### EPA/635/R-08/012A



# DEVELOPMENT OF A RELATIVE POTENCY FACTOR (RPF) APPROACH FOR POLYCYCLIC AROMATIC HYDROCARBON (PAH) MIXTURES

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September 2009

NOTICE

This document is an interagency science consultation draft . This information is distributed solely for the purpose of pre-dissemination peer re view 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 implic ations.

U.S. Environmental Protection Agency

Washington, DC

### ii DRAFT – DO NOT CITE OR QUOTE DISCLAIMER

This document is a preliminary draft for review pur poses only. This information is distributed solely for the purpose of pre-dissemina tion peer review under applicable information quality guidelines. It has not been formally disse minated by EPA. It does not represent and should not be construed to represent any Agency det ermination or policy. Mention of trade names or commercial products does not constitute en dorsement or recommendation for use.

### iii DRAFT – DO NOT CITE OR QUOTE EXECUTIVE SUMMARY

The U.S. Environmental Protection Agency’s (U.S. EP A’s) Integrated Risk Information System (IRIS) Program is releasing for scientific r eview a relative potency factor (RPF) approach for polycyclic aromatic hydrocarbon (PAH) mixtures as one approach to assessing cancer risk from exposure to PAH mixtures. The RPF approach 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 d oses (with RPFs) relative to the potency of an index PAH (i.e., benzo[a]pyrene). The cancerrisk is then estimated using the doseresponse curve for the index PAH. RPFs for seven i ndividual PAHs were developed in the U.S. EPA (1993) Provisional Guidance for Quantitative Risk Assessme nt of PAHs ( Provisional

Guidance ) and are utilized extensively within U.S. EPA prog ram offices and other regulatory agencies. The Provisional Guidance , however, does not reflect the most recent researc h, nor does it consider additional PAHs with carcinogenic potential (such as fjord-region PAHs). The Supplemental Guidance for Conducting Health Ris k Assessment of Chemical

Mixtures (U.S. EPA, 2000) highlights that approaches based on whole mixtures are preferred to component approaches, such as the RPF approach. Ri sk assessment approaches based on toxicity evaluations of whole mixtures inherently a ddress 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 co mponents (e.g., dose additivity or response additivity). While whole mixture assessment is pre ferred, there are challenges associated with using these approaches. There are very few toxicit y data available for whole PAH mixtures and, in most cases, chemical analyses of the composition of mixtures are limited. In addition, PAHcontaining mixtures tend to be very complex; the co mposition of these mixtures appears to vary across sources releasing these mixtures to the envi ronment and in various environmental media in which they occur. For these reasons, a whole mi xtures approach may not always be practicable for risk assessment purposes. This report provides recommendations for developmen t of the RPF approach for PAH

mixtures health risk assessment and includes:

(1) A rationale for recommending an RPF approach (S ection 2);

(2) A summary of previous approaches for developing the RPF approach for PAHs (Section 3);

(3) An evaluation of the carcinogenic potential of individual PAHs (Section 4);

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(4) Methods for dose response assessment and indivi dual study RPF calculation (Section 5);

(5) Selection of PAHs for inclusion in the RPF appr oach (Section 6);

(6) Derivation of RPFs for selected PAHs (Section 7 ); and

(7) Characterization of strengths, weaknesses, and uncertainties associated with the RPF approach to PAH cancer risk assessment (Section 8).

The RPF approach involves two key assumptions: (1) similar toxicological action of PAH components in the mixture; and (2) interactions among PAH mixture components do not occur at low levels of exposure typically encounter ed in the environment (that is, additivity is assumed). Mechanistic studies indicate that the mu tagenic and tumor-initiating activity of carcinogenic PAHs requires metabolic activation to reactive intermediates (e.g., dihydrodiol epoxides, quinones, radical cations), which covalen tly modify 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 gen e expression or a cell proliferation response to metabolite cytotoxicity (see Section 2.4, Simila rities in Carcinogenic Mode of 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 similar toxicological action is reasonable; however, the carcinogenic process for P AHs is likely to be related to some unique combination of multiple molecular events resulting from formation of several reactive species. The second assumption of no interactions at low lev els of exposure is reasonable, but evidence of toxicological interactions among PAHs at higher dos e levels has been observed (see Section 2.7, Additivity of PAHs in Combined Exposures). Several approaches have been used previously for th e determination of RPFs for PAHs (see Section 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 R PF values are generally comparable for most PAHs across studies. The RPF approach provided in the current report makes use of more recent data on genotoxicity and tumorigenicity of PAHs. There is a large PAH database on carcinogenicity in animal bioassays, genotoxicity in various test systems, and bioactivation to tumorige nic and/or genotoxic metabolic intermediates. The RPF analysis presented here includes only unsub stituted 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 stu dy types that were considered most useful for RPF derivation were rodent carcinogenicity bioa ssays (all routes) in which one or more PAHs was tested at the same time as benzo[a]pyrene. In addition, in vivo and in vitro data for

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cancer-related endpoints in which one or more PAHs and benzo[a]pyrene were tested simultaneously were obtained, including studies on the formation of DNA adducts, mutagenicity, chromosomal aberrations, sister chromatid exchange frequency, aneuploidy, DNA damage/repair/recombination, unscheduled DNA synthe sis, and cell transformation. Although it would be possible to calculate RPFs from studies wh ere a PAH and benzo[a]pyrene were tested by the same laboratory using the same test system b ut at different times, this approach was not

considered because it could introduce differences i n the dose-response information that are unrelated to the chemical (e.g., variability associ ated 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 consider ed for use in deriving RPFs because there is evidence indicating that 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]flu oranthene (Machala et al., 2001), are only weakly carcinogenic. In addition, some studies hav e demonstrated the formation of DNA

adducts in the liver of AhR knock-out mice followin g intraperitoneal or oral exposure to benzo[a]pyrene (Sagredo et al., 2006; Uno et al., 2 006; Kondraganti et al., 2003), indicating that Ah responsiveness is not a prerequisite to genotoxi city. 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 pr edictor of carcinogenic potency. Several study types were excluded from the database because they did not provide carcinogenicity or cancer-related endpoint informat ion 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 im portant information on human exposure to

PAH mixtures and the mode of action for PAH toxicit y, they generally do not contain doseresponse information that would be useful for calcu lation of RPF estimates. A database of primary literature relevant to the RP F 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 PAH s. The search identified over 900 individual publications for a target list of 74 PAHs (see Tabl e 2-1) that have been identified in environmental media or for which toxicological data are available. Review of these publications resulted in the identification of more than 600 pap ers that included carcinogenicity or cancerrelated 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 endpoi nts, and in vitro studies of cancer-related endpoints. These categories were further sorted by route (for bioassays) or by endpoint (for

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cancer-related endpoints). Each study was reviewed , and critical study details were extracted into tables for each individual endpoint (see Secti on 4). The tables also include an initial determination of whether the data from each study m eet selection criteria for use in the RPF analysis. Studies with data on selected PAHs and b enzo[a]pyrene were considered for RPF determination, even if a particular PAH has not bee n classified by U.S. EPA or International Agency for Research on Cancer (IARC) as a carcinoge n. Studies were included in the analysis if the following selection criteria were met:

• Benzo[a]pyrene was tested simultaneously with anoth er PAH;

• A statistically increased incidence of tumors was o bserved 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 be nzo[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 multipl icity findings; and

• There were no study quality concerns or potential c onfounding factors that precluded use (e.g., no concurrent control, different vehicles, s trains, 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 select ed for use in the analysis regardless of whether positive or negative results were reported. Studies with positive findings were used for calculation of RPFs. Studies with negative finding s were used in a weight of evidence evaluation of potential carcinogenicity (discussed in Section 6.1). Dose-response data were extracted from studies with positive findings that met selection criteria. For studies that reported results graphi cally, individual data points were extracted using digitizing software. In all, over 300 data sets we re extracted, reflecting dose-response data from at least one study for 50 of the 74 PAHs included i n the analysis. All of the extracted data are presented in Appendix C of this report. Statistical analyses were performed on tumor bioass ay data to determine whether the tumor incidence or multiplicity observed at a parti cular dose represented a statistically significant increase over controls. If statistical analyses we re not described in the original report, incidence data were analyzed using Fisher’s Exact test and th e Cochran-Armitage trend test. Positive

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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 equi valent test) or a significant dose-response trend (Cochran-Armitage or equivalent) for multi-do se studies. For tumor bioassay data reported as tumor count, a t-test was conducted (when varian ce 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 A ppendix C. Statistical analyses of the cancer-related endpoint data were not conducted; th e study author’s conclusions as to response (positive or negative) was used.

Section 5 describes both the methods used for dose- response assessment and RPF calculation in detail. The general equation for es timating an RPF was the ratio of the slope of the dose-response curve for the subject PAH to the slop e of the dose-response curve for benzo[a]pyrene. For bioassay data, tumor incidence s 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, b enchmark response (BMR) values of 10% for quantal data and 1 standard deviation 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 instance s, as described in Section 5.3. For data sets that included only a single dose, or those for whic h no model fit was achieved with the selected models, a point estimate RPF was calculated.

The RPFs calculated from individual studies for eac h PAH were used in a weight of evidence evaluation to assess the potential carcino genicity of each compound (see Section 6) and in the derivation of a final RPF for each compound (Section 7). The selection of PAHs to be included in the RPF approach began with an evaluati on of whether the available data were adequate to assess the potential carcinogenicity of each compound. At least one RPF value was

calculated for each of 50 PAHs. For 16 of these co mpounds, 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 selecte d for inclusion in the RPF approach. For the remaining 34 PAHs, a weight of evidence eva luation (see Figure 6-1) was

conducted to assess the evidence that each PAH coul d induce a carcinogenic response. This evaluation did not constitute a formal weight of ev idence evaluation of carcinogenic potential; rather, an expedited approach was developed using t he data collected to determine whether the available information for each PAH was adequate to draw a conclusion regarding carcinogenic potential. When the data were considered adequate for a given PAH, it was selected for

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inclusion in the RPF approach; if the data were not considered adequate to assess potential carcinogenicity, the PAH was excluded. In vivo tum or bioassays that included benzo[a]pyrene were given the greatest weight in assessing the pot ential carcinogenicity of a given PAH; data from other bioassays and cancer-related endpoint st udies were used to supplement the weight of evidence when the bioassay data that included benzo [a]pyrene were conflicting or negative. Structural alerts for PAH carcinogenicity or mutage nicity (as defined in Section 2.5 as the presence of a classic bay region 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 (Section 6) indic ated that the available data were adequate to determine that 23 of the 34 PAHs were p otentially carcinogenic, that three PAHs (anthracene, phenanthrene, and pyrene) exhibited li ttle or no carcinogenic potential, and that data were inadequate to evaluate the carcinogenic potent ial for eight 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 the PAH had minima l carcinogenic potential (i.e., robust negative tumor bioassay data and cancer-related endpoint dat a), a final RPF of 0 was recommended. While there is little quantitative difference betwe en selecting a final RPF of 0 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 0, there is evidence to suggest that these compounds are of little or no carcinogenic potentia l, and the uncertainty associated with the cancer risk for these compounds is markedly reduced

. For each of the remaining 23 compounds, a final non zero RPF was derived. A number of options were considered for deriving an RPF from am ong the numerous values calculated for each individual PAH. These options included: prio ritizing bioassay RPFs from different exposure routes based on relevance to environmental ly-relevant routes; prioritizing bioassay RPFs based on target organs considered relevant to human susceptibility to PAH carcinogenesis; prioritizing RPFs based on quality of the underlyin g study; prioritizing cancer-related endpoints by their correlation with bioassay potency (i.e., a bility to predict bioassay potency); and aggregating RPFs across all bioassays, all cancer-r elated endpoints, or across all endpoints. In
the end, it was concluded that the available data d id not provide a clear scientific basis for prioritizing RPFs except for a preference for bioas say 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.

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For each potentially carcinogenic PAH with bioassay data, the average RPF was calculated from bioassays with positive results. F or those PAHs that did not have any estimated RPF based on a bioassay, but for which the weight o f evidence evaluation indicated a potential for carcinogenic response (e.g., dibenz[a,c]anthrac ene), the final RPF was calculated from all cancer-related endpoint studies with positive resul ts. In both cases, nonpositive results were not

included in the calculation. The final RPF for eac h PAH was reported to one significant figure. The range of RPF values was also reported. Present ing the RPFs in this manner provides an average and maximum estimate for each PAH that has data from multiple studies. All tumor bioassay RPFs (across all exposure routes , species, sexes, and including both tumor incidence and tumor multiplicity RPFs) were c ombined to estimate the mean and range, except as follows. In some cases, two separate RPF s were calculated in the same group of

animals. There were two situations in which this oc curred: RPFs for different target organs in
the same animals, and RPFs based on incidence of tu mors and tumor count in the same animals In these instances, the higher value of the two RPF s was included in the average and range, and the lower value was dropped from the combined data. Several options were considered for the determinati on of a 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 a pproach to describing the calculated RPF

values available for each PAH. Other statistical m easures (i.e., geometric mean, weighted average) 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 (19/26, 73%) had ≤3 calculated RPF values and the range of RPF values was greater than an order of magnitude for several compounds (6/26 PAHs). The variability in RPF esti mates is likely due to differences in study design parameters (e.g., route, species/strain, exp osure duration, exposure during sensitive time periods, initiation vs. promotion and complete carc inogenesis protocols, tumor incidence vs. multiplicity reporting) and dose-response methods ( modeled vs. point estimates). Calculation of a weighted average was not possible because there i s no clear biological rationale for choosing among study types or tumor data outcomes. Providin g order of magnitude estimates, as has been previously done for estimating RPFs for PAHs, was n ot 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 r esulting 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 databa se. For final RPFs based on tumor bioassay data, confidence ratings considered both the availa ble tumor bioassays and the size and

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consistency of the cancer-related endpoint database . The most important factors that were considered included the availability of in vivo dat a and whether multiple exposure routes were represented. Other database characteristics that w ere considered important included the strength of evidence of genotoxicity data and SAR informatio n, the availability of more than one in vivo study, and whether effects were evident in more tha n 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 ap plied if the data implied high or medium relative confidence

. Table 1 shows the average RPFs based on tumor bioas say 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, se xes 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 rati ng, and an overview of the database for this compound.xi DRAFT – DO NOT CITE OR QUOTETable 1. PAHs with final RPFsbased on tumor bioassay dataPAHAverageRPFRange ofRPFsRelativeconfidenceNumber ofdatasets Exposure routes testedSpeciestested Sexes testedAnthanthrene 0.4 0.2–0.5 Medium 2 Dermal, lung implantation Mouse, ratFAnthracene 0 0 Medium 1 (Negative) Dermal Mouse FBenz[a]anthracene 0.2 0.02–0.4 Medium 3 Dermal, intraperitoneal Mouse F, MBenz[b,c]aceanthrylene, 11H- 0.05 0.05 Low 1 DermalMouse FBenzo[b]fluoranthene 0.5 0.1–2 High 5 Dermal, intraperitoneal, lung implantation Mouse, rat F, MBenz[e]aceanthrylene 0.9 0.5–1 Low 2 Dermal Mouse F, MBenzo[g,h,i]perylene 0.009 0.009 Low 1 Lung implantation Rat FBenz[j]aceanthrylene 60 60 Low 1 Intraperitoneal Mouse FBenzo[j]fluoranthene 0.3 0.01–1 High 5 Dermal, intraperitoneal, lung implantation Mouse, rat F, MBenzo[k]fluoranthene 0.03 0.03–0.03 Medium 2 Dermal, lung implantation Mouse, rat FBenz[l]aceanthrylene 5 4–7 Low 2 Dermal Mouse F, MChrysene 0.1 0.04–0.2 High 7 Dermal, intraperitoneal, lung implantation Mouse, rat F, MCyclopenta[c,d]pyrene 0.4 0.07–1 Medium 5 Dermal, intraperitoneal Mouse F, MCyclopenta[d,e,f]chrysene, 4H- 0.3 0.2–0.5 Low 2 Dermal Mouse FDibenzo[a,e]fluoranthene 0.9 0.7–1 Low 2 Dermal Mouse FDibenzo[a,e]pyrene 0.4 0.3–0.4 Low 2 Dermal Mouse FDibenz[a,h]anthracene 6 1–10 High 3 Dermal, intraperitoneal, lung implantation Mouse, rat F, MDibenzo[a,h]pyrene 0.9 0.9 Low 1 Dermal Mouse FDibenzo[a,i]pyrene 0.6 0.5–0.7 Low 2 Dermal Mouse FDibenzo[a,l]pyrene 30 10–40 Medium 3 Dermal, intraperitoneal Mouse F, MFluoranthene 0.08 0.009–0.2 Low 6 Intraperitoneal Mouse F, MIndeno[1,2,3-c,d]pyrene 0.07 0.07 Low 1 Lung implantation Rat FNaphtho[2,3-e]pyrene 0.3 0.3 Low 1 Dermal Mouse FPhenanthrene 0 0 High 3 (Negative) Dermal, intraperitoneal, lung implantation Mouse, rat F, MPyrene 0 0 High 7 (Negative) Dermal, intraperitoneal Mouse F, MNA = not applicable; M = male; F = female

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Table 2. PAHs with final RPFs based on cancer-related endpoint data (no tumor bioassay data available)

PAH Average
RPF Range of

RPFs Relative

confidence Types of studies Multiple dose studies Dibenz[a,c]anthracene 4 0.04–50 Very low 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 Total = 6 studies Four in vitro bacterial mutagenicity One in vitro DNA damage One in vitro DNA adduct



### 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. The PAH compounds for which a RPF value was derived are also considered to be carcinogenic by a mutagenic mode of action (see Section 2.4 for discussion of similarities in mode of action for PAHs). When assessing PAH cance r risks for life-stages under 16 years of age, or for lifetime exposures that include early-l ife exposures, the RPF values should be applied with specific exposure information to the benzo[a]p yrene cancer risk estimates including adjustment for early-life susceptibility, through t he application of age-dependent adjustment factors (ADAFs). A description of uncertainties and limitations is c rucial to interpretation of the RPF approach for PAH mixtures risk assessment (see Sect ion 8). Many of the general uncertainties related to chemical-specific risk assessment are al so applicable to the proposed RPF approach for PAHs (e.g., appropriateness of animal models, low-d ose and interspecies extrapolation, variability within the human population). Use of a component-based approach for mixtures risk

assessment leads to additional uncertainties relate d to adequate characterization of the mixture and the potential interactions that may occur betwe en 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, assumptions regarding mode of action and dose addit ivity, and cross-route extrapolation. Specific uncertainties that are related to dose-res ponse assessment (i.e., calculation of RPFs) and the selection of single RPF values for each PAH are also discussed in Section 8. In summary, the current analysis represents a signi ficant improvement upon the previous component-based approaches for PAH mixtures risk as sessment. One of the most important

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improvements is the consideration of data from a co mprehensive review of the scientific literature dating from the 1950s through 2009 on th e carcinogenicity and genotoxicity of PAHs. The search identified over 900 individual publicati ons for a target list of 74 PAHs that have been

identified in environmental media and for which tox icological data are available. Review of these publications resulted in the identification o f 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 50 individual PAHs. A weight of evidence

evaluation was conducted to evaluate the evidence f or potential carcinogenicity of 34 of these PAHs; data were inadequate to conduct such an evalu ation for the remaining 16 compounds. A

final RPF was derived for each PAH based on tumor b ioassay data (if available) or cancerrelated endpoint data (if no tumor bioassay RPFs we re available). Final RPFs were derived for 26 PAHs, significantly increasing the number of PAH s that can be addressed through this approach. Each RPF was assigned a relative confide nce rating reflecting the nature of the tumor bioassay or cancer-related endpoint database that w as used to derive the final RPF for that PAH.

### xiv DRAFT – DO NOT CITE OR QUOTE CONTENTS

LIST OF TABLES .....................................

................................................... ............................. xvii
LIST OF FIGURES....................................

................................................... ................................. 1
LIST OF ABBREVIATIONS AND ACRONYMS.................

................................................... ... 4 AUTHORS, CONTRIBUTORS, AND REVIEWERS...............

................................................... 5

1. BACKGROUND FOR THE DEVELOPMENT OF A PAH MIXTURE S HEALTH ASSESSMENT.........................................

................................................... .............................. 7

2. RATIONALE FOR RECOMMENDING AN RPF APPROACH ..... ....................................... 8
2.1. PAHs AS A CHEMICAL CLASS .....................

................................................... ............ 11 2.2. THE TOXICOLOGICAL DATABASE FOR PAHs .......... .............................................. 27 2.3. BENZO[A]PYRENE AS AN INDEX CHEMICAL.......... ............................................... 28
2.4. SIMILARITIES IN MODE OF CARCINOGENIC ACTION F OR PAHs ...................... 31 2.5. STRUCTURAL ALERTS FOR PAH CARCINOGENESIS..... ....................................... 42
2.6. SIMILARITIES IN RELATIVE POTENCY ACROSS ENDPO INTS............................ 43 2.7. SIMILARITIES IN RELATIVE POTENCY ESTIMATES AC ROSS SPECIES AND EXPOSURE ROUTES................................

................................................... ......... 45
2.8. DOSE ADDITIVITY OF PAHs IN COMBINED EXPOSURES .................................... 46

3. DISCUSSION OF PREVIOUSLY PUBLISHED RPF APPROACH ES ............................... 50 3.1. PREVIOUS EFFORTS TO VALIDATE RPF APPROACH .... ....................................... 58

4. EVALUATION OF THE CARCINOGENICITY OF INDIVIDUAL PAHs......................... 60 4.1. DATABASE OF STUDIES ON PAH CARCINOGENICITY AN D CANCERRELATED ENDPOINTS ..................................

................................................... ............ 60 4.2. STUDIES IN HUMANS ............................

................................................... .................... 62
4.3. STUDIES IN ANIMALS ...........................

................................................... .................... 62 4.3.1. In Vivo Cancer Bioassays in Animals ........

................................................... .............. 89 4.3.1.1. Dermal Exposure ..........................

................................................... ................... 89 4.3.1.2. Intraperitoneal Exposure.................

................................................... ................. 92 4.3.1.3. Subcutaneous Injection Exposure..........

................................................... .......... 93 4.3.1.4. Oral Exposure ............................

................................................... ...................... 94 4.3.1.5. Other Routes .............................

................................................... ....................... 95 4.3.2. In Vivo Studies of Cancer-Related Endpoints

................................................... .......... 95 4.3.2.1. DNA Adducts..............................

................................................... ..................... 96 4.3.2.2. Clastogenicity or Sister Chromatid Exchan ge Frequency .................................. 98 4.3.2.3. In Vivo Mutagenicity.....................

................................................... .................. 99 4.3.3. In Vitro Studies of Cancer-Related Endpoint s.................................................. ......... 100 4.3.3.1. Bacterial Mutagenicity...................

................................................... ................ 100 4.3.3.2. Mammalian Mutagenicity...................

................................................... ........... 101 4.3.3.3. Morphological/Malignant Cell Transformati on................................................ 1 02 4.3.3.4. DNA Adducts..............................

................................................... ................... 103 4.3.3.5. DNA Damage/Repair........................

................................................... ............. 104 4.3.3.6. Clastogenicity or Sister Chromatid Exchan ge Frequency ................................ 104 4.4. SUMMARY OF INFORMATION AVAILABLE TO DEVELOP R PFS FOR INDIVIDUAL PAHs ....................................

................................................... ............... 106

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5. METHODS FOR DOSE-RESPONSE ASSESSMENT AND RPF CA LCULATION ........ 106
5.1. CHOICE OF DOSE-RESPONSE DATA .................

................................................... ... 106 5.2. OVERALL FORM OF RPF ESTIMATE .................

................................................... ... 108 5.3. RPF CALCULATION FOR MULTIDOSE DATASETS ....... ....................................... 108
5.4. RPF CALCULATION FOR SINGLE DOSE DATASETS..... ....................................... 110 5.5. DOSE CONVERSION FOR RPF CALCULATION.......... ............................................ 111
5.6. SPECIAL CONSIDERATIONS FOR RPF CALCULATION US ING TUMOR BIOASSAY DATA ......................................

................................................... ............... 112 5.7. SPECIAL CONSIDERATIONS FOR RPF CALCULATION US ING CANCERRELATED ENDPOINT DATA ..............................

................................................... .... 112

6. SELECTION OF PAHS FOR INCLUSION IN RELATIVE POT ENCY APPROACH ..... 113 6.1. METHOD FOR SELECTING PAHS FOR INCLUSION IN RE LATIVE POTENCY APPROACH...................................

................................................... .......... 115
6.2. WEIGHT OF EVIDENCE EVALUATION FOR 34 INDIVIDU AL PAHS.................. 118

7. CONCLUSIONS ....................................

................................................... ............................ 189 7.1. METHODS FOR DERIVING FINAL RPFs ..............

................................................... . 189 7.2. CONFIDENCE RATINGS FOR FINAL RPFs ............ .................................................. 1 91
7.3. SUSCEPTIBILITY FROM EARLY LIFE EXPOSURE TO CA RCINOGENS ............ 196

8. UNCERTAINTIES ASSOCIATED WITH RPF APPROACHES ... .................................... 197 8.1. UNCERTAINTY IN DOSE-RESPONSE ASSESSMENT FOR I NDIVIDUAL
PAHS ...............................................

................................................... ............................ 198 8.2. UNCERTAINTY IN SELECTING PAHs FOR INCLUSION I N RPF APPROACH ...........................................

................................................... ..................... 200
8.3. UNCERTAINTY IN DERIVING A FINAL RPF FOR EACH PAH............................. 202 8.4. UNCERTAINTY IN USE OF ANIMAL DATA TO PREDICT HUMAN
CANCER RISK ........................................

................................................... ................... 206 8.5. UNCERTAINTY IN THE ASSUMPTIONS OF COMMON MODE OF ACTION AND DOSE ADDITIVITY ................................

................................................... ......... 208
8.6. UNCERTAINTY IN EXTRAPOLATING RPFs ACROSS EXPO SURE ROUTES..... 209

9. REFERENCES.....................................

................................................... .............................. 215

APPENDIX A. SECONDARY SOURCES REVIEWED FOR IDENTIF ICATION OF
PRIMARY LITERATURE.................................

................................................... ................... 1

APPENDIX B. BIBLIOGRAPHY OF STUDIES WITHOUT BENZO[ A]PYRENE AS A REFERENCE COMPOUND.................................

................................................... ............ B-1 B.1. BIBLIOGRAPHY OF BIOASSAYS WITHOUT BENZO[A]PYR ENE ...................... B-4
B.2. BIBLIOGRAPHY OF STUDIES ON CANCER-RELATED END POINTS WITHOUT BENZO[A]PYRENE .............................

................................................... B-10

APPENDIX C. DOSE-RESPONSE DATA FOR POTENCY CALCULA TIONS.................. C-1

### xvi DRAFT – DO NOT CITE OR QUOTE

APPENDIX D. BENCHMARK DOSE MODELING OUTPUTS ....... .................................... D-1 D.1. DERMAL BIOASSAYS .............................

................................................... ................ D-1
D.2. INTRAPERITONEAL BIOASSAYS ....................

................................................... ... D-45 D.3. LUNG IMPLANTATION BIOASSAYS..................

................................................... D-67 D.4. BACTERIAL MUTAGENICITY .......................

................................................... ...... D-87
D.5. MAMMALIAN MUTAGENICITY.......................

................................................... ... D-96 D.6. MALIGNANT TRANSFORMATION ..................... ................................................. D- 124
D.7. IN VITRO DNA DAMAGE..........................

................................................... .......... D-156

APPENDIX E. CALCULATION OF RPFs...................

................................................... .........E-1

APPENDIX F. EXAMPLE CALCULATION OF RPF DETECTION L IMIT.........................F-1

APPENDIX G: EVALUATION OF ALTERNATIVES FOR RANKING RPFs ..................... G-1 G.1. OPTIONS FOR RANKING TUMOR BIOASSAY RPFS ...... ...................................... G-1
G.2. RANKING NON-BIOASSAY DATA....................

................................................... .... G-6

### xvii DRAFT – DO NOT CITE OR QUOTE LIST OF TABLES

2-1. PAHs evaluated in the RPF analysis ...........

................................................... ...................... 12

2-2. Studies of binary mixtures of PAHs and tumori genicity........................................... ........... 47

3-1. Comparison among various relative potency est imates for PAHs from the published literature and regulatory agencies (1984–2004).....

................................................... ........... 51

4-1. Study summaries: dermal bioassays of benzo[a ]pyrene and at least one other PAH.......... 64

4-2. Study summaries: intraperitoneal bioassays o f benzo[a]pyrene and at least one other PAH..........................................

................................................... ................................ 68

4-3. Study summaries: subcutaneous bioassays of b enzo[a]pyrene and at least one other PAH..........................................

................................................... ................................ 69

4-4. Study summaries: oral bioassays of benzo[a]p yrene and at least one other PAH............... 70

4-5. Study summaries: other route bioassays of be nzo[a]pyrene and at least one other
PAH................................................

................................................... ................................... 71

4-6. Study summaries: in vivo DNA adducts with be nzo[a]pyrene and at least one other PAH..........................................

................................................... ................................ 72

4-7. Study summaries: in vivo clastogenicity or s ister chromatid exchange with benzo[a]pyrene and at least one other PAH ..........

................................................... ............ 74

4-8. Study summaries: in vivo mutagenicity with b enzo[a]pyrene and at least one other PAH..........................................

................................................... ................................ 75

4-9. Study summaries: in vitro bacterial mutageni city with benzo[a]pyrene and at least one other PAH......................................

................................................... ............................. 76

4-10. Study summaries: in vitro mammalian mutagen icity assays with benzo[a]pyrene and at least one other PAH.............................

................................................... ........................ 79

4-11. Study summaries: in vitro morphological/mal ignant cell transformation with benzo[a]pyrene and at least one other PAH ..........

................................................... .......... 82

4-12. Study summaries: in vitro DNA adducts with benzo[a]pyrene and at least one other PAH..........................................

................................................... .............................. 84

4-13. Study summaries: in vitro DNA damage, repai r, or synthesis with benzo[a]pyrene and at least one other PAH.........................

................................................... ..................... 85

4-14. Study summaries: in vitro clastogenicity or sister chromatid exchange with benzo[a]pyrene and at least one other PAH ..........

................................................... .......... 87

### xviii DRAFT – DO NOT CITE OR QUOTE

5-1 Comparison between molar and mass-based RPF...

................................................... ......... 111

6-1. PAHs with only one RPF from in vitro cancer-r elated endpoint study and excluded from RPF approach ..................................

................................................... ....................... 114

6-2. Results of weight of evidence evaluation of 3 4 PAHs.......... Error! Bookmark not defined.

7-1. Final RPFs based on tumor bioassay data ......

................................................... ................. 193

7-2. Final RPFs based on cancer-related endpoint d ata (no tumor bioassay data available) ....................................

................................................... ............................. 195

8-1. Results of simple linear regression of log–tr ansformed average genotoxicity RPF vs. log average tumor bioassay RPF.....................

................................................... ................ 204

8-2. PAHs with RPFs of varying confidence.........

................................................... ................. 205

8-3. Comparisons among average tumor bioassay RPF values by exposure route and target organ.......................................

................................................... ............................... 211

B-1. Bioassays with and without benzo[a]pyrene by PAH................................................ ........ B-2

C-1. Dermal bioassays: dose response information f or incidence data .................................. ... C-2

C-2. Dermal bioassays: dose-response information f or tumor multiplicity.............................. . C-8

C-3. Intraperitoneal bioassays: dose response info rmation for incidence data........................ C -13

C-4. Intraperitoneal bioassays: dose-response info rmation for tumor multiplicity ................. C-22

C-5. Lung implantation bioassays: dose response in formation for incidence data.................. C-26

C-6. In vitro bacterial mutagenicity: data use....

................................................... ................... C-30

C-7. In vitro bacterial mutagenicity: dose respons e data............................................. ............ C-33

C-8. In vitro mammalian mutagenicity: data use....

................................................... .............. C-51

C-9. In vitro mammalian mutagenicity: dose respons e data............................................. ....... C-53

C-10. In vitro malignant/morphological cell transf ormation: data use................................. ... C-61

C-11. In vitro malignant/morphological cell transf ormation: dose response data................... C-63

C-12. In vitro DNA adducts: data use..............

................................................... ..................... C-71

C-13. In vitro DNA adducts: dose response data...........

................................................... ....... C-72

### xix DRAFT – DO NOT CITE OR QUOTE

C-14. In vitro DNA damage: data use ...............

................................................... ................... C-75

C-15. In vitro DNA damage: dose response data .....

................................................... ............ C-76

C-16. In vitro clastogenicity: data use ...........

................................................... ....................... C-80

C-17. In vitro clastogenicity: dose response data .

................................................... ................ C-81

C-18. In vivo DNA adducts: data use ..............................

................................................... ..... C-82

C-19. In vivo DNA adducts: dose response data ............

................................................... ...... C-83

C-20. In vivo clastogenicity: data use............

................................................... ....................... C-90

E-1. Dermal bioassays: RPF calculations for incide nce data ........................................... ..........E-2

E-2. Dermal bioassays: RPF calculations for multip licity data ........................................ ..........E-5

E-3. Intraperitoneal bioassays: RPF calculations for incidence data................................. ........E-7

E-4. Intraperitoneal bioassays: RPF calculations for multiplicity data.............................. ........E-9

E-5. Lung implantation bioassays: RPF calculation s (incidence data)................................. ...E-10

E-6. In vivo DNA adducts: RPF calculations ........

................................................... ................E-11

E-7. In vivo clastogenicity or sister chromatid ex change: RPF calculation ........................... .E-13

E-8. In vitro bacterial mutagenicity: RPF calcula tions.............................................. ..............E-15

E-9. In vitro mammalian mutagenicity: RPF calcula tions.............................................. .........E-21

E-10. In vitro morphological/malignant transformat ion: RPF calculation .............................. E-24

E-11. In vitro DNA adducts: RPF calculations......

................................................... ................E-27

E-12. In vitro DNA damage: RPF calculations ......

................................................... ..............E-29

E-13. In vitro clastogenicity or sister chromatid exchange: RPF calculations ......................... E-31

F-1. Example data for calculation of RPF detection limit ............................................. .............F-1

G-1. Average RPF value by exposure route ..........

................................................... ................. G-3

G-2. Comparisons among average tumor bioassay-base d RPF values by data availability or calculation method ..............................

................................................... ....................... G-5

G-3. Correlation between LED and TD 25 

### by endpoint ....................................... ....................... G-7

### xx DRAFT – DO NOT CITE OR QUOTE

G-4. Correlation between tumor potency (log 1/TD 50 

### ) and mutagenic potency........................ G-7

G-5. Results of simple linear regression of log–tr ansformed average genotoxicity RPF vs. log average tumor bioassay RPF.....................

................................................... ............... G-9

### 1 DRAFT – DO NOT CITE OR QUOTE LIST OF FIGURES 1

2

3 

### 2-1. Structural features of PAHs..................

................................................... ............................. 27 4

5 

### 2-2. Metabolic pathways for benzo[a]pyrene ........

................................................... ................... 32 6

7 

### 2-3. Overview of the proposed key events in the mo de of action for PAH 8 

### carcinogenicity. ...................................

................................................... .............................. 34 9

10 

### 2-4. Structures of the four stereoisomeric adduct moieties, anti -[BaP]- N 2 

### -dG, derived from 11 

### the trans - or cis -covalent binding of (+)- anti -BaP diol epoxide or (-)- anti -BaPdiol 12 

### epoxide to dG residues in DNA. .....................

................................................... .................. 35 13

14 

### 2-5. Depurinating adducts of benzo[a]pyrene formed by one-electron oxidation....................... 36 15

16 

### 2-6. Spectrum of DNA adducts anticipated with PAH o-quinones. ........................................ .... 37 17

18 

### 2-7. Interaction of PAHs with the Ah receptor – re gulation of genes related to induction of 19 

### metabolism and cell differentiation and proliferati on. ................................................ ......... 39 20

21 

### 6-1. Weight of evidence assessment of potential ca rcinogenicity. ..................................... ....... 116 22

23 

### 6-2. 2,3-Acepyrene (ACEP) RPFs....................

................................................... ...................... 121 24

25 

### 6-3. Anthanthrene (AA) RPFs. ......................

................................................... ......................... 123 26

27 

### 6-4. Anthracene (AC) RPFs.........................

................................................... ........................... 125 28

29 

### 6-5. Benz[a]anthracene (BaA) RPFs.................

................................................... ...................... 127 30

31 

### 6-6. Benz[b,c]aceanthrylene (BbcAC) RPFs..........

................................................... ................ 129 32

33 

### 6-7. Benzo[b]fluoranthene (BbF) RPFs..............

................................................... .................... 131 34

35 

### 6-8. 11H-Benzo[b]fluorene (BbFE) RPFs. ............

................................................... ................. 133 36

37 

### 6-9. Benz[e]aceanthrylene (BeAC) RPFs.............

................................................... .................. 135 38

39 

### 6-10. Benzo[e]pyrene (BeP) RPFs...................

................................................... ....................... 137 40

41 

### 6-11. Benzo[g,h,i]fluoranthene (BghiF) RPFs.......

................................................... ................. 139 42

43 

### 6-12. Benzo[g,h,i]perylene (BghiP) RPFs. ..........

................................................... ................... 141 44

45 

### 6-13. Benz[j]aceanthrylene (BjAC) RPFs ............

................................................... .................. 143 46

47 

### 6-14. Benzo[j]fluoranthene (BjF) RPFs.............

................................................... ..................... 145 48

49

### 2 DRAFT – DO NOT CITE OR QUOTE

6-15. Benzo[k]fluoranthene (BkF) RPFs.............

................................................... ................... 147 1

2 

### 6-16. Benz[l]aceanthrylene (BlAC) RPFs. ...........

................................................... .................. 149 3

4 

### 6-17. Chrysene (CH) RPFs. .........................

................................................... ........................... 151 5

6 

### 6-18. Coronene (CO) RPFs..........................

................................................... ........................... 153 7

8 

### 6-19. Cyclopenta[c,d]pyrene (CPcdP) RPFs. .........

................................................... ................ 155 9

10 

### 6-20. Cyclopenta[d,e,f]chrysene (CPdefC) RPFs.....

................................................... .............. 157 11

12 

### 6-21. Dibenz[a,c]anthracene (DBacA) RPFs..........

................................................... ................ 159 13

14 

### 6-22. Dibenzo[a,e]fluoranthene (DBaeF) RPFs.......

................................................... ............... 161 15

16 

### 6-23. Dibenzo[a,e]pyrene (DBaeP) RPFs.............

................................................... .................. 163 17

18 

### 6-24. Dibenz[a,h]anthracene (DBahA) RPFs. .........

................................................... ............... 165 19

20 

### 6-25. Dibenzo[a,h]pyrene (DBahP) RPFs. ............

................................................... ................. 167 21

22 

### 6-26. Dibenzo[a,i]pyrene (DbaiP) RPFs.............

................................................... .................... 169 23

24 

### 6-27. Dibenzo[a,l]pyrene (DBalP) RPFs. ............

................................................... ................... 172 25

26 

### 6-28. Fluoranthene (FA) RPFs......................

................................................... .......................... 174 27

28 

### 6-29. Fluorene (FE) RPFs..........................

................................................... ............................. 176 29

30 

### 6-30. Indeno[1,2,3-c,d]pyrene (IP) RPFs...........

................................................... ..................... 178 31

32 

### 6-31. Naphtho[2,3-e]pyrene (N23eP) RPFs...........

................................................... ................. 180 33

34 

### 6-32. Perylene (Pery) RPFs........................

................................................... ............................. 182 35

36 

### 6-33. Phenanthrene (PH) RPFs......................

................................................... ......................... 184 37

38 

### 6-34. Pyrene (Pyr) RPFs. ..........................

................................................... .............................. 186 39

40 

### 6-35. Triphenylene (Tphen) RPFs ...................

................................................... ....................... 188 41

42 

### 8-1. Correlation between incidence and multiplicit y RPFs ............................................. .......... 203 43

44 

### G-1. Average bioassay RPF vs. average in vivo DNA adduct RPF ........................................ G -10 45

46 

### G-2. Average bioassay RPF vs. average in vivo nonb ioassay RPF........................................ . G-10 47

48 

### G-3. Average bioassay RPF vs. average nonbioassay RPF................................................ ..... G-11 49

### 3 DRAFT – DO NOT CITE OR QUOTE

1 

### G-4. Average bioassay RPF vs. average in vitro non -bioassay RPF ...................................... . G-11 2

3

### 4 DRAFT – DO NOT CITE OR QUOTE LIST OF ABBREVIATIONS AND ACRONYMS * 1

2

3 

### AEL acceptable exposure level 4 

### Ah aryl hydrocarbon 5 

### AhR Ah receptor 6 

### AhRE DNA 

### Ah-responsive elements of DNA 7 

### ARNT Ah-receptor nuclear translocator 8 

### ATSDR

Agency for Toxic Substances and Disease Registry 9 

### AUC area under the curve 10 

### BMD benchmark dose 11 

### BMR

benchmark response 12 

### CASRN Chemical Abstract Service Registry Number 13 

### CHO Chinese hamster ovary 14 

### CYP cytochrome P450 15 

### dG deoxyguanosine 16 

### EAL environmental assessment level 17 

### EOPP estimated order of potential potency 18 

### EROD ethoxyresorufin O-deethylase 19 

### HPRT hypoxanthine-guanine phosphoribosyl transferase ge ne 20 

### Hsp90 heat shock protein 90 21 

### IARC International Agency for Research on Cancer 22 

### IRIS Integrated Risk Information System 23 

### LED lowest effective dose 24 

### MGP manufactured gas plant 25 

### MVK

Moolgavkar-Venson-Knudsen two-stage model 26 

### OEHHA Office of Environmental Health Hazard Assessment, California EPA 27 

### PAC polycyclic aromatic compound 28 

### PAH polycyclic aromatic hydrocarbon 29 

### PCR

polymerase chain reaction 30 

### PEF potency equivalency factor 31 

### RTECS Registry of Toxic Effects of Chemical Substances 32 

### RPF relative potency factor 33 

### RTD relative tumor dose 34 

### SMART somatic mutation and recombination test 35 

### TK thymidine kinase locus 36 

### TIDAL time-integrated DNA adduct level 37 

### TEF toxicity equivalency factor 38 

### TPA 12-O-tetra-decanoylphorbol-13-acetate 39 

### U.S. EPA U.S. Environmental Protection Agency 40 

### WHO World Health Organization 41

42

* 

### Abbreviations for PAH chemical names are provided i n Table 2-1. 43

### 5 DRAFT – DO NOT CITE OR QUOTE AUTHORS, CONTRIBUTORS, AND REVIEWERS 1

2 

### CHEMICAL MANAGERS 3

4 

### Lynn Flowers, Ph.D., DABT 5 

### National Center for Environmental Assessment 6 

### Office of Research and Development 7 

### U.S. Environmental Protection Agency 8 

### Washington, DC 9

10 

### Martin Gehlhaus, III 11 

### National Center for Environmental Assessment, IRIS Program 12 

### Office of Research and Development 13 

### U.S. Environmental Protection Agency 14 

### Washington, DC 15

16 

### AUTHORS 17

18 

### Lynn Flowers, Ph.D., DABT 19 

### National Center for Environmental Assessment 20 

### Office of Research and Development 21 

### U.S. Environmental Protection Agency 22 

### Washington, DC 23

24 

### Martin Gehlhaus, III 25 

### National Center for Environmental Assessment, IRIS Program 26 

### Office of Research and Development 27 

### U.S. Environmental Protection Agency 28 

### Washington, DC 29

30 

### Karen Hogan 31 

### National Center for Environmental Assessment, IRIS Program 32 

### Office of Research and Development 33 

### U.S. Environmental Protection Agency 34 

### Washington, DC 35

36 

### Channa Keshava, Ph.D. 37 

### National Center for Environmental Assessment, IRIS Program 38 

### Office of Research and Development 39 

### U.S. Environmental Protection Agency 40 

### Washington, DC 41

42 

### Glenn Rice, Ph.D. 43 

### National Center for Environmental Assessment 44 

### Office of Research and Development 45 

### U.S. Environmental Protection Agency 46 

### Cincinnati, OH 47

48

### 6 DRAFT – DO NOT CITE OR QUOTE

Jamie Strong, Ph.D. 1 

### National Center for Environmental Assessment, IRIS Program 2 

### Office of Research and Development 3 

### U.S. Environmental Protection Agency 4 

### Washington, DC 5

6 

### Linda Teuschler, Ph.D. 7 

### National Center for Environmental Assessment 8 

### Office of Research and Development 9 

### U.S. Environmental Protection Agency 10 

### Cincinnati, OH 11

12 

### Stephen Nesnow, Ph.D. 13 

### Environmental Carcinogenesis Division 14 

### National Health and Environmental Effects Research Laboratory 15 

### Office of Research and Development 16 

### Research Triangle Park, NC 17

18 

### Chao Chen, Ph.D. 19 

### National Center for Environmental Assessment 20 

### Office of Research and Development 21 

### Washington, DC 22

23 

### Heather Carlson-Lynch, S.M. 24 

### Syracuse Research Corporation, Inc. 25 

### Syracuse, NY 26

27 

### Julie Stickney, Ph.D., DABT 28 

### Syracuse Research Corporation, Inc. 29 

### Syracuse, NY 30

31 

### Peter R. McClure, Ph.D., DABT 32 

### Syracuse Research Corporation, Inc. 33 

### Syracuse, NY 34

35 

### Amber Bacom 36 

### Syracuse Research Corporation, Inc. 37 

### Syracuse, NY 38

39

40

### 7 DRAFT – DO NOT CITE OR QUOTE

1. BACKGROUND FOR THE DEVELOPMENT OF A PAH MIXTURE S HEALTH 1



### ASSESSMENT 2

3

4 

### The U.S. Environmental Protection Agency (U.S. EPA) IRIS Program is undertaking a 5 

### project to develop a PAH Mixtures Health Assessment . This assessment focuses on the RPF 6 

### approach which is based on component PAHs in the mi xture. In preparation for the development 7



### of the PAH Mixtures Health Assessment, U.S. EPA held a peer consultation workshop to outl ine 8 

### some of the important issues. These issues are dis cussed in Peer Consultation Workshop on 9 

### Approaches to Polycyclic Aromatic Hydrocarbon (PAH) Health Assessment (U.S. EPA, 2002) 10 

### and the accompanying discussion document. 11 

### Health assessments for 15 unsubstituted, nonheteroc yclic polycyclic aromatic 12 

### hydrocarbons (PAHs) with three or more rings are cu rrently entered on EPA’s Integrated Risk 13 

### Information System (IRIS) database. Benzo[a]pyrene is the only PAH for which quantitative 14 

### oral, dermal, and inhalation data are available. 15 

### In 1993, U.S. EPA published the Provisional Guidance for Quantitative Risk Assessme nt 16 

### of PAHs (

Provisional Guidance ). The Provisional Guidance recommended estimated orders of 17 

### potential potency (EOPP) for individual PAHs that c ould be used in a component-based 18 

### approach to PAH mixtures risk assessment. The Provisional Guidance recommended EOPPs for 19 

### 7 PAHs categorized as Group B2 (probable human carc inogens) under 1986 U.S. EPA Cancer 20 

### Guidelines: benzo[a]pyrene, benz[a]anthracene, ben zo[b]fluoranthene, benzo[k]fluoranthene, 21 

### chrysene, dibenz[a,h]anthracene, and indeno[1,2,3-c ,d]pyrene (U.S. EPA, 1993). The current 22 

### analysis extends the 1993 Provisional Guidance and provides recommendations for further 23 

### development of this approach to PAH mixtures risk a ssessment. The assessment includes the 24 

### following: 25

26 

### (1) A rationale for recommending an order of potency, o r relative potency factor (RPF), 27 

### approach; 28

29 

### (2) A summary of previous approaches for developing the RPF approach for PAHs; 30

31 

### (3) Identification of individual carcinogenic PAHs that could be included in the RPF 32 

### approach; 33

34 

### (4) Identification of potential index chemicals; 35

36 

### (5) Presentation of the available literature for in viv o carcinogenicity and both in vivo and in 37 

### vitro cancer-related endpoint assays for individual PAHs; 38

39 

### (6) Development of a recommendation for the RPF approac h for PAH mixtures; and 40

41

### 8 DRAFT – DO NOT CITE OR QUOTE

(7) Characterization of strengths, weaknesses, and unce rtainties associated with the 1 

### recommended approaches. 2

3

4



### 2. RATIONALE FOR RECOMMENDING AN RPF APPROACH 5

6

7 

### PAHs are a concern as human health hazards, because many PAHs are demonstrated 8 

### tumorigenic agents in animal bioassays and are acti ve in in vivo or in vitro tests for genotoxicity 9 

### or DNA damage. PAHs do not occur in the environmen t as isolated entities; they primarily 10 

### occur in complex mixtures generated from the combus tion or pyrolysis of substances containing 11 

### carbon and hydrogen. Several complex mixtures of P AHs have been classified as possibly 12 

### carcinogenic, probably carcinogenic, or carcinogeni c to humans (Straif et al., 2005; U.S. EPA, 13 

### 2002; Bostrom et al., 2002; WHO, 1998; ATSDR, 1995; IARC, 1985, 1984a, b, 1983). 14 

### In concordance with U.S. EPA (2000, 1986) guidance for health risk assessment of 15 

### chemical mixtures, assessment of the cancer risk fr om long-term human exposure to a particular 16 

### PAH mixture would best be conducted with quantitati ve information on the dose-response 17 

### relationship for cancer from chronic exposure to th e mixture of concern. When data for the 18 

### mixture of concern are not available, U.S. EPA (200 0, 1986) guidance recommends using 19 

### toxicity data on a “sufficiently similar” mixture. However, quantitative cancer dose-response 20 

### information exists only for a few complex mixtures generated from the combustion or pyrolysis 21 

### of organic matter; for example, tobacco smoke, coke oven emissions, and emissions from roofing 22 

### tar pots (see Bostrom et al., 2002; Albert et al., 1983). U.S. EPA’s IRIS database currently 23 

### includes assessments for only three PAH-containing mixtures: coke oven emissions, creosote, 24 

### and diesel emissions. The availability of oral car cinogenicity bioassays of manufactured gas 25 

### plant (MGP) residue (Weyand et al., 1995) and coal tar preparations (Culp et al., 1998; Gaylor et 26 

### al., 1998) has expanded the PAH mixture cancer data base. 27 

### Component-based approaches, involving an analysis o f the toxicity of components of the 28 

### mixture, are recommended when appropriate toxicity data on a complex mixture of concern, or 29 

### on a “sufficiently similar” mixture, are unavailabl e (U.S. EPA, 2000, 1986). Component-based 30 

### approaches involving dose addition (such as the RPF approach) are recommended when 31 

### components in the mixture are judged to act in a to xicologically similar manner. In the RPF 32 

### approach, doses of component chemicals that act in a toxicologically similar manner are added 33 

### together, after scaling the doses relative to the p otency of an index chemical (U.S. EPA, 2000, 34 

### 1986). Then, using the dose-response curve of the index chemical, the response to the total 35 

### equivalent dose in the mixture is estimated. The i ndex compound is typically the best-studied 36 

### member of the class with the largest body of availa ble data describing exposure and health 37 

### effects. The index chemical should have a quantita tive dose-response assessment of acceptable 38

### 9 DRAFT – DO NOT CITE OR QUOTE

scientific quality and must have (or be expected to have) similar toxic effects to the rest of the 1



### members of the class. 2 

### For chemicals that act independently (e.g., by diff erent modes of action), U.S. EPA 3 

### guidance (2000, 1986) recommends a component-based approach involving response addition. 4 

### In this approach, response is defined as the percen tage or fraction of exposed individuals that 5 

### show the effect of concern (i.e., the risk of havin g the effect). To apply a response-addition 6 

### approach to a complex mixture, information on the d ose-response relationships for the effect of 7 

### concern from exposure to individual components must be available. Based on an analysis of the 8 

### amount of each component in the mixture and its dos e-response relationship, risks from exposure 9 

### to the individual components are calculated and add ed together to estimate the risk for the effect 10 

### from exposure to the complex mixture. 11 

### Component-based approaches, either involving dose a ddition or response addition, 12 

### include a general assumption that interaction effec ts at low dose levels either do not occur or are 13 

### small enough to be neglected (U.S. EPA, 2000, 1986) . However, when information on 14 

### interactions among the components is available, U.S . EPA guidance recommends incorporating 15 

### this information into the risk assessment, either a s a part of a quantitative approach or as a 16 

### qualitative evaluation. 17



### The assessment of cancer risk from chronic oral or inhalation exposure to complex PAH 18 

### mixtures using component-based approaches is restri cted by the limited availability of cancer 19 

### dose-response data for individual PAHs. Benzo[a]py rene is the only PAH that has dose-response 20 

### data for cancer from chronic oral, inhalation or de rmal exposure (WHO, 1998; ATSDR, 1995). 21 

### The IRIS Program developed cancer assessments for 1 5 PAHs in the early 1990s, but a 22 

### quantitative cancer assessment was only developed f or benzo[a]pyrene. Six other PAHs were 23 

### qualitatively assessed as B2, probable human carcin ogens, but the available data were 24 

### characterized as inadequate to develop oral or inha lation risk estimates. Thus, data are 25 

### insufficient to use a component-based approach invo lving response addition for assessing cancer 26 

### risks from PAH mixtures. Although a response addit ion approach does not require an 27 

### assumption of similar toxicological action, it does require dose-response data for individual 28



### components. 29 

### For exposure situations in which dose-response data for the PAH mixture or a sufficiently 30 

### similar mixture are not available (e.g., the source of the PAH contamination may be mixed or 31 

### unknown), there are at least three practical advant ages of an RPF approach that uses 32 

### benzo[a]pyrene as the index PAH: 33

34 

### (1) Benzo[a]pyrene is routinely assayed and detecte d in environmental media contaminated 35 

### with PAH mixtures; 36

37 

### (2) Benzo[a]pyrene is the only PAH for which cancer dose-response data involving chronic 38 

### exposures are available; and 39

### 10 DRAFT – DO NOT CITE OR QUOTE

1 

### (3) There is a large database of studies in which t he potency of benzo[a]pyrene is compared 2 

### with the potency of other PAHs in various assays. 3

4 

### The database includes animal tumorigenicity 1 

### assays involving dermal or parenteral 5 

### administration, and in vivo and in vitro assays of cancer-related endpoints (e.g., various 6 

### genotoxic endpoints). Thus, RPFs for a number of P AHs can be derived. 7 

### The RPF approach involves two key assumptions: (1) the assumption of similar 8 

### toxicological action as required by dose addition; and (2) the assumption that interactions among 9 

### PAH mixture components do not occur at low levels o f exposure typically encountered in the 10



### environment. 11 

### Mechanistic studies indicate that the mutagenic and tumor-initiating activity of most 12 

### carcinogenic PAHs requires metabolic activation to reactive intermediates (e.g., stereospecific 13 

### dihydrodiol epoxides). For several PAHs, (e.g., be nzo[a]pyrene, dibenz[a,h]anthracene, 14 

### dibenzo[a,l]pyrene) there is evidence that DNA dama ge associated with metabolism can lead to 15 

### mutations in cancer-related genes. Tumor promotion and progression by PAHs may involve 16 

### parent compound binding to the aryl hydrocarbon (Ah ) receptor and subsequent alterations of 17 

### gene expression, as well as by cell proliferation i n response to cytotoxic effects from metabolites 18 

### (see Section 2.4, Similarities in Carcinogenic Mode of Action for PAHs). Thus, there is some 19 

### evidence that an assumption of similar toxicologica l action is reasonable, but some aspects of the 20 

### diversity of biological activities among PAHs are u nexplained. The second assumption of no 21 

### interactions at low levels of exposure may be reaso nable, but some evidence of toxicological 22 

### interactions among PAHs and among PAHs and other ch emicals is available (see Section 2.8, 23 

### Additivity of PAHs in Combined Exposures). 24 

### Other key limitations to the RPF approach, relativ e to whole mixture approaches, are: 25 

### (1) RPFs have been derived for a limited number of PAHs; and (2) cancer risks from non-PAH 26 

### components, unidentified PAHs, and heterocyclic and substituted PAHs in PAH mixtures are not 27 

### estimated. The first of these limitations is being addressed, to the degree allowable by available 28 

### data, by the derivation of RPFs for numerous PAHs a s discussed in Sections 4 and 5 of this 29 

### report. If non-PAH carcinogenic components are ide ntified and quantified in the complex 30 

### mixture of concern and appropriate dose-response da ta are available, the second limitation can be 31 

### addressed by using a response addition approach (i. e., adding the cancer risk from PAH 32 

### components estimated by the RPF approach to cancer risks estimated for the non-PAH 33 

### carcinogenic components of the mixture). Previous efforts to validate the RPF approach using 34



### data for PAH mixtures are discussed in Section 6.7. These validation efforts compared the 35 

### cancer risk of a PAH mixture measured experimentall y with the cancer risk that was predicted 36 

### using the RPF method but were limited by the small number of compounds for which RPFs and 37

1 Throughout this report, the term “tumorigenicity” i s used to describe the production of either benign or malignant

tumors.

### 11 DRAFT – DO NOT CITE OR QUOTE

analytical data were available (Muller et al., 1997 ; McClure, 1996; Goldstein et al., 1994; 1 

### Clement Associates, 1990, 1988; Krewski et al., 198 9). Validation of the updated approach 2 

### presented here wold be of value, either using prev ious data on PAH mixtures (human and 3 

### animal) or using new data collected with the main p urpose of evaluating the validity of the 4 

### approach. 5

6



### 2.1. PAHs AS A CHEMICAL CLASS 7 

### The PAH chemical class has been variously defined t o include organic compounds 8 

### containing either two or more, or three or more, fu sed rings made up of carbon and hydrogen 9 

### atoms (i.e., unsubstituted parent PAHs and their al kyl-substituted derivatives) (WHO, 1998). 10 

### Most PAHs are high-melting, high-boiling point, lip ophilic compounds, predominately generated 11 

### from the combustion or pyrolysis of organic matter. The PAH chemical class includes alkylated 12 

### PAHs (e.g., 1,4-dimethylphenanthrene and 5-methylch rysene), but not heterocyclic compounds 13 

### containing N, S, or O or PAHs substituted with N-, S-, or O-containing groups; these are 14 

### included in a larger chemical class, often referred to as polycyclic aromatic compounds (PACs) 15 

### (WHO, 1998). The number of chemicals that comprise the PAHs class is unknown; however, 16 

### there are thought to be hundreds of individual PAHs present as components of complex mixtures 17 

### (WHO, 1998). The analysis presented here is limite d in focus to include only unsubstituted 18 

### PAHs with three or more fused aromatic rings contai ning only carbon and hydrogen atoms, 19 

### because these are the most widely studied members o f the PAH chemical class. Naphthalene is a 20 

### widely studied 2-ring PAH compound; however, a sepa rate toxicological review and 21 

### carcinogenicity assessment is being developed by th e IRIS Program for this compound and it is 22 

### not included in this RPF approach. The list of PAH compounds that were considered for 23 

### inclusion in this analysis is presented in Table 2- 1 along with the Chemical Abstracts Service 24 

### Registry Numbers (CASRNs) and the abbreviations tha t are utilized in tables throughout the 25


### report. 26

### 12 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Benzo[a]pyrene 50-32-8 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

### 13 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF 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

Benz[a]anthracene 56-55-3 BaA 228.29

Benzo[a]fluoranthene 203-33-8 BaF 252.32

### 14 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF 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

### 15 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF 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 BbPery 302.38

Benzo[c]chrysene 194-69-4 BcC 278.35

Benzo[c]fluorene 205-12-9 BcFE 216.28

### 16 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Benzo[c]phenanthrene 195-19-7 BcPH 228.29

Benz[e]aceanthrylene 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]perylene 191-24-2 BghiP 276.34

### 17 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Benzo[g]chrysene 196-78-1 BgC 278.35

Benz[j]aceanthrylene 202-33-5 BjAC 252.32

Benzo[j]fluoranthene 205-82-3 BjF 252.32

Benzo[k]fluoranthene 207-08-9 BkF 252.32

Benz[l]aceanthrylene 211-91-6 BlAC 252.32

Benzophenanthrene 65777-08-4 BPH 228.29

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Table 2-1. PAHs evaluated in the RPF 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 RPF 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-1,2-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

### 20 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Dibenzo[a,e]fluoranthene 5385-75-1 DBaeF 302.38

Dibenzo[a,e]pyrene 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

### 21 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Dibenzo[a,h]pyrene 189-64-0 DBahP 302.38

Dibenzo[a,i]pyrene 189-55-9 DBaiP 302.38

Dibenzo[a,l]pyrene 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

### 22 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Dibenzo[h,rst]pentaphene 192-47-2 DBhrstPent 352.43

Dibenz[j,mno]acephenanthrylene 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 RPF 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[1,2,3-mno]acephenanthrylene 113779-16-1 N123mnoAPH 276.34

### 24 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Naphtho[1,2-b]fluoranthene 111189-32-3 N12bF 302.38

Naphtho[2,1-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

### 25 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF 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

### 26 DRAFT – DO NOT CITE OR QUOTE

Table 2-1. PAHs evaluated in the RPF analysis

PAH (common synonyms) CASRN Abbreviation Structure Molecular weight (g/mol) Pyrene 129-00-0 Pyr 202.26

Tribenzofluoranthene 3,4-10, 11-12,13- 13579-05-0 T BF 352.43

Triphenylene 217-59-4 Tphen 228.29

1 

### Unsubstituted PAHs have been further classified int o alternant and nonalternant 2 

### compounds. Alternant PAHs are those compounds comp osed solely of fused benzene rings, 3



### while nonalternant PAHs contain both benzene and fi ve carbon rings. Among alternant PAHs, 4 

### important structural features related to enhanced m utagenicity and carcinogenicity include the 5 

### presence of at least four rings (Bostrom et al., 20 02). Common structural features of PAH 6 

### compounds are illustrated in Figure 2-1. 7

### 27 DRAFT – DO NOT CITE OR QUOTE

1 

### Figure 2-1. Structural features of PAHs. 2

3



### 2.2. THE TOXICOLOGICAL DATABASE FOR PAHs 4 

### Over the last 30- to 50-years, a large PAH database has been generated including studies 5 

### of carcinogenicity in animal bioassays, genotoxicit y in various test systems, and metabolism 6 

### (bioactivation) to tumorigenic and/or genotoxic int ermediates. Carcinogenicity and genotoxicity 7 

### data are sufficient to classify a number of individ ual PAHs as possibly carcinogenic to humans 8 

### (WHO, 1998; U.S. EPA, 1993; IARC, 1989, 1986, 1985, 1984a, b, 1983). Other PAHs have 9 

### been tested for tumorigenicity and/or genotoxicity, but either negative or equivocal results were 10 

### obtained; for many positive results were only obser ved in genotoxicity assays (e.g., pyrene). 11 

### Many studies have been performed to provide further understanding about the carcinogenic 12 

### mode of action of PAHs (see Bostrom et al., 2002; W HO, 1998; ATSDR, 1995). Therefore, the 13



### PAH database contains studies that evaluate: 14

15 C H 2 Benzo[a]pyrene Pyrene

Examples of Alternant PAHs

Examples of Nonalternant PAHs Fluorene Fluoranthene

Bay-region and Fjord-regions of PAHs Chrysene Benzo[c]phenanthrene Bay-region Fjord-region

### 28 DRAFT – DO NOT CITE OR QUOTE

• Metabolism to reactive intermediates; 1

2



### • Characterization of PAH-DNA adducts; 3

4



### • Mutagenicity of PAHs in bacterial and mammalian cel ls; 5

6



### • Mutation spectra in identified oncogene and tumor s uppressor genes; 7

8



### • Clastogenic effects; 9

10



### • Cell transformation; and 11

12



### • Initiation and promotion of carcinogenicity. 13

14 

### A significant limitation to the database is the lac k of data from long-term oral or 15



### inhalation cancer studies for most individual PAH c ompounds. In addition, benzo[a]pyrene is 16 

### the only chemical for which long-term animal studie s have been conducted by multiple exposure 17 

### routes (Kroese et al., 2001; Culp et al., 1998, 199 6a, b; Thyssen et al., 1981, 1980; Rigdon et al., 18 

### 1969; Rigdon and Neal, 1969, 1966; Neal and Rigdon, 1967). Furthermore, most of the 19 

### toxicological data available for PAHs relate to can cer or genotoxicity. Available information on 20 

### the systemic, noncarcinogenic effects of PAHs is li mited although immunological, neurotoxic, 21 

### and developmental effects have been noted in animal studies (for earlier reviews see WHO, 22 

### 1998; ATSDR, 1995). As a result, the relative potency methodology descr ibed here is applied 23 

### only to cancer risk assessment for PAHs. 24

25 

### 2.3. BENZO[A]PYRENE AS AN INDEX CHEMICAL 26 

### Because long-term animal studies are not available for many individual PAHs, it is 27 

### necessary to choose an appropriate index chemical f or comparison of relative carcinogenic 28 

### potency. The index compound is typically the best- studied member of the class, with the largest 29 

### body of available data describing exposure and heal th effects. The index chemical should have a 30 

### quantitative dose-response assessment of acceptable scientific quality and must have (or be 31 

### expected to have) similar toxic effects to the rest of the members of the class. 32 

### Although the PAH composition of complex mixtures va ries, benzo[a]pyrene is 33 

### considered to be present in significant amounts in certain occupational environments and urban 34 

### settings (WHO, 1998; Petry et al., 1996; ATSDR, 199 5). Benzo[a]pyrene is one of the most 35 

### potent of the carcinogenic PAHs and has, therefore, been proposed to contribute significantly to 36 

### the carcinogenicity of a PAH mixture, even when pre sent in low concentrations (Petry, 1996). 37 

### Benzo[a]pyrene is also the best-studied PAH compoun d, with carcinogenicity bioassay data 38

### 29 DRAFT – DO NOT CITE OR QUOTE

available for several routes of exposure and a cons iderable number of studies on carcinogenic 1



### mode of action. 2 

### The laboratory animal database for benzo[a]pyrene i s robust. Benzo[a]pyrene has been 3



### shown to induce tumors at the site of administratio n and at distal sites in numerous studies. 4 

### Dose-response data for tumors are available for the oral, inhalation, and dermal routes of 5 

### administration in multiple species. There are meth odological limitiations associated with the 6 

### inhalation data (Thyssen et al., 1981), although po sitive findings in intratracheal instillation 7 

### studies support the observed positive response. Li mited dermal exposure studies with several 8 

### strains of mice also provide data on dose-related t umor incidences (Albert et al., 1991; 9 

### Warshawsky and Barkley, 1987; Habs et al., 1984, 19 80; Nesnow et al., 1983; Wynder et al., 10 

### 1957). 11 

### The animal carcinogenicity database for benzo[a]pyr ene includes several well-conducted 12 

### oral cancer bioassays. Kroese et al. (2001) conduc ted a well-designed gavage study of 13 

### benzo[a]pyrene carcinogenicity and found that benzo [a]pyrene induced tumors at multiple sites 14 

### in rats of both sexes, specifically in the liver, f orestomach, auditory canal, and oral cavity. In 15 

### another well-conducted study, using Ah-responsive B 6C3F 1 

### female mice exposed in the diet 16 

### (Beland and Culp, 1998; Culp et al., 1998), only po rtal-of-entry tumors were found, including 17 

### papillomas and/or carcinomas of the forestomach, es ophagus, tongue, and larynx. Earlier, Neal 18 

### and Rigdon conducted a number of related studies ev aluating the carcinogenicity of 19 

### benzo[a]pyrene in feed in Ah-responsive white Swiss mice (Rigdon and Neal, 1969, 1966; Neal 20 

### and Rigdon, 1967). These latter studies were not c onducted using standard, modern 21 

### toxicological methods and have limitations, includi ng: inconsistent dosing protocols; varying 22 

### ages of the animals; use of benzene as a solvent; s mall numbers of animals; and evaluation of 23 

### only a limited number of tissues. However, the Nea l and Rigdon studies provide useful dose24 

### response information on benzo[a]pyrene carcinogenic ity. Following oral administration via 25 

### feeding of benzo[a]pyrene, site-of-contact tumors, both papillomas and carcinomas, were 26 

### induced in the forestomach, esophagus, and larynx o f mice (Culp et al., 1998; Neal and Rigdon, 27 

### 1967) and rats (Brune et al., 1981). The results f ollowing inhalation, dermal, or oral exposure 28 

### are further supported by numerous mechanistic studi es or assays using infant mice, susceptible 29 

### transgenic strains, or Ah-receptor knockout mice. 30 

### Benzo[a]pyrene is a complete carcinogen and likely acts by initiating tumors through 31 

### direct DNA damage as well as by promoting tumor gro wth. Benzo[a]pyrene has been shown to 32 

### be mutagenic in multiple assay systems. Several mo des of carcinogenic action are possible. 33



### These include: 34

35 

### (1) Alteration of pathways regulating cell proliferatio n and survival (Tannheimer et al., 36 

### 1998); 37

38

### 30 DRAFT – DO NOT CITE OR QUOTE

(2) Inhibition of intracellular communication (Sharovsk aia et al., 2003; Blaha et al., 2002; 1 

### Rummel et al., 1999); 2

3 

### (3) Altered intracellular Ca 2+ 

### signaling (Tannheimer et al., 1998); 4

5 

### (4) Modulation of cell survival, cell proliferation, an d altered growth via generation of 6 

### oxidative stress and activation of oxidant stress s ignaling (Burdick et al., 2003; Miller 7 

### and Ramos, 2001); 8

9 

### (5) Altered apoptosis processes (Chen et al., 2003); 10

11 

### (6) Dysregulation of normal circulating hormone levels or activity affecting tumorigenesis in 12 

### reproductive tissues (Safe and Wormke, 2003; Archib ong et al., 2002) or the central 13 

### nervous system (Dasgupta and Lahiri, 1992); 14

15 

### (7) Disruption of cell cycle kinetics in breast cancer cells (Jeffy et al., 2002, 2000); and 16

17 

### (8) Disruption of DNA repair through alteration of RNA polymerase activity (Shah and 18 

### Bhattacharya, 1989). 19

20 

### Oral (dietary) carcinogenicity bioassays are availa ble that compare manufactured gas 21 

### plant (MGP) residue (Weyand et al., 1995) or coal t ar preparations (Culp et al., 1998; Gaylor et 22 

### al., 1998) with benzo[a]pyrene. In both cases, the re were significant differences in target organ 23 

### distribution of tumors between benzo[a]pyrene and c omplex mixtures of PAHs. Following 24 

### dietary administration, benzo[a]pyrene-induced tumo rs were observed primarily at the point of 25 

### contact (i.e., the forestomach), while MGP residue and coal tar produced tumors in the lung, 26 

### liver, forestomach, skin, and other organs. Tissue -specific differences in metabolic activation 27 

### and DNA binding of PAHs may contribute to the obser ved differences in target organ sensitivity 28 

### (Weyand and Wu, 1995; Culp and Beland, 1994). A re cent gavage study in rats (Kroese et al., 29 

### 2001) demonstrated that oral exposure to benzo[a]py rene could induce tumors at distal sites (i.e., 30 

### liver, auditory canal); however, no lung tumors wer e observed. The lung appears to be a 31 

### sensitive target organ for complex mixture carcinog enicity, but is insensitive to benzo[a]pyrene32 

### induced tumorigenicity via oral and dermal exposure s. The existing data limitations for other 33 

### PAHs, however, necessitate the use of benzo[a]pyren e as the only appropriate index chemical for 34



### PAHs. 35 

### In summary, benzo[a]pyrene is the most appropriate compound to use as an index 36 

### chemical for carcinogenic PAHs. It is well-studied , with a robust database of both bioassay data 37 

### and mode of action information. Benzo[a]pyrene is a complete carcinogen with both initiating 38 

### and promoting properties, is among the most potent PAH carcinogens, and is prevalent in many 39 

### complex environmental mixtures. No alternative ind ex chemical was identified from the list of 40 

### target PAHs. 41

42

### 31 DRAFT – DO NOT CITE OR QUOTE

2.4. SIMILARITIES IN MODE OF CARCINOGENIC ACTION F OR PAHs 1



### A common mode of action for chemicals is the basis for the assumption of dose additivity 2 

### that underlies the RPF approach (U.S. EPA, 1990). The carcinogenic mode of action for PAHs 3 

### has been extensively reviewed (Ramesh, 2004; CCME, 2003; Bostrom et al., 2002; Larsen and 4 

### Larsen, 1998; WHO, 1998; Muller et al., 1997; Sjogr en et al., 1996; ATSDR, 1995; Malcolm 5 

### and Dobson, 1994; U.S. EPA, 1990). The major key e vents that have been associated with PAH 6 

### carcinogenicity include: 7

8



### • Oxidative metabolism to reactive intermediates that covalently bind to DNA, RNA, and 9 

### proteins (benzo[a]pyrene metabolism is illustrated in Figure 2-2); 10

11



### • Formation of DNA adducts; 12

13



### • Tumor initiation due to mutations in cancer-related genes (e.g., tumor suppressor genes 14 

### or oncogenes); and 15

16



### • Tumor promotion related to cytotoxicity and formati on of reactive oxygen species, and 17 

### Ah receptor (AhR) affinity and upregulation of gene s related to biotransformation, 18 

### growth, and differentiation. 19

20

### 32 DRAFT – DO NOT CITE OR QUOTE

1
2

3 

### Source: Miller and Ramos (2001). 4

5 

### Figure 2-2. Metabolic pathways for benzo[a]pyrene. 6

### 33 DRAFT – DO NOT CITE OR QUOTE

Formation of reactive intermediates and DNA adducts 1 

### Each of the key events identified above is affected by the chemical structure of the 2



### individual PAH. At least three distinct molecular mechanisms have been proposed to explain the 3 

### tumor initiation process of PAHs (Xu et al., 2009; Jiang et al., 2007, 2005; Xue and 4 

### Warshawsky, 2005; Bolton et al., 2000; Penning et a l., 1999; Harvey, 1996; Cavalieri and 5 

### Rogan, 1995). These modes of action include the fo rmation of diol epoxides, radical cations, 6 

### and o-quinones (Figure 2-3). Diol epoxide formatio n leads to stable and unstable DNA adducts, 7 

### mainly at guanine and adenine, which can lead to mu tations in proto-oncogenes and tumor8 

### suppressor genes. Radical cation formation may lea d to the generation of unstable adducts at 9 

### guanine and adenine, leading to apurinic sites and mutation in HRAS . Orthoquinone formation 10 

### could lead to stable and unstable DNA adducts and g eneration of reactive oxygen species, 11 

### inducing mutations in P53. The evidence supporting the role of these reactive metabolites in 12



### tumor initiation includes a characterization of the specific DNA adducts arising from PAH 13 

### metabolism and observations of mutagenesis resultin g from direct exposure. Figure 2-3 14 

### illustrates the proposed key steps in the mode of a ction for PAH carcinogenesis. These include 15



### the interaction of reactive metabolites with DNA to form adducts, induction of depurination, 16 

### transversion mutations (e.g., GC→TA or AT→TA), and oxidative damage to DNA, and tumor 17 

### promotion mediated by AhR-mediated effects on gene regulation. 18

### 34 DRAFT – DO NOT CITE OR QUOTE

1

2

3 

### Figure 2-3. Overview of the proposed key events in the mode of action for 4 

### PAH carcinogenicity. 5

6 

### The formation of diol epoxides is a proposed key st ep in the most established mode of 7 

### action for PAH-induced carcinogenicity. Extensive studies of the metabolism of carcinogenic 8 

### PAHs suggest that bay-region and fjord-region diol epoxides are some of the ultimate reactive 9 

### metabolites of PAHs (Jerina et al., 1978; Jerina an d Lehr, 1977). These metabolites are 10 

### generally formed through cytochrome P450 (CYP) oxid ation to form epoxides and epoxide 11 

### hydrolase cleavage resulting in diol formation. CY P1A1 appears to be the primary isozyme 12 

### involved in diol epoxide formation; however, other isozymes may also contribute to PAH 13 

### metabolism (i.e., CYPIA2, CYP1B1, CYP3A4) (Bostrom et al., 2002; ATSDR, 1995). Non14 

### alternant PAHs, composed of fused benzenoid and fiv e-membered rings, may be metabolized 15 

### through other pathways resulting in the formation o f reactive intermediates that bind to DNA. 16 

### Classic bay- and fjord-region diol epoxides may be formed from these compounds; however, 17 

### epoxide formation at cyclopenta-ring structures has also been demonstrated to result in DNA 18 

### adduct formation (Bostrom et al., 2002). 19 

### Many studies have been performed to evaluate the fo rmation of DNA adducts following 20 

### in vivo or in vitro exposure to PAHs. Diol epoxide metabolites interact preferentially with the 21 PAH diol epoxide

radical cation

o

- quinone and
ROS DNA Adducts

DNA Adducts

DNA Adducts
and oxidative base damage Mutation ( transversion )

Mutation ( depurination )

Mutation ( depurination

, oxidative damage, and strand scission) Proliferation of initiated cells Neoplasm

Inflammatory

response

Cytotoxicity OH OH O

+ Exposure Metabolism

O O Initiation Pro motion Progression Key Events in the Mode of Action for PAH Carcinogen icity

Binding to Ah Receptor

Upregulation of genes related to biotransformation, growth, and differentiation

### 35 DRAFT – DO NOT CITE OR QUOTE

exocyclic amino groups of deoxyguanine and deoxyade nine (Geacintov et al., 1997; Jerina et al., 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 stereois omeric adducts arising from the interaction of 5 

### benzo[a]pyrene diol epoxide metabolites with the de oxyguanosine (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 fo llowing diol epoxide exposure (Bostrom 8 

### et al., 2002). 9

10 

### Source: Geacintov et al. (1997). 11

12 

### Figure 2-4. Structures of the four stereoisomeric adduct moieties, 13 

### anti -[BaP]-

N 2 

### -dG, derived from the trans - or cis -covalent binding of 14 

### (+)- anti

-BaP diol epoxide or (-)- anti -BaP diol epoxide to dG residues in DNA. 15

16



### Radical cation formation involves a one-electron ox idation that produces electrophilic 17 

### radical cation intermediates (Cavalieri and Rogan, 1995, 1992). Oxidation of this type can occur 18 

### by CYP or peroxidase enzymes (i.e., horseradish per oxidase, prostaglandin H synthetase). 19 

### O

H

O H

OH NH NH

N N

N O

R O H

O H

OH NH NH

N N

N O

R

O H

O H

OH NH NH

N N

N O

R O H

O H

OH NH NH

N N

N O

R 10S β ' α '

10R

10R 10S



### 10 R (+)- cis

- anti

-[BaP]- N

2 

### -dG 10 S (-)- cis

- anti

-[BaP]- N

2 

### -dG 10 S (+)- trans

- anti

-[BaP]- N

2 

### -dG 10 R

(-)- trans

- anti

-[BaP]- N

2 

### -dG

### 36 DRAFT – DO NOT CITE OR QUOTE

Radical cations can be further metabolized to pheno ls and quinones (Cavalieri et al., 1988a), or 1 

### they can form unstable adducts with DNA that ultima tely result in depurination (Cavalieri et al., 2 

### 2005, 1993; Rogan et al., 1993). Radical cations h ave been shown to play a major role in 3 

### formation of DNA adducts for several carcinogenic P AHs (e.g., 7,12-dimethylbenzanthracene, 4 

### benzo[a]pyrene, dibenzo[a,l]pyrene). The predomina nt depurinating adducts occur at the N-3 5 

### and N-7 positions of adenine and the C-8 and N-7 po sitions of guanine (Cavalieri and Rogan, 6 

### 1995; Li et al., 1995). Figure 2-5 illustrates thr ee depurinating adducts of benzo[a]pyrene 7 

### formed by one-electron oxidation. Abasic sites res ulting from base depurination undergo error8 

### prone excision repair to induce mutations. In the case of DBalP-treated mouse skin, repair error 9 

### from abasic sites resulted in H-ras oncogene mutati ons that underwent rapid clonal expansion 10 

### and regression (Chakravarti et al., 2000). H-ras m utations in mouse skin papillomas also 11 

### corresponded to adenine and guanine depurinating ad ducts resulting from exposure to DBalP, 12 

### 7,12-dimethyl-benz[a]anthracene, benzo[a]pyrene, an d benzo[a]pyrene-7,8-dihydrodiol 13 

### (Chakravarti et al., 2008). 14

15

16 

### Source. Cavalieri and Rogan (1995). 17

18 

### Figure 2-5. Depurinating adducts of benzo[a]pyrene formed by one-electron 19 

### oxidation. 20

21 

### o-Quinone metabolites of PAHs are formed by enzymat ic dehydrogenation of 22 

### dihydrodiols (Bolton et al., 2000; Penning et al., 1999; Harvey, 1996; ATSDR, 1995). 23 

### Dihydrodiol dehydrogenase enzymes are members of th e α-keto reductase gene superfamily. 24 

### o-Quinone metabolites are potent cytotoxins, are we akly mutagenic, and are capable of 25 

### producing a broad spectrum of DNA damage. These me tabolites can interact directly with DNA 26 

### and also result in production of reactive oxygen sp ecies (i.e., hydroxyl and superoxide radicals) 27 

### that may produce further cytotoxicity and DNA damag e. The DNA damage caused by 28 

### o-quinones may include the formation of stable addu cts (Balu et al., 2006), N-7 depurinating 29 

### adducts (McCoull et al., 1999), oxidative base dama ge (i.e., 8-oxo-2’-dG or 8-oxo-dG) (Park et 30 N

H

N N

H N

N H 2 O

N H

N N

N

N H 2 O

N

N N N NH 2

BaP-6-C8-guanine BaP-6-N7-guanine BaP-6-N7-adenine

### 37 DRAFT – DO NOT CITE OR QUOTE

al., 2006, 2005), and strand scission (Flowers-Gear y et al., 1997). The reactive oxygen species 1 

### generated by the o-quinone of benzo[a]pyrene and ot her PAH o-quinones have been shown to 2 

### induce mutation in the p53 tumor suppressor gene (P ark et al., 2008; Shen et al., 2006; Yu et al., 3 

### 2002). Figure 2-6 illustrates the spectrum of DNA adducts associated with PAH o-quinones. 4

5

6 

### Source: Bolton et al. (2000). 7

8 

### Figure 2-6. Spectrum of DNA adducts anticipated wi th PAH o-quinones. 9

10 

### The cytotoxicity of o-quinone metabolites may also contribute to tumor promotion via 11 

### inflammatory responses leading to cell proliferatio n (Burdick et al., 2003). 12

13 

### Genotoxicity and mutagenicity 14 

### The genotoxicity and mutagenicity of PAHs have been demonstrated in various bacterial 15 

### and mammalian assays (see Section 4.3.2 below) (rev iewed in WHO, 1998; ATSDR, 1995). 16 

### Mutagenesis of PAHs in the Ames assay ( Salmonella typhimurium ) as well as other bacterial 17 N

H N

N

N O OPO 3 R

N H 2

O R 3 OPO

N H N

N

N O OPO 3 R

N H 2

O R 3 OPO

O O O

O O N H

N N

N O

N H 2

OPO 3 R RO 3 PO OH

O N H

N N

N O

N H 2

OPO 3 R RO 3 PO

HO

O B

O

OH OPO 3 R RO 3 PO

B

O O N H

N N

N O

N H 2

OPO 3 R OH

RO 3 PO N H N

N

N

O N H 2

O

O NH

O O N H N N

N O

O o-Quinone adducts ROS modifications

H

base propenals 8'-oxo-dG base pair mismatch G to T transversions depurinating adducts apurinic sites G to T transversions stable adducts translesional synthesis G to T transversions RO 3 PO

RO 3 PO

### 38 DRAFT – DO NOT CITE OR QUOTE

assays requires the presence of a mammalian metabol ic enzyme system. In most cases, this is 1 

### supplied by postmitochondrial supernatant (S9) from the liver of rodents treated with an enzyme 2 

### inducer. Mammalian cell mutagenesis in Chinese ham ster V79 cells and mouse lymphoma 3 

### L5178Y cells also requires metabolic activation in the form of a rodent S9 mix or co-cultivation 4 

### with metabolically active rodent cells (i.e., cell- mediated assay). Several studies have noted a 5 

### correlation between mutagenic potency and tumor ini tiation potency in the 2-stage dermal 6 

### carcinogenicity assay for multiple PAH compounds (L aVoie et al., 1985, 1979; Raveh et al., 7 

### 1982). 8

9 

### Tumor promotion and the AhR 10 

### The ability of certain PAHs to act as tumor promote rs as well as initiators may increase 11 

### their carcinogenic potency (Andrews et al., 1978). The promotional effects of PAHs appear to 12 

### be related to AhR affinity and the upregulation of genes related to biotransformation (i.e., 13 

### induction of CYP1A1), growth, and differentiation ( Bostrom et al., 2002). Figure 2-7 illustrates 14 

### the function of the AhR and depicts the genes regul ated by this receptor as belonging to two 15 

### major functional groups (i.e., induction of metabol ism or regulation cell differentiation and 16 

### proliferation). PAHs bind to the cytosolic AhR in complex with heat shock protein 90 (Hsp90). 17 

### The ligand-bound receptor is then transported to nu cleus in complex with the AhR nuclear 18 

### translocator protein (ARNT). The AhR complex inter acts with the Ah responsive elements of 19 

### the DNA (AhRE DNA 

### ) to increase the transcription of proteins associa ted with induction of 20 

### metabolism and regulation of cell differentiation a nd proliferation. 21

22

### 39 DRAFT – DO NOT CITE OR QUOTE AHR AHR Hsp90

Hsp90

AHR Hsp90

Hsp90 ARNT

ARNT Hsp90

Hsp90

AHR

ARNT AHRE DNA PAH

nucleus Enhanced specific mRNA production

Increased synthesis of PAH metabolizing
enzymes Increased synthesis of proteins that regulate cell differentiation and proliferation

1 

### AhRAhRSource: Okey et al. (1994). 2

3 

### Figure 2-7. Interaction of PAHs with the Ah recept or – regulation of genes 4 

### related to induction of metabolism and cell differe ntiation and proliferation. 5

6 

### In general, it has been demonstrated that fjord-reg ion PAHs are strong mutagens and 7 

### carcinogens, but have a low binding affinity to the AhR. Conversely bay-region PAHs possess a 8 

### greater affinity for AhR binding, and are better tu mor promoters in carcinogenicity bioassays 9 

### (Bostrom et al., 2002). CYP1A1 induction by PAHs i s considered to contribute to tumorigenesis 10 

### by increasing the production of DNA-reactive metabo lites (Ayrton et al., 1990). However, 11



### several recent studies indicate that CYP1A1 inducti on potency does not correlate well with 12 

### carcinogenic potency. These studies compared CYP1A 1 induction potency for several PAHs 13 

### using assays to measure ethoxyresorufin O-deethylas e (EROD) activity, CYP1A1 protein, and 14 

### mRNA levels, or chemical-activated luciferase repor ter gene expression (Bosveld et al., 2002; 15 

### Machala et al., 2001; Bols et al., 1999; Till et al ., 1999; Willett et al., 1997). 16

17 

### Tumor promotion and cytotoxicity 18 

### PAHs are metabolized to o-quinones, which are cyto toxic and can generate reactive 19 

### oxygen species (Bolton et al., 2000; Penning, 1999) . PAH o-quinones reduce the viability and 20 

### survival of rat and human hepatoma cells (Flowers-G eary et al., 1996, 1993). Inflammatory 21

### 40 DRAFT – DO NOT CITE OR QUOTE

responses to cytotoxicity may contribute to the tum or promotion process. For example, 1 

### benzo[a]pyrene quinones (1,6-, 3,6-, and 6,12-benzo [a]pyrene-quinone) generated reactive 2 

### oxygen species and increased cell proliferation by enhancing the epidermal growth factor 3 

### receptor pathway in cultured breast epithelial cell s (Burdick et al., 2003). Dermal exposure of 4 

### mice to DBalP and dimethyl-benz[a]anthracene result ed in an inflammatory response that was 5 

### correlated with epidermal hyperplasia and skin tumo r promotion (Casale et al., 2000, 1997). The 6 

### extent of epidermal hyperplasia was correlated with the cytokine mRNA response in lymph 7 

### nodes and skin of treated mice (Casale et al., 2000 ). 8

9 

### Genetic targets and tumor formation 10 

### DNA adducts and oncogenes/tumor suppressor gene mu tations have been demonstrated 11 

### in tumor tissue from humans and laboratory animals. DeMarini et al. (2001) demonstrated 12 

### mutations in the p53 tumor suppressor gene and the K-ras oncogene in the lung tumors of 13 

### nonsmokers, whose tumors were associated with expos ure to smoky coal. Lung tumors were 14 

### obtained from 24 nonsmoking women from China (age 3 0–63, mean age 48.5 + 8.8 years) who 15 

### used smoky coal in their homes without chimneys. B ronchioloalveolar adenocarcinoma and 16



### acinar adenocarcinoma were observed in 54 and 46% o f the women studied, respectively. The 17 

### observed mutations in lung tumors were primarily G→ T transversions at either K-ras or p53. 18 

### Mutation hotspots in the lung tumors examined corre sponded with hot spots for PAH adducts 19 

### (codon 154), cigarette smoke associated mutations ( codon 249), and both of these events 20 

### together (codon 273). The mutation spectrum was de scribed as unique and consistent with 21 

### exposure to PAHs in the absence of cigarette smoke. 22 

### Mutations in the K-ras, H-ras, and p53 genes were a ssessed in forestomach tumors 23 

### (n = 31) of mice fed benzo[a]pyrene in the diet (0, 5, 25, or 100 ppm) for 2 years (Culp et al., 24 

### 2000). Forestomach tumors had K-ras mutations (68% of tumors), which were G→T or 25 

### C transversions in codon 12 or 13. H-ras (codon 13 ) and p53 mutations characterized as G→T 26



### or C transversions were also each found in 10% of f orestomach tumors. [ 32 

### P]-postlabeling of 27 

### forestomach DNA of benzo[a]pyrene-treated mice reve aled one major adduct characterized as 28



### dG-N 2 

### -BPDE. There was a linear relationship between the amount of benzo[a]pyrene consumed 29



### and the concentration of dG-N 2 

### -BPDE in the forestomach of mice. For benzo[a]pyre ne, 30 

### forestomach tumor incidence increased sharply with adduct concentrations between 50 and 31 

### 140 fmol/mg DNA and in coal-tar fed mice. Tumor in cidence increased sharply with 32



### dG-N 2 

### -BPDE adduct levels between 20 and 60 fmol/mg DNA. The same levels of adduct were 33 

### present in lung and liver of benzo[a]pyrene-treated mice, although only the forestomach 34 

### exhibited benzo[a]pyrene-induced tumors (Goldstein et al., 1998). The presence of adducts in 35 

### tumor-free tissue suggests that DNA adduct levels a lone are not necessarily predictors of tumor 36



### outcome. 37

### 41 DRAFT – DO NOT CITE OR QUOTE

A series of experiments designed to evaluate the me chanistic relationship between PAH 1 

### DNA adducts, oncogene mutations, and lung tumorigen esis were performed in the A/J mouse 2 

### lung model (Nesnow et al., 1998a, 1996, 1995; Mass et al., 1993). Tumorigenic potency in the 3 

### lung of A/J mice varied over 2 orders of magnitude following a single intraperitoneal injection of 4 

### seven PAHs of varying structure (benzo[a]pyrene, be nzo[b]fluoranthene, benz[j]aceanthrylene, 5 

### dibenz[a,h]anthracene, dibenzo[a,l]pyrene, cyclopen ta[c,d]pyrene, and 5-methylchrysene). 6 

### When considering the non-alkylated PAHs, the number of lung adenomas per mouse was highest 7 

### for benz[j]aceanthrylene and cyclopenta[c,d]pyrene, each of which contain a pentacyclic ring 8 

### feature. The major DNA adducts identified in the m ouse lung included: 9

10 

### (1) Bay region diol epoxide adducts for benzo[a]pyrene, dibenz[a,h]anthracene, and 11 

### 5-methylcholanthrene; 12

13 

### (2) Phenolic diol epoxide adducts for benzo[b]fluoranth ene; 14

15 

### (3) Cyclopenta-ring adducts for cyclopenta[c,d]pyrene a nd benz[j]aceanthrylene; 16

17 

### (4) Bisdihydrodiol epoxide adducts for dibenz[a,h]anthr acene; and 18

19 

### (5) Fjord-region diol epoxide adducts for dibenzo[a,l]p yrene (Nesnow et al., 1998a, 1996, 20 

### 1995; Mass et al., 1993). 21

22 

### Guanine adducts were most common for all PAHs; howe ver, adenine adducts were also 23 

### demonstrated for dibenzo[a,l]pyrene and benz[j]acea nthrylene. Quantitative analysis of DNA 24 

### adducts by [ 32 

### P]-postlabeling illustrates the importance of measu ring DNA adduct levels over 25 

### time. A time-integrated DNA adduct level (TIDAL) w as linearly related to the dose of a 26 

### particular PAH. The relationship of TIDAL level to tumor formation was similar for PAHs that 27 

### produce different types of adducts and different mu tations in the Ki-ras oncogene. This suggests 28 

### that the probability of tumor formation for these P AHs may be related to the extent of overall 29 

### DNA damage and repair rather than the formation of specific adduct at specific sites. 30 

### The DNA sequence analysis of Ki-ras mutations in lu ng adenomas at codons 12 and 61 31 

### was generally consistent with the DNA adduct data i n that PAHs that produced guanine adducts 32 

### also produced Ki-ras guanine mutations (Nesnow et a l., 1998a, 1996, 1995; Mass et al., 1993). 33 

### Cyclopenta[c,d]pyrene, benz[j]aceanthrylene, and 5- methylchrysene produced large numbers of 34 

### adenomas per mouse (>90) and also produced a large proportion of tumors with CGT mutations 35 

### at Ki-ras codon 12. Cyclopenta-ring adduct formati on by cyclopenta[c,d]pyrene and 36 

### benz[j]aceanthrylene was correlated with the format ion of GGT→CGT mutations at Ki-ras 37 

### codon 12. The primary mutation type for benzo[a]py rene, benzo[b]fluoranthene, and 38 

### dibenzo[a,l]pyrene was the GGT→TGT mutation, which is associated with the formation of diol 39 

### epoxide guanine adducts. Dibenz[a,h]anthracene did not induce mutations in Ki-ras codons 12 40

### 42 DRAFT – DO NOT CITE OR QUOTE

or 61; however, diol epoxide guanine adducts and lu ng adenomas in A/J mice were observed. 1 

### This suggests that a different genetic target may b e involved in carcinogenicity of this 2 

### compound. 3 

### H-ras mutations were studied in skin papillomas of SENCAR mice resulting from dermal 4 

### initiation by benzo[a]pyrene or benzo[a]pyrene-7,8- dihydrodiol (400 nmol) followed by 12-O5 

### tetra-decanoylphorbol-acetate (TPA) promotion (Chak ravarti et al., 2008). PCR amplification of 6



### the H- ras gene and sequencing revealed that codon 13 (GGC to GTC) and codon 61 (CAA to 7 

### CTA) mutations in papillomas corresponded to the re lative levels of depurinating adducts of 8 

### guanine and adenine, despite the formation of signi ficant amounts of stable DNA adducts. 9 

### Other studies also suggest that multiple genetic ta rgets may be involved in PAH 10 

### mutagenicity and carcinogenicity (Conney et al., 20 01; Smith et al., 2000). Smith et al. (2000) 11 

### indicated that diol epoxide adducts and mutations w ere observed in the p53 tumor suppressor 12 

### gene following in vitro exposure of cultured human bronchial epithelial cells to metabolites of 13 

### benzo[a]pyrene, chrysene, benzo[c]phenanthrene, and benzo[g]chrysene. PAH adducts and 14 

### corresponding mutations preferentially formed at lu ng mutational hot spots (codons 154, 157, 15 

### 158, 245, 248, and 273), suggesting that PAHs may c ontribute to the mutation spectrum 16 

### observed in human lung cancer. Conney et al. (2001 ) provided evidence that dose-dependent 17 

### differences may exist for the mutation spectra seen in PAH-induced tumors. Skin papillomas 18 

### induced by benzo[a]pyrene in female mice were exami ned for mutations in the c-Ha-ras proto19 

### oncogene. The major difference between high- and l ow-dose groups was mutations at exon 2 of 20 

### the c-Ha-ras gene, with the proportion of AT base p air mutations higher in the low-dose group. 21 

### Dose-dependent changes in mutation profile were als o evident in Chinese hamster V79 cells 22 

### exposed to the diol epoxides of benzo[a]pyrene and benzo[c]phenanthrene (i.e., the proportion of 23 

### AT mutations decreased with increasing concentratio n). 24 

### In conclusion, the available data indicate that the re may be multiple mechanisms of 25 

### PAH-induced carcinogenicity. However, a common mod e of action involving oxidative 26 

### metabolism to reactive intermediates, DNA adduct fo rmation, and subsequent mutagenic events 27 

### is considered to be the primary mode of carcinogeni c action. For these reasons, the use of a RPF 28 

### approach to estimate cancer risk associated with PA H exposure is considered appropriate. The 29



### uncertainties and limitations related to the mode o f action assumption for PAH-induced cancer 30



### are further discussed in Section 8.5. 31

32



### 2.5. STRUCTURAL ALERTS FOR PAH CARCINOGENESIS 33 

### The carcinogenic activity of PAH compounds is influ enced by specific structural 34 

### features. For example, alternant PAHs having four or more benzene rings exhibit greater 35 

### carcinogenic potency than PAHs with two or three be nzene rings (Bostrom et al., 2002). The 36 

### carcinogenic activity of PAHs is also related to th e specific arrangement of the benzene rings. 37 

### As described in Section 2.4, PAHs that form bay- an d fjord-region diol or dihydrodiol epoxides 38

### 43 DRAFT – DO NOT CITE OR QUOTE

are more potent carcinogens compared with linear PA Hs that lack this structural feature 1 

### (Bostrum et al., 2002). These metabolites are resi stant to detoxification due to stereochemical 2 

### effects and, consequently, are more likely to be mu tagenic and cause cancer (Flesher et al., 1976; 3 

### Buterin et al., 2000; Chang et al., 1981; Buening e t al., 1979; MacLeod et al., 1979). 4 

### Dihydrodiol epoxides formed at other positions on t he PAH molecule (i.e., not the bay or fjord5 

### regions) are more accessible to glutathione transfe rase detoxification and are less potent 6 

### mutagens and carcinogens (Flesher et al., 1976; Mac Leod et al., 1979). Nonalternant PAHs 7 

### containing fused benzenoid and five-membered rings, can also be metabolized to bay- and fjord8 

### region diol epoxides (Bostrum et al., 2002); howeve r, epoxide formation at the cyclopenta-ring 9 

### structure may also contribute to carcinogenicity (B ostrum et al., 2002; Nyholm et al., 1996). 10 

### PAHs with at least four rings and a classic bay- or fjord-region (formed entirely by 11 

### benzene rings; see Figure 2-1) may be characterized as containing structural alerts for 12 

### carcinogenesis. However, this structural character ization is likely to be overly simplistic and 13 

### other features may be important to carcinogenesis. Recent studies have applied quantitative 14 

### structure activity relationship (QSAR) methods to e valuate the relationship between specific 15



### PAH structural features and mechanistic events rela ted to carcinogenesis (Bruce et al., 2008; 16 

### Vijayalakshmi et al., 2008). 17

18



### 2.6. SIMILARITIES IN RELATIVE POTENCY ACROSS ENDPO INTS 19



### Studies that have evaluated the association between cancer-related endpoints and 20 

### tumorigencity of PAHs are briefly summarized below. 21 

### Several studies have been performed that compare th e bacterial or mammalian cell 22 

### mutagenicity of various PAHs with tumor initiating activity or complete carcinogenesis (LaVoie 23 

### et al., 1985, 1981, 1979; Raveh et al., 1982; Andre ws et al., 1978). In general, mutagenicity 24 

### appears to correlate best with tumor initiation. C omplete carcinogenicity is not well-predicted by 25 

### positive findings in short-term mutagenicity assays . Andrews et al. (1978) tested 24 PAHs for 26 

### bacterial mutagenicity in the Ames test and compare d these findings to evidence of 27 

### carcinogenicity (parent and metabolites) from previ ously published studies. Positive findings of 28 

### both mutagenicity and carcinogenicity were only rep orted for 14 of the 24 PAHs evaluated. 29 

### Eight of the 10 remaining PAHs were found to be mut agenic in the Ames assay, but were not 30 

### carcinogenic in animal studies. LaVoie et al. (197 9) compared the mutagenicity, tumor-initiating 31 

### activity, and complete carcinogenicity of several s eries of structurally related PAHs. Tumor32 

### initiating activity was found to correspond with co mplete carcinogenicity. Quantitation of 33 

### mutagenicity in the Ames assay for structurally rel ated PAHs failed to provide a reliable 34 

### indication of tumor-initiating activity or complete carcinogenicity. In addition, mutagenicity 35 

### results could not be used to predict which PAHs wou ld be noncarcinogenic. Many PAHs were 36 

### active mutagens, but were not shown to be carcinoge nic. Studies using methylated derivatives of 37 

### anthracene demonstrated a correlation between mutag enicity of specific metabolites and tumor 38

### 44 DRAFT – DO NOT CITE OR QUOTE

initiating activity in mouse skin (LaVoie et al., 1 985). Raveh et al. (1982) reported that the 1 

### mutagenic response to PAHs in Chinese hamster V79 c ells was similar to the skin tumor 2 

### initiating activity observed in SENCAR mice. Benzo [a]pyrene was demonstrated to be a more 3 

### potent mutagen and skin tumor initiator than cyclop enta[c,d]pyrene. 4 

### Sjogren et al. (1996) performed a multivariate anal ysis to evaluate the relevance of 5 

### different biological assays to the tumor initiating and promoting properties of PAHs. This 6 

### analysis considered carcinogenicity (strength of ev idence), bacterial mutagenicity, inhibition or 7 

### enhancement of bacterial mutagenicity, AhR affinity , and enzyme induction. A principle 8 

### components analysis indicated that bacterial mutage nicity data were poorly correlated with 9 

### cancer bioassay data (regression coefficients range d from -0.1 to 0.15). Variables describing 10 

### AhR affinity showed the highest correlation with ca ncer data. A partial least squares regression 11 

### analysis showed that all of the AhR affinity variab les analyzed in the report were statistically 12 

### relevant to describe cancer potency (regression coe fficients ranged from 0.1 to 0.25). The 13 

### structural requirements for AhR affinity are the sa me as those required for enzyme induction and 14 

### bioactivation of PAHs. This analysis suggests that different chemical species (i.e., parent 15 

### compounds or metabolites) may be responsible for th e initiating and promoting properties of 16 

### PAHs. Sjogren et al. (1996) proposed that mutageni city reflects the cancer initiation potency, 17 

### which may be more relevant at lower environmental e xposure levels, and AhR affinity reflects 18 

### the promoting effect of some PAHs that occur primar ily at high doses in animal bioassays. 19 

### Bostrom et al. (2002) suggested that the ability fo r a PAH to act as a promoter strongly increases 20 

### its carcinogenic potency in animal studies. Howeve r, highly mutagenic fjord-region PAHs are 21 

### potent carcinogens, despite a lower AhR affinity (B ostrom et al., 2002; Jerina et al., 1991). 22 

### CYP1A1 induction by PAHs is considered to contribut e to tumorigenesis by increasing 23 

### the production of DNA-reactive metabolites (Ayrton et al., 1990). However, CYP1A1 induction 24 

### potency alone does not appear to correlate well wit h carcinogenic potency of PAHs. EROD 25 

### activity was evaluated as a measure of CYP1A1 induc tion in rat hepatocytes (Bosveld et al., 26 

### 2002; Till et al., 1999; Willett et al., 1997) and trout liver cells (Bols et al., 1999). Till et al. 27 

### (1999) additionally measured levels of CYP1A1 prote in and mRNA. Machala et al. (2001) 28 

### measured PAH activation of the AhR using a chemical -activated luciferase reporter gene assay. 29 

### Comparable results were observed across studies and benzo[k]fluoranthene was consistently 30 

### demonstrated to be the most potent inducer of CYP1A 1. Chrysene, benzo[b]fluoranthene, 31 

### dibenz[a,h]anthracene, and indeno[1,2,3-c,d]pyrene were also demonstrated to be more potent 32 

### inducers of CYP1A1 than benzo[a]pyrene. However, m ost of these PAH compounds (except 33 

### dibenz[a,h]anthracene) are considerably less potent as carcinogens in animal bioassays. 34 

### Ross et al. (1995) evaluated the relationship betwe en TIDAL values for DNA adduct 35 

### formation and lung adenoma formation in A/J mice. The TIDAL value vs. tumor relationship 36 

### was similar for five different PAHs, suggesting a c orrelation between adduct levels and tumor 37 

### formation (regression analysis was not performed). As described above, the relationship of 38

### 45 DRAFT – DO NOT CITE OR QUOTE

TIDAL level to tumor formation was similar for PAHs that produce different types of adducts 1 

### and different mutations in the Ki-ras oncogene, sug gesting that the probability of tumor 2 

### formation may be related to the extent of overall D NA damage and repair (Nesnow et al., 1998a, 3 

### 1996, 1995; Mass et al., 1993). 4 

### To summarize, various cancer-related endpoints have been associated with PAH 5 

### carcinogenicity. Tumor initiation ability and AhR affinity were shown to correspond well with 6 

### complete carcinogenicity, while bacterial mutagenes is was not highly correlated with tumor 7 

### formation (Sjogren et al., 1996; Lavoie et al., 197 9). DNA adduct formation corresponded with 8 

### lung adenoma formation in A/J mice for several PAHs (Sjogren et al., 1996; Ross et al., 1995; 9 

### LaVoie et al., 1979). The development of RPFs in t his analysis considered both tumorigenicity 10 

### and cancer-related endpoints (e.g., mutagenicity, c lastogenicity, morphological transformation). 11

12



### 2.7. SIMILARITIES IN RELATIVE POTENCY ESTIMATES AC ROSS SPECIES AND 13



### EXPOSURE ROUTES 14 

### Available studies suggest that the potency of indiv idual PAHs is generally consistent 15 

### across species and study protocols. The consistenc y of potency estimates based on in vivo 16 

### tumorigenicity studies conducted using different st udy protocols and exposure routes in varying 17 

### species/strains of test animals is summarized below

. 18 

### Nisbet and LaGoy (1992) and Clement Associates (198 8) reported that RPFs for PAHs 19 

### are reasonably consistent across different study pr otocols using varying species/strains of 20 

### laboratory animals. RPF estimates were calculated in multiple test systems including mouse skin 21 

### complete carcinogenesis studies, mouse skin tumor i nitiation studies, studies in rat lung 22 

### (implantation), other rat studies (intrapulmonary i njection, subcutaneous injection), and newborn 23 

### mouse (intraperitoneal injection). The RPF estimat es for specific PAHs calculated from 24 

### different assay systems varied by less than an orde r of magnitude. The relative potency of 25 

### individual PAHs to benzo[a]pyrene was also shown to be very similar when based on data in 26 

### different strains of mice using different mouse tum or initiation models (Slaga and Fisher, 1983). 27 

### Muller et al. (1997) compared the relative potency of benzo[a]pyrene and 3-methylcholanthrene 28 

### from data generated in three species (rat, mouse, a nd hamster). Similar RPF values (i.e., within a 29 

### factor of 2) were derived for oral exposures in mic e, rats, and hamsters. In their comparison 30 

### across different exposure routes (oral, respiratory , and dermal), Muller et al. (1997) reported 31 

### similar relative potencies for benzo[a]pyrene and 3 -methylcholanthrene (within a factor of 2) for 32 

### data from rats exposed via oral and respiratory rou tes, and for mice exposed via oral and dermal 33 

### routes. The relative potency for respiratory expos ure in mice was an order of magnitude lower 34 

### than relative potencies for the other two exposure routes. 35 

### Schneider et al. (2002) performed a more recent eva luation of the impact of exposure 36 

### route on the determination of RPFs. Potency ratios were calculated for several carcinogenicity 37 

### bioassays by dividing the carcinogenic potency of a PAH mixture by the carcinogenic potency of 38

### 46 DRAFT – DO NOT CITE OR QUOTE

benzo[a]pyrene as a single substance. The potency ratios were observed to vary by exposure 1 

### route and target organ. For example, potency ratio s associated with forestomach tumors from 2 

### oral exposure ranged from 0.7 to 1.2 (i.e., the pot encies of the PAH mixtures and benzo[a]pyrene 3 

### to induce forestomach tumors were approximately equ al). This suggested that these tumors may 4 

### be attributable to the benzo[a]pyrene content of th e mixture. Potency ratios for skin tumor 5 

### production from dermal exposure ranged from 2 to 11 , whereas RPFs calculated for lung tumors 6 

### from oral exposure, pulmonary implantation, or inha lation were greater than 20. These results 7 

### suggested that the benzo[a]pyrene content of PAH mi xtures may be only slightly responsible for 8 

### lung and dermal carcinogenicity. Schneider et al. (2002) suggested that RPF estimates should be 9 

### derived separately for oral, dermal, and inhalation exposure using studies with the relevant 10 

### exposure pathway. 11 

### To summarize, there is some consistency within the in vivo carcinogenicity database for 12 

### relative potency estimates derived from different s pecies and strains exposed by various routes, 13 

### although this is an area for which further research is needed. However, Schneider et al. (2002) 14 

### have cautioned that potency ratios appear to cluste r by exposure route and target organ and have 15 

### suggested that route-specific RPFs be developed. T here is also some concern regarding the use 16 

### of benzo[a]pyrene as an index chemical to estimate lung cancer from PAH mixtures, considering 17 

### that the lung is relatively insensitive to benzo[a] pyrene-induced tumorigenicity following oral 18 

### exposure (Gaylor et al., 1998). Section 8.6 provid es a comparison of RPF values calculated in 19 

### this report, using bioassay data from different exp osure routes and study designs. RPF values 20 

### were comparable across most exposure routes, with t he exception of the newborn mouse 21 

### intraperitoneal injection studies. 22

23



### 2.8. DOSE ADDITIVITY OF PAHs IN COMBINED EXPOSURES 24 

### Use of the RPF approach assumes that doses of compo nent chemicals that act in a similar 25 

### manner can be added together, after scaling the pot encies relative to the index chemical, and that 26 

### interaction effects do not occur (U.S. EPA, 2000, 1 986). The level of confidence in the RPF 27 

### approach is increased if additivity can be demonstr ated experimentally, even with simple 28 

### mixtures. For PAHs, the assumption of additivity c annot be confirmed or refuted based on the 29 

### available experimental data. It appears that risks may be generally additive for complex 30 

### mixtures, while binary mixtures can exhibit antagon ism, synergism, or additivity as discussed 31



### below. 32 

### The complexity of potential interactions for tumori genesis of binary mixtures of PAHs is 33



### illustrated in Table 2-2. The nature of the intera ction varies with the PAHs evaluated and the 34 

### study conditions (e.g., vehicle used, dose selectio n, study method). Many studies were designed 35



### to evaluate the combined administration of a known carcinogen with either a weak carcinogen or 36 

### a noncarcinogenic PAH. The true nature of the inte raction (i.e., additive, synergistic, or 37 

### antagonistic) can be difficult to determine in stud ies wherein the tumorigenic response is not 38

### 47 DRAFT – DO NOT CITE OR QUOTE

measured for both PAHs given alone and in combinati on. These studies can distinguish between 1 

### an enhanced or cocarcinogenic response and an inhib itory response, but a further classification 2



### cannot be made. The interactions described as coca rcinogenic in Table 2-2 may be either 3 

### additive or synergistic in nature. 4

5 

### Table 2-2. Studies of binary mixtures of PAHs and tumorigenicity

Reference Endpoint Findings Net effect Cavalieri et al., 1983 Mouse skin

carcinogenicity BaP and CPcdP given together resulted in 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. S

DiGiovanni et al., 1982 Skin tumor initiation in

mice BeP increased BaP tumor initiation (30%↑), inhibited tumor initiation by DMBA (84%↓) and DBahA (48%↓) and produced no change in combination with 3-MC; DBacA inhibited tumor initiation by DMBA (92%↓), DBahA (39%↓), and 3-MC (61%↓) and produced no change in combination with BaP. Co, I

Falk et al., 1964 Sarcoma induction in

mice by subcutaneous injection PH inhibited tumor response of DBahA in ethyl laurate vehicle (approximately 30%↓, estimated from graph); tumor response was enhanced in triethylene glycol vehicle (approximately 50%↑ to 100% tumor-bearing animals, estimated from graph). Co, I

Lavik et al., 1942 Mouse skin tumors 3-MC and BaP, DBahA, or BaA essentially additive. A

Pfeiffer, 1973 Sarcoma induction in

mice by subcutaneous injection BaP and DBahA less than additive; tumor response for combined treatment was within 10% of DBahA

response. I

Slaga et al., 1979 Skin tumor initiation in

mice 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). Co, I

Steiner, 1955; Steiner and Falk, 1951 Sarcoma induction in

mice by subcutaneous injection DBahA and 3-MC in combination roughly additive; BaA and CH in combination resulted in synergistic effect (9%↑ above additive response); BaA and DBahA in combination resulted in inhibition (48%↓ below additive response). A, S, and I

Van Duuren and Goldschmidt, 1976; Goldschmidt et al.,
1973 Mouse skin

carcinogenicity BeP, BghiP, Pyr, or FA and BaP increased tumors over BaP alone (>50% increase in incidence, also ↑multiplicity); no tumors were observed for PAHs without BaP. S

Van Duuren et al.,
1973 Mouse skin

carcinogenicity BaP and BghiP had cocarcinogenic effect (23%↑ over BaP response alone). Co

Warshawsky et al.,
1993 Mouse skin

carcinogenicity Nontumorigenic dose of BaP increased tumor incidence produced by CH (16%↑), anthracene (8%↑), and FA (8%↑). S

3-MC = 3-methylchloanthrene; A = additive; Co = coc arcinogenic (enhanced tumorigenicity, study design does not allow for determination of A or S); DMBA = 7,12-dim ethyl-benz[a]anthracene; I = inhibitory; S = synerg istic

### 48 DRAFT – DO NOT CITE OR QUOTE

1 

### Slooff et al. (1989) reviewed the available data ad dressing the carcinogenicity of 2



### individual PAHs and in combination. It was conclud ed that a generally additive effect was 3 

### observed following administration of more than two different PAHs in weight ratios similar to 4 

### those occurring in ambient air or in various emissi ons. Combinations of only two PAHs 5 

### produced either additive, synergistic, or inhibitor y effects. The complexity of the interaction 6 

### among single PAH compounds is thought to be related to effects on metabolic enzyme systems 7 

### including induction processes and competitive inhib ition. The generlaly additive response noted 8 

### for a more complex mixture may reflect the balance between inhibitory and synergistic 9



### processes. 10 

### Additivity has been observed in carcinogenicity stu dies of complex mixtures of PAHs. 11 

### Schmähl et al. (1977) evaluated the production of s kin tumors following combined dermal 12



### treatment with 11 PAHs found as constituents of aut omobile exhaust. Tumor findings were 13 

### presented separately for two groups of PAHs. High potency carcinogens (Group 1) included 14 

### benzo[a]pyrene, dibenz[a,h]anthracene, benz[a]anthr acene, and benzo[b]fluoranthene. Lower 15 

### potency PAHs (Group 2) included anthracene, benzo[e ]pyrene, benzo[g,h,i]perylene, chrysene, 16 

### fluoranthene, phenanthrene, and pyrene. Chronic de rmal exposure to PAHs in both groups 17 

### resulted in an additive response when compared to t he tumor response for each group alone. 18 

### Nesnow et al. (1998b) evaluated lung tumor formatio n in A/J mice following combined 19 

### administration of five carcinogenic PAH compounds ( benzo[a]pyrene, benzo[b]fluoranthene, 20 

### dibenz[a,h]anthracene, 5-methylchrysene, and cyclop enta[c,d]pyrene). High and low doses were 21 

### selected for each PAH in this study based on toxici ty, survival, range of response, and predicted 22 

### tumor yield. The ratio of PAH doses was designed t o simulate PAH ratios found in 23 

### environmental air and emissions samples. PAHs were administered to mice in a 2 5 

### factorial 24 

### study design yielding 32 dose groups (combination o f five PAHs at high and low doses). The 25 

### formation of lung adenomas was evaluated 8 months f ollowing intraperitoneal injection of PAH 26 

### mixtures. A response surface model was used to eva luate specific interactions among PAHs. 27 

### The results of the study indicated that greater-tha n-additive effects were seen at low doses, while 28 

### less-than-additive effects were observed at high do ses. However, the magnitude of the 29 

### interactions was relatively small (twofold), sugges ting that potential interactions are limited in 30



### extent. 31 

### Dermal application of binary mixtures of PAHs has a lso been shown to produce additive, 32 

### synergistic, and inhibitory effects on DNA binding in mouse skin (Hughes and Phillips, 1993, 33 

### 1990). Hermann (1981) demonstrated that many PAHs could both enhance and inhibit the 34 

### bacterial mutagenicity of benzo[a]pyrene depending on the relative concentrations in the binary 35 

### mixture. Binary mixtures of benzo[a]pyrene and ben zo[e]pyrene produced a synergistic 36 

### response in the TA98 strain of S. typhimurium (which detects frameshift mutations), and 37 

### antagonistic and additive effects in strain TA100 ( which detects a broad spectrum of mutations) 38

### 49 DRAFT – DO NOT CITE OR QUOTE

depending on the concentration (Hass et al., 1981). Binary mixtures of PAHs have also been 1 

### shown to produce antagonistic or less than additive effects in the Ames assay of bacterial 2 

### mutagenicity (Barrai et al., 1992; Salamone et al., 1979a). Vaca et al. (1992) demonstrated an 3 

### additive effect for sister chromatid exchange induc tion by combined administration of 4 

### benzo[a]pyrene and fluoranthene in human peripheral lymphocytes cocultured with 5



### PCB-induced rodent liver cells. 6 

### The effects of binary PAH mixtures on gene expressi on, DNA adduct formation, 7 

### apoptosis, and cell cycle are additive compared to the effects of the individual compounds in 8 

### human hepatoma cells (HepG2) (Staal et al., 2007). Equimolar and equitoxic mixtures of 9 

### benzo[a]pyrene with either dibenzo[a,l]pyrene, dibe nz[a,h]anthracene, benzo[b]fluoranthene, 10 

### fluoranthene, or 1-methylphenanthrene were studied. PAH mixtures showed an additive effect 11 

### on apoptosis and on cell cycle blockage. The effec ts of binary mixtures of PAHs on gene 12 

### expression were generally additive or slightly anta gonistic. 13 

### Additivity has also been observed for the mutagenic ity of PAHs administered as a 14 

### complex mixture (Bostrom et al., 1998; Kaden et al. , 1979). Kaden et al. (1979) evaluated the 15 

### bacterial mutagenicity of the PAH fraction of keros ene soot using resistance to 8-azaguanine as a 16 

### genetic marker for forward mutation in S. typhimurium . Approximately half of the PAHs tested 17 

### (34 of 70) produced a significant increase in the m utant fraction in this assay system. The 18 

### mutagenicity of the complex soot mixture was demons trated to be approximately equal to the 19 

### additive mutagenicity of the individual components. Bostrom et al. (1998) reported additivity in 20 

### the Ames test of bacterial mutagenesis (i.e., rever sion to histidine independence) for a mixture of 21 

### four PAHs (benzo[a]pyrene, benz[a]anthracene, fluor ene, and pyrene) using four different strains 22



### of S. typhimurium. 23 

### Mechanistic studies have suggested that the outcome of the interaction between two 24 

### PAHs in a binary mixture is dependent on changes in metabolism. PAHs can act as both 25 

### inducers and competitive inhibitors of the CYP enzy mes that are responsible for generation of 26 

### reactive metabolites. Benzo[e]pyrene has been show n to alter the oxidative metabolism of 27 

### benzo[a]pyrene, which may be related to the cocarci nogenic effect seen in skin tumor initiation 28 

### studies (Baird et al., 1984). Alterations in the t ypes and amounts of benzo[a]pyrene metabolites 29 

### suggest that benzo[e]pyrene-induced changes may be isozyme specific (Smolarek and Baird, 30 

### 1984). An increase in the formation of benzo[a]pyr ene DNA adducts has also been 31 

### demonstrated for coadministration of benzo[e]pyrene in Sencar mouse skin (Smolarek et al., 32 

### 1987). Fluoranthene and pyrene have been shown to increase the formation of benzo[a]pyrene33 

### DNA adducts in mouse skin following a combined trea tment (Rice et al., 1988, 1984). 34 

### Enhancement of the metabolism of benzo[a]pyrene to diol epoxide metabolites and subsequent 35 

### DNA binding may explain the increased carcinogenic effect in this case. Phenanthrene did not 36 

### increase the formation of benzo[a]pyrene-DNA adduct s and was not shown to be cocarcinogenic 37 

### following combined administration with benzo[a]pyre ne in this study. Cherng et al. (2001) 38

### 50 DRAFT – DO NOT CITE OR QUOTE

demonstrated that benzo[g,h,i]perylene increased th e formation of benzo[a]pyrene adducts in 1 

### hepatoma cells (HepG2) by enhancing benzo[a]pyrene induction of CYP1A1. Benzo[g,h,i] 2 

### perylene increased the nuclear accumulation of the AHR and/or the activation of the AhR to a 3 

### DNA-binding form (Cherng et al., 2001). Benzo[k]fl uoranthene altered the metabolic profile of 4 

### benz[a]anthracene by increasing the activity of CYP 1A1 (Schmoldt et al., 1981). The bacterial 5 

### mutagenicity of benz[a]anthracene was enhanced by u se of a rodent liver S9 that was obtained 6 

### from animals previously exposed to other PAHs (Norp oth et al., 1984). Coadministration of 7 

### benzo[a]pyrene and benz[a]anthracene to hamster emb ryo cell cultures resulted in a decrease in 8 

### the metabolism of benzo[a]pyrene, a decrease in the level of DNA binding, and a decrease in 9 

### mutation frequency in hamster V79 cells (Smolarek e t al., 1986). 10 

### In summary, combined administration of binary mixtu res of PAHs can result in several 11 

### types of joint action (i.e., additive, synergistic, or antagonistic). The nature of the joint action 12 

### appears to be dependent on the characteristics of t he individual PAHs, related changes in 13 

### metabolism and possibly the test species/strain. P AHs can act as both inducers and competitive 14 

### inhibitors of the CYP enzymes that are responsible for generation of reactive metabolites. 15 

### Additivity has been observed for some complex mixtu res of PAHs, suggesting a balance in the 16 

### relative metabolism of individual PAHs. For the pu rposes of this analysis, an assumption is 17



### made that the combination of individual PAHs result s in additive effects. Additional research is 18 

### needed to characterize the validity of this assumpt ion. 19

20

21



### 3. DISCUSSION OF PREVIOUSLY PUBLISHED RPF APPROACH ES 22

23

24 

### There are multiple analyses available for the deriv ation of relative potency estimates for 25 

### individual PAHs. All of these analyses utilize ben zo[a]pyrene as the index chemical. Table 3-1 26 

### compares relative cancer potency values for PAHs pr esented by several authors. A review of the 27 

### derivation of these relative potency values follows

. 28

29

3051 DRAFT – DO NOT CITE OR QUOTE1Table 3-1. Comparison among various relative potency estimates for PAHs from the published literatureandregulatory agencies (1984–2004)PAH AbbrU.S.EPA(1993)ChuandChen(1984)Clement(1988)Clement(1990)Rugen etal. (1989)Slooff etal. (1989)Kroeseet al.(2001)NisbetandLaGoy(1992)MalcolmandDobson(1994)Meeket al.(1994)Mulleret al.(1997)Larsen andLarsen(1998)Collinset al.(1998)CaliforniaEPA(2004)Acenaphthene AN 0.001 0.001Acenaphthylene ANL 0.001 0.001Anthanthrene AA 0.32 0.316 0.28 0.3Anthracene AC 0 0 0.01 0.01 0.0005Benzo[a]pyrene BaP 1 1 1 1 1 1 1 1 1 1 1 1 1Benz[a]anthracene BaA 0.1 0.013 0.145 0.004–0.0060–0.04 <0.1 0.1 0.1 0.014 0.005 0.1Benzo[b]fluorantheneBbF 0.1 0.08 0.14 0.1228 0.0235 0.1 0.1 0.06 0.110.1 0.1 0.62Benzo[c]phenanthreneBcPH 0.023 0.023Benzo[e]pyrene BeP 0.004 0.007 0.01 0 0.002Benzo[g,h,i]perylene BghiP 0.022 0.0212 0.01–0.030.03 0.01 0.01 0.012 0.02Benzo[j]fluoranthene BjF 0.061 0.0523 0.0763 0.1 0.05 0.045 0.05 0.1 0.52Benzo[k]fluorantheneBkF 0.01 0.004 0.066 0.0523 0.03–0.09<0.1 0.1 0.1 0.04 0.037 0.05 0.1Chrysene CH 0.0010.001 0.0044 0.05–0.890.1–0.030.01 0.01 0.026 0.03 0.01 0.17Coronene CO 0.001Cyclopenta[c,d]pyreneCPcdP 0.023 0.1 0.012 0.02Dibenzo[a,h]anthraceneDBahA 1 0.69 1.11 0.599 5 1 0.89 1.1Dibenz[a,c]anthraceneDBacA 0.1Dibenzo[a,e]pyrene DBaeP 0.2 1Dibenzo[a,h]pyrene DBahP 1.2 1 10 11Dibenzo[a,i]pyrene DBaiP 1.1 0.1 10 12Dibenzo[a,l]pyrene DBalP 1 10Fluoranthene FA 0–0.06 0.01 0.001 0.001 0.05Fluorene FE 0.001 0.001Indeno[1,2,3-c,d]pyreneIP 0.1 0.017 0.232 0.278 0.00599 0–0.08 0.1 0.1 0.10.12 0.067 0.1 0.1Perylene Pery 0.00152 DRAFT – DO NOT CITE OR QUOTETable 3-1. Comparison among various relative potency estimates for PAHs from the published literatureandregulatory agencies (1984–2004)PAH AbbrU.S.EPA(1993)ChuandChen(1984)Clement(1988)Clement(1990)Rugen etal. (1989)Slooff etal. (1989)Kroeseet al.(2001)NisbetandLaGoy(1992)MalcolmandDobson(1994)Meeket al.(1994)Mulleret al.(1997)Larsen andLarsen(1998)Collinset al.(1998)CaliforniaEPA(2004)Phenanthrene PH 0.01 <0.01 0.001 0.001 0.000640.0005Pyrene Pyr 0.081 0.001 0.001 0 0.001Abbr = abbreviation

### 53 DRAFT – DO NOT CITE OR QUOTE

U.S. EPA (1993) presented RPFs (termed EOPPs) for s even PAHs (benzo[a]pyrene, 1 

### benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fl uoranthene, chrysene, 2 

### dibenz[a,h]anthracene, indeno[1,2,3-c,d]pyrene) as Provisional Guidance for the risk evaluation 3 

### of PAHs at hazardous waste sites. On IRIS (U.S. EP A, 2009), all seven of these compounds 4 

### were assigned a cancer weight of evidence classific ation of Group B2 (probable human 5 

### carcinogen, based on sufficient evidence of carcino genicity in animals) under the U.S. EPA 6 

### (1986) Guidelines for Carcinogen Risk Assessment . U.S. EPA (1993) indicated that the data for 7 

### PAHs did not meet the criteria for the development of toxicity equivalency factors (TEFs). In 8 

### particular, the existing database was limited prima rily to studies of metabolism, genotoxicity, 9 

### and cancer, and the assumption of additivity was no t proven or refuted. The EOPP terminology 10 

### was used because this approach was limited to skin painting data and was based on 11 

### benzo[a]pyrene exposure from a single (oral) pathwa y (for the derivation of the slope factor). 12 

### This analysis considered only a small subset of PAH s routinely measured in PAH mixtures at 13



### hazardous waste sites. The EOPP values were based on previous evaluations conducted by Chu 14 

### and Chen (1984) and Clement Associates (1988) and w ere calculated for various test systems 15 

### (i.e., mouse skin carcinogenesis, subcutaneous inje ction in mice, intrapulmonary administration 16 

### to rats, tumor initiation on mouse skin, and intrap eritoneal injection in newborn mice) (Clement 17 

### Associates, 1988). Various statistical methods for combining data sets were considered; 18 

### however, final EOPP values were based on a single t est system (skin painting) and were rounded 19 

### to the closest order of magnitude. The EOPPs were recommended for the oral exposure route 20 

### only, because the quantitative dose-response assess ment for benzo[a]pyrene was from an oral 21 

### carcinogenicity bioassay (i.e., an oral cancer slop e factor). This recommendation was, however, 22 

### complicated by the fact that the EOPPs were derived from comparisons based on dermal 23



### exposure. 24 

### Chu and Chen (1984) presented RPF values for the se ven PAH compounds described in 25



### the Provisional Guidance described above (U.S. EPA, 1993) (see Table 3-1). These values were 26 

### calculated using mouse skin painting data only. Tu mor incidence data were modeled using the 27 

### linearized multistage model and the resulting ED 10 

### and q1* (upper confidence limit of the linear 28 

### slope) were presented for target PAHs and benzo[a]p yrene. The RPFs listed in Table 3-1 29 

### represent the ratio of the q1* value for a PAH comp ound to the q1* value for benzo[a]pyrene 30 

### (i.e., q1* PAH 

### ÷ q1* BaP 

### ). 31 

### Clement Associates (1988) identified 11 published studies that concurrently compared 32 

### the carcinogenicity of benzo[a]pyrene with one or m ore other PAHs, and used the data to derive 33 

### relative cancer potencies for 13 PAHs, including be nzo[a]pyrene. Test protocols used in this 34 

### analysis included mouse skin complete carcinogenesi s, initiation-promotion on mouse skin, 35 

### subcutaneous injection into mice, lung implantation in rats, and intraperitoneal injection into 36



### newborn mice. Tumor incidence data were fit to a s implified version of the Moolgavkar37 

### Venson-Knudsen (MVK) two-stage model and to the lin earized multistage model to obtain low- 38

### 54 DRAFT – DO NOT CITE OR QUOTE

dose cancer potency values (transition rates and lo w-dose slope factors, respectively). Most of 1 

### the estimates were derived using data for multiple exposure levels and controls, but some were 2 

### based on a single exposure level and a control. RP Fs were calculated as the ratio of the 3 

### estimated transition rate or slope factor for a par ticular PAH to the corresponding values for 4 

### benzo[a]pyrene from the same study. Clement Associ ates (1988) selected representative RPFs 5



### for each of the studied PAHs based on evaluations o f the quality of the studies from which the 6



### estimates were obtained. 7 

### Clement Associates (1990) also derived relative can cer potencies for eight PAHs based 8 

### on tumor incidence data from rat lung implantation data only (Deutsch-Wenzel, 1983). The data 9 

### were restricted to a single group of studies using a defined experimental protocol in order to 10 

### address issues of questionable data quality associa ted with other studies. Data quality concerns 11



### cited for other studies include variation in surviv al, saturation of the carcinogenic effect, 12 

### outmoded pathological classification, and inadequat e controls. The RPF values based on rat lung 13 

### implantation data were comparable to those original ly derived by Clement Associates (1988) 14 

### (see Table 3-1). 15 

### Rugen et al. (1989) proposed a relative potency app roach to establish acceptable 16 

### exposure levels (AELs) for six carcinogenic PAHs in drinking water (listed in Table 3-1). These 17 

### authors reviewed mouse skin painting studies in whi ch the cancer potency of benzo[a]pyrene 18 

### was compared with those of other PAHs (Bingham and Falk, 1969; Wynder and Hoffmann, 19 

### 1961, 1959a, b). The following relationship was us ed to calculate conversion factors (“relative 20 

### tumor dose” = RTD) to derive AELs for these PAHs fr om the AEL for benzo[a]pyrene: RTD = 21 

### (d 1 

### /n 1 

### )/(d 2 

### /n 2 

### ); where d 1 

### and n 1 

### represented a dosage level and associated tumor in cidence after a 22 

### given exposure duration to a certain PAH, PAH 1 

### , and d 2 

### and n 2 

### represented similar quantities for 23 

### exposure to the index PAH, benzo[a]pyrene, for the same exposure duration. The AEL for a 24 

### particular PAH was then derived with the following relationship: AEL (PAHi) 

### = AEL (benzo[a]pyrene) 

### × 25



### RTD (PAHi) 

### . In this approach, RTDs for PAHs more potent than benzo[a]pyrene were less 26 

### than 1 and RTDs for PAHs less potent than benzo[a]p yrene were greater than 1. The reciprocal 27 

### of the RTDs derived by Rugen et al. (1989) were com parable to the RPFs presented by other 28 

### authors and are presented as such in Table 3-1. 29 

### The Netherlands (RIVM) proposed RPF values for 10 P AHs (naphthalene, anthracene, 30 

### phenanthrene, fluoranthene, chrysene, benz[a]anthra cene, benzo[k]fluoranthene, benzo[a]pyrene, 31 

### benzo[g,h,i]perylene, and indeno[1,2,3-c,d]pyrene) (Slooff et al., 1989). RPFs were calculated 32



### as a ratio of ED 50 

### values that were calculated using a simple linear model. For dermal studies in 33 

### which the latency period was determined, the tumor incidence was divided by latency and 34 

### concentration, and the values were averaged for the different concentrations. Kroese et al. 35 

### (2001) provided an update of the RPF values calcula ted by Slooff et al. (1989) by incorporating 36 

### more recent evaluations conducted by other authors (Larsen and Larsen, 1998; Nesnow et al., 37

### 55 DRAFT – DO NOT CITE OR QUOTE

1998b; Muller, 1997; Nisbet and LaGoy, 1992). The RPF values for chrysene and fluoranthene 1 

### were decreased, while other values remained similar to those originally proposed (see Table 3-1). 2 

### Nisbet and LaGoy (1992) proposed toxicity equivalen ce factors for 17 PAHs commonly 3



### found at hazardous waste sites. These authors revi ewed published studies in which the 4 

### tumorigenic potencies of one or more PAHs were comp ared with benzo[a]pyrene (essentially the 5 

### same as those reviewed by Clement Associates, 1988) and rounded, to an order of magnitude, the 6 

### estimates presented by Clement Associates (1988) fo r seven carcinogenic PAHs 7 

### (dibenz[a,h]anthracene, benz[a]anthracene, benzo[b] fluoranthene, benzo[k]fluoranthene, 8 

### indeno[1,2,3-c,d]pyrene, benzo[g,h,i]perylene, and chrysene) (see Table 3-1). Nisbet and LaGoy 9 

### (1992) argued that the rounded estimates more accur ately reflected the uncertainty in the 10 

### estimates than the values presented by Clement Asso ciates (1988). Nisbet and LaGoy (1992) 11 

### stated that Clement Associates (1988) proposed a TE F of 0.32 for anthracene (CASRN 12 

### 120-12-7), but examination of the original report s hows that Clement Associates (1988) 13 

### proposed this value for anthanthrene (CASRN 191-26- 4) and did not propose a value for 14 

### anthracene. Nisbet and LaGoy (1992) assigned a val ue of 0.01 to anthracene. In addition, 15 

### Nisbet and LaGoy (1992) arbitrarily assigned TEFs o f 0.001 to eight other PAHs for which 16 

### adequate evidence of carcinogenicity in animals was not available (acenaphthene, 17 

### acenaphthylene, fluoranthene, fluorene, 2-methylnap hthalene, naphthalene, phenanthrene, and 18 

### pyrene). In defense of this assignment, the argume nt was made that some of these PAHs have 19 

### been shown to have some, albeit limited, evidence f or carcinogenic or genotoxic activity in some 20 

### studies (e.g., phenanthrene and naphthalene 2 

### ). The RPF value proposed for 21 

### dibenz[a,h]anthracene was substantially higher than that proposed by Clement Associates (1988). 22 

### Nisbet and LaGoy (1992) indicate that their analysi s of the dose-response data suggests that an 23 

### RPF value of 5 is more appropriate for environmenta l exposures where the chemically-related 24 

### tumor incidence rate would be approximately <25%. 25 

### Malcolm and Dobson (1994) used RPFs for 23 PAHs to calculate environmental 26 

### assessment levels (EALs) for atmospheric PAHs (spon sored by the Great Britain Department of 27 

### the Environment). The RPFs were derived from previ ously reported review papers (Nisbet and 28 

### LaGoy, 1992; Rugen et al., 1989; Clement Associates , 1988; Chu and Chen, 1984), as well as the 29 

### primary literature describing pulmonary implant, sk in painting, subcutaneous injection, and 30 

### mouse skin DNA binding studies. No information was provided regarding the methodology used 31 

### to derive RPFs from specific experimental studies. The proposed RPF values for individual 32 

### PAHs were the highest values reported in the litera ture. Many of the RPF values are similar to 33 

### those reported by Nisbet and LaGoy (1992). RPFs we re additionally reported for 34 

### benzo[e]pyrene, coronene, cyclopenta[c,d]pyrene, di benz[a,c]anthracene, and perylene. The 35 

### benzo[e]pyrene and cyclopenta[c,d]pyrene RPFs were apparently calculated directly from mouse 36

2 It should be noted that a recent bioassay for napht halene has shown increased incidence of nasal tumor s in exposed rats (NTP, 2000).

