United States Environmental Protection Agency Office of Research and Development Washington, DC 20460 EPA/600/R-92ra45 December 1991 &EPA Dose-Response Analysis of Ingested Benzo[a]Pyrene (CAS No. 50-32-8) ------- ------- EPA/600/R-92/045 December 1991 DOSE-RESPONSE ANALYSIS OF INGESTED BENZO[a]PYREIME {CAS No. 50-32-8) Human Health Assessment Group Office of Health and Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Washington, DC Printed on Recycled Paper ------- DISCLAIMER This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ------- CONTENTS List of Tables iv Preface v Authors and Reviewers vi 1. INTRODUCTION 1 2. DATA AND IMPLICATIONS FOR DOSE-RESPONSE MODELING 2 2.1. Biological and Mechanistic Data 2 2.2. Empirical Observations 3 3. CALCULATION OF POTENCY SLOPES 4 3.1. Brune et al., 1981 . . .'.. 5 3.2. Neil and Rigdon, 1967 5 3.3. Chouroulinkov et al., 1967 9 4. EVALUATION OF DOSE-RESPONSE MODELS CONSTRUCTED ON THE BASIS OF THE NEIL AND RIGDON DATA . 10 5. DISCUSSION AND SUMMARY . . 15 6. OUTSTANDING ISSUES . 17 7. CONTINUING RISK ASSESSMENT WORK AND RESEARCH NEEDS . 20 Appendix A: Other B[a]P Data . 22 Appendix B: Derivation of Dose-Response Model to Fit Neil and Rigdon Data 26 References . . 28 HI ------- LIST OF TABLES 1. Incidence rates of forestomach and total contact sites (forestomach, larynx, esophagus) tumors in Sprague-Dawley rats exposed to B[a]P in caffeine solution by intubation and in diet mix 6 2. Predicted vs. observed tumor incidence and data used to calculate the dose-response model • • • • 7 3. Potency slopes per (mg/kg/day) for humans calculated from different studies 10 4. Using the Weibull-type model to predict other mouse studies in additional to those in Tables - . 12 5. Using Clement's dose-response model to predict other mouse studies 14 IV ------- PREFACE This report was prepared by the Human Health Assessment Group within the Office of Health and Environmental Assessment (OHEA) to support OHEA's preparation of a Drinking Water Criteria Document (DWCD) on Polycyclic Aromatic Hydrocarbons (PAHs). The DWCDs are prepared for the Office of Ground Water and Drinking Water and the Office of Science and Technology within the Office of Water. The dose-response assessment of PAHs will comprise three steps. The first two steps involve assessing the dose-response relationships of benzo[a]pyrene (B[a]P), one of the PAHs, via ingestion and inhalation. The third step involves the analysis of PAHs other than B[a]P that are commonly found in the environment. This report focuses on the dose-response analysis of ingested B[a]P as there is more information available on B[a]P that is suitable for quantitative assessment than is available for the other PAHs. ------- AUTHORS AND REVIEWERS AUTHORS Chao W. Chen Carcinogen Assessment Statistics and Epidemiology Branch Human Health Assessment Group Office of Health and Environmental Assessment U.S. Environmental Protection Agency Margaret M.L Chu Genetic Toxicology Assessment Branch Human Health Assessment Group Office of Health and Environmental Assessment U.S. Environmental Protection Agency REVIEWERS V. James Cogliano Carcinogen Assessment Statistics and Epidemiology Branch Human Health Assessment Group Office of Health and Environmental Assessment U.S. Environmental Protection Agency Vicki L. Dellarco Genetic Toxicology Assessment Branch Human Health Assessment Group Office of Health and Environmental Assessment U.S. Environmental Protection Agency vi ------- Robert E. McGaughy Human Health Assessment Group Office of Health and Environmental Assessment U.S. Environmental Protection Agency Rita S. Schoeny Methods Evaluation and Development Staff Office of Health and Environmental Assessment Environmental Criteria and Assessment Office U.S. Environmental Protection Agency VII ------- ------- 1. INTRODUCTION This report represents the first part of the Office of Health and Environmental Assessment's dose-response analysis of polycyclic aromatic hydrocarbons (PAHs). Because of the tremendous amount of information bearing on approaches that can be developed to estimate risks from exposures to PAHs, the task is divided into three steps beginning with an analysis of benzo[a]pyrene (BAP), one of the PAHs. The steps are: (1) determining the oral potency slope for benzo[a]pyrene (B[a]P), (2) determining the inhalation potency slope for B[a]P, and (3) determining the potency slopes for other PAHs using a relative potency approach. The focus of this report is on estimating the carcinogenic potency slope of B[a]P by ingestion. The potency slope is used to provide an upper-bound estimate for risk from ingesting low doses of B[a]P. It should be noted that only information relevant to analyzing dose-response relationships by ingestion is analyzed in this report. The determination of potency slopes for B[a]P is chosen as the first two steps because there is more information on B[a]P than other PAHs that are found in environmental media. Furthermore, few PAHs have even minimal information for performing dose-response analysis of oral or inhalation routes of exposure. However, results from skin painting experiments on PAHs where B[a]P is used as positive controls are available. The potential of using B[a]P as a reference point in deriving oral and inhalation potency slopes for other PAHs can be developed. The validity of calculating relative potency between two compounds based on data from one route of exposure and then extrapolated to another route of exposure needs verification. In 1987, ICF-Clement Associates (now known as Clement International Corporation, hereafter referred to as Clement) was contracted to prepare a quantitative risk assessment of PAHs. The report was peer-reviewed at an EPA-sponsored peer-review workshop on May 28- 29, 1988. A substantial part of the Clement report concentrated on constructing a dose- response model using data from Neil and Rigdon (1967) for B[a]P. At the peer-review workshop, a number of recommendations were made by the expert review panel relevant to the estimation of an oral potency for B[a]P. These included: • As the multistage model is currently used by the EPA, it should also be used to calculate potency, and the results should be compared to those calculated by Clement. ------- • Include other B[a]P studies and B[a]P data from studies using B[a]P as a positive control. • Other tumor sites should also be considered. In this report, 14 studies on B[a]P-induced forestomach tumors via the oral route of exposure are reviewed and evaluated. In addition to the potency slope derived from the Clement report, several other estimates are provided, using either different dose-response models on the same data (from the Neil and Rigdon, 1967 study used by Clement) or data from different oral studies. 