-17-5
205-99-2
243- 17-4 or
30777-19-6
197-70-6
194-69-4
205-12-9 or
30777-20-9
195-19-7
192-97-2
196-78-1
203-12-3
191-24-2
205-82-3
207-08-9
65777-08-4
218-01-9
191-07-1
27208-37-3
202-98-2
203-64-5
114959-37-4
Bioassays with benzo[a]pyrene
Dermal
Initiation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Complete
X
X
X
X
X
X
X
X
X
X
X
X
X
Intra-
peritoneal
X
X
X
X
X
X
X
Sub-
cutaneous
X
X
X
Oral
X
Other
X
X
X
X
X
X
X
X
Bioassays without benzo[a]pyrene
Dermal
Initiation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Complete
X
X
X
X
X
Intra-
peritoneal
X
X
X
X
X
X
X
X
X
Sub-
cutaneous
X
X
X
X
Oral
X
X
Other
X
X
X
X
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Table B-l. Bioassays with and without benzo[a]pyrene by PAH
PAH"
Cyclopenta[h,i]aceanthrylene
Cyclopentaphenanthrene
Cyclopenteno-l,2-benzanthracene, 5,6-
Dibenz[a,c]anthracene
Dibenzo [a,e]fluoranthene
Dibenz[a,j]anthracene
Dibenzo [b,e]fluoranthene
Dibenzo [a,c]fluorene, 13H-
Dibenzo [a,e]pyrene
Dibenzo [a,f]fluoranthene
Dibenzo[a,g]fluorene, 13H-
Dibenz[a,h]anthracene
Dibenzo [a,h]pyrene
Dibenzo [a,i]pyrene
Dibenzo [a,l]pyrene
Dibenzo [e,l]pyrene
Dibenzo [h,rst]pentaphene
Dibenz[k,mno]acephenanthrylene
Dibenzo [j ,mno]acephenanthrylene
Dihydroaceanthrylene, 1,2-
Fluoranthene
Fluorene
Indeno [ 1 ,2,3-c,d]fluoranthene
Indeno [ 1 ,2,3-c,d]pyrene
Naphtho [1 ,2-b] fluoranthene
Naphtho[l,2,3,-nmo]acephenanthrylene
Naphtho [2,1 -a] fluoranthene
Naphtho[2,3-a]pyrene
Naphtho[2,3-e]pyrene
Pentacene
Pentaphene
Perylene
Phenanthrene
Picene
Pyrene
Tribenzofluoranthene 3,4-10, 11-12,13-
Triphenylene
CASRN
131581-33-4
219-08-9
7099-43-6
215-58-7
5385-75-1
224-41-9
2997-45-7
201-65-0
192-65-4
203-11-2
207-83-0
53-70-3
189-64-0
189-55-9
191-30-0
192-51-8
192-47-2
153043-81-3
153043-82-4
641-48-5
206-44-0
86-73-7
193-43-1
193-39-5
111189-32-3
113779-16-1
203-20-3
196-42-9
193-09-9
135-48-8
222-93-5
198-55-0
85-01-8
213-46-7
129-00-0
13579-05-0
217-59-4
Bioassays with benzo[a]pyrene
Dermal
Initiation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Complete
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Intra-
peritoneal
X
X
X
X
X
X
Sub-
cutaneous
X
X
Oral
X
X
Other
X
X
X
X
Bioassays without benzo[a]pyrene
Dermal
Initiation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Complete
X
X
X
X
X
X
X
X
X
Intra-
peritoneal
X
X
X
X
X
X
X
X
Sub-
cutaneous
X
X
X
X
X
X
X
X
Oral
X
X
Other
X
X
X
X
X
"PAHs in bold have at least one bioassay without benzo[a]pyrene and no bioassays with benzo[a]pyrene.
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B.I. BIBLIOGRAPHY OF BIOASSAYS WITHOUT BENZO[A]PYRENE
Amin, S; Huie, K; Hecht, SS; (1985) Mutagenicity and tumor-initiating activity of methylated
benzo[b]fluoranthenes. Carcinogenesis 6:1023-1025.
Amin, S; Hussain, N; Balanikas, G; et al. (1985) Mutagenicity and tumor initiating activity of methylated benzo[k]
fluoranthenes. Cancer Lett 26:343-347.
Amin, S; Misra, B; Braley, J; et al. (1991) Comparative tumorigenicity in newborn mice of chrysene and
5-alkylchrysene-l,2-diol-3,4-epoxides. Cancer Lett 58:115-118.
Amin, S; Weyand, EH; Huie, K; etal. (1991) Effects of fluorene substitution on benzo[b]fluoranthene tumorgenicity
and DNA adduct formation in mouse skin. In: Cooke, M; Loening, K; Merritt, J, eds. Polynuclear aromatic
hydrocarbons: measurements, means and metabolism. Columbus, OH, Battelle Press, pp. 25-35.
Amin, S; Desai, D; Dai, W; et al. (1995) Tumorigenicity in newborn mice of fjord region and other sterically
hindered diol epoxides of benzo[g]chrysene, dibenzo[a,l]pyrene (dibenzo[def,p]chrysene),
4H-cyclopenta[def]chrysene and fluoranthene. Carcinogenesis 16:2813-2817.
Barry, G; Cook, CW; Amin, S; et al. (1934) A comparison of the action of some polycyclic aromatic hydrocarbons
in producing tumours of connective tissue. Am J Cancer 20:58-69.
Bhatt, TS; Coombs, MM. (1990) The carcinogenicity of cyclopenta[a]phenanthrene and chrysene derivatives in the
Sencar mouse. Polycycl Aromat Compd 1:51-58.
Bock, FG; King, DW. (1959) A study of the sensitivity of the mouse forestomach toward certain polycyclic
hydrocarbons. J Natl Cancer Inst 23:833-838.
Bottomley, AC; Twort, CC. (1934) The carcinogenicity of chrysene andoleic acid. Am J Cancer 21:781-786.
Boyland, E; Burrows, H. (1935) The experimental production of sarcoma in rats and mice by a colloidal aqueous
solution of l:2:5:6-dibenzanthracene. J Pathol Bacteriol 41:231-238.
Boyland, E; Sims, P. (1967) The carcinogenic activities in mice of compounds related to benz[a]anthracene. Int J
Cancer 2:500-504.
Buening, MK; Levin, W; Karle, JM; et al. (1979) Tumorigenicity of bay-region epoxides and other derivatives of
chrysene and phenanthrene in newborn mice. Cancer Res 39:5063-5068.
Buening, MK; Levin, W; Wood, A; et al. (1979) Tumorigenicity of the dihydrodiols of dibenz[a,h]anthracene on
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B.2. BIBLIOGRAPHY OF STUDIES ON CANCER-RELATED ENDPOINTS
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B-20 DRAFT - DO NOT CITE OR QUOTE
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APPENDIX C. DOSE-RESPONSE DATA FOR POTENCY CALCULATIONS
C-1 DRAFT - DO NOT CITE OR QUOTE
-------
Table C-l. Dermal bioassays: dose-response information for incidence data
Record
number
Reference
Study
type
Species
Tumor
type
PAH
Sex
Dose
of
PAH
Dose
units
Number of
animals
with tumors
Number
of
animals
in group
%
Tumor-
bearing
animals
Results of
authors'
statistical
analysis
(p-value)
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
Comments
Complete carcinogenicity studies
600
13640
Habsetal.,
1980
Cavalieri et
al., 1983
Complete
Complete
Mice
Mice
Sum of
Papilloma,
carcinoma,
sarcoma
Papilloma,
adenoma,
carcinoma
Acetone
DMSO
BaP
BaP
BaP
BbF
BbF
BbF
BjF
BjF
BjF
BkF
BkF
BkF
CPcdP
CPcdP
CPcdP
IP
IP
IP
CO
CO
Acetone
BaP
BaP
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
0
0
1.7
2.8
4.6
3.4
5.6
9.2
3.4
5.6
9.2
3.4
5.6
9.2
1.7
6.5
27.2
3.4
5.6
9.2
5.6
15
0
2.2
6.6
Hg/animal
Hg/animal
Hg/animal
jig/animal
Hg/animal
Hg/animal
jig/animal
Hg/animal
Hg/animal
jig/animal
(ig/animal
Hg/animal
jig/animal
(ig/animal
Hg/animal
jig/animal
(ig/animal
Hg/animal
jig/animal
(ig/animal
Hg/animal
jig/animal
nmol
nmol
nmol
0
0
8
24
22
2
5
20
1
1
2
1
0
0
0
0
3
1
0
0
1
2
0
2
2
35
36
34
35
36
38
34
37
38
35
38
39
38
38
34
35
38
36
37
37
39
40
29
30
28
0
0
24
69
61
5
15
54
3
3
5
3
0
0
0
0
8
3
0
0
3
5
0
7
7
1.92x 10'3
1.67 x 10'11
2.1 x 10'9
2.6 x 10'1
2.3 x 10'2
3.7 x 10'8
5.1 x 10'1
4.9 x 10'1
2.6 x 10'1
5.2 x 10'1
1.3 x 10'1
5 x 10'1
0.52
0.27
0.25
0.24
2.15 x 10'9
1.33 x 10'9
1.77x 10'1
6.36 x 10'2
1.83 x 10'1
C-2
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-l. Dermal bioassays: dose-response information for incidence data
Record
number
620
17660
Reference
Hoffmann
and Wynder
1966
Cavalieri et
al., 1977
Study
type
Complete
Complete
Species
Mice
Mice
Tumor
type
Papilloma
Papilloma,
kerato-
acanthoma,
carcinoma
PAH
BaP
CPcdP
CPcdP
CPcdP
Dioxane
BaP
BaP
DBaeP
DBaeP
DBahP
DBahP
DBaiP
DBaiP
DBaeF
DBaeF
Acetone
BaP
DBahP
AA
BaA
Sex
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Dose
of
PAH
20
22.2
66.6
200
0
0.05
0.1
0.05
0.1
0.05
0.1
0.05
0.1
0.05
0.1
0
0.396
0.396
0.396
0.396
Dose
units
nmol
nmol
nmol
nmol
%
%
%
%
%
%
%
%
%
%
%
|imol/ap-
plication
|imol/ap-
plication
|imol/ap-
plication
|imol/ap-
plication
|imol/ap-
plication
Number of
animals
with tumors
17
2
2
24
0
17
19
16
9
16
15
16
16
17
18
0
30
35
18
1
Number
of
animals
in group
30
29
29
29
20
20
20
30
17
17
18
19
19
19
19
29
38
39
38
39
%
Tumor-
bearing
animals
57
7
7
83
0
85
95
53
53
94
83
84
84
89
95
0
79
90
47
3
Results of
authors'
statistical
analysis
(p-value)
Fisher's
exact
p-value
4.32 x 10'7
0.25
0.25
9.25 x 10'12
1.28x 10'8
1.5 x 10'10
3.31 x 10'5
1.95 x 10'4
1.32x 10'9
5.27 x 10'8
2.58 x 10'9
2.58 x 10'9
3.35 x 10'9
3.05 x 10'10
4.9 x 10'12
2.98 x 10'15
3.59 x 10'6
0.66
Cochran-
Armitage
trend test
p-value
2.96 x 10'1
1.39 x 10'16
8.7 x 10'10
5.69 x 10'4
1.29x 10'7
9.81 x 10'8
1.13 x 10'9
Comments
C-3
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-l. Dermal bioassays: dose-response information for incidence data
Record
number
Reference
Study
type
Species
Tumor
type
PAH
Sex
Dose
of
PAH
Dose
units
Number of
animals
with tumors
Number
of
animals
in group
%
Tumor-
bearing
animals
Results of
authors'
statistical
analysis
(p-value)
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
Comments
Initiation studies
630
18570
24800
LaVoie et
al., 1982
Hecht et al.,
1974
Nesnow et
al., 1984
Initiation
Initiation
Initiation
Mice
Mice
Mice
Primarily
squamous
cell
papilloma
Unspeci-
fied
Papilloma
Acetone/
TPA
BaP
BbF
BbF
BbF
BjF
BjF
BjF
BkF
BkF
BkF
Acetone
BaP
CH
Acetone
Acetone
BaP
BaP
B1AC
B1AC
B1AC
B1AC
F
F
F
F
F
F
F
F
F
F
F
F
F
F
M
F
M
F
M
M
M
M
0
30
10
30
100
30
100
1,000
30
100
1,000
0
0.05
1
0
0
200
200
50
100
250
500
Hg/mouse
Hg/mouse
Hg/mouse
jig/mouse
Hg/mouse
Hg/mouse
jig/mouse
Hg/mouse
Hg/mouse
jig/mouse
Hg/mouse
mg/mouse
mg/mouse
mg/mouse
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
0
17
9
12
16
6
11
19
1
5
15
0
6
11
0
1
13
10
12
16
21
16
20
20
20
20
20
20
20
20
20
20
20
20
20
19
20
19
18
19
20
17
21
16
0
85
45
60
80
30
55
95
5
25
75
0
30
58
0
5
67
53
60
94
100
100
<0.005
0.005
O.005
<0.005
0.005
O.005
1.28x 10'8
6.14 x 10'4
2.25 x 10'5
7.7 x 10'8
0.01
7.27 x 10'5
1.52x 10'10
0.01
0.02
3.85 x 10'7
0.01
4.51 x 10'5
1.46x 10'5
4.67 x 10'8
4.51 x lO'9
Number of
surviving not
reported for
controls; initial
group size used
here
Data at 30 wks
C-4
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-l. Dermal bioassays: dose-response information for incidence data
Record
number
21420
Reference
Slagaetal.,
1980
Study
type
Initiation
Species
Mouse
Tumor
type
Papilloma
PAH
B1AC
B1AC
B1AC
B1AC
B1AC
B1AC
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
Control
Control
Control
Control
Control
pooled
BaP
BeP
CH
DBacA
DBahA
Sex
M
F
F
F
F
F
M
M
M
M
M
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Dose
of
PAH
1,000
50
100
250
500
1,000
50
100
250
500
1,000
50
100
250
500
1,000
0
0
0
0
0
200
2,000
2,000
2,000
100
Dose
units
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
jimol
(imol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
Number of
animals
with tumors
19
13
18
19
20
20
4
4
12
15
16
4
7
10
8
18
2
3
3
2
10
20
5
21
8
15
Number
of
animals
in group
20
20
19
21
21
20
20
20
20
20
18
20
19
19
18
20
30
30
30
29
119
30
29
29
28
29
%
Tumor-
bearing
animals
95
65
95
91
95
100
20
20
60
75
89
20
37
53
44
90
6
10
10
6
8
67
17
73
27
50
Results of
authors'
statistical
analysis
(p-value)
O.005
<0.005
<0.005
O.005
<0.005
<0.005
<0.005
0.005
<0.005
0.005
0.005
O.005
0.005
Fisher's
exact
p-value
1.41 x 10'6
0.33
8.38 x 10'7
0.07
3.52 x 10'6
Cochran-
Armitage
trend test
p-value
Comments
Different
controls used for
each chemical
except DBacA
and BeP
C-5
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-l. Dermal bioassays: dose-response information for incidence data
Record
number
15640
620
13650
Reference
Raveh et al.,
1982
Hoffmann
and Wynder
1966
Cavalieri et
al., 1981b
Study
type
Initiation
Initiation
Initiation
Species
Mice
Mice
Mice
Tumor
type
Papilloma
Papilloma
Papilloma
PAH
Control
BaP
BaP
BaP
BaP
BaP
CPcdP
CPcdP
CPcdP
Croton oil
control
BaP
DBaeF
DBaeP
DBelP
DBahP
DBaiP
AA
BghiP
N23eP
IP
Acetone/
TPA
BaP
CPcdP
CPcdP
CPcdP
ACEP
ACEP
Sex
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Dose
of
PAH
0
10
25
50
100
200
10
100
200
0
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0
0.2
0.2
0.6
1.8
0.2
0.6
Dose
units
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
(imol
(imol
(imol
jimol
(imol
(imol
jimol
Number of
animals
with tumors
3
17
21
24
27
26
3
11
16
2
24
18
10
0
21
12
2
2
9
5
3
12
1
9
6
0
1
Number
of
animals
in group
29
29
28
28
27
26
30
29
28
30
30
30
27
29
29
30
29
27
30
30
29
30
30
29
29
30
30
%
Tumor-
bearing
animals
10
58
76
87
100
100
11
39
57
7
80
60
37
0
72
40
7
7
30
17
10
40
3
31
21
0
3
Results of
authors'
statistical
analysis
(p-value)
Fisher's
exact
p-value
1.11 x 10'4
5.96 x 10'7
5.43 x 10'9
5.50 x 10'13
1.03 x lO'12
0.65
0.01
1.90x 10'4
3.80 x 10'9
9.40 x 10'6
0.006
0.25
1.30x 10'7
0.002
0.68
0.65
0.02
0.21
0.009
0.29
0.05
0.24
0.11
0.29
Cochran-
Armitage
trend test
p-value
2.78 x 10'10
2.75 x lO'6
0.14
Comments
C-6
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-l. Dermal bioassays: dose-response information for incidence data
Record
number
15700
Reference
Rice et al,
1988
Study
type
Initiation
Species
Mice
Tumor
type
Unspeci-
fied
PAH
ACEP
Acetone
BaP
CH
CH
CH
CPdefC
(4,5-MC)
CPdefC
(4,5-MC)
CPdefC
(4,5-MC)
BbcAC
(1,12-
MBA)
BbcAC
(1,12-
MBA)
BbcAC
(1,12-
MBA)
Sex
F
F
F
F
F
F
F
F
F
F
F
F
Dose
of
PAH
1.8
0
0.1
0.15
0.5
1.5
0.15
0.5
1.5
0.5
2
4
Dose
units
(imol
(imol
jimol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
Number of
animals
with tumors
4
1
17
5
18
19
13
19
19
15
18
18
Number
of
animals
in group
30
20
19
20
20
20
20
19
19
20
20
20
%
Tumor-
bearing
animals
13
5
89
25
90
95
65
100
100
75
90
90
Results of
authors'
statistical
analysis
(p-value)
<0.005
0.05
O.005
<0.005
0.005
0.005
O.005
O.005
0.005
0.005
Fisher's
exact
p-value
0.52
Cochran-
Armitage
trend test
p-value
0.18
6.39 x 10'9
1.90x 10'7
3.03 x 10'6
Comments
C-7
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-2. Dermal bioassays: dose-response information for tumor multiplicity
Record
number
Reference
Study
type
Species
Tumor type
PAH
Sex
Dose of
PAH
Dose units
Number of
animals
with
tumors
Number
of
animals
in
group
%
Tumor-
bearing
animals
Results of
authors'
statistical
analysis
(p-value)
Results of
SRC
statistical
analysis
Fisher's
exact p-value
Mean
number
tumors/
animal
Comments
Complete carcinogenicity
13640
13650
Cavalierietal.,
1983
Cavalieri etal.,
1981b
Complete
Complete
Mice
Mice
Papilloma,
adenoma,
carcinoma
Primarily
squamous
cell
carcinoma
Acetone
BaP
BaP
BaP
CPcdP
CPcdP
CPcdP
Acetone
BaP
CPcdP
CPcdP
CPcdP
ACEP
ACEP
ACEP
F
F
F
F
F
F
F
US
US
US
US
US
US
US
US
0
2.2
6.6
20
22.2
66.6
200
0
0.2
0.2
0.6
1.8
0.2
0.6
1.8
nmol
nmol
nmol
nmol
nmol
nmol
nmol
|imol/
application
(imol/
application
(imol/
application
(imol/
application
|imol/
application
|imol/
application
|imol/
application
(imol/
application
0
2
2
17
2
2
24
0
30
17
11
7
0
1
1
29
30
28
30
29
29
29
30
30
30
30
30
30
30
30
0
7
7
57
7
7
83
0
100
57
37
23
0
3
3
>0.05
>0.05
0.001
>0.05
>0.05
0.001
0.001
0.001
0.001
0.0053
>0.05
>0.05
>0.05
0
0.07
0.07
1.5
0.07
0.07
2.45
0
1.5
0.8
0.5
0.4
0
0.03
0.03
Number tumors per
animal at risk
calculated
Number tumors per
animal at risk
calculated
Initiation
630
LaVoie etal.,
1982
Initiation
Mice
Primarily
squamous
cell
papilloma
Acetone/
TPA
BaP
BbF
BbF
BbF
F
F
F
F
F
0
30
10
30
100
|xg/mouse
|ig/mouse
|xg/mouse
|ig/mouse
|ig/mouse
0
17
9
12
16
20
20
20
20
20
0
85
45
60
80
0.001
O.001
0.001
O.001
0
4.9
0.9
2.3
7.1
C-8
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-2. Dermal bioassays: dose-response information for tumor multiplicity
Record
number
18570
21420
15640
Reference
Hecht et al,
1974
Slagaetal.,
1980
Raveh et al.,
1982
Study
type
Initiation
Initiation
Initiation
Species
Mice
Mouse
Mice
Tumor type
Unspecified
Papilloma
Papilloma
PAH
BjF
BjF
BjF
BkF
BkF
BkF
Acetone
BaP
CH
Control
Control
Control
Control
Control
pooled
BaP
BeP
CH
DBacA
DBahA
Control
BaP
BaP
BaP
BaP
BaP
CPcdP
Sex
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Dose of
PAH
30
100
1,000
30
100
1,000
0
0.05
1
0
0
0
0
0
200
2,000
2,000
2,000
100
0
10
25
50
100
200
10
Dose units
|ig/mouse
|ig/mouse
|ig/mouse
|xg/mouse
jig/mouse
|ig/mouse
mg/animal
mg/animal
mg/animal
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
re
re
re
re
re
re
re
Number of
animals
with
tumors
6
11
19
1
5
15
0
6
11
2
3
3
2
10
20
5
21
8
15
3
17
21
24
27
26
3
Number
of
animals
in
group
20
20
20
20
20
20
20
20
19
29
30
30
29
119
30
29
29
28
29
29
29
28
28
27
26
30
%
Tumor-
bearing
animals
30
55
95
5
25
75
0
30
61
6
10
10
6
8
67
17
73
27
50
10
58
76
87
100
100
11
Results of
authors'
statistical
analysis
(p-value)
Results of
SRC
statistical
analysis
Fisher's
exact p-value
0.01
O.001
0.001
>0.05
0.02
0.001
0.01
0.001
O.001
>0.05
O.001
>0.05
0.001
O.001
O.001
0.001
O.001
0.001
>0.05
Mean
number
tumors/
animal
0.6
1.9
7.2
0.1
0.4
2.8
0
0.5
1
0.1
0.2
0.1
0.1
0.13
2.2
0.2
1.6
0.5
1.4
0.2
1.3
3.8
6.2
8.8
9
0.1
Comments
Number surviving
not reported for
controls; initial
group size used
here; number
tumors per animal
at risk calculated
Different controls
used for each
chemical except
DBacA and BeP
C-9
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-2. Dermal bioassays: dose-response information for tumor multiplicity
Record
number
13650
21410
16310
10200
24300
Reference
Cavalierietal.,
1981
Slagaetal.,
1978
Weyandetal,
1992
El-Bayoumy et
al., 1982
Rice et al.,
1985
Study
type
Initiation
Initiation
Initiation
Initiation
Initiation
Species
Mice
Mice
Mice
Mice
Mice
Tumor type
Papilloma
Papilloma
Unspecified
Primarily
squamous
cell
papilloma
Unspecified
PAH
CPcdP
CPcdP
Acetone/
TPA
BaP
CPcdP
CPcdP
CPcdP
ACEP
ACEP
ACEP
Acetone/
TPA
BaP
BaA
Acetone
BaP
BjF
BjF
BjF
Acetone
BaP
CH
Pery
Pyr
Acetone
BaP
CH
Sex
F
F
F
F
F
F
F
F
F
F
F
F
F
US
US
US
US
US
F
F
F
F
F
F
F
F
Dose of
PAH
100
200
0
0.2
0.2
0.6
1.8
0.2
0.6
1.8
0
0.2
2
0
0.01
0.3
1
2
0
0.05
1
1
1
0
0.3
1
Dose units
re
re
(imol
(imol
|imol
|imol
(imol
(xmol
jimol
|imol
(xmol
(xmol
(imol
(imol
(imol
(xmol
(imol
|imol
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
mg/mouse
Number of
animals
with
tumors
11
16
3
12
1
9
6
0
1
4
2
27
17
1
24
11
21
24
1
18
20
1
4
2
24
23
Number
of
animals
in
group
29
28
29
30
30
29
29
30
30
30
29
29
30
21
24
20
24
24
20
20
20
20
20
25
25
25
%
Tumor-
bearing
animals
39
57
10
40
3
31
21
0
3
13
6
92
57
5
100
55
88
100
5
90
100
5
20
8
96
92
Results of
authors'
statistical
analysis
(p-value)
0.01
O.01
<0.01
0.01
O.01
O.01
Results of
SRC
statistical
analysis
Fisher's
exact p-value
0.01
O.001
0.009
>0.05
0.05
>0.05
>0.05
>0.05
>0.05
O.001
0.001
O.001
0.001
Mean
number
tumors/
animal
0.4
0.9
0.14
1.2
0.03
0.31
0.31
0
0.03
0.13
0.1
5.3
1.2
0.05
4.08
1.75
4.08
7.17
0.1
7.1
7.7
0.1
0.2
0.12
8.04
5
Comments
Mean number of
tumors/animal
digitally estimated
from Figure 2 and
rounded to even
number tumors
C-10
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-2. Dermal bioassays: dose-response information for tumor multiplicity
Record
number
13660
13660
16440
18680
24800
Reference
Cavalierietal.,
1991
Cavalierietal.,
1991
Wood et al.,
1980
Hoffmann et
al., 1972
Nesnowet al.,
1984
Study
type
Initiation
Initiation
Initiation
Initiation
Initiation
Species
Mice
Mice
Mice
Mice
Mice
Tumor type
Primarily
papilloma
Primarily
papilloma
Papilloma
Papilloma
Papilloma
PAH
CPdefC
Acetone
BaP
BaP
BaP
DBalP
DBalP
DBalP
Acetone
BaP
BaP
BaP
DBalP
DBalP
DBalP
Acetone
BaP
BaP
Pyr
Pyr
CPcdP
CPcdP
Acetone
BaP
FA
Acetone
Acetone
BaP
BaP
BeAC
BeAC
Sex
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
M
F
M
F
M
F
Dose of
PAH
1
0
33.3
100
300
33.3
100
300
0
4
20
100
4
20
100
0
0.1
0.4
0.1
0.4
0.1
0.4
0
0.05
1
0
0
200
200
50
50
Dose units
mg/mouse
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
(xmol
jimol
|imol
|imol
(imol
(xmol
jimol
mg
mg
mg
nmol
nmol
nmol
nmol
nmol
nmol
Number of
animals
with
tumors
24
0
10
17
21
23
22
24
0
1
10
22
22
20
20
3
20
22
4
3
3
6
1
19
1
0
1
12
10
4
4
Number
of
animals
in
group
24
24
23
24
23
24
24
24
24
24
24
24
24
24
24
30
30
30
30
30
30
30
30
29
29
20
19
18
19
20
20
%
Tumor-
bearing
animals
100
0
43
71
91
96
92
100
0
4
42
92
92
83
83
10
68
73
14
10
10
21
3
66
3
0
5
67
53
20
20
Results of
authors'
statistical
analysis
(p-value)
O.05
<0.05
>0.05
>0.05
>0.05
>0.05
Results of
SRC
statistical
analysis
Fisher's
exact p-value
0.001
0.001
0.001
O.001
0.001
O.001
0.001
>0.05
O.001
0.001
0.001
O.001
0.001
0.001
>0.05
0.001
0.0015
>0.05
>0.05
Mean
number
tumors/
animal
5.63
0
0.65
2.75
5.22
6.75
7.92
8.5
0
0.04
0.75
3.42
6.96
5.29
3.29
0.1
2
4.6
0.14
0.1
0.1
0.29
0.03
2.3
0.03
0
0.05
1.4
1.5
0.25
0.25
Comments
Number reported in
text
16-Wk experiment
27- Wk experiment
Number tumors per
animal at risk
calculated
C-ll
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-2. Dermal bioassays: dose-response information for tumor multiplicity
Record
number
Reference
Study
type
Species
Tumor type
PAH
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
BeAC
B1AC
B1AC
B1AC
B1AC
B1AC
B1AC
B1AC
B1AC
B1AC
B1AC
Sex
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Dose of
PAH
100
100
250
250
500
500
1,000
1,000
50
50
100
100
250
250
500
500
1,000
1,000
Dose units
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
Number of
animals
with
tumors
4
7
12
10
15
8
16
18
12
13
16
18
21
19
16
20
19
20
Number
of
animals
in
group
20
19
20
19
20
18
18
20
20
20
17
19
21
21
16
21
20
20
%
Tumor-
bearing
animals
20
37
60
53
75
44
89
90
60
65
94
95
100
91
100
95
95
100
Results of
authors'
statistical
analysis
(p-value)
Results of
SRC
statistical
analysis
Fisher's
exact p-value
>0.05
0.02
0.001
O.001
O.001
0.007
O.001
0.001
O.001
O.001
0.001
O.001
0.001
O.001
O.001
0.001
O.001
0.001
Mean
number
tumors/
animal
0.4
0.53
1.3
1.1
1.9
1.2
3.1
2.2
1.4
1.1
2.3
3.1
8.4
4.7
10.8
6.6
8.7
10.8
Comments
C-12
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
17560
Reference
Busby etal.,
1989
Species
Mice
Expo-
sure
route
Intra-
periton-
eal
Target
organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Tumor type
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
PAH
DMSO
DMSO
BaP
BaP
Pyr
Pyr
Pyr
Pyr
FA
FA
Sex
M
F
M
F
M
F
M
F
M
F
Dose
0
0
59.5
59.5
86.1
86.1
1,750
1,750
257.6
257.6
Dose
units
Hg
(total)
Mg
(total)
Hg
(total)
Hg
(total)
Hg
(total)
Hg
(total)
Hg
(total)
Hg
(total)
Hg
(total)
Hg
(total)
Number of
animals with
tumors
13
7
13
19
4
1
2
3
5
9
Number of
animals in
group
91
101
28
27
23
28
27
26
23
29
% Tumor bear-
ing animals
0.14
0.07
0.46
0.70
0.17
0.04
0.07
0.12
0.22
0.31
Results of
authors'
statistical
analysis
(p-value)
SRC Statistical
Analysis
Fisher's
exact
p-value
7.2 x 10'4
3.96 x 10'11
4.60 x 10'1
4.50 x 10'1
2.80 x 10'1
3.30 x 10'1
2.80 x 10'4
1.65 x lO'3
Cochran-
Armitage
trend test
p-value
3.13X10'1
3.50 x 10'1
Comments
Stats reported for
combined M and F
only for each dose
and treatment
compared to control
not individual sexes
C-13
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
640
Reference
LaVoie et
al., 1987
Species
Mice
Expo-
sure
route
Intra-
periton-
eal
Target
organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Tumor type
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma +
adeno-
carcinoma
Adenoma
Adenoma
Adenoma
Adenoma
Adenoma
Adenoma
Adenoma
Adenoma
Adenoma
Adenoma
PAH
CH
CH
CH
CH
DMSO
DMSO
BaP
BaP
BbF
BbF
BjF
BjF
BkF
BkF
Sex
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Dose
6.3
6.3
210
210
0
0
1.1
1.1
0.5
0.5
1.1
1.1
2.1
2.1
Dose
units
Mg
(total)
Hg
(total)
Hg
(total)
Hg
(total)
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
jimol/
mouse
jimol/
mouse
jimol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
Number of
animals with
tumors
2
3
3
0
0
0
14
9
2
3
11
4
1
3
Number of
animals in
group
27
29
20
29
17
18
17
14
15
17
21
18
16
18
% Tumor bear-
ing animals
0.07
0.10
0.15
0.00
0
0
0.82
0.64
0.13
0.18
0.52
0.22
0.06
0.17
Results of
authors'
statistical
analysis
(p-value)
<0.005
>0.05
>0.05
<0.005
0.05
>0.05
>0.05
SRC Statistical
Analysis
Fisher's
exact
p-value
2.80 x 10'1
3.90 x 10'1
5.85 x 10'1
1.60 x lO'1
Cochran-
Armitage
trend test
p-value
8.03 x 10'1
1.28X10'1
Comments
C-14
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
Reference
Species
Expo-
sure
route
Target
organ
Lung
Lung
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver or
lung
Tumor type
Adenoma
Adenoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
PAH
IP
IP
DMSO
DMSO
BaP
BaP
BbF
BbF
BjF
BjF
BkF
BkF
IP
IP
DMSO
Sex
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Dose
2.1
2.1
0
0
1.1
1.1
0.5
0.5
1.1
1.1
2.1
2.1
2.1
2.1
0
Dose
units
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
jimol/
mouse
jimol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
jimol/
mouse
Number of
animals with
tumors
1
0
1
0
13
0
8
0
11
0
3
0
0
0
1
Number of
animals in
group
11
9
17
18
17
14
15
17
21
18
16
18
11
9
17
% Tumor bear-
ing animals
0.09
0
0.06
0
0.76
0
0.53
0
0.52
0
0.19
0
0
0
0.06
Results of
authors'
statistical
analysis
(p-value)
<0.005
<0.005
0.005
>0.05
SRC Statistical
Analysis
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
Comments
Adenoma and
hepatoma also
reported separately;
none of animals
surviving 35 wks
C-15
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
7510
Reference
LaVoie et
al., 1994
Species
Mice
Expo-
sure
route
Intra-
periton-
eal
Target
organ
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Liver or
lung
Lung
Lung
Lung
Lung
Tumor type
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Adenoma +
hepatoma
Total
Total
Total
Total
PAH
DMSO
BaP
BaP
BbF
BbF
BjF
BjF
BkF
BkF
IP
IP
DMSO
DMSO
BaP
BaP
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Dose
0
1.1
1.1
0.5
0.5
1.1
1.1
2.1
2.1
2.1
2.1
0
0
1.1
1.1
Dose
units
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
jimol/
mouse
jimol/
mouse
jimol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
Number of
animals with
tumors
0
13
9
8
3
17
4
3
3
1
0
5
4
24
17
Number of
animals in
group
18
17
14
15
17
21
18
16
18
11
9
29
34
32
20
% Tumor bear-
ing animals
0
0.76
0.64
0.53
0.18
0.81
0.22
0.19
0.17
0.09
0
0.17
0.12
0.75
0.85
Results of
authors'
statistical
analysis
(p-value)
0.001
O.001
SRC Statistical
Analysis
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
Comments
Survival to 1 yr
C-16
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
Reference
Species
Expo-
sure
route
Target
organ
Lung
Lung
Lung
Lung
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Tumor type
Total
Total
Total
Total
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
PAH
FA
FA
FA
FA
DMSO
DMSO
BaP
BaP
FA
FA
FA
FA
Sex
M
F
M
F
M
F
M
F
M
F
M
F
Dose
3.46
3.46
17.3
17.3
0
0
1.1
1.1
3.46
3.46
17.3
17.3
Dose
units
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
(imol/
mouse
jimol/
mouse
jimol/
mouse
jimol/
mouse
(imol/
mouse
Number of
animals with
tumors
12
11
11
25
5
2
27
2
18
0
17
2
Number of
animals in
group
28
31
17
29
29
34
32
20
28
31
17
29
% Tumor bear-
ing animals
0.43
0.35
0.65
0.86
0.17
0.06
0.84
0.10
0.64
0
1.00
0.07
Results of
authors'
statistical
analysis
(p-value)
0.05
0.05
O.005
O.001
0.001
>0.05
O.001
O.001
SRC Statistical
Analysis
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
2.84 x 10'3
2.18 x 10'9
5.10 x 10'7
5.47 x 10'1
Comments
Foci, adenomas,
carcinomas also
reported separately
C-17
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
24590
24590
24801
Reference
Nesnow et
al., 1998b
Nesnow et
al., 1998b
Weyand et
al., 2004
Species
Mice
Mice
Mouse
Expo-
sure
route
Intra-
periton-
eal
Intra-
periton-
eal
Intra-
periton-
eal
Target
organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Tumor type
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Adenoma
PAH
Control
BaP
BaP
BaP
BaP
BaP
BbF
BbF
BbF
BbF
CPcdP
CPcdP
CPcdP
CPcdP
DBahA
DBahA
DBahA
DBahA
Control
DBalP
DBalP
DBalP
DBalP
Tri-
caprylin
Sex
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
F
Dose
0
5
10
50
100
200
10
50
100
200
10
50
100
200
1.25
2.5
5
10
0
0.3
1.5
3
6
0
Dose
units
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Number of
animals with
tumors
6
6
7
19
16
24
9
16
20
19
8
20
19
19
12
18
20
19
15
13
33
35
30
14
Number of
animals in
group
20
20
17
19
16
24
18
20
20
19
20
20
19
19
18
19
20
19
30
33
34
35
30
29
% Tumor bear-
ing animals
0.30
0.30
0.41
1.00
1.00
1.00
0.50
0.80
1.00
1.00
0.40
1.00
1.00
1.00
0.67
0.95
1.00
1.00
0.50
0.39
0.97
1.00
1.00
0.48
Results of
authors'
statistical
analysis
(p-value)
SRC Statistical
Analysis
Fisher's
exact
p-value
>0.05
>0.05
0.001
0.0018
0.001
>0.05
>0.05
0.001
0.001
>0.05
0.001
O.001
O.001
0.05
0.0053
O.001
0.001
>0.05
0.001
O.001
O.001
Cochran-
Armitage
trend test
p-value
Comments
Data provided by S.
