------- DRAFT EPA-600/8-82-004FA DO NOT QUOTE OR CITE Review Draft ADDENDUM TO THE HEALTH ASSESSMENT DOCUMENT FOR DICHLOROMETHANE (METHYLENE CHLORIDE) Updated Carcinogen Assessment of Dichloromethane (Methylene Chloride) NOTICE THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by the U.S. Environmental Protection Agency and should not at this stage be construed to represent Agency policy. It is being circulated for comment on its technical accuracy and policy implications. Office of Health and Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Washington, D.C. ------- ------- CONTENTS Authors, Contributors, and Reviewers •• ... • ,iv 1. SUMMARY AND CONCLUSIONS. . . . ..... . . 1 1.1. SUMMARY. .. .... . . . . . . . . . . . . . 1 1.1.1. Qualitative Assessment ... 1 1.1.2. Pharmacokinetics/Metabolism. . 4 1.1.3. Quantitative Assessment 5 1,2. CONCLUSIONS. . . . . . . . . . . . 8 2. INTRODUCTION ........ 10 3. CARCINOGENICITY .11 3.1. NATIONAL TOXICOLOGY PROGRAM INHALATION BIOASSAY (1985, DRAFT). . 11 3.1.1. Rat Study . . . . . . . . . . 12 3.1.2. Mouse Study. . . . . ;. . . . 23 3.1.3. Summary . . . . . . ... 32 3.2. PHARMACOKINETICS/METABOLISM . . . . . . ....... 35 3.2.1. In Vitro Metabolism/Pathways . . ... ... . .35 3.2.2. Tissue Distribution . 38 3.2.3. In Vivo Metabolism/Effect of Dose 39 .3.2.4. Human Studies . . . . ... . . ... . . .48 3.2.5. Summary 51 4. .QUANTITATIVE ESTIMATION (USING THE NTP INHALATION BIOASSAY) 57 4.1. SUMMARY OF NTP FINDINGS USED FOR QUANTITATIVE ANALYSIS .... 57 4.2. DOSE-RESPONSE MODEL SELECTION 60 4.3. APPLICATION OF THE MULTISTAGE MODEL TO NTP BIOASSAY DATA ... 61 4.4. RISK ANALYSIS CONSIDERING TIME-TO-TUMOR INFORMATION 67 4.5. COMPARISON OF RISKS ESTIMATED WITH OTHER DOSE-RESPONSE MODELS V 73 4.6. COMPARISON OF NTP (1985) RESULTS WITH OTHER BIOASSAYS . . . .81 4.7. DERIVATION OF HUMAN UNIT RISK ESTIMATES FOR INHALATION OF DCM . . . . . . / 85 4.8. HUMAN UNIT RISK ESTIMATE FOR INGESTION OF DCM .89 4.9. COMPARISON OF ANIMAL AND HUMAN DATA RELEVANT TO CANCER RISK . . 92 REFERENCES . . . . . . . . . 95 iii ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS The Carcinogen Assessment Group within the Office of Health and Environ- mental Assessment was responsible for preparing this document. PRINCIPAL AUTHORS Dharm V. Singh, Ph.D. Chapters 1 and 3 Hugh L. Spitzer, B.S. Chapters 1, 2, and 3 Paul D. White, B.A.1 Chapters 1 and 4 PARTICIPATING MEMBERS Roy E. Albert, M.U., Chairman Steven Bayard, Ph.D. David L. Bayliss, M.S. Robert P. Beliles, Ph.D. Chao W. Chen, Ph.D. Arthur Chiu, Ph.D., M.D. Margaret M.L. Chu, Ph.D. Herman J. Gibb, B.S., M.P.H. Bernard H. Haberman, D.V.M., M.S. Charalingayya B. Hiremath, Ph.D. James W. Holder, Ph.D. Robert E. McGaughy, Ph.D., Acting Technical Director Jean C. Parker, Ph.D. William E. Pepelko, Ph.D. Charles H. Ris, P.E., Acting Executive Director Todd W. Thorslund, Ph.D. ^•Exposure Assessment Group, Office of Health and Environmental Assessment, IV ------- .1. SUMMARY AND CONCLUSIONS 1.1. SUMMARY 1.1.1. Qualitative Assessment There have been eight chronic studies in which dichloromethane (methy- lene chloride, DCM) was administered to animals: five in rats, two in mice, and one in hamsters. The Dow Chemical Company (1980) reported the results of chronic inhalation studies in rats and hamsters. There was a statistically significant increased incidence of ventral cervical sarcomas, probably of the salivary gland, consisting of sarcomas only, and appearing in males but not in females. In addition, the study showed a small increase in the number of benign mammary tumors compared to controls in female rats at all doses and in male rats at the highest dose. In hamsters, there was an increased incidence of lymphosarcoma in females which was not statistically significant after correction for survival. In a second inhalation study, the Dow Chemical Com- pany (1982) reported that there was no increase in compound-related tumors in rats; however, the highest dose used in this study was far below that of the previous study. The National Coffee Association (1982a, b) conducted a study in which Fischer 344 rats and B6C3F1 mice were exposed to DCM in drinking water. The results indicated that Fischer 344 rats had an increased incidence of neoplastic nodules and/or hepatocellular carcinomas in female rats, which was significant with respect to matched controls; however, the incidence was within the range of historical control values at that laboratory. The National Coffee Association (1983) drinking water study in B6C3F1 mice also showed a borderline response of combined neoplastic nodules and hepatocellular carci- nomas. The National Toxicology Program (1982) draft gavage study on rats and mice has not been published due to data discrepanci.es; however, usable infor- 1 ------- mation from the gavage studies has been incorporated by the NTP into the inha- lation bioassay (1985, draft). The recently released NTP (1985) inhalation bioassay concluded that "there was some evidence of carcinogenicity of dichloromethane for male F344/N rats as shown by increased incidence of benign neoplasms of the mammary gland. There was clear evidence of carcinogenicity of dichloromethane for female F344/N rats as shown by increased incidence of benign neoplasms of the mammary gland. There was clear evidence of carcinogenicity of dichloromethane for male and female B6C3F1 mice, as shown by increased incidences of alveolar/ bronchiolar neoplasms and of hepatocellular neoplasms." There are some other inadequate animal studies in the literature. One study (Theiss et al., 1977) reported a marginally positive pulmonary adenoma response in strain A mice injected intraperitoneally with DCM. Two negative animal inhalation studies were judged to be inadequate because they were not carried out for the full lifetime of the animals (Heppel et al., 1944; MacEwen et al., 1972). Positive results in a rat embryo cell transformation study were reported by Price et al. (1978). The significance of their findings with regard to carcinogenicity is not well understood at the present time. The epidemiologic data consist of two studies: Friedlander et al. (1978), updated by Hearne and Friedlander (1981), and Ott.et al. (1983a, b, c, d, e). Although neither study showed excessive risk, both showed sufficient deficien- cies to prevent them from being judged negative studies. The Friedlander et al. study (1978) lacked a large enough exposure (based on animal cancer potency estimates) to provide sufficient statistical power to detect a potential carci- nogenic effect. The Ott et al. (1983a, b, c, d, e) study, among other defici- encies, lacked a sufficient latency period for site-specific cancer. ------- The NTP (1985) inhalation