### 56 DRAFT – DO NOT CITE OR QUOTE

skin painting studies (Habs et al., 1980; Hoffmann and Wynder, 1966; Wynder and Hoffmann, 1 

### 1959a, b). Coronene and perylene were arbitrarily assigned RPF values of 0.001 given the 2 

### International Agency for Research on Cancer (IARC) and U.S. EPA designation as “not 3 

### classifiable as to human carcinogenicity” (similar approach to Nisbet and LaGoy, 1992). 4 

### Dibenz[a,c]anthracene was assigned an RPF value of 0.1 based on the IARC designation of 5 

### “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[1,2,3-cd]pyrene) 8 

### based on the results of multistage modeling of inci dence data in Osborne-Mendel rats treated by 9 

### lung implantation (Deutsch-Wenzel et al., 1983). V alues were based on a comparison of the 10



### doses that caused a 5% increase in tumor incidence (ED 05 

### ). RPFs were calculated as the ratio of 11



### the ED 05 

### for benzo[a]pyrene to the ED 05 

### for a specific PAH compound. 12 

### The Ontario Ministry of Environment and Energy (Mul ler 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 a nalysis are listed in Table 3-1. Muller et al. 16 

### (1997) derived a standard time of observation in or der to account for varying study duration 17 

### across experiments. Several dose-response models w ere considered for the evaluation of tumor 18 

### incidence and multiplicity, and linear regression w as selected as the preferable method. 19 

### Tumorigenic potency (i.e., the slope of incidence/m g) was determined separately for each data 20 

### set based on the following order of preference rega rding study type: tumor initiation in 21 

### CD-1 mice, tumor initiation in SENCAR mice, rat lun g implantation, and complete 22 

### carcinogenicity in C57BL mice. RPFs were determine d as the ratio of PAH potency to the 23 

### potency of benzo[a]pyrene. RPF values derived by M uller 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 ora l, pulmonary, and skin application of PAHs. 27 

### The authors indicated that these values represent a n entirely subjective estimate of relative 28 

### potency; however, further detail regarding the deri vation of RPF estimates was not provided. 29 

### Collins et al. (1998) developed RPFs (termed potenc y equivalency factors [PEFs]) for 30 

### 21 PAHs, 10 of these were either methyl- or nitro-s ubstituted or heterocyclic PAHs. A hierarchy 31 

### of data types was utilized to provide an order of p reference 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, a nd subcutaneous or intraperitoneal injection. 35 

### Genotoxicity and structure activity data were consi dered the least-preferred data type for 36 

### calculation of RPFs. Collins et al. (1998) noted t hat a wide range of PEFs were observed for 37 

### individual chemicals using different types of data (e.g., mutagenicity versus tumor data). The 38

### 57 DRAFT – DO NOT CITE OR QUOTE

basis for the derivation of individual RPF values w as presented in a California EPA (2002) 1 

### technical support document. RPF values for benz[a] anthracene, benzo[b]fluoranthene, 2 

### benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[ 1,2,3-c,d]pyrene, and chrysene were similar 3 

### to those described by Clement Associates (1988). A dditional 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 relativ e potency of dibenz[a,h]anthracene was 8 

### estimated to be 0.1, when compared to the oral pote ncy 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 re presented the upper 95 th 

### percent confidence 13 

### limit on the linear term of the multistage model fi t for the newborn mouse dose-response data. 14 

### Because benzo[a]pyrene was demonstrated to be 75 ti mes more toxic in newborn mouse 15 

### intraperitoneal assays than in adult oral studies, oral equivalent potencies for individual PAHs 16 

### were derived by adjusting the cancer potency downwa rd by a factor of 75. The RPFs listed in 17



### Table 3-1 were calculated as the ratio of the oral equivalent potency for a PAH to the oral 18 

### potency estimate for benzo[a]pyrene. This methodol ogy resulted in a significant increase in RPF 19 

### values for benzo[b]fluoranthene, benzo[j]fluoranthe ne, and chrysene when compared with other 20 

### approaches. 21 

### In summary, several approaches are available for th e 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 acro ss 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, chry sene, and indeno[1,2,3-c,d]pyrene. RPF 28 

### values were also highly variable for dibenzo[a,e]py rene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, 29 

### and dibenzo[a,l]pyrene; however, these were only pr esented in a few recent studies. As 30 

### indicated above, the recent California EPA (2004) a pproach to estimating RPFs provides 31 

### considerably higher RPF values for benzo[b]fluorant hene, 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 understan ding of PAH toxicology. A great deal of 35



### scientific research on PAHs has been conducted sinc e 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 do es not meet the criteria for the derivation of 38

### 58 DRAFT – DO NOT CITE OR QUOTE

TEFs. U.S. EPA (2000) defines TEFs as special type s of RPFs that are derived when there are 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 cert ain or is known for only a subset of all health 3 

### endpoints. The major differences in the use of TEF s and RPFs is that TEFs are applied to all 4 

### health endpoints, exposure routes, and exposure dur ations (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 appli cable 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 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; Gol dstein et al., 1994; Clement Associates, 15 

### 1990, 1988; Krewski et al., 1989). These studies p rovide semi-quantitative information on the 16 

### overall uncertainty in using a component-based appr oach. 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 oth ers 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. Schmähl et al. (1977) exposed groups of 22 

### mice to multiple doses of benzo[a]pyrene alone or t o one of two defined mixtures of PAHs. The 23 

### first of these mixtures was comprised of benzo[a]py rene, dibenz[a,h]anthracene, 24 

### benz[a]anthracene, and benzo[b]fluoranthene. The s econd mixture contained seven PAHs: 25 

### phenanthrene, anthracene, fluoranthene, pyrene, chr ysene, benzo[e]pyrene, and 26 

### benzo[g,h,i]perylene. The predicted tumor incidenc es 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 Schmähl et al. (1977) data for benzo[a]pyrene a lone. Predicted tumor incidences for the first 29 

### mixture were comparable to observed tumor incidence s, 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 wer e 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-f ired 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 c omplex mixtures were calculated using a 37 

### simplified version of the MVK two-stage model and t umor incidence data from a series of 38

### 59 DRAFT – DO NOT CITE OR QUOTE

published rat lung implantation studies that examin ed the carcinogenicity of each complex 1 

### mixture, various sub-fractions of the mixtures, and benzo[a]pyrene (Grimmer et al., 1988, 2 

### 1987a, b, 1984). Lung implantation data (Deutsch-W enzel, 1983) were used to calculate RPFs 3 

### for benzo[b]fluoranthene, benzo[e]pyrene, benzo[j]f luoranthene, benzo[k]fluoranthene, 4 

### indeno[1,2,3-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 eig ht PAHs (i.e., the sum of the products of the 6 

### relative cancer potencies of the eight PAHs multipl ied by their concentrations in the respective 7 

### complex mixtures) accounted for only minor fraction s of the total carcinogenicity of each of the 8 

### four complex mixtures. When the assumption was mad e that each of the eight PAHs was as 9 

### potent as benzo[a]pyrene, the sum of the benzo[a]py rene equivalents still accounted for only 10 

### minor fractions of the carcinogenicity of each mixt ure. 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 fur ther 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 which may be carcinogenic. In addition, complex PAH 15 

### mixtures found in the environment contain other pot ential carcinogens including substituted and 16 

### heterocyclic PAHs and non-PAH components. 17 

### Krewski et al. (1989) compared the observed tumor r esponse 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 c arcinogenicity of a MGP residue to 23 

### the predicted cancer risk using the Nisbet and LaGo y (1992) RPF scheme. The RPF method 24 

### underestimated the potential carcinogenicity of the mixture. The lack of correspondence was 25 

### suggested to be related to the presence of unidenti fied carcinogens in the mixture or possible 26 

### synergistic interactions between PAHs. 27 

### McClure et al. (1996) compared the tumor response p redicted using U.S. EPA’s 1993 28 

### provisional values (i.e., EOPPs) to the observed re sponse reported in studies of mice exposed to 29 

### synthetic and complex mixtures of PAHs. The result s of this analysis were mixed. EOPP values 30 

### closely predicted the mouse tumor response to subcu taneous or dermal application of synthetic 31 

### mixtures containing relatively potent carcinogens, while overestimating the response to synthetic 32 

### mixtures containing only relatively weak carcinogen s (similar to findings of Clement Associates, 33 

### 1988). Mouse skin tumor initiation with several co al liquids was closely predicted by the EOPP 34 

### approach; however, this method underestimated the t umor response from lung implantation of 35



### coal furnace emission condensate and its PAH-contai ning neutral fraction. 36 

### The validation analyses that were performed by Mul ler et al. (1997) consisted of 37 

### component versus whole mixture risk comparisons usi ng data for smoky coal and coke oven 38

### 60 DRAFT – DO NOT CITE OR QUOTE

emissions. The human lung cancer risks that were e stimated using the RPF approach were 1 

### compared to the whole mixture cancer risk derived f rom 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 estimate d as a standard mixture assumed to 4 

### represent an average PAH profile. The RPF method p roduced PAH cancer risk estimates that 5 

### were significantly lower than the risk estimates de rived from epidemiology studies. 6

7

8



### 4. EVALUATION OF THE CARCINOGENICITY OF INDIVIDUAL PAHs 9

10

11 

### 4.1. DATABASE OF STUDIES ON PAH CARCINOGENICITY AN D CANCER12



### RELATED ENDPOINTS 13 

### A database of primary literature relevant to the RP F approach for PAHs was developed. 14 

### This was accomplished through the following means: 15

16



### • Definition of the study types that were considered relevant to relative potency 17 

### development; 18

19



### • Review of reference lists from review articles and other secondary sources; 20

21



### • Identification of selected PAHs to be included in s earch of open literature; 22

23



### • Performance of targeted searches of open literature on selected PAHs; and 24

25



### • Population of the database with references and mean ingful keywords. 26

27 

### The study types that were considered most useful fo r RPF derivation were rodent 28 

### carcinogenicity bioassays (all routes) in which one or more PAHs was tested at the same time as 29 

### benzo[a]pyrene. In addition, in vivo and in vitro data for cancer-related endpoints (in which one 30 

### or more PAHs and benzo[a]pyrene were tested simulta neously) were obtained, including studies 31 

### on the formation of DNA adducts, mutagenicity, chro mosomal aberrations, aneuploidy, DNA 32 

### damage/repair/recombination, unscheduled DNA synthe sis, and cell transformation. Although it 33 

### would be possible to calculate RPFs from studies wh ere a PAH and benzo[a]pyrene were tested 34 

### by the same laboratory using the same test system b ut at different times, this approach was not 35



### considered because it could introduce differences i n the dose-response information that are 36 

### unrelated to the chemical (e.g., variability associ ated with laboratory environment conditions, 37 

### animal handling, food supply). Thus, studies in wh ich benzo[a]pyrene was not tested 38 

### simultaneously with another PAH were not considered in this analysis. 39 

### Studies of AhR binding/activation were not consider ed for use in deriving RPFs because 40 

### there is evidence indicating that highly mutagenic fjord-region PAHs are potent carcinogens, 41

### 61 DRAFT – DO NOT CITE OR QUOTE

despite a lower AhR affinity (reviewed by Bostrom e t al., 2002). Likewise, some PAHs that 1 

### strongly activate the AhR, such as benzo[k]fluorant hene (Machala et al., 2001), are only weakly 2 

### carcinogenic. In addition, some studies have demon strated the formation of DNA adducts in the 3 

### liver of AhR knockout mice following intraperitonea l or oral exposure to benzo[a]pyrene 4 

### (Sagredo et al., 2006; Uno et al., 2006; Kondragant i et al., 2003). These findings suggest that 5 

### there may be alternative (i.e., non-AhR mediated) m echanisms of benzo[a]pyrene activation in 6 

### the mouse liver, and the AhR affinity would not be a good predictor of carcinogenic potency. 7 

### Several study types were initially excluded from th e database because they did not 8 

### provide carcinogenicity or cancer-related endpoint information for individual PAHs. These 9 

### include biomarker studies measuring DNA adducts in humans, studies of PAH metabolism, and 10 

### studies of PAH mixtures. Although these studies co ntain important information on human 11 

### exposure to PAH mixtures and the mode of action for PAH toxicity, they generally do not 12 

### contain dose-response information that would be use ful for calculation of RPF estimates. In 13 

### addition to the primary bioassay and cancer-related endpoint studies described above, the RPF 14



### database also includes information on PAH mode of c arcinogenic action, interactions among 15 

### PAHs in mixtures, and the influence of exposure rou te on carcinogenic action of PAHs. 16 

### Primary studies were identified through review of a vailable secondary sources and 17 

### review articles, supplemented by a targeted literat ure search. A complete list of the secondary 18 

### sources that were reviewed is contained in Appendix A. A literature search strategy was 19 

### developed by first constructing a list of the indiv idual PAHs to be included. The list of PAHs 20



### was restricted to unsubstituted PAHs with three or more fused aromatic rings containing only 21 

### carbon and hydrogen atoms, because these are the mo st widely studied members of the PAH 22 

### chemical class. Heterocyclic PACs or PAHs with sub stituted groups (e.g., alkyl, hydroxyl, 23 

### sulfhydryl, amino, or nitro groups) were not includ ed. An initial search yielded a list of PAHs 24 

### for which toxicological data are available. Indivi dual PAHs were then chosen for the literature 25 

### search because they were known to have toxicologica l information relevant to cancer, and in 26 

### most cases, their presence in environmental sources of PAH exposure was known. Using these 27 

### criteria and excluding benzo[a]pyrene, 74 PAHs were identified from primary and secondary 28 

### sources (see Table 2-1 in Section 2). 29 

### A search of the open literature was conducted in th e Medline (PUBMED) database for 30 

### the PAHs identified. This database encompasses man y of the studies that would also be found in 31 

### TOXLINE and Cancer Lit (the latter is no longer ava ilable as a separate database). Medline 32 

### (PUBMED) was searched by CASRN in conjunction with cancer and cancer-related endpoint 33 

### keywords. The search was not limited by publicatio n date to ensure that all relevant studies were 34 

### identified. A few compounds did not show any resul t when searched by CASRN. For these 35 

### PAHs, an additional search by name was conducted. Search results, including Medline 36 

### keywords, were downloaded directly into the working RPF database. 37

### 62 DRAFT – DO NOT CITE OR QUOTE

In addition to Medline, computer searches of the fo llowing databases and websites were 1 

### conducted: IARC, World Health Organization (WHO), Agency for Toxic Substances and 2 

### Disease Registry (ATSDR), Health Canada, NTP, Calif ornia EPA’s Office of Environmental 3 

### Health Hazard Assessment (OEHHA), the Substance Reg istry System, CCRIS, TSCATS, and 4



### DSSTOX. 5 

### Primary and secondary studies were entered in the R PF database and relevant keywords 6 

### (identifying study type, whether benzo[a]pyrene was included, route of administration, target 7 

### organ, etc.) were identified for each study. The l ist of keywords was developed in order to 8 

### facilitate database searching for references on a s pecific topic. Quality assurance procedures 9 

### were employed to ensure that database references we re properly keyword-coded for retrieval. 10

11



### 4.2. STUDIES IN HUMANS 12



### Numerous studies have evaluated cancer outcomes in PAH-exposed individuals 13 

### (reviewed in Bostrom et al., 2002; WHO, 1998; ATSDR , 1995; IARC, 1987, 1983, 1973). 14 

### However, since these exposures were to complex mixt ures containing multiple PAH 15 

### carcinogens, they did not provide adequate data to evaluate the human carcinogenicity of 16 

### individual PAH compounds. Epidemiology studies hav e focused on occupational exposure to 17 

### PAH mixtures. Emissions from coke production, coal gasification, aluminum production, iron 18 

### and steel founding, coal tars, coal tar pitches, an d soot have produced lung cancer in humans 19 

### (Bostrom et al., 2002). Skin and scrotal cancers h ave resulted from exposure to coal tar, coal tar 20 

### pitches, nonrefined mineral oils, shale oils, and s oot (Larsen and Larsen, 1998; WHO, 1998; 21 

### ATSDR, 1995). Occupational studies clearly demonst rate exposure-response relationships for 22 

### PAH mixtures; however, quantitative estimates of ri sk are limited primarily to lung cancer in 23 

### coke oven workers (Bostrom et al., 2002; Larsen and Larsen, 1998; ATSDR, 1995). 24 

### Biomonitoring of exposure to PAHs includes measurem ent of DNA and protein adducts 25 

### and measurement of urinary metabolites of PAHs, stu dies on genetic polymorphisms of CYP450 26 

### and other enzymes, and changes in PAH metabolism (B ostrom et al., 2002; Larsen and Larsen, 27 

### 1998; ATSDR, 1995). While these studies demonstrat e the degree of exposure to PAHs from 28 

### various settings, quantitative dose-response data f or humans exposed to individual PAHs are not 29 

### available. Cancer-related endpoint studies that we re performed using human cell lines are 30 

### presented with similar assays in other mammalian sp ecies in Section 4.3. 31

32



### 4.3. STUDIES IN ANIMALS 33 

### The database of studies investigating cancer or can cer-related endpoints in animals 34 

### exposed to PAHs is extensive. For the purpose of d eveloping relative potency estimates, only 35



### those studies that included at least one selected P AH and benzo[a]pyrene as a reference 36 

### compound were reviewed. Studies were excluded if P AH potency comparisons were not 37 

### conducted in the same laboratory in concurrent expe riments. Studies without benzo[a]pyrene are 38

### 63 DRAFT – DO NOT CITE OR QUOTE

listed in two separate bibliographies in Appendix B . Table B-1 of the appendix shows PAHs that 1 

### were assayed with or without benzo[a]pyrene. Table B-1 shows that 32 of the 74 PAHs were 2 

### assayed with benzo[a]pyrene; an additional 14 PAHs were not tested in the same study as 3 

### benzo[a]pyrene. The remaining 28 PAHs either have only cancer-related endpoint data, or have 4 

### neither bioassays nor cancer-related endpoint data. Bioassays without benzo[a]pyrene were 5 

### considered in the weight of evidence evaluation for individual PAHs (Section 6.1). Studies that 6 

### provided only information on PAH mixtures or PAH me tabolites were not reviewed or 7 

### summarized for this analysis. 8 

### References in the database were sorted by keyword i nto the following major categories: 9 

### cancer bioassays, in vivo studies of cancer-relatedendpoints, and in vitro studies of cancer10 

### related endpoints. These categories were further d ivided by route (for bioassays) or by endpoint 11 

### (for cancer-related endpoints). Each study was rev iewed, and critical study details were 12 

### extracted into tables (Tables 4-1 through 4-14) for each individual endpoint. Studies with data 13 

### on selected PAHs and benzo[a]pyrene were used, even if a particular PAH has not been 14 

### evaluated by U.S. EPA or IARC for carcinogenicity. Studies were included in the analysis if the 15 

### following selection criteria were met: 16

17



### • Benzo[a]pyrene was tested simultaneously with anoth er PAH; 18

19



### • A statistically increased incidence of tumors was o bserved with benzo[a]pyrene 20 

### administration; 21

22



### • Benzo[a]pyrene produced a statistically significant change in a cancer-related endpoint 23 

### finding; 24

25



### • Quantitative results were presented; 26

27



### • The carcinogenic response observed in either the be nzo[a]pyrene- or other PAH-treated 28 

### animals at the lowest dose level was not saturated (i.e., tumor incidence at the lowest 29 

### dose was <90%); and 30

31



### • There were no study quality concerns or potential c onfounding factors that precluded use 32 

### (e.g., no concurrent control, different vehicles, s trains, etc. were used for the tested PAH 33 

### and benzo[a]pyrene; use of cocarcinogenic vehicle; PAHs of questionable purity; 34 

### unexplained mortality in treated or control animals ). 35

3664 DRAFT – DO NOT CITE OR QUOTETable 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAHRecordnumber ReferenceMouseastrain Exposure Follow up VehiclePromoter Tumor typePositiveresult Negative resultMeets selectioncriteria? CommentsComplete carcinogenicity studies480 Bingham and Falk,1969CH3/He 3 times/wk 50 wk Tolueneor n-dodecaneNone Malignantand benignBaA No BaP administered in different vehicle.n-Dodecane cocarcinogenic with BaA.No concurrent untreated, toluene or ndodecane control.600 Habs et al., 1980 NMRI 2 times/wk(4 times forCO) for lifeuntilmoribund ordeadAcetone(DMSOfor CO)None Papilloma,carcinoma,sarcomaBbFBkF, BjF, CPcdP,CO, IPYes22390 Wynder andHoffmann, 1959aSwiss 3 times/wk 6–14 mo cyclohexaneNone Papilloma,carcinomaBbF, BjF BghiF, BkF NoDeaths prior to first tumor appearance.No concurrent control.19320 LaVoie et al., 1979 HA/ICRSwissalbino3 times/wk Unspecified Acetone None Unspecified CH,BbF,BjF,DBaeP,DBahP,DBaiPAC, Pyr, BghiF,BkF, AA, BeP,DBelP, IP,BghiP, N23ePNo Reiterates data published elsewhere.22400 Wynder andHoffmann, 1959bSwiss 3 times/wk 10–22 mo Acetone None Papilloma,carcinomaCH,DBahA,DBaiPAC, BeP, Pyr, FANoDeaths prior to first tumor appearance.Not clear if BaP administeredsimultaneously. No concurrentcontrol.13640 Cavalieri et al., 1983Swiss 2 times/wkfor 48 wkUntil 2 cmtumor or61 wkAcetone None Papilloma,adenoma,carcinomaCPcdP Yes Reports both incidence andmultiplicity.13650 Cavalieri et al.,1981bSwiss 2 times/wkfor 30 wkUntil 2 cmtumor,moribund or57 wkAcetone None PrimarilysquamouscellcarcinomaCPcdP ACEP Yes Tumor incidence not useable becauseBaP tumor incidence was 100%.Tumor multiplicity data available fordose-response assessment.620 Hoffmann andWynder, 1966Ha/ICR/Mil Swiss3 times/wkfor 12 moUp to 15 moDioxaneNone Papillomas DBaeP,DBahP,DBaiP,DBaeFYes Paper in German. Paper reportscompound as DBalP; LaCassagne etal. (1968) state that it is actuallyDBaeF. DBahP incidence ≥ 90% atlowest dose.17660 Cavalieri et al., 1977Swiss 2 times/wkfor 30 wkUntilmoribund,dead, orafter 70 wkAcetone None Papilloma,keratoacanthoma,carcinomaDBahP,AABaA Yes DBahP incidence ≥ 90% at lowestdose.610 Higginbotham et al.,1993Swiss 2 times/wk 40 wk Acetone None Papilloma,carcinomaDBalP No No tumors with BaP.19760 Masuda andKagawa, 1972Ha/ICR/Mil Swiss3 times/wkfor60 applications7 months DioxaneNone Unspecified DBalP No No concurrent untreatedor vehiclecontrol; lowest dose DBalP gave100% incidence.18570 Hecht et al., 1974 Ha/ICR/Mil Swiss3 times/wkfor 17 mo72 wk Acetone None Unspecified CH No BaP dose notreported.65 DRAFT – DO NOT CITE OR QUOTETable 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAHRecordnumber ReferenceMouseastrain Exposure Follow up VehiclePromoter Tumor typePositiveresult Negative resultMeets selectioncriteria? Comments21310 Shubik et al., 1960 Syriangoldenhamster2 times/wkfor 10 wk75 wk MineraloilNone None DBahA, BaA No Small number of animals (5/sex/dose).23310 Pfeiffer and Allen,1948Rhesusmonkeyvarious Various SesameoilNone Various Multiple No Sequential exposure to multiplecompounds; no concurrent untreatedcontrol.23840 Barry et al., 1935 Unspecified2 times/wk 1–2+ yr BenzeneNone Epithelioma,papillomaMultiple No Test compounds from various sourcesgave differing results; purity may besuspect; use of benzene vehicleconfounds tumorigenicity results; nobenzene or untreated control.Initiation studies24800 Nesnow et al., 1984 SENCARSingle 31 wk Acetone 12-O-tetradecanoylphorbol-13-acetate(TPA) 2 μg2 times/wkfor 30 wkPapilloma BeAC,BlACYes Reports both incidence andmultiplicity.21410 Slaga et al., 1978 CD-1 Single 27 wk AcetoneTPA 10 μg2 times/wkfor 26 wkPapilloma BaA Yes Tumor incidence data not useablebecause BaP gave 93% tumorincidence. Tumor multiplicity dataavailable for dose-responseassessment.630 LaVoie et al., 1982 Crl:CD1[ICR]BR10 subdosesevery other dUnspecified Acetone TPA 3times/wk20 wkPrimarilysquamouscellpapillomaBbF, BjF,BkFYes Reports both incidence andmultiplicity.16310 Weyand et al., 1992 Crl:CD-15 or10 applications givenevery other dUntilpromotioncompleteAcetone TPA 2.5 μg3 times/wkfor 20 wkUnspecified BjF Yes Tumor incidence data not useablebecause BaP gave 100% tumorincidence. Tumor multiplicity dataavailable for dose-responseassessment. DNA adducts,mutagenicity also evaluated.10200 El-Bayoumy et al.,1982Crl:CD1[ICR]BR10 subdosesevery other dUnspecified Acetone TPA 2.5 μg3 times/wkfor 25 wkPrimarilysquamouscellpapillomaCH Pery, Pyr Yes Tumor incidence data not useablebecause single dose CH gave 100%tumor incidence; BaP gave 90% tumorincidence. Tumor multiplicity dataavailable for dose-responseassessment.18570 Hecht et al., 1974 Ha/ICR/Mil Swiss10 subdosesevery other dUntilpromotioncompleteAcetone TPA 2.5 μg3 times/wkfor 20 wkUnspecified CH Yes Reports both incidence andmultiplicity.66 DRAFT – DO NOT CITE OR QUOTETable 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAHRecordnumber ReferenceMouseastrain Exposure Follow up VehiclePromoter Tumor typePositiveresult Negative resultMeets selectioncriteria? Comments22500 Van Duuren et al.,1966ICR/HA Single 63 wk Acetone Croton resin,25 μg3 times/wkPapilloma,carcinomaCH, BbF BghiF No BaP gave 100% tumor incidence.Corollary data with acetone only aspromotion agent not included.24300 Rice et al., 1985 CD-1 10 subdosesevery other dUntilpromotioncompleteAcetone TPA0.0025%3 times/wkfor 20 wkUnspecified CH,CPdefCYes Tumor incidence data not useablebecause all compounds gave >90%tumor incidence. Tumor multiplicitydata available for dose-responseassessment.19320 LaVoie et al., 1979 HA/ICRSwissalbino10 subdosesevery other dUntilpromotioncompleteAcetoneordioxaneTPA 2.5 μg3 times/wkfor 20 wk orcroton oil2.5%3 times/wkUnspecified CH,DBaeP,DBahP,DBaiP,N23ePFA, AA, DBelP,BghiP, IPNo Reiterates data published elsewhere.21420 Slaga, et al., 1980 Sencar Single 15 wk Acetone TPA 2 μg2 times/wkPapilloma CH,DBahA,BeP, DBacA Yes Not clear if BaP done simultaneouslybut protocol, vehicle, follow up same.Reports both incidence andmultiplicity.15640 Raveh et al., 1982 Sencar Single 25 wk UnspecifiedTPA 2 μg2 times/wkfor 25 wkPapilloma CPcdP Yes Reports both incidence andmultiplicity.620 Hoffmann andWynder, 1966Ha/ICR/Mil SwissSingle 6 mo DioxaneCroton oil Papillomas DBaeF,DBaeP,DBahP,DBaiP,N23ePIP, AA, BghiP,DBelPYes Paper reports compound as DBalP;LaCassagne et al. (1968) state that it isactually DBaeF.610 Higginbotham et al.,1993Sencar Single 27 wk Acetone TPA2.6 nmol,2 times/wkPapillomas,fewcarcinomasDBalP No No tumors with BaP.13660 Cavalieri et al., 1991Sencar Single 16 wk and27 wk (twoexperiments)Acetone TPA3.24 nmol2 times/wkfor 11 wkPrimarilypapillomaDBalP Yes Tumor incidence data not useablebecause lowest dose DBalP gave>90% tumor incidence. Tumormultiplicity data from bothexperiments available for doseresponse assessment.19360 LaVoie et al., 1985 Crl:CD/1(ICR)BR10 subdosesevery other dUnspecified Acetone TPA 2.5 μg3 times/wkfor 20 wkUnspecified AC Yes13650 Cavalieri et al.,1981bCD-1 10 subdosesevery other d57 wk Acetone TPA0.017 μmol2 times/wkfor 40 wkPapilloma CPcdP ACEP Yes Reports both incidence andmultiplicity.67 DRAFT – DO NOT CITE OR QUOTETable 4-1. Study summaries: dermal bioassays of benzo[a]pyrene and at least one other PAHRecordnumber ReferenceMouseastrain Exposure Follow up VehiclePromoter Tumor typePositiveresult Negative resultMeets selectioncriteria? Comments20830 Roe, 1962 Albino Single UntilpromotioncompleteAcetone Croton oilonce/wk for20 wkPapilloma PH No BaP not simultaneous.16440 Wood et al., 1980 CD-1 Single 27 wk AcetoneTPA 16 nmol2 times/wkfor 26 weeksUnspecified Pyr, CPcdP Yes17450 Brune et al., 1978 NMRI Unspecified Unspecified UnspecifiedTPA Unspecified AC No Study design not reported.Resultsreported qualitatively.18680 Hoffmann et al.,1972Ha/ICR/Mil Swiss10 subdosesevery other dUntilpromotioncompleteAcetone Croton oil2.5% for20 wkUnspecified FA Yes19420 LaVoie et al., 1981 HA/ICRSwissalbino10 subdosesevery other dUnspecified Acetone TPA 2.5 μg3 times/wkfor 20 wkUnspecified PH Yes13660 Cavalieri et al., 1991SENCARSingle 27 wk Acetone None PrimarilypapillomaDBalP Yes Initiating dose only; no promoter.Tumor incidence data not useablebecause lowest dose DBalP gave>90% tumor incidence. Tumormultiplicity data available for doseresponse assessment.15700 Rice et al., 1988 CD-1 10 subdosesevery other d24 wk Acetone TPA 2.5 μg3 times/wkfor 20 wkUnspecified CH,BbcAC,CPdefCYes Not clear if BaP done simultaneouslyfor all PAHs.Cocarcinogenicity studies18700 Horton andChristian, 1974C3H 2 times/wkfor 80 wk82 wk n-Dodecane/decalinmixtureNone Carcinoma,papillomaDBacA,PyrCH, FA, Tphen,Pery,No Not clear if BaP done simultaneously.Experiments with decalin(noncarcinogen) and 50/50 decalin/dodecane mix (cocarcinogenic). Nodata for BaP in 50/50 mix. No vehiclecontrol in decalin.21430 Slaga et al., 1979 CD-1 Single 30 wk AcetoneTPA 10 μg2 times/wkfor 30 wkPapilloma BeP NoNo concurrent control. Study aimed atexploring interactions; not clear if BaPdone simultaneously.21840 Van Duuren andGoldschmidt, 1976ICR/HaSwiss3 times/wk 368 or 440 dAcetone None Papilloma Pyr, BghiP, BeP,FAYes21850 Van Duuren et al.,1973ICR/HA 3 times/wkfor 52 wk52 wk Acetone None None Pyr, BghiP, BeP No Qualitative results reported.21920 Warshawsky et al.,1993C3H/HEJ2 times/wk Until lesiondeveloped or104 wkTolueneor n-dodecaneNone Unspecified AC,CH, Pyr, FA,PHNo No tumors with BaP.aExcept where noted, all studies were conducted in mice.68 DRAFT – DO NOT CITE OR QUOTE1Table 4-2. Study summaries: intraperitoneal bioassays of benzo[a]pyrene and at least one other PAHRecordnumberReference Mouse strainaExposureFollowupVehicleTargetorgan(s)Tumor type(s)PositiveresultNegativeresultMeets selectioncriteria?CommentsNewborn mouse studies13610 Busby et al.,1984SwissWebsterBLU:Ha(ICR)1st, 8th, 15th d 26 wk DMSO Lung Adenoma,adenocarcinomaFA Yes Tumor incidence data not useablebecause lowest dose BaP gave >90%tumor incidence. Tumor multiplicitydata available for dose-responseassessment.17560 Busby et al.,1989SwissWebsterBLU:Ha(ICR)1st, 8th, 15th d 26 wk DMSO Lung Adenoma,adenocarcinomaFA Pyr, CH Yes Reports both incidence and multiplicity.640 LaVoie et al.,1987CD-1 1st, 8th, 15th d 52 wk DMSO Lung,liverAdenoma,hepatomaBbF, BjF BkF, IP Yes7510 LaVoie et al.,1994CD-1 1st, 8th, 15th d 12 mo DMSO Lung,liverFoci, adenoma,carcinomaFA Yes Reports both incidence and multiplicity.22040 Weyand andLaVoie, 1988CD-1 1st, 8th, 15th d NotreportedDMSO Lung,liverUnspecified NotreportedNo Abstract only; dose-response informationnot included.22510 Wislocki et al.,1986CD-1 1st, 8th, 15th d 12 mo DMSO Lung,liver,lymphaticsystemAdenoma,carcinoma,lymphomaCH, BaA Pyr Yes Reports both incidence and multiplicity.Studies in adult A/J mice11190 Mass et al., 1993A/J Single 8 mo TricaprylinLung Adenoma,carcinomaBjAC No Reiterates data reported elsewhere(Record 24590).23960 and23450Nesnow et al.,1998a, 1995A/J Single 8 mo TricaprylinLung Adenoma BbF,DBahA,CPcdPNo Reiterates data reported elsewhere(Record 24590).22670 Nesnow et al.,1996A/J Single 8 mo TricaprylinLung Adenoma BbF,DBahA,CPcdPNo (Reiterates data reported elsewhere(Record 24590).)24590 Nesnow et al.,1998bA/J Single 8 mo TricaprylinLung Adenoma CPcdP,BbF,DBahA,BjAC,DBalPYes Raw data obtained courtesy of S.Nesnow. Tumor incidence for BaP was100% at lowest dose with significantincrease over control; tumor multiplicitydata available.20920 Ross et al., 1995A/J Single 240 d TricaprylinLung Adenoma BbF,DBahA,CPcdPPyr No Reiterates data reported elsewhere(Record 24590).aAll studies were conducted in mice.Comment [JS1]:HCL: Indicatetwhich studies reported incidence andmultiplicity data.69 DRAFT – DO NOT CITE OR QUOTE1Table 4-3. Study summaries: subcutaneous bioassays of benzo[a]pyrene and at least one other PAHRecordnumber Reference Species StrainExposuresite ExposureFollowup VehicleTargetorgan(s)Tumortype(s)PositiveresultNegativeresultMeets selectioncriteria? Comments23840 Barry et al., 1935 Mouse UnspecifiedUnspecified Single 1–2+ yr Lard InjectionsiteSarcoma Multiple No Test compounds fromvarious sources gavediffering results; puritymay be suspect; nountreated control.220 Bryan andShimkin, 1943Mouse C3H Right axilla Single until20 mmtumorTricaprylin InjectionsiteUnspecified DBahA NoNo concurrent untreatedcontrol.18350 Grant and Roe,1963Mouse Albino Neck 1st d afterbirth52–62 wkAqueousgelatinLung Adenoma PH Yes23200 Homburger et al.,1972Hamster Various Groin Single 52 wk Tricaprylin Injectionsite; lungVarious BaA No Study aimed atevaluating strainspecificity oftumorigenicity. BaAresults equivocal. Notclear if BaP treatmentsimultaneous. "Aged"mice used as controls;aged mice allowed tolive 16 weeks longer.660 Pfeiffer, 1977 Mouse NMRI Neck Single 114 wk Tricaprylin InjectionsiteSarcoma DBahA No Less than 10% of100 control mice aliveat 114 wk; control datanot provided.23310Pfeiffer and Allen,1948Monkey Rhesus Various Various variable Sesame oil Various Various Multiple No Sequential exposure tomultiple compounds; noconcurrent untreatedcontrol.24290 Rask-Nielson,1950Mouse Street Thymus,lung,mammaryareaSingle 30 mo Paraffin Various Various DBahA No Number of control andexposed varies bytumor type reported;BaP nontumorigenic;DBahA resultsequivocal; resultsunclear.24310 Roe and Waters,1967Mouse SwissalbinoNotspecified1st d afterbirth50–60 wkAqueousgelatinLiver Hepatoma PH No Study methodology andresults not detailed; PHresults equivocal.21560 Steiner, 1955 Mouse C57BL InterscapularSingle 22–28 moTricaprylin InjectionsiteSarcoma DBahA,BaA, CHAC, PH No No concurrent untreatedcontrol; study aimed atevaluating interactions.270 DRAFT – DO NOT CITE OR QUOTE1Table 4-4. Study summaries: oral bioassays of benzo[a]pyrene and at least one other PAHRecordnumberReference SpeciesStrainExposurerouteExposure Follow upTargetorgan(s)Tumortype(s)PositiveresultNegativeresultMeets selectioncriteria?Comments17280 Biancifiori and Caschera,1962Mouse BALB/c Gavage 2 times/wk,15 wkVariable;50–60 wkMammaryglandCarcinomasandsarcomasDBahANo Tumors observed after DBahA onlyin pseudopregnant mice, not virginmice.23880 Huggins and Yang, 1962 Rat SpragueDawleyGavage Single NotreportedMammaryglandUnspecifiedBaA, PH No Untreated control information notincluded.271 DRAFT – DO NOT CITE OR QUOTE1Table 4-5. Study summaries: other route bioassaysof benzo[a]pyrene and at least one other PAHRecordnumberReference Species Strain Exposure route Exposure Follow upVehicleTargetorgan(s)Tumor type(s)PositiveresultNegativeresultMeetsselectioncriteria?Comments21750 Topping et al., 1981 Rat F344 Implantation intransplantedtracheasReleasefrom pellet28 mo BeeswaxpelletTrachealepitheliumCarcinoma,sarcomaBeP No Interactioninformationincluded.17620 Cavalieri et al.,1988bRat SpragueDawleyIntramammillary Single 20 wk None Mammary Adeno-carcinoma,adenofibroma,fibrosarcomaDBahA,BaANo Control datafrom untreatedmammaryglands of samerats.13660 Cavalieri et al., 1991Rat SpragueDawleyIntramammillary Single Until 2 cmtumor or24 wkTrioctanoinMammary,otherAdenocarcinoma,adenofibroma,fibrosarcoma,squamous cellcarcinomaDBalP No DBalPproducedtumors in allanimals at thelowest dose.21620 Sugiyama, 1973 Rat Long EvansIntramuscular Single 9 mo Sesame oil InjectionsiteSarcoma BaA NoBaP gave 100%tumorincidence.20280 Pataki and Huggins,1969Rat SpragueDawleyIntravenous 3 doses 3 dapart98 d LipidemulsionMammary Unspecified BaA No No controlgroup.17940 Deutsch-Wenzel etal., 1983Rat OsborneMendelLung implantation Releasefrom pelletUntilmoribundor deadBeeswax/trioctanoinLung Carcinoma,sarcomaBbF,BjF,BkF,IP, AA,BghiPBeP Yes22000 Wenzel-Hartung etal., 1990Rat OsborneMendelLung implantation Releasefrom pelletUntilmoribundor deadBeeswax/trioctanoinLung Carcinoma CH,DBahAPH Yes21500 Solt et al., 1987 Hamster SyriangoldenPainting buccalpouch2 times/wkfor 20 wkUp to44 wkParaffin oilBuccalpouchCarcinoma BaA No Fewer than 20animals pergroup; negativeresult.23910 Nikonova, 1977 Mouse A Subcutaneous (F0)and transplacental(F1)GD 18 or191 yr SunfloweroilLung,mammary,liver,injectionsiteAdenoma Pyr No Transplacentalexposure notquantified.272 DRAFT – DO NOT CITE OR QUOTE1Table 4-6. Study summaries: in vivo DNA adducts with benzo[a]pyrene and at least one other PAHRecordnumberReferenceRoute ofadministrationExposurefrequencyHours betweendosing andsacrificeTissue analyzedMethod ofanalysisPAHs evaluatedaMeets selectioncriteria?Comments6210 Arif et al., 1997Intramammillary Single dose 48 Mammaryepithelium, lung[32P] postlabelingDBalP Yes17420 Brookes andLawley, 1964Dermal Single dose various to ~12 d Skin [3H]- prelabeling DBacA, DBahA No Data on individualcompounds notreported.17630 Cavalieri et al.,1981aDermal Single dose 4, 24 Skin [3H] or [14C]prelabelingCPcdP, ACEP Yes18810 Hughes andPhillips, 1990Dermal Single dose 0.5, 1, 2, 4, 7, 21,84 dSkin, lung [32P] postlabelingDBalP, DBaeP,DBahP, DBaiPYes 24-hr experiment with DBaeP andDBalP; 84-d experiment with all.18790 Hughes andPhillips, 1991Dermal Single dose 24 Skin [32P] postlabelingDBaeP No No quantitative information; abstractonly.10900 Koganti et al.,2000Oral-diet 14 d not stated Lung [32P] postlabelingBcFE, BaFE, BbFE No Not quantified.13200 Li et al., 2002 Oral-gavage ororal-diet1 time/d for 1–4 d; diet 14 dMammary glandand liver; lung[32P] postlabelingBcFE No Not quantified; BaP administered bygavage, BcFE admin in diet.11190 Mass et al.,1993Intraperitoneal Single dose 24, 48, 72 Lung [32P] postlabelingBjAC Yes8010 Nesnow et al.,1993bIntraperitoneal Single dose 1, 3, 7, 14, 28, 56 d Lung, liver,peripheral bloodlymphocytes[32P] postlabelingBbF Yes Peaks differ temporally; study alsocorrelates number of adducts inorgans.22670 Nesnow et al.,1996Intraperitoneal Single dose 7 d Lung [32P] postlabelingBbF, DBahA,CPcdPNo Not quantified.23960 Nesnow et al.,1995Intraperitoneal Single dose 7 d Lung [32P] postlabelingBbF, DBahA,CPcdPNo Not quantified.24590 Nesnow et al.,1998aIntraperitoneal Single dose various to 21 d Lung [32P] postlabelingBbF, CPcdP,DBahA, DBalPYes Used data from Ross et al., 1995 (ref20920) to calculate slope.22810 Phillips et al.,1979Dermal Single dose 19, 24, 48, 72, 96,120, 144Skin [3H]-Prelabeling BaA, DBacA,DBahAYes20650 Reddy et al.,1984Dermal 4 doses (0, 6,30, 54 hr24 Skin [32P] postlabelingAC, BaA, BghiP,BeP, CH, DBacA,DBahA, Pery, PyrNo Semiquantitative data only.20920 Ross et al.,1995Intraperitoneal Single dose 0, 1, 3, 5, 7, 14, 21 dLung [32P] postlabelingBbF, CPcdP,DBahANo Reiterates data published elsewhere(see 24590)16310 Weyand et al.,1992Dermal Single dose 24 Skin [32P] postlabelingBjF No Not quantified.22040 Weyand andLaVoie, 1988Intraperitoneal Postnatal d 1,8, 1524 Lung, liver [32P] postlabelingBbF, BjF, BkF No No quantitative data; abstract only.73 DRAFT – DO NOT CITE OR QUOTETable 4-6. Study summaries: in vivo DNA adducts with benzo[a]pyrene and at least one other PAHRecordnumber ReferenceRoute ofadministrationExposurefrequencyHours betweendosing andsacrifice Tissue analyzedMethod ofanalysis PAHs evaluatedaMeets selectioncriteria? Comments24790 Kligerman etal., 2002Intraperitonealand oralSingle dose 7 d Peripheral bloodlymphocytes[32P] postlabelingBaA, BbF, CH Yes Data in both rats and mice.aPositive findings were reported for all PAHs evaluated.74 DRAFT – DO NOT CITE OR QUOTE1Table 4-7. Study summaries: in vivo clastogenicity or sister chromatid exchange with benzo[a]pyreneand atleast one other PAHRecordnumberReference Species StrainRoute ofadministrationVehicle ExposureHours betweendosing andsacrificeTissueanalyzedClastogenicendpointPositiveresultsNegativeresultsMeetsselectioncriteria? Comments24740 Allen et al., 1999Mice A/J orp53 +/+,+/-, and-/-Intraperitoneal Tricaprylin Single 48 or 72 hr Bonemarrow orperipheralbloodMicronucleiDBalP Yes14270 He and Baker,1991Mice HRA/SkhhairlessDermal Acetone Single 24 hr KeratinocytesMicro-nucleiCH Pyr Yes17190 Bayer, 1978 Hamsters Chinese IntraperitonealTricaprylin Single 24 hr foraberrations; 30 hrfor micronucleiBonemarrowGaps,breaks,micronuclei,SCEsPH (highdoseonly)Yes19030 Katz et al., 1981 Mice B6C3F1/BRIntraperitoneal DMSO At 0 and24 hrvarious; 24, 30,48, 72 hr after lastdoseBonemarrowmicronucleiDBaiP,AC,BghiP, PyrNo No quantitative data.24720 Kligerman et al.,1986Mice C57BL6 Gavage Corn oil Single 23.5–25 hr PeripheralbloodSCEs BlAC Yes24790 Kligerman et al.,2002Mice andratsCD-1Swissmice; CDratsOral andintraperitonealSunflowerseed oilSingle 7 dWhole bloodor mononuclearleukocytesSCE,micronucleiBaA,BbF, CHYes All positive for SCEvia intraperitonealadministration;mixed results for oraladministration.20200 Oshiro et al.,1992Mice CD-1 Peroral PEG 1 time/d,4 d24 hr after 2ndand 4th treatmentPeripheralbloodMicronucleiPyr, AC No No quantitative data;published as abstract.20230 Paika et al., 1981Mice CBA/J Intraperitoneal DMSO single 16–20 hr BonemarrowSCEs Pyr No No quantitative data.20950 RoszinskyKocher et al.,1979Hamsters Chinese Intraperitoneal Tricapryline2 doses24 hrapart24 hr after 2ndtreatmentBonemarrowSCEs,aberrationsPH, CH,DBahA,BaA,BbF, BePAC Yes Positive results forSCEs, notaberrations.21050 Salamone et al.,1981Mice B6C3F1Intraperitoneal Notspecified2 doses24 hrapart24, 48, 72 hr after2nd treatmentBonemarrowMicronucleiAC, Pyr Yes21770 Tsuchimoto andMatter, 1981Mice CD-1 Intraperitoneal DMSO 2 doses24 hrapart6 hr after 2ndtreatmentBonemarrowMicronucleiPyr Yes21390 Sirianni andHuang, 1978Mice C3H/St V79 cells in diffusion chamberimplanted inperitonealcavity of miceChinesehamster V79cellsSCEs AC, Pyr,PeryYes21620 Sugiyama, 1973 Rats LongEvansIntravenous LipidemulsionSingle 12, 24 hr BonemarrowGaps,breaksBaA Yes75 DRAFT – DO NOT CITE OR QUOTE1Table 4-8. Study summaries: in vivo mutagenicitywith benzo[a]pyrene and at least one other PAHRecordnumberReference Species/strainRoute ofadministrationExposurefrequency/follow upMutagenic endpointPositiveresultNegativeresultMeetsselectioncriteria? Comments18130 Fahmy and Fahmy,1980DrosophilamelanogasterSuspension inmedia48–72 hr Somatic mutation; eye colormosaicismBaA Yes13980 Frolich and Wurgler,1990D. melanogasterSuspension inmedia48–72 hr Somatic mutation andrecombination test(SMART); wing spotsBaA No Inconsistent results for BaA; significanteffects only seen with cross-breeding ofstrains selected for enhanced metabolicactivity (not standard strains).11190 Mass et al., 1993 A/J mice Intraperitoneal 3d/8 mo Mutations in codon 12 ofthe Ki-ras oncogene;polymerase chain reaction(PCR) and DNA sequencingof lung tumor DNABjAC No Quantitative dose-response data werenot available. Different mutationsequences observed; GGT→TGT forBaP and GGT→CGT for BjAC;mutation sequence for BjAC maycorrelate with cyclopenta-adductformation.23960 Nesnow et al., 1995 A/J mice IntraperitonealSingle injection/8 moMutations in codon 12 ofthe Ki-ras oncogene; PCRand DNA sequencing oflung tumor DNABbF,DBahA,CPcdPNo Quantitative dose-response data werenot available. GGT→TGT mutationsfor BaP and BbF; GGT→CGT forCPcdP; no mutations seen for DBahA.22670 Nesnow et al., 1996 A/J mice IntraperitonealSingle injection/8 moMutations in codon 12 ofthe Ki-ras oncogene; PCRand DNA sequencing oflung tumor DNABbF,DBahA,CPcdPNoQuantitative dose response data were notavailable. GGT→TGT mutations forBaP and BbF; GGT→CGT for CPcdP;no mutations seen for DBahA.24590 Nesnow et al., 1998bA/J mice Intraperitoneal Single injection/8 moMutations in codons 12 and61 of the Ki-ras oncogene;PCR and DNA sequencingof lung tumor DNABbF,DBahA,CPcdP,BjAC,DBalPNo Quantitative dose-response data werenot available. Mutations in codon 12,GGT→TGT for BaP, BbF, and DBalP;GGT→CGT for CPcdP and BjAC; nomutations seen for DBahA; GTTmutations seen for all other PAHs. OnlyDBalP caused mutations in codon 61.21370 Simmon et al., 1979 Swiss WebstermicePAHsintramuscular orperoral;microorganismsintraperitonealSingle injection/4 hr Intraperitoneal hostmediated assay;mutagenicity inS.typhimuriumandS.cerevisiaeof recoveredmicroorganismsAC, BaA,BeP, CH,PHNo Assay was not considered sensitiveenough for detecting carcinogens.21830 Valencia andHoutchens, 1981D. melanogasterFilter feeding 48–72 hr Sex-linked recessive lethaltestPyr No Results were negative for BaP.22450 Zijlstra and Vogel,1984D. melanogasterAbdominalinjectionNot applicable Sex-linked recessive lethaltest; 2–3 translocation andring-X lossBaA No Results were negative for BaP.76 DRAFT – DO NOT CITE OR QUOTE1Table 4-9. Study summaries: in vitro bacterial mutagenicity with benzo[a]pyrene and at least one other PAHRecordnumberReference Salmonella strain(s) Activation system Positive result Negative resultMeetsselectioncriteria?Comments17030 Andrews et al.,1978TA100, TA1527,TA1538Ar S9 and others AA, DBahA, DBajA, DBacA, BghiP,BePYes TA100 results include BaP.23830 Baker et al., 1980 TA100 Guinea pig MC S9and othersDBaiP, BaA, DBacA, DBahA Yes23660 Bartsch et al., 1980 TA100, TA1535,TA98Rat MC S9 BaA Yes17380 Bos et al., 1988 TA98, TA100 Rat Ar S9 PH, Pyr Yes Qualitative data for other PAHs (no BaP);quantitative data with BaP comparison forPH and Pyr in TA100.9560 Carver et al., 1985 TA98, TA100 S9 Pery No The response varied at differentconcentrations of S9; BaP was more potentat low S9 while Pery was more potent athigh S9.17590 Carver et al., 1986 TA100Ar rat and Ar hamsterS9BaA, BghiF, Pery Yes Qualitative data also presented for otherPAHs. S9 concentration varied;400 μL/plate optimal.17630 Cavalieri et al.,1981aTM677 Ar S9 CPcdP, ACEP, Pyr Yes BaP data from previous publication used.Dose-response data not provided for Pyr.9620 Chang et al., 2002 TA100 Rat Ar S9 BghiF, BcPHYes24030 De Flora et al., 1984TA1535, TA1537,TA1538, TA98,TA100Rat AR S9 BaA, Pery, BeP AC Yes13860 Devanesan et al.,1990TA100, TA98 Rat Ar S9 DBaeP, DBalP No No concurrent control.18030 Dunkel et al., 1984 TA1535, TA1537,TA1538, TA98,TA100Rat, mouse, hamsterAr S9BaA, BeP, PH, Pyr AC No Dose-response data not provided.18050 Eisenstadt andGold, 1978TA1537, TA100 Rat Ar S9 CPcdP Yes18180 Florin et al., 1980 TA98, TA100 Rat Ar and MCS9 BaA, CH, Pery, CO Yes24080 Gibson et al., 1978 TA1535, TA1537,TA1538, TA98Nonenzymatic(gamma radiation)BaA, BghiP, CH, FE, Pyr DBahA, AC,Pic, TphenYes AN, PH also tested; toxicity interfered withmutagenicity testing.14080 Gold andEisenstadt, 1980TA100 Rat MC S9 CPcdP Yes BaP and CPcdP maximal responsesoccurred at different S9 levels.14170 Guthrie et al., 1982 TA98, TA100 Rat Ar S9 compare toPGS from ramseminal vesiclesBaA, CH No BaP tested in TA98, BaA and CH tested inTA100.14260 Hass et al., 1981 TA98, TA100 Rat Ar S9BeP Yes77 DRAFT – DO NOT CITE OR QUOTETable 4-9. Study summaries: in vitro bacterial mutagenicity with benzo[a]pyrene and at least one other PAHRecordnumberReference Salmonella strain(s) Activation system Positive result Negative resultMeetsselectioncriteria? Comments18650 Hermann, 1981 TA98 Rat Ar S9 BbA, BaA, CH, FA, Tphen, BeP,DBacA, DBahA, BbF, Pery, DBalP,DBaiP, AA, COAC, PH, FE,Pyr, BbFEYes10670 Johnsen et al., 1997 TA98 Rat control or PB S9 BjAC, BlAC Yes19000 Kaden et al., 1979 TM677 Rat Ar or PB S9 AN,ANL, Pyr, BbFE, CPcdP, BaA, CH,Tphen, FA, BeP, Pery, BghiP, AA,DBacA, DBahA, DBbeFFE, AC, PH, Pic,COYes Mutagenic activity relative to BaP reported.24680 Lafleur et al., 1993 TM677 Ar PMS CPcdP, APA,ACEA, CPhiAPA,CPhiACEAYes19320 LaVoie et al., 1979 TA98, TA100 Rat Ar S9 BeP, Pery Yes Several other PAHs were evaluated, butnotconcurrent with BaP.19360 LaVoie et al., 1985 TA98, TA100 Rat Ar S9AC Yes23650 McCann et al., 1975TA1535, TA1537,TA98, TA100Rat Ar S9 DBaiP, BeP, DBacA, DBahA, CH, BaA Pyr, AC, PH, FEYes15170 Norpoth et al., 1984TA100 Rat and mouse S9;induction by ClophenA50 and 18 PAHsBaA No S9 composition was different for BaA andBaP; result cannot be compared.20220 Pahlman andPelkonen, 1987TA100 S9 from control, MC,or TCDD treated ratsand miceBaA, CH, Tphen, DBacA, DBahA AN, AC, PH,FE, Pyr, BeP,Pery, PCEYes20530 Penman et al., 1980 TM677 Rat Ar or PB S9 Pery, CPcdP, DBacA No No concurrent control values were reported.20450 Phillipson andIoannides, 1989TA100 S9 isolated frommouse, hamster, rat,pig, and humanBaA, DBaiP, DBahA Yes20490 Poncelet et al., 1978TA1530, TA1535,TA1537, TA1538,TA98, TA100S9 (origin unknown) CO, Tphen, FA, BghiP BbF No Qualitative data reported in publishedabstract.20560 Probst et al., 1981 TA1530, TA1535,TA1537, TA1538,TA98, TA100Rat Ar S9 BbA, DBacA AC, DBahA,PH, Pyr, DBaiPNo Data reported as minimum mutagenicconcentration (nmol/mL).20880 Rosenkranz andPoirier, 1979TA1530, TA1535 Uninduced rat S9AC, BaA, BeP,CH, PHYes21000 Sakai et al., 1985 TA97, TA98, TA100 Rat Ar S9 FE (equiv.), AC, PH, FA, CH, Pyr, BeP,Pery, BghiP, COYes21040 Salamone et al.,1979aTA1535, TA1537,TA1538, TA98,TA100Rat Ar S9 BaA, BeP (equiv), BghiP, DBaiP, BPH,CH, CO, DBacA, PCEAC, BaFE,BbFE, FA, Pery,PyrNo Increase in spontaneous mutation rate wasindicated, but dose data were not provided.78 DRAFT – DO NOT CITE OR QUOTETable 4-9. Study summaries: in vitro bacterial mutagenicity with benzo[a]pyrene and at least one other PAHRecordnumberReference Salmonella strain(s) Activation system Positive result Negative resultMeetsselectioncriteria? Comments13260 Salamone et al.,1979bTA98, TA100 Rat Ar S9 DBaiP No Dose-response datawere not completelyreported; maximal response information(dose and number of revertants) waspresented in text; BaP max response atdifferent S9 than DBaiP.11860 Sangaiah et al.,1983TA1535, TA1537,TA1538, TA98,TA100Rat Ar S9 BjAC Yes Dose-response data for BaP waspresentedfor TA98 only.21360 Simmon, 1979a TA1535, TA1536,TA1537, TA1538,TA98, TA100Rat Ar S9 BaA, BeP AC, CH, PH Yes21640 Teranishi et al.,1975TA1535, TA1536,TA1537, TA1538S9 from rats treatedwith PB and MC orDBahADBaiP, DBaeP DBahA, BaA,BePYes16180 Utesch et al., 1987 TA100 Intact orhomogenizedhepatocytes from Artreated ratsBaA Yes16440 Wood et al., 1980 TA98, TA100 Rat Ar S9 andpurified MFOenzymes systemCPcdP YesAr = Arochlor 1254-treated; MC = 3-methylcholanthrene-treated; PB = phenobarbital-treated; PMS = postmitochondrial supernatant79 DRAFT – DO NOT CITE OR QUOTE1Table 4-10. Study summaries: in vitro mammalian mutagenicity assays with benzo[a]pyrene and at leastoneother PAHRecordnumberReference Cell type Metabolic activation Mutagenesis assayPositiveresultNegativeresultMeets selectioncriteria? Comments16900 Allen-Hoffmann andRheinwald, 1984Human epidermalkeratinocyteNone 6-Thioguanine resistance (HPRT) BaA Yes16920 Amacher and Paillet,1982Mouse lymphoma cells(L5178Y)Syrian golden hamster S9 mix orcocultivated hamster hepatocytesTrifluorothymidine resistance(thymidine kinase locus [TK])BaA Yes16930 Amacher and Paillet,1983Mouse lymphoma cells(L5178Y)Cocultivated rat hepatocytes Trifluorothymidine resistance (TK) BaA Yes16940 Amacher and Turner,1980Mouse lymphoma cells(L5178Y)S9 from eight rodent species orstrain; one rat strain induced by ArTrifluorothymidine resistance (TK) AC, BaA Yes ACdata notuseable; BaPnotsimultaneous16910 Amacher et al., 1980 Mouse lymphoma cells(L5178Y)Rat Ar and noninduced S9 Trifluorothymidine resistance (TK) BaA AC, Pyr Yes13440 Baird et al., 1984 V79 Chinese hamster cellsHamster embryo cells 6-Thioguanine resistance (HPRT) BeP Yes17140 Barfknecht et al., 1982TK6 human lymphoblast cellsRat Ar S9 Trifluorothymidine resistance (TK) FA, BaA,CH, Tphen,CPcdPPH, AC,ACEPYes24670 Durant et al., 1999 H1A1v2 humanlymphoblastoid cellsTransfected with cyp1a1 cDNA Trifluorothymidine resistance (TK) BaPery,BbPery,DBaeF,DBafF,DBahP,DBaiP,DBelP,N23aP,N23ePDBjlF,N12bFYes18260 Gehly et al., 1982 C3H/10T1/2 clone 8 mousefibroblast cellsNone Ouabain resistance (HPRT) BeP Yes14250 Hass et al., 1982 V79 Chinese hamster cells Hamster embryo cells Ouabain and 6-thioguanineresistance (HPRT)DBaiP,DBahPYes18750 Huberman, 1975 V79 Chinese hamster cells Hamster cells 8-Azaguanine resistance (HPRT) BaA, PyrYes18740 Huberman and Sachs,1976V79 Chinese hamster cells Hamster embryo cells Ouabain and 8-azaguanineresistance (HPRT)DBacA,DBahA(both weak)Pyr, PH,CH, BaAYes24120 Huberman and Sachs,1974V79 Chinese hamster cells Hamster embryo cells 8-Azaguanine resistance (HPRT) BaA Yes18990 Jotz and Mitchell,1981Mouse lymphoma cells(L5178Y)Rat Ar S9 Trifluorothymidine resistance (TK) Pyr Yes24720 Kligerman et al., 1986 Mouse lymphoma cells(L5178Y)Rat Ar S9 Trifluorothymidine resistance (TK) BlACYes80 DRAFT – DO NOT CITE OR QUOTETable 4-10. Study summaries: in vitro mammalian mutagenicity assays with benzo[a]pyrene and at leastoneother PAHRecordnumberReference Cell type Metabolic activation Mutagenesis assayPositiveresultNegativeresultMeets selectioncriteria? Comments19180 Krahn andHeidelberger, 1977V79 Chinese hamster cells Rat MC S9 6-Thioguanine resistance (HPRT) BaA,DBacA,DBahAYes DBacA andDBahA datanot useable;treatmentdifferent thanBaP.24680 Lafleur et al., 1993 MCL-3 humanlymphoblastoid cellsTransfected with cyp1a2 andcyp2a6 cDNATrifluorothymidine resistance (TK) CPcdP,ACEA,CPhiACEAAPA,CPhiAPA, BghiFYes24170 Langenbach et al.,1983V79 Chinese hamster cells Cocultivation with primary rodentcells from liver, lung, kidney, andbladderOuabain resistance (HPRT) AC Yes7550 Li and Lin, 1996 HS1 HeLa cells (humanepithelial cells)None 6-Thioguanine resistance (HPRT) BaA Yes19870 Mishra et al., 1978 Fischer rat embryo cellsinfected with Rauscherleukemia virusRat Ar S9 Ouabain resistance (HPRT) AC, PH,Pyr, BePYes20040 Myhr and Caspary,1988Mouse lymphoma cells(L5178Y)Rat Ar and noninduced S9 Trifluorothymidine resistance (TK) AC, BaA,BePNo Resultsreported asranges.11450 Nesnow et al., 1984 V79 Chinese hamster cellsRat Ar S9 6-Thioguanine resistance (HPRT) BlAC,BeAC,BjACYes15630 Raveh and Huberman,1983V79 Chinese hamster cells Hamster embryo fibroblasts 6-Thioguanine resistance(HPRT);phorbol myristate acetate used toenhance recoveryCPcdP BaA Yes15640 Raveh et al., 1982 V79 Chinese hamster cellsHamster embryo fibroblasts Ouabain and 6-thioguanineresistance (HPRT)CPcdP Yes Mutagenicitycorrelated withskin tumorinitiation.21410 Slaga et al., 1978 V79 Chinese hamster cellsHamster embryo cells Ouabain resistance (HPRT) BaA(weak)Yes21720 Tong et al., 1983 Rat liver epithelial cells(ARL-18)6-Thioguanine resistance (HPRT) BaA,BeP, PyrNo Repeats datafrom 21730Tong et al.,1981b21730 Tong et al., 1981b Rat liver epithelial cells(ARL-18)None 6-Thioguanine resistance (HPRT) BeP, Pyr,BaAYes81 DRAFT – DO NOT CITE OR QUOTETable 4-10. Study summaries: in vitro mammalian mutagenicity assays with benzo[a]pyrene and at leastoneother PAHRecordnumberReference Cell type Metabolic activation Mutagenesis assayPositiveresultNegativeresultMeets selectioncriteria? Comments16190 Vaca et al., 1992 UV-sensitive Chinese hamsterovary (CHO) cellsRat Ar S9 6-Thioguanine resistance (HPRT) FA Yes21900 Wangenheim andBolcsfoldi, 1988Mouse lymphoma cells(L5178Y)Rat Ar S9 Trifluorothymidine resistance (TK) Pyr, FE YesHPRT = hypoxanthine-guanine phosphoribosyl transferase mutagenicity assay (resistance to 6-thioguanine, 8-azaguanine, or ouabain); TK = thymidine kinasemutagenicity assay (resistance totrifluorothymidine)82 DRAFT – DO NOT CITE OR QUOTE1Table 4-11. Study summaries: in vitro morphological/malignant cell transformation with benzo[a]pyrene andat least one other PAHRecordnumberReference Cell type Metabolic activation systemPositive result Negative resultMeets selectioncriteria? Comments13390 Atchison et al., 1985 BALB/3T3 mouse embryo fibroblasts None FA, Pyr Yes17610 Casto, 1979 Syrian golden hamster embryo cells None DBahA Pyr Yes17730 Chen andHeidelberger, 1969Adult C3H mouse ventral prostate cells Cocultivatedirradiated C3Hmouse embryonic fibroblastsDBahA DBacA, Pyr No Control data not provided.24750 Davis, 1999 C3H10T1/2 cells None DBalP, DBaeP,BcC, BgC, BcPHNo Control data not provided.17970 DiPaolo et al., 1969 Syrian golden hamster embryo cells Cocultivated irradiatedSprague-Dawley rat fetalcellsDBahA, BaA,BeP, DBacAPyr, PH Yes17990 DiPaolo et al., 1972 BALB/3T3 None AC, Pyr Yes23630 DiPaolo et al., 1973 Syrian golden hamster embryo cells In vivo (transplacental)exposureAC, PH, Pyr No No quantitative information.18020 Dunkel et al., 1981 Balb/3T3, Syrian golden hamster embryo,and Rauscher murine leukemia virusinfected F344 rat embryo cellsNone BaA BeP, PH, AC Yes Qualitative data only forR-MuLVRE cells. BaA positive in SHEM,equivocal in Balb/3T3.18080 Emura et al., 1980 Syrian golden hamster fetal lung cells None BbF, BaA, IP BkF, BeP Yes23640 Evans and DiPaolo,1975Strain 2 guinea pig fetal cells None AC, Pyr, PH No No quantitative information.18260 Gehly et al., 1982 C3H10T1/2CL8 mouse embryofibroblastsNone BeP Yes14130 Greb et al., 1980 BHK 21/CL 13 Rat Ar S9 CH,BaA, BbF,DBahA, BePPH, AC Yes23890 Kakunaga, 1973 BALB/3T3 subclone A31-714 NonePH, Pyr No Not clear if BaP adminsimultaneously.14640 Krolewski et al., 1986C3H10T1/2CL8 mouse embryo fibroblastsNone CPcdP Yes14700 Laaksonen et al., 1983Newborn NMRI nu/nu nude mouse skinfibroblastsNone BaA AC Yes14850 Lubet et al., 1983 C3H10T1/2CL8 mouse embryofibroblastsNone BeP AC, DBahA, PHYes19870 Mishra et al., 1978 Rauscher leukemia virus-infected Fischerrat embryoNone AC, PH, Pyr,BePNo No quantitative information.24710 Mohapatra et al., 1987C3H10T1/2CL8 mouse embryo fibroblastsNone BeAC, BjAC,BlACBkAC Yes24700 Nesnow et al., 1990 Human neonatal foreskin fibroblasts None BlAC Yes7980 Nesnow et al., 1997 C3H10T1/2CL8 mouse embryofibroblastsNone DBalP Yes7990 Nesnow et al., 1994 C3H10T1/2CL8 mouse embryofibroblastsNone DBahA Yes8000 Nesnow et al., 1993a C3H10T1/2CL8 mouse embryofibroblastsNone DBkmnoAPH DBjmnoAPH,N123mnoAPHYes20120 Nesnow et al., 1991 C3H10T1/2CL8 mouse embryofibroblastsNone ACEA Yes83 DRAFT – DO NOT CITE OR QUOTETable 4-11. Study summaries: in vitro morphological/malignant cell transformation with benzo[a]pyrene andat least one other PAHRecordnumberReference Cell type Metabolic activation systemPositive result Negative resultMeets selectioncriteria? Comments23720 Pienta et al., 1977 Syrian golden hamster embryo Cocultivated X-irradiatedcells of same typeBaA, DBahA CH, BeP, Pyr,AC, DBacA, PHYes8490 Sheu et al., 1994 BALB/3T3 A31-1-1 None Pyr,BaA, CH Yes84 DRAFT – DO NOT CITE OR QUOTE1Table 4-12. Study summaries: in vitro DNA adductswith benzo[a]pyrene and at least one other PAHRecordnumberReference Cell type or DNA sourceIncubationtime Activation system Method of analysisPAHsevaluatedaMeets selectioncriteria? Comments16890 Allen and Coombs,1980Mouse embryo cells from TO mice24 hr None [3H] prelabeling BaA Yes6300 Binkova et al., 2000 Human diploid lung fibroblastcellsVarious up to24 hrNone [32P] postlabeling DBalP Yes9510 Bryla and Weyand,1992Calf thymus DNA 1 hr None [32P] postlabeling BaA, DBacA,PHYes PH did not form measurableDNA adducts. Adductformation enhanced whenreacted under white light.6570 Cherng et al., 2001 Human hepatoma HepG2 cells24 hr None [32P] postlabeling BghiP Yes BghiP did not form measurableDNA adducts.13780 Cooper et al., 1982 Fibroblasts and epithelial cellsfrom Wistar rat mammary tissue24 hr None [3H] prelabeling BaA Yes BaA formed little or nomeasurable DNA adducts.22800 Grover and Sims, 1968Salmon testes DNA Not specified Rat liver microsomes [3H] prelabeling DBahA, DBacA,BaA, Pyr, PHYes10660 Johnsen et al., 1998 Human lymphocytes and humanpromyelocytic HL-60 cells24 hr None [32P] postlabeling BjAC, BlAC Yes10670 Johnsen et al., 1997 Rat lung Clara cells, Type 2 cells,and macrophages2 hr PCB pretreatment ofwhole animals[32P] postlabeling BjAC, BlAC Yes13200 Li et al., 2002 MCF-7 cells or rat lung DNA 7–24 hr Human mammarymicrosomes with ratlung DNA[32P] postlabeling DBalP, BcPH,DBahANo No quantitative results.7870 Melendez-Colon et al.,2000Human mammary carcinomaMCF-7 cells and leukemia HL-60cells4 or 24 hr None [32P] postlabeling DBalP Yes No adducts formed in HL-60cells that lack significant P450activity.7990 Nesnow et al., 1994 C3H10T1/2CL8 fibroblasts 24 hr None32P] postlabeling DBahA No No quantitative results.20120 Nesnow et al., 1991 C3H10T1/2 cells 24 hr None32P] postlabeling ACEA NoMeasures repair of adducts only,not synthesis.21200 Segerback andVodicka, 1993Calf thymus DNA 3 hr Rat Ar S932P] postlabeling,3HbindingCH, BaA, BbF,DBahA, FA,BghiP, PyrYes24810 Baird et al., 2002 MCF-7 cells 24 hr Morpholinosinhibition (antisenseoligomer that blocksprotein synthesis ofCYPIA1)[32P] postlabeling DBalP No Confounded by CYP1A1inhibition by morpholinos.aExcept where noted, positive findings were reportedfor all PAHs evaluated.85 DRAFT – DO NOT CITE OR QUOTE1Table 4-13. Study summaries: in vitro DNA damage,repair, or synthesis with benzo[a]pyrene and at least oneother PAHRecordnumberReference Cell type Metabolic activationEndpoint Assay Positive resultNegativeresultMeets selectioncriteria?Comments16840 Agrelo and Amos,1981Human fibroblasts Rat Ar S9 Unscheduled DNAsynthesis[3H] ThymidineuptakePyr Yes17610 Casto, 1979 Syrian golden hamsterembryoIntrinsic Unscheduled DNAsynthesis[3H] ThymidineuptakeDBahA, Pyr,PHYes24030 De Flora et al., 1984Escherichia coliWP2,WP67, and CM871Rat Ar S9 DNA damage Differentialkilling repairdeficient strainsAC, BaA Pery, BeP No Semiquantitativedata.18030 Dunkel et al., 1984E. coliWP-2uvrARat, mouse, hamsterAr S9DNA damage Differentialkilling repairdeficient strainsBaA, BeP, PH, Pyr AC No Dose-responsedata notprovided.23790 Ichinotsubo et al.,1977E. coliRec BCS9 (origin unknown) DNA damage DBaiP, DBahA Yes10670 Johnsen et al., 1997 Rat lungClara cells, Type 2cells, and macrophagesPCB pretreatment ofwhole animalsDNA damage Alkaline elution BjAC, BlAC No No untreatedcontrol.10660 Johnsen et al., 1998 Human lymphocytes andhuman promyelocytic HL60 cellsRat or human livermicrosomesDNA damage Alkaline elution BjAC, BlAC Yes19270 Lake et al., 1978 Human foreskin epithelialcellsNone Unscheduled DNAsynthesis[3H] ThymidineuptakeDBahA AC, BeP, PH,PyrNo Doses reportedas ranges.19680 Mamber et al., 1983E. coliWP2 and WP100 Rat Ar S9 DNA damage Growthinhibition ofrepair deficientstrainsAC, FE, Pyr Yes19690 Mane et al., 1990 Human and rat mammaryepithelial cellsNone Inhibition of DNAsynthesis[3H] ThymidineuptakeBaA (in human MEConly)BeP NoPositive responsefor BaA notobservedconsistently.19730 Martin andMcDermid, 1981HeLa S3 cells PB-induced rat liverpostmitochondrialsupernatantUnscheduled DNAsynthesis[3H] ThymidineuptakePyr (authors:“dubious” result)AC No No quantitativeinformation.19740 Martin et al., 1978 HeLa S3 cells 3-MC induced ratliverpostmitochondrialsupernatantUnscheduled DNAsynthesis[3H] ThymidineuptakeBeP, BaA, DBacA,DBahAPyr, AC Yes23800 McCarroll et al.,1981E. coliWP2, WP2 uvrA,WP67, CM611, WP100,W3110polA+, andp3478polaRat Ar S9 DNA damage Differentialkilling repair-deficient strainsAC, PH Yes86 DRAFT – DO NOT CITE OR QUOTETable 4-13. Study summaries: in vitro DNA damage,repair, or synthesis with benzo[a]pyrene and at least oneother PAHRecordnumberReference Cell type Metabolic activationEndpoint Assay Positive resultNegativeresultMeets selectioncriteria? Comments19830 MerschSundermann et al.,1992E. coliPQ37 Rat Ar S9 Induction of SOSsystemSOS chromotest AA, BaA, BbF, BghiF,BjF, BbFE, BghiP,BeP, CH, DBacA,DBahA, DBalP,DBahP, DBaiP, FA,IP, PH, TphenAC, BaFE,CO, FE, Pery,PyrYes19850 Milo et al., 1978 Human skin fibroblast NFand Detroit 550 cellsNone DNA damage Alkaline elution AC, Pyr, PH,BePYes20050 Nagabhushan et al.,1990Hamster buccal pouchepithelial cells and tissuefragmentsNot specified Inhibition of DNAsynthesis[3H] ThymidineuptakeBaA No Abstract only.BaA inhibitedsynthesis 4%.20560 Probst et al., 1981 Rat hepatocyte primarycultureNone Unscheduled DNAsynthesis[3H] ThymidineuptakeBbA, DBacA AC, DBahA,PH, Pyr,DBaiP, FE,BePNo Artifact ofcounting methodresulted incontrol responsesreported asnegative values.20810 Robinson andMitchell, 1981Human fibroblasts WI-38cellsRat Ar S9 Unscheduled DNAsynthesis[3H] ThymidineuptakePyr (with activation) Yes23900 Rosenkranz andLeifer, 1980E colipol A1- Rat liver S9 DNA damage Differentialkilling repairdeficient strainsAC, BaA, BeP,CH, PHYes20880 Rosenkranz andPoirier, 1979E. colipol A1-Uninduced rat S9 DNA damage Differentialkilling repairdeficient strainsAC, BaA, BeP,CH, PHYes20940 Rossman et al.,1991E coliWP2s(λ) Rat liver S9 DNA damage Λ prophageinductionAC, DBacA, DBahA,PHBeP, FA, Pyr Yes21380 Simmon, 1979bSaccharomyes cerevisiaeD3Rat Ar S9 inducedrecombinationColonypigmentation onadenine mediumAC, BaA, BeP,CH, PHYes21720 Tong et al., 1983 Rat hepatocyte primarycultureNone Unscheduled DNAsynthesis[3H] ThymidineuptakeBaA BeP, AC, CH,PyrNo Repeats datafrom 21730 Tonget al., 1981b.21730 Tong et al., 1981b Rat hepatocyte primarycultureNone Unscheduled DNAsynthesis[3H] ThymidineuptakeBaA BeP, AC, CH,PyrYes21790 Tweats, 1981E. coliWP2, WP67(uvrApolA), CM871 (uvrA lexArecA)Rat Ar S9 DNA damage Differentialkilling repairdeficient strainsPyr, AC No No quantitativeinformation.16190 Vaca et al., 1992 CHO cells Rat Ar S9 DNA damage Alkaline elution FA No No untreated orvehicle control.22260 Williams et al.,1982Rat hepatocyte primarycultureNone Unscheduled DNAsynthesis[3H] ThymidineuptakePyr, BeP No No quantitativeinformation.87 DRAFT – DO NOT CITE OR QUOTE1Table 4-14. Study summaries: in vitro clastogenicity or sister chromatid exchange with benzo[a]pyrene and atleast one other PAHRecordnumberReference Cell type Metabolic activationClastogenicendpoint(s)PositiveresultsNegativeresultsMeets selectioncriteria? Comments16740 Abe and Sasaki, 1977Pseudodiploid ChineseHamster D-6None Aberrations and SCEsAC, Pyr Yes17890 Dean, 1981Near-diploid epithelialtype rat liver RL1None Various aberrations AC, Pyr No Semiquantitative results.17930 DeSalvia et al., 1988Male Chinese hamsterliver epithelial cells(CHEL)None SCEs Pyr, FA Yes18120 Evans and Mitchell,1981CHO Rat Ar S9 SCEs Pyr (withactivation)No No untreated or vehicle control.23640 Evans, and DiPaolo,1975Diploid strain 2 guineapig fetal cellsNone Aneuploidy AC No No quantitative data. Pyr,PH alsoevaluated using different protocol withoutBaP reference.18260 Gehly et al., 1982CH3/10T1/2 Clone 8mouse fibroblastsNone SCEs BeP Yes14620 Kochhar, 1982Chinese hamster V79 None Aberrations includinggaps, rings, breaks,fragments, exchangesBaA Yes Dose-dependent increase in % cells withaberrations.14640 Krolewski et al., 1986CH3/10T1/2 Clone 8mouse embryo cellsNone SCEs CPcdP Yes CPcdP appears to increase SCEsin dosedependent fashion (two doses).19690 Mane et al., 1990Chinese hamster V79cellsWith and without rat mammary epithelial cell cocultureSCEs BaA BeP Yes19770 Matsuoka et al., 1979Male Chinese hamsterlung (CHL)Rat Ar S9 Aberrations and SCEsPH No Not clear if BaP administered simultaneously. No untreated control.20020 Murison, 1988P3 clonal isolate fromhuman epithelialteratocarcinomaBJ-015 human breastepithelial cell cocultureSCEs CPcdP BeP No Not clear if BaP administeredsimultaneously; no concurrent control.20340 Perry and Thomson,1981CHO cells Rat Ar S9 SCEs Pyr AC No No untreated control.88 DRAFT – DO NOT CITE OR QUOTETable 4-14. Study summaries: in vitro clastogenicity or sister chromatid exchange with benzo[a]pyrene and atleast one other PAHRecordnumberReference Cell type Metabolic activationClastogenicendpoint(s)PositiveresultsNegativeresultsMeets selectioncriteria?Comments20500 Popescu et al., 1977Chinese hamsterV79-4 cellsWith or without irradiatedSyrian golden hamstersecondary embryo feeder cellsAberrations and SCEsPery, Pyr PH No BaP increased SCEs but Pyr and Peryincreased aberrations. Pery increased aberrations w/o activation. 60% of Pyr treatedcells (activated) polyploid. Increasedaberrations in polyploid cells.21710 Tong et al., 1981aAdult rat liverepithelial (ARL 18)cellsNone SCEs BaA BeP, Pyr,ACYes21720 Tong et al., 1983Adult rat liverepithelial (ARL 18)cellsNone SCEs BaA BeP, Pyr,ACNo Repeats data from 21710 Tong et al.,1981a8780 Vienneau et al., 1995UDP-Glucuronosyltransferases-deficientrat (RHA-J/J) skinfibroblastsNone Micronuclei BeP Yes8850 Warshawsky et al.,1995Human lymphocytes None Micronuclei and SCEsBaA Yes21980 Weinstein et al., 1977Human diploidfibroblasts (WI-38)With or without rat Ar s9 Chromosomaldamage, mitoticindex, abnormalmetaphasesPyr Yes

### 89 DRAFT – DO NOT CITE OR QUOTE

If the above criteria were met, studies were select ed for use in the analysis regardless of 1 

### whether positive or negative results were reported. Studies with positive findings were used for 2 

### calculation of RPFs. Studies with negative finding s were used in a weight of evidence 3 

### evaluation of potential carcinogenicity (discussed later in Section 6.1). To be considered 4 

### adequate for use in the analysis, nonpositive bioas says were selected only if two additional 5 

### conditions were met: (1) at least 20 animals were used per dose group, and (2) animals were 6



### observed for at least 6 months. More strict criter ia were applied to nonpositive studies due to the 7 

### difficulty in demonstrating the absence of an effec t. For example, if a positive tumor response 8 

### (i.e., statistically significant increase in incide nce) was observed after 3 months of treatment with 9 

### a given PAH, the positive finding is clear; however , if no response (or a nonsignificant response) 10 

### was observed after 3 months, the absence of respons e might reflect a lack of carcinogenic action, 11 

### but might also have resulted from inadequate follow -up time. The use of these additional criteria 12 

### for nonpositive studies served to ensure that PAHs would not be treated as noncarcinogenic 13 

### based on inadequate nonpositive bioassays. 14 

### Study design details, findings, limitations, and a determination of whether the study met 15 

### selection criteria are presented in Tables 4-1 thro ugh 4-14 for each study reviewed in each 16 

### category. Positive and negative findings as report ed in the table are based on the author’s 17 

### determination except where noted. When statistical analysis of tumor bioassay data was not 18 

### included in the pertinent publication, statistical analysis was conducted to determine whether the 19 

### response differed from control. In the sections th at follow, overviews of the data available in 20 

### each category are presented. The overviews address the nature of the studies available, concise 21 

### information on general study methods, general findi ngs for the tested compounds, and key 22 

### strengths and limitations of the available data for relative potency development. 23

24 

### 4.3.1. In Vivo Cancer Bioassays in Animals 25 

### The PAH database contained a large number of cancer bioassay studies in which one or 26 

### more PAHs was evaluated along with benzo[a]pyrene. The vast majority of the tumor bioassay 27 

### studies were mouse skin painting studies (n = 43). In addition, there were 11 intraperitoneal 28 

### studies, 9 subcutaneous exposure studies, 2 oral st udies, and 9 studies using miscellaneous 29



### exposure routes. 30

31



### 4.3.1.1. Dermal Exposure 32 

### A summary of the 43 dermal bioassays is provided in Table 4-1. These studies were all 33 

### conducted in mice. Fifteen studies tested the comp lete carcinogenicity of PAHs, while 34



### 23 studies tested PAHs as initiators in initiation- promotion protocols. In some cases, both 35 

### complete and initiation-promotion studies were repo rted in the same reference. For these 36 

### references, two entries are included in the table. 37

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Complete carcinogenicity studies were conducted in mice using either dropper or 1 

### paintbrush application. Swiss mice were typically preferred for these studies. PAHs, usually in 2 

### acetone, were applied to the shaved interscapular s kin 2 or 3 times/week. The duration of 3 

### exposure varied from 10 weeks up to about 70 weeks; most studies continued exposure for at 4



### least 30 weeks. Skin tumor counts were recorded on a weekly basis, and animals were sacrificed 5 

### when tumors reached a minimum size (e.g., 2 cm) or when the animals were moribund. These 6 

### studies generally focused exclusively on skin papil lomas and carcinomas. Skin tumor data were 7 

### reported as incidence (i.e., number of animals with tumors) and/or tumor count (mean number of 8 

### tumors per animal) (indicated in Table 4-1). 9 

### Several PAHs consistently (in two or more studies) proved to be complete carcinogens in 10 

### mouse skin painting assays, including benzo[b]fluor anthene, benzo[j]fluoranthene, 11 

### cyclopenta[c,d]pyrene, dibenzo[a,e]pyrene, dibenzo[ a,h]pyrene, dibenzo[a,i]pyrene, and 12 

### dibenzo[a,l]pyrene. Chrysene gave positive results in two complete carcinogenicity studies 13 

### (LaVoie et al., 1979; Wynder and Hoffmann, 1959) an d equivocal results in a third (Hecht et al., 14 

### 1974). Anthanthrene, dibenzo[a,e]fluoranthene, and dibenz[a,h]anthracene each gave positive 15 

### tumorigenicity results in a single assay (Cavalieri et al., 1977; Hoffmann and Wynder, 1966; and 16 

### Wynder and Hoffmann, 1959; respectively). Negative or equivocal results were reported for 17 

### benzo[k]fluoranthene, benzo[g,h,i]fluoranthene, dib enzo[e,l]pyrene, indeno[1,2,3-c,d]pyrene, 18 

### benzo[g,h,i]perylene, naphtho[2,3-e]pyrene, anthrac ene, pyrene, fluoranthene, 2,3-acepyrene, 19 

### benz[a]anthracene, coronene, and benzo[e]pyrene (se e Table 4-1). 20 

### According to LaCassagne et al. (1968), in studies c onducted prior to 1966, the compound 21 

### reported as dibenzo[a,l]pyrene was actually dibenzo [a,e]fluoranthene. In the text and tables of 22 

### this report, data from Hoffmann and Wynder (1966) a re reported as dibenzo[a,e]fluoranthene in 23



### Table 4-1. 24 

### The initiation studies in Table 4-1 were performed under a generally consistent protocol, 25 

### as follows. During the early part of the second te logen phase of the hair cycle (at about 7–8 26 

### weeks of age), PAHs in acetone were applied to the shaved interscapular skin of mice. In 27 

### general, female Swiss, CD-1, or SENCAR mice were us ed. Some studies used dropper 28 

### administration, but the majority employed a paintin g method using a camel’s hair brush. About 29 

### half of the initiation studies used a single initia tion dose, while the other half administered the 30 

### initiating compound in 10 subdoses given every othe r day. One to 2 weeks after the final 31 

### initiating dose, promotion was begun with twice or thrice weekly applications of a promoting 32 

### agent, usually TPA (12-0-tetra-decanoylphorbol-13-a cetate) or croton oil. The dose of the 33 

### promoting agent varied by study. Promotion usually continued for about 20 weeks (with a range 34 

### across studies from 11 to 26 weeks). The incidence of skin papillomas was recorded on a weekly 35 

### basis until the promotion period was ended. Papill omas were removed at random for histological 36 

### verification. Some studies reported the number of tumors per animal; some reported only the 37



### incidence. 38

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The initiation studies in Table 4-1 consistently sh owed positive tumorigenicity across two 1 

### or more studies for the following compounds: benzo [j]fluoranthene, benzo[b]fluoranthene, 2 

### chrysene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, d ibenzo[a,i]pyrene, dibenzo[a,l]pyrene, and 3 

### cyclopenta[d,e,f]chrysene. In at least one study, benzo[k]fluoranthene, benz[l]aceanthrylene, 4 

### benz[e]aceanthrylene, naphtho[2,3-e]pyrene, dibenz[ a,h]anthracene, dibenz[a,c]anthracene, and 5 

### benz[b,c]aceanthrylene showed positive initiating a ctivity. Negative results were reported for 6 

### pyrene, perylene, benzo[g,h,i]fluoranthene, fluoran thene, anthanthrene, dibenzo[e,l]pyrene, 7 

### benzo[g,h,i]perylene, indeno[1,2,3-c,d]pyrene, benz o[e]pyrene, anthracene, 2,3-acepyrene, and 8 

### phenanthrene. Cyclopenta[c,d]pyrene gave negative results in one study—Wood et al. (1980)— 9 

### and positive results in two— Cavalieri et al. (1981 b) and Raveh et al. (1982) (see Table 4-1). 10 

### The vast majority of the initiation and complete ca rcinogenicity studies were conducted 11 

### in female mice, so data on gender differences in sk in tumor susceptibility are not available. 12 

### A few studies using dermal application (Warshawsky et al., 1993; Slaga et al., 1979; Van 13 

### Duuren and Goldschmidt, 1976; Horton and Christian, 1974; Van Duuren et al., 1973) were 14 

### designed to evaluate the cocarcinogenicity of two o r more PAHs, or of a single PAH with 15 

### dodecane as a vehicle. These were primarily comple te carcinogenicity studies, wherein PAHs 16 

### were administered together over a chronic time peri od, although Slaga et al. (1979) used an 17 

### initiation-promotion design. Study design was simi lar to other complete carcinogenicity 18 

### experiments. In these studies, the carcinogenicity of single PAHs was evaluated for comparison 19



### with the results obtained when the PAHs were admini stered with a cocarcinogen. Data on single 20 

### PAHs (without a cocarcinogen) were generally limite d to single dose levels. In the 21 

### cocarcinogenesis studies, only dibenz[a,c]anthracen e, benzo[e]pyrene, and pyrene gave positive 22 

### results when administered without a cocarcinogen; r esults for pyrene were judged to be 23 

### equivocal in the absence of statistical confirmatio n. The PAHs chosen for cocarcinogenesis 24 

### studies were often those traditionally understood t o be nontumorigenic or weakly tumorigenic 25 

### when administered alone (e.g., perylene, pyrene, be nzo[e]pyrene, benzo[g,h,i]perylene, 26 

### phenanthrene, fluoranthene). 27 

### Several issues relating to the potential use of the dermal bioassay data for relative 28 

### potency development were identified during study re view. Several studies did not include a 29 

### concurrent untreated or vehicle-treated control gro up (Masuda and Kagawa, 1972; Bingham and 30 

### Falk, 1969; Wynder and Hoffmann, 1959a, b). In a n umber of reports, it appears that bioassays 31 

### were done in batches and reported in a single publi cation. In these cases, it appears that 32 

### benzo[a]pyrene treatment may not have been undertak en concurrently with all of the compounds 33 

### in the report. For some of these studies (Horton a nd Christian, 1974; Bingham and Falk, 1969), 34 

### there are differences in the choice of vehicle or p romoter, or other issues that argue against using 35 

### the benzo[a]pyrene data for direct comparison. In several others studies, however (Rice et al., 36 

### 1988; Slaga et al., 1980; Van Duuren and Goldschmid t, 1976; Wynder and Hoffmann, 1959), the 37 

### protocols (including vehicle and promoting agent) a ppear to have been the same. 38

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Among the dermal tumor bioassay studies in Table 4- 1, 24 studies met the selection 1 

### criteria for use in this analysis. 2

3



### 4.3.1.2. Intraperitoneal Exposure 4 

### Eleven cancer bioassays in the literature used intr aperitoneal injection. Six of these 5 

### studies were carried out in newborn mice, while the other five used adult A/J mice. All of the 6 

### studies focused on lung and liver tumorigenicity af ter PAH exposure. Study summaries for all of 7 

### these references are reported in Table 4-2. Tumor data were reported as incidence (i.e., number 8 

### of animals with tumors) and/or tumor count (mean nu mber of tumors per animal) (indicated in 9 

### Table 4-2). 10



### Newborn mouse studies. Six cancer bioassays in newborn mice were identifie d (LaVoie 11 

### et al., 1994, 1987; Busby et al., 1989, 1984; Weyan d and LaVoie, 1988; Wislocki et al., 1986). 12 

### In general, PAHs were administered intraperitoneall y to newborn mice (usually of the Swiss or 13 

### CD-1 strains). The dosing schedule called for 1/7 th 

### , 2/7 ths 

### , and 4/7 ths 

### of the total dose to be 14



### administered on the 1 st 

### , 8 th 

### , and 15 th 

### days of life. Typically, the mice were sacrificed at either 6 15 

### months or 1 year, and lung and/or liver tumors were identified and classified. 16 

### The studies in newborn mice showed a distinct gende r difference in liver tumorigenicity. 17 

### Male mice appear to be substantially more susceptib le to liver tumor induction than females. In 18 

### contrast, both male and female mice developed lung tumors after exposure. Three studies 19 

### (LaVoie et al., 1994; Busby et al., 1989, 1984) rep orted that fluoranthene induced lung tumors in 20 

### both male and female mice, while one study reported that fluoranthene induced liver tumors in 21 

### male mice only (LaVoie et al., 1994). LaVoie et al . (1987) reported that benzo[b]fluoranthene 22 

### and benzo[j]fluoranthene induced lung adenomas in b oth male and female mice, but induced 23 

### liver tumors only in males. Wislocki et al. (1986) reported that treatment with benz[a]anthracene 24 

### resulted in a significant increase in liver tumors in male mice. In this study, benz[a]anthracene 25



### treatment resulted in an increased incidence of lun g tumors in both males and females, although 26 

### the tumor incidence was significantly increased onl y for females. The same authors (Wislocki et 27 

### al., 1986) reported a significant increase in liver tumors in male mice treated with chrysene, but 28 

### no increase in lung tumorigenicity. The lack of lu ng tumorigenicity in mice treated with 29 

### chrysene was also reported by Busby et al. (1989). 30 

### Negative tumorigenicity results in newborn mouse as says were reported for pyrene, 31 

### chrysene, benzo[k]fluoranthene, and indeno[1,2,3-c, d]pyrene (Busby et al., 1989; LaVoie et al., 32 

### 1987). 33 

### Most of the data from the newborn mouse assays met the criteria for relative potency 34 

### development, although Weyand and LaVoie (1988) is a n abstract and does not provide dose35 

### response information. LaVoie et al. (1994) noted t hat liver tumorigenicity in newborn mice 36 

### exposed to weak tumorigenic agents may not be fully realized for 12 months; thus, the failure to 37

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observe liver tumors in studies of shorter duration (Busby et al., 1989, 1984) may result from the 1 

### longer latency and should be taken into considerati on in using these data. 2 

### Lung adenoma A/J mouse studies. Five studies (Nesnow et al., 1998a, b, 1996, 1995; 3 

### Ross et al., 1995; Mass et al., 1993) were carried out in 6- to 8-week-old A/J mice by the same 4 

### laboratory using a standard protocol (Table 4-2). Mice were given a single intraperitoneal 5 

### injection of PAH in tricaprylin and followed for 8 months. Upon sacrifice, the lungs were 6 

### removed and adenomas were counted. Tumor multiplic ity was reported, while tumor incidence 7



### was not. Several of these studies include estimate s of relative potency based on statistical 8 

### analysis of the tumor multiplicity data. 9 

### These studies report positive tumor findings (repor ted as an increase in the number of 10 

### tumors per animal) for all of the PAHs tested (benz [j]aceanthrylene, benzo[b]fluoranthene, 11 

### dibenz[a,h]anthracene, cyclopenta[c,d]pyrene, and d ibenzo[a,l]pyrene). 12 

### Among the intraperitoneal tumor bioassay studies in Table 4-2, eight studies met the 13 

### selection criteria for use in this analysis. 14

15



### 4.3.1.3. Subcutaneous Injection Exposure 16 

### Nine studies employing a subcutaneous exposure desi gn were identified. All of the 17 

### subcutaneous exposure studies are more than 25 year s old; the most recent is Pfeiffer (1977). 18 

### Study descriptions are presented in Table 4-3. 19 

### Two studies utilized newborn mice (Roe and Waters, 1967; Grant and Roe, 1963). In 20 

### these studies, phenanthrene was administered subcut aneously to newborn albino mice on the first 21 

### day of life. Ten mice of each group were sacrifice d after 52 weeks, and the remaining animals 22 

### were sacrificed at 62 weeks. Grant and Roe (1963) reported lung tumorigenicity, while Roe and 23 

### Waters (1967) reported liver tumors in the same gro up of mice. Roe and Waters (1967) reported 24



### an elevated incidence of liver tumors in male mice exposed subcutaneously to phenanthrene; 25 

### however, it is not clear whether the difference was significant. Roe and Waters (1967) is a brief 26 

### communication with limited details of the study des ign and results. 27 

### In most of the remaining studies, single subcutaneo us doses of one or more PAHs and 28 

### benzo[a]pyrene were administered to mice, followed 1–2.5 years later by an evaluation of 29 

### injection site and other tumors. Tumors at the inj ection site were most commonly reported; 30 

### however, in some studies, investigators also examin ed other organs for tumors (Homburger et 31 

### al., 1972; Roe and Waters, 1967; Grant and Roe, 196 3; Rask-Nielsen, 1950; Pfeiffer and Allen, 32 

### 1948). 33 

### Most of the subcutaneous bioassays suffer from crit ical shortcomings in design or 34 

### reporting. One study used “aged” mice for controls , allowing these animals to live 16 weeks 35 

### longer than the treated group (Homburger et al., 19 72). Three studies gave apparently positive 36 

### results for dibenz[a,h]anthracene (i.e., substantia l tumor induction; Pfeiffer, 1977; Steiner, 1955; 37 

### Bryan and Shimkin, 1943). However, neither Bryan a nd Shimkin (1943) nor Steiner (1955) 38

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included untreated control groups. Pfeiffer (1977) included an untreated control group in which 1 

### there was 90% mortality prior to sacrifice of the t reated animals; data on tumor incidence in 2 

### controls were not reported. Several other studies (Pfeiffer and Allen, 1948; Barry et al., 1935) 3



### also did not include a concurrent untreated or vehi cle-treated control group. These studies were 4 

### not used for dose-response assessment due to the la ck of appropriate controls. 5



### Fundamental flaws were observed in two older studie s. Pfeiffer and Allen (1948) 6 

### examined the effects of PAHs in Rhesus monkeys. In dividual animals were exposed 7 

### sequentially to several PAHs via multiple exposure routes; thus, the effect of any individual PAH 8 

### or benzo[a]pyrene cannot be discerned. Barry et al . (1935) treated mice with PAHs from varying 9 

### sources and of varying purity. Given the age of th e study and the attendant issues with 10 

### nomenclature, purity, and analysis of the treatment compounds, data from this study are excluded 11 

### from use in relative potency development. 12 

### Among the subcutaneous tumor bioassay studies in Ta ble 4-3, only a single study met 13 

### selection criteria for use in this analysis. 14

15



### 4.3.1.4. Oral Exposure 16 

### The literature search identified two oral bioassays that included benzo[a]pyrene and at 17 

### least one other PAH. Critical aspects of the study design for these studies are reported in 18



### Table 4-4. 19 

### Biancifiori and Caschera (1962) compared the induct ion of mammary tumors in virgin 20 

### and pseudopregnant mice (female mice mated with vas ectomized males) after gavage exposure 21 

### to dibenz[a,h]anthracene or benzo[a]pyrene. Tumor incidence was increased in pseudopregnant 22 

### mice given 1 mg/week of either compound for 15 week s, but not in virgin mice given the same 23 

### dose. The relevance of the positive findings in ps eudopregnant mice is uncertain given that an 24



### increased incidence of tumors was not observed in v irgin mice treated at the same dose. One 25 

### possible explanation for the disparate findings is that circulating hormones in pseudopregnant 26 

### mice differed from those in virgin mice and interac ted with the PAH to enhance tumor 27 

### formation. Huggins and Yang (1962) also evaluated mammary tumor incidence after a single 28 

### oral PAH exposure. Sprague-Dawley rats were given gavage doses of benzo[a]pyrene, 29 

### benz[a]anthracene, or phenanthrene. This study did not include an untreated or vehicle-treated 30 

### control group. No tumors were observed in the rats treated with either benz[a]anthracene or 31 

### phenanthrene, while mammary tumors were observed in eight of the nine benzo[a]pyrene-treated 32



### animals. 33 

### Among the oral tumor bioassay studies in Table 4-4, none met the selection criteria for 34 

### use in this analysis. 35

36

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4.3.1.5. Other Routes 1 

### Nine bioassays were available that did not fit into other exposure route categories (i.e., 2 

### dermal, intraperitoneal, subcutaneous, or oral) (se e Table 4-5). Among these were studies using 3 

### intramammillary, intramuscular, and intravenous inj ection as well as lung implantation, tracheal 4 

### implantation, and transplacental exposure after sub cutaneous injection. Seven studies were in 5 

### rats, with one each in mice and hamsters. 6 

### Deutsch-Wenzel et al. (1983) and Wenzel-Hartung et al. (1990) implanted 7 

### PAH-containing pellets (consisting of beeswax and t rioctanoin) into the lungs of inbred female 8 

### Osborne-Mendel rats. Lung tumor incidence was repo rted for a total of 10 PAHs and 9 

### benzo[a]pyrene. The authors reported relative pote ncy estimates based on the lung tumor data. 10 

### Lung tumors were induced by benzo[b]fluoranthene, b enzo[j]fluoranthene, 11 

### benzo[k]fluoranthene, benzo[g,h,i]perylene, indeno[ 1,2,3-c,d]pyrene, anthanthrene, chrysene, 12 

### and dibenz[a,h]anthracene. Negative findings were reported for benzo[e]pyrene and 13 

### phenanthrene. 14 

### Cavalieri et al. (1991) treated Sprague-Dawley rats with single intramammillary 15 

### injections of dibenzo[a,l]pyrene into the left mamm ary glands and followed them for up to 24 16 

### weeks. Tumors of the mammary gland, mesenchymal ti ssue, or skin were recorded. 17 

### Dibenzo[a,l]pyrene produced tumors in all animals a t both doses. 18 

### In six studies, tumors were not induced after expos ure to any target PAH. 19 

### Intramammillary injection of dibenz[a,h]anthracene and benz[a]anthracene did not induce 20 

### mammary tumors in rats (Cavalieri et al., 1988b). Pregnant mice receiving subcutaneous 21 

### injection of pyrene did not develop tumors, nor did their offspring (Nikonova, 1977). Rats 22 

### treated either intravenously or intramuscularly wit h benz[a]anthracene did not develop either 23 

### mammary or injection site tumors (Pataki and Huggin s, 1969). Similarly, benz[a]anthracene was 24 

### not tumorigenic after intramuscular injection in ra ts (Sugiyama, 1973) or buccal pouch painting 25 

### in hamsters (Solt et al., 1987). Finally, benzo[e] pyrene was not tumorigenic when it was 26 

### implanted into tracheas transplanted subcutaneously into isogenic rats (Topping et al., 1981). 27 

### Among the tumor bioassays that used alternative exp osure routes in Table 4-5, four 28



### studies met the selection criteria for use in this analysis. 29

30 

### 4.3.2. In Vivo Studies of Cancer-Related Endpoints 31 

### The database of cancer-related endpoints measured a fter in vivo exposure to PAHs is 32 

### much smaller than the in vitro database. Endpoints examined after in vivo exposure include 33 

### mutagenicity, DNA adducts, and clastogenicity or si ster chromatid exchange. As with the in 34 

### vitro database, only studies of selected PAHs that included benzo[a]pyrene as a reference 35 

### compound were reviewed. Each study that was review ed for consideration in relative potency 36 

### development is presented in tabular format in subse quent sections. The tables summarize study37 

### specific information and indicate whether a particu lar study is considered useful for dose- 38

### 96 DRAFT – DO NOT CITE OR QUOTE

response assessment. The text provides an overall description of the available studies, including 1 

### a general description of the methodology used for e ach study type, the results, and the 2 

### weaknesses or problems associated with specific stu dies or study types. 3

4



### 4.3.2.1. DNA Adducts 5 

### Eighteen studies evaluating DNA adduct formation fo r PAHs and benzo[a]pyrene were 6 

### identified in the database (Table 4-6). Nine studi es presented quantitative data for DNA adduct 7 

### formation and are discussed below. Among studies w ith data potentially useful for RPF 8 

### derivation, the route of exposure was intramammilla ry injection in one study (Arif et al., 1997), 9 

### intraperitoneal injection in five studies (Kligerma n et al., 2002; Nesnow et al., 1998a, 1996, 10 

### 1995; Ross et al., 1995; Mass et al., 1993), dermal in three studies (Hughes and Phillips, 1990; 11 

### Cavalieri et al., 1981b; Phillips et al., 1979), an d oral in one study (Kligerman et al., 2002). 12 

### Adducts were identified by [ 32 

### P]-postlabeling in all of the studies except for tw o by Phillips et al. 13 

### (1979) and Cavalieri et al. (1981b), which utilized [ 3 

### H]- or [ 14 

### C]-radiolabeled PAHs. Two 14 

### papers described experiments with a single time poi nt(s) at 24 or 48 hours (Arif et al., 1997, 15 

### Hughes and Phillips, 1990), whereas the rest had mu ltiple time points. The duration of exposure 16 

### was as short as 4 hours (Cavalieri et al., 1981b), although 24 hours was usually the first time 17 

### point(s) in time course studies. The longest durat ion for a time course study was 84 days 18 

### (Hughes and Phillips, 1990), but most were 3 weeks or less. The tissues evaluated included 19 

### mammary epithelium (Arif et al., 1997), skin (Hughe s and Phillips, 1990; Cavalieri et al., 1981b; 20 

### Phillips et al., 1979), liver and peripheral blood lymphocytes (Kligerman et al., 2002; Nesnow et 21 

### al., 1993b), and lung (Nesnow et al., 1998a, 1993b; Arif et al., 1997; Ross et al., 1995; Mass et 22 

### al., 1993; Hughes and Phillips, 1990). 23 

### Dermal-exposure studies typically involved applicat ion of the chemical in solution to the 24 

### shaved dorsal skin of mice (Hughes and Phillips, 19 90; Cavalieri et al., 1981b; Phillips et al., 25 

### 1979). After the scheduled sacrifice, the treated skin was excised and frozen; a scalpel was used 26 

### to scrape away the dermis from the epidermis that w as subsequently powdered in liquid nitrogen. 27 

### In one study, the lung was also excised and frozen in liquid nitrogen (Hughes and Phillips, 28 

### 1990). DNA was isolated from the frozen epidermis or lung. Liquid scintillation counting was 29 

### used to quantify DNA adducts to PAH labeled with [ 3 

### H] or [ 14 

### C] (Cavalieri et al., 1981b; Phillips 30 

### et al., 1979). For [ 32 

### P]-postlabeling, DNA was treated to selectively dep hosphorylated 31 

### nonadducted nucleotides; after postlabeling, adduct s were resolved by sequential anion-exchange 32 

### thin layer chromatography on PEI-cellulose plates i n several directions using three solvents 33 

### (Hughes and Phillips, 1990). Adduct spots on chrom atograms were located by autoradiography, 34 

### after which the spots were excised and radioactivit y levels were determined by Cerenkov 35 

### counting. 36

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Compounds administered by intraperitoneal or intram ammillary injection were also 1 

### delivered in solution. As in dermal-exposure studi es, DNA was isolated from frozen tissues and 2 

### adducts were identified by [ 32 

### P]-postlabeling and quantified via autoradiography. 3 

### Most studies reported the mean number of adducts fo rmed within a tissue per unit of 4 

### DNA, with time-course data displayed graphically. Peak values were sometimes called out 5 

### specifically in the text or tables. As the shapes of dose-response curves differ among different 6 

### PAHs, the peak value is an imprecise measure for co mparing the relative adduct-forming 7 

### potency of the different compounds. The TIDAL has also been used for reporting results for a 8 

### time-course study (Ross et al., 1995). The TIDAL v alue is the area under the curve (AUC) for 9 

### adduct persistence (based on the rate of adduct for mation and repair) for the duration of the 10 

### study. The TIDAL value expresses the total DNA add uct burden experienced by the tissue from 11 

### the time of treatment to the end of the study. The TIDAL versus administered dose curve 12 

### provides a convenient way to compare adduct-forming potency for different PAHs in time13 

### course experiments. An important limitation of the TIDAL approach is the inherent assumption 14 

### that the ratios of specific adducts are relatively constant across dose and time course. Ross et al. 15 

### (1995) demonstrated that this assumption was valid for several different PAHs; however, it was 16 

### also noted that two adducts of benzo[a]pyrene in ra t liver did not conform to this general pattern. 17 

### Ross et al. (1995) presented data for lung adenoma incidence (measured at 8 months) in 18 

### several ways: as a function of administered dose, as a function of adduct levels per dose 19 

### measured 24 hours after dosing (results for 3 days postdosing were mentioned but not shown), as 20 

### a function of TIDAL values measured over 21 days (d uring which period adduct levels were 21 

### specifically quantified), and as a function of TIDA L values extrapolated to 8 months. The 22 

### relative tumor induction potencies of the studied P AHs were similar for each assay for a single 23



### PAH when described as functions of administered dos e, the adduct levels per dose at 3 days, the 24 

### TIDAL values over 21 days, or the TIDAL values extr apolated to 8 months. The relative 25 

### potencies for tumor incidence as a function of addu ct levels at 24 hours were not similar to those 26 

### associated with the other measures of exposure. Ro ss et al. (1995) suggested that 27 

### pharmacokinetic differences in adduct formation amo ng the PAHs were responsible for the 28 

### discrepancy, but suggested that peak levels could b e used to compare the potencies of different 29



### PAHs if adduct formation for those PAHs followed si milar kinetics. 30 

### DNA adduct experiments were carried out in replicat e and were usually analyzed 31 

### statistically. It should be noted that, based on t he work of Ross et al. (1995), relative potencies 32 

### determined from studies that administered a single dose level and measured adducts at a single 33 

### time point will be less reliable unless the shapes of the adduct formation curves are similar. 34 

### However, the single dose and single measurement stu dies were also used for dose-response 35



### assessment. 36 

### Among the in vivo DNA adduct studies shown in Table 4-6, nine studies met the 37 

### selection criteria for use in this analysis. 38

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1



### 4.3.2.2. Clastogenicity or Sister Chromatid Exchange Frequen cy 2 

### The database included 13 studies in which clastogen ic effects or frequency of sister 3 

### chromatid exchanges of benzo[a]pyrene and at least one other PAH were tested in whole animal 4 

### systems. Table 4-7 lists the studies along with im portant study design details. The clastogenic 5 

### endpoints measured in these studies were micronucle i, chromosome gaps and breaks, and 6 

### nonspecific aberrations; sister chromatid exchanges were also measured. These studies were all 7 

### conducted in rodents, including mice, rats, and ham sters. 8 

### Eight of the studies evaluated micronuclei, sister chromatid exchanges, or chromosome 9 

### gaps or breaks in bone marrow from treated mice or hamsters (Allen et al., 1999; Katz et al., 10 

### 1981; Paika et al., 1981; Salamone et al., 1981; Ts uchimoto and Matter, 1981; Roszinsky-Kocher 11 

### et al., 1979; Bayer, 1978; Sugiyama, 1973). In the se studies, one or two doses of PAH were 12 

### injected intraperitoneally into the animals, and sa crifice occurred at various time points thereafter 13 

### (typically 24 hours after). Bone marrow smears wer e examined microscopically and scored for 14 

### micronuclei, sister chromatid exchanges, gaps, or b reaks. 15 

### He and Baker (1991) applied multiple dose levels of chrysene or phenanthrene to the skin 16 

### of hairless mice and harvested keratinocytes upon s acrifice 24 hours later. The keratinocytes 17 

### were incubated for 2 days and treated with cytochal asin B to identify binucleated cells. After 18 

### 4 days in vitro, cells were mounted on slides and e xamined microscopically for micronuclei. 19 

### Results were reported as the percent of binucleated cells with one or more micronuclei among 20 

### the total number of binucleated cells scored. Chry sene treatment resulted in a dose-related 21 

### increase in micronuclei, while pyrene did not. 22 

### Kligerman et al. (2002, 1986) measured sister chrom atid exchanges and/or micronuclei in 23 

### the blood of mice or rats given a single dose of PA H either orally or intraperitoneally. The study 24 

### by Oshiro et al. (1992) involved two or four oral d oses of pyrene or anthracene in mice. Blood 25



### obtained from the tail 24 hours after the last trea tment was examined microscopically and 26 

### micronuclei were scored in polychromatic erythrocyt es. In an unusual study design, Sirianni and 27 

### Huang (1978) measured sister chromatid exchanges in V79 cells placed in a diffusion chamber 28 

### implanted in the peritoneal cavity of mice. 29



### Thirteen individual PAHs were evaluated in these st udies. Only chrysene gave positive 30 

### results for more than one endpoint (for sister chro matid exchange and micronucleus frequency; 31 

### He and Baker, 1991; Roszinsky-Kocher et al., 1979). Five other PAHs (phenanthrene, 32 

### dibenz[a,h]anthracene, benz[a]anthracene, benzo[b]f luoranthene, and benzo[e]pyrene) increased 33 

### the frequency of sister chromatid exchange in hamst er bone marrow after intraperitoneal 34 

### administration (Roszinsky-Kocher et al., 1979). Ba yer (1978) also reported an increase in sister 35 

### chromatid exchange frequency in hamster bone marrow after phenanthrene administration (high 36 

### dose only). Anthracene and pyrene consistently gav e negative results in several studies (Oshiro 37 

### et al., 1992; He and Baker, 1991; Katz et al., 1981 ; Paika et al., 1981; Salamone et al., 1981; 38

### 99 DRAFT – DO NOT CITE OR QUOTE

Tsuchimoto and Matter, 1981; Roszinsky-Kocher et al ., 1979; Sirianni and Huang, 1978). 1 

### Dibenzo[a,i]pyrene and benzo[g,h,i]perylene each ga ve negative results in an assay for bone 2 

### marrow micronuclei (Katz et al., 1981). 3 

### Among studies with positive results, only He and Ba ker (1991), Kligerman et al. (1986), 4 

### and Bayer (1978) administered PAHs at multiple dose levels. Bayer (1978) observed a positive 5 

### response only with the highest dose of phenanthrene . Of the single dose studies, only 6 

### Roszinsky-Kocher et al. (1979) reported responses c learly differing from controls. 7 

### Among the in vivo clastogenicity or sister chromati d exchange studies shown in 8 

### Table 4-7, 10 studies met the selection criteria fo r use in this analysis. 9

10



### 4.3.2.3. In Vivo Mutagenicity 11



### The PAH database contains several studies that eval uate specific mutagenic end points 12 

### following in vivo exposure to PAHs (see Table 4-8). These studies include mutagenicity 13 

### experiments in Drosophila melanogaster , an intraperitoneal host-mediated assay using 14 

### Salmonella strains or yeast, and DNA sequence analy sis of specific codons in the Ki-ras 15 

### oncogene in mouse lung tumors. 16 

### Most Drosophila studies administered PAH compounds to either the suspension media or 17 

### to the diet for 48–72 hours prior to cross-mating a nd analysis of mutations (Frolich and Wurgler, 18 

### 1990; Valencia and Houtchens, 1981; Fahmy and Fahmy , 1980). One study used abdominal 19 

### injection as an exposure pathway (Zijlstra and Voge l, 1984). The mutagenic endpoints evaluated 20 

### included somatic mutations (i.e., eye color mosaici sm, wing spots) (Frolich and Wurgler, 1990; 21 

### Fahmy and Fahmy, 1980) or sex-linked recessive leth al mutations (Zijlstra and Vogel, 1984; 22 

### Valencia and Houtchens, 1981). Only two PAHs were evaluated in the Drosophila studies in 23 

### addition to benzo[a]pyrene (benz[a]anthracene and p yrene), and the results were either negative 24 

### or inconsistent in all studies (Frolich and Wurgler , 1990; Zijlstra and Vogel, 1984; Valencia and 25 

### Houtchens, 1981; Fahmy and Fahmy, 1980). A signifi cant effect was seen for benz[a]anthracene 26 

### only with cross-breeding of strains selected for en hanced metabolic activity (Frolich and 27 

### Wurgler, 1990). No effect was observed using the s tandard strains. 28 

### An intraperitoneal host-mediated assay was describe d by Simmon et al. (1979). Five 29 

### PAHs (anthracene, benz[a]anthracene, benzo[e]pyrene , chrysene, and phenanthrene) were 30 

### administered to Swiss Webster mice by gavage or int ramuscular injection (single dose only). 31 

### Microorganisms ( S. typhimurium and Saccharomyces cerevisiae ) were injected intraperitoneally 32 

### into exposed mice and were recovered 4 hours later for mutation analysis. Negative results were 33 

### observed and the host-mediated assay system was con sidered insensitive for detecting 34 

### carcinogenic PAHs. 35 

### A series of studies have investigated the mutation sequence in codons 12 and 61 of the 36 

### Ki-ras oncogene from PAH-induced lung adenomas in A /J mice (Nesnow et al., 1998a, 1996, 37 

### 1995; Mass et al., 1993). As discussed in Section 2.4 (Similarities in Mode of Action for PAHs), 38

### 100 DRAFT – DO NOT CITE OR QUOTE

the purpose of these studies was to correlate the t umorigenic potency of specific PAHs with the 1 