Nesnow
Data provided by S.
Nesnow
C-18
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
22510
Reference
Wislocki et
al., 1986
Species
Mice
Expo-
sure
route
Intra-
periton-
eal
Target
organ
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Tumor type
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
PAH
BaP
BcFE
DMSO
DMSO
DMSO
DMSO
DMSO
pooled
DMSO
pooled
BaP
BaP
CH
CH
CH
CH
BaA
Sex
F
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Dose
100
100
0
0
0
0
0
0
560
560
700
700
2,800
2,800
2,800
Dose
units
mg/kg
mg/kg
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
Number of
animals with
tumors
27
26
2
0
5
0
7
0
18
0
10
0
14
0
31
Number of
animals in
group
30
28
28
31
45
34
73
65
37
27
35
33
34
24
39
% Tumor bear-
ing animals
0.90
0.92
0.07
0
0.11
0
0.09
0
0.49
0
0.29
0
0.41
0
0.79
Results of
authors'
statistical
analysis
(p-value)
<0.05
<0.05
<0.05
<0.05
SRC Statistical
Analysis
Fisher's
exact
p-value
0.0005
0.0002
Cochran-
Armitage
trend test
p-value
6 x 10'3
1
Comments
Animals surviving
through weaning
0
This group started
10 wks after other
groups
This group started
10 wks after other
groups
This group started
10 wks after other
groups
This group started
10 wks after other
groups
C-19
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
Reference
Species
Expo-
sure
route
Target
organ
Liver
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Tumor type
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
PAH
BaA
DMSO
DMSO
DMSO
DMSO
DMSO
pooled
DMSO
pooled
BaP
BaP
CH
CH
CH
CH
BaA
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Dose
2,800
0
0
0
0
0
0
560
560
700
700
2,800
2,800
2,800
Dose
units
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
Number of
animals with
tumors
0
1
0
4
2
5
2
13
13
6
2
7
1
6
Number of
animals in
group
32
28
31
45
34
73
65
37
27
35
33
34
24
39
% Tumor bear-
ing animals
0
0.04
0
0.09
0.06
0.07
0.03
0.35
0.48
0.17
0.06
0.21
0.04
0.15
Results of
authors'
statistical
analysis
(p-value)
O.05
O.05
O.05
SRC Statistical
Analysis
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
1.1 x 10'1
5.6 x 10'1
Comments
This group started
10 wks after other
groups
This group started
10 wks after other
groups
This group started
10 wks after other
groups
This group started
10 wks after other
groups
C-20
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
Reference
Species
Expo-
sure
route
Target
organ
Lung
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Lymph-
atic
system
Tumor type
Adenoma +
carcinoma
Lymphoma
Lymphoma
Lymphoma
Lymphoma
Lymphoma
Lymphoma
Lymphoma
Lymphoma
Lymphoma
Lymphoma
PAH
BaA
DMSO
DMSO
DMSO
DMSO
BaP
BaP
CH
CH
CH
CH
Sex
F
M
F
M
F
M
F
M
F
M
F
Dose
2,800
0
0
0
0
560
560
700
700
2,800
2,800
Dose
units
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
Number of
animals with
tumors
6
1
1
0
0
2
4
3
1
0
0
Number of
animals in
group
32
28
31
45
34
37
27
35
33
34
24
% Tumor bear-
ing animals
0.19
0.04
0.03
0
0
0.05
0.15
0.09
0.03
0
0
Results of
authors'
statistical
analysis
(p-value)
0.05
O.05
SRC Statistical
Analysis
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
2.2 x 10'1
3.9 x 10'1
Comments
This group started
10 wks after other
groups
This group started
10 wks after other
groups
This group started
10 wks after other
groups
This group started
10 wks after other
groups
C-21
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-3. Intraperitoneal bioassays: dose-response information for incidence data
Record
number
Reference
Species
Expo-
sure
route
Target
organ
Lymph-
atic
system
Lymph-
atic
system
Tumor type
Adenoma +
carcinoma
Adenoma +
carcinoma
PAH
BaA
BaA
Sex
M
F
Dose
2,800
2,800
Dose
units
nmol
nmol
Number of
animals with
tumors
1
3
Number of
animals in
group
39
32
% Tumor bear-
ing animals
0.03
0.09
Results of
authors'
statistical
analysis
(p-value)
SRC Statistical
Analysis
Fisher's
exact
p-value
Cochran-
Armitage
trend test
p-value
Comments
C-22
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicity
Record
number
17560
7510
Reference
Busby etal., 1989
LaVoieetal., 1994
Species
Mice
Mice
Exposure
route
Intra-
peritoneal
Intra-
peritoneal
Target
organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Tumor type
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adenocarcin
oma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Adenoma+
adeno-
carcinoma
Total
Total
Total
Total
Total
PAH
DMSO
DMSO
BaP
BaP
Pyr
Pyr
Pyr
Pyr
FA
FA
CH
CH
CH
CH
DMSO
DMSO
BaP
BaP
FA
Sex
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Dose
0
0
59.5
59.5
86.1
86.1
1,750
1,750
257.6
257.6
6.3
6.3
210
210
0
0
1.1
1.1
3.46
Dose units
Hg (total)
Hg (total)
ug (total)
ug (total)
Ug (total)
ug (total)
ug (total)
ug (total)
ug (total)
ug (total)
ug (total)
ug (total)
ug (total)
ug (total)
umol/mouse
umol/mouse
umol/mouse
umol/mouse
umol/mouse
Number
of animals
with
tumors
13
7
13
19
4
1
2
-*
5
9
2
3
-^
0
5
4
24
17
12
Number
of animals
in group
91
101
28
27
23
28
27
26
23
29
27
29
20
29
29
34
32
20
28
%
Tumor-
bearing
animals
0.14
0.07
0.46
0.70
0.17
0.04
0.07
0.12
0.22
0.31
0.07
0.10
0.15
0.00
0.17
0.12
0.75
0.85
0.43
Results of
authors'
statistical
analysis
(p-value)
<0.001
<0.001
<0.05
Results of
SRC
statistical
analysis
(Fisher's
exact
p-value)
<0.001
<0.001
>0.05
>0.05
>0.05
>0.05
>0.05
0.00165
>0.05
>0.05
>0.05
>0.05
Mean
number
tumors/
animal
0.15
0.08
0.71
1.19
0.17
0.04
0.07
0.12
0.22
0.41
0.07
0.1
0.15
0
0.17
0.15
4.3
3.55
0.64
SDof
mean
0.38
0.30
1.01
1.09
0.38
0.21
0.26
0.31
0.43
0.70
0.26
0.32
0.36
0.00
Results of
SRC
statistical
analysis
(t-test
p-value)
<0.001
<0.001
>0.05
>0.05
>0.05
>0.05
>0.05
<0.0001
>0.05
>0.05
>0.05
>0.05
Comments
Stats
reported for
combined M
and F
Survived to
lyr
C-23
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicity
Record
number
22510
Reference
Wislocki et al., 1986
Species
Mice
Exposure
route
Intra-
peritoneal
Target
organ
Lung
Lung
Lung
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Tumor type
Total
Total
Total
FOCI +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
FOCI +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
Foci +
adenoma +
carcinoma
FOCI +
adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
PAH
FA
FA
FA
DMSO
DMSO
BaP
BaP
FA
FA
FA
FA
DMSO
DMSO
DMSO
DMSO
DMSO
pooled
DMSO
pooled
BaP
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Dose
3.46
17.3
17.3
0
0
1.1
1.1
3.46
3.46
17.3
17.3
o
o
0
0
0
0
560
Dose units
umo I/mouse
umo I/mouse
umo I/mouse
umo I/mouse
umo I/mouse
umo I/mouse
umo I/mouse
umo I/mouse
umol/mouse
umo I/mouse
umol/mouse
nmol
nmol
nmol
nmol
nmol
nmol
nmol
Number
of animals
with
tumors
11
11
25
5
2
27
2
18
0
17
0
2
0
5
0
1
0
18
Number
of animals
in group
31
17
29
29
34
32
20
28
31
17
29
28
31
45
34
73
65
37
%
Tumor-
bearing
animals
0.35
0.65
0.86
0.17
0.06
0.84
0.10
0.64
0
1.00
0.07
0.07
0
0.11
0
0.09
0
0.49
Results of
authors'
statistical
analysis
(p-value)
<0.05
<0.005
<0.001
<0.001
>0.05
<0.001
<0.001
<0.05
Results of
SRC
statistical
analysis
(Fisher's
exact
p-value)
Mean
number
tumors/
animal
0.35
1.12
2.45
0.41
0.06
4.53
0.3
1.86
0
7.53
0.07
0.07
0
0.11
0
0.096
0
1.46
SDof
mean
Results of
SRC
statistical
analysis
(t-test
p-value)
Comments
Tumor
count
appears to
be error in
publication
Animals
surviving
through
weaning
This group
started
lOwks after
other groups
This group
started
1 0 wks after
other groups
C-24
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicity
Record
number
13610
Reference
Busby etal., 1984
Species
Mice
Exposure
route
Intra-
peritoneal
Target
organ
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Tumor type
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
Adenoma +
carcinoma
PAH
BaP
Pyr
Pyr
Pyr
Pyr
Pyr
Pyr
CH
CH
CH
CH
BaA
BaA
DMSO
DMSO
BaP
BaP
BaP
BaP
FA
FA
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Dose
560
200
200
700
700
2,800
2,800
700
700
2,800
2,800
2,800
2,800
0
0
0.28
0.28
1.4
1.4
0.7
0.7
Dose units
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
mg (total)
mg (total)
mg (total)
mg (total)
mg (total)
mg (total)
mg (total)
mg (total)
Number
of animals
with
tumors
0
0
0
0
3
0
10
0
14
0
31
0
1
4
24
25
16
21
7
3
Number
of animals
in group
27
29
31
25
49
14
18
35
33
34
24
39
32
27
28
25
27
20
24
31
20
%
Tumor-
bearing
animals
0
0
0
0.12
0
0.21
0
0.29
0
0.41
0
0.79
0
0.04
0.14
0.96
0.93
0.80
0.88
0.23
0.15
Results of
authors'
statistical
analysis
(p-value)
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
<0.05
>0.05
<0.05
>0.05
<0.05
>0.05
Results of
SRC
statistical
analysis
(Fisher's
exact
p-value)
<0.001
<0.001
<0.001
<0.001
0.0412
>0.05
Mean
number
tumors/
animal
0
0
0
0.12
0
0.21
0
0.86
0
1.03
0
2.38
0
0.04
0.14
4.32
3.7
10.15
4.25
0.29
0.15
SDof
mean
0.21
0.37
3.5
3.10
13.0
4.70
0.84
0.49
Results of
SRC
statistical
analysis
(t-test
p-value)
<0.001
<0.001
<0.001
<0.001
>0.05
>0.05
Comments
This group
started
1 0 wks after
other groups
This group
started
1 0 wks after
other groups
This group
started
10 wks after
other groups
This group
started
10 wks after
other groups
No model fit
No model fit
C-25
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicity
Record
number
24590
24590
11190
24801
Reference
Nesnowetal., 1998b
Nesnowetal., 1998b
Massetal., 1993
Weyand et al., 2004
Species
Mice
Mice
Mice
Mice
Exposure
route
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Target
organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Tumor type
Adenoma +
carcinoma
Adenoma +
carcinoma
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Adenoma
Adenoma
Adenoma
PAH
FA
FA
Control
BaP
BaP
BaP
BaP
BaP
BbF
BbF
BbF
BbF
CPcdP
CPcdP
CPcdP
CPcdP
DBabA
DBahA
DBahA
DBahA
Control
DBalP
DBalP
DBalP
DBalP
Control
BaP
BaP
BaP
BjAC
BjAC
BjAC
Tn-
caprylin
BaP
BcFE
Sex
M
F
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
F
F
F
Dose
3.5
3.5
0
5
10
50
100
200
10
50
100
200
10
50
100
200
1.25
2.5
5
10
0
0.3
1.5
3
6
0
20
50
100
20
50
100
0
100
100
Dose units
mg (total)
mg (total)
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Number
of animals
with
tumors
20
8
6
6
7
19
16
24
9
16
20
19
8
20
19
19
12
18
20
19
15
13
33
35
30
19
10
15
14
12
13
14
14
27
26
Number
of animals
in group
27
21
20
20
17
19
16
24
18
20
20
19
20
20
19
19
18
19
20
19
30
33
34
35
30
34
16
16
14
12
13
14
29
30
28
%
Tumor-
bearing
animals
0.74
0.38
0.30
0.30
0.41
1.00
1.00
1.00
0.50
0.80
1.00
1.00
0.40
1.00
1.00
1.00
0.67
0.95
1.00
1.00
0.50
0.39
0.97
1.00
1.00
0.56
0.63
0.94
1.00
1.00
1.00
1.00
0.48
0.9
0.92
Results of
authors'
statistical
analysis
(p-value)
Results of
SRC
statistical
analysis
(Fisher's
exact
p-value)
<0.001
>0.05
>0.05
>0.05
<0.001
0.0018
<0.001
>0.05
<0.05
<0.001
<0.001
>0.05
<0.001
<0.001
<0.001
<0.05
0.0053
<0.001
<0.001
>0.05
<0.001
<0.001
<0.001
>0.05
0.0065
0.0017
0.0036
0.0025
0.0017
0.0005
0.0002
Mean
number
tumors/
animal
1.52
0.52
0.53
0.45
0.53
4.37
12.75
32.96
0.67
2.00
5.30
6.95
0.55
4.75
32.21
97.68
1.44
3.05
13.05
32.16
0.67
0.42
4.32
7.49
16.10
0.85
1
3.9
5.9
60.3
140.6
97.6
0.6
6.7
4
SDof
mean
1.66
0.82
0.72
0.80
0.78
2.74
4.28
10.23
0.75
1.82
3.21
3.52
0.80
2.12
15.15
28.68
1.46
1.90
5.99
10.78
0.80
0.56
2.86
3.79
7.26
0.9
1
2.9
3.3
14.6
21.5
28.2
0.75
5.26
2.8
Results of
SRC
statistical
analysis
(t-test
p-value)
<0.001
0.0343
>0.05
>0.05
<0.001
<0.001
<0.001
>0.05
0.0022
<0.001
<0.001
>0.05
<0.001
<0.001
<0.001
0.0229
<0.001
<0.001
<0.001
>0.05
<0.001
<0.001
<0.001
>0.05
<0.001
<0.001
<0.001
<0.001
<0.001
<0.01
<0.01
Comments
Nonconstant
variance
NS
incidence;
nonconstant
variance
Pooled
controls
from data
provided by
Nesnow
C-26
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-5. Lung implantation bioassays: dose-response information for incidence data
Record
number
17940
Reference
Deutsch-Wenzel et
al., 1983
Species
Rat
Target
organ
Lung
Lung
Tumor type
Epidermoid
carcinoma
Pleomorphic
sarcoma
PAH
Untreated
control
Vehicle
control
BaP
BaP
BaP
BbF
BbF
BbF
BeP
BeP
BeP
BjF
BjF
BjF
BkF
BkF
BkF
IP
IP
IP
AA
AA
BghiP
BghiP
BghiP
Untreated
control
Vehicle
control
Dose
0
0
0.1
0.3
1
0.1
0.3
1
0.2
1
5
0.2
1
5
0.16
0.83
4.15
0.16
0.83
4.15
0.16
0.83
0.16
0.83
4.15
0
0
Dose
units
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
Number
of
animals
with
tumors
0
0
4
21
33
0
1
9
0
0
1
1
3
18
0
3
12
3
8
21
1
19
0
1
4
0
0
Number
of
animals
in group
35
35
35
35
35
35
35
35
35
30
35
35
35
35
35
31
27
35
35
35
35
35
35
35
34
35
35
% Tumor-
bearing
animals
0.00
0.00
0.11
0.60
0.94
0.00
0.03
0.26
0.00
0.00
0.03
0.03
0.09
0.51
0.00
0.10
0.44
0.09
0.23
0.60
0.03
0.54
0.00
0.03
0.12
0.00
0.00
SRC statistical analysis
Fisher's
exact p-value
5.70 x 10'2
6.02 x 10'9
5.93 x 10'18
5 x 10'1
1 x 10'3
5 x 10'1
5 x 10'1
1.2 x 10'1
1.96x 10'7
1 x lO'1
8.05 x 10'6
1.20x 10'1
2 x lO'3
6.02 x 10'9
5 x 10'1
6.4 x 10'8
1.2 x 10'1
5.4 x 10'2
Cochran-
Armitage trend
testp-value
1.57 x 10'17
5.12 x 10'7
9.49 x 10'2
1.28 x 10'11
1.03 x 10'9
2.09 x 10'10
1.13 xlO'10
2.47 x 10'3
Comments
C-27
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-5. Lung implantation bioassays: dose-response information for incidence data
Record
number
Reference
Species
Target
organ
Lung
Tumor type
Carcinoma+
sarcoma
PAH
BaP
BaP
BaP
BbF
BbF
BbF
BeP
BeP
BeP
BjF
BjF
BjF
BkF
BkF
BkF
IP
IP
IP
AA
AA
BghiP
BghiP
BghiP
Untreated
control
Vehicle
control
BaP
BaP
BaP
Dose
0.1
0.3
1
0.1
0.3
1
0.2
1
5
0.2
1
5
0.16
0.83
4.15
0.16
0.83
4.15
0.16
0.83
0.16
0.83
4.15
0
0
0.1
0.3
1
Dose
units
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
Number
of
animals
with
tumors
6
2
0
1
2
4
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
10
23
33
Number
of
animals
in group
35
35
35
35
35
35
35
30
35
35
35
35
35
31
27
35
35
35
35
35
35
35
34
35
35
35
35
35
% Tumor-
bearing
animals
0.17
0.06
0.00
0.03
0.06
0.11
0.00
0.03
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.03
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.29
0.66
0.94
SRC statistical analysis
Fisher's
exact p-value
1.2 x 10'2
2.5 x 10'1
1.2 x 10'1
2.5 x 10'1
6. x 10'2
1.2 x 10'1
4.63 x 10'4
4.7 x 10'10
5.9 x ID'19
Cochran-
Armitage trend
testp-value
1.36x 10'1
7.55 x 10'3
3.66 x 10'9
Comments
C-28
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-5. Lung implantation bioassays: dose-response information for incidence data
Record
number
22000
Reference
Wenzel-Hartung et
al., 1990
Species
Rat
Target
organ
Lung
Tumor type
Carcinoma
PAH
BbF
BbF
BbF
BeP
BeP
BeP
BjF
BjF
BjF
BkF
BkF
BkF
IP
IP
IP
AA
AA
BghiP
BghiP
BghiP
Untreated
control
Vehicle
control
BaP
BaP
BaP
Dose
0.1
0.3
1
0.2
1
5
0.2
1
5
0.16
0.83
4.15
0.16
0.83
4.15
0.16
0.83
0.16
0.83
4.15
0
0
0.03
0.1
0.3
Dose
units
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg/
animal
mg/
animal
mg/
animal
mg/
animal
mg/
animal
Number
of
animals
with
tumors
1
3
13
0
1
1
1
3
18
0
3
12
4
8
21
1
19
0
1
4
0
0
3
11
27
Number
of
animals
in group
35
35
35
35
30
35
35
35
35
35
31
27
35
35
35
35
35
35
35
34
35
35
35
35
35
% Tumor-
bearing
animals
0.03
0.09
0.37
0.00
0.03
0.03
0.03
0.09
0.51
0.00
0.10
0.44
0.11
0.23
0.60
0.03
0.54
0.00
0.03
0.12
0.00
0.00
0.09
0.31
0.77
SRC statistical analysis
Fisher's
exact p-value
1.2 x 10'1
1.2 x 10'1
3.1 x 10'5
1.2 x 10'1
1.2 x 10'1
1.20x 10'1
1.96x 10'7
1 x 10'1
8.05 x 10'4
6 x 10'2
2 x 10'3
6.02 x 10'9
6.4 x 10'8
5.4 x 10'2
1.2 x 10'1
1.93 x 10'4
1.29E x 10'12
Cochran-
Armitage trend
testp-value
9.63 x 10'8
3.23 x 10'1
1.28 x 10'11
1.03 x 10'9
7.56 x 10'10
1.13 x 10'10
2.47 x 10'3
8.85 x 10'15
Comments
ED10, relative
potencies reported
C-29
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-5. Lung implantation bioassays: dose-response information for incidence data
Record
number
Reference
Species
Target
organ
Tumor type
PAH
PH
PH
PH
CH
CH
DBahA
Dose
1
3
10
1
3
0.1
Dose
units
mg/
animal
mg/
animal
mg/
animal
mg/
animal
mg/
animal
mg/
animal
Number
of
animals
with
tumors
0
0
1
5
10
20
Number
of
animals
in group
35
35
35
35
35
35
% Tumor-
bearing
animals
0.00
0.00
0.03
0.14
0.29
0.57
SRC statistical analysis
Fisher's
exact p-value
5 x 10'1
2.7 x 10'2
4.63 x lO'4
2.01 x 10'8
Cochran-
Armitage trend
testp-value
1
7.96 x 10'4
Comments
C-30
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-6. Oral bioassays: dose-response information for incidence data
Record
number
24801
Reference
Weyand et al., 2004
Species
Mouse
Target
organ
Lung
Fore-
stomach
Tumor type
Adenoma
Squamous
cell
carcinoma
PAH
Control
BaP
BcFE
BcFE
Control
BaP
BcFE
BcFE
Dose
0
230
13.6
197
0
230
13.6
197
Dose units
Hg/mouse/
day
Hg/mouse/
day
Hg/mouse/
day
Hg/mouse/
day
Hg/mouse/
day
Hg/mouse/
day
Hg/mouse/
day
Hg/mouse/
day
Number
of
animals
with
tumors
7
21
13
29
0
10
0
0
Number
of
animals
in group
29
27
28
29
29
27
28
29
% Tumor-
bearing
animals
0.24
0.77
0.46
1
0
0.36
0
0
SRC statistical analysis
Fisher's
exact p-value
>0.0001
0.0684
>0.0001
Cochran-
Armitage trend
testp-value
Comments
Table C-7. Oral bioassays: dose-response information for tumor multiplicity
Reference
24801
Species
Weyand et
al., 2004
Exposure
route
Mouse
Target
organ
Lung
Tumor type
Adenoma
PAH
Control
BaP
BcFE
BcFE
Sex
F
F
F
F
Dose
0
230
13.6
197
Dose units
ug/mouse/
day
ug/mouse/
day
ug/mouse/
day
ug/mouse/
day
Number
of animals
with
tumors
7
21
13
29
Number
of animals
in group
29
27
28
29
°/o Tumor-
bearing
animals
0.24
0.77
0.46
1
Results of
authors'
statistical
analysis
(p-value)
Results of
SRC
statistical
analysis
(Fisher's
exact
p-value)
>0.0001
0.0684
>0.0001
Mean
number
tumors/
animal
0.31
1.4
0.57
46
SDof
mean
0.59
1.14
0.69
15.1
Results of
SRC
statistical
analysis
(t-test
p-value)
>0.0001
0.13
>0.0001
Comments
C-31
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-8. In vitro bacterial mutagenicity: data use
Record
number
17030
23830
23660
17380
17590
17630
9620
24030
18050
Reference
Andrews et
al., 1978
Baker et
al., 1980
Bartsch et
al., 1980
Bosetal.,
1988
Carver et
al., 1986
Cavalieri et
al., 1981a
Chang et
al., 2002
De Flora et
al., 1984
Eisenstadt
and Gold,
1978
Data
source
Figure 1
Table 2
Appendix
table
Table 1
Figure 1
Figure 1
Figure 7
Table 2
Figure 2B
Data points
Dose (ug) and number of
revertant colonies for DBacA,
DBajA, DBahA, AA, BghiP,
BeP, BaP
Use data for guinea pig-MC S9
only (column D); dose in
ug/plate and number of
revertant colonies; BaP,
DBaiP, BaA, DBacA, DBahA
Use data for BaA and BaP;
dose in umol/plate and
mutagenic activity in
revertants/umol
Use TA100 strain only; dose
(ug/plate) and number of
revertant colonies/plate for PH,
Pyr, BaP
Use curves for BaP, BaA,
BghiF, and Pery; use 400 uL
S9 per plate (last data point on
x-axis); each curve is different
dose in ug/plate, use hamster
data; revertants per plate is
y-axis
Dose-response curves for BaP,
CPcdP (CPEP in figure), and
ACEP (CPAP in figure); dose
as uM, response as mutant
fraction x 105
Dose-response curves for
BghiF, BcPH, and BaP; dose
(ug/plate) and revertants/plate
Table provides potency
estimates as revertants/nmol
for BaA, Pery, BaP, and BeP
Use TA100 data for BaP and
CPcdP (open circles); dose is
1 ug for CPcdP and 2 ug for
BaP (legend); use the same S9
concentration (20 uL/plate)
Basis for RPF approach
Point estimate
Point estimate Table 2
Point estimate
Derive point estimate for
BaP (use PH control as
background); continuous
model PH and Pyr using the
BaP response as the BMR
Point estimate; use highest
dose in hamster, except for
perylene (use 10 ug/plate);
this is maximal response in
hamsters
Model as quanta! data
(mutant fraction reported)
Point estimate; use
5 ug/plate dose for BghiF
and BaP; use 10 ug/plate for
BcPH
Calculate the RPF ratio
using the potency estimates
provided
Point estimate; single point
data (20 uL S9/plate)
Comments
TA100withArS9
TA100 with guinea
pig-MC S9; Table 1
data not used,
different S9 mix
used for each of
three experiments
TA100 rat MC S9
TA100 with rat Ar
S9
TA100 with hamster
Ar S9; multidose
data but not SD was
reported
TM677withArS9
TA100 with rat Ar
S9; SD not available
from graph (reported
for some data points,
but not all)
Determine strain
used to calculate
potencies; rat Ar S9
TA100 with rat Ar
S9; uL S9 that
maximizes the BaP
response does not
produce maximal
response for CPcdP
C-32
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-8. In vitro bacterial mutagenicity: data use
Record
number
18180
24080
14080
18650
10670
19000
24680
19320
Reference
Florin et
al., 1980
Gibson et
al., 1978
Gold and
Eisenstadt,
1980
Hermann,
1981
Johnsen et
al., 1997
Kaden et
al., 1979
Lafleur et
al., 1993
LaVoie et
al., 1979
Data
source
Table III
Table 1
(BaP)
Table 3
(PAHs)
Table 2
Table 1
Figure 2
Table 1
Figures 3
and 4
Table VI
Data points
Use TA100 data for BaA, CH,
and BaP, use TA98 data for
Pery, CO, and BaP; dose is
indicated as optimal dose
(umol/plate) and number
revertants/plate
Use data for TA98; in Table 1
use Expt. No.l for BaP; in
Table 3 use data for DBahA,
Tphen, BaA, BghiP, CH, FE,
Pyr; dose as ug/plate, response
as increase in revertants
Use data for 3-MC induction at
50 uL S9/plate; dose is 4 nmol
for BaP and CPcdP, results as
revertants/plate
Table provides potency
estimates as revertants/nmol
forBbA,BaA, CH, FA,
Tphen, BeP, DBacA, DBahA,
BbF, Pery, DBalP, DBaiP, AA,
CO; potency of BaP in legend
as 100 revertants/nmol
Use data for PCB microsomes
for BaP, BjAC, B1AC; dose as
ug/plate, response as revertants
RPFs calculated for AN, ANL,
Pyr, BbFE, CPcdP, BaA, CH,
Tphen, FA, BeP, Pery, BghiP,
AA, DBacA, DBahA, DBbeF
Use dose-response curves for
BaP, BghiF, CPcdP,
CPhiACEA (CPAA), ACEA
(AA), CPhiAPA (CPAP), APA
(AP); dose as ug/mL, response
as mutant fraction (* 105)
Use data for TA98 for BaP,
BeP, and Pery; 10 ug dose and
response as revertants/plate
Basis for RPF approach
Point estimate; please note
that reported response
includes subtraction of
spontaneous revertants
(control); need to use
formula for added risk; make
sure to flag in comments
Point estimate; use the dose
associated with the max-
imum response (if reported
as a range, do not use);
controls were reported as
negative (no mutagenic or
toxic response)
Point estimate
Calculate the RPF ratio
using the potency estimates
provided
Model to derive BMDsdl;
need to extract SDs from
graph; control response is
1 13 ± 9 revertants per plate
(see legend); add control
response to each response
for modeling (it was
subtracted prior to graphing)
Not applicable
Model as quanta! data
(mutant fraction reported)
Point estimate; use 20 ug for
BaP; 10 ug for BeP; and
20 ug for Pery
Comments
Note that data for
both TA100 and
TA98 strains were
used; BaP results
were provided for
each; rat MC S9
TA98 with non-
enzymatic induction
(gamma irradiation);
multidose data but
not SD reported
TA100 using 50 uL
ofratMCS9;
important to note
that maximal
response for CPcdP
occurred at much
lower dose of S9
(5 uL/plate)
TA98 with rat Ar
S9; potency
estimates were
calculated from the
linear portion of the
dose-response curve
TA98 with PCB
microsomes
TM677 with Ar S9
and PB S9
Forward mutation to
8-azaguanine
resistance in TM677
with rat AR S9
TA98 with rat Ar
S9; for BeP and
Pery the maximal
response was in
TA100
C-33
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-8. In vitro bacterial mutagenicity: data use
Record
number
23650
20220
20450
21000
11860
21360
21640
16180
16440
Reference
McCann et
al., 1975
Pahlman
and
Pelkonen,
1987
Phillipson
and
loannides,
1989
Sakaiet
al., 1985
Sangaiah et
al., 1983
Simmon,
1979a
Teranishi
etal., 1975
Utesch et
al., 1987
Wood et
al., 1980
Data
source
Table 1
Table 1
Figures 2
and 3
Table 3
Figure 2
Table 1
Table I
and
Figure 3
Figures 2
and 3
Chart 3A
Data points
Table provides potency
estimates as revertants/nmol
forDBaiP, BaP, BeP, DBacA,
DBahA, CH, BaA
Use data for rat-MC induced
(last column); potency
estimates are provided as
revertants/nmol for BaA, CH,
Tphen, DBacA, DBahA
Use the curve for hamster S9
(open triangles); data for BaP,
DBaiP, BaA, and DBahA, dose
as ug/plate, revertants/plate
Use data for TA97 +S9 for FE,
AC, PH, FA, Ch, Pyr, BaP,
BeP, Pery, BghiP, CO; dose
ug, response as revertants per
plate
Use data for Bj AC and BaP;
dose as ug/plate, response as
revertants/plate
Use data for TA100 for BaA,
BaP, BeP; dose as ug, response
as revertants/plate after
subtracting background
Use data for TA1538 for
DBaiP and BaP; use data in
Figure 3 for TA 1538, PB and
DBahA-induced S9 (open
circles) for DBaeP
Use data for homogenized
hepatocytes (open circles) for
BaA and BaP; dose as
ug/plate, response as
revertants/plates
Use dose-response curves for
BaP and CPcdP; dose as nmol,
response as revertants/plate
Basis for RPF approach
Calculate the RPF ratio
using the potency estimates
provided
Calculate the RPF ratio
using the potency estimates
provided
Point estimate; use
10 ug/plate for BaP,
DBahA; 20 ug/plate BaA,
DBaiP
Point estimate; use 10 ug for
AC, PH, FA, BaP, BeP; use
5 ug for FE; use 20 ug for
CH, Pyr, BghiP; use 4 ug for
Pery; use 100 ug for CO
Point estimate; use
10 ug/plate forBjAC; use
6 ug/plate for BaP
Point estimate
Point estimate
Point estimate; use
12.5 ug/plate for BaP; use
25 ug/plate for BaA
Point estimate; use 15 nmol
forBaPandCPcdP
Comments
Multiple strains, rat
ArS9
TA100 with rat MC
S9
TA100 with hamster
S9; multidose data
but not SD reported
TA97 with rat Ar
S9; multidose data
but not SD reported
TA98 with rat Ar
S9; multidose data
but not SD was
reported
TA100 with rat Ar
S9
TA1538 with rat PB
S9 for DBaiP;
TA1538withPB
and DBahA S9 for
DBaeP
TA100 with homo-
genized hepatocytes
from Ar-treated rats;
multidose data but
not SD reported
TA98 with purified
microsomal P450;
multidose data but
not SD reported
C-34
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
17030
23830
23660
17380
Reference
Andrews etal.,
1978
Baker etal., 1980
Bartsch et al, 1980
Bos etal., 1988
Cell type
TA100
TA100
TA100
TA100
Activation
system
ArS9
Guinea pig-
MC
Rat MC S9
RatArS9
PAH
Control
BaP
DBacA
DBajA
DBahA
AA
BghiP
BeP
Control
BaP
DBaiP
BaA
DBacA
DBahA
BaP
BaA
BaP
Control
Dose
0
250
10
10
25
250
100
1,000
0
2.5
5
10
2.5
5
0.027
0.067
7.5
0
Dose units
Hg
Mg
Hg
Hg
Hg
Hg
Hg
Mg
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
(imol/plate
(imol/plate
Hg/plate
Hg/plate
Response
150
1,681
2,957
843
617
1,796
793
643
134
1,278
737
947
1,738
1,331
29,000
6,000
824
85
Response
units
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertant
colonies
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
10
10
10
10
10
3
3
Units
Replic-
ates
Replic-
ates
% Resp-
onse
SD
18
97
73
47
88
98
21
12
SE
12
7
Comments
Control response
subtracted
Control response
subtracted
C-35
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
17590
Reference
Carver etal., 1986
Cell type
TA100
Activation
system
Hamster
ArS9
PAH
PH
PH
PH
Control
Pyr
Pyr
Pyr
Control
BaP
BaP
BaP
BaA
BaA
BaA
BghiF
BghiF
Dose
1
5
25
0
1
5
25
0
1
10
50
15
40
50
10
25
Dose units
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
jig/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Response
108
167
240
86
93
164
279
140
141
482
1,035
346
892
1,263
333
727
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
3
3
3
3
3
3
3
Units
Replic-
ates
Replic-
ates
Replic-
ates
Replic-
ates
Replic-
ates
Replic-
ates
Replic-
ates
% Resp-
onse
SD
10
5
10
7
9
23
10
SE
6
3
6
4
5
13
6
Comments
Control curves
difficult to
digitize; control
value estimated
from BaP graph
and used for all
Continuous data,
noSD
C-36
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
17630
9620
Reference
Cavalieri etal.,
1981a
Chang et al., 2002
Cell type
TM677
TA100
Activation
system
ArS9
RatArS9
PAH
BghiF
Perylene
Perylene
Perylene
Control
BaP
BaP
BaP
BaP
CPcdP
CPcdP
ACEP
ACEP
ACEP
Control
BaP
BghiF
Dose
50
5
10
15
0
10
20
40
60
20
40
10
40
120
0
5
5
Dose units
Mg/plate
Hg/plate
Hg/plate
Mg/plate
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
Mg/plate
Mg/plate
Mg/plate
Response
985
195
993
922
5
15
26
84
131
34
133
11
25
55
326
2,543
1,630
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Revertants/
plate!