bioassay of DCM was conducted in male and female F344/N rats and B6C3F1 mice. The animals were exposed at concentra- tions of 0, 1000, 2000, and 4000 ppm for rats and 0, 2000, and 4000 ppm for mice, 6 hours/day, 5 days/week, for 102 weeks. There was an increased inci- dence of benign mammary gland neoplasms, primarily fibroadenomas, in both male and female rats. In female rats there was a significant increase in hepato- cellular neoplastic nodules and hepatocellular carcinomas (combined) by the trend test only. There was also a statistically significant increase of mono- nuclear cell leukemias in female rats by age adjustment. In male rats there was a significant increase in mesotheliomas, primarily in the tunica vagina- lis. Lastly, a marginally significant increase was noted in adrenal pheo- chromocytomas and interstitial cell tumors in male rats and pituitary gland adenomas and carcinomas combined, in male and female rats by the trend test only. In the study using B6C3F1 mice, there was a highly significant increase in alveolar/bronchiolar adenoma and/or carcinoma in both sexes of mice. The incidence of hepatocellular adenoma and hepatocellular carcinoma combined was increased in the high-dose male group and in both dosed groups of female mice. It should be noted that there was also a dose-related increase in the number of mice bearing multiple lung and liver tumors. The control mice had no more than one lung tumor per mouse, whereas 38% of all dosed males and 42% of all dosed females had multiple lung tumors. The incidence of multiple hepatocellular tumors in the exposed groups increased in both sexes in a dose-related manner. Multiple hepatocellular tumors were found in only 4% of the male controls, and none were found in the female controls. In contrast, 28% of the males and 32% of the exposed females exhibited multiple liver tumors. The NTP concluded that, under the conditions of this bioassay, there was ------- some evidence of the carcinogenicity of DCM for male F344/N rats as shown by an increased incidence of benign neoplasms of the mammary gland. There was suffi- cient or clear evidence of the carcinogenicity of DCM for female F344/N rats as shown by an increased incidence of benign neoplasms of the mammary gland. There was sufficient or clear evidence of carcinogenicity in male and female B6C3F1 mice as shown by increased incidences of lung and liver tumors. 1.1.2. Pharmacoki neti cs/Metabo.1 i sm The available data have been analyzed to determine if there are quali- tative or quantitative metabolic differences or similarities between species which may alter the assumptions '.used in estimating the carcinogenic risk arising from exposure to DCM. The results of both ui vitro and in vivo studies indicate that DCM is metabolized via two pathways. One pathway yields carbon monoxide as an end product, and the other pathway yields carbon dioxide as an end product with formaldehyde and formic acid as metabolic intermediates. Each pathway involves formation of a metabolically active intermediate that is theoretically capable of irreversibly binding to cellular macromolecules. A comparative analysis of the capability of various tissues to metabolize DCM indicates that the liver is the primary site of metabolism, with some metabolism taking place in the lung and kidney. An analysis of the available in vivo data suggests that when rats or mice are exposed to high concentrations of DCM they exhale more carbon dioxide and excrete more formic acid than carbon monoxide. At exposure to low concentrations of DCM, both pathways appear to be utilized about equally. At the present time the implications of these observations in assessing the car- cinogenic potency of DCM are unclear. A comparative analysis of the data from in vivo studies in mice, rats, and humans indicates that all three species metabolize DCM to carbon monoxide. ------- Both mice and rats metabolize DCM to carbon dioxide. There are no human data on the metabolism of DCM to carbon dioxide. However, based on uptake data, some investigators have speculated that this pathway is functional in humans. At present, the available data are insufficient for the purpose of esti- mating doses at which metabolism is saturated. The data indicate that, at low doses, little unmetabolized DCM is exhaled. At high doses there is a significant exhalation of DCM immediately post-exposure. The available data do suggest that at high doses more DCM is taken up into the body. Currently, the data are insufficient to determine the relationship between exposure con- centration and uptake. Based on this analysis it is concluded that the avail- able data do not offer useful parameters for modifying the assumptions used in the calculation of the carcinogenic unit risk of DCM. 1.1.3. Quantitative Estimation In the previous carcinogenicity evaluation of DCM, a quantitative estimate for the upper-bound incremental unit risk was developed on the basis of sali- vary gland region tumors seen in an inhalation study with male rats (U.S. EPA, 1985). The upper-bound estimate of incremental unit risk has been re-evaluated using the results of the NTP inhalation bioassay (NTP, 1985). The risk calcu- lations presented here are based primarily on the NTP findings of carcinogeni- city to the liver and lung in male and female mice. The elevated mammary tumor incidence in female rats and mammary and subcutaneous tumor incidence in male rats were also used for risk analysis. In mice, both separate and combined analyses were conducted for benign and.malignant tumor types. Risk calcula- tions were made for mice developing either the lung or liver tumors in order to indicate the total risk associated with tumors of these two organs. There were no adequate metabolic or pharmacokinetic data to support any modifications to ------- the experimentally applied doses. Thus, risk calculations were based on the experimentally applied doses (in ppm using the study dose schedule), with sub- sequent adjustment to estimate human equivalent doses and risks. The multi- stage dose-response model, as incorporated in the GLOBAL83 computer program (with the number of terms restricted to the number of experimental dose groups minus one), is the primary model utilized in the analysis. Both maximum like- lihood estimates (MLE) and 95% upper confidence limit (UCL) values for risk are given. The multistage model was found to provide an adequate fit to the experi- mental data for the tumor sites, tumor pathology types, sexes, and species groups examined. The highest estimate of risk was obtained from the UCL value for combined adenoma and carcinoma response in the lung and/or liver of female mice. To provide comparison with the basic multistage risk