### formation of DNA adducts and the mutation of specific codons in the Ki-ras oncogene. Six non2 

### alkylated PAHs were utilized in these studies (benz o[a]pyrene, benz[j]aceanthrylene, 3 

### benzo[b]fluoranthene, dibenz[a,h]anthracene, cyclop enta[c,d]pyrene, and dibenzo[a,l]pyrene). 4 

### Mutation analysis of the Ki-ras oncogene at codons 12 and 61 was carried out in PAH-induced 5 

### lung adenomas using polymerase chain reaction (PCR) amplification and dideoxy nucleotide 6 

### sequencing methods. The primary mutation type for benzo[a]pyrene, benzo[b]fluoranthene, and 7 

### dibenzo[a,l]pyrene was the GGT→TGT mutation. This guanine mutation was correlated with 8 

### the formation of diol epoxide guanine adducts. The GGT→CGT mutation was the primary 9 

### mutation type for benz[j]aceanthrylene and cyclopen ta[c,d]pyrene. The CGT mutation was 10 

### associated with the formation of cyclopenta-guanine adducts and increased tumorigenic potency 11 

### (i.e., >90 adenomas per mouse) in A/J mice. Dibenz [a,h]anthracene was the only PAH evaluated 12



### that did not induce mutations in Ki-ras codons 12 o r 61. This compound produced diol epoxide 13 

### guanine adducts and lung adenomas in A/J mice, sugg esting a possible interaction at a different 14 

### genetic target. The Ki-ras mutation analysis data were presented as percent of tumors with a 15 

### specific mutation at either codon 12 or 61. No dos e-response data were provided. 16 

### Among the in vivo mutagenicity studies shown in Tab le 4-8, only one study met the 17 

### selection criteria for use in this analysis. 18

19 

### 4.3.3. In Vitro Studies of Cancer-Related Endpoint s 20 

### Many in vitro studies of cancer-related endpoints a re present in the PAH database. As 21 

### previously discussed, only those studies that inclu ded at least one selected PAH and 22 

### benzo[a]pyrene as a reference compound were reviewe d. Each study that was reviewed for the 23 

### purpose of RPF development is included in Tables 4- 9 through 4-14. The tables summarize 24 

### study-specific information and indicate whether a p articular study is considered useful for dose25 

### response assessment. The text provides an overall description of the available studies, including 26 

### a general description of the methodology used for e ach study type, the results, and the 27 

### weaknesses or problems associated with specific stu dies or study types. 28

29



### 4.3.3.1. Bacterial Mutagenicity 30 

### The bacterial mutagenicity of many PAHs has been ex tensively studied (39 studies with 31 

### benzo[a]pyrene; see Table 4-9). All of the studies used the Ames assay in S. typhimurium . A 32



### total of 38 PAHs have been evaluated for their abil ity to induce mutations in bacterial systems. 33 

### The Ames Salmonella assay is a bacterial reverse mu tation assay, which measures the 34 

### frequency at which histidine-independent bacteria a rise from histidine-requiring bacterial strains 35 

### in the presence of a chemical mutagen. The results are generally expressed as either the number 36 

### of revertant colonies per plate or the number of re vertants/nmol of the test compound (calculated 37 

### from the linear portion of the dose-response curve) . Several strains of S. typhimurium have been 38

### 101 DRAFT – DO NOT CITE OR QUOTE

used to evaluate specific PAH mutation types; for e xample TA98, TA1537, and TA1538 detect 1 

### various frameshift mutations, TA1535 responds to ba se-pair substitution and TA100 responds to 2 

### a broad spectrum of mutations. Metabolism to react ive intermediates is required for PAH 3 

### mutagenicity in Salmonella and many metabolic activ ation systems have been employed. Rat 4 

### liver postmitochondrial supernatant (known as S9) f rom Aroclor-induced rats is most often used, 5 

### although other rodent species and enzyme inducers a re sometimes employed. Isolated rat 6 

### hepatocytes or purified mixed-function oxidase enzy mes were occasionally utilized for metabolic 7



### activation of PAHs. 8 

### Of the PAHs tested for bacterial mutagenicity, most were considered positive in at least 9 

### one study under optimal study conditions. Compound s that produced negative results in multiple 10 

### studies include anthracene, fluorene, phenanthrene, and pyrene. The primary weakness of the 11 

### bacterial mutagenicity database for PAHs is the lim ited amount of multiple-dose data for many 12 

### PAHs. Many studies report findings at a single dos e level for several PAHs. 13 

### Among the in vitro bacterial mutagenicity studies s hown in Table 4-9, 29 studies met the 14 

### selection criteria for use in this analysis. 15

16



### 4.3.3.2. Mammalian Mutagenicity 17 

### Studies that evaluate the mutagenicity of target PA Hs in mammalian cells are described 18 

### in Table 4-10 (29 studies). The most common cell t ypes used in these studies were the 19 

### V79 Chinese hamster cells and the L5178Y mouse lymp homa cells. Other cell types include 20 

### human epidermal keratinocytes, TK6 human lymphoblas ts, human epithelial cells (HS1 HeLa), 21 

### human foreskin fibroblasts (D-550), mouse fibroblas ts, rat embryo cells, rat liver epithelial cells 22 

### (ARL-18), and Chinese hamster ovary (CHO) cells. A total of 14 PAHs have been evaluated for 23 

### their ability to induce mutations in mammalian cell systems. 24 

### Each of the mammalian cell assays detects forward m utations that confer resistance to a 25 

### toxic chemical. Mutations in the hypoxanthine-guan ine phosphoribosyl transferase gene (HPRT) 26 

### result in resistance to purine analogs such as 6-th ioguanine, 8-azaguanine, and ouabain. HPRT 27 

### mutations induced by PAHs were most often measured in V79 Chinese hamster cells, but have 28 

### also been detected in human, rat, and mouse cell li nes. Forward mutation at the thymidine 29 

### kinase locus (TK) is measured as colony growth in t he presence of thymidine analogs (e.g., 30 

### trifluorothymidine or 5-bromo-2’-deoxyuridine). PA H-induced TK mutations were measured in 31 

### mouse lymphoma cells (L5178Y) and human lymphoblast s. Forward mutation assays are 32 

### considered to respond to a variety of mutation type s (including frameshift, base-pair substitution, 33 

### deletions and rearrangements or complex mutations). Exogenous metabolic activation is 34 

### required for PAH mutagenicity in most mammalian cel l assays. This was accomplished using a 35



### rat liver S9 mix or cocultivation with other rodent cells able to metabolize PAHs to reactive 36 

### intermediates (i.e., hamster embryo cells, fibrobla sts, or hepatocytes; rat hepatocytes). The 37

### 102 DRAFT – DO NOT CITE OR QUOTE

results of forward mutation assays in mammalian cel l lines are generally expressed as mutant 1 

### frequency/10 x



### survivors. 2 

### Of the 26 PAHs tested for mammalian cell mutagenici ty, all were considered positive in 3 

### at least one study under optimal study conditions. Compounds that produced negative results in 4 

### some studies include anthracene, benzo[e]pyrene, ph enanthrene, and pyrene. Benzo[a]- 5 

### anthracene produced positive findings in seven stud ies and negative findings in four studies. The 6 

### mammalian mutagenicity studies generally provide mo re multi-dose data than the bacterial 7 

### mutagenicity studies. 8 

### Among the in vitro mammalian mutagenicity studies s hown in Table 4-10, 27 studies met 9 

### the selection criteria for use in this analysis. 10

11



### 4.3.3.3. Morphological/Malignant Cell Transformation 12 

### Twenty-five studies examined the capacity of benzo[ a]pyrene and other PAHs to 13 

### transform cells in culture (Table 4-11). All of th ese studies were conducted using mammalian 14 

### cells, most commonly mouse or hamster embryo cells. A few studies added feeder cells or rat 15 

### liver homogenate to enhance metabolic activation in the test system; however, the majority relied 16 

### on the intrinsic metabolic capacity of the cells. The general test protocol involved seeding the 17 

### cultured cells in Petri dishes followed by exposure to a solution of the test compound, usually for 18 

### a period of 24 hours. The cells were then cultured for about 6 weeks before being fixed and 19 

### stained. Transformed colonies (foci) were scored b ased on characteristics such as cell piling, 20 

### criss-crossing, basophilic staining, and/or invasio n of surrounding (nontransformed) cell 21 

### monolayer. In studies conducted by some laboratori es, foci were classified as Type II or 22 

### Type III; the latter category included those with i nvasion of the surrounding monolayer, highly 23 

### criss-crossed arrays, and deep staining. Data were generally reported as the number of foci 24 

### (colony of transformed cells) per dish or per survi ving cells and/or the percent of dishes with 25



### foci. 26 

### In a few cases (e.g., Greb et al., 1980), transform ation was assessed by growth of treated 27 

### cells in soft agar. Transformed cell colonies grow ing in semi-solid agar are capable of 28 

### anchorage-independent growth. 29 

### Three studies (Evans and DiPaolo, 1975; Kakunaga, 1 973; DiPaolo et al., 1972) 30 

### confirmed the identification of malignant cells by injecting the transformed cells into rodents and 31 

### following tumor induction in the animals. In all t hree cases, cells identified as transformed gave 32 

### rise to tumors, while the cells without these chara cteristics did not. 33 

### Cell transformation assays were identified that inc luded 22 individual PAHs other than 34 

### benzo[a]pyrene. Dibenz[a,h]anthracene consistently gave rise to transformed cells in all but one 35 

### of the seven studies in which it was tested. Cyclo penta[c,d]pyrene, indeno[1,2,3-c,d]pyrene, 36 

### benzo[j]aceanthralene, benz[e]aceanthrylene, and di benz[k,mno]acephenanthrylene were each 37 

### tested in a single study and gave positive results. Benz[a]anthracene, pyrene, phenanthrene, 38

### 103 DRAFT – DO NOT CITE OR QUOTE

benzo[e]pyrene, and anthracene each gave negative r esults in a number of studies, while 1 

### fluoranthene, benzo[k]fluoranthene, dibenz[j,mno]ac ephenanthrylene, naphth[1,2,3-mno]ace2 

### phenanthrylene, and aceanthrylene were each tested in a single study and gave negative results. 3 

### Only a single dose of the target PAH was applied in 8 of the 26 studies of in vitro morphological/ 4 

### malignant cell transformation. 5 

### Among the in vitro morphological/malignant transfor mation studies shown in Table 4-11, 6



### 19 studies met the selection criteria for use in th is analysis. 7

8



### 4.3.3.4. DNA Adducts 9 

### Several studies (14) were identified in which DNA a dducts were measured after either 10



### whole cells or extracted DNA were incubated with be nzo[a]pyrene and at least one other PAH. 11 

### Table 4-12 shows general study details for these st udies. Most of the studies involved 12



### measurement of DNA adducts in whole mammalian cells , while some measured adducts formed 13



### when PAHs were incubated with extracted DNA. Whole cells were usually incubated with 14 

### PAHs for about 24 hours, while extracted DNA was ex posed to PAH solutions for a shorter time 15 

### period (1–3 hours). Some of the studies added meta bolic activation (usually rat liver 16 

### microsomes) to the incubation solution. Melendez-C olon et al. (2000) evaluated DNA adduct 17 

### formation after dibenzo[a,l]pyrene exposure in two cell types: one having significant CYP450 18 

### activity (MCF-7 cells) and one lacking significant CYP450 activity (HL-60). The authors 19 

### reported that adducts were formed in the cells havi ng CYP450 activity, but no adducts were 20 

### formed in the cells lacking such activity. 21 

### Identification and quantification of adducts was ge nerally done using a [ 32 

### P]-postlabeling 22 

### assay as follows. After exposure, DNA was isolated and digested to mononucleotides. 23 

### Mononucleotides were radiolabeled with [ 32 

### P]-ATP, separated with thin layer chromatography, 24 

### and visualized by autoradiography. Relative adduct labeling was measured using a scintillation 25 

### counter. A few early studies used [ 3 

### H]-labeled PAHs to identify and quantify adducts. In some 26 

### cases, adducts were identified by high-performance liquid chromatography and GC-MS. 27



### The 14 studies reviewed examined 15 PAHs other than benzo[a]pyrene. Apart from 28 

### phenanthrene, which did not result in measurable DN A adducts when incubated with calf thymus 29 

### DNA under various conditions (Bryla and Weyand, 199 2), each of the PAHs produced 30 

### measurable DNA adducts in at least one study. 31 

### Major limitations associated with some of the in vi tro DNA adduct data for relative 32 

### potency development include the lack of data at mul tiple PAH exposure levels, the use of 33 

### extracted DNA rather than whole cell assays, and th e inconsistent use of extrinsic metabolic 34 

### activation sources. Only three studies with positi ve adduct findings reported adduct 35 

### measurements at multiple doses (concentrations) of PAH (Binkova et al., 2000; Melendez-Colon, 36 

### 2000; Bryla and Weyand, 1992). Three studies used extracted DNA rather than whole cells to 37 

### measure DNA binding (Segerback and Vodicka, 1993; B ryla and Weyand, 1992; Grover and 38

### 104 DRAFT – DO NOT CITE OR QUOTE

Sims, 1968). Finally, the available studies on DNA adduct formation use cell types with varying 1 

### degrees of PAH metabolic capacity, with and without added metabolic activation sources. Both 2 

### the types and the quantities of DNA adducts formed are likely to depend on the level of 3



### metabolic activation for most PAHs. 4 

### Among the in vitro DNA adduct studies shown in Tabl e 4-12, 10 studies met the 5 

### selection criteria for use in this analysis. 6

7



### 4.3.3.5. DNA Damage/Repair 8 

### Twenty-four reports in the database evaluated the e ffects of one or more PAHs on DNA 9 

### damage, repair, or synthesis. Table 4-13 summarize s the study design information and results of 10



### these studies. Studies included measures of unsche duled DNA synthesis and DNA damage. 11 

### Unscheduled DNA synthesis was generally measured by increased radiolabeled ( 3 

### H) thymidine 12 

### uptake in treated cells versus untreated cells. DN A damage was measured either using the 13 

### alkaline elution assay for DNA strand breakage in m ammalian cells, or using the differential 14 

### killing of DNA repair-deficient bacterial strains. Metabolic activation of PAHs was most often 15 

### accomplished using a rat liver S9 mix. 16 

### Twenty-eight different PAHs have been tested for ef fects on DNA in one or more assays. 17 

### In general, pyrene, anthracene, phenanthrene, peryl ene, fluorene, and benzo[e]pyrene gave 18 

### negative results in multiple studies. Chrysene gav e negative results in four assays and positive 19 

### results in one assay (Mersch-Sundermann et al., 199 2). More positive than negative results were 20 

### reported for benz[a]anthracene, dibenz[a,h]anthrace ne, and dibenz[a,c]anthracene. Other PAHs 21 

### were tested only once, or gave roughly an equal fre quency of positive and negative responses in 22 

### these assays. 23 

### Although a large number of PAHs have been tested fo r DNA damage/repair, the database 24



### includes both bacterial and mammalian cells and sev eral different genotoxic endpoints. In 25 

### addition, the use of external metabolic activation, or cell types with intrinsic metabolic capacity, 26



### was inconsistent across these studies. These limit ations make it difficult to compare studies 27 

### using the same target PAHs. 28 

### Among the in vitro DNA damage/repair studies shown in Table 4-13, 15 studies met the 29 

### selection criteria for use in this analysis. 30

31



### 4.3.3.6. Clastogenicity or Sister Chromatid Exchange Frequen cy 32 

### The database contains 18 studies in which clastogen icity or sister chromatid exchange 33 

### frequency was measured in cultured cells after expo sure to benzo[a]pyrene and at least one other 34 

### PAH (Table 4-14). A wide variety of cell types was used in these assays, including hamster 35 

### liver, lung, CHO and V79 cells; rat liver epithelia l cells; human teratocarcinoma epithelial cells; 36 

### rat and human mammary epithelial cells; mouse, rat, and human fibroblasts; human 37 

### lymphocytes; and guinea pig fetal cells. A number of the studies used a metabolic activation 38

### 105 DRAFT – DO NOT CITE OR QUOTE

system, typically either rat liver S9 or coculture with a cell type able to metabolize PAHs. While 1 

### laboratory methods varied widely, the general appro ach involved treating the cultured cells with 2 

### a solution of the test compound, either with or wit hout metabolic activation. Usually, 3 

### bromodeoxyuridine was added to the growth medium to provide a means of staining metaphase 4 

### chromosomes, and colcemid was used to arrest mitoti c cells. Chromosomes were examined 5 

### microscopically and aberrations or exchanges were s cored visually. In most cases, the endpoint 6 

### examined was frequency of sister chromatid exchange s. Other endpoints included frequency of 7 

### micronuclei and scoring of chromosomal aberrations such as breaks, gaps, deletions, etc. 8 

### Only eight PAHs (anthracene, benz[a]anthracene, ben zo[e]pyrene, cyclopenta9 

### [c,d]pyrene, fluoranthene, perylene, phenanthrene, and pyrene) have been tested for clastogenic 10 

### effects in vitro. In many cases, the available stu dies were aimed at evaluating the validity of a 11 

### given test system to predict carcinogenicity. In t hese studies, a range of compounds of known or 12 

### believed carcinogenicity were used. Often, benzo[a ]pyrene was included as a known carcinogen, 13 

### and other PAHs were chosen because they were known or believed to be noncarcinogenic or 14 

### weakly carcinogenic. 15 

### Among the tested compounds, four gave positive resu lts in at least one study. With few 16 

### exceptions, PAHs administered without metabolic act ivation gave negative responses in these 17 

### assays. Cyclopenta[c,d]pyrene was reported to incr ease the frequency of sister chromatid 18 

### exchanges in two assays, one with and one without m etabolic activation (Murison, 1988; 19 

### Krolewski et al., 1986). Benz[a]anthracene gave po sitive results in three studies of sister 20 

### chromatid exchange induction (Mane et al., 1990; To ng et al., 1983; 1981a) and negative results 21 

### in a fourth (Warshawsky et al., 1995). Kochhar (19 82) reported a dose-dependent increase in 22



### chromosomal aberrations in V79 cells treated with b enz[a]anthracene in the absence of metabolic 23 

### activation. Perylene increased aberrations in one system (Popescu et al., 1977), but did not 24 

### increase sister chromatid exchanges in another (Sir ianni and Huang, 1978). Likewise, pyrene 25 

### gave positive results in a number of studies that i ncluded metabolic activation (Evans and 26 

### Mitchell, 1981; Perry and Thomson, 1981; Popescu et al., 1977) and negative results in several 27 

### that did not include activation (DeSalvia et al., 1 988; Tong et al., 1983, 1981a; Dean, 1981; Abe 28 

### and Sasaki, 1977). 29 

### The clastogenicity and sister chromatid exchange da ta for PAHs are variable with respect 30 

### to cell type and use of extrinsic metabolic activat ion. Some cells have intrinsic metabolic 31 

### activity, while others require activation from an e xternal source. The degree to which metabolic 32 

### activation is required for PAHs to exert a clastoge nic effect in cell cultures is not well 33



### established. Another limitation of these data stem s from the fact that a small number of PAHs, 34 

### many traditionally believed to be noncarcinogenic o r weakly carcinogenic, have been tested for 35 

### clastogenic effects in vitro. 36 

### Among the in vitro clastogenicity/sister chromatid exchange studies shown in Table 4-14, 37



### 10 studies met the selection criteria for use in th is analysis. 38

### 106 DRAFT – DO NOT CITE OR QUOTE

1



### 4.4. SUMMARY OF INFORMATION AVAILABLE TO DEVELOP R PFS FOR 2



### INDIVIDUAL PAHs 3 

### The PAH database contains several different types o f data that may be used to estimate 4 

### relative potencies of individual PAHs. The data we re summarized in Section 4.3 and include in 5 

### vivo tumor bioassays using various routes of exposu re and data for cancer-related endpoints 6



### from both in vivo and in vitro studies. As discuss ed above, the concurrent testing of 7 

### benzo[a]pyrene as a reference compound was consider ed essential to allow for RPF calculation. 8



### The introduction to Section 4.3 lists criteria for selecting studies or data sets for use in the 9 

### analysis. Studies that met these criteria were use d in the development of the RPF approach. 10 

### Section 5 discusses methods used for dose response assessment and RPF calculation from each 11 

### study or dataset, and Section 6 discusses the selec tion of PAHs to be included in the RPF 12 

### approach using a weight of evidence evaluation of t he available data. Section 7 describes the 13



### derivation of final RPFs for each PAH included in t he analysis. 14

15

16



### 5. METHODS FOR DOSE-RESPONSE ASSESSMENT AND RPF CA LCULATION 17

18

19 

### A discussion of the available data on PAH carcinoge nicity and cancer-related endpoints 20 

### and criteria for selection of studies was presented in Section 4. This section describes the 21 

### selection of dose-response data and methods for dos e-response assessment and RPF calculation 22 

### from the selected datasets. The dose-response data extracted from each study with positive 23 

### results and the results of the statistical analyses are shown in Appendix C. Appendix C also 24 

### contains information regarding the source of the do se-response data (i.e., the figure or table 25 

### number from the study and the particular data point s that were used in the dose-response 26 

### assessment) and additional comments on the use of t he data for dose-response assessment and 27



### RPF calculation. The results of the RPF calculations are shown in ta bles in Appendix E. These 28 

### tables provide summary information for each study, including the PAHs that were tested, the 29 

### data used to estimate the slopes (point estimate or BMD model result), the calculated RPF value, 30 

### and any specific comments related to the data analy sis. 31

32



### 5.1. CHOICE OF DOSE-RESPONSE DATA 33 

### For each of the endpoints evaluated in Section 4 (d ermal, intraperitoneal, subcutaneous, 34 

### and other route bioassays; in vivo DNA adducts; in vivo clastogenicity or sister chromatid 35 

### exchange frequency; in vitro bacterial and mammalia n mutagenicity; in vitro morphological/ 36 

### malignant transformation; in vitro clastogenicity o r sister chromatid exchange frequency; and 37 

### other in vitro endpoints [DNA adducts, unscheduled DNA synthesis, DNA damage, etc.]) there 38

### 107 DRAFT – DO NOT CITE OR QUOTE

was at least one study that met selection criteria. For those studies with positive findings, dose1 

### response data were extracted for dose-response asse ssment and calculation of RPFs. Data that 2 

### were reported in graphical format in published stud ies were digitized (Grab It!TM Graph 3 

### Digitizer, Datatrend Software) to identify the dose -response data points. 4 

### As discussed in Section 4.3, statistics were used f or tumor bioassay data to determine 5 

### whether the tumor incidence or multiplicity observe d at a particular dose represented a 6 

### statistically significant increase over controls. If statistical analyses were not described in the 7 

### original report, incidence data were analyzed using Fisher’s Exact test and the Cochran-Armitage 8 

### trend test. Positive findings were indicated by a significant ( p < 0.05) difference for at least one 9 

### dose group by comparison to control (in Fisher’s Ex act or an equivalent test) or a significant 10 

### dose-response trend (Cochran-Armitage or equivalent ) for multi-dose studies. For tumor 11 

### bioassay data reported as tumor count, a t-test was conducted (when variance data were 12 

### available) to determine whether the count was signi ficantly different from control ( p < 0.05). 13 

### The results of the statistical analyses are shown w ith the dose-response data in Appendix C. 14 

### The tumor bioassays that reported both incidence a nd tumor count were unique in 15 

### offering two different datasets for the same study. For each dose of each PAH in the tumor 16 

### bioassays, the decision to calculate an RPF, and in some instances, the selection of the point of 17 

### departure, was based on whether the tumor incidence or count was statistically significantly 18 

### increased over the control; if there was a signific ant increase, an RPF was calculated. There 19



### were a few instances where the statistical tests fo r tumor incidence and tumor number were 20 

### inconsistent (i.e., the incidence of tumors was sta tistically significantly increased, but the tumor 21 

### count was not, or vice versa). Sometimes, this cir cumstance existed only at a low dose, with 22 

### consistent findings at higher doses, so the conclus ion as to whether there was treatment-related 23 

### tumorigenicity (and whether an RPF should be calcul ated) was clear. In one case, however, the 24



### conclusion as to whether there was treatment-relate d tumorigenicity was not clear. In female 25 

### mice exposed at the high dose of fluoranthene in th e study reported by Busby et al. (1984), the 26 

### lung tumor count was significantly increased (albei t borderline, p = 0.0343) while the incidence 27 

### was not, and neither was statistically significantl y increased at the lower dose. For the purpose 28 

### of this analysis, the multiplicity data were treate d as an independent measure of carcinogenic 29 

### potency, and an RPF was calculated for the statisti cally increased tumor count irrespective of the 30 

### analysis of incidence. It should be noted that ave rage tumor count can be skewed by an unusual 31 

### response in a single animal, and no information was available to determine whether such 32 

### response represented an anomaly unrelated to exposu re or an unusual susceptibility to the 33 

### exposure. Thus, reliance on statistical analysis o f mean tumor count alone as a measure of 34 

### carcinogenic response may be subject to additional uncertainty. 35 

### For cancer-related endpoint data, each study author s’ conclusions regarding a positive or 36 

### negative response for each PAH were accepted, and R PFs were calculated when positive results 37 

### were reported. 38

### 108 DRAFT – DO NOT CITE OR QUOTE

In a few cases, the only data in a given publicatio n were given as relative potency 1 

### (relative to benzo[a]pyrene). For these publicatio ns, which included only in vitro cancer-related 2 

### endpoint data (primarily mutagenicity), the relativ e potency estimates calculated by the authors 3 

### were used without modification (except for dose adj ustment where appropriate; see Section 5.5). 4

5



### 5.2. OVERALL FORM OF RPF ESTIMATE 6 

### The overall goal of the dose-response analysis was to calculate ratios representing the 7 

### relative potency of a given PAH compared with benzo [a]pyrene (i.e., RPFs). For all datasets, the 8 

### RPF was defined as the ratio (PAH i 

### :BaP) of the slopes of the dose-response curves in the low9 

### dose region, following the equation (eq 5-1) below: 10

11 

### RPF = slope PAH i 

### ÷slope BaP (5-1) 12

13



### Data available for calculation of RPFs consisted of both quantal and continuous 14 

### endpoints. Quantal endpoints included tumor incide nce or incidence of cancer-related endpoints 15 

### (including frequency of mutations). Continuous end point datasets included tumor counts 16 

### (number of tumors per animal) or cancer-related end points of a continuous-variable nature (e.g., 17 

### number of sister chromatid exchanges, number of morphologically transformed colonies). Dose18 

### response assessment methods were specific to each t ype of endpoint (quantal or continuous) and 19 

### differed depending on whether there were multiple d ose groups or a single dose group in the 20 

### dataset. Methods for multidose and single dose qua ntal and continuous data are described below. 21

22



### 5.3. RPF CALCULATION FOR MULTIDOSE DATASETS 23 

### Dose-response modeling using U.S. EPA’s Benchmark D ose (BMD) Software (Version 24 

### 1.3.2) was conducted on multiple-dose data sets to estimate potency for both the target PAHs and 25 

### benzo[a]pyrene. Modeled estimates consider informa tion about the shape of the dose-response 26 

### curve and are thus preferred over using a single do se group as the point of departure. 27 

### Dose-response modeling. For multidose quantal data, the multistage model wa s used and 28 

### the degree of the polynomial was assumed to equal t he number of dose groups minus 2 (extra 29 

### risk with background subtracted). The multistage m odel was selected because it is the preferred 30 

### model for cancer risk assessment of animal bioassay data, and it provided a consistent model 31



### form for all of the datasets. For multidose contin uous data, the linear model was selected for all 32 

### datasets, as it is the simplest model form for cont inuous data. For both quantal and continuous 33 

### datasets, the goodness-of-fit criteria were used to evaluate model fit. If the model did not 34 

### provide adequate fit to the data, high-dose groups were sequentially eliminated in an effort to 35 

### achieve adequate fit. The focus of the modeling ef fort is on the low dose and response region, so 36 

### doses and responses much higher than the benchmark response (BMR) are not as informative 37 

### and can be eliminated to improve model fit. If dos e-group elimination did not improve the 38

### 109 DRAFT – DO NOT CITE OR QUOTE

model fit, a point-estimate ratio approach was used (see Section 5.4). The BMD modeling 1 

### output for all datasets that were successfully mode led are shown in Appendix D. 2 

### Selection of BMR: Multidose data for both PAH and benzo[a]pyrene. For tumor 3 

### incidence data, the BMR used in estimating the poin t of departure was a 10% increase in tumor 4 

### incidence over controls (extra risk form). For can cer-related endpoints such as frequency of 5 

### mutations, endpoint-specific points of departure we re selected based on the background/control 6 

### frequency of the endpoint and the detection limit o f the assay. For example, a 1% frequency was 7 

### selected for a control mutation frequency of 1/10,0 00 and a detection limit of two- to threefold 8 

### above background. 9 

### For multidose continuous data, the BMR used in esti mating the point of departure was a 10 

### change of 1 standard-deviation (1 SD) from the cont rol mean. In the event that multiple-dose 11 

### continuous data were reported in the absence of SD values, a point estimate ratio approach was 12 

### employed to calculate the slope (see Section 5.4). 13 

### Selection of BMR: Multidose data for PAH, single d ose benzo[a]pyrene. Some studies 14 

### included only one dose of benzo[a]pyrene as a posit ive control, while providing multiple-dose 15 

### data for a selected PAH. In these cases, dose-resp onse modeling was performed for the selected 16 

### PAH and the BMR used for modeling was the observed response for benzo[a]pyrene adjusted for 17 

### background response. For tumor incidence data, for example, if the benzo[a]pyrene dose was 18 

### associated with a 60% extra risk for tumors, the BM R chosen for modeling the data for the PAH 19



### was 60% extra risk. RPFs were then calculated usin g a ratio of the slope factors calculated with 20 

### equivalent points of departure (e.g., ED 60 

### ). The goal of this approach was to compare PAH 21 

### potencies at similar response locations on the dose -response curve. There is uncertainty 22 

### associated with relative potency estimates calculat ed at the high end of the dose-response curves 23 

### and using the resultant RPF for low-exposure scenar ios, because the relative potency relationship 24 

### between any two PAHs may be different at the low en d, compared with the high end, of the 25 

### dose-response curves. The uncertainties and limita tions associated with the use of high-dose 26 

### data to estimate relative potency are further discu ssed in Section 7. Data sets for which tumor 27 

### incidence was ≥90% in the lowest dose group were no t used to calculate potency estimates and 28 

### RPFs, because the response is near plateau and such data provide insufficient information on the 29 

### slope of the dose-response relationship. 30 

### For continuous data, when a point estimate was use d to estimate the slope for 31 

### benzo[a]pyrene and modeling was used to estimate th e slope for a given PAH, the BMR used for 32 

### BMD modeling was a point value set at the response (e.g., mean number of tumors per animal 33 

### for tumor multiplicity data) observed in the benzo[ a]pyrene group, adjusted for response in the 34 

### control group. This approach is consistent with th e BMR used for quantal data when only a 35 

### single benzo[a]pyrene dose group was available. Pr ovided that a linear model is fit to continuous 36 

### data, the choice of a higher BMR would not apprecia bly change the RPF. 37

### 110 DRAFT – DO NOT CITE OR QUOTE

Selection of point of departure. The point of departure selected for slope estimatio n was 1



### the BMD estimate rather than the lower confidence l imit on the benchmark dose (BMDL). The 2 

### BMD, as the central or “best” estimate of the dose associated with the selected BMR, was 3 

### considered a more stable basis for comparison betwe en the potency of the selected PAH and 4 

### benzo[a]pyrene, and thus for calculation of relativ e potency, than the lower confidence limit. 5 

### Extrapolation from point of departure. The slopes of the dose-response curves in the 6 

### low-dose regions were calculated by linear extrapol ation to the origin from the model-predicted 7 

### points of departure. Equation 5-2 below shows the calculation of slope from multidose quantal 8



### data. 9

10 

### Slope = [0.1/ED 10 

### ] (5-2) 11

12 

### Equation 5-3 below shows the calculation of slope f rom multidose continuous data. 13

14 

### Slope = [1SDchange]/[ED 1SD 

### ] (5-3) 15

16



### 5.4. RPF CALCULATION FOR SINGLE DOSE DATASETS 17 

### A number of studies reported data for only single doses of benzo[a]pyrene and other 18 

### PAHs; for these studies, a point estimate approach was used to calculate the RPF. A point 19 

### estimate approach was also used to calculate RPFs f or multidose datasets when model fit was not 20 

### achieved, when variance data were not available for continuous data, or when problems with 21 

### model implementation were encountered. 22 

### Selection of point of departure. When only one dose of each compound was used, the re 23 

### was only one choice for the point of departure. Ho wever, when multidose data were available, 24 

### but a point estimate approach was used, the point o f departure was chosen as follows. For tumor 25 

### bioassay data, the lowest dose associated with a st atistically significant increase in tumor 26 

### incidence or multiplicity over control values was s elected as the point of departure. Variance 27 

### was not reported for tumor multiplicity data in any of the dermal studies and for some of the 28 

### intraperitoneal studies, so the corresponding incid ence data were used to determine the dose at 29 

### which a significant difference from control was obs erved. 30 

### The benzo[a]pyrene dose chosen in most instances wa s the lowest dose associated with a 31 

### significant increase in tumor count or incidence. For tumor multiplicity data, the PAH dose 32 

### chosen for the point estimate RPF calculation was t he lowest dose associated with a tumor count 33 

### similar to that observed at the selected benzo[a]py rene dose (similar to selecting a BMR similar 34 

### to the benzo[a]pyrene incidence). In the case of t wo dermal initiation studies conducted by 35 

### Cavalieri et al. (1991), however, the tumor count a t the lowest dose of DBalP was much higher 36 

### than the tumor count at the lowest benzo[a]pyrene d ose associated with statistical significance. 37 

### In order to compare the doses associated with simil ar tumor counts (i.e., at a similar place on the 38

### 111 DRAFT – DO NOT CITE OR QUOTE

dose-response curve), a higher benzo[a]pyrene dose was chosen for the RPF calculation. A 1 

### comparison of the RPFs calculated using this approa ch with RPFs calculated using the lowest 2 

### dose associated with a statistically significant in crease over controls for both dibenzo[a,l]pyrene 3 

### and benzo[a]pyrene showed only small differences in the RPF values (9 vs. 10 in the 16-week 4 

### study and 39 vs. 42 in the 27-week study). A simil ar approach was used to calculate the RPF for 5 

### BjAC using the intraperitoneal multiplicity data fr om Mass et al. (1993). 6 

### For cancer-related endpoint data, statistical analy sis was not always available for each 7 

### dose group. For these data, the lowest dose that p roduced a near maximal change in the assay of 8 

### concern was selected as the point of departure. Th at is, the highest dose in the linear portion of 9 

### the dose-response curve (identified by visual displ ay of the data) was selected in these cases. 10 

### Extrapolation from point of departure. As with multiple dose slope estimations, point 11 

### estimate slope calculations also used the extra ris k form. Thus, for single dose quantal data, the 12 

### slope was calculated by linear extrapolation to the origin after an extra risk adjustment of the 13 

### observed response (eq 5-4): 14

15 

### Slope = [(Response at dose -Control Response)÷(1- Control Response)]/Dose (5-4) 16

17 

### For single dose continuous data, the slope was calc ulated by linear extrapolation to the 18 

### origin after adjustment of the observed response in the PAH-treated animals for the control 19 

### response (eq 5-5). 20

21 

### Slope = [(Value of variable at dose) - (Value of variable) control 

### ]/Dose (5-5) 22

23



### 5.5. DOSE CONVERSION FOR RPF CALCULATION 24 

### Some of the studies used to calculate RPFs reporte d doses or test concentrations on a 25 

### molar basis (e.g., μmol per mouse, μmol/L), rather than a mass basis (mg or μg). The molar 26 

### ratio differs from the mass ratio for any PAH with a molecular weight that differs from that of 27 

### benzo[a]pyrene; thus, for these compounds, an RPF e xpressed on a mass basis will differ from 28 

### that expressed on a molar basis. Table 5-1 below s hows a hypothetical example for 29 

### fluoranthene, a PAH with a molecular weight that di ffers from benzo[a]pyrene by 20%. As the 30 

### table shows, the RPF differs depending on which dos e units are used. 31

32 

### Table 5-1 Comparison between molar and mass-based RPF

Response Dose in mol Molecular weight (g/mol) Dose in g Molar RPF Mass RPF FA 0.1 5 202.26 1,011 0.20 0.25
BaP 0.1 1 252.32 252 1 1

33

### 112 DRAFT – DO NOT CITE OR QUOTE

In order to ensure that comparisons across endpoin ts used consistent units, the doses used 1



### to calculate RPFs were converted to mass-based unit s using the molecular weight of the relevant 2 

### PAH prior to estimating the RPF. The mass-based RP F was selected to be consistent with dose 3 

### metrics used to calculate cancer risk; RPFs are use d with oral slope factors and inhalation unit 4 

### risks reported on a mass basis (e.g., [mg/kg-day] -1 

### ; [μg/m 3 

### ] -1 

### ). 5

6



### 5.6. SPECIAL CONSIDERATIONS FOR RPF CALCULATION US ING TUMOR 7



### BIOASSAY DATA 8 

### Several dermal bioassays reported significant morta lity prior to the appearance of the first 9 

### skin tumor. For these data sets, an assumption was made that the number of animals at risk for 10 

### tumor development was equal to the total number of animals alive at the time of the appearance 11 

### of the first tumor. Benign and malignant tumor typ es within the same target organ were 12



### combined for calculation of the RPF. The total inc idence of animals with either a benign or 13 

### malignant lesion was directly reported in each stud y (i.e., the number of animals with adenoma 14 

### or carcinoma). 15 

### Tumor incidence data reported for different target organs within the same group of 16 

### animals were analyzed separately unless the joint i ncidence (incidence of either tumor type in 17 

### each dose group) was reported in the publication. Liver and lung tumors were reported in 18 

### newborn mice exposed to PAHs by intraperitoneal inj ection (LaVoie et al., 1994, 1987; Busby et 19 

### al., 1989, 1984; Weyand and LaVoie, 1988; Wislocki et al., 1986). In most studies, tumor 20 

### incidence was reported separately for the different target organs and could not be combined as 21 

### the joint incidence was unknown. A gender differen ce was observed in the newborn mouse 22 

### studies, with liver tumors observed in male mice on ly, and lung tumors reported for both male 23 

### and female mice. The tumor incidence data were, th erefore, evaluated separately for male and 24 

### female mice. RPF values were calculated separately for male and female mice and for lung 25



### tumor incidence and liver tumor incidence in these studies. 26

27



### 5.7. SPECIAL CONSIDERATIONS FOR RPF CALCULATION US ING CANCER28



### RELATED ENDPOINT DATA 29 

### The in vitro studies of cancer-related endpoints in cluded measurements of bacterial 30 

### mutagenicity, mammalian mutagenicity, morphological /malignant cell transformation, DNA 31 

### adduct formation, DNA damage or repair, and clastog enicity or sister chromatid exchange 32 

### frequency. Many of the studies describing in vitro cancer-related endpoints provide dose33 

### response data under varying study conditions. For example, bacterial mutagenesis studies used 34 

### multiple strains, different metabolic activation pr ocesses, and/or varying assay systems. In order 35 

### to limit the number of datasets used for dose-respo nse analysis of in vitro mutagenicity studies, 36 

### and to provide a consistent basis for comparing RPF s for differenct PAHs, data associated with 37 

### the conditions that maximized the benzo[a]pyrene re sponse within a particular study were used 38

### 113 DRAFT – DO NOT CITE OR QUOTE

for the dose-response assessment of PAHs. It shoul d be noted that in several studies, test 1 

### conditions that were optimal for benzo[a]pyrene wer e not necessarily optimal for the selected 2 

### PAH (see Appendix C for specific studies). The unc ertainties and limitations associated with 3 

### this approach are discussed further in Section 8. 4 

### For time-course studies of DNA adducts, results wer e reported as either AUC or peak 5 

### formation of adducts. AUC was considered preferabl e for dose-response assessment, because 6



### this measure considers both adduct formation and re pair. Adducts measured in more than one 7 

### organ were summed to derive a total measure of addu ct formation (standardized per unit amount 8 

### of DNA). 9 

### The data for bacterial and mammalian cell mutagenic ity and malignant cell 10 

### transformation were sometimes expressed as a mutati on or transformation frequency (i.e., 11



### mutants/total cell count or transformed cells/total cells). For multiple-dose studies, these quantal 12 

### variables were evaluated using the multistage model as described above. Problems were 13 

### sometimes encountered when using the multistage mod el for incidence data of this type. In some 14 

### cases, modifying the initial parameters in the mult istage algorithm facilitated convergence. In a 15 

### select few cases, the quantal linear model was used when the multistage model would not 16 

### converge. If neither the multistage nor quantal li near models provided adequate fit, a point 17 

### estimate approach was used. If possible, the point estimates for both benzo[a]pyrene and the 18 

### target PAH were chosen at a comparable response lev el (e.g., the doses of benzo[a]pyrene and 19 

### the target PAH that both gave two mutants in 10 5 

### cells). However, in many cases, a comparable 20 

### response rate was not available. In these instance s, the RPF was derived from slopes calculated 21 

### by linear extrapolation from the peak response. 22 

### As noted earlier, for studies that included only on e dose of benzo[a]pyrene and multiple 23 

### dose data for a selected PAH, the BMR selected for dose-response modeling for the selected 24 

### PAH was the benzo[a]pyrene response with the backgr ound or control response subtracted. In 25 

### some instances, when the benzo[a]pyrene response le vel greatly exceeded the response at the 26 

### highest dose of the selected PAH, the software woul d fail to calculate the ED at the 27 

### benzo[a]pyrene response level. In these instances, a point estimate approach using the peak 28 

### response for the selected PAH was used. 29 

### The individual study RPFs calculated for each PAH w ere used in a weight of evidence 30 

### evaluation to assess the potential carcinogenicity of each compound (see Section 6) and in the 31 

### derivation of a final RPF for each compound (Sectio n 7). 32

33

34



### 6. SELECTION OF PAHS FOR INCLUSION IN RELATIVE POT ENCY APPROACH 35

36

37

### 114 DRAFT – DO NOT CITE OR QUOTE

The selection of PAHs to be included in the RPF app roach began with an evaluation of 1 

### whether the available data were adequate to assess the potential carcinogenicity of each 2 

### compound. At least one RPF value was calculated fo r each of 50 PAHs. For 16 of these 3 

### compounds, only a single RPF value derived from an in vitro cancer-related endpoint (primarily 4 

### mutagenicity assays) was available. These PAHs are shown in Table 6-1. Due to the limited 5 

### data available for these 16 compounds, no further e valuation of these PAHs was conducted, and 6 

### they were not selected for inclusion in the RPF app roach. 7

8


### Table 6-1. PAHs with only one RPF from in vitro ca ncer-related endpoint

study and excluded from RPF approach

PAH CASRN Abbreviation Aceanthrylene 202-03-09 ACEA Acenaphthene 83-32-9 AN Acenaphthylene 208-96-8 ANL Acephenanthrylene 201-06-9 APA Benzo[a]perylene 191-85-5 BaPery Benz[b]anthracene 92-24-9 BbA Benzo[b]perylene 197-70-6 BbPery Benzo[c]phenanthrene 195-19-7 BcPH Cyclopent[h,i]aceanthrylene 131581-33-4 CPhiACEA
Cyclopent[h,i]acephenanthrylene 114959-37-4 CPhiAPA
Dibenzo[a,f]fluoranthene 203-11-2 DBafF Dibenz[a,j]anthracene 224-41-9 DBajA
Dibenzo[b,e]fluoranthene 2997-45-7 DBbeF Dibenzo[e,l]pyrene 192-51-8 DBelP Dibenz[k,mno]acephenanthrylene 153043-81-3 DBkmnoAP H Naphtho[2,3-a]pyrene 196-42-9 N23aP

9 

### The remaining 34 PAHs had RPF values calculated fro m at least one in vivo dataset or at 10 

### least two in vitro cancer-related endpoint datasets . For these compounds, a weight of evidence 11 

### approach was used to determine whether the availabl e data (including the calculated RPFs as 12 

### well as negative studies that met selection criteri a) were adequate to assess the carcinogenic 13 

### potential. Using the calculated RPFs in the weight of evidence evaluation allowed consideration 14 

### of the magnitude of calculated RPFs in assessing po tential carcinogenicity. When data were not 15 

### considered adequate, the PAH was excluded from the RPF approach. When data were 16 

### considered adequate for a given PAH, it was selecte d for inclusion. 17 

### A PAH with adequate evidence to suggest that it has little or no carcinogenic potential 18 

### was selected for inclusion in the RPF approach and assigned an RPF of 0. While there is little 19 

### quantitative difference between selecting a final R PF of zero for a given PAH and excluding that 20 

### PAH from the RPF approach, this is an important dis tinction for uncertainty analysis. There is 21

### 115 DRAFT – DO NOT CITE OR QUOTE

substantial uncertainty in the risk associated with a PAH that is excluded from the RPF approach 1 

### due to inadequate data; this compound could be of l ow or high potency. However, for a PAH 2 

### with an RPF of 0, there is evidence to suggest that this compound exhibits little or no 3 

### carcinogenic potential, and the uncertainty associa ted with the cancer risk for this compounds is 4 

### markedly reduced. For anthracene, phenanthrene, an d pyrene, it has been determined that the 5 

### available data support a practical RPF of zero. It is possible that the studies available may not 6 

### provide sufficient sensitivity to compare the poten cy of the PAHs of interest to benzo[a]pyrene, 7 

### and thus, the RPF of zero should not be considered a characterization of the inherent 8 

### carcinogenicity of anthracene, phenanthrene, or pyr ene. The weight of evidence analysis is 9



### outlined in Section 6.1 and the results are describ ed in narratives for each of the 34 individual 10 

### PAHs (Section 6.2). Section 7 describes how the RP Fs from multiple datasets were used to 11



### derive final RPFs for those PAHs selected for inclu sion in the approach, and reports the final 12



### RPF information for each PAH. 13

14



### 6.1. METHOD FOR SELECTING PAHS FOR INCLUSION IN RE LATIVE POTENCY 15



### APPROACH 16 

### For each of the 34 PAHs, a weight of evidence evalu ation was conducted to assess the 17 

### evidence that each PAH could induce a carcinogenic response. This evaluation did not constitute 18 

### a formal weight of evidence evaluation of carcinoge nic potential; rather, an approach was 19 

### developed using the data collected for this analysi s to determine whether the available 20 

### information for each PAH was adequate to draw a con clusion regarding carcinogenic potential. 21 

### When the data were considered adequate for a given PAH, it was selected for inclusion in the 22 

### RPF approach. Figure 6-1 shows the decision tree t hat was used to evaluate the data for each 23



### PAH and to determine whether it should be included in the RPF approach. The weight of 24 

### evidence evaluation concluded with one of two possi ble outcomes: 25

26



### 1. The data reviewed are adequate to evaluate potentia l carcinogenicity and the PAH should 27 

### be included in the RPF analysis, or 28

29 

### 2. The data reviewed are inadequate to assess carcinog enic potential and the PAH should be 30 

### excluded from the RPF analysis. 31

32

### 116 DRAFT – DO NOT CITE OR QUOTE Has PAH been tested in tumor bioassay with
BaP a ?

Did PAH give positive result in any tumor bioassays with BaP?

Did PAH give positive result in all tumor bioassays with
BaP? Can conflicting tumor bioassay results be explained by differences in study design?

Do other tumor bioassays b

and/or cancer-related endpoint data c provide adequate data to assess carcinogenic potential? Data are adequate to

assess carcinogenic potential of
PAH:
Include in RPF approach

Data are inadequate to assess carcinogenic potential of PAH:
Exclude from RPF approach No

No No

No

No

Yes

Yes Yes

Yes Yes

1 2

a 

### Bioassays with benzo[a]pyrene that met study qualit y criteria (includes studies with 3 

### negative results). 4
b 

### Other bioassays include those that did not test ben zo[a]pyrene and/or those that were not 5 

### suitable for RPF derivation (e.g., incidence at low est dose exceeded 90%). 6

c 

### Cancer-related endpoint data examined in this proce ss included studies of DNA adducts, 7 

### clastogenicity or sister chromatid exchange, mutage nicity, morphological transformation, 8 

### DNA damage, unscheduled DNA synthesis, etc. that in cluded the selected PAH and 9 

### benzo[a]pyrene. 10

11 

### Figure 6-1. Weight of evidence analysis of potenti al carcinogenicity. 12

13 

### In vivo tumor bioassays that included benzo[a]pyren e were given the greatest weight in 14 

### assessing the potential carcinogenicity of a given PAH; data from other bioassays and cancer- 15

### 117 DRAFT – DO NOT CITE OR QUOTE

related endpoint studies were used to supplement th e weight of evidence when the bioassay data 1 

### that included benzo[a]pyrene were conflicting or ne gative. Structural alerts for PAH 2 

### carcinogenicity or mutagenicity (specifically, at l east four aromatic rings, or the presence of a 3 

### classic bay region or fjord region formed entirely by aromatic rings) were noted in the evaluation 4 

### for each PAH, but were not used explicitly in the w eight of evidence evaluation. 5 

### When there were bioassays including benzo[a]pyrene with positive findings, and none 6 

### with negative findings for a given PAH, that compou nd was selected for inclusion in the RPF 7 

### approach, and no further evaluation of cancer-relat ed endpoint data was conducted. However, 8 

### the cancer-related endpoint findings for these comp ounds were noted in the individual PAH 9 

### narratives (Section 6.2). Among the PAHs included in this analysis, there were none with 10 

### positive bioassay data and robust negative cancer-r elated endpoint data. Were this instance to 11 

### arise, it would require special consideration, as i t might imply a different mode of carcinogenic 12



### action than the PAHs addressed herein. 13 

### Bioassays that met selection criteria (see Section 4.3) were included in the weight of 14 

### evidence analysis, regardless of whether positive o r nonpositive (i.e., negative) results were 15 

### found. However, the weight of evidence evaluation assumed that a given compound may be 16 

### active in one system (e.g., newborn mouse), and ina ctive or weakly active in another (e.g., 17 

### dermal initiation). Thus, when conflicting results were observed in different test systems, 18 

### different species, or different genders, the PAH wa s assumed to be potentially carcinogenic 19 

### based on the positive findings and was included in the RPF approach. 20 

### In order to compare the results of bioassays with p ositive and nonpositive results in the 21 

### same test system, an “RPF detection limit” was conc eptualized as a means of approximating the 22



### minimum RPF that could be determined under the cond itions of the study. The “RPF detection 23 

### limit” was defined as the ratio of the dose-respons e slopes 3 

### , using the lowest statistically 24 

### significant response that could be calculated for t he subject PAH and the actual benzo[a]pyrene 25 

### response, as points of departure for the slope calc ulation. The lowest statistically significant 26 

### response was calculated using the incidence of tumo rs in the control group, number of animals in 27 

### the group treated with the subject PAH, and Fisher’ s exact test 4 

### (employing a one-sided 28



### p- value ≤ 0.05). Appendix F shows an example calcula tion of an “RPF detection limit.” The 29 

### utility of this concept is in weighing positive and nonpositive bioassay results. If all of the 30 

### nonpositive studies had “RPF detection limits” in e xcess of what is observed in the positive 31 

### studies, then it is plausible that the nonpositive studies may not have been sufficiently sensitive 32 

### to estimate the low RPF appropriate to the subject PAH. In this event, the PAH was considered 33 

### potentially carcinogenic and included in the RPF ap proach. 34

3 The standard RPF equation is RPF = slope PAH i ÷ slope BaP = [response/dose] PAHi ÷ [response/dose] BaP.

4 This calculation was implemented using trial and er ror within the Fisher’s exact test in the online st atistical calculator GraphPad ©

.

### 118 DRAFT – DO NOT CITE OR QUOTE

If there were no bioassays with benzo[a]pyrene for a given compound, all of the selected 1 

### bioassays gave nonpositive results, or inconsistent results could not be explained by test system 2 

### or “RPF detection limit”, then the results of other bioassays (those without benzo[a]pyrene, or 3 

### those rejected from dose-response assessment exclus ively because of concerns associated with 4 

### benzo[a]pyrene) and cancer-related endpoint data we re evaluated. The weight of evidence 5 

### analysis then considered all of the following infor mation: bioassays with benzo[a]pyrene, other 6 

### bioassays, and cancer-related endpoint data. If th ese data were determined to be inadequate to 7 

### assess the carcinogenic potential for a given PAH, then that compound was excluded from the 8 

### RPF approach. If the data were considered adequate to assess the carcinogenic potential, the 9 

### compound was retained and a final RPF was derived. Section 6.2 below describes the weight of 10



### evidence evaluation for each of the 34 PAHs. Secti on 7.1 describes how final RPFs were 11



### derived for the 26 PAHs selected for inclusion in t he RPF approach. 12

13



### 6.2. WEIGHT OF EVIDENCE EVALUATION FOR 34 INDIVIDU AL PAHS 14 

### For each PAH, the structure is shown along with a b rief reference to any structural alerts 15 

### for potential carcinogenicity (specifically, more t han three aromatic rings and/or bay or fjord 16 

### region in alternant PAH). Next, a brief narrative describing the weight of evidence evaluation is 17 

### given, with a graphical representation of the data that were available for RPF calculation 18 

### (Figures 6-2 to 6-35). The graph for each compound provides a visual representation of the 19 

### database of studies that included both the subject PAH and benzo[a]pyrene. The solid bars show 20



### the values of the RPFs calculated from all studies with positive findings. The x-axis label shows 21 

### the reference for the pertinent study. The RPFs ar e color-coded to distinguish among in vivo 22 

### tumor bioassays based on incidence data, in vivo tu mor bioassays based on multiplicity data, in 23 

### vivo cancer-related endpoint studies, and in vitro cancer-related endpoint studies. Within these 24 

### categories, the RPFs are ordered (left to right in the graph) from highest to lowest, with positive 25 

### results shown before nonpositive results. 26 

### For each nonpositive bioassay, an empty, dotted bar shows what is termed the “RPF 27 

### detection limit” (see Section 6.1 for description). Missing bars designate cancer-related studies 28 

### that resulted in nonpositive findings. An RPF dete ction limit for nonpositive cancer-related 29 

### studies was not included, because comparisons betwe en nonpositive and positive studies were 30 

### complicated by the wide variety of study conditions (e.g., test species and strains, metabolic 31 

### activation sources, assay systems). 32



### Each narrative concludes with a statement as to whe ther the subject PAH was selected for 33 

### inclusion in the PAH RPF approach. The weight of e vidence evaluation for the 34 PAHs with at 34



### least one in vivo RPF or at least 2 in vitro cancer -related endpoint RPFs resulted in the selection 35 

### of 26 PAHs for inclusion in the RPF approach and th e exclusion of 8 PAHs from the approach 36 

### (see Table 6-2). 37

38

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1



### Table 6-2. Results of weight of evidence evaluatio n of 34 PAHs

Adequate data: selected for inclusion in RPF appro ach

PAH CASRN Abbreviation PAH CASRN Abbreviation

Benzo[a]pyrene 50-32-8 BaP Cyclopenta[d,e,f]chrysene,
4H- 202-98-2 CPdefC

Anthanthrene 191-26-4 AA Dibenz[a,c]anthracene 215- 58-7 DBacA Anthracene 120-12-7 AC Dibenzo[a,e]fluoranthene 538 5-75-1 DBaeF Benz[a]anthracene 56-55-3 BaA Dibenzo[a,e]pyrene 19 2-65-4 DBaeP Benz[b,c]aceanthrylene,
11H- 202-94-8 BbcAC Dibenz[a,h]anthracene 53-70-3 DBahA

Benzo[b]fluoranthene 205-99-2 BbF Dibenzo[a,h]pyren e 189-64-0 DBahP Benz[e]aceanthrylene 199-54-2 BeAC Dibenzo[a,i]pyre ne 189-55-9 DBaiP Benzo[g,h,i]perylene 191-24-2 BghiP Dibenzo[a,l]pyr ene 191-30-0 DBalP Benz[j]aceanthrylene 202-33-5 BjAC Fluoranthene 206 -44-0 FA Benzo[j]fluoranthene 205-82-3 BjF Indeno[1,2,3-c,d] pyrene 193-39-5 IP Benzo[k]fluoranthene 207-08-9 BkF Naphtho[2,3-e]pyr ene 193-09-9 N23eP Benz[l]aceanthrylene 211-91-6 BlAC Phenanthrene 85- 01-8 PH Chrysene 218-01-9 CH Pyrene 129-00-0 Pyr Cyclopenta[c,d]pyrene 27208-37-3 CPcdP
Inadequate data

PAH CASRN Abbreviation PAH CASRN Abbreviation Acepyrene, 2,3- 25732-74-5 ACEP Coronene 191-07-1 C O Benzo[b]fluorene, 11H- 243-17-4 BbFE Fluorene 86-73 -7 FE Benzo[e]pyrene 192-97-2 BeP Perylene 198-55-0 Pery Benzo[g,h,i]fluoranthene 203-12-3 BghiF Triphenylen e 217-59-4 Tphen

2

### 120 DRAFT – DO NOT CITE OR QUOTE

1 

### 2,3-Acepyrene (ACEP) 2

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 do es not contain a classic bay or fjord region 6



### in its structure. 7 

### Five datasets for 2,3-acepyrene met selection crite ria and included benzo[a]pyrene 8 

### (shown in Figure 6-2). Dermal initiation and compl ete carcinogenicity bioassays in mice both 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 fo rmation, one positive bacterial mutagenicity 12 

### dataset (both published by Cavalieri et al., 1981a) , and one negative mammalian mutagenicity 13 

### dataset (Barfknecht et al., 1982). There are no bi oassays of 2,3-acepyrene without 14 

### benzo[a]pyrene. Overall, the database for 2,3-acep yrene is both limited and inconsistent. The 15 

### database for 2,3-acepyrene does not provide adequat e information with which to assess potential 16 

### carcinogenicity; this PAH was not selected for incl usion in the RPF approach. 17

18121 DRAFT – DO NOT CITE OR QUOTE1Figure 6-2. 2,3-Acepyrene (ACEP) RPFs*.20.0010.010.1110100Cavalieri et al., 1981b Cavalieri et al., 1981b Cavalieri et al., 1981a Cavalieri et al., 1981a Barfknechtet al., 1982

Value of RPFReferenceRPF detection limitsfor nonpositivebioassays* Missing bar indicates nonpositive cancer-relatedendpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 122 DRAFT – DO NOT CITE OR QUOTE Anthanthrene (AA) 1

2

3

4 

### Anthanthrene (CASRN 191-26-4) is an alternant PAH c omprised of six fused aromatic 5 

### rings. Anthanthrene does not have a bay or fjord r egion 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 D NA damage dataset. Statistically increased 9 

### tumor incidences were reported in both a rat lung i mplantation bioassay (Deutsch-Wenzel et al., 10 

### 1983) and a dermal complete carcinogenicity bioassa y 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 co nflicting bioassay data can be explained by 14 

### differences in study design (initiation versus comp lete dermal carcinogenicity), anthanthrene was 15 

### considered potentially carcinogenic and selected fo r inclusion in the RPF approach. 16

17123 DRAFT – DO NOT CITE OR QUOTE1Figure 6-3. Anthanthrene (AA) RPFs.20.0010.010.1110100Cavalieri et al., 1977 Deutsch-Wenzel et al.,1983Hoffman and Wynder,1966Andrews et al., 1978 Hermann, 1981 Mersch-Sundermannetal., 1992Kaden et al., 1979

Value of RPFReferenceRPF detection limitfor nonpositivebioassayPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 124 DRAFT – DO NOT CITE OR QUOTE Anthracene (AC) 1

2

3

4 

### Anthracene (CASRN 120-12-7) is an alternant PAH com prised of three fused aromatic 5 

### rings. Anthracene does not have a bay or fjord reg ion 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 datase ts, 4 mammalian mutagenicity datasets, 10 

### 6 morphological/malignant cell transformation datas ets, and 13 in vitro DNA adduct, DNA 11 

### damage or clastogenicity datasets (Figure 6-4). Th e single dermal initiation bioassay gave a 12 

### nonpositive result, with a RPF detection limit of 0 .2 (LaVoie et al., 1985). Only two datasets 13 

### gave positive results: an in vitro bacterial mutag enicity assay and an in vitro study of DNA 14 

### damage. The remaining 35 datasets reported nonposi tive findings. To confirm the negative 15 

### findings in the one tumor bioassay that included be nzo[a]pyrene, other bioassays and cancer16 

### related endpoint data for anthracene were considere d in the weight of evidence evaluation. In 17 

### bioassays without benzo[a]pyrene, anthracene did no t induce a statistically significant increase in 18 

### tumor incidence in two dermal initiation studies (L aVoie 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 μmol 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 negative for 24 

### anthracene (13/14 studies). Studies of morphologic al/malignant cell transformation were all 25 

### negative. Finally, in numerous in vitro studies of DNA damage or clastogenicity, anthracene has 26 

### given nonpositive results (12/13). Sakai et al. (1 985) reported a mutagenic response in bacteria 27 

### treated with anthracene, and Rossman et al. (1991) observed evidence of unscheduled DNA 28 

### synthesis in E. coli treated with anthracene. Overall, the weight of ev idence suggests that 29 

### anthracene is not carcinogenic or is of very low ca rcinogenic potential. In addition, anthracene 30 

### lacks all three known structural alerts (at least f our rings, bay region or fjord region) for PAH 31 

### carcinogenicity and/or mutagenicity. Because the w eight of evidence evaluation suggests that 32 

### the data are adequate to assess the carcinogenic po tential of anthracene, this compound was 33 

### selected for inclusion in the RPF approach and assi gned a RPF of 0. 34125 DRAFT – DO NOT CITE OR QUOTE1Figure 6-4. Anthracene (AC) RPFs*.20.0010.010.1110100

Value of RPFReferenceRPF detection limitsfor nonpositivebioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 126 DRAFT – DO NOT CITE OR QUOTE Benz[a]anthracene (BaA) 1

2

3 

### Benz[a]anthracene (CASRN 56-55-3) is an alternant P AH comprised of four fused 4 

### aromatic rings. Benz[a]anthracene contains a bay r egion but no fjord region in its structure. 5 

### There are 65 datasets for benz[a]anthracene that me t selection criteria and included 6 

### benzo[a]pyrene (Figure 6-5). Included in the datab ase are tumor bioassays (5), in vivo DNA 7 

### adduct studies (4), in vivo clastogenicity studies (4), an in vivo mutagenicity study (1), bacterial 8 

### mutagenicity (15), mammalian mutagenicity (14), mor phological/malignant cell transformation 9 

### assays (6), and in vitro studies of DNA damage, add ucts, or clastogenicity (16). There are five 10 

### tumor bioassay datasets of benz[a]anthracene that i ncluded benzo[a]pyrene; four gave positive 11 

### results, and one gave a nonpositive result. The po sitive findings were in different genders tested 12 

### in a newborn mouse study using intraperitoneal inje ction (Wislocki et al., 1986); the datasets 13 

### included both tumor incidence and multiplicity data for both sexes. Positive results were also 14 

### reported in a dermal initiation study (Slaga et al. , 1978). The one nonpositive bioassay (Cavalieri 15 

### et al., 1977) was a dermal complete carcinogenicity study with an RPF detection limit of 0.2. 16 

### Benz[a]anthracene was shown to form DNA adducts whe n administered in vivo in both rats and 17 

### mice via injection and gavage (Kligerman et al., 20 02). Mutagenicity and morphological/ 18 

### malignant cell transformation assays of benz[a]anth racene were predominantly positive, as were 19 

### studies of other cancer-related endpoints. 20 

### Given that the differing bioassay results can be at tributed to different test systems and 21 

### study design, benz[a]anthracene was considered pote ntially carcinogenic and was selected for 22 

### inclusion in the RPF approach. 23

24127 DRAFT – DO NOT CITE OR QUOTE1Figure 6-5. Benz[a]anthracene (BaA) RPFs*.20.0010.010.1110100

Value of RPFReferenceRPF detection limitfor nonpositivebioassay* Missing bar indicates nonpositive cancer-relatedendpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 128 DRAFT – DO NOT CITE OR QUOTE 11H-Benz[b,c]aceanthrylene (BbcAC) 1

2

3 

### 11H-Benz[b,c]aceanthrylene (CASRN 202-94-8) is a no nalternant PAH comprised of 4 

### four aromatic rings and one five-membered ring. 11H -Benz[b,c]aceanthrylene does not contain a 5 

### classic bay or fjord region in its structure. 6 

### There was only one dataset for benz[b,c]aceanthryle ne that met selection criteria and 7 

### included benzo[a]pyrene (Figure 6-6). This multi-d ose dermal initiation study resulted in an 8 

### RPF estimate of 0.05 (Rice et al., 1988). Benz[b,c ]aceanthrylene has not been tested in any 9 

### bioassay without benzo[a]pyrene. There are no canc er-related endpoint data for 10 

### benz[b,c]aceanthrylene. As the only available bioa ssay of this PAH was positive, 11 

### benz[b,c]aceanthrylene was considered potentially c arcinogenic and was selected for inclusion in 12 

### the RPF approach. 13

14129 DRAFT – DO NOT CITE OR QUOTE1Figure 6-6. 11H-Benz[b,c]aceanthrylene (BbcAC) RPFs.20.0010.010.1110100Rice et al., 1988

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 130 DRAFT – DO NOT CITE OR QUOTE Benzo[b]fluoranthene (BbF) 1

2

3 

### Benzo[b]fluoranthene (CASRN 205-99-2) is a nonalter nant PAH comprised of four 4 

### aromatic rings and one five-membered ring. Benzo[b ]fluoranthene contains one classic bay 5 

### region but no fjord region in its structure. 6 

### There were 21 datasets of benzo[b]fluoranthene that met selection criteria and included 7 

### benzo[a]pyrene (Figure 6-7). Included in the datab ase are in vivo tumor bioassay datasets (7), in 8 

### vivo DNA adduct datasets (7), in vivo clastogenicit y datasets (3), mutagenicity and 9 

### morphological/malignant cell transformation dataset s (3), and an in vitro DNA damage dataset 10 

### (1). Statistically significant increases in tumor incidence and/or multiplicity were reported in 11 

### male mice tested in two newborn mouse bioassays usi ng intraperitoneal injection (Nesnow et al., 12 

### 1998b; LaVoie et al., 1987), in dermal initiation ( LaVoie et al., 1982) and dermal complete 13 

### carcinogenicity (Habs et al., 1980) bioassays, and in a rat lung implantation bioassay (Deutsch14 

### Wenzel et al., 1983). The one nonpositive result w as in female mice tested in the newborn 15 

### mouse bioassay; the RPF detection limit was 0.8 (La Voie et al., 1987). A number of studies 16 

### showed that benzo[b]fluoranthene forms DNA adducts when administered in vivo to rats or mice 17 

### via injection or gavage (Kligerman et al., 2002; Ne snow et al., 1998b, 1993b). One mutagenicity 18 

### assay and two morphological/malignant cell transfor mation assays of benzo[b]fluoranthene were 19 

### positive, as were studies of other cancer-related e ndpoints; there were no negative studies of 20 

### cancer-related endpoints. Given that the differein g bioassay results can be attributed to different 21 

### gender, benz[a]anthracene was considered potentiall y carcinogenic and was selected for 22 

### inclusion in the RPF approach. 23131 DRAFT – DO NOT CITE OR QUOTE1Figure 6-7. Benzo[b]fluoranthene (BbF) RPFs.20.0010.010.1110100

Value of RPFReferenceRPF detection limitfor nonpositivebioassayPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 132 DRAFT – DO NOT CITE OR QUOTE 11H-Benzo[b]fluorene (BbFE) 1

2 

### 11H-Benzo[b]fluorene (CASRN 243-17-4) is a nonalter nant PAH comprised of three 3 

### aromatic rings and one five-membered ring. 11H-Benz o[b]fluorene does not contain a classic 4 

### bay or fjord region in its structure. 5 

### There were three datasets for 11H-benzo[b]fluorene that met selection criteria and 6 

### included benzo[a]pyrene (Figure 6-8): two mutageni city datasets and an in vitro DNA damage 7 

### dataset. There are no bioassays of 11H-benzo[b]flu orene that included benzo[a]pyrene, so 8 

### bioassays without benzo[a]pyrene and cancer-related endpoint data were considered. LaVoie et 9 

### al. (1981) conducted a study of skin tumor initiati on in mice treated with 1.0 mg 11H-benzo[b] 10 

### fluorene followed by 20 weeks of treatment with TPA . The incidence of tumor-bearing animals 11 

### (4/20) was not significantly increased over control s (1/20) (LaVoie et al., 1981). The limited 12 

### cancer-related endpoint data were mixed, with one p ositive mutagenicity study (Kaden et al., 13 

### 1979), one negative mutagenicity study (Hermann, 19 81), and one positive in vitro study of 14 

### DNA damage (Mersch-Sundermann et al., 1992). Overa ll, the database for 11H-benzo[b] 15



### fluorene is both limited and inconsistent. Because the database for 11H-benzo[b]fluorene does 16 

### not provide adequate information with which to asse ss potential carcinogenicity, this PAH was 17 

### not selected for inclusion in the RPF approach. 18

19133 DRAFT – DO NOT CITE OR QUOTE1Figure 6-8. 11H-Benzo[b]fluorene (BbFE) RPFs*.20.0010.010.1110100Kaden et al., 1979 Mersch-Sundermann et al., 1992 Hermann, 1981

Value of RPFReference* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 134 DRAFT – DO NOT CITE OR QUOTE Benz[e]aceanthrylene (BeAC. 1

2 

### Benz[e]aceanthrylene (CASRN 199-54-2) is a nonalter nant PAH comprised of four 3 

### aromatic rings and one five-membered ring. Benz[e] aceanthrylene contains a classic bay region 4 

### but no fjord region in its structure. 5 

### There were six datasets for benz[e]aceanthrylene th at met selection criteria and included 6 

### benzo[a]pyrene (Figure 6-9); all gave positive resu lts. The database includes an in vivo tumor 7 

### bioassay in two sexes (each reporting both incidenc e and multiplicity), a mammalian 8 

### mutagenicity study, and a morphological/malignant c ell transformation study. Significantly 9 

### increased tumor incidence and tumor multiplicity we re reported for both male and female mice 10 

### in a dermal initiation bioassay in mice (Nesnow et al., 1984). As the available bioassay that 11 

### included benzo[a]pyrene was positive, benz[e]aceant hrylene was considered potentially 12 

### carcinogenic and was selected for inclusion in the RPF approach. 13

14135 DRAFT – DO NOT CITE OR QUOTE1Figure 6-9. Benz[e]aceanthrylene (BeAC) RPFs.20.0010.010.1110100Nesnow et al., 1984 Nesnow et al., 1984 Nesnow et al., 1984 Nesnow et al., 1984 Nesnow et al., 1984b Mohapatra et al., 1987

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 136 DRAFT – DO NOT CITE OR QUOTE Benzo[e]pyrene (BeP) 1

2

3 

### Benzo[e]pyrene (192-97-2) is an alternant PAH compr ised of five fused aromatic rings. 4 

### Benzo[e]pyrene contains two bay regions and no fjor d region in its structure. 5 

### Thirty-seven datasets for benzo[e]pyrene met select ion criteria and included 6 

### benzo[a]pyrene: 2 tumor bioassays, 1 in vivo clast ogenicity dataset, 12 bacterial mutagenicity 7 

### datasets, 4 mammalian mutagenicity datasets, 7 morp hological/malignant cell transformation 8 

### datasets, and 11 in vitro DNA damage or clastogenic ity datasets (Figure 6-10). No increase in 9 

### tumor incidence was observed when benzo[e]pyrene wa s tested alone as part of a dermal 10 

### cocarcinogenicity bioassay (Van Duuren and Goldschm idt, 1976). When tested in a lung 11 

### implantation bioassay in rats, benzo[e]pyrene expos ure did not result in a significant increase in 12 

### tumor incidence (Deutsch-Wenzel et al., 1983). The RPF detection limits of these studies were 13 

### approximately 0.01 and 0.1. To confirm the negativ e findings in the available tumor bioassays 14 

### that included benzo[a]pyrene, other bioassays and c ancer-related endpoint data were considered. 15 

### In bioassays without benzo[a]pyrene, benzo[e]pyrene gave negative results in a dermal initiation 16 

### bioassay (1 mg/mouse; Van Duuren et al., 1968) and a newborn mouse bioassay (0.7 μmol; 17 

### Chang et al., 1981). A significant increase in tum or incidence was reported in a single18 

### concentration dermal initiation study in mice; 11/1 3 surviving mice (20 were treated) had 19 

### papillomas by week 35 after dermal treatment with 1 0 μmol benzo[e]pyrene in benzene 20 

### ( p < 0.0001), followed by twice weekly treatment with TPA; no control mice had papillomas 21 

### (Scribner, 1973). 22 

### In vitro assays of mutagenicity (both bacterial and mammalian) and morphological/ 23 

### malignant cell transformation give inconsistent res ults for benzo[e]pyrene; 11/23 studies were 24 

### positive and the rest were negative. Positive stud ies include a mix of bacterial mutagenicity and 25 

### morphological/malignant cell transformation assays; four mammalian mutagenicity assays were 26 

### negative. One study of in vivo clastogenicity and two studies of in vitro DNA damage were 27 

### positive, while nine studies of in vitro DNA damage or clastogenicity were negative. 28 

### While the database for benzo[e]pyrene is quite larg e, the results are inconsistent; as a 29 

### result, no conclusion can be drawn as to potential carcinogenicity. This PAH was not selected 30 

### for inclusion in the RPF approach. 31137 DRAFT – DO NOT CITE OR QUOTE1Figure 6-10. Benzo[e]pyrene (BeP) RPFs*.20.0010.010.1110100

Value of RPFReferenceRPF detectionlimits fornonpositivebioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 138 DRAFT – DO NOT CITE OR QUOTE Benzo[g,h,i]fluoranthene (BghiF) 1

2

3 

### Benzo[g,h,i]fluoranthene (CASRN 203-12-3) is a nona lternant PAH comprised of four 4 

### aromatic rings and one five-membered ring. Benzo[g ,h,i]fluoranthene does not contain a classic 5 

### bay or fjord region in its structure. 6 

### There were six datasets for benzo[g,h,i]fluoranthen e that met selection criteria and 7 

### included benzo[a]pyrene (Figure 6-11). A dermal in itiation bioassay in mice (Van Duuren et al., 8 

### 1966) did not result in a statistically significant increase in tumor incidence; the RPF detection 9 

### limit was 0.06. There were no other bioassays that met selection criteria. There were three 10 

### positive bacterial mutagenicity studies (Chang et a l., 2002; Lafleur et al., 1993; Carver et al., 11 

### 1986), one positive study of in vitro DNA damage (M ersch-Sundermann et al., 1992), and a 12 

### mammalian mutagenicity study with negative results (Lafleur et al., 1993). The RPF values for 13 

### the positive cancer-related endpoint datasets range d from 0.6 to 1. Overall, the database for 14 

### benzo[g,h,i]fluroanthene is both limited and incons istent. Because the database for 15 

### benzo[g,h,i]fluoranthene does not provide adequate information with which to assess potential 16 

### carcinogenicity, this PAH was not selected for incl usion in the RPF approach. 17

18139 DRAFT – DO NOT CITE OR QUOTE1Figure 6-11. Benzo[g,h,i]fluoranthene (BghiF) RPFs*.20.0010.010.1110100Van Duuren et al., 1966 LaFleur et al., 1993 Carver et al., 1986 Chang et al., 2002 Mersch-Sundermann et al.,1992LaFleur et al., 1993

Value of RPFReferenceRPF detection limitsfor nonpositivebioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 140 DRAFT – DO NOT CITE OR QUOTE Benzo[g,h,i]perylene (BghiP) 1

2

3 

### Benzo[g,h,i]perylene (CASRN 191-24-2) is an alterna nt PAH comprised of six fused 4 

### aromatic rings. Benzo[g,h,i]perylene contains a ba y region but no fjord region in its structure. 5 

### There were 10 datasets for benzo[g,h,i]perylene tha t met selection criteria and included 6 

### benzo[a]pyrene (Figure 6-12). The database include s three in vivo tumor bioassays, four 7 

### bacterial mutagenicity datasets, an in vitro DNA da mage dataset, and two in vitro DNA adduct 8 

### datasets. Of the three bioassays, positive finding s were only reported in one: a rat lung 9 

### implantation bioassay (Deutsch-Wenzel et al., 1983) that resulted in a RPF estimate of 0.009. In 10 

### a dermal initiation bioassay (Hoffmann and Wynder, 1966) and a dermal cocarcinogenicity 11 

### bioassay (Van Duuren and Goldschmidt, 1976), there was no statistically significant increase in 12 

### tumor incidence, but these studies had relatively i nsensitive RPF detection limits (around 0.1) 13 

### compared with the positive study. There were four positive mutagenicity studies; all were 14 

### conducted in bacterial systems. Studies of in vitr o DNA adducts and DNA damage were 15 

### positive. Because the inconsistent bioassay result s can be attributed to different test systems 16 

### (different species and route), benzo[g,h,i]perylene was considered potentially carcinogenic and 17 

### was selected for inclusion in the RPF approach. 18141 DRAFT – DO NOT CITE OR QUOTE1Figure 6-12. Benzo[g,h,i]perylene (BghiP) RPFs*.20.0010.010.1110100Deutsch-Wenzelet al., 1983Hoffmann andWynder, 1966Van Duuren andGoldschmidt,1976Andrews et al.,1978Sakai et al., 1985 Gibson et al.,1978Kaden et al.,1979MerschSundermann etal., 1992Segerback andVodicka, 1993Cherng et al.,2001

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 142 DRAFT – DO NOT CITE OR QUOTE Benz[j]aceanthrylene (BjAC) 1

2

3

4 

### Benz[j]aceanthrylene (CASRN 202-33-5) is a nonalter nant 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 tha t met selection criteria and included 8 

### benzo[a]pyrene (Figure 6-13); all of the studies ga ve positive results. The database includes one 9 

### in vivo tumor bioassay dataset, one in vivo DNA add uct dataset, four mutagenicity or 10 

### morphological/malignant cell transformation dataset s, and six in vitro DNA damage or DNA 11 

### adduct datasets. In a bioassay of benz[j]aceanthry lene that used intraperitoneal injection in an 12 

### A/J mouse system (Mass et al., 1993), all mice trea ted 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 de rivation of an RPF using incidence data. 15 

### However, tumor multiplicity (average number of tumo rs per animal) data were available for 16 

### dose-response modeling and resulted in an RPF estim ate of 60. Benz[j]aceanthrylene treatment 17 

### resulted in a pronounced increase in the average nu mber of tumors per animal (59.45 tumors per 18 

### animal at 20 mg/kg), much higher than benzo[a]pyren e treatment (5.05 tumors per animal at 19 

### 100 mg/kg), indicating that this compound is very p otent in this test system. In a dermal 20 

### initiation bioassay that did not include benzo[a]py rene, benz[j]aceanthrylene induced papillomas 21 

### in 90% of mice treated with an initiating dose of 4 0 μg (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]aceanthry lene was considered potentially carcinogenic 24 

### and was selected for inclusion in the RPF approach. 25

26143 DRAFT – DO NOT CITE OR QUOTE1Figure 6-13. Benz[j]aceanthrylene (BjAC) RPFs.20.0010.010.1110100Mass et al.,1993Mass et al.,1993Johnsen et al.,1997Johnsen et al.,1997Johnsen et al.,1997Sangaiah etal., 1983Nesnow et al.,1984Mohapatra etal., 1987Johnsen et al.,1998Johnsen et al.,1998Johnsen et al.,1998Johnsen et al.,1998

Value RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 144 DRAFT – DO NOT CITE OR QUOTE Benzo[j]fluoranthene (BjF) 1

2 

### Benzo[j]fluoranthene (CASRN 205-82-3) is a nonalter nant PAH comprised of four 3 

### aromatic rings and one five-membered ring. Benzo[j ]fluoranthene does not contain a classic bay 4 

### or fjord region in its structure. 5 

### There were eight datasets for benzo[j]fluoranthene that met selection criteria and 6 

### included benzo[a]pyrene (Figure 6-14): seven in vi vo tumor bioassay datasets and one in vitro 7 

### study of DNA damage. Of the seven bioassay dataset s, significant increases in tumor incidence 8 

### or count were observed in all but one. Significant increases in tumor incidence were reported in 9



### both male and female mice tested in a newborn mouse bioassay using intraperitoneal injection of 10 

### single doses (LaVoie et al., 1987), a mouse dermal initiation study (LaVoie et al., 1982), and a 11 

### rat lung implantation bioassay (Deutsch-Wenzel et a l., 1983). Significant increases in tumor 12 

### multiplicity were reported in two mouse dermal init iation studies (Weyand et al., 1992; LaVoie 13 

### et al., 1982). The one nonpositive bioassay was a mouse dermal complete carcinogenicity 14 

### bioassay with an RPF detection limit of 0.1 (Habs e t al., 1980). The in vitro study of DNA 15 

### damage gave positive results (Mersch-Sundermann et al., 1992). Because the inconsistent 16 

### bioassay results can be attributed to different tes t systems or study design, benzo[j]fluroanthene 17 

### was considered potentially carcinogenic and was sel ected for inclusion in the RPF approach. 18

19145 DRAFT – DO NOT CITE OR QUOTE1Figure 6-14. Benzo[j]fluoranthene (BjF) RPFs.20.0010.010.1110100LaVoie et al., 1987 LaVoie et al., 1987 LaVoie et al., 1982 LaVoie et al., 1982 Deutsch-Wenzel etal., 1983Weyand et al., 1992 Habs et al., 1980 MerschSundermann et al.,1992

Value of RPFReferenceRPF detectionlimit fornonpositivebioassayPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 146 DRAFT – DO NOT CITE OR QUOTE Benzo[k]fluoranthene (BkF) 1

2

3

4 

### Benzo[k]fluoranthene (CASRN 207-08-9) is a nonalter nant 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 t hat met selection criteria and included 8 

### benzo[a]pyrene (Figure 6-15). The database include s four in vivo tumor bioassay datasets and 9 

### one morphological/malignant cell transformation dat aset. 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 (Deutsch12 

### Wenzel et al., 1983). No significant increase in t umor incidence was observed in a dermal 13 

### complete carcinogenicity study with an RPF detectio n limit of 0.1 (Habs et al., 1980). The 14 

### morphological/malignant cell transformation study ( Emura et al., 1980) was negative. Because 15 

### the inconsistent bioassay results can be attributed to different test systems or study design 16 

### (dermal initiation vs. dermal complete carcinogenic ity), benzo[k]fluroanthene was considered 17 

### potentially carcinogenic and was selected for inclu sion in the RPF approach. 18

19147 DRAFT – DO NOT CITE OR QUOTE1Figure 6-15. Benzo[k]fluoranthene (BkF) RPFs*.20.0010.010.1110100Deutsch-Wenzel et al.,1983LaVoie et al., 1982 LaVoie et al., 1982 Habs et al.,1980 Emura et al., 1980

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 148 DRAFT – DO NOT CITE OR QUOTE Benz[l]aceanthrylene (BlAC) 1

2

3 

### Benz[l]aceanthrylene (CASRN 211-91-6) is a nonalter nant PAH comprised of four 4 

### aromatic rings and one five-membered ring. Benz[l] aceanthrylene does not contain a classic bay 5 

### or fjord region in its structure. 6 

### There were 16 datasets for benz[l]aceanthrylene tha t met selection criteria and included 7 

### benzo[a]pyrene (Figure 6-16); all of the studies ga ve positive results. The database includes four 8 

### in vivo tumor bioassay datasets, five mutagenicity or morphological/malignant cell 9 

### transformation datasets, one in vivo clastogenicity dataset, and six in vitro DNA adduct or DNA 10 

### damage datasets. Significant increases in tumor co unt and multiplicity were reported in both 11



### male and female mice in a dermal initiation bioassa y (Nesnow et al., 1984). All of the cancer12 

### related endpoint studies were positive as well. Re lative potency estimates for most of the 13 

### available datasets were ≥1.0, suggesting equivalent or greater potency than benzo[a]pyrene. As 14 

### the available bioassays that included benzo[a]pyren e were positive, benz[l]aceanthrylene was 15 

### considered potentially carcinogenic and was selecte d for inclusion in the RPF approach. 16149 DRAFT – DO NOT CITE OR QUOTE1Figure 6-16. Benz[l]aceanthrylene (BlAC) RPFs.20.0010.010.1110100

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 150 DRAFT – DO NOT CITE OR QUOTE Chrysene (CH) 1

2

3 

### Chrysene (CASRN 218-01-9) is an alternant PAH compr ised of four fused aromatic 4 

### rings. Chrysene contains two bay regions but no fj ord region in its structure. 5 

### There were 40 datasets for chrysene that met select ion criteria and included 6 

### benzo[a]pyrene (Figure 6-17). Included in the data base are 13 in vivo tumor bioassay datasets, 7 

### 4 in vivo DNA adduct datasets, 3 in vivo clastogeni city datasets, 11 mutagenicity datasets, 8 

### 3 morphological/malignant cell transformation datas ets, and 6 in vitro studies of DNA damage, 9 

### adducts, or clastogenicity. Among the bioassays th at included benzo[a]pyrene, 11 reported 10 

### significant increases in tumor incidence or tumor m ultiplicity, and 3 did not. Significant 11 

### increases in tumor incidence and/or multiplicity we re reported in three dermal initiation studies 12 

### in mice (Rice et al., 1988; Slaga et al., 1980; Hec ht et al., 1974), a newborn mouse study in 13 

### males (Wislocki et al., 1986), and a rat lung impla ntation bioassay (Wenzel-Hartung et al., 14 

### 1990). Female mice tested in the newborn mouse ass ay published by Wislocki et al. (1986) did 15 

### not have a significant increase in tumor incidence, resulting in one of the three nonpositive 16 

### studies. The other two nonpositive findings were i n males and females tested in another 17 

### newborn mouse bioassay (Busby et al., 1989). The b ioassays with nonpositive findings had RPF 18 

### detection limits between 0.06 and 0.2. Conflicting results in male mice were reported in the two 19 

### newborn mouse bioassays (Busby et al., 1989; Wisloc ki et al., 1986). The major difference 20



### between the two studies is the duration of follow-u p; Busby et al. (1989) sacrificed the mice at 21 

### 26 weeks, while Wislocki et al. (1986) followed the mice for a full year. LaVoie et al. (1994) 22



### observed that liver tumor induction in the newborn mouse bioassay is not fully realized until the 23 

### mice have reached 1 year of age, and the positive f indings by Wislocki et al. (1986) indeed 24 

### reflect liver tumors in the male mice. Chrysene wa s shown to form DNA adducts when 25 

### administered in vivo in both rats and mice via inje ction and gavage (Kligerman et al., 2002). 26 

### Bacterial and mammalian mutagenicity and morphologi cal/malignant cell transformation assays 27 

### of chrysene were all positive, as were studies of c lastogenicity tested in vivo. In contrast, results 28 

### from in vitro studies of DNA adducts, DNA damage, a nd clastogenicity were not consistent. 29 

### Because the inconsistent bioassay results can be at tributed to different study designs 30 

### (gender, follow-up time), chrysene was considered p otentially carcinogenic and was selected for 31 

### inclusion in the RPF approach. 32

33151 DRAFT – DO NOT CITE OR QUOTE1Figure 6-17. Chrysene (CH) RPFs*.20.0010.010.1110100

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 152 DRAFT – DO NOT CITE OR QUOTE Coronene (CO) 1

2 

### Coronene (CASRN 191-07-1) is an alternant PAH compr ised of seven fused aromatic 3 

### rings. Coronene contains no bay or fjord regions i n its structure. 4



### There were six datasets for coronene that met selec tion criteria and included 5 

### benzo[a]pyrene (Figure 6-18). A dermal complete ca rcinogenicity bioassay in mice did not 6 

### result in a statistically significant increase in t umor incidence (Habs et al., 1980); the RPF 7 

### detection limit was 0.06. To confirm the nonpositi ve findings in the one tumor bioassay that 8 

### included benzo[a]pyrene, other bioassays and cancer -related endpoint data were considered. 9 

### There was one bioassay of coronene that did not inc lude benzo[a]pyrene. Van Duuren et al. 10 

### (1968) conducted a dermal initiation bioassay of co ronene using groups of 20 mice (0.5 mg 11 

### coronene in 0.5 mL benzene, followed by croton resi n treatment until death). Although the 12



### authors characterized coronene as a weak tumor init iator, the incidence of tumors was not 13 

### significantly increased over concurrent controls. The limited cancer-related endpoint data were 14 

### mixed, with three positive bacterial mutagenicity s tudies (with RPFs ranging from 0.01 to 0.5), 15 

### one negative bacterial mutagenicity study, and a ne gative in vitro DNA damage study. 16 

### Overall, the database for coronene is both limited and inconsistent. Because the database 17 

### for coronene does not provide adequate information with which to assess potential 18 

### carcinogenicity, this PAH was not selected for incl usion in the RPF approach. 19153 DRAFT – DO NOT CITE OR QUOTE1Figure 6-18. Coronene (CO) RPFs*.20.0010.010.1110100Habs et al., 1980 Hermann, 1981 Sakai et al., 1985 Florin et al., 1980 Kaden et al., 1979 Mersch-Sundermannetal., 1992

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 154 DRAFT – DO NOT CITE OR QUOTE Cyclopenta[c,d]pyrene (CPcdP) 1

2 

### Cyclopenta[c,d]pyrene (CASRN 27208-37-3) is a nonal ternant PAH comprised of four 3 

### aromatic rings and one five-membered ring. Cyclope nta[c,d]pyrene does not contain a classic 4 

### bay or fjord region in its structure. 5 

### There were 24 datasets for cyclopenta[c,d]pyrene th at met selection criteria and included 6 

### benzo[a]pyrene (Figure 6-19). The database include s 10 in vivo tumor bioassay datasets, 2 in 7 

### vivo DNA adduct datasets, 11 studies of mutagenicit y or morphological/malignant cell 8 

### transformation, and a single study of in vitro clas togenicity. Eight of the 10 tumor bioassay 9 

### datasets and all of the cancer-related endpoint stu dies gave positive results. Statistically 10 

### significant increases in tumor incidence and/or mul tiplicity were reported in two dermal 11 

### complete carcinogenicity bioassay (Cavalieri et al. , 1983, 1981b), two dermal initiation 12 

### bioassays (Raveh et al., 1982; Cavalieri et al., 19 81b), and an intraperitoneal study using adult 13 

### A/J mice (Nesnow et al., 1998b). Bioassays in whic h no significant increase in tumorigenicity 14 

### was observed included a dermal initiation (Wood et al., 1980) and complete carcinogenicity 15 

### study (Habs et al., 1980); these studies had RPF de tection limits of 0.1 and 0.03, respectively. 16 

### After obtaining nonpositive results for low initiat ing doses of cyclopenta[c,d]pyrene, Wood et al. 17 

### (1980) repeated their experiment with higher doses and observed statistically significant 18 

### increases in tumor incidence. In the latter experi ment, benzo[a]pyrene was not included, so an 19



### RPF could not be calculated from these data. The s tudy design of the nonpositive complete 20 

### carcinogenicity bioassay was quite similar to that of the two positive studies of this type, with the 21 

### exception of the mouse strain used; Habs et al. (19 80) used NMRI mice, while Cavalieri et al. 22 

### (1983, 1981b) used Swiss mice. Although the differ ing results in dermal complete 23 

### carcinogenicity studies may be explained by slight differences in strain susceptibility, these two 24 

### strains are of common origin, which argues against this explanation. 25 

### The available cancer-related endpoint data indicat e that cyclopenta[c,d]pyrene is 26 

### mutagenic and capable of morphological/malignant ce ll transformation in vitro; a single study of 27 

### in vitro clastogenicity was also positive. Overall , the data supporting a finding of potential 28 

### carcinogenicity for cyclopenta[c,d]pyrene are very consistent, and this compound was selected 29 

### for inclusion in the RPF approach. 30155 DRAFT – DO NOT CITE OR QUOTE1Figure 6-19. Cyclopenta[c,d]pyrene (CPcdP) RPFs.20.0010.010.1110100

Value of RPFReferenceRPF detection limits fornonpositive bioassaysPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 156 DRAFT – DO NOT CITE OR QUOTE 4H-Cyclopenta[d,e,f]chrysene (CPdefC) 1

2

3 

### 4H-Cyclopenta[d,e,f]chrysene (CASRN 202-98-2) is a nonalternant PAH comprised of 4 

### four aromatic rings and one five-membered ring. 4H -Cyclopenta[d,e,f]chrysene contains a 5 

### classic bay region but no fjord region in its struc ture. 6 

### There were two datasets for 4H-cyclopenta[d,e,f]chr ysene that met selection criteria and 7 

### included benzo[a]pyrene (Figure 6-20): both were m ulti-dose dermal initiation datasets (Rice et 8 

### al., 1988, 1985). Rice et al. (1988) reported a st atistically significant increase in tumor incidence 9 

### in a multi-dose dermal initiation study. In the se cond study, the incidence of tumors after 10 

### treatment with cyclopenta[d,e,f]chrysene exceeded 9 0%, precluding RPF derivation from 11 

### incidence data, but tumor multiplicity data were av ailable for RPF calculation (Rice et al., 1985). 12 

### Cyclopenta[d,e,f]chrysene has not been tested in a bioassay without benzo[a]pyrene; however, 13 

### sterically hindered diol epoxides of this compound have given positive results in a newborn 14 

### mouse assay (Amin et al., 1995). Because the bioas say of cyclopenta[d,e,f]chrysene was 15 

### positive, this PAH was considered potentially carci nogenic and was selected for inclusion in the 16 

### RPF approach. 17157 DRAFT – DO NOT CITE OR QUOTE1Figure 6-20. Cyclopenta[d,e,f]chrysene (CPdefC) RPFs.20.0010.010.1110100Rice et al., 1988Rice et al., 1985

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 158 DRAFT – DO NOT CITE OR QUOTE Dibenz[a,c]anthracene (DBacA) 1

2

3 

### Dibenz[a,c]anthracene (CASRN 215-58-7) is an altern ant PAH comprised of five fused 4 

### aromatic rings. Dibenz[a,c]anthracene contains thr ee bay regions but no fjord region in its 5



### structure. 6 

### There were 15 datasets for dibenz[a,c]anthracene th at met selection criteria and included 7 

### benzo[a]pyrene (Figure 6-21). The database include s a single in vivo study of DNA adducts, 8 

### nine mutagenicity or morphological/malignant cell t ransformation studies, and five studies of in 9 

### vitro DNA damage or adducts. One morphological/mal ignant cell transformation assay gave 10 

### nonpositive results, while the remaining studies we re positive. In the absence of positive 11 

### bioassays with benzo[a]pyrene, other bioassays and cancer-related data were considered to 12 

### evaluate the potential carcinogenicity of dibenz[a, c]anthracene. 13 

### Conflicting results were reported in three dermal i nitiation bioassays of 14 

### dibenz[a,c]anthracene in which benzo[a]pyrene was n ot included. Van Duuren et al. (1970) 15 

### observed a tumor incidence of 95% (19/20, compared to 1/20 controls) when mice were treated 16 

### with an initiating dose of 1 mg dibenz[a,c]anthrace ne in benzene followed by thrice weekly 17 

### treatment with phorbol myristate acetate. In contr ast, there was no significant increase in tumor 18 

### formation when the same initiating dose was followe d by thrice weekly application of croton 19 

### resin (Van Duuren et al., 1968); however, the laten cy to first tumor was substantially reduced 20 

### (65 vs. 150 days in controls). Latency was also su bstantially reduced in the study by Van 21 

### Duuren et al. (1970), in which the first tumor appe ared after 74 days, compared with 338 days in 22



### controls. 23 

### Cancer-related endpoint data for dibenz[a,c]anthrac ene are predominantly positive 24 

### (8/9 mutagenicity or morphological/malignant cell t ransformation studies and 5/5 studies of in 25 

### vitro DNA adducts or DNA damage). Although the con flicting bioassay data are not easily 26 

### explained, the high incidence of tumors (19/20) in the study by Van Duuren et al. (1970), and the 27 

### reduced latency to tumor formation in both studies, coupled with predominantly positive cancer28 

### related endpoint data, suggest that dibenz[a,c]anth racene is potentially carcinogenic. 29 

### Contributing to this conclusion is the observation that dibenz[a,c]anthracene is an alternant PAH 30 

### with known structural alerts for carcinogenicity (m ore than three rings, and three bay regions). 31 

### Thus, dibenz[a,c]anthracene was selected for inclus ion in the RPF approach. 32159 DRAFT – DO NOT CITE OR QUOTE1Figure 6-21. Dibenz[a,c]anthracene (DBacA) RPFs*.20.0010.010.1110100

Value of RPFReference* Missing bar indicates nonpositive cancer-relatedendpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 160 DRAFT – DO NOT CITE OR QUOTE Dibenzo[a,e]fluoranthene (DBaeF) 1

2 

### Dibenzo[a,e]fluoranthene (CASRN 5385-75-1) is a non alternant PAH comprised of five 3 

### aromatic rings and one five-membered ring. Dibenzo [a,e]fluoranthene contains a classic bay 4 

### region but no fjord region in its structure. 5 

### There were three datasets for dibenzo[a,e]fluoranth ene that met selection criteria and 6 

### included benzo[a]pyrene (Figure 6-22); all gave pos itive results. The database includes two in 7 

### vivo tumor bioassays and one mammalian mutagenicity study. Statistically significant increases 8 

### in tumor incidence were reported in dermal initiati on and complete carcinogenicity bioassays in 9 

### mice (both reported by Hoffmann and Wynder, 1966). As the available bioassays for 10 

### dibenzo[a,e]fluoranthene were positive, this compou nd was considered potentially carcinogenic 11 

### and was selected for inclusion in the RPF approach. 12

13161 DRAFT – DO NOT CITE OR QUOTE1Figure 6-22. Dibenzo[a,e]fluoranthene (DBaeF) RPFs.20.0010.010.1110100Hoffmann and Wynder 1966 Hoffmann and Wynder 1966 Durant et al., 1999

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 162 DRAFT – DO NOT CITE OR QUOTE Dibenzo[a,e]pyrene (DBaeP) 1

2

3 

### Dibenzo[a,e]pyrene (CASRN 192-65-4) is an alternant PAH comprised of six fused 4 

### aromatic rings. Dibenzo[a,e]pyrene contains three bay regions but no fjord region in its 5



### structure. 6 

### There were three datasets for dibenzo[a,e]pyrene th at met selection criteria and included 7 

### benzo[a]pyrene (Figure 6-23). The database include s two in vivo tumor bioassay datasets and 8 

### one in vitro bacterial mutagenicity dataset, all of which gave positive results. Statistically 9 

### significant increases in tumor incidence were repor ted in dermal initiation and complete 10 

### carcinogenicity bioassays in mice (Hoffmann and Wyn der, 1966). The complete carcinogenicity 11 

### bioassay was confounded by significant toxicity-rel ated mortality unrelated to tumors (Hoffmann 12 

### and Wynder, 1966). The one bacterial mutagenicity study reported positive results. Because the 13 

### available bioassays with benzo[a]pyrene were both p ositive, dibenzo[a,e]pyrene was considered 14 

### potentially carcinogenic and was selected for inclu sion in the RPF approach. 15

16163 DRAFT – DO NOT CITE OR QUOTE1Figure 6-23. Dibenzo[a,e]pyrene (DBaeP) RPFs.20.0010.010.1110100Hoffmann and Wynder 1966 Hoffmann and Wynder 1966 Teranishi et al., 1975

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 164 DRAFT – DO NOT CITE OR QUOTE Dibenz[a,h]anthracene (DBahA) 1

2 

### Dibenz[a,h]anthracene (CASRN 53-70-3) is an alterna nt PAH comprised of five fused 3 

### aromatic rings. Dibenz[a,h]anthracene contains two bay regions and no fjord region in its 4



### structure. 5 

### There were 30 datasets for dibenz[a,h]anthracene th at met selection criteria and included 6 

### benzo[a]pyrene (Figure 6-24). Included in the data base are in vivo tumor bioassay datasets (4), 7 

### in vivo DNA adduct datasets (2), an in vivo clastog enicity dataset, mutagenicity datasets (10) 8 

### morphological/malignant cell transformation dataset s (6), and in vitro DNA damage, adducts, or 9 

### clastogenicity datasets (7). There were three tumo r bioassays for dibenz[a,h]anthracene that 10 

### included benzo[a]pyrene, and all resulted in statis tically significant increases in tumor incidence 11 

### and/or multiplicity. The bioassays were in three d ifferent test systems: a rat lung implantation 12 

### study (Wenzel-Hartung et al., 1990), a mouse dermal initiation study reporting both incidence 13 

### and multiplicity (Slaga et al., 1980), and an intra peritoneal study in A/J mice (Nesnow et al., 14 

### 1998b). Dibenz[a,h]anthracene was shown to form DN A adducts when administered in vivo to 15 

### mice via intraperitoneal injection (Nesnow et al., 1998b) and dermal application (Phillips et al., 16 

### 1979). Mutagenicity and morphological/malignant ce ll transformation assays of 17 

### dibenz[a,h]anthracene were predominantly positive ( 13/16), as were studies of other cancer18 

### related endpoints. Because the available bioassays with benzo[a]pyrene were positive, 19 

### dibenz[a,h]anthracene was considered potentially ca rcinogenic and was selected for inclusion in 20 

### the RPF approach. 21

22165 DRAFT – DO NOT CITE OR QUOTE1Figure 6-24. Dibenz[a,h]anthracene (DBahA) RPFs*.20.0010.010.1110100

Value of RPFReference* Missing bar indicates nonpositive cancer-relatedendpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 166 DRAFT – DO NOT CITE OR QUOTE Dibenzo[a,h]pyrene (DBahP) 1

2 

### Dibenzo[a,h]pyrene (CASRN 189-64-0) is an alternant PAH comprised of six fused 3 

### aromatic rings. Dibenzo[a,h]pyrene contains two ba y regions and no fjord region in its structure. 4 

### There were five datasets for dibenzo[a,h]pyrene tha t met selection criteria and included 5 

### benzo[a]pyrene (Figure 6-25); all gave positive res ults. The database includes one in vivo 6 

### bioassay dataset, one in vivo DNA adduct dataset, t wo in vitro mammalian mutagenicity 7 

### datasets, and one in vitro DNA damage dataset. A s tatistically significant increase in tumor 8 

### incidence was reported in a dermal initiation bioas say in mice (Hoffmann and Wynder, 1966). 9 

### In addition, two dermal studies of complete carcino genicity that included benzo[a]pyrene gave 10 

### positive results, but no RPF could be calculated be cause the incidence of tumors in the mice 11 

### exposed to dibenzo[a,h]pyrene was ≥90% at the lowes t dose tested (Cavalieri et al., 1977; 12 

### Hoffmann and Wynder, 1966) and tumor multiplicity w as not reported. As all of the available 13 

### bioassays that included benzo[a]pyrene showed expos ure-related tumorigenic responses, 14 

### dibenzo[a,h]pyrene was considered potentially carci nogenic and was selected for inclusion in the 15 

### RPF approach. 16

17167 DRAFT – DO NOT CITE OR QUOTE1Figure 6-25. Dibenzo[a,h]pyrene (DBahP) RPFs.20.0010.010.1110100Hoffmann and Wynder 1966 Hughes and Phillips 1990 Durant et al., 1999 Hass et al., 1982 Mersch-Sundermannet al.,1992

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 168 DRAFT – DO NOT CITE OR QUOTE Dibenzo[a,i]pyrene (DBaiP) 1

2 

### Dibenzo[a,i]pyrene (CASRN 189-55-9) is an alternant PAH comprised of six fused 3 

### aromatic rings. Dibenzo[a,i]pyrene contains two ba y regions and no fjord region in its structure. 4 

### There were 12 datasets for dibenzo[a,i]pyrene that met selection criteria and included 5 

### benzo[a]pyrene (Figure 6-26); all gave positive res ults. The database includes two in vivo 6 

### bioassay datasets, one in vivo DNA adduct dataset, seven in vitro mutagenicity datasets, and two 7 

### in vitro DNA damage datasets. Statistically signif icant increases in tumor incidence were 8 

### reported in dermal initiation and complete carcinog enicity bioassays in mice, both published by 9 

### Hoffmann and Wynder (1966). The cancer-related end point studies were all positive. As the 10 

### available bioassays that included benzo[a]pyrene we re both positive, dibenzo[a,i]pyrene was 11 

### considered potentially carcinogenic and was selecte d for inclusion in the RPF approach. 12

13169 DRAFT – DO NOT CITE OR QUOTE1Figure 6-26. Dibenzo[a,i]pyrene (DbaiP) RPFs*.20.0010.010.1110100Hoffmann andWynder 1966Hoffmann andWynder 1966Hughes andPhillips 1990Durant et al.,1999Hass et al.,1982Teranishi etal., 1975Hermann,1981MerschSundermann etal., 1992)Phillipson andIoannides 1989Baker et al.,1980Ichinotsubo etal., 1977McCann et al.,1975

Value of RPFReference* Missing bar indicates nonpositive genotoxicity studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 170 DRAFT – DO NOT CITE OR QUOTE Dibenzo[a,l]pyrene (DBalP). 1

2 

### Dibenzo[a,l]pyrene (CASRN 191-30-0) is an alternan t PAH comprised of six fused 3 

### aromatic rings. Dibenzo[a,l]pyrene contains both a bay region and a fjord region in its structure. 4 

### There were 15 datasets for dibenzo[a,l]pyrene that met selection criteria and included 5 

### benzo[a]pyrene (Figure 6-27); all of the studies ga ve positive results. The database includes 6 

### three in vivo tumor bioassay datasets, three in viv o DNA adduct datasets, one bacterial 7 

### mutagenicity dataset, one morphological/malignant c ell transformation dataset, four in vivo 8 

### clastogenicity datasets, and three in vitro DNA add uct or DNA damage datasets. 9 

### In three bioassays of dibenzo[a,l]pyrene included b enzo[a]pyrene, RPFs could not be 10 

### calculated using incidence data, because the incide nce of tumors associated with the lowest dose 11 

### of dibenzo[a,l]pyrene exceeded 90% (two dermal init iation experiments in mice and an 12 

### intramammilary injection study in rats, both report ed by Cavalieri et al., 1991); however, tumor 13 

### multiplicity data were reported for the dermal init iation experiments and were used to calculate 14 

### RPFs of 10 and 40. Nesnow et al. (1998b) reported tumor multiplicity, but not tumor incidence 15 

### in A/J mice exposed intraperitoneally; an RPF of 30 was calculated. Because the available 16 

### studies indicated that dibenzo[a,l]pyrene may be mu ch more potent benzo[a]pyrene, other studies 17 

### were also examined to confirm the potency of this c ompound. 18 

### Dibenzo[a,l]pyrene treatment resulted in significan t increases in tumor incidence in seven 19 

### bioassays that did not include benzo[a]pyrene, incl uding two dermal initiation studies (Gill et al., 20 

### 1994; Cavalieri et al., 1989), a dermal complete ca rcinogenicity study (Nakatsuru et al., 2004), 21 

### an intramammilary injection study in rats (Cavalier i et al., 1989), a newborn mouse bioassay 22 

### (Platt et al., 2004), an intraperitoneal bioassay u sing A/J mice (Prahalad et al., 1997), and a 23 

### gavage bioassay comparing the responses of cyp1B1 w ild-type and null mice (Buters et al., 24 

### 2002). In several of these studies, there was sign ificant toxicity associated with 25 

### dibenzo[a,l]pyrene treatment. Tumor incidences wer e very high in most of the studies, including 26 

### the gavage study (Buters et al., 2002), which repor ted an overall tumor incidence of 100% in 27 

### cyp1B1 wild-type mice treated with a single dose of dibenzo[a,l]pyrene. A recent study 28 

### examining in utero and/or lactational exposure to d ibenzo[a,l]pyrene showed that mouse pups 29 

### exposed during late gestation develop T-cell lympho mas between 3 and 6 months of age, as well 30 

### multiple lung and liver tumors (Castro et al., 2008 ). All of the cancer-related data for 31 

### dibenzo[a,l]pyrene were positive and resulted in hi gh RPF estimates, including in vivo and in 32 

### vitro studies of DNA adducts, in vivo clastogenicit y studies, morphological/malignant cell 33 

### transformation, bacterial mutagenicity, and in vitr o DNA damage or DNA adduct studies. 34

### 171 DRAFT – DO NOT CITE OR QUOTE

The weight of evidence supporting a finding of pote ntial carcinogenicity for 1 

### dibenzo[a,l]pyrene is strong and suggests that this compound is very potent; thus, it was selected 2 

### for inclusion in the RPF approach. 3

4172 DRAFT – DO NOT CITE OR QUOTE1Figure 6-27. Dibenzo[a,l]pyrene (DBalP) RPFs.20.0010.010.1110100

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 173 DRAFT – DO NOT CITE OR QUOTE Fluoranthene (FA) 1

2 

### Fluoranthene (CASRN 206-44-0) is a nonalternant PAH comprised of three aromatic 3 

### rings and one five-membered ring. Fluoranthene doe s not contain a classic bay or fjord region in 4



### its structure. 5



### There were 21 datasets for fluoranthene that met se lection criteria and included 6 

### benzo[a]pyrene (Figure 6-28). Included in the data base are in vivo tumor bioassay datasets (11), 7 

### bacterial and mammalian mutagenicity datasets (5), a morphological/malignant cell 8 

### transformation assay, and in vitro studies of DNA d amage, DNA adducts, or clastogenicity (4). 9 

### Of the bioassay datasets that included benzo[a]pyre ne, nine gave positive results and two gave 10 

### nonpositive results. Statistically significant inc reases in tumor incidence and tumor multiplicity 11 

### were reported in newborn mouse bioassays (in male a nd female mice [LaVoie et al., 1994] and in 12 

### female mice [Busby et al., 1989]). The tumor incid ence was not significantly increased by 13 

### fluoranthene in a mouse dermal initiation study wit h an RPF detection limit of 0.01 (Hoffman et 14 

### al., 1972) and when fluoranthene was tested alone i n a dermal cocarcinogenicity bioassay with 15 

### an RPF detection limit of 0.1 (Van Duuren and Golds chmidt, 1976). In another newborn mouse 16 

### bioassay (Busby et al., 1984) that reported both in cidence and multiplicity, the lowest dose of 17 

### benzo[a]pyrene resulted in a tumor incidence of >90 %, precluding RPF calculation from the 18 

### incidence data; however, multiplicity data were ava ilable. Statistical analysis of the data for 19 

### fluoranthene demonstrated positive findings for bot h incidence and multiplicity in male mice, but 20 

### the results for the two endpoints were inconsistent in females. In female mice exposed at the 21 

### high dose of fluoranthene in a newborn mouse bioass ay reported by Busby et al. (1984), the lung 22 

### tumor count was significantly increased (albeit bor derline, p = 0.0343) while the incidence was 23 

### not (

p > 0.05), and neither was statistically significant ly increased at the lower dose. For the 24 

### purpose of this analysis, the multiplicity data wer e treated as an independent measure of 25 

### carcinogenic potency, and an RPF was calculated for the statistically increased tumor count in 26



### female mice. 27 

### The mutagenicity studies of fluoranthene were all p ositive, but in vitro studies of DNA 28 

### damage, DNA adducts, and clastogenicity gave incons istent results. Because the inconsistent 29 

### bioassay results can be attributed to different tes t systems (different exposure route and/or 30 

### gender) or study design, fluoranthene was considere d potentially carcinogenic and was selected 31 

### for inclusion in the RPF approach. 32

33174 DRAFT – DO NOT CITE OR QUOTE1Figure 6-28. Fluoranthene (FA) RPFs*.20.0010.010.1110100

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 175 DRAFT – DO NOT CITE OR QUOTE Fluorene (FE) 1

2 

### Fluorene (CASRN 86-73-7) is a nonalternant PAH comp rised of two aromatic rings and 3 

### one five-membered ring. Fluorene does not contain a classic bay or fjord region in its structure. 4



### There were nine datasets for fluorene that met sele ction criteria and included 5 

### benzo[a]pyrene (Figure 6-29). There were no tumor bioassays of fluorene that included 6 

### benzo[a]pyrene, so other bioassays and cancer-relat ed endpoint data were considered. LaVoie et 7 

### al. (1980) conducted a study of skin tumor initiati on in mice treated with 1.0 mg fluorene 8 

### followed by 20 weeks of treatment with TPA; the stu dy did not include benzo[a]pyrene. The 9 

### incidence of tumor-bearing animals (5%) was not sig nificantly increased over controls (0%) 10 

### (LaVoie et al.,1980). The limited cancer-related e ndpoint data were mixed, with three positive 11 

### and four negative mutagenicity datasets, and two ne gative in vitro DNA damage datasets. 12 

### Overall, the database for fluorene is both limited and inconsistent. Because the database for 13 

### fluorene does not provide adequate information with which to assess potential carcinogenicity, 14



### this PAH was not selected for inclusion in the RPF approach. 15

16

17176 DRAFT – DO NOT CITE OR QUOTE1Figure 6-29. Fluorene (FE) RPFs*.20.0010.010.1110100Sakai et al., 1985 Gibson et al., 1978 Wangenheim andBolcsfoldi 1988Hermann, 1981 Kaden et al., 1979 McCann et al.,1975Pahlman andPelkonen, 1987Mamber et al.,1983MerschSundermann et al.,1992

Value of RPFReference* Missing bar indicates nonpositive cancer-relatedendpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 177 DRAFT – DO NOT CITE OR QUOTE Indeno[1,2,3-c,d]pyrene (IP) 1

2 

### Indeno[1,2,3-c,d]pyrene (CASRN 193-39-5) is a nonal ternant PAH comprised of five 3 

### aromatic rings and one five-membered ring. Indeno[ 1,2,3-c,d]pyrene does not contain a classic 4 

### bay or fjord region in its structure. 5 

### There were five datasets for indeno[1,2,3-c,d]pyren e that met selection criteria and 6 

### included benzo[a]pyrene (Figure 6-30). There are t hree tumor bioassays, one in vitro study of 7 

### morphological/malignant cell transformation (Emura et al., 1980), and one in vitro study of DNA 8 

### damage (Mersch-Sundermann et al., 1992). Of the th ree tumor bioassays, only one, a rat lung 9 

### implantation study (Deutsch-Wenzel et al., 1983), r eported a statistically significant increase in 10 

### tumor incidence or multiplicity; the RPF was 0.07. Nonpositive findings were reported in mouse 11 

### dermal initiation (Hoffmann and Wyner, 1966) and co mplete carcinogenicity (Habs et al., 1980) 12 

### studies with RPF detection limits in the range of 0 .1–0.3. Because the inconsistent bioassay 13 

### results can be attributed to different test systems (different species and route), and the 14 

### nonpositive studies may not have been sufficiently sensitive to detect an effect, indeno[1,2,3-c,d] 15 

### pyrene was considered potentially carcinogenic and was selected for inclusion in the RPF 16 

### approach. 17

18178 DRAFT – DO NOT CITE OR QUOTE1Figure 6-30. Indeno[1,2,3-c,d]pyrene (IP) RPFs.20.0010.010.1110100Deutsch-Wenzel et al., 1983Hoffmann and Wynder 1966Habs et al., 1980Mersch-Sundermann et al., 1992Emura et al., 1980

Value of RPFReferenceRPF detection limits fornonpositive bioassaysPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 179 DRAFT – DO NOT CITE OR QUOTE Naphtho[2,3-e]pyrene (N23eP) 1

2 

### Naphtho[2,3-e]pyrene (CASRN 193-09-9) is an alterna nt PAH comprised of six fused 3 

### aromatic rings. Naphtho[2,3-e]contains two bay reg ions and no fjord region in its structure. 4 

### There were two datasets for naphtho[2,3-e]pyrene th at met selection criteria and included 5 

### benzo[a]pyrene (Figure 6-31): a tumor bioassay dat aset and an in vitro mammalian mutagenicity 6 

### dataset (both were positive). The tumor bioassay w as a single dose dermal initiation bioassay 7 

### (Hoffmann and Wynder, 1966). As the available bioa ssay reported a statistically significant 8 

### increase in tumor incidence, naphtho[2,3-e]pyrene w as considered potentially carcinogenic, and 9 

### was selected for inclusion in the RPF approach. 10

11180 DRAFT – DO NOT CITE OR QUOTE1Figure 6-31. Naphtho[2,3-e]pyrene (N23eP) RPFs.20.0010.010.1110100Hoffmann and Wynder 1966Durant et al., 1999

Value of RPFReferencePositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 181 DRAFT – DO NOT CITE OR QUOTE Perylene (Pery) 1

2 

### Perylene (CASRN 198-55-0) is an alternant PAH compr ised of five fused aromatic rings. 3 

### Perylene contains two bay regions and no fjord regi on in its structure. 4 

### There were 11 datasets for perylene that met select ion criteria and included 5 

### benzo[a]pyrene (Figure 6-32). The database include s an in vivo tumor bioassay dataset, an in 6 

### vivo clastogenicity dataset, eight bacterial mutage nicity datasets, and an in vitro DNA damage 7 

### dataset. The single tumor bioassay, a dermal initi ation study, gave nonpositive results for 8 

### perylene (El-Bayoumy et al., 1982); the RPF detecti on limit was 0.01. To confirm the 9 

### nonpositive bioassay findings, other bioassays and cancer-related endpoint data were considered. 10 

### In a study that did not include benzo[a]pyrene, Van Duuren et al. (1970) did not observe an 11



### increase in tumor incidence over controls when mice were treated by dermal application with an 12 

### initiating dose of 0.8 mg perylene in benzene follo wed by thrice weekly treatment with phorbol 13 

### myristate acetate for 58 weeks. However, seven of the eight bacterial mutagenicity studies gave 14 

### positive results, while perylene tested negative in one bacterial mutagenicity study, the 15 

### clastogenicity study, and the DNA damage study. Ov erall, the database for perylene is both 16



### limited and inconsistent. Because the database for perylene does not provide adequate 17 

### information with which to assess potential carcinog enicity, this PAH was not selected for 18 

### inclusion in the RPF approach. 19

20182 DRAFT – DO NOT CITE OR QUOTE1Figure 6-32. Perylene (Pery) RPFs*.20.0010.010.1110100El-Bayoumy etal., 1982Sirianni andHuang, 1978Kaden et al.,1979Carver et al.,1986Sakai et al., 1985 Hermann, 1981 LaVoie et al.,1979De Flora et al.,1984Florin et al.,1980Pahlman andPelkonen, 1987MerschSundermann etal., 1992

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 183 DRAFT – DO NOT CITE OR QUOTE Phenanthrene (PH) 1

2 

### Phenanthrene (CASRN 85-01-8) is an alternant PAH co mprised of three fused aromatic 3 

### rings. Phenanthrene contains a bay region in its s tructure, but has less than four aromatic rings. 4 

### There were 34 datasets for phenanthrene that met se lection criteria and included 5 

### benzo[a]pyrene, including 3 in vivo tumor bioassay datasets, 2 in vivo clastogenicity datasets, 6 

### 11 mutagenicity datasets, 6 morphological/malignant cell transformation datasets, and 12 in vitro 7 

### studies of DNA adducts, DNA damage, or clastogenici ty (Figure 6-33). Only seven studies 8 

### reported positive results; the remaining 27 studies reported nonpositive findings, including all 9 

### three bioassays. Nonpositive findings were reporte d in the three bioassays that included 10 

### benzo[a]pyrene, including a lung implantation study in rats (Wenzel-Hartung et al., 1990), a 11 

### dermal initiation study in mice (LaVoie et al., 198 1), and a subcutaneous study in mice (Grant 12 

### and Roe, 1963). To confirm the nonpositive finding s, other bioassays and cancer-related 13 

### endpoint data were considered. In bioassays withou t benzo[a]pyrene, phenanthrene did not 14 

### induce significant increases in tumors in a newborn mouse assay using a total dose of 1.4 μmol 15 

### (Buening et al., 1979) or in two dermal initiation assays (Wood et al., 1979; Salaman and Roe, 16 

### 1956) using doses of 10 μmol and 540 mg, respective ly. However, 12/30 mice developed 17 

### papillomas by week 35 after dermal treatment with 1 0 μmol phenanthrene (in benzene) followed 18 

### by twice weekly treatment with TPA; no control mice had papillomas (Scribner, 1973). The 19 

### response was statistically significantly increased over controls ( p < 0.01). 20 

### In vitro assays of mutagenicity and morphological/m alignant cell transformation were 21 

### predominantly negative for phenanthrene. One of th e two positive studies (Sakai et al., 1988) 22 

### reported a poor dose-response relationship for phen anthrene. Two studies found evidence of 23 

### clastogenicity after in vivo administration of phen anthrene (Roszinsky-Kocher et al., 1979; 24 

### Bayer, 1978). However, in the study by Bayer (1978 ), only the high dose gave a significant 25 

### response, and there was not a significant dose-resp onse trend. When phenathrene was tested in 26 

### in vitro studies of DNA adducts, DNA damage, and cl astogenicity, the results were 27 

### predominantly negative (9/12 studies). Overall, th e database for phenanthrene is substantial, and 28 

### the weight of evidence suggests that this PAH is no t carcinogenic or is of very low carcinogenic 29 

### potential. Based on the large number of negative b ioassays and the abundant evidence that 30 

### phenanthrene lacks genotoxic action, this compound was selected for inclusion in the RPF 31 

### approach and assigned an RPF of 0. 32

33184 DRAFT – DO NOT CITE OR QUOTE1Figure 6-33. Phenanthrene (PH) RPFs*.20.0010.010.1110100

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 185 DRAFT – DO NOT CITE OR QUOTE Pyrene (Pyr) 1

2

3 

### Pyrene (CASRN 129-00-0) is an alternant PAH compris ed of four fused aromatic rings. 4 

### Pyrene does not contain a bay or fjord region in it s structure. 5 

### There were 49 datasets for pyrene that met study qu ality criteria and included 6 

### benzo[a]pyrene (Figure 6-34). Included in the data base are in vivo tumor bioassay datasets (7), 7 

### in vivo clastogenicity datasets (5), bacterial and mammalian mutagenicity datasets (14), 8 

### morphological/malignant cell transformation dataset s (7), and in vitro DNA damage, DNA 9 

### adducts, or clastogenicity datasets (16). There we re seven bioassays of pyrene that included 10 

### benzo[a]pyrene; all gave nonpositive results. Nonp ositive results were reported in two newborn 11 

### mouse bioassays in which both males and females wer e tested (Busby et al., 1989; Wislocki et 12 

### al., 1986), two studies of dermal initiation (El-Ba youmy et al., 1982; Wood et al., 1980), and a 13 

### dermal cocarcinogenesis bioassay (Van Duuren and Go ldschmidt, 1976). RPF detection limits in 14 

### these studies ranged from about 0.01 to 0.1 (see Fi gure 6-34). In an intraperitoneal bioassay 15 

### using A/J mice that included benzo[a]pyrene, the au thors reported that pyrene treatment did not 16 

### induce lung adenomas (Ross et al., 1995); data were not reported, so an RPF detection limit 17 

### could not be estimated. In bioassays without benzo [a]pyrene, pyrene did not induce a significant 18 

### increase in tumors in a dermal initiation bioassay (Salaman and Roe, 1956). Scribner (1973) 19 

### reported a weak tumorigenic response in a dermal in itiation study in mice (5/29 mice developed 20 

### papillomas 35 weeks after dermal treatment with 10 μmol pyrene in benzene followed by twice 21 

### weekly treatment with TPA as compared with 0/30 con trol mice, p = 0.02). 22 

### In vitro assays of bacterial and mammalian mutageni city and morphological/malignant 23 

### cell transformation were predominantly negative for pyrene. In five studies of clastogenicity in 24 

### animals exposed in vivo to pyrene, no evidence of c lastogenic effects was reported. Further, in 25 

### vitro studies of DNA adducts, DNA damage, and clast ogenicity using pyrene also largely 26 

### reported negative results. Overall, the database f or pyrene is substantial, and the weight of 27 

### evidence suggests that this PAH is not carcinogenic or is of very low carcinogenic potential. 28 

### Based on the large number of negative bioassays and the abundant evidence that pyrene lacks 29 

### genotoxic action, this compound was selected for in clusion in the RPF approach and assigned an 30



### RPF of 0. 31

32186 DRAFT – DO NOT CITE OR QUOTE1Figure 6-34. Pyrene (Pyr) RPFs*.20.0010.010.1110100

Value of RPFReferenceRPF detection limits fornonpositive bioassays* Missing bar indicates nonpositive cancer-related endpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 187 DRAFT – DO NOT CITE OR QUOTE Triphenylene (TPhen) 1

2 

### Triphenylene (CASRN 217-59-4) is an alternant PAH c omprised of four fused aromatic 3 

### rings. Triphenylene contains several bay regions a nd no fjord region in its structure. 4 

### There were six datasets for triphenylene that met s election criteria and included 5 

### benzo[a]pyrene (Figure 6-35); all but one of the st udies gave positive results. The database 6 

### includes five mutagenicity studies (four positive a nd one negative) and a study of in vitro DNA 7 

### damage. There were no bioassays of triphenylene th at met selection criteria, and no bioassays 8 

### without benzo[a]pyrene. Although all of the availa ble cancer-related endpoint studies for 9 

### triphenylene gave positive results, the database is very limited, consisting of only a few in vitro 10 

### mutagenicity and DNA damage studies. The RPFs for cancer-related endpoints ranged from 11 

### 0.02 to 0.4. Because the database for triphenylene does not provide adequate information with 12 

### which to assess potential carcinogenicity, this PAH was not selected for inclusion in the RPF 13 

### approach. 14

15

16188 DRAFT – DO NOT CITE OR QUOTE1Figure 6-35. Triphenylene (Tphen) RPFs*.20.0010.010.1110100Mersch-Sundermann etal., 1992Hermann, 1981Kaden et al., 1979Barfknecht et al., 1982Pahlman and Pelkonen1987Gibson et al., 1978

Value of RPFReference* Missing bar indicates nonpositive cancer-relatedendpoint studyPositive bioassay (incidence)Positive bioassay (multiplicity)In vivo cancer-related endpointIn vitro cancer-related endpoint