2. DATA AND IMPLICATIONS FOR DOSE-RESPONSE MODELING Since humans are exposed to mixtures of PAHs, information from human studies for dose-response analysis of B[a]P alone is not available. Tumor induction data from animal studies on B[a]P by ingestion are also limited. Fourteen studies are reviewed here for applicability in the derivation of an oral potency slope. Current reviews concerning biological and mechanistic data were consulted to guide the effort. 2.1. BIOLOGICAL AND MECHANISTIC DATA Biological and mechanistic data that impact the dose-response analysis for determining human risk from ingesting B[a]P include: quantitative biological data (e.g., specific DMA • '• adduct(s) as measures of internal dose; comparative species sensitivity data to guide cross- species extrapolation; absorption, distribution, and elimination kinetics to determine if high dose response is predictive of low dose response; and data from other routes of administration (e.g., skin painting) to derive the ingestion response. Reviews of B[a]P published in the literature (Cooper et al., 1983; Baird et al., 1988; Graslund and Jernstrom, 1989; DiGiovanni, 1989; Nebert, 1988, 1989; McKay et al., 1988; IARC, 1983; National Research Council, 1983; Santodonato et al., 1981) suggest that many factors modulate the biological conversion of B[a]P to its ultimate carcinogenic form. Further, the expression of the carcinogenic potential of B[a]P depends on factors such as species, strain, diet, route and method of administration, and other host-related factors such as the age, sex, and previous and concurrent exposure of the organism to other environmental agents. More than one mechanism may be involved in B[a]P induced tumors. However, the ------- effect of dose, route of entry, and tissue or genetic factors of the organism, which determines if one mechanism predominates or if mechanisms work jointly, is not completely understood. Even though the volume of information on the mechanism of carcinogenic action of B[a]P is large, it does not seem to provide quantitative data that are directly applicable or that can be linked to deriving oral potency estimates. Definitive conclusions can only be made upon a more comprehensive evaluation of the data and integrating the data from other routes of administration. It is clear, however, that the key limitations of the data for quantitative risk assessment are the lack of a quality bioassay for the ingestion route and the lack of quantitative biological data linked to the bioassay. These limitations cannot be bridged by a thorough analysis of the existing data alone. 2.2. EMPIRICAL OBSERVATIONS Fourteen B[a]P ingestion studies (1 in rats and 13 in mice) were reviewed and evaluated for quantitative risk assessment. These studies included long- and short-term B[a]P bioassays and using B[a]P bioassays of other compounds in positive control groups. These data are used either for calculating potency slopes or for evaluating the validity of the model constructed on the basis of the Neil and Rigdon (1967) data. Although its experimental protocol is unconventional and somewhat complicates mathematical modeling, the Neil and Rigdon (1967) study provides useful empirical information on B[a]P because of its variation in exposure duration, dosing pattern, and large number of animals studied. It provides empirical information that usually is not available from long-term bioassays. The main drawback of the Neil and Rigdon study is that, for some dosed groups, only ranges of the ages when exposure began or was terminated are reported, and the interval of these ranges vary. To use these data in dose-response modeling, the midpoint of the range is used as a surrogate for the age exposure began or was terminated. Although only 3 of the 14 studies are appropriate for calculating a B[a]P potency slope, two interesting observations can be made from these data when they are taken together: 1. B[a]P is capable of inducing papillomas from a single oral dose (see Appendix A: Berenblum and Haran, 1955; Neil and Rigdon, 1967; Field and Roe, 1965; Fedorenko and Yansheva, 1967; Roe et a!., 1970; Robinson et al., 1987). However, to induce carcinomas, multiple dosing may be necessary. It should be emphasized here that these observations ------- should not be construed as a real biological mechanism; it is only an empirical observation of animals exposed to B[a]P via the oral route. 2. In the studies cited above, the proportion of animals with tumors (papillomas and carcinomas combined) appears to depend less on the duration of exposure (or total dose) but more on the dose rate and duration of study, irrespective of animals being exposed to B[a]P either from single or multiple doses (at an identical dose rate). The difference between the single exposure and the multiple (longer) exposure is that almost all carcinomas were induced only from multiple dose exposure; single exposure induced only papillomas. For instance, in the Berenblum and Haran (1955) study, 15 animals (out of 19) had carcinomas when animals were exposed to 1.5 mg of B[a]P weekly for 20 to 30 weeks, while no animals (out of 17) had carcinomas when animals were exposed to a single dose (1.5 mg) of B[a]P. However, despite the great difference in the exposure duration and totaldose, the proportion of animals with tumors (papillomas and carcinomas combined) in both experiments are comparable (16/19 vs. 16/17). Based on these empirical observations and the multistage theory of careinogenesis (i.e., initiation-promotion-conversion), the available data seem to suggest that B[a]P has potential for initiation and conversion (from initiated cells to malignancy), without excluding the possibility of tumor promotion, and that a continuous exposure to B[a]P may not be required for initiated cells to grow into detectable tumors. However, the presence of B[a]P may be required for progression to carcinomas. A mathematical model that is consistent with the empirical evidence is presented in Appendix B and is used for dose-response modeling based on the Neil and Rigdon data. 3. CALCULATION OF POTENCY SLOPES Only data from three dietary studies (Brune et al., 1981 [Table 1]; Neil and Rigdon, 1967 [Table 2]; Chouroulinkov et al., 1967) are considered adequate for estimating potency slopes. Other studies are used to evaluate the validity of the dose-response model constructed on the basis of the Neil and Rigdon data. The procedures and data for calculating slopes from each of the three studies are described below. In the calculations, the body surface equivalence assumption is used for interspecies conversion; namely, the carcinogenic effect due to a dose expressed in units of ------- mg per body surface area is assumed to be equivalent between animals and humans. This assumption is recommended in EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986). In the actual calculations, a slope for humans can be derived by multiplying a factor, (70 kg/Wa)1/3, to the animal slope, where Wa is animal body weight in kg, 70 kg is the assumed human body weight, and the animal slope is expressed in terms of (mg/kg/day)"1. 