estimates, addi- tional calculations were made with other risk estimation approaches and models using the data on mice having lung or liver tumors. An analysis which excluded animals that died before the first tumors developed produced similar risk estimates (results were within 10% for female mice with lung and/or liver adenomas and carcinomas combined). A time-to-tumor analysis using the multistage model, as formulated in the WEIBULL82 computer program, was also applied to the data to determine if the inclusion of a time term would influence risk estimates. The time-to-tumor estimates for the UCL of risk at low dose were generally in good agreement with the multistage model. The probit and dichotomous Weibull models, in both background-independent and background-additive formulations (using the RISK81 computer program), were applied to the mouse data for comparison with the multistage model. For combined lung and liver tumors in female mice, the background-additive formula- ------- tions of both the probit and Weibull models are in good agreement with the multistage model. The background-independent formulations of the probit and Weibull models lead to much lower risk estimates. The quantitative risk estimates developed from the NTP inhalation bio- assay data were compared for consistency with findings in earlier long-term bioassays of DCM conducted by the Dow Chemical Company and the National Coffee Association. These studies provide some evidence of DCM-induced tumors con- sistent with the NTP findings. Multistage model UCL calculations using the results from these studies are comparable to, and in some cases exceed, estimates for respective tumor sites in the NTP study. In addition, the Dow inhalation study in rats showed an increase in tumors of the salivary gland region; the multistage UCL risk estimates for mammary tumors in the NTP female rats (the highest risk finding for the NTP rats) exceed the corresponding risk estimate based on the salivary tumors by a factor of three. Equivalent human dose and upper-bound incremental unit risk estimates were developed using the standard assumptions of the Carcinogen Assessment Group on the inhalation rates of rodents and humans, and use of the body weight, to the two-thirds power interspecies extrapolation factor. Using the multistage UCL estimates for female mice with either adenomas or carcinomas of the lung and/or liver, the upper-limit incremental unit risk from exposure over a lifetime to 1 mg/kg/day DCM is 1.4.x 10~2. Equivalently, the unit risk for inhaling 1'yg/m^ DCM over a lifetime, is 4.1 x 10"^; the unit risk for exposure to 1 ppm DCM is 1.4 x 10~2. Estimates of the incremental unit risk from exposure to DCM in drinking water were made using two approaches: first, based on the findings of liver, but not lung, tumors in the NTP inhalation bioassay with mice, and secondly, using.the suggestively positive finding of liver tumors in the National Coffee 7 ------- Association (1983) ingestion study in mice. Since the risk estimates from these two studies are roughly comparable, the mean of the derived risk values is chosen for the unit risk estimate via ingestion. Using the mean of the UCL risk calculations from these two studies, consumption of drinking water con- taining 1 \i g/L DCM over a lifetime has an associated upper-bound incremental unit risk estimate of 2.1 x 10'?. The upper-bound incremental unit risk for inhalation exposure estimated using the NTP bioassay was compared with the findings of the strongest epide- miologic study of workers exposed to DCM. While the epidemiologic study did not show any evidence for the carcinogenicity of DCM, power calculations showed that the study also did not have the power to detect the estimated increase with any degree of confidence. 1.2. CONCLUSIONS Animal studies showed a statistically positive salivary gland sarcoma re- sponse in male rats (Dow Chemical Company, 1980) and a borderline hepatocell- ular neoplastic nodule response in female rats (National Coffee Association, 1982a, b). There is some evidence of the carcinogenicity of DCM in male rats, as shown by an increased incidence of benign mammary gland neoplasms; and clear evidence in female rats (Dow Chemical Company, 1980; Burek et al., 1984; NTP, 1985). There is clear evidence for the carcinogenicity of DCM in male and female mice, as shown by statistically significant increased incidences of alveolar/bronchiolar neoplasms and hepatocellular neoplasms (NTP, 1985 draft). There is also evidence that DCM is weakly mutagenic. Using the pro- posed EPA guidelines for carcinogen risk assessment (U.S. EPA, 1984), the weight-of-evidence ranking for the carcinogenicity of DCM in experimental animals is "sufficient," and for human evidence the ranking is "inadequate." Overall, an EPA category of B2 is assigned to DCM, meaning that DCM is to be 8 ------- considered a "probable" human carcinogen. According to the criteria of the International Agency for Research on Cancer (IARC), the weight-of-evidence for the carcinogenicity of DCM in animals is "sufficient," placing it in Group 2B. The upper-bound incremental unit risk for the inhalation of air contaminated with DCM is 4.1 x 10~6 (u g/m3)"1 -[B2]. The upper-bound incremental unit risk for drinking water is 0.21 x 10~6 (ug/L)"1 -[B2]. The CA6 potency index for DCM is 1.2 (mmol/kg/day)"1, which places DCM in the lowest quartile of the ranking of the chemicals that the CAG has evaluated as suspect carcinogens. ------- 2. INTRODUCTION In February of 1985 the Office of Health and Environmental Assessment published a Health Assessment Document on Dichloromethane (Methylene Chloride). The document, which contained an analysis of evidence for the carcinogenic potential of dichloromethane (DCM), concluded: "Using the criteria of the International Agency for Research on Cancer (IARC), the weight of evidence for carcinogenicity in animals is judged to be limited. . . . based upon the statistically positive salivary gland sarcoma response in male rats (Dow Chemical Company, 1980) and the borderline hepatocell.ular neoplastic nodule response in the rat and hepatocellular adenoma and/or carcinoma in male mice (National Coffee Association, 1982-1983)." It was further concluded: "When the absence of epidemiological evidence is considered along with the limited animal evidence, as well as the potential for DCM to cause gene mutations in mammalian systems, DCM is judged to be in IARC Group 3 ... ." Because the National Toxicology Program (NTP) inhalation bioassay has been completed (NTP, 1985, draft) and the report has been reviewed and approved by the NTP Board of Scientific Councillors, it is necessary to update the February 1985 Health Assessment Document for Dichloromethane (Methylene Chloride). The purpose of this addendum is to: o Review and integrate the data obtained in the NTP inhalation bioassay, » Analyze the pharmacokinetic/metabolic data presented in Chapter 4 of the Health Assessment Document and determine its usefulness in the quantitative estimation of carcinogenic risk, and 0 Revise the estimated carcinogenic potency for DCM using the data from the NTP bioassay and pharmacokinetic data if appropriate. 