### 189 DRAFT – DO NOT CITE OR QUOTE 7. DERIVATION OF FINAL RPFs FOR SELECTED PAHs 1

2

3 

### The weight of evidence evaluation (Section 6) indic ates that the available data are 4 

### adequate to suggest that 23 of the 26 PAHs are pote ntially carcinogenic, three PAHs (anthracene, 5 

### phenanthrene, and pyrene) exhibited little or no ca rcinogenic potential, and data are inadequate 6 

### to evaluate the carcinogenic potential for eight PA Hs. The eight PAHs with inadequate data are 7 

### excluded from the RPF analysis. 8



### For the three PAHs for which there were sufficient data to conclude that the PAH had 9 

### minimal carcinogenic potential (i.e., robust negati ve tumor bioassay data and cancer-related 10 

### endpoint data), a final RPF of 0 was recommended. While there is little quantitative difference 11 

### between selecting a final RPF of 0 for a given PAH and excluding that PAH from the RPF 12 

### approach, this is an important distinction for unce rtainty analysis. There is substantial 13 

### uncertainty in the risk associated with PAHs that a re excluded from the RPF analysis due to 14 

### inadequate data, as these compounds could be of low or high potency. However, for PAHs with 15 

### an RPF of 0, there is evidence to suggest that thes e compounds are of little or no carcinogenic 16 

### potential, and the uncertainty associated with the cancer risk for these compounds is markedly 17



### reduced. 18 

### For each of the remaining 23 compounds, a final non zero RPF was derived. A number of 19 

### options were considered for deriving a final RPF fr om among the numerous values calculated for 20 

### each individual PAH. These options included: prio ritizing bioassay RPFs from different 21 

### exposure routes based on relevance to environmental ly-relevant routes; prioritizing bioassay 22 

### RPFs based on target organs considered relevant to human susceptibility to PAH carcinogenesis; 23 

### prioritizing RPFs based on quality of the underlyin g study; prioritizing cancer-related endpoints 24 

### by their correlation with bioassay potency (i.e., a bility to predict bioassay potency); and 25 

### combining RPFs across all bioassays, all cancer-rel ated endpoints, or across all endpoints. 26 

### Appendix G details analyses that were undertaken to assess various options for ranking or 27 

### prioritizing RPFs. It was concluded that the avail able data did not provide a basis for prioritizing 28 

### RPFs except for a preference for bioassay data over cancer-related endpoints. As a consequence, 29 

### final RPFs were derived from bioassay data for any PAH that had at least one RPF based on a 30 

### bioassay. For potentially carcinogenic PAHs withou t bioassay data, final RPFs were calculated 31 

### from all cancer-related endpoint datasets with posi tive results (see next section). 32

33



### 7.1. METHODS FOR DERIVING FINAL RPFs 34 

### For each potentially carcinogenic PAH with bioassay data, the average RPF was 35 

### calculated from bioassay datasets with positive res ults (nonpositive bioassay results were not 36 

### included in the calculation). For those PAHs that did not have any RPF based on a bioassay, but 37 

### for which the weight of evidence evaluation indicat ed a potential for carcinogenic response (e.g., 38

### 190 DRAFT – DO NOT CITE OR QUOTE

dibenz[a,c]anthracene), the average RPF was calcula ted from all cancer-related endpoint datasets 1 

### with positive results (again, nonpositive results w ere not included in the calculation). The range 2 

### of RPF values was also reported. Presenting the av erage and the range provides an average and 3



### maximum estimate for each PAH that has data from mu ltiple studies. 4 

### Several options were considered for the estimation of a final RPF (e.g., arithmetic mean, 5 

### geometric mean, weighted average, maximum, or order of magnitude estimates). The arithmetic 6 

### mean and range were chosen as a simple approach to describing the calculated RPF values 7



### available for each PAH. Other statistical measures (i.e., geometric mean, weighted average) 8



### were not considered due to the limited number of RP F values calculated for most PAHs and the 9 

### variability in the RPF estimates. There were usual ly not enough data (3 or fewer RPFs for 17/23 10 

### PAHs with nonzero RPFs) to assess the shape of the RPF distribution for any given PAH , so a 11 

### geometric mean was not considered. Further, the ra nge of RPF values from tumor bioassays was 12 

### greater than an order of magnitude for several comp ounds (6/23 PAHs). The variability in RPF 13 

### estimates is likely due to differences in study des ign parameters (e.g., route, species/strain, 14 

### exposure duration, exposure during sensitive time p eriods, initiation vs. complete carcinogenesis 15 

### protocol, tumor incidence vs. tumor multiplicity re porting) and dose response methods (modeled 16 

### vs. point estimates). Calculation of a weighted av erage was considered, but without a rationale 17 

### for assigning weights among study types or tumor da ta outcomes, using a weighting approach 18 

### might increase uncertainty. Several previous appro aches for generating RPF values for PAHs 19 

### have used order of magnitude estimates (Collins et al., 1998; Malcolm and Dobson, 1994; U.S. 20 

### EPA, 1993; Nisbet and LaGoy, 1992, see Section 3). Providing order of magnitude estimates 21 

### was not considered to be superior to calculating si mple means. The presentation of the 22 

### arithmetic mean and range of RPFs for each PAH was considered to be more transparent and 23



### more reflective of the available data than an order -of-magnitude approach. Including the range 24



### in the estimated RPFs was considered to be informat ive to the user for characterizing 25 

### uncertainty. 26 

### The range was reported as a measure of variability instead of a confidence interval on the 27 

### average RPF. The input data for the average RPF (b ioassay RPFs of different route, species, sex, 28 

### and target organ, or cancer-related endpoint data a cross a wide variety of assays and test 29 

### conditions) are likely to be correlated in unquanti fiable and variable ways. There may be a high 30 

### degree of correlation between RPFs calculated for a given PAH from dermal initiation and 31 

### complete carcinogenicity studies, or between RPFs c alculated from incidence and multiplicity 32 

### data reported for the same study, but lower correla tion between RPFs from dermal initiation and 33 

### intraperitoneal injection. In addition, there are differences between male and female target 34 

### organs after exposure to PAHs in newborn mouse tumo r bioassays; RPFs from these datasets 35 

### may have little or no correlation. As a result, a confidence interval on the average RPF was not 36 

### calculated, but rather the range of calculated valu es was used as a means of expressing 37 

### variability. 38

### 191 DRAFT – DO NOT CITE OR QUOTE

All tumor bioassay RPFs (across all exposure route s, species, sexes, and including both 1 

### tumor incidence and tumor multiplicity RPFs) were c ombined to estimate the mean and range, 2 

### except as follows. When separate RPFs were calcula ted for different endpoints in the same 3 

### group of animals, the higher value of the two RPFs was included in the average and range, and 4 

### the lower value was dropped from the combined data. There were two situations in which this 5 

### occurred: RPFs for different target organs in the same animals, and RPFs based on incidence of 6



### tumors and tumor count in the same animals. Differ ent RPFs were calculated for liver and lung 7 

### tumors in male mice (females did not develop liver tumors) in newborn mouse studies that 8 

### reported incidences or tumor counts separately. Th is occurrence applied only to 9 

### benz[a]anthracene, chrysene, and fluoranthene teste d in studies reported by LaVoie et al. (1994) 10 

### and Wislocki et al. (1986). Likewise, when both in cidence and multiplicity RPFs were 11 

### calculated from the same experiment, the higher of the two values was included in the combined 12 

### data, and the lower value was excluded. A comparis on between RPFs calculated from incidence 13 

### and tumor multiplicity data from the same experimen t showed these values to be highly 14 

### correlated (r 2 

### = 0.8; see further discussion in Section 8), so RP Fs from the two endpoints could 15 

### not be treated as independent measures of relative potency. 16

17



### 7.2. CONFIDENCE RATINGS FOR FINAL RPFs 18 

### Once a final RPF was derived for a given PAH, the r esulting value was assigned a 19 

### relative confidence rating of high, medium, low, or very low confidence . The relative confidence 20 

### rating characterized the nature of the database upo n which the final RPF was based. Confidence 21 

### rankings were based on the robustness of the databa se. For final RPFs based on tumor bioassay 22 

### data, confidence ratings considered both the availa ble tumor bioassays and the size and 23 

### consistency of the cancer-related endpoint database . The most important factors that were 24 

### considered included the availability of in vivo dat a and whether multiple exposure routes were 25 

### represented. Other database characteristics that w ere considered important included the strength 26 

### of evidence of genotoxicity data and SAR informatio n, the availability of more than one in vivo 27 

### study, and whether effects were evident in more tha n one sex or species. Very low relative 28 

### confidence was used to describe final RPFs based on cancer-rel ated endpoint data only (e.g., 29 

### dibenz[a,c]anthracene). 30 

### For RPFs of zero, the confidence rating considered both the available tumor bioassays 31 

### and the size and consistency of the cancer-related endpoint database. An RPF of zero was only 32 

### applied if the data implied high or medium relative confidence . For anthracene, phenanthrene, 33 

### and pyrene, it has been determined that the availab le data supports a practical RPF of zero. It is 34 

### possible that the studies available may not provide sufficient sensitivity to compare the potency 35 

### of the PAHs of interest to benzo[a]pyrene, and thus , the RPF of zero should not be considered a 36 

### characterization of the inherent carcinogenicity of anthracene, phenanthrene, or pyrene. 37

### 192 DRAFT – DO NOT CITE OR QUOTE

Table 7-1 shows the average RPFs based on tumor bio assay data with their associated 1 

### range and relative confidence ratings, and an overv iew of the tumor bioassay database (total 2 

### number of studies, exposure routes tested, species tested, sexes tested, and number of RPFs 3 

### derived from BMD modeling) for each PAH. Table 7-2 shows the average RPF for 4 

### dibenz[a,c]anthracene, the only RPF based on cancer -related endpoint data, with its associated 5 

### range, relative confidence rating, and an overview of the database for this compound. 6

7

8193 DRAFT – DO NOT CITE OR QUOTETable 7-1. Final RPFs based on tumor bioassay dataPAHAverageRPFRange ofRPFsRelativeconfidence No. datasets Exposure routes testedSpeciestestedSexestestedRPFs based onBMDmodelingAnthanthrene 0.4 0.2–0.5 Medium 2 Dermal, lung implantation Mouse, rat F 1Anthracene 0 0 Medium 1 (Negative) Dermal Mouse F NABenz[a]anthracene 0.2 0.02–0.4 Medium 3 Dermal, intraperitoneal Mouse F, M 0Benz[b,c]aceanthrylene, 11H- 0.05 0.05 Low 1 DermalMouse F 0Benzo[b]fluoranthene0.5 0.1–2 High 5Dermal, intraperitoneal, lungimplantation Mouse, rat F, M 3Benz[e]aceanthrylene 0.9 0.5–1 Low 2 Dermal Mouse F, M 2Benzo[g,h,i]perylene 0.009 0.009 Low 1 Lung implantation Rat F 1Benz[j]aceanthrylene 60 60 Low 1 Intraperitoneal Mouse F 0Benzo[j]fluoranthene0.3 0.01–1 High 5Dermal, intraperitoneal, lungimplantation Mouse, rat F, M 2Benzo[k]fluoranthene 0.03 0.03–0.03 Medium 2 Dermal, lung implantation Mouse, rat F 2Benz[l]aceanthrylene 5 4–7 Low 2 Dermal Mouse F, M2Chrysene0.1 0.04–0.2 High 7Dermal, intraperitoneal, lungimplantationMouse, rat F, M 3Cyclopenta[c,d]pyrene 0.4 0.07–1 Medium 5 Dermal, intraperitoneal Mouse F, M 2Cyclopenta[d,e,f]chrysene, 4H- 0.3 0.2–0.5 Low 2 Dermal Mouse F 1Dibenzo[a,e]fluoranthene 0.9 0.7–1 Low 2 Dermal Mouse F 1Dibenzo[a,e]pyrene 0.4 0.3–0.4 Low 2 Dermal Mouse F1Dibenz[a,h]anthracene6 1–10 High 3Dermal, intraperitoneal, lungimplantation Mouse, rat F, M 1Dibenzo[a,h]pyrene 0.9 0.9 Low 1 Dermal Mouse F 0Dibenzo[a,i]pyrene 0.6 0.5–0.7 Low 2 Dermal Mouse F1Dibenzo[a,l]pyrene 30 10–40 Medium 3 Dermal, intraperitoneal Mouse F, M 0Fluoranthene 0.08 0.009–0.2 Low 6 Intraperitoneal Mouse F, M 5Indeno[1,2,3-c,d]pyrene 0.07 0.07 Low 1 Lung implantation Rat F 1Naphtho[2,3-e]pyrene 0.3 0.3 Low 1 Dermal Mouse F 0Phenanthrene0 0 High 3 (Negative)Dermal, intraperitoneal, lungimplantation Mouse, rat F, M NAPyrene 0 0 High 7 (Negative) Dermal, intraperitoneal Mouse F, M NA194 DRAFT – DO NOT CITE OR QUOTENA = not applicable, M = male, F = female

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Table 7-2. Final RPFs based on cancer-related endp oint data (no tumor bioassay data available)

PAH Average

RPF Range of

RPFs Relative

confidence Types of studies Multiple dose studies Dibenz[a,c]anthracene 4 0.04–50 Very low 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 Total = 6 studies Four in vitro bacterial mutagenicity One in vitro DNA damage One in vitro DNA adduct

1
2

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7.3. SUSCEPTIBILITY FROM EARLY LIFE EXPOSURE TO CA RCINOGENS 1 

### According to the Supplemental Guidance for Assessing Susceptibility from Early Life 2 

### Exposure to Carcinogens (U.S. EPA, 2005b), benzo[a]pyrene is carcinogenic by a mutagenic 3 

### mode of action. For example, an acute dosing studyusing benzo[a]pyrene suggests that early4 

### lifestage exposure would lead to an increased incid ence of tumors compared with adult 5 

### exposures of a similar dose and duration (EPA 2005b ). Mice that were treated with 6 

### benzo[a]pyrene (75 or 150 μg/g body weight intraper itoneal) within 24 hours of birth or at 15 7 

### days of age developed hepatomas at a higher inciden ce than similarly treated animals at 42 days 8 

### of age (Vesselinovitch et al., 1975, as cited in EP A 2005b. 9



### The Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to 10 

### Carcinogens establishes ADAFs for three specific age groups. T he current ADAFs and their age 11 

### groupings are 10 for <2 years, 3 for 2 to <16 years , and 1 for 16 years and above (U.S. EPA, 12 

### 2005b). The 10-fold and 3-fold adjustments in slop e factor are to be combined with age specific 13 

### exposure estimates when estimating cancer risks fro m early life (<16 years age) exposure to 14



### PAHs. 15 

### Because a mutagenic mode of action for benzo[a]pyre ne carcinogenicity is sufficiently 16 

### supported in laboratory animals and relevant to hum ans, and in the absence of chemical-specific 17 

### data to evaluate differences in susceptibility, inc reased early-life susceptibility is assumed and 18 

### the age-dependent adjustment factors (ADAFs) should be applied, as appropriate. 19 

### The 23 PAH compounds for which a RPF value was dete rmined (see Table 7-2) are also 20 

### considered to be carcinogenic by a mutagenic mode o f action (see Section 2.4 for discussion of 21 

### similarities in mode of action for PAHs). In the a bsence of chemical-specific data to evaluate 22 

### differences in susceptibility, increased early-life susceptibility to the 23 PAHs (for which RPFs 23 

### were derived) in this analysis is assumed and the a ge-dependent adjustment factors (ADAFs) 24 

### should be applied, along with exposure information, as appropriate. When assessing PAH cancer 25 

### risks, the RPF values should be applied to the benz o[a]pyrene risk estimates with adjustment for 26 

### early life susceptibility (See Table 7-3 for exampl e). 27

28 

### Table 7-3. Sample calculation of estimated cancer r isk for

benz[a]anthracene (BaA) with the application of ADA Fs

age
group ADAF B[a]P oral slope factor (per mg/kg-day) adjusted

B[a]P cancer risk estimate RPF BaA estimated

cancer risk (per mg/kg-day)

0 < 2 10 7.3 73 0.2 15

2<16 3 7.3 24 0.2 4.8

16+ 1 7.3 7.3 0.2 1.5

29

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1



### 8. UNCERTAINTIES ASSOCIATED WITH RPF APPROACHES 2

3

4 

### A description of uncertainties and limitations is a n important component of the RPF 5 

### approach for PAH mixtures risk assessment. Many of the general uncertainties related to 6 

### chemical-specific risk assessment are also applicab le to the proposed RPF approach for PAHs. 7 

### These include issues related to selection of an app ropriate animal model, low-dose and 8 

### interspecies extrapolation, and variability within the human population. Use of a component9 

### based approach to mixtures risk assessment leads to additional uncertainties, e.g., the lack of 10 

### experimental data on potential interactions among i ndividual components within the mixture 11 

### (i.e., among PAHs and with other chemicals). 12 

### The feasibility of conducting a robust component-ba sed approach for PAH mixtures 13 

### (RPF approach) was evaluated by a PAH mixtures peer consultation workshop (U.S. EPA, 14 

### 2002). Included in the discussion was a general ev aluation of U.S. EPA’s Provisional Guidance 15 

### (U.S. EPA, 1993). Workshop participants highlighte d the following limitations of the 1993 16 

### guidance: 17

18 

### (1) The approach only considered a small subset of PAHs (that is unsubstituted PAHs only, 19 

### no heterocyclic compounds or nitro- or alkyl-substi tuted PAHs); 20

21 

### (2) There are no human toxicity data for any individual PAH; 22

23 

### (3) The assumption of additivity may not be valid, and there may be interactions among 24 

### PAHs or between PAHs and other components of a mixt ure (e.g., metals); 25

26 

### (4) PAHs may generally have a common mode of action (i. e., mutagenicity), but multiple 27 

### modes of action for carcinogenesis are possible; an d 28

29 

### (5) The estimated order of potency (EOPP) approach was limited to the oral exposure route 30 

### (i.e., a recommendation was made not to apply the f actors to dermal and inhalation 31 

### exposures). 32

33 

### The current analysis represents a significant impr ovement upon the previous component34 

### based approach to PAH mixtures risk assessment. On e of the most important improvements is a 35 

### comprehensive review of the scientific literature d ating from the 1950s through 2009 on the 36 

### carcinogenicity and genotoxicity of PAHs. The sear ch 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 include d 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 50 42

### 198 DRAFT – DO NOT CITE OR QUOTE

individual PAHs. A weight of evidence evaluation w as conducted to evaluate the evidence for 1 

### potential carcinogenicity of 34 of these PAHs; data were inadequate to conduct such an 2 

### evaluation for the remaining 16 compounds. A final RPF was derived for each PAH based on 3 

### tumor bioassay data (if available) or cancer-relate d endpoint data if no tumor bioassay RPFs 4



### were available. Final RPFs were derived for 26 PAH s (see Table 7-2 in Section 7), significantly 5 

### increasing the number of PAHs that can be addressed through this approach. Each RPF was 6 

### assigned a relative confidence rating reflecting th e size and diversity of the tumor bioassay or 7 

### cancer-related endpoint database that was used to d erive the final RPF for that PAH. 8 

### Despite these improvements, many of the uncertainti es highlighted during the 2002 peer 9 

### consultation workshop (U.S. EPA, 2002) also apply t o the current analysis. The following 10 

### sections describe some specific uncertainties and l imitations associated with the development 11



### and use of RPFs for PAHs. The uncertainties that a re specific to the approach presented herein 12



### are discussed below in Sections 8.1 and 8.2. The r emaining sections (8.3–8.6) discuss the 13 

### general uncertainties associated with a component-b ased approach to PAH mixtures risk 14



### assessment. These include the number of PAHs inclu ded in the approach, human relevance of 15 

### animal data, assumptions regarding mode of action a nd dose additivity, and cross-route 16 

### extrapolation. 17

18



### 8.1. UNCERTAINTY IN DOSE-RESPONSE FOR INDIVIDUAL PAHS 19 

### Several uncertainties and limitations are specifica lly associated with the dose-response 20 

### assessment methodology used in this analysis to der ive RPFs for PAHs. Uncertainties are 21 

### associated with the following decisions: 22

23



### • Use of a single dose-response model for quantal or continuous data; 24

25



### • Use of varying BMR levels; 26

27



### • Use of tumor incidence data at the upper end of the dose-response curve (e.g., greater that 28 

### 75% incidence) to calculate some RPFs; 29
30



### • Use of tumor multiplicity data to calculate some RP Fs; 31

32



### • Use of single-dose point estimates to calculate som e RPFs; 33

34



### • Reliance on data from cancer-related endpoint studi es in the absence of bioassays; and 35

36



### • Use of cancer-related data from assay conditions th at maximize the benzo[a]pyrene 37 

### response, even though these conditions were not nec essarily optimal for other PAHs. 38

39 

### The decision was made to employ a single dose-respo nse model for either quantal or 40 

### continuous data due to the large number of data set s that needed be analyzed from the PAH 41

### 199 DRAFT – DO NOT CITE OR QUOTE

database. The multistage model for incidence data and the linear model for continuous data were 1 

### considered to be broadly applicable to different ty pes of data as simple curve-fitting models. In 2 

### some cases, the goodness-of-fit criteria indicated that the selected model did not fit the data. In 3 

### these cases, high-dose groups were sequentially eli minated until an adequate fit was achieved, 4 

### but other model structures (e.g., gamma, probit, lo gistic, etc.) were not considered. 5 

### Tumor bioassay data were modeled at a BMR of 10% (e xtra risk above control) in order 6 

### to target the low end of the dose-response curve as the point of departure for slope estimation. 7 

### When this was not feasible, usually because only a single dose was used for benzo[a]pyrene, an 8 

### attempt was made to match individual target PAH res ponse levels to the benzo[a]pyrene 9 

### response chosen for the point estimate. This assum es that the shape of the dose-response curve 10 

### is similar for the target PAH and benzo[a]pyrene (a lso a necessary assumption of dose additivity) 11 

### and that the slope is constant across the dose-resp onse curve. These assumptions may not hold, 12 

### especially in studies of tumor incidence where the point estimate benzo[a]pyrene response was 13 

### very high or near maximal. In many cases, the dose of benzo[a]pyrene selected as the positive 14 

### control produced near maximal tumor incidence in ex posed animals (i.e., >75%). There is 15 

### uncertainty associated with comparing potency estim ates at the high end of the dose-response 16 

### curves and using the resultant RPF to estimate risk s associated with low environmental 17 

### exposures. The relative potency relationship betwe en any two PAHs may be different at the low 18 

### end, compared with the high end, of the dose-respon se curves. 19 

### It is not clear whether relative potency values est imated at the high end of the dose20 

### response curve are reasonably predictive of relativ e potency at low environmental exposure 21 

### levels. For this reason, additional uncertainty is involved in using RPFs that are not based on a 22 

### BMR of 10% (especially those RPFs that are based on responses exceeding 75%) to estimate 23 

### risks associated with low exposures. 24 

### If model fit was not achieved, then a point-estimat e ratio approach was used. Point 25



### estimate ratios were also used for several other re asons: 26

27 

### (1) Only a single dose group was tested; 28

29 

### (2) When the standard deviation or number of replicates were not reported for continuous 30 

### data sets; or 31

32 

### (3) High-dose groups from multiple dose data sets were not usable due to a saturated tumor 33 

### response (>90% incidence in the lowest exposure gro up). 34

35 

### The point estimate approach is most reliable when t he chosen point is in the linear 36 

### portion of the dose-response curve. In many cases, however, especially for single-dose data, it 37 

### was not possible to determine whether the chosen po int was in a linear or nonlinear portion of 38 

### the dose-response curve. The dose-response relatio nship observed in many studies of cancer- 39

### 200 DRAFT – DO NOT CITE OR QUOTE

related endpoints was nonlinear at high doses. Whe never possible, the point estimate was chosen 1 

### from the linear portion of the dose-response curve (i.e., before the response plateau that occurs at 2 

### high doses). Of 50 individual RPFs calculated from tumor incidence data, 19 were calculated 3 

### using a point of departure incidence ≤25%, 21 were calculated using a point of departure 4 

### incidence between 25 and 75%, and the remaining 10 were calculated using a point of departure 5 

### incidence between 75 and 90%. Thus, only 20% of th e individual RPFs for tumor incidence data 6 

### were calculated from a point high (>75 and <90% inc idence) on the dose-response curve. 7 

### For a few PAHs tested in older dermal bioassays , t he authors reported mortality prior to 8 

### the appearance of the first tumor. For these data sets, an assumption was made that the number 9 

### of animals at risk for tumor development was equal to the total number of animals alive at the 10 

### time of the appearance of the first tumor. This ap proach ensures that the incidence is not 11 

### underestimated by including animals that did not su rvive long enough to develop tumors. As this 12 

### assumption applied to a small number of RPFs (speci fically, individual RPFs for chrysene, 13 

### dibenzo[a,e]pyrene, dibenzo[a,e]fluoranthene, and d ibenzo[a,h]pyrene calculated from data 14 

### reported by Hecht et al. [1974] and Hoffmann and Wy nder [1966]), it had little impact on the 15 

### overall analysis. However, as the final RPFs for d ibenzo[a,e]pyrene, dibenzo[a,e]fluoranthene, 16 

### and dibenzo[a,h]pyrene are based exclusively on the data reported by Hoffmann and Wynder 17 

### (1966), there is additional uncertainty in these va lues stemming from the occurrence of early 18 

### mortality. 19 

### RPFs were calculated for many cancer-related endpoi nts. Many of the studies describing 20 

### in vitro cancer-related endpoints provided dose-res ponse data under varying study conditions. 21 

### For example, bacterial mutagenesis studies utilized multiple strains, different metabolic 22 

### activation processes, and varying assay systems. I n order to minimize the amount of data used 23 

### for dose-response analysis of in vitro mutagenicity studies, and to provide a consistent basis for 24 

### comparing RPFs for differenct PAHs, the data from c onditions that maximize the 25 

### benzo[a]pyrene response within a particular study w ere used for the dose-response assessment. 26 

### In several studies, the conditions that were optima l for benzo[a]pyrene were not necessarily 27 

### optimal for the target PAH. For example, the conce ntration of S9 mix that produced the highest 28 

### mutation rate for benzo[a]pyrene did not produce a maximal response for perylene or 29 

### cyclopenta[c,d]pyrene (Carver et al., 1986; Eisenst adt and Gold, 1978). In vitro data were only 30 

### used in the derivation of a single final RPF (for d ibenz[a,c]anthracene; see Table 7-2); thus, the 31



### uncertainties associated with the use of cancer-rel ated endpoint data are important for 32 

### dibenz[a,c]anthracene but have minimal impact on th e proposed RPFs for the other 25 PAHs. 33

34



### 8.2. UNCERTAINTY IN SELECTING PAHs FOR INCLUSION I N RPF APPROACH 35 

### One of the uncertainties highlighted by the peer co nsultation workshop (U.S. EPA, 2002) 36 

### stemmed from the fact that U.S. EPA’s 1993 provisio nal EOPP approach only considered a small 37 

### subset of PAHs (i.e., unsubstituted PAHs only, no h eterocyclic compounds or nitro- or alkyl- 38

### 201 DRAFT – DO NOT CITE OR QUOTE

substituted PAHs), and EOPPs were available for onl y seven PAHs. Although the present report 1 

### considered a larger number of PAHs than previous an alyses (the toxicological literature was 2



### searched for data on 74 individual PAHs identified in environmental media or for which there 3 

### were toxicological data), the focus of this analysi s remains limited to unsubstituted PAHs with 4 

### three or more fused aromatic rings containing only carbon and hydrogen atoms. Thus, the RPF 5 

### analysis presented here does not account for the po ssible carcinogenicity of substituted or 6 

### heterocyclic PAHs that may be present in complex mi xtures. This may result in an 7



### underestimation of PAH mixture cancer risk. 8



### Of the 74 unsubstituted PAHs with three or more aro matic rings, there were studies 9 

### including benzo[a]pyrene that were suitable for RPF calculation for 50 compounds. The 10 

### methodology for selecting PAHs for inclusion in the RPF approach from among these 50 PAHs 11 

### is described in Section 6. At the outset, 16 PAHs were excluded because only one or two in vitro 12 

### cancer-related endpoint RPFs were available. The r emaining 34 were evaluated using a weight 13 

### of evidence approach. The primary uncertainties as sociated with the selection process relate to: 14

15 

### (1) The use of a weight of evidence approach that focus ed on tumor bioassays including 16 

### benzo[a]pyrene as opposed to a comprehensive cancer assessment to select PAHs for 17 

### inclusion in the approach; and 18

19 

### (2) The exclusion of PAHs with limited or inconclusive data. 20

21 

### The weight of evidence approach was used due to the large number of compounds that 22 

### were under consideration. The approach was structu red as a decision tree that focused primarily 23 

### on cancer bioassays that included benzo[a]pyrene, a nd only considered other data (e.g., bioassays 24 

### that did not include benzo[a]pyrene, or cancer-rela ted data) when cancer bioassays with 25 

### benzo[a]pyrene were unavailable, nonpositive, or in consistent (see Figure 6-1). The data 26 

### collection for this analysis was centered on studie s that included benzo[a]pyrene, as these studies 27 

### would be most useful for RPF calculation. Conseque ntly, information from bioassays that 28 

### included benzo[a]pyrene were readily available for use in the weight of evidence determinations. 29 

### Bioassays that did not include benzo[a]pyrene and c ancer-related endpoint data were considered 30 

### only when there were conflicting or negative result s in the studies that did include 31 

### benzo[a]pyrene. There is uncertainty in drawing co nclusions as to potential carcinogenicity 32



### based on a narrow subset of the available database. Other elements of a more comprehensive 33 

### weight of evidence determination that were not cons idered include: cancer-related endpoint data 34 

### from studies that did not include benzo[a]pyrene; i nformation on tumorigenicity of metabolites; 35 

### information on formation of reactive metabolites; o ther mechanistic data (e.g., Ah reactivity, 36 

### inhibition of gap junction intercellular communicat ion, etc.); and quantitative structure-activity 37



### assessment. 38

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A number of PAHs (24 of 50 PAHs that had at least o ne RPF value) were excluded from 1 

### the relative potency approach because the available data were inadequate to draw a conclusion as 2 

### to potential carcinogenicity (see Tables 6-1 and 6- 2). All of these PAHs had at least one RPF, 3 

### indicating that the compounds were active in at lea st one cancer-related endpoint assay. 4 

### Excluding these PAHs from the approach increases th e uncertainty in assessing risks from a 5 

### mixture that includes them, particularly if the exc luded PAHs constitute a large fraction of the 6



### mixture. 7 

### In final, RPFs were proposed for only 26 of the 74 PAHs initially considered, because the 8 

### remaining 48 compounds did not have adequate data. Thus, even among the subset of PAHs 9 

### upon which this analysis was focused, RPFs were onl y recommended for only about one-third of 10 

### the compounds. Because only a fraction of any give n PAH mixture can be evaluated using the 11 

### RPF approach, it will be important to provide an ev aluation of the proportion of the total mixture 12 

### (i.e., mass fraction) that is comprised of compound s that are not considered in the component13 

### based approach as part of the uncertainty evaluatio n of a risk assessment using these RPFs. 14

15



### 8.3. UNCERTAINTY IN DERIVING A FINAL RPF FOR EACH PAH 16 

### The methodology for deriving a final RPF value and assigning a relative confidence 17 

### rating is described in Section 6.1. The primary un certainties associated with RPF derivation 18



### relate to: 19

20 

### (1) Combining RPFs across multiple exposure routes, spe cies, sexes, tumor types, and 21 

### studies; 22

23 

### (2) Inclusion of RPFs based on tumor multiplicity data in the combined data; 24

25 

### (3) Use of an arithmetic mean to derive final RPFs; and 26

27 

### (4) Use of cancer-related endpoint data to derive final RPFs for compounds without tumor 28 

### bioassay RPFs. 29

30 

### A variety of options were considered for prioritizi ng and/or combining RPFs. Appendix 31 

### G describes analyses that were undertaken to assess options for prioritizing RPFs. As the 32 

### appendix indicates, the current state of knowledge does not suggest a clear biological basis for 33 

### prioritizing RPFs. As a result, RPFs were combined across exposure routes, species, sexes, 34 

### tumor types, dose-response methods, and studies. 35 

### In addition to tumor incidence data, tumor multipli city data were used to calculate RPFs. 36 

### In some instances, tumor incidence data could not b e used for RPF derivation (e.g., the incidence 37 

### at the lowest dose was in the plateau region of the dose-response curve; ≥90% incidence), while 38 

### tumor multiplicity data were available. The relati onship between tumor incidence RPFs and 39 

### tumor multiplicity RPFs is not known; however, this analysis resulted in the calculation of both 40

### 203 DRAFT – DO NOT CITE OR QUOTE

incidence and multiplicity RPFs for a number of stu dies. These data were plotted, and a linear 1 

### regression analysis was performed to assess the cor relation between these two relative potency 2 

### estimates. Figure 8-1 shows the results. 3

4

0.01

0.1 1 10

0.01 0.1 1 10Incidence RPF

Multiplicity RPF 1:1 Correspondence Line

5

6 

### Figure 8-1. Correlation between incidence and mult iplicity RPFs. 7

8 

### As shown in Figure 8-1, there is a high degree of c orrelation between incidence and 9 

### multiplicity RPFs calculated from results in the sa me animals. The regression analysis indicated 10



### an r

2 

### of 0.80 for the correlation. The figure also show s that multiplicity RPFs exhibit a slight 11 

### tendency to underestimate the RPF from incidence da ta (more points are to the right of the 12 

### 1:1 correspondence line). Nevertheless, the correl ation plot and regression results provide 13 

### support for the use of RPFs from tumor multiplicity data when incidence data were not available 14



### or not useable. 15 

### As the incidence and multiplicity RPFs from the sa me study were highly correlated, only 16



### one of the two metrics was included in the combined RPFs. Specifically, the higher of the 17 

### incidence or multiplicity RPF from the same study w as included in the average and range. 18 

### Consistent with the figure, the higher value was us ually calculated from incidence data. 19



### Final RPFs were calculated as the arithmetic mean a nd range of RPFs from tumor 20 

### bioassay data when such data were available. Prese nting the average and the range provides both 21 

### an average and a maximum estimate for each PAH that has data from multiple studies. Other 22

### 204 DRAFT – DO NOT CITE OR QUOTE

options for deriving a central tendency RPF include geometric mean, median, weighted average, 1 

### and order of magnitude estimates. The arithmetic m ean represents a simple approach to 2 

### describing the calculated RPF values available for each PAH. There were usually not enough 3 

### data (≤3 RPFs for 17/23 PAHs with nonzero RPFs) to assess the shape of the RPF distribution 4 

### for any given PAH, so a geometric mean was not cons idered. Calculation of a weighted average 5 

### was considered, but without a clear biological rati onale for assigning weights among study types 6 

### or tumor data outcomes, using a weighting approach might increase uncertainty. Finally, 7 

### providing order of magnitude estimates, as has been previously done for estimating RPFs for 8 

### PAHs, was not considered to be superior to calculat ing simple means. Including the range in the 9



### estimated RPFs was considered to be informative to the user for characterizing uncertainty. 10 

### Cancer-related endpoint data were relied upon for t he derivation of a RPF for only one 11 

### PAH (dibenz[a,c]anthracene). For this compound, th ere were no tumor bioassay data suitable for 12 

### the determination of an RPF. However, cancer-relat ed endpoint data provided qualitative 13 

### support for the finding of potential carcinogenicit y for this compound (see individual narrative 14 

### for this compound in Section 6.2). Although the mo de of action for PAHs suggests that, in 15 

### general, these endpoints may be relevant to PAH car cinogenicity, the predictive value of a 16 

### positive response in these tests has not been concl usively demonstrated. Thus, there is 17 

### considerable uncertainty in an RPF based on cancer- related endpoint data. Appendix G includes 18 

### analysis of the correlation between average RPFs ca lculated from cancer-related endpoint data 19 

### and tumor bioassay data. As shown in Table 8-1, an d further discussed in Appendix G, cancer20 

### related endpoint RPFs are reasonably predictive of tumor bioassay RPFs; however, the 21 

### relationship between these RPFs and the relative po tency of a given PAH in humans exposed via 22 

### environmentally-relevant routes is unknown. 23

24


### Table 8-1. Results of simple linear regression of log–transformed average

genotoxicity RPF vs. log average tumor bioassay RPF

Genotoxicity endpoint R 2 Slope p -Value n

All in vivo DNA adducts 0.64 1.24 <0.01 9 All in vivo non-bioassays 0.54 1.05 0.016 10 All non-bioassay endpoints (in vitro and in vivo) 0 .43 1.03 <0.01 19 All in vitro non-bioassays 0.39 0.91 <0.01 19

All in vivo micronuclei and sister chromatid exchan ges 0.58 0.83 >0.05 (NS) 6 All in vitro mutagenicity 0.047 0.39 >0.05 (NS) 17

25 

### For three PAHs (anthracene, phenanthrene, and pyren e), a final RPF of 0 was 26 

### recommended. As noted earlier in Section 6, there is little quantitative difference between 27 

### selecting a final RPF of 0 for a given PAH and excl uding that PAH from the RPF approach. 28 

### However, excluding PAHs from the RPF approach impli es substantial uncertainty (these 29 

### compounds could be of low or high potency), while a ssigning an RPF of 0 suggests lower 30

### 205 DRAFT – DO NOT CITE OR QUOTE

uncertainty because there is evidence to suggest th at these compounds are of little or no 1 

### carcinogenic potential. Nevertheless, there remain s uncertainty in the RPFs for these three 2 

### compounds, as all of them included one or more stud ies suggesting activity in cancer-related 3 

### endpoint assays. 4 

### In the present analysis, RPFs for individual PAHs w ere based on data of varying quality 5 

### and reproducibility, so there is additional uncerta inty in risks estimated for mixtures containing 6 

### differing concentrations of individual PAHs. Confi dence ratings were assigned to each RPF to 7 

### qualitatively characterize the uncertainty in each individual RPF. Table 8-2 shows the 8



### distribution of PAHs with RPFs of each confidence r ating. As the table indicates, there are 9 

### 6 PAHs with RPFs of high confidence, 6 PAHs with RP Fs of medium confidence, 14 PAHs with 10 

### RPFs of low confidence, and 1 PAH with an RPF of ve ry low confidence. The confidence 11 

### ratings assigned to the RPFs may be used to qualita tively assess the uncertainty in a mixtures 12 

### risk assessment that utilizes the RPFs. For exampl e, if a high proportion of the total cancer risk 13 

### predicted for a given mixture is attributable to be nzo[a]pyrene and other PAHs with RPFs of 14 

### high or medium confidence, then the confidence in t he overall cancer risk assessment will be 15 

### relatively high. If, in contrast, benzo[a]pyrene c ontributes a relatively small fraction of the 16 

### overall risk, and/or the mixture consists primarily of PAHs with RPFs of low confidence, then 17



### the confidence in the overall cancer risk assessmen t will be correspondingly lower. Thus, it will 18 

### be important to consider the relative contribution of benzo[a]pyrene to the total risk, as well as 19 

### the relative confidence ratings of the RPF values f or component PAHs, in the uncertainty 20 

### evaluation for cancer risk assessments that employ these RPFs. 21

22


### Table 8-2. PAHs with RPFs of varying confidence

High confidence RPF Medium confidence RPF Low confidence RPF Very low confidence
RPF Benzo[b]fluoranthene Anthanthrene Benz[b,c]aceanthr ylene, 11H- Dibenz[a,c]anthracene Benzo[j]fluoranthene Anthracene Benz[e]aceanthrylen e Chrysene Benz[a]anthracene Benzo[g,h,i]perylene Dibenz[a,h]anthracene Benzo[k]fluoranthene Benz[j]a ceanthrylene Phenanthrene Cyclopenta[c,d]pyrene Benz[l]aceanthry lene Pyrene Dibenzo[a,l]pyrene Cyclopenta[d,e,f]chrysene , 4H- Dibenzo[a,e]fluoranthene Dibenzo[a,e]pyrene Dibenzo[a,h]pyrene Dibenzo[a,i]pyrene

Fluoranthene Indeno[1,2,3-c,d]pyrene Naphtho[2,3-e]pyrene

23

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8.4. UNCERTAINTY IN USE OF ANIMAL DATA TO PREDICT HUMAN CANCER 1



### RISK 2 

### Section 4.2 briefly summarizes the epidemiology and human biomarker data related to 3 

### exposure to PAH mixtures and carcinogenicity. Expo sure to certain PAH mixtures is clearly 4 

### associated with cancer in humans. Epidemiology stu dies 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 be tween exposure and increased risk of lung 7 

### cancer in humans (see review of Bostrom et al., 200 2). Skin and scrotal cancers have been 8 

### associated with exposure to coal tar, coal tar pitc hes, non-refined mineral oils, shale oils, and 9 

### soot (Larsen and Larsen, 1998; WHO, 1998; ATSDR, 19 95). While human epidemiology data 10 

### may be sufficient for the purpose of quantifying th e cancer risks associated with exposure to a 11 

### few PAH mixtures, there are no data for many mixtur es; hence the need for other approaches 12 

### including surrogate-mixture and component-based app roaches. As noted by the peer 13 

### consultation workshop (U.S. EPA, 2002), there are n o 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]pyre ne, 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 toxicokine tics, toxicodynamics, and mode of action 22 

### contribute to the uncertainty. Cancer-related endp oint data are available using human cells (e.g., 23 

### epidermal keratinocytes, lymphoblasts, human epithe lial cells) for the evaluation of 24 

### mutagenicity, DNA adducts, unscheduled DNA synthesi s, 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; how ever, whether this finding of consistency 27 

### extends to effects in vivo, and specifically to for mation 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 direc tly relevant to expected routes of exposure 30



### for humans. It is difficult to determine whether t he 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 exp osure routes (see further discussion of 33 

### exposure-route uncertainties in Section 6.6). An a dditional 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 en vironment. Further discussion of this issue 36 

### as it relates to dose-response modeling is provided in Section 6.1. 37

### 207 DRAFT – DO NOT CITE OR QUOTE

Mechanistic data, primarily obtained using benzo[a] pyrene, provide support for the 1 

### human relevance of PAH tumorigenicity in animals. There is evidence linking three pathways 2 

### activating benzo[a]pyrene to DNA-reactive agents [( +)-anti-BPDE, radical cations, 3 

### benzo[a]pyrene-7,8-dione, and reactive oxygen speci es] with key mutational events in genes 4 

### (p53 tumor suppressor gene and H-ras or K-ras oncog enes) that can lead to tumor initiation. 5 

### Results in support of mutagenic modes of action via the diol epoxide and radical cation pathways 6



### include in vivo results in animals. All of these a ctivation pathways occur in human tissues, and 7 

### associations have been made between spectra of muta tions in the p53 tumor suppressor gene or 8 

### ras oncogenes induced by benzo[a]pyrene metabolites with spectra of mutations in these genes in 9 

### tumor tissue from benzo[a]pyrene-exposed animals or tumor tissue in humans. 10 

### Support for the association between the diol epoxid e pathway and tumor initiation 11 

### includes observation that: (+)-anti-BPDE activated the H-ras-1 proto-oncogene to transform 12 

### NIH/3T3 cells via G→T point mutations in the 12 th

### codon (Marshall et al., 1984); (+)-anti13 

### BPDE reacts with the p53 tumor suppressor gene at s everal hotspots mutated in lung cancer 14 

### patients (Denissenko et al., 1996; Puisieux et al., 1991); the spectra of p53 and K-ras mutations 15 

### in lung tumors of nonsmoking patients, chronically exposed to smoky coal emissions, was 16 

### consistent with (+)-anti-BPDE mutations in these ge nes (DeMarini et al., 2001); elevated BPDE17



### DNA adducts have been observed in coke oven workers and chimney sweepers (Pavanello et al., 18 

### 1999); and the spectra of mutation in the K-ras, H- ras, and p53 genes in forestomach tumors of 19 

### mice fed benzo[a]pyrene in the diet for 2 years wer e consistent with (+)-anti-BPDE DNA 20 

### reactions (Culp et al., 2000). 21 

### Support for the radical cation pathway includes obs ervations that depurinated adducts, 22 

### (expected products from reactions of benzo[a]pyrene radical cations with DNA) accounted for 23 

### 74% of identified DNA adducts in mouse skin exposed to benzo[a]pyrene (Rogan et al., 1993) 24 

### and 9/13 examined tumors from mice exposed to derma l applications of benzo[a]pyrene had H25 

### ras oncogene mutations attributed to depurinated DN A adducts from benzo[a]pyrene radical 26 

### cations (Chakravarti et al., 1995). 27 

### Support for the AKR pathway includes in vitro demon stration that several types of DNA 28 

### damage can occur from o-quinones and reactive oxyge n species (Park et al., 2006; Balu et al., 29 

### 2004; McCoull et al., 1999; Flowers-Geary et al., 1 997, 1996), benzo[a]pyrene-7,8-dione can 30 

### induce mutations in the p53 tumor suppressor gene u sing an in vitro yeast reporter gene assay 31 

### (Park et al., 2008; Shen et al., 2006; Yu et al., 2 002), and dominant p53 mutations induced by 32 

### benzo[a]pyrene,7,8-dione in this system corresponde d with p53 mutation hotspots observed in 33 

### human lung cancer tissue (Park, 2008). 34 

### All three activation pathways are expected to occur in human tissues (Jiang et al., 2007), 35 

### and associations have been made between spectra of mutations in the p53 tumor suppressor gene 36 

### or ras oncogenes induced by benzo[a]pyrene metaboli tes with spectra of mutations in these genes 37 

### in tumor tissue from benzo[a]pyrene-exposed animals or humans. In particular, DeMarini et al. 38

### 208 DRAFT – DO NOT CITE OR QUOTE

(2001) demonstrated mutations in the p53 tumor supp ressor gene and the K-ras oncogene in the 1 

### lung tumors of nonsmokers, whose tumors were associ ated with exposure to smoky coal. 2 

### The available information supporting these actions for benzo[a]pyrene is consistent with 3



### what is known about the mode of action for other PA Hs demonstrated to induce cancer in 4 

### animals, including cyclopenta[cd]pyrene, dibenz[a,h ]anthracene, and dibenzo[a,l]pyrene 5 

### (Cogliano et al., 2008; Straif et al., 2005). All PAHs that have been studies require metabolic 6 

### activation to produce carcinogenic responses in ani mals and there is evidence for activation to 7 

### DNA reactive intermediates via several pathways (St raif et al., 2005; Xue and Warshawsky, 8 

### 2005; WHO, 1998; Cavalieri and Rogan, 1995). For e xample, incubation of rat liver 9 

### microsomes with dibenzo[a,l]pyrene, a PAH that is m ore tumorigenically potent than 10 

### benzo[a]pyrene in mouse skin and rat mammary tissue , formed depurinated DNA adducts from 11 

### the radical cation pathway, as well as DNA adducts from the diol epoxide pathway (Cavalieri 12 

### and Rogan, 1995). 13 

### In summary, the relevance of animal bioassay data t o the prediction of human 14 

### carcinogenic potency remains a significant area of uncertainty in the use of this and other 15 

### approaches to PAH cancer risk assessment. However, mechanistic data on benzo[a]pyrene and 16 

### other PAHs provide evidence that the molecular even ts leading to PAH-induced tumor formation 17



### in animals are relevant to humans. 18

19



### 8.5. UNCERTAINTY IN THE ASSUMPTIONS OF COMMON MODE OF ACTION 20



### AND DOSE ADDITIVITY 21 

### A discussion of the potential modes of action for P AH carcinogenicity is presented in 22 

### Section 2.4. Individual carcinogenic PAHs are link ed by a common effect (i.e., tumorigenicity), 23 

### which may occur through multiple mechanisms. React ive metabolites produced during 24 

### metabolic transformations of PAHs include diol epox ides, reactive oxygen species, radical 25 

### cations, and o-quinones. The formation of these me tabolites is not mutually exclusive, and the 26 

### carcinogenic process for PAHs is likely to be relat ed to some combination of molecular events 27 

### resulting from formation of several reactive specie s. Reactive metabolites of PAHs interact with 28 

### DNA to form adducts and produce DNA damage resultin g in mutations in cancer-related genes 29 

### such as tumor suppressor genes or oncogenes. These events appear to reflect the initiation 30 

### potency of an individual PAH (e.g., strong mutagens are generally potent initiators) (Sjogren et 31 

### al., 1996). Certain PAHs exhibit promotional effec ts that may be related to cytotoxicity and the 32 

### formation of reactive oxygen species, AHR affinity and the upregulation of genes related to 33 

### biotransformation (i.e., induction of CYP1A1), grow th, and differentiation (Bostrom et al., 34 

### 2002). The inhibition of gap junctional intracellu lar communication is also related to tumor 35 

### promotion by PAHs (Bostrom et al., 2002). The abil ity of certain PAHs to act as tumor 36 

### promoters as well as initiators may increase their carcinogenic potency in animal bioassays 37 

### conducted at high doses. Initiation potency may be more relevant to low level environmental 38

### 209 DRAFT – DO NOT CITE OR QUOTE

exposure in humans (Bostrom et al., 2002; Sjogren e t al., 1996); however, the proposed RPF 1 

### approach is not unduly affected by this as it relie s largely on high dose animal bioassay data for 2 

### selecting RPF values. This represents an uncertain ty in the use of the RPF approach in 3 

### estimating human cancer risks from PAHs. 4 

### Conceptually, the uncertainty related to relative p otency for initiation versus promotion 5 

### could be reduced by using separate RPF schemes for each part of the carcinogenic process. This 6 

### would require selection of indicator compounds that best represent the initiation and promotion 7 

### processes, and use of mechanistic data to determine relative potency for each process (i.e., 8 

### mutagenicity for initiation, AhR binding or enzyme induction for promotion). There are several 9 

### problems with this approach, including the lack of data to support the selection of indicator 10 

### compounds and the complete carcinogenic nature of m any PAHs (i.e., they act as both initiators 11 

### and promoters). The initiation and promotion poten cy of an individual PAH is determined by its 12 

### chemical structure. Some PAHs are strong mutagens, but have low affinity for the AHR (e.g., 13 

### fjord region PAHs) (Bostrum et al., 2002; Sjogren e t al., 1996). Other PAHs are complete 14 

### carcinogens, with initiating properties (i.e., muta genesis) and AhR affinity leading to tumor 15 

### promotion (e.g., benzo[a]pyrene, dibenz[a,h]anthrac ene) (Bostrum et al., 2002; Sjogren et al., 16 

### 1996). Benzo[a]pyrene is considered a good indicat or compound for similar PAHs with 17 

### complete carcinogenic activity. However, the relat ive potency of other PAHs, especially those 18 

### that act primarily via either initiation or promoti on, may be over- or underestimated. 19 

### The absence of a clearly-defined common mode of act ion increases the level of 20 

### uncertainty associated with the use of an RPF appro ach. It is not possible to determine whether 21 

### cancer risks would be under- or overestimated by us ing a PAH RPF approach that assumes a 22 

### common mode of action. The assumption of dose addi tivity inherent in the RPF approach may 23



### not be valid for a class of chemicals for which var ying mechanisms of action occur to produce a 24 

### common effect. A response addition methodology wou ld be used to assess the combined risks 25 

### from compounds with distinct mechanisms of action. For subgroups of PAHs with a common 26 

### mechanism of action, an integrated RPF with a respo nse addition approach may be applicable 27 

### (U.S. EPA, 2000). 28 

### The assumption of additivity cannot be confirmed or refuted based on evidence available 29 

### in the peer-reviewed literature. The experimental data relating to dose additivity for PAH 30 

### carcinogenicity are discussed in Section 2.7. Base d on the available data, it appears that risks 31 

### may be generally additive for complex mixtures, whi le binary mixtures can exhibit antagonism, 32 

### synergism, or additivity. The level of confidence in the RPF approach would be increased if 33 

### additivity could be demonstrated experimentally, ev en with simple mixtures. This remains a 34 

### significant uncertainty in the proposed RPF approac h. 35

36



### 8.6. UNCERTAINTY IN EXTRAPOLATING RPFs ACROSS EXPO SURE ROUTES 37

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The peer consultation workshop (U.S. EPA, 2002) als o identified uncertainty in 1 

### extrapolation of RPFs across exposure routes. As w ith the 1993 Provisional Guidance , RPFs 2 

### proposed in this analysis are also based on in vivo bioassay data collected using various routes of 3 

### administration (e.g., dermal, intraperitoneal, subc utaneous, intramammillary, intramuscular, or 4 

### intravenous injection, as well as lung implantation , tracheal implantation, and transplacental 5 

### exposure after subcutaneous injection). The propos ed RPF approach considers each bioassay 6 

### type equivalent for the purpose of determining rela tive potency to benzo[a]pyrene. 7 

### Table 8-3 compares the average RPFs (calculated fr om raw numbers and rounded to one 8 

### significant digit) based on tumor bioassay data for each PAH across exposure routes. Dermal 9 

### studies are shown collectively as well as separated by study type (complete or initiation). 10

11

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Table 8-3. Comparisons among average tumor bioassa y RPF values by exposure route and target organ

Dermal Dermal

complete Dermal

initiation Intraperitoneal Intraperitoneal, target organ = lung Intraperitoneal, target o rgan = liver Lung

implantation

PAH N Average N Average N Average N Average N Average N Average N Average

AA 1 0.5 1 0.5 – – – – – – – – 1 0.2

AC – – – – – – – – – – – – – – BaA 1 0.02 – – 1 0.02 3 0.3 a 1 0.08 2 0.4 – – BbcAC (1,12-MBA) 1 0.05 – – 1 0.05 – – – – – – – – BbF 3 0.3 1 0.2 2 0.4 3 b 1 c 1 0.4 1 2 1 0.1

BeAC 4 0.7 – – 4 0.7 – – – – – – – – BghiP –
– – – – – – – – – – – 1 0.009 BjAC –
– – – – – 1 60 d 1 60 – – – – BjF 3 0.06 – – 3 0.06 5 b 0.6 a 2 0.5 1 0.7 1 0.03

BkF 2 0.02 – – 2 0.02 – – – – – – 1 0.03

BlAC 4 4 – – 4 4 – – – – – – – – CH 7 0.1 – – 7 0.1 3 0.1 a 1 0.1 2 0.2 1 0.04

CPcdP 7 0.2 3 0.3 4 0.1 1 1 d 1 1 – – – – CPdefC 2 0.3 – – 2 0.3 – – – – – – – – DBacA – – – – – – – – – – – – – – DBaeF 2 0.9 1 1 1 0.7 – – – – – – – – DBaeP 2 0.4 1 0.3 1 0.4 – – – – – – – – DBahA 2 1 – – 2 1 1 10 d 1 10 – – 1 2

DBahP 1 0.9 – – 1 0.9 – – – – – – – – DBaiP 2 0.6 1 0.7 1 0.5 – – – – – – – – DBalP 2 30 – – 2 30 1 30 d 1 30 – – – – FA – – – – – – 10 0.08 a 8 0.05 2 0.2 – – IP – – – – – – – – – – – – 1 0.07

N23eP 1 0.3 – – 1 0.3 – – – – – – – –
PH – – – – – – – – – – – – – – Pyr – – – – – – – – – – – – – –

a Newborn mouse model.

b Number of intraperitoneal RPFs includes those calcu lated for combined lung and liver incidence; these are not included in numbers of RPFs with lung or li ver tumors.

c Includes both newborn mouse and adult A/J mouse mod els.

d Adult A/J mouse model.

1 

### Likewise, intraperitoneal studies are shown groupe d as well as separated by target organ 2 

### (lung and liver). In general, the table shows that RPFs calculated from lung implantation and 3 

### dermal studies are similar, while RPFs calculated f rom intraperitoneal studies tend to be higher 4 

### for most compounds. However, intraperitoneal RPFs for chrysene (CH) and dibenz[a,l]pyrene 5 

### (DBalP) are similar to dermal RPFs for these compou nds. 6

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One possible explanation for the higher intraperito neal RPFs calculated from newborn 1 

### mouse assays (footnoted “a” in the table) might be that the newborn mouse is more sensitive to 2 

### the carcinogenic action of PAHs than an adolescent or adult mouse. Likewise, the adult 3 

### A/J mouse is considered to be particularly sensitiv e to PAH lung tumorigenicity (Nesnow et al., 4 

### 1995), which may result in higher RPFs with this mo del (in Table 8-3, the intraperitoneal RPFs 5 

### based on the A/J mouse model are footnoted “d”). T here is little information to evaluate whether 6



### the newborn mouse is more or less sensitive than th e adult A/J mouse model. Only one 7 

### compound, benzo[b]fluoranthene (BbF), had RPFs calc ulated from both newborn mouse and 8 

### adult A/J mouse models; the newborn mouse RPF was 2 , while the A/J mouse RPF was 0.4. In 9 

### summary, it is not clear whether the intraperitonea l RPFs are higher than dermal or lung 10 

### implantation RPFs due to route-specific differences or animal model differences in susceptibility. 11 

### Cross-route extrapolation of relative potency esti mates is a necessary, though uncertain, 12 

### aspect of the RPF approach. It is difficult to det ermine which of the available study types (e.g., 13 

### dermal, intraperitoneal, intratracheal) is most pre dictive of potential risks from oral and 14 

### inhalation exposure in humans. In order to priorit ize bioassays by exposure route, data are 15 

### needed on relative potencies through environmentall y relevant exposure routes (oral, inhalation, 16 

### dermal) with relative potencies based on experiment al exposure routes. 17 

### The inhalation RPF scheme used by the California E PA (2004) employed a hierarchy of 18 

### bioassay data based on exposure route (inhalation s tudies were preferred followed by 19 

### intratracheal or intrapulmonary instillation, oral administration, skin-painting, and subcutaneous 20 

### or intraperitoneal injection). Apart from the obvi ous preference for exposure routes that targeted 21 

### the respiratory tract (inhalation, intratracheal, i ntrapulmonary), the basis for prioritizing the othe r 22 

### exposure routes is not evident. Pufulete et al. (2 004), who were also focused on PAHs as air 23 

### contaminants, suggested that the clearance of PAHs after intratracheal instillation may be similar 24 

### to clearance after inhalation exposure. The author s acknowledged that the high concentrations of 25 

### PAHs used in intratracheal and intrapulmonary insti llation studies may lead to major differences 26 

### in pharmacokinetics, compared with inhalation expos ure (Pufulete et al., 2004). Nevertheless, 27 

### the authors suggested that intratracheal instillati on of low doses of PAHs might be an appropriate 28 

### surrogate exposure model for assessing relative pot ency of inhalation exposure. It is important 29



### to note that no intratracheal instillation studies were identified in the search for studies from 30 

### which to calculate RPFs; thus, the information prov ided by Pufulete et al. (2004) is not directly 31 

### useful for suggesting route-specific RPFs. Pufulet e et al. (2004) did not provide any specific 32 

### information on the relevance of intrapulmonary admi nistration (a route used in several of the 33 

### bioassays used to calculate RPFs) to inhalation exp osure. 34 

### As noted by U.S. EPA (2004), cross-route extrapolat ion would be contraindicated if there 35 

### were convincing toxicokinetic evidence that absorpt ion of PAHs does not occur by one or more 36 

### exposure routes. However, available data on the ab sorption of PAHs indicates that, in general, 37 

### PAHs are readily absorbed via ingestion, inhalation , and dermal exposure routes; however, the 38

### 213 DRAFT – DO NOT CITE OR QUOTE

rate of uptake varies with route and other factors (e.g., matrix, intake of fats and oils) (ATSDR, 1 

### 1995). Evidence for absorption of PAHs through the se routes includes measurement of PAH2 

### DNA adducts at sites distal from the route of entry , measurement of urinary metabolites, and 3 

### radiotracer studies in animals (ATSDR, 1995). U.S. EPA (2004) indicated that demonstration of 4 

### any degree of uptake for each of the routes of inte rest is sufficient to allow the qualitative 5 

### judgment to apply the route-to-route extrapolation; thus, cross-route extrapolation is supported 6 

### by current data on the bioavailability of PAHs acro ss several exposure routes. 7 

### U.S. EPA (1994, 2004) also noted that point-of-entr y toxicity may be considered contrary 8 

### evidence for cross-route extrapolation. With respe ct to PAHs, available information on this issue 9



### is both limited and mixed. The one inhalation bioa ssay of benzo[a]pyrene, which suffered from 10 

### several methodological limitations, identified the upper respiratory tract as the site of tumor 11 

### formation, suggesting a point-of-entry effect (Thys sen et al., 1981). Dermal bioassays of 12 

### benzo[a]pyrene have generally evaluated only skin t umors, precluding their use in determining 13



### whether distal tumors are induced. A number of ear ly oral cancer bioassays of benzo[a]pyrene 14 

### suggested that tumor formation was limited to point -of-entry sites (Rigdon and Neal, 1969, 1966; 15 

### Neal and Rigdon, 1967). More recent oral carcinoge nicity bioassays comparing MGP residue 16 

### (Weyand et al., 1995) or coal tar preparations (Cul p et al., 1998; Gaylor et al., 1998) with 17 

### benzo[a]pyrene showed significant differences in ta rget organ distribution of tumors between 18 

### benzo[a]pyrene and complex mixtures of PAHs. Benzo [a]pyrene-induced tumors were observed 19 

### primarily at the point of contact (i.e., the forest omach), while MGP residue and coal tar produced 20 

### tumors in the lung, liver, forestomach, skin, and o ther organs. Other PAHs (e.g., 21 

### benzo[c]fluorine) are proposed to be responsible fo r tumors at distal sites such as the lung 22 

### (Koganti et al., 2000; Culp et al., 1998). However , a recent gavage study in rats (Kroese et al., 23 

### 2001) demonstrated that oral exposure to benzo[a]py rene could induce tumors at distal sites, 24 

### including the liver and auditory canal. Tissue-spe cific differences in metabolic activation and 25 

### DNA binding of PAHs may contribute to the observed differences in target organ sensitivity 26 

### (Weyand and Wu, 1995; Culp and Beland, 1994). 27 

### In summary, available information provides some sup port for cross-route extrapolation. 28 

### Absorption of PAHs across oral, inhalation, and der mal routes is evident and, while many of the 29 

### cancer bioassays of benzo[a]pyrene suggested tumor formation limited to the point-of-entry, at 30 

### least one recent study (Kroese et al., 2001) sugges ts that tumors may also be induced at distal 31 

### sites. Furthermore, there is evidence that other P AHs (e.g., benzo[c]fluorene) may induce 32 

### tumors at distal sites after oral exposure to coal tar preparations (Koganti et al., 2000; Culp et al.

, 33 

### 1998). However, cross-route extrapolation of RPFs is a significant source of uncertainty in this 34 

### approach. 35 

### Another approach to the issue of route-to-route ext rapolation would be to prefer RPFs 36 

### derived from particular target tissues deemed relev ant to the exposure route of interest. For 37 

### example, RPFs based on lung tumor data might be pre ferred for use in inhalation risk 38

### 214 DRAFT – DO NOT CITE OR QUOTE

assessment. To examine whether lung tumor RPFs wer e consistent across routes, RPFs 1 

### calculated from lung tumor potency in intraperitone al studies (both newborn mouse and adult 2 

### A/J mouse models) were compared with RPFs from lung implantation studies in Table 8-3. 3 

### RPFs for both intraperitoneal-lung and lung implant ation studies were available for only four 4 

### compounds (BbF, BjF, CH, and DBahA); for each of th ese, the intraperitoneal lung tumor RPF 5 

### exceeded the lung implantation RPF. No information assessing the concordance between lung 6 

### tumor potency after intraperitoneal administration and inhalation cancer potency was identified 7



### in the literature. The use of the final RPFs derive d in this analysis across all routes of exposure is 8 

### recommended given the information outlined above an d in the absence of data to indicate 9



### otherwise. 10

### 215 DRAFT – DO NOT CITE OR QUOTE 9. REFERENCES

Abe, S; Sasaki, M. (1977) Chromosome aberrations an d sister chromatid exchanges in Chinese hamster cel ls exposed to various chemicals. J Natl Cancer Inst 5 8:1635–1641.

Agrelo, C; Amos, H. (1981) DNA repair in human fibr oblasts. In: De Serres, FJ; Ashby, J, eds. Evaluati on of shortterm tests for carcinogens. Prog Mutat Res 1:528–5 32.

Albert, RE; Lewtas, J; Nesnow, S; et al. (1983) Com parative potency method for cancer risk assessment: Application to diesel particulate emissions. Risk Anal 3:101–117.

Albert, RE; Miller, ML; Cody TE; et al. (1991) Benz o[a]pyrene-induced skin damage and tumor promotion in the mouse. Carcinogenesis 12:1273–1280.

Allen, JA; Coombs, MM. (1980) Covalent binding of p olycyclic aromatic hydrocarbons to mitochondrial an d nuclear DNA. Nature 287:244–245.

Allen, CCR; Boyd, DR; Hempenstall, F; et al. (1999) Contrasting effects of a nonionic surfactant on th e biotransformation of polycyclic aromatic hydrocarbo ns to cis-dihydrodiols by soil bacteria. Appl Envi ron Microbiol
65:1335–1339.

Allen-Hoffmann, BL; Rheinwald, JG. (1984) Polycycli c aromatic hydrocarbon mutagenesis of human epiderm al keratinocytes in culture. Proc Natl Acad Sci USA 8 1:7802–7806.

Amacher, DE; Paillet, SC. (1982) Hamster hepatocyte -mediated activation of procarcinogens to mutagens in the L5178Y/TK mutation assay. Mutat Res 106:305–316.

Amacher, DE; Paillet, SC. (1983) The activation of procarcinogens to mutagens by cultured rat hepatocy tes in the L5178Y/TK mutation assay. Mutat Res 113:77–88.

Amacher, DE; Turner, GN. (1980) Promutagen activati on by rodent-liver postmitochondrial fractions in t he L5178Y/TK cell mutation assay. Mutat Res 74:485–50 1.

Amacher, DE; Paillet, SC; Turner, GN; et al. (1980) Point mutations at the thymidine kinase locus in L 5178Y mouse lymphoma cells. II. Test validation and inter pretation. Mutat Res 72:447–474.

Amin, S; Desai, D; Dai, W; et al. (1995) Tumorigeni city in newborn mice of fjord region and other ster ically hindered diol epoxides of benzo[g]chrysene, dibenzo [a,l]pyrene (dibenzo[def,p]chrysene), 4H-cyclopenta[def]chrysene and fluoranthene. Carci nogenesis 16:2813–2817.

Andrews, AW; Thibault, LH; Lijinsky, W. (1978) The relationship between carcinogenicity and mutagenici ty of some polynuclear hydrocarbons. Mutat Res 51:311–31 8.

Archibong, AE; Inyang, F; Ramesh, A; et al. (2002) Alteration of pregnancy related hormones and fetal survival in F-344 rats exposed by inhalation to benzo[a]pyrene. Reprod Toxicol 16:801–808.

Arif, JM; Smith, WA; Gupta, RC. (1997) Tissue distr ibution of DNA adducts in rats treated by intramamm illary injection with dibenzo[a,l]pyrene, 7,12-dimethylben z[a]anthracene and benzo[a]pyrene. Mutat Res 378:3 1–39.

Atchison, M; Atchison, ML; Van Duuren, BL. (1985) C ocarcinogenesis in vitro using Balb/3T3 cells and a romatic hydrocarbon cocarcinogens. Cell Biol Toxicol 1:323 –331.

ATSDR (Agency for Toxic Substances and Disease Regi stry). (1995) Toxicological profile for polycyclic aromatic Hydrocarbons (PAHs). Public Health Service, U.S. D epartment of Health and Human Services. Available o nline at http://www.atsdr.cdc.gov/toxprofiles.

### 216 DRAFT – DO NOT CITE OR QUOTE

Ayrton, AD; McFarlane, M; Walker, R; et al. (1990) Induction of the P-450 I family of proteins by poly cyclic aromatic hydrocarbons: possible relationship to the ir carcinogenicity. Toxicology 60:173–186.

Baird, WM; Salmon, CP; Diamond, L. (1984) Benzo[e]p yrene-induced alterations in the metabolic activati on of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene b y hamster embryo cells. Cancer Res 44:1445–1452.

Baird, WM; Hooven, LA; Mahadevan, B; et al. (2002) Responses of human cells to PAH-induced DNA damage. Polycyclic Aromatic Compounds 22:771–780.

Baker, RS; Bonin, AM; Stupans, I; et al. (1980) Com parison of rat and guinea pig as sources of the S9 fraction in the Salmonella/mammalian microsome mutagenicity tes t. Mutat Res 71:43–52.

Balu, N; Padgett, WT, Lambert, GR; et al. (2004) Id entification and characterization of novel stable d eoxyguanosine and deoxyadenosine adducts of benzo[a]pyrene-7,8-qu inone from reactions of physiological pH. Chem Res Toxicol 17(6):827–838.

Balu, N; Padgett, WT; Nelson, GB. (2006) Benzo[a]py rene-7,8-quinone-3’-mononucleotide adduct standards for 32 P postlabeling analysis: detection of benzo[a]pyrene- 7,8-quinone-calf thymus DNA adducts. Anal Biochem 15(2):213–223.

Barfknecht, TR; Hites, RA; Cavaliers, EL; et al. (1 982) Human cell mutagenicity of polycyclic aromatic hydrocarbon components of diesel emissions. Dev To xicol Environ Sci 10:277–294.

Barrai, I; Barale, R; Scapoli, C; et al. (1992) The analysis of the joint effect of substances on reve rsion systems and the assessment of antimutagenicity. Mutat Res 267: 173–182.

Barry, G; Cook, JW; Haslewood, GAD; et al. (1935) T he production of cancer by pure hydrocarbons. Part III. Proc Royal Soc London 117:318–351.

Bartsch, H; Malaveille, C; Camus, AM; et al. (1980) Validation and comparative studies on 180 chemical s with S. typhimurium strains and V79 Chinese hamster cells in the prese nce of various metabolizing systems. Mutat Res
76:1–50.

Bayer, U. (1978) In vivo induction of sister chroma tid exchanges by three polyaromatic hydrocarbons. Carcinogenesis 3:423–428.

Beland, F; Culp, S. (1998) Chronic bioassay of two composite samples from selected manufactured gas pl ant waste sites. Jefferson, AK: Division of Biochemical Toxi cology, National Center for Toxicological Research. Technical Report 6722.02. Unpublished report.

Biancifiori, C; Caschera, F. (1962) The relation be tween pseudopregnancy and the chemical induction by four carcinogens of mammary and ovarian tumours in BALB/ c mice. Br J Cancer 16:722–730.

Bingham, E; Falk, HL. (1969) The modifying effects of carcinogens on the threshold response. Arch Env iron Health 19:779–783.

Binkova, B; Giguere, Y; Rossner, P, Jr; et al. (200 0) The effect of dibenzo[a,1]pyrene and benzo[a]pyr ene on human diploid lung fibroblasts: the induction of DNA addu cts, expression of p53 and p21(WAF1) proteins and c ell cycle distribution. Mutat Res 471:57–70.

Blaha, L; Kapplova, P; Vondracek, J; et al. (2002) Inhibition of gap-junctional intercellular communic ation by environmentally occurring polycyclic aromatic hydro carbons. Toxicol Sci 65:43–51.

Bols, NC; Schirmer, K; Joyce, EM; et al. (1999) Abi lity of polycyclic aromatic hydrocarbons to induce 7-ethoxyresorufin-o-deethylase activity in a trout liver cell line. Ecotoxicol Environ Saf 44:118–128

.

Bolton, JL; Trush, MA; Penning, TM; et al. (2000) R ole of quinones in toxicology. Chem Res Toxicol 13 (3):2–17.

### 217 DRAFT – DO NOT CITE OR QUOTE

Bos, RP; Theuws, JLG; Jongeneelen, FJ; et al. (1988 ) Mutagenicity of bi-,tri- and tetra-cyclic aromati c hydrocarbons in the 'taped-plate assay' and in the conventional Salmonella mutagenicity assay. Mutat Res 204:203–
206.

Bostrom, E; Engen, S; Eide, I. (1998) Mutagenicity testing of organic extracts of diesel exhaust parti cles after spiking with polycyclic aromatic hydrocarbons (PAH) . Arch Toxicol 72:645–649.

Bostrom, CC; Gerde, P; Hanberg, A; et al. (2002) Ca ncer risk assessment, indicators, and guidelines fo r polycyclic aromatic hydrocarbons in the ambient air. Environ Health Perspect 110(Suppl 3):451–488.

Bosveld, AT; de Bie, PA; van den Brink, NW; et al. (2002) In vitro EROD induction equivalency factors for the 10 PAHs generally monitored in risk assessment stud ies in The Netherlands. Chemosphere 49:75–83.

Brookes, P; Lawley, PD. (1964) Evidence of the bind ing of polynuclear aromatic hydrocarbons to the nuc leic acids of mouse skin: relation between carcinogenic power of hydrocarbons and their binding to deoxyribonucle ic acid.
Nature 202:781–784.

Bruce, ED; Austenrieth, RL; Burghardt, RC; et al. ( 2008) Using quantitative structure-activity relatio nships (QSAR) to predict toxic endpoints for polycyclic aromatic hydrocarbons (PAH). J Toxicol Environ Health Part A 71:1073–
1084.

Brune, K; Kalin, H; Schmidt, R; et al. (1978) Infla mmatory, tumor initiating and promoting activities of polycyclic aromatic hydrocarbons and diterpene esters in mouse skin as compared with their prostaglandin releasin g potency in vitro. Cancer Lett 4:333–342.

Brune, H; Deutsch-Wenzel, RP; Habs, M; et al. (1981 ) Investigation of the tumorigenic response to benz o[a]pyrene in aqueous caffeine solution applied orally to Spra gue-Dawley rats. J Cancer Res Clin Oncol 102:153–1 57.

Bryan, WR; Shimkin, MB. (1943) Quantitative analysi s of dose-response data obtained with three carcino genic hydrocarbons in strain C3H male mice. J Natl Cance r Inst 3:503–531.

Bryla, P; Weyand, EH. (1992) Detection of PAH:DNA a dducts from auto-oxidation using 32 P -postlabeling. Cancer
Lett 65:35–41.

Buening, MK; Levin, W; Wood, A; et al. (1979) Tumor igenicity of the dihydrodiols of dibenz(a,h)anthrac ene on mouse skin and in newborn mice. Cancer Res 39:1310 –1314.

Burdick, AD; Davis, JW; Liu, KJ; et al. (2003) Benz o[a]pyrene quinones increase cell proliferation, ge nerate reactive oxygen species, and transactivate the epid ermal growth factor receptor in breast epithelial c ells. Cancer Res
63:7825–7833.

Busby, WFJ; Goldman, ME; Newberne, PM; et al. (1984 ) Tumorigenicity of fluoranthene in a newborn mouse lung adenoma bioassay. Carcinogenesis 5:1311–1316.

Busby, WFJ; Stevens, EK; Martin, CN; et al. (1989) Comparative lung tumorigenicity of parent and monon itropolynuclear aromatic hydrocarbons in the BLU: Ha ne wborn mouse assay. Toxicol Appl Pharmacol 99:555–5 63.

Buterin, T; Hess, MT; Luneva, N; et al. (2000) Unre paired fjord region polycyclic aromatic hydrocarbon -DNA adducts in ras codon 61 mutational hot spots. Cancer Res 60:1849– 1856.

Buters, JT; Mahadevan, B; Quintanilla-Martinez, L; et al. (2002) Cytochrome P450 1B1 determines suscep tibility to dibenzo[a,l]pyrene-induced tumor formation. Chem R es Toxicol 15:1127–1135.

California EPA (California Environmental Protection Agency). (2002) Air toxics hot spots program risk assessment guidelines. Part II: Technical support document for describing available cancer potency factors. Offi ce of Environmental Health Hazard Assessment, Air Toxicol ogy and Epidemiology Section, Oakland, CA.

### 218 DRAFT – DO NOT CITE OR QUOTE

California EPA (California Environmental Protection Agency). (2004) No Significant Risk Levels (NSRLs) for the Proposition 65 carcinogens benzo[b]fluoranthene, be nzo[j]fluoranthene, chrysene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and 5-methylchrysene by the ora l route. Office of Environmental Health Hazard Ass essment, Reproductive and Cancer Hazard Assessment Section, Oakland, CA.

Carver, JH; Machado, ML; MacGregor, JA. (1985) Petr oleum distillates suppress in vitro metabolic activ ation: higher (S-9) required in the Salmonella/microsome m utagenicity assay. Environ Mutagen 7:369–379.

Carver, JH; Machado, ML; MacGregor, JA. (1986) Appl ication of modified Salmonella/microsome prescreen to petroleum-derived complex mixtures and polynuclear aromatic hydrocarbons (PAH). Mutat Res 174:247–253

.

Casale, GP; Higginbotham, S; Johansson, SL; et al. (1997) Inflammatory response of mouse skin exposed to the very potent carcinogen dibenzo[a,l]pyrene: a model for tumor promotion. Fundam Appl Toxicol 36(1):71– 78.

Casale, GP; Cheng, Z; Liu, J; et al. (2000) Profile s of cytokine mRNAs in the skin and lymph nodes of SENCAR mice treated epicutaneously with dibenzo[a,l]pyrene or dimethylbenz[a]anthracene reveal a direct corre lation between carcinogen-induced contact hypersensitivity and epidermal hyperplasia. Mol Carcinog 27(2):125 –140.

Casto, BC. (1979) Polycyclic hydrocarbons and Syria n hamster embryo cells: cell transformation, enhanc ement of viral transformation and analysis of DNA-damage. I n: Jones, PW; Leber, P, eds. Polynuclear aromatic hydrocarbons. Ann Arbor, MI: Ann Arbor Science Publ ishers; pp. 51–66.

Castro, DJ; Lohr, CV; Fischer, KA; et al. (2008) Ly mphoma and lung cancer in offspring born to pregnan t mice dosed with dibenzo[a,l]pyrene: the importance of in utero vs. lactational exposure. Toxicol Appl Phar macol
233:454–458.

Cavalieri, EL; Rogan, EG. (1992) The approach to un derstanding aromatic hydrocarbon carcinogenesis. Th e central role of radical cations in metabolic activation. P harmacol Ther 55:183–199.

Cavalieri, EL; Rogan, EG. (1995) Central role of ra dical cations in metabolic activation of polycyclic aromatic hydrocarbons. Xenobiotica 25:677–688.

Cavalieri, EL; Mailander, P; Pelfrene, A. (1977) Ca rcinogenic activity of anthanthrene on mouse skin. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol 89:113 –118.

Cavalieri, E; Rogan, E; Thilly, WG. (1981a) Carcino genicity, mutagenicity and binding studies of the e nvironmental contaminant cyclopenteno(c,d]pyrene and some of its derivatives. In: Cook, M; Dennis, AJ, eds. Chemic al analysis and biological fate: polynuclear aromatic hydrocarb ons. Columbus, OH: Battelle; pp. 487–499.

Cavalieri, E; Rogan, E; Toth, B; et al. (1981b) Car cinogenicity of the environmental pollutants cyclop enteno- [cd]pyrene and cyclopentano[cd]pyrene in mouse skin . Carcinogenesis 2:277–281.

Cavalieri, E; Munhall, A; Rogan, E; et al. (1983) S yncarcinogenic effect of the environmental pollutan ts cyclopenteno[cd]pyrene and benzo[a]pyrene in mouse skin. Carcinogenesis 4:393–397.

Cavalieri, EL; Rogan, EG; Cremonesi, P; et al. (198 8a) Radical cations as precursors in the metabolic formation of quinones from benzo[a]pyrene and 6-fluorobenzo[a]py rene. Fluoro substitution as a probe for one-electr on oxidation in aromatic substrates. Biochem Pharmacol 37(11):2 173–2182.

Cavalieri, E; Rogan, E; Sinha, D. (1988b) Carcinoge nicity of aromatic hydrocarbons directly applied to rat mammary gland . Cancer Res Clin Oncol 114:3–9.

Cavalieri, EL; Rogan, EG; Higginbotham, S; et al. ( 1989) Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a]p yrenes: the very potent environmental carcinogen dibenzo[a,l]pyrene. J Cancer Res Clin Oncol 115:67 –72.

Cavalieri, EL; Higginbotham, S; RamaKrishna, NV; et al. (1991) Comparative dose-response tumorigenicit y studies of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]an thracene, benzo[a]pyrene and two dibenzo[a,l]pyrene dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis 12:1939–1944.

### 219 DRAFT – DO NOT CITE OR QUOTE

Cavalieri, EL; Rogan, EG; Ramakrishna NVS; et al. ( 1993) Mechanisms of benzo(a)pyrene and 7,12-diemthylbenz(a)antrhacene activation: qualitat ive aspects of the stable and depurination DNA addu cts obtained from radical cations and diol epoxides. In: Polycy clic aromatic hydrocarbons: synthesis, properties, analytical measurements, occurrence and biological effects. Bo rdeaux, France: Gordon and Breach Science Publisher s, pp. 725–732.

Cavalieri, EL; Rogan, EG; Li, KM; et al. (2005) Ide ntification and quantification of the depurinating DNA adducts formed in mouse skin treated with dibenzo[a,l]pyren e (DB[a,l]P) or its metabolites and in rat mammary gland treated with DB[a,l]P. Chem Res Toxicol 18(6):976– 983.

CCME (Canadian Council of the Ministers of the Envi ronment). (2003) Canadian soil quality guidelines f or potentially carcinogenic and higher molecular weigh t polycyclic aromatic hydrocarbons (environmental a nd human health aspects). Scientific supporting document. U MA Group, Ltd., Victoria, British Columbia.

Chakravarti, D; Pelling, JC; Cavalieri, EL; et al. (1995) Relating aromatic hydrocarbon-induced DNA ad ducts and c-H-ras mutations in mouse skin papillomas: the rol e of apurinic sites. Proc Natl Acad Sci USA 92(22) :10422–
10426.

Chakravarti, D; Mailander, PC; Cavalieri, EL; et al . (2000) Evidence that error-prone DNA repair conve rts dibenzo[a,l]pyrene-induced depurinating lesions int o mutations: formation, clonal proliferation and re gression of initiated cells carrying H-ras oncogene mutations i n early preneoplasia. Mutat Res 456(1-2):17–32.

Chakravarti, D; Venugopal D; Mailander PC; et al. ( 2008) The role of polycyclic aromatic hydrocarbon-D NA adducts in inducing mutations in mouse skin. Mutat Res 649(1–2):161–178.

Chang, RL; Levin, W; Wood, AW; et al. (1981) Tumori genicity of the diastereomeric bay-region benzo(e)p yrene 9,10-diol-11,12-epoxides in newborn mice. Cancer R es 41:915–918.

Chang, HF; Huffer, DM; Chiarelli, MP; et al. (2002) Characterization of DNA adducts derived from synbenzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide and comparative DNA binding studies with structura llyrelated anti-diolepoxides of benzo[ghi]fluoranthene and benzo[c]phenanthrene. Chem Res Toxicol 15:198 –208.

Chen, TT; Heidelberger, C. (1969) Quantitative stud ies on the morphological/malignant cell transformat ion of mouse prostate cells by carcinogenic hydrocarbons i n vitro. Int J Cancer 4:166–178.

Chen, S; Nguyen, N; Tamura, K; et al. (2003) The ro le of the Ah receptor and p38 in benzo[a]pyrene7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol- 9,10-epoxide-induced apoptosis. J Biol Chem 278:19 526–
19533.

Cherng, SH; Lin, P; Yang, JL; et al. (2001) Benzo[g ,h,i]perylene synergistically transactivates benzo[ a]pyreneinduced CYP1A1 gene expression by aryl hydrocarbon receptor pathway. Toxicol Appl Pharmacol 170:63–68

.

Chu, ML; Chen, CW. (1984) Evaluation and estimation of potential carcinogenic risks of polynuclear aro matic hydrocarbons. In: Polynuclear aromatic hydrocarbon s in the workplace: proceedings of a symposium; 198 4. Chemical Congress of Pacific Basin Societies.

Clement Associates. (1988) Comparative potency appr oach for estimating the cancer risk associated with exposure to mixtures of polycyclic aromatic hydrocarbons. I CF Clement Associates, Fairfax, VA.

Clement Associates. (1990) Development of relative potency estimates for PAHs and hydrocarbon combusti on product fractions compared to benzo[a]pyrene and th eir use in carcinogenic risk assessments. ICF Clem ent Associates, Fairfax, VA.

Cogliano, VJ: Baan, RA; Straif, K; et al. (2008) Us e of mechanistic data in IARC evaluations. Environ Mol Mutagen 49(2):100–109.

Collins JF; Brown, JP; Alexeeff, GV; et al. (1998) Potency equivalency factors for some polycyclic aro matic hydrocarbons and polycyclic aromatic hydrocarbon de rivatives. Regul Toxicol Pharmacol 28:45–54.

### 220 DRAFT – DO NOT CITE OR QUOTE

Conney, AH; Chang, RL; Cui, XX; et al. (2001) Dose- dependent differences in the profile of mutations i nduced by carcinogenic (R,S,S,R) bay- and fjord-region diol e poxides of polycyclic aromatic hydrocarbons. Adv E xp Med Biol 500:697–707.

Cooper, CS; Pal, K; Hewer, A; et al. (1982) The met abolism and activation of polycyclic aromatic hydro carbons in epithelial cell aggregates and fibroblasts prepared from rat mammary tissue. Carcinogenesis 3:203–210

.

Culp, SJ; Beland, FA. (1994) Comparison of DNA addu ct formation in mice fed coal tar or benzo[a]pyrene

. Carcinogenesis 15:247–252.

Culp, SJ; Gaylor, DW; Sheldon, WG; et al. (1996a) D NA adduct measurements in relation to tumor inciden ce during the chronic feeding of coal tar or benzo[a]p yrene to mice. Polycyclic Aromat Compd 11:161–168.

Culp, SJ; Gaylor, DW; Sheldon, WG; et al. (1996b) R elationship between DNA adduct levels and tumor inc idence in mice fed coal tar or benzo[a]pyrene for two year s. Proc Am Assoc Cancer Res 37:101.

Culp, SJ; Gaylor, DW; Sheldon, WG; et al. (1998) A comparison of the tumors induced by coal tar and benzo[a]pyrene in a 2-year bioassay. Carcinogenesi s 19:117–124.

Culp, SJ; Warbritton, AR; Smith, BA; et al. (2000) DNA adduct measurements, cell proliferation and tum or mutation induction in relation to tumor formation i n B6C3F1 mice fed coal tar or benzo[a]pyrene. Carc inogenesis 21(7):1433–1440.

Dasgupta, PS; Lahiri, T. (1992) Alteration of brain catecholamines during growth of benzo[a]pyrene ind uced murine fibrosarcoma. Neoplasma 39:163–165.

Davis, C; Desai, D; Amin, S; et al. (1999) Comparis on of the morphological transforming activities of fjord-region PAHs with dibenzo[a,e]pyrene and benzo[a]pyrene. P olycyclic Aromatic Compounds 16:141–149.

Dean, BJ. (1981) Activity of 27 coded compounds in the RLl chromosome assay. In: De Serres, FJ; Ashby, J, eds. Evaluation of short-term tests for carcinogens. Pr og Mutat Res 1:570–579.

De Flora, S; Zanacchi, P; Camoirano, A; et al. (198 4) Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair t est. Mutat Res 133:161–198.

DeMarini, DM; Landi, S; Tian, D; et al. (2004) Lung tumor KRAS and TP53 mutations in nonsmokers reflect exposure to PAH-rich coal combustión emissions. Ca ncer Res 61:6679–6681.

Denissenko, MF; Pao, A; Tang, M; et al. (1996) Pref erential formation of benzo[a]pyrene adducts at lun g cáncer mutational hotspots in P53. Science 274(5286):430– 432.

DeSalvia, R; Meschini, R; Fiore, M; et al. (1988) I nduction of sister-chromatid exchanges by procarcin ogens in metabolically competent Chinese hamster epithelial liver cells. Mutat Res 207:69–75.

Deutsch-Wenzel, RP; Brune, H; Grimmer, G; et al. (1 983) Experimental studies in rat lungs on the carci nogenicity and dose- response relationships of eight frequentl y occurring environmental polycyclic aromatic hydro carbons. J Natl Cancer Inst 71:539–544.

Devanesan, PD; Cremonesi, P; Nunnally, JE; et al. ( 1990) Metabolism and mutagenicity of dibenzo[a,e]py rene and the very potent environmental carcinogen dibenzo[a, l]pyrene. Chem Res Toxicol 3:580–586.

DiGiovanni, J; Rymer, J; Slaga, TJ; et al. (1982) A nticarcinogenic and cocarcinogenic effects of benzo [e]pyrene and dibenz[u,c]anthracene on skin tumor initiation by p olycyclic hydrocarbons. Carcinogenesis 3:371–375.

DiPaolo, JA; Donovan, JP; Nelson, RL. (1969) Quanti tative studies of in vitro transformation by chemic al carcinogens. J Natl Cancer Inst 42:867–874.

### 221 DRAFT – DO NOT CITE OR QUOTE

DiPaolo, JA; Takano, K; Popescu, NC. (1972) Quantit ation of chemically induced neoplastic transformati on of BALB/3T3 cloned cell lines. Cancer Res 35:2686–269 5.

DiPaolo, JA; Nelson, RL; Donovan, PJ; et al. (1973) Host-mediated in vivo-in vitro assay for chemical carcinogenesis. Arch Pathol 95:380–385.

Dunkel, VC; Pienta, RJ; Sivak, A; et al. (1981) Com parative neoplastic transformation responses of Bal b 3T3 cells, Syrian hamster embryo cells, and Rauscher mm-me leu kemia virus-infected Fischer 344 rat embryo cells t o chemical carcinogens. J Natl Cancer Inst 67:1303–1 315.

Dunkel, VC; Zeiger, E; Brusick, D; et al. (1984) Re producibility of microbial mutagenicity assays: Tes ts with Salmonella typhimurium and Escherichia coli using a standardized protocol. Environ Mutagen 6: 1–251.

Durant, JL; Lafleur, AL; Busby, WF, Jr; et al. (199 9) Mutagenicity of C24H14 PAH in human cells expres sing
CYP1A1. Mutat Res 446:1–14.

Eisenstadt, E; Gold, A. (1978) Cyclopenta[c,d]pyren e: A highly mutagenic polycyclic aromatic hydrocarb on. Proc Natl Acad Sci USA 75:1667–1669.

El-Bayoumy, K; Hecht, SS; Hoffmann, D. (1982) Compa rative tumor initiating activity on mouse skin of 6-nitrobenzo[a]pyrene, 6-nitrochrysene, 3-nitropery lene, 1-nitropyrene and their parent hydrocarbons. Cancer Lett
16:333–337.

Emura, M; Richter-Reichhelm, HB; Schneider, P; et a l. (1980) Sensitivity of Syrian golden hamster feta l lung cells to benzo[a]pyrene and other polycyclic hydrocarbons in vitro. Toxicology 17:149–155.

Evans, CH; DiPaolo, JA. (1975) Neoplastic transform ation of guinea pig fetal cells in culture induced by chemical carcinogens. Cancer Res 35:1035–1044.

Evans, EL; Mitchell, AD. (1981) Effects of 20 coded chemicals on sister chromatid exchange frequencies in cultured Chinese hamster cells. In: De Serres FJ; Ashby J, eds. Evaluation of short-term tests for ca rcinogens. Prog
Mutat Res 1:538–550.

Fahmy, M; Fahmy, OG. (1980) Altered control of gene activity in the soma by carcinogens. Mutat Res 72 :165–172.

Falk, HL; Kotin, P; Thompson, S. (1964) Inhibition of carcinogenesis. The effect of hydrocarbons and r elated compounds. Arch Environ Health 13:169–179.

Flesher, JW; Harvey, RG; Sydnor, KL. (1976) Oncogen icity of K-region epoxides of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene. Int J Cancer 18:35 1–353.

Florin, I; Rutberg, L; Curvall, M; et al. (1980) Sc reening of tobacco smoke constituents for mutagenic ity using the Ames' test. Toxicology 18:219–232.

Flowers, L; Ohnishi, T; Penning, TM. (1997) DNA str and scission by polycyclic aromatic hydrocarbon o-q uinones: role of reactive oxygen species, Cu(II)/Cu(I) redox cycling and o-semiquinone anion radicals. Biochem istry
36:8640–8648.

Flowers-Geary, L; Harvey, RG; Penning, TM. (1993) C ytotoxicity of polycyclic aromatic hydrocarbon o-qu inones in rat and human hepatoma cells. Chem Res Toxicol 6(3):252–260.

Flowers-Geary, L; Bleczinki, W; Harvey RG; et al. ( 1996) Cytotoxicity and mutagenicity of polycyclic a romatic hydrocarbon ortho-quinones produced by dihydrodiol dehydrogenase. Chem Biol Interact 99(1–3):55–72.

Frolich, A; Wurgler, FE. (1990) Drosophila wing-spo t test: improved detectability of genotoxicity of p olycyclic aromatic hydrocarbons. Mutat Res 234:71–80.

Gaylor, DW; Moolgavkar, S; Krewski, D; et al. (1998 ) Recent bioassay results on coal tars and benzo[a] pyrene: implications for risk assessment. Regul Toxicol Ph armacol 28:178–179.

### 222 DRAFT – DO NOT CITE OR QUOTE

Geacintov, NE; Cosman, M; Hingerty, BE; et al. (199 7) NMR solution structures of stereoisomeric covale nt polycyclic aromatic carcinogen – DNA adducts: princ iples, patterns, and diversity. Chem Res Toxicol 1 0(2):111–
146.

Gehly, EB; Landolph, JR; Heidelberger, C; et al. (1 982) Induction of cytotoxicity, mutation, cytogenet ic changes and neoplastic transformation by benzo[a]pyrene and derivatives in C3H/10T 1/2 clone 8 mouse fibroblas ts. Cancer
Res 42:1866–1875.

Gibson, TL; Smart, VB; Smith, LL. (1978) Non-enzymi c activation of polycyclic aromatic hydrocarbons as mutagens. Mutat Res 49:153–161.

Gill, HS; Kole, PL; Wiley, JC; et al. (1994) Synthe sis and tumor-initiating activity in mouse skin of dibenzo[a,l]pyrene syn- and anti-fjord-region diole poxides. Carcinogenesis 15:2455–2460.

Gold, A; Eisenstadt, E. (1980) Metabolic activation of cyclopenta[cd]pyrene to 3,4-epoxycyclopenta[cd] pyrene by rat liver microsomes. Cancer Res 40:3940–3944.

Goldschmidt, BM; Katz, C; Van Duuren, BL. (1973) Th e cocarcinogenic activity of noncarcinogenic aromat ic hydrocarbons. Proc Am Assoc Cancer Res 14:84.

Goldstein, LS; Safe, S; Weyand, EH. (1994) Carcinog enicity of coal tars: a multidisciplinary approach. Polycyclic Aromatic Compounds 7:161–174.

Goldstein, LS; Weyland, EH; Safe, S; et al. (1998) Tumors and DNA adducts in mice exposed to benzo[a]p yrene and coal tars: implications for risk assessment. E nviron Health Perspect 106(Suppl 6):1325–1330.

Grant, G; Roe, FJC. (1963) The effect of phenanthre ne on tumour induction by 3,4-benzopyrene administe red to newly born mice. Br J Cancer 17:261–265.

Greb,W; Strobel, R; Rohrborn, G. (1980) Transformat ion of BHK 21/CL 13 cells by various polycyclic aro matic hydrocarbons using the method of Styles. Toxicol L ett 7:143–148.

Grimmer, G; Brune, H; Deutsch-Wenzel, R; et al. (19 84) Contribution of polycyclic aromatic hydrocarbon s to the carcinogenic impact of gasoline engine exhaust cond ensate evaluated by implantation into the lungs of rats. J Natl
Cancer Inst 72:733–739.

Grimmer, G; Brune, H; Deutsch-Wenzel, R; et al. (19 87a) Contribution of polycyclic aromatic hydrocarbo ns and nitro-derivatives to the carcinogenic impact of die sel engine exhaust condensate evaluated by implanta tion into the lungs of rats. Cancer Lett 37:173–180.

Grimmer, G; Brune, H; Deutsch-Wenzel, R; et al. (19 87b) Contribution of polycyclic aromatic hydrocarbo ns and polar polycyclic aromatic compounds to the carcinog enic impact of flue gas condensate from coal-fired residential furnaces evaluated by implantation into the rat lun g. J Natl Cancer Inst 78:935–942.

Grimmer, G; Brune, H; Dettbarn, G; et al. (1988) Co ntribution of polycyclic aromatic compounds to the carcinogenicity of sidestream smoke of cigarettes e valuated by implantation into the lungs of rats. C ancer Lett 43:173–177.

Grover, PL; Sims, P. (1968) Enzyme-catalysed reacti ons of polycyclic hydrocarbons with deoxyribonuclei c acid and protein in vitro. Biochem J 110:159–160.

Guthrie, J; Robertson, IG; Zeiger, E; et al. (1982) Selective activation of some dihydrodiols of sever al polycyclic aromatic hydrocarbons to mutagenic products by pros taglandin synthetase. Cancer Res 42:1620–1623.

Habs, M; Schmähl, D; Misfeld, J. (1980) Local carci nogenicity of some environmentally relevant polycyc lic aromatic hydrocarbons after lifelong topical applic ation to mouse skin. Arch Geschwulstforsch 50:266– 274.

### 223 DRAFT – DO NOT CITE OR QUOTE

Habs, M; Jahn, SA; Schmahi, D. (1984) Carcinogenic activity of condensate from coloquint seeds ( Citrullus colocynthis ) after chronic epicutaneous administration to mice . J Cancer Res Clin Oncol 108:154–156.

Harvey, RG. (1996) Mechanisms of carcinogenesis of polycyclic aromatic hydrocarbons. Polycyclic Aroma tic Compounds 9:1–23.

Hass, BS; Brooks, EE; Schumann, KE; et al. (1981) S ynergistic, additive, and antagonistic mutagenic re sponses to binary mixtures of benzo[a]pyrene and benzo[e]pyren e as detected by strains TA98 and TA100 in the Salmonella/microsome assay. Environ Mutagen 3:159– 166.

Hass, BS; McKeown, CK; Sardella, DJ; et al. (1982) Cell-mediated mutagenicity in Chinese hamster V79 c ells of dibenzopyrenes and their bay-region fluorine-substi tuted derivatives. Cancer Res 42:1646–1649.

He, SL; Baker, R. (1991) Micronuclei in mouse skin cells following in vivo exposure to benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, chrysene, pyrene an d urethane. Environ Mol Mutagen 17:163–168.

Hecht, SS; Bondinell, WE; Hoffman, D. (1974) Chryse ne and methylchrysenes: presence on tobacco smoke a nd carcinogenicity. J Natl Cancer Inst 53:1121–1133.

Hermann, M. (1981) Synergistic effects of individua l polycyclic aromatic hydrocarbons on the mutagenic ity of their mixtures. Mutat Res 90:399–409.

Higginbotham, S; RamaKrishna, NV; Johansson, SL; et al. (1993) Tumor-initiating activity and carcinoge nicity of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthr acene and benzo[a]pyrene at low doses in mouse skin

. Carcinogenesis 14:875–878.

Hoffmann, D; Wynder, EL. (1966) [Contribution on th e carcinogenic effect of dibenzopyrenes]. Z Krebsf orsch
68:137–149.

Hoffmann, D; Rathkamp, G; Nesnow, S; et al. (1972) Fluoranthenes: quantitative determination in cigare tte smoke, formation by pyrolysis and tumor initiating activit y. J Natl Cancer Inst 49:1165–1175.

Homburger, F; Hsueh, SS; Kerr, CS et al. (1972) Inh erited susceptibility of inbred strains of Syrian h amsters to induction of subcutaneous sarcomas and mammary and gastrointestinal carcinomas by subcutaneous and gas tric administration of polynuclear hydrocarbons. Cancer Res 32:360–366.

Horton, AW; Christian, GM. (1974) Cocarcinogenic ve rsus incomplete carcinogenic activity among aromati c hydrocarbons: Contrast between chrysene and benzo[b ]-triphenylene. J Natl Cancer Inst 53:1017–1020.

Huberman, E. (1975) Mammalian cell transformation a nd cell-mediated mutagenesis by carcinogenic polycy clic hydrocarbons. Mutat Res 29:285–291.

Huberman, E; Sachs, L. (1974) Cell-mediated mutagen esis of mammalian cells with chemical carcinogens. Int J Cancer 13:326–333.

Huberman, E; Sachs, L. (1976) Mutability of differe nt genetic loci in mammalian cells by metabolically activated carcinogenic polycyclic hydrocarbons. Proc Natl Ac ad Sci USA 73:188–192.

Huggins, C; Yang, NC. (1962) Induction and extincti on of mammary cancer. A striking effect of hydrocar bons permits analysis of mechanisms of causes and cure o f breast cancer. Science 137:257–262.

Hughes, NC; Phillips, DH. (1990) Covalent binding o f dibenzpyrenes and benzo[a]pyrene to DNA: evidence for synergistic and inhibitory interactions when applie d in combination to mouse skin. Carcinogenesis 11: 161l–1620.

Hughes, NC; Phillips, DH. (1991) Dependence on dose of initial and persistent levels of benzo[a]pyrene and dibenzo[a,e]pyrene DNA adducts in mouse tissues. P roc Am Assoc Cancer Res 32:98.

Hughes, NC; Phillips, DH. (1993) 32 P -postlabeling analysis of the covalent binding of b enzo[ghi]perylene to DNA in vivo and in vitro. Carcinogenesis 14:127–133.

### 224 DRAFT – DO NOT CITE OR QUOTE

IARC (International Agency for Research on Cancer). (1973) In: IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Certain p olycyclic aromatic hydrocarbons and heterocyclic co mpounds. Vol. 3. Lyon, France.

IARC. (1983) In: IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Polynu clear aromatic compounds. Part 1. Chemical, environmental and experimental data. Vol. 32, Lyon, France.

IARC. (1984a) IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Polynucle ar aromatic compounds. Part 2. Carbon black, mineral o ils (lubricant base oils and derived products) and some nitroarenes. Lyon, France. pp. 87–168.

IARC. (1984b) IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Polynucle ar aromatic compounds. Part 3. Industrial exposures in aluminum production, coal gasification, coke produ ction, and iron and steel founding. Lyon, France, pp. 37–111,

IARC. (1985) IARC monographs on the evaluation of c arcinogenic risk of chemicals to humans. Polynuclea r aromatic compounds. Part 4. Bitumens, coal-tars and derived products, shale-oils and soots. Lyon, Fra nce. pp. 65–
159.

IARC. (1986) IARC monographs on the evaluation of c arcinogenic risk of chemicals to humans. Some halog enated hydrocarbons and pesticide exposures. Lyon, France

.

IARC. (1987) IARC monographs on the evaluation of c arcinogenic risks to humans. Overall evaluation of carcinogenicity: an updating of IARC Monographs vol umes 1 to 42. Suppl. 7. Lyon, France.

IARC. (1989) IARC monographs on the evaluation of c arcinogenic risk of chemicals to humans. Occupation al exposures in petroleum refining; crude oil and majo r petroleum fuels. Vol. 45. Lyon, France, pp. 239– 270.

Ichinotsubo, D; Mower, HF; Setliff, J; et al. (1977 ) The use of rec-bacteria for testing of carcinogen ic substances.
Mutat Res 46:53–56.

Jeffy, BD; Chen, EJ; Gudas, JM; et al. (2000) Disru ption of cell cycle kinetics by benzo[a]pyrene: inv erse expression patterns of BRCA-1 and p53 in MCF-7 cell s arrested in S and G2. Neoplasia 2:460–470.

Jeffy, BD; Chirnomas, RB; Chen, EJ; et al. (2002) A ctivation of the aromatic hydrocarbon receptor path way is not sufficient for transcriptional repression of BRCA-1 : requirements for metabolism of benzo[a]pyrene to 7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenz o[a]pyrene. Cancer Res 62:113–121.

Jerina, DM; Lehr, RE. (1977) The bay-region theory: a quantum mechanical approach to aromatic hydrocarboninduced carcinogenicity. In: Microsomes and drug o xidations. Oxford: Pergamon Press; pp. 709–720.

Jerina, DM; Yagi, H; Lehr, RE; et al. (1978) The ba y-region theory of carcinogenesis by polycyclic aro matic hydrocarbons. In: Polycyclic hydrocarbons and canc er: environment, chemistry and metabolism. Boca Rat on, Florida: CRC Press; pp. 173–188.

Jerina, DM; Chadha, A; Cheh, AM; et al. (1991) Cova lent bonding of bay-region diol epoxides to nucleic acids. Adv Exp Med Biol 283:533–553.

Jiang, H; Shen, YM; Quinn, AM; et al. (2005) Compet ing roles of cytochrom P450 1A1/1B1 and aldo-keto reductase 1A1 in the metabolic activation of (±)-7, 8-dihydroxy-7,8-dihydro-benzo[a]pyrene in human bronchoalveolar cell extracts. Chem Res Toxicol 18 :365–374.

Jiang, H; Gelhaus, SL; Mangal, D; et al. (2007) Met abolism of benzo[a]pyrene in human bronchoalveolar H358 cells using liquid chromatography-mass spectrometry . Chem Res Toxicol 20:1331–1341.

Johnsen, NM; Schwarze, PE; Nyholm, SH; et al. (1997 ) Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in different types of isolated rat lun g cells. Carcinogenesis 18:193–199.

### 225 DRAFT – DO NOT CITE OR QUOTE

Johnsen, NM; Brunborg; G, Haug, K; et al. (1998) Me tabolism and activation of cyclopenta polycyclic ar omatic hydrocarbons in isolated human lymphocytes, HL-60 c ells and exposed rats. Chem Biol Interact 114:77–9 5.

Jotz, MM; Mitchell, AD. (1981) Effects of 20 coded chemicals on the forward mutation frequency at the thymidine kinase locus in L5178Y mouse lymphoma cells. In: D e Serres, FJ; Ashby, J, eds. Evaluation of short-te rm tests for carcinogens. Prog Mutat Res 1:580–593.

Kaden, DA; Hites, RA; Thilly, WG. (1979) Mutagenici ty of soot and associated polycyclic aromatic hydro carbons

to Salmonella typhimurium . Cancer Res 39:4152–4159.

Kakunaga, T. (1973) A quantitative system for assay of morphological/malignant cell transformation by chemical carcinogens using a clone derived from BALB-3T3. I nt J Cancer 12(2):463–473.

Katz, M; Heddle, JA; Salamone, MF. (1981) Mutagenic activity of polycyclic aromatic hydrocarbons and o ther environmental pollutants. Columbus, OH: Battelle P ress; pp. 519–528.

Kligerman, AD; Moore, MM; Erexson, GL; et al. (1986 ) Genotoxicity studies of benz[l]aceanthrylene

. Cancer Lett
31:123–131.

Kligerman, AD; Nelson, GB; Ross, JA; et al. (2002) Effect of the route of administration on the induct ion of cytogenetic damage and DNA adducts in peripheral bl ood lymphocytes of rats and mice by polycyclic arom atic hydrocarbons. Polycyclic Aromatic Compounds 22:814 –851.

Kochhar, TS. (1982) Effects of polycyclic hydrocarb ons on the induction of chromosomal aberrations in absence of an exogenous metabolic activation system in culture d hamster cells. Experientia 38:845–846.

Koganti, A; Singh, R; Rozett, K; et al. (2000) 7H-b enzo[c]fluorene: a major DNA adduct-forming compone nt of coal tar. Carcinogenesis 21:1601–1609.

Kondraganti, SR; Fernandez-Salguerro, P; Gonzalez, FJ; et al. (2003) Polycyclic aromatic hydrocarbon-i nducible DNA adducts: evidence by 32 P -postlabeling and use of knockout mice for AH recep tor-independent mechanisms of metabolic activation in vivo. Int J Cancer 103:5–1 1.

Krahn, DF; Heidelberger, C. (1977) Liver homogenate -mediated mutagenesis in Chinese hamster V79 cells by polycyclic aromatic hydrocarbons and aflatoxins. M utat Res 46:27–44.

Krewski, D; Thorslund, T; Withey, J. (1989) Carcino genic risk assessment of complex mixtures. Toxicol Ind Health
5:851–867.

Kroese, ED; Muller, JJA; Mohn, GR; et al. (2001) Tu morigenic effects in Wistar rats orally administere d benzo[a]pyrene for two years (gavage studies). Impl ications for human cancer risks associated with ora l exposure to polycyclic aromatic hydrocarbons. National Institu te of Public Health and the Environment, Bilthoven, Netherlands.

Krolewski, B; Nagasawa, H; Little, JB. (1986) Effec t of aliphatic amides on oncogenic transformation, sister chromatid exchanges, and mutations induced by cyclo penta[cd]-pyrene and benzo[a]pyrene. Carcinogenesi s 7:1647–1650.

Laaksonen, AM; Mantyjarvi, RA; Hanninen, OO. (1983) Fibroblast cultures of nude mouse skin as targets for transformation by chemical carcinogens. Med Biol 6 1:59–64.

Lacassagne, A; Buu-Hoi, NP; Zajdela, F; et al. (196 8) The true dibenzo[a,l]pyrene, a new, potent carci nogen. Naturwissenschaften 55:43.

Lafleur, AL; Longwell, JP; Marr, JA; et al. (1993) Bacterial and human cell mutagenicity study of some C18H10 cyclopenta-fused polycyclic aromatic hydrocarbons a ssociated with fossil fuels combustion. Environ He alth Perspect 101:146–153.

Lake, RS; Kropko, ML; Pezzutti, MR; et al. (1978) C hemical induction of unscheduled DNA synthesis in h uman skin epithelial cell cultures. Water Res 38:2091–2 098.

### 226 DRAFT – DO NOT CITE OR QUOTE

Langenbach, R; Hix, C; Oglesby, L; et al. (1983) Ce ll-mediated mutagenesis of Chinese hamster V79 cell s and Salmonella typhimurium . Ann NY Acad Sci 407:258–266.

Larsen, JC; Larsen, PB. (1998) Chemical carcinogens . In: Air pollution and health. Cambridge, UK: The Royal Society of Chemistry, pp. 33–56.

Lavik, PS; Moore, PR; Rusch, HP; et al. (1942) Some additive effects of carcinogenic hydrocarbons. Ca ncer Res 2:189–192.

LaVoie, EJ; Bedenko, V; Hirota, N; et al. (1979) A comparison of the mutagenicity, tumor-initiating ac tivity and complete carcinogenicity of polynuclear aromatic hy drocarbons. In: Jones, PW; Leber, P, eds. Polynucl ear aromatic hydrocarbons. Ann Arbor, MI; Ann Arbor Science Publ ishers; pp. 705–721.

LaVoie, EJ; Tulley, L; Bedenko, V; et al. (1980) Mu tagenicity, tumor initiating activity, and metaboli sm of tricyclic polynuclear aromatic hydrocarbons. In: Bjorseth, A ; Dennis, AJ, eds. Polynuclear aromatic hydrocarbon s: chemistry and biological effects. Columbus, Ohio: B attelle Press; pp. 1041–1057.

LaVoie, EJ; Tulley, L; Bedenko, V; et al. (1981) Mu tagenicity, tumor-initiating activity and metabolis m of methylphenanthrenes. Cancer Res 41:3441–3447.

LaVoie, EJ; Amin, S; Hecht, SS; et al. (1982) Tumou r initiating activity of dihydrodiols of benzo[b]fl uoranthene, benzo[j]fluoranthene, and benzo[k]fluoranthene. Ca rcinogenesis 3:49–52.

LaVoie, EJ; Coleman, DT; Tonne, RL; et al. (1983) M utagenicity, tumor initiating activity and metaboli sm of methylated anthracenes. In: Cooke, M; Dennis, AJ, eds. Proceedings of the seventh international sympo sium. Columbus, OH: Battelle Press; pp. 785–798.

LaVoie, EJ; Coleman, DT; Rice, JE; et al. (1985) Tu mor-initiating activity, mutagenicity, and metaboli sm of methylated anthracenes. Carcinogenesis 6:1483–1488

.

LaVoie, EJ; Braley, J; Rice, JE; et al. (1987) Tumo rigenic activity of non-alternant polynuclear aroma tic hydrocarbons in newborn mice. Cancer Lett 34:15–20

.

LaVoie, EJ; Cai, ZW; Meschter, CL; et al. (1994) Tu morigenic activity of fluoranthene, 2-methylfluoran thene and 3-methylfluoranthene in newborn CD-1 mice. Carcino genesis 15:2131–2135.

Li, CS; Lin, RH. (1996) Evaluation of low-dosage en vironmental mutagens with a long-term, cultured epi thelial cell line. Bull Environ Contam Toxicol 56:919–925.

Li, KM; Todorovic, R; Rogan, EG; et al. (1995) Iden tification and quantitation of dibenzo[a,l]pyrene-- DNA adducts formed by rat liver microsomes in vitro: prepondera nce of depurinating adducts. Biochemistry 34(25):8 043–8049.

Li, D; Wang, M; Firozi, PF; et al. (2002) Character ization of a major aromatic DNA adduct detected in human breast tissues. Environ Mol Mutagen 39:193–200.

Lubet, RA; Kiss, E; Gallagher, MM; et al. (1983) In duction of neoplastic transformation and DNA single -strand breaks in C3H/10T1/2 clone 8 cells by polycyclic hy drocarbons and alkylating agents. J Natl Cancer In st 71:991–
997.

Machala, M; Vondracek, J; Blaha, L; et al. (2001) A ryl hydrocarbon receptor-mediated activity of mutag enic polycyclic aromatic hydrocarbons determined using i n vitro reporter gene assay. Mutat Res 497:49–62.

MacLeod, MC; Cohen, GM; Selkirk, JK. (1979) Metabol ism and macromolecular binding of the carcinogen benzo(a)pyrene and its relatively inert isomer benz o(e)pyrene by hamster embryo cells. Cancer Res 39: 3463–3470.

Malcolm, HM; Dobson S. (1994) The calculation of an environmental assessment level (EAL) for atmospher ic PAHs using relative potencies. Department of the E nvironment, London, England; Report No. DoE/HMIP/RR/94/041.

### 227 DRAFT – DO NOT CITE OR QUOTE

Mamber, SW; Bryson, V; Katz, SE. (1983) The Esherichia coli WP2/WP100 ret assay for detection of potential chemical carcinogens. Mutat Res 119:135–144.

Mane, SS; Purnell, DM; Hsu, IC. (1990) Genotoxic ef fects of five polycyclic aromatic hydrocarbons in h uman and rat mammary epithelial cells. Environ Mol Mutagen 15:78–82.

Marshall, CJ; Vousden, KH; Phillips, DH. (1984) Act ivation of c-Ha-ras-1 proto-oncogene by in vitro mo dification with a chemical carcinogen, benzo(a)pyrene diol-epo xide. Nature 310(5978):586–589.

Martin, CN; McDermid, AC. (1981) Testing of 42 code d compounds for their ability to induce unscheduled DNA repair synthesis in HeLa cells. In: De Serres, FJ; Ashby, J, eds. Evaluation of short-term tests for carcinogens. Prog
Mutat Res 1:533–537.

Martin, CN; McDermid, AC; Garner, RC. (1978) Testin g of known carcinogens and noncarcinogens for their ability to induce unscheduled DNA synthesis in HeLa cells. Cancer Res 38:2621–2627.

Mass, MJ; Jeffers, AJ; Ross, JA; et al. (1993) Ki-r as oncogene mutations in tumors and DNA adducts for med by benz[j]aceanthrylene and benzo[a]pyrene in the lung s of strain A/J mice. Mol Carcinog 8:186–192.

Masuda, Y; Kagawa, R. (1972) A novel synthesis and carcinogenicity of dibenzo[a,l]pyrene. Chem Pharm Bull
20:2736–2737.

Matsuoka, A; Hayashi, M; Ishidate, MJ. (1979) Chrom osomal aberration tests on 29 chemicals combined wi th S9 mix in vitro. Mutat Res 66:277–290.

Matthews E.J; Kruhlak, NL; Cimino, MC; et al. (2006 a) An analysis of genetic toxicity, reproductive an d developmental toxicity, and carcinogenicity data: I . Identification of carcinogens using surrogate end points. Regul Toxicol Pharmacol 44:83–96.

Matthews E.J; Kruhlak, NL; Cimino, MC; et al. (2006 b) An analysis of genetic toxicity, reproductive an d developmental toxicity, and carcinogenicity data: I I. Identification of genotoxicants, reprotoxicants, and carcinogens using in silico methods. Regul Toxicol Pharmacol 4 4:97–110.

McCann, J; Choi, E; Yamasaki, E; et al. (1975) Dete ction of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals. Proc Natl Acad Sci U SA 72:5135–5139.

McCarroll, NE; Piper, CE; Keech, BH. (1981) An E co li microsuspension assay for the detection of DNA d amage induced by direct-acting agents and promutagens. E nviron Mutagen 3:429–444.

McClure, PR. (1996) Evaluation of a component-based relative potency approach to cancer risk assessmen t for exposure to PAHs. Toxicologist 30(1, Part 2):8.

McCoull, KD; Rindgen, D; Blair, IA; et al. (1999) S ynthesis and characterization of polycyclic aromati c hydrocarbon o-quinone depurinating N7-guanine adduc ts. Chem Res Toxicol 12:237–246.

Meek, ME; Chan, PKL; Bartlett, S. (1994) Polycyclic aromatic hydrocarbons: evaluation of risks to heal th from environmental exposures in Canada. Environ Carcino g Ecotoxicol Rev C 12:443–452.

Melendez-Colon, VJ; Luch, A; Seidel, A; et al. (200 0) Formation of stable DNA adducts and apurinic sit es upon metabolic activation of bay and fjord region polycy clic aromatic hydrocarbons in human cell cultures. Chem Res Toxicol 13:10–17.

Mersch-Sundermann, V; Mochayedi, S; Kevekordes, S. (1992) Genotoxicity of polycyclic aromatic hydrocar bons in Escherichia coli PQ37. Mutat Res 278:1–9.

Miller, KP; Ramos, KS. (2001) Impact of cellular me tabolism on the biological effects of benzo[a]pyren e and related hydrocarbons. Drug Metab Rev 33:1–35.

### 228 DRAFT – DO NOT CITE OR QUOTE

Milo, GE; Blakeslee, J; Yohn, DS; et al. (1978) Bio chemical activation of aryl hydrocarbon hydroxylase activity, cellular distribution of polynuclear hydrocarbon me tabolites, and DNA damage by polynuclear hydrocarbo n products in human cells in vitro. Cancer Res 38:16 38–1644.

Mishra, NK; Wilson, CM; Pant, KJ; et al. (1978) Sim ultaneous determination of cellular mutagenesis and transformation by chemical carcinogens in Fischer r at embryo cells. J Toxicol Environ Health 4:79–91.

Mohapatra, N; MacNair, P; Bryant, BJ; et al. (1987) Morphological transforming activity and metabolism of cyclopenta-fused isomers of benz[a]anthracene in ma mmalian cells. Mutat Res 188:323–334.

Muller, P; Leece, B; Raha, D. (1997) Scientific cri teria document for multimedia standards development . Polycyclic aromatic hydrocarbons (PAHs). Part 1: Hazard identi fication and dose-response assessment. Ontario Mini stry of the Environment, Standards Development Branch.

Murison, GL. (1988) Induction of sister-chromatid e xchanges by direct and indirect agents in a human t eratoma cell line. Mutat Res 203:347–354.

Myhr, BC; Caspary, WJ. (1988) Evaluation of the L51 78Y mouse lymphoma cell mutagenesis assay: intralab oratory results for sixty-three coded chemicals tested at L itton Bionetics, Inc. Environ Mol Mutag 12 (Suppl 13):103–194.

Nagabhushan, M; Hussong, J; Polverini, PJ; et al. ( 1990) Inhibition of hamster buccal pouch epithelial cell replication during in vitro exposure to polycyclic aromatic hydrocarbons. Proc Am Assoc Cancer Res 31 :86.

Nakatsuru, Y; Wakabayashi, K; Fujii-Kuriyama, Y; et al. (2004) Dibenzo[a,l]pyrene-induced genotoxic an d carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor-deficient mice. Int J Cancer
112:179–183.

Neal, J; Rigdon, RH. (1967) Gastric tumors in mice fed benzo[a]pyrene: a quantitative study. Tex Rep Biol Med
25:553–557.

Nesnow, S; Triplett, LL; Slaga, TJ. (1983) Mouse sk in tumor initiation-promotion and complete carcinog enesis bioassays: mechanisms and biological activities of emission samples. Environ Health Perspect 47:255–2 68.

Nesnow, S; Gold, A; Sangaiah, R; et al. (1984) Mous e skin tumor-initiating activity of benz[e]aceanthr ylene and benz[l]aceanthrylene in Sencar mice. Cancer Lett 2 2:263–268.

Nesnow, S; Milo, G; Kurian, P; et al. (1990) Induct ion of anchorage-independent growth in human diploi d fibroblasts by the cyclopenta-polycyclic aromatic h ydrocarbon, benz[l]aceanthrylene. Mutat Res 244:22 1–225.

Nesnow, S; Ross, J; Mohapatra, N; et al. (1991) Gen otoxicity and identification of the major DNA-adduc ts of aceanthrylene. In: Cooke M LKMJe, eds. Polynuclear aromatic hydrocarbons: Measurements, means, and metabolism. Columbus, OH: Battelle Press; pp. 629–6 39.

Nesnow, S; Beck, S; Ball, LM; et al. (1993a) Morpho logical transformation of C3H10T1/2CL8 cells by cyc lopentafused derivatives of benzo[a]pyrene and benzo[e]pyr ene. Cancer Lett 74:25–30.

Nesnow, S; Ross, J; Nelson, G; et al. (1993b) Quant itative and temporal relationships between DNA addu ct formation in target and surrogate tissues: implicat ions for biomonitoring. Environ Health Perspect 10 1(Suppl 6)
3:37–42.

Nesnow, S; Ross, J; Beck, S; et al. (1994) Morpholo gical transformation and DNA adduct formation by dibenz[a,h]anthracene and its metabolites in C3H10T 1/2CL8 cells. Carcinogenesis 15:2225–2231.

Nesnow, S; Ross, JA; Stoner, GD; et al. (1995) Mech anistic linkage between DNA adducts, mutations in o ncogenes and tumorigenesis of carcinogenic environmental pol ycyclic aromatic hydrocarbons in strain A/J mice. Toxicology
105:403–413.

### 229 DRAFT – DO NOT CITE OR QUOTE

Nesnow, S; Ross, JA; Stoner, GD; et al. (1996) Tumo rigenesis of carcinogenic environmental polycyclic aromatic hydrocarbons in strain A/J mice: linkage to DNA add ucts and mutations in oncogenes. Polycyclic Aromat ic Hydrocarbons 10:259–266.

Nesnow, S; Davis, C; Nelson, G; et al. (1997) Compa rison of the morphological transforming activities of dibenzo[a,l]pyrene and benzo[a]pyrene in C3H10T1/2C L8 cells and characterization of the dibenzo[a,l]py rene-DNA adducts. Carcinogenesis 18:1973–1978.

Nesnow, S; Mass, MJ; Ross, JA; et al. (1998a) Lung tumorigenic interactions in strain A/J mice of five environmental polycyclic aromatic hydrocarbons. En viron Health Perspect 106(Suppl 6):1337–1346.

Nesnow, S; Ross, JA; Mass, MJ; et al. (1998b) Mecha nistic relationships between DNA adducts, oncogene mutations, and lung tumorigenesis in strain A mice. Exp Lung Res 24:395–405.

Nikonova, TV. (1977) Transplacental effect of benz[ a]pyrene and pyrene. Bull Exp Biol Med 84:1025–102 7.

Nisbet, ICT; LaGoy, PK. (1992) Toxic equivalency fa ctors (TEFs) for polycyclic aromatic hydrocarbons ( PAHs). Regul Toxicol Pharmacol 16:290–300.

Norpoth, K; Kemena, A; Jacob, J; et al. (1984) The influence of 18 environmentally relevant polycyclic aromatic hydrocarbons and Clophen A50, as liver monooxygenas e inducers, on the mutagenic activity of benz[a]ant hracene in the Ames test. Carcinogenesis 5:747–752.

NTP (National Toxicology Program). (2000) Toxicolog y and carcinogenesis studies of naphthalene (CAS no . 91- 20-3) in F344/N rats (inhalation studies). National Toxicology Program. U.S. Department of Health and Human Services, National Institutes of Health, Rockville, MD. Technical report series no. 500.

Nyholm, SH; Alexander, J; Lundanes, E; et al. (1996 ) Biotransformation of the cyclopenta-fused polycyc lic aromatic hydrocarbon benz[j]aceanthrylene in isolat ed rat liver cells: identification of nine new meta bolites. Carcinogenesis 17(5):1111–1120.

Oshiro, Y; Balwierz, PS; Soelter, SG; et al. (1992) Evaluation of mouse peripheral blood micronucleus assay. Environ Mol Mutag 19(Suppl 20):47.

Pahlman, R; Pelkonen, O. (1987) Mutagenicity studie s of different polycyclic aromatic hydrocarbons: th e significance of enzymatic factors and molecular str ucture. Carcinogenesis 8:773–778.

Paika, IJ; Beauchesne, MT; Randall, M; et al. (1981 ) In vivo SCE analysis of 20 coded compounds. In: D e Serres, FJ; Ashby, J, eds. Evaluation of short-term tests f or carcinogens. Prog Mutat Res 1:672–681.

Park, JH; Gopishetty, S; Szewczuk, LM; et al. (2005 ) Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) by PAH o-quinones: involvement of reactive ox ygen species and copper(II)/copper(I) redox cycling . Chem Res Toxicol 18(6):1026–1037.

Park, JH; Troxel, AB; Harvey, RG; et al. (2006) Pol ycyclic aromatic hydrocarbon (PAH) o-quinones produ ced by the aldo-keto-reductases (AKRs) generate abasic sit es, oxidized pyrimidines, and 8-oxo-dGuo via reacti ve oxygen species. Chem Res Toxicol 19(5):719–728.

Park, JH; Gelhaus, S; Vedantam, S; et al. (2008) Th e pattern of p53 mutations caused by PAH o-quinones is driven by 8-oxo-dGuo formation while the spectrum of mutat ions is determined by biological selection for domi nance. Chem Res Toxicol 21(5):1039–1049.

Pataki, J; Huggins, C. (1969) Molecular site of sub stituents of benz[a]anthracene related to carcinoge nicity. Cancer
Res 29:506–509.

Pavanello, S; Favretto, D; Brugnone, F; et al. (199 9) HPLC/fluorescence determination of anti-BPDE-DNA adducts in mononuclear white blood cells from PAH-exposed h umans. Carcinogenesis 20(3):431–435.

### 230 DRAFT – DO NOT CITE OR QUOTE

Penman, BW; Kaden, DA; Liber, HL; et al. (1980) Per ylene is a more potent mutagen than benzo[a]pyrene for Salmonella typhimurium . Mutat Res 77:271–277.