3.1. BRUNEETAL, 1981 The study by Brune et al. (1981) is the only 2-year ingestion study reviewed. B[a]P was administered to Sprague-Dawley rats either as an admixture to the diet or by gavage in an aqueous 1.5% caffeine solution. Combined data from male and female rats are given in Table 1. The linearized multistage (LMS) model was used to calculate the potency slope for B[a]P, assuming that the average body weight for the animals was 0.4 kg. The results are given in Table 3. The study with caffeine solution by intubation produced a larger slope estimate. It is not clear whether or not the larger slope estimate is due to the effects of caffeine or intubation. As will be discussed, there is some possibility that animals exposed to B[a]P by intubation could produce more tumors than animals exposed to B[ai]P in the diet. In the diet mix study (but not in the caffeine solution study), more total contact-site tumors (larynx, esophagus, and forestomach) were induced in males than in females. Although it is possible to separate total tumor incidence rates between males and females, not enough information is provided in the paper to separate the forestomach tumor incidence rates between males and females. In order to derive a male-based potency slope based on the forestomach tumors, a factor of 1.5 is multiplied to the slopes calculated on the basis of combined (male and female) forestomach data. The 50% increase of slope values represents the cancer risk increase when male data (total tumors) are used, instead of combined data. 3.2. NEIL AND RIGDON, 1967 Since animals in the Neil and Rigdon (1967) study were exposed (partial lifetime) to B[a]P during different periods of their lifetimes, a model that reflects this exposure pattern and is consistent with the empirical evidence discussed previously must be developed to fit the dose-response data and to calculate the potency slopes for B[a]P. A Weibutl-type of dose-response model, which can incorporate partial-lifetime exposure, is used to fit the Neil and Rigdon data (Table 2). ------- Table 1. Incidence rates of fbrestomach and total contact sites (forestomach, larynx, esophagus) tumors in Sprague-Dawley rats exposed to B[a]P in caffeine solution by intubation and in diet mix Dose (mg/kg/year) Forestomach tumors Total tumors Median survival Time (days) In caffeine solution (via intubation) 0 6 18 39 3/64 11/64 25/64 14/64 6/64 13/64 26/64 14/64 102 112 113 87a In diet mix 0 6 39 2/64 1/64 9/64 3/64" 3/64b 10/64b 129 128 131 • Data from this group were not used in the calculation because the median survival time was much shorter than for the other groups. No adjustment is possible because time to death data were not available. "The incidence of total tumors in males was 3/32, 3/32, and 8/32, respectively, for control-, low-, and high-dose groups. No sex-specific forestomach tumor data were available. Source: Brune etal., 1981. The probability of observing tumors by age t (days) when animals are administered d (mg/g diet) of B[a]P during the age interval (t^) is given by t>tr Using the above model and data from Table 2, the parameters are estimated by the maximum likelihood method as follows: -q^O, q2 = 2.10x10-5, k = 3.13. ------- Table 2. Predicted vs. observed tumoY incidence and data used to calculate the dose-response model3 ;, : Dose (mg/g diet) 0.000 0.001 0.01 0.02 0.03 0.04 0.045 0.05 0.10 0.25 to (day) 0 30 30 116 50 67 50 20 22 19 tt (day) 300 140 140 226 160 177 160 172 132 137 - ' , t , ..-„•" (day) , 300 140 140 226 160 177 162 172 132 137 Observed" 0/289 (0.00) 0/25 (0.00) 0/24 (0.00) 1/23 (0.05) 0/37 (0.00) 1/40 (0.03) 4/40 (0.10) 24/34 (0.70) 19/23 (0.82) 66/73 (0.90) Predicted incidence rate 0.00 0.00 0.00 0.02 0.04 0.08 0.11 0.30C 0.40° 0.98 a ^ = age of mice (in days) when exposure began, t, = age of mice (in days) when exposure was terminated, t = age of mice (in days) when the study was terminated. Data on the control and the first nine low-dosed groups were taken from Table 1 of Neil and Rigdon (1967). Data from other groups were not included because animals in these groups were exposed to high doses of B[a]P and/or for only very short periods. b Observed number of animals with tumors (incidence rate). 0 The predicted value lies outside the 95% confidence interval calculated from the observed response. This crude test is used to determine whether or not the model reasonably predicts the observed response. This model is equivalent to the linearized-muitistage model that would be used when animals are exposed to B|a]P for their entire lifetimes. The derivation of the model is given in Appendix B. The resultant dose-response model is also used to evaluate the other 12 mouse studies (Table 4). -'.••<...• As indicated in Table 2, the model fits the Neil and Rigdon data adequately for all dose groups except for the two treatment groups (0.05 and 0.1 mg/g B[a]P in the diet). There may be some underlying biological, rather than statistical, reasons to explain the poor fit of these two treatment groups. It is interesting to observe that animals in the three highest-dosed ------- groups, including the two indicated above, were exposed to B[a]P at a much younger age (about 20 weeks) than animals in other groups. The higher than expected tumor response might be due to age-dependent physiological parameters (e.g., a difference in cell division rates in the forestomach tissue between younger and older animals). Further research is needed to confirm or deny this hypothesis. As indicated in Table 4, the model appears to adequately predict the tumor response in mice in most of the studies in which the animals were exposed to B[a]P via dietary intake, but the model does not adequately predict the tumor response in mice exposed to B[a]P via gavage. The gavage studies (most of which are also single-dose experiments) tend to produce a higher tumor incidence than predicted by the model. The Weibull-type model is used to calculate the potency slope