10 ------- 3. CARCINOGENICITY 3.1 NATIONAL TOXICOLOGY PROGRAM INHALATION BIOASSAY (1985, DRAFT) A 2-year carcinogenesis study of DCM (99% pure) was conducted at Battelle Pacific Northwest Laboratories by inhalation exposure to groups of 50 male and female F344/N rats and B6C3F1 mice (6 hours/day, 5 days/week) for 102 weeks. The exposure concentrations used were 0, 1000, 2000, or 4000 ppm for rats and 0, 2000, or 4000 ppm for mice. These doses were selected on the basis of re- sults obtained from a 13-week subchronic inhalation study in which animals were exposed to concentrations of 525 to 8400 ppm 6 hours/day, 5 days/week. The maximum exposure concentration of 4000 ppm was selected because mimimal histopathologic changes were found after exposure to 4000 ppm. The second dose was 2000 ppm for both species. The third dose, 1000 ppm, was added for rats because in an earlier inhalation study in male and female Sprague-Dawley rats (Dow Chemical Company, 1980; Burek et al., 1984) reduced survival was observed in the highest exposure group, 3500 ppm. All animals used in this experiment were produced under strict barrier conditions at Charles River Breeding Laboratories under a contract to the car- cinogenesis program of the National Toxicology Program (NTP). The rats were placed in the study at 7 to 8 weeks of age and mice at 8 to 9 weeks. All animals were housed individually. Food and water were available ad libitum except during exposure periods, when only water was available. All animals were observed twice a day for signs of moribundity or mortality. Clinical signs were recorded every week. Body weight was recorded once a week. A comp.lete quality-controlled environment was maintained during the experiment. The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Tests of significance included pair-wise comparisons 11 ------- of high-dose and low-dose groups with controls and tests for overall dose- response trends. Life table analysis, incidental tumor analysis, and the Fisher Exact Test were used to evaluate tumor incidence. 3.1.1. Rat Study The mean body weights of experimental and control rats of each sex were similar throughout the studies (Figure 1). Rats exposed to 4000 ppm, the highest dose, were restless and pawed at the eyes and muzzle during the expo- sure period. The survival of male and female rats exposed to DCM is shown in Figure 2. The survival of female rats was significantly lower than that of the controls after week 100, and the survival in all groups of male rats at the termination of the study was low (Table 1). There were many deaths of males in the final 16 weeks of the study. The decreased survival is believed to be related to high incidence of leukemias. There was. a significant positive trend for mammary gland fibroadenoma and adenomas or fibroma (combined) in male and female rats. The incidence in high- dosed males (0/50, 0/50, 2/50, and 5/50) and in females (7/50, 13/50, 14/50, and 23/50) were significantly (p < 0.001) higher than the controls (Tables 2 and 3). Also, subcutaneous fibroma or sarcoma (combined), located in the mam- mary area in male rats, occurred with a significant positive trend (p = 0.008), and the incidence in the high-dose group was significantly (p < 0.05) greater than in the controls (Table 2). The subcutaneous tumors all occurred in the area of the mammary chain; therefore, the subcutaneous tumors were combined by the NTP for comparative purposes (male rats 1/50, 1/50, 4/50, and 9/50). The incidence of subcutaneous tumors in the highest dose group was significantly (p = 0.002) higher in than the controls. The historical incidence of mammary gland tumors at the same laboratory is 0% in males and 16% in females, and the NTP historical control incidence is 3% in males and 28% in females for the 12 ------- 4t M WOKS ON STUDY § -8- * WOKS ON STUDY Figure 1. Growth curves for rats exposed to dichloromethane by inhalation for 2 years. SOURCE: NTP, 1985. 13 ------- H a 2.000 PPU A -4,000 PPU WOKS ON STUDY FEMALE RATS UNTREATED O-1.000 PPVI 2.000 PPU 4,000 PPM WEEKS ON STUDY Figure 2. Kaplan-Meier survival curves for rats exposed to dicfiloromethane by inhalation for 2 years. SOURCE: NTP, 1985. 14 ------- TABLE 1. SURVIVAL OF RATS IN THE 2-YEAR INHALATION STUDY OF DICHLOROMETHANE Control. 1,000 ppm 2,000 ppm 4,000 ppm \ Male* Animals initially in study Nonacci dental deaths before termination15 Killed at termination Survival p values0 Female3 Animals initially in study Nonacci dental deaths before termination^5 Killed at termination Survival p values0 50 34 16 0.116 50 20 30 0.006 50 34 16 0.945 50 28 22 0.223 . 50 33 17 0.935 - . 50 28 22 0.118 50 41 9 0.163 50 35 15 0.006 aTerminal kill period: week 104. ^Includes animals killed in a moribund condition. cThe results of the life table trend test are in the control comparisons with the controls are in the dosed columns. SOURCE: NTP, 1985. column, and those of the life table pairwise ------- TABLE ANALYSIS OF PRIMARY TUMORS IN MALE RATS IN THE TWO-YEAR INHALATION STUDY OF OICHLOROMETHANE Control 1,000 ppm 2,000 ppm 4,000 ppm Subcutaneous tissue: Fibroma Overall rates8 Adjusted rates" Terminal rates0 Week of first observation Life table testsd. Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd Subcutaneous tissue: Fibroma or sarcoma Overall rates8 Adjusted rates0 Terminal rates0 Week of first observation Life table testsd Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd Hematopoietic system: Hononuclear cell leukemia Overall rates* Adjusted rates0 Terminal rates0 Week of first observation Life table tests'1 Incidental tumor testsd Cochran-Armitage Trend Test"1 Fisher Exact Testd Adrenal : Pheochromoeytoma Overall rates8 Adjusted ratesb Terminal rates0 Week of first observation Life table testsd Incidental tumor tests'1 Cocnran-Armltage Trend Testd Fisher Exact Testd Adrenal: Pheochromoeytoma or pheochromocytoma, Overall rates8 Adjusted rates0 Terminal rates0 Week of first observation Life table tests'1 Incidental tumor tests'1 Cochran-Armltage Trend Testd Fisher Exact Testd 1/50(2%) 6.3% 1/16(6%) 104 p- 0.024 p=0.064 p=0.072 1/50(2%) 6.3% 1/16(6%) 104 P'0.008 p-0.026 p-0.029 34/50(68%) 80.31 8/16(50%) 57 p-0.045 p-0.399 p-0.251 . 