Penning, TM; Burczynski, ME; Hung, CF; et al. (1999 ) Dihydrodiol dehydrogenases and polycyclic aromati c hydrocarbon activation: generation of generation of reactive and redox active o-quinones. Chem Res To xicol 12:1–
18.

Perry, PE; Thomson, EJ. (1981) Evaluation of the si ster chromatid exchange method in mammalian cells a s a screening system for carcinogens. In: De Serres FJ ; Ashby J, eds. Prog Mutat Res 1:560–569.

Petry, T; Schmid, P; Schlatter, C. (1996) The use o f toxic equivalency factors in assessing occupation al and environmental health risk associated with exposure to airborne mixtures of polycyclic aromatic hydroca rbons (PAHs). Chemosphere 32:639–648.

Pfeiffer, EH (1973) Investigations on the carcinoge nic burden by air pollution in man. VII. Studies on the oncogenetic interaction of polycyclic aromatic hydr ocarbons. Zbl Bakt Hyg, J Abt Org B158:69–83.

Pfeiffer, EH. (1977) Oncogenic interaction of carci nogenic and non-carcinogenic polycyclic aromatic hy drocarbons in mice. IARC Sci Publ 16:69–77.

Pfeiffer, CA; Allen, E. (1948) Attempts to produce cancer in rhesus monkeys with carcinogenic hydrocar bons and estrogens. Cancer Res 8:97–127.

Phillips, DH; Grover, PL; Sims, P. (1979) A quantit ative determination of the covalent binding of a se ries of polycyclic hydrocarbons to DNA in mouse skin. Int J Cancer 23:201–208.

Phillipson, CE; Ioannides, C. (1989) Metabolic acti vation of polycyclic aromatic hydrocarbons to mutag ens in the Ames test by various animal species including man. Mutat Res Mar 211:147–151.

Pienta, RJ; Poiley, JA; Lebherz, WB 3rd. (1977) Mor phological transformation of early passage golden S yrian hamster embryo cells derived from cryopreserved pri mary cultures as a reliable in vitro bioassay for i dentifying diverse carcinogens. Int J Cancer 19:642–655.

Platt, KL; Dienes, HP; Tommasone, M; et al. (2004) Tumor formation in the neonatal mouse bioassay indi cates that the potent carcinogen dibenzo[def,p]chrysene (diben zo[a,l]pyrene) is activated in vivo via its trans

11,12-dihydrodiol. Chem Biol Interact 148:27–36.

Poncelet, F; Massanda, K; Fouassin, A; et al. (1978 ) Mutagenic study of some polycyclic aromatic hydro carbons present in smoked fishes from Africa. Arch Int Phy s Biochem 86:954–955.

Popescu, NC; Turnbull, D; DiPaolo, JA. (1977) Siste r chromatid exchange and chromosome aberration anal ysis with the use of several carcinogens and noncarcinog ens: brief communication. J Natl Cancer Inst 59:28 9–293.

Prahalad, AK; Ross, JA; Nelson, GB; et al. (1997) D ibenzo[a,l]pyrene-induced DNA adduction, tumorigeni city, and Ki-ras oncogene mutations in strain A/J mouse lung. Carcinogenesis 18:1955–1963.

Probst, GS; McMahon, RE; Hill, LE; et al. (1981) Ch emically-induced unscheduled DNA synthesis in prima ry rat hepatocyte cultures: a comparison with bacterial mu tagenicity using 218 compounds. Environ Mutagen 3: 11–32.

Pufulete M.; Battershill, J; Boobis, A; et al.(2004 ) Approaches to carcinogenic risk assessment for po lycylic aromatic hydrocarbons: a UK perspective. Regul Tox icol Pharmacol 40:54–66.

Puisieux, A; Lim, S; Groopman, J; et al. (1991) Sel ective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Canc er Res 51(22):6185–6189.

Ramesh, A; Walker, SA; Hood, DB; et al. (2004) Bioa vailability and risk assessment of orally ingested polycyclic aromatic hydrocarbons. Int J Toxicol 23:301–333.

### 231 DRAFT – DO NOT CITE OR QUOTE

Rask-Nielson, R. (1950) The susceptibility of the t hymus, lung, subcutaneous and mammary tissues in st rain St mice to direct application of small doses of four differ ent carcinogenic hydrocarbons. Br J Cancer 4:108–1 16.

Raveh, D; Huberman, E. (1983) A microtiter plate as say for the selection of 6-thioguanine-resistant mu tants in Chinese hamster V79 cells in the presence of phorbo l-12-myristate-13-acetate. Mutat Res 113:499–506.

Raveh, D; Slaga, TJ; Huberman, E. (1982) Cell-media ted mutagenesis and tumor-initiating activity of th e ubiquitous polycyclic hydrocarbon, cyclopenta[c,d]pyrene. Car cinogenesis 3:763–766.

Reddy, MV; Gupta, RC; Randerath, E; et al. (1984) 32 P -Postlabeling test for covalent DNA binding of chem icals in vivo: application to a variety of aromatic carcinog ens and methylating agents. Carcinogenesis 5:231–2 43.

Rice, JE; Hosted, TJ, Jr; LaVoie, EJ. (1984) Fluora nthene and pyrene enhance benzo[a]pyrene--DNA adduc t formation in vivo in mouse skin. Cancer Lett 24:32 7–333.

Rice, JE; Makowski, GS; Hosted, TJ, Jr; et al. (198 5) Methylene-bridged bay region chrysene and phenan threne derivatives and their keto-analogs: mutagenicity in Salmonella typhimurium and tumor-initiating activi ty on mouse skin. Cancer Lett 27:199–206.

Rice, JE; Jordan, K; Little, P; et al. (1988) Compa rative tumor-initiating activity of methylene-bridg ed and bayregion methylated derivatives of benz[a]anthracene and chrysene. Carcinogenesis 9:2275–2278.

Rigdon, RH; Neal, J. (1966) Gastric carcinomas and pulmonary adenomas in mice fed benzo[a]pyrene. Tex Rep Biol Med 24:195–207.

Rigdon, RH; Neal, J. (1969) Relationship of leukemi a to lung and stomach tumors in mice fed benzo[a]py rene. Proc Soc Exp Biol Med 130:146–148.

Rigdon, RH; Benge, MC; Kirchoff, H; et al. (1969) L eukemia in mice fed benzo (a) pyrene: a clinical, p athologic and hematologic study. Tex Rep Biol Med 27:803–820

.

Robinson, DE; Mitchell, AD. (1981) Unscheduled DNA synthesis response of human fibroblasts, WI-38 cell s, to 20 coded chemicals. In: De Serres, FJ; Ashby, J, e ds. Evaluation of short-term tests for carcinogens. Prog Mutat Res
1:517–527.

Roe, FJC. (1962) Effect of phenanthrene on tumour-i nitiation by 3,4-benzpyrene. Br J Cancer 16:503–50 6.

Roe, FJ; Waters, MA. (1967) Induction of hepatoma i n mice by carcinogens of the polycyclic hydrocarbon type. Nature 214:299–300.

Rogan, EG; Cavalieri, EL; Ramakrishna NVS; et al. ( 1993) Mechanisms of benzo(a)pyrene and 7,12-diemthylbenz(a)antrhacene activation: qualitat ive aspects of the stable and depurination DNA addu cts obtained from radical cations and diol epoxides. In: Polycy clic aromatic hydrocarbons: synthesis, properties, analytical measurements, occurrence and biological effects. Go rdon and Breach Science Publishers: Bordeaux, Franc e, pp. 733–740.

Rosenkranz, HS; Leifer, Z. (1980) Determining the D NA-modifying activity of chemicals using DNA polyme rasedeficient Escherichia coli . Chemical mutagens: principles and methods for the ir detection. New York, NY: Plenum Press; pp. 109–147.

Rosenkranz, HS; Poirier, LA. (1979) Evaluation of t he mutagenicity and DNA-modifying activity of carci nogens and noncarcinogens in microbial systems. J Natl Ca ncer Inst 62:873–891.

Ross, JA; Nelson, GB; Wilson, KH; et al. (1995) Ade nomas induced by polycyclic aromatic hydrocarbons i n strain A/J mouse lung correlate with time-integrated DNA a dduct levels. Cancer Res 55:1039–1044.

Rossman, TG; Molina, M; Meyer, L; et al. (1991) Per formance of 133 compounds in the lambda prophage in duction endpoint of the microscreen assay and a comparison with Salmonella typhimurium mutagenicity and rodent carcinogenicity assays. Mutat Res 260:349–367.

### 232 DRAFT – DO NOT CITE OR QUOTE

Roszinsky-Kocher, G; Basler, A; Rohrborn, G. (1979) Mutagenicity of polycyclic hydrocarbons. V. Induct ion of sister-chromatid exchanges in vivo. Mutat Res 66:6 5–67.

Rugen, PJ; Stern, CD; Lamm, SH. (1989) Comparative carcinogenicity of the PAHs as a basis for acceptab le exposure levels (AELs) in drinking water. Regul To xicol Pharmacol 9:273–283.

Rummel, AM; Trosko, JE; Wilson, MR; et al. (1999) P olycyclic aromatic hydrocarbons with bay-like regio ns inhibited gap junctional intercellular communicatio n and stimulated MAPK activity. Toxicol Sci 49:232 –240.

Safe, S; Wormke, M. (2003) Inhibitory aryl hydrocar bon receptor-estrogen receptor alpha cross-talk and mechanisms of action. Chem Res Toxicol 16:807–816.

Sagredo, C; Øvrebø, S; Haugen, A; et al. (2006) Qua ntitative analysis of benzo[a]pyrene biotransformat ion and adduct formation in AhR knockout mice. Toxicol Let t 167:173–182.

Sakai, M; Yoshida, D; Mizusaki, S. (1985) Mutagenic ity of polycyclic hydrocarbons and quinones on Salmonella thyphimurium TA97. Mutat Res 156:61–67.

Salaman, MH; Roe, FJC. (1956) Further tests for tum our-initiating activity: N,N-di(2-chloroethyl)-p-am inophenylbutic acid (CB1348) as an initiator of skin t umour formation in the mouse. Br J Cancer 10:363–3 78.

Salamone, MF; Heddle, JA; Katz, M. (1979a) The muta genic activity of thirty polycyclic aromatic hydroc arbons (PAH) and oxides in urban airborne particulates. E nviron Int 2:37–43.

Salamone, MF; Heddle, JA; Katz, M. (1979b) The use of the Salmonella/microsomal assay to determine mutagenicity in paired chemical mixtures. Can J Ge net Cytol 21:101–107.

Salamone, MF; Heddle, JA; Katz, M. (1981) Mutagenic activity of 41 compounds in the in vivo micronucle us assay. In: De Serres, FJ; Ashby, J, eds. Evaluation of sho rt-term tests for carcinogens. Prog Mutat Res 1:68 6–697.

Sangaiah, R; Gold, A; Toney, GE; et al. (1983) Benz [j]aceanthrylene: a novel polycyclic aromatic hydro carbon with bacterial mutagenic activity. Mutat Res 119:259–26 6.

Sanner T; Dybing, E. (2005) Comparison of carcinoge nic and in vivo genotoxic potency estimates. Basic Clin Pharmacol Toxicol 96:131–139.

Schmähl, D; Schmidt, KG; Habs, M. (1977) Syncarcino genic action of polycyclic hydrocarbons in automobi le exhaust gas condensates. IARC Sci Publ 16:53–59.

Schmoldt, A; Jacob, J; Grimmer, G. (1981) Dose-depe ndent induction of rat liver microsomal aryl hydroc arbon monooxygenase by benzo[k]fluoranthene. Cancer Lett 13:249–257.

Schneider, K; Roller, M; Kalberlah, F; et al. (2002 ) Cancer risk assessment for oral exposure to PAH m ixtures. J Appl Toxicol 22:73–83.

Scribner, JD. (1973) Brief communication: Tumor ini tiation by apparently noncarcinogenic polycyclic ar omatic hydrocarbons. J Natl Cancer Inst 50:1717–1719.

Segerback, D; Vodicka, P. (1993) Recoveries of DNA adducts of polycyclic aromatic hydrocarbons in the

32 P -postlabeling assay. Carcinogenesis 14:2463–2469.

Shah, GM; Bhattacharya, RK. (1989) Alteration in he patic nuclear RNA polymerase activity following benzo[a]pyrene administration in rat. In Vivo 3:12 5–127.

Sharovskaia, I; Rokitskaia, TI; Kobliakov, VA. (200 3) [Effect of some polycyclic aromatic hydrocarbons on gap junction intercellular communication in hepatoma He p G2 cell culture]. Tsitologiia 45:51–58.

### 233 DRAFT – DO NOT CITE OR QUOTE

Shen, YM; Troxel, AB; Vedantam, S; et al. (2006) Co mparison of p53 mutations induced by PAH o-quinones with those caused by anti-benzo[a]pyrene diol epoxide in vitro: role of reactive oxygen and biological sele ction. Chem Res Toxicol 19(11):1441–1450.

Sheu, CW; Dobras, SN; Rodriguez, I; et al. (1994) T ransforming activity of selected polycyclic aromati c hydrocarbons and their nitro-derivatives in BALB/3T 3 A31-1-1 cells. Food Chem Toxicol 32:611–615.

Shubik, P; Pietra, G; Della Porta, G. (1960) Studie s of skin carcinogenesis in the Syrian golden hamst er. Cancer Res 20:l00–105.

Simmon, VF. (1979a) In vitro mutagenicity assays of chemical carcinogens and related compounds with Salmonella typhimurium . J Natl Cancer Inst 62:893–899.

Simmon, VF. (1979b) In vitro assays for recombinoge nic activity of chemical carcinogens and related co mpounds with Saccharomyces cerevisiae D3. J Natl Cancer Inst 62:901–910.

Simmon, VF; Rosenkranz, HS; Zeiger, E; et al. (1979 ) Mutagenic activity of chemical carcinogens and re lated compounds in the intraperitoneal host-mediated assa y. J Natl Cancer Inst 62:911–918.

Sirianni, SR; Huang, CC. (1978) Sister chromatid ex change induced by promutagens/carcinogens in Chines e hamster cells cultured in diffusion chambers in mic e. Proc Soc Exp Biol Med 158:269–274.

Sjogren, M; Ehrenberg, L; Rannug, U. (1996) Relevan ce of different biological assays in assessing init iating and promoting properties of polycyclic aromatic hydroca rbons with respect to carcinogenic potency. Mutat Res 358:97–
112.

Slaga, TJ; Fischer, SM. (1983) Strain differences a nd solvent effects in mouse skin carcinogenesis exp eriments using carcinogens, tumor initiators and promoters. Prog Exp Tumor Res 26:85–109.

Slaga, TJ; Hubermann, E; Selkirk, JK; et al. (1978) Carcinogenicity and mutagenicity of benz[a]anthrac ene diols and diol-epoxides. Cancer Res 38:1699–1704.

Slaga, TJ; Jecker, L; Bracken, WM; et al. (1979) Th e effects of weak or noncarcinogenic polycyclic hyd rocarbons on 7,12-dimethylbenz[a]anthracene and benzo [a]pyre ne skin tumor-initiation. Cancer Lett 7:51–59.

Slaga, TJ; Iyer, RP; Lyga, W; et al. (1980) Compari son of the skin tumor-initiating activities of dihy drodiols, diolepoxides, and methylated derivatives of various pol ycyclic aromatic hydrocarbons. In: Bjorseth, A; De nnis, AJ, eds. Polynuclear aromatic hydrocarbons: chemistry and bi ological effects. Columbus, OH: Battelle Press; pp. 753–769.

Slooff, W; Janus, JA; Matthijsen, AJCM; et al. (198 9) Integrated criteria document PAHs (PDF includes addendum by Montizaan). National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherl ands.

Smith, LE; Denissenko, MF; Bennett, WP; et al. (200 0) Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons. J Natl Cancer Inst 92:803–8 11.

Smolarek, TA; Baird, WM. (1984) Benzo[e]pyrene-indu ced alterations in the binding of benzo[a]pyrene to DNA in hamster embryo cell cultures. Carcinogenesis 5:106 5–1069.

Smolarek, TA; Moynihan, CG; Salmon, CP; et al. (198 6) Benz[a]anthracene-induced alterations in the met abolic activation of benzo[a]pyrene by hamster embryo cell cultures. Cancer Lett 30:243–249.

Smolarek, TA; Baird, WM; Fisher, EP; et al. (1987) Benzo[e]pyrene-induced alterations in the binding o f benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene t o DNA in Sencar mouse epidermis. Cancer Res 47:370 1–
3706.

Snell, KC; Stewart, HL. (1962) Pulmonary adenomatos is induced in DBA/2 mice by oral administration of dibenz(a,h)anthracene. J Natl Cancer Inst 28:1043– 1049.

### 234 DRAFT – DO NOT CITE OR QUOTE

Solt, DB; Polverini, PJ; Calderon, L. (1987) Carcin ogenic response of hamster buccal pouch epithelium to 4 polycyclic aromatic hydrocarbons. J Oral Pathol 16:294–302.

Staal, YCM; Hebels DGAJ; van Herwijnen, MHM; et al. (2007) Binary PAH-mixture cause additive or antago nistic effects on gene expression but synergistic effects on DNA adduct formation. Carcinogenesis 28:2632–26 40.

Stanton, MF; Miller, E; Wrench, C; et al. (1972) Ex perimental induction of epidermoid carcinoma in the lungs of rats by cigarette smoke condensate. J Nat1 Cancer Inst 49:867–877.

Steiner, PF. (1955) Carcinogenicity of multiple che micals simultaneously administered. Cancer Res 15: 632–635.

Steiner, PF; Falk, HL. (1951) Summation and inhibit ion effects of weak and strong carcinogenic hydroca rbons: 1:2-benzanthracene, chrysene, 1:2:5:6-dibenzanthrac ene, and 20-methylcholanthrene. Cancer Res 11:56–6 3.

Straif K; Baan, R; Grosse, Y; et al. (2005) Carcino genicity of polycyclic aromatic hydrocarbons. Lan cet 6:931–
932.

Sugiyama, T. (1973) Chromosomal aberrations and car cinogenesis by various benz(a)-anthracene derivativ es. Gann
64:637–639.

Tannheimer, SL; Ethier, SP; Caldwell, KK; et al. (1 998) Benzo[a]pyrene- and TCDD-induced alterations i n tyrosine phosphorylation and insulin-like growth factor sign aling pathways in the MCF-10A human mammary epithel ial cell line. Carcinogenesis 19:1291–1297.

Teranishi, K; Hamada, K; Watanabe, H. (1975) Quanti tative relationship between carcinogenicity and mut agenicity of polyaromatic hydrocarbons in Salmonella typhimur ium mutants. Mutat Res 31:97–102.

Thyssen, J; Althoff, J; Kimmerle, G; et al. (1980) Investigations on the carcinogenic burden of air po llution in man. XIX. Effect of inhaled benzo[a]pyrene in Syrian gol den hamsters: a pilot study. Zentralbl Bakteriol H yg I Abt Orig
B 171:441–444.

Thyssen, J; Althoff, J; Kimmerle, G; et al. (1981) Inhalation studies with benzo[a]pyrene in Syrian go lden hamsters. J Natl Cancer Inst 66:575–577.

Till, M; Riebniger, D’ Schmitz, HJ; et al. (1999) P otency of various polycyclic aromatic hydrocarbons as inducers of CYP1A1 in rat hepatocyte cultures. Chem Biol Inter act 117:135–150.

Tong, C; Brat, SV; Williams, GM. (1981a) Sister-chr omatid exchange induction by polycyclic aromatic hydrocarbons in an intact cell system of adult rat- liver epithelial cells. Mutat Res 91:467–473.

Tong, C; Laspia, MF; Telang, S; et al. (1981b) The use of adult rat liver cultures in the detection of the genotoxicity of various polycyclic aromatic hydrocarbons. Envir on Mutagen 3:477–487.

Tong, C; Brat, VS; Telang, S; et al. (1983) Effects of genotoxic polycyclic aromatic hydrocarbons in r at liver culture systems. In: Cooke, M; Dennis, AJ, eds. Polynuclea r aromatic hydrocarbons: formation; metabolism, and measurement. Columbus, OH: Battelle Press; pp. 1189 –1203.

Topping, DC; Martin, DH; Nettesheim, P. (1981) Dete rmination of cocarcinogenic activity of benzo[e]pyr ene for respiratory tract mucosa. Cancer Lett 11:315–321.

Travis C.C, Saulsbury, AW; Richter Pack, SA. (1990) Prediction of cancer potency using a battery of mu tation and toxicity data. Mutagenesis 5:213–219.

Tsuchimoto, T; Matter, BE. (1981) Activity of coded compounds in the micronucleus test. In: De Serres, FJ; Ashby, J, eds. Evaluation of short-term tests for carcinog ens. Prog Mutat Res 1:705–711.

Tweats, DJ. (1981) Activity of 42 coded compounds i n a differential killing test using Escherichia coli strains WP2, WP67 (uvrA polA), and CM871 (uvrA 1exA recA). In: De Serres, FJ; Ashby, J, eds. Evaluation of short-t erm tests for carcinogens. Prog Mutat Res 1:199–209.

### 235 DRAFT – DO NOT CITE OR QUOTE

Uno, S; Dalton, TP; Dragin, N; et al. (2006) Oral b enzo[a]pyrene in Cyp1 knockout mouse lines: CYP1A1 important in detoxication, CYP1B1 metabolism required for imm une damage independent of total-body burden and cle arance rate. Mol Pharmacol 69:1103–1114.

U.S. EPA (United States Environmental Protection Ag ency). (1986) Guidelines for the health risk assess ment of chemical mixtures. Federal Register 51(185):34014– 34025.

U.S. EPA. (1990) Drinking water criteria document f or polycyclic aromatic hydrocarbons. Prepared by t he Office of Health and Environmental Assessment, Environment al Criteria and Assessment Office, Cincinnati, Ohio , for the Office of Drinking Water, Washington, DC.

U.S. EPA. (1993) Provisional guidance for quantitat ive risk assessment of polycyclic aromatic hydrocar bons. Office of Health and Environmental Assessment, Envi ronmental Criteria and Assessment Office, Cincinnat i, Ohio.

U.S. EPA. (2000) Supplementary guidance for conduct ing health risk assessment of chemical mixtures. EPA/630/R-00/002. Available online at http://www.ep a.gov/iris/backgr-d.htm.

U.S. EPA. (2002) Peer consultation workshop on appr oaches to polycyclic aromatic hydrocarbon (PAH) hea lth assessment. National Center for Environmental Asse ssment, Office of Research and Development, Washing ton, DC; EPA/635/R-02/005.

U.S. EPA. (2004) An examination of EPA risk assessm ent principles and practices. Staff paper prepared for the U.S. EPA by members of the Risk Assessment Task For ce at the request of the EPA science advisor. Avai lable online at http://www.epa.gov/osa/ratf.htm.

U.S. EPA. (2005a) Guidelines for carcinogen risk as sessment. Risk Assessment Forum, Washington, DC; EPA/630/P-03/001B. Available online at http://www.e pa.gov/iris/backgr-d.htm (accessed January 15, 2009 ).

U.S. EPA. (2005b) Supplemental guidance for assessi ng susceptibility from early-life exposure to carci nogens. Risk Assessment Forum, Washington, DC; EPA/630/R-03/003F . Available online at http://www.epa.gov/iris/backg rd.htm (accessed January 15, 2009).

U.S. EPA. (2008) Polycyclic aromatic hydrocarbons ( PAHs). Integrated Risk Information System (IRIS). National Center for Environmental Assessment, Washington, DC . Available online at http://www.epa.gov/iris.

Utesch, D; Glatt, H; Oesch, F. (1987) Rat hepatocyt e-mediated bacterial mutagenicity in relation to th e carcinogenic potency of benz[a]anthracene, benzo[a]pyrene, and t wenty-five methylated derivatives. Cancer Res 47(6 ):1509–
1515.

Vaca, C; Tornqvist, M; Rannug, U; et al. (1992) On the bioactivation and genotoxic action of fluoranth ene. Arch Toxicol 66:538–545.

Valencia, R; Houtchens, K. (1981) Mutagenic activit y of 10 coded compounds in the Drosophila sex-linke d recessive lethal test. In: De Serres, FJ; Ashby, J , eds. Evaluation of short-term tests for carcinoge ns. Prog Mutat Res 1:652–659.

Van Duuren, BL; Sivak, A; Segal, A; et al. (1966) T he tumor-promoting agents of tobacco leaf and tobac co smoke condensate. J Natl Cancer Inst 37:519–526.

Van Duuren, BL; Sivak, A; Langseth, L; et al. (1968 ) Initiators and promoters in tobacco carcinogenesi s. Natl Cancer Inst Monogr 28:173–80.

Van Duuren, BL; Sivak, A; Goldschmidt, B.M; et al. (1970) Initiating activity of aromatic hydrocarbons in twostage carcinogenesis. J Natl Cancer Inst 44:1167–1 173.

Van Duuren, BL; Katz, C; Goldschmidt, BM; et al. (1 973) Brief communication: cocarcinogenic agents in tobacco carcinogenesis. J Natl Cancer Inst 51:703–705.

### 236 DRAFT – DO NOT CITE OR QUOTE

Van Duuren, BL; Goldschmidt, BM. (1976) Cocarcinoge nic and tumor-promoting agents in tobacco carcinoge nesis. J Natl Cancer Inst 56:1237–1242.

Vesselinovitch, SD; Kyriazis, AP; Mihailovich, N; e t al. (1975) Factors influencing and/or acceleratio n of lymphoreticula tumors in mice by benzo[a]pyrene tre atment. Cancer Res 35:1963–1969.

Vienneau, DS; DeBoni, U; Wells, PG. (1995) Potentia l genoprotective role for UDP-glucuronosyltransfera ses in chemical carcinogenesis: initiation of micronuclei by benzo[a]pyrene and benzo[e]pyrene in UDPglucuronosyltransferase-deficient cultured rat skin fibroblasts. Cancer Res 55:1045–1051.

Vijayalakshmi, KP; Suresh, CH. (2008) Theoretical s tudies on the carcinogenicity of polycyclic aromati c hydrocarbons. J Comput Chem 29:1108–1117.

Wangenheim, J; Bolcsfoldi, G. (1988) Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compound s. Mutagenesis 3:193–205.

Warshawsky, D; Barkley, W. (1987) Comparative carci nogenic potencies of 7H-dibenzo[c,g]carbazole, dibenz[a,j]acridine and benzo[a]pyrene in mouse ski n. Cancer Lett 37:337–344.

Warshawsky, D; Barkley, W; Bingham, E. (1993) Facto rs affecting carcinogenic potential of mixtures. F undam Appl Toxicol 20:376–382.

Warshawsky, D; Livingston, GK; Fonouni-Fard, M; et al. (1995) Induction of micronuclei and sister chro matid exchanges by polycyclic and N-heterocyclic aromatic hydrocarbons in cultured human lymphocytes. Envir on Mol Mutagen 26:109–118.

Weinstein, D; Katz, ML; Kazmer, S. (1977) Chromosom al effects of carcinogens and noncarcinogens on WI- 38 after short term exposures with and without metabol ic activation. Mutat Res 46:297–304.

Wenzel-Hartung, R; Brune, H; Grimmer, G; et al. (19 90) Evaluation of the carcinogenic potency of four environmental polycyclic aromatic compounds followi ng intrapulmonary application in rats. Exp Pathol 40:221–
227.

Weyand, EH; LaVoie, EJ. (1988) Comparison of PAH DN A adduct formation and tumor initiating activity in newborn mice. Proc Am Assoc Cancer Res 29:98.

Weyand, EH; Wu, Y. (1995) Covalent binding of polyc yclic aromatic hydrocarbon components of manufactur ed gas plant residue to mouse lung and forestomach DNA. C hem Res Toxicol 8:955–962.

Weyand, EH; He, ZM; Ghodrati, F; et al. (1992) Effe ct of fluorine substitution on benzo[j]fluoranthene genotoxicity. Chem Biol Interact 84:37–53.

Weyand, EH; Chen, YC; Wu, Y; et al. (1995) Differen ces in the tumorigenic activity of a pure hydrocarb on and a complex mixture following ingestion: benzo[a]pyrene vs, manufactured gas plant residue. Chem Res Toxi col
8:949–954.

WHO (World Health Organization). (1998) Selected no n-heterocyclic polycyclic aromatic hydrocarbons Environmental health criteria. Vol. 202. Internati onal Programme on Chemical Safety, Geneva, Switzerl and.

Willett, KL; Gardinali, PR; Sericano, JL; et al. (1 997) Characterization of the H4IIE rat hepatoma cel l bioassay for evaluation of environmental samples containing poly nuclear aromatic hydrocarbons (PAHs). Arch Environ Contam Toxicol 32:442–448.

Williams, GM; Laspia, MF; Dunkel, VC. (1982) Reliab ility of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens. M utat Res 97:359–370.

Wislocki, PG; Bagan, ES; Lu, AY; et al. (1986) Tumo rigenicity of nitrated derivatives of pyrene, benz[ a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse as say. Carcinogenesis 7:1317–1322.

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Wood, AW; Chang, RL; Levin, W; et al. (1979) Mutage nicity and tumorigenicity of phenanthrene and chrys ene epoxides and diol epoxides. Cancer Res 39:4069–407 7.

Wood, AW; Levin, W; Chang, RL; et al. (1980) Mutage nicity and tumor-initiating activity of cyclopenta[ c,d]pyrene and structurally related compounds. Cancer Res 40: 642–649.

Wynder, EL; Hoffmann, D. (1959a) The carcinogenicit y of benzofluoranthenes. Cancer 12:1194–1199.

Wynder, EL; Hoffmann, D. (1959b) A study of tobacco carcinogenesis: VII. The role of higher polycyclic hydrocarbons. Cancer 12:1079–1086.

Wynder, EL; Hoffmann, D. (1961) Carcinogenicity of dibenzo[a,1]pyrene. Nature 192:1092–1093.

Wynder, EL; Fritz, L; Furth, N. (1957) Effect of co ncentration of benzopyrene in skin carcinogenesis. J Natl Cancer
Inst 19:361–370.

Xu, D; Penning, TM; Blair, IA; et al. (2009) Synthe sis of phenol and quinine metabolites of benzo[a]py rene, a carcinogenic component of tobacco smoke implicated in lung cancer. J Org Chem 74:597–604.

Xue, W; Warshawsky, D. (2005) Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbon s and DNA damage: a review. Toxicol Appl Pharmacol 206:7 3–93.

Yu, C; Xu, S; Chen, S; et al. (2002) Investigation of photobleaching of hypocrellin B in non-polar org anic solvent and in liposome suspension. J Photochem Photobiol B 68:73–78.

Zijlstra, JA; Vogel, EW. (1984) Mutagenicity of 7,1 2-dimethylbenz[a]anthracene and some other aromatic mutagens in Drosophila melanogaster. Mutat Res 125:243–261.

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APPENDIX A. SECONDARY SOURCES REVIEWED FOR IDENTIF ICATION OF

PRIMARY LITERATURE

ATSDR (Agency for Toxic Substances and Disease Regi stry). (1995) Toxicological profile for polycyclic aromatic hydrocarbons (PAHs). Atlanta, GA, Agency for Toxic Substances and Disease Registry.

Bostrom, CC; Gerde, P; Hanberg, A; et al. (2002) Ca ncer risk assessment, indicators, and guidelines fo r polycyclic aromatic hydrocarbons in the ambient air. Environ Health Perspect 110[Suppl. 3]:451–488.

CalEPA. (2002) Air toxics hot spots program risk a ssessment guidelines Part II: technical support doc ument for describing available cancer potency factors. Calif ornia Environmental Protection Agency, Office of En vironmental Health Hazard Assessment.

CalEPA. (2004) No Significant Risk Levels (NSRLs) for the Proposition 65 carcinogens benzo[b]fluorant hene, benzo[j]fluoranthene, chrysene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and 5-methyl chrysene by the o ral route. California Environmental Protection Agency, Office of Environmental Health Hazard Assessment, Reproduc tive and Cancer Hazard Assessment Section.

CCME. (2003) Canadian soil quality guidelines for p otentially carcinogenic and higher molcular weight polycyclic aromatic hydrocarbons (environmental and human heal th aspects). Scientific Supporting Document. UMA G roup Ltd. Victoria, British Columbia

Clement Associates. (1988) Comparative potency appr oach for estimating the cancer risk associated with exposure to mixtures of polycyclic aromatic hydrocarbons. R eport No. 68-02-4403.

Clement Associates. (1990) Development of relative potency estimates for PAHs and hydrocarbon combusti on product fractions compared to benzo[a]pyrene and th eir use in carcinogenic risk assessments. Draft Re port, prepared for the U.S.EPA. September 30, 1990.

Collins, JF; Brown, JP; Alexeeff, GV; et al. (1998) Potency equivalency factors for some polycyclic ar omatic hydrocarbons and polycyclic aromatic hydrocarbon de rivatives. Regul Toxicol Pharmacol 28:45–54.

Health Canada. (1994) Canadian Environmental Protec tion Act; Priority substances list assessment repor t: polycyclic aromatic hydrocarbons. Government of Ca nada, Environment Canada, Health Canada.

IARC (International Agency for Research on Cancer). (1983) Polynuclear aromatic compounds. Part 1. Che mical, environmental and experimental data. International Agency for Research on Cancer. IARC Monographs on t he Evaluation of Carcinogenic Risk of Chemicals to Hum ans, Vol 32. Lyon, France: World Health Organizatio n.

IARC. (1984a) Polynuclear aromatic compounds. Part 2. Carbon black, mineral oils (lubricant base oils and derived products) and some nitroarenes. International Agenc y for Research on Cancer. Monographs on the Evaluat ion of the Carcinogenic Risk of Chemicals to Humans, Lyon, Fra nce: World Health Organization; pp. 87–168.

IARC. (1984b) Polynuclear aromatic compounds. Part 3. Industrial exposures in aluminum production, coa l gasification, coke production, and iron and steel f ounding. International Agency for Research on Cance r. IARC Monographs on the Evaluation of the Carcinogenic Ri sk of Chemicals to Humans, Lyon, France: World Heal th Organization; pp. 37–111.

IARC. (1985) Polynuclear aromatic compounds. Part 4 . Bitumens, coal-tars and derived products, shale-o ils and soots. International Agency for Research on Cancer. IARC Monographs on the Evaluation of the Carcinoge nic Risk of Chemicals to Humans, Lyon, France: World Health Organization; pp. 65–159.

IARC. (1989) In: Occupational exposures in petroleu m refining; crude oil and major petroleum fuels, Vo l. 45. International Agency for Research on Cancer. Monogr aphs on the evaluation of the carcinogenic risk of chemicals to humans. Lyon, France: World Health Organization; pp. 239–270.

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IARC. (1996) Printing processes and printing inks, carbon black and some nitro compounds. Internation al Agency for Research on Cancer. In: IARC Monographs on th e Evaluation of Carcinogenic Risks to Humans. Vol. 65. World Health Organization: Lyon, France.

IPCS/WHO. (1998) Environmental health criteria 202. International Programme on Chemical Safety. Sele cted nonheterocyclic polycyclic aromatic hydrocarbons. Ava ilable online at http://www.inchem.org/documents/ehc/ehc202.htm.

Krewski, D; Thorslund, T; Withey, J. (1989) Carcin ogenic risk assessment of complex mixtures. Toxico l Ind Health 5:851–867.

Larsen, JC; Larsen, PB. (1998) Chemical carcinogens . Air Pollution and Health. Cambridge, UK: The Roy al Society of Chemistry; pp. 33–56.

Malcolm, HM; Dobson S. (1994) The calculation of an environmental assessment level (EAL) for atmospher ic PAHs using relative potencies. Report No. DoE/HMIP/ RR/94/041. London, Department of the Environment.

McClure, P; Shoeny, R. (1995) Evaluation of a compo nent-based relative potency approach to cancer risk assessment for exposure to PAH. In: Fifteenth inte rnational symposium on polycyclic aromatic compound s: Chemistry, biology and environmental impact. Belgi rate, Italy, 19-22 September 1995. Ispra, Joint Re search Centre European Commission; pp. 161.

Meek, ME; Chan, PKL; Bartlett, S. (1994) Polycyclic aromatic hydrocarbons: evaluation of risks to heal th from environmental exposures in Canada. Environ Carcino g Ecotoxicol Rev C 12(2):443–452.

Muller, P. (1997) Scientific criteria document for multimedia standards development polycyclic aromati c hydrocarbons (PAH). Part 1: hazard identification and dose-response assessment. Ontario Ministry of Environment and Energy, Standards Development Branch. Ontario.

Nisbet, ICT; LaGoy, PK. (1992) Toxic equivalency fa ctors (TEFs) for polycyclic aromatic hydrocarbons ( PAHs). Regul Toxicol Pharmacol 16:290–300.

Petry, T; Schmid, P; Schlatter, C. (1996) The use o f toxic equivalency factors in assessing occupation al and environmental health risk associated with exposure to airborne mixtures of polycyclic aromatic hydroca rbons (PAHs). Chemosphere 32(4):639–648.

Rugen, PJ; Stern, CD; Lamm, SH. (1989) Comparative carcinogenicity of the PAHs as a basis for acceptab le exposure levels (AELs) in drinking water. Regul To xicol Pharmacol 9(3):273–283.

Schneider, K; Roller, M; Kalberlah, F; et al. (2002 ) Cancer risk assessment for oral exposure to PAH m ixtures. J Appl Toxicol 22(1):73–83.

Sjogren, M; Ehrenberg, L; Rannug, U. (1996) Relevan ce of different biological assays in assessing init iating and promoting properties of polycyclic aromatic hydroca rbons with respect to carcinogenic potency. Mutat Res 358(1):97–112.

Slooff, W; Janus, JA; Matthijsen, AJCM; et al. (198 9) Integrated criteria document PAHs (PDF includes addendum by Montizaan). Bilthoven, The Netherlands, Nationa l Institute of Public Health and the Environment (R IVM).

SRC (Syracuse Research Corporation). (1993) Estima ting cancer risk from exposure to PAHs: a relative potency approach. SRC TR-93-045. Draft report prepared fo r U.S. EPA, Environmental Criteria and Assessment O ffice, Cincinnati, OH.

U.S. EPA (Environmental Protection Agency). (1990) Drinking water criteria document for polycyclic aro matic hydrocarbons. Cincinnati, Ohio, Office of Health a nd Environmental Assessment, Environmental Criteria and Assessment Office.

U.S. EPA. (1993) Provisional guidance for quantitat ive risk assessment of polycyclic aromatic hydrocar bons. Cincinnati, Ohio, Office of Health and Environmenta l Assessment, Environmental Criteria and Assessment Office.

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APPENDIX B. BIBLIOGRAPHY OF STUDIES WITHOUT BENZO[ A]PYRENE AS A 1



### REFERENCE COMPOUND 2

3
4
5 6B-2 DRAFT – DO NOT CITE OR QUOTETable B-1. Bioassays with and without benzo[a]pyrene by PAHBioassays with benzo[a]pyreneBioassays without benzo[a]pyreneDermalDermalPAH CASRNInitiation CompleteIntraperitonealSub-cutaneousOral OtherInitiation CompleteIntra-peritonealSub-cutaneousOral OtherAceanthrylene 202-03-09Acenaphthene 83-32-9Acenaphthylene 208-96-8Acephenanthrylene 201-06-9Acepyrene, 2,3- 25732-74-5 x xAnthanthrene 191-26-4 x x x x xAnthracene 120-12-7 x x x x x x x x xBenz[a]anthracene 56-55-3 x x x x x x x x x x x xBenz[b]anthracene 92-24-9Benz[b,c]aceanthrylene, 11H- 202-94-8 xBenz[e]aceanthrylene 199-54-2Benz[j]aceanthrylene 202-33-5 x xBenz[l]aceanthrylene 211-91-6 xBenzacenaphthylene 76774-50-0Benzo[a]fluoranthene203-33-8 xBenzo[a]fluorene238-84-6 or30777-18-5xBenzo[a]perylene 191-85-5Benzo[b]chrysene214-17-5 xBenzo[b]fluoranthene 205-99-2 x x x x x xx11H-Benzo[b]fluorene243-17-4 or30777-19-6 xBenzo[b]perylene 197-70-6Benzo[c]chrysene 194-69-4Benzo[c]fluorene205-12-9 or30777-20-9 xBenzo[c]phenanthrene195-19-7 x x x xBenzo[e]pyrene 192-97-2 x x x x xBenzo[g]chrysene 196-78-1Benzo[g,h,i]fluoranthene 203-12-3 x xBenzo[g,h,i]perylene 191-24-2 x x x xBenzo[j]fluoranthene 205-82-3 x x x x x xxBenzo[k]fluoranthene 207-08-9 x x x x xBenzophenanthrene 65777-08-4Chrysene 218-01-9 x x x x x x x x xCoronene 191-07-1 x xCyclopenta[c,d]pyrene 27208-37-3 x x xxCyclopenta[d,e,f]chrysene, 4H- 202-98-2 xxCyclopenta[d,e,f]phenanthrene, 4H- 203-64-5Cyclopenta[h,i]acephenanthrylene 114959-37-4B-3 DRAFT – DO NOT CITE OR QUOTETable B-1. Bioassays with and without benzo[a]pyrene by PAHBioassays with benzo[a]pyreneBioassays without benzo[a]pyreneDermalDermalPAH CASRNInitiation CompleteIntraperitonealSub-cutaneousOral OtherInitiation CompleteIntra-peritonealSub-cutaneousOral OtherCyclopenta[h,i]aceanthrylene 131581-33-4Cyclopentaphenanthrene 219-08-9Cyclopenteno-1,2-benzanthracene, 5,6-7099-43-6 xDibenz[a,c]anthracene 215-58-7 x x x x xxDibenzo[a,e]fluoranthene 5385-75-1 x x xDibenz[a,j]anthracene224-41-9 xDibenzo[b,e]fluoranthene 2997-45-7Dibenzo[a,c]fluorene, 13H- 201-65-0Dibenzo[a,e]pyrene 192-65-4 x x xDibenzo[a,f]fluoranthene 203-11-2 x x x xDibenzo[a,g]fluorene, 13H-207-83-0 xDibenz[a,h]anthracene 53-70-3 x x x x x x x x x xx xDibenzo[a,h]pyrene 189-64-0 x x x xDibenzo[a,i]pyrene 189-55-9 x x x x x xxDibenzo[a,l]pyrene 191-30-0 x x x x x x x xDibenzo[e,l]pyrene 192-51-8 x xDibenzo[h,rst]pentaphene 192-47-2Dibenz[k,mno]acephenanthrylene 153043-81-3Dibenzo[j,mno]acephenanthrylene 153043-82-4Dihydroaceanthrylene, 1,2-641-48-5 xFluoranthene 206-44-0 x x x x xFluorene86-73-7 x xIndeno[1,2,3-c,d]fluoranthene 193-43-1Indeno[1,2,3-c,d]pyrene 193-39-5 x x x x xNaphtho[1,2-b]fluoranthene111189-32-3 xNaphtho[1,2,3,-mno]acephenanthrylene 113779-16-1Naphtho[2,1-a]fluoranthene203-20-3 xNaphtho[2,3-a]pyrene 196-42-9Naphtho[2,3-e]pyrene 193-09-9 x xPentacene 135-48-8Pentaphene 222-93-5Perylene 198-55-0 x x xPhenanthrene 85-01-8 x x x x x x x x x x xPicene213-46-7 x x x xPyrene 129-00-0 x x x x x xTribenzofluoranthene 3,4-10,11-12,13- 13579-05-0Triphenylene 217-59-4 xPAHs in bold have at least one bioassay without BaPand no bioassays with BaP.1

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B.1. BIBLIOGRAPHY OF BIOASSAYS WITHOUT BENZO[A]PYR ENE

Amin, S; Huie, K; Hecht, SS; (1985) Mutagenicity an d tumor-initiating activity of methylated benzo[b]fluoranthenes. Carcinogenesis 6:1023–1025.

Amin, S; Hussain, N; Balanikas, G; et al. (1985) Mu tagenicity and tumor initiating activity of methyla ted benzo[k] fluoranthenes. Cancer Lett 26:343–347.

Amin, S; Misra, B; Braley, J; et al. (1991) Compara tive tumorigenicity in newborn mice of chrysene and 5-alkylchrysene-1,2-diol-3,4-epoxides. Cancer Lett 58:115–118.

Amin, S; Weyand, EH; Huie, K; et al. (1991) Effects of fluorine substitution on benzo[b]fluoranthene t umorgenicity and DNA adduct formation in mouse skin. In: Cooke, M; Loening, K; Merritt, J, eds. Polynuclear aroma tic hydrocarbons: Measurements, means and metabolism. Columbus, OH, Battelle Press pp. 25–35.

Amin, S; Desai, D; Dai, W; et al. (1995) Tumorigeni city in newborn mice of fjord region and other ster ically hindered diol epoxides of benzo[g]chrysene, dibenzo [a,l]pyrene (dibenzo[def,p]chrysene), 4H-cyclopenta[def]chrysene and fluoranthene. Carci nogenesis 16:2813–2817.

Barry, G; Cook, CW; Amin, S; et al. (1934) A compar ison of the action of some polycyclic aromatic hydr ocarbons in producing tumours of connective tissue. Am J Ca ncer 20:58–69.

Bhatt, TS; Coombs, MM. (1990) The carcinogenicity o f cyclopenta[a]phenanthrene and chrysene derivative s in the Sencar mouse. Polycyclic Aromat Compd 1:51–58.

Bock, FG; King, DW. (1959) A study of the sensitivi ty of the mouse forestomach toward certain polycycl ic hydrocarbons. J Natl Cancer Inst 23:833–838.

Bottomley, AC; Twort, CC. (1934) The carcinogenicit y of chrysene and oleic acid. Am J Cancer 21:781– 786.

Boyland, E; Burrows, H. (1935) The experimental pro duction of sarcoma in rats and mice by a colloidal aqueous solution of 1:2:5:6-dibenzanthracene. J Pathol Bac teriol 41:231–238.

Boyland, E; Sims, P. (1967) The carcinogenic activi ties in mice of compounds related to benz[a]anthrac ene. Int J Cancer 2:500–504.

Buening, MK; Levin, W; Karle, JM; et al. (1979) Tum origenicity of bay-region epoxides and other deriva tives of chrysene and phenanthrene in newborn mice. Cancer Res 39:5063–5068.

Buening, MK; Levin, W; Wood, A; et al. (1979) Tumor igenicity of the dihydrodiols of dibenz[a,h]anthrac ene on mouse skin and in newborn mice. Cancer Res 39:1310 –1314.

Buters, JT; Mahadevan, B; Quintanilla-Martinez, L; et al. (2002) Cytochrome P450 1B1 determines suscep tibility to dibenzo[a,l]pyrene-induced tumor formation. Chem R es Toxicol 15:1127–1135.

Cavalieri, EL; Rogan, EG; Higginbotham, S; et al. ( 1989) Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a]p yrenes: the very potent environmental carcinogen di benzo[a, l]pyrene. J Cancer Res Clin Oncol 115:67–72.

Chang, RL; Levin, W; Wood, AW; et al. (1981) Tumori genicity of the diastereomeric bay-region benzo[e]p yrene 9,10-diol-11,12-epoxides in newborn mice. Cancer R es 41:915–918.

Chang, RL; Levin, W; Wood, AW; et al. (1982) Tumori genicity of bay-region diol-epoxides and other benz o-ring derivatives of dibenzo[a,h]pyrene and dibenzo[a,i]p yrene on mouse skin and in newborn mice. Cancer Re s 42:25–
29.

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Chang, RL; Levin, W; Wood, AW; et al. (1983) Tumori genicity of enantiomers of chrysene 1,2-dihydrodiol and of the diastereomeric bay-region chrysene 1,2-diol-3,4 -epoxides on mouse skin and in newborn mice. Cance r Res 43:192–196.

Chouroulinkov, I; Coulomb, H; MacNicoll, AD; et al. (1983) Tumour-initiating activities of dihydrodiol s of dibenz[a,c]anthracene. Cancer Lett 19:21–26.

Danz, M; Hartmann, A; Otto, M; et al. (1991) Hither to unknown additive growth effects of fluorene and 2-acetylaminofluorene on bile duct epithelium and h epatocytes in rats. Arch Toxicol Suppl 14:71–74.

Flesher, JW; Horn, J; Lehner, AF. (2002) Comparativ e carcinogenicity of picene and dibenz[a,h]anthrace ne in the rat. Biochem Biophys Res Commun 290:275–279.

Forbes, PD; Davies, RE; Urbach, F. (1976) Phototoxi city and photocarcinogenesis: Comparative effects o f anthracene and 8-methoxypsoralen in the skin of mic e. Food Cosmet Toxicol 14:303–306.

Geddie, JE; Amin, S; Huie, K; et al. (1987) Formati on and tumorigenicity of benzo[b]fluoranthene metab olites in mouse epidermis. Carcinogenesis 8:1579–1584.

Gill, HS; Kole, PL; Wiley, JC; et al. (1994) Synthe sis and tumor-initiating activity in mouse skin of dibenzo[a,l]pyrene syn- and anti-fjord-region diole poxides. Carcinogenesis 15:2455–2460.

Hecht, SS; LaVoie, E; Amin, S; et al. (1980) On the metabolic activation of the benzofluoranthenes. P olynucl Arom Hydrocarb 417–433.

Hecht, SS; LaVoie, EJ; Bedenko, V; et al. (1981a) O n the metabolic activation of dibenzo[a,i]pyrene an d dibenzo [a,h]pyrene. Chem Analy & Biol Fate: Polynucl Arom Hydrocarb 43–45.

Hecht, SS; LaVoie, EJ; Bedenko, V; et al. (1981b) R eduction of tumorigenicity and of dihydrodiol forma tion by fluorine substitution in the angular rings of diben zo[a,i]pyrene. Cancer Res 41:4341–4345.

Hecht, SS; Amin, S; Lin, JM; et al. (1995) Mammary carcinogenicity in female CD rats of a diol epoxide metabolite of fluoranthene, a commonly occurring environmental pollutant. Carcinogenesis 16:1433–1435.

Hecht, SS; Rivenson, A; Amin, S; et al. (1996) Mamm ary carcinogenicity of diol epoxide metabolites of benzo[j]fluoranthene in female CD rats. Cancer Let t 106:251–255.

Hermann, M. (1981) Synergistic effects of individua l polycyclic aromatic hydrocarbons on the mutagenic ity of their mixtures. Mutat Res 90:399–409.

Hill, WT; Stanger, DW; Pizzo, A; et al. (1951) Inhi bition of 9,10-dimethyl-1,2-benzanthracene skin car cinogenesis in mice by polycyclic hydrocarbons. Cancer Res 11: 892–897.

Homburger, F; Treger, A. (1970) Transplantation tec hnique for acceleration of carcinogenesis by benz[a ]anthracene or 3,4,9,10-dibenzpyrene. J Natl Cancer Inst 44:35 7–360.

Homburger, F; Treger, A; Boger, E. (1971) Inhibitio n of murine subcutaneous and intravenous benzo[rst]pentaphene. Carcinogenesis by sweet orang e oils and d-limonene. Oncology 25:1–10.

Jerina, DM; Sayer, JM; Yagi, H; et al. (1981) Highl y tumorigenic bay-region diol epoxides from the wea k carcinogen benzo[c]phenanthrene. Adv Exp Med Biol 136 Pt A:501–523.

Johnson, S. (1968) Effect of thymectomy on the indu ction of skin tumours by dibenzanthracene, and of b reast tumours by dimethylbenzanthracene in mice of the IF strain. Br J Cancer 22:755–761.

Klein, M. (1952) Effect of croton oil on induction of tumors by 1,2-benzanthracene, deoxychloric or lo w doses of 20-methylcholanthrene in mice. J Nat1 Cancer Inst 13:333–341.

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Klein, M. (1960) A comparison of the initiating and promoting actions of 9,10-dimethyl-1,2-benzanthrac ene and 1,2,5,6-dibenzanthracene in skin tumorigenesis. Ca ncer Res 20:1179–1183.

Klein, M. (1963) Susceptibility of strain B6AF1/J h ybrid infant mice to tumorigenesis with 1,2-benzant hracene, deoxycholic acid, and 3-methylcholanthrene. II. Tum ours called forth by painting the skin with dibenzp yrene. Cancer Res 23:1701–1707.

Kouri, RE; Connolly, GM; Nebert, DW; et al. (1983) Association between susceptibility to dibenzanthrac ene induced fibrosarcoma formation and the Ah locus. I nt J Cancer 32:765–768.

Lacassagne, A; Buu-Hoi, NP; Zajdela, F; et al. (196 8) The true dibenzo[a,l]pyrene, a new, potent carci nogen. Naturwissenschaften 55:43.

LaVoie, EJ; Tulley L; Bedenko, V; et al. (1980) Mut agenicity, tumor initiating activity, and metabolis m of tricyclic polynuclear aromatic hydrocarbons. In: Bjorseth, A ; Dennis, AJ, eds. Polynuclear aromatic hydrocarbo ns: Chemistry and biological effects. Columbus, OH: Bat telle Press; pp. 1041–1057.

LaVoie, EJ; Tulley-Freiler, L; Bedenko, V; et al. ( 1981) Comparative studies on the tumor initiating a ctivity and metabolism of methylfluorenes and methylbenzofluore nes. In: Cooke, M; Dennis, AJ, eds. Chemical analy sis and biological fate: Polynuclear hydrocarbons. Columbus , OH: Battelle Press; pp. 417–427.

LaVoie, EJ; Coleman, DT; Tonne, RL; et al. (1983) M utagenicity, tumor initiating activity and metaboli sm of methylated anthracenes. In: Cooke, M; Dennis, AJ, eds. Proceedings of the Seventh International Symp osium. Columbus, OH: Battelle Press pp. 785–798.

LaVoie, EJ; Cai, ZW; Meegalla, RL; et al. (1993a) E valuation of the tumor-initiating activity of 4-, 5 -, 6-, and 7-fluorobenzo[b]fluoranthene in mouse skin. Chem B io Interact 89:129–139.

LaVoie, EJ; He, ZM; Meegalla, RL; et al. (1993b) Ex ceptional tumor-initiating activity of 4-fluorobenz o[j]- fluoranthene on mouse skin: comparison with benzo[j ]-fluoranthene, 10-fluoro-benzo[j]fluoranthene, benzo[a]pyrene, dibenzo[a,l]pyrene and 7,12-dimethy lbenz[a]anthracene. Cancer Lett 70:7–14.

LaVoie, EJ; He, ZM; Wu, Y; et al. (1994) Tumorigeni c activity of the 4,5- and 9,10-dihydrodiols of benzo[j]fluoranthene and their syn- and anti-diol e poxides in newborn mice. Cancer Res 54:962–968.

Levin, W; Wood, AW; Chang, RL; et al. (1978) Eviden ce for bay region activation of chrysene 1,2-dihydr odiol to an ultimate carcinogen. Cancer Res 38:1831–1834.

Levin, W; Wood, AW; Chang, RL; et al. (1980) Except ionally high tumor-initiating activity of benzo[c]phenanthrene bay-region diol-epoxides on mo use skin. Cancer Res 40:3910–3914.

Levin, W; Chang, RL; Wood, AW; et al. (1984) High s tereoselectivity among the optical isomers of the diastereomeric bay-region diolepoxides of benz[a]an thracene in the expression of tumorigenic activity in murine tumor models. Cancer Res 44:929–933.

Levin, W; Chang, RL; Wood, AW; et al. (1986) Tumori genicity of optical isomers of the diastereomeric b ay-region 3,4-diol-1,2-epoxides of benzo[c]phenanthrene in mu rine tumor models. Cancer Res 46:2257–2261.

Lijinsky, W; Garcia, H. (1972) Skin carcinogenesis tests of hydrogenated derivatives of anthanthrene a nd other polynuclear hydrocarbons. Z Krebsforsch 77:226–230

.

Lijinsky, WH; Garcia, B; Terrracini, B. (1965) Tumo rigenic activity of hydrogenated derivatives of dibenz[a,h]anthracene. J Natl Cancer Inst 34:1–6.

Lijinsky, W; Garcia, H; Saffiotti, U. (1970) Struct ure-activity relationships among some polynuclear h ydrocarbons and their hydrogenated derivatives. J Natl Cancer Inst 44:641–649.

Lorenz, E; Stewart, HL. (1947) Tumors of the alimen tary tract induced in mice by feeding olive oil emu lsions containing carcinogenic hydrocarbons. J Natl Cance r Inst 7:227–238.

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Lorenz, E; Stewart, HL. (1948) Tumors of alimentary tract in mice fed carcinogenic hydrocarbons in min eral-oil emulsions. J Natl Cancer Inst 9:173–180.

Lubet, RA; Connolly, GM; Nebert, DW; et al. (1983) Dibenz[a,h]anthracene-induced subcutaneous tumors i n mice. Strain sensitivity and the role of carcinogen metab olism. Carcinogenesis 4:513–517.

Malament, DS; Shklar, G. (1981) Inhibition of DMBA carcinogenesis of hamster buccal pouch by phenanthr ene and dimethylnaphthalene. Carcinogenesis 2:723–729.

Mass, MJ; Abu-Shakra, A; Roop, BC; et al. (1996) Be nzo[b]fluoranthene: tumorigenicity in strain A/J mo use lungs, DNA adducts and mutations in the Ki-ras oncogene. C arcinogenesis 17:1701–1704.

Nakatsuru, Y; Wakabayashi, K; Fujii-Kuriyama, Y; et al. (2004) Dibenzo[a,l]pyrene-induced genotoxic an d carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor-deficient mice. Int J Cancer
112:179–183.

Nesnow, S; Gold, A; Sangaiah, R; et al. (1993) Mous e skin tumor-initiating activity of benz[j]aceanthr ylene in SENCAR mice. Cancer Lett 73:73–76.

Nesnow, S; Ross, JA; Nelson, G; et al. (1994) Cyclo penta[cd]pyrene-induced tumorigenicity, Ki-ras codo n 12 mutations and DNA adducts in strain A/J mouse lung. Carcinogenesis 15:601–606.

O'Gara, RW; Kelly, MG; Brown, J; et al. (1965) Indu ction of tumors in mice given a minute single dose of dibenz[a,h]anthracene or 3-methylcholanthrene as ne wborns: A dose-response study. J Natl Cancer Inst 35(6):1027–1042.

Platt, KL; Pfeiffer, EH; Glatt, HR; et al. (1983) B acterial mutagenicity and carcinogenicity of potent ial metabolites of dibenz[a,h]anthracene. J Cancer Res Clin Oncol 105:A23.

Platt, KL; Pfeiffer, E; Petrovic, P; et al. (1990) Comparative tumorigenicity of picene and dibenz[a,h ]anthracene in the mouse. Carcinogenesis 11:1721–1726.

Platt, KL; Dienes, HP; Tommasone, M; et al. (2004) Tumor formation in the neonatal mouse bioassay indi cates that the potent carcinogen dibenzo[def,p]chrysene (diben zo[a,l]pyrene) is activated in vivo via its trans-11,12-dihydrodiol. Chem Biol Interact 148:27 –36.

Pollia, JA. (1939) Investigations on the possible c arcinogenic effect of anthracene and chrysene and s ome of their compounds. I. The effect of skin painting on the sk in of mice. J Ind Hyg Toxicol 23:449–451.

Pollia, JA. (1941) Investigation on the possible ca rcinogenic effect of anthracene and chrysene and so me of their compounds. II. The effect of subcutaneous injection in rats. J Ind Hyg Toxicol 223:449–451.

Prahalad, AK; Ross, JA; Nelson, GB; et al. (1997) D ibenzo[a,l]pyrene-induced DNA adduction, tumorigeni city, and Ki-ras oncogene mutations in strain A/J mouse lung. Carcinogenesis 18:1955–1963.

Ranadive, KJ; Karande, KA. (1963) Studies on 1,2,5, 6-dibenzanthracene-induced mammary carcinogenesis i n mice.
Br J Cancer 17:272–280.

Rice, JE; Coleman, DT; Hosted, TJJ; et al. (1985) O n the metabolism, mutagenicity, and tumor-initiatin g activity of indeno[1,2,3-cd]pyrene. pp. 1097–1109.

Rice, JE; Hosted, TJ Jr; DeFloria, MC; et al. (1986 ) Tumor-initiating activity of major in vivo metabo lites of indeno[1,2,3-cd]pyrene on mouse skin. Carcinogenes is 7:1761–1764.

Rice, JE; Weyand, EH; Geddie, NG; et al. (1987) Ide ntification of tumorigenic metabolites of benzo[j]f luoranthene formed in vivo in mouse skin. Cancer Res 47:6166–6 170.

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Rice, JE; Weyand, EH; Burrill, C; et al. (1990) Flu orine probes for investigating the mechanism of act ivation of indeno[1,2,3-cd]pyrene to a tumorigenic agent. Car cinogenesis 11:1971–1974.

Riegel, B; Watman, WB; Hill, WT. (1951) Delay of me thylcholanthrene skin carcinogenesis in mice by 1,2,5,6-dibenzofluorene. Cancer Res 11:301–306.

Salaman, MH; Roe, FJC. (1956) Further tests for tum our-initiating activity: N,N-di(2-chloroethyl)- paminophenylbutic acid (CB1348) as an initiator of skin tumour formation in the mouse. Br J Cancer 10 :363–378.

Sardella, DJ; Boger, E; Ghoshal, PK. (1981) Active sites in hexacyclic carcinogens probed by the fluor ine substitution methodology. Columbus, OH: Battelle P ress; pp. 529–538.

Schmähl, D. (1955) [Testing of naphthalene and anth racene for carcinogenic effects in rats.] (in Germa n). Z Krebsforsch 60:697–710.

Schoental, R. (1959) Carcinogenic activity of 3:4:9 :10-dibenzopyrene. Acta Unio Int Contra Cancrum 15 :216–219.

Schoket, B; Hewer, A; Grover, PL; et al. (1988) Cov alent binding of components of coal-tar, creosote a nd bitumen to the DNA of the skin and lungs of mice following topical application. Carcinogenesis 9:1253–1258.

Scribner, JD. (1973) Brief communication: Tumor ini tiation by apparently noncarcinogenic polycyclic ar omatic hydrocarbons. J Natl Cancer Inst 50:1717–1719.

Sellakumar, A; Shubik, P. (1974) Carcinogenicity of different polycyclic hydrocarbons in the respirato ry tract of hamsters. J Natl Cancer Inst 53:1713–1719.

Shear, MJ. (1938) Studies in carcinogenesis. V. Met hyl derivatives of 1,2-benzanthracene. Am J Cancer 33:499–
537.

Shear, MJ; Leiter, J. (1941) Studies in carcinogene sis. XVI. Production of subcutaneous tumors in mice by miscellaneous polycyclic compounds. J Natl Cancer Inst 2:241–259.

Siebert, D; Marquardt, H; Friesel, H; et al. (1981) Polycyclic aromatic hydrocarbons and possible meta bolites: Convertogenic activity in yeast and tumor initiatin g activity in mouse skin. J Cancer Res Clin Oncol 102:127–139.

Slaga, TJ; Gleason, GL; Mills, C; et al. (1980) Com parison of the tumour-initiating activities of dihy drodiols and diol-epoxides of various polycyclic aromatic hydroc arbons. Cancer Res 40:1981–1984.

Snell, KC; Stewart, HL. (1962) Pulmonary adenomatos is induced in DBA/2 mice by oral administration of dibenz(a,h]anthracene. J Natl Cancer Inst 28:1043– 1049.

Snell, KC; Stewart, HL. (1963) Induction of pulmona ry adenomatoses in DBA/2 mice by the oral administr ation of dibenz[a,h]anthracene. Acta Un Int Cancer 19:692–6 94.

Stanton, MF; Miller, E; Wrench, C; et al. (1972) Ex perimental induction of epidermoid carcinoma in the lungs of rats by cigarette smoke condensate. J Nat1 Cancer Inst 49:867–877.

Steiner, PE; Edgcomb, JH. (1952) Carcinogenicity of 1,2-benzanthracene. Cancer Res 12:657–659.

Steiner, PF; Falk, HL. (1951) Summation and inhibit ion effects of weak and strong carcinogenic hydroca rbons: 1:2-Benzanthracene, chrysene, 1:2:5:6-dibenzanthrac ene, and 20-methylcholanthrene. Cancer Res 11:56–6 3.

Stenbk, F; Sellakumar, A. (1974) Lung tumor inducti on by dibenz(a,i]pyrene in the Syrian golden hamste r. Z Krebsforsch 82:175–182.

Stevenson, JL; Von Haam, E. (1965) Carcinogenicity of benz[a]anthracene and benzo[c]phenanthrene deriv atives. Am Ind Hyg Assoc J 26:475–478.

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Tawfic, HN. (1965) Studies on ear duct tumors in ra ts. Part II: Inhibitory effect of methylcholanthren e and 1,2-benzanthracene on tumor formation by 4-dimethyl amino-stilbene. Acta Pathol Jpn 15:255–260.

Van Duuren, BL; Langseth, L; Goldschmidt, BM. (1967 ) Carcinogenicity of epoxides, lactones and peroxy compounds: VI. Structure and carcinogenic activity. J Nat1 Cancer Inst 39:1217–1227.

Van Duuren, BL; Sivak, A; Langseth, L; et al. (1968 ) Initiators and promoters in tobacco carcinogenesi s. Natl Cancer Inst Monogr 28:173–180.

Van Duuren, BL; Sivak, A; Goldschmidt, BM; et al. ( 1970) Initiating activity of aromatic hydrocarbons in two-stage carcinogenesis. J Natl Cancer Inst 44:1167–1173.

Vulimiri, SV; Baer-Dubowska, W; Harvey, RG; et al. (1999) Analysis of highly polar DNA adducts formed in SENCAR mouse epidermis following topical applicatio n of dibenz[a,j]anthracene. Chem Res Toxicol 12:60 –67.

Wang, JS; Busby, WF. Jr. (1993) Induction of lung a nd liver tumors by fluoranthene in a preweanling CD -1 mouse bioassay. Carcinogenesis 14:1871–1874.

Waravdekar, SS; Ranadive, KJ. (1958) Biologic testi ng of 3,4,9,10-dibenzpyrene. J Natl Cancer Inst 21 :1151–1159.

Weyand, EH; Amin, S; Huie, K; et al. (1989) Effects of fluorine substitution on the DNA binding and tumorigenicity of benzo[b]fluoranthene in mouse epi dermis. Chem Biol Interact 71:279–290.

Weyand, EH; Patel, S; LaVoie, EJ; et al. (1990) Rel ative tumor initiating activity of benzo[a]fluorant hene, benzo[b]fluoranthene, naphtho[1,2-b]fluoranthene an d naphtho[2,1-a]fluoranthene on mouse skin. Cancer Lett
52:229–233.

Weyand, EH; Cai, ZW; Wu, Y; et al. (1993) Detection of the major DNA adducts of benzo[b]fluoranthene i n mouse skin: role of phenolic dihydrodiols. Chem Res Toxi col 6:568–577.

White, FR; Eschenbrenner, AB. (1945) Note on the oc currence of hepatomas in rats following the ingesti on of 1,2-benzoanthracene. J Natl Cancer Inst 6:19–21.

Wislocki, PG; Buening, MK; Levin, W; et al. (1979) Tumorigenicity of the diastereomeric benz[a]anthrac ene 3,4-diol-1,2-epoxides and the (+)-and (-)-enantiome rs of benz [a]anthracene 3,4-dihydrodiol in newborn mice. J Natl Cancer Inst 63:201–204.

Wodinsky, I; Helinski, A; Kensler, CJ. (1964) Susce ptibility of Syrian hamsters to induction of fibros arcomas with a single injection of 3,4,9,10-dibenzpyrene. Nature 203:308–309.

Wood, AW; Chang, RL; Levin, W; et al. (1979) Mutage nicity and tumorigenicity of phenanthrene and chrys ene epoxides and diol epoxides. Cancer Res 39:4069–407 7.

Wynder, EL.; Hoffmann, D. (1961) Carcinogenicity of dibenzo (a,1]pyrene. Nature 192:1092–1093.

Zajdela, F; Perin-Roussel, O; Saguem, S. (1987) Mar ked differences between mutagenicity in Salmonella and tumour-initiating activities of dibenzo[a,e]fluoran thene proximate metabolites; initiation inhibiting activity of norharman. Carcinogenesis 8:461–464.

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B.2. BIBLIOGRAPHY OF STUDIES ON CANCER-RELATED END POINTS
WITHOUT BENZO[A]PYRENE

Abe, S; Sasaki, M. (1977) Studies on chromosomal ab errations and sister chromatid exchanges induced by chemicals. Proc Jpn Acad 53:46–49.

Agarwal, SK; Sayer, JM; Yeh, HJC; et al. (1987) Che mical characterization of DNA adducts derived from the configurationally isomeric benzo[c]phenanthrene-3,4 -diol 1,2-epoxides. J Am Chem Soc 109:2497–2504.

Agarwal, R; Canella, KA; Yagi, H; et al. (1996) Ben zo[c]phenanthrene-DNA adducts in mouse epidermis in relation to the tumorigenicities of four configurationally i someric 3,4-dihydrodiol 1,2-epoxides. Chem Res Tox icol 9:586–
592.

Agarwal, R; Coffing, SL; Baird, WM; et al. (1997) M etabolic activation of benzo[g]chrysene in the huma n mammary carcinoma cell line MCF-7. Cancer Res 57:4 15–419.

Amin, S; Desai, D; Hecht, SS. (1993) Tumor-initiati ng activity on mouse skin of bay region diol-epoxid es of 5,6-dimethylchrysene and benzo[c]phenanthrene. Car cinogenesis 14:2033–2037.

Arif, JM; Gupta, RC. (1997) Microsome-mediated bioa ctivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling. Carcinogenesis 18:199 9–2007.

Arif, JM; Smith, WA; Gupta, RC. (1999) DNA adduct f ormation and persistence in rat tissues following e xposure to the mammary carcinogen dibenzo[a,l]pyrene. Carcino genesis 20:1147–1150.

Ayrton, AD; McFarlane, M; Walker, R; et al. (1990) Induction of the P-450 I family of proteins by poly cyclic aromatic hydrocarbons: possible relationship to the ir carcinogenicity. Toxicology 60:173–186.

Babson, JR; Russo-Rodriguez, SE; Rastetter, WH; et al. (1986) In vitro DNA-binding of microsomally-act ivated fluoranthene: evidence that the major product is a fluoranthene N2-deoxyguanosine adduct. Carcinogene sis 7, 859–
65.

Babson, JR; Russo-Rodriguez, SE; Wattley, RV; et al . (1986) Microsomal activation of fluoranthene to m utagenic metabolites. Toxicol Appl Pharmacol 85:355–366.

Baer-Dubowska, W; Nair, RV; Cortez, C; et al. (1995 ) Covalent DNA adducts formed in mouse epidermis fr om dibenz[a,j]anthracene: evidence for the formation o f polar adducts. Chem Res Toxicol 8:292–301.

Ball, LM; Warren, SH; Sangaiah, R; et al. (1989) Ba cterial mutagenicity of new cyclopenta-fused cata-a nnelated polycyclic aromatic hydrocarbons, and identificatio n of the major metabolites of benz[j]acephenanthryl ene formed by Aroclor-treated rat liver microsomes. Mutat Res 224:115–25.

Barfknecht, TR: Andon, BM: Thilly, WG; et al. (1981 ) Soot and mutation in bacteria and human cells. I n: Cooke, M; Dennis, AJ, eds. Chemical Analysis and Biologica l Fate: Polynuclear Aromatic Hydrocarbons. pp. 231– 242.

Barrai, I; Barale, R; Scapoli, C; et al. (1992) The analysis of the joint effect of substances on reve rsion systems and the assessment of antimutagenicity. Mutat Res 267: 173–182.

Barratt, RW; Tatum, EL (1958) Carcinogenic mutagens . Ann NY Acad Sci 71:1072–1084.

Bartczak, AW; Sangaiah, S; Ball, LM; et al. (1987) Synthesis and bacterial mutagenicity of the cyclope nta oxides of the four cyclopenta-fused isomers of benzanthracene . Mutagenesis 2:101–105.

Basler, A; Herbold, B; Peter, S; et al. (1977) Muta genicity of polycyclic hydrocarbons. II. Monitoring genetical hazards of chrysene in vitro and in vivo. Mutat Re s 48:249–254.

Baum, M; Amin, S; Guengerich, FP; et al. (2001) Met abolic activation of benzo[c]phenanthrene by cytoch rome P450 enzymes in human liver and lung. Chem Res Tox icol 14:686–693.

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Beach, AC; Gupta, RC. (1991) Analysis of cyclopenta [CP)-fused and 'pseudo-CP' polycyclic aromatic hydr ocarbon (PAH)-DNA adducts by 32P-postlabeling. Proc Am Ass oc Cancer Res 32:98.

Beach, AC; Gupta, RC. (1994) DNA adducts of the ubi quitous environmental contaminant cyclopenta[cd]pyr ene. Carcinogenesis 15:1065–1072.

Beach, AC; Agarwal, SC; Lamberg, GR; et al. (1993) Reaction of cyclopenta (c,d]pyrene-3,4-epoxide with DNA and desoxynucleotides. Carcinogenesis 14:767–771.

Bos, RP; Prinsen, WJ; van Rooy, JG; et al. (1987) F luoranthene, a volatile mutagenic compound, present in creosote and coal tar. Mutat Res 187:119–125.

Boutwell, RK. (1989) Model systems for defining ini tiation, promotion, and progression of skin neoplas ms. Prog Clin Biol Res 298:3–15.

Bryant, MF; Kwanyuen, P; Atwater, AL; et al. (1991) Cytogenetic effects of benzo-b-fluoranthene in Spr agueDawley rat peripheral blood lymphocytes after in vi vo exposure (Abstract 33). Environ Mol Mutag 17 (s uppl 19):13.

Bu-Abbas, A; Ioannides, C; Walker, R. (1994) Evalua tion of the antimutagenic potential of anthracene: in vitro and ex vivo studies. Mutat Res 309:101–107.

Budunova, IV; Mittleman, LA; Safaev, RD; et al. (19 93) The carcinogen benzo[e]pyrene is metabolized by DM15 cells without an uncoupling effect on their gap jun ctions. Cell Biol Toxicol 9:131–140.

Carmichael, PL; Platt, KL; She, MN; et al. (1993) E vidence for the involvement of a bis-diol-epoxide i n the metabolic activation of dibenz[a,h]anthracene to DN A-binding species in mouse skin. Cancer Res 53:944 –948.

Cary, PD; Turner, CH; Cooper, CS; et al. (1980) Met abolic activation of benz[a]anthracene in hamster e mbryo cells: the structure of a guanosine-anti-BA-8,9-diol 10,11 -oxide adduct. Carcinogenesis 1:505–512.

Casale, GP; Higginbotham, S; Johansson, SL; et al. (1997) Inflammatory response of mouse skin exposed to the very potent carcinogen dibenzo[a,l]pyrene: a model for tumor promotion. Fundam Appl Toxicol 36:71–78.

Casto, BC. (1973) Enhancement of adenovirus transfo rmation by treatment of hamsters with ultraviolet i rradiation, DNA base analogs, and dibenz(a,h]anthracene. Cance r Res 33:402–407.

Chakravarti, D; Mailander, P; Franzen, J; et al. (1 998) Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic Sencar mouse skin usi ng a new PCR-RFLP method. Oncogene 16:3203–3210.

Chakravarti, D; Mailander, PC; Cavalieri, EL; et al . (2000) Evidence that error-prone DNA repair conve rts dibenzo[a,l]pyrene-induced depurinating lesions int o mutations: formation, clonal proliferation and re gression of initiated cells carrying H-ras oncogene mutations i n early preneoplasia. Mutat Res 456:17–32.

Chaloupka, K; Santostefano, M; Goldfarb, IS; et al. (1994) Aryl hydrocarbon (Ah) receptor-independent induction of Cyp1a2 gene expression by acenaphthylene and rel ated compounds in B6C3F1 mice. Carcinogenesis 15:2 835–
2840.

Chiarelli, MP; Chang, HF; Olsen, KW; et al. (2003) Structural differentiation of diastereomeric benzo[ghi]fluoranthene adducts of deoxyadenosine by matrix-assisted laser desorption/ionization time-o f-flight mass spectrometry and postsource decay. Chem Res T oxicol 16:1236–1241.

Chroust, K; Jowett, T; Farid-Wajidi, MF; et al. (20 01) Activation or detoxification of mutagenic and c arcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase. Mutat Res 498:169–179.

Cizmas, L; Zhou, GD; Safe, SH; et al. (2004) Compar ative in vitro and in vivo genotoxicities of 7H-benzo[c]fluorene, manufactured gas plant residue (MGP), and MGP fractions. Environ Mol Mutagen 43: 159–
168.

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Clayson, DB; Iverson, F; Nera, EA; et al. (1990) Th e significance of induced forestomach tumors. Annu Rev Pharmacol Toxicol 30:441–463.

Collin, G; H"ke, H. (1985) Anthracene. In: Elvers, B; Hawkins, S; Schulz, G, eds. Ullmann's encyclope dia of industrial chemistry, 5th ed.,Volume A2: Weinheim, Verlagsgesellschaft; pp. 343–345.

Collins, JF; Brown, JP; Alexeeff, GV; et al. (1998) Potency equivalency factors for some polycyclic ar omatic hydrocarbons and polycyclic aromatic hydrocarbon de rivatives. Regul Toxicol Pharmacol 28:45–54. (Abst ract)

Coombs, MM; Bhatt, TS, eds. (1987) [chapters 6 & 7] In: Cyclopenta[a]phenanthrenes. Polycyclic aromati c compounds structurally related to steroids; pp. 132 –210.

Coombs, MM; Dixon, C; Kissonerghis, AM; et al. (197 6) Evaluation of the mutagenicity of compounds of k nown carcinogenicity, belonging to the benz[a]anthracene , chrysene, and cyclopenta [a]phenanthrene series, using Ames'
test. Cancer Res 36:4525–4529.

Dai, Q. (1980) Researches on chemical carcinogens a nd mechanism of chemical carcinogenesis. DI-region theory: A quantitative molecular orbital model of carcinogeni c activity for polycyclic aromatic hydrocarbons. S ci Sin 23:453–
470.

Danz, M; Hartmann, A; Blaszyk, H. (1998) Mitogenic short-term effects on hepatocytes and adrenocortica l cells: phenobarbital and reserpine compared to carcinogeni c and non-carcinogenic fluorene derivatives. Exp T oxicol Pathol 50:416–424.

Devanesan, P; Ariese, F; Jankowiak, R; et al. (1999 ) A novel method for the isolation and identificati on of stable DNA adducts formed by dibenzo[a,l]pyrene and dibenz o[a,l]pyrene 11,12-dihydrodiol 13,14-epoxides in vi tro. Chem Res Toxicol 12:796–801.

DeVito, MJ; Maier, WE; Diliberto, JJ; et al. (1993) Comparative ability of various PCBs, PCDFs, and TC DD to induce cytochrome P450 1A1 and 1A2 activity followi ng 4 weeks of treatment. Fundam Appl Toxicol 20:12 5–130.

Diamond, L; Cherian, K; Harvey, RG; et al. (1984) M utagenic activity of methyl- and fluoro-substituted derivatives of polycyclic aromatic hydrocarbons in a human hepa toma (HepG2) cell-mediated assay. Mutat Res 136:65 –72.

Dipple, A; Pigott, MA; Agarwal, SK; et al. (1987) O ptically active benzo[c]phenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327:535–536.

Dong, S; Fu, PP; Shirsat, RN; et al. (2002) UVA lig ht-induced DNA cleavage by isomeric methylbenz[a]anthracenes. Chem Res Toxicol 15:400– 407.

Einolf, HJ; Amin, S; Yagi, H; et al. (1996) Benzo[c ]phenanthrene is activated to DNA-binding diol epox ides in the human mammary carcinoma cell line MCF-7 but only li mited activation occurs in mouse skin. Carcinogene sis
17:2237–2244.

Einolf, HJ; Story, WT; Marcus, CB; et al. (1997) Ro le of cytochrome P450 enzyme induction in the metab olic activation of benzo[c]phenanthrene in human cell li nes and mouse epidermis. Chem Res Toxicol 10:609–6 17.

Ensell, MX; Hubbs, A; Zhou, G; et al. (1999) Neopla stic potential of rat tracheal epithelial cell line s induced by 1-nitropyrene and dibenzo[a,i]pyrene. Mutat Res 44 4:193–199.

Ensell, MX; Whong, WZ; Heng, ZC; et al. (1998) In v itro and in vivo transformation in rat tracheal epi thelial cells exposed to diesel emission particles and related co mpounds. Mutat Res 412:283–291.

Fahmy, OG; Fahmy, MJ. (1973) Oxidative activation o f benz(a)anthrazene and methylated derivatives in mutagenesis and carcinogenesis. Cancer Res 33:2354– 2361.

Fuchs, J; Mlcoch, J; Platt, KL; et al. (1993) Chara cterization of highly polar bis-dihydrodiol epoxide --DNA adducts formed after metabolic activation of dibenz[a,h]ant hracene. Carcinogenesis 14:863–867.

### B-13 DRAFT – DO NOT CITE OR QUOTE

Gatehouse, D. (1980) Mutagenicity of 1,2 ring-fused acenaphthenes against S. typhimurium TA1537 and TA 1538: structure-activity relationship. Mutat Res 78:121– 135.

Giles, AS; Seidel, A; Phillips, DH. (1995) In vitro reaction with DNA of the fjord-region diol epoxide s of benzo[g]chrysene and benzo[c]phenanthrene as studie d by 32P-postlabeling. Chem Res Toxicol 8:591–599.

Giles, AS; Seidel, A; Phillips, DH. (1996) Covalent DNA adducts formed in mouse epidermis by benzo[g]c hrysene. Carcinogenesis 17:1331–1336.

Glatt, H; Seidel, A; Bochnitschek, W; et al. (1986) Mutagenic and cell-transforming activities of trio l-epoxides as compared to other chrysene metabolites. Cancer Res 46:4556–4565.

Glatt, H; Abu-Shqara, E; Harvey, RG; et al. (1994) Mutagenicity of K-region oxides and imines of chrys ene, benzo[c]phenanthrene and benzo[g]chrysene in Salmon ella typhimurium. Mutat Res 308:135–141.

Gold, A; Nesnow, S; Moore, M; et al. (1980) Mutagen esis and morphological transformation of mammalian cells by a non-bay-region polycyclic cyclopenta[cd]pyrene an d its 3,4-oxide. Cancer Res 40:4482–4484.

Gorelick, NJ; Wogan, GN. (1989) Fluoranthene-DNA ad ducts: identification and quantification by an HPLC-32P-postlabeling method. Carcinogenesis 10:15 67–1577.

Gorelick, NJ; Hutchins, DA; Tannenbaum, SR; et al. (1989) Formation of DNA and hemoglobin adducts of fluoranthene after single and multiple exposures. Carcinogenesis 10:1579–1587.

Goshman, LM; Heidelberger, C. (1967) Binding of tri tium-labeled polycyclic hydrocarbons to DNA of mous e skin.
Cancer Res 27:1678–1688.

Hall, M; Parker, DK; Hewer, AJ; et al. (1988) Furth er metabolism of diol-epoxides of chrysene and dibenz[a,c]anthracene to DNA binding species as evi denced by 32P-postlabeling analysis. Carcinogenesi s 9:865–
868.

Harvey, RG. (1996) Mechanisms of carcinogenesis of polycyclic aromatic compounds. Polycyclic Aromat C ompd
9:1–23.

Herner, HA; Trosko, JE; Masten, SJ. (2001) The epig enetic toxicity of pyrene and related ozonation byp roducts containing an aldehyde functional group. Environ S ci Technol 35:3576–3583.

Hermann, M. (1981) Synergistic effects of individua l polycyclic aromatic hydrocarbons on the mutagenic ity of their mixtures. Mutat Res 90:399–409.

Hewer, A; Cooper, CS; Ribeiro, O; et al. (1981) The metabolic and activation of dibenz[a,c]anthracene. Carcinogenesis 2:1345–1352.

Holme, JA; Bjorge, C; Soderlund, EJ; et al. (1993) Genotoxic effects of cyclopenta-fused polycyclic ar omatic hydrocarbons in isolated rat hepatocytes and rabbit lung cells. Carcinogenesis 14:1125–1131.

Huberman, E; Kuroki, T; Marquardt, H; et al. (1972) Transformation of hamster embryo cells by epoxides and other derivatives of polycyclic hydrocarbons. Cancer Res 32:1391–1396.

Hughes, NC; Phillips, DH. (1993) 32P-postlabeling a nalysis of the covalent binding of benzo[ghi]peryle ne to DNA in vivo and in vitro. Carcinogenesis 14:127–133.

Ishidate, M; Odashima, S. (1977) Chromosome tests w ith 134 compounds on Chinese hamster cells i n vitro : A screening for chemical carcinogens. Mutat Res 48:3 37–354.

Isu, Y; Nagashima, U; Aoyama, T; et al. (1996) Deve lopment of neural network simulator for structure-- activity correlation of molecules (NECO). Prediction of endo /exo substitution of norbornane derivatives and of carcinogenic activity of PAHs from 13C-NMR shifts. J Chem Inf C omput Sci 36:286–293.

### B-14 DRAFT – DO NOT CITE OR QUOTE

Jankowiak, R; Ariese, F; Hewer, A; et al. (1998) St ructure, conformations, and repair of DNA adducts f rom dibenzo[a,l]pyrene: 32P-postlabeling and fluorescen ce studies. Chem Res Toxicol 11:674–685.

Jerina, DM; Lehr, RE. (1977) The bay-region theory: A quantum mechanical approach to aromatic hydrocarboninduced carcinogenicity. pp. 709–720.

Jerina, DM; Yagi, H; Lehr, RE; et al. (1978) The ba y-region theory of carcinogenesis by polycyclic aro matic hydrocarbons. pp. 173–188.

Juhasz, AL; Stanley, GA; Britz, ML. (2000) Microbia l degradation and detoxification of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomona s maltophilia strain VUN 10,003. Lett Appl Microbi ol
30:396–401.

Kemena, A; Norpoth, KH; Jacob, J. (1988) Differenti al induction of the monooxygenase isoenzymes in mou se liver microsomes by polycyclic aromatic hydrocarbons. In : Cooke, M; Dennis, AJ, eds. Polynuclear aromatic hydrocarbons: A decade of progress. Columbus, OH: B attelle Press; pp. 449–460.

Keohavong, P; Melacrinos, A; Shukla, R. (1995) In v itro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene. Carcinogenesis 16:855–860.

King, LC; Adams, L; Allison, J; et al. (1999) A qua ntitative comparison of dibenzo[a,l]pyrene-DNA addu ct formation by recombinant human cytochrome P450 micr osomes. Mol Carcinog 26:74–82.

Knaap, AGAC; Goze, C; Simons, JWIM (1981) Mutagenic activity of seven coded samples in V79 Chinese ham ster cells. In: De Serres, FJ; Ashby, J, eds. Evaluatio n of short-term tests for carcinogens. Report of th e international collaborative programme. New York: Elsevier/North H olland; pp. 608–613 (Progress in Mutation Research, Volume 1).

Kumar, S; Kole, PL; Sikka, HC. (1990) Mutagenicity of dibenz[a,c]anthracene and its derivatives in Sal monella typhimurium TA100. Mutat Res 242:337–343.

Laryea, A; Cosman, M; Lin, JM; et al. (1995) Direct synthesis and characterization of site-specific ad enosyl adducts derived from the binding of a 3,4-dihydroxy-1,2-epo xybenzo[c]phenanthrene stereoisomer to an 11-mer oligodeoxyribonucleotide. Chem Res Toxicol 8:444–4 54.

Lasley, J; Curti, S; Ross, J; et al. (1991) Morphol ogical cell transformation and DNA adduction by benz(j]aceanthrylene and its presumptive reaction m etabolites in C3H10T1/2CL8 cells. Adv Exp Med Biol
283:759–762.

LaVoie, EJ; Hecht, SS; Amin, S; et al. (1980) Ident ification of mutagenic dihydrodiols as metabolites of benzo[j]fluoranthene and benzo[k]fluoranthene. Can cer Res 40:4528–4532.

LaVoie, EJ; Tulley, L; Bedenko, V; et al. (1981) Mu tagenicity of methylated fluorenes and benzofluoren es. Mutat Res 91:167–176.

LaVoie, EJ; Hecht, SS; Bedenko, V; et al. (1982) Id entification of the mutagenic metabolites of fluora nthene, 2-methylfluoranthene, and 3-methylfluoranthene. Ca rcinogenesis 3:841–846.

Lecoq, S; Perin, F; Plessis, MJ; et al. (1989) Comp arison of the in vitro metabolisms and mutageniciti es of dibenz[a,c]anthracene, dibenz[a,h]anthracene and di benz[a,j]anthracene: influence of norharman. Carci nogenesis
10:461–469.

Lecoq, S; Ni She, M; Grover, PL; et al. (1991a) The in vitro metabolic activation of dibenz[a,h]anthra cene, catalyzed by rat liver microsomes and examined by 3 2P-postlabeling. Cancer Lett 57:261–269.

Lecoq, S; She, MN; Hewer, A; et al. (1991b) The met abolic activation of dibenz[a,h]anthracene in mouse skin examined by 32P-postlabeling: minor contribution of the 3,4-diol 1,2-oxides to DNA binding. Carcinoge nesis
12:1079–1083.

### B-15 DRAFT – DO NOT CITE OR QUOTE

Lecoq, S; Pfau, W; Grover, PL; et al. (1992) HPLC s eparation of 32P-postlabelled DNA adducts formed fr om dibenz[a,h]anthracene in skin. Chem Biol Interact 85:173–185.

Levin, W; Wood, A; Chang, R; et al. (1982) Oxidativ e metabolism of polycyclic aromatic hydrocarbons to ultimate carcinogens. Drug Metab Rev 13:555–580.

Lewtas, J. (1985) Development of a comparative pote ncy method for cancer risk assessment of complex mi xtures using short-term in vivo and in vitro bioassays. T oxicol Ind Health 1:193–203.

Lewtas, J. (1988) Genotoxicity of complex mixtures: strategies for the identification and comparative assessment of airborne mutagens and carcinogens from combustion s ources. Fundam Appl Toxicol 10:571–589.

Li, KM; Todorovic, R; Rogan, EG; et al. (1995) Iden tification and quantitation of dibenzo[a,l]pyrene-- DNA adducts formed by rat liver microsomes in vitro: prepondera nce of depurinating adducts. Biochemistry 34:8043– 8049.

Li, KM; Byun, J; Gross, ML; et al. (1999) Synthesis and structure determination of the adducts formed by electrochemical oxidation of Dibenzo[a,l]pyrene in the presence of adenine. Chem Res Toxicol 12:749–7 57.

Lloyd, DR; Hanawalt, PC. (2002) p53 controls global nucleotide excision repair of low levels of struct urally diverse benzo[g]chrysene-DNA adducts in human fibroblasts. Cancer Res 62:5288–5294.

Luch, A; Coffing, SL; Tang, YM; et al. (1998) Stabl e expression of human cytochrome P450 1B1 in V79 Ch inese hamster cells and metabolically catalyzed DNA adduc t formation of dibenzo[a,l]pyrene. Chem Res Toxico l
11:686–695.

Luch, A; Kishiyama, S; Seidel, A; et al. (1999) The K-region trans-8,9-diol does not significantly con tribute as an intermediate in the metabolic activation of dibenzo [a,l]pyrene to DNA-binding metabolites by human cyt ochrome
P450 1A1 or 1B1. Cancer Res 59:4603–4609.

Luch, A; Kudla, K; Seidel, A; et al. (1999) The lev el of DNA modification by (+)-syn-(11S,12R,13S,14R) - and (-)-anti-(11R,12S,13S,14R)-dihydrodiol epoxides of dibenzo[a,l]pyrene determined the effect on the pro teins p53 and p21WAF1 in the human mammary carcinoma cell lin e MCF-7. Carcinogenesis 20:859–865.

Lupp, A; Tralls, M; Fuchs, U; et al. (1999) Transpl antation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of various mitogens and cyto toxins on cytochrome P450 (P450) isoforms expressio n and on P450 mediated monooxygenase functions. Exp Toxicol Pathol 51:375–388.

Malaveille, C; Hautefeuille, A; Bartsch, H; et al. (1980) Liver microsome-mediated mutagenicity of dih ydrodiols derived from dibenz(a,c]anthracene in S. typhimuriu m TA 100. Carcinogenesis 1:287–289.

Malaveille, C; Hautefeuille, A; Perin-Roussel, O; e t al. (1984) Possible involvement of a vicinal, non -bay-region dihydrodiol-epoxide in the activation of dibenzo[a, e]fluoranthene into bacterial mutagens. Carcinogen esis 5:1263–
1266.

Marquardt, H; Heidelberger, C. (1972) Influence of 'feeder cells' and inducers and inhibitors of micro somal mixedfunction oxidases on hydrocarbon-induced malignant transformation of cells derived from C3H mouse pros tate. Cancer Res 32:721–725.

Marquardt, H; Kuroki, T; Huberman, E; et al. (1972) Malignant transformation of cells derived from mou se prostate by epoxides and other derivatives of polycyclic hyd rocarbons. Cancer Res 32:716–720.

Marrocchi, A; Minuti, L; Morozzi, G; et al. (1996) Synthesis and mutagenicity of some cyclopenta[c]phe nanthrenes and indeno[c]phenanthrenes. Carcinogenesis 17:2009 –2012.

Marsch, GA; Jankowiak, R; Small, GJ; et al. (1992) Evidence of involvement of multiple sites of metabo lism in the in vivo covalent binding of dibenzo[a,h]pyrene to D NA. Chem Res Toxicol 5:765–772.

### B-16 DRAFT – DO NOT CITE OR QUOTE

Marshall, MV; He, ZM; Weyand, EH; et al. (1993) Mut agenic activity of the 4,5- and 9,10-dihydrodiols o f benzo[j]fluoranthene and their syn- and anti-dihydr odiol epoxides in Salmonella typhimurium. Environ Mol Mutagen 22:34–45.

Matijasevic, Z; Zeiger, E. (1985) Mutagenicity of p yrene in Salmonella. Mutat Res 142:149–152.

Matsuoka, A; Sofuni, T; Miyata, N; et al. (1991) Cl astogenicity of 1-nitropyrene, dinitropyrenes, fluo rene and mononitrofluorenes in cultured Chinese hamster cell s. Mutat Res 259:103–110.

Meek, ME; Chan, PKL; Bartlett, S. (1994) Polycyclic aromatic hydrocarbons: evaluation of risks to heal th from environmental exposures in Canada. Environ Carcino g Ecotoxicol Rev C 12:443–452.

Melendez-Colon, VJ; Smith, CA; Seidel, A; et al. (1 997) Formation of stable adducts and absence of dep urinating DNA adducts in cells and DNA treated with the poten t carcinogen dibenzo[a,l]pyrene or its diol epoxide s. Proc Natl Acad Sci U S A 94:13542–13547.

Melendez-Colon, VJ; Luch, A; Seidel, A; et al. (199 9) Comparison of cytochrome P450- and peroxidase-de pendent metabolic activation of the potent carcinogen diben zo[a,l]pyrene in human cell lines: formation of sta ble DNA adducts and absence of a detectable increase in apu rinic sites. Cancer Res 59:1412–1416.

Mlcoch, J; Fuchs, J; Oesch, F; et al. (1993) Charac terization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro. Carcinogene sis 14:469–473.

Mori, Y; Goto, S; Onodera, S; et al. (1993) Changes in mutagenic properties and chemical fate of benz[ a]anthracene in chlorine-treated water with and without bromide ion. Chemosphere 27(11):2155–2162.

Moyer, SR; Jurs, PC. (1990) An SRA study of the mut agenicity of PAH compounds in Salmonella typhimuriu m. In: Mendelsohn, ML; Albertini, J, eds. Progress in clinical and biological research: Vol. 340. Mutatio n and the environment: Part B. Metabolism, testing methods, a nd chromosomes. New York, NY: Wiley-Liss; pp. l–10.

Nair, RV; Gill, RD; Nettikumara, AN; et al. (1991) Characterization of covalently modified deoxyribonu cleosides formed from dibenz[a,j]anthracene in primary cultur es of mouse keratinocytes. Chem Res Toxicol 4:115– 122.

Nesnow, S; Leavitt, S; Easterling, R; et al. (1984) Mutagenicity of cyclopenta-fused isomers of benz[a ]anthracene in bacterial and rodent cells and identification of th e major rat liver microsomal metabolites. Cancer R es 44:4993–
5003.

Nesnow, S; Lasley, J; Curti, S; et al. (1991) Morph ological transformation and DNA adduct formation by benz[j]aceanthrylene and its metabolites in C3H10T1 /2CL8 cells: evidence for both cyclopenta-ring and bay-region metabolic activation pathways. Cancer Res 51:6163– 6169.

Nesnow, S; Davis, C; Padgett, W; et al. (1998) Meta bolic activation of racemic and enantiomeric trans- 8, 9-dihydroxy-8,9-dihydrodibenzo[a,l]pyrene (dibenzo[ def,p]chrysene) to dibenzo[a,l]pyrene-bis-dihydrodi ols by induced rat liver microsomes and a recombinant huma n P450 1A1 system: the role of the K-region-derived metabolic intermediates in the formation of dibenzo [a,l]pyrene-DNA adducts. Chem Res Toxicol 11:1596– 1607.

Newcomb, KO; Sangaiah, R; Gold, A; et al. (1993) Ac tivation and metabolism of benz[j]aceanthrylene9,10-dihydrodiol, the precursor to bay-region metab olism of the genotoxic cyclopenta-PAH benz[j]aceant hrylene.
Mutat Res 287:181–190.

Oesch, F; Bücker, M; Glatt, HR. (1981) Activation o f phenanthrene to mutagenic metabolites and evidenc e for at least two different activation pathways. Mutat Res 81:1–10.

Oshiro, Y; Balwierz, PS. (1982) Morphological trans formation of C3H/10T1/2 CL8 cells by procarcinogens

. Environ Mutagen 4:105–108.

Otero-Lobato, MJ; Jenneskens, LW; Seinen, W. (2004) Bacterial mutagenicity of the three isomeric dicyc lopentafused pyrenes: the effects of dicyclopenta topology . Mutat Res 559:105–119.

### B-17 DRAFT – DO NOT CITE OR QUOTE

Pal, K. (1981) The induction of sister-chromatid ex changes in Chinese hamster ovary cells by K-region epoxides and some dihydrodiols derived from benz[a]anthracen e, dibenz [a,c]anthracene and dibenz [a,h]anthracen e. Mutat Res 84:389–398.

Palitti, F; Cozzi, R; Fiore, M; et al. (1986) An in vitro and in vivo study on mutagenic activity of f luoranthene: comparison between cytogenetic studies and HPLC ana lysis. Mutat Res 174:125–130.

Perin-Roussel, O; Saguem, S; Ekert, B; et al. (1983 ) Binding to DNA of bay region and pseudo bay regio n diolepoxides of dibenzo[a,e]fluoranthene and comparison with adducts obtained with dibenzo[a,e]fluoranthen e or its dihydrodiols in the presence of microsomes. Carcin ogenesis 4:27–32.

Perin-Roussel, O; Croisy, A; Ekert, B; et al. (1984 a) The metabolic activation of dibenzo[a,e]fluorant hene in vitro. Evidence that its bay-region and pseudo-bay-region diol-epoxides react preferentially with guanosine. Cancer Lett
22:289–298.

Perin-Roussel, O; Ekert, B; Barat, N; et al. (1984b ) DNA-protein crosslinks induced by exposure of cul tured mouse fibroblasts to dibenzo[a,e]fluoranthene and its bay - and pseudo-bay region dihydrodiols. Carcinogenes is 5:379–383.

Perin-Roussel, O; Barat, N; Zajdela, F. (1985) Form ation and removal of dibenzo[a,e]fluoranthene-DNA a dducts in mouse embryo fibroblasts. Carcinogenesis 6:1791–17 96.

Perin-Roussel, O; Barat, N; Zajdela, F. (1988) Non- random distribution of dibenzo[a,e]fluoranthene-ind uced DNA adducts in DNA loops in mouse fibroblast nuclei. C arcinogenesis 9:1383–1388.

Perin-Roussel, O; Perin, F; Zajdela, F. (1990) 32P- post-labeling analysis of DNA adducts in mouse embr yo fibroblasts treated with dibenzo[a,e]fluoranthene a nd its major metabolites. Carcinogenesis 11:301–30 6.

Peter, S; Palme, GE; R"hrborn, G. (1979) Mutagenici ty of polycyclic hydrocarbons. III. Monitoring gene tic hazards of benz[a]anthracene. Acta Morphol Acad Sci Hung 2 7:199–204.

Pfau, W; Hughes, NC; Grover, PL; et al. (1992) HPLC separation of 32P-postlabelled benzo[b]fluoranthen e-DNA adducts. Cancer Lett 65:159–167.

Pfau, W; Lecoq, S; Hughes, NC; et al. (1993) Separa tion of 32P-labelled nucleoside 3',5'-bisphosphate adducts by HPLC. IARC Sci Publ pp. 233–242.

Phillips, DH. (1997) Detection of DNA modifications by the 32P-postlabeling assay. Mutat Res 378:1–12

.

Platt, KL; Bucker, M; Golan, M; et al. (1982) The m utagenicity of dibenz(a,h]anthracene activated by phenobarbital-inducible mouse-liver mono-oxygenase is potentiated by the presence of hydrophilic resid ues at the K-region of the molecule. Mutat Res 96:1–13.

Platt, KL; Schollmeier, M; Frank, H; et al. (1990) Stereoselective metabolism of dibenz(a,h]anthracene to transdihydrodiols and their activation to bacterial muta gens. Environ Health Perspect 88:37–41.

Polcaro, C; Nicoletti, I; Ossicini, L; et al. (1988 ) Chromatographic and cytogenetic analysis of in vi vo metabolites of fluoranthene. J Chromatogr 448:127–133.

Pruess-Schwartz, D; Baird, WM; Yagi, H; et al. (198 7) Stereochemical specificity in the metabolic acti vation of benzo[c]phenanthrene to metabolites that covalently bind to DNA in rodent embryo cell cultures. Cance r Res
47:4032–4037.

Purchase, IFH; Longstaff, E; Ashby, J; et al. (1976 ) Evaluation of six short term tests for detecting organic chemical carcinogens and recommendations for their use. Nat ure 264:624–627.

Ralston, SL; Lau, HH; Seidel, A; et al. (1994) The potent carcinogen dibenzo[a,l]pyrene is metabolical ly activated to fjord-region 11,12-diol 13,14-epoxides in human mammary carcinoma MCF-7 cell cultures. Cancer Res 54:887–
890.

### B-18 DRAFT – DO NOT CITE OR QUOTE

Ralston, SL; Seidel, A; Luch, A; et al. (1995) Ster eoselective activation of dibenzo[a,l]pyrene to (-) -anti (11R,12S,13S,14R)- and (+)-syn(11S,12R,13S,14R)-11, 12-diol-13,14-epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary carcinoma cell line MCF-7. Carcinogenesis 16:2899

– 2907.

Ralston, SL; Coffing, SL; Seidel, A; et al. (1997) Stereoselective activation of dibenzo[a,l]pyrene an d its trans11,12-dihydrodiol to fjord region 11,12-diol 13,14- epoxides in a human mammary carcinoma MCF-7 cell-me diated V79 cell mutation assay. Chem Res Toxicol 10:687–6 93.

RamaKrishna, NV; Padmavathi, NS; Cavalieri, EL; et al. (1993) Synthesis and structure determination of the adducts formed by electrochemical oxidation of the potent carcinogen dibenzo[a,I]pyrene in the presenc e of nucleosides. Chem Res Toxicol 6:554–560.

Rastetter, WH; Nachbar, RB; Russo-Rodriguez, S; et al. (1982) Fluoranthene: Synthesis and mutagenicity of fluor diol epoxides. J Org Chem 47:4873–4878.

Reznikoff, CA; Bertram, JS; Brankow, DW; et al. (19 73) Quantitative and qualitative studies of chemica l transformation of cloned C3H mouse embryo cells sen sitive to postconfluence inhibition of cell divisio n. Cancer
Res 33:3239–3249.

Rice, JE; Coleman, DT; Hosted, TJ Jr; et al. (1985) Identification of mutagenic metabolites of indeno[ 1,2,3- cd]pyrene formed in vitro with rat liver enzymes. Cancer Res 45:5421–5425.

Rice, JE; Geddie, NG; Defloria, MC; et al. (1988) S tructural requirements favoring mutagenic activity among methylated pyrenes in S. typhimurium. In: Cooke, M ; Dennis, AJ, eds. Polynuclear aromatic hydrocarbon s: A decade of progress. Columbus, OH: Battelle Press; p p. 773–785.

Ridler, P.; Jennings, B. (1984) The binding of poly cyclic aromatic hydrocarbon diol-epoxides to DNA. Cancer Lett
22:95–98.

Ross, JA; Nelson, GB; Holden, KL; et al. (1992) DNA adducts and induction of sister chromatid exchange s in the rat following benzo[b]fluoranthene administration. Carcinogenesis 13:1731–1734.

Rugen, PJ; Stern, CD; Lamm, SH. (1989) Comparative carcinogenicity of the PAHs as a basis for acceptab le exposure levels (AELs) in drinking water. Regul To xicol Pharmacol 9:273–283.

Safe, S. (1990) Polychlorinated biphenyls (PCBs), d ibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), an d related compounds: environmental and mechanistic considerat ions which support the development of toxic equival ency factors (TEFs). Crit Rev Toxicol 21:51–88.

Saffiotti, U. (1969) Experimental respiratory tract carcinogenesis. Prog Exp Tumor Res 11:302–333.

Sangaiah, R; Gold, A; Newcomb, KO; et al. (1991) Sy nthesis and biological activity of bay-region metab olites of a cyclopenta-fused polycyclic aromatic hydrocarbon: b enz[j]aceanthrylene. J Med Chem 34:546–549.

Schneider, K; Roller, M; Kalberlah, F; et al. (2002 ) Cancer risk assessment for oral exposure to PAH m ixtures. J Appl Toxicol 22:73–83.

Slaga, TJ; Gleason, GL; Mills, C; et al. (1980) Com parison of the tumour-initiating activities of dihy drodiols and diol-epoxides of various polycyclic aromatic hydroc arbons. Cancer Res 40:1981–1984.

Snell, KC; Stewart, HL. (1962) Pulmonary adenomatos is induced in DBA/2 mice by oral administration of dibenz(a,h]anthracene. J Natl Cancer Inst 28:1043– 1049.

Snell, KC; Stewart, HL. (1963) Induction of pulmona ry adenomatoses in DBA/2 mice by the oral administr ation of dibenz[a,h]anthracene. Acta Un Int Cancer 19:692–6 94.

Stanton, MF; Miller, E; Wrench, C; et al. (1972) Ex perimental induction of epidermoid carcinoma in the lungs of rats by cigarette smoke condensate. J Nat1 Cancer Inst 49:867–877.

### B-19 DRAFT – DO NOT CITE OR QUOTE

Stocker, KJ; Howard, WR; Statham, J; et al. (1996) Assessment of the potential in vivo genotoxicity of fluoranthene. Mutagenesis 11:493–496.

Upham, BL; Weis, LM; Rummel, AM; et al. (1996) The effects of anthracene and methylated anthracenes on gap junctional intercellular communication in rat liver epithelial cells. Fundam Appl Toxicol 34:260–264.

U.S. EPA (1989) Health and environmental effects pr ofile for benzo[g,h,i]perylene. Cincinnati, OH: En vironmental Protection Agency, Environmental Criteria and Asses sment Office. Contract Number EPA-600-X-87-395.

Wang, JS; Busby, WF; Wogan, GN. (1995) Tissue distr ibution of DNA adducts in pre-weanling BLU:Ha mice treated with a tumorigenic dose of fluoranthene. C ancer Lett 92:9–19.

Wang, JS; Busby, WF Jr; Wogan, GN. (1995) Formation and persistence of DNA adducts in organs of CD-1 m ice treated with a tumorigenic dose of fluoranthene. C arcinogenesis 16:2609–2616.

Weis, LM; Rummel, AM; Masten, SJ; et al. (1998) Bay or baylike regions of polycyclic aromatic hydrocar bons were potent inhibitors of Gap junctional intercellu lar communication. Environ Health Perspect 106:17– 22.

Wester, PW; Kroes, R. (1988) Forestomach carcinogen s: pathology and relevance to man. Toxicol Pathol 16:165–
171.

Weyand, EH; Rice, JE; Hussain, N; et al. (1987a) De tection of DNA adducts of tumorigenic nonalternant polycyclic aromatic hydrocarbons by 32P-postlabeling. Proc Am Assoc Cancer Res 28:102.

Weyand, EH; Rice, JE; LaVoie, EJ. (1987b) 32P-postl abeling analysis of DNA adducts from non-altemant P AH using thin-layer and high performance liquid chroma tography. Cancer Lett 37:257–266.

Weyand, EH; Geddie, N; Rice, JE; et al. (1988) Meta bolism and mutagenic activity of benzo[k]fluoranthe ne and 3-, 8- and 9-fluorobenzo[k]fluoranthene. Carcinogenesi s 9:1277–1281.

Weyand, EH; Bryla, P; Wu, Y; et al. (1993) Detectio n of the major DNA adducts of benzo[j]fluoranthene in mouse skin: nonclassical dihydrodiol epoxides. Chem Res Toxicol 6:117–124.

Whong, WZ; Stewart, JD; Cutler, D; et al. (1992) Co mparative study of DNA adduct formation and cytogen ic effects of two constituents in coke oven emissions with an in vivo rat lung cell system. Environ Mol Mutag 19(Suppl 20):70.

Whong, WZ.; Stewart, JD; Cutler, D; et al. (1994) I nduction of in vivo DNA adducts by 4 industrial by- products in the rat-lung-cell system. Mutat Res 312:165–172.

Wigley, CB; Newbold, RF; Amos, J; et al. (1979) Cel l-mediated mutagenesis in cultured Chinese hamster cells by polycyclic hydrocarbons: mutagenicity and DNA react ion related to carcinogenicity in a series of compo unds. Int J Cancer 23, 691–696.

Willett, KL; Randerath, K; Zhou, GD; et al. (1998) Inhibition of CYP1A1-dependent activity by the poly nuclear aromatic hydrocarbon (PAH) fluoranthene. Biochem P harmacol 55:831–839.

Williams, GM. (1977) Detection of chemical carcinog ens by unscheduled DNA synthesis in rat liver prima ry cell cultures. Cancer Res 37:1845–1851.

Wood, AW; Levin, W; Ryan, D; et al. (1977) High mut agenicity of metabolically activated chrysene 1,2-dihydrodiol: Evidence for bay region activation of chrysene. Biochem Biophys Res Commun 78:847–85 4.

Wood, AW Levin, W; Thomas, PE; et al. (1978) Metabo lic activation of dibenz[a,h]anthracene and its dih ydrodiols to bacterial mutagens. Cancer Res 38:1967–1973.

Wood, AW; Chang, RL; Huang, MT; et al. (1980a) Muta genicity of benzo[e]pyrene and triphenylene tetrahydroepoxides and diol-epoxides in bacterial a nd mammalian cells. Cancer Res 40:1985–1989.

### B-20 DRAFT – DO NOT CITE OR QUOTE

Wood, AW; Chang, RL; Levin, W; et al. (1980b) Mutag enicity of the dihydrodiols and bay-region diol-epo xides of benzo[c]phenanthrene in bacterial and mammalian cel ls. Cancer Res 40:2876–2883.

Wood, AW; Chang, RL; Levin, W; et al. (1981) Mutage nicity of the bay-region diol-epoxides and other be nzo-ring derivatives of dibenzo[a,h]pyrene and dibenzo[a,i]p yrene. Cancer Res 41:2589–2597.

Wood, AW; Chang, RL; Levin, W; et al. (1983) Mutage nicity of the enantiomers of the diastereomeric bay -region benz[a]anthracene 3,4-diol-1,2-epoxides in bacteria l and mammalian cells. Cancer Res 43:5821–5825.

Wu, J; Zhu, BB; Yu, J; et al. (2003) In vitro and i n vivo modulations of benzo[c]phenanthrene-DNA addu cts by DNA mismatch repair system. Nucleic Acids Res 31:6 428–6434.

Yamaguchi, K; Near, R.; Shneider, A; et al. (1996) Fluoranthene-induced apoptosis in murine T cell hyb ridomas is independent of the aromatic hydrocarbon receptor. Toxicol Appl Pharmacol 139:144–152.

Zhong, BZ; Gu, ZW; Stewart, J; et al. (1995) Micron ucleus formation induced by three polycyclic aromat ic hydrocarbons in rat bone marrow and spleen erythroc ytes following intratracheal instillation. Mutat R es 326:147–
153.