at the 10% point by setting ^=0, ^=730, and t=730 days in the model. The 10% point corresponds to d=2.4 ppm diet (equivalent to 0.3 mg/kg/day). The Neil and Rigdon data were also used by Clement to calculate potency slopes. The two slopes proposed in the Clement report are 2.9/(mg/kg/day) (point estimate) and 5.9/(mg/kg/day) (95% upper-bound estimate). The linear term (slope), 2.9/(mg/kg/day), proposed by Clement (1990a), is not used in Table 3 because it is the slope (tangent) at dose zero. When the dose-response curve is not linear at low doses, the tangent at dose zero should not be used as a potency slope for low-dose extrapolation because it is a lower bound, not an upper bound, for the dose-response curve at low doses. In order to obtain the 95% upper bound, Clement found that the point estimate of the cell proliferation parameter b must be reduced (rather than increased or held constant as one would expect) by more than 50% from the point estimate. The value 5.9/(mg/kg/day) is not recommended for two reasons: (1) Not all parameters are included in searching for an upper bound; only two dose-related parameters are included. If background parameters are included, the upper-bound value may be different. (2) It is not biologically reasonable to haye to reduce the cell growth rate by more than 50% in order to get an upper bound. The upper bound for risk is supposed to be calculated by increasing the value of parameters starting from their maximum likelihood estimates until the log-likelihood ratio statistic exceeds a given critical value (which depends on the confidence level specified). From the approach that an upper bound is calculated, one would not expect to obtain an upper bound by decreasing a parameter from the value where the point estimate of the tumor response is obtained. Being unable to obtain an upper bound 8 ------- by starting from parameter values associated with the point estimate of dose, response suggests the peculiarity of the model. It can be demonstrated that a 50% decrease of cell- growth rate can significantly alter the shape of the dose-response model. Note also that the background tumor incidence is estimated to be greater than zero, a consequence of using a nonzero historic background rate of a different strain of mice (SWR/J). CFW mice were used in the Neil and Rigdon study. In this report, the slope is obtained by extrapolating linearly from the 10% point to the background on the fitted dose-response curve. Using this gipproach, two slopes are obtained: 4.5/(mg/kg/day), based on the Weibull-type model, and 9.3/(mg/kg/day), based on the Clement model. These two slopes are presented in Table 3. When the fitted dose-response curve is not linear at low doses, the potency slope can be defined as a secant from a point on the dose-response curve to the point at zero dose. This is a reasonable way to find an upper bound for risk at low doses. A similar concept and approach have been proposed by Krewski et al. (1986, 1991) and Gaylor and Kodell (1980). These investigators found that the potency slopes calculated from this approach are comparable to those obtained from the linearized multistage model for most of the compounds they have studied. 3.3. CHOUROULINKOV ET AL, 1967 This study was selected for slope calculation because it is a long-term study. Mice (strain unknown) were given 8 mg of B[a]P in olive oil mixed in the diet over 14 months. The following gastric tumors were observed; • Dose (mg/kg/day) 0 0.63 Incidence rate 0/81 5/81 (0.062) Since there is only one dosed group, the slope is calculated by direct extrapolation from the observed response (0.062) at 0.63 mg/kg/day to the background rate (0.00): ------- Table 3. Potency slopes per (mg/kg/day) for humans calculated from different studies0 Study Neil and Rigdon (1967) Bruneetal. (1981) Chouroulinkov et al. (1967) Potency slope 4.5 9.3 Remarks Weibull-type Clement (calculated at the 10% point) In caffeine solution via intubation 67.8b 68.7" total tumors forestomach tumor only In diet mix 11.7, (17.8)° 9.5, (14.2)c 6.5 total tumors forestomach tumor only linearly extrapolated from the observed response a The potency slope is used to calculate an upper-bound estimate for risk due to ingesting low doses of B[a]P. b B[a]P was given to animals via intubation. There is some evidence that intubation may increase tumor response: therefore, this value is excluded from the final recommended slopes. c Values in parentheses represent the potency slope estimates based on male rat data alone, by adjusting upward 50% of the slopes calculated on the basis of combined data (male and female) data. See discussion in the text under calculation of potency slopes. (0.062/0.63) x (70 kg/0.03 kg)1/3 x (24 months/14 months)3 = 6.5/(mg/kg/day), where (24/14)3 is a factor to adjust for less-than-lifetime follow-up. 4. EVALUATION OF DOSE-RESPONSE MODELS CONSTRUCTED ON THE BASIS OF THE NEIL AND RIGDON DATA Data from 12 mouse studies are used to evaluate the two dose-response models (the Weibull-type and the Clement models) calculated on the basis of the Neil and Rigdon data. 10 ------- As shown in Table 4, the Weibull-type model appears to adequately predict the tumor response in most of the dietary studies, but is inadequate for most of the intubation studies. In those animals exposed to B[a]P by intubation, B[a]P induced greater tumor incidence than predicted by the model. There are two possible explanations for the discrepancy observed between these two types of studies (dietary vs. gavage): (1) B[a]P by gavage intake induces more tumor responses than dietary intake (i.e., the gavage effect), and (2) the effect of B[a]P continues after termination of exposure (i.e., the B[a]P effect is longer than that implied by the term ^ in the model). Note that it is impossible to separate the two possible effects (i.e., the gavage effect and the long-lasting effect of B[a]P) because all of the gavage studies (except for those by Brune et al. [1981] and Robinson et al. [1987]) are also single dose studies. Considering the empirical evidence observed previously that a single dose of B[a]P is sufficient to induce tumors, one is tempted to conclude that the second reason explains all the discrepancy observed between the two sets of data. However, the second reason cannot totally explain the discrepancy because there are cases in the gavage studies showing comparable to or less than the predicted values. It is possible that both effects are operating. Pharmacokinetic information on B[a]P during and after exposure will be useful in answering the questions about the effect of B[a]P after exposure