5/50(10%) 23.5% 2/16(13%) 75 p-0.035 p-0.131 p-0.192 malignant 5/50(10%) 23.5% 2/16(13%) 75 p-0.034 ' p-0.134 p-0.186 1/50(2%) 6.3% 1/16(6%) 104 p=0.764 p=0.764 p-0.753 1/50(2%) 6.3% 1/16(6%) 104 p=0.764 p»0.764 p-0.753 26/50(52%) 77.0% 9/16(56%) 82 P-0.147N p*0.049N p=0.076N 11/50(22%) 46.4% 5/16(31%) 89 p=0.094 p-0.093 p-0.086 11/50(22%) 46.4% 5/16(31%) 89 p- 0.094 p-0.093 P'0.086 2/50(4%) 9.2% 1/17(6%) 96 p-0.523 p-0.505 p-0.500 2/50(4%) 9.2% 1/17(6%) 96 p=0.523 p=0.505 p-0.500 32/50(64%) 80.2% 10/17(59%) 71 ' P-0.400N p=0,434N p=0.417N 10/50(20%) 45.4% 6/17(35%) 89 p-0.149' p»0.131 p»0.131 11/50(20%) 47.0% 6/17(35%) 89 p«0.104 p«0.087 p-0.086 4/50(8%) 19.5% 0/9(0%) 89 p-0.095 P'0.204 p=0.181 5/50(10%) 22.7% 0/9(0%) 89 p=0.050 p=0.125 p=0.102 35/50(70%) 89.4% 6/9(67%) 75 p=0.134 p=0.487N P"0.500 10/50(20%) 52.9% 3/9(33%) 80 p«0.039 p«0.108 p»0.131 10/50(20%) 52.9% 3/9(33%) 80 p* 0.039 p-0.108 p=0.131 ('coh'f 1 nued on the following page) 16 ------- TABLE 2. (continued) Mammary gland: Fibroadenoma Overall rates3 Adjusted rates6 Terminal ratesc Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Mammary gland: Adenoma or f ibroadenoma Overall rates3 Adjusted ratesb Terminal rates0 Week of first observation Life table tests'1 Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Mammary gland or subcutaneous tissue: Adenoma, Overall rates3 Adjusted ratesb Terminal rates0 Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Testis: Interstitial cell tumor Overall rates3 Adjusted rates'5 Terminal ratesc Week of first observation Life table testsd Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd Tunica vaginalis: Malignant mesothelioma Overall rates3 Adjusted ratesb Terminal rates0 Week of first observation Life table tests" Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd Control 0/50(0%) 0.0% 0/16(0%) p<0.001 p<0.003 p-0.009 0/50(0%) 0.0% 0/16(0%) p<0.001 p<0.001 p=0.003 f ibroadenoma, or 1/50(2%) 6.3% 1/16(6%) 104 p<0.001 p=0.003 p<0.001 39/50(78%) 94.9% 14/16(88%) 65 p=0.009 p=0.114 p=0.129 0/50(0%) 0.0% 0/16(0%) p-0.025 p»0.060 p=0.044 1,000 ppm 0/50(0%) 0.0% 6/16(0%) e e e 0/50(0%) 0.0% 0/16(0%) e e e fibroma 1/50(2%) 6.3% 1/16(6%) 104 p=0.764 P'0.764 p=0.753N 37/49(76%) 97.3% 15/16(94%) 69 p*0.420N p«0.385N ' p=0.478N 1/50(2%) 2.2% 0/16(0%) . 69 p=0.496 p*0.473 p=0.500 2,000 ppm 2/50(4%) 11.8% 2/17(12%) 104 p<=0.250 p=0.250 p=0.247 2/50(4%) 11.8% 2/17(12%) 104 p=0.250 p*0.250 p=0.247 4/50(8%) 20.6% 3/17(18%) 96 p=0.196 p=0.186 p-0.181 41/60(82%) 95.2% 15/17(88%) 75 p*0.512 p=0.387 p=0.401 0/50(0%) 0.0% 0/17(0%) e e e 4,000 ppp 4/50(8%) 34.0% 2/9(221) 101 p=0.020 p=0.040 p=0.059 5/50(10%) 36.6% 2/9(22%) 93 p=0.010 p=0.023 p=0.028 9/50(18%) 49.0'* 2/9(22%) 89 p=0.002 p=0.008 p= 0.008 43/50(86%) 97.7% 8/9(89%) 75 p=0.029 p=0.253 p=0.218 3/50(6%) 19.6% 0/9(0%) 92 p= 0.068 p=0.172 p=0.121 (continued on the following page) 17 ------- TABLE 2. (continued) Control 1,000 ppm 2,000 ppm 4,000 ppm Tunica vaginalis: Mesothelioma (all types) Overall rates* 0/50(0%) 1/50(2%) 4/50(8%) 4/50(8%)' Adjusted rates1* 0.0% 2.2% 19.2% 24.4% Terminal ratesc 0/16(0%) 0/16(0%) 2/17(12%) 0/9(0%) Week of first observation ' 69 .> 96 92 Life table testsd . p=0.009 p=0.496 p=0.070 p=0.031 Incidental tumor testsd p«0.030 p=0.473 p=0.062 p=0.097 Cochran-Armitage Trend Testd p=0.029 Fisher Exact Testd p=0.500 p=0.059 p=0.059 All sites: Malignant mesothelioma Overall rates9 0/50(0%) 2/50(4%) 0/50(0%) 3/50(6%) Adjusted ratesb 0.0% 4.4% 0.0% 19.6% Terminal ratesc 0/16(0%) 0/16(0%) 0/17(0%) 0/9(0%) Week of first observation 69 92 Life table tests" p=0.066 p*0.243 e p=0.068 Incidental tumor testsd p=0.136 p=0.225 e p=0.172 Cochran-Armitage Trend Testd p=0.097 Fisher Exact Testd p=0.247 e p=0.121 All sites: Mesothelioma (all types) Overall rates9 0/50(0%) 2/50(4%) 5/50(10%) 4/50(8%) Adjusted ratesb 0.0% 4.4% 22.8% 24.4% Terminal rates^ 0/16(0%) 0/16(0%) 2/17(12%) 0/9(0%) Week of first observation 69 96 92 Life table testsd p=0.020 p=0.243 p=0.038 p=0.031 Incidental tumor testsd p=0.063 p=0.225 p=0.030 p=0.097 Cochran-Armitage Trend Testd p=0.052 Fisher Exact Testd p=0.247 ' p=0.028 p=0.059 ^Number of tumor-bearing animals/number of animals examined at the site. ^Kaplan-Meier estimated tumor incidences at the end of the study after adjusting for intercurrent mortality. C0bserved tumor incidence at terminal kill. dBeneath the control incidence are the p values associated with the trend test. Beneath the dosed group incidence are the p values corresponding to pairwise comparisons between the dosed group and the controls. The life table analysis regards tumors in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The incidental tumor test regards these lesions as nonfatal. The Cochran-Armitage Trend Test and the Fisher Exact Test directly compare the overall incidence rates. A negative trend or lower incidence in a dose group is indicated by the letter N. eNo p value is presented because no tumors were observed in the dosed and control groups. Mammary gland fibroadenoma: Historical incidence at testing laboratory 0/100 (0%); historical incidence in NTP studies 51/1,727 (3%) t 3%. Mesothelioma--all sites: Historical incidence at testing laboratory 4/100 (4%); historical incidence in NTP studies 44/1,727 (3%) ±2%. Mononuclear cell leukemia: Historical incidence at testing laboratory 36/100 (36%); historical incidence in NTP studies 458/1,727 (27%) ± 9%. SOURCE: NTP, 1985. 18 ------- TABLE 3. ANALYSIS OF PRIMARY TUMORS IN FEMALE RATS IN THE TWO-YEAK INHALATION STUDY OF DICHLOROMETHANE Control' 1,000 ppm 2,000 ppm 4,000 ppm Hematopo1et1c system: Mononuclear cell leukemia Overall rates8 Adjusted ratesb Terminal ratesc Week of first observation Life table tests'1 Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd Liver: Neoplastic nodule Overall rates3 Adjusted rates'" Terminal rates' Meek of first observation Life table testsd Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd Liver: Neoplastic nodule or hepatocellular Overall rates' Adjusted ratesb Terminal rates0 Week of first observation Life table tests'1 Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Mammary gland: Flbroadenoraa Overall rates8 Adjusted ratesb Terminal rates0 Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Mammary gland: Adenoma or fibroadenoma Overall rates8 Adjusted ratesb Terminal rates0 Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Test"3 17/50(34%) 41.11 8/30(271) 73 p= 0.009 p-0.273 p«0.086 2/50(41) 6.71 2/30(71) 104 p-0.030 p=0.097 p-0.078 carcinoma 2/50(4%) 6.71 2/30(71) 104 p-0.027 p-0.086 P'0.079 5/50(101) 15.71 4/30(131) 96 p<0.001 p<0.001 p<0.001 5/50(101) 15.71 4/30(131) 96 p<0.001 p<0.001 p<0.001 17/50(341) 44.41 4/22(181) 76 p=0.402 P-0.425N P-O.S84N 1/50(2%) 2.4% 0/22(0%) 61 p»0.5'69N P-0.494N P-0.500N 1/50(2%) 