### C-1 DRAFT – DO NOT CITE OR QUOTE

APPENDIX C. DOSE-RESPONSE DATA FOR POTENCY CALCULA TIONS 1

2
3C-2 DRAFT – DO NOT CITE OR QUOTETable C-1. Dermal bioassays: dose response information for incidence dataRecordnumberReferenceStudytype SpeciesTumortype PAH SexDoseofPAHDoseunitsNumber ofanimalswith tumorsNumberofanimalsin group% tumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Fisher'sexactp-valueCochranArmitagetrend testp-value CommentsComplete carcinogenicity studies600 Habs et al.,1980Complete Mice Sum ofPapilloma,carcinoma,sarcomaAcetone F 0 pg/animal 0 35 0%DMSO F 0 pg/animal 0 36 0%BaP F 1.7 pg/animal 8 34 24% 1.92 × 10-3BaP F 2.8 pg/animal 24 35 69% 1.67 × 10-11BaP F 4.6 pg/animal 22 36 61% 2.1 × 10-92.15 × 10-9BbF F 3.4 pg/animal 2 38 5% 2.6 × 10-1BbF F 5.6 pg/animal 5 34 15% 2.3 × 10-2BbF F 9.2 pg/animal 20 37 54% 3.7 × 10-81.33 × 10-9BjF F 3.4 pg/animal 1 38 3% 5.1 × 10-1BjF F 5.6 pg/animal 1 35 3% 4.9 × 10-1BjF F 9.2 pg/animal 2 38 5% 2.6 × 10-11.77 × 10-1BkF F 3.4 pg/animal 1 39 3% 5.2 × 10-1BkF F 5.6 pg/animal 0 38 0%BkF F 9.2 pg/animal 0 38 0%CPcdP F 1.7 pg/animal 0 34 0%CPcdP F 6.5 pg/animal 0 35 0%CPcdP F 27.2 pg/animal 3 38 8% 1.3 × 10-16.36 × 10-2IP F 3.4 pg/animal 1 36 3% 5 × 10-1IP F 5.6 pg/animal 0 37 0%IP F 9.2 pg/animal 0 37 0%CO F 5.6 pg/animal 1 39 3% 0.52CO F 15 pg/animal 2 40 5% 0.27 1.83 × 10-113640 Cavalieri etal., 1983Complete Mice Papilloma,adenoma,carcinomaAcetone F 0 nmol 0 29 0%BaP F 2.2 nmol 2 30 7% 0.25BaP F 6.6 nmol 2 28 7% 0.24C-3 DRAFT – DO NOT CITE OR QUOTETable C-1. Dermal bioassays: dose response information for incidence dataRecordnumberReferenceStudytype SpeciesTumortype PAH SexDoseofPAHDoseunitsNumber ofanimalswith tumorsNumberofanimalsin group% tumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Fisher'sexactp-valueCochranArmitagetrend testp-value CommentsBaP F 20 nmol 17 30 57% 4.32 × 10-72.96 × 10-1CPcdP F 22.2 nmol 2 29 7% 0.25CPcdP F 66.6 nmol 2 29 7% 0.25CPcdP F 200 nmol 24 29 83% 9.25 × 10-121.39 × 10-16620 Hoffmannand Wynder1966Complete Mice PapillomasDioxane F 0 % 0 20 0%BaP F 0.05 % 17 20 85% 1.28 × 10-8BaP F 0.1 % 19 20 95% 1.5 × 10-108.7 × 10-10DBaeP F 0.05 % 16 30 53% 3.31 × 10-5DBaeP F 0.1 % 9 17 53% 1.95 × 10-45.69 × 10-4DBahP F 0.05 % 16 17 94% 1.32 × 10-9DBahP F 0.1 % 15 18 83% 5.27 × 10-81.29 × 10-7DBaiP F 0.05 % 16 19 84% 2.58 × 10-9DBaiP F 0.1 % 16 19 84% 2.58 × 10-99.81 × 10-8DBaeF F 0.05 % 17 19 89% 3.35 × 10-9DBaeF F 0.1 % 18 19 95% 3.05 × 10-101.13 × 10-917660 Cavalieri etal., 1977Complete Mice Papilloma,keratoacanthoma,carcinomaAcetone F 0 μmol/application0 29 0%BaP F 0.396μmol/application30 38 79% 4.9 × 10-12DBahP F 0.396μmol/application35 39 90% 2.98 × 10-15AA F 0.396μmol/application18 38 47% 3.59 × 10-6BaA F 0.396μmol/application1 39 3% 0.66C-4 DRAFT – DO NOT CITE OR QUOTETable C-1. Dermal bioassays: dose response information for incidence dataRecordnumberReferenceStudytype SpeciesTumortype PAH SexDoseofPAHDoseunitsNumber ofanimalswith tumorsNumberofanimalsin group% tumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Fisher'sexactp-valueCochranArmitagetrend testp-value CommentsInitiation studies630 LaVoie etal., 1982Initiation Mice PrimarilysquamouscellPapillomaAcetone/TPAF 0 μg/mouse 0 20 0%BaP F 30 μg/mouse 17 20 85% 1.28 × 10-8BbF F 10 μg/mouse 9 20 45% 6.14 × 10-4BbF F 30 μg/mouse 12 20 60% 2.25 × 10-5BbF F 100 μg/mouse 16 20 80% 7.7 × 10-81.46 × 10-5BjF F 30 μg/mouse 6 20 30% 0.01BjF F 100 μg/mouse 11 20 55% 7.27 × 10-5BjF F 1,000μg/mouse 19 20 95% 1.52 × 10-104.67 × 10-8BkF F 30 μg/mouse 1 20 5% 0.01BkF F 100 μg/mouse 5 20 25% 0.02BkF F 1,000μg/mouse 15 20 75% 3.85 × 10-74.51 × 10-918570 Hecht et al.,1974Initiation Mice UnspecifiedAcetone F 0 mg/ 0 20 0% No. survivingnot reported forcontrols; initialgroup size usedhereBaP F 0.05 mg/ 6 20 30% 0.01CH F 1 mg/ 11 19 58% 4.51 × 10-524800 Nesnow etal., 1984Initiation Mice Papilloma Acetone M 0 nmol 0 20 0%Data at 30 wksAcetone F 0 nmol 1 19 5%BaP M 200 nmol 13 18 67% <0.005BaP F 200 nmol 10 19 53% <0.005BlAC M 50 nmol 12 20 60% <0.005BlAC M 100 nmol 16 17 94% <0.005BlAC M 250 nmol 21 21 100% <0.005BlAC M 500 nmol 16 16 100% <0.005BlAC M 1,000nmol 19 20 95% <0.005C-5 DRAFT – DO NOT CITE OR QUOTETable C-1. Dermal bioassays: dose response information for incidence dataRecordnumberReferenceStudytype SpeciesTumortype PAH SexDoseofPAHDoseunitsNumber ofanimalswith tumorsNumberofanimalsin group% tumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Fisher'sexactp-valueCochranArmitagetrend testp-value CommentsBlAC F 50 nmol 13 20 65% <0.005BlAC F 100 nmol 18 19 95% <0.005BlAC F 250 nmol 19 21 91% <0.005BlAC F 500 nmol 20 21 95% <0.005BlAC F 1,000nmol 20 20 100% <0.005BeAC M 50 nmol 4 20 20%BeAC M 100 nmol 4 20 20%BeAC M 250 nmol 12 20 60% <0.005BeAC M 500 nmol 15 20 75% <0.005BeAC M 1,000nmol 16 18 89% <0.005BeAC F 50 nmol 4 20 20%BeAC F 100 nmol 7 19 37% <0.005BeAC F 250 nmol 10 19 53% <0.005BeAC F 500 nmol 8 18 44% <0.005BeAC F 1,000nmol 18 20 90% <0.00521420 Slaga et al.,1980Initiation Mouse Papilloma Control F 0 nmoles 2 306% Differentcontrols used foreach chemicalexcept DBacAand BePControl F 0 μmoles 3 30 10%Control F 0 μmoles 3 30 10%Control F 0 nmoles 2 29 6%ControlPOOLEDF 0 nmoles 10 119 8%BaP F 200 nmoles 20 30 67% 1.41 × 10-6BeP F 2,000nmoles 5 29 17% 0.33CH F 2,000nmoles 21 29 73% 8.38 × 10-7DBacA F 2,000nmoles 8 28 27% 0.07DBahA F 100 nmoles 15 29 50% 3.52 × 10-6C-6 DRAFT – DO NOT CITE OR QUOTETable C-1. Dermal bioassays: dose response information for incidence dataRecordnumberReferenceStudytype SpeciesTumortype PAH SexDoseofPAHDoseunitsNumber ofanimalswith tumorsNumberofanimalsin group% tumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Fisher'sexactp-valueCochranArmitagetrend testp-value Comments15640 Raveh et al.,1982Initiation Mice Papilloma Control F 0 μg 3 29 10%BaP F 10 μg 17 29 58% 1.11 × 10-4BaP F 25 μg 21 28 76% 5.96 × 10-7BaP F 50 μg 24 28 87% 5.43 × 10-9BaP F 100 μg 27 27 100% 5.50 × 10-13BaP F 200 μg 26 26 100% 1.03 × 10-122.78 × 10-10CPcdP F 10 μg 3 30 11% 0.65CPcdP F 100 μg 11 29 39% 0.01CPcdP F 200 μg 16 28 57% 1.90 × 10-42.75 × 10-6620 Hoffmannand Wynder1966Initiation Mice PapillomasCroton oilcontrolF 0 mg/mouse2 30 7%BaP F 0.25 mg/mouse24 30 80% 3.80 × 10-9DBaeF F 0.25 mg/mouse18 30 60% 9.40 × 10-6DBaeP F 0.25 mg/mouse10 27 37% 0.006DBelP F 0.25 mg/mouse0 29 0% 0.25DBahP F 0.25 mg/mouse21 29 72% 1.30 × 10-7DBaiP F 0.25 mg/mouse12 30 40% 0.002AA F 0.25 mg/mouse2 29 7% 0.68BghiP F 0.25 mg/mouse2 27 7% 0.65N23eP F 0.25 mg/mouse9 30 30% 0.02IP F 0.25 mg/mouse5 30 17% 0.2113650 Cavalieri etal., 1981bInitiation Mice Papilloma Acetone/TPAF 0 μmol 3 29 10%BaP F 0.2 μmol 12 30 40% 0.009CPcdP F 0.2 μmol 1 30 3% 0.29CPcdP F 0.6 μmol 9 29 31% 0.05CPcdP F 1.8 μmol 6 29 21% 0.24 0.14ACEP F 0.2 μmol 0 30 0% 0.11ACEP F 0.6 μmol 1 30 3% 0.29C-7 DRAFT – DO NOT CITE OR QUOTETable C-1. Dermal bioassays: dose response information for incidence dataRecordnumberReferenceStudytype SpeciesTumortype PAH SexDoseofPAHDoseunitsNumber ofanimalswith tumorsNumberofanimalsin group% tumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Fisher'sexactp-valueCochranArmitagetrend testp-value CommentsACEP F 1.8 μmol 4 30 13% 0.52 0.1815700 Rice et al.,1988Initiation Mice UnspecifiedAcetone F 0 μmol 1 20 5%BaP F 0.1 μmol 17 19 89% <0.005CH F 0.15 μmol 5 20 25% <0.05CH F 0.5 μmol 18 20 90% <0.005CH F 1.5 μmol 19 20 95% <0.005 6.39 × 10-9CPdefC(4,5-MC)F 0.15 μmol 13 20 65% <0.005CPdefC(4,5-MC)F 0.5 μmol 19 19 100% <0.005CPdefC(4,5-MC)F 1.5 μmol 19 19 100% <0.005 1.90 × 10-7BbcAC(1,12-MBA)F 0.5 μmol 15 20 75% <0.005BbcAC(1,12-MBA)F 2 μmol 18 20 90% <0.005BbcAC(1,12-MBA)F 4 μmol 18 20 90% <0.005 3.03 × 10-61C-8 DRAFT – DO NOT CITE OR QUOTE1Table C-2. Dermal bioassays: dose-response information for tumor multiplicityRecordnumber ReferenceStudytype SpeciesTumortype PAH SexDose ofPAH Dose unitsNumber ofanimalswithtumorsNumberofanimalsingroup%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysisFisher'sexactp-valueMeannumbertumors/animalCommentsComplete carcinogenicity13640 Cavalieri et al.,1983CompleteMice Papilloma,adenoma,carcinomaAcetoneF 0 nmol 0 29 0% 0 Number tumors peranimal at riskcalculatedBaP F 2.2 nmol 2 30 7% >0.05 0.07BaP F 6.6 nmol 2 28 7% >0.05 0.07BaP F 20 nmol 17 30 57% <0.001 1.5CPcdP F 22.2 nmol 2 29 7% >0.05 0.07CPcdP F 66.6 nmol 2 29 7% >0.05 0.07CPcdP F 200 nmol 24 29 83% <0.001 2.4513650 Cavalieri et al.,1981bCompleteMice PrimarilysquamouscellcarcinomaAcetoneUS 0 μmol/application0 30 0% 0 Number tumors peranimal at riskcalculatedBaP US 0.2 μmol/application30 30 100% <0.001 1.5CPcdP US 0.2 μmol/application17 30 57% <0.001 0.8CPcdP US 0.6 μmol/application11 30 37% <0.001 0.5CPcdP US 1.8 μmol/application7 30 23% 0.0053 0.4ACEP US 0.2 μmol/application0 30 0% >0.05 0ACEP US 0.6 μmol/application1 30 3% >0.05 0.03ACEP US 1.8 μmol/application1 30 3% >0.05 0.03Initiation630 LaVoie et al.,1982Initiation Mice PrimarilysquamouscellpapillomaAcetone/TPAF 0 μg/mouse 0 20 0% 0BaP F 30 μg/mouse 17 20 85% <0.001 4.9BbF F 10 μg/mouse 9 20 45% <0.001 0.9BbF F 30 μg/mouse 12 20 60% <0.001 2.3C-9 DRAFT – DO NOT CITE OR QUOTETable C-2. Dermal bioassays: dose-response information for tumor multiplicityRecordnumber ReferenceStudytype SpeciesTumortype PAH SexDose ofPAH Dose unitsNumber ofanimalswithtumorsNumberofanimalsingroup%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysisFisher'sexactp-valueMeannumbertumors/animalCommentsBbF F 100 μg/mouse 16 20 80% <0.001 7.1BjF F 30 μg/mouse 6 20 30% 0.01 0.6BjF F 100 μg/mouse 11 20 55% <0.001 1.9BjF F 1,000 μg/mouse 19 20 95% <0.001 7.2BkF F 30 μg/mouse 1 20 5% >0.05 0.1BkF F 100 μg/mouse 5 20 25% 0.02 0.4BkF F 1,000 μg/mouse 15 20 75% <0.001 2.818570 Hecht et al., 1974Initiation Mice UnspecifiedAcetoneF 0 mg/animal 0 20 0% 0 Number survivingnot reported forcontrols; initialgroup size usedhere. Numbertumors per animalat risk calculatedBaP F 0.05 mg/animal 6 20 30% 0.01 0.5CH F 1 mg/animal 11 19 61% <0.001 121420 Slaga et al., 1980Initiation Mouse Papilloma Control F 0 nmol 2 29 6%0.1 Different controlsused for eachchemical exceptDBacA and BePControl F 0 nmol 3 30 10% 0.2Control F 0 nmol 3 30 10% 0.1Control F 0 nmol 2 29 6% 0.1ControlPOOLEDF 0 nmol 10 119 8% 0.13BaP F 200 nmol 20 30 67% <0.001 2.2BeP F 2,000 nmol 5 29 17% >0.05 0.2CH F 2,000 nmol 21 29 73% <0.001 1.6DBacA F 2,000 nmol 8 28 27% >0.05 0.5DBahA F 100 nmol 15 29 50% <0.001 1.415640 Raveh et al.,1982Initiation Mice Papilloma Control F 0 μg 3 29 10%0.2BaP F 10 μg 17 29 58% <0.001 1.3BaP F 25 μg 21 28 76% <0.001 3.8BaP F 50 μg 24 28 87% <0.001 6.2BaP F 100 μg 27 27 100% <0.001 8.8C-10 DRAFT – DO NOT CITE OR QUOTETable C-2. Dermal bioassays: dose-response information for tumor multiplicityRecordnumber ReferenceStudytype SpeciesTumortype PAH SexDose ofPAH Dose unitsNumber ofanimalswithtumorsNumberofanimalsingroup%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysisFisher'sexactp-valueMeannumbertumors/animalCommentsBaP F 200 μg 26 26 100% <0.001 9CPcdP F 10 μg 3 30 11% >0.05 0.1CPcdP F 100 μg 11 29 39% 0.01 0.4CPcdP F 200 μg 16 28 57% <0.001 0.913650 Cavalieri et al.,1981Initiation Mice PapillomaAcetone/TPAF 0 μmol 3 29 10% 0.14BaP F 0.2 μmol 12 30 40% 0.009 1.2CPcdP F 0.2 μmol 1 30 3% >0.05 0.03CPcdP F 0.6 μmol 9 29 31% 0.05 0.31CPcdP F 1.8 μmol 6 29 21% >0.05 0.31ACEP F 0.2 μmol 0 30 0% >0.05 0ACEP F 0.6 μmol 1 30 3% >0.05 0.03ACEP F 1.8 μmol 4 30 13% >0.05 0.1321410 Slaga et al., 1978Initiation Mice PapillomasAcetone/TPAF 0 μmol 2 29 6% 0.1BaP F 0.2 μmol 27 29 92% <0.001 5.3BaA F 2 μmol 17 30 57% <0.001 1.216310 Weyand et al.,1992Initiation Mice UnspecifiedAcetoneUS 0 μmol 1 21 5% 0.05BaP US 0.01 μmol 24 24 100% <0.01 4.08BjF US 0.3 μmol 11 20 55% <0.01 1.75BjF US 1 μmol 21 24 88% <0.01 4.08BjF US 2 μmol 24 24 100% <0.01 7.1710200 El-Bayoumy etal., 1982Initiation Mice PrimarilysquamouscellpapillomaAcetoneF 0 mg/mouse 1 20 5% 0.1BaP F 0.05 mg/mouse 18 20 90% <0.01 7.1CH F 1 mg/mouse 20 20 100% <0.01 7.7Pery F 1 mg/mouse 1 20 5% 0.1Pyr F 1 mg/mouse 4 20 20% 0.224300 Rice et al., 1985 Initiation Mice UnspecifiedAcetoneF 0 mg/mouse 2 25 8% 0.12 Mean number oftumors/ animaldigitally estimatedfrom Figure 2 androunded to evennumber tumorsBaP F 0.3 mg/mouse 24 25 96% <0.001 8.04C-11 DRAFT – DO NOT CITE OR QUOTETable C-2. Dermal bioassays: dose-response information for tumor multiplicityRecordnumber ReferenceStudytype SpeciesTumortype PAH SexDose ofPAH Dose unitsNumber ofanimalswithtumorsNumberofanimalsingroup%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysisFisher'sexactp-valueMeannumbertumors/animalCommentsCH F 1 mg/mouse 23 25 92% <0.001 5CPdefC F 1 mg/mouse 24 24 100% <0.001 5.63 Number reported intext13660 Cavalieri et al.,1991Initiation Mice PrimarilypapillomaAcetoneF 0 nmol 0 24 0% 0 16 Wk experimentBaP F 33.3 nmol 10 23 43% <0.001 0.65BaP F 100 nmol 17 24 71% <0.001 2.75BaP F 300 nmol 21 23 91% <0.001 5.22DBalP F 33.3 nmol 23 24 96% <0.001 6.75DBalP F 100 nmol 22 24 92% <0.001 7.92DBalP F 300 nmol 24 24 100% <0.001 8.513660 Cavalieri et al.,1991Initiation Mice PrimarilypapillomaAcetoneF 0 nmol 0 24 0% 0 27 Wk experimentBaP F 4 nmol 1 24 4% >0.05 0.04BaP F 20 nmol 10 24 42% <0.001 0.75BaP F 100 nmol 22 24 92% <0.001 3.42DBalP F 4 nmol 22 24 92% <0.001 6.96DBalP F 20 nmol 20 24 83% <0.001 5.29DBalP F 100 nmol 20 24 83% <0.001 3.2916440 Wood et al., 1980Initiation Mice PapillomasAcetoneF 0 μmol 3 30 10% 0.1 Number tumors peranimal at riskcalculatedBaP F 0.1 μmol 20 30 68% <0.05 2BaP F 0.4 μmol 22 30 73% <0.05 4.6Pyr F 0.1 μmol 4 30 14% >0.05 0.14Pyr F 0.4 μmol 3 30 10% >0.05 0.1CPcdP F 0.1 μmol 3 30 10% >0.05 0.1CPcdP F 0.4 μmol 6 30 21% >0.05 0.2918680 Hoffmann et al.,1972Initiation Mice PapillomasAcetoneF 0 mg 1 30 3% 0.03BaP F 0.05 mg 19 29 66% <0.001 2.3FA F 1 mg 1 29 3% >0.05 0.0324800 Nesnow et al.,1984Initiation Mice PapillomasAcetoneM 0 nmol 0 20 0% 0AcetoneF 0 nmol 1 19 5% 0.05BaP M 200 nmol 12 18 67% <0.001 1.4BaP F 200 nmol 10 19 53% 0.0015 1.5BeAC M 50 nmol 4 20 20% >0.05 0.25C-12 DRAFT – DO NOT CITE OR QUOTETable C-2. Dermal bioassays: dose-response information for tumor multiplicityRecordnumber ReferenceStudytype SpeciesTumortype PAH SexDose ofPAH Dose unitsNumber ofanimalswithtumorsNumberofanimalsingroup%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysisFisher'sexactp-valueMeannumbertumors/animalCommentsBeAC F 50 nmol 4 20 20% >0.05 0.25BeAC M 100 nmol 4 20 20% >0.05 0.4BeAC F 100 nmol 7 19 37% 0.02 0.53BeAC M 250 nmol 12 20 60% <0.001 1.3BeAC F 250 nmol 10 19 53% <0.001 1.1BeAC M 500 nmol 15 20 75% <0.001 1.9BeAC F 500 nmol 8 18 44% 0.007 1.2BeAC M 1,000 nmol 16 18 89% <0.001 3.1BeAC F 1,000 nmol 18 20 90% <0.001 2.2BlAC M 50 nmol 12 20 60% <0.001 1.4BlAC F 50 nmol 13 20 65% <0.001 1.1BlAC M 100 nmol 16 17 94% <0.001 2.3BlAC F 100 nmol 18 19 95% <0.001 3.1BlAC M 250 nmol 21 21 100% <0.001 8.4BlAC F 250 nmol 19 21 91% <0.001 4.7BlAC M 500 nmol 16 16 100% <0.001 10.8BlAC F 500 nmol 20 21 95% <0.001 6.6BlAC M 1,000 nmol 19 20 95% <0.001 8.7BlAC F 1,000 nmol 20 20 100% <0.001 10.81C-13 DRAFT – DO NOT CITE OR QUOTE1Table C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueComments17560 Busby et al.,1989Mice IntraperitonealLung Adenoma +adeno-carcinomaDMSO M 0 μg(total)13 91 0.14 Stats reported forcombined M and Fonly for each doseand treatmentcompared to controlnot individual sexes.Lung Adenoma +adenocarcinomaDMSO F 0 μg(total)7 101 0.07Lung Adenoma +adenocarcinomaBaP M 59.5 μg(total)13 28 0.46 7.2 × 10-4Lung Adenoma +adenocarcinomaBaP F 59.5 μg(total)19 27 0.70 3.96 × 10-11Lung Adenoma +adenocarcinomaPyr M 86.1 μg(total)4 23 0.17 4.60 × 10-1Lung Adenoma +adenocarcinomaPyr F 86.1 μg(total)1 28 0.04 4.50 × 10-1Lung Adenoma +adenocarcinomaPyr M 1,750μg(total)2 27 0.07 2.80 × 10-13.13 × 10-1Lung Adenoma +adenocarcinomaPyr F 1,750μg(total)3 26 0.12 3.30 × 10-13.50 × 10-1Lung Adenoma +adenocarcinomaFA M 257.6μg(total)5 23 0.22 2.80 × 10-4Lung Adenoma +adenocarcinomaFA F 257.6μg(total)9 29 0.31 1.65 × 10-3C-14 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLung Adenoma +adenocarcinomaCH M 6.3 μg(total)2 27 0.07 2.80 × 10-1Lung Adenoma +adenocarcinomaCH F 6.3 μg(total)3 29 0.10 3.90 × 10-1Lung Adenoma +adenocarcinomaCH M 210 μg(total)3 20 0.15 5.85 × 10-18.03 × 10-1Lung Adenoma +adenocarcinomaCH F 210 μg(total)0 29 0.00 1.60 × 10-11.28 × 10-1640 LaVoie etal., 1987Mice IntraperitonealLung Adenoma DMSO M 0 μmol/mouse0 17 0Lung Adenoma DMSO F 0 μmol/mouse0 18 0Lung Adenoma BaP M 1.1 μmol/mouse14 17 0.82 <0.005Lung Adenoma BaP F 1.1 μmol/mouse9 14 0.64Lung Adenoma BbF M 0.5 μmol/mouse2 15 0.13 >0.05Lung Adenoma BbF F 0.5 μmol/mouse3 17 0.18 >0.05Lung Adenoma BjF M 1.1 μmol/mouse11 21 0.52 <0.005Lung Adenoma BjF F 1.1 μmol/mouse4 18 0.22 <0.05Lung Adenoma BkF M 2.1 μmol/mouse1 16 0.06 >0.05Lung Adenoma BkF F 2.1 μmol/mouse3 18 0.17 >0.05C-15 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLung Adenoma IP M 2.1 μmol/mouse1 11 0.09Lung Adenoma IP F 2.1 μmol/mouse0 9 0Liver Adenoma +hepatomaDMSO M 0 μmol/mouse1 17 0.06 Adenoma andhepatoma alsoreported separately.None of animalssurviving 35 wksLiver Adenoma +hepatomaDMSO F 0 μmol/mouse0 18 0Liver Adenoma +hepatomaBaP M 1.1 μmol/mouse13 17 0.76 <0.005Liver Adenoma +hepatomaBaP F 1.1 μmol/mouse0 14 0Liver Adenoma +hepatomaBbF M 0.5 μmol/mouse8 15 0.53 <0.005Liver Adenoma +hepatomaBbF F 0.5 μmol/mouse0 17 0Liver Adenoma +hepatomaBjF M 1.1 μmol/mouse11 21 0.52 <0.005Liver Adenoma +hepatomaBjF F 1.1 μmol/mouse0 18 0Liver Adenoma +hepatomaBkF M 2.1 μmol/mouse3 16 0.19 >0.05Liver Adenoma +hepatomaBkF F 2.1 μmol/mouse0 18 0Liver Adenoma +hepatomaIP M 2.1 μmol/mouse0 11 0Liver Adenoma +hepatomaIP F 2.1 μmol/mouse0 9 0Liver orlungAdenoma +hepatomaDMSO M 0 μmol/mouse1 17 0.06C-16 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLiver orlungAdenoma +hepatomaDMSO F 0 μmol/mouse0 18 0Liver orlungAdenoma +hepatomaBaP M 1.1 μmol/mouse13 17 0.76Liver orlungAdenoma +hepatomaBaP F 1.1 μmol/mouse9 14 0.64Liver orlungAdenoma +hepatomaBbF M 0.5 μmol/mouse8 15 0.53Liver orlungAdenoma +hepatomaBbF F 0.5 μmol/mouse3 17 0.18Liver orlungAdenoma +hepatomaBjF M 1.1 μmol/mouse17 21 0.81Liver orlungAdenoma +hepatomaBjF F 1.1 μmol/mouse4 18 0.22Liver orlungAdenoma +hepatomaBkF M 2.1 μmol/mouse3 16 0.19Liver orlungAdenoma +hepatomaBkF F 2.1 μmol/mouse3 18 0.17Liver orlungAdenoma +hepatomaIP M 2.1 μmol/mouse1 11 0.09Liver orlungAdenoma +hepatomaIP F 2.1 μmol/mouse0 9 07510 LaVoie etal., 1994mice intraperitonealLung Total DMSO M 0 μmol/mouse5 29 0.17 Surv to 1 yrLung Total DMSO F 0 μmol/mouse4 34 0.12Lung Total BaP M 1.1 μmol/mouse24 32 0.75 <0.001Lung Total BaP F 1.1 μmol/mouse17 20 0.85 <0.001Lung Total FA M 3.46 μmol/mouse12 28 0.43 <0.05C-17 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLung Total FA F 3.46 μmol/mouse11 31 0.35 <0.05Lung Total FA M 17.3 μmol/mouse11 17 0.65 <0.005 2.84 × 10-3Lung Total FA F 17.3 μmol/mouse25 29 0.86 <0.001 2.18 × 10-9Liver Foci +adenoma +carcinomaDMSO M 0 μmol/mouse5 29 0.17 Foci, adenomas,carcinomas alsoreported separatelyLiver Foci +adenoma +carcinomaDMSO F 0 μmol/mouse2 34 0.06Liver Foci +adenoma +carcinomaBaP M 1.1 μmol/mouse27 32 0.84 <0.001Liver Foci +adenoma +carcinomaBaP F 1.1 μmol/mouse2 20 0.10 >0.05Liver Foci +adenoma +carcinomaFA M 3.46 μmol/mouse18 28 0.64 <0.001Liver Foci +adenoma +carcinomaFA F 3.46 μmol/mouse0 31 0Liver Foci +adenoma +carcinomaFA M 17.3 μmol/mouse17 17 1.00 <0.001 5.10 × 10-7Liver Foci +adenoma +carcinomaFA F 17.3 μmol/mouse2 29 0.07 5.47 × 10-122510 Wislocki etal., 1986Mice IntraperitonealLiver Adenoma +carcinomaDMSO M 0 nmol 2 28 0.07 Animals survivingthru weaningC-18 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLiver Adenoma +carcinomaDMSO F 0 nmol 0 31 0 0Liver Adenoma +carcinomaDMSO M 0 nmol 5 45 0.11 This group started10 wks after othergroupsLiver Adenoma +carcinomaDMSO F 0 nmol 0 34 0 This group started10 wks after othergroupsLiver Adenoma +carcinomaDMSOPOOLEDM 0 nmol 7 73 0.09Liver Adenoma +carcinomaDMSOPOOLEDF 0 nmol 0 65 0Liver Adenoma +carcinomaBaP M 560 nmol 18 37 0.49 <0.05Liver Adenoma +carcinomaBaP F 560 nmol 0 27 0Liver Adenoma +carcinomaCH M 700 nmol 10 35 0.29 <0.05 This group started10 wks after othergroupsLiver Adenoma +carcinomaCH F 700 nmol 0 33 0 This group started10 wks after othergroupsLiver Adenoma +carcinomaCH M 2,800nmol 14 34 0.41 <0.05 6 × 10-3Liver Adenoma +carcinomaCH F 2,800nmol 0 24 0 1Liver Adenoma +carcinomaBaA M 2,800nmol 31 39 0.79 <0.05Liver Adenoma +carcinomaBaA F 2,800nmol 0 32 0C-19 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLung Adenoma +carcinomaDMSO M 0 nmol 1 28 0.04Lung Adenoma +carcinomaDMSO F 0 nmol 0 31 0Lung Adenoma +carcinomaDMSO M 0 nmol 4 45 0.09 This group started10 wks after othergroupsLung Adenoma +carcinomaDMSO F 0 nmol 2 34 0.06 This group started10 wks after othergroupsLung Adenoma +carcinomaDMSOpooledM 0 nmol 5 73 0.07Lung Adenoma +carcinomaDMSOpooledF 0 nmol 2 65 0.03Lung Adenoma +carcinomaBaP M 560 nmol 13 37 0.35 <0.05Lung Adenoma +carcinomaBaP F 560 nmol 13 27 0.48 <0.05Lung Adenoma +carcinomaCH M 700 nmol 6 35 0.17 This group started10 wks after othergroupsLung Adenoma +carcinomaCH F 700 nmol 2 33 0.06 This group started10 wks after othergroupsLung Adenoma +carcinomaCH M 2,800nmol 7 34 0.21 <0.05 1.1 × 10-1Lung Adenoma +carcinomaCH F 2,800nmol 1 24 0.04 5.6 × 10-1Lung Adenoma +carcinomaBaA M 2,800nmol 6 39 0.15Lung Adenoma +carcinomaBaA F 2,800nmol 6 32 0.19 <0.05C-20 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLymphaticsystemLymphoma DMSO M 0 nmol 1 28 0.04LymphaticsystemLymphoma DMSO F 0 nmol 1 31 0.03LymphaticsystemLymphoma DMSO M 0 nmol 0 45 0 This group started10 wks after othergroupsLymphaticsystemLymphoma DMSO F 0 nmol 0 34 0 This group started10 wks after othergroupsLymphaticsystemLymphoma BaP M 560 nmol 2 37 0.05LymphaticsystemLymphoma BaP F 560 nmol 4 27 0.15LymphaticsystemLymphoma CH M 700 nmol 3 35 0.09 <0.05 This groupstarted10 wks after othergroupsLymphaticsystemLymphoma CH F 700 nmol 1 33 0.03 This group started10 wks after othergroupsLymphaticsystemLymphoma CH M 2,800nmol 0 34 0 2.2 × 10-1LymphaticsystemLymphoma CH F 2,800nmol 0 24 0 3.9 × 10-1LymphaticsystemAdenoma +carcinomaBaA M 2,800nmol 1 39 0.03C-21 DRAFT – DO NOT CITE OR QUOTETable C-3. Intraperitoneal bioassays: dose response information for incidence dataSRC StatisticalAnalysisRecordnumberReference SpeciesExposurerouteTargetorganTumor typePAH SexDoseDoseunits No. of animals with tumors No. of animals in group % Tumor bearing animalsResults ofauthors'statisticalanalysis(p-value)Fisher'sExactp-valueCochranArmitagetrend testp-valueCommentsLymphaticsystemAdenoma +carcinomaBaA F 2,800nmol 3 32 0.091C-22 DRAFT – DO NOT CITE OR QUOTE1Table C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicityRecordnumber Reference SpeciesExposurerouteTargetorgan Tumor typePAH Sex Dose Dose unitsNumberof animalswithtumorsNumberof animalsin group%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysis(Fisher'sexactp-value)Meannumbertumors/animalStddeviationof meanResults ofSRCstatisticalanalysis(t-testp-value)Comments17560 Busby et al., 1989 Mice IntraperitonealLung Adenoma+adenocarcinomaDMSO M 0 μg (total) 13 91 0.14 0.15 0.38 Statsreported forcombined Mand FLung Adenoma+adenocarcinomaDMSO F 0 μg (total) 7 101 0.07 0.08 0.30Lung Adenoma+adenocarcinomaBaP M 59.5 μg (total) 13 28 0.46 <0.001 0.71 1.01<0.001Lung Adenoma+adenocarcinomaBaP F 59.5 μg (total) 19 27 0.70 <0.001 1.19 1.09<0.001Lung Adenoma+adenocarcinomaPyr M 86.1 μg (total) 4 23 0.17 >0.05 0.17 0.38 >0.05Lung Adenoma+adenocarcinomaPyr F 86.1 μg (total) 1 28 0.04 >0.05 0.04 0.21 >0.05Lung Adenoma+adenocarcinomaPyr M 1,750 μg (total) 2 27 0.07 >0.05 0.07 0.26 >0.05Lung Adenoma+adenocarcinomaPyr F 1,750 μg (total) 3 26 0.12 >0.05 0.12 0.31 >0.05Lung Adenoma+adenocarcinomaFA M 257.6 μg (total) 5 23 0.22 >0.05 0.22 0.43 >0.05Lung Adenoma+adenocarcinomaFA F 257.6 μg (total) 9 29 0.31 0.00165 0.41 0.70<0.0001Lung Adenoma+adenocarcinomaCH M 6.3 μg (total) 2 27 0.07 >0.05 0.07 0.26 >0.05Lung Adenoma+adenocarcinomaCH F 6.3 μg (total) 3 29 0.10 >0.05 0.1 0.32 >0.05Lung Adenoma+adenocarcinomaCH M 210 μg (total) 3 20 0.15 >0.05 0.15 0.36 >0.05Lung Adenoma+adenocarcinomaCH F 210 μg (total) 0 29 0.00 >0.05 0 0.00 >0.057510 LaVoie et al., 1994 Mice intraperitonealLung Total DMSO M 0 μmol/mouse 5 29 0.17 0.17 Survived to1 yrLung Total DMSO F 0 μmol/mouse 4 34 0.12 0.15Lung Total BaP M 1.1 μmol/mouse 24 32 0.75 <0.001 4.3Lung Total BaP F 1.1 μmol/mouse 17 20 0.85 <0.001 3.55Lung Total FA M 3.46 μmol/mouse 12 28 0.43 <0.05 0.64C-23 DRAFT – DO NOT CITE OR QUOTETable C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicityRecordnumber Reference SpeciesExposurerouteTargetorgan Tumor typePAH Sex Dose Dose unitsNumberof animalswithtumorsNumberof animalsin group%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysis(Fisher'sexactp-value)Meannumbertumors/animalStddeviationof meanResults ofSRCstatisticalanalysis(t-testp-value)CommentsLung Total FA F 3.46 μmol/mouse 11 31 0.35 <0.05 0.35Lung Total FA M 17.3 μmol/mouse 11 17 0.65 <0.005 1.12Lung Total FA F 17.3 μmol/mouse 25 29 0.86 <0.001 2.45Liver Foci +adenoma +carcinomaDMSO M 0 μmol/mouse 5 29 0.17 0.41Liver Foci +adenoma +carcinomaDMSO F 0 μmol/mouse 2 34 0.06 0.06 Tumorcountappears tobe error inpublicationLiver Foci +adenoma +carcinomaBaP M 1.1 μmol/mouse 27 32 0.84 <0.001 4.53Liver Foci +adenoma +carcinomaBaP F 1.1 μmol/mouse 2 20 0.10 >0.05 0.3Liver Foci +adenoma +carcinomaFA M 3.46 μmol/mouse 18 28 0.64 <0.001 1.86Liver Foci +adenoma +carcinomaFA F 3.46 μmol/mouse 0 31 0 0Liver Foci +adenoma +carcinomaFA M 17.3 μmol/mouse 17 17 1.00 <0.001 7.53Liver Foci +adenoma +carcinomaFA F 17.3 μmol/mouse 2 29 0.07 0.0722510 Wislocki et al., 1986 Mice intraperitonealLiver Adenoma +carcinomaDMSO M 0 nmol 2 28 0.07 0.07 AnimalssurvivingthruweaningLiver Adenoma +carcinomaDMSO F 0 nmol 0 31 0 0Liver Adenoma +carcinomaDMSO M 0 nmol 5 45 0.11 0.11 This groupstarted10 wks afterother groupsLiver Adenoma +carcinomaDMSO F 0 nmol 0 34 0 0 This groupstarted10 wks afterother groupsLiver Adenoma +carcinomaDMSOPOOLEDM 0 nmol 7 73 0.09 0.096Liver Adenoma +carcinomaDMSOPOOLEDF 0 nmol 0 65 0 0C-24 DRAFT – DO NOT CITE OR QUOTETable C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicityRecordnumber Reference SpeciesExposurerouteTargetorgan Tumor typePAH Sex Dose Dose unitsNumberof animalswithtumorsNumberof animalsin group%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysis(Fisher'sexactp-value)Meannumbertumors/animalStddeviationof meanResults ofSRCstatisticalanalysis(t-testp-value)CommentsLiver Adenoma +carcinomaBaP M 560 nmol 18 37 0.49 <0.05 1.46Liver Adenoma +carcinomaBaP F 560 nmol 0 27 0 >0.05 0Liver Adenoma +carcinomaPyr M 200 nmol 0 29 0 >0.05 0Liver Adenoma +carcinomaPyr F 200 nmol 0 31 0 >0.05 0Liver Adenoma +carcinomaPyr M 700 nmol 3 25 0.12 >0.05 0.12 This groupstarted10 wks afterother groupsLiver Adenoma +carcinomaPyr F 700 nmol 0 49 0 >0.05 0 This groupstarted10 wks afterother groupsLiver Adenoma +carcinomaPyr M 2,800 nmol 3 14 0.21 >0.05 0.21Liver Adenoma +carcinomaPyr F 2,800 nmol 0 18 0 >0.05 0Liver Adenoma +carcinomaCH M 700 nmol 10 35 0.29 <0.05 0.86 This groupstarted10 wks afterother groupsLiver Adenoma +carcinomaCH F 700 nmol 0 33 0 >0.05 0 This groupstarted10 wks afterother groupsLiver Adenoma +carcinomaCH M 2,800 nmol 14 34 0.41 <0.05 1.03Liver Adenoma +carcinomaCH F 2,800 nmol 0 24 0 >0.05 0Liver Adenoma +carcinomaBaA M 2,800 nmol 31 39 0.79 <0.05 2.38Liver Adenoma +carcinomaBaA F 2,800 nmol 0 32 0 >0.05 013610 Busby et al., 1984 mice intraperitonealLung Adenoma +carcinomaDMSO M 0 mg (total) 1 27 0.04 0.04 0.21Lung Adenoma +carcinomaDMSO F 0 mg (total) 4 28 0.14 0.14 0.37Lung Adenoma +carcinomaBaP M 0.28 mg (total) 24 25 0.96 <0.001 4.32 3.5 <0.001Lung Adenoma +carcinomaBaP F 0.28 mg (total) 25 27 0.93 <0.001 3.7 3.10 <0.001Lung Adenoma +carcinomaBaP M 1.4 mg (total) 16 20 0.80 <0.001 10.15 13.0<0.001 No model fitLung Adenoma +carcinomaBaP F 1.4 mg (total) 21 24 0.88 <0.001 4.25 4.70 <0.001 No model fitLung Adenoma +carcinomaFA M 0.7 mg (total) 7 31 0.23 0.0412 0.29 0.84 >0.05C-25 DRAFT – DO NOT CITE OR QUOTETable C-4. Intraperitoneal bioassays: dose-response information for tumor multiplicityRecordnumber Reference SpeciesExposurerouteTargetorgan Tumor typePAH Sex Dose Dose unitsNumberof animalswithtumorsNumberof animalsin group%TumorbearinganimalsResults ofauthors'statisticalanalysis(p-value)Results ofSRCstatisticalanalysis(Fisher'sexactp-value)Meannumbertumors/animalStddeviationof meanResults ofSRCstatisticalanalysis(t-testp-value)CommentsLung Adenoma +carcinomaFA F 0.7 mg (total) 3 20 0.15 >0.05 0.15 0.49 >0.05Lung Adenoma +carcinomaFA M 3.5 mg (total) 20 27 0.74 <0.001 1.52 1.66 <0.001 NonconstantvarianceLung Adenoma +carcinomaFA F 3.5 mg (total) 8 21 0.38 >0.05 0.52 0.82 0.0343 NSincidence;nonconstantvariance24590 Nesnow et al., 1998b mice intraperitonealLung NS Control M 0 mg/kg 6 20 0.30 0.53 0.72 Pooledcontrolsfrom dataprovided byNesnow.Lung NS BaP M 5 mg/kg 6 20 0.30 >0.05 0.45 0.80 >0.05Lung NS BaP M 10 mg/kg 7 17 0.41 >0.05 0.53 0.78 >0.05Lung NS BaP M 50 mg/kg 19 19 1.00 <0.001 4.372.74 <0.001Lung NS BaP M 100 mg/kg 16 16 1.00 0.0018 12.75 4.28 <0.001Lung NS BaP M 200 mg/kg 24 24 1.00 <0.001 32.96 10.23 <0.001Lung NS BbF M 10 mg/kg 9 18 0.50 >0.05 0.67 0.75 >0.05Lung NS BbF M 50 mg/kg 16 20 0.80 >0.05 2.00 1.82 0.0022 NSincidenceLung NS BbF M 100 mg/kg 20 20 1.00 <0.001 5.303.21 <0.001Lung NS BbF M 200 mg/kg 19 19 1.00 <0.001 6.953.52 <0.001Lung NS CPcdP M 10 mg/kg 8 20 0.40 >0.05 0.550.80 >0.05Lung NS CPcdP M 50 mg/kg 20 20 1.00 <0.001 4.75 2.12 <0.001Lung NS CPcdP M 100 mg/kg 19 19 1.00 <0.001 32.21 15.15 <0.001Lung NS CPcdP M 200 mg/kg 19 19 1.00 <0.001 97.68 28.68 <0.001Lung NS DBahA M 1.25 mg/kg 12 18 0.67 >0.05 1.44 1.46 0.0229 NSincidenceLung NS DBahA M 2.5 mg/kg 18 19 0.95 0.0053 3.05 1.90 <0.001Lung NS DBahA M 5 mg/kg 20 20 1.00 <0.001 13.05 5.99 <0.001Lung NS DBahA M 10 mg/kg 19 19 1.00 <0.001 32.16 10.78 <0.00124590 Nesnow et al., 1998b mice intraperitonealLung NS Control M 0 mg/kg 15 30 0.50 0.67 0.80Lung NS DBalP M 0.3 mg/kg 13 33 0.39 >0.05 0.42 0.56 >0.05Lung NS DBalP M 1.5 mg/kg 33 34 0.97 <0.001 4.32 2.86 <0.001Lung NS DBalP M 3 mg/kg 35 35 1.00 <0.001 7.493.79 <0.001Lung NS DBalP M 6 mg/kg 30 30 1.00 <0.001 16.10 7.26 <0.00111190 Mass et al., 1993 mice intraperitonealLung NS Control US 0 mg/kg 19 34 0.56 0.85 0.9NS BaP US 20 mg/kg 10 16 0.63 >0.05 1 1 >0.05NS BaP US 50 mg/kg 15 16 0.94 0.0065 3.9 2.9<0.001NS BaP US 100 mg/kg 14 14 1.00 0.0017 5.9 3.3<0.001NS BjAC US 20 mg/kg 12 12 1.00 0.0036 60.3 14.6 <0.001NS BjAC US 50 mg/kg 13 13 1.00 0.0025 140.6 21.5 <0.001NS BjAC US 100 mg/kg 14 14 1.00 0.0017 97.6 28.2 <0.00112C-26 DRAFT – DO NOT CITE OR QUOTETable C-5. Lung implantation bioassays: dose response information for incidence dataSRC statistical analysisRecordnumberReference SpeciesTargetorganTumor typePAH DoseDoseunitsNumberofanimalswithtumorsNumberofanimalsin group% TumorbearinganimalsFisher'sexactp-valueCochranArmitage trendtestp-valueComments17940 Deutsch-Wenzel etal., 1983Rat Lung EpidermoidcarcinomaUntreatedcontrol0 mg 0 35 0.00Vehiclecontrol0 mg 0 35 0.00BaP 0.1 mg 4 35 0.11 5.70 × 10-2BaP 0.3 mg 21 35 0.60 6.02 × 10-9BaP 1 mg 33 35 0.94 5.93 × 10-181.57 × 10-17BbF 0.1 mg 0 35 0.00BbF 0.3 mg 1 35 0.03 5 × 10-1BbF 1 mg 9 35 0.26 1 × 10-35.12 × 10-7BeP 0.2 mg 0 35 0.00BeP 1 mg 0 30 0.00BeP 5 mg 1 35 0.03 5 × 10-19.49 × 10-2BjF 0.2 mg 1 35 0.03 5 × 10-1BjF 1 mg 3 35 0.09 1.2 × 10-1BjF 5 mg 18 35 0.51 1.96 × 10-71.28 × 10-11BkF 0.16 mg 0 35 0.00BkF 0.83 mg 3 31 0.10 1 × 10-1BkF 4.15 mg 12 27 0.44 8.05 × 10-61.03 × 10-9IP 0.16 mg 3 35 0.09 1.20 × 10-1IP 0.83 mg 8 35 0.23 2 × 10-3IP 4.15 mg 21 35 0.60 6.02 × 10-92.09 × 10-10AA 0.16 mg 1 35 0.03 5 × 10-1AA 0.83 mg 19 35 0.54 6.4 × 10-81.13 × 10-10BghiP 0.16 mg 0 35 0.00BghiP 0.83 mg 1 35 0.03 1.2 × 10-1BghiP 4.15 mg 4 34 0.12 5.4 × 10-22.47 × 10-3Lung PleomorphicsarcomaUntreatedcontrol0 mg 0 35 0.00Vehiclecontrol0 mg 0 35 0.00C-27 DRAFT – DO NOT CITE OR QUOTETable C-5. Lung implantation bioassays: dose response information for incidence dataSRC statistical analysisRecordnumberReference SpeciesTargetorganTumor typePAH DoseDoseunitsNumberofanimalswithtumorsNumberofanimalsin group% TumorbearinganimalsFisher'sexactp-valueCochranArmitage trendtestp-valueCommentsBaP 0.1 mg 6 35 0.17 1.2 × 10-2BaP 0.3 mg 2 35 0.06 2.5 × 10-1BaP 1 mg 0 35 0.00 1.36 × 10-1BbF 0.1 mg 1 35 0.03 1.2 × 10-1BbF 0.3 mg 2 35 0.06 2.5 × 10-1BbF 1 mg 4 35 0.11 6. × 10-27.55 × 10-3BeP 0.2 mg 0 35 0.00BeP 1 mg 1 30 0.03BeP 5 mg 0 35 0.00BjF 0.2 mg 0 35 0.00BjF 1 mg 0 35 0.00BjF 5 mg 0 35 0.00BkF 0.16 mg 0 35 0.00BkF 0.83 mg 0 31 0.00BkF 4.15 mg 0 27 0.00IP 0.16 mg 1 35 0.03 1.2 × 10-1IP 0.83 mg 0 35 0.00IP 4.15 mg 0 35 0.00AA 0.16 mg 0 35 0.00AA 0.83 mg 0 35 0.00BghiP 0.16 mg 0 35 0.00BghiP 0.83 mg 0 35 0.00BghiP 4.15 mg 0 34 0.00Lung Carcinoma+sarcomaUntreatedcontrol0 mg 0 35 0.00Vehiclecontrol0 mg 0 35 0.00BaP 0.1 mg 10 35 0.29 4.63 × 10-4BaP 0.3 mg 23 35 0.66 4.7 × 10-10BaP 1 mg 33 35 0.94 5.9 × 10-193.66 × 10-9C-28 DRAFT – DO NOT CITE OR QUOTETable C-5. Lung implantation bioassays: dose response information for incidence dataSRC statistical analysisRecordnumberReference SpeciesTargetorganTumor typePAH DoseDoseunitsNumberofanimalswithtumorsNumberofanimalsin group% TumorbearinganimalsFisher'sexactp-valueCochranArmitage trendtestp-valueCommentsBbF 0.1 mg 1 35 0.03 1.2 × 10-1BbF 0.3 mg 3 35 0.09 1.2 × 10-1BbF 1 mg 13 35 0.37 3.1 × 10-59.63 × 10-8BeP 0.2 mg 0 35 0.00BeP 1 mg 1 30 0.03BeP 5 mg 1 35 0.03 1.2 × 10-13.23 × 10-1BjF 0.2 mg 1 35 0.03 1.2 × 10-1BjF 1 mg 3 35 0.09 1.20 × 10-1BjF 5 mg 18 35 0.51 1.96 × 10-71.28 × 10-11BkF 0.16 mg 0 35 0.00BkF 0.83 mg 3 31 0.10 1 × 10-1BkF 4.15 mg 12 27 0.44 8.05 × 10-41.03 × 10-9IP 0.16 mg 4 35 0.11 6 × 10-2IP 0.83 mg 8 35 0.23 2 × 10-3IP 4.15 mg 21 35 0.60 6.02 × 10-97.56 × 10-10AA 0.16 mg 1 35 0.03AA 0.83 mg 19 35 0.54 6.4 × 10-81.13 × 10-10BghiP 0.16 mg 0 35 0.00BghiP 0.83 mg 1 35 0.03BghiP 4.15 mg 4 34 0.12 5.4 × 10-22.47 × 10-322000 Wenzel-Hartung etal., 1990Rat Lung Carcinoma Untreatedcontrol0 mg/animal0 35 0.00 ED10, relativepotencies reportedVehiclecontrol0 mg/animal0 35 0.00BaP 0.03 mg/animal3 35 0.09 1.2 × 10-1BaP 0.1 mg/animal11 35 0.31 1.93 × 10-4BaP 0.3 mg/animal27 35 0.77 1.29E × 10-128.85 × 10-15C-29 DRAFT – DO NOT CITE OR QUOTETable C-5. Lung implantation bioassays: dose response information for incidence dataSRC statistical analysisRecordnumberReference SpeciesTargetorganTumor typePAH DoseDoseunitsNumberofanimalswithtumorsNumberofanimalsin group% TumorbearinganimalsFisher'sexactp-valueCochranArmitage trendtestp-valueCommentsPH 1 mg/animal0 35 0.00PH 3 mg/animal0 35 0.00PH 10 mg/animal1 35 0.03 5 × 10-11CH 1 mg/animal5 35 0.14 2.7 × 10-2CH 3 mg/animal10 35 0.29 4.63 × 10-47.96 × 10-4DBahA 0.1 mg/animal20 35 0.57 2.01 × 10-81

### C-30 DRAFT – DO NOT CITE OR QUOTE

Table C-6. In vitro bacterial mutagenicity: data u se

Record
number Reference Data

source Data points Basis for RPF approach Comments

17030 Andrews et al., 1978 Figure 1 Dose (μg) and # of revertant

colonies for DBacA, DBajA, DBahA, AA, BghiP, BeP, BaP Point estimate TA100 with Ar S9

23830 Baker et al., 1980 Table 2 Use data for guinea pig- MC S9

only (column D); dose in μg/plate and # of revertant colonies; BaP, DBaiP, BaA, DBacA, DBahA Point estimate Table 2 TA100 with guinea pig-MC S9; Table 1 data not used, different S9 mix used for each of three experiments

23660 Bartsch et al., 1980 Appendix

table Use data for BaA and BaP;

dose in μmol/plate and mutagenic activity in revertants/μmol. Point estimate. TA100 rat MC S9

17380 Bos et al.,
1988 Table 1 Use TA100 strain only; dose

(μg/plate) and # of revertant colonies/plate for PH, Pyr, BaP Derive point estimate for BaP (use PH control as background); continuous model PH and Pyr using the BaP response as the BMR TA100 with rat Ar
S9

17590 Carver et al., 1986 Figure 1 Use curves for BaP, BaA,

BghiF, and Pery; use 400 μL S9 per plate (last data point on x-axis); each curve is different dose in μg/plate, use hamsterdata; revertants per plate is yaxis Point estimate; use highest dose in hamster, except for perylene (use 10 μg/plate); this is maximal response in hamsters TA100 with hamster Ar S9; multi-dose data but not SD was reported

17630 Cavalieri et al., 1981a Figure 1 DR curves for BaP, CPcdP

(CPEP in fig.), and ACEP (CPAP in fig.); dose as μM, response as mutant fraction x
105 Model as quantal data (mutant fraction reported) TM677 with Ar S9

9620 Chang et al., 2002 Figure 7 DR curves for BghiF, BcPH,

and BaP; dose (μg/plate) and revertants/plate Point estimate; use 5 μg/plate dose for BghiF and BaP; use 10 μg/plate for
BcPH TA100 with rat Ar S9; SD not available from graph (reported for some data points, but not all)

24030 De Flora et al., 1984 Table 2 Table provides potency

estimates as revertants/nmol for BaA, Pery, BaP and BeP Calculate the RPF ratio using the potency estimates provided Determine strain used to calculate potencies; rat Ar S9

18050 Eisenstadt and Gold,
1978 Figure 2B Use TA100 data for BaP and

CPcdP (open circles); dose is 1 μg for CPcdP and 2 μg for BaP (legend); use the same S9 concentration (20 μL/plate) Point estimate; single point data (20 μL S9/plate) TA100 with rat Ar S9; μL S9 that maximizes the BaP response does not produce maximal response for CPcdP

18180 Florin et al., 1980 Table III Use TA100 data for BaA, CH,

and BaP, use TA98 data for Pery, CO, and BaP; dose is indicated as optimal dose (μmoles/plate) and # revertants/ plate 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 Note that data for both TA100 and TA98 strains were used; BaP results were provided for each; rat MC S9

### C-31 DRAFT – DO NOT CITE OR QUOTE

Table C-6. In vitro bacterial mutagenicity: data u se

Record
number Reference Data

source Data points Basis for RPF approach Comments

24080 Gibson et al., 1978 Table 1

(BaP) Table 3 (PAHs) Use data for TA98; in Table 1

use Expt. No.1 for BaP; in Table 3 use data for DBahA, Tphen, BaA, BghiP, CH, FE, Pyr; dose as μg/plate, response as increase in revertants Point estimate; use the doseassociated with the maximum response (if reported as a range, do not use); controls were reported as negative (no mutagenic or toxic response) TA98 with nonenzymatic induction (gamma irradiation); multi-dose data but not SD was reported

14080 Gold and Eisenstadt,
1980 Table 2 Use data for 3- MC induction at

50 μL S9/plate; dose is 4 nmol for BaP and CPcdP, results as revertants/plate Point estimate TA100 using 50 μL of rat MC S9; important to note that maximal response for CPcdP occurred at much lower dose of S9 (5 μL/plate)
18650 Hermann,
1981 Table 1 Table provides potency

estimates as revertants/nmol for BbA, BaA, CH, FA, Tphen, BeP, DBacA, DBahA, BbF, Pery, DBalP, DBaiP, AA, CO; potency of BaP in legend as 100 revertants/nmol Calculate the RPF ratio using the potency estimates provided TA98 with rat Ar S9; potency estimates were calculated from the linear portion of the
DR curve

10670 Johnsen et al., 1997 Figure 2 Use data for PCB microsomes

for BaP, BjAC, BlAC; dose as μg/plate, response as revertants Model to derive EDsd1; need to extract SDs from graph; control response is 113 ± 9 revertants per plate (see legend); add control response to each response for modeling (it was subtracted prior to graphing) TA98 with PCB microsomes

19000 Kaden et al., 1979 Table 1 RPFs calculated for AN, ANL,

Pyr, BbFE, CPcdP, BaA, CH, Tphen, FA, BeP, Pery, BghiP, AA, DBacA, DBahA, DBbeF NA TM677 with Ar S9 and PB S9

24680 Lafleur et al., 1993 Figures

3 and 4 Use DR curves for BaP,

BghiF, CPcdP, CPhiACEA (CPAA), ACEA (AA), CPhiAPA (CPAP), APA (AP); dose as μg/mL, response as mutant fraction (×10 5 ) Model as quantal data (mutant fraction reported) Forward mutation to 8-azaguanine resistance in TM677 with rat AR S9

19320 LaVoie et al., 1979 Table VI Use data for TA98 for BaP,

BeP, and Pery; 10 μg dose and response as revertants/plate Point estimate; use 20 μg for BaP; 10 μg for BeP; and 20 μg for Pery TA98 with rat Ar S9; for BeP and Pery the maximal response was in
TA100

23650 McCann et al., 1975 Table 1 Table provides potency

estimates as revertants/nmol for DBaiP, BaP, BeP, DBacA, DBahA, CH, BaA Calculate the RPF ratio using the potency estimates provided Multiple strains, rat
Ar S9

### C-32 DRAFT – DO NOT CITE OR QUOTE

Table C-6. In vitro bacterial mutagenicity: data u se

Record
number Reference Data

source Data points Basis for RPF approach Comments

20220 Pahlman and Pelkonen,
1987 Table 1 Use data for rat-MC induced

(last column); potency estimates are provided as revertants/nmol for BaA, CH, Tphen, DBacA, DBahA Calculate the RPF ratio using the potency estimates provided TA100 with rat MC
S9

20450 Phillipson and Ioannides,
1989 Figures

2 and 3 Use the curve for hamster S9

(open triangles); data for BaP, DBaiP, BaA, and DBahA, dose as μg/plate, revertants/plate Point estimate; use 10 μg/plate for BaP, DBahA; 20 μg/plate BaA, DBaiP TA100 with hamster S9; multi-dose data but not SD was reported

21000 Sakai et al., 1985 Table 3 Use data for TA97 +S9 for FE,

AC, PH, FA, Ch, Pyr, BaP, BeP, Pery, BghiP, CO; dose μg, response as revertants per plate Point estimate; use 10 μg for AC, PH, FA, BaP, BeP; use 5 μg for FE; use 20 μg for CH, Pyr, BghiP; use 4 μg for Pery; use 100 μg for CO TA97 with rat Ar S9; multi-dose data but not SD was reported

11860 Sangaiah et al., 1983 Figure 2 Use data for BjAC and BaP;

dose as μg/plate, response as revertants/plate Point estimate; use 10 μg/plate for BjAC; use 6 μg/plate for BaP TA98 with rat Ar S9; multi-dose data but not SD was reported
21360 Simmon,
1979a Table 1 Use data for TA100 for BaA,

BaP, BeP; dose as μg, response as revertants/plate after subtracting background Point estimate TA100 with rat Ar
S9

21640 Teranishi et al., 1975 Table I

and Figure 3 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 Point estimate TA1538 with rat PB S9 for DBaiP; TA1538 with PB and DBahA S9 for
DBaeP

16180 Utesch et al., 1987 Figures

2 and 3 Use data for homogenized

hepatocytes (open circles) for BaA and BaP; dose as μg/plate, response as revertants/plates Point estimate; use 12.5 μg/plate for BaP; use 25 μg/plate for BaA TA100 with homogenized hepatocytes from Ar treated rats; multi-dose data but not SD was reported

16440 Wood et al., 1980 Chart 3A Use DR curves for BaP and

CPcdP; dose as nmol, response as revertants/plate Point estimate; use 15 nmol for BaP and CPcdP TA98 with purified microsomal P450; multi-dose data but not SD was reportedC-33 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorComments17030 Andrews et al.,1978TA100 ArS9 Control 0 μg 150 RevertantcoloniesBaP 250 μg 1,681 RevertantcoloniesDBacA 10 μg 2,957 RevertantcoloniesDBajA 10 μg 843 RevertantcoloniesDBahA 25 μg 617 RevertantcoloniesAA 250 μg 1,796 RevertantcoloniesBghiP 100 μg 793 RevertantcoloniesBeP 1,000 μg 643 Revertantcolonies23830 Baker et al., 1980 TA100 Guinea pigMCControl 0 μg/plate 134 Revertantcolonies18BaP 2.5 μg/plate 1,278 Revertantcolonies10 97DBaiP 5 μg/plate 737 Revertantcolonies10 73BaA 10 μg/plate 947 Revertantcolonies10 47DBacA 2.5 μg/plate 1,738 Revertantcolonies10 88DBahA 5 μg/plate 1,331 Revertantcolonies10 9823660 Bartsch et al., 1980TA100 Rat MC S9BaP 0.027 μmol/plate29,000 Revertants/plateControl responsesubtracted.BaA 0.067 μmol/plate6,000 Revertants/plateControl responsesubtracted.17380 Bos et al., 1988 TA100 Rat ArS9 BaP 7.5 μg/plate 824 Revertants/plate3 Replicates21 12Control 0 μg/plate 85 Revertants/plate3 Replicates12 7C-34 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsPH 1 μg/plate 108 Revertants/plate3 Replicates10 6PH 5 μg/plate 167 Revertants/plate3 Replicates5 3PH 25 μg/plate 240 Revertants/plate3 Replicates10 6Control 0 μg/plate 86 Revertants/plate3 Replicates7 4Pyr 1 μg/plate 93 Revertants/plate3 Replicates9 5Pyr 5 μg/plate 164 Revertants/plate3 Replicates23 13Pyr 25 μg/plate 279 Revertants/plate3 Replicates10 617590 Carver et al., 1986 TA100 HamsterArS9Control 0 μg/plate 140 Revertants/plateControl curvesdifficult todigitize; controlvalue estimatedfrom BaP graphand used for all.BaP 1 μg/plate 141 Revertants/plateContinuous data,no SDBaP 10 μg/plate 482 Revertants/plateBaP 50 μg/plate 1,035 Revertants/plateBaA 15 μg/plate 346 Revertants/plateBaA 40 μg/plate 892 Revertants/plateBaA 50 μg/plate 1,263 Revertants/plateBghiF 10 μg/plate 333 Revertants/plateBghiF 25 μg/plate 727 Revertants/plateC-35 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBghiF 50 μg/plate 985 Revertants/platePerylene 5 μg/plate 195 Revertants/platePerylene 10 μg/plate 993 Revertants/platePerylene 15 μg/plate 922 Revertants/plate17630 Cavalieri et al.,1981aTM677 Ar S9 Control 0 μM 5 Mutants 1 × 105Survivors0.000050Control valueestimatedBaP 10 μM 15 Mutants 1 × 105Survivors0.000150BaP 20 μM 26 Mutants 1 × 105Survivors0.000256BaP 40 μM 84 Mutants 1 × 105Survivors0.000839BaP 60 μM 131 Mutants 1 × 105Survivors0.001308CPcdP 20 μM 34 Mutants 1 × 105Survivors0.000337CPcdP 40 μM 133 Mutants 1 × 105Survivors0.001330ACEP 10 μM 11 Mutants 1 × 105Survivors0.000110ACEP 40 μM 25 Mutants 1 × 105Survivors0.000248ACEP 120 μM 55 Mutants 1 × 105Survivors0.0005519620 Chang et al., 2002 TA100 Rat ArS9 Control 0 μg/plate 326 Revertants/plateSD notconsistentlyplotted; extractedonly point estimatedataBaP 5 μg/plate 2,543 Revertants/plateBghiF 5 μg/plate 1,630 Revertants/plateC-36 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBcPH 10 μg/plate 1,043 Revertants/plate24030 De Flora et al.,1984Rat ARS9BaP 185 Revertants/nmol(potency)BaA 12 Revertants/nmol(potency)Pery 21 Revertants/nmol(potency)BeP 1.6 Revertants/nmol(potency)18050 Eisenstadt andGold, 1978TA100 Rat ArS9 BaP 2 μg 1,705 Revertants/plateBackgroundsubtracted fromdata reportedCPcdP 1 μg 134 Revertants/plate18180 Florin et al., 1980TA100 Rat MC S9BaP 0.0030μmol/plate255 Revertants/plateBackgroundsubtracted fromdata reportedTA100 BaA 0.10 μmol/plate326 Revertants/plateOnly peakresponse reportedTA100 CH 0.0050μmol/plate196 Revertants/plateTA98 BaP 0.0030μmol/plate235 Revertants/plateTA98 Pery 0.025 μmol/plate91 Revertants/plateTA98 CO 0.070 μmol/plate82 Revertants/plate24080 Gibson et al., 1978 TA9860Cogammaradiation,for 7 d(2.5x107rad)Control 0 μg/plate 0 Increase inrevertantsContinuous data,no SDC-37 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBaP 10 μg/plate 1.5 Increase inrevertantsBaP 20 μg/plate 3 Increase inrevertantsBaP 50 μg/plate 10 Increase inrevertantsBaP 100 μg/plate 15 Increase inrevertantsBaP 200 μg/plate 21 Increase inrevertantsBaP 300 μg/plate 35 Increase inrevertantsBaA 150 μg/plate 1.8 Increase inrevertantsBaA 250 μg/plate 6.4 Increase inrevertantsBghiP 400 μg/plate 4.2 Increase inrevertantsCH 500 μg/plate 6.1 Increase inrevertantsCH 1,000 μg/plate 6.7 Increase inrevertantsFE 200 μg/plate 1.1 Increase inrevertantsFE 360 μg/plate 2.2 Increase inrevertantsPyr 160 μg/plate 28 Increase inrevertants14080 Gold andEisenstadt, 1980TA100 50ul ratMC S9BaP 4 nmol 1,103 Revertants/plateBackgroundsubtracted fromdata reportedCPcdP 4 nmol 281 Revertants/plate18650 Hermann, 1981 TA98 Rat Ar S9 BaP 100 Revertants/nmol(potency)C-38 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBbA 8 Revertants/nmol(potency)BaA 4 Revertants/nmol(potency)CH 2 Revertants/nmol(potency)FA 3 Revertants/nmol(potency)Tphen 13 Revertants/nmol(potency)BeP 15 Revertants/nmol(potency)DBacA 42 Revertants/nmol(potency)DBahA 8 Revertants/nmol(potency)BbF 15 Revertants/nmol(potency)Pery 31 Revertants/nmol(potency)DBalP 21 Revertants/nmol(potency)DBaiP 38 Revertants/nmol(potency)AA 62 Revertants/nmol(potency)C-39 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsCO 60 Revertants/nmol(potency)10670 Johnsen et al., 1997TA98 PCBmicrosomesControl 0 μg/plate 113 Revertants/plate3 8.54 Control responseadded back to eachresponse formodelingBaP 10 μg/plate 128 Revertants/plate3 3.66BaP 20 μg/plate 123 Revertants/plate3 13.41BjAC 10 μg/plate 192 Revertants/plate3 10.98BjAC 20 μg/plate 213 Revertants/plate3 9.76BIAC 10 μg/plate 204 Revertants/plate3 13.41BIAC 20 μg/plate 207 Revertants/plate3 43.9019000 Kaden et al., 1979 TM677 ArS9 andPB S9BaP 1 RPF Mutagenic activityrelative to that ofthe 80 μmol BaPpositive controlperformedsimultaneouslywith testcompound.AN NA 0.010 RPFANL NA 0.070 RPFPyr NA 0.070 RPFBbFE NA 0.080 RPFCPcdP NA 1.5 RPFBaA NA 0.14 RPFCH NA 0.20 RPFTphen NA 0.070 RPFFA NA 1.0 RPFBeP NA 0.11 RPFC-40 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsPery NA 6 RPFBghiP NA 0.080 RPFAA NA 0.080 RPFDBacA NA 0.77 RPFDBahA NA 0.080 RPFDBbeF NA 0.88 RPF24680 Lafleur et al., 1993TM677 Rat AR S9 BaP 0 μg/mL 7 Mutants 100,000 Survivors0.000070BaP 0.5 μg/mL 8 Mutants 100,000 Survivors0.000080BaP 1 μg/mL 10 Mutants 100,000 Survivors0.000101BaP 2 μg/mL 18 Mutants 100,000 Survivors0.000175BaP 4 μg/mL 22 Mutants 100,000 Survivors0.000220BaP 8 μg/mL 33 Mutants 100,000 Survivors0.000327BghiF 0 μg/mL 11 Mutants 100,000 Survivors0.00011BghiF 1 μg/mL 10 Mutants 100,000 Survivors0.00010BghiF 3 μg/mL 14 Mutants 100,000 Survivors0.00014BghiF 10 μg/mL 55 Mutants 100,000 Survivors0.00055CPcdP 0 μg/mL 12 Mutants 100,000 Survivors0.000120CPcdP 0.5 μg/mL 15 Mutants 100,000 Survivors0.000146CPcdP 1 μg/mL 13 Mutants 100,000 Survivors0.000130CPcdP 2 μg/mL 17 Mutants 100,000 Survivors0.000172CPcdP 4 μg/mL 27 Mutants 100,000 Survivors0.000274C-41 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsCPcdP 8 μg/mL 60 Mutants 100,000 Survivors0.000597CPhiACEA0 μg/mL 8 Mutants 100,000 Survivors0.000084CPhiACEA0.5 μg/mL 10 Mutants 100,000 Survivors0.000103CPhiACEA1 μg/mL 16 Mutants 100,000 Survivors0.000157CPhiACEA2 μg/mL 29 Mutants 100,000 Survivors0.000286CPhiACEA4 μg/mL 67 Mutants 100,000 Survivors0.000670CPhiAPA0 μg/mL 9 Mutants 100,000 Survivors0.000090CPhiAPA10 μg/mL 12 Mutants 100,000 Survivors0.000117CPhiAPA30 μg/mL 21 Mutants 100,000 Survivors0.000210CPhiAPA100 μg/mL 26 Mutants 100,000 Survivors0.000263ACEA 0 μg/mL 9 Mutants 100,000 Survivors0.000092ACEA 10 μg/mL 21 Mutants 100,000 Survivors0.000214ACEA 35 μg/mL 69 Mutants 100,000 Survivors0.000686APA 0 μg/mL 16 Mutants 100,000 Survivors0.000160APA 10 μg/mL 37 Mutants 100,000 Survivors0.000375APA 30 μg/mL 42 Mutants 100,000 Survivors0.000416APA 100 μg/mL 22 Mutants 100,000 Survivors0.00022019320 LaVoie et al., 1979TA98 Rat Ar S9 BaP 10 μg 450 Revertants/plateBackgroundsubtracted fromdata reportedC-42 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBaP 20 μg 480 Revertants/plateBeP 10 μg 20 Revertants/plateBeP 20 μg 20 Revertants/platePery 20 μg 70 Revertants/plate23650 McCann et al.,1975MultiplestrainsRat Ar S9 BaP NA 121 Revertants/nmol(potency)Paper states thatcomparison ofpotency estimatesshould be donewith caution (nonlinear doseresponse) see tablefootnotesDBaiP NA 20 Revertants/nmol(potency)BeP NA 0.6 Revertants/nmol(potency)DBacA NA 175 Revertants/nmol(potency)DBahA NA 11 Revertants/nmol(potency)CH NA 38 Revertants/nmol(potency)BaA NA 11 Revertants/nmol(potency)20220 Pahlman andPelkonen, 1987TA100 Rat MC S9BaP NA 272 Revertants/nmol(potency)C-43 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBaA NA 10.4 Revertants/nmol(potency)CH NA 9.7 Revertants/nmol(potency)Tphen NA 4 Revertants/nmol(potency)DBacA NA 35 Revertants/nmol(potency)DBahA NA 4.4 Revertants/nmol(potency)20450 Phillipson andIoannides, 1989TA100 Hamster S9BaP 0 μg/plate 0.000 Revertants/plateBaP 5 μg/plate 68.833 Revertants/plateBaP 10 μg/plate 118.948 Revertants/plateBaP 15 μg/plate 99.744 Revertants/plateBaP 20 μg/plate 96.101 Revertants/plateBaA 0 μg/plate 0.000 Revertants/plateBaA 20 μg/plate 109.877 Revertants/plateBaA 40 μg/plate 115.248 Revertants/plateBaA 60 μg/plate 114.430 Revertants/plateBaA 100 μg/plate 98.846 Revertants/plateDBaiP 0 μg/plate 0.000 Revertants/plateC-44 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsDBaiP 20 μg/plate 64.638 Revertants/plateDBaiP 40 μg/plate 75.747 Revertants/plateDBaiP 60 μg/plate 80.394 Revertants/plateDBaiP 100 μg/plate 63.880 Revertants/plateDBahA 0 μg/plate 0.000 Revertants/plateDBahA 10 μg/plate 50.899 Revertants/plateDBahA 20 μg/plate 56.886 Revertants/plateDBahA 30 μg/plate 52.419 Revertants/plateDBahA 50 μg/plate 34.980 Revertants/plate21000 Sakai et al., 1985 TA97 Rat Ar S9 Control 0μg 177 Revertants/plateBaP 1 μg 1,208 Revertants/plateBaP 5 μg 1,432 Revertants/plateBaP 10 μg 1,742 Revertants/plateControl 0 μg 189 Revertants/plateFE 5 μg 254 Revertants/plateFE 10 μg 240 Revertants/plateFE 50 μg 240 Revertants/plateFE 250 μg 232 Revertants/plateC-45 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsControl 0 μg 189 Revertants/plateAC 5 μg 360 Revertants/plateAC 10 μg 509 Revertants/plateAC 50 μg 293 Revertants/plateAC 250 μg 279 Revertants/plateControl 0 μg 189 Revertants/platePH 5 μg 454 Revertants/platePH 10 μg 534 Revertants/platePH 50 μg 321 Revertants/platePH 250 μg T Revertants/plateControl 0 μg 177 Revertants/plateFA 5 μg 652 Revertants/plateFA 10 μg 1,012 Revertants/plateFA 50 μg 1,042 Revertants/plateFA 250 μg 518 Revertants/plateControl 0 μg 177 Revertants/plateCH 5 μg 640 Revertants/plateCH 10 μg 815 Revertants/plateC-46 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsCH 20 μg 888 Revertants/plateCH 50 μg 723 Revertants/plateControl 0 μg 177 Revertants/platePyr 2 μg 929 Revertants/platePyr 4 μg 1,582 Revertants/platePyr 6 μg 2,057 Revertants/platePyr 10 μg 2,577 Revertants/platePyr 20 μg 2,832 Revertants/platePyr 50 μg 2,296 Revertants/plateControl 0 μg 177 Revertants/plateBeP 5 μg 944 Revertants/plateBeP 10 μg 1,100 Revertants/plateBeP 50 μg 606 Revertants/plateBeP 250 μg 640 Revertants/plateControl 0 μg 177 Revertants/platePery 1 μg 1,516 Revertants/platePery 2 μg 2,236 Revertants/platePery 4 μg 2,784 Revertants/plateC-47 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsPery 10 μg 2,550 Revertants/platePery 50 μg 1,808 Revertants/plateControl 0 μg 177 Revertants/plateBghiP 10 μg 896 Revertants/plateBghiP 20 μg 991 Revertants/plateBghiP 50 μg 896 Revertants/plateBghiP 250 μg 612 Revertants/plateControl 0 μg 177 Revertants/plateCO 5 μg 362 Revertants/plateCO 10 μg 400 Revertants/plateCO 50 μg 405 Revertants/plateCO 100 μg 490 Revertants/plateCO 200 μg 479 Revertants/plate11860 Sangaiah et al.,1983TA98 Rat Ar S9 Control 0 μg/plate 35.43 Revertants/plateBaP 2 μg/plate 177.37 Revertants/plateBaP 3 μg/plate 266.02 Revertants/plateBaP 6 μg/plate 419.68 Revertants/plateBaP 10 μg/plate 312.76 Revertants/plateC-48 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBaP 30 μg/plate 358.41 Revertants/plateBaP 50 μg/plate 350.92 Revertants/plateBaP 100 μg/plate 323.12 Revertants/plateControl 0 μg/plate 53.15 Revertants/plateBjAC 2 μg/plate 124.15 Revertants/plateBjAC 3 μg/plate 331.10 Revertants/plateBjAC 6 μg/plate 674.11 Revertants/plateBjAC 10 μg/plate 993.21 Revertants/plateBjAC 30 μg/plate 1,027.06 Revertants/plateBjAC 50 μg/plate 883.45 Revertants/plateBjAC 100 μg/plate 1,021.36 Revertants/plate21360 Simmon, 1979a TA100 Rat Ar S9 BaP 5 μg 1,141Revertants/plateBackgroundsubtracted fromdata reportedBaA 50 μg 280 Revertants/plateBeP 50 μg 57 Revertants/plate21640 Teranishi et al.,1975TA1538 Rat PB S9 Control 0 μg/plate 38 Revertantcolonies/plateBaP 50 μg/plate 77 Revertantcolonies/plateC-49 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsDBaiP 50 μg/plate 102 Revertantcolonies/plateTA1538 Rat PB andDBahA S9Control 0 μg/plate 25 Revertantcolonies/plateBaP 50 μg/plate 279 Revertantcolonies/plateDBaeP 50 μg/plate 88 Revertantcolonies/plate16180 Utesch et al., 1987 TA100 Withhomogenizedhepatocytesfrom Artreated ratsControl 0 μg/plate 159 Revertants/platesBaP 6.3 μg/plate 998 Revertants/plateBaP 12.5 μg/plate 1,079 Revertants/plateBaP 25 μg/plate 1,178 Revertants/plateBaP 50 μg/plate 1,141 Revertants/plateBaP 100 μg/plate 1,114 Revertants/plateControl 0 μg/plate 199 Revertants/plateBaA 6.3 μg/plate 861 Revertants/plateBaA 12.5 μg/plate 2,583 Revertants/plateBaA 25 μg/plate 3,546 Revertants/plateBaA 50 μg/plate 3,786 Revertants/plateC-50 DRAFT – DO NOT CITE OR QUOTETable C-7. In vitro bacterial mutagenicity: dose response dataRecordnumberReference Cell typePAH Dose Dose unitsResponseResponseunits n Units% responseStddevStderrorCommentsBaA 100 μg/plate 3,406 Revertants/plate16440 Wood et al., 1980 TA98 PurifiedmicrosomalP450Control 0 nmol 0 Revertants/plateBackgroundsubtracted fromdata reportedBaP 3.75 nmol 45 Revertants/plateBaP 7.5 nmol 63 Revertants/plateBaP 15 nmol 99 Revertants/plateBaP 30 nmol 103 Revertants/plateControl 0 nmol 0 Revertants/plateCPcdP 3.75 nmol 303 Revertants/plateCPcdP 7.5 nmol 491 Revertants/plateCPcdP 15 nmol 685 Revertants/plateCPcdP 30 nmol 776 Revertants/plate1

### C-51 DRAFT – DO NOT CITE OR QUOTE

Table C-8. In vitro mammalian mutagenicity: data u se

Record
number Reference Data source Data points Basis for RPF approach Comments

16920 Amacher and Paillet, 1982 Figure 1 Use lines for BaP (open circles) and BaA (closed triangles; dose is μg/mL and response is mutation frequency (MF)/10 6 survivors Model; quantal data Thymidine kinase assay (resistance to trifluorothymidine) in mouse lymphoma cells (L5178Y) with Syrian golden hamster S9 mix or cocultivated hamster hepatocytes

16940 Amacher and Turner, 1980 Figure 3 Use bars for SM2 S9 activation for BaP and BaA; dose is 1.25 × 10 -5 M for BaP and 3.22 × 10 -5 M for BaP; response is IMF/10 4 survivors Point estimate Thymidine kinase assay(resistance to trifluorothymidine) in mouse lymph oma cells (L5178Y) with mouse S9 mix

16910 Amacher et al., 1980 Table 3 Use DR data for BaA and BaP; dose as concentration (M), response as mutants per
10 4 survivors Model; quantal data Thymidine kinase assay

(resistance to trifluorothymidine) in mouse lymphoma cells (L5178Y) with mouse S9 mix

17140 Barfknecht et al., 1982 Figure 2

(BaP, FA); Figure 4 (BaA, CH, Tphen); Figure 6 (CPcdP) Dose is μM and mutant

fraction ×10 6 Model; quantal data Thymidine kinase assay

(resistance to trifluorothymidine) in human lymphoblast cells with rat Ar S9 mix

14250 Hass et al.,

1982 Table 1 DR data for DBaiP, DBahP, and BaP; dose is μg/mL; use response data for TG mutants only (mutants/10 6 cells); control value is 4 ± 1 mutants/10 6 cells Model; quantal data Hypoxanthine-guanine phosphoribosyl transferase assay (resistance to 6-thioguanine) in V79 Chinese hamster cells with rat MC S9

18740 Huberman and Sachs,
1976 Table 2 Use data for BaP, DBacA, DBahA; 8-azaguanine resistance only; use 1μg/mL dose for all (*), response as mutants per 10 5 survivors Point estimate Hypoxanthine-guanine phosphoribosyl transferase assay (resistance to 8-azaguanine) in V79 Chinese hamster cells with hamster embryo cells

18990 Jotz and Mitchell,
1981 Table 2 Use data for BaP and Pyr with metabolic activation; subtract negative control, dose as μg/mL, response as MF × 10 -6 Point estimate Thymidine kinase assay(resistance to trifluorothymidine) in mouse lymph oma cells (L5178Y) with rat Ar S9 24720 Kligerman et

al., 1986 Figure 1 Use DR dat for BaP and BlAC; dose as μg/mL, response as mutant frequency/10 6 survivors; average data from two experiments Model; quantal data Thymidine kinase assay

(resistance to trifluorothymidine) in mouse lymph oma cells (L5178Y) with rat Ar S9

19180 Krahn and Heidelberger,
1977 Table II Use data for BaP, DBahA, DBacA, and BaA; cell survival @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/10 5 cells Point estimate Hypoxanthine-guanine phosphoribosyl transferaseassay (resistance to 6-thioguanine) in V79 Chinese hamster cells with hamster embryo cells

### C-52 DRAFT – DO NOT CITE OR QUOTE

Table C-8. In vitro mammalian mutagenicity: data u se

Record
number Reference Data source Data points Basis for RPF approach Comments
24680 Lafleur et al.,

1993 Figures 5

and 6 Use DR curves for BaP,

CPcdP (CPP), CPhiACEA (CPAA), ACEA (AA); dose as μg/mL, response as mutant fraction (ppm) Model as quantal data (mutant fraction reported) Thymidine kinase assay (resistance to trifluorothymidine) inMCL-3 cells (human Blymphoblastoid cells)

7550 Li and Lin,

1996 Text Mutant frequency of controls
2 × 10 -5 ; 10 ng/mL BaP = 5 ×
10 -5 ; BaA = 5.6 × 10 -5 Point estimate Hypoxanthine-guanine phosphoribosyl transferase assay (resistance to 6-thioguanine) in HS1 HeLa cells (human epithelial cells)

11450 Nesnow et al., 1984 Chart 9 Use data for BaP, BlAC, BeAC, and BjAC; dose as μg/mL, response as 6TG-resistant mutants/
10 6 survivors Model; quantal data Hypoxanthine-guanine phosphoribosyl transferase assay (resistance to 6-thioguanine) in V79 Chinese hamster cells with rat AR S9

15630 Raveh and Huberman,
1983 Table 1 Use data for CPcdP and BaP, with PMA only; dose in μg/mL, response in mutants/10 5 cells Model; quantal data Hypoxanthine-guanine

phosphoribosyl transferaseassay (resistance to 6-thioguanine) in V79 Chinese hamster cells with hamster embryo cells
15640 Raveh et al.,

1982 Figure 4 Use DR data for CPcdP and BaP (ouabain resistance only); dose in μg/mL, response in mutants/10 6 cells Model; quantal data Hypoxanthine-guanine phosphoribosyl transferase assay (resistance to ouabain) in V79 Chinese hamster cells with hamster embryo cells 21410 Slaga et al.,

1978 Table 3 Use DR data for BaA and BaP; dose as μM, response as ouabain resistant mutants/10 4 survivors Model; quantal data Hypoxanthine-guanine phosphoribosyl transferase assay (resistance to ouabain) in V79 Chinese hamster cells with hamster embryo cells
16190 Vaca et al.,

1992 Figure 5 DR data for FA and BaP; dose as μM, response as 6-Tg resistant cells/100,000 Model; quantal data Hypoxanthine-guanine

phosphoribosyl transferaseassay (resistance to 6-thioguanine) in UV-sensitive CHO cells with rat Ar S9 21900 Wangenheim

and Bolcsfoldi,
1988 Table 1 Use +S9 DR data for Pyr, BaP, and FE; dose as mol/L, response as mutation frequency Model; quantal data Thymidine kinase assay

(resistance to trifluorothymidine) in mouse lymphoma cells (L5178Y) with rat
Ar S9
24670 Durant et al.,

1999 Table 1 Use DR 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) Model; quantal data Thymidine kinase assay

(resistance to trifluorothymidine) in human h1Alv2 cellsC-53 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response Comments16920 Amacher andPaillet, 1982Control 0 μg/mL 39 1 × 106Survivors 0.000039BaP 2.5 μg/mL 119 1 × 106Survivors 0.00012BaP 5 μg/mL 170 1 × 106Survivors 0.00017BaP 7.5 μg/mL 196 1 × 106Survivors 0.00020BaP 10 μg/mL 267 1 × 106Survivors 0.00027Control 0 μg/mL 20 1 × 106Survivors 0.000020BaA 2.5 μg/mL 65 1 × 106Survivors 0.000065BaA 5 μg/mL 62 1 × 106Survivors 0.000062BaA 10 μg/mL 88 1 × 106Survivors 0.000088BaA 15 μg/mL 89 1 × 106Survivors 0.00008916940 Amacher andTurner, 1980Control 0 M 0.4 1 × 104Survivors 0.000040 Control w/o S9 treatmentBaP 1.25 × 10-5M 2.85 1 × 104Survivors 0.000285BaA 3.22 × 10-5M 3.12 1 × 104Survivors 0.00031216910 Amacher et al.,1980Control 0 M 0.680 1 × 104Survivors 0.000068BaP 5.30 × 10-6M 1.360 1 × 104Survivors 0.000136BaP 7.00 × 10-6M 1.790 1 × 104Survivors 0.000179BaP 9.40 × 10-6M 1.470 1 × 104Survivors 0.000147BaP 1.25 × 10-5M 1.870 1 × 104Survivors 0.000187BaP 1.67 × 10-5M 2.600 1 × 104Survivors 0.000260BaP 2.23 × 10-5M 2.490 1 × 104Survivors 0.000249BaP 2.97 × 10-5M 2.650 1 × 104Survivors 0.000265BaP 3.96 × 10-5M 3.970 1 × 104Survivors 0.000397Control 0 M 0.770 1 × 104Survivors 0.000077BaA 1.36 × 10-5M 0.810 1 × 104Survivors 0.000081BaA 1.81 × 10-5M 0.840 1 × 104Survivors 0.000084BaA 2.42 × 10-5M 1.000 1 × 104Survivors 0.000100BaA 3.22 × 10-5M 1.230 1 × 104Survivors 0.000123BaA 4.30 × 10-5M 1.470 1 × 104Survivors 0.000147BaA 5.47 × 10-5M NS 1 × 104Survivors NS = no survivorsBaA 7.65 × 10-5M NS 1 × 104SurvivorsC-54 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response CommentsBaA 1.02 × 10-4M NS 1 × 104Survivors17140 Barfknecht et al.,1982Control 0 μM 0 1 × 106Survivors 0.000000BaP 10 μM 51 1 × 106Survivors 0.000051BaP 20 μM 120 1 × 106Survivors 0.000120BaP 30 μM 155 1 × 106Survivors 0.000155Control 0 μM 0 1 × 106Survivors 0.000000FA 10 μM 27 1 × 106Survivors 0.000027FA 20 μM 50 1 × 106Survivors 0.000050FA 40 μM 62 1 × 106Survivors 0.000062Control 0 μM 0 1 × 106Survivors 0.000000BaA 20 μM 12 1 × 106Survivors 0.000012BaA 50 μM 29 1 × 106Survivors 0.000029BaA 100 μM 34 1 × 106Survivors 0.000034BaA 150 μM 64 1 × 106Survivors 0.000064Control 0 μM 0 1 × 106Survivors 0.000000CH 20 μM 17 1 × 106Survivors 0.000017CH 50 μM 26 1 × 106Survivors 0.000026CH 100 μM 30 1 × 106Survivors 0.000030Control 0 μM 0 1 × 106Survivors 0.000000Tphen 50 μM 10 1 × 106Survivors 0.000010Tphen 100 μM 20 1 × 106Survivors 0.000020Tphen 200 μM 35 1 × 106Survivors 0.000035Control 0 μM 3 1 × 106Survivors 0.000003CPcdP 23 μM 11 1 × 106Survivors 0.000011CPcdP 47 μM 24 1 × 106Survivors 0.000024CPcdP 88 μM 27 1 × 106Survivors 0.00002724670 Durant et al., 1999 BaP 1,000 ng/mL 170 1 × 106Survivors 0.00017BaP 1,000 ng/mL 170 1 × 106Survivors 0.00017BaP 1,000 ng/mL 200 1 × 106Survivors 0.00020BaP 1,000 ng/mL 200 1 × 106Survivors 0.00020BaP 1,000 ng/mL 160 1 × 106Survivors 0.00016C-55 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response CommentsBaP 1,000 ng/mL 170 1 × 106Survivors 0.00017BaP 1,000 ng/mL 190 1 × 106Survivors 0.00019BaP 1,000 ng/mL 200 1 × 106Survivors 0.00020BaP 1,000 ng/mL 210 1 × 106Survivors 0.00021AveragedBaP1,000 ng/mL 186 1 × 106Survivors 0.00019Averagedcontrols0 ng/mL 20 1 × 106Survivors 0.00002Control 0 ng/mL 18 1 × 106Survivors 0.000018BaPery 0.1 ng/mL 21 1 × 106Survivors 0.000021BaPery 0.3 ng/mL 23 1 × 106Survivors 0.000023BaPery 1 ng/mL 28 1 × 106Survivors 0.000028BaPery 3 ng/mL 50 1 × 106Survivors 0.000050BaPery 10 ng/mL 82 1 × 106Survivors 0.000082BaPery 100 ng/mL 200 1 × 106Survivors 0.00020Control 0 ng/mL 18 1 × 106Survivors 0.000018BbPery 1 ng/mL 19 1 × 106Survivors 0.000019BbPery 3 ng/mL 22 1 × 106Survivors 0.000022BbPery 10 ng/mL 32 1 × 106Survivors 0.000032BbPery 100 ng/mL 54 1 × 106Survivors 0.000054Control 0 ng/mL 21 1 × 106Survivors 0.000021DBaeF 1 ng/mL 29 1 × 106Survivors 0.000029DBaeF 10 ng/mL 72 1 × 106Survivors 0.000072DBaeF 100 ng/mL 190 1 × 106Survivors 0.00019DBaeF 1,000 ng/mL np 1 × 106Survivors Not plated due toexcessive toxicityControl 0 ng/mL 21 1 × 106Survivors 0.000021DBafF 1 ng/mL 21 1 × 106Survivors 0.000021DBafF 10 ng/mL 37 1 × 106Survivors 0.000037DBafF 100 ng/mL 81 1 × 106Survivors 0.000081DBafF 1,000 ng/mL 190 1 × 106Survivors 0.00019Control 0 ng/mL 19 1 × 106Survivors 0.000019DBahP 0.1 ng/mL 24 1 × 106Survivors 0.000024C-56 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response CommentsDBahP 1 ng/mL 24 1 × 106Survivors 0.000024DBahP 10 ng/mL 46 1 × 106Survivors 0.000046DBahP 100 ng/mL 80 1 × 106Survivors 0.000080Control 0 ng/mL 20 1 × 106Survivors 0.000020DBaiP 0.3 ng/mL 20 1 × 106Survivors 0.000020DBaiP 1 ng/mL 35 1 × 106Survivors 0.000035DBaiP 10 ng/mL 88 1 × 106Survivors 0.000088DBaiP 100 ng/mL 150 1 × 106Survivors 0.00015Control 0 ng/mL 21 1 × 106Survivors 0.000021DBelP 10 ng/mL 28 1 × 106Survivors 0.000028DBelP 100 ng/mL 34 1 × 106Survivors 0.000034DBelP 1,000 ng/mL 55 1 × 106Survivors 0.000055Control 0 ng/mL 21 1 × 106Survivors 0.000021N23aP 0.1 ng/mL 23 1 × 106Survivors 0.000023N23aP 1 ng/mL 44 1 × 106Survivors 0.000044N23aP 10 ng/mL 84 1 × 106Survivors 0.000084N23aP 100 ng/mL 94 1 × 106Survivors 0.000094N23aP 1,000 ng/mL 73 1 × 106Survivors 0.000073Control 0 ng/mL 19 1 × 106Survivors 0.000019N23eP 1 ng/mL 20 1 × 106Survivors 0.000020N23eP 10 ng/mL 41 1 × 106Survivors 0.000041N23eP 100 ng/mL 74 1 × 106Survivors 0.000074N23eP 1,000 ng/mL 98 1 × 106Survivors 0.0001014250 Hass et al., 1982 Control 0 μg/mL 4 1 × 106CFC 0.0000040BaP 0.30 μg/mL 267 1 × 106CFC 0.00027BaP 1.00 μg/mL 293 1 × 106CFC 0.00029DBaiP 0.03 μg/mL 124 1 × 106CFC 0.00012DBaiP 0.10 μg/mL 289 1 × 106CFC 0.00029DBaiP 0.30 μg/mL 1211 1 × 106CFC 0.00121DBahP 0.03 μg/mL 110 1 × 106CFC 0.00011DBahP 0.10 μg/mL 264 1 × 106CFC 0.00026DBahP 0.30 μg/mL 668 1 × 106CFC 0.00067C-57 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response Comments18740 Huberman andSachs, 1976Control 0 μg/mL 6 1 × 105Survivors 0.000060BaP 1 μg/mL 425 1 × 105Survivors 0.00425DBacA 1 μg/mL 22 1 × 105Survivors 0.00022DBahA 1 μg/mL 17 1 × 105Survivors 0.0001718990 Jotz and Mitchell,1981Control 0 μg/mL 80 1 × 106Survivors 0.000080BaP 4.5 μg/mL 224 1 × 106Survivors 0.00022 With metabolicactivationControl 0 μg/mL 116 1 × 106Survivors 0.00012Pyr 10.6 μg/mL 150 1 × 106Survivors 0.00015 With metabolicactivation24720 Kligerman et al.,1986Control 0 nmol/mL 92 1 × 106Survivors 0.00009 Avg. of 2 experimentsBaP 2.0 nmol/mL 258 1 × 106Survivors 0.00026BaP 3.0 nmol/mL 417 1 × 106Survivors 0.00042BaP 4.0 nmol/mL 557 1 × 106Survivors 0.00056Control 0 nmol/mL 90 1 × 106Survivors 0.00009BlAC 0.5 nmol/mL 93 1 × 106Survivors 0.00009BlAC 2.5 nmol/mL 197 1 × 106Survivors 0.00020BlAC 5.0 nmol/mL 374 1 × 106Survivors 0.0003719180 Krahn andHeidelberger, 1977Control 0 nmol/mL 1.7 1 × 105Survivors 0.000017BaP 15.9 nmol/mL 14 1 × 105Survivors 0.000136 3-MC S9; 40% survivalControl 0 nmol/mL 1.5 1 × 105Survivors 0.000015BaA 46.5 nmol/mL 6.5 1 × 105Survivors 0.000065 3-MC S9; 40% survival24680 Lafleur et al., 1993Control 0 μg/mL 1.2 1 × 106Survivors 0.0000012BaP 0.02 μg/mL 4.8 1 × 106Survivors 0.0000048BaP 0.06 μg/mL 24 1 × 106Survivors 0.000024BaP 0.2 μg/mL 25 1 × 106Survivors 0.000025BaP 1 μg/mL 39 1 × 106Survivors 0.000039BaP 5 μg/mL 56 1 × 106Survivors 0.000056Control 0 μg/mL 1.8 1 × 106Survivors 0.0000018ACEA 1 μg/mL 6.0 1 × 106Survivors 0.0000060C-58 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response CommentsACEA 3 μg/mL 15 1 × 106Survivors 0.000015ACEA 8 μg/mL 21 1 × 106Survivors 0.000021Control 0 μg/mL 2.5 1 × 106Survivors 0.0000025CPcdP 0.03 μg/mL 4.2 1 × 106Survivors 0.0000042CPcdP 0.06 μg/mL 4.9 1 × 106Survivors 0.0000049CPcdP 0.2 μg/mL 5.9 1 × 106Survivors 0.0000059CPcdP 0.6 μg/mL 10 1 × 106Survivors 0.000010CPcdP 2 μg/mL 17 1 × 106Survivors 0.000017Control 0 μg/mL 2.8 1 × 106Survivors 0.0000028CPhiACEA0.1 μg/mL 12 1 × 106Survivors 0.000012CPhiACEA0.3 μg/mL 25 1 × 106Survivors 0.000025CPhiACEA0.8 μg/mL 31 1 × 106Survivors 0.0000317550 Li and Lin, 1996 Control 0 ng/mL 2 1 × 105Survivors 0.000020BaP 10 ng/mL 5 1 × 105Survivors 0.000050BaA 10 ng/mL 5.6 1 × 105Survivors 0.00005611450 Nesnow et al., 1984Control 0 μg/mL 16 1 × 106Survivors 0.000016BaP 0.5 μg/mL 10 1 × 106Survivors 0.000010BaP 1.0 μg/mL 46 1 × 106Survivors 0.000046BaP 2.5 μg/mL 72 1 × 106Survivors 0.000072BaP 5.0 μg/mL 206 1 × 106Survivors 0.000206BaP 10.0 μg/mL 215 1 × 106Survivors 0.000215BaP 20.0 μg/mL 293 1 × 106Survivors 0.000293BeAC 1.0 μg/mL 17 1 × 106Survivors 0.000017BeAC 2.5 μg/mL 53 1 × 106Survivors 0.000053BeAC 5.0 μg/mL 435 1 × 106Survivors 0.000435BeAC 10.0 μg/mL 235 1 × 106Survivors 0.000235BeAC 20.0 μg/mL 349 1 × 106Survivors 0.000349BjAC 1.0 μg/mL 24 1 × 106Survivors 0.000024BjAC 2.5 μg/mL 94 1 × 106Survivors 0.000094BjAC 5.0 μg/mL 268 1 × 106Survivors 0.000268BjAC 10.0 μg/mL 225 1 × 106Survivors 0.000225BjAC 20.0 μg/mL 215 1 × 106Survivors 0.000215C-59 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response CommentsBlAC 1.0 μg/mL 31 1 × 106Survivors 0.000031BlAC 2.5 μg/mL 454 1 × 106Survivors 0.000454BlAC 5.0 μg/mL 320 1 × 106Survivors 0.000320BlAC 10.0 μg/mL 704 1 × 106Survivors 0.000704BlAC 20.0 μg/mL 769 1 × 106Survivors 0.00076915630 Raveh andHuberman, 1983Control 0 μg/mL 3 1 × 105Survivors 0.000030BaP 0.3 μg/mL 25 1 × 105Survivors 0.00025BaP 1 μg/mL 103 1 × 105Survivors 0.0010CPcdP 0.3 μg/mL 9 1 × 105Survivors 0.000090CPcdP 1 μg/mL 20 1 × 105Survivors 0.0002015640 Raveh et al., 1982 BaP 0 μg/mL 7 1 × 106CFC 0.0000070BaP 0.3 μg/mL 20 1 × 106CFC 0.000020BaP 1 μg/mL 74 1 × 106CFC 0.000074BaP 3 μg/mL 74 1 × 106CFC 0.000074CPcdP 0 μg/mL 1 1 × 106CFC 0.0000010CPcdP 0.3 μg/mL 5 1 × 106CFC 0.0000047CPcdP 1 μg/mL 10 1 × 106CFC 0.000010CPcdP 3 μg/mL 28 1 × 106CFC 0.00002821410 Slaga et al., 1978 Control 0 μM 0.7 1 × 104Survivors 0.000070BaA 4.4 μM 0.9 1 × 104Survivors 0.000090BaA 44.0 μM 2.1 1 × 104Survivors 0.00021BaP 0.4 μM 11.0 1 × 104Survivors 0.0011BaP 1.3 μM 25.0 1 × 104Survivors 0.0025BaP 4.0 μM 99.0 1 × 104Survivors 0.009916190 Vaca et al., 1992 BaP 0 μM 3 1 × 105Survivors 0.000032BaP 2 μM 10 1 × 105Survivors 0.000102BaP 4 μM 23 1 × 105Survivors 0.000229BaP 10 μM 31 1 × 105Survivors 0.000306FA 0 μM 10 1 × 105Survivors 0.000105FA 5 μM 20 1 × 105Survivors 0.000203FA 7.5 μM 27 1 × 105Survivors 0.000274C-60 DRAFT – DO NOT CITE OR QUOTETable C-9. In vitro mammalian mutagenicity: dose response dataRecordnumberReference PAH Dose Dose unitsMutants In number Units % response Comments10 μM 32 1 × 105Survivors 0.00031821900 Wangenheim andBolcsfoldi, 1988Control 0 mol/L 61 1 × 106Survivors 0.000061Control 0 mol/L 62 1 × 106Survivors 0.000062 Used average of controlsAverage 0 mol/L 62 1 × 106Survivors 0.000062BaP 0.000001 mol/L 65 1 × 106Survivors 0.000065BaP 0.000005 mol/L 243 1 × 106Survivors 0.000243BaP 0.000010 mol/L 858 1 × 106Survivors 0.00086Control 0 mol/L 68 1 × 106Survivors 0.00007FE 0.0000195 mol/L 92 1 × 106Survivors 0.00009FE 0.0000389 mol/L 91 1 × 106Survivors 0.00009FE 0.0000681 mol/L 114 1 × 106Survivors 0.00011FE 0.000122 mol/L 154 1 × 106Survivors 0.00015FE 0.000170 mol/L 147 1 × 106Survivors 0.00015Control 0 mol/L 125 1 × 106Survivors 0.00013Control 0 mol/L 106 1 × 106Survivors 0.00011Average 0 mol/L 116 1 × 106Survivors 0.00012Pyr 0.0000101 mol/L 162 1 × 106Survivors 0.00016Pyr 0.0000151 mol/L 228 1 × 106Survivors 0.00023Pyr 0.0000202 mol/L 345 1 × 106Survivors 0.00035Pyr 0.0000252 mol/L 418 1 × 106Survivors 0.00042Pyr 0.0000302 mol/L 650 1 × 106Survivors 0.000651C-61 DRAFT – DO NOT CITE OR QUOTETable C-10. In vitro malignant/morphological celltransformation: data useRecordnumberReference PageTablenumberFigurenumberPAHs Data to be extractedBasis forRPF Comment Notes:17610 Casto, 1979 54 I and IVBaP, DBahA TF in number foci per 105survivingcells and dose (μg/mL)Ratio ofslopesData on enhancement ofviral transformation notused; no straightforwardway to model doseresponseModel as incidence datausing multistage17970 DiPaolo et al.,1969871 3 BaP, DBahA,BaA, BeP,DBacATotal transformants, total no.colonies, and dose (μg/mL)PointestimateDo not use % transformants;appears to be error forDBahA18020 Dunkel et al.,1981Use data as reported in 23720 Pienta1977; report under that record18080 Emura et al.,1980153,154I and II BaP, BbF,BaA, IPT, number of transformedcolonies/1,000 survivals in 10 dishesand dose (μg/mL)Ratio ofslopesModel as incidence datausing multistage14130 Greb et al.,1980147 1 BaP, CH, BaA,BbF, DBahA,BePRelative transformation rate(potency) in %/mmolRatio ofslopesRelative transformationpotency at LC50. Slopealready calculated.14640 Krolewski etal., 19861,648 1 BaP, CPcdP Transformation frequency per viablecell × 10-3; single dose (5 μM)PointestimateUse only BaP and CPcdPalone (not with IVA/AIA)14700 Laaksonen etal., 198362 4 BaP, BaA Transformation frequency (no.foci/105surviving cells) and dose(μM)Ratio ofslopesInverse dose responserelationship poss. due tocytotoxicity. Use peak.14850 Lubet et al.,1983992 1 BaP, BeP DwT-III/td (dishes with Type III foci/total dishes) and dose (μg/mL)Ratio ofslopesControl data in caption (notransformants). Model asincidence data.24710 Mohapatra etal., 1987327 1 BaP, BeAC,BjAC, BlACNumber of dishes scored and percentof dishes with Type II or Type IIIfoci and dose (μg/mL)Ratio ofslope toBaP pointestimateUse BaP incidence asBMRConvert percent into numberof dishes and model asincidence data.24700 Nesnow et al.,1990224 1 BaP, BlAC Anchorage independentcolonies/50,000 cells and dose(μg/mL)Ratio ofslopesContinuous data, no SD forcontrols; use peak.7980 Nesnow et al.,19971,975 I BaP, DBalP Type II and III foci/dish (meanandSD) and dose (μM)Ratio ofslopesModel as continuous data7990 Nesnow et al.,19942,227 I BaP, DBahA Type II and III Foci/dish and dose.Use 1 μg/mL dose for DBahA andmean foci/dish (in parentheses).Single dose for BaPPointestimateC-62 DRAFT – DO NOT CITE OR QUOTETable C-10. In vitro malignant/morphological celltransformation: data useRecordnumberReference PageTablenumberFigurenumberPAHs Data to be extractedBasis forRPF Comment Notes:8000 Nesnow et al.,1993a28 I DBkmnoAPHPeak of Type II and III foci/dish. Use5 μg/mL dose for DBkmnoAPH and3 μg/mL dose for BaP. Averagenumber foci/ dish across the twoexperimentsPointestimatePeak transformation for eachcompound. DBkmnoAPHreported in paper asCP(3,4)B[a]P23720 Pienta et al.,1977648 IV BaP, BaA,DBahATransformed colonies/survivingcolonies and dose (μg/mL, in rowacross)Ratio ofslopesModel as incidence datausing multistage1C-63 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotes17610 Casto, 1979 Control 0 μg/mL 0 Foci 100,000Surviving cells0BaP 0.62 μg/mL 8 Foci 100,000 Surviving cells0.00008BaP 1.25 μg/mL 10 Foci 100,000 Surviving cells0.0001DBahA 1.2 μg/mL 0.5 Foci 100,000 Surviving cells0.000005DBahA 2.5 μg/mL 1 Foci 100,000 Surviving cells0.0000117970 DiPaolo et al.,1969Control 0 μg/mL 0 Transformants 354 No. surviving0BaP 10 μg/mL 8 Transformants 138 No. surviving0.058DBahA 10 μg/mL 11 Transformants 354 No. surviving 0.031BaA 10 μg/mL 2 Transformants 190 No. surviving0.011BeP 10 μg/mL 1 Transformants 172 No. surviving0.0058DBacA 10 μg/mL 2 Transformants 181 No. surviving 0.01118080 Emura et al.,1980Control 0 μg/mL 0 Transformedcolonies1,000 Survivals 0Expt 1 BaP 0.01 μg/mL 0 Transformedcolonies1,000 Survivals 0BaP 0.05 μg/mL 1.1 Transformedcolonies1,000 Survivals 0.0011BaP 0.1 μg/mL 2.9 Transformedcolonies1,000 Survivals 0.0029BaP 0.25 μg/mL 5.3 Transformedcolonies1,000 Survivals 0.0053BaP 0.5 μg/mL 6.8 Transformedcolonies1,000 Survivals 0.0068BbF 0.025μg/mL 0 Transformedcolonies1,000 Survivals 0BbF 0.1 μg/mL 0.4 Transformedcolonies1,000 Survivals 0.00040BbF 0.25 μg/mL 0.3 Transformedcolonies1,000 Survivals 0.00030BbF 0.5 μg/mL 0.6 Transformedcolonies1,000 Survivals 0.00060BbF 1 μg/mL 1.2 Transformedcolonies1,000 Survivals 0.0012C-64 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotesBaA 0.025μg/mL 0 Transformedcolonies1,000 Survivals 0BaA 0.1 μg/mL 0.3 Transformedcolonies1,000 Survivals 0.00030BaA 0.25 μg/mL 0.3 Transformedcolonies1,000 Survivals 0.00030BaA 0.5 μg/mL 0.6 Transformedcolonies1,000 Survivals 0.00060BaA 1 μg/mL 1 Transformedcolonies1,000 Survivals 0.0010Expt 2 Control 0 μg/mL 0 Transformedcolonies1,000 Survivals 0BaP 0.01 μg/mL 0.4 Transformedcolonies1,000 Survivals 0.00040BaP 0.05 μg/mL 1 Transformedcolonies1,000 Survivals 0.0010BaP 0.1 μg/mL 2.9 Transformedcolonies1,000 Survivals 0.0029BaP 0.25 μg/mL 4.6 Transformedcolonies1,000 Survivals 0.0046BaP 0.5 μg/mL 7.8 Transformedcolonies1,000 Survivals 0.0078IP 0.025μg/mL 0 Transformedcolonies1,000 Survivals 0IP 0.1 μg/mL 0.3 Transformedcolonies1,000 Survivals 0.00030IP 0.25 μg/mL 0.3 Transformedcolonies1,000 Survivals 0.00030IP 0.5 μg/mL 0.7 Transformedcolonies1,000 Survivals 0.00070IP 1 μg/mL 1 Transformedcolonies1,000 Survivals 0.001014130 Greb et al., 1980BaP NA 277 %/mmolCH NA 37 %/mmolBaA NA 13.9 %/mmolC-65 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotesBbF NA 11.5 %/mmolDBahA NA 0.3 %/mmolBeP NA 3.1 %/mmol14640 Krolewski et al.,1986Control 0 μM 0 Transformationfrequency1,000 Viable cells 0BaP 5 μM 5.5 0.7 Transformationfrequency1,000 Viable cells 0.0055CPcdP 5 μM 1.7 0.3 Transformationfrequency1,000 Viable cells 0.001714700 Laaksonen et al.,1983Control 0 μM 0 Foci 1 × 105Surviving cells0BaP 5 μM 0.8 Foci 1 × 105Surviving cells0.0000080 Inverse dose responserelationship poss. due tocytotoxicity. Use peak.BaP 10 μM 0.9 Foci 1 × 105Surviving cells0.0000090BaP 20 μM 0.3 Foci 1 × 105Surviving cells0.0000030BaP 40 μM 0.4 Foci 1 × 105Surviving cells0.0000040Control 0 0 Foci 1 × 105Surviving cells0BaA 11 μM 1.8 Foci 1 × 105Surviving cells0.000018 Inverse dose responserelationship poss. due tocytotoxicity. Use peak.BaA 22 μM 1.5 Foci 1 × 105Surviving cells0.000015BaA 44 μM 1.1 Foci 1 × 105Surviving cells0.000011BaA 88 μM 0.8 Foci 1 × 105Surviving cells0.000008014850 Lubet et al.,1983Control 0 μg/mL 0 Dishes withType III fociTotal dishes 0BaP 1 μg/mL 1 Dishes withType III foci15 Total dishes 0.067BaP 3 μg/mL 4 Dishes withType III foci15 Total dishes 0.267BaP 10 μg/mL 5 Dishes withType III foci15 Total dishes 0.333BeP 10 μg/mL 0 Dishes withType III foci15 Total dishes 0C-66 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotesBeP 30 μg/mL 1 Dishes withType III foci15 Total dishes 0.067BeP 100 μg/mL 7 Dishes withType III foci15 Total dishes 0.46724710 Mohapatra et al.,1987Control 0 μg/mL 0 Dishes withType II or III foci48 Dishes scored 0BaP 1 μg/mL 44 Dishes withType II or III foci48 Dishes scored 0.92BjAC 0.01 μg/mL 2 Dishes withType II or III foci48 Dishes scored 0.04BjAC 0.05 μg/mL 5 Dishes withType II or III foci48 Dishes scored 0.1BjAC 0.5 μg/mL 34 Dishes withType II or III foci48 Dishes scored 0.71BjAC 1 μg/mL 45 Dishes withType II or III foci48 Dishes scored 0.94BjAC 2 μg/mL 48 Dishes withType II or III foci48 Dishes scored 1Control 0 μg/mL 0 Dishes withType II or III foci60 Dishes scored 0BaP 1 μg/mL 50 Dishes withType II or III foci60 Dishes scored 0.83BlAC 0.5 μg/mL 8 Dishes withType II or III foci60 Dishes scored 0.13BlAC 1 μg/mL 14 Dishes withType II or III foci60 Dishes scored 0.26BlAC 2.5 μg/mL 31 Dishes withType II or III foci60 Dishes scored 0.52BlAC 5 μg/mL 42 Dishes withType II or III foci60 Dishes scored 0.7BlAC 10 μg/mL 51 Dishes withType II or III foci60 Dishes scored 0.85Control 0 μg/mL 0 Dishes withType II or III foci36 Dishes scored 0BaP 1 μg/mL 31 Dishes withType II or III foci36 Dishes scored 0.86C-67 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotesBeAC 0.5 μg/mL 4 Dishes withType II or III foci36 Dishes scored 0.11BeAC 1 μg/mL 6 Dishes withType II or III foci36 Dishes scored 0.17BeAC 2.5 μg/mL 13 Dishes withType II or III foci36 Dishes scored 0.36BeAC 5 μg/mL 15 Dishes withType II or III foci36 Dishes scored 0.42BeAC 10 μg/mL 21 Dishes withType II or III foci36 Dishes scored 0.5824700 Nesnow et al.,1990Acetone 0 μg/mL 25 Anchorageindependentcolonies/50,000cellsBaP 0.1 μg/mL 43 14.7 Anchorageindependentcolonies/50,000cellsBaP 0.5 μg/mL 42 20.7 Anchorageindependentcolonies/50,000cellsBaP 2.5 μg/mL 39 19.5 Anchorageindependentcolonies/50,000cellsBaP 10 μg/mL 72 23.1 Anchorageindependentcolonies/50,000cellsAcetone 0 μg/mL 30 Anchorageindependentcolonies/50,000cellsBlAC 0.1 μg/mL 74 5.2 Anchorageindependentcolonies/50,000cellsC-68 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotesBlAC 0.5 μg/mL 68 14.4 Anchorageindependentcolonies/50,000cellsBlAC 2.5 μg/mL 123 15.6 Anchorageindependentcolonies/50,000cellsBlAC 10 μg/mL 150 16.8 Anchorageindependentcolonies/50,000cells7980 Nesnow et al.,1997Control 0 μM 0 0 Type II and IIIfoci/dishBaP 0.4 μM 0.44 0.24 Type II and IIIfoci/dishBaP 1.2 μM 1.25 0.15 Type II and IIIfoci/dishBaP 4 μM 2.54 0.56 Type II and IIIfoci/dishDBalP 0.0033μM 0.14 0.35 Type II and IIIfoci/dishDBalP 0.1 μM 1 0.24 Type II and IIIfoci/dishDBalP 0.33 μM 1.74 0.78 Type II and IIIfoci/dish7990 Nesnow et al.,1994Control 0 μg/mL 0.06 0.10 Type II and IIIfoci/dishBaP 1 μg/mL 1 0.43 Type II and IIIfoci/dishDBahA 0.25 μg/mL 0.23 0.21 Type II and IIIfoci/dishDBahA 0.5 μg/mL 0.25 0.33 Type II and IIIfoci/dishDBahA 1 μg/mL 0.43 0.11 Type II and IIIfoci/dishC-69 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotesDBahA 2.5 μg/mL 0.29 0.085 Type II and IIIfoci/dish8000 Nesnow et al.,1993aControl 0 μg/mL 0 Type II and IIIfoci/dishBaP 0.3 μg/mL 0.48 Type II and IIIfoci/dishBaP 1 μg/mL 0.665 Type II and IIIfoci/dishBaP 3 μg/mL 1.4 Type II and IIIfoci/dishControl 0 μg/mL 0 Type II and IIIfoci/dishDBkmnoAPH0.5 μg/mL 0.23 Type II and IIIfoci/dishDBkmnoAPH1 μg/mL 0.52 Type II and IIIfoci/dishDBkmnoAPH2.5 μg/mL 0.605 Type II and IIIfoci/dishDBkmnoAPH5 μg/mL 1.085 Type II and IIIfoci/dish23720 Pienta et al.,1977Control 0 μg/mL 0 Transformedcolonies504 Survivingcolonies0 BaP and BaA data alsoreported in 18020 Dunkel1981BaP 1 μg/mL 1 Transformedcolonies393 Survivingcolonies0.0025BaP 5 μg/mL 2 Transformedcolonies406 Survivingcolonies0.0049BaP 10 μg/mL 3 Transformedcolonies434 Survivingcolonies0.0069BaP 20 μg/mL 5 Transformedcolonies410 Survivingcolonies0.0122BaP 40 μg/mL 4 Transformedcolonies427 Survivingcolonies0.0094Control 0 μg/mL 0 Transformedcolonies229 Survivingcolonies0C-70 DRAFT – DO NOT CITE OR QUOTETable C-11. In vitro malignant/morphological celltransformation: dose response dataTransformation measureRecordnumberReference PAH DoseDoseunitsMeanStandarddeviationStandarderror Unitsn units % ResponseNotesBaA 0.1 μg/mL 1 Transformedcolonies225 Survivingcolonies0.0044BaA 0.5 μg/mL 2 Transformedcolonies252 Survivingcolonies0.0079BaA 1 μg/mL 2 Transformedcolonies193 Survivingcolonies0.0104BaA 5 μg/mL 1 Transformedcolonies312 Survivingcolonies0.0032BaA 10 μg/mL 7 Transformedcolonies250 Survivingcolonies0.028Control 0 μg/mL 0 Transformedcolonies229 Survivingcolonies0DBahA 0.1 μg/mL 0 Transformedcolonies219 Survivingcolonies0DBahA 0.5 μg/mL 4 Transformedcolonies233 Survivingcolonies0.0172DBahA 1 μg/mL 4 Transformedcolonies217 Survivingcolonies0.0184DBahA 5 μg/mL 5 Transformedcolonies270 Survivingcolonies0.0185DBahA 10 μg/mL 0 Transformedcolonies232 Survivingcolonies0

### C-71 DRAFT – DO NOT CITE OR QUOTE

Table C-12. In vitro DNA adducts: data use

Record
number Reference Page Table
number Figure
number PAHs Data to be

extracted Basis for

RPF Comment Notes:

16890 Allen and Coombs,
1980 245 1 BaP, BaA μmol compound/mol
DNA P Point

estimate Adducts in

nuclear and mitochondrial
DNA Calculate

separate RPFs for nuclear andmitochondrial DNA

6300 Binkova et

al., 2000 62 3 BaP, DBalP Adducts at

each dose level Ratio of

slopes Slope of adduct

vs. dose curve May need to

drop high dose data for adequate fit 9510 Bryla and

Weyand,
1992 39 1 BaP, BaA,
DBacA Adducts at

each dose level Ratio of

slopes Slope of adduct

vs. dose curve under light conditions (max response for all compounds)

22800 Grover and

Sims, 1968 160 1 BaP, DBahA, DBacA, BaA, Pyr,
PH Reaction

with DNA Point

estimate

10660 Johnsen et al., 1998 80 2 BjAC, BlAC, BaP Total adduct

levels in human lymphocytes and HL-60 cells Point

estimate Total adducts

formed in human lymphocytes or HL-60 cells Calc RPFs

separately by cell type

10670 Johnsen et al., 1997 196 II BjAC, BlAC, BaP DNA adduct

levels in PCB-treated rat lung cells Point

estimate Adducts in

PCB-treated rat lung Clara and Type 2 cells Calc RPFs

separately by cell type

7870 MelendezColon et al., 2000 13 2 BaP, DBalP Stable DNA

adducts at each dose level Ratio of

slopes Slope of adduct

vs. dose curve at two doses

21200 Segerback

and Vodicka,
1993 2,465 3 Pyr, BghiP, FA, DBahA, BbF, BaP, BaA, CH Total adduct

levels Point

estimate Total adduct

level in optimized nuclease P1 adduct enrichment procedureC-72 DRAFT – DO NOT CITE OR QUOTETable C-13.In vitro DNA adducts: dose response dataDNA AdductsRecordnumberReferencePAH DoseDoseunitsMeanStandarddeviation Adduct unitsn Units Notes16890 Allen andCoombs,1980BaP 0.235 μg/mL 7.5 1.9 μmol/mol DNAPNuclear DNABaA 0.644 μg/mL 0.44 0.11 μmol/mol DNAPNuclear DNABaP 0.235 μg/mL 413 164 μmol/mol DNAPMitochondrial DNABaA 0.644 μg/mL 104 40.2 μmol/mol DNAPMitochondrial DNA6300 Binkova etal., 2000BaP 0.010 μM 1.8 1.16 Adducts 1 × 108Nucleotides0.10 μM 18 7.18 Adducts 1 × 108Nucleotides0.40 μM 95 39.4 Adducts 1 × 108Nucleotides1.0 μM 258 115 Adducts 1 × 108Nucleotides4.0 μM 205 81.9 Adducts 1 × 108Nucleotides10 μM 69 21.9 Adducts 1 × 108Nucleotides40 μM 37 10.8 Adducts 1 × 108NucleotidesDBalP 0.010 μM 179 55.3 Adducts 1 × 108Nucleotides0.020 μM 534 52.6 Adducts 1 × 108Nucleotides0.040 μM 1,304 375 Adducts 1 × 108Nucleotides0.080 μM 1,696 644 Adducts 1 × 108Nucleotides0.10 μM 2,317 774 Adducts 1 × 108Nucleotides0.40 μM 1,971 729 Adducts 1 × 108Nucleotides1.0 μM 632 170 Adducts 1 × 108Nucleotides9510 Bryla andWeyand,1992BaP 0.12 nmol 0.17 Adducts 1 × 107Nucleotides Light conditions; max for BaP and othersBaP 12 nmol 1.37 Adducts 1 × 107NucleotidesBaP 120 nmol 2.21 Adducts 1 × 107NucleotidesBaP 600 nmol 5.45 Adducts 1 × 107NucleotidesBaA 0.12 nmol 0.15 Adducts 1 × 107NucleotidesBaA 12 nmol 0.09 Adducts 1 × 107NucleotidesBaA 120 nmol 0.8 Adducts 1 × 107NucleotidesC-73 DRAFT – DO NOT CITE OR QUOTETable C-13.In vitro DNA adducts: dose response dataDNA AdductsRecordnumberReferencePAH DoseDoseunitsMeanStandarddeviation Adduct unitsn Units NotesBaA 600 nmol 0.95 Adducts 1 × 107NucleotidesDBacA 0.12 nmol 0 Adducts 1 × 107NucleotidesDBacA 12 nmol 0.06 Adducts 1 × 107NucleotidesDBacA 120 nmol 0.57 Adducts 1 × 107NucleotidesDBacA 600 nmol 1.76 Adducts 1 × 107Nucleotides22800 Grover andSims, 1968BaP 5 μg 1.41 μmol/g-atom ofDNA PDBahA 5 μg 0.44 μmol/g-atom ofDNA PDBacA5 μg 0.56 μmol/g-atom ofDNA PBaA 5 μg 0.7 μmol/g-atom ofDNA PPyr 5 μg 0.31 μmol/g-atom ofDNA PPH 5 μg 0.05 μmol/g-atom ofDNA P10670 Johnsen etal., 1997BaP 30 μg/mL 0.05 fmol adducts/μgDNAClara cellsBjAC 30 μg/mL 0.15 fmol adducts/μgDNAClara cellsBlAC 30 μg/mL 0.24 fmol adducts/μgDNAClara cellsBaP 30 μg/mL 0.02 fmol adducts/μgDNAType 2 cellsBjAC 30 μg/mL 0.06 fmol adducts/μgDNAType 2 cellsBlAC 30 μg/mL 0.03 fmol adducts/μgDNAType 2 cells10660 Johnsen etal., 1998BaP 30 μg/mL 0.333 0.093 fmol adducts/μgDNA3 Human lymphocytesBjAC 30 μg/mL 0.110 0.026 fmol adducts/μgDNA3 Human lymphocytesBlAC 30 μg/mL 1.089 0.595 fmol adducts/μgDNA3 Human LymphocytesC-74 DRAFT – DO NOT CITE OR QUOTETable C-13.In vitro DNA adducts: dose response dataDNA AdductsRecordnumberReferencePAH DoseDoseunitsMeanStandarddeviation Adduct unitsn Units NotesBaP 30 μg/mL 0.239 0.172 fmol adducts/μgDNA3 HL-60 CellsBjAC 30 μg/mL 0.149 0.146 fmol adducts/μgDNA3 HL-60 CellsBlAC 30 μg/mL 0.942 0.344 fmol adducts/μgDNA3 HL-60 Cells7870 MelendezColon et al.,2000BaP 1 μm 18 8.07 Stable adducts 1 × 106NucleotidesBaP 2 μm 34 6.46 Stable adducts 1 × 106NucleotidesDBalP 1 μm 254 4.30 Stable adducts 1 × 106NucleotidesDBalP 2 μm 348 17.20 Stable adducts 1 × 106Nucleotides21200 SegerbackandVodicka,1993BaP 100 mM 15 μmol adducts permol dNpPyr 100 mM 0.14 μmol adducts permol dNpBghiP 100 mM 0.50 μmol adducts permol dNpFA 100 mM 1.5 μmol adducts permol dNpDBahA 100 mM 2.8 μmol adducts permol dNpBbF 100 mM 3.7 μmol adducts permol dNpBaA 100 mM 30 μmol adducts permol dNpCH 100 mM 50 μmol adducts permol dNp12C-75 DRAFT – DO NOT CITE OR QUOTETable C-14. In vitro DNA damage: data useRecordnumberReference PageTablenumberFigurenumberPAHs Data to be extracted Basis for RPF Comment Notes:16840 Agrelo andAmos, 1981531 2 BaP, Pyr Hydroxyurea inhibited3H Thymidineincorporation into cells (dpm) and dose(μg/mL). Use 10 μg/mL dose for BaP and100 μg/mL dose for pyrene.Point estimate23790Ichinotsuboet al., 197756Table IIBaP, DBaiP, DBahAUse column designated JC5519 +S9for BaP, DBaiP, and DBahA; dose asμg/well and response as diameter ofzone of inhibition (mm). The controlis wild type strain AB1157Point estimateE. coliRec BC, S9identificationunknown10660 Johnsen etal., 199882 4 BaP, BjAC, BlAC DNA damage (NAAC, 10-3h-1), std devand dose (μg/mL) for both humanlymphocytes and HL-60 cells. Use 24 h +1 h AraC/HU data (crosshatched bars)Ratio of slopes(human lymphocytes); point estimates (HL-60 cells)Model ascontinuousdata.19740 Martin et al.,19782,6241 BaP, BeP, BaA,DBacA, DBahAMaximum dpm/μg DNA abovebackground and dose (M). Dose is incolumn marked "M".Point estimate Background alreadysubtracted19830 MerschSundermannet al., 19923-6 2 BaP, AA, BaA, BbF,BghiF, BjF, BbFE,BghiP, BeP, CH,DBacA, DBahA,DBalP, DBahP,DBaiP, FA, IP, PH,TphenSOS induction potential (SOSIP) for assay(+S9) for each compound (alreadyincorporates dose)Ratio of SOSIPsSOSIP reported in textas slope of steepestportion of theinduction factor (IF)dose-response curve.No modelingnecessary;slopesreported intext.20810 Robinsonand Mitchell,1981520 1 BaP, Pyr Maximum3H-TDR incorporation anddose (test concentration in μg/mL inparentheses after maximum) for rows withmetabolic activation (+). Use compoundspecific background3H -TDRincorporation in same row.Point estimate20940 Rossman etal., 1991354 2 BaP, AC, DBacA,DBahA, PHMax enhancement of prophage inductionover background and dose (amount atmax, in μg/well) for those rows with S9 (+rows).Point estimate Background alreadyaddressed21730 Tong et al.,1981b480 I BaP, BaA DNA repair grains/nucleus, std dev,anddose (M). Four doses BaA, three dosesBaP and DMSO control.Ratio of slopes Model ascontinuousdata.1C-76 DRAFT – DO NOT CITE OR QUOTE1Table C-15. In vitro DNA damage: dose response dataDNA DamageRecordnumberReference PAH DoseDoseunits EndpointMean SD Unitsn Notes16840 Agrelo andAmos, 1981Control0 μg/mL Unscheduled DNA synthesis177 dpm HU inhibitedBaP 0.001 μg/mL Unscheduled DNA synthesis195 dpm HU inhibitedBaP 0.01 μg/mL Unscheduled DNA synthesis126 dpm HU inhibitedBaP 0.1 μg/mL Unscheduled DNA synthesis262 dpm HU inhibitedBaP 1 μg/mL Unscheduled DNA synthesis818 dpm HU inhibitedBaP 10 μg/mL Unscheduled DNA synthesis2,270 dpm HU inhibitedBaP 100 μg/mL Unscheduled DNA synthesis819 dpm HU inhibitedBaP 1,000 μg/mL Unscheduled DNA synthesis373 dpm HU inhibitedControl0 μg/mL Unscheduled DNA synthesis1,168 dpm HU inhibitedPyr 0.032 μg/mL Unscheduled DNA synthesis1,293 dpm HU inhibitedPyr 0.16 μg/mL Unscheduled DNA synthesis1,192 dpm HU inhibitedPyr 0.8 μg/mL Unscheduled DNA synthesis1,367 dpm HU inhibitedPyr 4 μg/mL Unscheduled DNA synthesis1,510 dpm HU inhibitedPyr 20 μg/mL Unscheduled DNA synthesis1,694 dpm HU inhibitedPyr 100 μg/mL Unscheduled DNA synthesis1,716 dpm HU inhibited23790Ichinotsubo et al.,1977Control0DNA damage0Diameter of zoneof inhibition mmBaP70μg/wellDNA damage6Diameter of zoneof inhibition mmControl0DNA damage0Diameter of zoneof inhibition mmDBaiP600μg/wellDNA damage10Diameter of zoneof inhibition mmControl0DNA damage0Diameter of zoneof inhibition mmDBahA25μg/wellDNA damage10Diameter of zoneof inhibition mm10660 Johnsen et al.,1998DMSO0 μg/mL DNA damage 4.4 1.3 NAAC, 10-3h-13 Human lymphocytes withAraC/HUC-77 DRAFT – DO NOT CITE OR QUOTETable C-15. In vitro DNA damage: dose response dataDNA DamageRecordnumberReference PAH DoseDoseunits EndpointMean SD Unitsn NotesBaP 3 μg/mL DNA damage 12 3.2 NAAC, 10-3h-63 Human lymphocytes withAraC/HU. No continuous linearmodel fit30 μg/mL DNA damage 15 2.7 NAAC, 10-3h-73 Human lymphocytes withAraC/HUBjAC 3 μg/mL DNA damage 6.0 2.1 NAAC, 10-3h-23 Human lymphocytes withAraC/HU30 μg/mL DNA damage 9.4 3.4 NAAC, 10-3h-33 Human lymphocytes withAraC/HUBIAC 3 μg/mL DNA damage 8.2 3.2 NAAC, 10-3h-43 Human lymphocytes withAraC/HU. No continuous linearmodel fit30 μg/mL DNA damage 9.3 2.1 NAAC, 10-3h-53 Human lymphocytes withAraC/HUDMSO0 μg/mL DNA damage 7.8 3.1 NAAC, 10-3h-53 HL-60 cells with AraC/HUBaP 30 μg/mL DNA damage 13.2 9.5 NAAC, 10-3h-53 HL-60 cells with AraC/HUBjAC 30 μg/mL DNA damage 9.6 3.0 NAAC, 10-3h-53 HL-60 cells with AraC/HUBIAC 30 μg/mL DNA damage 11.6 5.5 NAAC, 10-3h-53 HL-60 cells with AraC/HU19740 Martin et al.,1978BaP 1 × 10-5M Unscheduled DNA synthesis210 Max dpm/μgDNAIncrease above backgroundBeP 1 × 10-6M Unscheduled DNA synthesis256 Max dpm/μgDNAIncrease above backgroundBaA 1 × 10-7M Unscheduled DNA synthesis59 Max dpm/μgDNAIncrease above backgroundDBacA1 × 10-5M Unscheduled DNA synthesis97 Max dpm/μgDNAIncrease above backgroundDBahA1 × 10-5M Unscheduled DNA synthesis96 Max dpm/μgDNAIncrease above background19830 MerschSundermann etal., 1992BaP NA SOS induction potential 0.605 NA Steepestslope of induction factordose-response curve; + S9AA NA SOS induction potential 0.142 NA Steepest slope of induction factordose-response curve; + S9C-78 DRAFT – DO NOT CITE OR QUOTETable C-15. In vitro DNA damage: dose response dataDNA DamageRecordnumberReference PAH DoseDoseunits EndpointMean SD Unitsn NotesBaA NA SOS induction potential 0.1 NA Steepestslope of induction factordose-response curve; + S9BbF NA SOS induction potential 0.045 NA Steepest slope of induction factordose-response curve; + S9BghiF NA SOS induction potential 0.34 NA Steepest slope of induction factordose-response curve; + S9BjF NA SOS induction potential 0.254 NA Steepest slope of induction factordose-response curve; + S9BbFE NA SOS induction potential 0.024 NA Steepest slope of induction factordose-response curve; + S9BghiP NA SOS induction potential 0.033 NA Steepest slope of induction factordose-response curve; + S9BeP NA SOS induction potential 0.032 NA Steepest slope of induction factordose-response curve; + S9CH NA SOS induction potential 0.221 NA Steepest slope of induction factordose-response curve; + S9DBacANA SOS induction potential 0.104 NA Steepest slope of induction factordose-response curve; + S9DBahANA SOS induction potential 0.039 NA Steepest slope of induction factordose-response curve; + S9DBalP NA SOS induction potential 2.1 NA Steepest slope of induction factordose-response curve; + S9DBahPNA SOS induction potential 0.117 NA Steepest slope of induction factordose-response curve; + S9DBaiP NA SOS induction potential 0.174 NA Steepest slope of induction factordose-response curve; + S9FA NA SOS induction potential 0.412 NA Steepest slope of induction factordose-response curve; + S9IP NA SOS induction potential 0.036 NA Steepest slope of induction factordose-response curve; + S9PH NA SOS induction potential 0.053 NA Steepest slope of induction factordose-response curve; + S9C-79 DRAFT – DO NOT CITE OR QUOTETable C-15. In vitro DNA damage: dose response dataDNA DamageRecordnumberReference PAH DoseDoseunits EndpointMean SD Unitsn NotesTphen NA SOS induction potential 0.26 NA Steepest slope of induction factordose-response curve; + S920810 Robinson andMitchell, 1981Control0 μg/mL Unscheduled DNA synthesis53 43H-TdRincorporationMax 3H-TdR incorporationBaP 10 μg/mL Unscheduled DNA synthesis142 73H-TdRincorporationMax 3H-TdR incorporationControl0 μg/mL Unscheduled DNA synthesis52 23H-TdRincorporationMax 3H-TdR incorporationPyr 7.2 μg/mL Unscheduled DNA synthesis115 93H-TdRincorporationMax 3H-TdR incorporation20940 Rossman et al.,1991BaP 12.5 μg/mL DNA damage 10.4 Lambda prophage inductionMax enhancement overbackgroundAC 12.5 μg/mL DNA damage 4.8 Lambda prophage inductionMax enhancement overbackgroundDBacA1.44 μg/mL DNA damage 8 Lambda prophage inductionMax enhancement overbackgroundDBahA2 μg/mL DNA damage 4 Lambda prophage inductionMax enhancement overbackgroundPH 25 μg/mL DNA damage 4.5 Lambda prophage inductionMax enhancement overbackground21730 Tong et al.,1981bControl0 M Unscheduled DNA synthesis0.1 0.1 Grains/nucleusBaP 1 × 10-4M Unscheduled DNA synthesis45.1 3.7 Grains/nucleusBaP 5 × 10-4M Unscheduled DNA synthesis47.7 3.7 Grains/nucleusBaP 1 × 10-3M Unscheduled DNA synthesis65.6 17.8 Grains/nucleusBaA 5 × 10-5M Unscheduled DNA synthesis0.6 Grains/nucleusBaA 1 × 10-4M Unscheduled DNA synthesis14.8 2.6 Grains/nucleusBaA 5 × 10-4M Unscheduled DNA synthesis17.2 6 Grains/nucleusBaA 1 × 10-3M Unscheduled DNA synthesisToxic Grains/nucleus1

### C-80 DRAFT – DO NOT CITE OR QUOTE

Table C-16. In vitro clastogenicity: data use

Record
number Reference Page Table
number PAHs Data to be used Basis for
RPF Comment
14620 Kochhar, 1982 846 Not numbered BaP,

BaA % cells with aberrations and dose (μg/mL) Ratio of

slopes Model as incidence

data.

14640 Krolewski et al.,

1986 1,648 II BaP, CPcdP Mean no. SCE/ chromosome, std dev, and dose (μM) Ratio of

slopes Use first column of

data; not data with AIA or IVA. Model as continuous data.
19690 Mane et al., 1990 81 III BaP,
BaA SCE frequencies/for V79 cell + rat MEC and dose Point

estimates Use SCE data for V79 +

rat MEC only.

21710 Tong et al.,

1981a 469 1 BaP,
BaA SCE/cell, std dev, and dose Point

estimates Continuous data, no n

provided in study.