Revertants/
plate]
Revertants/
plate]
n
1 x 105
1 x 105
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
Units
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
% Resp-
onse
0.000050
0.000150
0.000256
0.000839
0.001308
0.000337
0.001330
0.000110
0.000248
0.000551
SD
SE
Comments
Control value
estimated
SDnot
consistently
plotted; extracted
only point estimate
data
C-37
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
24030
18050
18180
24080
Reference
De Flora etal.,
1984
Eisenstadt and
Gold, 1978
Florin etal., 1980
Gibson etal., 1978
Cell type
RatAR
S9
TA100
TA100
TA100
TA100
TA98
TA98
TA98
TA98
Activation
system
RatArS9
RatMCS9
[60Co]
gamma
radiation,
for7d
(2.5 x
107rad)
PAH
BcPH
BaP
BaA
Pery
BeP
BaP
CPcdP
BaP
BaA
CH
BaP
Pery
CO
Control
Dose
10
2
1
0.0030
0.10
0.0050
0.0030
0.025
0.070
0
Dose units
Hg/plate
Mg
Mg
(imol/plate
(imol/plate
(imol/plate
(imol/plate
(imol/plate
(imol/plate
Hg/plate
Response
1,043
185
12
21
1.6
1,705
134
255
326
196
235
91
82
0
Response
units
Revertants/
plate!
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Increase in
revertants
n
Units
% Resp-
onse
SD
SE
Comments
Background
subtracted from
data reported
Background
subtracted from
data reported
Only peak
response reported
Continuous data,
noSD
C-38
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
14080
18650
Reference
Gold and
Eisenstadt, 1980
Hermann, 1981
Cell type
TA100
TA98
Activation
system
50 (iL rat
MCS9
RatArS9
PAH
BaP
BaP
BaP
BaP
BaP
BaP
BaA
BaA
BghiP
CH
CH
FE
FE
Pyr
BaP
CPcdP
BaP
Dose
10
20
50
100
200
300
150
250
400
500
1,000
200
360
160
4
4
Dose units
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
nmol
nmol
Response
1.5
3
10
15
21
35
1.8
6.4
4.2
6.1
6.7
1.1
2.2
28
1,103
281
100
Response
units
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Increase in
revertants
Revertants/
plate!
Revertants/
plate!
Revertants/
nmol
(potency)
n
Units
% Resp-
onse
SD
SE
Comments
Background
subtracted from
data reported
C-39
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
Reference
Cell type
Activation
system
PAH
BbA
BaA
CH
FA
Tphen
BeP
DBacA
DBahA
BbF
Pery
DBalP
DBaiP
AA
Dose
Dose units
Response
8
4
2
3
13
15
42
8
15
31
21
38
62
Response
units
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
n
Units
% Resp-
onse
SD
SE
Comments
C-40
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
10670
19000
Reference
Johnsen et al., 1997
Kadenetal., 1979
Cell type
TA98
TM677
Activation
system
PCB
micro-
somes
ArS9 and
PBS9
PAH
CO
Control
BaP
BaP
BjAC
BjAC
BIAC
BIAC
BaP
AN
ANL
Pyr
BbFE
CPcdP
BaA
CH
Tphen
FA
BeP
Dose
0
10
20
10
20
10
20
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Dose units
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Response
60
113
128
123
192
213
204
207
1
0.010
0.070
0.070
0.080
1.5
0.14
0.20
0.070
1.0
0.11
Response
units
Revertants/
nmol
(potency)
Revertants/
plate!
Revertants/
plate]
Revertants/
plate]
Revertants/
plate]
Revertants/
plate]
Revertants/
plate]
Revertants/
plate]
RPF
RPF
RPF
RPF
RPF
RPF
RPF
RPF
RPF
RPF
RPF
n
3
3
3
3
3
3
3
Units
% Resp-
onse
SD
8.54
3.66
13.41
10.98
9.76
13.41
43.90
SE
Comments
Control response
added back to each
response for
modeling
Mutagenic activity
relative to that of
the 80 |imol BaP-
positive control
performed
simultaneously
with test
compound
C-41
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
24680
Reference
Lafleuretal, 1993
Cell type
TM677
Activation
system
RatARS9
PAH
Pery
BghiP
AA
DBacA
DBahA
DBbeF
BaP
BaP
BaP
BaP
BaP
BaP
BghiF
BghiF
BghiF
BghiF
CPcdP
CPcdP
CPcdP
CPcdP
CPcdP
Dose
NA
NA
NA
NA
NA
NA
0
0.5
1
2
4
8
0
1
3
10
0
0.5
1
2
4
Dose units
Hg/mL
Hg/mL
Hg/mL
|ig/mL
Hg/mL
Hg/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Response
6
0.080
0.080
0.77
0.080
0.88
7
8
10
18
22
33
11
10
14
55
12
15
13
17
27
Response
units
RPF
RPF
RPF
RPF
RPF
RPF
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
n
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
Units
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
% Resp-
onse
0.000070
0.000080
0.000101
0.000175
0.000220
0.000327
0.00011
0.00010
0.00014
0.00055
0.000120
0.000146
0.000130
0.000172
0.000274
SD
SE
Comments
C-42
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
19320
Reference
LaVoieetal., 1979
Cell type
TA98
Activation
system
RatArS9
PAH
CPcdP
CPhiACE
A
CPhiACE
A
CPhiACE
A
CPhiACE
A
CPhiACE
A
CPhiAPA
CPhiAPA
CPhiAPA
CPhiAPA
ACEA
ACEA
ACEA
APA
APA
APA
APA
BaP
Dose
8
0
0.5
1
2
4
0
10
30
100
0
10
35
0
10
30
100
10
Dose units
Hg/mL
Hg/mL
Hg/mL
Hg/mL
Hg/mL
|ig/mL
|ig/mL
|ig/mL
Hg/rnL
Hg/rnL
|ig/mL
Hg/mL
|ig/mL
Hg/mL
Hg/mL
|ig/mL
|ig/mL
Mg
Response
60
8
10
16
29
67
9
12
21
26
9
21
69
16
37
42
22
450
Response
units
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Mutants
Revertants/
plate}
n
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
100,000
Units
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
Surviv-
ors
% Resp-
onse
0.000597
0.000084
0.000103
0.000157
0.000286
0.000670
0.000090
0.000117
0.000210
0.000263
0.000092
0.000214
0.000686
0.000160
0.000375
0.000416
0.000220
SD
SE
Comments
Background
subtracted from
data reported
C-43
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
23650
20220
Reference
McCannet al.,
1975
Pahlman and
Pelkonen, 1987
Cell type
Multiple
strains
TA100
Activation
system
RatArS9
Rat MC S9
PAH
BaP
BeP
BeP
Pery
BaP
DBaiP
BeP
DBacA
DBahA
CH
BaA
BaP
Dose
20
10
20
20
NA
NA
NA
NA
NA
NA
NA
NA
Dose units
Mg
US
US
W
Response
480
20
20
70
121
20
0.6
175
11
38
11
272
Response
units
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Revertants/
plate!
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
n
Units
% Resp-
onse
SD
SE
Comments
Paper states that
comparison of
potency estimates
should be done
with caution (non-
linear dose-
response), see
table footnotes
C-44
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
20450
Reference
Phillipson and
loannides, 1989
Cell type
TA100
Activation
system
Hamster S9
PAH
BaA
CH
Tphen
DBacA
DBahA
BaP
BaP
BaP
BaP
BaP
BaA
BaA
BaA
BaA
BaA
DBaiP
Dose
NA
NA
NA
NA
NA
0
5
10
15
20
0
20
40
60
100
0
Dose units
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Response
10.4
9.7
4
35
4.4
0.000
68.833
118.948
99.744
96.101
0.000
109.877
115.248
114.430
98.846
0.000
Response
units
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
nmol
(potency)
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
Units
% Resp-
onse
SD
SE
Comments
C-45
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
21000
Reference
Sakaietal., 1985
Cell type
TA97
Activation
system
RatArS9
PAH
DBaiP
DBaiP
DBaiP
DBaiP
DBahA
DBahA
DBahA
DBahA
DBahA
Control
BaP
BaP
BaP
Control
FE
FE
FE
FE
Dose
20
40
60
100
0
10
20
30
50
0
1
5
10
0
5
10
50
250
Dose units
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg
Mg
Mg
Mg
Hg
Hg
Hg
Mg
Mg
Response
64.638
75.747
80.394
63.880
0.000
50.899
56.886
52.419
34.980
177
1,208
1,432
1,742
189
254
240
240
232
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
Units
% Resp-
onse
SD
SE
Comments
C-46
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
Reference
Cell type
Activation
system
PAH
Control
AC
AC
AC
AC
Control
PH
PH
PH
PH
Control
FA
FA
FA
FA
Control
CH
CH
Dose
0
5
10
50
250
0
5
10
50
250
0
5
10
50
250
0
5
10
Dose units
Hg
Mg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Mg
Mg
Response
189
360
509
293
279
189
454
534
321
T
177
652
1,012
1,042
518
177
640
815
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
Units
% Resp-
onse
SD
SE
Comments
C-47
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
Reference
Cell type
Activation
system
PAH
CH
CH
Control
Pyr
Pyr
Pyr
Pyr
Pyr
Pyr
Control
BeP
BeP
BeP
BeP
Control
Pery
Pery
Pery
Dose
20
50
0
2
4
6
10
20
50
0
5
10
50
250
0
1
2
4
Dose units
Hg
Mg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Mg
Mg
Hg
Mg
Hg
Mg
Mg
Response
888
723
177
929
1,582
2,057
2,577
2,832
2,296
177
944
1,100
606
640
177
1,516
2,236
2,784
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
Units
% Resp-
onse
SD
SE
Comments
C-48
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
11860
Reference
Sangaiahetal.,
1983
Cell type
TA98
Activation
system
RatArS9
PAH
Pery
Pery
Control
BghiP
BghiP
BghiP
BghiP
Control
CO
CO
CO
CO
CO
Control
BaP
BaP
BaP
BaP
Dose
10
50
0
10
20
50
250
0
5
10
50
100
200
0
2
3
6
10
Dose units
Hg
Mg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Hg
Mg
Mg
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Response
2,550
1,808
177
896
991
896
612
177
362
400
405
490
479
35.43
177.37
266.02
419.68
312.76
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
Units
% Resp-
onse
SD
SE
Comments
C-49
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
21360
21640
Reference
Simmon, 1979a
Teranishi et al,
1975
Cell type
TA100
TA1538
Activation
system
RatArS9
RatPBS9
PAH
BaP
BaP
BaP
Control
BjAC
BjAC
BjAC
BjAC
BjAC
BjAC
BjAC
BaP
BaA
BeP
Control
BaP
Dose
30
50
100
0
2
3
6
10
30
50
100
5
50
50
0
50
Dose units
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Mg
Mg
Mg
Hg/plate
Hg/plate
Response
358.41
350.92
323.12
53.15
124.15
331.10
674.11
993.21
1,027.06
883.45
1,021.36
1,141
280
57
38
77
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertant
colonies/
plate
Revertant
colonies/
plate
n
Units
% Resp-
onse
SD
SE
Comments
Background
subtracted from
data reported
C-50
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
16180
Reference
Uteschetal., 1987
Cell type
TA1538
TA100
Activation
system
Rat PB and
DBahA S9
With
homogen-
ized
hepatocytes
from Ar-
treated rats
PAH
DBaiP
Control
BaP
DBaeP
Control
BaP
BaP
BaP
BaP
BaP
Control
BaA
BaA
BaA
BaA
Dose
50
0
50
50
0
6.3
12.5
25
50
100
0
6.3
12.5
25
50
Dose units
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Hg/plate
Response
102
25
279
88
159
998
1,079
1,178
1,141
1,114
199
861
2,583
3,546
3,786
Response
units
Revertant
colonies/
plate
Revertant
colonies/
plate
Revertant
colonies/
plate
Revertant
colonies/
plate
Revertants/
plates
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
Units
% Resp-
onse
SD
SE
Comments
C-51
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. In vitro bacterial mutagenicity: dose-response data
Record
number
16440
Reference
Wood etal., 1980
Cell type
TA98
Activation
system
Purified
microsomal
P450
PAH
BaA
Control
BaP
BaP
BaP
BaP
Control
CPcdP
CPcdP
CPcdP
CPcdP
Dose
100
0
3.75
7.5
15
30
0
3.75
7.5
15
30
Dose units
Hg/plate
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
Response
3,406
0
45
63
99
103
0
303
491
685
776
Response
units
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
Revertants/
plate
n
Units
% Resp-
onse
SD
SE
Comments
Background
subtracted from
data reported
C-52
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-10. In vitro mammalian mutagenicity: data use
Record
number
16920
16940
16910
17140
14250
18740
18990
24720
Reference
Amacher and
Paillet, 1982
Amacher and
Turner, 1980
Amacher et
al., 1980
Barfknecht et
al., 1982
Hass et al.,
1982
Huberman
and Sachs,
1976
Jotz and
Mitchell,
1981
Kligerman et
al., 1986
Data
source
Figure 1
Figure 3
Table 3
Figure 2
(BaP, FA);
Figure 4
(BaA, CH,
Tphen);
Figure 6
(CPcdP)
Table 1
Table 2
Table 2
Figure 1
Data points
Use lines for BaP (open
circles) and BaA (closed
triangles; dose is ug/mL and
response is mutation
frequency (MF)/106 survivors
Use bars for SM2 S9
activation for BaP and BaA;
dose is 1.25 x 10"5MforBaP
and 3.22 x 10"5MforBaP;
response is IMF/104 survivors
Use dose-response data for
BaA and BaP; dose as
concentration (M), response
as mutants per 104 survivors
Dose is uM and mutant
fraction xlO6
Dose-response data for
DBaiP, DBahP, and BaP; dose
is ug/mL; use response data
for TG mutants only
(mutants/106 cells); control
value is 4 ± 1 mutants/
106 cells
Use data for BaP, DBacA,
DBahA; 8-azaguanine
resistance only; use 1 ug/mL
dose for all (*), response as
mutants per 105 survivors
Use data for BaP and Pyr with
metabolic activation; subtract
negative control, dose as
ug/mL, response as MF x 10"6
Use dose-response data for
BaP and B1AC; dose as
ug/mL, response as mutant
frequency/106 survivors;
average data from two
experiments
Basis for RPF
approach
Model;
quanta! data
Point estimate
Model;
quanta! data
Model;
quanta! data
Model;
quanta! data
Point estimate
Point estimate
Model;
quanta! data
Comments
Thymidine kinase assay
(resistance to trifluorothymi-
dine) in mouse lymphoma cells
(L5178Y) with Syrian golden
hamster S9 mix or cocultivated
hamster hepatocytes
Thymidine kinase assay
(resistance to trifluorothymi-
dine) in mouse lymphoma cells
(L5178Y) with mouse S9 mix
Thymidine kinase assay
(resistance to trifluorothymi-
dine) in mouse lymphoma cells
(L5178Y) with mouse S9 mix
Thymidine kinase assay
(resistance to trifluorothymi-
dine) in human lymphoblast
cells with rat Ar S9 mix
Hypoxanthine-guanine phos-
phoribosyl transferase assay
(resistance to 6-thioguanine) in
V79 Chinese hamster cells
with rat MC S9
Hypoxanthine-guanine phos-
phoribosyl transferase assay
(resistance to 8-azaguanine) in
V79 Chinese hamster cells
with hamster embryo cells
Thymidine kinase assay
(resistance to trifluorothymi-
dine) in mouse lymphoma cells
(L5178Y) with rat ArS9
Thymidine kinase assay
(resistance to trifluorothymi-
dine) in mouse lymphoma cells
(L5178Y) with rat ArS9
C-53
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-10. In vitro mammalian mutagenicity: data use
Record
number
19180
24680
7550
11450
15630
15640
21410
16190
21900
Reference
Krahn and
Heidelberger,
1977
Lafleur etal.,
1993
Li and Lin,
1996
Nesnow et
al., 1984
Raveh and
Huberman,
1983
Raveh etal.,
1982
Slagaetal.,
1978
Vacaetal.,
1992
Wangenheim
and
Bolcsfoldi,
1988
Data
source
Table II
Figures 5
and 6
Text
Chart 9
Table 1
Figure 4
Table 3
Figure 5
Table 1
Data points
Use data for BaP, DBahA,
DBacA, and BaA; cell
survival at 40% control
(column 3), controls are 100%
survival group (column 1);
use 3-MC S9 data only; dose
as nmol/mL, response as
6-TG/105 cells
Use dose-response curves for
BaP, CPcdP (CPP),
CPhiACEA (CPAA), ACEA
(AA); dose as ug/mL,
response as mutant fraction
(ppm)
Mutant frequency of controls
2 x lO'5; 10 ng/mL BaP = 5 x
10'5; BaA =5.6 x 10'5
Use data for BaP, B1AC,
BeAC, and BjAC; dose as
ug/mL, response as
6TG-resistant mutants/
106 survivors
Use data for CPcdP and BaP,
with PMA only; dose in
ug/mL, response in
mutants/105 cells
Use dose-response data for
CPcdP and BaP (ouabain
resistance only); dose in
ug/mL, response in
mutants/106 cells
Use dose-response data for
BaA and BaP; dose as uM,
response as ouabain resistant
mutants/104 survivors
Dose-response data for FA
and BaP; dose as uM,
response as 6-Tg resistant
cells/100,000
Use +S9 dose-response data
for Pyr, BaP, and FE; dose as
mol/L, response as mutation
frequency
Basis for RPF
approach
Point estimate
Model as
quanta! data
(mutant
fraction
reported)
Point estimate
Model;
quanta! data
Model;
quanta! data
Model;
quanta! data
Model;
quanta! data
Model;
quanta! data
Model;
quanta! data
Comments
Hypoxanthine-guanine
phosphoribosyl transferase
assay (resistance to 6-thio-
guanine) in V79 Chinese
hamster cells with hamster
embryo cells
Thymidine kinase assay
(resistance to trifluorothymi-
dine) in MCL-3 cells (human
B-lymphoblastoid cells)
Hypoxanthine-guanine phos-
phoribosyl transferase assay
(resistance to 6-thioguanine) in
HS1 HeLa cells (human
epithelial cells)
Hypoxanthine-guanine phos-
phoribosyl transferase assay
(resistance to 6-thioguanine) in
V79 Chinese hamster cells
with rat AR S9
Hypoxanthine-guanine
phosphoribosyl transferase
assay (resistance to 6-thio-
guanine) in V79 Chinese
hamster cells with hamster
embryo cells
Hypoxanthine-guanine phos-
phoribosyl transferase assay
(resistance to ouabain) in V79
Chinese hamster cells with
hamster embryo cells
Hypoxanthine-guanine phos-
phoribosyl transferase assay
(resistance to ouabain) in V79
Chinese hamster cells with
hamster embryo cells
Hypoxanthine-guanine
phosphoribosyl transferase
assay (resistance to 6-thio-
guanine) in UV-sensitive CHO
cells with rat Ar S9
Thymidine kinase assay
(resistance to trifluoro-
thymidine) in mouse lymph-
oma cells (L5 178Y) with rat
ArS9
C-54
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-10. In vitro mammalian mutagenicity: data use
Record
number
24670
Reference
Durantetal.,
1999
Data
source
Table 1
Data points
Use dose-response data for
BaPery, BbPery, DBaeF,
DBafF, DBahP, DBaiP,
DBelP, N23aP, N23eP;
positive control is reported as
1,000 ng/mL BaP (reported
separately for each PAH)
Basis for RPF
approach
Model;
quanta! data
Comments
Thymidine kinase assay
(resistance to trifluoro-
thymidine) in human hi Alv2
cells
C-55
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
16920
16940
16910
Reference
Amacher and
Paillet, 1982
Amacher and
Turner, 1980
Amacher et al,
1980
PAH
Control
BaP
BaP
BaP
BaP
Control
BaA
BaA
BaA
BaA
Control
BaP
BaA
Control
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
Control
BaA
BaA
BaA
BaA
BaA
BaA
BaA
Dose
0
2.5
5
7.5
10
0
2.5
5
10
15
0
1.25 x 10'5
3.22 x 10'5
0
5.30 x 10'6
7.00 x 10'6
9.40 x 10'6
1.25 x 10'5
1.67x 10'5
2.23 x 10'5
2.97 x 10'5
3.96 x 10'5
0
1.36x 10'5
1.81 x 10'5
2.42 x 10'5
3.22 x 10'5
4.30 x 10'5
5.47 x 10'5
7.65 x 10'5
Dose units
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
Mutants
39
119
170
196
267
20
65
62
88
89
0.4
2.85
3.12
0.680
1.360
1.790
1.470
1.870
2.600
2.490
2.650
3.970
0.770
0.810
0.840
1.000
1.230
1.470
NS
NS
In number
1 x 106
1 x 106
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x 106
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
% Response
0.000039
0.00012
0.00017
0.00020
0.00027
0.000020
0.000065
0.000062
0.000088
0.000089
0.000040
0.000285
0.000312
0.000068
0.000136
0.000179
0.000147
0.000187
0.000260
0.000249
0.000265
0.000397
0.000077
0.000081
0.000084
0.000100
0.000123
0.000147
Comments
Control without S9
treatment
NS = no survivors
C-56
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
17140
24670
Reference
Barfknechtet al,
1982
Durantetal., 1999
PAH
BaA
Control
BaP
BaP
BaP
Control
FA
FA
FA
Control
BaA
BaA
BaA
BaA
Control
CH
CH
CH
Control
Tphen
Tphen
Tphen
Control
CPcdP
CPcdP
CPcdP
BaP
BaP
BaP
BaP
BaP
Dose
1.02x 10'4
0
10
20
30
0
10
20
40
0
20
50
100
150
0
20
50
100
0
50
100
200
0
23
47
88
1,000
1,000
1,000
1,000
1,000
Dose units
M
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
Mutants
NS
0
51
120
155
0
27
50
62
0
12
29
34
64
0
17
26
30
0
10
20
35
3
11
24
27
170
170
200
200
160
In number
1 x 104
1 x 106
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x IQ6
1 x IQ6
1 x IQ6
1 x 1Q6
1 x IQ6
1 x IQ6
1 x 1Q6
1 x 106
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
% Response
0.000000
0.000051
0.000120
0.000155
0.000000
0.000027
0.000050
0.000062
0.000000
0.000012
0.000029
0.000034
0.000064
0.000000
0.000017
0.000026
0.000030
0.000000
0.000010
0.000020
0.000035
0.000003
0.000011
0.000024
0.000027
0.00017
0.00017
0.00020
0.00020
0.00016
Comments
C-57
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
Reference
PAH
BaP
BaP
BaP
BaP
Averaged
BaP
Averaged
controls
Control
BaPery
BaPery
BaPery
BaPery
BaPery
BaPery
Control
BbPery
BbPery
BbPery
BbPery
Control
DBaeF
DBaeF
DBaeF
DBaeF
Control
DBafF
DBafF
DBafF
DBafF
Control
DBahP
Dose
1,000
1,000
1,000
1,000
1,000
0
0
0.1
0.3
1
3
10
100
0
1
3
10
100
0
1
10
100
1,000
0
1
10
100
1,000
0
0.1
Dose units
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
Mutants
170
190
200
210
186
20
18
21
23
28
50
82
200
18
19
22
32
54
21
29
72
190
np
21
21
37
81
190
19
24
In number
1 x 106
1 x 106
1 x 106
1 x 1Q6
1 x 106
1 x 106
1 x IQ6
1 x IQ6
1 x IQ6
1 x IQ6
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x 106
1 x 106
1 x 106
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x 106
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
% Response
0.00017
0.00019
0.00020
0.00021
0.00019
0.00002
0.000018
0.000021
0.000023
0.000028
0.000050
0.000082
0.00020
0.000018
0.000019
0.000022
0.000032
0.000054
0.000021
0.000029
0.000072
0.00019
0.000021
0.000021
0.000037
0.000081
0.00019
0.000019
0.000024
Comments
Not plated due to
excessive toxicity
C-58
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
14250
Reference
Hassetal., 1982
PAH
DBahP
DBahP
DBahP
Control
DBaiP
DBaiP
DBaiP
DBaiP
Control
DBelP
DBelP
DBelP
Control
N23aP
N23aP
N23aP
N23aP
N23aP
Control
N23eP
N23eP
N23eP
N23eP
Control
BaP
BaP
DBaiP
DBaiP
DBaiP
DBahP
DBahP
DBahP
Dose
1
10
100
0
0.3
1
10
100
0
10
100
1,000
0
0.1
1
10
100
1,000
0
1
10
100
1,000
0
0.30
1.00
0.03
0.10
0.30
0.03
0.10
0.30
Dose units
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
Hg/mL
Hg/mL
Hg/mL
Hg/mL
Hg/mL
Hg/mL
Hg/mL
|ig/mL
Hg/rnL
Mutants
24
46
80
20
20
35
88
150
21
28
34
55
21
23
44
84
94
73
19
20
41
74
98
4
267
293
124
289
1211
110
264
668
In number
1 x 106
1 x 106
1 x 106
1 x 1Q6
1 x 106
1 x 106
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 1Q6
1 x IQ6
1 x IQ6
1 x 1Q6
1 x IQ6
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x IQ6
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
CFC
CFC
CFC
CFC
CFC
CFC
CFC
CFC
CFC
% Response
0.000024
0.000046
0.000080
0.000020
0.000020
0.000035
0.000088
0.00015
0.000021
0.000028
0.000034
0.000055
0.000021
0.000023
0.000044
0.000084
0.000094
0.000073
0.000019
0.000020
0.000041
0.000074
0.00010
0.0000040
0.00027
0.00029
0.00012
0.00029
0.00121
0.00011
0.00026
0.00067
Comments
C-59
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
18740
18990
24720
19180
24680
Reference
Huberman and
Sachs, 1976
Jotz and Mitchell,
1981
Kligerman et al,
1986
Krahn and
Heidelberger, 1977
Lafleur et al., 1993
PAH
Control
BaP
DBacA
DBahA
Control
BaP
Control
Pyr
Control
BaP
BaP
BaP
Control
B1AC
B1AC
B1AC
Control
BaP
Control
BaA
Control
BaP
BaP
BaP
BaP
BaP
Control
ACEA
Dose
0
1
1
1
0
4.5
0
10.6
0
2.0
3.0
4.0
0
0.5
2.5
5.0
0
15.9
0
46.5
0
0.02
0.06
0.2
1
5
0
1
Dose units
Hg/mL
Hg/mL
Hg/mL
Hg/mL
|ig/mL
Hg/mL
Hg/mL
|ig/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
nmol/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Mutants
6
425
22
17
80
224
116
150
92
258
417
557
90
93
197
374
1.7
14
1.5
6.5
1.2
4.8
24
25
39
56
1.8
6.0
In number
1 x 105
1 x 105
1 x IQ5
1 x IQ5
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x IQ6
1 x 1Q6
1 x IQ6
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
% Response
0.000060
0.00425
0.00022
0.00017
0.000080
0.00022
0.00012
0.00015
0.00009
0.00026
0.00042
0.00056
0.00009
0.00009
0.00020
0.00037
0.000017
0.000136
0.000015
0.000065
0.0000012
0.0000048
0.000024
0.000025
0.000039
0.000056
0.0000018
0.0000060
Comments
With metabolic
activation
With metabolic
activation
Average of two
experiments
3-MC S9; 40% survival
3-MC S9; 40% survival
C-60
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
7550
11450
Reference
Li and Lin, 1996
Nesnowetal., 1984
PAH
ACEA
ACEA
Control
CPcdP
CPcdP
CPcdP
CPcdP
CPcdP
Control
CPhiACEA
CPhiACEA
CPhiACEA
Control
BaP
BaA
Control
BaP
BaP
BaP
BaP
BaP
BaP
BeAC
BeAC
BeAC
BeAC
BeAC
BjAC
BjAC
BjAC
BjAC
BjAC
Dose
3
8
0
0.03
0.06
0.2
0.6
2
0
0.1
0.3
0.8
0
10
10
0
0.5
1.0
2.5
5.0
10.0
20.0
1.0
2.5
5.0
10.0
20.0
1.0
2.5
5.0
10.0
20.0
Dose units
Hg/mL
Hg/mL
Hg/mL
Hg/mL
Hg/mL
|ig/mL
Hg/rnL
|ig/mL
|ig/mL
Hg/mL
|ig/mL
|ig/mL
ng/mL
ng/mL
ng/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Mutants
15
21
2.5
4.2
4.9
5.9
10
17
2.8
12
25
31
2
5
5.6
16
10
46
72
206
215
293
17
53
435
235
349
24
94
268
225
215
In number
1 x 106
1 x 106
1 x 106
1 x 1Q6
1 x 106
1 x 106
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ6
1 x 1Q6
1 x IQ6
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x IQ6
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
% Response
0.000015
0.000021
0.0000025
0.0000042
0.0000049
0.0000059
0.000010
0.000017
0.0000028
0.000012
0.000025
0.000031
0.000020
0.000050
0.000056
0.000016
0.000010
0.000046
0.000072
0.000206
0.000215
0.000293
0.000017
0.000053
0.000435
0.000235
0.000349
0.000024
0.000094
0.000268
0.000225
0.000215
Comments
C-61
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
15630
15640
21410
16190
Reference
Raveh and
Huberman, 1983
Raveh etal., 1982
Slagaetal., 1978
Vaca etal., 1992
PAH
B1AC
B1AC
B1AC
B1AC
B1AC
Control
BaP
BaP
CPcdP
CPcdP
BaP
BaP
BaP
BaP
CPcdP
CPcdP
CPcdP
CPcdP
Control
BaA
BaA
BaP
BaP
BaP
BaP
BaP
BaP
BaP
FA
FA
FA
Dose
1.0
2.5
5.0
10.0
20.0
0
0.3
1
0.3
1
0
0.3
1
3
0
0.3
1
3
0
4.4
44.0
0.4
1.3
4.0
0
2
4
10
0
5
7.5
Dose units
Mg/mL
Mg/mL
Hg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Hg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
Mutants
31
454
320
704
769
3
25
103
9
20
7
20
74
74
1
5
10
28
0.7
0.9
2.1
11.0
25.0
99.0
3
10
23
31
10
20
27
In number
1 x 106
1 x 106
1 x 106
1 x 1Q6
1 x 106
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ4
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
CFC
CFC
CFC
CFC
CFC
CFC
CFC
CFC
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
% Response
0.000031
0.000454
0.000320
0.000704
0.000769
0.000030
0.00025
0.0010
0.000090
0.00020
0.0000070
0.000020
0.000074
0.000074
0.0000010
0.0000047
0.000010
0.000028
0.000070
0.000090
0.00021
0.0011
0.0025
0.0099
0.000032
0.000102
0.000229
0.000306
0.000105
0.000203
0.000274
Comments
C-62
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. In vitro mammalian mutagenicity: dose-response data
Record
number
21900
Reference
Wangenheim and
Bolcsfoldi, 1988
PAH
Control
Control
Average
BaP
BaP
BaP
Control
FE
FE
FE
FE
FE
Control
Control
Average
Pyr
Pyr
Pyr
Pyr
Pyr
Dose
10
0
0
0
0.000001
0.000005
0.000010
0
0.0000195
0.0000389
0.0000681
0.000122
0.000170
0
0
0
0.0000101
0.0000151
0.0000202
0.0000252
0.0000302
Dose units
HM
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
mol/L
Mutants
32
61
62
62
65
243
858
68
92
91
114
154
147
125
106
116
162
228
345
418
650
In number
1 x 105
1 x 106
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x 1Q6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
1 x IQ6
1 x IQ6
1 x 106
Units
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
Survivors
% Response
0.000318
0.000061
0.000062
0.000062
0.000065
0.000243
0.00086
0.00007
0.00009
0.00009
0.00011
0.00015
0.00015
0.00013
0.00011
0.00012
0.00016
0.00023
0.00035
0.00042
0.00065
Comments
Used average of controls
C-63
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-12. In vitro malignant/morphological cell transformation: data use
Record
number
17610
17970
18020
18080
14130
14640
14700
14850
24710
24700
7980
7990
Reference
Casto, 1979
DiPaolo et al.,
1969
Dunkel etal.,
1981
Emura et al.,
1980
Greb et al.,
1980
Krolewski et
al., 1986
Laaksonen et
al., 1983
Lubetetal.,
1983
Mohapatra et
al., 1987
Nesnowetal.,
1990
Nesnowetal.,
1997
Nesnowetal.,
1994
Page
54
871
153,
154
147
1,648
62
992
327
224
1,975
2,227
Table
number
I and IV
3
I and II
1
1
4
1
1
1
I
I
Figure
number
PAHs
BaP, DBahA
BaP, DBahA,
BaA, BeP,
DBacA
BaP, BbF,
BaA, IP
BaP, CH, BaA,
BbF, DBahA,
BeP
BaP, CPcdP
BaP, BaA
BaP, BeP
BaP, BeAC,
BjAC, B1AC
BaP, B1AC
BaP, DBalP
BaP, DBahA
Data to be extracted
TF in number foci per 105 surviving
cells and dose (jig/mL)
Total transformants, total number of
colonies, and dose (jig/mL)
Use data as reported in 23720 Pienta
1 977; report under that record
T, number of transformed
colonies/1,000 survivals in 10 dishes
and dose (jig/mL)
Relative transformation rate
(potency) in percent/mmol
Transformation frequency per viable
cell x 10"3; single dose (5 |iM)
Transformation frequency (number of
foci/105 surviving cells) and dose
(uM)
DwT-III/td (dishes with Type III foci/
total dishes) and dose (jig/mL)
Number of dishes scored and percent
of dishes with Type II or Type III
foci and dose (jig/mL)
Anchorage independent
colonies/50,000 cells and dose
(Hg/mL)
Type II and III foci/dish (mean and
SD) and dose (|iM)
Type II and III Foci/dish and dose;
use 1 ng/mL dose for DBahA and
mean foci/dish (in parentheses);
single dose for BaP
Basis for
RPF
Ratio of
slopes
Point
estimate
Ratio of
slopes
Ratio of
slopes
Point
estimate
Ratio of
slopes
Ratio of
slopes
Ratio of
slope to
BaP point
estimate
Ratio of
slopes
Ratio of
slopes
Point
estimate
Comment