2.0% 0/22(0%) 61 P-0.569N P-0.494N p-O.SOON 11/50(221) 41.21 8/22(36%) 74 p- 0.028 p»0.04Q p*0.086 11/50(22%) 41.2% 8/22(36%) 74 p- 0.028 p- 0.049 p-0.086 23/50(46%) 63.6% 10/22(45%) 73 p-0.049 p-0.189 p*0.154 3/50(6%) 10.2% 1/22(51) 85 p= 0.382 p-0.482 p=0.500 4/50(8%) 14.41 2/22(91) 85 P'0.223 p=0.297 p-0.339 13/50(26%) 43.6% 7/22(32%) 65 p=0.009 p=0.025 p'0.033 13/50(26%) 43.6% 7/22(32%) 65 p»0.009 p«0.025 p-0.033 23/50(46%) 53.1% 1/15(71) 63 p=0.028 p«0.579 P'0.154 5/50(10%) 19.6% 1/15(7%) 73 p= 0.080 p=0.229 ?'0.218 5/50(10%) 19.6% 1/15(7%) 73 p=0.080 p«0.229 P'0.218 22/50(44%) 79.4% 10/15(67%) 73 p<0.001 p<0.001 p<0.001 23/50(46%) 83.51 11/15(731) 73 p<0.001 p<0.001 p<0.001 (continued on the following page) 19 ------- TABLE 3. (continued) Control 1,000 ppm 2,000 ppm 4,000 ppm Mammary gland: Adenoma, fibroadenoma. Overall ratesa Adjusted rates'5 Terminal ratesc Week of first observation Life table testsd Incidental tumor tests'1 Cochran-Armitage Trend Test'1 Fisher Exact Testd Mammary gland: Adenoma, fibroadenoma. Overall rates3 Adjusted rates'5 Terminal ratesc Week of first observation Life table tests'1 Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd or adenocarcinoma 6/50(12%) 17.8% 4/30(13%) 92 p<0.001 p<0.001 p<0.001 13/50(26%) 44.4% 8/22(36%) 74 p=0.023 p=0.053 p=0.062 adenocarcinoma, or mixed tumor, malignant 7/50(14%) 20.0% 4/30(13%) 92 p<0.001 p<0.001 p<0.001 13/50(26%) 44.4% 8/22(36%) 74 p=0.045 p=0.092 p*0.105 14/50(28%) 44.9% 7/22(32%) 65 p=0.012 p=0.043 p=0.039 e!4/50(28%) 44.9% 7/22(32%) 65 p=0.022 • p=0.083 p=0.070 23/50(46%) 83.5% 11/15(73%) 73 p<0.001 p<0.001 p<0.001 23/50(46%) 83.5% 11/15(73%) 73 p<0.001 p<0.001 p<0.001 aNunber of tumor-bearing animals/number of animals examined at the site. bKaplan-Meier estimated tumor incidences at the end of the study after adjusting for intercurrent mortality. C0bserved tumor incidence at terminal kill. ^Beneath the control incidence are the p .values associated with the trend test. Beneath the dosed group incidence are the p values corresponding to pairwise comparisons between the dosed group and the controls. The life table analysis regards tumors in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The incidental tumor test regards these lesions as nonfatal. The Cochran-Armitage Trend Test and the Fisher Exact Test directly compare the overall incidence rates.. A negative trend or lower incidence in a dose group is indicated by the letter N. eA carcinoma was also present in one of the animals that had a fibroadenoma. Mammary gland fibroadenoma: Historical incidence at testing laboratory 16/99 (16%); historical incidence in NTP studies 492/1,772 (28%) * 10%. Mononuclear cell leukemia: Historical incidence at testing laboratory 27/99 (27%); historical incidence in NTP studies 307/1,772 (17%) ± 6. SOURCE: NTP, 1985. 20 ------- same strain of rats. The increased incidence of mammary gland tumors is consistent with the results reported by Dow Chemical Company (1980) and Burek et al. (1984) in Sprague-Dawley rats (U.S. EPA, 1985). This study has been reviewed previously. Sprague-Dawley rats have a spontaneous incidence of mammary gland tumors, about 80% in female and 10% in males. In males (Burek et al., 1984) the mammary tumors increased in the highest dose group to 14/97, as compared to 7/92 in the controls. In females the number of tumors per rat increased with dose. The increased incidence of benign mammary gland tumors in males and females provides some supportive evidence for mammary gland carcinogenesis. Maltoni (1984) also reported at the Food Solvent Workshop (March 8-9, 1984) on a study in which DCM was administered by gavage at 500 mg/kg/day, 5 days/week for 64 weeks, followed by an observation period until spontaneous death. Maltoni (1984) observed an increased incidence of mammary gland tumors in Sprague-Dawley rats. Thus, the incidence of mammary gland benign tumors in female rats in the present NTP study is consistent with the reports of Burek et al. (1984), Maltoni (1984), and Nitschke et al. (1982). The incidence of liver neoplastic nodules or hepatocellular carcinomas (combined) in female rats (2/50, 1/50, 4/50, and 5/50) occurred with positive trends (p = 0.030) by life table analysis only (Table 3). The incidence in the high-dose group was not significantly greater than in the controls; this result is consistent with observations made by other investigators (Maltoni, 1984; NTP, 1982, unpublished; National Coffee Association, 1982a, b). Maltoni (1984) reported that gavage administration of 100 or 500 mg/kg/day for 64 weeks induced a dose-related increase in the incidence of nodular hyperplasia of the liver in Sprague-Dawley rats. The earlier gavage study (NTP, 1982, unpublished) indicated that administration of 500 or 1000 mg/kg/day increased the incidence of hepatocellular nodules in both male and female F344/N rats. 21 ------- Furthermore, there was a significant increase in liver tumors in female rats dosed at 250 mg/kg/day in the drinking water study (National Coffee Associa- tion, 1982), although the number of tumors were within the range of the his- torical control values of the laboratory. The pharmacokinetic data presented by Dr. Kirshman at the Food Solvent Workshop (1984, page 41) indicated that 250 my/kg/day in the drinking water study was equivalent to a 750 ppm inhalation level, which is 1/5 of the MTD used in the Burek et al. (1984) study and in the NTP (1985) study. The NTP reported that the highest exposure concentration (4000 ppm) in the inhalation study has been estimated to be equivalent to 1300 mg/kg/day from an oral dose. In male rats the incidence of mesothelioma arising from tunica vaginalis (0/5, 2/50, 5/50, and 4/50) occurred with a significant positive trend (p = 0.02); the incidence in the mid- and high-dose groups was significantly higher (p = 0.038, p = 0.30) than in the controls (Table 2). This increased incidence may not be due to administration of DCM because the concurrent controls in the same laboratory were low in comparison with earlier inhalation studies (4/100). Mononuclear cell leukemia in male (Table 2) and female (Table 3) rats occurred with a significant positive trend by life table analysis only. The incidence (17/50, 17/50, 23/50, 23/50) in females was significantly greater than in the controls at the mid-dose (p = 0.049) and high-dose (p = 0.028) levels. Some other tumor incidences were increased marginally in experimental groups as compared to tnc controls. These increases were characterized by a significant trend only. These tumors included adrenal gland pheochromocytoma and interstitial cell tumors in males (Table 2) and pituitary gland adenoma or carcinoma (combined) in males and females (Tables 2 and 3). The squamous cell metaplasia of the nasal cavity in female rats (1/50, 2/50, 3/50, and 9/50) was increased significantly in the high-dose group, but no nasal tumors were 22 ------- found in this group. 