1

### C-81 DRAFT – DO NOT CITE OR QUOTE

Table C-17. In vitro clastogenicity: dose response data

Clastogenicity

Record
number Reference PAH Dose Dose units n Mean Standard deviation Units Notes

14620 Kochhar,

1982 Control 0 μg/mL 100 0.06 Fraction cells with aberrations
BaP 0.6 μg/mL 100 0.23 Fraction cells with
aberrations
BaP 1.25 μg/mL 100 0.32 Fraction cells with aberrations
BaP 2.5 μg/mL 100 0.45 Fraction cells with aberrations
BaP 5 μg/mL 100 0.56 Fraction cells with aberrations
BaA 0.6 μg/mL 100 0.17 Fraction cells with aberrations
BaA 1.25 μg/mL 100 0.23 Fraction cells with
aberrations
BaA 2.5 μg/mL 100 0.3 Fraction cells with
aberrations
BaA 5 μg/mL 100 0.38 Fraction cells with aberrations

14640 Krolewski et al., 1986 Control 0 μM 30 0.147 0.059 SCE/

BaP 1 μM 30 0.874 0.275 SCE/
BaP 5 μM 30 0.932 0.266 SCE/
CPcdP 1 μM 30 0.348 0.119 SCE/
CPcdP 5 μM 30 0.432 0.15 SCE/

19690 Mane et al.,

1990 Control 0 μg/mL 0.3 1 SCE frequency/ For V79 cell + rat MEC
BaP 1 μg/mL 3 1 SCE frequency/ For V79 cell + ra t MEC
BaA 1 μg/mL 0.7 0.5 SCE frequency/ For V79 cell + rat MEC
21710 Tong et al.,

1981a Control 0 M 11.15 3.81 SCE/cell

BaP 1 × 10 -6 M 16.15 3.83 SCE/cell

BaP 1 × 10 -5 M 59.75 16.96 SCE/cell

BaP 1 × 10 -4 M 103.3 22.75 SCE/cell

Control 0 M 15.75 5.18 SCE/cell

BaA 1 × 10 -5 M 21.2 9.59 SCE/cell

BaA 1 × 10 -4 M 29.15 9.93 SCE/cell

BaA 1 × 10 -3 M 26.2 6.96 SCE/cellC-82 DRAFT – DO NOT CITE OR QUOTETable C-18.In vivoDNA adducts: data useRecordnumberReferencePageTablenumberFigurenumberPAHsData to beextractedBasis forRPF Comment Notes6210 Arif et al.,199736 4 DBalP and BaP Mean adduct levelsfor heart, pancreas,bladder, liverPointestimateMean adduct levels summed acrossmammary epithelial, lung, heart,pancreas, bladder, liver17630Cavalieri etal., 1981a4913 CPcdP, ACEP(reported in paperas CPAP), BaPDone PointestimateDNA-bound PAH in mouse skinafter 4 hr or 24 hr treatmentCalculate separateRPFs for 4 hr and24 hr treatment18810 HughesandPhillips,19901,6143 DBalP, DBaeP,DBahP, DBaiP,BaPAUC for skin andlung through 84 dPointestimateSum of AUCs for skin and lung0–84 d11190Mass et al.,19931881 BjAC, BaP Done Ratio ofSlopesAUC (adduct-time curve) vs. dosefor lung adducts 24–72 hr8010 Nesnow etal., 1993b39 1 and 2 BbF, BaPAUC for lung, liver,and PBL through56 dPointestimateSum of AUCs for lung, liver, andlymphocytes 0–56 d24590/20920Nesnow etal., 1998b;Ross et al.,19954022 BaP, BbF,DBahA, CPcdP,DBalPDone Ratio ofSlopesSlope of TIDAL/dose (slope reportedin 24590 based on data from 20920).DBalP data reported in separatestudy w/o BaP concurrent22810 Phillips etal., 1979205I DBahA, DBacA,BaPDone PointestimatePeak binding in mouse skin. BaAdropped; not clear if reported level ispeak.24790 Kligermanet al., 20028461 BaA, BaP, BbF,CHDone PointestimateAdducts in mouse or rat PBLs atsingle time point after eitherintraperitoneal or gavageadministrationCalculate separateRPFs forintraperitoneal andgavage, rat andmouse12C-83 DRAFT – DO NOT CITE OR QUOTETable C-19.In vivo DNA adducts: dose response dataDNA adductsRecordnumberReferencePAH SpeciesDoseDose units Organ TimeMeanStandarddeviationStandarderror Adduct unitsSlope ofAUC vs.dose Comments6210 Arif et al.,1997ControlRat 0 μmol/ mammary glandLiver 0 Adducts/109nucleotidesBaP Rat 0.25μmol/mammary glandMammarygland300 45 Adducts/109nucleotidesBaP Rat 0.25μmol/mammary glandLung 11 1.3 Adducts/109nucleotidesBaP Rat 0.25μmol/mammary glandHeart 9.5 Adducts/109nucleotidesBaP Rat 0.25μmol/mammary glandPancreas 0 Adducts/109nucleotidesBaP Rat 0.25μmol/mammary glandBladder 0 Adducts/109nucleotidesBaP Rat 0.25μmol/mammary glandLiver 4.5 Adducts/109nucleotidesSum 324.74DBalP Rat 0.25μmol/mammary glandMammarygland1,878 378 Adducts/109nucleotidesDBalP Rat 0.25μmol/mammary glandLung 85 24 Adducts/109nucleotidesDBalP Rat 0.25μmol/mammary glandHeart 64 Adducts/109nucleotidesDBalP Rat 0.25μmol/mammary glandPancreas 32 Adducts/109nucleotidesDBalP Rat 0.25μmol/mammary glandBladder 69 Adducts/109nucleotidesDBalP Rat 0.25μmol/mammary glandLiver 116 Adducts/109nucleotidesSum 2,244.6317630Cavalieri etal., 1981aBaP 0.2 μmol/mouse Skin 4 hr 16.3 1μmol adduct/molDNACPcdP 0.2 μmol/mouse Skin 4 hr 2.3 0.2μmol adduct/molDNAACEP 0.2 μmol/mouse Skin 4 hr 2.2 0.1μmol adduct/molDNAC-84 DRAFT – DO NOT CITE OR QUOTETable C-19.In vivo DNA adducts: dose response dataDNA adductsRecordnumberReferencePAH SpeciesDoseDose units Organ TimeMeanStandarddeviationStandarderror Adduct unitsSlope ofAUC vs.dose CommentsBaP 0.2 μmol/mouse Skin 24 hr 6.7 1.6μmol adduct/molDNACPcdP 0.2 μmol/mouse Skin 24 hr 8.8 1μmol adduct/molDNAACEP 0.2 μmol/mouse Skin 24 hr 0.30 0.1μmol adduct/molDNA18810 HughesandPhillips,1990BaP 1 μmol Skin 1 d 7.8 fmol adducts/μgDNAOnly peak extracted;interrupted scaleprecluded digitizingBaP 1 μmol Lung 2 d 1.2 fmol adducts/μgDNABaP 1 μmol Sum skinand lung9.0 fmol adducts/μgDNADBaeP1 μmol Skin 2 d 0.50 fmol adducts/μgDNADBaeP1 μmol Lung 7 d Cannotdeterminefmol adducts/μgDNADBaeP1 μmol Sum skinand lungCannotdeterminefmol adducts/μgDNADBahP1 μmol Skin 2 d 3.1 fmol adducts/μgDNADBahP1 μmol Lung 2 d 0.14 fmol adducts/μgDNADBahP1 μmol Sum skinand lung3.2 fmol adducts/μgDNADBaiP 1 μmol Skin 2 d 0.75 fmol adducts/μgDNADBaiP 1 μmol Lung 2 d 0.10 fmol adducts/μgDNADBaiP 1 μmol Sum skinand lung0.85 fmol adducts/μgDNADBalP 1 μmol Skin 1 d 62 fmol adducts/μgDNADBalP 1 μmol Lung 2 d 2.3 fmol adducts/μgDNAC-85 DRAFT – DO NOT CITE OR QUOTETable C-19.In vivo DNA adducts: dose response dataDNA adductsRecordnumberReferencePAH SpeciesDoseDose units Organ TimeMeanStandarddeviationStandarderror Adduct unitsSlope ofAUC vs.dose CommentsDBalP 1 μmol Sum skinand lung65 fmol adducts/μgDNA11190 Mass et al.,1993BaP 20 mg/kg bw Lung 24 hr 116 53 amol adducts/μgDNAAUC calculated usingtrapezoid ruleBaP 20 mg/kg bw Lung 48 hr 122 25 amol adducts/μgDNABaP 20 mg/kg bw Lung 72 hr 181 101 amol adducts/μgDNABaP 50 mg/kg bw Lung 24 hr 120 20 amol adducts/μgDNABaP 50 mg/kg bw Lung 48 hr 201 170 amol adducts/μgDNABaP 50 mg/kg bw Lung 72 hr 432 274 amol adducts/μgDNABaP 100mg/kg bw Lung 24 hr 427 140 amol adducts/μgDNABaP 100mg/kg bw Lung 48 hr 407 197 amol adducts/μgDNABaP 100mg/kg bw Lung 72 hr 2,004 314 amol adducts/μgDNABaP 20 mg/kg bw Lung AUC 7,884 469.73BaP 50 mg/kg bw Lung AUC 12,888BaP 100mg/kg bw Lung AUC 44,064BjAC 20 mg/kg bw Lung 24 hr 63 34 amol adducts/μgDNAAUC calculated usingtrapezoid ruleBjAC 20 mg/kg bw Lung 48 hr 97 101 amol adducts/μgDNABjAC 20 mg/kg bw Lung 72 hr 255 392 amol adducts/μgDNABjAC 50 mg/kg bw Lung 24 hr 116 121 amol adducts/μgDNABjAC 50 mg/kg bw Lung 48 hr 402 237 amol adducts/μgDNABjAC 50 mg/kg bw Lung 72 hr 1,954 1,921 amol adducts/μgDNAC-86 DRAFT – DO NOT CITE OR QUOTETable C-19.In vivo DNA adducts: dose response dataDNA adductsRecordnumberReferencePAH SpeciesDoseDose units Organ TimeMeanStandarddeviationStandarderror Adduct unitsSlope ofAUC vs.dose CommentsBjAC 100mg/kg bw Lung 24 hr 180 133 amol adducts/μgDNABjAC 100mg/kg bw Lung 48 hr 532 559 amol adducts/μgDNABjAC 100mg/kg bw Lung 72 hr 2,439 2,242 amol adducts/μgDNABjAC 20 mg/kg bw Lung AUC 6,900 464.25BjAC 50 mg/kg bw Lung AUC 35,880BjAC 100mg/kg bw Lung AUC 46,3568010 Nesnow etal., 1993bBaP 100mg/kg Lung d 1 453 AUC calculated usingtrapezoid ruleBaP 100mg/kg Lung d 3 1,001BaP 100mg/kg Lung d 7 574BaP 100mg/kg Lung d 14 386BaP 100mg/kg Lung d 28 381BaP 100mg/kg Lung d 56 143BaP 100mg/kg Lung AUC 20,892BaP 100mg/kg Liver d 1 398BaP 100mg/kg Liver d 3 1,317BaP 100mg/kg Liver d 7 931BaP 100mg/kg Liver d 14 537BaP 100mg/kg Liver d 28 394BaP 100mg/kg Liver d 56 116BaP 100mg/kg Liver AUC 25,207BaP 100mg/kg PBL d 1 158BaP 100mg/kg PBL d 3 273BaP 100mg/kg PBL d 7 162BaP 100mg/kg PBL d 14 187BaP 100mg/kg PBL d 28 72BaP 100mg/kg PBL d 56 41BaP 100mg/kg PBL AUC 5,985C-87 DRAFT – DO NOT CITE OR QUOTETable C-19.In vivo DNA adducts: dose response dataDNA adductsRecordnumberReferencePAH SpeciesDoseDose units Organ TimeMeanStandarddeviationStandarderror Adduct unitsSlope ofAUC vs.dose CommentsBaP 100mg/kg Sum ofAUCs52,084BbF 100mg/kg Lung d 1 21 AUC calculated usingtrapezoid ruleBbF 100mg/kg Lung d 3 184BbF 100mg/kg Lung d 5 233BbF 100mg/kg Lung d 7 211BbF 100mg/kg Lung d 14 229BbF 100mg/kg Lung d 28 145BbF 100mg/kg Lung d 56 106BbF 100mg/kg Lung AUC 8,763BbF 100mg/kg Liver d 1 12BbF 100mg/kg Liver d 3 35BbF 100mg/kg Liver d 5 51BbF 100mg/kg Liver d 7 61BbF 100mg/kg Liver d 14 21BbF 100mg/kg Liver d 28 15BbF 100mg/kg Liver d 56 12BbF 100mg/kg Liver AUC 1,173BbF 100mg/kg PBL d 1 12BbF 100mg/kg PBL d 3 29BbF 100mg/kg PBL d 5 59BbF 100mg/kg PBL d 7 57BbF 100mg/kg PBL d 14 40BbF 100mg/kg PBL d 28 15BbF 100mg/kg PBL d 56 13BbF 100mg/kg PBL AUC 1,378BbF 100mg/kg Sum ofAUCs11,31424590/20920Nesnow etal., 1998b;Ross, 1995BaP NALung >21 d 3.9 113 Slope of dose vs.TIDAL value (in fmold/μg DNA)C-88 DRAFT – DO NOT CITE OR QUOTETable C-19.In vivo DNA adducts: dose response dataDNA adductsRecordnumberReferencePAH SpeciesDoseDose units Organ TimeMeanStandarddeviationStandarderror Adduct unitsSlope ofAUC vs.dose CommentsBbF NALung >21 d 5 37.5 Slope of dose vs.TIDAL value (in fmold/μg DNA)CPcdP NALung >21 d 3.69 148 Slope of dose vs.TIDAL value (in fmold/μg DNA)DBahANALung >21 d 19.1 219 Slope of dose vs.TIDAL value (in fmold/μg DNA)DBalP NALung >21 d 267 1,390 Slope of dose vs.TIDAL value (in fmold/μg DNA)22810 Phillips etal., 1979BaP 1 μmol/mouse Skin 19 hr 27pmol adducts/mgDNApeakDBacA1 μmol/mouse Skin 24 hr 10 pmol adducts/mgDNApeakDBahA1 μmol/mouse Skin 72 hr 15pmol adducts/mgDNApeak24790 Kligermanet al., 2002BaP Mice 100mg/kg PBL d 7 4,186 273 amol adducts/μgDNAIntraperitonealBaA Mice 100mg/kg PBL d 7 93 8 amol adducts/μgDNAIntraperitonealBbF Mice 100mg/kg PBL d 7 516 7 amol adducts/μgDNAIntraperitonealCH Mice 100mg/kg PBL d 7 81 11 amol adducts/μgDNAIntraperitonealControlMice 0 mg/kg PBL d 7 0 amol adducts/μgDNAIntraperitonealBaP Mice 100mg/kg PBL d 7 143 17 amol adducts/μgDNAGavageBaA Mice 100mg/kg PBL d 7 32 2 amol adducts/μgDNAGavageBbF Mice 100mg/kg PBL d 7 39 4 amol adducts/μgDNAGavageCH Mice 100mg/kg PBL d 7 37 1 amol adducts/μgDNAGavageC-89 DRAFT – DO NOT CITE OR QUOTETable C-19.In vivo DNA adducts: dose response dataDNA adductsRecordnumberReferencePAH SpeciesDoseDose units Organ TimeMeanStandarddeviationStandarderror Adduct unitsSlope ofAUC vs.dose CommentsControlMice 0 mg/kg PBL d 7 0 amol adducts/μgDNAGavageBaP Rat 100mg/kg PBL d 7 755 56 amol adducts/μgDNAIntraperitonealBaA Rat 100mg/kg PBL d 7 38 3 amol adducts/μgDNAIntraperitonealBbF Rat 100mg/kg PBL d 7 63 1 amol adducts/μgDNAIntraperitonealCH Rat 100mg/kg PBL d 7 24 2 amol adducts/μgDNAIntraperitonealControlRat 0 mg/kg PBL d 7 0 amol adducts/μgDNAIntraperitonealBaP Rat 100mg/kg PBL d 7 177 30 amol adducts/μgDNAGavageBaA Rat 100mg/kg PBL d 7 20 2 amol adducts/μgDNAGavageBbF Rat 100mg/kg PBL d 7 17 1 amol adducts/μgDNAGavageCH Rat 100mg/kg PBL d 7 10 4 amol adducts/μgDNAGavageControlRat 0 mg/kg PBL d 7 0 amol adducts/μgDNAGavage

### C-90 DRAFT – DO NOT CITE OR QUOTE

Table C-20. In vivo clastogenicity: data use

Record
number Reference Page Table
number Figure

number PAHs Data to be extracted Basis for
RPF Comment

24740 Allen et

al., 1999 I and III BaP, DBalP Total micronuleated polychromatic erythrocytes (MNPCE)/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) Point estimate Incidence data.

Single dose
BaP.

14270 He and Baker,
1991 166 1 BaP, CH MN cells/1,000 binucleated and dose (μg/mouse) Ratio of slopes Incidence data

17190 Bayer,

1978 426 3 BaP,
PH SCE/cells and dose (mg/kg) Point estimate Continuous

data. Only one dose PH significant. BaP given as 3,4-BaP.20950 RoszinskyKocher et al., 1979 66 1 BaP, DBah A, CH, PH, BeP, BbF,
BaA SCEs/metaphase and dose (mg/kg) Point estimate

24720 Kligerman

et al., 1986 129 3 BaP, BlAC SCEs/metaphase and dose (mg/kg) Point estimate Continuous

data, no SD for control; use lowest dose approaching peak 24790 Kligerman

et al., 2002 846 1 BaP, BaA, BbF,
CH SCEs/metaphase, intraperitoneal, for BaP, BaA, BbF, and CH. SCEs/, gavage, for BaP and BaA (use 17.91 value for BaP). Also use MNbn/1,000 bn, gavage, for BaP and BbF. Dose in mg/kg. Point estimates Separate RPFs

for SCEs and micronuclei, oral and intraperitonealC-91 DRAFT – DO NOT CITE OR QUOTETable C-21. In vivo clastogenicity: dose responsedataClastogenicityRecordnumberReference PAHRoute ofadministrationDose Dose unitsMeanStandarddeviationUnits n%ResponseUnitsp<0.05 Notes24740 Allen et al., 1999 TricaprylinIntra-peritoneal0 mg/kg 2.6 MN-PCEs 1,000 0.0026 PCEs A/J mice, bone marrowBaP Intraperitoneal200 mg/kg 11.2 MN-PCEs 1,000 0.0112 PCEs xDBalP Intraperitoneal0.3 mg/kg 2 MN-PCEs 1,000 0.0020 PCEsDBalP Intraperitoneal1.5 mg/kg 3.9 MN-PCEs 1,000 0.0039 PCEs xDBalP Intraperitoneal3 mg/kg 3.4 MN-PCEs 1,000 0.0034 PCEsDBalP Intraperitoneal6 mg/kg 3.8 MN-PCEs 1,000 0.0038 PCEsTricaprylinIntra-peritoneal0 mg/kg 2.8 MN-PCEs 1,000 0.0028 PCEs A/J mice, peripheralbloodBaP Intraperitoneal200 mg/kg 9.5 MN-PCEs 1,000 0.0095 PCEs xDBalP Intraperitoneal0.3 mg/kg 2.8 MN-PCEs 1,000 0.0028 PCEsDBalP Intraperitoneal1.5 mg/kg 2.9 MN-PCEs 1,000 0.0029 PCEsDBalP Intraperitoneal3 mg/kg 4 MN-PCEs 1,000 0.0040 PCEsDBalP Intraperitoneal6 mg/kg 4.3 MN-PCEs 1,000 0.0043 PCEs xTricaprylinIntra-peritoneal0 mg/kg 3.2 MN-PCEs 1,000 0.0032 PCEs p53 +/+ wtmice, bonemarrowBaP Intraperitoneal200 mg/kg 5.1 MN-PCEs 1,000 0.0051 PCEs xDBalP Intraperitoneal9 mg/kg 4.3 MN-PCEs 1,000 0.0043 PCEsDBalP Intraperitoneal12 mg/kg 7.4 MN-PCEs 1,000 0.0074 PCEs xDBalP Intraperitoneal18 mg/kg 6.1 MN-PCEs 1,000 0.0061 PCEs xC-92 DRAFT – DO NOT CITE OR QUOTETable C-21. In vivo clastogenicity: dose responsedataClastogenicityRecordnumberReference PAHRoute ofadministrationDose Dose unitsMeanStandarddeviationUnits n%ResponseUnitsp<0.05 NotesTricaprylinIntra-peritoneal0 mg/kg 3.5 MN-PCEs 1,000 0.0035 PCEs p53 +/+ wtmice, peri-pheral bloodBaP Intraperitoneal200 mg/kg 5.7 MN-PCEs 1,000 0.0057 PCEs xDBalP Intraperitoneal9 mg/kg 3.1 MN-PCEs 1,000 0.0031 PCEsDBalP Intraperitoneal12 mg/kg 3.1 MN-PCEs 1,000 0.0031 PCEsDBalP Intraperitoneal18 mg/kg 4.6 MN-PCEs 1,000 0.0046 PCEs14270 He and Baker, 1991 Control Dermal 0 μg/mouse13.3 2.8 MN cells 1,000 0.013 BinucleatedBaP Dermal 0.5 μg/mouse 50.5 11.5 MN cells 1,0000.051 BinucleatedxBaP Dermal 5 μg/mouse 66.8 4.1 MN cells 1,000 0.067 BinucleatedxBaP Dermal 50 μg/mouse 76 2.8 MN cells 1,000 0.076 BinucleatedxBaP Dermal 100 μg/mouse 64.3 5.4 MN cells 1,000 0.064 BinucleatedxBaP Dermal 500 μg/mouse 55.8 13 MN cells 1,000 0.056 BinucleatedxControl Dermal 0 μg/mouse 12.8 2.2 MN cells 1,0000.013 BinucleatedCH Dermal 50 μg/mouse 43.3 2.2 MN cells 1,000 0.043 BinucleatedxCH Dermal 100 μg/mouse 56 4.9 MN cells 1,000 0.056 BinucleatedxCH Dermal 500 μg/mouse 62 8.6 MN cells 1,000 0.062 BinucleatedxCH Dermal 1,000 μg/mouse 47.3 3.8 MN cells 1,0000.047 Binucleatedx17190 Bayer, 1978 pooledcontrolsIntraperitoneal0 mg/kg 3.2 0.07 SCE/cellsBaP Intraperitoneal2.5 mg/kg 3.4 0.8 SCE/cellsBaP Intraperitoneal25 mg/kg 3.5 0.2 SCE/cellsBaP Intraperitoneal40 mg/kg 3.9 0.2 SCE/cells xBaP Intraperitoneal50 mg/kg 6.4 0.2 SCE/cells xBaP Intraperitoneal75 mg/kg 6.4 0.3 SCE/cells xC-93 DRAFT – DO NOT CITE OR QUOTETable C-21. In vivo clastogenicity: dose responsedataClastogenicityRecordnumberReference PAHRoute ofadministrationDose Dose unitsMeanStandarddeviationUnits n%ResponseUnitsp<0.05 NotesBaP Intraperitoneal100 mg/kg 7.4 0.2 SCE/cells xPH Intraperitoneal25 mg/kg 3.5 0.2 SCE/cells Only one dose significantPH Intraperitoneal50 mg/kg 3.4 0.2 SCE/cellsPH Intraperitoneal75 mg/kg 3.5 0.2 SCE/cellsPH Intraperitoneal100 mg/kg 4.1 0.2 SCE/cells x20950 Roszinsky-Kocher etal., 1979Control Intraperitoneal0 mg/kg 3.9 0.9 SCEs/metaphaseBaP Intraperitoneal900 mg/kg 10.6 1.6 SCEs/metaphasexDBahA Intraperitoneal900 mg/kg 4.9 0.7 SCEs/ xCH Intraperitoneal900 mg/kg 5.1 1 SCEs/ xPH Intraperitoneal900 mg/kg 5.5 0.7 SCEs/ xBeP Intraperitoneal900 mg/kg 5.5 0.7 SCEs/ xBbF Intraperitoneal900 mg/kg 5.6 0.5 SCEs/ xBaA Intraperitoneal900 mg/kg 6.1 0.4 SCEs/ x24720 Kligerman et al.,1986Control Gavage 0 mg/kg 11.9 SCEs/metaphaseBaP Gavage 63 mg/kg 19.4 0.0 SCEs/metaphaseBaP Gavage 252 mg/kg 21.5 1.4 SCEs/metaphaseBaP Gavage 504 mg/kg 21.7 1.4 SCEs/metaphaseC-94 DRAFT – DO NOT CITE OR QUOTETable C-21. In vivo clastogenicity: dose responsedataClastogenicityRecordnumberReference PAHRoute ofadministrationDose Dose unitsMeanStandarddeviationUnits n%ResponseUnitsp<0.05 NotesControl Gavage 0 mg/kg 11.0 SCEs/metaphaseBlAC Gavage 32 mg/kg 16.5 3.6 SCEs/metaphaseBlAC Gavage 63 mg/kg 20.5 1.6 SCEs/metaphaseBlAC Gavage 126 mg/kg 27.8 2.6 SCEs/metaphase24790 Kligerman et al.,2002Control Intraperitoneal0 mg/kg 8.79 1.26 SCEs/BaP Intraperitoneal100 mg/kg 21.212.93 SCEs/ xBaA Intraperitoneal100 mg/kg 14.8 3.16 SCEs/ xBbF Intraperitoneal100 mg/kg 22.251.45 SCEs/ xCH Intraperitoneal100 mg/kg 11.961.8 SCEs/ xControl Gavage 0 mg/kg 11.121.5 SCEs/BaP Gavage 100 mg/kg 17.911.49 SCEs/ xBaA Gavage 100 mg/kg 13.381.53 SCEs/ xControl Gavage 0 mg/kg 6.6 0.9 MN bn 1,000 0.007BinucleatedBaP Gavage 100 mg/kg 9.1 1.8 MN bn 1,000 0.009 BinucleatedxBbF Gavage 100 mg/kg 8.3 0.9 MN bn 1,000 0.008 Binucleatedx

### D-1 DRAFT – DO NOT CITE OR QUOTE APPENDIX D. BENCHMARK DOSE MODELING OUTPUTS 1

2 

### D.1. DERMAL BIOASSAYS 3

Cav 1983 bap dermal.out.txt 4

================================================== ================== 5 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 6 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 7 RPS\MODELING\UNSAVED1.(d) 8 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 9 DOCUMENTS\PAH RPS\MODELING\UNSAVED1.plt 10

Tue Jul 05 10:10:03 2005 11

================================================== ================== 12 13

BMDS MODEL RUN 14

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 15
16 The form of the probability function is: 17
18 P[response] = background + (1-background)*[1-EXP ( 19 -beta1*dose^1-beta2*dose^2)] 20
21 The parameter betas are restricted to be positiv e 22
23 24 Dependent variable = COLUMN2 25 Independent variable = COLUMN1 26 27

Total number of observations = 4 28 Total number of records with missing values = 0 29 Total number of parameters in model = 3 30 Total number of specified parameters = 0 31 Degree of polynomial = 2 32
33 34

Maximum number of iterations = 250 35 Relative Function Convergence has been set to: 1e- 008 36 Parameter Convergence has been set to: 1e-008 37
38 39 40

Default Initial Parameter Values 41 Background = 0.0155298 42 Beta(1) = 0 43 Beta(2) = 0.00204447 44 45
46 Asymptotic Correlation Matrix of Paramet er Estimates 47 48 ( *** The model parameter(s) -Backgroun d 49 have been estimated at a boundary point, or have been 50 specified by the user, 51 and do not appear in the correlati on matrix ) 52
53 Beta(1) Beta(2) 54
55 Beta(1) 1 -0.96 56
57 Beta(2) -0.96 1 58

### D-2 DRAFT – DO NOT CITE OR QUOTE

1 2
3

Parameter Estimates 4
5
Variable Estimate Std. Err. 6 Background 0 N A 7 Beta(1) 0.0126577 0.046 0858 8 Beta(2) 0.00134916 0.0024 5743 9 10 NA - Indicates that this parameter has hit a bound 11 implied by some inequality constraint and thus 12

has no standard error. 13
14
15 16 Analysis of Deviance Table 17 18 Model Log(likelihood) Deviance Test D F P-value 19

Full model -35.0798 20
Fitted model -36.0272 1.89478 2 0.3878 21

Reduced model -55.062 39.9644 3 <.0001 22
23

AIC: 76.0543 24 25 26

Goodness of Fit 27
28 Dose Est._Prob. Expected Observed Size Chi^2 Res. 29

------------------------------------------------- ---------------------- 30

i: 1 31

0.0000 0.0000 0.000 0 29 0.000 32

i: 2 33

2.2000 0.0338 1.014 2 30 1.007 34

i: 3 35

6.6000 0.1326 3.714 2 28 -0.532 36

i: 4 37

20.0000 0.5474 16.423 17 30 0.078 38 39 Chi-square = 1.95 DF = 2 P-value = 0.3772 40
41
42 Benchmark Dose Computation 43 44 Specified effect = 0.1 45 46 Risk Type = Extra risk 47 48

Confidence level = 0.95 49
50

BMD = 5.31398 51

BMDL = 2.86439 52

### D-3 DRAFT – DO NOT CITE OR QUOTE

CAVALIERI1983BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\SETS\CAVALIERI1983BAP.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\SETS\CAVALIERI1983BAP.plt 7

Thu Jun 02 11:12:07 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.062817 39 Beta(1) = 0.0817095 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-4 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0.0898383 0.018 6734 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -41.1202 13

Fitted model -44.3027 6.36496 3 0.09514 14

Reduced model -76.9419 71.6434 3 <.0001 15 16

AIC: 90.6054 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 29 0.000 25

i: 2 26

2.2000 0.1793 5.380 2 30 -0.766 27

i: 3 28

6.6000 0.4473 12.524 17 28 0.647 29

i: 4 30

20.0000 0.8342 25.025 24 30 -0.247 31 32 Chi-square = 5.73 DF = 3 P-value = 0.1253 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.1 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 1.17278 44
45

BMDL = 0.902296 46

### D-5 DRAFT – DO NOT CITE OR QUOTE

CAVALIERI1983CPcdP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\SETS\CAVALIERI1983BAP.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\SETS\CAVALIERI1983BAP.plt 7

Thu Jun 02 11:16:02 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceCPcdP 22 Independent variable = doseCPcdP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0 40 Beta(2) = 4.42193e-005 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.93 53 54 Beta(2) -0.93 1 55 56
57 58

Parameter Estimates 59 60

### D-6 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 0.000525849 0.0047 7908 3 Beta(2) 3.60995e-005 2.68444e -005 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -27.8865 15

Fitted model -30.0799 4.38685 2 0.1115 16

Reduced model -64.1091 72.4452 3 <.0001 17 18

AIC: 64.1598 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 29 0.000 27

i: 2 28

22.2000 0.0290 0.842 2 29 1.416 29

i: 3 30

66.6000 0.1773 5.141 2 29 -0.743 31

i: 4 32

200.0000 0.7876 22.840 24 29 0.239 33 34 Chi-square = 4.25 DF = 2 P-value = 0.1194 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.1 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 47.2296 46 47

BMDL = 30.0553 48 49

### D-7 DRAFT – DO NOT CITE OR QUOTE

HABS1980BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\SETS\HABS1980BAP.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\SETS\HABS1980BAP.plt 7 Thu May 26 14:32:01 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 4 25 Total number of records with missing values = 1 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0 40 Beta(2) = 0.151094 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(1) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(2) 51 52 Beta(2) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-8 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0 N A 1 Beta(2) 0.127547 0.031 4898 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -40.3373 13

Fitted model -41.0055 1.33647 2 0.5126 14

Reduced model -64.1931 47.7118 2 <.0001 15 16

AIC: 84.011 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 35 0.000 25

i: 2 26

1.7000 0.3083 10.482 8 34 -0.342 27

i: 3 28

2.8000 0.6321 22.124 24 35 0.231 29
30 Chi-square = 1.28 DF = 2 P-value = 0.5267 31 32 33 Benchmark Dose Computation 34
35 Specified effect = 0.1 36 37 Risk Type = Extra risk 38 39

Confidence level = 0.95 40
41

BMD = 0.908873 42
43

BMDL = 0.484748 44

### D-9 DRAFT – DO NOT CITE OR QUOTE

HABS1980BBF.OUT.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\HABS1980BBF.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\HABS1980BBF.plt 7 Wed May 25 13:00:07 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBbF 22 Independent variable = doseBbF 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0 40 Beta(2) = 0.00945627 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(1) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(2) 51 52 Beta(2) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-10 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0 N A 1 Beta(2) 0.00748156 0.0023 3324 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -47.5575 13

Fitted model -48.6255 2.13602 3 0.5447 14

Reduced model -69.4912 43.8674 3 <.0001 15 16

AIC: 99.251 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 35 0.000 25

i: 2 26

3.4000 0.0829 3.148 2 38 -0.398 27

i: 3 28

5.6000 0.2091 7.110 5 34 -0.375 29

i: 4 30

9.2000 0.4691 17.358 20 37 0.287 31 32 Chi-square = 2.01 DF = 3 P-value = 0.5711 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.1 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 3.75269 44
45

BMDL = 2.91511 46 47

### D-11 DRAFT – DO NOT CITE OR QUOTE

hoff 1966 dermal bap for dbaep.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\UNSAVED1.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\UNSAVED1.plt 7

Tue Jul 05 10:20:14 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.124609 39 Beta(1) = 29.9573 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 34.3074 7.9 8663 60

### D-12 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -12.4245 11

Fitted model -12.5735 0.297928 2 0.8616 12

Reduced model -40.3807 55.9124 2 <.0001 13
14

AIC: 27.1469 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 20 -1.000 23

i: 2 24

0.0500 0.8201 16.402 17 20 0.203 25

i: 3 26

0.1000 0.9676 19.353 19 20 -0.563 27
28 Chi-square = 0.32 DF = 1 P-value = 0.5717 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 0.73 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 0.0381647 40
41

BMDL = 0.026721 42

### D-13 DRAFT – DO NOT CITE OR QUOTE

HOFFMANWYNDER966DBAIP.OUT.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\HOFFMANWYNDER966DBAIP.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\HOFFMANWYNDER966DBAIP.plt 7 Wed May 25 15:24:49 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceDBaiP 22 Independent variable = doseDBaiP 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.264818 39 Beta(1) = 18.4583 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 25.3832 5.8 3589 60

### D-14 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -16.5742 11

Fitted model -18.019 2.88957 2 0.2358 12

Reduced model -39.8916 46.6349 2 <.0001 13
14

AIC: 38.0379 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 20 0.000 23

i: 2 24

0.0500 0.7189 13.660 16 19 0.610 25

i: 3 26

0.1000 0.9210 17.499 16 19 -1.084 27
28 Chi-square = 3.05 DF = 2 P-value = 0.2174 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 0.1 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 0.00415079 40
41

BMDL = 0.00298234 42

### D-15 DRAFT – DO NOT CITE OR QUOTE

HOFFMANWYNDER1966BAP.OUT.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\HOFFMANWYNDER1966BAP.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\HOFFMANWYNDER1966BAP.plt 7 Wed May 25 15:13:08 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.124609 39 Beta(1) = 29.9573 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 34.3074 7.9 8663 60

### D-16 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -12.4245 11

Fitted model -12.5735 0.297928 2 0.8616 12

Reduced model -40.3807 55.9124 2 <.0001 13
14

AIC: 27.1469 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 20 -1.000 23

i: 2 24

0.0500 0.8201 16.402 17 20 0.203 25

i: 3 26

0.1000 0.9676 19.353 19 20 -0.563 27
28 Chi-square = 0.32 DF = 1 P-value = 0.5717 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 0.1 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 0.00307107 40
41

BMDL = 0.00215021 42

### D-17 DRAFT – DO NOT CITE OR QUOTE

HOFFMANWYNDER1966DBAEF.OUT.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\SETS\HOFFMANWYNDER1996DBAEP.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\SETS\HOFFMANWYNDER1996DBAEP.plt 7

Thu Jun 02 11:25:43 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceDBaeF 22 Independent variable = doseDBaeF 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.22871 39 Beta(1) = 29.4444 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 37.3037 9.0 4943 60

### D-18 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -10.3111 11

Fitted model -10.7582 0.894194 2 0.6395 12

Reduced model -38.9521 57.2822 2 <.0001 13
14

AIC: 23.5163 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 20 0.000 23

i: 2 24

0.0500 0.8451 16.058 17 19 0.379 25

i: 3 26

0.1000 0.9760 18.544 18 19 -1.224 27
28 Chi-square = 1.02 DF = 2 P-value = 0.5995 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 0.1 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 0.0028244 40
41

BMDL = 0.00193834 42

### D-19 DRAFT – DO NOT CITE OR QUOTE

HOFFMANWYNDER1996DBAEP.OUT.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\BMDS\HOFF_WYND_DBAEP_COMPL ETE.(d) 4 Gnuplot Plotting File: C:\BMDS\HOFF_WYND_DBAE P_COMPLETE.plt 5

Wed Jul 27 16:17:30 2005 6

================================================== ================== 7 8

BMDS MODEL RUN 9

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 10
11 The form of the probability function is: 12 13 P[response] = background + (1-background)*[1-EXP ( 14 -beta1*dose^1)] 15 16 The parameter betas are restricted to be positiv e 17 18 19 Dependent variable = COLUMN2 20 Independent variable = COLUMN1 21
22

Total number of observations = 3 23 Total number of records with missing values = 0 24 Total number of parameters in model = 2 25 Total number of specified parameters = 0 26 Degree of polynomial = 1 27
28 29

Maximum number of iterations = 250 30 Relative Function Convergence has been set to: 1e- 008 31 Parameter Convergence has been set to: 1e-008 32 33 34
35
Default Initial Parameter Values 36 Background = 0.120514 37 Beta(1) = 7.53772 38 39 40 Asymptotic Correlation Matrix of Paramet er Estimates 41
42 ( *** The model parameter(s) -Backgroun d 43 have been estimated at a boundary point, or have been 44 specified by the user, 45 and do not appear in the correlati on matrix ) 46 47 Beta(1) 48 49 Beta(1) 1 50 51 52
53

Parameter Estimates 54
55
Variable Estimate Std. Err. 56 Background 0 N A 57 Beta(1) 11.2084 3.2 1468 58 59 NA - Indicates that this parameter has hit a bound 60

### D-20 DRAFT – DO NOT CITE OR QUOTE

implied by some inequality constraint and thus 1

has no standard error. 2
3
4
5 Analysis of Deviance Table 6
7 Model Log(likelihood) Deviance Test D F P-value 8

Full model -32.4818 9

Fitted model -33.903 2.84251 2 0.2414 10

Reduced model -44.2604 23.5572 2 <.0001 11
12

AIC: 69.8061 13
14
15

Goodness of Fit 16 17 Dose Est._Prob. Expected Observed Size Chi^2 Res. 18

------------------------------------------------- ---------------------- 19

i: 1 20

0.0000 0.0000 0.000 0 20 0.000 21

i: 2 22

0.0500 0.4290 12.871 16 30 0.426 23

i: 3 24

0.1000 0.6740 11.458 9 17 -0.658 25 26 Chi-square = 2.95 DF = 2 P-value = 0.2288 27
28 29 Benchmark Dose Computation 30 31 Specified effect = 0.1 32 33 Risk Type = Extra risk 34
35
Confidence level = 0.95 36 37

BMD = 0.00940018 38 39

BMDL = 0.00681373 40

### D-21 DRAFT – DO NOT CITE OR QUOTE

LAVOIE1982BbF.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\LAVOIE1982.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\LAVOIE1982.plt 7 Wed May 25 16:18:48 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBbF 22 Independent variable = doseBbF 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.253748 39 Beta(1) = 0.0139485 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-22 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0.0256902 0.0062 4424 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -37.2311 13

Fitted model -41.3599 8.25761 3 0.04098 14

Reduced model -55.2266 35.991 3 <.0001 15 16

AIC: 84.7197 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 20 0.000 25

i: 2 26

10.0000 0.2266 4.531 9 20 1.275 27

i: 3 28

30.0000 0.5373 10.746 12 20 0.252 29

i: 4 30

100.0000 0.9234 18.468 16 20 -1.744 31 32 Chi-square = 10.32 DF = 3 P-value = 0.0160 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.85 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 73.8461 44
45

BMDL = 55.1641 46

### D-23 DRAFT – DO NOT CITE OR QUOTE

LAVOIE1982BjF.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\SETS\LAVOIE1982.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\SETS\LAVOIE1982.plt 7 Thu May 26 15:18:06 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBjF 22 Independent variable = doseBjF 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0505665 39 Beta(1) = 0.00768856 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 0.00907208 0.0033 0195 60

### D-24 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -25.9801 11

Fitted model -26.2675 0.574796 2 0.7502 12

Reduced model -35.7644 19.5688 2 <.0001 13
14

AIC: 54.5349 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 20 0.000 23

i: 2 24

30.0000 0.2383 4.765 6 20 0.340 25

i: 3 26

100.0000 0.5964 11.927 11 20 -0.193 27
28 Chi-square = 0.60 DF = 2 P-value = 0.7414 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 0.85 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 209.116 40
41

BMDL = 142.347 42

### D-25 DRAFT – DO NOT CITE OR QUOTE

LAVOIE1982BkF.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\LAVOIE1982.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\LAVOIE1982.plt 7 Wed May 25 16:21:19 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBkF 22 Independent variable = doseBkF 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0504814 39 Beta(1) = 0.00134342 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-26 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0.00163117 0.00050 3161 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -26.4637 13

Fitted model -27.3094 1.69146 3 0.6388 14

Reduced model -46.0525 39.1775 3 <.0001 15 16

AIC: 56.6189 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 20 0.000 25

i: 2 26

30.0000 0.0478 0.955 1 20 0.049 27

i: 3 28

100.0000 0.1505 3.010 5 20 0.778 29

i: 4 30

1000.0000 0.8043 16.086 15 20 -0.345 31 32 Chi-square = 1.93 DF = 3 P-value = 0.5881 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.85 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 1163.04 44
45

BMDL = 802.998 46 47

### D-27 DRAFT – DO NOT CITE OR QUOTE

RAVEH1982CPCDP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\RAVEH1982CPCDP.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\RAVEH1982CPCDP.plt 7 Wed May 25 16:10:56 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.086614 39 Beta(1) = 0.00379482 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 Background Beta(1) Beta(2) 46 47 Background 1 -0.51 0.37 48 49 Beta(1) -0.51 1 -0.96 50 51 Beta(2) 0.37 -0.96 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0.0898028 0.13 4663 59 Beta(1) 0.0034393 0.0054 7992 60

### D-28 DRAFT – DO NOT CITE OR QUOTE

Beta(2) 1.91358e-006 2.86516e -005 1 2
3
4 Analysis of Deviance Table 5 6 Model Log(likelihood) Deviance Test D F P-value 7

Full model -57.7672 8

Fitted model -57.8738 0.213129 1 0.6443 9

Reduced model -69.2679 23.0015 3 <.0001 10
11

AIC: 121.748 12 13
14

Goodness of Fit 15 16 Dose Est._Prob. Expected Observed Size Chi^2 Res. 17

------------------------------------------------- ---------------------- 18

i: 1 19

0.0000 0.0898 2.604 3 29 0.167 20

i: 2 21

10.0000 0.1207 3.622 3 30 -0.195 22

i: 3 23

100.0000 0.3669 10.641 11 29 0.053 24

i: 4 25

200.0000 0.5762 16.134 16 28 -0.020 26 27 Chi-square = 0.21 DF = 1 P-value = 0.6472 28 29
30 Benchmark Dose Computation 31 32 Specified effect = 0.1 33 34 Risk Type = Extra risk 35 36

Confidence level = 0.95 37
38

BMD = 30.1292 39 40

BMDL = 19.4197 41

### D-29 DRAFT – DO NOT CITE OR QUOTE

RAVEH_1982BaP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\RAVEH_1982.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\RAVEH_1982.plt 7 Wed May 25 16:06:59 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 6.01899e+017 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Beta(2) -Beta(3) 48 have been estimated at a boundary point, or have been 49 specified by the user, 50 and do not appear in the correlati on matrix ) 51 52 Background Beta(1) Beta(4) 53 54 Background 1 -0.66 0.27 55 56 Beta(1) -0.66 1 -0.52 57 58 Beta(4) 0.27 -0.52 1 59 60

### D-30 DRAFT – DO NOT CITE OR QUOTE

1 2

Parameter Estimates 3
4

Variable Estimate Std. Err. 5 Background 0.132052 0.16 8781 6 Beta(1) 0.0479561 0.013 3452 7 Beta(2) 0 N A 8 Beta(3) 0 N A 9 Beta(4) 4.58924e-009 3.25141e -008 10
11 NA - Indicates that this parameter has hit a bound 12 implied by some inequality constraint and thus 13

has no standard error. 14
15 16 17 Analysis of Deviance Table 18 19 Model Log(likelihood) Deviance Test D F P-value 20

Full model -56.5419 21

Fitted model -58.376 3.66814 3 0.2996 22

Reduced model -101.065 89.0461 5 <.0001 23
24

AIC: 122.752 25 26 27

Goodness of Fit 28 29 Dose Est._Prob. Expected Observed Size Chi^2 Res. 30

------------------------------------------------- ---------------------- 31

i: 1 32

0.0000 0.1321 3.830 3 29 -0.250 33

i: 2 34

10.0000 0.4627 13.419 17 29 0.497 35

i: 3 36

25.0000 0.7388 20.685 21 28 0.058 37

i: 4 38

50.0000 0.9233 25.853 24 28 -0.935 39

i: 5 40

100.0000 0.9955 26.878 27 27 1.005 41

i: 6 42

200.0000 1.0000 26.000 26 26 1.000 43 44 Chi-square = 3.86 DF = 3 P-value = 0.2771 45 46 47 Benchmark Dose Computation 48 49 Specified effect = 0.1 50 51 Risk Type = Extra risk 52
53

Confidence level = 0.95 54
55

BMD = 2.19702 56
57

BMDL = 1.66278 58 59

### D-31 DRAFT – DO NOT CITE OR QUOTE

RICE1988INITIATIONBbcAC.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ DERMAL 4 STUDIES\RICE1988INITIATION.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\DERMAL 6 STUDIES\RICE1988INITIATION.plt 7 Thu May 26 12:50:09 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBbcAC 22 Independent variable = doseBbcAC 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.515248 39 Beta(1) = 0.484044 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Background Beta(1) 51 52 Background 1 -0.68 53 54 Beta(1) -0.68 1 55 56
57 58

Parameter Estimates 59 60

### D-32 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0.110219 0.24 3438 2 Beta(1) 1.01652 0.31 9132 3 Beta(2) 0 N A 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -28.2203 15

Fitted model -34.2145 11.9884 2 0.002493 16

Reduced model -51.7957 47.1508 3 <.0001 17 18

AIC: 72.4291 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.1102 2.204 1 20 -0.614 27

i: 2 28

0.5000 0.4648 9.295 15 20 1.147 29

i: 3 30

2.0000 0.8835 17.670 18 20 0.160 31

i: 4 32

4.0000 0.9847 19.695 18 20 -5.641 33 34 Chi-square = 16.90 DF = 2 P-value = 0.0002 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.89 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 2.1714 46 47

BMDL = 1.54343 48

### D-33 DRAFT – DO NOT CITE OR QUOTE

Rice 1988CH.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\BMDS\UNSAVED1.(d) 4 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 5 Thu May 26 12:43:53 2005 6

================================================== ================== 7 8

BMDS MODEL RUN 9

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 10
11 The form of the probability function is: 12 13 P[response] = background + (1-background)*[1-EXP ( 14 -beta1*dose^1-beta2*dose^2)] 15 16 The parameter betas are restricted to be positiv e 17 18 19 Dependent variable = incidenceCH 20 Independent variable = doseCH 21
22

Total number of observations = 4 23 Total number of records with missing values = 0 24 Total number of parameters in model = 3 25 Total number of specified parameters = 0 26 Degree of polynomial = 2 27
28 29

Maximum number of iterations = 250 30 Relative Function Convergence has been set to: 1e- 008 31 Parameter Convergence has been set to: 1e-008 32 33 34
35
Default Initial Parameter Values 36 Background = 0.305186 37 Beta(1) = 1.94458 38 Beta(2) = 0 39 40
41 Asymptotic Correlation Matrix of Paramet er Estimates 42
43 ( *** The model parameter(s) -Beta(2) 44 have been estimated at a boundary point, or have been 45 specified by the user, 46 and do not appear in the correlati on matrix ) 47 48 Background Beta(1) 49
50 Background 1 -0.51 51 52 Beta(1) -0.51 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0.0435025 0.18 2669 60

### D-34 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 2.7068 0.7 4964 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -25.689 13

Fitted model -28.1343 4.89062 2 0.0867 14

Reduced model -55.2266 59.0752 3 <.0001 15 16

AIC: 60.2686 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0435 0.870 1 20 0.156 25

i: 2 26

0.1500 0.3627 7.254 5 20 -0.488 27

i: 3 28

0.5000 0.7529 15.058 18 20 0.791 29

i: 4 30

1.5000 0.9835 19.670 19 20 -2.065 31 32 Chi-square = 4.83 DF = 2 P-value = 0.0894 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.89 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 0.815455 44
45

BMDL = 0.584044 46

### D-35 DRAFT – DO NOT CITE OR QUOTE

RICE_CPDEFC.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\RICE_DERMAL_CPDEFC.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\RICE_DERMAL_CPDEFC.plt 7

Thu Jun 30 20:30:27 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 1 39 Beta(1) = 6.76726e+019 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Beta(1) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Background Beta(2) 51 52 Background 1 -0.52 53 54 Beta(2) -0.52 1 55 56
57 58

Parameter Estimates 59 60

### D-36 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0.0499932 0.21 7763 2 Beta(1) 0 N A 3 Beta(2) 44.3918 19. 5918 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -16.9192 15

Fitted model -16.9195 0.000547543 2 0.9997 16

Reduced model -49.6481 65.4577 3 <.0001 17 18

AIC: 37.839 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0500 1.000 1 20 0.000 27

i: 2 28

0.1500 0.6501 13.002 13 20 -0.000 29

i: 3 30

0.5000 1.0000 19.000 19 19 1.000 31

i: 4 32

1.5000 1.0000 19.000 19 19 0.000 33 34 Chi-square = 0.00 DF = 2 P-value = 0.9999 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.88 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 0.218546 46 47

BMDL = 0.172781 48 49

### D-37 DRAFT – DO NOT CITE OR QUOTE

NESNOW_1984_DERMAL_BLAC_MALE.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\_PAH 4 RPS\MODELING\NESNOW_1984_DERMAL_BLAC_MALE.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\_PAH RPS\MODELING\NESNOW_1984_DERMAL_BLAC _MALE.plt 7

Thu Feb 08 09:10:48 2007 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0.0283321 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 0.0219722 0.0053 4523 60

### D-38 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -17.2634 11

Fitted model -17.7362 0.945584 2 0.6233 12

Reduced model -39.5006 44.4744 2 <.0001 13
14

AIC: 37.4725 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 20 0.000 23

i: 2 24

50.0000 0.6667 13.333 12 20 -0.300 25

i: 3 26

100.0000 0.8889 15.111 16 17 0.529 27
28 Chi-square = 0.87 DF = 2 P-value = 0.6471 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 0.67 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 50.4574 40
41

BMDL = 35.8134 42
43

### D-39 DRAFT – DO NOT CITE OR QUOTE

NESNOW_1984_DERMAL_BLAC_FEMALE.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\_PAH 4 RPS\MODELING\NESNOW_1984_DERMAL_BLAC_FEMALE.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\_PAH RPS\MODELING\NESNOW_1984_DERMAL_BLAC _FEMALE.plt 7

Thu Feb 08 09:13:51 2007 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0.0289037 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 Background Beta(1) 45 46 Background 1 -0.49 47 48 Beta(1) -0.49 1 49
50 51 52

Parameter Estimates 53 54

Variable Estimate Std. Err. 55 Background 0.05051 0.2 1268 56 Beta(1) 0.0234714 0.0064 8098 57 58 59 60

### D-40 DRAFT – DO NOT CITE OR QUOTE

Analysis of Deviance Table 1 2 Model Log(likelihood) Deviance Test D F P-value 3

Full model -20.7842 4

Fitted model -21.1281 0.687832 1 0.4069 5

Reduced model -39.8916 38.2148 2 <.0001 6
7

AIC: 46.2563 8 9 10

Goodness of Fit 11
12 Dose Est._Prob. Expected Observed Size Chi^2 Res. 13

------------------------------------------------- ---------------------- 14

i: 1 15

0.0000 0.0505 0.960 1 19 0.044 16

i: 2 17

50.0000 0.7064 14.127 13 20 -0.272 18

i: 3 19

100.0000 0.9092 17.275 18 19 0.462 20 21 Chi-square = 0.64 DF = 1 P-value = 0.4224 22
23
24 Benchmark Dose Computation 25 26 Specified effect = 0.51 27
28 Risk Type = Extra risk 29
30

Confidence level = 0.95 31 32

BMD = 30.3924 33 34

BMDL = 21.4681 35

### D-41 DRAFT – DO NOT CITE OR QUOTE

NESNOW_1984_DERMAL_BEAC_MALE.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\_PAH 4 RPS\MODELING\NESNOW_1984_DERMAL_BEAC_MALE.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\_PAH RPS\MODELING\NESNOW_1984_DERMAL_BEAC _MALE.plt 7

Fri Feb 09 10:09:40 2007 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.121669 39 Beta(1) = 0.00219353 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 48 -Beta(4) 49 have been estimated at a boundary point, or have been 50 specified by the user, 51 and do not appear in the correlati on matrix ) 52
53 Beta(1) 54
55 Beta(1) 1 56
57 58 59

Parameter Estimates 60

### D-42 DRAFT – DO NOT CITE OR QUOTE

1
Variable Estimate Std. Err. 2 Background 0 N A 3 Beta(1) 0.00282301 0.0005 6435 4 Beta(2) 0 N A 5 Beta(3) 0 N A 6 Beta(4) 0 N A 7 8 NA - Indicates that this parameter has hit a bound 9 implied by some inequality constraint and thus 10

has no standard error. 11
12 13
14 Analysis of Deviance Table 15 16 Model Log(likelihood) Deviance Test D F P-value 17

Full model -51.002 18

Fitted model -52.1858 2.36767 5 0.7963 19

Reduced model -80.7033 59.4025 5 <.0001 20 21

AIC: 106.372 22
23
24

Goodness of Fit 25 26 Dose Est._Prob. Expected Observed Size Chi^2 Res. 27

------------------------------------------------- ---------------------- 28

i: 1 29

0.0000 0.0000 0.000 0 20 0.000 30

i: 2 31

50.0000 0.1316 2.633 4 20 0.598 32

i: 3 33

100.0000 0.2460 4.919 4 20 -0.248 34

i: 4 35

250.0000 0.5063 10.125 12 20 0.375 36

i: 5 37

500.0000 0.7562 15.124 15 20 -0.034 38

i: 6 39

1000.0000 0.9406 16.930 16 18 -0.925 40
41 Chi-square = 2.61 DF = 5 P-value = 0.7594 42
43 44 Benchmark Dose Computation 45 46 Specified effect = 0.67 47 48 Risk Type = Extra risk 49
50

Confidence level = 0.95 51 52

BMD = 392.724 53 54

BMDL = 305.587 55

### D-43 DRAFT – DO NOT CITE OR QUOTE

NESNOW_1984_DERMAL_BEAC_FEMALE.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\_PAH 4 RPS\MODELING\NESNOW_1984_DERMAL_BEAC_FEMALE.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\_PAH RPS\MODELING\NESNOW_1984_DERMAL_BEAC _FEMALE.plt 7

Fri Feb 09 10:12:31 2007 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0667811 39 Beta(1) = 0.00288357 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-44 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0.00366005 0.0012 6417 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -35.6556 13

Fitted model -36.1032 0.895174 3 0.8266 14

Reduced model -45.1184 18.9255 3 0.0002833 15 16

AIC: 74.2064 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 19 0.000 25

i: 2 26

50.0000 0.1672 3.345 4 20 0.235 27

i: 3 28

100.0000 0.3065 5.824 7 19 0.291 29

i: 4 30

250.0000 0.5995 11.390 10 19 -0.305 31 32 Chi-square = 0.92 DF = 3 P-value = 0.8205 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.51 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 194.902 44
45

BMDL = 137.872 46

### D-45 DRAFT – DO NOT CITE OR QUOTE

D.2. INTRAPERITONEAL BIOASSAYS 1

lavoie 1994 female lung FA.txt 2

================================================== ================== 3 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 4 Input Data File: C:\BMDS\UNSAVED1.(d) 5 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 6

Thu Jun 30 15:55:31 2005 7

================================================== ================== 8 9

BMDS MODEL RUN 10

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 11
12 The form of the probability function is: 13 14 P[response] = background + (1-background)*[1-EXP ( 15 -beta1*dose^1)] 16 17 The parameter betas are restricted to be positiv e 18 19
20 Dependent variable = COLUMN2 21 Independent variable = COLUMN1 22 23

Total number of observations = 3 24 Total number of records with missing values = 0 25 Total number of parameters in model = 2 26 Total number of specified parameters = 0 27 Degree of polynomial = 1 28 29
30

Maximum number of iterations = 250 31 Relative Function Convergence has been set to: 1e- 008 32 Parameter Convergence has been set to: 1e-008 33 34
35 36

Default Initial Parameter Values 37 Background = 0.0929049 38 Beta(1) = 0.108473 39 40
41 Asymptotic Correlation Matrix of Paramet er Estimates 42 43 Background Beta(1) 44 45 Background 1 -0.48 46
47 Beta(1) -0.48 1 48 49
50 51

Parameter Estimates 52 53

Variable Estimate Std. Err. 54 Background 0.112497 0.13 7421 55 Beta(1) 0.103015 0.029 1539 56
57 58 59

### D-46 DRAFT – DO NOT CITE OR QUOTE

Analysis of Deviance Table 1 2 Model Log(likelihood) Deviance Test D F P-value 3

Full model -44.1118 4

Fitted model -44.1689 0.114322 1 0.7353 5

Reduced model -64.1094 39.9952 2 <.0001 6
7

AIC: 92.3379 8 9 10

Goodness of Fit 11
12 Dose Est._Prob. Expected Observed Size Chi^2 Res. 13

------------------------------------------------- ---------------------- 14

i: 1 15

0.0000 0.1125 3.825 4 34 0.052 16

i: 2 17

3.4600 0.3786 11.737 11 31 -0.101 18

i: 3 19

17.3000 0.8507 24.669 25 29 0.090 20 21 Chi-square = 0.11 DF = 1 P-value = 0.7366 22
23
24 Benchmark Dose Computation 25 26 Specified effect = 0.83 27
28 Risk Type = Extra risk 29
30

Confidence level = 0.95 31 32

BMD = 17.201 33 34

BMDL = 12.2186 35

### D-47 DRAFT – DO NOT CITE OR QUOTE

LAVOIEETAL1994LIVERmale.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD 4 ANALYSIS\BIOASSAY\INTRAPERITONEAL\SETS\LAVOIEETAL19 94LIVER.(d) 5 Gnuplot Plotting File: C:\PAH\BMD 6 ANALYSIS\BIOASSAY\INTRAPERITONEAL\SETS\LAVOIEETAL19 94LIVER.plt 7

Wed Jun 01 09:15:04 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = response 22 Independent variable = dose 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 6.19323e+018 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 Background Beta(1) 45 46 Background 1 -0.47 47 48 Beta(1) -0.47 1 49
50 51 52

Parameter Estimates 53 54

Variable Estimate Std. Err. 55 Background 0.168707 0.16 0946 56 Beta(1) 0.259821 0.090 2001 57 58 59 60

### D-48 DRAFT – DO NOT CITE OR QUOTE

Analysis of Deviance Table 1 2 Model Log(likelihood) Deviance Test D F P-value 3

Full model -31.5803 4

Fitted model -31.7622 0.363803 1 0.5464 5

Reduced model -51.0494 38.9382 2 <.0001 6
7

AIC: 67.5244 8 9 10

Goodness of Fit 11
12 Dose Est._Prob. Expected Observed Size Chi^2 Res. 13

------------------------------------------------- ---------------------- 14

i: 1 15

0.0000 0.1687 4.892 5 29 0.026 16

i: 2 17

3.4600 0.6617 18.527 18 28 -0.084 18

i: 3 19

17.3000 0.9907 16.842 17 17 1.009 20 21 Chi-square = 0.21 DF = 1 P-value = 0.6496 22
23
24 Benchmark Dose Computation 25 26 Specified effect = 0.81 27
28 Risk Type = Extra risk 29
30

Confidence level = 0.95 31 32

BMD = 6.39183 33 34

BMDL = 4.18834 35

### D-49 DRAFT – DO NOT CITE OR QUOTE

LAVOIEETAL1994LUNGmale.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD 4 ANALYSIS\BIOASSAY\INTRAPERITONEAL\SETS\LAVOIEETAL19 94LUNG.(d) 5 Gnuplot Plotting File: C:\PAH\BMD 6 ANALYSIS\BIOASSAY\INTRAPERITONEAL\SETS\LAVOIEETAL19 94LUNG.plt 7

Wed Jun 01 09:49:11 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = responsemale 22 Independent variable = dosemale 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.24757 39 Beta(1) = 0.0451334 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 Background Beta(1) 45 46 Background 1 -0.57 47 48 Beta(1) -0.57 1 49
50 51 52

Parameter Estimates 53 54

Variable Estimate Std. Err. 55 Background 0.209483 0.14 2769 56 Beta(1) 0.0559823 0.029 7979 57 58 59 60

### D-50 DRAFT – DO NOT CITE OR QUOTE

Analysis of Deviance Table 1 2 Model Log(likelihood) Deviance Test D F P-value 3

Full model -43.4897 4

Fitted model -44.1071 1.23476 1 0.2665 5

Reduced model -49.0816 11.1837 2 0.003728 6
7

AIC: 92.2143 8 9 10

Goodness of Fit 11
12 Dose Est._Prob. Expected Observed Size Chi^2 Res. 13

------------------------------------------------- ---------------------- 14

i: 1 15

0.0000 0.2095 6.075 5 29 -0.224 16

i: 2 17

3.4600 0.3487 9.763 12 28 0.352 18

i: 3 19

17.3000 0.6999 11.898 11 17 -0.251 20 21 Chi-square = 1.25 DF = 1 P-value = 0.2629 22
23
24 Benchmark Dose Computation 25 26 Specified effect = 0.7 27
28 Risk Type = Extra risk 29
30

Confidence level = 0.95 31 32

BMD = 21.5063 33 34

BMDL = 12.5156 35

### D-51 DRAFT – DO NOT CITE OR QUOTE

WISLOCKI_CHRYSENE_MALE_LIVER.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\BMDS\WISLOCKI_CHRYSENE_MAL E_LIVER.(d) 4 Gnuplot Plotting File: C:\BMDS\WISLOCKI_CHRYS ENE_MALE_LIVER.plt 5

Wed Jun 15 13:20:42 2005 6

================================================== ================== 7 8

BMDS MODEL RUN 9

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 10
11 The form of the probability function is: 12 13 P[response] = background + (1-background)*[1-EXP ( 14 -beta1*dose^1)] 15 16 The parameter betas are restricted to be positiv e 17 18 19 Dependent variable = COLUMN2 20 Independent variable = COLUMN1 21
22

Total number of observations = 3 23 Total number of records with missing values = 0 24 Total number of parameters in model = 2 25 Total number of specified parameters = 0 26 Degree of polynomial = 1 27
28 29

Maximum number of iterations = 250 30 Relative Function Convergence has been set to: 1e- 008 31 Parameter Convergence has been set to: 1e-008 32 33 34
35
Default Initial Parameter Values 36 Background = 0.147839 37 Beta(1) = 0.000139419 38 39 40 Asymptotic Correlation Matrix of Paramet er Estimates 41
42 Background Beta(1) 43 44 Background 1 -0.57 45 46 Beta(1) -0.57 1 47 48 49
50

Parameter Estimates 51 52

Variable Estimate Std. Err. 53 Background 0.109703 0.1 0278 54 Beta(1) 0.000173669 9.88799e -005 55 56
57 58 Analysis of Deviance Table 59 60

### D-52 DRAFT – DO NOT CITE OR QUOTE

Model Log(likelihood) Deviance Test D F P-value 1

Full model -67.0392 2

Fitted model -67.7628 1.44719 1 0.229 3

Reduced model -74.516 14.9536 2 0.0005661 4
5

AIC: 139.526 6
7 8

Goodness of Fit 9 10 Dose Est._Prob. Expected Observed Size Chi^2 Res. 11

------------------------------------------------- ---------------------- 12

i: 1 13

0.0000 0.1097 8.008 7 73 -0.141 14

i: 2 15

700.0000 0.2116 7.407 10 35 0.444 16

i: 3 17

2800.0000 0.4525 15.387 14 34 -0.165 18 19 Chi-square = 1.52 DF = 1 P-value = 0.2172 20 21
22 Benchmark Dose Computation 23
24 Specified effect = 0.44 25 26 Risk Type = Extra risk 27
28
Confidence level = 0.95 29
30

BMD = 3338.63 31 32

BMDL = 2098.51 33

### D-53 DRAFT – DO NOT CITE OR QUOTE

WISLOCKI_CHRYSENE_MALE_LUNG.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\BMDS\WISLOCKI_CHRYSENE_MAL E_LUNG.(d) 4 Gnuplot Plotting File: C:\BMDS\WISLOCKI_CHRYS ENE_MALE_LUNG.plt 5

Wed Jun 15 13:21:42 2005 6

================================================== ================== 7 8

BMDS MODEL RUN 9

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 10
11 The form of the probability function is: 12 13 P[response] = background + (1-background)*[1-EXP ( 14 -beta1*dose^1)] 15 16 The parameter betas are restricted to be positiv e 17 18 19 Dependent variable = COLUMN2 20 Independent variable = COLUMN1 21
22

Total number of observations = 3 23 Total number of records with missing values = 0 24 Total number of parameters in model = 2 25 Total number of specified parameters = 0 26 Degree of polynomial = 1 27
28 29

Maximum number of iterations = 250 30 Relative Function Convergence has been set to: 1e- 008 31 Parameter Convergence has been set to: 1e-008 32 33 34
35
Default Initial Parameter Values 36 Background = 0.101102 37 Beta(1) = 4.85056e-005 38 39 40 Asymptotic Correlation Matrix of Paramet er Estimates 41
42 Background Beta(1) 43 44 Background 1 -0.6 45 46 Beta(1) -0.6 1 47 48 49
50

Parameter Estimates 51 52

Variable Estimate Std. Err. 53 Background 0.0806675 0.10 3469 54 Beta(1) 6.36834e-005 8.767e -005 55 56
57 58 Analysis of Deviance Table 59 60

### D-54 DRAFT – DO NOT CITE OR QUOTE

Model Log(likelihood) Deviance Test D F P-value 1

Full model -51.5522 2

Fitted model -52.0709 1.03747 1 0.3084 3

Reduced model -53.9858 4.86735 2 0.08771 4
5

AIC: 108.142 6
7 8

Goodness of Fit 9 10 Dose Est._Prob. Expected Observed Size Chi^2 Res. 11

------------------------------------------------- ---------------------- 12

i: 1 13

0.0000 0.0807 5.889 5 73 -0.164 14

i: 2 15

700.0000 0.1207 4.226 6 35 0.477 16

i: 3 17

2800.0000 0.2308 7.848 7 34 -0.140 18 19 Chi-square = 1.11 DF = 1 P-value = 0.2917 20 21
22 Benchmark Dose Computation 23
24 Specified effect = 0.3 25 26 Risk Type = Extra risk 27
28
Confidence level = 0.95 29
30

BMD = 5600.76 31 32

BMDL = 2691.64 33

### D-55 DRAFT – DO NOT CITE OR QUOTE

Busby 1984 i.p. multiplicity 1

FA male 2

Linear 3

Nonconstant variance 4 BMR = lowest statistically significant response in BaP treated animals (after 5 control subtracted) 6
7

================================================== ================== 8 Polynomial Model. (Version: 2.12; Date: 02/2 0/2007) 9 Input Data File: C:\BMDS\UNSAVED1.(d) 10 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 11 Mon May 11 21:08:40 2009 12

================================================== ================== 13
14

BMDS MODEL RUN 15

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 16 17 The form of the response function is: 18 19 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 20 21
22 Dependent variable = MEAN 23 Independent variable = COLUMN1 24 The polynomial coefficients are restricted to be positive 25 The variance is to be modeled as Var(i) = exp(la lpha + log(mean(i)) * rho) 26 27 Total number of dose groups = 3 28 Total number of records with missing values = 0 29

Maximum number of iterations = 250 30 Relative Function Convergence has been set to: 1 e-008 31 Parameter Convergence has been set to: 1e-008 32 33 34
35
Default Initial Parameter Values 36 lalpha = 0.136152 37

rho = 0 38 beta_0 = 0.0180952 39 beta_1 = 0.427551 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 lalpha rho beta_0 beta_1 45 46 lalpha 1 0.65 0.015 0.00041 47 48

rho 0.65 1 0.22 -0.061 49
50 beta_0 0.015 0.22 1 -0.24 51 52 beta_1 0.00041 -0.061 -0.24 1 53 54
55 56

Parameter Estimate s 57 58

95.0% Wald 59

Confidence Interval 60

### D-56 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. Lower Conf. Limit 1 Upper Conf. Limit 2 lalpha 0.634298 0.204652 0.233188 3

1.03541 4

rho 0.923372 0.0876305 0.751619 5

1.09512 6 beta_0 0.0170376 0.0434041 -0.0680328 7

0.102108 8 beta_1 0.426604 0.0861283 0.257796 9

0.595413 10
11
12 13

Table of Data and Estimated Values of Interest 14
15

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled 16

Res. 17

------ --- -------- -------- ---------- - ----------- --------- 18

- 19
20

0 27 0.04 0.017 0.21 0.21 0.57 21

0.7 31 0.29 0.316 0.84 0.806 -0.177 22

3.5 27 1.52 1.51 1.66 1.66 0.0308 23
24 25 26 Model Descriptions for likelihoods calculated 27
28 29 Model A1: Yij = Mu(i) + e(ij) 30 Var{e(ij)} = Sigma^2 31 32 Model A2: Yij = Mu(i) + e(ij) 33 Var{e(ij)} = Sigma(i)^2 34
35 Model A3: Yij = Mu(i) + e(ij) 36 Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 37 Model A3 uses any fixed variance parameters th at 38 were specified by the user 39 40 Model R: Yi = Mu + e(i) 41 Var{e(i)} = Sigma^2 42
43 44

Likelihoods of Interest 45 46 Model Log(likelihood) # Param's AIC 47

A1 -46.759351 4 101.518703 48

A2 -7.114400 6 26.228800 49

A3 -7.317284 5 24.634569 50

fitted -7.329046 4 22.658093 51

R -59.984569 2 123.969139 52
53 54 Explanation of Tests 55 56 Test 1: Do responses and/or variances differ amon g Dose levels? 57 (A2 vs. R) 58 Test 2: Are Variances Homogeneous? (A1 vs A2) 59 Test 3: Are variances adequately modeled? (A2 vs. A3) 60

### D-57 DRAFT – DO NOT CITE OR QUOTE

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 1 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 2
3

Tests of Interest 4
5 Test -2*log(Likelihood Ratio) Test df p-value 6
7

Test 1 105.74 4 <.0001 8

Test 2 79.2899 2 <.0001 9

Test 3 0.405769 1 0.5241 10

Test 4 0.0235238 1 0.8781 11
12 The p-value for Test 1 is less than .05. There app ears to be a 13 difference between response and/or variances among the dose levels 14 It seems appropriate to model the data 15 16 The p-value for Test 2 is less than .1. A non-homo geneous variance 17 model appears to be appropriate 18 19 The p-value for Test 3 is greater than .1. The mod eled variance appears 20 to be appropriate here 21
22 The p-value for Test 4 is greater than .1. The mod el chosen seems 23 to adequately describe the data 24 25 26 Benchmark Dose Computation 27
28 Specified effect = 4.28 29
30 Risk Type = Point risk 31 32

Confidence level = 0.95 33 34

BMD = 9.99278 35 36 37

BMDL = 7.55762 38

### D-58 DRAFT – DO NOT CITE OR QUOTE

Busby 1984 i.p. multiplicity 1

FA female 2

Linear 3

Nonconstant variance 4 BMR = lowest statistically significant response in BaP treated animals (after 5 control subtracted) 6
7

================================================== ================== 8 Polynomial Model. (Version: 2.12; Date: 02/2 0/2007) 9 Input Data File: C:\BMDS\UNSAVED1.(d) 10 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 11 Mon May 11 21:14:08 2009 12

================================================== ================== 13
14

BMDS MODEL RUN 15

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 16 17 The form of the response function is: 18 19 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 20 21
22 Dependent variable = MEAN 23 Independent variable = COLUMN1 24 The polynomial coefficients are restricted to be positive 25 The variance is to be modeled as Var(i) = exp(la lpha + log(mean(i)) * rho) 26 27 Total number of dose groups = 3 28 Total number of records with missing values = 0 29

Maximum number of iterations = 250 30 Relative Function Convergence has been set to: 1 e-008 31 Parameter Convergence has been set to: 1e-008 32 33 34
35
Default Initial Parameter Values 36 lalpha = -1.11206 37

rho = 0 38 beta_0 = 0.108571 39 beta_1 = 0.115306 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 lalpha rho beta_0 beta_1 45 46 lalpha 1 0.94 0.036 -0.047 47 48

rho 0.94 1 0.04 -0.052 49
50 beta_0 0.036 0.04 1 -0.46 51 52 beta_1 -0.047 -0.052 -0.46 1 53 54
55 56

Parameter Estimate s 57 58

95.0% Wald 59

Confidence Interval 60

### D-59 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. Lower Conf. Limit 1 Upper Conf. Limit 2 lalpha 0.353344 0.480274 -0.587974 3

1.29466 4

rho 1.1315 0.292904 0.557421 5

1.70558 6 beta_0 0.123135 0.0618608 0.00189039 7

0.24438 8 beta_1 0.106469 0.0535364 0.00153987 9

0.211399 10
11
12 13

Table of Data and Estimated Values of Interest 14
15

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled 16

Res. 17

------ --- -------- -------- ---------- - ----------- --------- 18

- 19
20

0 28 0.14 0.123 0.37 0.365 0.245 21

0.7 20 0.15 0.198 0.49 0.477 -0.447 22

3.5 21 0.52 0.496 0.82 0.802 0.138 23
24 25 26 Model Descriptions for likelihoods calculated 27
28 29 Model A1: Yij = Mu(i) + e(ij) 30 Var{e(ij)} = Sigma^2 31 32 Model A2: Yij = Mu(i) + e(ij) 33 Var{e(ij)} = Sigma(i)^2 34
35 Model A3: Yij = Mu(i) + e(ij) 36 Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 37 Model A3 uses any fixed variance parameters th at 38 were specified by the user 39 40 Model R: Yi = Mu + e(i) 41 Var{e(i)} = Sigma^2 42
43 44

Likelihoods of Interest 45 46 Model Log(likelihood) # Param's AIC 47

A1 5.399546 4 -2.799091 48

A2 13.307908 6 -14.615816 49

A3 13.189903 5 -16.379806 50

fitted 13.167852 4 -18.335705 51

R 2.264796 2 -0.529591 52
53 54 Explanation of Tests 55 56 Test 1: Do responses and/or variances differ amon g Dose levels? 57 (A2 vs. R) 58 Test 2: Are Variances Homogeneous? (A1 vs A2) 59 Test 3: Are variances adequately modeled? (A2 vs. A3) 60

### D-60 DRAFT – DO NOT CITE OR QUOTE

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 1 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 2
3

Tests of Interest 4
5 Test -2*log(Likelihood Ratio) Test df p-value 6
7

Test 1 22.0862 4 0.0 001927 8

Test 2 15.8167 2 0.0 003677 9

Test 3 0.23601 1 0.6271 10

Test 4 0.0441012 1 0.8337 11
12 The p-value for Test 1 is less than .05. There app ears to be a 13 difference between response and/or variances among the dose levels 14 It seems appropriate to model the data 15 16 The p-value for Test 2 is less than .1. A non-homo geneous variance 17 model appears to be appropriate 18 19 The p-value for Test 3 is greater than .1. The mod eled variance appears 20 to be appropriate here 21
22 The p-value for Test 4 is greater than .1. The mod el chosen seems 23 to adequately describe the data 24 25 26 Benchmark Dose Computation 27
28 Specified effect = 3.56 29
30 Risk Type = Point risk 31 32

Confidence level = 0.95 33 34

BMD = 32.2804 35 36 37

BMDL = 18.094 38

### D-61 DRAFT – DO NOT CITE OR QUOTE

Nesnow 1998b i.p. multiplicity 1

BbF 2 Drop 2 high doses 3

Linear 4

Nonconstant variance 5 BMR = lowest statistically significant response in BaP treated animals (after 6 control subtracted) 7 8

================================================== ================== 9 Polynomial Model. (Version: 2.12; Date: 02/2 0/2007) 10 Input Data File: C:\BMDS\UNSAVED1.(d) 11 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 12 Mon May 11 20:47:24 2009 13

================================================== ================== 14
15

BMDS MODEL RUN 16

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 17 18 The form of the response function is: 19
20 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 21
22
23 Dependent variable = MEAN 24 Independent variable = COLUMN1 25 The polynomial coefficients are restricted to be positive 26 The variance is to be modeled as Var(i) = exp(la lpha + log(mean(i)) * rho) 27
28 Total number of dose groups = 3 29 Total number of records with missing values = 0 30

Maximum number of iterations = 250 31 Relative Function Convergence has been set to: 1 e-008 32 Parameter Convergence has been set to: 1e-008 33 34
35 36

Default Initial Parameter Values 37 lalpha = 0.21205 38

rho = 0 39 beta_0 = 0.453571 40 beta_1 = 0.0305714 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 lalpha rho beta_0 beta_1 46 47 lalpha 1 0.38 0.032 -0.058 48 49

rho 0.38 1 -0.032 -0.017 50 51 beta_0 0.032 -0.032 1 -0.39 52
53 beta_1 -0.058 -0.017 -0.39 1 54
55 56
57

Parameter Estimate s 58 59

### D-62 DRAFT – DO NOT CITE OR QUOTE 95.0% Wald 1
Confidence Interval 2

Variable Estimate Std. Err. Lower Conf. Limit 3 Upper Conf. Limit 4 lalpha 0.158349 0.173664 -0.182027 5

0.498724 6

rho 1.42233 0.285984 0.861815 7

1.98285 8 beta_0 0.516257 0.101994 0.316353 9

0.71616 10 beta_1 0.0272062 0.00781084 0.0118972 11

0.0425152 12 13
14
15

Table of Data and Estimated Values of Interest 16 17

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled 18

Res. 19

------ --- -------- -------- ---------- - ----------- --------- 20

- 21
22

0 40 0.525 0.516 0.72 0.676 0.0818 23

10 18 0.67 0.788 0.75 0.914 -0.549 24

50 20 2 1.88 1.82 1.69 0.326 25 26 27
28 Model Descriptions for likelihoods calculated 29
30 31 Model A1: Yij = Mu(i) + e(ij) 32 Var{e(ij)} = Sigma^2 33 34 Model A2: Yij = Mu(i) + e(ij) 35 Var{e(ij)} = Sigma(i)^2 36 37 Model A3: Yij = Mu(i) + e(ij) 38 Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 39 Model A3 uses any fixed variance parameters th at 40 were specified by the user 41
42 Model R: Yi = Mu + e(i) 43 Var{e(i)} = Sigma^2 44
45 46

Likelihoods of Interest 47 48 Model Log(likelihood) # Param's AIC 49

A1 -45.740331 4 99.480662 50

A2 -31.124575 6 74.249150 51

A3 -31.233847 5 72.467694 52

fitted -32.276084 4 72.552168 53

R -56.886387 2 117.772774 54
55 56 Explanation of Tests 57 58 Test 1: Do responses and/or variances differ amon g Dose levels? 59 (A2 vs. R) 60

### D-63 DRAFT – DO NOT CITE OR QUOTE

Test 2: Are Variances Homogeneous? (A1 vs A2) 1 Test 3: Are variances adequately modeled? (A2 vs. A3) 2 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 3 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 4
5

Tests of Interest 6
7 Test -2*log(Likelihood Ratio) Test df p-value 8 9

Test 1 51.5236 4 <.0001 10

Test 2 29.2315 2 <.0001 11

Test 3 0.218544 1 0.6402 12

Test 4 2.08447 1 0.1488 13
14 The p-value for Test 1 is less than .05. There app ears to be a 15 difference between response and/or variances among the dose levels 16 It seems appropriate to model the data 17 18 The p-value for Test 2 is less than .1. A non-homo geneous variance 19 model appears to be appropriate 20 21 The p-value for Test 3 is greater than .1. The mod eled variance appears 22 to be appropriate here 23
24 The p-value for Test 4 is greater than .1. The mod el chosen seems 25 to adequately describe the data 26 27
28 Benchmark Dose Computation 29
30 Specified effect = 3.85 31 32 Risk Type = Point risk 33 34

Confidence level = 0.95 35 36

BMD = 122.536 37
38 39

BMDL = 84.4572 40

### D-64 DRAFT – DO NOT CITE OR QUOTE

Nesnow 1998b i.p. multiplicity 1

DBahA 2 Drop 2 high doses 3

Linear 4

Nonconstant variance 5 BMR = lowest statistically significant response in BaP treated animals (after 6 control subtracted) 7 8

================================================== ================== 9 Polynomial Model. (Version: 2.12; Date: 02/2 0/2007) 10 Input Data File: C:\BMDS\UNSAVED1.(d) 11 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 12 Mon May 11 20:55:01 2009 13

================================================== ================== 14
15

BMDS MODEL RUN 16

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 17 18 The form of the response function is: 19
20 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 21
22
23 Dependent variable = MEAN 24 Independent variable = COLUMN1 25 The polynomial coefficients are restricted to be positive 26 The variance is to be modeled as Var(i) = exp(la lpha + log(mean(i)) * rho) 27
28 Total number of dose groups = 3 29 Total number of records with missing values = 0 30

Maximum number of iterations = 250 31 Relative Function Convergence has been set to: 1 e-008 32 Parameter Convergence has been set to: 1e-008 33 34
35 36

Default Initial Parameter Values 37 lalpha = 0.495312 38

rho = 0 39 beta_0 = 0.409167 40 beta_1 = 1.01 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 lalpha rho beta_0 beta_1 46 47 lalpha 1 -0.039 -0.0077 0.0076 48 49

rho -0.039 1 0.042 -0.057 50 51 beta_0 -0.0077 0.042 1 -0.37 52
53 beta_1 0.0076 -0.057 -0.37 1 54
55 56
57

Parameter Estimate s 58 59

### D-65 DRAFT – DO NOT CITE OR QUOTE 95.0% Wald 1
Confidence Interval 2

Variable Estimate Std. Err. Lower Conf. Limit 3 Upper Conf. Limit 4 lalpha 0.090155 0.16129 -0.225967 5

0.406277 6

rho 1.13256 0.215446 0.710291 7

1.55482 8 beta_0 0.509019 0.111758 0.289977 9

0.728061 10 beta_1 0.936099 0.156639 0.629093 11

1.24311 12 13
14
15

Table of Data and Estimated Values of Interest 16 17

Dose N Obs Mean Est Mean Obs Std De v Est Std Dev Scaled 18

Res. 19

------ --- -------- -------- ---------- - ----------- --------- 20

- 21
22

0 40 0.525 0.509 0.72 0.714 0.142 23

1.25 18 1.44 1.68 1.46 1.4 -0.723 24

2.5 19 3.05 2.85 1.9 1.89 0.462 25 26 27
28 Model Descriptions for likelihoods calculated 29
30 31 Model A1: Yij = Mu(i) + e(ij) 32 Var{e(ij)} = Sigma^2 33 34 Model A2: Yij = Mu(i) + e(ij) 35 Var{e(ij)} = Sigma(i)^2 36 37 Model A3: Yij = Mu(i) + e(ij) 38 Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 39 Model A3 uses any fixed variance parameters th at 40 were specified by the user 41
42 Model R: Yi = Mu + e(i) 43 Var{e(i)} = Sigma^2 44
45 46

Likelihoods of Interest 47 48 Model Log(likelihood) # Param's AIC 49

A1 -56.039525 4 120.079049 50

A2 -42.832497 6 97.664993 51

A3 -43.013192 5 96.026383 52

fitted -43.223844 4 94.447689 53

R -75.955323 2 155.910645 54
55 56 Explanation of Tests 57 58 Test 1: Do responses and/or variances differ amon g Dose levels? 59 (A2 vs. R) 60

### D-66 DRAFT – DO NOT CITE OR QUOTE

Test 2: Are Variances Homogeneous? (A1 vs A2) 1 Test 3: Are variances adequately modeled? (A2 vs. A3) 2 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 3 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 4
5

Tests of Interest 6
7 Test -2*log(Likelihood Ratio) Test df p-value 8 9

Test 1 66.2457 4 <.0001 10

Test 2 26.4141 2 <.0001 11

Test 3 0.36139 1 0.5477 12

Test 4 0.421305 1 0.5163 13
14 The p-value for Test 1 is less than .05. There app ears to be a 15 difference between response and/or variances among the dose levels 16 It seems appropriate to model the data 17 18 The p-value for Test 2 is less than .1. A non-homo geneous variance 19 model appears to be appropriate 20 21 The p-value for Test 3 is greater than .1. The mod eled variance appears 22 to be appropriate here 23
24 The p-value for Test 4 is greater than .1. The mod el chosen seems 25 to adequately describe the data 26 27
28 Benchmark Dose Computation 29
30 Specified effect = 3.85 31 32 Risk Type = Point risk 33 34

Confidence level = 0.95 35 36

BMD = 3.56905 37
38 39

BMDL = 2.82758 40
41
42

### D-67 DRAFT – DO NOT CITE OR QUOTE

D.3. LUNG IMPLANTATION BIOASSAYS 1

DEUTSCH-WENZEL1983AA.OUT.txt 2

================================================== ================== 3 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 4 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\DEUTSCH5 WENZEL1983.(d) 6 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 7 ROUTE\DEUTSCH-WENZEL1983.plt 8 Fri May 27 10:51:53 2005 9

================================================== ================== 10
11

BMDS MODEL RUN 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 13 14 The form of the probability function is: 15 16 P[response] = background + (1-background)*[1-EXP ( 17 -beta1*dose^1)] 18 19 The parameter betas are restricted to be positiv e 20
21
22 Dependent variable = incidenceAA 23 Independent variable = doseAA 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: 1e- 008 34 Parameter Convergence has been set to: 1e-008 35 36 37 38

Default Initial Parameter Values 39 Background = 0 40 Beta(1) = 0.996523 41
42 43 Asymptotic Correlation Matrix of Paramet er Estimates 44 45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59

### D-68 DRAFT – DO NOT CITE OR QUOTE

Background 0 N A 1 Beta(1) 0.773841 0.26 0605 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -28.6723 13

Fitted model -30.8245 4.30422 2 0.1162 14

Reduced model -51.1258 44.907 2 <.0001 15 16

AIC: 63.6489 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 35 0.000 25

i: 2 26

0.1600 0.1165 4.076 1 35 -0.854 27

i: 3 28

0.8300 0.4739 16.587 19 35 0.277 29
30 Chi-square = 3.29 DF = 2 P-value = 0.1926 31 32 33 Benchmark Dose Computation 34
35 Specified effect = 0.1 36 37 Risk Type = Extra risk 38 39

Confidence level = 0.95 40
41

BMD = 0.136153 42
43

BMDL = 0.0956191 44

### D-69 DRAFT – DO NOT CITE OR QUOTE

DEUTSCH-WENZEL1983BaP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\DEUTSCH4 WENZEL1983.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\DEUTSCH-WENZEL1983.plt 7 Fri May 27 10:42:10 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0757681 39 Beta(1) = 2.82425 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-70 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 3.25323 0.59 3548 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -51.1075 13

Fitted model -51.3412 0.467435 3 0.926 14

Reduced model -96.8119 91.4088 3 <.0001 15 16

AIC: 104.682 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 35 0.000 25

i: 2 26

0.1000 0.2777 9.720 10 35 0.040 27

i: 3 28

0.3000 0.6232 21.811 23 35 0.145 29

i: 4 30

1.0000 0.9614 33.647 33 35 -0.498 31 32 Chi-square = 0.51 DF = 3 P-value = 0.9177 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.1 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 0.0323864 44
45

BMDL = 0.0255063 46

### D-71 DRAFT – DO NOT CITE OR QUOTE

DEUTSCH-WENZEL1983BbF.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\DEUTSCH4 WENZEL1983.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\DEUTSCH-WENZEL1983.plt 7 Fri May 27 10:46:47 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBbF 22 Independent variable = doseBbF 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.00149382 39 Beta(1) = 0.226374 40 Beta(2) = 0.236366 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.97 53 54 Beta(2) -0.97 1 55 56
57 58

Parameter Estimates 59 60

### D-72 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 0.24518 0.78 1411 3 Beta(2) 0.217701 0.83 0304 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -37.8686 15

Fitted model -37.8743 0.0112712 2 0.9944 16

Reduced model -51.7666 27.796 3 <.0001 17 18

AIC: 79.7485 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 35 0.000 27

i: 2 28

0.1000 0.0263 0.922 1 35 0.087 29

i: 3 30

0.3000 0.0889 3.113 3 35 -0.040 31

i: 4 32

1.0000 0.3705 12.969 13 35 0.004 33 34 Chi-square = 0.01 DF = 2 P-value = 0.9943 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.1 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 0.33191 46 47

BMDL = 0.184961 48

### D-73 DRAFT – DO NOT CITE OR QUOTE

DEUTSCH-WENZEL1983BghiP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\DEUTSCH4 WENZEL1983.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\DEUTSCH-WENZEL1983.plt 7 Fri May 27 10:49:54 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBghiP 22 Independent variable = doseBghiP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0.0304801 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.98 53 54 Beta(2) -0.98 1 55 56
57 58

Parameter Estimates 59 60

### D-74 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 0.0277423 0.23 2348 3 Beta(2) 0.000645059 0.057 4865 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -16.8561 15

Fitted model -17.033 0.353756 2 0.8379 16

Reduced model -21.5342 9.35614 3 0.02491 17 18

AIC: 38.0659 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 35 0.000 27

i: 2 28

0.1600 0.0044 0.156 0 35 -1.004 29

i: 3 30

0.8300 0.0232 0.812 1 35 0.237 31

i: 4 32

4.1500 0.1186 4.032 4 34 -0.009 33 34 Chi-square = 0.20 DF = 2 P-value = 0.9043 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.1 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 3.51117 46 47

BMDL = 1.82558 48

### D-75 DRAFT – DO NOT CITE OR QUOTE

DEUTSCH-WENZEL1983BjF.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\DEUTSCH4 WENZEL1983.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\DEUTSCH-WENZEL1983.plt 7 Fri May 27 10:47:32 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBjF 22 Independent variable = doseBjF 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.00616121 39 Beta(1) = 0.0709095 40 Beta(2) = 0.0144537 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.98 53 54 Beta(2) -0.98 1 55 56
57 58

Parameter Estimates 59 60

### D-76 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 0.0929144 0.22 6076 3 Beta(2) 0.0101278 0.046 6964 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -39.0246 15

Fitted model -39.1336 0.218103 2 0.8967 16

Reduced model -60.8862 43.7233 3 <.0001 17 18

AIC: 82.2673 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 35 0.000 27

i: 2 28

0.2000 0.0188 0.658 1 35 0.529 29

i: 3 30

1.0000 0.0979 3.427 3 35 -0.138 31

i: 4 32

5.0000 0.5122 17.926 18 35 0.009 33 34 Chi-square = 0.24 DF = 2 P-value = 0.8868 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.1 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 1.02045 46 47

BMDL = 0.580958 48

### D-77 DRAFT – DO NOT CITE OR QUOTE

DEUTSCH-WENZEL1983BkF.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\DEUTSCH4 WENZEL1983.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\DEUTSCH-WENZEL1983.plt 7 Fri May 27 10:48:11 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceBkF 22 Independent variable = doseBkF 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0.126747 40 Beta(2) = 0.00410997 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.97 53 54 Beta(2) -0.97 1 55 56
57 58

Parameter Estimates 59 60

### D-78 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 0.0842968 0.25 1118 3 Beta(2) 0.0142917 0.063 2842 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -28.404 15

Fitted model -28.9719 1.1357 2 0.5667 16

Reduced model -46.2443 35.6806 3 <.0001 17 18

AIC: 61.9437 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 35 0.000 27

i: 2 28

0.1600 0.0138 0.482 0 35 -1.014 29

i: 3 30

0.8300 0.0767 2.378 3 31 0.283 31

i: 4 32

4.1500 0.4490 12.122 12 27 -0.018 33 34 Chi-square = 0.67 DF = 2 P-value = 0.7165 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.1 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 1.05954 46 47

BMDL = 0.557079 48

### D-79 DRAFT – DO NOT CITE OR QUOTE

DEUTSCH-WENZEL1983IP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\DEUTSCH4 WENZEL1983.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\DEUTSCH-WENZEL1983.plt 7 Fri May 27 10:49:04 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = incidenceIP 22 Independent variable = doseIP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0539703 39 Beta(1) = 0.20919 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Background Beta(1) 51 52 Background 1 -0.55 53 54 Beta(1) -0.55 1 55 56
57 58

Parameter Estimates 59 60

### D-80 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0.0224449 0.11 3638 2 Beta(1) 0.241452 0.079 7033 3 Beta(2) 0 N A 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -54.8079 15

Fitted model -56.5662 3.5166 2 0.1723 16

Reduced model -76.4525 43.2893 3 <.0001 17 18

AIC: 117.132 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0224 0.786 0 35 -1.023 27

i: 2 28

0.1600 0.0595 2.082 4 35 0.979 29

i: 3 30

0.8300 0.2000 6.999 8 35 0.179 31

i: 4 32

4.1500 0.6411 22.439 21 35 -0.179 33 34 Chi-square = 3.12 DF = 2 P-value = 0.2104 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.1 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 0.436361 46 47

BMDL = 0.309504 48

### D-81 DRAFT – DO NOT CITE OR QUOTE

WENZEL-HARTUNG1990BaP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\WENZEL4 HARTUNG1990.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\WENZEL-HARTUNG1990.plt 7 Fri May 27 10:58:05 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = responseBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 3.21631 40 Beta(2) = 5.7325 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.93 53 54 Beta(2) -0.93 1 55 56
57 58

Parameter Estimates 59 60

### D-82 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 3.01149 2.7 9594 3 Beta(2) 6.44644 10. 7674 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -50.8389 15

Fitted model -50.8521 0.0264626 2 0.9869 16

Reduced model -84.6566 67.6355 3 <.0001 17 18

AIC: 105.704 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 35 0.000 27

i: 2 28

0.0300 0.0917 3.208 3 35 -0.072 29

i: 3 30

0.1000 0.3062 10.718 11 35 0.038 31

i: 4 32

0.3000 0.7732 27.062 27 35 -0.010 33 34 Chi-square = 0.03 DF = 2 P-value = 0.9870 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.1 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 0.0326976 46 47

BMDL = 0.0198862 48

### D-83 DRAFT – DO NOT CITE OR QUOTE

WENZEL-HARTUNG1990BaPforDBahA.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER 4 ROUTE\SETS\WENZEL-HARTUNG1990.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\SETS\WENZEL-HARTUNG1990.plt 7

Thu Jun 02 09:02:58 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = responseBaP 22 Independent variable = doseBaP 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 3.21631 40 Beta(2) = 5.7325 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.93 53 54 Beta(2) -0.93 1 55 56
57 58

Parameter Estimates 59 60

### D-84 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 3.01149 2.7 9594 3 Beta(2) 6.44644 10. 7674 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -50.8389 15

Fitted model -50.8521 0.0264626 2 0.9869 16

Reduced model -84.6566 67.6355 3 <.0001 17 18

AIC: 105.704 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 35 0.000 27

i: 2 28

0.0300 0.0917 3.208 3 35 -0.072 29

i: 3 30

0.1000 0.3062 10.718 11 35 0.038 31

i: 4 32

0.3000 0.7732 27.062 27 35 -0.010 33 34 Chi-square = 0.03 DF = 2 P-value = 0.9870 35 36 37 Benchmark Dose Computation 38 39 Specified effect = 0.57 40
41 Risk Type = Extra risk 42
43
Confidence level = 0.95 44
45

BMD = 0.197095 46 47

BMDL = 0.157781 48

### D-85 DRAFT – DO NOT CITE OR QUOTE

WENZEL-HARTUNG1990CH.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\PAH\BMD ANALYSIS\BIOASSAY\ OTHER ROUTE\WENZEL4 HARTUNG1990.(d) 5 Gnuplot Plotting File: C:\PAH\BMD ANALYSIS\BI OASSAY\OTHER 6 ROUTE\WENZEL-HARTUNG1990.plt 7 Fri May 27 10:58:53 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = responseCH 22 Independent variable = doseCH 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0178361 39 Beta(1) = 0.109158 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 0.123432 0.064 7008 60

### D-86 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -35.2935 11

Fitted model -35.455 0.323044 2 0.8508 12

Reduced model -43.0622 15.5374 2 0.0004228 13
14

AIC: 72.9101 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 35 0.000 23

i: 2 24

1.0000 0.1161 4.064 5 35 0.261 25

i: 3 26

3.0000 0.3095 10.831 10 35 -0.111 27
28 Chi-square = 0.34 DF = 2 P-value = 0.8453 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 0.1 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 0.853595 40
41

BMDL = 0.57298 42
43

### D-87 DRAFT – DO NOT CITE OR QUOTE

D.4. BACTERIAL MUTAGENICITY 1

Hass 1981 bact mut bap.out.txt 2

================================================== ================== 3 Polynomial Model. Revision: 2.2 Date: 9/12/2 002 4 Input Data File: C:\BMDS\UNSAVED1.(d) 5 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 6

Wed Jul 06 11:29:07 2005 7

================================================== ================== 8 9

BMDS MODEL RUN 10

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 11
12 The form of the response function is: 13 14 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 15 16 17 Dependent variable = MEAN 18 Independent variable = COLUMN1 19

rho is set to 0 20 Signs of the polynomial coefficients are not res tricted 21

A constant variance model is fit 22 23 Total number of dose groups = 4 24 Total number of records with missing values = 0 25

Maximum number of iterations = 250 26 Relative Function Convergence has been set to: 1 e-008 27 Parameter Convergence has been set to: 1e-008 28 29
30 31

Default Initial Parameter Values 32 alpha = 194.5 33 rho = 0 Sp ecified 34 beta_0 = 121.8 35 beta_1 = 297.029 36 37 38 39

Parameter Estimate s 40
41

95.0% Wald 42

Confidence Interval 43

Variable Estimate Std. Err. Lower Conf. Limit 44 Upper Conf. Limit 45 alpha 132.71 54.1784 26.5217 46

238.897 47 beta_0 121.8 5.15188 111.702 48

131.898 49 beta_1 297.029 8.99387 279.401 50

314.656 51 52 53 Asymptotic Correlation Matrix of Paramet er Estimates 54
55 alpha beta_0 beta_1 56 alpha 1 -1.4e-009 -1.1e-008 57 beta_0 -1.4e-009 1 -0.76 58 beta_1 -1.1e-008 -0.76 1 59

### D-88 DRAFT – DO NOT CITE OR QUOTE

1 2

Table of Data and Estimated Values of Interest 3
4

Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev Chi^2 5

Res. 6

------ --- -------- ----------- -------- ----------- --------- 7

- 8 9

0 3 124 8 122 11.5 0.331 10

0.25 3 194 16 196 11.5 -0.309 11

0.5 3 269 13 270 11.5 -0.198 12

1 3 420 17 419 11.5 0.176 13
14
15 16 Model Descriptions for likelihoods calculated 17 18 19 Model A1: Yij = Mu(i) + e(ij) 20 Var{e(ij)} = Sigma^2 21
22 Model A2: Yij = Mu(i) + e(ij) 23 Var{e(ij)} = Sigma(i)^2 24 25 Model R: Yi = Mu + e(i) 26 Var{e(i)} = Sigma^2 27
28 29

Likelihoods of Interest 30 31 Model Log(likelihood) DF AIC 32

A1 -35.189802 5 80 .379605 33

A2 -34.317788 8 84 .635576 34

fitted -35.328976 2 74 .657952 35

R -62.974684 2 129 .949369 36 37 Test 1: Does response and/or variances differ amo ng dose 38

levels 39 (A2 vs. R) 40 Test 2: Are Variances Homogeneous (A1 vs A2) 41 Test 3: Does the Model for the Mean Fit (A1 vs. f itted) 42
43

Tests of Interest 44
45 Test -2*log(Likelihood Ratio) Test df p- value 46 47

Test 1 57.3138 6 <.0001 48

Test 2 1.74403 3 0.6272 49

Test 3 0.278348 2 0.8701 50 51 The p-value for Test 1 is less than .05. There app ears 52

to be a 53 difference between response and/or variances among the 54

dose levels. 55 It seems appropriate to model the data 56
57 The p-value for Test 2 is greater than .05. A 58 homogeneous variance 59 model appears to be appropriate here 60

### D-89 DRAFT – DO NOT CITE OR QUOTE

1 2 The p-value for Test 3 is greater than .05. The mo del 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 deviation s from the control mean 12 13
14

Confidence level = 0.95 15 16

BMD = 0.038784 17 18 19

BMDL = 0.0286028 20 21

### D-90 DRAFT – DO NOT CITE OR QUOTE

HASS_1981_BACT_MUT_BEP.OUT.txt 1

================================================== ================== 2 Polynomial Model. Revision: 2.2 Date: 9/12/2 002 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\HASS_1981_BACT_MUT_BEP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\HASS_1981_BACT_MUT_BEP.p lt 7

Wed Jul 06 13:42:38 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the response function is: 14
15 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 16 17 18 Dependent variable = MEAN 19 Independent variable = COLUMN1 20
rho is set to 0 21 Signs of the polynomial coefficients are not res tricted 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: 1 e-008 28 Parameter Convergence has been set to: 1e-008 29
30 31 32

Default Initial Parameter Values 33 alpha = 117.5 34 rho = 0 Sp ecified 35 beta_0 = 120.75 36 beta_1 = 77.5 37
38 39 40

Parameter Estimate s 41
42

95.0% Wald 43
Confidence Interval 44

Variable Estimate Std. Err. Lower Conf. Limit 45 Upper Conf. Limit 46 alpha 98.6458 40.272 19.7142 47

177.577 48 beta_0 120.75 4.19706 112.524 49

128.976 50 beta_1 77.5 7.66275 62.4813 51

92.5187 52
53 54 Asymptotic Correlation Matrix of Paramet er Estimates 55 56 alpha beta_0 beta_1 57 alpha 1 -8e-012 1.1e-011 58 beta_0 -8e-012 1 -0.73 59 beta_1 1.1e-011 -0.73 1 60

### D-91 DRAFT – DO NOT CITE OR QUOTE

1 2

Table of Data and Estimated Values of Interest 3
4

Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev Chi^2 5

Res. 6

------ --- -------- ----------- -------- ----------- --------- 7

- 8 9

0 3 124 8 121 9.93 0.567 10

0.2 3 129 6 136 9.93 -1.26 11

0.4 3 156 9 152 9.93 0.741 12

1 3 198 17 198 9.93 -0.0436 13
14
15 16 Model Descriptions for likelihoods calculated 17 18 19 Model A1: Yij = Mu(i) + e(ij) 20 Var{e(ij)} = Sigma^2 21
22 Model A2: Yij = Mu(i) + e(ij) 23 Var{e(ij)} = Sigma(i)^2 24 25 Model R: Yi = Mu + e(i) 26 Var{e(i)} = Sigma^2 27
28 29

Likelihoods of Interest 30 31 Model Log(likelihood) DF AIC 32

A1 -32.165839 5 74 .331679 33

A2 -30.272126 8 76 .544252 34

fitted -33.549216 2 71 .098432 35

R -47.594288 2 99 .188576 36 37 Test 1: Does response and/or variances differ amo ng dose 38

levels 39 (A2 vs. R) 40 Test 2: Are Variances Homogeneous (A1 vs A2) 41 Test 3: Does the Model for the Mean Fit (A1 vs. f itted) 42
43

Tests of Interest 44
45 Test -2*log(Likelihood Ratio) Test df p- value 46 47

Test 1 34.6443 6 <.0001 48

Test 2 3.78743 3 0.2854 49

Test 3 2.76675 2 0.2507 50 51 The p-value for Test 1 is less than .05. There app ears 52

to be a 53 difference between response and/or variances among the 54

dose levels. 55 It seems appropriate to model the data 56
57 The p-value for Test 2 is greater than .05. A 58 homogeneous variance 59 model appears to be appropriate here 60

### D-92 DRAFT – DO NOT CITE OR QUOTE

1 2 The p-value for Test 3 is greater than .05. The mo del 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 deviation s from the control mean 12 13
14

Confidence level = 0.95 15 16

BMD = 0.128156 17 18 19

BMDL = 0.0923937 20 21

### D-93 DRAFT – DO NOT CITE OR QUOTE

JOHNSEN_1997_BAC_MUT_BAP.OUT.txt 1

================================================== ================== 2 Polynomial Model. Revision: 2.2 Date: 9/12/2 002 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\JOHNSEN_1997_BAC_MUT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\JOHNSEN_1997_BAC_MUT_BAP .plt 7

Fri Jul 08 09:02:29 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the response function is: 14
15 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 16 17 18 Dependent variable = MEAN 19 Independent variable = COLUMN1 20
rho is set to 0 21 Signs of the polynomial coefficients are not res tricted 22

A constant variance model is fit 23
24 Total number of dose groups = 3 25 Total number of records with missing values = 0 26

Maximum number of iterations = 250 27 Relative Function Convergence has been set to: 1 e-008 28 Parameter Convergence has been set to: 1e-008 29
30 31 32

Default Initial Parameter Values 33 alpha = 70.2768 34 rho = 0 Sp ecified 35 beta_0 = 115.5 36 beta_1 = 0.65 37
38 39 40

Parameter Estimate s 41
42

95.0% Wald 43
Confidence Interval 44

Variable Estimate Std. Err. Lower Conf. Limit 45 Upper Conf. Limit 46 alpha 59.3512 27.9784 4.51449 47

114.188 48 beta_0 115.5 4.06035 107.542 49

123.458 50 beta_1 0.65 0.314513 0.0335651 51

1.26643 52
53 54 Asymptotic Correlation Matrix of Paramet er Estimates 55 56 alpha beta_0 beta_1 57 alpha 1 -7.9e-010 -3.4e-012 58 beta_0 -7.9e-010 1 -0.77 59 beta_1 -3.4e-012 -0.77 1 60

### D-94 DRAFT – DO NOT CITE OR QUOTE

1 2

Table of Data and Estimated Values of Interest 3
4

Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev Chi^2 5

Res. 6

------ --- -------- ----------- -------- ----------- --------- 7

- 8 9

0 3 113 9.68 115 7.7 -0.562 10

10 3 127 4.84 122 7.7 1.12 11

20 3 126 9.68 128 7.7 -0.562 12 13
14
15 Model Descriptions for likelihoods calculated 16 17 18 Model A1: Yij = Mu(i) + e(ij) 19 Var{e(ij)} = Sigma^2 20 21 Model A2: Yij = Mu(i) + e(ij) 22 Var{e(ij)} = Sigma(i)^2 23
24 Model R: Yi = Mu + e(i) 25 Var{e(i)} = Sigma^2 26 27
28
Likelihoods of Interest 29
30 Model Log(likelihood) DF AIC 31

A1 -21.811395 4 51 .622790 32

A2 -21.026523 6 54 .053045 33

fitted -22.875626 2 49 .751251 34

R -24.653317 2 53 .306634 35 36 Test 1: Does response and/or variances differ amo ng dose 37

levels 38 (A2 vs. R) 39 Test 2: Are Variances Homogeneous (A1 vs A2) 40 Test 3: Does the Model for the Mean Fit (A1 vs. f itted) 41
42

Tests of Interest 43 44 Test -2*log(Likelihood Ratio) Test df p- value 45 46

Test 1 7.25359 4 0.0266 47

Test 2 1.56974 2 0.4562 48

Test 3 2.12846 1 0.1446 49
50 The p-value for Test 1 is less than .05. There app ears 51

to be a 52 difference between response and/or variances among the 53

dose levels. 54 It seems appropriate to model the data 55 56 The p-value for Test 2 is greater than .05. A 57 homogeneous variance 58 model appears to be appropriate here 59 60

### D-95 DRAFT – DO NOT CITE OR QUOTE

1 The p-value for Test 3 is greater than .05. The mo del 2 chosen appears 3 to adequately describe the data 4
5 6
7 Benchmark Dose Computation 8 Specified effect = 1 9 10 Risk Type = Estimated standard deviation s from the control mean 11
12 13

Confidence level = 0.95 14
15

BMD = 11.8523 16 17 18

BMDL = 6.27094 19
20 21
22

### D-96 DRAFT – DO NOT CITE OR QUOTE

D.5. MAMMALIAN MUTAGENICITY 1

BARF_MUT_BAA.OUT.txt 2

================================================== ================== 3 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 5 RPS\MODELING\BARF_MUT_BAA.(d) 6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 7 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_BAA.plt 8

Thu Jun 30 12:46:38 2005 9

================================================== ================== 10
11

BMDS MODEL RUN 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 13 14 The form of the probability function is: 15 16 P[response] = background + (1-background)*[1-EXP ( 17 -beta1*dose^1-beta2*dose^2-beta3*dose^3)] 18 19 The parameter betas are restricted to be positiv e 20
21
22 Dependent variable = COLUMN2 23 Independent variable = COLUMN1 24 25

Total number of observations = 5 26 Total number of records with missing values = 0 27 Total number of parameters in model = 4 28 Total number of specified parameters = 0 29 Degree of polynomial = 3 30 31
32
Maximum number of iterations = 250 33 Relative Function Convergence has been set to: 1e- 008 34 Parameter Convergence has been set to: 1e-008 35 36 37 38

Default Initial Parameter Values 39 Background = 3.89426e-006 40 Beta(1) = 3.46216e-007 41 Beta(2) = 0 42 Beta(3) = 1.93939e-012 43 **** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 44 pont  45 46
47 48 Asymptotic Correlation Matrix of Paramet er Estimates 49
50 ( ** * The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 51 have been estimated at a boundary point, or have been 52 specified by the user, 53 and do not appear in the correlati on matrix ) 54
55 Beta(1) 56
57 Beta(1) 1 58 59

### D-97 DRAFT – DO NOT CITE OR QUOTE

1 2

Parameter Estimates 3
4

Variable Estimate Std. Err. 5 Background 0 N A 6 Beta(1) 4.34385e-007 5.43792e -006 7 Beta(2) 0 N A 8 Beta(3) 0 N A 9 10 NA - Indicates that this parameter has hit a bound 11 implied by some inequality constraint and thus 12

has no standard error. 13
14
15 16 Analysis of Deviance Table 17 18 Model Log(likelihood) Deviance Test D F P-value 19

Full model -1545.82 20
Fitted model -1548.6 5.57201 4 0.2335 21

Reduced model -1597.17 102.713 4 <.0001 22
23

AIC: 3099.21 24 25 26

Goodness of Fit 27
28 Dose Est._Prob. Expected Observed Size Chi^2 Res. 29

------------------------------------------------- ---------------------- 30

i: 1 31

0.0000 0.0000 0.000 0 1 000000 0.000 32

i: 2 33

20.0000 0.0000 8.688 12 1 000000 0.381 34

i: 3 35

50.0000 0.0000 21.719 29 1 000000 0.335 36

i: 4 37

100.0000 0.0000 43.438 34 1 000000 -0.217 38

i: 5 39

150.0000 0.0001 65.156 64 1 000000 -0.018 40
41 Chi-square = 5.77 DF = 4 P-value = 0.2166 42
43 44 Benchmark Dose Computation 45 46 Specified effect = 1e-005 47 48 Risk Type = Extra risk 49
50

Confidence level = 0.95 51 52

BMD = 23.0212 53 54 **** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 55 point  56
57 ** ** WARNING 0: Completion code = -2 trying new st art  58 59 ** ** WARNING 1: Completion code = -2 trying new st art  60

### D-98 DRAFT – DO NOT CITE OR QUOTE

1 ** ** WARNING 2: Completion code = -2 trying new st art  2
3 ** ** WARNING 3: Completion code = -2 trying new st art  4
5 ** ** WARNING 4: Completion code = -2 trying new st art  6
7 ** ** WARNING 5: Completion code = -2 trying new st art  8 9 ** ** WARNING 6: Completion code = -2 trying new st art  10
11 ** ** WARNING 7: Completion code = -2 trying new st art  12 13 ** ** WARNING 8: Completion code = -2 trying new st art  14
15 ** ** WARNING 9: Completion code = -2 trying new st art  16 17 ** ** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 18 point  19
20 ** ** WARNING 0: Completion code = -2 trying new st art  21
22 ** ** WARNING 1: Completion code = -3 trying new st art  23
24 ** ** WARNING 2: Completion code = -3 trying new st art  25 26 ** ** WARNING 3: Completion code = -3 trying new st art  27
28 ** ** WARNING 4: Completion code = -3 trying new st art  29
30 ** ** WARNING 5: Completion code = -3 trying new st art  31 32 ** ** WARNING 6: Completion code = -2 trying new st art  33 34 ** ** WARNING 7: Completion code = -3 trying new st art  35 36 ** ** WARNING 8: Completion code = -3 trying new st art  37
38 ** ** WARNING 9: Completion code = -3 trying new st art**** 39 40
41 Warning: completion code still negative 42 BMDL did not converge for BMR = 0.000010 43 44 Program execution is stopped 45

### D-99 DRAFT – DO NOT CITE OR QUOTE

BARF_MUT_BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\BARF_MUT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_BAP.plt 7

Thu Jun 30 12:40:17 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 1.39884e-006 39 Beta(1) = 5.34042e-006 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-100 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 5.43367e-006 2.68102e -005 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -3273.08 13

Fitted model -3273.96 1.75092 3 0.6257 14

Reduced model -3395.25 244.327 3 <.0001 15 16

AIC: 6549.92 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 1 000000 0.000 25

i: 2 26

10.0000 0.0001 54.335 51 1 000000 -0.061 27

i: 3 28

20.0000 0.0001 108.668 120 1 000000 0.104 29

i: 4 30

30.0000 0.0002 162.997 155 1 000000 -0.049 31 32 Chi-square = 1.78 DF = 3 P-value = 0.6195 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 1e-005 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 1.84039 44
45   WARNING: Completion code = -3. Optimum not f ound. Trying new starting 46 point** ** 47 48   WARNING 0: Completion code = -3 trying new st art** ** 49
50   WARNING 1: Completion code = -3 trying new st art** ** 51 52   WARNING 2: Completion code = -3 trying new st art** ** 53 54   WARNING 3: Completion code = -3 trying new st art** ** 55 56   WARNING 4: Completion code = -3 trying new st art** ** 57 58   WARNING 5: Completion code = -3 trying new st art** ** 59 60

### D-101 DRAFT – DO NOT CITE OR QUOTE

  WARNING 6: Completion code = -3 trying new st art** ** 1 2   WARNING 7: Completion code = -3 trying new st art** ** 3
4   WARNING 8: Completion code = -3 trying new st art** ** 5 6   WARNING 9: Completion code = -3 trying new st art** ** 7 8   WARNING: Completion code = -3. Optimum not f ound. Trying new starting 9 point** ** 10
11   WARNING 0: Completion code = -1 trying new st art** ** 12 13   WARNING 1: Completion code = -1 trying new st art** ** 14
15   WARNING 2: Completion code = -1 trying new st art** ** 16 17

BMDL = 1.68248 18

### D-102 DRAFT – DO NOT CITE OR QUOTE

BARF_MUT_CH.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\BARF_MUT_CH.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_CH.plt 7

Thu Jun 30 12:48:57 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 2.60526e-006 39 Beta(1) = 5.02638e-007 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 6.14293e-007 1.93539e -005 60

### D-103 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -504.191 11

Fitted model -505.38 2.37752 2 0.3046 12

Reduced model -522.575 36.7681 2 <.0001 13
14

AIC: 1012.76 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 1 000000 0.000 23

i: 2 24

20.0000 0.0000 12.286 17 1 000000 0.384 25

i: 3 26

50.0000 0.0000 30.714 26 1 000000 -0.153 27
28 Chi-square = 2.53 DF = 2 P-value = 0.2819 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 1e-005 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 16.279 40
41   WARNING: Completion code = -1. Optimum not f ound. Trying new starting 42 point** ** 43 44   WARNING 0: Completion code = -1 trying new st art** ** 45 46   WARNING 1: Completion code = -1 trying new st art** ** 47 48   WARNING 2: Completion code = -1 trying new st art** ** 49
50   WARNING 3: Completion code = -1 trying new st art** ** 51 52   WARNING 4: Completion code = -1 trying new st art** ** 53 54   WARNING 5: Completion code = -1 trying new st art** ** 55 56   WARNING 6: Completion code = -1 trying new st art** ** 57 58   WARNING 7: Completion code = -1 trying new st art** ** 59 60

### D-104 DRAFT – DO NOT CITE OR QUOTE

  WARNING 8: Completion code = -1 trying new st art** ** 1 2   WARNING 9: Completion code = -1 trying new st art** ** 3
4   WARNING: Completion code = -1. Optimum not f ound. Trying new starting 5 point** ** 6
7   WARNING 0: Completion code = -3 trying new st art** ** 8 9   WARNING 1: Completion code = -3 trying new st art** ** 10
11   WARNING 2: Completion code = -3 trying new st art** ** 12 13   WARNING 3: Completion code = -3 trying new st art** ** 14
15   WARNING 4: Completion code = -3 trying new st art** ** 16 17   WARNING 5: Completion code = -3 trying new st art** ** 18 19   WARNING 6: Completion code = -3 trying new st art** ** 20 21   WARNING 7: Completion code = -3 trying new st art** ** 22
23   WARNING 8: Completion code = -3 trying new st art** ** 24 25   WARNING 9: Completion code = -3 trying new st art** ** 26 27
28 Warning: completion code still negative 29 BMDL did not converge for BMR = 0.000010 30 31 Program execution is stopped 32

### D-105 DRAFT – DO NOT CITE OR QUOTE

BARF_MUT_FA.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\BARF_MUT_FA.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_FA.plt 7

Thu Jun 30 12:43:11 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 6.6658e-007 39 Beta(1) = 2.50006e-006 40
41
42 Asymptotic Correlation Matrix of Paramet er Estimates 43 44 ( *** The model parameter(s) -Backgroun d 45 have been estimated at a boundary point, or have been 46 specified by the user, 47 and do not appear in the correlati on matrix ) 48 49 Beta(1) 50 51 Beta(1) 1 52
53 54
55
Parameter Estimates 56
57

Variable Estimate Std. Err. 58 Background 0 N A 59 Beta(1) 2.56672e-006 4.49565e -005 60

### D-106 DRAFT – DO NOT CITE OR QUOTE

1 NA - Indicates that this parameter has hit a bound 2 implied by some inequality constraint and thus 3

has no standard error. 4
5 6
7 Analysis of Deviance Table 8 9 Model Log(likelihood) Deviance Test D F P-value 10

Full model -856.204 11

Fitted model -856.255 0.103 2 0.9498 12

Reduced model -890.913 69.419 2 <.0001 13
14

AIC: 1714.51 15 16 17

Goodness of Fit 18 19 Dose Est._Prob. Expected Observed Size Chi^2 Res. 20

------------------------------------------------- ---------------------- 21

i: 1 22

0.0000 0.0000 0.000 0 1 000000 0.000 23

i: 2 24

10.0000 0.0000 25.667 27 1 000000 0.052 25

i: 3 26

20.0000 0.0001 51.333 50 1 000000 -0.026 27
28 Chi-square = 0.10 DF = 2 P-value = 0.9494 29
30 31 Benchmark Dose Computation 32 33 Specified effect = 1e-005 34
35 Risk Type = Extra risk 36 37

Confidence level = 0.95 38 39

BMD = 3.89604 40
41   WARNING: Completion code = -1. Optimum not f ound. Trying new starting 42 point** ** 43 44   WARNING 0: Completion code = -1 trying new st art** ** 45 46   WARNING 1: Completion code = -5 trying new st art** ** 47 48

BMDL = 0 49
50

### D-107 DRAFT – DO NOT CITE OR QUOTE

BARF_MUT_TPHEN.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\BARF_MUT_TPHEN.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\BARF_MUT_TPHEN.plt 7

Thu Jun 30 12:52:56 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 9.99937e-007 39 Beta(1) = 1.74289e-007 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-108 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 1.85717e-007 4.42148e -006 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -755.63 13

Fitted model -755.773 0.2868 3 0.9625 14

Reduced model -781.782 52.3039 3 <.0001 15 16

AIC: 1513.55 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 1 000000 0.000 25

i: 2 26

50.0000 0.0000 9.286 10 1 000000 0.077 27

i: 3 28

100.0000 0.0000 18.572 20 1 000000 0.077 29

i: 4 30

200.0000 0.0000 37.143 35 1 000000 -0.058 31 32 Chi-square = 0.29 DF = 3 P-value = 0.9622 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 1e-005 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 53.8457 44
45   WARNING: Completion code = -2. Optimum not f ound. Trying new starting 46 point** ** 47 48   WARNING 0: Completion code = -2 trying new st art** ** 49
50   WARNING 1: Completion code = -2 trying new st art** ** 51 52   WARNING 2: Completion code = -2 trying new st art** ** 53 54   WARNING 3: Completion code = -2 trying new st art** ** 55 56   WARNING 4: Completion code = -2 trying new st art** ** 57 58   WARNING 5: Completion code = -2 trying new st art** ** 59 60

### D-109 DRAFT – DO NOT CITE OR QUOTE

  WARNING 6: Completion code = -2 trying new st art** ** 1 2   WARNING 7: Completion code = -2 trying new st art** ** 3
4   WARNING 8: Completion code = -2 trying new st art** ** 5 6   WARNING 9: Completion code = -2 trying new st art** ** 7 8   WARNING: Completion code = -2. Optimum not f ound. Trying new starting 9 point** ** 10
11   WARNING 0: Completion code = -2 trying new st art** ** 12 13   WARNING 1: Completion code = -5 trying new st art** ** 14
15   WARNING 2: Completion code = -2 trying new st art** ** 16 17   WARNING 3: Completion code = -2 trying new st art** ** 18 19   WARNING 4: Completion code = -2 trying new st art** ** 20 21   WARNING 5: Completion code = -2 trying new st art** ** 22
23   WARNING 6: Completion code = -2 trying new st art** ** 24 25   WARNING 7: Completion code = -5 trying new st art** ** 26 27   WARNING 8: Completion code = -2 trying new st art** ** 28 29   WARNING 9: Completion code = -5 trying new st art** ** 30 31 32 Warning: completion code still negative 33 BMDL did not converge for BMR = 0.000010 34
35 Program execution is stopped 36

### D-110 DRAFT – DO NOT CITE OR QUOTE

RAVEH_HUB_MUT_BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\RAVEH_HUB_MUT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\RAVEH_HUB_MUT_BAP.plt 7

Wed Jun 29 12:15:41 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0.00102082 40 **** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 41 pont  42
43 ** ** WARNING 0: Completion code = -2 trying new st art  44
45 ** ** WARNING 1: Completion code = -2 trying new st art  46 47 ** ** WARNING 2: Completion code = -2 trying new st art  48 49 ** ** WARNING 3: Completion code = -2 trying new st art  50 51 ** ** WARNING 4: Completion code = -2 trying new st art  52
53 ** ** WARNING 5: Completion code = -2 trying new st art  54
55 ** ** WARNING 6: Completion code = -2 trying new st art  56
57 ** ** WARNING 7: Completion code = -2 trying new st art  58 59 ** ** WARNING 8: Completion code = -2 trying new st art  60

### D-111 DRAFT – DO NOT CITE OR QUOTE

1 ** ** WARNING 9: Completion code = -2 trying new st art  2
3 ** ** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 4 point  5 6 ** ** WARNING 0: Completion code = -2 trying new st art  7 8 ** ** WARNING 1: Completion code = -2 trying new st art  9 10 ** ** WARNING 2: Completion code = -2 trying new st art  11
12 ** ** WARNING 3: Completion code = -2 trying new st art**** 13
14
15 16 Asymptotic Correlation Matrix of Paramet er Estimates 17 18 Background Beta(1) 19
20 Background 1 -0.71 21
22 Beta(1) -0.71 1 23
24 25 26

Parameter Estimates 27
28
Variable Estimate Std. Err. 29 Background 2.6399e-005 0.0025 7721 30 Beta(1) 0.000947187 0.0041 9869 31 32 33 34 Analysis of Deviance Table 35 36 Model Log(likelihood) Deviance Test D F P-value 37

Full model -1077.99 38

Fitted model -1078.81 1.63811 1 0.2006 39

Reduced model -1144.43 132.88 2 <.0001 40
41

AIC: 2161.62 42
43 44

Goodness of Fit 45 46 Dose Est._Prob. Expected Observed Size Chi^2 Res. 47

------------------------------------------------- ---------------------- 48

i: 1 49

0.0000 0.0000 2.640 3 100000 0.136 50

i: 2 51

0.3000 0.0003 31.051 25 100000 -0.195 52

i: 3 53

1.0000 0.0010 97.311 103 100000 0.059 54
55 Chi-square = 1.56 DF = 1 P-value = 0.2115 56
57 58 Benchmark Dose Computation 59 60

### D-112 DRAFT – DO NOT CITE OR QUOTE

Specified effect = 0.0001 1 2 Risk Type = Extra risk 3
4

Confidence level = 0.95 5 6

BMD = 0.105581 7 8

BMDL = 0.0908465 9

### D-113 DRAFT – DO NOT CITE OR QUOTE

RAVEH_HUB_MUT_cpcdp.OUT.txt 1

================================================== ================== 2 Quantal Linear Model $Revision: 2.2 $ $Date: 2000/03/17 22:27:16 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\RAVEH_HUB_MUT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\RAVEH_HUB_MUT_BAP.plt 7

Wed Jun 29 12:09:01 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP (-slope*dose)] 16 17 18 Dependent variable = COLUMN2 19 Independent variable = COLUMN1 20 21

Total number of observations = 3 22 Total number of records with missing values = 0 23

Maximum number of iterations = 250 24 Relative Function Convergence has been set to: 1 e-008 25 Parameter Convergence has been set to: 1e-008 26 27
28 29 Default Initial (and Specified) P arameter Values 30 Background = 3.49997e-005 31 Slope = 0.000170019 32 Power = 1 Sp ecified 33 34
35 Asymptotic Correlation Matrix of Paramet er Estimates 36 37 ( *** The model parameter(s) -Power 38 have been estimated at a boundary point, or have been 39 specified by the user, 40 and do not appear in the correlati on matrix ) 41
42 Background Slope 43 44 Background 1 -0.51 45 46 Slope -0.51 1 47 48 49
50

Parameter Estimates 51 52

Variable Estimate Std. Err. 53 Background 3.16959e-005 1.69176e -005 54 Slope 0.000173022 4.78826e -005 55 56
57 58 Analysis of Deviance Table 59 60

### D-114 DRAFT – DO NOT CITE OR QUOTE

Model Log(likelihood) Deviance Test D F P-value 1

Full model -317.426 2

Fitted model -317.46 0.0679084 1 0.7944 3

Reduced model -324.664 14.4766 2 0.0007185 4
5

AIC: 638.919 6
7 8

Goodness of Fit 9 10

Scaled 11 Dose Est._Prob. Expected Observed Size Residual 12

------------------------------------------------- ----------------------- 13

0.0000 0.0000 3.170 3 100000 -0.09526 14

0.3000 0.0001 8.360 9 100000 0.2214 15

1.0000 0.0002 20.470 20 100000 -0.1038 16 17 Chi-square = 0.07 DF = 1 P-value = 0.7930 18 19
20 Benchmark Dose Computation 21
22 Specified effect = 0.0001 23
24 Risk Type = Extra risk 25 26