Data on enhancement of
viral transformation not
used; no straightforward
way to model dose-
response
Use BaP incidence as
BMR
Notes
Model as incidence data
using multistage
Do not use percent
transformants; appears to be
error for DBahA
Model as incidence data
using multistage
Relative transformation
potency at LC50; slope
already calculated
Use only BaP and CPcdP
alone (not with IVA/AIA)
Inverse dose-response
relationship possible due to
cytotoxicity; use peak
Control data in caption (no
transformants); model as
incidence data
Convert percent into number
of dishes and model as
incidence data
Continuous data, no SD for
controls; use peak
Model as continuous data
C-64
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-12. In vitro malignant/morphological cell transformation: data use
Record
number
8000
23720
Reference
Nesnowet al.,
1993a
Pienta et al.,
1977
Page
28
648
Table
number
I
IV
Figure
number
PAHs
DBkmnoAPH
BaP, BaA,
DBahA
Data to be extracted
Peak of Type II and III foci/dish; use
5 ng/mL dose for DBkmnoAPH and
3 ng/mL dose for BaP; average
number foci^dish across the two
experiments
Transformed colonies/surviving
colonies and dose (jig/mL, in row
across)
Basis for
RPF
Point
estimate
Ratio of
slopes
Comment
Notes
Peak transformation for each
compound; DBkmnoAPH
reported in paper as
CP(3,4)B[a]P
Model as incidence data
using multistage
C-65
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
17610
17970
18080
Reference
Casto, 1979
DiPaolo et al.,
1969
Emura et al.,
1980
Expt 1
PAH
Control
BaP
BaP
DBahA
DBahA
Control
BaP
DBahA
BaA
BeP
DBacA
Control
BaP
BaP
BaP
BaP
BaP
BbF
BbF
BbF
Dose
0
0.62
1.25
1.2
2.5
0
10
10
10
10
10
0
0.01
0.05
0.1
0.25
0.5
0.025
0.1
0.25
Dose
units
Hg/mL
Hg/mL
Hg/mL
|ig/mL
Hg/rnL
|ig/mL
Hg/mL
Hg/rnL
Hg/rnL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Transformation measure
Mean
0
8
10
0.5
1
0
8
11
2
1
2
0
0
1.1
2.9
5.3
6.8
0
0.4
0.3
SD
SE
Units
Foci
Foci
Foci
Foci
Foci
Transformants
Transformants
Transformants
Transformants
Transformants
Transformants
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
n
100,000
100,000
100,000
100,000
100,000
354
138
354
190
172
181
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
units
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Number of
surviving
Number of
surviving
Number of
surviving
Number of
surviving
Number of
surviving
Number of
surviving
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
% Response
0
0.00008
0.0001
0.000005
0.00001
0
0.058
0.031
0.011
0.0058
0.011
0
0
0.0011
0.0029
0.0053
0.0068
0
0.00040
0.00030
Notes
C-66
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
Reference
Expt2
PAH
BbF
BbF
BaA
BaA
BaA
BaA
BaA
Control
BaP
BaP
BaP
BaP
BaP
IP
IP
IP
IP
IP
Dose
0.5
1
0.025
0.1
0.25
0.5
1
0
0.01
0.05
0.1
0.25
0.5
0.025
0.1
0.25
0.5
1
Dose
units
Hg/mL
Hg/mL
Hg/mL
Hg/mL
Hg/rnL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Transformation measure
Mean
0.6
1.2
0
0.3
0.3
0.6
1
0
0.4
1
2.9
4.6
7.8
0
0.3
0.3
0.7
1
SD
SE
Units
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
n
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
units
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
Survivals
% Response
0.00060
0.0012
0
0.00030
0.00030
0.00060
0.0010
0
0.00040
0.0010
0.0029
0.0046
0.0078
0
0.00030
0.00030
0.00070
0.0010
Notes
C-67
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
14130
14640
14700
14850
Reference
Greb etal., 1980
Krolewski etal.,
1986
Laaksonen et al.,
1983
Lubet etal.,
1983
PAH
BaP
CH
BaA
BbF
DBahA
BeP
Control
BaP
CPcdP
Control
BaP
BaP
BaP
BaP
Control
BaA
BaA
BaA
BaA
Control
BaP
BaP
BaP
Dose
NA
NA
NA
NA
NA
NA
0
5
5
0
5
10
20
40
0
11
22
44
88
0
1
3
10
Dose
units
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Transformation measure
Mean
277
37
13.9
11.5
0.3
3.1
0
5.5
1.7
0
0.8
0.9
0.3
0.4
0
1.8
1.5
1.1
0.8
0
1
4
5
SD
0.7
0.3
SE
Units
%/mmol
%/mmol
%/mmol
%/mmol
%/mmol
%/mmol
Transformation
frequency
Transformation
frequency
Transformation
frequency
Foci
Foci
Foci
Foci
Foci
Foci
Foci
Foci
Foci
Foci
Dishes with
Type III foci
Dishes with
Type III foci
Dishes with
Type III foci
Dishes with
Type III foci
n
1,000
1,000
1,000
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
1 x IQ5
15
15
15
units
Viable cells
Viable cells
Viable cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Surviving cells
Total dishes
Total dishes
Total dishes
Total dishes
% Response
0
0.0055
0.0017
0
0.0000080
0.0000090
0.0000030
0.0000040
0
0.000018
0.000015
0.000011
0.0000080
0
0.067
0.267
0.333
Notes
Inverse dose-response
relationship possible due
to cytotoxicity; use peak
Inverse dose-response
relationship possible due
to cytotoxicity; use peak
C-68
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
24710
Reference
Mohapatra et al.,
1987
PAH
BeP
BeP
BeP
Control
BaP
BjAC
BjAC
BjAC
BjAC
BjAC
Control
BaP
B1AC
B1AC
B1AC
B1AC
B1AC
Control
Dose
10
30
100
0
1
0.01
0.05
0.5
1
2
0
1
0.5
1
2.5
5
10
0
Dose
units
Hg/mL
Hg/mL
Hg/mL
Hg/rnL
Hg/rnL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Transformation measure
Mean
0
1
7
0
44
2
5
34
45
48
0
50
8
14
31
42
51
0
SD
SE
Units
Dishes with
Type III foci
Dishes with
Type III foci
Dishes with
Type III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
n
15
15
15
48
48
48
48
48
48
48
60
60
60
60
60
60
60
36
units
Total dishes
Total dishes
Total dishes
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
% Response
0
0.067
0.467
0
0.92
0.04
0.1
0.71
0.94
1
0
0.83
0.13
0.26
0.52
0.7
0.85
0
Notes
C-69
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
24700
Reference
Nesnowetal.,
1990
PAH
BaP
BeAC
BeAC
BeAC
BeAC
BeAC
Acetone
BaP
BaP
BaP
BaP
Acetone
Dose
1
0.5
1
2.5
5
10
0
0.1
0.5
2.5
10
0
Dose
units
Hg/mL
Hg/mL
Hg/mL
Hg/rnL
Hg/rnL
|ig/mL
|ig/mL
Hg/mL
Hg/rnL
|ig/mL
Hg/rnL
Hg/rnL
Transformation measure
Mean
31
4
6
13
15
21
25
43
42
39
72
30
SD
14.7
20.7
19.5
23.1
SE
Units
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Dishes with
Type II or III foci
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
n
36
36
36
36
36
36
units
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
Dishes scored
% Response
0.86
0.11
0.17
0.36
0.42
0.58
Notes
C-70
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
7980
7990
Reference
Nesnowetal.,
1997
Nesnowetal.,
1994
PAH
B1AC
B1AC
B1AC
B1AC
Control
BaP
BaP
BaP
DBalP
DBalP
DBalP
Control
BaP
DBahA
DBahA
Dose
0.1
0.5
2.5
10
0
0.4
1.2
4
0.0033
0.1
0.33
0
1
0.25
0.5
Dose
units
Hg/mL
Hg/mL
Mg/mL
Hg/mL
MM
MM
MM
MM
MM
MM
MM
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Transformation measure
Mean
74
68
123
150
0
0.44
1.25
2.54
0.14
1
1.74
0.06
1
0.23
0.25
SD
5.2
14.4
15.6
16.8
0
0.24
0.15
0.56
0.35
0.24
0.78
0.10
0.43
0.21
0.33
SE
Units
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
Anchorage
independent
colonies/
50,000 cells
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
n
units
% Response
Notes
C-71
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
8000
23720
Reference
Nesnowetal.,
1993a
Pienta et al,
1977
PAH
DBahA
DBahA
Control
BaP
BaP
BaP
Control
DBkmno
APH
DBkmno
APH
DBkmno
APH
DBkmno
APH
Control
BaP
BaP
BaP
BaP
BaP
Control
Dose
1
2.5
0
0.3
1
3
0
0.5
1
2.5
5
0
1
5
10
20
40
0
Dose
units
Hg/mL
Hg/mL
Hg/mL
Hg/mL
Hg/rnL
|ig/mL
|ig/mL
Hg/mL
Hg/rnL
Hg/rnL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Transformation measure
Mean
0.43
0.29
0
0.48
0.665
1.4
0
0.23
0.52
0.605
1.085
0
1
2
3
5
4
0
SD
0.11
0.085
SE
Units
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Type II and III
foci/dish
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
n
504
393
406
434
410
427
229
units
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
% Response
0
0.0025
0.0049
0.0069
0.0122
0.0094
0
Notes
BaP and BaA data also
reported in Record 18020
Dunkel 1981
C-72
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-13. In vitro malignant/morphological cell transformation: dose-response data
Record
number
Reference
PAH
BaA
BaA
BaA
BaA
BaA
Control
DBahA
DBahA
DBahA
DBahA
DBahA
Dose
0.1
0.5
1
5
10
0
0.1
0.5
1
5
10
Dose
units
Hg/mL
Hg/mL
Hg/mL
Hg/rnL
Hg/rnL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
Transformation measure
Mean
1
2
2
1
7
0
0
4
4
5
0
SD
SE
Units
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
Transformed
colonies
n
225
252
193
312
250
229
219
233
217
270
232
units
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
Surviving
colonies
% Response
0.0044
0.0079
0.0104
0.0032
0.028
0
0
0.0172
0.0184
0.0185
0
Notes
C-73
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-14. In vitro DNA adducts: data use
Record
number
16890
6300
9510
22800
10660
10670
7870
21200
Reference
Allen and
Coombs,
1980
Binkova et
al., 2000
Bryla and
Weyand,
1992
Grover and
Sims, 1968
Johnsen et
al., 1998
Johnsen et
al., 1997
Melendez-
Colon et
al., 2000
Segerback
and
Vodicka,
1993
Page
245
62
39
160
80
196
13
2,465
Table
number
1
1
1
II
Figure
number
o
5
2
2
o
3
PAHs
BaP, BaA
BaP,
DBalP
BaP, BaA,
DBacA
BaP,
DBahA,
DBacA,
BaA, Pyr,
PH
BjAC,
B1AC, BaP
BjAC,
B1AC, BaP
BaP,
DBalP
Pyr, BghiP,
FA,
DBahA,
BbF, BaP,
BaA, CH
Data to be
extracted
umol com-
pound/mol
DNA PI
Adducts at
each dose
level
Adducts at
each dose
level
Reaction
with DNA
Total adduct
levels in
human
lymphocytes
andHL-60
cells
DNA adduct
levels in
PCB-treated
rat lung cells
Stable DNA
adducts at
each dose
level
Total adduct
levels
Basis for
RPF
Point
estimate
Ratio of
slopes
Ratio of
slopes
Point
estimate
Point
estimate
Point
estimate
Ratio of
slopes
Point
estimate
Comment
Adducts in
nuclear and
mitochondrial
DNA
Slope of adduct
versus dose
curve
Slope of adduct
versus dose
curve under
light conditions
(maximum
response for all
compounds)
Total adducts
formed in
human
lymphocytes or
HL-60 cells
Adducts in
PCB-treated rat
lung Clara and
Type 2 cells
Slope of adduct
versus dose
curve at two
doses
Total adduct
level in
optimized
nuclease PI
adduct
enrichment
procedure
Notes
Calculate
separate
RPFs for
nuclear and
mitochon-
drial DNA
May need to
drop high-
dose data
for adequate
fit
Calculate
RPFs
separately
by cell type
Calculate
RPFs
separately
by cell type
C-74
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-15. In vitro DNA adducts: dose-response data
Record
number
16890
6300
9510
Reference
Allen and
Coombs,
1980
Binkova et
al., 2000
Bryla and
Weyand,
1992
PAH
BaP
BaA
BaP
BaA
BaP
DBalP
BaP
BaP
BaP
BaP
BaA
BaA
BaA
BaA
Dose
0.235
0.644
0.235
0.644
0.010
0.10
0.40
1.0
4.0
10
40
0.010
0.020
0.040
0.080
0.10
0.40
1.0
0.12
12
120
600
0.12
12
120
600
Dose
units
Hg/mL
Hg/mL
Hg/mL
Mg/mL
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
MM
nmol
nmol
nmol
nmol
nmol
nmol
nmol
nmol
DNA adducts
Mean
7.5
0.44
413
104
1.8
18
95
258
205
69
37
179
534
1,304
1,696
2,317
1,971
632
0.17
1.37
2.21
5.45
0.15
0.09
0.8
0.95
SD
1.9
0.11
164
40.2
1.16
7.18
39.4
115
81.9
21.9
10.8
55.3
52.6
375
644
774
729
170
Adduct units
Mmol/mol DNA
P
Mmol/mol DNA
P
Mmol/mol DNA
P
Mmol/mol DNA
P
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
Adducts
n
1 x 108
1 x 108
1 x 108
IxlQ8
1 x 108
1 x 108
IxlQ8
1 x IQ8
1 x 108
IxlQ8
1 x IQ8
1 x 108
IxlQ8
1 x IQ8
1 x IQ7
1 x IQ7
1 x IQ7
1 x IQ7
1 x IQ7
1 x IQ7
1 x IQ7
1 x IQ7
Units
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Notes
Nuclear DNA
Nuclear DNA
Mitochondrial DNA
Mitochondrial DNA
Light conditions; max for BaP and others
C-75
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-15. In vitro DNA adducts: dose-response data
Record
number
22800
10670
10660
Reference
Grover and
Sims, 1968
Johnsen et
al, 1997
Johnsen et
al., 1998
PAH
DBacA
DBacA
DBacA
DBacA
BaP
DBahA
DBacA
BaA
Pyr
PH
BaP
BjAC
B1AC
BaP
BjAC
B1AC
BaP
BjAC
B1AC
BaP
Dose
0.12
12
120
600
5
5
5
5
5
5
30
30
30
30
30
30
30
30
30
30
Dose
units
nmol
nmol
nmol
nmol
Hg
Mg
Hg
Hg
Hg
Hg
Hg/mL
|ig/mL
Hg/mL
Hg/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
|ig/mL
DNA adducts
Mean
0
0.06
0.57
1.76
1.41
0.44
0.56
0.7
0.31
0.05
0.05
0.15
0.24
0.02
0.06
0.03
0.333
0.110
1.089
0.239
SD
0.093
0.026
0.595
0.172
Adduct units
Adducts
Adducts
Adducts
Adducts
jimol/g-atom of
DNAP
|imol/g-atom of
DNAP
|imol/g-atom of
DNAP
limol/g-atom of
DNAP
limol/g-atom of
DNAP
limol/g-atom of
DNAP
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
fmol adducts/ng
DNA
n
1 x 107
1 x 107
1 x IQ7
1 x IQ7
3
3
3
3
Units
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Notes
Clara cells
Clara cells
Clara cells
Type 2 cells
Type 2 cells
Type 2 cells
Human lymphocytes
Human lymphocytes
Human lymphocytes
HL-60 cells
C-76
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-15. In vitro DNA adducts: dose-response data
Record
number
7870
21200
Reference
Melendez-
Colonetal.,
2000
Segerback
and
Vodicka,
1993
PAH
BjAC
B1AC
BaP
BaP
DBalP
DBalP
BaP
Pyr
BghiP
FA
DBahA
BbF
BaA
CH
Dose
30
30
1
2
1
2
100
100
100
100
100
100
100
100
Dose
units
Hg/mL
Hg/mL
\im
\im
|im
|im
mM
mM
mM
mM
mM
mM
mM
mM
DNA adducts
Mean
0.149
0.942
18
34
254
348
15
0.14
0.50
1.5
2.8
3.7
30
50
SD
0.146
0.344
8.07
6.46
4.30
17.20
Adduct units
fmol adducts/jig
DNA
fmol adducts/jig
DNA
Stable adducts
Stable adducts
Stable adducts
Stable adducts
(imol adducts per
mol dNp
(imol adducts per
mol dNp
(imol adducts per
mol dNp
(imol adducts per
mol dNp
(imol adducts per
mol dNp
(imol adducts per
mol dNp
(imol adducts per
mol dNp
|imol adducts per
mol dNp
n
3
3
1 x 106
1 x 106
1 x 106
1 x 106
Units
Nucleotides
Nucleotides
Nucleotides
Nucleotides
Notes
HL-60 cells
HL-60 cells
C-77
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-16. In vitro DNA damage: data use
Record
number
16840
23790
10660
19740
19830
20810
20940
21730
Reference
Agrelo and
Amos, 1981
Ichinotsubo
etal., 1977
Johnsen et
al, 1998
Martinet al,
1978
Mersch-
Sundennann
etal., 1992
Robinson
and Mitchell,
1981
Rossman et
al., 1991
long etal.,
1981b
Page
531
56
82
2,624
3-6
520
354
480
Table
number
2
Table II
1
2
1
2
I
Figure
number
4
PAHs
BaP, Pyr
BaP, DBaiP, DBahA
BaP, BjAC, B1AC
BaP, BeP, BaA,
DBacA, DBahA
BaP, AA, BaA, BbF,
BghiF, BjF, BbFE,
BghiP, BeP, CH,
DBacA, DBahA,
DBalP, DBahP,
DBaiP, FA, IP, PH,
Tphen
BaP, Pyr
BaP, AC, DBacA,
DBahA, PH
BaP, BaA
Data to be extracted
Hydroxyurea inhibited [3H]-thymidine
incorporation into cells (dpm) and dose
(Hg/mL); use 10 ng/mL dose for BaP and
100 ng/mL dose for pyrene
Use column designated JC5519 +S9 for
BaP, DBaiP, and DBahA; dose as ng/well
and response as diameter of zone of
inhibition (mm); the control is wild type
strain AB 11 57
DNA damage (NAAC, 10'V), SDand
dose (ng/mL) for both human
lymphocytes and HL-60 cells; use 24 h +
1 h AraC/HU data (crosshatched bars)
Maximum dpm/jig DNA above
background and dose (M); dose is in
column marked "M"
SOS induction potential for assay (+S9)
for each compound (already incorporates
dose)
Maximum [3H]-TDR incorporation and
dose (test concentration in ng/rnL in
parentheses after maximum) for rows with
metabolic activation (+); use compound-
specific background [ HJ-TDR
incorporation in same row
Max enhancement of prophage induction
over background and dose (amount at
max, in jig/well) for those rows with
S9 (+ rows).
DNA repair grains/nucleus, SD, and dose
(M); four doses BaA, three doses BaP and
DMSO control
Basis for RPF
Point estimate
Point estimate
Ratio of slopes
(human lympho-
cytes); point esti-
mates (HL-60 cells)
Point estimate
Ratio of SOS
induction potentials
Point estimate
Point estimate
Ratio of slopes
Comment
E. coftRecBC, S9
identification
unknown
Background already
subtracted
SOSIP reported in text
as slope of steepest
portion of the
induction factor dose-
response curve
Background already
addressed
Notes
Model as
continuous
data
No modeling
necessary;
slopes
reported in
text
Model as
continuous
data
C-78
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-17. In vitro DNA damage: dose-response data
Record
number
16840
23790
Reference
Agrelo and
Amos, 1981
Ichinotsubo et
al., 1977
PAH
Control
BaP
BaP
BaP
BaP
BaP
BaP
BaP
Control
Pyr
Pyr
Pyr
Pyr
Pyr
Pyr
Control
BaP
Control
DBaiP
Control
Dose
0
0.001
0.01
0.1
1
10
100
1,000
0
0.032
0.16
0.8
4
20
100
0
70
0
600
0
Dose
units
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/well
ug/well
Endpoint
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
Mean
177
195
126
262
818
2,270
819
373
1,168
1,293
1,192
1,367
1,510
1,694
1,716
0
6
0
10
0
SD
Units
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
dpm
Diameter of
zone of
inhibition mm
Diameter of
zone of
inhibition mm
Diameter of
zone of
inhibition mm
Diameter of
zone of
inhibition mm
Diameter of
zone of
inhibition mm
n
Notes
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
HU inhibited
C-79
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-17. In vitro DNA damage: dose-response data
Record
number
10660
19740
Reference
Johnsen et al,
1998
Martin etal.,
1978
PAH
DBahA
DMSO
BaP
BjAC
BIAC
DMSO
BaP
BjAC
BIAC
BaP
BeP
BaA
DBacA
DBahA
Dose
25
0
3
30
3
30
3
30
0
30
30
30
1 x lO'5
1 x lO'6
1 x lO'7
1 x lO'5
1 x lO'5
Dose
units
ug/well
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
M
M
M
M
M
Endpoint
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
DNA damage
Mean
10
4.4
12
15
6.0
9.4
8.2
9.3
7.8
13.2
9.6
11.6
210
256
59
97
96
SD
1.3
3.2
2.7
2.1
3.4
3.2
2.1
3.1
9.5
3.0
5.5
Units
Diameter of
zone of
inhibition mm
NAAC, 10'3 h'1
NAAC, 10'3 h'6
NAAC, 10'3 h'7
NAAC, 10'3 h'2
NAAC, 10'3 h'3
NAAC, 10'3 h'4
NAAC, 10"3 h"5
NAAC, 10"3 h"5
NAAC, 10"3 h"5
NAAC, 10'3 h'5
NAAC, 10'3 h'5
Maximum
dpm/ug DNA
Maximum
dpm/ug DNA
Maximum
dpm/ug DNA
Maximum
dpm/ug DNA
Maximum
dpm/ug DNA
n
3
3
3
3
3
3
3
3
3
3
3
Notes
Human lymphocytes with
AraC/HU
Human lymphocytes with
AraC/HU; no continuous linear
model fit
Human lymphocytes with
AraC/HU
Human lymphocytes with
AraC/HU
Human lymphocytes with
AraC/HU
Human lymphocytes with
AraC/HU; no continuous linear
model fit
Human lymphocytes with
AraC/HU
HL-60 cells with AraC/HU
HL-60 cells with AraC/HU
HL-60 cells with AraC/HU
HL-60 cells with AraC/HU
Increase above background
Increase above background
Increase above background
Increase above background
Increase above background
C-80
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-17. In vitro DNA damage: dose-response data
Record
number
19830
Reference
Mersch-
Sundermann et
al., 1992
PAH
BaP
AA
BaA
BbF
BghiF
BjF
BbFE
BghiP
BeP
CH
DBacA
DBahA
DBalP
DBahP
DBaiP
FA
Dose
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Dose
units
Endpoint
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
SOS induction potential
DNA damage
Mean
0.605
0.142
0.1
0.045
0.34
0.254
0.024
0.033
0.032
0.221
0.104
0.039
2.1
0.117
0.174
0.412
SD
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Units
n
Notes
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
C-81
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-17. In vitro DNA damage: dose-response data
Record
number
20810
20940
Reference
Robinson and
Mitchell, 1981
Rossmanetal.,
1991
PAH
IP
PH
Tphen
Control
BaP
Control
Pyr
BaP
AC
DBacA
DBahA
PH
Dose
NA
NA
NA
0
10
0
7.2
12.5
12.5
1.44
2
25
Dose
units
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
ug/mL
Endpoint
SOS induction potential
SOS induction potential
SOS induction potential
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
Mean
0.036
0.053
0.26
53
142
52
115
10.4
4.8
8
4
4.5
SD
NA
NA
NA
4
7
2
9
Units
[3H]-TdR
incorporation
[3H]-TdR
incorporation
[3H]-TdR
incorporation
[3H]-TdR
incorporation
Lambda pro-
phage induction
Lambda pro-
phage induction
Lambda pro-
phage induction
Lambda pro-
phage induction
Lambda pro-
phage induction
n
Notes
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Steepest slope of induction factor
dose-response curve; + S9
Maximum [3H]-TdR incorporation
Maximum [3H]-TdR incorporation
Maximum [3H]-TdR incorporation
Maximum [3H]-TdR incorporation
Maximum enhancement over
background
Maximum enhancement over
background
Maximum enhancement over
background
Maximum enhancement over
background
Maximum enhancement over
background
C-82
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-17. In vitro DNA damage: dose-response data
Record
number
21730
Reference
Tong et al.,
1981b
PAH
Control
BaP
BaP
BaP
BaA
BaA
BaA
BaA
Dose
0
1 x lO'4
5 x 1Q-4
1 x ID'3
5 x ID'5
1 x ID'4
5 x 1Q-4
1 x ID'3
Dose
units
M
M
M
M
M
M
M
M
Endpoint
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
Unscheduled DNA synthesis
DNA damage
Mean
0.1
45.1
47.7
65.6
0.6
14.8
17.2
Toxic
SD
0.1
3.7
3.7
17.8
2.6
6
Units
Grains/nucleus
Grains/nucleus
Grains/nucleus
Grains/nucleus
Grains/nucleus
Grains/nucleus
Grains/nucleus
Grains/nucleus
n
Notes
C-83
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-18. In vitro clastogenicity: data use
Record
number
14620
14640
19690
21710
Reference
Kochhar, 1982
Krolewski et al.,
1986
Maneetal., 1990
Tongetal.,
1981a
Page
846
1,648
81
469
Table
number
Not
numbered
II
III
1
PAHs
BaP,
BaA
BaP,
CPcdP
BaP,
BaA
BaP,
BaA
Data to be used
Percentage of cells
with aberrations and
dose (ug/mL)
Mean number sister
chromatid exchange/
chromosome, SD, and
dose (uM)
Sister chromatid
exchange frequencies/
for V79 cell + rat
MEC and dose
Sister chromatid
exchange/cell, SD, and
dose
Basis for
RPF
Ratio of
slopes
Ratio of
slopes
Point
estimates
Point
estimates
Comment
Model as incidence data
Use first column of
data; not data with AIA
or IVA; model as
continuous data
Use sister chromatid
exchange data for V79 +
rat MEC only
Continuous data, no n
provided in study
C-84
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-19. In vitro clastogenicity: dose-response data
Record
number
14620
14640
19690
21710
Reference
Kochhar,
1982
Krolewski et
al., 1986
Mane etal.,
1990
Tong etal.,
1981a
PAH
Control
BaP
BaP
BaP
BaP
BaA
BaA
BaA
BaA
Control
BaP
BaP
CPcdP
CPcdP
Control
BaP
BaA
Control
BaP
BaP
BaP
Control
BaA
BaA
BaA
Dose
0
0.6
1.25
2.5
5
0.6
1.25
2.5
5
0
1
5
1
5
0
1
1
0
1 x 10'6
1 x 10'5
1 x 10'4
0
1 x 10'5
1 x 10'4
1 x 10'3
Dose
units
Hg/mL
Hg/mL
Mg/mL
Hg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
Mg/mL
MM
MM
MM
MM
MM
Mg/mL
Mg/mL
Mg/mL
M
M
M
M
M
M
M
M
n
100
100
100
100
100
100
100
100
100
30
30
30
30
30
Clastogenicity
Mean
0.06
0.23
0.32
0.45
0.56
0.17
0.23
0.3
0.38
0.147
0.874
0.932
0.348
0.432
0.3
3
0.7
11.15
16.15
59.75
103.3
15.75
21.2
29.15
26.2
SD
0.059
0.275
0.266
0.119
0.15
1
1
0.5
3.81
3.83
16.96
22.75
5.18
9.59
9.93
6.96
Units
Fraction cells with
aberrations
Fraction cells with
aberrations
Fraction cells with
aberrations
Fraction cells with
aberrations
Fraction cells with
aberrations
Fraction cells with
aberrations
Fraction cells with
aberrations
Fraction cells with
aberrations
Fraction cells with
aberrations
Sister chromatid
exchange
Sister chromatid
exchange
Sister chromatid
exchange
Sister chromatid
exchange
Sister chromatid
exchange
Sister chromatid
exchange frequency
Sister chromatid
exchange frequency
Sister chromatid
exchange frequency
Sister chromatid
exchange/cell
Sister chromatid
exchange/cell
Sister chromatid
exchange/cell
Sister chromatid
exchange/cell
Sister chromatid
exchange/cell
Sister chromatid
exchange/cell
Sister chromatid
exchange/cell
Sister chromatid
exchange/cell
Notes
For V79 cell + rat
MEC
For V79 cell + rat
MEC
For V79 cell + rat
MEC
C-85
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-20. In vivo DNA adducts: data use
Record
number
6210
17630
18810
11190
8010
24590/
20920
22810
24790
24801
Reference
Arif etal.,
1997
Cavalieri et
al., 1981a
Hughes
and
Phillips,
1990
Mass et al.,
1993
Nesnow et
al., 1993b
Nesnow et
al., 1998b;
Ross et al.,
1995
Phillips et
al., 1979
Kligerman
etal., 2002
Weyand et
al., 2004
Page
36
491
1,614
188
39
402
205
846
12,
14
Table
number
o
J
1
2
I
1
Figure
number
4
o
6
land 2
4 and 6
PAHs
DBalP and BaP
CPcdP, ACEP
(reported in
paper as CPAP),
BaP
DBalP, DBaeP,
DBahP, DBaiP,
BaP
BjAC, BaP
BbF, BaP
BaP, BbF,
DBahA, CPcdP,
DBalP
DBahA, DBacA,
BaP
BaA, BaP, BbF,
CH
BcFE, BaP
Data to be
extracted
Mean adduct levels
for heart, pancreas,
bladder, liver
Done
AUC for skin and
lung through 84 d
Done
AUC for lung, liver,
and PEL through
56 d
Done
Done
Done
Mean adduct levels
for lung and
forestomach
Basis for
RPF
Point
estimate
Point
estimate
Point
estimate
Ratio of
Slopes
Point
estimate
Ratio of
Slopes
Point
estimate
Point
estimate
Point
estimate
Comment
Mean adduct levels summed across
mammary epithelial, lung, heart,
pancreas, bladder, liver
DNA-bound PAH in mouse skin
after 4-hr or 24-hr treatment
Sum of AUCs for skin and lung
0-84 d
AUC (adduct-time curve) versus
dose for lung adducts 24-72 hr
Sum of AUCs for lung, liver, and
lymphocytes 0-56 d
Slope of TID AL/dose (slope reported
in Record 24590 based on data from
Record 20920); DBalP data reported
in separate study without BaP
concurrent
Peak binding in mouse skin; BaA
dropped; not clear if reported level is
peak
Adducts in mouse or rat PBLs at
single time point after either
intraperitoneal or gavage
administration
Adducts in mouse lung and
forestomach at single time point after
either intraperitoneal or dietary
administration
Notes
Calculate separate
RPFs for 4-hr and
24-hr treatment
Calculate separate
RPFs for
intraperitoneal and
gavage, rat and
mouse
Calculate separate
RPFs for lung and
forestomach after
oral exposure and
for lung after
intraperitoneal
exposure
C-86
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
6210
17630
Reference
Arif etal,
1997
Cavalieri et
al., 1981a
PAH
Control
BaP
BaP
BaP
BaP
BaP
BaP
DBalP
DBalP
DBalP
DBalP
DBalP
DBalP
BaP
CPcdP
ACEP
BaP
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dose
0
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.2
0.2
0.2
0.2
Dose units
(imol^mammary
gland
|imo I/mammary
gland
|imo I/mammary
gland
(imol/mammary
gland
(imol/mammary
gland
|imo I/mammary
gland
|imo I/mammary
gland
|imo I/mammary
gland
|imo I/mammary
gland
(imol/mammary
gland
(imol/mammary
gland
(imol/mammary
gland
|imo I/mammary
gland
(imol/mouse
(imol/mouse
(imol/mouse
(imol/mouse
Organ
Liver
Mammary
gland
Lung
Heart
Pancreas
Bladder
Liver
Sum
Mammary
gland
Lung
Heart
Pancreas
Bladder
Liver
Sum
Skin
Skin
Skin
Skin
Time
4hr
4hr
4hr
24 hr
DNA adducts
Mean
0
300
11
9.5
0
0
4.5
324.74
1,878
85
64
32
69
116
2,244.63
16.3
2.3
2.2
6.7
SD
45
1.3
378
24
SE
1
0.2
0.1
1.6
Adduct units
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
Adducts/109
nucleotides
(imol adduct/mol
DNA
(imol adduct/mol
DNA
(imol adduct/mol
DNA
(imol adduct/mol
DNA
Slope of
AUC
versus dose
Comments
C-87
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
18810
Reference
Hughes
and
Phillips,
1990
PAH
CPcdP
ACEP
BaP
BaP
BaP
DBaeP
DBaeP
DBaeP
DBahP
DBahP
DBahP
DBaiP
DBaiP
DBaiP
DBaiP
DBaiP
DBaiP
Species
Dose
0.2
0.2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Dose units
(imol/mouse
(imol/mouse
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
(imol
Organ
Skin
Skin
Skin
Lung
Sum skin
and lung
Skin
Lung
Sum skin
and lung
Skin
Lung
Sum skin
and lung
Skin
Lung
Sum skin
and lung
Skin
Lung
Sum skin
and lung
Time
24 hr
24 hr
Id
2d
2d
7d
2d
2d
2d
2d
Id
2d
DNA adducts
Mean
8.8
0.30
7.8
1.2
9.0
0.50
Cannot
determine
Cannot
determine
3.1
0.14
3.2
0.75
0.10
0.85
62
2.3
65
SD
SE
1
0.1
Adduct units
(imol adduct/mol
DNA
(imol adduct/mol
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
fmol adducts/ jig
DNA
Slope of
AUC
versus dose
Comments
Only peak extracted;
interrupted scale
precluded digitizing
C-88
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
11190
Reference
Mass et al.,
1993
PAH
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BjAC
BjAC
BjAC
BjAC
BjAC
BjAC
BjAC
Species
Dose
20
20
20
50
50
50
100
100
100
20
50
100
20
20
20
50
50
50
100
Dose units
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
Organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Time
24 hr
48hr
72hr
24hr
48 hr
72 hr
24 hr
48 hr
72 hr
AUC
AUC
AUC
24hr
48 hr
72 hr
24 hr
48 hr
72 hr
24 hr
DNA adducts
Mean
116
122
181
120
201
432
427
407
2,004
7,884
12,888
44,064
63
97
255
116
402
1,954
180
SD
53
25
101
20
170
274
140
197
314
34
101
392
121
237
1,921
133
SE
Adduct units
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
Slope of
AUC
versus dose
469.73