3.1.2. Mouse Study The mean initial body weight of males in the 4000 ppm group was 15% lower than that of controls (Figure 3). The mean body weights were comparable in the high-dose and control groups until week 90, but after week 90 the body weights were 8% to 11% lower than those of controls. During the exposure period, the mice were hyperactive. The probabilities of survival of male and female mice are shown in Figure 4. The survival in both male and female high- dose groups decreased significantly compared with controls (Table 4). The reduced survival may have been due to chemically-induced lung and liver tumors in both male and female mice. The incidences of lung tumors increased significantly (p = 0.0001) in both males (Table 5) and females (Table 6). The latent period for tumor induction was significantly (trend analysis) decreased in the high-dose groups as compared to controls. The tumors observed were alveolar/bronchiolar adenomas (males, 3/50, 19/50, and 24/50; females, 2/50, 23/48, and 28/48) and alveolar/bronchiolar carcinomas (males, 2/50, 10/50, and 28/50; females, 1/50, 13/48, 29/48, and 29/48). In addition to the dose-related increase in lung tumors in male and female mice, there were dose-related increases in multiple lung tumor-bearing mice (Table 7). The multiplicity of tumors included both alveolar/bronchiolar adenoma and carcinoma. Only one lung tumor per mouse was found in the controls, whereas 70% of the high-dose males and females had multiple tumors (males, 0/50, 10/50, and 28/bO: females, 0/50, 11/48, and 29/48). In the experimental groups, 38% of the dosed male mice and 42% of the dosed female mice had multiple lung tumors. These results are consistent with the data obtained in other studies. In the earlier NTP (1982, unpublished) gavage study, DCM produced a significant increase in lung tumors in female mice. Maltoni (1984) also 23 ------- I 2 * A O I "" D A A : UNTREATED I "i O=2.000 PPM r 4» M WEEKS ON STUDY t I • AA 4$r w~ Q ^r f &*" ) i O (5 a 9 • " • *22 O I 1 ! .6 J ! ] e ^ 2 k- 6 . A A A - g O - e ( A A • > 0 o a ^ 1 i A - ! i i FEMALE MCE • -UNTREATED " " O= 2.000 Wiii A» 4.000 PPU i i j > a M 4i M n M K> WEEKS ON 51UDY Figure 3. Growth curves for mice exposed to dichloromethane by inhalation for 2 years. SOURCE: NTP, 1985. 24 ------- M I JALE UICE = UNTREATED I IO=2.000 PPUI WEEKS ON STUDY FEMALE UICE • a UNTREATED O» 2.000 PPM 4.000 PPM WEEKS ON STUDY Figure 4. Kaplan-Meier survival curves for mice exposed to dichloromethane by inhalation for 2 years. SOURCE: NTP, 1985. 25 ------- TABLE 4. SURVIVAL OF MICE IN THE 2-YEAR INHALATION STUDY OF DICHLOROMETHANE Control 2,000 ppm 4,000 ppm ro CTl Male3 Animals initially in study Nonaccidental deaths before termination^ Accidentally killed Killed at termination Died during termination period Survival p values0 Female9 Animals initially in study Nonaccidental deaths before termination'3 Accidentally killed Killed.at termination Died during termination period Survival p values0 50 11 0 39 0 <0.001 50 24 1 0 25 0.002 50 24 2 24 0 <0.010 50 22 2 1 25 0.678 50 38 1 9 . 2 <0.001 50 40 1 1 8 0.004 aTerminal kill period: week 104. ^Includes animals killed in a moribund condition. cThe results of the life table trend test are in the control column, and those of the life table pairwise comparisons with the controls are in the dosed columns. SOURCE: NTP, 1985. ------- TABLE 5. ANALYSIS OF PRIMARY TUMORS IN MALE MICE IN THE TWO-YEAR INHALATION STUDY OF D1CHLOROMETHANE Lung: Al veolar/bronchlolar adenoma Overall rates8 Adjusted rates6 Terminal rates0 Week of first observation Life table tests'1 Incidental tumor testbd Cochran-Annitaye Trend Testd Fisher Exact Testd Luny: Al veolar/bronchfolar carcinoma Overall rates8 Adjusted ratesb Tenninal ratesc Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Lung: Al veolar/bronchlolar adenoma or Overall rates3 Adjusted rates'3 Tenninal rates0 Week of first observation Life table tests'1 Incidental tumor tests'1 Cochran-Armitaye Trend Testd Fisher Exact Testd Circulatory system: Hemangiosarcoma Overall rates8 Adjusted rates6 Terminal ratesc We»K of first observation Life table tests'1 Incidental tumor tests'1 Cochran-Armitaye Trend Testd Fisher Exact Testd Control 3/50(6%) 7.7% 3/39(8%) 104 p<0.001 p<0.001 p<0.001 2/50(,«) 4.9% 1/39(3%) 94 p<0.001 p<0.001 p<0.001 carcinoma 5/50(10%) 12.4% 4/39(10%) 94 p<0.001 p<0.001 p<0.001 1/50(2%) 2.6% 1/39(3%) 104 p-0.007 p-0.083 p=0.06Q 2,000 ppm 19/50(38%) 55.6% 10/24(42%) 71 p<0.001 p<0.001 p<0.001 10/50(20%) 34.0% 6/24(25%) 78 p<0.002 p<0.016 p<0.014 27/50(54%) 74.2% 15/24(63%) 71 p<0.001 p<0.001 p<0.001 2/50(4%) 7.6% 1/24(4%) 101 p=0.352 p=0.495 p=0.500 4,000 ppm 24/50(48i) 78.5% 6/11(55%) 70 p<0.001 p<0.001 p<0.001 28/50(56%) 92.9% 9/11(82%) 72 p<0.001 p<0.001 p<0.001 40/50(80%) . 100.0% 11/11(100%) 70 p<0.001 p<0.001 p<0.001 5/50(10%) 21.4% 1/11(9%) 70 p=0.017 p*0.142 p=0.102 Circulatory system: Hemangioma or hemangiosarcoma Overall rates* Adjusted rates6 Terminal rates0 Week of first observation Life table tests'1 Incidental tumor tests'1 Cochran-Armitage Trend Test11 Fisher Exact Testd 2/50(4%) 4.8% 1/39(3%) 87 p-0.010 p<=0.170 p«0.080 2/50(4%) 7.6% 1/24(4%) 101 p=0.558 p*0.643N p=0.691 6/50(12%) 25.8% 1/11(9<) 70 p-0.022 p='!.^U p=0.134 27 ------- TABLE 5. (continued) Control 2,000 ppm 4,000 ppm Liver: Hepatocellular adenoma Overall rates3 Adjusted ratesb Terminal ratesc Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd 10/50(20%) 23.0% 7/39(181) 73 p<0.001 p<0.075 p-0.194 14/49(29%) 46.9% 9/24(38%) 71 p=0.041 p«0.161 p=0.224 14/49(29%) 68.3% 6/11(55%) 80 p=0.001 p=0.095 p=0.224 Liver: Hepatocellular carcinoma Overall rates3 13/50(26%) Adjusted ratesb 29.7% Terminal rates^ 9/39(23%) Week of first observation 73 Life table tests'1 p<0.001 Incidental tumor tests'1 p=0.016 Cochran-Armitage Trend Testd p=0.004 Fisher Exact Testd Liver: Hepatocellular adenoma or carcinoma Overall rates3 22/50(44%) Adjusted ratesb 48.3% Terminal rates^ 16/39(41%) Week of first observation 73 Life table testsd p<0.001 Incidental tumor tests'1 p=0.010 Cochran-Armitage Trend Testd p=0.013 Fisher Exact Testd 15/49(31%) 43.7% 7/24(29%) 72 p-=0.111 p=0.422 p«0.387 22/49(49%) 66.8% 13/24(54%) 71 p=0.048 p=0.305 p=0.384 26/49(53%) 76.4% 5/11(45%) 61 p<0.001 p=0.042 p=0.005 33/49(67%) 93.0% 9/11(82%) 61 p<0.001 p=0.020 p=0.016 aNumber of tumor-bearing animals/number of animals examined at the site. bKaplan-Meier estimated tumor incidences at the end of the study after adjusting for intercurrent mortality. 