is terminated. For the Clement model, two approaches are used to calculate predicted values. Despite the fact that it is not reasonable to substitute a different parameter (Approach II in Table 5) after the model has been derived, we have followed the Clement model using two approaches (Clement, 1990a; [Table 8]) to obtain predicted values in Table 5. The first approach assumes that the growth rate for initiated cells during the nonexposed period is the same as the background growth rate. The second approach assumes that the growth rate for initiated cells during the nonexposed period is the same as that during the exposed period. As shown in Table 5, the Clement model (i.e., under Approach I) has predicted values equal to 0 for almost all of the cases where ^ is less than t (i.e., when animals were not sacrificed immediately after exposure was terminated). There seems to be no clear pattern for Approach II. 11 ------- Table 4. Using the Weibull-type model to predict other mouse studies in addition to those in Table 3. Study Dose (mg/g diet) V (days) ti (days) t (days) Observed forestomach tumors Predicted Dietary Studies: ; ; Trioloetal. (1977) Wattenberg (1972) Wattenberg (1974) Chouroulinkov et al. (1967) 0.2 0.3 0.1 0.3 1.0 0.3 0.0032 63 63 70 70 63 63 70 147 147 196 287 147 105 495 147 147 196 287 147 203 495 6/9 (0.66) 9/19 (1.00) 11/20 (0.55) 13/19 (0.68) 12/12 (1.00) 8/20 (0.40) 5/81 (0.06) 0.59 0.86 0.55 0.99a 1.00 ;1.00a 0.04 Gavage Studies: , Berenblum and Haran (1955) Single dose Ei-Bayoumy (1985) Biancifiori et al. (1967) Field and Roe (1965) Single dose Fedorenko and Yansheva (1967) 0.054 0.375 0.14 - 0.023 0.00313 0.0313 0.05 3.6x1 Q-5 3.6x1 0"4 3.6x1 Q-3 3.6x1 0'2 3.6x1 0'1 92 92 63 56 63 63 63 76 76 76 76 76 302 93 91 161 64 64 64 146 146 146 146 146 302 302 175 301 569 569 569 578 578 578 578 578 16/19 (0.84) 16/17 (0.94) 17/20, (0.85) 5/25 (0.20) 5/35 (0.14) 17/109 (0.16) 8/17 (0.47) 0/16 (0.00) 2/26 (0.08) 5/24 (0.20) 23/30 (0.77) 23/27 (0.85) 0.68 0.60a 0.47a 0.24 0.00a 0.01a 0.09a 0.00 0.00 0.03a 0.94a 1.00a 12 ------- Study Robinson et al. (1987) Roe et al. (1970) Benjamin etal. (1988) Dose (mg/g diet) 0.027 0.0125 0.11 to (days) 63 70 71 V (days) 119 71 97 t (days) 274 548 211 Observed forestomach tumoirs 24/36 (0.67) 21/61 (0.34) 42/44 (0.95) Predicted 0.1 6a .0.0.1 a 0.47a a The predicted incidence rate Hes outside the 95% confidence interval constructed from the observed tumor response. 13 ------- Table 5. Using Clement's dose-response models to predict other mouse studies Study Dose (mg/g diet) to (days) V (days) t (days) Observed (days) Predicted8 1 1" Dietary Studies: Trioloetal. (1977) Wattenberg (1972) Wattenberg (1974) Chouroulinkov et al. (1967) 0.2 0.3 0.1 0.3 1.0 0.3 0.0032 63 63 70 70 63 63 70 147 147 196 287 147 105 495 147 147 196 287 147 203 495 6/9 (0.66) 9/19 (1.00) 11/20 (0.55) 13/19 (0.68) 12/12 (1.00) 8/20 (0.40) 5/81 (0.06) 0.98" 1.00 1 .00b 1.00b 1.00 0.13b 0.03 Gavage Studies: Berenblum and Haran (1955) Single dose Ei-Bayoumy (1985) Biancifiori et al. (1967) Field and Roe (1965) Single dose Fedorenko and Yansheva(1967) 0.054 0.375 0.14 0.023 0.00313 0.0313 0.05 3.6x1 0'5 3.6x1 Q-4 3.6x1 0'3 3.6x1 0'2 92 92 63 56 63 63 63 76 76 76 76 302 93 91 161 64 64 64 146 146 146 146 302 302 175 301 569 569 569 578 578 578 578 16/19 (0.84) 16/17 (0.94) 17/20 (0.85) 5/25 (0.20) 5/35 (0.14) 17/109 (0.16) 8/17 (0.47) 0/16 (0.00) 2/26 (0.08) 5/24 (0.20) 23/30 (0.77) 1.00 0.00b 0.00b 0.01b 0.00b 0.00b 0.00b 0.00 0.00 o.oob 0.12" NA NA NA NA NA 1.00b NA NA 1.00 0.1 2b 0.14 0.01 b 1.00b 1.00b 0.00 0.00 0.04b 1.00b 14 ------- Study Robinson et al. (1987) Roe etal. (1970) Benjamin et al. (1988) Dose (mg/g diet) 3.6x1 0'1 0.027 0.0125 0.11 t0 (days) 76 63 70 71 t! (days) 146 119 71 97 t (days) 578 274 548 211 Observed (days) 23/27 (0.85) 24/36 (0.67) 21/61 (0.34) 42/44 (0.95) Pred 1 1.00b 0.00" o.oob o.oob icted" II 1.00b 0.07"° 0.98b 0.46" a I and II are different approaches used to calculate predicted values under two different assumptions: (1) the growth rate for initiated cells during the nonexposed period is the same as the background rate, and (2) the growth rate during the nonexposed period is the same as that during the exposed period. b The predicted incidence rate lies outside the 95% confidence interval constructed from the observed tumor response. c The data (ages at which exposure began and was terminated) used for this prediction differ from that used in Table 8 of the Clement (1990a) report. It is not clear why the difference exists. When age last observed is the same as age last exposed, there is no issue of cell growth rate during the nonexposure period. NA = not applicable. 5. DISCUSSION AND SUMMARY Information from 14 oral studies was integrated to arrive at a quantitative risk assessment for B[a]P. Although most of the studies are not suitable for potency slope calculation, when taken together, they do provide usable information about the dose-response relationship of B[a]P. An important observation is that dose rate and the time elapsed since exposure began are more relevant to tumor (papillomas in the forestomach) occurrence than the total dose given to animals. However, available bioassay data indicate that multiple dosings are required to induce carcinomas in the forestomach. Seven potency slopes are provided in Table 3. The two largest slope estimates come from the Brune et al. (1981) study in which animals were given B[a]P in a caffeine solution via intubation. Since there is some evidence that intubation may increase tumor response, these two slope estimates are excluded from further consideration. The remaining five slope 15 ------- estimates are remarkably similar. If a single value must be adopted, we recommend that some form of mean slope be used. One approach would be to take the geometric mean of the four forestomach-based slope estimates (4.5, 9.3, 14.2, and 6.5; geometric mean = 7.9) calculated on the basis of forestomach tumors. The potency slope based on forestomach tumors is recommended because it is the common tumor site observed in all 14 studies reviewed in this report. In this report, an empirical model is used to fit the Neil and Rigdon data. This model is selected to reflect empirical evidence, rather than biological information. The linearized multistage model, which is also a curve-fitting procedure, is used to calculate potency slope on the basis of the Brune et al. (1981) study. These empirical/curve-fitting procedures are used because there is no sufficient biological information on B[a]P to construct a biologically based dose-response (BBDR) model. The Clement