Comments
AUC calculated using
trapezoid rule
AUC calculated using
trapezoid rule
C-89
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
8010
Reference
Nesnow et
al., 1993b
PAH
BjAC
BjAC
BjAC
BjAC
BjAC
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BaP
BbF
Species
Dose
100
100
20
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Dose units
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg bw
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Liver
Liver
Liver
Liver
Liver
Liver
Liver
PEL
PEL
PEL
PEL
PEL
PEL
PEL
Sum of
AUCs
Lung
Time
48 hr
72 hr
AUC
AUC
AUC
dl
d3
d7
d!4
d28
d56
AUC
dl
d3
d7
d!4
d28
d56
AUC
dl
d3
d7
d!4
d28
d56
AUC
dl
DNA adducts
Mean
532
2,439
6,900
35,880
46,356
453
1,001
574
386
381
143
20,892
398
1,317
931
537
394
116
25,207
158
273
162
187
72
41
5,985
52,084
21
SD
559
2,242
SE
Adduct units
amol adducts/jig
DNA
amol adducts/jig
DNA
Slope of
AUC
versus dose
464.25
Comments
AUC calculated using
trapezoid rule
AUC calculated using
trapezoid rule
C-90
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
24590/
20920
Reference
Nesnow et
al., 1998b;
Ross, 1995
PAH
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BbF
BaP
BbF
Species
Dose
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
NA
NA
Dose units
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Organ
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
PEL
PEL
PEL
PEL
PEL
PEL
PEL
PEL
Sum of
AUCs
Lung
Lung
Time
d3
d5
d7
d!4
d28
d56
AUC
dl
d3
d5
d7
d!4
d28
d56
AUC
dl
d3
d5
d7
d!4
d28
d56
AUC
>21 d
>21d
DNA adducts
Mean
184
233
211
229
145
106
8,763
12
35
51
61
21
15
12
1,173
12
29
59
57
40
15
13
1,378
11,314
SD
SE
3.9
5
Adduct units
Slope of
AUC
versus dose
113
37.5
Comments
Slope of dose versus
TIDAL value (in fmol-
d/ng DNA)
Slope of dose versus
TIDAL value (in fmol-
d/jjg DNA)
C-91
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
22810
24790
Reference
Phillips et
al., 1979
Kligerman
et al., 2002
PAH
CPcdP
DBahA
DBalP
BaP
DBacA
DBahA
BaP
BaA
BbF
CH
Control
BaP
BaA
BbF
CH
Control
Species
Mice
Mice
Mice
Mice
Mice
Mice
Mice
Mice
Mice
Mice
Dose
NA
NA
NA
1
1
1
100
100
100
100
0
100
100
100
100
0
Dose units
(imol/mouse
(imol/mouse
(imol/mouse
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Organ
Lung
Lung
Lung
Skin
Skin
Skin
PEL
PEL
PEL
PEL
PEL
PEL
PEL
PEL
PEL
PEL
Time
>21 d
>21d
>21d
19hr
24 hr
72 hr
d7
d7
d7
d7
d7
d7
d7
d7
d7
d7
DNA adducts
Mean
27
10
15
4,186
93
516
81
0
143
32
39
37
0
SD
273
8
7
11
17
2
4
1
SE
3.69
19.1
267
Adduct units
pmol adducts/mg
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
Slope of
AUC
versus dose
148
219
1,390
Comments
Slope of dose versus
TIDAL value (in finol-
d/ng DNA)
Slope of dose versus
TIDAL value (in finol-
d/ng DNA)
Slope of dose versus
TIDAL value (in finol-
d/ng DNA)
peak
peak
peak
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Gavage
Gavage
Gavage
Gavage
Gavage
C-92
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
24801
Reference
Weyand et
al., 2004
PAH
BaP
BaA
BbF
CH
Control
BaP
BaA
BbF
CH
Control
BaP
BcFE
BcFE
BaP
BcFE
BaP
BcFE
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Mice
Mice
Mice
Mice
Mice
Mice
Mice
Dose
100
100
100
100
0
100
100
100
100
0
230
13.6
197
230
197
230
13.6
Dose units
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg food
mg/kg food
mg/kg food
mg/kg food
mg/kg food
mg/kg food
mg/kg food
Organ
PEL
PEL
PEL
PEL
PEL
PEL
PEL
PEL
PEL
PEL
Lung
Lung
Lung
Forestomach
Forestomach
Sum of lung
and
forestomach
Sum of lung
and
forestomach
Time
d7
d7
d7
d7
d7
d7
d7
d7
d7
d7
d!4
d!4
d!4
d!4
d!4
d!4
d!4
DNA adducts
Mean
755
38
63
24
0
177
20
17
10
0
0.084
0.014
0.18
0.033
0.0092
0.117
0.014
SD
56
3
1
2
30
2
1
4
SE
0.009
0.002
0.023
0.005
0.001
Adduct units
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
amol adducts/ jig
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
Slope of
AUC
versus dose
Comments
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Gavage
Gavage
Gavage
Gavage
Gavage
Diet
Diet
Diet
Diet
Diet
Diet
Diet
C-93
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-21. In vivo DNA adducts: dose-response data
Record
number
Reference
PAH
BcFE
BaP
BcFE
Species
Mice
Mice
Mice
Dose
197
100
100
Dose units
mg/kg food
mg/kg bw
mg/kg bw
Organ
Sum of lung
and
forestomach
Lung
Lung
Time
d!4
24 h
24 h
DNA adducts
Mean
0.19
0.78
0.33
SD
SE
0.13
0.030
Adduct units
pmol adducts/mg
DNA
pmol adducts/mg
DNA
pmol adducts/mg
DNA
Slope of
AUC
versus dose
Comments
Diet
Intraperitoneal
Intraperitoneal
C-94
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-22. In vivo clastogenicity: data use
Record
number
24740
14270
17190
20950
24720
24790
Reference
Allen et
al., 1999
He and
Baker,
1991
Bayer,
1978
Roszinsky-
Kocher et
al., 1979
Kligerman
etal., 1986
Kligerman
etal., 2002
Page
166
426
66
129
846
Table
number
I and III
1
o
3
i
3
1
Figure
number
PAHs
BaP,
DBalP
BaP,
CH
BaP,
PH
BaP,
DBah
A,
CH,
PH,
BeP,
BbF,
BaA
BaP,
B1AC
BaP,
BaA,
BbF,
CH
Data to be extracted
Total micronuleated poly-
chromatic erythrocytes (MN-
PCEs) and dose (mg/kg);
extract data for bone marrow
and peripheral blood for both
A/J mice (Table 1) and p53+/+
(wild type) mice (Table III)
MN cells/ 1,000 binucleated
and dose (ug/mouse)
Sister chromatid exchange/cells
and dose (mg/kg)
Sister chromatid exchanges/
metaphase and dose (mg/kg)
Sister chromatid exchanges/
metaphase and dose (mg/kg)
Sister chromatid exchanges/
metaphase, intraperitoneal, for
BaP, BaA, BbF, and CH; sister
chromatid exchanges, gavage,
for BaP and BaA (use 17.91
value for BaP); also use MN
bn/1,000 bn, gavage, for BaP
and BbF; dose in mg/kg
Basis for
RPF
Point
estimate
Ratio of
slopes
Point
estimate
Point
estimate
Point
estimate
Point
estimates
Comment
Incidence data;
single dose
BaP
Incidence data
Continuous
data; only one
dose PH
significant;
BaP given as
3,4-BaP
Continuous
data, no SD for
control; use
lowest dose
approaching
peak
Separate RPFs
for sister
chromatid
exchanges and
micronuclei,
oral and
intraperitoneal
C-95
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-23. In vivo clastogenicity: dose-response data
Record
number
24740
Reference
Allen etal., 1999
PAH
Tri-
caprylin
BaP
DBalP
DBalP
DBalP
DBalP
Tri-
caprylin
BaP
DBalP
DBalP
DBalP
DBalP
Tri-
caprylin
BaP
DBalP
DBalP
DBalP
Tri-
caprylin
Route of
admini-
stration
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Clastogenicity
Dose
0
200
0.3
1.5
3
6
0
200
0.3
1.5
3
6
0
200
9
12
18
0
Dose units
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Mean
2.6
11.2
2
3.9
3.4
3.8
2.8
9.5
2.8
2.9
4
4.3
3.2
5.1
4.3
7.4
6.1
3.5
SD
Units
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
n
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
%
Response
0.0026
0.0112
0.0020
0.0039
0.0034
0.0038
0.0028
0.0095
0.0028
0.0029
0.0040
0.0043
0.0032
0.0051
0.0043
0.0074
0.0061
0.0035
Units
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
PCEs
P<
0.05
X
X
X
X
X
X
X
Notes
A/J mice, bone marrow
A/J mice, peripheral
blood
p53 +/+ wt mice, bone
marrow
p53 +/+ wt mice, peri-
pheral blood
C-96
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-23. In vivo clastogenicity: dose-response data
Record
number
14270
17190
Reference
He and Baker, 1991
Bayer, 1978
PAH
BaP
DBalP
DBalP
DBalP
Control
BaP
BaP
BaP
BaP
BaP
Control
CH
CH
CH
CH
Pooled
controls
BaP
BaP
BaP
Route of
admini-
stration
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Clastogenicity
Dose
200
9
12
18
0
0.5
5
50
100
500
0
50
100
500
1,000
0
2.5
25
40
Dose units
mg/kg
mg/kg
mg/kg
mg/kg
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
Hg/mouse
mg/kg
mg/kg
mg/kg
mg/kg
Mean
5.7
3.1
3.1
4.6
13.3
50.5
66.8
76
64.3
55.8
12.8
43.3
56
62
47.3
3.2
3.4
3.5
3.9
SD
2.8
11.5
4.1
2.8
5.4
13
2.2
2.2
4.9
8.6
3.8
0.07
0.8
0.2
0.2
Units
MN-PCEs
MN-PCEs
MN-PCEs
MN-PCEs
MN cells
MN cells
MN cells
MN cells
MN cells
MN cells
MN cells
MN cells
MN cells
MN cells
MN cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
n
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
%
Response
0.0057
0.0031
0.0031
0.0046
0.013
0.051
0.067
0.076
0.064
0.056
0.013
0.043
0.056
0.062
0.047
Units
PCEs
PCEs
PCEs
PCEs
Binucleated
Binucleated
Binucleated
Binucleated
Binucleated
Binucleated
Binucleated
Binucleated
Binucleated
Binucleated
Binucleated
P<
0.05
X
X
X
X
X
X
X
X
X
X
X
Notes
C-97
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-23. In vivo clastogenicity: dose-response data
Record
number
20950
Reference
Roszinsky-Kocher et
al., 1979
PAH
BaP
BaP
BaP
PH
PH
PH
PH
Control
BaP
Route of
admini-
stration
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Clastogenicity
Dose
50
75
100
25
50
75
100
0
900
Dose units
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Mean
6.4
6.4
7.4
3.5
3.4
3.5
4.1
3.9
10.6
SD
0.2
0.3
0.2
0.2
0.2
0.2
0.2
0.9
1.6
Units
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchange/
cells
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges/
meta-phase
n
%
Response
Units
P<
0.05
X
X
X
X
X
Notes
Only one dose significant
C-98
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-23. In vivo clastogenicity: dose-response data
Record
number
24720
Reference
Kligerman et al,
1986
PAH
DBahA
CH
PH
BeP
BbF
BaA
Control
BaP
BaP
BaP
Control
Route of
admini-
stration
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Gavage
Gavage
Gavage
Gavage
Gavage
Clastogenicity
Dose
900
900
900
900
900
900
0
63
252
504
0
Dose units
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Mean
4.9
5.1
5.5
5.5
5.6
6.1
11.9
19.4
21.5
21.7
11.0
SD
0.7
1
0.7
0.7
0.5
0.4
0.0
1.4
1.4
Units
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges/
meta-phase
n
%
Response
Units
P<
0.05
X
X
X
X
X
X
Notes
C-99
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-23. In vivo clastogenicity: dose-response data
Record
number
24790
Reference
Kligerman et al.,
2002
PAH
B1AC
B1AC
B1AC
Control
BaP
BaA
BbF
CH
Control
BaP
BaA
Control
Route of
admini-
stration
Gavage
Gavage
Gavage
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Intra-
peritoneal
Gavage
Gavage
Gavage
Gavage
Clastogenicity
Dose
32
63
126
0
100
100
100
100
0
100
100
0
Dose units
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
Mean
16.5
20.5
27.8
8.79
21.21
14.8
22.25
11.96
11.12
17.91
13.38
6.6
SD
3.6
1.6
2.6
1.26
2.93
3.16
1.45
1.8
1.5
1.49
1.53
0.9
Units
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges/
meta-phase
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
Sister
chromatid
exchanges
MNbn
n
1,000
%
Response
0.007
Units
Binucleated
P<
0.05
X
X
X
X
X
X
Notes
C-100
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-23. In vivo clastogenicity: dose-response data
Record
number
Reference
PAH
BaP
BbF
Route of
admini-
stration
Gavage
Gavage
Clastogenicity
Dose
100
100
Dose units
mg/kg
mg/kg
Mean
9.1
8.3
SD
1.8
0.9
Units
MNbn
MNbn
n
1,000
1,000
%
Response
0.009
0.008
Units
Binucleated
Binucleated
P<
0.05
X
X
Notes
C-101
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
o
J
4
5
6
APPENDIX D. BENCHMARK DOSE MODELING OUTPUTS
D.I. DERMAL BIOASSAYS
o
I
C
o
•*=
o
(0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
BMDL
BMD
10
dose
15
20
11:1412/282009
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Cav 1983 bap dermal.out.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\complete\Cavalieril983\BaP\msc_CavalieriBaP_MS_2.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\complete\Cavalieril983\BaP\msc_CavalieriBaP_MS_2.pit
Tue Dec 22 14:50:32 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
D-l
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The parameter betas are restricted
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing
Total number of parameters in model
Total number of specified parameters
Degree of polynomial = 2
Maximum number of iterations = 250
to be positive
values = 0
= 3
= 0
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates
which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background =
Beta(l) =
Beta(2) = 0
0.0155298
0
.00204447
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter (
have been estimated
specified by the user,
and do not appear in
Beta(l) Beta(2)
Beta(l) 1 -0.96
Beta(2) -0.96 1
s) -Background
at a boundary point, or have been
the correlation matrix )
Parameter Estimates
Confidence Interval
Variable Estimate
Upper Conf. Limit
Background 0
*
Beta(l) 0.0126577
*
Beta(2) 0.00134916
*
* - Indicates that this value is not
95.0% Wald
Std. Err. Lower Conf. Limit
* *
* *
* *
calculated.
D-2 DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
i «
lo
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
Model
Full model
Fitted model
0.3878
Reduced model
AIC:
Dose Est
0.0000 0.
2.2000 0.
6.6000 0.
20.0000 0.
ChiA2 = 1.95
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-35.0798 4
-36.0272 2 1.89478 2
-55.062 1 39.9644 3 <.0001
76.0543
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 29 0.000
0338 1.014 2.000 30 0.996
1326 3.714 2.000 28 -0.955
5474 16.423 17.000 30 0.212
d.f. = 2 P-value = 0.3772
Computation
0.1
= Extra risk
0.95
5.31398
2.86439
8.84432
2.86439, 8.84432) is a 90 % two-sided confidence
BMD
Slope Factor = 0.0349115
D-3
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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
T3
-------
1
2 Total number of observations = 4
3 Total number of records with missing values = 0
4 Total number of parameters in model = 3
5 Total number of specified parameters = 0
6 Degree of polynomial = 2
7
8
9 Maximum number of iterations = 250
10 Relative Function Convergence has been set to: 2.22045e-016
11 Parameter Convergence has been set to: 1.49012e-008
12
13 **** we are sorry but Relative Function and Parameter Convergence ****
14 **** are currently unavailable in this model. Please keep checking ****
15 **** the web sight for model updates which will eventually ****
15 **** incorporate these convergence criterion. Default values used. ****
17
18
19
20 Default Initial Parameter Values
21 Background = 0
22 Beta(l) = 0
23 Beta(2) = 4.42193e-005
24
25
26 Asymptotic Correlation Matrix of Parameter Estimates
27
28 ( *** The model parameter(s) -Background
29 have been estimated at a boundary point, or have been
30 specified by the user,
31 and do not appear in the correlation matrix )
32
33 Beta(l) Beta(2)
34
35 Beta(l) 1 -0.93
36
37 Beta(2) -0.93 1
38
39
40
41 Parameter Estimates
42
43 95.0% Wald
44 Confidence Interval
45 Variable Estimate Std. Err. Lower Conf. Limit
46 Upper Conf. Limit
47 Background 0 * *
48 *
49 Beta(l) 0.000525847 * *
50 *
51 Beta(2) 3.60995e-005 * *
52 *
53
54 * - Indicates that this value is not calculated.
55
56
57
58 Analysis of Deviance Table
59
60 Model Log(likelihood) # Param's Deviance Test d.f. P-value
D-5 DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
9
10
11
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
Full model
Fitted model
0.1115
Reduced model
AIC:
Dose Est
0.0000 0.
22.2000 0.
66.6000 0.
200.0000 0.
ChiA2 = 4.25
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
-27.8865 4
-30.0799 2 4.38685 2
-64.1091 1 72.4452 3 <.0
64.1598
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 29 0.000
0290 0.842 2.000 29 1.281
1773 5.141 2.000 29 -1.527
7876 22.840 24.000 29 0.527
d.f. = 2 P-value = 0.1194
Computation
0.1
= Extra risk
0.95
47.2296
30.0553
62.746
30.0553, 62.746 ) is a 90 % two-sided confidence
BMD
Slope Factor = 0.00332721
D-6
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
T3
0)
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0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
dose
1
2
3
4
5
6
7
8
9
10
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12
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30
12:3912/282009
HABS1980BBF.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\complete\Habsl980\BbF\msc_HabsBbF_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\complete\Habsl980\BbF\msc_HabsBbF_MS_2_10.plt
Thu Dec 24 10:03:13 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
D-7
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
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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
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence
**** are currently unavailable in this model. Please keep checking
**** the web sight for model updates which will eventually
**** incorporate these convergence criterion. Default values used.
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0.00945627
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta(2;
Beta (2)
1
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta (1)
*
Beta(2)
Estimate
0
0
0.00748156
Parameter Estimates
95.0% Wald
Std. Err. Lower Conf. Limit
- Indicates that this value is not calculated.
Model
Full model
Fitted model
0.5447
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-47.5575 4
-48.6255 1 2.13602 3
-69.4912
1
43.8674
P-value
<.0001
D-8
DRAFT - DO NOT CITE OR QUOTE
-------
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
AIC:
99.251
Goodness of Fit
Dose
0.0000
3.4000
5.6000
9.2000
Est. Prob.
0.0000
0.0829
0.2091
0.4691
Expected
0.000
3.148
7.110
17.358
Observed
0.000
2.000
5.000
20.000
Size
35
38
34
37
Scaled
Residual
0.000
-0.676
-0.890
0.870
ChiA2 =2.01
d.f. = 3
P-value = 0.5711
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.24
Extra risk
0.95
6.05655
5.19938
7.17099
Taken together, (5.19938, 7.17099) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.0461594
D-9
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
1
2
3
4
5
6
7
8
9
10
11
12
13
14
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12:4412/282009
HOFFMANWYNDER966DBAIP.OUT.txt
0.04 0.06
dose
0.08
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\DBaiP\msc_HoffWynDBaiP_MS_l.(d
)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\DBaiP\msc_HoffWynDBaiP_MS_l.pl
t
Tue Dec 22 14:50:33 2009
BMDS Model Run
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 = incidence
Independent variable = dose
Total number of observations = 3
D-10
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
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31
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.264818
Beta(l) = 18.4583
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta (1)
1
Parameter Estimates
Std. Err.
Confidence Interval
Variable Estimate
Upper Conf. Limit
Background 0
*
Beta(l) 25.3832
* - Indicates that this value is not calculated.
95.0% Wald
Lower Conf. Limit
Model
Full model
Fitted model
0.2358
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-16.5742 3
-18.019 1 2.88957 2
-39.8916
38.0379
1
46.6349
2
P-value
<.0001
D-ll
DRAFT - DO NOT CITE OR QUOTE
-------
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
Goodness of Fit
Dose Est. Prob. Expected Observed
0.0000 0.0000 0.000 0.000
0.0500 0.7189 13.660 16.000
0.1000 0.9210 17.499 16.000
ChiA2 = 3.05 d.f. = 2 P-value = 0.2174
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.00415079
BMDL = 0.00298234
BMDU = 0. 00587793
Taken together, (0.00298234, 0.00587793) is a 90
interval for the BMD
Multistage Cancer Slope Factor = 33.5308
Scaled
Size Residual
20 0.000
19 1.194
19 -1.275
% two-sided confidence
D-12 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
1
2
3
4
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7
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12:4512/282009
HOFFMANWYNDER1966BAP.OUT.txt
0.04 0.06
dose
0.08
0.1
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\BaP\msc_HoffWynBaP_MS_l.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\BaP\msc_HoffWynBaP_MS_l.plt
Tue Dec 22 14:50:32 2009
BMDS Model Run
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 = incidence
Independent variable = dose
Total number of observations = 3
Total number of records with missing values
Total number of parameters in model = 2
= 0
D-13
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.124609
Beta(l) = 29.9573
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta(l)
1
Parameter Estimates
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta (1)
Estimate
0
34.3074
Std. Err.
95.0% Wald
Lower Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
0.8616
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-12.4245 3
-12.5735 1 0.297928 2
-40.3807
27.1469
1
55.9124
2
P-value
<.0001
Goodness of Fit
D-14
DRAFT - DO NOT CITE OR QUOTE
-------
1 Scaled
2 Dose Est. Prob. Expected Observed Size Residual
3 1
4 0.0000 0.0000 0.000 0.000 20 0.000
5 0.0500 0.8201 16.402 17.000 20 0.348
6 0.1000 0.9676 19.353 19.000 20 -0.446
7
8 ChiA2 = 0.32 d.f. = 2 P-value = 0.8522
9
10
11 Benchmark Dose Computation
12
13 Specified effect = 0.1
14
15 Risk Type = Extra risk
16
17 Confidence level = 0.95
18
19 BMD = 0.00307107
20
21 BMDL = 0.00215021
22
23 BMDU = 0.00440601
24
25 Taken together, (0.00215021, 0.00440601) is a 90 % two-sided confidence
26 interval for the BMD
27
28 Multistage Cancer Slope Factor = 46.5071
29
30
31
D-15 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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12:4712/282009
HOFFMANWYNDER1966DBAEF.OUT.txt
0.04 0.06
dose
0.08
0.1
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\DBaeF\msc_HoffWynDBaeF_MS_l.(d
)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\DBaeF\msc_HoffWynDBaeF_MS_l.pl
t
Tue Dec 22 14:50:34 2009
BMDS Model Run
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 = incidence
Independent variable = dose
Total number of observations = 3
D-16
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.22871
Beta(l) = 29.4444
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta (1)
1
Parameter Estimates
Std. Err.
Confidence Interval
Variable Estimate
Upper Conf. Limit
Background 0
*
Beta(l) 37.3037
* - Indicates that this value is not calculated.
95.0% Wald
Lower Conf. Limit
Model
Full model
Fitted model
0.6395
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-10.3111 3
-10.7582 1 0.894194 2
-38.9521
23.5163
1
57.2822
2
P-value
<.0001
D-17
DRAFT - DO NOT CITE OR QUOTE
-------
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
Goodness of Fit
Dose Est. Prob. Expected Observed
0.0000 0.0000 0.000 0.000
0.0500 0.8451 16.058 17.000
0.1000 0.9760 18.544 18.000
ChiA2 = 1.02 d.f. = 2 P-value = 0.5995
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.0028244
BMDL = 0.00193834
BMDU = 0. 00411821
Taken together, (0.00193834, 0.00411821) is a 90
interval for the BMD
Multistage Cancer Slope Factor = 51.5905
Scaled
Size Residual
20 0.000
19 0.598
19 -0.816
% two-sided confidence
D-18 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
0.8
0.7
0.6
T3 0.5
0)
•5
^ 0.4
0
1 0.3
U-
0.2
0.1
0
: Multistage Ca
Linear extrapole
-
-
- //
//
• /
:T /
V
< •
BMDL BMD
0 0.02 0.04
1PPT
ILrCI
tjnn
LIUII
^-^^
'/^
0.06 0.08 0
-;
:
:
-
:
.1
dose
1 12:4812/282009
2
3 HOFFMANWYNDER1996DBAEP.OUT.txt
4
c
J
6 Multistage Cancer Model. (Version:
7 Input Data File:
1.7; Date: 05/16/2008)
8 C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\DBaeP\msc HoffWynDBaeP MS l.(d
9 )
10 Gnuplot Plotting File:
11 C:\USEPA\IRIS\PAH\dermal\complete\HoffWyndl966\DBaeP\msc HoffWynDBaeP MS l.pl
12 t
13 Tue Dec 22 14:50:32 2009
1 A
14
15
16 BMDS Model Run
1 7
18
19 The form of the probability function is :
20
21 P [response] = background + ( 1-background) * [ 1-EXP (
22 -betal*doseAl) ]
23
24 The parameter betas are restricted to be positive
25
26
27 Dependent variable = incidence
28 Independent variable = dose
29
30 Total number of observations = 3
D-19
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.120514
Beta(l) = 7.53772
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta (1)
1
Parameter Estimates
Std. Err.
Confidence Interval
Variable Estimate
Upper Conf. Limit
Background 0
*
Beta(l) 11.2084
* - Indicates that this value is not calculated.
95.0% Wald
Lower Conf. Limit
Model
Full model
Fitted model
0.2414
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-32.4818 3
-33.903 1 2.84251 2
-44.2604
69.8061
1
23.5572
2
P-value
<.0001
D-20
DRAFT - DO NOT CITE OR QUOTE
-------
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
Goodness of Fit
Dose Est. Prob. Expected Observed
0.0000 0.0000 0.000 0.000
0.0500 0.4290 12.871 16.000
0.1000 0.6740 11.458 9.000
ChiA2 = 2.95 d.f. = 2 P-value = 0.2288
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.00940018
BMDL = 0.00681373
BMDU = 0. 0134192
Taken together, (0.00681373, 0.0134192) is a 90
interval for the BMD
Multistage Cancer Slope Factor = 14.6763
Scaled
Size Residual
20 0.000
30 1.154
17 -1.272
% two-sided confidence
D-21 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
2
3
4
5
6
7
8
9
10
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0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
BMDL
BMi)
200
400
600
dose
800
1000
1200
12:5012/282009
LAVOIE1982BkF.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\initiation\LaVoiel982\BkF\msc_LaVoieBkF_MS_2_85.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\initiation\LaVoiel982\BkF\msc_LaVoieBkF_MS_2_85.plt
Thu Dec 24 10:09:52 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
D-22
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence
**** are currently unavailable in this model. Please keep checking
**** the web sight for model updates which will eventually
**** incorporate these convergence criterion. Default values used.
Default Initial Parameter Values
Background = 0.0504814
Beta(l) = 0.00134342
Beta(2) = 0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(2)
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta (1)
1
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta (1)
*
Beta(2)
Estimate
0
0.00163117
0
Parameter Estimates
95.0% Wald
Std. Err. Lower Conf. Limit
- Indicates that this value is not calculated.
Model
Full model
Fitted model
0.6388
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-26.4637 4
-27.3094 1 1.69146 3
-46.0525
1
39.1775
P-value
<.0001
D-23
DRAFT - DO NOT CITE OR QUOTE
-------
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
AIC:
56.6189
Goodness of Fit
Dose
0.0000
30.0000
100.0000
1000.0000
Est. Prob.
0.0000
0.0478
0.1505
0.8043
Expected
0.000
0.955
3.010
16.086
Observed
0.000
1.000
5.000
15.000
Size
20
20
20
20
Scaled
Residual
0.000
0.047
1.244
-0.612
ChiA2 = 1.93
d.f. = 3
P-value = 0.5881
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.85
Extra risk
0.95
1163.04
802.998
1836.46
Taken together, (802.998, 1836.46) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.00105853
D-24
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
2
3
4
5
6
7
8
9
10
11
12
13
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18
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30
T3
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I
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0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL BMD
50
100
dose
150
200
12:51 12/282009
RAVEH1982CPCDP.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\initiation\Ravehl982\CPcdP\msc_RavehCPcdP_MS_2.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\initiation\Ravehl982\CPcdP\msc_RavehCPcdP_MS_2.plt
Tue Dec 22 14:50:35 2009
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
D-25
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
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21
22
23
24
25
26
27
28
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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
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence
**** are currently unavailable in this model. Please keep checking
**** the web sight for model updates which will eventually
**** incorporate these convergence criterion. Default values used.
Default Initial Parameter Values
Background = 0.086614
Beta(l) = 0.00379482
Beta(2) = 0
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l) Beta(2)
Background 1 -0.51 0.37
Beta(l) -0.51 1 -0.96
Beta(2) 0.37 -0.96 1
Parameter Estimates
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta(l)
*
Beta (2)
Estimate
0.0898027
0.0034393
1.91358e-006
Std. Err.