Confidence level = 0.95 27
28

BMD = 0.577991 29
30

BMDL = 0.390507 31 32

### D-115 DRAFT – DO NOT CITE OR QUOTE

RAVEH_MUT_bap.OUT.txt 1

================================================== ================== 2 Quantal Linear Model $Revision: 2.2 $ $Date: 2000/03/17 22:27:16 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\RAVEH_MUT_CPCDP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\RAVEH_MUT_CPCDP.plt 7

Wed Jun 29 12:33:35 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP (-slope*dose)] 16 17 18 Dependent variable = COLUMN2 19 Independent variable = COLUMN1 20 21

Total number of observations = 3 22 Total number of records with missing values = 0 23

Maximum number of iterations = 250 24 Relative Function Convergence has been set to: 1 e-008 25 Parameter Convergence has been set to: 1e-008 26 27
28 29 Default Initial (and Specified) P arameter Values 30 Background = 7.49999e-006 31 Slope = 6.70027e-005 32 Power = 1 Sp ecified 33 34
35 Asymptotic Correlation Matrix of Paramet er Estimates 36 37 ( *** The model parameter(s) -Power 38 have been estimated at a boundary point, or have been 39 specified by the user, 40 and do not appear in the correlati on matrix ) 41
42 Background Slope 43 44 Background 1 -0.38 45 46 Slope -0.38 1 47 48 49
50

Parameter Estimates 51 52

Variable Estimate Std. Err. 53 Background 6.11766e-006 2.23574e -006 54 Slope 6.35766e-005 8.04156e -006 55 56
57 58 Analysis of Deviance Table 59 60

### D-116 DRAFT – DO NOT CITE OR QUOTE

Model Log(likelihood) Deviance Test D F P-value 1

Full model -1104.33 2

Fitted model -1105.09 1.53413 1 0.2155 3

Reduced model -1141.2 73.7415 2 <.0001 4
5

AIC: 2214.19 6
7 8

Goodness of Fit 9 10

Scaled 11 Dose Est._Prob. Expected Observed Size Residual 12

------------------------------------------------- ----------------------- 13

0.0000 0.0000 6.118 7 1000000 0.3567 14

0.3000 0.0000 25.190 20 1000000 -1.034 15

1.0000 0.0001 69.692 74 1000000 0.5161 16 17 Chi-square = 1.46 DF = 1 P-value = 0.2264 18 19
20 Benchmark Dose Computation 21
22 Specified effect = 1e-005 23
24 Risk Type = Extra risk 25 26

Confidence level = 0.95 27
28

BMD = 0.157291 29
30

BMDL = 0.12931 31 32

### D-117 DRAFT – DO NOT CITE OR QUOTE

RAVEH_MUT_CPCDP.OUT.txt 1

================================================== ================== 2 Quantal Linear Model $Revision: 2.2 $ $Date: 2000/03/17 22:27:16 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\RAVEH_MUT_CPCDP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\RAVEH_MUT_CPCDP.plt 7

Wed Jun 29 12:31:46 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP (-slope*dose)] 16 17 18 Dependent variable = COLUMN2 19 Independent variable = COLUMN1 20 21

Total number of observations = 4 22 Total number of records with missing values = 0 23

Maximum number of iterations = 250 24 Relative Function Convergence has been set to: 1 e-008 25 Parameter Convergence has been set to: 1e-008 26 27
28 29 Default Initial (and Specified) P arameter Values 30 Background = 1.5e-006 31 Slope = 9.00013e-006 32 Power = 1 Sp ecified 33 34
35 Asymptotic Correlation Matrix of Paramet er Estimates 36 37 ( *** The model parameter(s) -Power 38 have been estimated at a boundary point, or have been 39 specified by the user, 40 and do not appear in the correlati on matrix ) 41
42 Background Slope 43 44 Background 1 -0.43 45 46 Slope -0.43 1 47 48 49
50

Parameter Estimates 51 52

Variable Estimate Std. Err. 53 Background 1.26496e-006 1.07098e -006 54 Slope 9.05599e-006 1.68076e -006 55 56
57 58 Analysis of Deviance Table 59 60

### D-118 DRAFT – DO NOT CITE OR QUOTE

Model Log(likelihood) Deviance Test D F P-value 1

Full model -527.507 2

Fitted model -527.666 0.317201 2 0.8533 3

Reduced model -546.375 37.7352 3 <.0001 4
5

AIC: 1059.33 6
7 8

Goodness of Fit 9 10

Scaled 11 Dose Est._Prob. Expected Observed Size Residual 12

------------------------------------------------- ----------------------- 13

0.0000 0.0000 1.265 1 1000000 -0.2356 14

0.3000 0.0000 3.982 5 1000000 0.5103 15

1.0000 0.0000 10.321 10 1000000 -0.09989 16

3.0000 0.0000 28.433 28 1000000 -0.08112 17 18 Chi-square = 0.33 DF = 2 P-value = 0.8469 19
20 21 Benchmark Dose Computation 22
23 Specified effect = 1e-005 24 25 Risk Type = Extra risk 26 27

Confidence level = 0.95 28 29

BMD = 1.10425 30 31

BMDL = 0.835597 32 33

### D-119 DRAFT – DO NOT CITE OR QUOTE

SLAGA_MUT_BAA.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\SLAGA_MUT_BAA.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\SLAGA_MUT_BAA.plt 7

Thu Jul 07 15:25:30 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 7.29666e-005 39 Beta(1) = 3.12233e-006 40 **** WARNING: Completion code = 7. Optimum not fo und. Trying new starting 41 pont  42
43 ** ** WARNING 0: Completion code = 7 trying new sta rt  44
45 ** ** WARNING 1: Completion code = -2 trying new st art  46 47 ** ** WARNING 2: Completion code = -2 trying new st art  48 49 ** ** WARNING 3: Completion code = -2 trying new st art  50 51 ** ** WARNING 4: Completion code = 7 trying new sta rt  52
53 ** ** WARNING 5: Completion code = -2 trying new st art  54
55 ** ** WARNING 6: Completion code = -2 trying new st art  56
57 ** ** WARNING 7: Completion code = -2 trying new st art  58 59 ** ** WARNING 8: Completion code = -2 trying new st art  60

### D-120 DRAFT – DO NOT CITE OR QUOTE

1 ** ** WARNING 9: Completion code = 7 trying new sta rt  2
3 ** ** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 4 point  5 6 ** ** WARNING 0: Completion code = -2 trying new st art**** 7 8 9 10 Asymptotic Correlation Matrix of Paramet er Estimates 11
12 Background Beta(1) 13
14 Background 1 -0.63 15 16 Beta(1) -0.63 1 17 18 19
20
Parameter Estimates 21
22

Variable Estimate Std. Err. 23 Background 7.26607e-005 0.002 3585 24 Beta(1) 3.14129e-006 9.25599e -005 25 26 27
28 Analysis of Deviance Table 29
30 Model Log(likelihood) Deviance Test D F P-value 31

Full model -365.644 32

Fitted model -365.656 0.0243422 1 0.876 33

Reduced model -370.021 8.75326 2 0.01257 34
35

AIC: 735.312 36 37
38

Goodness of Fit 39 40 Dose Est._Prob. Expected Observed Size Chi^2 Res. 41

------------------------------------------------- ---------------------- 42

i: 1 43

0.0000 0.0001 7.266 7 100000 -0.037 44

i: 2 45

4.4000 0.0001 8.648 9 100000 0.041 46

i: 3 47

44.0000 0.0002 21.086 21 100000 -0.004 48 49 Chi-square = 0.02 DF = 1 P-value = 0.8758 50 51 52 Benchmark Dose Computation 53 54 Specified effect = 0.0001 55 56 Risk Type = Extra risk 57 58

Confidence level = 0.95 59 60

### D-121 DRAFT – DO NOT CITE OR QUOTE

BMD = 31.8356 1 2

BMDL = 19.0163 3

### D-122 DRAFT – DO NOT CITE OR QUOTE

SLAGA_MUT_BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\SLAGA_MUT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\SLAGA_MUT_BAP.plt 7

Wed Jun 29 13:01:31 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.000214668 39 Beta(1) = 0.00154564 40 Beta(2) = 0.00022152 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) Beta(2) 51 52 Beta(1) 1 -0.98 53 54 Beta(2) -0.98 1 55 56
57 58

Parameter Estimates 59 60

### D-123 DRAFT – DO NOT CITE OR QUOTE

Variable Estimate Std. Err. 1 Background 0 N A 2 Beta(1) 0.00207246 0.010 9511 3 Beta(2) 9.74689e-005 0.0028 6413 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 Warning: Likelihood for the fitted model larger th an the Likelihood for the 10

full model. 11 Error in computing chi-square; returning 2 12 13
14 Analysis of Deviance Table 15 16 Model Log(likelihood) Deviance Test D F P-value 17

Full model -823.498 18

Fitted model -816.691 -13.6145 2 2 19

Reduced model -907.084 167.172 3 <.0001 20 21

AIC: 1637.38 22
23
24

Goodness of Fit 25 26 Dose Est._Prob. Expected Observed Size Chi^2 Res. 27

------------------------------------------------- ---------------------- 28

i: 1 29

0.0000 0.0000 0.000 1 1000070000000.000 30

i: 2 31

0.4000 0.0008 8.442 11 10000 0.303 32

i: 3 33

1.3000 0.0029 28.548 25 10000 -0.125 34

i: 4 35

4.0000 0.0098 98.010 99 10000 0.010 36 37 Chi-square = 1.23 DF = 2 P-value = 0.5412 38 39 40 Benchmark Dose Computation 41
42 Specified effect = 0.0001 43 44 Risk Type = Extra risk 45 46

Confidence level = 0.95 47 48

BMD = 0.0481451 49
50

BMDL = 0.0370516 51 52

### D-124 DRAFT – DO NOT CITE OR QUOTE

D.6. MALIGNANT TRANSFORMATION 1

CASTO_MT_BAP.OUT.txt 2

================================================== ================== 3 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 5 RPS\MODELING\CASTO_MT_BAP.(d) 6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 7 DOCUMENTS\PAH RPS\MODELING\CASTO_MT_BAP.plt 8

Thu Jun 23 13:30:59 2005 9

================================================== ================== 10
11

BMDS MODEL RUN 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 13 14 The form of the probability function is: 15 16 P[response] = background + (1-background)*[1-EXP ( 17 -beta1*dose^1)] 18 19 The parameter betas are restricted to be positiv e 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: 1e- 008 34 Parameter Convergence has been set to: 1e-008 35 36 37 38

Default Initial Parameter Values 39 Background = 1.02144e-005 40 Beta(1) = 7.98743e-005 41
42 43 Asymptotic Correlation Matrix of Paramet er Estimates 44 45 ( *** The model parameter(s) -Backgroun d 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59

### D-125 DRAFT – DO NOT CITE OR QUOTE

Background 0 N A 1 Beta(1) 9.62612e-005 0.0023 4809 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -185.57 13

Fitted model -186.065 0.988828 2 0.6099 14

Reduced model -192.98 14.82 2 0.0006052 15 16

AIC: 374.13 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 100000 0.000 25

i: 2 26

0.6200 0.0001 5.968 8 100000 0.340 27

i: 3 28

1.2500 0.0001 12.032 10 100000 -0.169 29
30 Chi-square = 1.04 DF = 2 P-value = 0.5960 31 32 33 Benchmark Dose Computation 34
35 Specified effect = 1e-005 36 37 Risk Type = Extra risk 38 39

Confidence level = 0.95 40
41

BMD = 0.103885 42
43   WARNING: Completion code = -5. Optimum not f ound. Trying new starting 44 point** ** 45 46   WARNING 0: Completion code = -5 trying new st art** ** 47 48   WARNING 1: Completion code = -5 trying new st art** ** 49
50   WARNING 2: Completion code = -5 trying new st art** ** 51 52   WARNING 3: Completion code = -5 trying new st art** ** 53 54   WARNING 4: Completion code = -5 trying new st art** ** 55 56   WARNING 5: Completion code = -5 trying new st art** ** 57 58   WARNING 6: Completion code = -5 trying new st art** ** 59 60

### D-126 DRAFT – DO NOT CITE OR QUOTE

  WARNING 7: Completion code = -5 trying new st art** ** 1 2   WARNING 8: Completion code = -5 trying new st art** ** 3
4   WARNING 9: Completion code = -5 trying new st art** ** 5 6   WARNING: Completion code = -5. Optimum not f ound. Trying new starting 7 point** ** 8 9

BMDL = 0.0721753 10

### D-127 DRAFT – DO NOT CITE OR QUOTE

CASTO_MT_DBAHA.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\CASTO_MT_DBAHA.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\CASTO_MT_DBAHA.plt 7

Thu Jun 23 13:32:00 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 3 25 Total number of records with missing values = 0 26 Total number of parameters in model = 2 27 Total number of specified parameters = 0 28 Degree of polynomial = 1 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 6.92924e-008 39 Beta(1) = 3.99789e-006 40 **** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 41 pont  42
43 ** ** WARNING 0: Completion code = -2 trying new st art  44
45 ** ** WARNING 1: Completion code = -2 trying new st art  46 47 ** ** WARNING 2: Completion code = -2 trying new st art  48 49 ** ** WARNING 3: Completion code = -2 trying new st art  50 51 ** ** WARNING 4: Completion code = -2 trying new st art  52
53 ** ** WARNING 5: Completion code = -2 trying new st art  54
55 ** ** WARNING 6: Completion code = -2 trying new st art  56
57 ** ** WARNING 7: Completion code = -2 trying new st art  58 59 ** ** WARNING 8: Completion code = -2 trying new st art  60

### D-128 DRAFT – DO NOT CITE OR QUOTE

1 ** ** WARNING 9: Completion code = -2 trying new st art  2
3 ** ** WARNING: Completion code = -2. Optimum not f ound. Trying new starting 4 point  5 6
7 8 Asymptotic Correlation Matrix of Paramet er Estimates 9 10 ( ** * The model parameter(s) -Backgroun d 11 have been estimated at a boundary point, or have been 12 specified by the user, 13 and do not appear in the correlati on matrix ) 14
15 Beta(1) 16 17 Beta(1) 1 18 19
20 21

Parameter Estimates 22
23

Variable Estimate Std. Err. 24 Background 0 N A 25 Beta(1) 4.05407e-006 0.00036 1631 26 27 NA - Indicates that this parameter has hit a bound 28 implied by some inequality constraint and thus 29

has no standard error. 30 31 32 33 Analysis of Deviance Table 34
35 Model Log(likelihood) Deviance Test D F P-value 36

Full model -191.16 37

Fitted model -191.162 0.00552866 2 0.9972 38

Reduced model -198.091 13.863 2 0.0009765 39 40

AIC: 384.325 41
42
43
Goodness of Fit 44
45 Dose Est._Prob. Expected Observed Size Chi^2 Res. 46

------------------------------------------------- ---------------------- 47

i: 1 48

0.0000 0.0000 0.000 0 1 000000 0.000 49

i: 2 50

1.2000 0.0000 4.865 5 1 000000 0.028 51

i: 3 52

2.5000 0.0000 10.135 10 1 000000 -0.013 53 54 Chi-square = 0.01 DF = 2 P-value = 0.9972 55 56
57 Benchmark Dose Computation 58 59 Specified effect = 1e-005 60

### D-129 DRAFT – DO NOT CITE OR QUOTE

1 Risk Type = Extra risk 2
3

Confidence level = 0.95 4
5

BMD = 2.46667 6
7 **** WARNING: Completion code = -5. Optimum not f ound. Trying new starting 8 point  9 10 ** ** WARNING 0: Completion code = -1 trying new st art  11
12 ** ** WARNING 1: Completion code = -1 trying new st art  13
14 ** ** WARNING 2: Completion code = -1 trying new st art  15 16 ** ** WARNING 3: Completion code = -1 trying new st art  17 18 ** ** WARNING 4: Completion code = -1 trying new st art  19
20 ** ** WARNING 5: Completion code = -1 trying new st art  21
22 ** ** WARNING 6: Completion code = -1 trying new st art  23
24 ** ** WARNING 7: Completion code = -1 trying new st art  25 26 ** ** WARNING 8: Completion code = -1 trying new st art  27
28 ** ** WARNING 9: Completion code = -1 trying new st art  29
30 ** ** WARNING: Completion code = -1. Optimum not f ound. Trying new starting 31 point**** 32 33

BMDL = 1.65901 34

### D-130 DRAFT – DO NOT CITE OR QUOTE

EMURA_MT_Baa.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\EMURA_MT_BBF.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\EMURA_MT_BBF.plt 7

Thu Jun 23 15:46:49 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 6.24839e-005 39 Beta(1) = 0.000973789 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 48 -Beta(4) 49 have been estimated at a boundary point, or have been 50 specified by the user, 51 and do not appear in the correlati on matrix ) 52
53 Beta(1) 54
55 Beta(1) 1 56
57 58 59

Parameter Estimates 60

### D-131 DRAFT – DO NOT CITE OR QUOTE

1
Variable Estimate Std. Err. 2 Background 0 N A 3 Beta(1) 0.00117377 0.009 1424 4 Beta(2) 0 N A 5 Beta(3) 0 N A 6 Beta(4) 0 N A 7 8 NA - Indicates that this parameter has hit a bound 9 implied by some inequality constraint and thus 10

has no standard error. 11
12 13
14 Analysis of Deviance Table 15 16 Model Log(likelihood) Deviance Test D F P-value 17

Full model -184.252 18

Fitted model -185.671 2.83903 5 0.7248 19

Reduced model -196.039 23.575 5 0.000262 20 21

AIC: 373.342 22
23
24

Goodness of Fit 25 26 Dose Est._Prob. Expected Observed Size Chi^2 Res. 27

------------------------------------------------- ---------------------- 28

i: 1 29

0.0000 0.0000 0.000 0 10000 0.000 30

i: 2 31

0.0250 0.0000 0.293 0 10000 -1.000 32

i: 3 33

0.1000 0.0001 1.174 3 10000 1.556 34

i: 4 35

0.2500 0.0003 2.934 3 10000 0.023 36

i: 5 37

0.5000 0.0006 5.867 6 10000 0.023 38

i: 6 39

1.0000 0.0012 11.731 10 10000 -0.148 40
41 Chi-square = 3.40 DF = 5 P-value = 0.6392 42
43 44 Benchmark Dose Computation 45 46 Specified effect = 0.001 47 48 Risk Type = Extra risk 49
50

Confidence level = 0.95 51 52

BMD = 0.85238 53

BMDL = 0.611981 54 EMURA_MT_BBF.OUT.txt 55

================================================== ================== 56 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 57 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 58 RPS\MODELING\EMURA_MT_BBF.(d) 59

### D-132 DRAFT – DO NOT CITE OR QUOTE

Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 1 DOCUMENTS\PAH RPS\MODELING\EMURA_MT_BBF.plt 2

Thu Jun 23 15:37:20 2005 3

================================================== ================== 4
5

BMDS MODEL RUN 6

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 7 8 The form of the probability function is: 9 10 P[response] = background + (1-background)*[1-EXP ( 11

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 12 13 The parameter betas are restricted to be positiv e 14
15 16 Dependent variable = COLUMN2 17 Independent variable = COLUMN1 18 19

Total number of observations = 6 20 Total number of records with missing values = 0 21 Total number of parameters in model = 5 22 Total number of specified parameters = 0 23 Degree of polynomial = 4 24 25 26

Maximum number of iterations = 250 27 Relative Function Convergence has been set to: 1e- 008 28 Parameter Convergence has been set to: 1e-008 29
30 31 32

Default Initial Parameter Values 33 Background = 6.48647e-005 34 Beta(1) = 0.00111706 35 Beta(2) = 0 36 Beta(3) = 1.51794e-005 37 Beta(4) = 0 38 39 40 Asymptotic Correlation Matrix of Paramet er Estimates 41
42 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 43 -Beta(4) 44 have been estimated at a boundary point, or have been 45 specified by the user, 46 and do not appear in the correlati on matrix ) 47 48 Beta(1) 49
50 Beta(1) 1 51 52
53 54

Parameter Estimates 55 56

Variable Estimate Std. Err. 57 Background 0 N A 58 Beta(1) 0.00133391 0.0090 9075 59 Beta(2) 0 N A 60

### D-133 DRAFT – DO NOT CITE OR QUOTE

Beta(3) 0 N A 1 Beta(4) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -205.838 13

Fitted model -208.019 4.36272 5 0.4985 14

Reduced model -219.575 27.4752 5 <.0001 15 16

AIC: 418.038 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 10000 0.000 25

i: 2 26

0.0250 0.0000 0.333 0 10000 -1.000 27

i: 3 28

0.1000 0.0001 1.334 4 10000 1.999 29

i: 4 30

0.2500 0.0003 3.334 3 10000 -0.100 31

i: 5 32

0.5000 0.0007 6.667 6 10000 -0.100 33

i: 6 34

1.0000 0.0013 13.330 12 10000 -0.100 35 36 Chi-square = 5.90 DF = 5 P-value = 0.3164 37
38 39 Benchmark Dose Computation 40
41 Specified effect = 0.001 42
43 Risk Type = Extra risk 44
45

Confidence level = 0.95 46 47

BMD = 0.750052 48

BMDL = 0.54909 49

### D-134 DRAFT – DO NOT CITE OR QUOTE

EMURA_MT_I_BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\EMURA_MT_I_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\EMURA_MT_I_BAP.plt 7

Thu Jun 23 15:28:17 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2-beta3*dose^3)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 5 25 Total number of records with missing values = 0 26 Total number of parameters in model = 4 27 Total number of specified parameters = 0 28 Degree of polynomial = 3 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 6.51885e-005 39 Beta(1) = 0.021934 40 Beta(2) = 0 41 Beta(3) = 0 42
43 44 Asymptotic Correlation Matrix of Paramet er Estimates 45 46 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 47 have been estimated at a boundary point, or have been 48 specified by the user, 49 and do not appear in the correlati on matrix ) 50 51 Beta(1) 52
53 Beta(1) 1 54
55 56
57

Parameter Estimates 58 59

Variable Estimate Std. Err. 60

### D-135 DRAFT – DO NOT CITE OR QUOTE

Background 0 N A 1 Beta(1) 0.0227293 0.036 9378 2 Beta(2) 0 N A 3 Beta(3) 0 N A 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -614.919 15

Fitted model -618.123 6.40862 4 0.1706 16

Reduced model -677.621 125.404 4 <.0001 17 18

AIC: 1238.25 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 10000 0.000 27

i: 2 28

0.0100 0.0002 2.273 0 10000 -1.000 29

i: 3 30

0.0500 0.0011 11.358 11 10000 -0.032 31

i: 4 32

0.1000 0.0023 22.703 29 10000 0.278 33

i: 5 34

0.2500 0.0057 56.662 53 10000 -0.065 35 36 Chi-square = 4.27 DF = 4 P-value = 0.3703 37
38 39 Benchmark Dose Computation 40
41 Specified effect = 0.001 42
43 Risk Type = Extra risk 44
45

Confidence level = 0.95 46 47

BMD = 0.0440182 48 49

BMDL = 0.037291 50

### D-136 DRAFT – DO NOT CITE OR QUOTE

EMURA_MT_II_BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\EMURA_MT_II_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\EMURA_MT_II_BAP.plt 7

Thu Jun 23 15:54:16 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2-beta3*dose^3)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 5 25 Total number of records with missing values = 0 26 Total number of parameters in model = 4 27 Total number of specified parameters = 0 28 Degree of polynomial = 3 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0002687 39 Beta(1) = 0.0184676 40 Beta(2) = 0 41 Beta(3) = 0 42
43 44 Asymptotic Correlation Matrix of Paramet er Estimates 45 46 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 47 have been estimated at a boundary point, or have been 48 specified by the user, 49 and do not appear in the correlati on matrix ) 50 51 Beta(1) 52
53 Beta(1) 1 54
55 56
57

Parameter Estimates 58 59

Variable Estimate Std. Err. 60

### D-137 DRAFT – DO NOT CITE OR QUOTE

Background 0 N A 1 Beta(1) 0.021747 0.038 1969 2 Beta(2) 0 N A 3 Beta(3) 0 N A 4
5 NA - Indicates that this parameter has hit a bound 6 implied by some inequality constraint and thus 7

has no standard error. 8 9 10
11 Analysis of Deviance Table 12 13 Model Log(likelihood) Deviance Test D F P-value 14

Full model -606.226 15

Fitted model -608.64 4.82649 4 0.3056 16

Reduced model -652.392 92.3321 4 <.0001 17 18

AIC: 1219.28 19
20 21

Goodness of Fit 22
23 Dose Est._Prob. Expected Observed Size Chi^2 Res. 24

------------------------------------------------- ---------------------- 25

i: 1 26

0.0000 0.0000 0.000 0 10000 0.000 27

i: 2 28

0.0100 0.0002 2.174 4 10000 0.840 29

i: 3 30

0.0500 0.0011 10.868 10 10000 -0.080 31

i: 4 32

0.1000 0.0022 21.723 29 10000 0.336 33

i: 5 34

0.2500 0.0054 54.220 46 10000 -0.152 35 36 Chi-square = 5.30 DF = 4 P-value = 0.2581 37
38 39 Benchmark Dose Computation 40
41 Specified effect = 0.001 42
43 Risk Type = Extra risk 44
45

Confidence level = 0.95 46 47

BMD = 0.0460064 48 49

BMDL = 0.0388361 50

### D-138 DRAFT – DO NOT CITE OR QUOTE

EMURA_MT_IP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\EMURA_MT_IP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\EMURA_MT_IP.plt 7

Thu Jun 23 15:50:44 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 7.12074e-005 39 Beta(1) = 0.00099924 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 48 -Beta(4) 49 have been estimated at a boundary point, or have been 50 specified by the user, 51 and do not appear in the correlati on matrix ) 52
53 Beta(1) 54
55 Beta(1) 1 56
57 58 59

Parameter Estimates 60

### D-139 DRAFT – DO NOT CITE OR QUOTE

1
Variable Estimate Std. Err. 2 Background 0 N A 3 Beta(1) 0.00122714 0.0091 8598 4 Beta(2) 0 N A 5 Beta(3) 0 N A 6 Beta(4) 0 N A 7 8 NA - Indicates that this parameter has hit a bound 9 implied by some inequality constraint and thus 10

has no standard error. 11
12 13
14 Analysis of Deviance Table 15 16 Model Log(likelihood) Deviance Test D F P-value 17

Full model -191.591 18

Fitted model -193.089 2.99724 5 0.7004 19

Reduced model -203.928 24.6739 5 0.0001611 20 21

AIC: 388.178 22
23
24

Goodness of Fit 25 26 Dose Est._Prob. Expected Observed Size Chi^2 Res. 27

------------------------------------------------- ---------------------- 28

i: 1 29

0.0000 0.0000 0.000 0 10000 0.000 30

i: 2 31

0.0250 0.0000 0.307 0 10000 -1.000 32

i: 3 33

0.1000 0.0001 1.227 3 10000 1.445 34

i: 4 35

0.2500 0.0003 3.067 3 10000 -0.022 36

i: 5 37

0.5000 0.0006 6.134 7 10000 0.141 38

i: 6 39

1.0000 0.0012 12.264 10 10000 -0.185 40
41 Chi-square = 3.41 DF = 5 P-value = 0.6369 42
43 44 Benchmark Dose Computation 45 46 Specified effect = 0.001 47 48 Risk Type = Extra risk 49
50

Confidence level = 0.95 51 52

BMD = 0.815309 53

BMDL = 0.589412 54

### D-140 DRAFT – DO NOT CITE OR QUOTE

LUBET_MT_BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\LUBET_MT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\LUBET_MT_BAP.plt 7

Thu Jun 23 16:11:06 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0617408 39 Beta(1) = 0.0378355 40 Beta(2) = 0 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(2) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(1) 51 52 Beta(1) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-141 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0.056828 0.034 0172 1 Beta(2) 0 N A 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -21.9204 13

Fitted model -22.8416 1.84243 3 0.6057 14

Reduced model -27.0337 10.2266 3 0.01674 15 16

AIC: 47.6832 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 15 0.000 25

i: 2 26

1.0000 0.0552 0.829 1 15 0.219 27

i: 3 28

3.0000 0.1567 2.351 4 15 0.832 29

i: 4 30

10.0000 0.4335 6.503 5 15 -0.408 31 32 Chi-square = 2.02 DF = 3 P-value = 0.5679 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.1 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 1.85403 44
45

BMDL = 1.14367 46

### D-142 DRAFT – DO NOT CITE OR QUOTE

LUBET_MT_BeP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\LUBET_MT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\LUBET_MT_BAP.plt 7

Thu Jun 23 16:14:09 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16 -beta1*dose^1-beta2*dose^2)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 4 25 Total number of records with missing values = 0 26 Total number of parameters in model = 3 27 Total number of specified parameters = 0 28 Degree of polynomial = 2 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0.000632445 40 Beta(2) = 5.70088e-005 41
42
43 Asymptotic Correlation Matrix of Paramet er Estimates 44
45 ( *** The model parameter(s) -Backgroun d -Beta(1) 46 have been estimated at a boundary point, or have been 47 specified by the user, 48 and do not appear in the correlati on matrix ) 49
50 Beta(2) 51 52 Beta(2) 1 53 54
55 56

Parameter Estimates 57 58

Variable Estimate Std. Err. 59 Background 0 N A 60

### D-143 DRAFT – DO NOT CITE OR QUOTE

Beta(1) 0 N A 1 Beta(2) 6.35618e-005 3.53139e -005 2
3 NA - Indicates that this parameter has hit a bound 4 implied by some inequality constraint and thus 5

has no standard error. 6
7 8 9 Analysis of Deviance Table 10
11 Model Log(likelihood) Deviance Test D F P-value 12

Full model -14.0378 13

Fitted model -14.1501 0.224517 3 0.9735 14

Reduced model -23.5605 19.0453 3 0.0002676 15 16

AIC: 30.3001 17 18 19

Goodness of Fit 20 21 Dose Est._Prob. Expected Observed Size Chi^2 Res. 22

------------------------------------------------- ---------------------- 23

i: 1 24

0.0000 0.0000 0.000 0 15 0.000 25

i: 2 26

10.0000 0.0063 0.095 0 15 -1.006 27

i: 3 28

30.0000 0.0556 0.834 1 15 0.211 29

i: 4 30

100.0000 0.4704 7.056 7 15 -0.015 31 32 Chi-square = 0.13 DF = 3 P-value = 0.9878 33 34
35 Benchmark Dose Computation 36 37 Specified effect = 0.1 38 39 Risk Type = Extra risk 40
41

Confidence level = 0.95 42
43

BMD = 40.7137 44
45

BMDL = 18.2541 46

### D-144 DRAFT – DO NOT CITE OR QUOTE

MOHAPATRA_MT_BJAC.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\_PAH 4 RPS\MODELING\MOHAPATRA_MT_BJAC.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\_PAH RPS\MODELING\MOHAPATRA_MT_BJAC.plt 7

Thu Feb 08 10:11:06 2007 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0 39 Beta(1) = 0 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 6.31048e+018 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 48 have been estimated at a boundary point, or have been 49 specified by the user, 50 and do not appear in the correlati on matrix ) 51 52 Beta(1) Beta(4) 53 54 Beta(1) 1 -0.73 55 56 Beta(4) -0.73 1 57 58 59 60

### D-145 DRAFT – DO NOT CITE OR QUOTE

Parameter Estimates 1 2

Variable Estimate Std. Err. 3 Background 0 N A 4 Beta(1) 2.44509 0.56 8863 5 Beta(2) 0 N A 6 Beta(3) 0 N A 7 Beta(4) 0.332129 0.77 8407 8 9 NA - Indicates that this parameter has hit a bound 10 implied by some inequality constraint and thus 11

has no standard error. 12 13
14
15 Analysis of Deviance Table 16 17 Model Log(likelihood) Deviance Test D F P-value 18

Full model -64.5493 19

Fitted model -64.8387 0.578751 4 0.9654 20

Reduced model -198.931 268.764 5 <.0001 21
22

AIC: 133.677 23
24 25

Goodness of Fit 26 27 Dose Est._Prob. Expected Observed Size Chi^2 Res. 28

------------------------------------------------- ---------------------- 29

i: 1 30

0.0000 0.0000 0.000 0 48 0.000 31

i: 2 32

0.0100 0.0242 1.159 2 48 0.743 33

i: 3 34

0.0500 0.1151 5.524 5 48 -0.107 35

i: 4 36

0.5000 0.7116 34.155 34 48 -0.016 37

i: 5 38

1.0000 0.9378 45.014 45 48 -0.005 39

i: 6 40

2.0000 1.0000 47.998 48 48 1.000 41
42 Chi-square = 0.68 DF = 4 P-value = 0.9532 43 44
45 Benchmark Dose Computation 46 47 Specified effect = 0.92 48 49 Risk Type = Extra risk 50 51
Confidence level = 0.95 52
53

BMD = 0.930952 54
55

BMDL = 0.766826 56

### D-146 DRAFT – DO NOT CITE OR QUOTE

MOHAPATRA_MT_BLAC.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\_PAH 4 RPS\MODELING\MOHAPATRA_MT_BLAC.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\_PAH RPS\MODELING\MOHAPATRA_MT_BLAC.plt 7

Thu Feb 08 10:13:14 2007 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0997842 39 Beta(1) = 0.189801 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Backgroun d -Beta(2) -Beta(3) 48 -Beta(4) 49 have been estimated at a boundary point, or have been 50 specified by the user, 51 and do not appear in the correlati on matrix ) 52
53 Beta(1) 54
55 Beta(1) 1 56
57 58 59

Parameter Estimates 60

### D-147 DRAFT – DO NOT CITE OR QUOTE

1
Variable Estimate Std. Err. 2 Background 0 N A 3 Beta(1) 0.237265 0.027 8061 4 Beta(2) 0 N A 5 Beta(3) 0 N A 6 Beta(4) 0 N A 7 8 NA - Indicates that this parameter has hit a bound 9 implied by some inequality constraint and thus 10

has no standard error. 11
12 13
14 Analysis of Deviance Table 15 16 Model Log(likelihood) Deviance Test D F P-value 17

Full model -159.727 18

Fitted model -161.509 3.56545 5 0.6135 19

Reduced model -243.072 166.691 5 <.0001 20 21

AIC: 325.019 22
23
24

Goodness of Fit 25 26 Dose Est._Prob. Expected Observed Size Chi^2 Res. 27

------------------------------------------------- ---------------------- 28

i: 1 29

0.0000 0.0000 0.000 0 60 0.000 30

i: 2 31

0.5000 0.1119 6.712 8 60 0.216 32

i: 3 33

1.0000 0.2112 12.673 14 60 0.133 34

i: 4 35

2.5000 0.4474 26.845 31 60 0.280 36

i: 5 37

5.0000 0.6947 41.679 42 60 0.025 38

i: 6 39

10.0000 0.9068 54.406 51 60 -0.671 40
41 Chi-square = 3.91 DF = 5 P-value = 0.5620 42
43 44 Benchmark Dose Computation 45 46 Specified effect = 0.83 47 48 Risk Type = Extra risk 49
50

Confidence level = 0.95 51 52

BMD = 7.46828 53 54

BMDL = 6.45083 55

### D-148 DRAFT – DO NOT CITE OR QUOTE

MOHAPATRA_MT_BEAC.txt 1

=================================================== ================= 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\_PAH 4 RPS\MODELING\MOHAPATRA_MT_BEAC.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\_PAH RPS\MODELING\MOHAPATRA_MT_BEAC.plt 7

Fri Feb 09 10:49:12 2007 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.0946116 39 Beta(1) = 0.082434 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Beta(2) -Beta(3) -Beta(4) 48 have been estimated at a boundary point, or have been 49 specified by the user, 50 and do not appear in the correlati on matrix ) 51 52 Background Beta(1) 53 54 Background 1 -0.68 55 56 Beta(1) -0.68 1 57 58 59 60

### D-149 DRAFT – DO NOT CITE OR QUOTE

Parameter Estimates 1 2

Variable Estimate Std. Err. 3 Background 0.0246825 0.10 6613 4 Beta(1) 0.109348 0.032 1778 5 Beta(2) 0 N A 6 Beta(3) 0 N A 7 Beta(4) 0 N A 8 9 NA - Indicates that this parameter has hit a bound 10 implied by some inequality constraint and thus 11

has no standard error. 12 13
14
15 Analysis of Deviance Table 16 17 Model Log(likelihood) Deviance Test D F P-value 18

Full model -101.226 19

Fitted model -104.24 6.02698 4 0.1971 20

Reduced model -126.655 50.8576 5 <.0001 21
22

AIC: 212.479 23
24 25

Goodness of Fit 26 27 Dose Est._Prob. Expected Observed Size Chi^2 Res. 28

------------------------------------------------- ---------------------- 29

i: 1 30

0.0000 0.0247 0.889 0 36 -1.025 31

i: 2 32

0.5000 0.0766 2.757 4 36 0.488 33

i: 3 34

1.0000 0.1257 4.525 6 36 0.373 35

i: 4 36

2.5000 0.2580 9.287 13 36 0.539 37

i: 5 38

5.0000 0.4355 15.676 15 36 -0.076 39

i: 6 40

10.0000 0.6732 24.236 21 36 -0.409 41
42 Chi-square = 5.44 DF = 4 P-value = 0.2448 43 44
45 Benchmark Dose Computation 46 47 Specified effect = 0.86 48 49 Risk Type = Extra risk 50 51
Confidence level = 0.95 52
53

BMD = 17.9803 54
55

BMDL = 12.7064 56

### D-150 DRAFT – DO NOT CITE OR QUOTE

PIENTA_MT_BAA.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\PIENTA_MT_BAA.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\PIENTA_MT_BAA.plt 7

Tue Jul 05 13:52:46 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 6 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.00472474 39 Beta(1) = 0 40 Beta(2) = 0 41 Beta(3) = 2.31177e-005 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Beta(1) -Beta(2) -Beta(3) 48 have been estimated at a boundary point, or have been 49 specified by the user, 50 and do not appear in the correlati on matrix ) 51 52 Background Beta(4) 53 54 Background 1 -0.43 55 56 Beta(4) -0.43 1 57 58 59 60

### D-151 DRAFT – DO NOT CITE OR QUOTE

Parameter Estimates 1 2

Variable Estimate Std. Err. 3 Background 0.00480466 0.029 0234 4 Beta(1) 0 N A 5 Beta(2) 0 N A 6 Beta(3) 0 N A 7 Beta(4) 2.25394e-006 6.9765e -006 8 9 NA - Indicates that this parameter has hit a bound 10 implied by some inequality constraint and thus 11

has no standard error. 12 13
14
15 Analysis of Deviance Table 16 17 Model Log(likelihood) Deviance Test D F P-value 18

Full model -67.8785 19

Fitted model -69.9491 4.14115 4 0.3872 20

Reduced model -74.327 12.8971 5 0.02436 21
22

AIC: 143.898 23
24 25

Goodness of Fit 26 27 Dose Est._Prob. Expected Observed Size Chi^2 Res. 28

------------------------------------------------- ---------------------- 29

i: 1 30

0.0000 0.0048 1.100 0 229 -1.005 31

i: 2 32

0.1000 0.0048 1.081 1 225 -0.075 33

i: 3 34

0.5000 0.0048 1.211 2 252 0.655 35

i: 4 36

1.0000 0.0048 0.928 2 193 1.161 37

i: 5 38

5.0000 0.0062 1.936 1 312 -0.487 39

i: 6 40

10.0000 0.0270 6.746 7 250 0.039 41
42 Chi-square = 3.34 DF = 4 P-value = 0.5028 43 44
45 Benchmark Dose Computation 46 47 Specified effect = 0.01 48 49 Risk Type = Extra risk 50 51
Confidence level = 0.95 52
53

BMD = 8.17165 54
55   WARNING: Completion code = -2. Optimum not f ound. Trying new starting 56 point** ** 57 58

BMDL = 4.47767 59

### D-152 DRAFT – DO NOT CITE OR QUOTE

PIENTA_MT_BAP.OUT.txt 1

================================================== ================== 2 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 3 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 4 RPS\MODELING\PIENTA_MT_BAP.(d) 5 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 6 DOCUMENTS\PAH RPS\MODELING\PIENTA_MT_BAP.plt 7

Mon Jun 27 16:28:28 2005 8

================================================== ================== 9 10

BMDS MODEL RUN 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 12 13 The form of the probability function is: 14
15 P[response] = background + (1-background)*[1-EXP ( 16

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^ 4)] 17 18 The parameter betas are restricted to be positiv e 19
20 21 Dependent variable = COLUMN2 22 Independent variable = COLUMN1 23
24

Total number of observations = 5 25 Total number of records with missing values = 0 26 Total number of parameters in model = 5 27 Total number of specified parameters = 0 28 Degree of polynomial = 4 29
30 31

Maximum number of iterations = 250 32 Relative Function Convergence has been set to: 1e- 008 33 Parameter Convergence has been set to: 1e-008 34
35 36 37

Default Initial Parameter Values 38 Background = 0.00129459 39 Beta(1) = 0.00056154 40 Beta(2) = 0 41 Beta(3) = 0 42 Beta(4) = 0 43 44
45 Asymptotic Correlation Matrix of Paramet er Estimates 46 47 ( *** The model parameter(s) -Beta(2) -Beta(3) -Beta(4) 48 have been estimated at a boundary point, or have been 49 specified by the user, 50 and do not appear in the correlati on matrix ) 51 52 Background Beta(1) 53 54 Background 1 -0.72 55 56 Beta(1) -0.72 1 57 58 59 60

### D-153 DRAFT – DO NOT CITE OR QUOTE

Parameter Estimates 1 2

Variable Estimate Std. Err. 3 Background 0.000529694 0.031 0484 4 Beta(1) 0.000662444 0.0032 1227 5 Beta(2) 0 N A 6 Beta(3) 0 N A 7 Beta(4) 0 N A 8 9 NA - Indicates that this parameter has hit a bound 10 implied by some inequality constraint and thus 11

has no standard error. 12 13
14
15 Analysis of Deviance Table 16 17 Model Log(likelihood) Deviance Test D F P-value 18

Full model -64.5099 19

Fitted model -65.0987 1.17762 3 0.7584 20

Reduced model -68.985 8.95024 4 0.06236 21
22

AIC: 134.197 23
24 25

Goodness of Fit 26 27 Dose Est._Prob. Expected Observed Size Chi^2 Res. 28

------------------------------------------------- ---------------------- 29

i: 1 30

0.0000 0.0005 0.267 0 504 -1.001 31

i: 2 32

1.0000 0.0012 0.468 1 393 1.137 33

i: 3 34

5.0000 0.0038 1.557 2 406 0.286 35

i: 4 36

10.0000 0.0071 3.094 3 434 -0.031 37

i: 5 38

20.0000 0.0137 5.611 5 410 -0.110 39 40 Chi-square = 1.07 DF = 3 P-value = 0.7847 41
42
43 Benchmark Dose Computation 44
45 Specified effect = 0.01 46 47 Risk Type = Extra risk 48 49

Confidence level = 0.95 50 51

BMD = 15.1716 52
53

BMDL = 8.76437 54 PIENTA_MT_DBAHA.OUT.txt 55

================================================== ================== 56 Multistage Model. $Revision: 2.1 $ $Date: 200 0/08/21 03:38:21 $ 57 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 58 RPS\MODELING\PIENTA_MT_DBAHA.(d) 59

### D-154 DRAFT – DO NOT CITE OR QUOTE

Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 1 DOCUMENTS\PAH RPS\MODELING\PIENTA_MT_DBAHA.plt 2

Mon Jun 27 16:35:08 2005 3

================================================== ================== 4
5

BMDS MODEL RUN 6

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 7 8 The form of the probability function is: 9 10 P[response] = background + (1-background)*[1-EXP ( 11 -beta1*dose^1-beta2*dose^2)] 12 13 The parameter betas are restricted to be positiv e 14
15 16 Dependent variable = COLUMN2 17 Independent variable = COLUMN1 18 19

Total number of observations = 4 20 Total number of records with missing values = 0 21 Total number of parameters in model = 3 22 Total number of specified parameters = 0 23 Degree of polynomial = 2 24 25 26

Maximum number of iterations = 250 27 Relative Function Convergence has been set to: 1e- 008 28 Parameter Convergence has been set to: 1e-008 29
30 31 32

Default Initial Parameter Values 33 Background = 0.000660992 34 Beta(1) = 0.020798 35 Beta(2) = 0 36 37
38 Asymptotic Correlation Matrix of Paramet er Estimates 39 40 ( *** The model parameter(s) -Backgroun d -Beta(2) 41 have been estimated at a boundary point, or have been 42 specified by the user, 43 and do not appear in the correlati on matrix ) 44
45 Beta(1) 46 47 Beta(1) 1 48 49
50 51
Parameter Estimates 52
53

Variable Estimate Std. Err. 54 Background 0 N A 55 Beta(1) 0.0227021 0.061 8036 56 Beta(2) 0 N A 57 58 NA - Indicates that this parameter has hit a bound 59 implied by some inequality constraint and thus 60

### D-155 DRAFT – DO NOT CITE OR QUOTE

has no standard error. 1 2
3
4 Analysis of Deviance Table 5 6 Model Log(likelihood) Deviance Test D F P-value 7

Full model -40.1618 8

Fitted model -41.0551 1.78665 3 0.6178 9

Reduced model -45.7301 11.1367 3 0.01101 10
11

AIC: 84.1102 12 13
14

Goodness of Fit 15 16 Dose Est._Prob. Expected Observed Size Chi^2 Res. 17

------------------------------------------------- ---------------------- 18

i: 1 19

0.0000 0.0000 0.000 0 229 0.000 20

i: 2 21

0.1000 0.0023 0.497 0 219 -1.002 22

i: 3 23

0.5000 0.0113 2.630 4 233 0.527 24

i: 4 25

1.0000 0.0224 4.871 4 217 -0.183 26 27 Chi-square = 1.38 DF = 3 P-value = 0.7105 28 29
30 Benchmark Dose Computation 31 32 Specified effect = 0.01 33 34 Risk Type = Extra risk 35 36

Confidence level = 0.95 37
38

BMD = 0.442705 39 40

BMDL = 0.260515 41
42
43

### D-156 DRAFT – DO NOT CITE OR QUOTE

D.7. IN VITRO DNA DAMAGE 1

JOHNSEN_DNA_DAM_BJAC.OUT.txt 2

================================================== ================== 3 Polynomial Model. Revision: 2.2 Date: 9/12/2 002 4 Input Data File: C:\DOCUMENTS AND SETTINGS\HCL YNCH\MY DOCUMENTS\PAH 5 RPS\MODELING\JOHNSEN_DNA_DAM_BAP.(d) 6 Gnuplot Plotting File: C:\DOCUMENTS AND SETTI NGS\HCLYNCH\MY 7 DOCUMENTS\PAH RPS\MODELING\JOHNSEN_DNA_DAM_BAP.plt 8

Mon Jul 04 21:51:27 2005 9

================================================== ================== 10
11

BMDS MODEL RUN 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 13 14 The form of the response function is: 15 16 Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 +

... 17 18 19 Dependent variable = MEAN 20 Independent variable = COLUMN1 21

rho is set to 0 22 Signs of the polynomial coefficients are not res tricted 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: 1 e-008 29 Parameter Convergence has been set to: 1e-008 30 31
32 33

Default Initial Parameter Values 34 alpha = 5.88667 35 rho = 0 Sp ecified 36 beta_0 = 4.94396 37 beta_1 = 0.150549 38 39 40
41

Parameter Estimate s 42 43

95.0% Wald 44

Confidence Interval 45

Variable Estimate Std. Err. Lower Conf. Limit 46 Upper Conf. Limit 47 alpha 4.14606 1.95447 0.315366 48

7.97675 49 beta_0 4.94396 0.875754 3.22751 50

6.6604 51 beta_1 0.150549 0.0503107 0.0519422 52

0.249157 53 54
55 Asymptotic Correlation Matrix of Paramet er Estimates 56
57 alpha beta_0 beta_1 58 alpha 1 7.6e-015 1.7e-015 59

### D-157 DRAFT – DO NOT CITE OR QUOTE

beta_0 7.6e-015 1 -0.63 1 beta_1 1.7e-015 -0.63 1 2
3
4

Table of Data and Estimated Values of Interest 5 6

Dose N Obs Mean Obs Std Dev Est Mean Est Std Dev Chi^2 7

Res. 8

------ --- -------- ----------- -------- ----------- --------- 9

- 10
11

0 3 4.4 1.3 4.94 2.04 -0.463 12

3 3 6 2.1 5.4 2.04 0.514 13

30 3 9.4 3.4 9.46 2.04 -0.0514 14
15 16 17 Model Descriptions for likelihoods calculated 18 19
20 Model A1: Yij = Mu(i) + e(ij) 21 Var{e(ij)} = Sigma^2 22
23 Model A2: Yij = Mu(i) + e(ij) 24 Var{e(ij)} = Sigma(i)^2 25 26 Model R: Yi = Mu + e(i) 27 Var{e(i)} = Sigma^2 28 29
30

Likelihoods of Interest 31 32 Model Log(likelihood) DF AIC 33

A1 -10.652512 4 29 .305023 34

A2 -9.359638 6 30 .719276 35
fitted -10.899709 2 25 .799418 36

R -14.037484 2 32 .074967 37
38 Test 1: Does response and/or variances differ amo ng dose 39

levels 40 (A2 vs. R) 41 Test 2: Are Variances Homogeneous (A1 vs A2) 42 Test 3: Does the Model for the Mean Fit (A1 vs. f itted) 43 44

Tests of Interest 45 46 Test -2*log(Likelihood Ratio) Test df p- value 47 48

Test 1 9.35569 4 0. 009299 49

Test 2 2.58575 2 0.2745 50

Test 3 0.494395 1 0.482 51 52 The p-value for Test 1 is less than .05. There app ears 53

to be a 54 difference between response and/or variances among the 55

dose levels. 56 It seems appropriate to model the data 57 58 The p-value for Test 2 is greater than .05. A 59 homogeneous variance 60

### D-158 DRAFT – DO NOT CITE OR QUOTE

model appears to be appropriate here 1 2
3 The p-value for Test 3 is greater than .05. The mo del 4 chosen appears 5 to adequately describe the data 6
7 8 9 Benchmark Dose Computation 10 Specified effect = 7.6 11
12 Risk Type = Point risk 13
14

Confidence level = 0.95 15 16

BMD = 17.6423 17 18 19

BMDL = 9.58925 20 21

### E-1 DRAFT – DO NOT CITE OR QUOTE APPENDIX E. CALCULATION OF RPFs 1

2

3E-2 DRAFT – DO NOT CITE OR QUOTETable E-1. Dermal bioassays: RPF calculations forincidence dataRelative potency calculationRecordno. ReferenceTumortype(s) Sex PAHBMR BMDPointestimateextra riskresponsePointestimatedose Dose unitsConverteddoseConverteddoseunits RPF CommentsComplete carcinogenicity studies600 Habs et al.,1980Sum ofPapilloma,carcinoma,sarcomaF BaP 0.1 0.91 pg/animal 1 High dose droppedF BbF 0.1 3.8 pg/animal 0.2413640Cavalieri etal., 1983Papilloma,adenoma,carcinomaF BaP 0.1 5.3 nmol 0.001 mg 1F CPcdP 0.1 47 nmol 0.011 mg 0.13620 HoffmannandWynder,1966Papillomas F BaP 0.1 0.0031 % 1F DBaeP 0.1 0.0094 % 0.33 Toxicity resulted insignificant mortalityunrelated to tumorinduction.F DBaiP 0.1 0.0042 % 0.74F DBaeF 0.1 0.0028 % 1.117660Cavalieri etal., 1977Papilloma,keratoacanthoma,carcinomaF BaP 0.79 0.396 1F AA 0.47 0.396 μmol/application0.109 mg/application0.55Initiation studies630 LaVoie etal., 1982PrimarilysquamouscellpapillomaF BaP 0.85 30 μg/animal 1F BbF 0.8 100 μg/animal 0.28 No modelfit. Pointestimate using incidence/dose point closest to BaPincidence.F BjF 0.85 209 μg/animal 0.14 High dose droppedF BkF 0.85 1,163 μg/animal 0.0318570 Hecht etal., 1974UnspecifiedF BaP 0.3 0.05 mg/animal 1E-3 DRAFT – DO NOT CITE OR QUOTETable E-1. Dermal bioassays: RPF calculations forincidence dataRelative potency calculationRecordno. ReferenceTumortype(s) Sex PAHBMR BMDPointestimateextra riskresponsePointestimatedose Dose unitsConverteddoseConverteddoseunits RPF CommentsF CH 0.58 1 mg/animal 0.1021420 Slaga etal., 1980Papilloma F BaP 0.64 200 nmol 0.050 mg 1F CH 0.71 2,000 nmol 0.457 mg 0.12 Not clear if BaPadministeredsimultaneously. Controlgroups pooled for analysis.F DBahA 0.45 100 nmol 0.028 mg 1.2715640 Raveh etal., 1982Papilloma F BaP 0.1 2.2 μg 1F CPcdP 0.1 30 μg 0.07620 HoffmannandWynder,1966Papillomas F BaP 0.79 0.25 mg/animal 1F DBaeF 0.57 0.25 mg/animal 0.73F DBaeP 0.33 0.25 mg/animal 0.41F DBahP 0.7 0.25 mg/animal 0.90F DBaiP 0.36 0.25 mg/animal 0.45F N23eP 0.25 0.25 mg/animal 0.3213650Cavalieri etal., 1981bPapillomas F BaP 0.33 0.2 μmol 0.050 mg 1F CPcdP 0.23 0.6 μmol 0.136 mg 0.26 Mid dose borderlinesignificant, high dose not,trend not; no model fit;RPF uses mid dose forpoint estimate.15700 Rice et al.,1988UnspecifiedF BaP 0.89 0.1 μmol 0.025 mg 1F CH 0.89 0.5 μmol 0.114 mg 0.22 No modelfit. Pointestimate using pointclosest to BaP incidence.F CPdefC 0.88 0.22 μmol 0.053 mg 0.48F BbcAC 0.89 2 μmol 0.481 mg 0.05 No model fit. Pointestimate using pointclosest to BaP incidence.24800 Nesnow etal., 1984Papilloma M BaP 0.67 200 nmol 0.050 mg 1E-4 DRAFT – DO NOT CITE OR QUOTETable E-1. Dermal bioassays: RPF calculations forincidence dataRelative potency calculationRecordno. ReferenceTumortype(s) Sex PAHBMR BMDPointestimateextra riskresponsePointestimatedose Dose unitsConverteddoseConverteddoseunits RPF CommentsM BeAC 0.67 393 nmol 0.099 mg 0.51 Threehigh doses droppeddue to plateauM BlAC 0.67 50 nmol 0.013 mg 4.00 Three high doses droppeddue to plateauF BaP 0.51 200 nmol 0.050 mg 1F BeAC 0.51 195 nmol 0.049 mg 1.03Two high doses dropped toachieve model fitF BlAC 0.51 30 nmol 0.008 mg 6.671E-5 DRAFT – DO NOT CITE OR QUOTE1Table E-2. Dermal bioassays: RPF calculations formultiplicity dataRelative potency calculationRecord no.Reference Tumor type(s)SexPAHPointestimateextra riskresponsePointestimatedoseDoseunitsConverteddoseConverteddose units RPF CommentsComplete carcinogenicity studies13640Cavalieri et al.,1983Papilloma,adenoma,carcinomaF BaP 1.5 20 nmol 0.0050 mg 1 Variance not reportedF CPcdP 2.5 200 nmol 0.045 mg 0.18 Variance notreported13650Cavalieri et al.,1981bPrimarilysquamous cellcarcinomaUS BaP 1.5 0.2 μmol 0.050 mg 1US CPcdP 0.80 0.2 μmol 0.045 mg 0.59 Variance not reportedInitiation studies630 LaVoie et al.,1982Primarilysquamous cellpapillomaF BaP 4.9 30 μg 1F BbF 7.1 100 μg 0.43 Variance not reportedF BjF 7.2 1,000 μg 0.044 Variance not reportedF BkF 2.8 1,000 μg 0.017 Variance not reported18570 Hecht et al.,1974Unspecified F BaP 0.5 0.05 mg 1F CH 1.0 1 mg 0.1021420 Slaga et al.,1980Papilloma F BaP 2.1 200 nmol 0.050 mg 1F CH 1.5 2,000 nmol 0.46 mg 0.078F DBahA 1.3 100 nmol 0.028 mg 1.115640 Raveh et al.,1982Papilloma F BaP 1.1 10 μg 1 Variance not reportedF CPcdP 0.7 200 μg 0.032 Variance not reported13650Cavalieri et al.,1981Papillomas F BaP 1.1 0.2 μmol 0.050 mg 1F CPcdP 0.17 0.6 μmol 0.14 mg 0.060 Variance notreported21410 Slaga et al.,1978Papillomas F BaP 5.2 0.2 μmol 0.050 mg 1F BaA 1.1 2 μmol 0.46 mg 0.02316310 Weyand et al.,1992Unspecified US BaP 4.0 0.01 μmol 0.0025 mg 1US BjF 4.0 1 μmol 0.252 mg 0.010 Variance not reported10200 El-Bayoumyetal., 1982Primarilysquamous cellpapillomaF BaP 7.0 0.05 mg 1E-6 DRAFT – DO NOT CITE OR QUOTETable E-2. Dermal bioassays: RPF calculations formultiplicity dataRelative potency calculationRecord no.Reference Tumor type(s)SexPAHPointestimateextra riskresponsePointestimatedoseDoseunitsConverteddoseConverteddose unitsRPF CommentsF CH 7.6 1 mg 0.05424300 Rice et al.,1985Unspecified F BaP 7.9 0.3 mg 1F CH 4.9 1 mg 0.18F CPdefC 5.5 1 mg 0.2113660Cavalieri et al.,1991PrimarilypapillomaF BaP Expt I 5.2 300 nmol 0.0757 mg 1 16 Wk experiment; variance not reportedF DBalP Expt I 6.8 33.3 nmol 0.010 mg 9.713660Cavalieri et al.,1991PrimarilypapillomaF BaP Expt II 3.4 100 nmol 0.0252 mg 1 27 Wk experiment; variance not reportedF DBalP Expt II 7.0 4 nmol 0.0012 mg 4224800 Nesnow et al.,1984Papillomas M BaP 1.4 200 nmol 0.050 mg 1 Variance not reportedM BeAC 1.3 250 nmol 0.063 mg 0.74 Variance not reportedM BlAC 1.4 50 nmol 0.013 mg 4.0 Variance not reportedF BaP 1.5 200 nmol 0.050 mg 1 Variance not reportedF BeAC 1.1 250 nmol 0.063 mg 0.58 Variance not reportedF BlAC 1.1 50 nmol 0.013 mg 2.9 Variance notreportedE-7 DRAFT – DO NOT CITE OR QUOTE1Table E-3. Intraperitoneal bioassays: RPF calculations for incidence dataRelative potency calculationRecordno. ReferenceTargetorganTumortype(s) Sex PAHBMR BMDPointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverteddoseunits RPF Comments17560 Busby etal., 1989Lung Adenoma,adenocarcinomaF BaP 0.68 59.5 μg 1FA 0.26 257.6 μg 0.09640 LaVoie etal., 1987Lung Adenoma M BaP 0.82 1.1 μmol/mouse0.28 mg/mouse1BjF 0.52 1.1 μmol/mouse0.28 mg/mouse0.64 Do not use: useliver or lungRPF belowF BaP 0.64 1.1 μmol/mouse0.28 mg/mouse1BjF 0.22 1.1 umol/mouse0.28 mg/mouse0.35 Do not use: useliver or lungRPF belowLiver Adenoma,hepatomaM BaP 0.75 1.1 μmol/mouse0.28 mg/mouse1BbF 0.5 0.5 μmol/mouse0.13 mg/mouse1.50 Do not use: useliver or lungRPF belowBjF 0.49 1.1 μmol/mouse0.28 mg/mouse0.66 Do not use: useliver or lungRPF belowLiver orlungAdenoma,hepatomaM BaP 0.75 1.1 μmol/mouse0.28 mg/mouse1BbF 0.51 0.5 μmol/mouse0.13 mg/mouse1.50BjF 0.8 1.1 μmol/mouse0.28 mg/mouse1.10F BaP 0.64 1.1 μmol/mouse0.28 mg/mouse1BjF 0.22 1.1 μmol/mouse0.28 mg/mouse0.357510 LaVoie etal., 1994Lung Total M BaP 0.7 1.1 μmol/mouse0.28 mg/mouse1FA 0.7 22 μmol/mouse4.45 mg/mouse0.06 Do not use:male liver RPFis higherE-8 DRAFT – DO NOT CITE OR QUOTETable E-3. Intraperitoneal bioassays: RPF calculations for incidence dataRelative potency calculationRecordno. ReferenceTargetorganTumortype(s) Sex PAHBMR BMDPointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverteddoseunits RPF CommentsF BaP 0.83 1.1 μmol/mouse0.28 mg/mouse1FA 0.83 17 μmol/mouse3.44 mg/mouse0.08Liver Foci, adenoma,carcinomaM BaP 0.81 1.1 μmol 0.28 mg/mouse1FA 0.81 6.4 μmol 1.29 mg/mouse0.2122510Wislocki etal., 1986Liver Adenoma,carcinomaM BaP 0.44 560 nmol 0.14 mg 1CH 0.44 3,339 nmol 0.76 mg 0.19 Using pooledcontrolsBaA 0.77 2,800 nmol 0.64 mg 0.39Lung UnspecifiedM BaP 0.3 560 nmol 0.14 mg 1CH 0.3 5,601 nmol 1.28 mg 0.11 Do not use:male liver RPFis higher.Using pooledcontrolsF BaP 0.46 560 nmol 0.14 mg 1BaA 0.16 2,800 nmol 0.64 mg 0.0812E-9 DRAFT – DO NOT CITE OR QUOTE1Table E-4. Intraperitoneal bioassays: RPF calculations for multiplicity dataRPF CalculationRecordno. ReferenceTargetorgan(s) Tumor type(s) Sex PAHBMR BMDPointestimateextra riskresponsePointestimatedoseDoseunitsConverteddoseConverteddose units RPF Comments17560Busby et al.,1989Lung Adenoma,adenocarcinomaF BaP 1.11 59.5 μg 1FA 0.33 257.6 μg 0.0697510 LaVoie etal., 1994Lung Total M BaP 4.13 1.1 μmol 0.28 mg 1FA 0.95 17.30 μmol 3.50 mg 0.018 Do not use:male liverRPF is higherF BaP 3.40 1.1 μmol 0.28 mg 1FA 2.30 17.30 μmol 3.50 mg 0.054Liver Foci, adenoma,carcinomaM BaP 4.12 1.1 μmol 0.28 mg 1FA 1.45 3.46 μmol 0.700 mg 0.1422510 Wislocki etal., 1986Liver Adenoma,carcinomaM BaP 1.36 560 nmol 0.141 mg 1CH 0.93 2,800 nmol 0.639 mg 0.15 Using pooledcontrolsBaA 2.28 2,800 nmol 0.639 mg 0.3713610Busby et al.,1984Lung Adenoma,carcinomaM BaP 4.28 0.28 mg 1 No model fitFA 4.28 9.99 mg 0.028F BaP 3.56 0.28 mg 1 No model fitFA 3.56 32.28 mg 0.008624590 Nesnow etal., 1998bLung NS M BaP 3.85 50 mg/kg 1 No model fitBbF 3.85 123 mg/kg 0.41 BMR = BaPresponseCPcdP 4.15 50 mg/kg 1.1 No model fitDBahA 3.85 3.57 mg/kg 14 BMR = BaPresponseDBalP 3.66 1.5 mg/kg 32 No model fit11190 Mass et al.,1993Lung NS US BaP 5.05 100 mg/kg 1 No model fitBjAC 59.45 20 mg/kg 59 No model fit2E-10 DRAFT – DO NOT CITE OR QUOTETable E-5. Lung implantation bioassays: RPF calculations (incidence data)Relative potency calculationRecord no. Reference Target organ(s) Tumor type(s)PAHBMR BMDPointestimateresponsePointestimatedose Dose unitsRPF Comments17940 Deutsch-Wenzel et al., 1983 Lung Sum carcinoma + sarcoma BaP 0.1 0.032 mg 1AA 0.1 0.14 mg 0.24BbF 0.1 0.33 mg 0.10BghiP 0.1 3.5 mg 0.0092BjF 0.1 1.0 mg 0.032BkF 0.1 1.1 mg 0.031IP 0.1 0.44 mg 0.07422000 Wenzel-Hartung et al., 1990 Lung Carcinoma BaP 0.1 0.033 mg/animal1CH 0.1 0.85 mg/animal0.038BaP 0.57 0.20 mg/animal1DBahA 0.57 0.1 mg/animal2.0 Single dose1234E-11 DRAFT – DO NOT CITE OR QUOTE1Table E-6. In vivo DNA adducts: RPF calculationsRelative potency calculationRecordno. ReferenceTargetorgan(s)/route PAHAUCAUC vs.dosePointestimateextra riskresponsePointestimatedose Dose units Converted doseConverted doseunits RPF Comments6210 Arif et al.,1997Sum of adducts inmammary gland,lung, heart,pancreas, bladder,liverBaP 325 0.25 μmol/mammarygland0.063 mg/mammary gland1DBalP 2,245 0.25 μmol/mammarygland0.076 mg/mammarygland5.817630 Cavalieri etal., 1981aSkin 4-hr BaP 16 0.2 μmol/animal 0.050 mg/animal1 Higher of 2 valuesmeasured at 4 hrsACEP 2.2 0.2 μmol/animal 0.046 mg/animal 0.15 Higher of 2 valuesmeasured at 4 hrsCPcdP 8.8 0.2 μmol/animal 0.045 mg/animal 0.61 Higher of 2 valuesmeasured at 24 hrs18810Hughes andPhillips,1990Sum of skin andlungBaP 9 1 μmol 0.25 mg 1 RPFs based onpeaks; digitizingnot possible.Peaks reached atdifferent timespostdosing.DBaeP cannotdetermine1 μmol NADBahP 3.2 1 μmol 0.30 mg 0.30DBaiP 0.85 1 μmol 0.30 mg 0.079DBalP 65 1 μmol 0.30 mg 6.011190 Mass et al.,1993Lung BaP 470 mg/kg 1BjAC 464 mg/kg 0.99 Ratio of slopes ofAUC vs. dose.BjAC plot showscurvature8010 Nesnow etal., 1993bTotal of lung, liver,and peripheralblood lymphocytesBaP 52,084 100 mg/kg 1BbF 11,314 100 mg/kg 0.22 Ratio of (sum ofAUCs)/doseE-12 DRAFT – DO NOT CITE OR QUOTETable E-6. In vivo DNA adducts: RPF calculationsRelative potency calculationRecordno. ReferenceTargetorgan(s)/route PAHAUCAUC vs.dosePointestimateextra riskresponsePointestimatedose Dose units Converted doseConverted doseunitsRPF Comments24590 Nesnow etal., 1998bLung BaP 113 mg/kg 1 Ratio of slopes ofAUC vs. dose asreported byauthorsBbF 38 mg/kg 0.33CPcdP 148 mg/kg 1.3DBahA 219 mg/kg 1.9DBalP 1,390 mg/kg 1222810 Phillips etal., 1979Skin BaP 27 1 0.25 mg/animal 1 Ratio of peaklevels. Peaksreached atdifferent timesDBacA 10 1 μmol/animal 0.28 mg/animal 0.34DBahA 15 1 μmol/animal 0.28 mg/animal 0.5024790 Kligermanet al., 2002Mouse peripheralblood lymphocytes/intraperitonealBaP 4,186 100 mg/kg 1 Ratio of singlemeasure on d 7postdosingBaA 93 100 mg/kg 0.022BbF 516 100 mg/kg 0.12CH 81 100 mg/kg 0.019Mouse peripheralblood lymphocytes/gavageBaP 143 100 mg/kg 1BaA 32 100 mg/kg 0.22BbF 39 100 mg/kg 0.27CH 37 100 mg/kg 0.26Rat peripheralblood lymphocytes/intraperitonealBaP 755 100 mg/kg 1BaA 38 100 mg/kg 0.05BbF 63 100 mg/kg 0.083CH 24 100 mg/kg 0.032Rat peripheralblood lymphocytes/gavageBaP 177 100 mg/kg 1BaA 20 100 mg/kg 0.11BbF 17 100 mg/kg 0.1CH 10 100 mg/kg 0.0561E-13 DRAFT – DO NOT CITE OR QUOTE1Table E-7. In vivo clastogenicity or sister chromatid exchange: RPF calculationRelative potency calculationRecordno. Reference Route EndpointData type: quantalor continuous PAHBMR BMDPointestimateextra riskresponsePoint estimate doseDoseunits RPF Comments24740 Allen et al.,1999Intraperitoneal Micronucleatedpolychromaticerythrocytes in bonemarrow (A/J mouse)Q BaP 0.0086 200 mg/kg 1DBalP 0.0013 1.5 mg/kg 20 Model won't predictBaP BMR. RPF basedon peakIntraperitoneal Micronucleatedpolychromaticerythrocytes inperipheral blood(A/J mouse)Q BaP 0.0067 200 mg/kg 1DBalP 0.0015 6 mg/kg 7.5 Model won't predictBaP BMR. RPF basedon peakIntraperitoneal Micronucleatedpolychromaticerythrocytes in bonemarrow (p53 wtmouse)Q BaP 0.0019 200 mg/kg 1DBalP 0.0042 12 mg/kg 37 Model won't predictBaP BMR. RPF basedon peakIntraperitoneal Micronucleatedpolychromaticerythrocytes inperipheral blood(p53 wt mouse)Q BaP 0.0022 200 mg/kg 1DBalP 0.0011 18 mg/kg 5.6 BMD doesn't reflectselected BMR. RPFbased on peak.14270 He and Baker,1991Dermal micronuclei Q BaP 0.064 50 μg/animal1 No model fit. RPFbased on peak.CH 0.05 500 μg/animal0.078 No model fit. RPFbased on peak.E-14 DRAFT – DO NOT CITE OR QUOTETable E-7. In vivo clastogenicity or sister chromatid exchange: RPF calculationRelative potency calculationRecordno. Reference Route EndpointData type: quantalor continuous PAHBMR BMDPointestimateextra riskresponsePoint estimate doseDoseunitsRPF Comments17190 Bayer, 1978 Intraperitoneal Sister chromatidexchangesC BaP 4.2 100 mg/kg 1 No model fit. RPFbased on peak.PH 0.9 100 mg/kg 0.21 No model fit. RPFbased on peak.20950 RoszinskyKocher et al.,1979Intraperitoneal Sister chromatidexchangesC BaP 6.7 900 mg/kg 1DBahA1 900 mg/kg 0.15CH 1.2 900 mg/kg 0.18PH 1.6 900 mg/kg 0.24BeP 1.6 900 mg/kg 0.24BbF 1.7 900 mg/kg 0.25BaA 2.2 900 mg/kg 0.3324720 Kligerman etal., 1986Gavage Sister chromatidexchangesC BaP 8 63 mg/kg 1 No SD for controlBlAC 16 126 mg/kg 1.1 No SD for control; RPFbased on lowest doseapproaching peak24790 Kligerman etal., 2002Intraperitoneal Sister chromatidexchangesC BaP 12.42 100 mg/kg 1BaA 6.01 100 mg/kg 0.48BbF 13.46 100 mg/kg 1.1CH 3.17 100 mg/kg 0.26Gavage Sister chromatidexchangesC BaP 6.79 100 mg/kg 1BaA 2.26 100 mg/kg 0.33Gavage Micronuclei Q BaP 0.0025 100 mg/kg 1BbF 0.0017 100 mg/kg 0.6812E-15 DRAFT – DO NOT CITE OR QUOTE1Table E-8. In vitro bacterial mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDSlopePointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverted doseunits RPF Comments17030 Andrews etal., 1978BaP C 1,531 250 μg 1DBacA C 2,807 10 μg 46DBajA C 693 10 μg 11DBahA C 467 25 μg 3AA C 1,645 250 μg 1.1BghiP C 642 100 μg 1BeP C 492 1,000 μg 0.0823830 Baker et al.,1980BaP C 1,144 2.5 μg/plate 1DBaiP C 603 5 μg/plate 0.26BaA C 813 10 μg/plate 0.18DBacA C 1,604 2.5 μg/plate 1.4DBahA C 1,197 5 μg/plate 0.5223660 Bartsch etal., 1980BaP C 29,000 0.027 μmol/plate 0.007 mg/plate 1BaA C 6,000 0.067 μmol/plate 0.015 mg/plate0.09217380 Bos et al.,1988BaP C 739 7.5 μg/plate 1 RPF based on peakresponse. BaP responsewell above range forother data sets; model fitrequired dropping highdoses but not appropriategiven BMR target.PH C 155 25 μg/plate 0.063Pyr C 193 25 μg/plate 0.07817590Carver et al.,1986BaP C 895 50 μg/plate 1 Continuous data, no SD;RPF based on peak orlowest dose approachingpeakBaA C 1,123 50 μg/plate 1.3BghiF C 845 50 μg/plate 0.94Pery C 853 10 μg/plate 4.817630 Cavalieri etal., 1981aBaP Q 0.00126 60 μM 15.1 mg/L 1 RPF based on peak; nomodel fitCPcdP Q 0.0013 40 μM 9.1 mg/L 1.7 RPF basedon peak; nomodel fitE-16 DRAFT – DO NOT CITE OR QUOTETable E-8. In vitro bacterial mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDSlopePointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverted doseunitsRPF CommentsACEP Q 0.0005 120 μM 27.4 mg/L 0.22 RPF based on peak;BMD doesn't coincidewith selected BMR.9620 Chang et al.,2002BaP C 2,217 5 μg/plate 1 Continuous data, noSD;RPF based on peak orlowest dose approachingpeakBghiF C 1,304 5 μg/plate 0.59BcPH C 717 10 μg/plate 0.1624030 De Flora etal., 1984BaP NA 185 revertants/nmol733,196 revertants/mg 1 RPFs based on potencyestimates as reported byauthorsBaA NA 12 revertants/nmol52,565 revertants/mg 0.072BeP 1.6 revertants/nmol6,341 revertants/mg 0.009Pery NA 21 revertants/nmol83,229 revertants/mg 0.1118050 Eisenstadtand Gold,1978BaP C 1,705 2 μg 1 Uses S9 level with maxBaP response; CPcdPmax at much lower S9CPcdP C 134 1 μg 0.1618180 Florin et al.,1980BaP C 255 0.003 μmol/plate 0.001 mg/plate 1 TA100BaA C 326 0.1 μmol/plate 0.023 mg/plate 0.042CH C 196 0.005 μmol/plate 0.001 mg/plate 0.51BaP C 235 0.003 μmol/plate 0.001 mg/plate 1TA 98CO C 82 0.07 μmol/plate 0.021 mg/plate 0.013Pery C 91 0.025 μmol/plate 0.006 mg/plate 0.04624080 Gibson etal., 1978BaP C 35 300 μg/plate 1 Continuous data, no SD;RPF based on peak orlowest dose approachingpeak. Metabolicactivation by gammaradiation.BaA C 6.4 250 μg/plate 0.22BghiP C 4.2 400 μg/plate 0.090CH C 6.1 500 μg/plate 0.1Lowest dose approachingpeakFE C 2.2 360 μg/plate 0.052E-17 DRAFT – DO NOT CITE OR QUOTETable E-8. In vitro bacterial mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDSlopePointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverted doseunitsRPF CommentsPyr C 28 160 μg/plate 1.514080 Gold andEisenstadt,1980BaP C 1,103 4 nmol 0.001 mg 1CPcdP C 281 4 nmol 0.001 mg 0.2818650 Hermann,1981BaP NA 100 revertants/nmol396,322 revertants/mg 1 RPFs based on potencyestimates as reported byauthorsAA NA 62 revertants/nmol224,394 revertants/mg 0.57BaA NA 4 revertants/nmol17,522 revertants/mg 0.044BbA NA 8 revertants/nmol35,043 revertants/mg 0.088BbF NA 15 revertants/nmol59,448 revertants/mg 0.15BeP NA 15 revertants/nmol59,449 revertants/mg 0.15CH NA 2 revertants/nmol8,761 revertants/mg 0.022CO NA 60 revertants/nmol199,761 revertants/mg 0.50DBacA NA 42 revertants/nmol150,888 revertants/mg 0.38DBahA NA 8 revertants/nmol28,743 revertants/mg 0.073DBaiP NA 38 revertants/nmol125,661 revertants/mg 0.32DBalP NA 21 revertants/nmol69,451 revertants/mg 0.18FA NA 3 revertants/nmol14,832 revertants/mg 0.037Pery NA 31 revertants/nmol122,862 revertants/mg 0.31Tphen NA 13 revertants/nmol56,944 revertants/mg 0.1410670 Johnsen etal., 1997BaP C 128 10 μg/plate 1BjAC C 192 10 μg/plate 1.5 RPF based on peak; nomodel fitBlAC C 204 10 μg/plate 1.6 RPF based on peak; nomodel fit19000 Kaden et al.,1979BaP NA 1 RPFs as reported byauthorsAA NA 0.08AN NA 0.01ANL NA 0.07BaA NA 0.14BbFE NA 0.08BeP NA 0.11BghiP NA 0.08CH NA 0.2E-18 DRAFT – DO NOT CITE OR QUOTETable E-8. In vitro bacterial mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDSlopePointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverted doseunitsRPF CommentsCPcdP NA 1.5DBacA NA 0.77DBahA NA 0.08DBbeF NA 0.88FA NA 1Pery NA 6Pyr NA 0.07Tphen NA 0.0724680 Lafleur etal., 1993BaP Q 0.00026 8 μg/mL 1 RPF based on peak;BMD doesn't coincidewith selected BMR.BghiF Q 0.00044 10 μg/mL 1.4CPcdP Q 0.00048 8 μg/mL 1.9CPhiACEAQ 0.00059 4 μg/mL 4.6CPhiAPA Q 0.00017 100 μg/mL 0.05ACEA Q 0.00059 35 μg/mL 0.53APA Q 0.00026 30 μg/mL 0.2719320 LaVoie etal., 1979BaP C 480 20 μg 1 Continuous data, no SD;RPF based on peak orlowest dose approachingpeakBeP C 20 10 μg 0.08Pery C 70 20 μg 0.1523650 McCann etal., 1975BaP NA 121 revertants/nmol479,550 revertants/mg 1 RPFs based on potencyestimates as reported byauthors; authors cautionthat dose-responsenonlinearBaA NA 11 revertants/nmol48,184 revertants/mg 0.10BeP NA 0.6 revertants/nmol2,378 revertants/mg 0.005CH NA 38 revertants/nmol166,455 revertants/mg 0.35DBacA NA 175 revertants/nmol628,698 revertants/mg 1.3DBahA NA 11 revertants/nmol39,521 revertants/mg 0.082DBaiP NA 20 revertants/nmol66,138 revertants/mg 0.1420220 PahlmanandPelkonen,1987BaP NA 272 revertants/mg 1,077,996 revertants/mg 1 RPFs based on potencyestimates as reported byauthorsBaA NA 10 revertants/mg 43,804 revertants/mg 0.041E-19 DRAFT – DO NOT CITE OR QUOTETable E-8. In vitro bacterial mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDSlopePointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverted doseunitsRPF CommentsCH NA 9.7 revertants/mg 42,490 revertants/mg 0.039DBacA NA 35 revertants/mg 125,740 revertants/mg 0.12DBahA NA 4 revertants/mg 14,371 revertants/mg 0.013Tphen NA 4 revertants/mg 17,521 revertants/mg 0.01620450 PhillipsonandIoannides,1989BaP C 119 10 μg/plate 1 No SD; RPFs based onpeak or lowest doseapproaching peakBaA C 110 20 μg/plate 0.46DBaiP C 65 20 μg/plate 0.27DBahA C 51 10 μg/plate 0.4321000 Sakai et al.,1985BaP C 1,565 10 μg 1 No SD; RPFs based onpeak or lowest doseapproaching peakFE C 65 5 μg 0.083AC C 320 10 μg 0.2PH C 345 10 μg 0.22FA C 835 10 μg 0.53CH C 638 10 μg 0.41Pyr C 2,400 10 μg 1.5BeP C 923 10 μg 0.59Pery C 2,607 4 μg 4.2BghiP C 814 20 μg 0.26CO C 223 10 μg 0.1411860 Sangaiah etal., 1983BaP C 384 6 μg/plate 1 No SD; RPFs based onpeak or lowest doseapproaching peakBjAC C 940 10 μg/plate 1.421360 Simmon,1979aBaP C 1,141 5 μg 1BaA C 280 50 μg 0.025BeP C 57 50 μg 0.00521640 Teranishi etal., 1975BaP C 39 50 μg/plate 1DBaiP C 64 50 μg/plate 1.6BaP 254 50 μg/plate 1DBaeP 63 50 μg/plate 0.25E-20 DRAFT – DO NOT CITE OR QUOTETable E-8. In vitro bacterial mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDSlopePointestimateextra riskresponsePoint estimate doseDose unitsConverteddoseConverted doseunitsRPF Comments16180 Utesch etal., 1987BaP C 839 6 μg/plate 1 No SD; RPF based onpeak or lowest doseapproaching peakBaA C 3,347 25 μg/plate 116440 Wood et al.,1980BaP C 99 15 μg/plate 1 No SD; RPF based onpeak or lowest doseapproaching peakCPcdP C 685 15 μg/plate 6.91E-21 DRAFT – DO NOT CITE OR QUOTE1Table E-9. In vitro mammalian mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHBMR BMDPointestimateextra riskresponsePointestimate doseDose unitsConverteddoseConverteddoseunits RPF Comments16920 Amacher andPaillet, 1982BaP 0.00023 10 μg/mL 1 No model fit. RPF basedon peakBaA 0.000068 10 μg/mL 0.3 No model fit. RPF basedon peak16940 Amacher andTurner, 1980BaP 0.00025 1.25 × 10-5M 3.15 mg/L 1 Control w/o S9 treatmentBaA 0.00027 3.22 × 10-5M 7.35 mg/L 0.4616910 Amacher et al.,1980BaP 0.00033 3.96 × 10-5M 9.99 mg/L 1 No model fit. RPF basedon peakBaA 0.00007 4.3 × 10-5M 9.82 mg/L 0.22BMD doesn't coincide withselected BMR; RPF basedon peak17140 Barfknecht et al.,1982BaP 0.00001 1.8 μM 0.45 mg/L 1BaA 0.00001 23 μM 5.25 mg/L 0.09CH 0.00001 16 μM 3.65 mg/L 0.12CPcdP 0.0000083 23 μM 5.20 mg/L 0.07BMD doesn't coincide withselected BMR; RPF basedon response closest toBMR of 0.00001FA 0.00001 3.9 μM 0.79 mg/L 0.58Tphen 0.00001 54 μM 12.33 mg/L 0.0424670 Durant et al., 1999BaP 0.00017 1,000 ng/mL 1 RPF based on peakresponse. Single dose BaPresponse at upper end orabove data range for mostother data sets; model fitrequired dropping highdoses but not appropriategiven BMR target at BaPresponse levelBaPery 0.00018 100 ng/mL 11BbPery 0.000036 100 ng/mL 2.2DBaeF 0.00017 100 ng/mL 10DBafF 0.00017 1,000 ng/mL 1DBahP 0.000061 100 ng/mL 3.7E-22 DRAFT – DO NOT CITE OR QUOTETable E-9. In vitro mammalian mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHBMR BMDPointestimateextra riskresponsePointestimate doseDose unitsConverteddoseConverteddoseunits RPF CommentsDBaiP 0.00013 100 ng/mL 7.8DBelP 0.000034 1,000 ng/mL 0.21N23aP 0.000073 100 ng/mL 4.4N23eP 0.000079 1,000 ng/mL 0.4814250 Hass et al., 1982 BaP 0.00026 0.3 μg/mL1 No model fit. Response atlow dose (approachingpeak)DBaiP 0.0012 0.3 μg/mL 4.6 No model fit.RPF basedon peakDBahP 0.00066 0.3 μg/mL 2.5 No model fit.RPF basedon peak18740 Huberman andSachs, 1976BaP 0.0042 1 μg/mL 1DBacA 0.00016 1 μg/mL 0.04DBahA 0.00011 1 μg/mL 0.0318990 Jotz and Mitchell,1981BaP 0.00014 4.5 μg/mL 1 With metabolic activationPyr 0.000034 11 μg/mL 0.1 With metabolic activation24720 Kligerman et al.,1986BaP 0.00047 4 nmol/mL 0.001 mg/mL 1 No model fit.RPF basedon peakBlAC 0.00028 5 nmol/mL 0.0013 mg/mL 0.48 No model fit. RPF basedon peak19180 Krahn andHeidelberger, 1977BaP 0.00012 15.9 nmol/mL 0.004 mg/mL 1 3-MC S9; 40% survivalBaA 0.00005 46.5 nmol/mL 0.011 mg/mL 0.16 3-MC S9; 40% survival24680 Lafleur et al., 1993BaP 0.000024 0.2 μg/mL 1 No model fit.ACEA 0.000013 3 μg/mL 0.037 No model fit.CPcdP 0.000015 2 μg/mL 0.061 No model fit.CPhiACEA0.000022 0.3 μg/mL 0.62 No model fit.7550 Li and Lin, 1996 BaP 0.00003 10 ng/mL 1BaA 0.000036 10 ng/mL 1.211450 Nesnow et al.,1984BaP 0.00019 5 μg/mL 1E-23 DRAFT – DO NOT CITE OR QUOTETable E-9. In vitro mammalian mutagenicity: RPF calculationsRelative potency calculationRecordno. Reference PAHBMR BMDPointestimateextra riskresponsePointestimate doseDose unitsConverteddoseConverteddoseunits RPF CommentsBeAC 0.00042 5 μg/mL 2.2 No model fit; RPFbasedon lowest doseapproaching peakBjAC 0.00025 5 μg/mL 1.3 No model fit; RPFbasedon lowest doseapproaching peakBlAC 0.00044 2.5 μg/mL 4.6 No model fit; RPF basedon lowest doseapproaching peak15630 Raveh andHuberman, 1983BaP 0.0001 0.11 μg/mL 1CPcdP 0.0001 0.58 μg/mL 0.18 Uses QL; MS didn'tconverge15640 Raveh et al., 1982 BaP 0.00001 0.16 μg/mL1 Uses QL, hi dose dropped;MS didn't fitCPcdP 0.00001 1.1 μg/mL 0.14 Uses QL; MS didn'tconverge21410 Slaga et al., 1978 BaP 0.0001 0.048 μM 0.012 mg/L 1BaA 0.0001 32 μM 7.3 mg/L 0.00165816190 Vaca et al., 1992 BaP 0.00027 10 μM 2.5 mg/L 1BMD doesn't coincide withselected BMR; RPF basedon peakFA 0.00021 10 μM 2.02 mg/L 0.97BMD doesn't coincide withselected BMR; RPF basedon peak21900 Wangenheim andBolcsfoldi, 1988BaP 0.0008 0.00001 mol/L 2.5 mg/L 1BMD doesn't coincide withselected BMR; RPF basedon peakFE 0.000086 0.00012 mol/L 19.9 mg/L 0.014BMD doesn't coincide withselected BMR; RPF basedon peakPyr 0.00053 0.00003 mol/L 6.1 mg/L 0.28BMD doesn't coincide withselected BMR; RPF basedon peak1E-24 DRAFT – DO NOT CITE OR QUOTE1Table E-10. In vitro morphological/malignant transformation: RPF calculationRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDPointestimateextra riskresponsePointestimate doseSlope ofdoseresponsecurveDoseunits Converted doseConverted doseunits RPF Comments17610 Casto, 1979BaP Q 0.00001 0.1 μg/mL 1DBahA Q 0.00001 2.5 μg/mL 0.04217970 DiPaolo etal., 1969BaP Q 0.058 10 μg/mL 1DBahA Q 0.031 10 μg/mL 0.54BaA Q 0.011 10 μg/mL 0.18BeP Q 0.0058 10 μg/mL 0.1DBacA Q 0.011 10 μg/mL 0.1918080Emura et al.,1980BaP Expt I Q 0.001 0.044 μg/mL 1BbF Q 0.001 0.75 μg/mL 0.059BaA Q 0.001 0.85 μg/mL 0.052BaP Expt II Q 0.001 0.046 μg/mL 1IP Q 0.001 0.82 μg/mL 0.05614130 Greb et al.,1980BaP NA 277 %/mmol1.10 %/mg 1 Relativetransformationpotenciesreported. RPFsare ratio ofpotencies.BaA NA 13.9 %/mmol0.061 %/mg 0.055BbF NA 11.5 %/mmol0.046 %/mg 0.042BeP NA 3.1 %/mmol0.012 %/mg 0.011CH NA 37 %/mmol0.16 %/mg 0.15DBahA NA 0.3 %/mmol0.001 %/mg 0.00098214640 Krolewskiet al., 1986BaP Q 0.0055 5 μM 1.3 mg/L 1CPcdP Q 0.0017 5 μM 1.1 mg/L 0.3414700 Laaksonenet al., 1983BaP Q 0.000009 10 μM 2.5 mg/L 1 RPF based onpeak. Inversedose-responserelationshippossibly due tocytotoxicity.E-25 DRAFT – DO NOT CITE OR QUOTETable E-10. In vitro morphological/malignant transformation: RPF calculationRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDPointestimateextra riskresponsePointestimate doseSlope ofdoseresponsecurveDoseunitsConverted doseConverted doseunitsRPF CommentsBaA Q 0.000018 11 μM 2.5 mg/L 2.0 RPF basedonpeak. Inversedose-responserelationshippossibly due tocytotoxicity.14850 Lubet et al.,1983BaP Q 0.1 1.9 μg/mL 1BeP Q 0.1 41 μg/mL 0.04624710 Mohapatraet al., 1987BaP 0.92 1 μg/mL 1BjAC Q 0.92 0.93 μg/mL 1.1BaP 0.83 1 μg/mL 1BlAC Q 0.83 7.5 μg/mL 0.13BaP 0.86 1 μg/mL 1BeAC Q 0.86 18 μg/mL 0.05624700 Nesnow etal., 1990BaP C 47 10 μg/mL 1BlAC C 120 10 μg/mL 2.5 Based on peakresponse; no SDfor control7980 Nesnow etal., 1997BaP C 2.5 4 μM 1.01 mg/L 1DBalP C 1.7 0.33 μM 0.10 mg/L 6.9 Based on peakresponse; no SDfor control7990 Nesnow etal., 1994BaP C 0.94 1 μg/mL 1DBahA C 0.37 1 μg/mL 0.39 Based on peakresponse; nocontinuouslinear model fit8000 Nesnow etal., 1993aBaP C 1.4 3 μg/mL 1DBkmnoAPH C 1.1 5 μg/mL 0.47 Based on peakresponse; no SDfor control23720 Pienta et al.,1977BaP Q 0.01 15 μg/mL 1 High dosedroppedE-26 DRAFT – DO NOT CITE OR QUOTETable E-10. In vitro morphological/malignant transformation: RPF calculationRelative potency calculationRecordno. Reference PAHData type:quantal orcontinuousBMR BMDPointestimateextra riskresponsePointestimate doseSlope ofdoseresponsecurveDoseunitsConverted doseConverted doseunitsRPF CommentsBaA Q 0.01 8.2 μg/mL 1.9 Caution:changing slopein region ofBMRDBahA Q 0.01 0.4 μg/mL 34 Two highestdoses dropped1E-27 DRAFT – DO NOT CITE OR QUOTE1Table E-11. In vitro DNA adducts: RPF calculationsaRelative potency calculationRecord no.Reference PAHPointestimateextra riskresponsePointestimatedose Dose unitsConverteddoseConverteddoseunits RPF Comments16890 Allen and Coombs, 1980 BaP 7.5 0.24 μg/mL1 Nuclear DNABaA 0.44 0.64 μg/mL 0.021BaP 413 0.24 μg/mL 1 Mitochondrial DNABaA 104 0.64 μg/mL 0.0926300 Binkova et al., 2000 BaP 258 1 μM 0.25 mg/L 1DBalP 2,317 0.1 μM 0.03 mg/L 759510 Bryla and Weyand, 1992 BaP 5.5 600 nmol 0.15 mg 1 Light conditionsBaA 1 600 nmol 0.14 mg 0.20DBacA 1.8 600 nmol 0.17 mg 0.3022800 Grover and Sims, 1968 BaP 1.4 5 μg 1DBahA 0.44 5 μg 0.31DBacA 0.56 5 μg 0.40BaA 0.7 5 μg 0.50Pyr 0.31 5 μg 0.22PH 0.05 5 μg 0.04010670 Johnsen et al., 1997 BaP 0.05 30 μg/mL 1 Clara cellsBjAC 0.15 30 μg/mL 3BlAC 0.24 30 μg/mL 4.8BaP 0.02 30 μg/mL 1 Type 2 cellsBjAC 0.06 30 μg/mL 3BlAC 0.03 30 μg/mL 1.510660 Johnsen et al., 1998 BaP 0.33 30 μg/mL 1 Human lymphocytesBjAC 0.11 30 μg/mL 0.33BlAC 1.1 30 μg/mL 3.3BaP 0.24 30 μg/mL 1 HL-60 cellsBjAC 0.15 30 μg/mL 0.62BlAC 0.94 30 μg/mL 3.97870 Melendez-Colon et al., 2000 BaP 34 2 μM 0.50 mg/L 1DBalP 348 2 μM 0.60 mg/L 8.521200 Segerback and Vodicka, 1993 BaP 15 100 mM 25,232 mg/L 1BaA 30 100 mM 22,829 mg/L 2.2BbF 3.7 100 mM 25,232 mg/L 0.25BghiP 0.5 100 mM 27,634 mg/L 0.03CH 50 100 mM 22,829 mg/L 3.7DBahA 2.8 100 mM 27,833 mg/L 0.17E-28 DRAFT – DO NOT CITE OR QUOTETable E-11. In vitro DNA adducts: RPF calculationsaRelative potency calculationRecord no.Reference PAHPointestimateextra riskresponsePointestimatedose Dose unitsConverteddoseConverteddoseunits RPF CommentsFA 1.5 100 mM 20,226 mg/L 0.12Pyr 0.14 100 mM 20,226 mg/L 0.012aAll RPFs are point estimates based on peak responseas adequate model fit was not achieved for any multi-dose dataset.No control data were available for any of these studies.12345E-29 DRAFT – DO NOT CITE OR QUOTE1Table E-12. In vitro DNA damage: RPF calculationsRelative potency calculationRecordno. Reference PAHBMR BMDPointestimateextra riskresponsePointestimatedoseSlope ofdoseresponsecurve Dose unitsConverteddoseConverteddoseunits RPF Comments16840 Agrelo andAmos, 1981BaP 2,093 10 μg/mL 1 Control responses for BaP and Pyr differby 10x.Pyr 548 100 μg/mL 0.026 RPF based on peak; continuous datawithout SD23790 Ichinotsuboet al., 1977BaP 6 70 μg/well 1DBaiP 10 600 μg/well 0.19DBahA 10 25 μg/well 4.710660 Johnsen etal., 1998BaP 7.9 3 μg/mL 1 Human lymphocytes. No model fit.Lowest response point estimateBjAC 7.6 18 μg/mL 0.16 Human lymphocytes.BMR is BaP pointestimate responseBlAC 4.9 30 μg/mL 0.062 Human lymphocytes. No model fit.Response point estimate closest to BaPresponse.BaP 5.4 30 μg/mL 1 HL-60 cellsBjAC 1.8 30 μg/mL 0.33 HL-60 cellsBlAC 3.8 30 μg/mL 0.7 HL-60 cells19740 Martin etal., 1978BaP 210 1 × 10-5M 2.5 mg/L 1 Increase over backgroundBaA 59 1 × 10-7M 0.023 mg/L 31BeP 256 1 × 10-6M 0.25 mg/L 12DBacA 97 1 × 10-5M 2.8 mg/L 0.42DBahA 96 1 × 10-5M 2.8 mg/L 0.4119830 MerschSundermann et al.,1992BaP 0.61 μg/assay 1 SOSIP - slope of SOS induction doseresponse curve as reportedAA 0.14 μg/assay 0.23BaA 0.1 μg/assay 0.17BbF 0.045 μg/assay 0.074BghiF 0.34 μg/assay 0.56BjF 0.25 μg/assay 0.42BbFE 0.024 μg/assay 0.04BghiP 0.033 μg/assay 0.055BeP 0.032 μg/assay 0.053CH 0.22 μg/assay 0.37DBacA 0.10 μg/assay 0.17E-30 DRAFT – DO NOT CITE OR QUOTETable E-12. In vitro DNA damage: RPF calculationsRelative potency calculationRecordno. Reference PAHBMR BMDPointestimateextra riskresponsePointestimatedoseSlope ofdoseresponsecurve Dose unitsConverteddoseConverteddoseunits RPF CommentsDBahA 0.039 μg/assay 0.064DBalP 2.1 μg/assay 3.5DBahP 0.12 μg/assay 0.19DBaiP 0.17 μg/assay 0.29FA 0.41 μg/assay 0.68IP 0.036 μg/assay 0.06PH 0.053 μg/assay 0.088Tphen 0.26 μg/assay 0.4320810 RobinsonandMitchell,1981BaP 89 10 μg/mL 1Pyr 63 7.2 μg/mL 0.9820940 Rossman etal., 1991BaP 10.4 12.5 μg/mL 1 Enhancement over backgroundAC 4.8 12.5 μg/mL 0.46DBacA 8 1.44 μg/mL 6.7DBahA 4 2 μg/mL 2.4PH 4.5 25 μg/mL 0.2221730 Tong et al.,1981bBaP 65.5 0.001 M 252 mg/L 1BaA 17.1 0.0005 M 114 mg/L 0.58 Based on peak response; no model fit12E-31 DRAFT – DO NOT CITE OR QUOTE1Table E-13. In vitro clastogenicity or sister chromatid exchange: RPF calculationsRecord no.Reference PAH EndpointData type:quantal orcontinuousBMR BMDPointestimateextra riskresponsePointestimatedoseDoseunitsConverteddoseConverteddoseunitsRPF Comments14620 Kochhar,1982BaP Aberrations Q 0.53 5 μg/mL 1 BMD doesn't reflectselected BMR. RPFbased on peak.BaA 0.34 5 μg/mL 0.64 BMD doesn't reflectselected BMR. RPFbased on peak.14640 Krolewskiet al., 1986BaP SisterchromatidexchangesC 0.79 5 μM 1.3 mg/L 1CPcdP 0.29 5 μM 1.1 mg/L 0.41 No model fit.RPFbased on peakresponse19690 Mane et al.,1990BaP SisterchromatidexchangesC 2.7 1 μg/mL 1BaA 0.4 1 μg/mL 0.1521710 Tong et al.,1981aBaP SisterchromatidexchangesC 92 1 × 10-4M 25.2 mg/L 1BaA 13 1 × 10-4M 22.8 mg/L 0.16No n provided. RPFbased on peakresponse234

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APPENDIX F. EXAMPLE CALCULATION OF RPF DETECTION L IMIT 1

2

3 

### Table F-1. Example data for calculation of RPF det ection limit

Group Dose Number with tumors Number in group Incid ence Extra risk

response a Actual responses
Control 0 2 30 0.067 NA
Anthanthrene 0.25 2 29 0.069 NA Benzo[a]pyrene 0.25 24 30 0.800 0.786 Theoretical statistically significant response b

Anthanthrene 0.25 8 29 0.276 0.224

a Calculated as described below in Step 1.

b Calculated as described below in Step 2.

Source: Hoffmann and Wynder (1966).

4 

### Step 1 . Estimate the number of tumor-bearing animals tha t would represent a statistically5 

### significant response (one-sided p ≤ 0.05 using Fisher’s exact test) in the number of animals 6 

### exposed to anthanthrene (29) given the observed con trol response (2/30). In this case, 7 

### 8/29 tumor-bearing animals (incidence of 0.276) wou ld represent a statistically significant 8 

### response to anthanthrene. 9

10 

### Step 2 . Calculate the extra risk response associated wit h the theoretical statistically significant 11 

### incidence for anthanthrene and the observed benzo[a ]pyrene incidence as follows: 12

13 

### Extra risk response = P(d) – P(0) 14 

### [1-P(0)] 15

16 

### For the theoretical statistically significant respo nse to anthanthrene, 17

18 

### Extra risk response = (0.276 – 0.067)/(1 – 0.067) = 0.224 19

20 

### Step 3 . Calculate the RPF detection limit as the ratio o f the slopes associated with extra risk 21 

### response and the actual doses of anthanthrene and b enzo[a]pyrene as follows: 22

23 

### RPF Detection Limit = (Theoretical anthanthrene ext ra risk response/dose anthanthrene) 24 

### (benzo[a]pyrene extra risk response/dose benzo[a]pyrene) 25

26 

### RPF Detection Limit = (0.224/0.25)/(0.786/0.25) = 0.28 27

28

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APPENDIX G: EVALUATION OF ALTERNATIVES FOR RANKING RPFs 1

2

3 

### For many of the PAHs evaluated in this report, a n umber of datasets were available for 4 

### use in calculating RPFs. The resulting RPFs are de rived from tumor bioassays using different 5 

### exposure routes, species, sexes, or tumor endpoints (incidence or multiplicity) and/or from a 6 

### variety of different cancer-related endpoint assays . In addition to using different types of data, 7 

### the various RPFs reflect studies of varying quality (different numbers of animals, follow-up time, 8 

### single or multiple dose groups, response levels low or high on the dose-response curve, etc.). In 9 

### order to derive a single final RPF for each individ ual PAH, the various results from different 10 

### datasets must either be ranked/prioritized and/or c ombined. This appendix details the options 11 

### that were considered for ranking RPFs. 12 

### A series of options were considered for prioritizi ng RPFs for the purpose of selecting a 13 

### single RPF for each PAH or exposure route. An a pr iori decision was made to consider tumor 14 

### bioassay data to be preferable to cancer-related en dpoint data because the tumor bioassay data 15 

### are in whole animals and address the endpoint of in terest for RPFs (tumorigenicity). Thus, 16 

### options for ordering or combining tumor bioassays a nd for cancer-related endpoint data were 17 

### considered separately; Section G.1 below discusses options considered for use of tumor bioassay 18 

### RPFs and Section G.2 discusses options considered f or use of cancer-related endpoint RPFs. 19

20 

### G.1. OPTIONS FOR RANKING TUMOR BIOASSAY RPFS 21 

### Approaches considered for ordering tumor bioassay w ere: (1) ranking by exposure route, 22 

### (2) ranking by target organ, and (3) preference for modeled data over point estimates. 23 

### Ranking by exposure route. One option for ranking RPFs derived from tumor bi oassay 24 

### data would be to order the datasets by exposure rou tes that are considered most relevant to 25 

### environmental exposure routes (oral, dermal, and in halation). RPFs for many PAHs were 26 

### calculated from dermal tumor bioassays. While derm al exposure to PAHs in the environment 27 

### does occur, there is currently no dermal slope fact or for benzo[a]pyrene on the IRIS database. If 28 

### a dermal slope factor is derived for benzo[a]pyrene , then the RPFs derived from dermal tumor 29 

### bioassays would be more suitable than those from ot her exposure routes for use in calculating 30 

### cancer risk from dermal exposure to other PAHs. Th e available database for PAHs did not 31 

### include any oral or inhalation studies that were su itable for RPF calculation; thus, route-to-route 32 

### extrapolation is necessary to derive RPFs applicabl e to oral or inhalation exposures. 33 

### Some earlier RPF approaches, primarily in the cour se of assessing risks from inhalation 34 

### exposure to PAHs, have proposed hierarchies of bioa ssay types based on route of administration. 35 

### Collins et al. (1998) proposed a hierarchy for PAH cancer potencies or PEFs for use in assessing 36 

### air contaminants. The hierarchy for inhalation pot encies or PEFs proposed by Collins et al. 37 

### (1998) ordered the exposure routes as follows: intr atracheal or intrapulmonary 38 

### administration>oral administration>skin-painting st udies> subcutaneous or intraperitoneal 39

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administration. However, Collins et al. (1998) did not provide any empirical data supporting the 1 

### ordering of these exposure routes, other than the i ntuitive preference for intratracheal or 2 

### intrapulmonary administration as a surrogate for in halation. In another review of data available 3 

### for relative potency assessment for PAHs as air con taminants, Pufulete et al. (2004) suggested 4



### that intratracheal instillation of low doses of PAH s might be an appropriate surrogate exposure 5 

### model for assessing relative potency of inhalation exposure. The basis for this suggestion was 6



### the authors’ observation that clearance of PAHs adm inistered in solution via intratracheal 7 

### instillation exhibited a biphasic pattern similar t o that observed after inhalation exposure to 8 

### benzo[a]pyrene bound to particulates. However, the authors acknowledged that the high 9



### concentrations of PAHs used in intratracheal and in trapulmonary instillation studies may lead to 10 

### major differences in pharmacokinetics compared with inhalation exposure (Pufulete et al., 2004). 11 

### Further, the authors expressed this suggestion as a path for future research, rather than as a 12 

### means of examining available data on PAHs; no intra tracheal instillation studies were identified 13



### in the search for studies from which to calculate R PFs for PAHs. Pufulete et al. (2004) did not 14 

### provide any specific information on the relevance o f intrapulmonary administration (a route used 15 

### in several of the bioassays used to calculate RPFs) to inhalation exposure. 16 

### To assess exposure-route differences in RPFs calcul ated in this review, a table comparing 17 

### the average RPF for each PAH across exposure routes was prepared (Table G-1). The average 18



### values include RPFs calculated with both incidence and multiplicity data; each RPF is treated as 19 

### an independent measure of relative potency in calcu lating the averages. Dermal studies are 20 

### shown collectively as well as separated by study ty pe (complete carcinogenesis or initiation 21 

### only). Likewise, intraperitoneal studies are shown grouped as well as separated by target organ 22 

### (lung and liver). In general, the table shows that RPFs calculated from lung implantation and 23 

### dermal studies are similar, while RPFs calculated f rom intraperitoneal studies tend to be higher 24 

### for most compounds. Among PAHs with RPF s derived from intraperitoneal and dermal data, 25 

### 5/7 showed higher RPF values from intraperitoneal d ata, compared with dermal data 26 

### (benz[a]anthracene, benzo[b]fluoranthene, benzo[j]f luoranthene, cyclopenta[c,d]pyrene, 27 

### dibenz[a,h]anthracene; Table G-1). However, intrap eritoneal RPFs for chrysene (CH) and 28 

### dibenzo[a,l]pyrene (DBalP) are similar to dermal RP Fs for these compounds. 29

30

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1


### Table G-1. Average RPF value by exposure route

Dermal Dermal

complete Dermal

initiation Intraperitoneal Intraperitoneal, target organ = lung Intraperitoneal, target

organ = liver Lung implantation

PAH N Average N Average N Average N Average N Average N Average N Average

AA 1 0.5 1 0.5 – – – – – – – – 1 0.2

AC – – – – – – – – – – – – – – BaA 1 0.02 – – 1 0.02 3 0.3 a 1 0.08 2 0.4 – – BbcAC (1,12-MBA) 1 0.05 – – 1 0.05 – – – – – – – – BbF 3 0.3 1 0.2 2 0.4 3 b 1 c 1 0.4 1 2 1 0.1

BeAC 4 0.7 – – 4 0.7 – – – – – – – – BghiP –
– – – – – – – – – – – 1 0.009 BjAC –
– – – – – 1 60 d 1 60 – – – – BjF 3 0.06 – – 3 0.06 5 b 0.6 a 2 0.5 1 0.7 1 0.03

BkF 2 0.02 – – 2 0.02 – – – – – – 1 0.03

BlAC 4 4 – – 4 4 – – – – – – – – CH 7 0.1 – – 7 0.1 3 0.1 a 1 0.1 2 0.2 1 0.04

CPcdP 7 0.2 3 0.3 4 0.1 1 1 d 1 1 – – – – CPdefC 2 0.3 – – 2 0.3 – – – – – – – – DBacA – – – – – – – – – – – – – – DBaeF 2 0.9 1 1 1 0.7 – – – – – – – – DBaeP 2 0.4 1 0.3 1 0.4 – – – – – – – – DBahA 2 1 – – 2 1 1 10 d 1 10 – – 1 2

DBahP 1 0.9 – – 1 0.9 – – – – – – – – DBaiP 2 0.6 1 0.7 1 0.5 – – – – – – – – DBalP 2 30 – – 2 30 1 30 d 1 30 – – – – FA – – – – – – 10 0.08 a 8 0.05 2 0.2 – –
IP – – – – – – – – – – – – 1 0.07

N23eP 1 0.3 – – 1 0.3 – – – – – – – – PH – – – – – – – – – – – – – – Pyr – – – – – – – – – – – – – –

a Newborn mouse model.

b Number of intraperitoneal RPFs includes those calcu lated for combined lung and liver incidence; these are not included in number of RPFs with lung or liv er tumors.

c Includes both newborn mouse and adult A/J mouse mod els.

d Adult A/J mouse model.

2 

### One possible explanation for the higher intraperit oneal RPFs calculated from newborn 3 

### mouse assays (footnoted “a” in the table) might be that the newborn mouse is more sensitive to 4 

### the carcinogenic action of PAHs than an adolescent or adult mouse. Likewise, the adult A/J 5 

### mouse is considered to be particularly sensitive to PAH lung tumorigenicity (Nesnow et al., 6

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1995), which may result in higher RPFs with this mo del (in Table G-1, the intraperitoneal RPFs 1 

### based on the A/J mouse model are footnoted “d”). T here is little information to evaluate whether 2



### the newborn mouse is more or less sensitive than th e adult A/J mouse model. Only one 3 

### compound, benzo[b]fluoranthene (BbF), had RPFs calc ulated from both newborn mouse and 4 

### adult A/J mouse models; the newborn mouse RPF was 2 , while the A/J mouse RPF was 0.4. In 5 

### summary, it is not clear whether the intraperitonea l RPFs are higher than dermal or lung 6 

### implantation RPFs due to route-specific differences or animal model differences in susceptibility. 7 

### Ranking by target tissue. An alternative approach to ranking tumor bioassay R PFs would 8 

### be to prefer target tissue-specific RPFs (for examp le, to prefer RPFs derived from lung tumor 9 

### data for inhalation RPFs). An analysis was conduct ed to assess whether RPFs calculated from 10 

### lung tumor potency in intraperitoneal studies (both newborn mouse and adult A/J mouse models) 11 

### were consistent with RPFs from lung implantation st udies. Table G-1 shows RPFs calculated for 12 

### lung tumors (separate from liver tumors also observ ed in some intraperitoneal studies) after 13 

### intraperitoneal administration. Only four compound s (BbF, BjF, CH, and DBahA) had RPFs for 14 

### both intraperitoneal and lung implantation studies; for each of these, the intraperitoneal lung 15 

### tumor RPF exceeded the lung implantation RPF. No i nformation assessing the concordance 16 

### between lung tumor potency after intraperitoneal ad ministration and inhalation cancer potency 17



### was identified in the literature. 18 

### Ranking by use of BMD. A third approach considered for ranking of tumor bi oassay data 19 

### was to prefer data amenable to BMD modeling (of eit her quantal or continuous data, depending 20 

### on whether incidence or multiplicity was modeled) o ver an analysis of data based on point 21 

### estimates. Table G-2 compares the average of RPFs for all bioassays with RPFs calculated using 22 

### BMD modeling, and RPFs calculated using a point est imate approach. 23

24

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Table G-2. Comparisons among average tumor bioassa y-based RPF values by data availability or calculation method

All bioassays Multidose bioassays BMD model Point e stimate N Average RPF N Average RPF N Average RPF N Average RPF
AA 2 0.4 1 0.2 1 0.2 1 0.5
AC – – – – – – – –
BaA 4 0.2 – – – – 4 0.2
BbcAC 1 0.05 1 0.05 – – 1 0.05
BbF 7 0.6 5 0.3 3 0.2 4 0.9
BeAC 4 0.7 4 0.7 2 0.8 2 0.7 BghiP 1 0.009 1 0.009 1 0.009 – – BjAC 1 60 1 60 – – 1 60 BjF 9 0.4 4 0.06 2 0.09 7 0.5
BkF 3 0.02 3 0.02 2 0.03 1 0.02
BlAC 4 4 4 4 2 5 2 3
CH 11 0.1 5 0.1 3 0.1 8 0.1 CPcdP 8 0.3 8 0.3 2 0.1 6 0.4
CPdefC 2 0.3 – – 1 0.5 1 0.2
DBacA – – – – – – – –
DBaeF 2 0.9 1 1.1 1 1 1 0.7
DBaeP 2 0.4 1 0.3 1 0.3 1 0.4
DBahA 4 4 1 10 1 10 3 1
DBahP 1 0.9 – – – – 1 0.9
DBaiP 2 0.6 1 0.7 1 0.7 1 0.5
DBalP 3 30 3 30 – – 3 30
FA 10 0.08 8 0.08 5 0.08 5 0.07
IP 1 0.07 1 0.07 1 0.07 – –
N23eP 1 0.3 – – – – 1 0.3

PH – – – – – – – – Pyr – – – – – – – –

1 

### While this ranking could be justified based on a g eneral preference for multidose data and 2 

### modeling to identify a point of departure, there ar e important limitations to this approach. First, 3 

### RPFs based on BMD modeling may still use a point of departure high on the dose-response 4 

### curve, if a single benzo[a]pyrene dose with an elev ated response level (BMR) 5 

### was used to 5 

### calculate the RPF. In some cases, an RPF based on a point estimate approach from a point of 6 

### departure lower on the dose-response curve may be a better predictor of relative potency at 7 

### environmental exposure levels. Second, unless RPFs based on BMD modeling are available for 8 

### all of the relevant exposure routes (dermal initiat ion and complete carcinogenicity, lung 9 

### implantation, and intraperitoneal), there may be di fferences between the RPFs calculated from 10 

### BMD modeling and those calculated using a point est imate approach that are unrelated to study 11 

### quality (i.e., route, species, sex differences). Thus, ranking RPFs based on a preference for 12 

### modeled data over point estimate data would neglect other sources of variability in the estimates 13 

### (exposure route, species, sex, target organ, dosing intervals, etc.) 14

5 The BMR selected for multidose PAH data for studies with a single BaP dose was the response level obse rved in the BaP dose group.

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In summary, the analysis of options for ranking bi oassay RPFs did not suggest a clear 1 

### basis for selecting among the available data types. As a consequence, none of the available data 2 

### types was considered preferable to any other; all b ioassay RPFs were considered equally 3



### relevant. 4

5



### G.2. RANKING NON-BIOASSAY DATA 6 

### Two approaches to ranking non-bioassay study types were evaluated: a theoretical 7 

### approach and an empirical approach. 8 

### Theoretical ranking of cancer-related endpoint data

. To identify whether a theoretical 9 

### basis for ranking was available, a limited literatu re search was conducted in PubMed to identify 10 

### publications that addressed the relationship betwee n various genotoxicity endpoints and 11 

### carcinogenicity. The search was intended to identi fy recent papers that described a quantitative 12 

### correlation between particular cancer-related endpo ints and cancer potency. Several papers (e.g., 13 

### Matthews et al., 2006a, b) analyzed the concordance between genotoxicity tests and 14 

### carcinogenicity (i.e., whether positive genotoxicit y findings predicted positive carcinogenicity 15 

### findings) but did not assess whether potency measur ed in genotoxicity studies correlated with 16 

### cancer potency. Three publications (Sanner and Dyb ing, 2005; Ross et al., 1995; Travis et al., 17 

### 1990) provided quantitative associations between ca ncer potency and genotoxic potency. There 18 

### were no publications that related relative cancer p otency to relative genotoxic potency for any 19 

### classes of compounds; this would be the most releva nt comparison for use in ranking. 20 

### Ross et al. (1995) provided evidence that DNA addu ct formation expressed as TIDAL 21 

### values was correlated with the numbers of lung aden omas in adult A/J mice treated with PAHs 22 

### by intraperitoneal injection (TIDAL values were als o strongly correlated with dose). The 23



### correlations were demonstrated for five PAHs: benzo [a]pyrene, benzo[b]fluoranthene, 24 

### dibenz[a,h]anthracene, 5-methylcholanthrene, and CP P. This paper demonstrates a quantitative 25 

### relationship between TIDAL values and tumorigenicit y in the compound class of interest 26 

### (PAHs). 27 

### Examining data from 42 substances, Sanner and Dybi ng (2005) showed a linear 28 

### relationship (slope = 1.05 ± 0.12, r 2 

### = 0.67) between the lowest effective dose (LED) pr oducing 29 

### genotoxicity in vivo after oral or inhalation expos ure and the chronic daily dose estimated to 30 

### result in a 25% increase (over controls) in tumor f ormation (TD 25 

### ). Genotoxicity endpoints 31 

### included in the analysis were micronuclei, sister c hromatid exchanges, chromosomal aberrations, 32 

### DNA adducts, DNA strand breaks, and the comet assay for DNA breaks. The chemical 33 

### compounds included in the analysis included a large variety of compounds (VOCs, chlorinated 34 

### compounds, dioxin, etc.); only one PAH (naphthalene ) was included in the database. Analyzing 35 

### specific endpoints separately, the authors reported the correlations shown in Table G-3. 36

37

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Table G-3. Correlation between LED and TD 25 

### by endpoint

Endpoint Number of observations (compounds) Slope Correlation coefficient (r 2 )

Chromosomal aberrations 4 1.29 ± 0.25 a 0.93

DNA breaks 9 1.02 ± 0.17 a 0.84

DNA adduct 11 1.44 ± 0.36 a 0.64

Micronucleus 8 0.63 ± 0.21 a 0.61 Sister chromatid exchange 2 0.98 –

a Slope significantly different from zero.

Source: Sanner and Dybing (2005).

1 

### The slopes and correlation coefficients shown in T able G-3 suggest that LEDs for 2



### chromosomal aberrations and DNA breaks are reasonab ly good predictors of cancer TD 25 

### , and 3



### that DNA adducts and micronuclei are also correlate d with potency. 4 

### Travis et al. (1990) examined correlations between cancer potency estimates and 5 

### mutagenicity or toxicity data from the Registry of Toxic Effects of Chemical Substances 6 

### (RTECS). Using data from 146 compounds, the author s demonstrated correlations between 7 

### tumor potency and mutagenicity or toxic potency. C orrelations between toxicity and 8 

### carcinogenicity were not considered here, as they a re not pertinent to the ranking of genotoxicity 9 

### data for RPF selection. Correlations between mutag enicity and cancer potency are shown in 10



### Table G-4. 11

12


### Table G-4. Correlation between tumor potency (log 1/TD 50 

### )

and mutagenic potency

Endpoint Number of observations (compounds) Correlation coefficient (r 2 ) Standard

error

Ames test 82 0.37 0.93

All mutation tests 112 0.69 0.90 All mutation tests: lung carcinogens a 17 0.61 0.74 All mutation tests: liver carcinogens a 40 0.43 0.94

a Due to the magnitude of the tumor potency for tetra chlorodibenzo-p-dioxin (TCDD), it had a strong infl uence on the correlation; the authors conducted analyses wit hout dioxin, and these are the results shown here.

Source: Travis et al. (1990).

13 

### The available published studies did not suggest a clear basis for ordering RPFs calculated 14 

### from data on different cancer-related endpoints. B ased on the information provided in the three 15 

### published analyses relating cancer potency with gen otoxic potency, one may conclude that 16 

### TIDAL values, in vivo measures of clastogenicity, D NA adducts, and DNA damage, and in vitro 17

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measures of mutagenicity are correlated with some m easure of cancer potency. While it could be 1 

### argued that a stronger basis for the correlation ex ists for TIDAL values (based on Ross et al., 2 

### 1995), since the data for this endpoint were collec ted for the compound class of interest (PAHs), 3



### there is no information to determine whether TIDAL values are better predictors of cancer 4 

### potency in PAHs than the other available endpoints, because other endpoints were not examined 5 

### for PAHs as a class. In addition, the measures of cancer and genotoxicity potency used in the 6 

### three papers were different, making comparisons amo ng the three difficult. Based only on the 7 

### analysis conducted by Sanner and Dybing (2005), one might use the slopes and/or correlations 8 

### (Table 3) to rank in vivo genotoxicity endpoints, b ut it is not clear where TIDAL values would 9 

### fit, as these were not analyzed separately. TIDAL values could be categorized with other DNA 10 

### adduct measures assessed by Sanner and Dybing (2005 ); however, Ross et al. (1995) 11 

### demonstrated that adduct levels measured at a singl e time point were uncertain predictors of 12 

### potency compared with TIDAL values. 13 

### Empirical ranking of cancer-related endpoint data. In view of the fact that no published 14 

### studies comparing relative genotoxic potency to rel ative cancer potency were available, and that 15 

### the present work created a large database of RPFs f or multiple endpoints, an empirical approach 16 

### to assigning ranks was also explored. The database of PAH RPFs was analyzed to determine 17 

### whether any individual cancer-related endpoint was more closely correlated with RPFs based on 18 

### tumor bioassay data. The premise behind this analy sis is that RPFs based on bioassay data 19 

### represent the best available information, and that the genotoxicity endpoints that best predict 20 

### bioassay RPFs should be preferred over those that s how little relationship to tumor bioassay 21 

### RPFs. The semiquantitative analysis was, of necess ity, restricted to those PAHs for which at 22 

### least one RPF based on bioassay data was available. 23



### For each of the 22 PAHs with nonzero RPFs based on bioassay data, the average bioassay 24 

### RPF was compared with the average RPF for several e ndpoints that the literature review 25 

### suggested could be correlated with cancer potency ( in vivo DNA adducts, in vivo micronuclei 26 

### and sister chromatid exchanges together, and in vit ro mutagenicity). TIDAL values were not 27 

### analyzed separately from other measures of DNA addu cts because there were only four PAHs 28 

### with both TIDAL and bioassay RPFs; similarly, micro nuclei and sister chromatid exchange 29 

### endpoints were grouped to increase the number of ob servations in the regression. In addition to 30 

### analyzing these endpoints, an analysis of several e ndpoints grouped across class (e.g., all in vivo 31 

### non-bioassay endpoints, all in vitro endpoints, and all non-bioassay endpoints) was performed. 32 

### Linear regression was performed on the log-transfor med average RPF values to assess the 33 

### predictive power of each endpoint or grouping, and to assess whether there was a quantitative 34 

### basis for ordering them. 35 

### Table G-5 shows the results of regression analyses assessing how well the average RPFs 36 

### for several endpoints correlated with average bioas say RPFs. The table shows that neither in 37 

### vivo clastogenicity RPFs (micronuclei and sister ch romatid exchanges) nor in vitro mutagenicity 38

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RPFs were significantly correlated with bioassay RP Fs for the dataset examined here. Among 1 

### those showing a significant ( p < 0.05) linear relationship, in vivo DNA adducts p rovided the best 2 

### correlation (r 2 

### = 0.64), followed by all in vivo non-bioassay endp oints (r 2 

### = 0.54), all non3 

### bioassay endpoints (r 2 

### = 0.43), and all in vitro non-bioassay endpoints ( r 2 

### = 0.39). Although in 4 

### vivo DNA adducts provided the strongest correlation , the slope for this regression was 1.25, 5 

### indicating that RPFs for in vivo DNA adducts system atically underpredicted bioassay RPFs. 6 

### Figure G-1 demonstrates this underprediction; as th e figure shows, most of the average RPF 7 

### values are to the left of the 1:1 correspondence li ne. The slopes for both in vivo non-bioassays 8 

### and all non-bioassays are much closer to 1.0. Plot s showing the average RPF comparisons for all 9 

### in vivo non-bioassays, all non-bioassays, and all i n vitro non-bioassays are shown in Figures G-2 10 

### through G-4. These plots suggest that in vivo non- bioassay RPFs tend to underpredict bioassay 11 

### RPFs, while all in vitro non-bioassays tend toward overprediction. 12

13


### Table G-5. Results of simple linear regression of log–transformed average

genotoxicity RPF vs. log average tumor bioassay RPF

Genotoxicity endpoint R 2 Slope p -Value n

All in vivo DNA adducts 0.64 1.24 <0.01 9 All in vivo non-bioassays 0.54 1.05 0.016 10 All non-bioassay endpoints (in vitro and in vivo) 0 .43 1.03 <0.01 19 All in vitro non-bioassays 0.39 0.91 <0.01 19

All in vivo micronuclei and sister chromatid exchan ges 0.58 0.83 >0.05 (NS) 6 All in vitro mutagenicity 0.047 0.39 >0.05 (NS) 17

14

### G-10 DRAFT – DO NOT CITE OR QUOTE 0.001 0.01 0.1 1 10 100

0.001 0.01 0.1 1 10 100

Average in vivo DNA adduct RPFAverage bioassay RPF

1 

### Figure G-1. Average bioassay RPF vs. average in vi vo DNA adduct RPF. 2

0.001

0.01 0.1 1 10 100

0.001 0.01 0.1 1 10 100

Average in vivo nonbioassay RPFAverage bioassay RPF

3 

### Figure G-2. Average bioassay RPF vs. average in vi vo nonbioassay RPF. 4

5

### G-11 DRAFT – DO NOT CITE OR QUOTE 0.001 0.01 0.1 1 10 100

0.001 0.01 0.1 1 10 100

Average non-bioassay RPFAverage bioassay RPF

1 

### Figure G-3. Average bioassay RPF vs. average nonbi oassay RPF. 2

0.001

0.01 0.1 1 10 100

0.001 0.01 0.1 1 10 100

Average in vitro nonbioassay RPFAverage bioassay RPF

3 

### Figure G-4. Average bioassay RPF vs. average in vi tro non-bioassay RPF. 4

5 

### Based on the results of the linear regression anal yses comparing PAH RPFs calculated 6 

### for genotoxicity endpoints and RPFs calculated for bioassays (Table G-5), an argument could be 7

### G-12 DRAFT – DO NOT CITE OR QUOTE

made for the following ranking: (1) bioassays, (2) in vivo non-bioassays, and (3) in vitro non1 

### bioassays. However, the improvement in correlation that is achieved with subdividing all non2 

### bioassays into in vivo and in vitro endpoints is sm all (r 2 

### improves from 0.43 for all nonbioassays 3 

### to 0.54 for in vivo non-bioassays), and the plot fo r in vivo nonbioassay shows that this grouping 4 

### exhibits a slight tendency to underpredict bioassay RPFs. 5 

### In summary, as with the findings for tumor bioassa y data, the analysis of options for 6 

### ranking cancer-related endpoint RPFs did not sugges t any clear theoretical or empirical basis for 7 

### prioritizing the available data for the purpose of selecting RPFs. Thus, for PAHs without any 8 

### tumor bioassay RPFs but with adequate information t o suggest potential carcinogenicity, the 9 

### cancer-related endpoint data were combined to calcu late a final RPF as described in Section 7. 10

11

12