model was developed by adapting a simplified version of the two-stage carcinogenesis model (also known as the MVK model) proposed by Moolgavkar and Vehzon (1979) and Moolgavkar and Knudson (1981). The Clement model invokes an additional assumption that the relative initiation rate (B[a]P-induced divided by spontaneous rate) is equal to that of the second transition (from an initiated cell to a malignant cell) rate. A simplified MVK model can be obtained by assuming that the second transition rate in the exact MVK model is negligible (this implies that the rate is not dose-related). The Clement model, by assuming identical relative transition rates, thus implies that the initiation rate is also independent of dose. Clearly, this implication is not reasonable because B[a]P is known to have initiation potential in many tissues. In general, the use of the simplified MVK model is not recommended for animal data with high tumor response. Chen and Moini (1990) discussed in detail the problems associated with the use of the simplified MVK model. The Clement model is more appropriately described as a curve-fitting procedure. 16 ------- 6. OUTSTANDING ISSUES 1. The selection of tumor site and mouse-to-human dose conversion. Forestomach tumors were used to model the dose-response of B[a]P by ingestion for several reasons: the background incidence for forestomach tumors in mice is low (a rare occurrence), forestomach tumors are found in several studies in which different strains of mice were used, and the studies contained more quantitative information for modeling. Because forestomach tumors in mice are produced as a result of contact at the site of administration, we are concerned about the appropriateness of using this tumor site for extrapolation to human ingestion risks. We are concerned that projecting human oral risk from mouse forestomach tumor data is analogous to using dermal data. Adriaenssens et al. (1983) studied dose-response relationships for binding of B[a]P metabolites to DMA and protein in lung, liver, and forestomach of control and butylated hydroxyanisole-treated female A/HeJ mice. Over an oral dose range of 0.012 to 7.5 mg/mouse, no indication of saturation of DNA B[a]P metabolite binding was observed for forestomach even though it was indicated for lung and liver. Linear dose response for adduct formation was also observed by Pereira et al. (1979) for mouse skin following topical application which spans the range of B[a]P doses of 0.01 to 300 fig/mouse. These observations support the concern of possible similarity between the reaction of forestomach and skin of mice to B[a]P. Is the use of a body surface area scaling factor for extrapolating from mice (site of contact tumors) to humans (site unknown) appropriate? What is the alternative that can be explored? One possible alternative is to use data from other routes of exposure. However, Nebert's review (1989) and earlier studies (Nebert and Jensen, 1979; Legraverend et al., 1983) on lethality, marrow toxicity, and leukemia in Ahb/Ahb and Ahd/Ahd mice from oral exposure to B[a]P indicated the potential for a first-pass elimination kinetics for metabolism. Even though much more information on B[a]P can be obtained from dermal and intratracheal studies, this information suggests that using data from inhalation or dermal exposure to derive an oral potency estimate may be inappropriate. Gould other tumor sites be used? 2. Use of other tumor sites for dose-response analysis. In addition to forestomach tumor in mice, ingestion of B[a]P also induces tumors at distal sites ( e.g., lung and mammary gland). Qualitatively the observation of tumors other than forestomach strengthens the evidence that ingestion of B[a]P could present a 17 ------- carcinogenic hazard for humans. However, the cjuahtitatiye information available is inadequate for meaningful dose-response analysis. For example, the distal tumors observed in the Neil and Rigdon studies (1967) do not show a dose response. Individual animal data • . . •'--,:'• i' '• ... are not available for the forestomach to correct for competing risks. Many of the other studies are single-dose experiments frequently with high tumor incidence (approaching 100%), or the site is associated with high background incidence (e.g., one study showed mammary tumors in the control group of female LEW/Mai rats to be 30%). For these reasons no potency slope is calculated on the basis of these tumor sites. However, it should not be interpreted that other tumor sites are not biologically significant. On the contrary, if quantitative information is available, it may provide insight for risk estimation. For example, Nebert and Jensen's (1979) study of B[a]P-initiated leukemia in mice associated with allelic difference at the Ah locus may contain information for such an analysis. However, their study, as reported,.lacked the necessary detailed information to proceed at this point. 3. Selection of measures for dose in modeling. Based on a number of review articles (DiGiovanni, 1989; Graslund and Jernstrom, 1989; Thakker et al., 1988; Cooper et al., 1983; Conney, 1982; Baird and Preuss-Schwartz, 1988), it is apparent that the metabolism of B[a]P is well studied, and specific DMA adduct levels have been determined in various rodent and human tissues. B[a]P metabolites and DNA adduct formation in the forestomach have been studied (Adriaenssens et al., 1983; loannou et al., 1982) in mice. However, the strains used are different from the studies with quantitative data for dose-response analysis. Can other comparative, quantitative-specific DNA adduct data be used as a measure of internal dose for modeling? The information reviewed thus far seems to indicate that most of the data provide qualitative mechanistic insight but no quantitative measure for dose-response analysis. A large number of studies on PAHs have used dermal assay systems. As stated by DiGiovanni (1989), qualitative metabolic events associated with tumor initiation seem to be similar between human and mouse epidermal cells. But it is not clear how to relate a quantitative parameter, such as a specific DNA adduct level, to tumor formation in the two species. 