95.0% Wald
Lower Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
0.6443
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-57.7672 4
-57.8738 3 0.213129 1
-69.2679
121.748
1
23.0015
P-value
<.0001
D-26
DRAFT - DO NOT CITE OR QUOTE
-------
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
Goodness of Fit
Dose
0.0000
10.0000
100.0000
200.0000
Est. Prob.
0.0898
0.1207
0.3669
0.5762
Expected
2.604
3.622
10.641
16.134
Observed
3.000
3.000
11.000
16.000
Size
29
30
29
28
Scaled
Residual
0.257
-0.349
0.138
-0.051
ChiA2 = 0.21
d.f. =1
P-value = 0.6472
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
30.1292
19.4197
83.2495
Taken together, (19.4197, 83.2495) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.00514942
D-27
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
2
3
4
5
6
7
8
9
10
11
12
13
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30
T3
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0.8
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
BMDLBMD
50
100
dose
150
200
12:5212/282009
RAVEH 1982BaP.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\initiation\Ravehl982\BaP\msc_RavehBaP_MS_4.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\initiation\Ravehl982\BaP\msc_RavehBaP_MS_4.plt
Tue Dec 22 14:50:34 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2-beta3*doseA3-beta4*doseA4)
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 6
Total number of records with missing values
= 0
D-28
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
H
12
13
14
15
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18
19
20
21
22
23
24
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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
Total number of parameters in model = 5
Total number of specified parameters = 0
Degree of polynomial = 4
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** we are sorry but Relative Function and Parameter Convergence
**** are currently unavailable in this model. Please keep checking
**** the web sight for model updates which will eventually
**** incorporate these convergence criterion. Default values used.
Default Initial Parameter Values
Background = 0
Beta(l) = 6.01899e+017
Beta (2) = 0
Beta(3) = 0
Beta (4) = 0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter (s) -Beta (2) -Beta (3)
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Background
Background 1
Beta(l) -0.66
Beta(4) 0.27
Beta
-0
-0
(1)
.66
1
.52
Beta
0
-0
(4)
.27
.52
1
Parameter Estimates
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta(l)
*
Beta (2)
*
Beta (3)
*
Beta(4)
Estimate
0.132052
0.0479561
0
0
4.58928e-009
Std. Err.
95.0% Wald
Lower Conf. Limit
- Indicates that this value is not calculated.
D-29
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
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
Model
Full model
Fitted model
0.2996
Reduced model
AIC:
Dose Est
0.0000 0.
10.0000 0.
25.0000 0.
50.0000 0.
100.0000 0.
200.0000 1.
ChiA2 = 3.86
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-56.5419 6
-58.376 3 3.66814 3
-101.065 1 89.0461 5 <.0001
122.752
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
1321 3.829 3.000 29 -0.455
4627 13.419 17.000 29 1.334
7388 20.685 21.000 28 0.135
9233 25.853 24.000 28 -1.316
9955 26.878 27.000 27 0.351
0000 26.000 26.000 26 0.001
d.f. = 3 P-value = 0.2771
Computation
0.1
= Extra risk
0.95
2.19702
1.66278
3.30927
1.66278, 3.30927) is a 90 % two-sided confidence
BMD
Slope Factor = 0.0601403
D-30
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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30
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0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
BMDL BMD
0 0.2
12:5312/282009
RICE CPDEFC.OUT.txt
0.4
0.6 0.8
dose
1.2
1.4
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\initiation\Rice\CPdefC\msc_RiceCPdefC_MS_2
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\initiation\Rice\CPdefC\msc_RiceCPdefC_MS_2
Tue Dec 22 16:05:10 2009
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
!. (d)
! .pit
Total number of observations = 4
Total number of records with missing values
Total number of parameters in model = 3
= 0
D-31
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of specified parameters = 0
2 Degree of polynomial = 2
3
4
5 Maximum number of iterations = 250
6 Relative Function Convergence has been set to: 2.22045e-016
7 Parameter Convergence has been set to: 1.49012e-008
8
9 **** we are sorry but Relative Function and Parameter Convergence ****
10 **** are currently unavailable in this model. Please keep checking ****
H **** the wek sight for model updates which will eventually ****
12 **** incorporate these convergence criterion. Default values used. ****
13
14
15
16 Default Initial Parameter Values
17 Background = 1
18 Beta(l) = 6.76726e+019
19 Beta (2) = 0
20
21
22 Asymptotic Correlation Matrix of Parameter Estimates
23
24 ( *** The model parameter(s) -Beta(l)
25 have been estimated at a boundary point, or have been
26 specified by the user,
27 and do not appear in the correlation matrix )
28
29 Background Beta(2)
30
31 Background 1 -0.52
32
33 Beta(2) -0.52 1
34
35
36
37 Parameter Estimates
38
39 95.0% Wald
40 Confidence Interval
41 Variable Estimate Std. Err. Lower Conf. Limit
42 Upper Conf. Limit
43 Background 0.0499931 * *
44 *
45 Beta(l) 0 * *
46 *
47 Beta(2) 44.3919 * *
48 *
49
50 * - Indicates that this value is not calculated.
51
52
53
54 Analysis of Deviance Table
55
56 Model Log(likelihood) # Param's Deviance Test d.f. P-value
57 Full model -16.9192 4
58 Fitted model -16.9195 2 0.000547543 2
59 0.9997
60 Reduced model -49.6481 1 65.4577 3 <.0001
D-32 DRAFT - DO NOT CITE OR QUOTE
-------
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
AIC:
37.839
Goodness of Fit
Dose
0.0000
0.1500
0.5000
1.5000
Est. Prob.
0.0500
0.6501
1.0000
1.0000
Expected
1.000
13.002
19.000
19.000
Observed
1.000
13.000
19.000
19.000
Size
20
20
19
19
Scaled
Residual
0.000
-0.001
0.017
0.000
ChiA2 = 0.00
d.f. =2
P-value = 0.9999
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.88
Extra risk
0.95
0.218546
0.172781
0.384831
Taken together, (0.172781, 0.384831) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
5.09315
D-33
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
2
o
6
4
5
6
7
8
9
10
11
12
13
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16
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0.6
0.4
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Multistage Cancer
Linear extrapolation
BMDL
BMD
0 20
12:5612/282009
NESNOW 1984 DERMAL BLAC MALE.txt
40
60
80
100
dose
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\initiation\Nesnowl984\BIACmale\msc_NesnowBAICmale3HD
D_MS_1.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\initiation\Nesnowl984\BIACmale\msc_NesnowBAICmale3HD
D_MS_l.plt
Tue Dec 22 16:05:10 2009
BMDS Model Run
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 = incidence
Independent variable = dose
D-34
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0
Beta(l) = 0.0283321
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta(l)
Beta(l)
Parameter Estimates
Confidence Interval
Variable Estimate Std. Err.
Upper Conf. Limit
Background 0 *
*
Beta(l) 0.0219722 *
* - Indicates that this value is not calculated.
95.0% Wald
Lower Conf. Limit
Model
Full model
Fitted model
0.6233
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-17.2634 3
-17.7362 1 0.945584 2
-39.5006
37.4725
1
44.4744
2
P-value
<.0001
D-35
DRAFT - DO NOT CITE OR QUOTE
-------
Dose
0.0000
50.0000
100.0000
Est. Prob.
0.0000
0.6667
0.8889
Expected
0.000
13.333
15.111
Observed
0.000
12.000
16.000
Size
20
20
17
Scaled
Residual
0.000
-0.632
0.686
1
2
3 Goodness of Fit
4
5
6
7
8
9
10
11 ChiA2 = 0.87 d.f. = 2 P-value = 0.6471
12
13
14 Benchmark Dose Computation
15
16 Specified effect = 0.67
17
18 Risk Type = Extra risk
19
20 Confidence level = 0.95
21
22 BMD = 50.4574
23
24 BMDL = 35.8134
25
26 BMDU = 72.6771
27
28 Taken together, (35.8134, 72.6771) is a 90 % two-sided confidence
29 interval for the BMD
30
31 Multistage Cancer Slope Factor = 0.0187081
32
33
34
35
36
D-36 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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17
18
19
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0.8
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
BMDL
BMD
0 20 40
13:4612/282009
NESNOW 1984 DERMAL BLAC FEMALE.txt
60
80
100
dose
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\dermal\initiation\Nesnowl984\BIACfemale\msc_NesnowBlaCfemal
e3HDD_MS_4.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\dermal\initiation\Nesnowl984\BIACfemale\msc_NesnowBlaCfemal
e3HDD_MS_4.pit
Mon Dec 28 13:46:08 2009
BMDS Model Run
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 = incidence
Independent variable = dose
Total number of observations = 3
D-37
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of records with missing values = 0
2 Total number of parameters in model = 2
3 Total number of specified parameters = 0
4 Degree of polynomial = 1
5
6
7 Maximum number of iterations = 250
8 Relative Function Convergence has been set to: 2.22045e-016
9 Parameter Convergence has been set to: 1.49012e-008
10
11 **** We are Sorry but Relative Function and Parameter Convergence ****
12 **** are currently unavailable in this model. Please keep checking ****
13 **** the wek sight for model updates which will eventually ****
14 **** incorporate these convergence criterion. Default values used. ****
15
16
17
18 Default Initial Parameter Values
19 Background = 0
20 Beta(l) = 0.0289037
21
22
23 Asymptotic Correlation Matrix of Parameter Estimates
24
25 Background Beta(l)
26
27 Background 1 -0.49
28
29 Beta(l) -0.49 1
30
31
32
33 Parameter Estimates
34
35 95.0% Wald
36 Confidence Interval
37 Variable Estimate Std. Err. Lower Conf. Limit
38 Upper Conf. Limit
39 Background 0.0505105 * *
40 *
41 Beta(l) 0.0234713 * *
42 *
43
44 * - Indicates that this value is not calculated.
45
46
47
48 Analysis of Deviance Table
49
50 Model Log(likelihood) # Param's Deviance Test d.f. P-value
51 Full model -20.7842 3
52 Fitted model -21.1281 2 0.687832 1
53 0.4069
54 Reduced model -39.8916 1 38.2148 2 <.0001
55
56 AIC: 46.2563
57
58
59 Goodness of Fit
60 Scaled
D-3 8 DRAFT - DO NOT CITE OR QUOTE
-------
0.0000
50.0000
100.0000
0.0505
0.7064
0.9092
0.960
14.127
17.275
1.000
13.000
18.000
19
20
19
0.042
-0.553
0.579
1 Dose Est. Prob. Expected Observed Size Residual
2
3
4
5
6
7 ChiA2 = 0.64 d.f. = 1 P-value = 0.4224
8
9
10 Benchmark Dose Computation
11
12 Specified effect = 0.51
13
14 Risk Type = Extra risk
15
16 Confidence level = 0.95
17
18 BMD = 30.3924
19
20 BMDL = 21.4681
21
22 BMDU = 44.3165
23
24 Taken together, (21.4681, 44.3165) is a 90 % two-sided confidence
25 interval for the BMD
26
27 Multistage Cancer Slope Factor = 0.0237562
28
29
30
31
D-39 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
3
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T3
-------
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Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.0934237
Beta(l) = 0.00272909
Beta(2) = 0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(2)
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Background Beta(l)
Background 1 -0.7
Beta(l) -0.7 1
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta(l)
*
Beta(2)
Estimate
0.0601262
0.00312448
0
Parameter Estimates
Std. Err.
95.0% Wald
Lower Conf. Limit
- Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
D-41
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
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Full model -39.5733 4
Fitted model -39.7914 2 0.436272
0.804
Reduced model -46.0668 1 12.987
0.004665
AIC: 83.5828
Goodness of Fit
Dose Est. Prob. Expected Observed Size
0.0000 0.0601 1.142 1.000 19
50.0000 0.1961 3.921 4.000 20
100.0000 0.3123 5.934 7.000 19
250.0000 0.5696 10.823 10.000 19
ChiA2 = 0.44 d.f. = 2 P-value = 0.8007
Benchmark Dose Computation
Specified effect = 0.51
Risk Type = Extra risk
Confidence level = 0.95
BMD = 228.31
BMDL = 149. 811
BMDU = 436.477
Taken together, (149.811, 436.477) is a 90 % two-sided
interval for the BMD
Multistage Cancer Slope Factor = 0.00340429
2
3
Scaled
Residual
-0.137
0.044
0.527
-0.381
confidence
43
D-42 DRAFT - DO NOT CITE OR QUOTE
-------
D.2. INTRAPERITONEAL BIOASSAYS
T3
-------
1
2
3 Dependent variable = incidence
4 Independent variable = dose
5
6 Total number of observations = 3
7 Total number of records with missing values = 0
8 Total number of parameters in model = 2
9 Total number of specified parameters = 0
10 Degree of polynomial = 1
11
12
13 Maximum number of iterations = 250
14 Relative Function Convergence has been set to: 2.22045e-016
15 Parameter Convergence has been set to: 1.49012e-008
16
YJ **** ^6 are sorry but Relative Function and Parameter Convergence ****
18 **** are currently unavailable in this model. Please keep checking ****
19 **** the web sight for model updates which will eventually ****
20 **** incorporate these convergence criterion. Default values used. ****
21
22
23
24 Default Initial Parameter Values
25 Background = 0.0929049
26 Beta(l) = 0.108473
27
28
29 Asymptotic Correlation Matrix of Parameter Estimates
30
31 Background Beta(l)
32
33 Background 1 -0.48
34
35 Beta(l) -0.48 1
36
37
38
39 Parameter Estimates
40
41 95.0% Wald
42 Confidence Interval
43 Variable Estimate Std. Err. Lower Conf. Limit
44 Upper Conf. Limit
45 Background 0.112498 * *
46 *
47 Beta(l) 0.103015 * *
48 *
49
50 * - Indicates that this value is not calculated.
51
52
53
54 Analysis of Deviance Table
55
56 Model Log(likelihood) # Param's Deviance Test d.f. P-value
57 Full model -44.1118 3
58 Fitted model -44.1689 2 0.114322 1
59 0.7353
60 Reduced model -64.1094 1 39.9952 2 <.0001
D-44 DRAFT - DO NOT CITE OR QUOTE
-------
1
2 AIC: 92.3379
3
4
5 Goodness of Fit
6 Scaled
7 Dose Est. Prob. Expected Observed Size Residual
8 1
9 0.0000 0.1125 3.825 4.000 34 0.095
10 3.4600 0.3786 11.737 11.000 31 -0.273
11 17.3000 0.8507 24.669 25.000 29 0.172
12
13 ChiA2 = 0.11 d.f. = 1 P-value = 0.7366
14
15
16 Benchmark Dose Computation
17
18 Specified effect = 0.83
19
20 Risk Type = Extra risk
21
22 Confidence level = 0.95
23
24 BMD = 17.201
25
26 BMDL = 12.2186
27
28 BMDU = 25.6067
29
30 Taken together, (12.2186, 25.6067) is a 90 % two-sided confidence
31 interval for the BMD
32
33 Multistage Cancer Slope Factor = 0.067929
34
35
36
37
D-45 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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T3
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Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0
Beta(l) = 6.19323e+018
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.47
Beta(l) -0.47 1
Parameter Estimates
Std. Err.
Confidence Interval
Variable Estimate
Upper Conf. Limit
Background 0.168707
*
Beta(l) 0.259821
* - Indicates that this value is not calculated.
95.0% Wald
Lower Conf. Limit
Model
Full model
Fitted model
0.5464
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-31.5803 3
-31.7622 2 0.363803 1
-51.0494
67.5244
1
38.9382
P-value
<.0001
Goodness of Fit
Scaled
D-47
DRAFT - DO NOT CITE OR QUOTE
-------
0.0000
3.4600
17.3000
0.1687
0.6617
0.9907
4.893
18.527
16.842
5.000
18.000
17.000
29
28
17
0.053
-0.210
0.399
1 Dose Est. Prob. Expected Observed Size Residual
2
3
4
5
6
7 ChiA2 = 0.21 d.f. = 1 P-value = 0.6496
8
9
10 Benchmark Dose Computation
11
12 Specified effect = 0.81
13
14 Risk Type = Extra risk
15
16 Confidence level = 0.95
17
18 BMD = 6.39184
19
20 BMDL = 4.18834
21
22 BMDU = 10.3811
23
24 Taken together, (4.18834, 10.3811) is a 90 % two-sided confidence
25 interval for the BMD
26
27 Multistage Cancer Slope Factor = 0.193394
28
29
30
D-48 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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0.6
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t>
C
o
'
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Multistage Cancer
Linear extrapolation
BMDL
BMJ)
500
1000
07:4712/282009
1500 2000
dose
2500
3000
3500
WISLOCKI CHRYSENE MALE LIVER.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\IP\Wislockil986\CH\msc_WislockiCHliver_MS_l_44.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\IP\Wislockil986\CH\msc_WislockiCHliver_MS_l_44.plt
Wed Dec 23 11:10:41 2009
HMDS Model Run
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 = incidence
Independent variable = dose
D-49
DRAFT - DO NOT CITE OR QUOTE
-------
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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: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.147839
Beta(l) = 0.000139419
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.57
Beta(l) -0.57 1
Parameter Estimates
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta (1)
Estimate
0.109703
0.00017367
Std. Err.
95.0% Wald
Lower Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
0.229
Reduced model
0.0005661
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-67.0392 3
-67.7628 2 1.44719 1
P-value
-74.516
139.526
1
14.9536
2
D-50
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
A
4-
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6
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33
Goodness of Fit
Dose Est. Prob. Expected Observed Size
0.0000 0.1097 8.008 7.000 73
700.0000 0.2116 7.407 10.000 35
2800.0000 0.4525 15.387 14.000 34
ChiA2 = 1.52 d.f. = 1 P-value = 0.2172
Benchmark Dose Computation
Specified effect = 0.44
Risk Type = Extra risk
Confidence level = 0.95
BMD = 3338.63
BMDL = 2098.51
BMDU = 6591.77
Taken together, (2098.51, 6591.77) is a 90 % two-sided
interval for the BMD
Multistage Cancer Slope Factor = 0.000209673
Scaled
Residual
-0.378
1.073
-0.478
confidence
D-51 DRAFT - DO NOT CITE OR QUOTE
-------
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Multistage Cancer Model with 0.95 Confidence Level
O
I
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ro
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
Multistage Cancer
Linear extrapolation
0 0.5 1 1.5 2
dose
07:5812/282009
Nesnow et al. 1998b i.p DBalP male lung High dose dropped
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\DBalP\msc_NesnowDBalPHDD_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\DBalP\msc_NesnowDBalPHDD_MS_2_10.plt
Wed Dec 23 14:50:54 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
D-52
DRAFT - DO NOT CITE OR QUOTE
-------
1 Independent variable = dose
2
3 Total number of observations = 4
4 Total number of records with missing values = 0
5 Total number of parameters in model = 3
6 Total number of specified parameters = 0
7 Degree of polynomial = 2
8
9
10 Maximum number of iterations = 250
11 Relative Function Convergence has been set to: 2.22045e-016
12 Parameter Convergence has been set to: 1.49012e-008
13
14 **** ^6 are sorry but Relative Function and Parameter Convergence ****
15 **** are currently unavailable in this model. Please keep checking ****
16 **** the web sight for model updates which will eventually ****
Y1 **** incorporate these convergence criterion. Default values used. ****
18
19
20
21 Default Initial Parameter Values
22 Background = 0
23 Beta(l) = 0
24 Beta(2) = 1.14332e+019
25
26
27 Asymptotic Correlation Matrix of Parameter Estimates
28
29 ( *** The model parameter(s) -Beta(l)
30 have been estimated at a boundary point, or have been
31 specified by the user,
32 and do not appear in the correlation matrix )
33
34 Background Beta(2)
35
36 Background 1 -0.27
37
38 Beta(2) -0.27 1
39
40
41
42 Parameter Estimates
43
44 95.0% Wald
45 Confidence Interval
46 Variable Estimate Std. Err. Lower Conf. Limit
47 Upper Conf. Limit
48 Background 0.419864 * *
49 *
50 Beta(l) 0 * *
51 *
52 Beta(2) 1.23372 * *
53 *
54
55 * - Indicates that this value is not calculated.
56
57
58
59 Analysis of Deviance Table
60
D-53 DRAFT - DO NOT CITE OR QUOTE
-------
1
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32
33
34
35
36
37
38
39
40
41
42
Model
Full model
Fitted model
0.3993
Reduced model
AIC:
Dose Est
0.0000 0.
0.3000 0.
1.5000 0.
3.0000 1.
ChiA2 = 1.83
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Log (likelihood) # Param's Deviance Test d.f. P-value
-47.4317 4
-48.3498 2 1.83615 2
-77.3457 1 59.8281 3 <.0001
100.7
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
4199 12.596 15.000 30 0.889
4808 15.867 13.000 33 -0.999
9639 32.771 33.000 34 0.210
0000 35.000 35.000 35 0.017
d.f. = 2 P-value = 0.3998
Computation
0.1
= Extra risk
0.95
0.292233
0.125394
0.383954
0.125394, 0.383954) is a 90 % two-sided confidence
BMD
Slope Factor = 0.797488
D-54
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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T3
0)
•5
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Multistage Cancer
Linear extrapolation
BVIDL BMD
50
100
dose
150
200
08:01 12/282009
Nesnow et al. 1998b i.p BaP male lung
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\BaP\msc_NesnowBaP_MS_4_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\BaP\msc_NesnowBaP_MS_4_10.plt
Wed Dec 23 14:46:42 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2-beta3*doseA3-beta4*doseA4)
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
D-55
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of observations = 6
2 Total number of records with missing values = 0
3 Total number of parameters in model = 5
4 Total number of specified parameters = 0
5 Degree of polynomial = 4
6
7
8 Maximum number of iterations = 250
9 Relative Function Convergence has been set to: 2.22045e-016
10 Parameter Convergence has been set to: 1.49012e-008
11
12 **** we are sorry but Relative Function and Parameter Convergence ****
13 **** are currently unavailable in this model. Please keep checking ****
14 **** the web sight for model updates which will eventually ****
15 **** incorporate these convergence criterion. Default values used. ****
16
17
18
19 Default Initial Parameter Values
20 Background = 1
21 Beta(l) = 5.5061e+017
22 Beta (2) = 0
23 Beta (3) = 0
24 Beta (4) = 0
25
26
27 Asymptotic Correlation Matrix of Parameter Estimates
28
29 ( *** The model parameter(s) -Beta(l) -Beta (2)
30 have been estimated at a boundary point, or have been
31 specified by the user,
32 and do not appear in the correlation matrix )
33
34 Background Beta (3) Beta (4)
35
36 Background 1 -0.67 0.64
37
38 Beta(3) -0.67 1 -1
39
40 Beta(4) 0.64 -1 1
41
42
43
44 Parameter Estimates
45
46 95.0% Wald
47 Confidence Interval
48 Variable Estimate Std. Err. Lower Conf. Limit
49 Upper Conf. Limit
50 Background 0.29287 * *
51 *
52 Beta(l) 0 * *
53 *
54 Beta(2) 0 * *
55 *
56 Beta(3) 0.000178164 * *
57 *
58 Beta(4) 3.09556e-007 * *
59 *
60
D-56 DRAFT - DO NOT CITE OR QUOTE
-------
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1 Q
1 y
20
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33
34
35
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
* - Indicates tha
Model
Full model
Fitted model
0.9997
Reduced model
AIC:
Dose Est
0.0000 0.
5.0000 0.
10.0000 0.
50.0000 1.
100.0000 1.
200.0000 1.
ChiA2 = 0.01
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
t this value is not calculated.
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-35.952 6
-35.958 3 0.0120148 3
-73.3649 1 74.8258 5 <.0001
77.916
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
2929 5.857 6.000 20 0.070
3086 6.172 6.000 20 -0.083
4101 6.972 7.000 17 0.014
0000 19.000 19.000 19 0.000
0000 16.000 16.000 16 0.000
0000 24.000 24.000 24 0.000
d.f. = 3 P-value = 0.9997
Computation
0.1
= Extra risk
0.95
8.35346
2.00564
22.6111
2.00564, 22.6111) is a 90 % two-sided confidence
BMD
Slope Factor = 0.0498594
D-57
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
2
o
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O
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Multistage Cancer
Linear extrapolation
EMDL BMD
50
08:0412/282009
Nesnow et al. 1998b i.p BbF male lung
100
dose
150
200
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\BbF\msc_NesnowBbF_MS_3.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\BbF\msc_NesnowBbF_MS_3.plt
Wed Dec 23 14:46:42 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2-beta3*doseA3)
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 5
D-58
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of records with missing values = 0
2 Total number of parameters in model = 4
3 Total number of specified parameters = 0
4 Degree of polynomial = 3
5
6
7 Maximum number of iterations = 250
8 Relative Function Convergence has been set to: 2.22045e-016
9 Parameter Convergence has been set to: 1.49012e-008
10
11 **** We are Sorry but Relative Function and Parameter Convergence ****
12 **** are currently unavailable in this model. Please keep checking ****
13 **** the wek sight for model updates which will eventually ****
14 **** incorporate these convergence criterion. Default values used. ****
15
16
17
18 Default Initial Parameter Values
19 Background = 0
20 Beta(l) = 5.84708e+017
21 Beta(2) = 0
22 Beta(3) = 0
23
24
25 Asymptotic Correlation Matrix of Parameter Estimates
26
27 ( *** The model parameter(s) -Beta (2)
28 have been estimated at a boundary point, or have been
29 specified by the user,
30 and do not appear in the correlation matrix )
31
32 Background Beta(l) Beta (3)
33
34 Background 1 -0.56 0.31
35
36 Beta(l) -0.56 1 -0.8
37
38 Beta(3) 0.31 -0.8 1
39
40
41
42 Parameter Estimates
43
44 95.0% Wald
45 Confidence Interval
46 Variable Estimate Std. Err. Lower Conf. Limit
47 Upper Conf. Limit
48 Background 0.328834 * *
49 *
50 Beta(l) 0.0184355 * *
51 *
52 Beta(2) 0 * *
53 *
54 Beta(3) 3.37339e-006 * *
55 *
56
57 * - Indicates that this value is not calculated.
58
59
60
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Model
Full model
Fitted model
0.7654
Reduced model
AIC:
Dose Est
0.0000 0.
10.0000 0.
50.0000 0.
100.0000 0.
200.0000 1.
ChiA2 = 0.47
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-34.702 5
-34.9693 3 0.53462 2
-57.3647 1 45.3254 4 <.0001
75.9386
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
3288 6.577 6.000 20 -0.274
4437 7.987 9.000 18 0.481
8249 16.497 16.000 20 -0.293
9964 19.927 20.000 20 0.270
0000 19.000 19.000 19 0.000
d.f. = 2 P-value = 0.7925
Computation
0.1
= Extra risk
0.95
5.68153
2.40867
28.009
2.40867, 28.009 ) is a 90 % two-sided confidence
BMD
Slope Factor = 0.0415166
D-60
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Multistage Cancer Model with 0.95 Confidence Level
1
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T3
0)
•5
I
o
13
ro
0.8
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
BVIDL BMD
50
100
dose
150
200
08:0512/282009
Nesnow et al. 1998b i.p CPcdP male lung
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\CPcdP\msc_NesnowCPcdP_MS_3.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\CPcdP\msc_NesnowCPcdP_MS_3.plt
Wed Dec 23 14:46:43 2009
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2-beta3*doseA3)
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
D-61
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1 Total number of observations = 5
2 Total number of records with missing values = 0
3 Total number of parameters in model = 4
4 Total number of specified parameters = 0
5 Degree of polynomial = 3
6
7
8 Maximum number of iterations = 250
9 Relative Function Convergence has been set to: 2.22045e-016
10 Parameter Convergence has been set to: 1.49012e-008
11
12 **** we are sorry but Relative Function and Parameter Convergence ****
13 **** are currently unavailable in this model. Please keep checking ****
14 **** the web sight for model updates which will eventually ****
15 **** incorporate these convergence criterion. Default values used. ****
16
17
18
19 Default Initial Parameter Values
20 Background = 1
21 Beta(l) = 5.02249e+017
22 Beta (2) = 0
23 Beta (3) = 0
24
25
26 Asymptotic Correlation Matrix of Parameter Estimates
27
28 ( *** The model parameter(s) -Beta(l)
29 have been estimated at a boundary point, or have been
30 specified by the user,
31 and do not appear in the correlation matrix )
32
33 Background Beta(2) Beta(3)
34
35 Background 1 -0.13 0.025
36
37 Beta(2) -0.13 1 -0.99
38
39 Beta(3) 0.025 -0.99 1
40
41
42
43 Parameter Estimates
44
45 95.0% Wald
46 Confidence Interval
47 Variable Estimate Std. Err. Lower Conf. Limit
48 Upper Conf. Limit
49 Background 0.299994 * *
50 *
51 Beta(l) 0 * *
52 *
53 Beta(2) 0.000554719 * *
54 *
55 Beta(3) 9.86997e-005 * *
56 *
57
58 * - Indicates that this value is not calculated.
59
60
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1 A
ID
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46
Model
Full model
Fitted model
1
Reduced model
AIC:
Dose Est
0.0000 0.
10.0000 0.
50.0000 1.
100.0000 1.
200.0000 1.
ChiA2 = 0.00
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-25.6775 5
-25.6775 3 3.06836e-005 2
-56.6963 1 62.0376 4 <.0001
57.3551
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
3000 6.000 6.000 20 0.000
4000 8.000 8.000 20 -0.000
0000 20.000 20.000 20 0.004
0000 19.000 19.000 19 0.000
0000 19.000 19.000 19 0.000
d.f. = 2 P-value = 1.0000
Computation
0.1
= Extra risk
0.95
8.64922
1.95607
17.5713
1.95607, 17.5713) is a 90 % two-sided confidence
BMD
Slope Factor = 0.0511229
D-63
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Multistage Cancer Model with 0.95 Confidence Level
1
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30
T3
0)
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I
o
13
ro
0.8
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
BVIDL BMD
024
08:0512/282009
Nesnow et al. 1998b i.p DBahA male lung
10
dose
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\DBahA\msc_NesnowDBahA_MS_3.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\IP\Nesnowl998b\DBahA\msc_NesnowDBahA_MS_3.plt
Wed Dec 23 14:46:43 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2-beta3*doseA3)
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 5
D-64
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58
59
60
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters =
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been
Parameter Convergence has been set to:
**** We are sorry but Relative Function
**** are currently unavailable in this
0
set to: 2.22045e-016
1.49012e-008
and Parameter Convergence ****
model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background =
Beta(l) = 1.
Beta(2) =
Beta(3) =
Asymptotic Correlation Matrix
( *** The model parameter (s)
have been estimated at
specified by the user,
0
2e+019
0
0
of Parameter Estimates
-Beta (2)
a boundary point, or have been
and do not appear in the correlation matrix )
Background Beta(l)
Background 1 -0.48
Beta(l) -0.48 1
Beta(3) 0.2 -0.81
Beta (3)
0.2
-0.81
1
Parameter Estimates
Confidence Interval
Variable Estimate
Upper Conf. Limit
Background 0.300001
*
Beta(l) 0.446326
*
Beta(2) 0
*
Beta(3) 0.0942115
*
95. 0% Wald
Std. Err. Lower Conf. Limit
* *
* *
* *
* *
* - Indicates that this value is not calculated.
D-65 DRAFT - DO NOT CITE OR QUOTE
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45
Model
Full model
Fitted model
1
Reduced model
AIC:
Dose Est
0.0000 0.
1.2500 0.
2.5000 0.
5.0000 1.
10.0000 1.
ChiA2 = 0.00
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-27.5922 5
-27.5922 3 2.31121e-005 2
-50.4308 1 45.6773 4 <.0001
61.1844
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
3000 6.000 6.000 20 -0.000
6667 12.000 12.000 18 0.000
9474 18.000 18.000 19 -0.000
0000 20.000 20.000 20 0.003
0000 19.000 19.000 19 0.000
d.f. = 2 P-value = 1.0000
Computation
0.1
= Extra risk
0.95
0.233378
0.0933198
0.955315
0.0933198, 0.955315) is a 90 % two-sided confidence
BMD
Slope Factor = 1.07158
D-66
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30
Busby 1984 i.p. multiplicity
FA male
Linear
Nonconstant variance
BMR = lowest statistically significant response in BaP treated animals (after
control subtracted)
Linear Model with 0.95 Confidence Level
c
o
Q.
o:
c
(0
OJ
Linear
BMDL
BME)
10
dose
08:1212/282009
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\IPmult\Busbyl984\FAmale\lin_BusbyFAM_linear_4_28.(d)
Gnuplot Plotting File:
C:\IPmult\Busbyl984\FAmale\lin_BusbyFAM_linear_4_28.plt
Wed Dec 23 15:26:52 2009
BMDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2
Dependent variable = mean
D-67
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60
Independent variable = dose
The polynomial coefficients are restricted to be positive
The variance is to be modeled as Var(i) = exp (lalpha + log (mean (i) ) *
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
lalpha = 0.136152
rho = 0
beta 0 = 0.0180952
beta 1 = 0.427551
Asymptotic Correlation Matrix of Parameter Estimates
lalpha rho beta 0 beta 1
lalpha 1 0.65 0.015 0.00041
rho 0.65 1 0.22 -0.061
beta 0 0.015 0.22 1 -0.24
beta 1 0.00041 -0.061 -0.24 1
Parameter Estimates
95.0% Wald
Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit
Upper Conf. Limit
lalpha 0.634298 0.204652 0.233188
1.03541
rho 0.923372 0.0876305 0.751619
1.09512
beta 0 0.0170376 0.0434041 -0.0680328
0.102108
beta 1 0.426604 0.0861283 0.257796
0.595413
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Seal
Res .
_
0 27 0.04 0.017 0.21 0.21
0.7 31 0.29 0.316 0.84 0.806 -0
3.5 27 1.52 1.51 1.66 1.66 0.
rho)
ed
0.57
.177
0308
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59
60
Model Descriptions for
Model Al: Yij =
Var{e(ij) } =
Model A2: Yij =
Var{e(ij) } =
Model A3: Yij =
Var{e(ij) } =
Model A3 uses any
were specified by
Model R: Yi =
Var{e(i) } =
Model
Al
A2
A3
fitted
R
likelihoods calculated
Mu (i) + e (ij )
SigmaA2
Mu (i) + e (ij )
Sigma (i) A2
Mu (i) + e (ij )
exp(lalpha + rho*ln (Mu (i) ) )
fixed variance parameters that
the user
Mu + e ( i )
S i gma A 2
Likelihoods of Interest
Log (likelihood) # Param's AIC
-46.759351 4 101.518703
-7.114400 6 26.228800
-7.317284 5 24.634569
-7.329046 4 22.658093
-59.984569 2 123.969139
Explanation of Tests
Test 1: Do responses
(A2 vs. R)
Test 2: Are Variances
Test 3: Are variances
and/or variances differ among Dose levels?
Homogeneous? (Al vs A2 )
adequately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the
results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log (Likelihood Ratio) Test df p-value
Test 1
Test 2
Test 3 0
Test 4 0.
The p-value for Test 1
105.74 4 <.0001
79.2899 2 <.0001
.405769 1 0.5241
0235238 1 0.8781
is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to
The p-value for Test 2
model the data
is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3
to be appropriate here
The p-value for Test 4
is greater than .1. The modeled variance appears
is greater than .1. The model chosen seems
D-69
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1 to adequately describe the data
2
3
4 Benchmark Dose Computation
5
6 Specified effect = 4.28
7
8 Risk Type = Point risk
9
10 Confidence level = 0.95
11
12 BMD = 9.99278
13
14
15 BMDL = 7.55762
16
17
18
19
D-70 DRAFT - DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
Busby 1984 i.p. multiplicity
FA female
Linear
Nonconstant variance
BMR = lowest statistically significant response in BaP treated animals (after
control subtracted)
Linear Model with 0.95 Confidence Level
9
10
11
12
13
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3.5
3
2.5
en
1
en
0)
o:
i 1-5
0)
1
0.5
0
-
-
-
~
-
~~/
0
08:1412/282009
Polynomial
Input Data
inr*ir
/
X^
s/
/
-^—
BMDL
X^
,/
^
BMI
5 10 15 20 25 30
dose
Model. (Version: 2.13; Date: 04/08/2008)
File:
C:\IPmult\Busbyl984\FAfemale\lin BusbyFAF linear 3 56. (d)
Gnuplot Plotting File:
C:\IPmult\Busbyl984\FAfemale\lin BusbyFAF linear 3 56. pit
BMDS Model Run
The form of the
Y[dose] = beta 0
Wed Dec 23 15:26:52 2009
response function is:
+ beta l*dose + beta 2*doseA2 + . . .
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Dependent variable = mean
Independent variable = dose
The polynomial coefficients are restricted to be positive
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
lalpha = -1.11206
rho = 0
beta_0 = 0.108571
beta 1 = 0.115306
Asymptotic Correlation Matrix of Parameter Estimates
lalpha rho beta_0 beta_l
lalpha 1 0.94 0.036 -0.047
rho 0.94 1 0.04 -0.052
beta_0 0.036 0.04 1 -0.46
beta 1 -0.047 -0.052 -0.46 1
Confidence Interval
Variable Estimate
Upper Conf. Limit
lalpha 0.353344
1.29466
rho 1.1315
1.70558
beta_0 0.123135
0.24438
beta_l 0.106469
0.211399
Parameter Estimates
Std. Err.
0.480274
0.292904
0.0618608
0.0535364
95.0% Wald
Lower Conf. Limit
-0.587974
0.557421
0.00189039
0.00153987
Dose
Res .
Table of Data and Estimated Values of Interest
N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled
0
0.7
28
20
0.14
0.15
0.123
0.198
0.37
0.49
0.365
0.477
0.245
-0.447
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3.5 21 0.52
Model Descriptions for
Model Al: Yij =
Var{e(ij) } =
Model A2: Yij =
Var{e(ij) } =
Model A3: Yij =
Var{e(ij) } =
Model A3 uses any
were specified by
Model R: Yi =
Var{e(i) } =
Model
Al
A2
A3
fitted
R
0.496 0.82 0.802 0
likelihoods calculated
Mu (i) + e (ij )
S i gma A 2
Mu (i) + e (ij )
Sigma (i) A2
Mu (i) + e (ij )
exp(lalpha + rho*ln (Mu (i) ) )
fixed variance parameters that
the user
Mu + e ( i )
SigmaA2
Likelihoods of Interest
Log (likelihood) # Param's AIC
5.399546 4 -2.799091
13.307908 6 -14.615816
13.189903 5 -16.379806
13.167852 4 -18.335705
2.264796 2 -0.529591
Explanation of Tests
Test 1: Do responses
(A2 vs. R)
Test 2 : Are Variances
Test 3: Are variances
and/or variances differ among Dose levels?
Homogeneous? (Al vs A2 )
adequately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the
results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log (Likelihood Ratio) Test df p-value
Test 1
Test 2
Test 3
Test 4 0.
The p-value for Test 1
22.0862 4 0.0001927
15.8167 2 0.0003677
0.23601 1 0.6271
0441012 1 0.8337
is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to
The p-value for Test 2
model the data
is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3
to be appropriate here
is greater than .1. The modeled variance appears
0.138
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1 The p-value for Test 4 is greater than .1. The model chosen seems
2 to adequately describe the data
3
4
5 Benchmark Dose Computation
6
7 Specified effect = 3.56
8
9 Risk Type = Point risk
10
11 Confidence level = 0.95
12
13 BMD = 32.2804
14
15
16 BMDL = 18.094
17
18
19
D-74 DRAFT - DO NOT CITE OR QUOTE
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1 Nesnow 1998b i.p. multiplicity
2 BbF
3 Drop 2 high doses
4 Linear
5 Nonconstant variance
6 BMR = lowest statistically significant response in BaP treated animals (after
7 control subtracted)
Linear Model with 0.95 Confidence Level
10
11
12
13
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22
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3.5
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49
50
51
52
53
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58
59
60
Dependent variable = mean
Independent variable = dose
The polynomial coefficients are restricted to be positive
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
lalpha = 0.403617
rho = 0
beta_0 = 0.456667
beta 1 = 0.0305
Asymptotic Correlation Matrix of Parameter Estimates
lalpha rho beta_0 beta_l
lalpha 1 0.15 0.059 -0.07
rho 0.15 1 -0.059 0.006
beta_0 0.059 -0.059 1 -0.49
beta 1 -0.07 0.006 -0.49 1
Confidence Interval
Variable Estimate
Upper Conf. Limit
lalpha 0.123284
0.492576
rho 1.49465
2.12253
beta_0 0.511616
0.771396
beta_l 0.0272932
0.0435087
Parameter Estimates
Std. Err.
0.188418
0.320356
0.132543
0.00827339
95.0% Wald
Lower Conf. Limit
-0.246009
0.866761
0.251836
0.0110776
Dose
Res .
Table of Data and Estimated Values of Interest
N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled
0
10
20
18
0.53
0.67
0.512
0.785
0.72
0.75
0.645
0.887
0.128
-0.548
D-76
DRAFT - DO NOT CITE OR QUOTE
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60
50 20 2
Model Descriptions for
Model Al: Yij =
Var{e(ij) } =
Model A2: Yij =
Var{e(ij) } =
Model A3: Yij =
Var{e(ij) } =
Model A3 uses any
were specified by
Model R: Yi =
Var{e(i) } =
Model
Al
A2
A3
fitted
R
1.88 1.82 1.7 0
likelihoods calculated
Mu (i) + e (ij )
S i gma A 2
Mu (i) + e (ij )
Sigma (i) A2
Mu (i) + e (ij )
exp(lalpha + rho*ln (Mu (i) ) )
fixed variance parameters that
the user
Mu + e ( i )
SigmaA2
Likelihoods of Interest
Log (likelihood) # Param's AIC
-39.164718 4 86.329436
-27.688080 6 67.376160
-27.755992 5 65.511983
-28.699972 4 65.399945
-47.123187 2 98.246375
Explanation of Tests
Test 1: Do responses
(A2 vs. R)
Test 2 : Are Variances
Test 3: Are variances
and/or variances differ among Dose levels?
Homogeneous? (Al vs A2 )
adequately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the
results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log (Likelihood Ratio) Test df p-value
Test 1
Test 2
Test 3 0
Test 4
The p-value for Test 1
38.8702 4 <.0001
22.9533 2 <.0001
.135824 1 0.7125
1.88796 1 0.1694
is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to
The p-value for Test 2
model the data
is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3
to be appropriate here
is greater than .1. The modeled variance appears
0.325
D-77
DRAFT - DO NOT CITE OR QUOTE
-------
1 The p-value for Test 4 is greater than .1. The model chosen seems
2 to adequately describe the data
3
4
5 Benchmark Dose Computation
6
7 Specified effect = 3.85
8
9 Risk Type = Point risk
10
11 Confidence level = 0.95
12
13 BMD = 122.316
14
15
16 BMDL = 84.0259
17
18
19
20
D-78 DRAFT - DO NOT CITE OR QUOTE
-------
1 Nesnow 1998b i.p. multiplicity
2 DBahA
3 Drop 2 high doses
4 Linear
5 Nonconstant variance
6 BMR = lowest statistically significant response in BaP treated animals (after
7 control subtracted)
Linear Model with 0.95 Confidence Level
9
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0)
o:
c
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0.5
08:1712/282009
BM1)
3.5
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\IPmult\Nesnowl998b\DBahA\lin_NesnowDBahA_linear_3_85.(d)
Gnuplot Plotting File:
C:\IPmult\Nesnowl998b\DBahA\lin_NesnowDBahA_linear_3_85.plt
Wed Dec 23 15:26:52 2009
BMDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2
Dependent variable = mean
D-79
DRAFT - DO NOT CITE OR QUOTE
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Independent variable = dose
The polynomial coefficients are restricted to be positive
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
lalpha = 0.721148
rho = 0
beta_0 = 0.413333
beta 1 = 1.008
Asymptotic Correlation Matrix of Parameter Estimates
lalpha rho beta 0 beta 1
lalpha 1 -0.35 -0.035 0.037
rho -0.35 1 0.073 -0.083
beta_0 -0.035 0.073 1 -0.49
beta 1 0.037 -0.083 -0.49 1
Confidence Interval
Variable Estimate
Upper Conf. Limit
lalpha 0.0932028
0.484496
rho 1.12871
1.63166
beta_0 0.498826
0.803442
beta_l 0.941334
1.26796
Parameter Estimates
Std. Err.
0.199643
0.256611
0.155419
0.166649
95.0% Wald
Lower Conf. Limit
-0.29809
0.625764
0.19421
0.614709
Dose
Res .
0
1.25
2.5
Table of Data and Estimated Values of Interest
N Obs Mean Est Mean Obs Std Dev Est Std Dev
Scaled
20
18
19
0.53
1.44
3.05
0.499
1.68
2.85
0.72
1.46
1.9
0.708
1.4
1.89
0.197
-0.713
0.456
D-80
DRAFT - DO NOT CITE OR QUOTE
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Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Log (likelihood)
-47.511796
-39.396001
-39.581359
-39.787219
-60.336483
# Param' s
4
6
5
4
2
AIC
103.023592
90.792002
89.162719
87.574439
124.672966
Model
Al
A2
A3
fitted
R
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (Al vs A2)
Test 3: Are variances adequately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio) Test df
41.881
16.2316
0.370717
0.41172
4
2
1
1
p-value
<.0001
0.0002988
0.5426
0.5211
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1.
model appears to be appropriate
The p-value for Test 3 is greater than
to be appropriate here
A non-homogeneous variance
,1. The modeled variance appears
The p-value for Test 4 is greater than .1. The model chosen seems
D-81
DRAFT - DO NOT CITE OR QUOTE
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3
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8
9
10
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12
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20
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to adequately describe the data
Benchmark Dose Computation
Specified effect = 3.85
Risk Type = Point risk
Confidence level = 0.95
BMD = 3.56003
BMDL =
2.81986
D.3. LUNG IMPLANTATION BIOASSAYS
Multistage Cancer Model with 0.95 Confidence Level
O
I
O
13
ro
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
0.1 0.2 0.3 0.4 0.5
dose
0.6 0.7
0.8
10:4912/282009
DEUTSCH-WENZEL1983AA.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008;
D-82
DRAFT - DO NOT CITE OR QUOTE
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Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\AA\msc_DeutschAA_MS_l_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\AA\msc_DeutschAA_MS_l_10.plt
Wed Dec 23 11:48:09 2009
HMDS Model Run
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 = incidence
Independent variable = dose
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: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually
**** incorporate these convergence criterion. Default values used.
****
Default Initial Parameter Values
Background = 0
Beta(l) = 0.996523
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta(l)
Parameter Estimates
D-83
DRAFT - DO NOT CITE OR QUOTE
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47
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49
50
51
52
53
54
55
56
95 . 0% Wald
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta (1)
*
Estimate Std. Err. Lower Conf. Limit
0 * *
0.773841 * *
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
0.1162
Reduced model
AIC:
Dose Est
0.0000 0.
0.1600 0.
0.8300 0.
ChiA2 = 3.29
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Analysis of Deviance Table
Log (likelihood) # Param's Deviance Test d.f. P-value
-28.6723 3
-30.8245 1 4.30422 2
-51.1258 1 44.907 2 <.0001
63.6489
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 35 0.000
1165 4.076 1.000 35 -1.621
4739 16.587 19.000 35 0.817
d.f. = 2 P-value = 0.1926
Computation
0.1
= Extra risk
0.95
0.136153
0.0956191
0.202527
0.0956191, 0.202527) is a 90 % two-sided confidence
BMD
Slope Factor = 1.04582
D-84
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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T3
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0.6
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0.2
Multistage Cancer
Linear extrapolation
3MDLBMD
0.2
0.4 0.6
dose
0.8
10:5012/282009
DEUTSCH-WENZEL1983BaP.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BaP\msc_DeutschBaP_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BaP\msc_DeutschBaP_MS_2_10.plt
Wed Dec 23 11:48:08 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
D-85
DRAFT - DO NOT CITE OR QUOTE
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59
60
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.0757681
Beta(l) = 2.82425
Beta(2) = 0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(2)
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta(l)
Beta(l)
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta (1)
*
Beta (2)
Estimate
0
3.25323
0
Parameter Estimates
95.0% Wald
Std. Err. Lower Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
0.926
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-51.1075 4
-51.3412 1 0.467435 3
-96.8119
1
91.4088
P-value
<.0001
D-86
DRAFT - DO NOT CITE OR QUOTE
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1
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20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
AIC:
104.682
Goodness of Fit
Dose
0.0000
0.1000
0.3000
1.0000
Est. Prob.
0.0000
0.2777
0.6232
0.9614
Expected
0.000
9.720
21.811
33.647
Observed
0.000
10.000
23.000
33.000
Size
35
35
35
35
Scaled
Residual
0.000
0.106
0.415
-0.568
ChiA2 = 0.51
d.f. =3
P-value = 0.9177
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
0.0323864
0.0255063
0.0445507
Taken together, (0.0255063, 0.0445507) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
3.9206
D-87
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
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0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
0 0.2
10:51 12/282009
DEUTSCH-WENZEL1983BbF.OUT.txt
0.4
0.6
0.8
dose
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BbF\msc_DeutschBbF_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BbF\msc_DeutschBbF_MS_2_10.plt
Wed Dec 23 11:48:08 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
Total number of parameters in model = 3
= 0
D-88
DRAFT - DO NOT CITE OR QUOTE
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50
51
52
53
54
55
56
57
58
59
60
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.00149382
Beta(l) = 0.226374
Beta(2) = 0.236366
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta(l)
Beta(2)
Beta (1)
1
-0.97
Beta (2)
-0.97
1
Confidence Interval
Variable
Upper Conf. Limit
Background
*
Beta (1)
*
Beta (2)
Estimate
0
0.24518
0.217701
Parameter Estimates
95.0% Wald
Std. Err. Lower Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
0.9944
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-37.8686 4
-37.8743 2 0.0112712 2
-51.7666
1
27.796
P-value
<.0001
D-89
DRAFT - DO NOT CITE OR QUOTE
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1
2
3
4
5
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7
8
9
10
11
12
13
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
AIC:
79.7485
Goodness of Fit
Dose
0.0000
0.1000
0.3000
1.0000
Est. Prob.
0.0000
0.0263
0.0889
0.3705
Expected
0.000
0.922
3.113
12.969
Observed
0.000
1.000
3.000
13.000
Size
35
35
35
35
Scaled
Residual
0.000
0.082
-0.067
0.011
ChiA2 = 0.01
d.f. =2
P-value = 0.9943
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
0.33191
0.184961
0.544229
Taken together, (0.184961, 0.544229) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.540655
D-90
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
1
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16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
T3
0)
•5
I
o
13
ro
0.3
0.25
0.2
0.15
0.1
0.05
Multistage Cancer
Linear extrapolation
BMDL
BMD
0.5
1.5
2
dose
2.5
3.5
10:5212/282009
DEUTSCH-WENZEL1983BghiP.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BghiP\msc_DeutschBghiP_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BghiP\msc_DeutschBghiP_MS_2_10.plt
Wed Dec 23 11:48:09 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
D-91
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of parameters in model = 3
2 Total number of specified parameters = 0
3 Degree of polynomial = 2
4
5
6 Maximum number of iterations = 250
7 Relative Function Convergence has been set to: 2.22045e-016
8 Parameter Convergence has been set to: 1.49012e-008
9
10 **** we are sorry but Relative Function and Parameter Convergence ****
H **** are currently unavailable in this model. Please keep checking ****
12 **** the web sight for model updates which will eventually ****
13 **** incorporate these convergence criterion. Default values used. ****
14
15
16
17 Default Initial Parameter Values
18 Background = 0
19 Beta(l) = 0.0304801
20 Beta (2) = 0
21
22
23 Asymptotic Correlation Matrix of Parameter Estimates
24
25 ( *** The model parameter(s) -Background
26 have been estimated at a boundary point, or have been
27 specified by the user,
28 and do not appear in the correlation matrix )
29
30 Beta(l) Beta(2)
31
32 Beta(l) 1 -0.98
33
34 Beta(2) -0.98 1
35
36
37
38 Parameter Estimates
39
40 95.0% Wald
41 Confidence Interval
42 Variable Estimate Std. Err. Lower Conf. Limit
43 Upper Conf. Limit
44 Background 0 * *
45 *
46 Beta(l) 0.0277423 * *
47 *
48 Beta(2) 0.000645059 * *
49 *
50
51 * - Indicates that this value is not calculated.
52
53
54
55 Analysis of Deviance Table
56
57 Model Log(likelihood) # Param's Deviance Test d.f. P-value
58 Full model -16.8561 4
59 Fitted model -17.033 2 0.353756 2
60 0.8379
D-92 DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Reduced model
0.02491
AIC:
-21.5342
38.0659
9.35614
Goodness of Fit
Dose
0.0000
0.1600
0.8300
4.1500
Est. Prob.
0.0000
0.0044
0.0232
0.1186
Expected
0.000
0.156
0.812
4.032
Observed
0.000
0.000
1.000
4.000
Size
35
35
35
34
Scaled
Residual
0.000
-0.395
0.211
-0.017
ChiA2 = 0.20
d.f. = 2
P-value = 0.9043
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
3.51117
1.82558
8.33008
Taken together, (1.82558, 8.33008) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.0547771
D-93
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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
T3
0)
•5
I
o
13
ro
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
10:5312/282009
DEUTSCH-WENZEL1983BJ F.OUT.txt
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BjF\msc_DeutschBjF_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BjF\msc_DeutschBjF_MS_2_10.plt
Wed Dec 23 11:48:08 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
D-94
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of parameters in model = 3
2 Total number of specified parameters = 0
3 Degree of polynomial = 2
4
5
6 Maximum number of iterations = 250
7 Relative Function Convergence has been set to: 2.22045e-016
8 Parameter Convergence has been set to: 1.49012e-008
9
10 **** we are sorry but Relative Function and Parameter Convergence ****
H **** are currently unavailable in this model. Please keep checking ****
12 **** the web sight for model updates which will eventually ****
13 **** incorporate these convergence criterion. Default values used. ****
14
15
16
17 Default Initial Parameter Values
18 Background = 0.00616121
19 Beta(l) = 0.0709095
20 Beta(2) = 0.0144537
21
22
23 Asymptotic Correlation Matrix of Parameter Estimates
24
25 ( *** The model parameter(s) -Background
26 have been estimated at a boundary point, or have been
27 specified by the user,
28 and do not appear in the correlation matrix )
29
30 Beta(l) Beta(2)
31
32 Beta(l) 1 -0.98
33
34 Beta(2) -0.98 1
35
36
37
38 Parameter Estimates
39
40 95.0% Wald
41 Confidence Interval
42 Variable Estimate Std. Err. Lower Conf. Limit
43 Upper Conf. Limit
44 Background 0 * *
45 *
46 Beta(l) 0.0929144 * *
47 *
48 Beta(2) 0.0101278 * *
49 *
50
51 * - Indicates that this value is not calculated.
52
53
54
55 Analysis of Deviance Table
56
57 Model Log(likelihood) # Param's Deviance Test d.f. P-value
58 Full model -39.0246 4
59 Fitted model -39.1336 2 0.218103 2
60 0.8967
D-95 DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
Q
y
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
Reduced model
AIC:
Dose Est
0.0000 0.
0.2000 0.
1.0000 0.
5.0000 0.
ChiA2 = 0.24
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
-60.8862 1 43.7233 3 <.0
82.2673
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 35 0.000
0188 0.658 1.000 35 0.425
0979 3.427 3.000 35 -0.243
5122 17.926 18.000 35 0.025
d.f. = 2 P-value = 0.8868
Computation
0.1
= Extra risk
0.95
1.02045
0.580958
2.07945
0.580958, 2.07945) is a 90 % two-sided confidence
BMD
Slope Factor = 0.172129
D-96 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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
T3
0)
•5
I
o
13
ro
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
0 0.5 1
10:5412/282009
DEUTSCH-WENZEL1983BkF.OUT.txt
1.5
2
dose
2.5
3.5
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BkF\msc_DeutschBkF_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\BkF\msc_DeutschBkF_MS_2_10.plt
Wed Dec 23 11:48:09 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
Total number of parameters in model = 3
= 0
D-97
DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0
Beta(l) = 0.126747
Beta(2) = 0.00410997
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta(l)
Beta(2)
Beta (1)
1
-0.97
Beta (2)
-0.97
1
Confidence Interval
Variable Estimate
Upper Conf. Limit
Background 0
*
Beta(l) 0.0842968
*
Beta(2) 0.0142917
* - Indicates that this value is not calculated.
Parameter Estimates
95.0% Wald
Std. Err. Lower Conf. Limit
Model
Full model
Fitted model
0.5667
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-28.404 4
-28.9719 2 1.1357 2
-46.2443
1
35.6806
P-value
<.0001
D-98
DRAFT - DO NOT CITE OR QUOTE
-------
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
AIC:
61.9437
Goodness of Fit
Dose
0.0000
0.1600
0.8300
4.1500
Est. Prob.
0.0000
0.0138
0.0767
0.4490
Expected
0.000
0.482
2.378
12.122
Observed
0.000
0.000
3.000
12.000
Size
35
35
31
27
Scaled
Residual
0.000
-0.699
0.420
-0.047
ChiA2 =0.67
d.f. =2
P-value = 0.7165
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
1.05954
0.557079
1.79525
Taken together, (0.557079, 1.79525) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.179508
D-99
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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
T3
0)
•5
I
o
13
ro
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
0 0.5 1
10:5512/282009
DEUTSCH-WENZEL1983IP.OUT.txt
1.5
2
dose
2.5
3.5
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\IP\msc_DeutschIP_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Deutschl983\IP\msc_DeutschIP_MS_2_10.plt
Wed Dec 23 11:48:09 2009
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
D-100
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of parameters in model = 3
2 Total number of specified parameters = 0
3 Degree of polynomial = 2
4
5
6 Maximum number of iterations = 250
7 Relative Function Convergence has been set to: 2.22045e-016
8 Parameter Convergence has been set to: 1.49012e-008
9
10 **** we are sorry but Relative Function and Parameter Convergence ****
H **** are currently unavailable in this model. Please keep checking ****
12 **** the web sight for model updates which will eventually ****
13 **** incorporate these convergence criterion. Default values used. ****
14
15
16
17 Default Initial Parameter Values
18 Background = 0.0539703
19 Beta(l) = 0.20919
20 Beta (2) = 0
21
22
23 Asymptotic Correlation Matrix of Parameter Estimates
24
25 ( *** The model parameter(s) -Beta (2)
26 have been estimated at a boundary point, or have been
27 specified by the user,
28 and do not appear in the correlation matrix )
29
30 Background Beta(l)
31
32 Background 1 -0.55
33
34 Beta(l) -0.55 1
35
36
37
38 Parameter Estimates
39
40 95.0% Wald
41 Confidence Interval
42 Variable Estimate Std. Err. Lower Conf. Limit
43 Upper Conf. Limit
44 Background 0.0224449 * *
45 *
46 Beta(l) 0.241452 * *
47 *
48 Beta(2) 0 * *
49 *
50
51 * - Indicates that this value is not calculated.
52
53
54
55 Analysis of Deviance Table
56
57 Model Log(likelihood) # Param's Deviance Test d.f. P-value
58 Full model -54.8079 4
59 Fitted model -56.5662 2 3.5166 2
60 0.1723
D-101 DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
Q
y
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
Reduced model
AIC:
Dose Est
0.0000 0.
0.1600 0.
0.8300 0.
4.1500 0.
ChiA2 =3.12
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
-76.4525 1 43.2893 3 <.0
117.132
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0224 0.786 0.000 35 -0.896
0595 2.082 4.000 35 1.370
2000 6.999 8.000 35 0.423
6411 22.439 21.000 35 -0.507
d.f. = 2 P-value = 0.2104
Computation
0.1
= Extra risk
0.95
0.436361
0.309504
0.819969
0.309504, 0.819969) is a 90 % two-sided confidence
BMD
Slope Factor = 0.323098
D-102 DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Cancer Model with 0.95 Confidence Level
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
T3
0)
•5
I
o
13
ro
0.8
0.6
0.4
0.2
Multistage Cancer
Linear extrapolation
0 0.05
10:5612/282009
WENZEL-HARTUNG1990BaP.OUT.txt
0.1
0.15
dose
0.2
0.25
0.3
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Wenzell990\BaP\msc_WenzelBaP_MS_2_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Wenzell990\BaP\msc_WenzelBaP_MS_2_10.plt
Wed Dec 23 11:48:09 2009
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
D-103
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of parameters in model = 3
2 Total number of specified parameters = 0
3 Degree of polynomial = 2
4
5
6 Maximum number of iterations = 250
7 Relative Function Convergence has been set to: 2.22045e-016
8 Parameter Convergence has been set to: 1.49012e-008
9
10 **** we are sorry but Relative Function and Parameter Convergence ****
H **** are currently unavailable in this model. Please keep checking ****
12 **** the web sight for model updates which will eventually ****
13 **** incorporate these convergence criterion. Default values used. ****
14
15
16
17 Default Initial Parameter Values
18 Background = 0
19 Beta(l) = 3.21631
20 Beta(2) = 5.7325
21
22
23 Asymptotic Correlation Matrix of Parameter Estimates
24
25 ( *** The model parameter(s) -Background
26 have been estimated at a boundary point, or have been
27 specified by the user,
28 and do not appear in the correlation matrix )
29
30 Beta(l) Beta(2)
31
32 Beta(l) 1 -0.93
33
34 Beta(2) -0.93 1
35
36
37
38 Parameter Estimates
39
40 95.0% Wald
41 Confidence Interval
42 Variable Estimate Std. Err. Lower Conf. Limit
43 Upper Conf. Limit
44 Background 0 * *
45 *
46 Beta(l) 3.01149 * *
47 *
48 Beta(2) 6.44644 * *
49 *
50
51 * - Indicates that this value is not calculated.
52
53
54
55 Analysis of Deviance Table
56
57 Model Log(likelihood) # Param's Deviance Test d.f. P-value
58 Full model -50.8389 4
59 Fitted model -50.8521 2 0.0264626 2
60 0.9869
D-104 DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
8
Q
y
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
Reduced model
AIC:
Dose Est
0.0000 0.
0.0300 0.
0.1000 0.
0.3000 0.
ChiA2 = 0.03
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
-84.6566 1 67.6355 3 <.0
105.704
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 35 0.000
0917 3.208 3.000 35 -0.122
3062 10.718 11.000 35 0.103
7732 27.062 27.000 35 -0.025
d.f. = 2 P-value = 0.9870
Computation
0.1
= Extra risk
0.95
0.0326976
0.0198862
0.0559366
0.0198862, 0.0559366) is a 90 % two-sided confidence
BMD
Slope Factor = 5.02861
D-105 DRAFT - DO NOT CITE OR QUOTE
-------
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
Multistage Cancer Model with 0.95 Confidence Level
T3
-------
1
2 Total number of observations = 4
3 Total number of records with missing values = 0
4 Total number of parameters in model = 3
5 Total number of specified parameters = 0
6 Degree of polynomial = 2
7
8
9 Maximum number of iterations = 250
10 Relative Function Convergence has been set to: 2.22045e-016
11 Parameter Convergence has been set to: 1.49012e-008
12
13 **** we are sorry but Relative Function and Parameter Convergence ****
14 **** are currently unavailable in this model. Please keep checking ****
15 **** the web sight for model updates which will eventually ****
15 **** incorporate these convergence criterion. Default values used. ****
17
18
19
20 Default Initial Parameter Values
21 Background = 0
22 Beta(l) = 3.21631
23 Beta(2) = 5.7325
24
25
26 Asymptotic Correlation Matrix of Parameter Estimates
27
28 ( *** The model parameter(s) -Background
29 have been estimated at a boundary point, or have been
30 specified by the user,
31 and do not appear in the correlation matrix )
32
33 Beta(l) Beta(2)
34
35 Beta(l) 1 -0.93
36
37 Beta(2) -0.93 1
38
39
40
41 Parameter Estimates
42
43 95.0% Wald
44 Confidence Interval
45 Variable Estimate Std. Err. Lower Conf. Limit
46 Upper Conf. Limit
47 Background 0 * *
48 *
49 Beta(l) 3.01149 * *
50 *
51 Beta(2) 6.44644 * *
52 *
53
54 * - Indicates that this value is not calculated.
55
56
57
58 Analysis of Deviance Table
59
60 Model Log(likelihood) # Param's Deviance Test d.f. P-value
D-107 DRAFT - DO NOT CITE OR QUOTE
-------
1
2
3
4
5
6
7
9
10
11
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
Full model
Fitted model
0.9869
Reduced model
AIC:
Dose Est
0.0000 0.
0.0300 0.
0.1000 0.
0.3000 0.
ChiA2 = 0.03
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
-50.8389 4
-50.8521 2 0.0264626 2
-84.6566 1 67.6355 3 <.0
105.704
Goodness of Fit
Scaled
. Prob. Expected Observed Size Residual
0000 0.000 0.000 35 0.000
0917 3.208 3.000 35 -0.122
3062 10.718 11.000 35 0.103
7732 27.062 27.000 35 -0.025
d.f. = 2 P-value = 0.9870
Computation
0.57
= Extra risk
0.95
0.197095
0.157781
0.247357
0.157781, 0.247357) is a 90 % two-sided confidence
BMD
Slope Factor = 3.6126
D-108
DRAFT - DO NOT CITE OR QUOTE
-------
1 WENZEL-HARTUNG1990BaPforDBahA.OUT.txt
2 ====================================================================
3 Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
4 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\OTHER
5 ROUTE\SETS\WENZEL-HARTUNG1990.(d)
6 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BIOASSAY\OTHER
7 ROUTE\SETS\WENZEL-HARTUNG1990.plt
8 Thu Jun 02 09:02:58 2005
9 ====================================================================
10
11 BMDS MODEL RUN
19 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
13
14 The form of the probability function is:
15
16 P[response] = background + (1-background)*[1-EXP(
17 -betal*doseAl-beta2*doseA2)]
18
19 The parameter betas are restricted to be positive
20
21
22 Dependent variable = responseBaP
23 Independent variable = doseBaP
24
25 Total number of observations = 4
26 Total number of records with missing values = 0
27 Total number of parameters in model = 3
28 Total number of specified parameters = 0
29 Degree of polynomial = 2
30
31
32 Maximum number of iterations = 250
33 Relative Function Convergence has been set to: le-008
34 Parameter Convergence has been set to: le-008
35
36
37
38 Default Initial Parameter Values
39 Background = 0
40 Beta(l) = 3.21631
41 Beta(2) = 5.7325
42
43
44 Asymptotic Correlation Matrix of Parameter Estimates
45
46 ( *** The model parameter(s) -Background
47 have been estimated at a boundary point, or have been
48 specified by the user,
49 and do not appear in the correlation matrix )
50
51 Beta(l) Beta(2)
52
53 Beta(l) 1 -0.93
54
55 Beta(2) -0.93 1
56
57
58
59 Parameter Estimates
60
D-109 DRAFT - DO NOT CITE OR QUOTE
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Variable
Background
Beta(l)
Beta (2)
Estimate Std. Err.
0 NA
3.01149 2.79594
6.44644 10.7674
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Model
Full model
Fitted model
Reduced model
AIC:
Dose Est
i: 1
0.0000 0.
i: 2
0.0300 0.
i: 3
0.1000 0.
i: 4
0.3000 0.
Chi-square =
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
Analysis of Deviance Table
Log (likelihood) Deviance Test DF P-value
-50.8389
-50.8521 0.0264626 2
-84.6566 67.6355 3
105.704
Goodness of Fit
. Prob. Expected Observed Size
0000 0.000 0 35
0917 3.208 3 35
3062 10.718 11 35
7732 27.062 27 35
0.03 DF = 2 P-value = 0.9870
Computation
0.57
= Extra risk
0.95
0.197095
0.157781
0.9869
<.0001
ChiA2 Res.