4. Range of human susceptibility to B[a]P carcinogenesis. • Calabrese (1988) compared data on induced AHH activities and DNA binding of hydrocarbons between various tissues in humans and rodents. In general, humans have a 18 ------- wider range of individual variation on these parameters, but the values seem to fall within the range observed in rodents of different sensitivity. The results of Autrup's (1990) analysis of data from cultured human tissues and cells versus rodent tissue/cell culture seem to indicate no qualitative difference in the metabolism of humans compared to that of animals, but the binding of B[a]P to DNA is higher in humans. Nebert's (1989) review of the Ah locus and genetic differences in toxicity and cancer highlighted the importance of genetic determinants of PAH toxicity and carcinogenicity. The Ah locus, localized to mouse chromosome 12, encodes a cytosolic receptor which regulates cytochrome P1-450 induction that mediates hydrocarbon metabolism. In Nebert and Jensen's experiment (1979), mice that were homozygous for the high-affinity Ah receptor (with alleles Ahb/Ahb or an Ah responsive phenotype), when exposed to B[a]P orally, developed no leukemia. Mice that were homozygous for the low-affinity Ah receptor (with alleles Ahd/Ahd or an Ah nonresponsive phenotype), when similarly exposed, developed leukemia. An estimated oral dose of 120 mg/kg-day for 10 days is lethal to the (Ah"/Ahd) homozygote (100%). Time to death was shortened as dose increased. The responsive strains were unaffected for up to 6 months of exposure. Nonresponsive mice also developed marrow toxicity from oral B[a]P. Nebert suggest that these data, taken together, are consistent with "first-pass elimination" kinetics. Other experiments indicated that responsive mice develop site-of-contact tumors when exposed dermally or orally, while nonresponsive mice develop thymoma/leukemia. What are the contributions of the Ah locus-mediated pharmacokinetic factors and target organ susceptibility in the expression of carcinogenicity? What is the range of human susceptibility to B[a]P carcinogenicity and other toxicities when ingested? Humans are polymorphic with respect to the Ah locus. Some human studies (Kellermann et al., 1978) have implicated the involvement of the Ah locus and human lung cancers. Which animal models can be used to bound the range of human susceptibilities? 5. Duration of exposure and concurrent exposure to dietary PAHs and other factors. Thus far, this analysis raises questions about the pattern of exposure on the effect of B[a]P-induced tumor progression: the effect of terminating exposure, the effect of caffeine intake, and the effect of loading dose (e.g., of B[a]P intake by gavage or intubation). These patterns can potentially be mimicked by humans through differences in lifestyle. Dietary ingestion constitutes the major route of human exposure to PAHs. Estimates by Santodonato et al. (1981) suggest that dietary exposure could be more than 100 times 19 ------- higher than drinking water as a source of human exposure. What is the site of carcinogenic action of PAHs on humans when ingested? Humans are exposed to PAHs as a group of mixtures with variable composition. Skin painting studies have indicated interactions. A study by Robinson et al. (1987) in A/J mice of particulates of a coal tar paint used in potable water systems suggested the potential for interaction via the oral route. Should we set standards using data from individual PAHs one at a time? 7. CONTINUING RISK ASSESSMENT WORK AND RESEARCH NEEDS This quantitative risk assessment of B[a]P focuses on the use of available route-specific information to estimate potential human carcinogenic risks. Because of the multiple mechanisms in which B[a]P can induce cancers, and because of the genetically determined detoxification/toxification pathways that influence the ultimate site and magnitude of carcinogenic action, this analysis has brought to light the need for a more comprehensive analysis. Since other PAHs found in drinking water have a more limited data base than B[a]P, the Agency needs to develop other approaches (e.g., a relative potency approach) to assess the risks of PAHs other than B[a]P. Some preliminary work has been attempted (Chu and Chen, 1984; Clement, 1990a, 1990b; Richard and Woo, 1990), however, validation of any relative potency approach is needed before adaptation. This opinion was also expressed by the panelists at the 1988 EPA-sponsored workshop on PAHs. It became apparent during the literature search for preparing this report that much information is available on 3-methylcholanthrene (3-MC) and dimethylbenzanthracene (DMBA). Even though these two compounds are not included in the Drinking Water Criteria Document for Polycyclic Aromatic Hydrocarbons, evaluations of these compounds can serve to validate relative potency approaches. This is not to underscore the fact that a key limitation of the data is the lack of quality bioassay and biological/toxicological data which can be used for dose-response analysis. Research that focuses on providing such data may be needed. The Electric Power Research Institute is in the process of conducting bioassays by ingestion of manufactured gas plant residues using B[a]P as a positive control focusing on providing data for dose-response analysis. When the results of their study become available (most likely in about 5 years) it will 20 ------- provide additional dose-response information, which coupled to a comprehensive analysis of the large volume of mechanistic information, will help to reduce the uncertainties of extrapolating from animals to humans. From the viewpoint of dose-response modeling for B[a]P, a carcinogenesis bioassay (e.g., one that includes the study of the number and size of papiHomas and carcinomas over time with serial sacrifice) can be useful for constructing a dose-response model that reflects the dynamics of tumor formation and progression. Such a bioassay, when coupled with quantitative pharmacokinetic and biological information, would be useful for improving low- dose extrapolation. Furthermore, comparative quantitative metabolic and pharmacokinetic data on B[a]P between test species and humans by ingestion would be particularly useful. jf B[a]P were present in drinking water at 0.03 iig/L, the highest detection limit value reported in the National Organic Monitoring Survey (U.S. EPA, 1989), daily human exposure to B[a]P from water would be several orders of magnitude lower than levels used in bioassays. To probe the dose-response characteristics closer to levels of human exposure, molecular parameters linkable to tumor formation are needed; the use of specific DNA adducts has the potential. Using the enzyme-linked immunosorbent assay (ELISA) technique, increased specific DNA adduct levels have been identified in peripheral white blood cells of human volunteers ingesting charcoal-broiled beef (Rothman et a!., 1990). Autrup's (1990) commentary on carcinogen metabolism in cultured human tissues and cells suggested a weak association between the carcinogenic potency of PAHs in experimental animals and the level of PAH-binding to human bronchial DNA. The major B[a]P-DNA adducts were the same in all cultured tissues and cells from hurnans and experimental animals, and the same metabolic profile was seen when rodent and human tissues were incubated with B[a]P. The effects of diet on levels of PAH-DNA adducts were measured (Kwei and Bjeldanes, 1990; O'Neill et al., 1990a, b). The composition of the diet seems to modulate the type and level of specific adducts formed. For in vivo PAH-DNA adduct data to be useful, factors, such as diet as a modulating factor, has to be the same for carcinogenesis studies. 21 ------- APPENDIX A OTHER B[a]P DATA The following data are extracted from B[a]P bioassay studies and non-B[a]P bioassay studies (positive control groups). 1. Trioloetal., 1977. Nine-week-old Ha/ICR mice were fed B[a]P for 12 weeks and sacrificed. B[a]P (mg/g diet) 0 0.2 0.3 Estimated dose (mg/kg/day) 0.0 27.3 41.0 Proportion of mice with forestomach tumors (%) 0/9(0%) 6/9(66.6%) 9/9(100%) 2. Wattenberg, 1972. Ten-week-old Ha/ICR mice were fed B[a]P in the diet. B[a]P (mg/g diet) 0.1 0.3 1.0 Estimated dose (mg/kg/day) 14.3 41.0 Weeks in study 18 20 12.0 Proportion of mice with forestomach tumors (%) 1 1/20 (55%) 13/19 (68%) 12/12 (100%) 3. Ei-Bayoumy, 1985. Nine-week-old CD-1 mice were given 1 mg of B[a]P by gavage two times/week for 4 weeks and then followed for 12 weeks. Proportion of mice with forestomach tumors: 17/20 (0.85%). 4. Biancifiori et al., 1967. A positive control in a study of the action of oestrone in intact and ovariectomized BALB/c/CB/SE mice. B[a]P or DMBA dissolved in almond oil at a concentration of 0.5%: 0.1 22 ------- ml_ (0.5 mg) was administered by stomach tube twice weekly for 15 weeks when mice reached 8 weeks of age. Results in the B[aJP group: 5 out of 25 mice had squamous carcinomas of the forestomach. Tumors occurred 28 to 65 weeks post-treatment. Mean age was about 43 weeks. 5. Berenblum and Haran, 1955. Age study began: about 3 months old. Study 1: 0.3 mL of 0.5% of B[a]P in polyethylene glycol-400 was given by stomach tube to C3H male mice weekly for 30 weeks. Papillomas Carcinomas 1/19 15/19 Study 2: Single dose was given to male mice as in Study 1 above. Papillomas Carcinomas 16/17 0/17 6. Field and Roe, 1965. The following data are taken from positive control groups in a study that investigated the effect of citrus oils on forestomach tumors. A single dose of B[a]P was given by stomach tube to Albino mice. Only mice that survived 60 days were included in the following table. Animals were killed at 569 days. Forestomach* , Dose (|ig) (No. of tumors/animal) 12.5 50.0 200.0 all papillomas. 5/17 17/109 8/17 23 ------- 7. Wattenberg, 1974. This is a positive control group in a bioassay studying the effect of sulfur-containing compounds on B[a]P-induced neoplasms of the forestomach in Ha/ICR mice. Nine-week-old mice were given 0.3 mg/g of B[a]P in the diet for 6 weeks and were sacrificed after 20 weeks in the study. Eight out of 20 animals had forestomach tumors. 8. Fedorenkoetal., 1967 B[a]P was introduced directly (by syringe probe) into the stomach of animals (2- to 3- month-old white mice) weekly for 10 weeks in 0.2 mL B[a]P solution in triethyleneglycol. The experiment lasted for 19 months. Tumors in the antrum of the stomach (the denominator is the number of animals at risk at the occurrence of the first tumor in the study) are given below. Dose (mg) for 1 0 times 0.001 0.01 0.1 1.0 10.0 Carcinomas 0/16 0/26 0/24 11/30 16/27 Papillomas 0/16 2/26 5/24 12/30 7/27 9. Robinson etal., 1987. This is a positive control in a coal tar paint study. Animals (8-week-old A/J mice) were treated with B[a]P for a total of 6 mg in 8 weeks and sacrificed at 9 months of age. Dose (mg/mouse) total dose (mg) Forestomach Papillomas carcinomas Total Lung 0 6 0/36 24/36 0/36 22/36 33/36 10/36 22/36 24 ------- 10. Roeetal., 1970. Fifty jig of B[a]P in 0.2 mL of PEG-400 was given by intragastric instillation to female Swiss Albino mice (9 to 14 weeks of age). Tumor incidence for animals examined at autopsy at 18 months is given below. Dose (ng) (single) 0 50 Forestomach Pap Car 2/65 20/61 0/65 1/61 Lung 15/65 18/61 Hepatomas Maiignant lymphomas 5/65 9/61 3/65 0/61 11. Benjamin etal., 1988. These are two positive controls in a study of reduction of B[a]P-induced forestomach neoplasms in ICR mice given nitrite and dietary soy sauce. Mice were given eight doses of B[a]P (1.5 mg, twice weekly for 4 weeks) starting at age 71 days and killed at 211 days of age. Exp. 1: 23/24 (96% or 5.2+-0.7 neoplasms/mouse). Exp. 2: 19/20 (95% or 5.8+-1.1 neoplasms/mouse). 25 ------- APPENDIX B DERIVATION OF DOSE-RESPONSE MODEL TO FIT NEIL AND RIGDON DATA An empirical model used to fit data from Neil and Rigdon (1967) is presented below. This model is selected to reflect empirical evidence rather than biological information. There is no sufficient biological information to construct a biologically based dose-response model. Derivation of Empirical Dose-Response Model: Assume that the hazard rate for tumor occurrence is given by h(t) = h0(t) + Mt), where h0(t) = atk"1 is the background rate of tumor occurrence and the dose-induced hazard rate is t h,(l)= / (t-sf2 D(s)ds, 0 where D(s)-f(d) = a,d + a2d2 + ... + amdm for some positive integer m if to < s < t,, and D(s) = 0 otherwise: d is dose given to animal at time s. The dose-related hazard function h^t) implies that the earlier B[a]P exposure contributes more to the hazard rate than the later exposure. This assumption is consistent with the observation that a single dose is sufficient to induce tumors if the follow-up period is long enough. 26 ------- The cumulative hazard by time T for background is given by Hn(t) = / at Mdt The cumulative hazard by time T>^ for the B[a]P-induced hazard is given by T H.fT) = .h 0 ^ -• tj" T = J'h1(t)dt+ / hi(t)dtt'+ I h^tjdt 0 ".to . t, T t = f(d) (t-s)k-2dsdt + f(d) (t-s)k-2ds dt to t, to Therefore, the probability of getting cancer by time t given that animals are exposed to B[a]P only during the time interval [t, t has the form P(d,t) = 1 - expHdo + q,d + .:. + qmdm)[(t-gk - (t- A quadratic model with m = 2 is adequate to fit the Neil and Rigdon data. 27 ------- REFERENCES Adriaenssens, P.I.; White, C.M.; Anderson M.W. (1983) Dose-response relationships for the binding of benzo[a]pyrene metabolites to DNA and protein in lung, liver, and forestomach of control and butylated hydroxyanisole-treated mice. Cancer Res. 43:3712-3719. Autrup, H. 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