0.000
-0.072
0.038
-0.010
D-110
DRAFT - DO NOT CITE OR QUOTE
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Multistage Cancer Model with 0.95 Confidence Level
1
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T3
0)
•5
I
o
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ro
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
0 0.5 1
10:5912/282009
WENZ EL-HARTUNG19 9 OCH.OUT.txt
1.5
dose
2.5
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\PAH\lungimplant\Wenzell990\CH\msc_WenzelCH_MS_l_10.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\PAH\lungimplant\Wenzell990\CH\msc_WenzelCH_MS_l_10.plt
Wed Dec 23 11:48:10 2009
HMDS Model Run
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 = incidence
Independent variable = dose
Total number of observations = 3
Total number of records with missing values
Total number of parameters in model = 2
= 0
D-lll
DRAFT - DO NOT CITE OR QUOTE
-------
1 Total number of specified parameters = 0
2 Degree of polynomial = 1
3
4
5 Maximum number of iterations = 250
6 Relative Function Convergence has been set to: 2.22045e-016
7 Parameter Convergence has been set to: 1.49012e-008
8
9 **** we are sorry but Relative Function and Parameter Convergence ****
10 **** are currently unavailable in this model. Please keep checking ****
H **** the wek sight for model updates which will eventually ****
12 **** incorporate these convergence criterion. Default values used. ****
13
14
15
16 Default Initial Parameter Values
17 Background = 0.0178361
18 Beta(l) = 0.109158
19
20
21 Asymptotic Correlation Matrix of Parameter Estimates
22
23 ( *** The model parameter(s) -Background
24 have been estimated at a boundary point, or have been
25 specified by the user,
26 and do not appear in the correlation matrix )
27
28 Beta(l)
29
30 Beta(l) 1
31
32
33
34 Parameter Estimates
35
36 95.0% Wald
37 Confidence Interval
38 Variable Estimate Std. Err. Lower Conf. Limit
39 Upper Conf. Limit
40 Background 0 * *
41 *
42 Beta(l) 0.123432 * *
43 *
44
45 * - Indicates that this value is not calculated.
46
47
48
49 Analysis of Deviance Table
50
51 Model Log(likelihood) # Param's Deviance Test d.f. P-value
52 Full model -35.2935 3
53 Fitted model -35.455 1 0.323044 2
54 0.8508
55 Reduced model -43.0622 1 15.5374 2
56 0.0004228
57
58 AIC: 72.9101
59
60
D-l 12 DRAFT - DO NOT CITE OR QUOTE
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Dose Est
0.0000 0.
1.0000 0.
3.0000 0.
ChiA2 = 0.34
Benchmark Dose
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
Taken together, (
interval for the
Multistage Cancer
Goodness of Fit
. Prob. Expected Observed Size
0000 0.000 0.000 35
1161 4.064 5.000 35
3095 10.831 10.000 35
d.f. = 2 P-value = 0.8453
Computation
0.1
= Extra risk
0.95
0.853595
0.57298
1.36494
0.57298, 1.36494) is a 90 % two-sided
BMD
Slope Factor = 0.174526
Scaled
Residual
0.000
0.494
-0.304
confidence
D-113 DRAFT - DO NOT CITE OR QUOTE
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D.4. ORAL BIOASSAYS
Weyand et al. 2004 BcFE lung
T3
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The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
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: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence
**** are currently unavailable in this model. Please keep checking
•k-k-k-k the web sight for model updates which will eventually
•k-k-k-k incorporate these convergence criterion. Default values used.
Default Initial Parameter Values
Background = 0
Beta(l) = 5.23754e+017
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.45
Beta(l) -0.45 1
Parameter Estimates
•k-k-k-k
•k-k-k-k
Confidence Interval
Variable Estimate Std. Err.
Upper Conf. Limit
Background 0.233316 *
*
Beta(l) 0.0289518 *
* - Indicates that this value is not calculated.
95.0% Wald
Lower Conf. Limit
D-115
DRAFT - DO NOT CITE OR QUOTE
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Model
Full model
Fitted model
0.6567
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-35.3639 3
-35.4627 2 0.197606 1
-58.7707
74.9254
46.8136
Goodness of Fit
Dose
Est. Prob.
Expected
Observed
Size
0.0000
13.6000
197.0000
ChiA2 =0.12
0.2333
0.4829
0.9974
d.f. =1
Benchmark Dose Computation
Specified effect = 0.7
Risk Type = Extra risk
Confidence level = 0.95
BMD =
BMDL =
BMDU =
41.5854
22.3673
81.9344
P-value
<.0001
Scaled
Residual
6.766 7.000 29 0.103
13.520 13.000 28 -0.197
28.926 29.000 29 0.273
P-value = 0.7253
Taken together, (22.3673, 81.9344) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.0312958
D-116
DRAFT - DO NOT CITE OR QUOTE
-------
1 D.5. BACTERIAL MUTAGENICITY
2 Hass 1981 bact mut bap.out.txt
3 ====================================================================
4 Polynomial Model. Revision: 2.2 Date: 9/12/2002
5 Input Data File: C:\BMDS\UNSAVED1.(d)
6 Gnuplot Plotting File: C:\BMDS\UNSAVEDl.plt
7 Wed Jul 06 11:29:07 2005
8 ====================================================================
9
10 HMDS MODEL RUN
11 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
12
13 The form of the response function is:
14
15 Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
16
17
18 Dependent variable = MEAN
19 Independent variable = COLUMN1
20 rho is set to 0
21 Signs of the polynomial coefficients are not restricted
22 A constant variance model is fit
23
24 Total number of dose groups = 4
25 Total number of records with missing values = 0
26 Maximum number of iterations = 250
27 Relative Function Convergence has been set to: le-008
28 Parameter Convergence has been set to: le-008
29
30
31
32 Default Initial Parameter Values
33 alpha = 194.5
34 rho = 0 Specified
35 beta_0 = 121.8
36 beta_l = 297.029
37
38
39
40 Parameter Estimates
41
42 95.0% Wald
43 Confidence Interval
44 Variable Estimate Std. Err. Lower Conf. Limit
45 Upper Conf. Limit
46 alpha 132.71 54.1784 26.5217
47 238.897
48 beta_0 121.8 5.15188 111.702
49 131.898
50 beta_l 297.029 8.99387 279.401
51 314.656
52
53
54 Asymptotic Correlation Matrix of Parameter Estimates
55
56
57
58
59
D-117 DRAFT - DO NOT CITE OR QUOTE
alpha
beta 0
beta 1
alpha
1
-1.4e-009
-1. le-008
beta 0
-1.4e-009
1
-0.76
beta 1
-1. le-008
-0.76
1
-------
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Table of Data and Estimated Values of Interest
Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev
Res .
_
0 3 124 8 122 11.5
0.25 3 194 16 196 11.5
0.5 3 269 13 270 11.5
1 3 420 17 419 11.5
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij) } = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma (i)A2
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model Log (likelihood) DF AIC
Al -35.189802 5 80.379605
A2 -34.317788 8 84.635576
fitted -35.328976 2 74.657952
R -62.974684 2 129.949369
Test 1: Does response and/or variances differ among dose
levels
(A2 vs. R)
Test 2: Are Variances Homogeneous (Al vs A2 )
Test 3: Does the Model for the Mean Fit (Al vs. fitted)
Tests of Interest
Test -2*log (Likelihood Ratio) Test df p-value
Test 1 57.3138 6 <.0001
Test 2 1.74403 3 0.6272
Test 3 0.278348 2 0.8701
The p-value for Test 1 is less than .05. There appears
to be a
difference between response and/or variances among the
dose levels .
It seems appropriate to model the data
The p-value for Test 2 is greater than .05. A
homogeneous variance
model appears to be appropriate here
ChiA2
0.331
-0.309
-0.198
0.176
D-118
DRAFT - DO NOT CITE OR QUOTE
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1
2
3 The p-value for Test 3 is greater than .05. The model
4 chosen appears
5 to adequately describe the data
6
7
8
9 Benchmark Dose Computation
10 Specified effect = 1
11
12 Risk Type = Estimated standard deviations from the control mean
13
14
15 Confidence level = 0.95
16
17 BMD = 0.038784
18
19
20 BMDL = 0.0286028
21
22
D-119 DRAFT - DO NOT CITE OR QUOTE
-------
1 HASS_1981_BACT_MUT_BEP.OUT.txt
2 ====================================================================
3 Polynomial Model. Revision: 2.2 Date: 9/12/2002
4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
5 RPS\MODELING\HASS_1981_BACT_MUT_BEP.(d)
6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
7 DOCUMENTS\PAH RPS\MODELING\HASS_1981_BACT_MUT_BEP.pit
8 Wed Jul 06 13:42:38 2005
9 ====================================================================
10
11 BMDS MODEL RUN
19 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
13
14 The form of the response function is:
15
16 Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
17
18
19 Dependent variable = MEAN
20 Independent variable = COLUMN1
21 rho is set to 0
22 Signs of the polynomial coefficients are not restricted
23 A constant variance model is fit
24
25 Total number of dose groups = 4
26 Total number of records with missing values = 0
27 Maximum number of iterations = 250
28 Relative Function Convergence has been set to: le-008
29 Parameter Convergence has been set to: le-008
30
31
32
33 Default Initial Parameter Values
34 alpha = 117.5
35 rho = 0 Specified
36 beta_0 = 120.75
37 beta_l = 77.5
38
39
40
41 Parameter Estimates
42
43 95.0% Wald
44 Confidence Interval
45
46 U
47 alpha 98.6458 40.272 19.7142
48 1
49 beta0 120.75 4.19706 112.524
50 1
51 betal 77.5 7.66275 62.4813
52 9
53
54
55
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57
58
59
60
D-120 DRAFT - DO NOT CITE OR QUOTE
Variable
;r Conf. Limit
alpha
577
beta 0
976
beta 1
i!87
Estimate
98.6458
120.75
77.5
Asymptotic Correlation Matrix
alpha
alpha 1
beta 0 -8e-012
beta 1 l.le-011
beta 0
-8e-012
1
-0.73
Std. Err.
40.272
4.19706
7.66275
of Parameter
beta 1
l.le-011
-0.73
1
Lower Con
1
1
6
Estimates
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Table of Data and Estimated Values of Interest
Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev
Res .
_
0 3 124 8 121 9.93
0.2 3 129 6 136 9.93
0.4 3 156 9 152 9.93
1 3 198 17 198 9.93
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij) } = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma (i)A2
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model Log (likelihood) DF AIC
Al -32.165839 5 74.331679
A2 -30.272126 8 76.544252
fitted -33.549216 2 71.098432
R -47.594288 2 99.188576
Test 1: Does response and/or variances differ among dose
levels
(A2 vs. R)
Test 2: Are Variances Homogeneous (Al vs A2 )
Test 3: Does the Model for the Mean Fit (Al vs. fitted)
Tests of Interest
Test -2*log (Likelihood Ratio) Test df p-value
Test 1 34.6443 6 <.0001
Test 2 3.78743 3 0.2854
Test 3 2.76675 2 0.2507
The p-value for Test 1 is less than .05. There appears
to be a
difference between response and/or variances among the
dose levels .
It seems appropriate to model the data
The p-value for Test 2 is greater than .05. A
homogeneous variance
model appears to be appropriate here
ChiA2
0.567
-1.26
0.741
-0.0436
D-121
DRAFT - DO NOT CITE OR QUOTE
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1
2
3 The p-value for Test 3 is greater than .05. The model
4 chosen appears
5 to adequately describe the data
6
7
8
9 Benchmark Dose Computation
10 Specified effect = 1
11
12 Risk Type = Estimated standard deviations from the control mean
13
14
15 Confidence level = 0.95
16
17 BMD = 0.128156
18
19
20 BMDL = 0.0923937
21
22
D-122 DRAFT - DO NOT CITE OR QUOTE
-------
1 JOHNSEN_1997_BAC_MUT_BAP.OUT.txt
2 ====================================================================
3 Polynomial Model. Revision: 2.2 Date: 9/12/2002
4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
5 RPS\MODELING\JOHNSEN_1997_BAC_MUT_BAP.(d)
6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
7 DOCUMENTS\PAH RPS\MODELING\JOHNSEN_1997_BAC_MUT_BAP.pit
8 Fri Jul 08 09:02:29 2005
9 ====================================================================
10
11 BMDS MODEL RUN
19 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
13
14 The form of the response function is:
15
16 Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
17
18
19 Dependent variable = MEAN
20 Independent variable = COLUMN1
21 rho is set to 0
22 Signs of the polynomial coefficients are not restricted
23 A constant variance model is fit
24
25 Total number of dose groups = 3
26 Total number of records with missing values = 0
27 Maximum number of iterations = 250
28 Relative Function Convergence has been set to: le-008
29 Parameter Convergence has been set to: le-008
30
31
32
33 Default Initial Parameter Values
34 alpha = 70.2768
35 rho = 0 Specified
36 beta_0 = 115.5
37 beta_l = 0.65
38
39
40
41 Parameter Estimates
42
43 95.0% Wald
44 Confidence Interval
45 Variable Estimate Std. Err. Lower Conf. Limit
46 Upper Conf. Limit
47 alpha 59.3512 27.9784 4.51449
48 114.188
49 beta_0 115.5 4.06035 107.542
50 123.458
51 beta_l 0.65 0.314513 0.0335651
52 1.26643
53
54
55 Asymptotic Correlation Matrix of Parameter Estimates
56
57
58
59
60
D-123 DRAFT - DO NOT CITE OR QUOTE
alpha
beta 0
beta 1
alpha
1
-7.9e-010
-3.4e-012
beta 0
-7.9e-010
1
-0.77
beta 1
-3.4e-012
-0.77
1
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Dose
Res .
Table of Data and Estimated Values of Interest
N Obs Mean Obs Std Dev Est Mean Est Std Dev ChiA2
0
10
20
113
127
126
9.68
4.84
9.68
115
122
128
7.7
7.7
7.7
-0.562
1.12
-0.562
Model Descriptions for likelihoods calculated
Model Al: Yij
Var{e(ij)}
Model A2: Yij
Var{e(ij) }
Model R: Yi
Var{e(i) }
Model
Al
A2
fitted
R
Mu (i) + e (ij )
SigmaA2
Mu (i) + e (ij )
Sigma(i)A2
Mu + e (i)
SigmaA2
Likelihoods of Interest
Log(likelihood) DF
-21.811395 4
-21.026523 6
-22.875626 2
-24.653317 2
AIC
51.622790
54.053045
49.751251
53.306634
Test 1: Does response and/or variances differ among dose
levels
(A2 vs. R)
Test 2: Are Variances Homogeneous (Al vs A2)
Test 3: Does the Model for the Mean Fit (Al vs. fitted)
Test
Test 1
Test 2
Test 3
Tests of Interest
-2*log(Likelihood Ratio) Test df
7.25359
1.56974
2.12846
p-value
0.0266
0.4562
0.1446
The p-value for Test 1 is less than .05. There appears
to be a
difference between response and/or variances among the
dose levels.
It seems appropriate to model the data
The p-value for Test 2 is greater than .05. A
homogeneous variance
model appears to be appropriate here
D-124
DRAFT - DO NOT CITE OR QUOTE
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1
2 The p-value for Test 3 is greater than .05. The model
3 chosen appears
4 to adequately describe the data
5
6
7
8 Benchmark Dose Computation
9 Specified effect = 1
10
11 Risk Type = Estimated standard deviations from the control mean
12
13
14 Confidence level = 0.95
15
16 BMD = 11.8523
17
18
19 BMDL = 6.27094
20
21
22
23
D-125 DRAFT - DO NOT CITE OR QUOTE
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1 D.6. MAMMALIAN MUTAGENICITY
2 BARF_MUT_BAA.OUT.txt
3 ====================================================================
4 Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
5 Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
6 RPS\MODELING\BARF_MUT_BAA.(d)
7 Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
8 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_BAA.pit
9 Thu Jun 30 12:46:38 2005
10 ====================================================================
11
12 BMDS MODEL RUN
13 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
14
15 The form of the probability function is:
16
17 P[response] = background + (1-background)*[1-EXP(
18 -betal*doseAl-beta2*doseA2-beta3*doseA3)]
19
20 The parameter betas are restricted to be positive
21
22
23 Dependent variable = COLUMN2
24 Independent variable = COLUMN1
25
26 Total number of observations = 5
27 Total number of records with missing values = 0
28 Total number of parameters in model = 4
29 Total number of specified parameters = 0
30 Degree of polynomial = 3
31
32
33 Maximum number of iterations = 250
34 Relative Function Convergence has been set to: le-008
35 Parameter Convergence has been set to: le-008
36
37
38
39 Default Initial Parameter Values
40 Background = 3.89426e-006
41 Beta(l) = 3.46216e-007
42 Beta(2) = 0
43 Beta(3) = 1.93939e-012
44 **** WARNING: Completion code = -2. Optimum not found. Trying new starting
45 pont****
46
47
48
49 Asymptotic Correlation Matrix of Parameter Estimates
50
51 ( *** The model parameter(s) -Background -Beta(2) -Beta(3)
52 have been estimated at a boundary point, or have been
53 specified by the user,
54 and do not appear in the correlation matrix )
55
56 Beta(l)
57
58 Beta(l) 1
59
D-126 DRAFT - DO NOT CITE OR QUOTE
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Parameter Estimates
Variable Estimate Std. Err.
Background 0 NA
Beta(l) 4.34385e-007 5.43792e-006
Beta (2) 0 NA
Beta (3) 0 NA
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Model Log (likelihood) Deviance Test DF
Full model -1545.82
Fitted model -1548.6 5.57201 4
Reduced model -1597.17 102.713 4
AIC: 3099.21
Goodness of Fit
Dose Est. Prob. Expected Observed Size
i: 1
0.0000 0.0000 0.000 0 1000000
i: 2
20.0000 0.0000 8.688 12 1000000
i: 3
50.0000 0.0000 21.719 29 1000000
i: 4
100.0000 0.0000 43.438 34 1000000
i: 5
150.0000 0.0001 65.156 64 1000000
Chi-square = 5.77 DF = 4 P-value = 0.21
Benchmark Dose Computation
Specified effect = le-005
Risk Type = Extra risk
Confidence level = 0.95
BMD = 23. 0212
**** WARNING: Completion code = -2. Optimum not found.
point****
**** WARNING 0: Completion code = -2 trying new start***
**** WARNING 1: Completion code = -2 trying new start***
P-value
0.2335
<.0001
ChiA2 Res.
0.000
0.381
0.335
-0.217
-0.018
66
Trying new starting
*
*
D-127
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1
2 **** WARNING 2: Completion code = -2 trying new start****
3
4 **** WARNING 3: Completion code = -2 trying new start****
5
6 **** WARNING 4: Completion code = -2 trying new start****
7
8 **** WARNING 5: Completion code = -2 trying new start****
9
10 **** WARNING 6: Completion code = -2 trying new start****
11
12 **** WARNING 7: Completion code = -2 trying new start****
13
14 **** WARNING 8: Completion code = -2 trying new start****
15
16 **** WARNING 9: Completion code = -2 trying new start****
17
18 **** WARNING: Completion code = -2. Optimum not found. Trying new starting
19 point****
20
21 **** WARNING 0: Completion code = -2 trying new start****
22
23 **** WARNING 1: Completion code = -3 trying new start****
24
25 **** WARNING 2: Completion code = -3 trying new start****
26
27 **** WARNING 3: Completion code = -3 trying new start****
28
29 **** WARNING 4: Completion code = -3 trying new start****
30
31 **** WARNING 5: Completion code = -3 trying new start****
32
33 **** WARNING 6: Completion code = -2 trying new start****
34
35 **** WARNING 7: Completion code = -3 trying new start****
36
37 **** WARNING 8: Completion code = -3 trying new start****
38
39 **** WARNING 9: Completion code = -3 trying new start****
40
41
42 Warning: completion code still negative
43 BMDL did not converge for BMR = 0.000010
44
45 Program execution is stopped
46
D-128 DRAFT - DO NOT CITE OR QUOTE
-------
1 BARF_MUT_BAP.OUT.txt
2 ====================================================================
3 Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
5 RPS\MODELING\BARF_MUT_BAP.(d)
6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
7 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_BAP.pit
8 Thu Jun 30 12:40:17 2005
9 ====================================================================
10
11 BMDS MODEL RUN
19 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
13
14 The form of the probability function is:
15
16 P[response] = background + (1-background)*[1-EXP(
17 -betal*doseAl-beta2*doseA2)]
18
19 The parameter betas are restricted to be positive
20
21
22 Dependent variable = COLUMN2
23 Independent variable = COLUMN1
24
25 Total number of observations = 4
26 Total number of records with missing values = 0
27 Total number of parameters in model = 3
28 Total number of specified parameters = 0
29 Degree of polynomial = 2
30
31
32 Maximum number of iterations = 250
33 Relative Function Convergence has been set to: le-008
34 Parameter Convergence has been set to: le-008
35
36
37
38 Default Initial Parameter Values
39 Background = 1.39884e-006
40 Beta(l) = 5.34042e-006
41 Beta(2) = 0
42
43
44 Asymptotic Correlation Matrix of Parameter Estimates
45
46 ( *** The model parameter(s) -Background -Beta(2)
47 have been estimated at a boundary point, or have been
48 specified by the user,
49 and do not appear in the correlation matrix )
50
51 Beta(l)
52
53 Beta(l) 1
54
55
56
57 Parameter Estimates
58
59 Variable Estimate Std. Err.
60 Background 0 NA
D-129 DRAFT - DO NOT CITE OR QUOTE
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Beta(l) 5.43367e-006
Beta(2) 0
NA - Indicates that this parameter
2.68102e-005
NA
has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of
Model Log (likelihood)
Full model -3273.08
Fitted model -3273.96
Reduced model -3395.25
AIC: 6549.92
Goodness of
Deviance Table
Deviance Test DF
1.75092 3
244.327 3
Fit
Dose Est. Prob. Expected Observed Size
i: 1
0.0000 0.0000 0.000
i: 2
10.0000 0.0001 54.335
i: 3
20.0000 0.0001 108.668
i: 4
30.0000 0.0002 162.997
Chi-square = 1.78 DF = 3
Benchmark Dose Computation
Specified effect = le-005
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1. 84039
**** WARNING: Completion code = -3
point****
**** WARNING 0: Completion code =
**** WARNING 1: Completion code =
**** WARNING 2: Completion code =
**** WARNING 3: Completion code =
**** WARNING 4: Completion code =
**** WARNING 5: Completion code =
0 1000000
51 1000000
120 1000000
155 1000000
P-value
0.6257
<.0001
ChiA2 Res.
0.000
-0.061
0.104
-0.049
P-value = 0.6195
Optimum not found.
-3 trying new start***
-3 trying new start***
-3 trying new start***
-3 trying new start***
-3 trying new start***
-3 trying new start***
Trying new starting
*
*
*
*
*
*
D-130
DRAFT - DO NOT CITE OR QUOTE
-------
1 **** WARNING 6: Completion code = -3 trying new start****
2
3 **** WARNING 7: Completion code = -3 trying new start****
4
5 **** WARNING 8: Completion code = -3 trying new start****
6
7 **** WARNING 9: Completion code = -3 trying new start****
8
9 **** WARNING: Completion code = -3. Optimum not found. Trying new starting
10 point****
11
12 **** WARNING 0: Completion code = -1 trying new start****
13
14 **** WARNING 1: Completion code = -1 trying new start****
15
16 **** WARNING 2: Completion code = -1 trying new start****
17
18 BMDL = 1.68248
19
D-131 DRAFT - DO NOT CITE OR QUOTE
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BARF MUT CH.OUT.txt
Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
RPS\MODELING\BARF_MUT_CH.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
DOCUMENTS\PAH RPS\MODELING\BARF_MUT_CH.pit
Thu Jun 30 12:48:57 2005
BMDS MODEL RUN
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 = COLUMN2
Independent variable = COLUMN1
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 = 2.60526e-006
Beta(l) = 5.02638e-007
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta (1)
1
Parameter Estimates
Variable
Background
Beta(l)
Estimate
0
6.14293e-007
Std. Err.
NA
1.93539e-005
D-132
DRAFT - DO NOT CITE OR QUOTE
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50
51
52
53
54
55
56
57
58
59
60
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-504.191
-505.38
-522.575
1012.76
Deviance Test DF
P-value
2.37752
36.7681
0.3046
<.0001
Dose
Goodness of Fit
Est._Prob. Expected Observed Size ChiA2 Res,
i: 1
0.0000
i: 2
20.0000
i: 3
50.0000
Chi-square =
0.0000
0.0000
0.0000
2.53
0.000
12.286
30.714
DF = 2
0 1000000 0.000
17 1000000 0.384
26 1000000 -0.153
P-value = 0.2819
Benchmark Dose Computation
Specified effect = le-005
Risk Type = Extra risk
Confidence level = 0.95
BMD = 16.279
**** WARNING: Completion code = -1. Optimum not found. Trying new starting
point****
**** WARNING 0: Completion code = -1 trying new start****
**** WARNING 1: Completion code = -1 trying new start****
**** WARNING 2: Completion code = -1 trying new start****
**** WARNING 3: Completion code = -1 trying new start****
**** WARNING 4: Completion code = -1 trying new start****
**** WARNING 5: Completion code = -1 trying new start****
**** WARNING 6: Completion code = -1 trying new start****
**** WARNING 7: Completion code = -1 trying new start****
D-133
DRAFT - DO NOT CITE OR QUOTE
-------
1 **** WARNING 8: Completion code = -1 trying new start****
2
3 **** WARNING 9: Completion code = -1 trying new start****
4
5 **** WARNING: Completion code = -1. Optimum not found. Trying new starting
6 point****
7
8 **** WARNING 0: Completion code = -3 trying new start****
9
10 **** WARNING 1: Completion code = -3 trying new start****
11
12 **** WARNING 2: Completion code = -3 trying new start****
13
14 **** WARNING 3: Completion code = -3 trying new start****
15
16 **** WARNING 4: Completion code = -3 trying new start****
17
18 **** WARNING 5: Completion code = -3 trying new start****
19
20 **** WARNING 6: Completion code = -3 trying new start****
21
22 **** WARNING 7: Completion code = -3 trying new start****
23
24 **** WARNING 8: Completion code = -3 trying new start****
25
26 **** WARNING 9: Completion code = -3 trying new start****
27
28
29 Warning: completion code still negative
30 BMDL did not converge for BMR = 0.000010
31
32 Program execution is stopped
33
D-134 DRAFT - DO NOT CITE OR QUOTE
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BARF MUT FA.OUT.txt
Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
RPS\MODELING\BARF_MUT_FA.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
DOCUMENTS\PAH RPS\MODELING\BARF_MUT_FA.pit
Thu Jun 30 12:43:11 2005
BMDS MODEL RUN
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 = COLUMN2
Independent variable = COLUMN1
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 = 6.6658e-007
Beta(l) = 2.50006e-006
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
Beta (1)
Beta (1)
1
Parameter Estimates
Variable
Background
Beta(l)
Estimate
0
2.56672e-006
Std. Err.
NA
4.49565e-005
D-135
DRAFT - DO NOT CITE OR QUOTE
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41
42
43
44
45
46
47
48
49
50
51
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-856.204
-856.255
-890.913
1714.51
Deviance Test DF
P-value
0.103
69.419
0.9498
<.0001
Dose
Goodness of Fit
Est._Prob. Expected Observed Size ChiA2 Res,
i: 1
0.0000
i: 2
10.0000
i: 3
20.0000
Chi-square =
0.0000
0.0000
0.0001
0.10
0.000
25.667
51.333
DF = 2
0 1000000 0.000
27 1000000 0.052
50 1000000 -0.026
P-value = 0.9494
Benchmark Dose Computation
Specified effect = le-005
Risk Type = Extra risk
Confidence level = 0.95
BMD = 3. 89604
**** WARNING: Completion code = -1. Optimum not found. Trying new starting
point****
**** WARNING 0: Completion code = -1 trying new start****
**** WARNING 1: Completion code = -5 trying new start****
BMDL = 0
D-136
DRAFT - DO NOT CITE OR QUOTE
-------
1 BARF_MUT_TPHEN.OUT.txt
2 ====================================================================
3 Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
5 RPS\MODELING\BARF_MUT_TPHEN.(d)
6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
7 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_TPHEN.pit
8 Thu Jun 30 12:52:56 2005
9 ====================================================================
10
11 HMDS MODEL RUN
19 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
13
14 The form of the probability function is:
15
16 P[response] = background + (1-background)*[1-EXP(
17 -betal*doseAl-beta2*doseA2)]
18
19 The parameter betas are restricted to be positive
20
21
22 Dependent variable = COLUMN2
23 Independent variable = COLUMN1
24
25 Total number of observations = 4
26 Total number of records with missing values = 0
27 Total number of parameters in model = 3
28 Total number of specified parameters = 0
29 Degree of polynomial = 2
30
31
32 Maximum number of iterations = 250
33 Relative Function Convergence has been set to: le-008
34 Parameter Convergence has been set to: le-008
35
36
37
38 Default Initial Parameter Values
39 Background = 9.99937e-007
40 Beta(l) = 1.74289e-007
41 Beta(2) = 0
42
43
44 Asymptotic Correlation Matrix of Parameter Estimates
45
46 ( *** The model parameter(s) -Background -Beta(2)
47 have been estimated at a boundary point, or have been
48 specified by the user,
49 and do not appear in the correlation matrix )
50
51 Beta(l)
52
53 Beta(l) 1
54
55
56
57 Parameter Estimates
58
59 Variable Estimate Std. Err.
60 Background 0 NA
D-137 DRAFT - DO NOT CITE OR QUOTE
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Beta(l) 1.85717e-007
Beta(2) 0
NA - Indicates that this parameter
4.42148e-006
NA
has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of
Model Log (likelihood)
Full model -755.63
Fitted model -755.773
Reduced model -781.782
AIC: 1513.55
Goodness of
Deviance Table
Deviance Test DF
0.2868 3
52.3039 3
Fit
Dose Est. Prob. Expected Observed Size
i: 1
0.0000 0.0000 0.000
i: 2
50.0000 0.0000 9.286
i: 3
100.0000 0.0000 18.572
i: 4
200.0000 0.0000 37.143
Chi-square = 0.29 DF = 3
Benchmark Dose Computation
Specified effect = le-005
Risk Type = Extra risk
Confidence level = 0.95
BMD = 53. 8457
**** WARNING: Completion code = -2
point****
**** WARNING 0: Completion code =
**** WARNING 1: Completion code =
**** WARNING 2: Completion code =
**** WARNING 3: Completion code =
**** WARNING 4: Completion code =
**** WARNING 5: Completion code =
0 1000000
10 1000000
20 1000000
35 1000000
P-value
0.9625
<.0001
ChiA2 Res.
0.000
0.077
0.077
-0.058
P-value = 0.9622
Optimum not found.
-2 trying new start***
-2 trying new start***
-2 trying new start***
-2 trying new start***
-2 trying new start***
-2 trying new start***
Trying new starting
*
*
*
*
*
*
D-138
DRAFT - DO NOT CITE OR QUOTE
-------
1 **** WARNING 6: Completion code = -2 trying new start****
2
3 **** WARNING 7: Completion code = -2 trying new start****
4
5 **** WARNING 8: Completion code = -2 trying new start****
6
7 **** WARNING 9: Completion code = -2 trying new start****
8
9 **** WARNING: Completion code = -2. Optimum not found. Trying new starting
10 point****
11
12 **** WARNING 0: Completion code = -2 trying new start****
13
14 **** WARNING 1: Completion code = -5 trying new start****
15
16 **** WARNING 2: Completion code = -2 trying new start****
17
18 **** WARNING 3: Completion code = -2 trying new start****
19
20 **** WARNING 4: Completion code = -2 trying new start****
21
22 **** WARNING 5: Completion code = -2 trying new start****
23
24 **** WARNING 6: Completion code = -2 trying new start****
25
26 **** WARNING 7: Completion code = -5 trying new start****
27
28 **** WARNING 8: Completion code = -2 trying new start****
29
30 **** WARNING 9: Completion code = -5 trying new start****
31
32
33 Warning: completion code still negative
34 BMDL did not converge for BMR = 0.000010
35
36 Program execution is stopped
37
D-139 DRAFT - DO NOT CITE OR QUOTE
-------
1 RAVEH_HUB_MUT_BAP.OUT.txt
2 ====================================================================
3 Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY DOCUMENTS\PAH
5 RPS\MODELING\RAVEH_HUB_MUT_BAP.(d)
6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\HCLYNCH\MY
7 DOCUMENTS\PAH RPS\MODELING\RAVEH_HUB_MUT_BAP.pit
8 Wed Jun 29 12:15:41 2005
9 ====================================================================
10
11 BMDS MODEL RUN
19 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
13
14 The form of the probability function is:
15
16 P[response] = background + (1-background)*[1-EXP(
17 -betal*doseAl)]
18
19 The parameter betas are restricted to be positive
20
21
22 Dependent variable = COLUMN2
23 Independent variable = COLUMN1
24
25 Total number of observations = 3
26 Total number of records with missing values = 0
27 Total number of parameters in model = 2
28 Total number of specified parameters = 0
29 Degree of polynomial = 1
30
31
32 Maximum number of iterations = 250
33 Relative Function Convergence has been set to: le-008
34 Parameter Convergence has been set to: le-008
35
36
37
38 Default Initial Parameter Values
39 Background = 0
40 Beta(l) = 0.00102082
41 **** WARNING: Completion code = -2. Optimum not found. Trying new starting
42 pont****
43
44 **** WARNING 0: Completion code = -2 trying new start****
45
46 **** WARNING 1: Completion code = -2 trying new start****
47
48 **** WARNING 2: Completion code = -2 trying new start****
49
50 **** WARNING 3: Completion code = -2 trying new start****
51
52 **** WARNING 4: Completion code = -2 trying new start****
53
54 **** WARNING 5: Completion code = -2 trying new start****
55
56 **** WARNING 6: Completion code = -2 trying new start****
57
58 **** WARNING 7: Completion code = -2 trying new start****
59
60 **** WARNING 8: Completion code = -2 trying new start****
D-140 |