'Observed tumor incidence at terminal kill. ^Beneath the control incidence are the p values associated with the trend test. Beneath the dosed group incidence are the p values corresponding to pairwise comparisons between the dosed group and the controls. The life table analysis regards tumors in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The incidental tumor test regards these lesions as nonfatal. The Cochran-Armitage Trend Test and the Fisher Exact Test directly compare the overall incidence rates. A negative trend or lower incidence in a dose group is indicated by the letter N. Alveolar/bronchiolar adenoma or carcinoma: Historical incidence at testing laboratory 31/100 (31%); historical incidence in NTP studies 296/1,780 (17%) t 8%. Hepatocellular adenoma or carcinoma: Historical incidence at testing laboratory 28/100 (28%); historical inci- dence in NTP studies 540/1,784 (30%) ± 8%. Hemangioma or hemangiosarcoma: Historical incidence at testing laboratory 2/100 (2%); historical incidence in NTP studies 78/1,791 (4%) ± 4%. SOURCE: NTP, 1985. 28 ------- TABLE 6. ANALYSIS OF PRIMARY TUMORS IN FEMALE MICE IN THE TWO-YEAR INHALATION STUDY OF IHCHLOKOMETHANE Lung: Al veolar/bronchiolar adenoma Overall rates3 Adjusted ratesh Terminal ratesc Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Lung: Al veolar/bronchiolar carcinoma Overall rates3 Adjusted rates6 Terminal rates0 Week of first observation Life table testsd Incidental tumor tests'1 Cochran-Armitage Trend Testd Fisher Exact Testd Lung: Al veolar/bronchiolar adenoma or Overall rates9 Adjusted rates'" Terminal rates0 Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Liver: Hepatocel lular adenoma Overall rates3 Adjusted ratesb Terminal rates0 Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitaije Trend Testd ' Fisher Exact Testd Liver: Hepatocel lular carcinoma Overall rates3 Adjusted rates'5 Terminal rates0 Week of first observation Life table testsd Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd Control 2/50(4%) 6.7% 1/25(4%) 87' p<0.001 p<0.001 p<0.001 1/50(4%) 4.0% 1/25(4%) 104 p<0.001 p<0.001 p<0.001 carcinoma 3/50(6%) 10.6% 2/25(8%) 87 p<0.001 p<0.001 p<0.001 2/50(4%) 6.5% 1/25(4%) 84 p<0.001 p<0.001 p<0.001 1/50(2%) 4.0% 1/25(4%) 104 p<0.001 p<0.001 p<0.001 2,000 ppro 23/48(48%) 66.5% 14/25(56%) 83 p<0.001 ' p<0.001 p<0.001 13/48(27%) 45.9% 10/25(40%) 89 p<0.001 p<0.001 p<0.001 30/48(63%) 82.9% 19/25(76%) 83 p<0.001 p<0.001 p<0.001 6/48(13%) •• 21.3% 4/25(16%) 96 p=0.151 p=0.155 p=0.121 11/48(23%) 34.0% .6/25(24%) 83 p=0.005 p=0.004 p=0.001 4,000 ppm 28/48(58%) 91.1% 6/a'v7S'il 68 p<0.001 p<0.001 p<0.001 29/48(60%) 92.2% 6/8(75S) 68 p<0.001 p<0.001 p<0.001 41/48(85%) 100.0% 8/8(100%) 63 p<0.001 p<0.001 p<0.001 22/48(46%) 83.0% 5/3(63%) 63 p<0.001 p<0.001 p<0.001 32/48(67%) 96.5% 7/8(88%) 68 p<0.001 p<0.001 p<0.001 (continued on the following page) 29 ------- TABLE 6. (continued) Control Thyroid gland: Follicular cell adenoma Overall rates8 1/48(21) Adjusted ratesb 4.2% Terminal rates0 1/24(4%) Week of first observation 104 Life table testsd ' p*0.012 Incidental tumor testsd p«0.040 Cochran-Armitaye Trend Testd p-0.093 Fisher Exact Testd 2,000 ppm 1/47(2%) 4.0% 1/25(4%) 104 p=0.754N p=0.754N p=0.747 4,000 ppm Liver: Hepatocellular adenoma or carcinoma Overall rates9 Adjusted ratesb Terminal rates0 Week of first observation Life table tests'1 Incidental tumor testsd Cochran-Armitage Trend Testd Fisher Exact Testd 3/50(6%) 10.4% 2/25(8%) 84 p<0.001 p<0.001 p<0.001 16/48(33%) 48.0% 9/25(36%) 83 p=0.002 p=0.002 p<0.001 40/48(83%) 100.0% 8/8(100%) 68 p<0.001 p<0.001 p<0.001 4/46(9%) 35.0% 2/8(25%) 77 p=0.022 p=0.069 p=0.168 aNumber of tumor-bearing animals/number of animals examined at the site. bKaplan-Meier estimated tumor incidences at the end of the study after adjusting for intercurrent mortality. C0bserved tumor incidence at terminal kill. dBeneath the control incidence are the p values associated with the trend test. Beneath the dosed group incidence are the p values corresponding to pairwise comparisons between the dosed group and the controls. The life table analysis regards tumors in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The incidental tumor test regards these lesions as nonfatal. The Cochran-Armitage Trend Test and the Fisher Exact Test directly compare the overall incidence rates. A negative trend or lower incidence in a dose group is indicated by the letter N. Alveolar/bronchiolar adenoma or carcinoma: Historical incidence at testing laboratory 10/100 (10%); historical incidence in NTP studies 122/1,777 (7%) ± 4%. Hepatocellular adenoma or carcinoma: Historical incidence at testing laboratory 5/100 (5%); historical incidence in NTP studies 147/1,781 (8%) ±5%. SOURCE: NTP, 1985. 30 ------- TABLE 7. MULTIPLICITY OF PULMONARY TUMORS IN MICE EXPOSED TO DICHLOROMETHANE Exposure groups (ppm) Diagnoses 2,000 4,000 Male One adenoma and one carcinoma Multiple adenomas Multiple carcinomas Multiple adenomas and multiple carcinomas One adenoma and multiple carcinomas Multiple adenomas and one carcinoma Incidence of mice with multiple tumors No. of mice with multiple tumors/no, of mice with pulmonary tumors Female One adenoma and one carcinoma Multiple adenomas Multiple carcinomas Multiple adenomas and multiple carcinomas One adenoma and multiple carcinomas Multiple adenomas and one carcinoma Incidence of mice with multiple tumors No. of mice with multiple tumors/no, of mice with pulmonary tumors 0/50 0/50 0/50 0/50 0/50 0/50 0/50(0%) 0/5(0%) 0/50 0/50 0/50 0/50 0/50 0/50 0/50(0%) 0/3(0%) 1/50 5/50 ,3/50 0/50 1/50 0/50 10/50(20%) 10/27(37%) 2/48 4/48 1/48 0/48 2/48 2/48 11/48(23%) 11/30(37%) 3/50 4/50 12/50 3/50 3/50 3/50 28/50(56%) 28/40(70%) 4/48 5/48 8/48 2/48 7/48 3/48 29/48(60%) 29/41(71%) SOURCE: NTP, 1985. 31 ------- reported an increased incidence of lung tumors (one and one-half times) in male mice dosed with 100 to 500 mg/kg/day DCM by gavage. In male mice (Table 5) the hepatocellular adenoma or carcinoma (combined) (22/50, 24/49, and 33/49) and hepatocellular carcinoma (13/50, 15/49, and 26/49) were increased significantly (p < 0.001), especially at 4000 ppm. In female mice (Table 6), DCM produced dose-related increases in both hepatocellular adenoma (2/50, 6/48, and 22/48) and hepatocellular carcinoma (1/50, 11/48, and 32/48), which were highly significant by any statistical test. The incidence of these tumors in the controls was consistent with the historical control values of this laboratory. The multiplicity of the hepatocellular neoplasms was common in the male and female dosed mice (Table 8). It should be noted that only 4% of the male control mice and none of the female control mice had multiplicity of liver tumors. The multiplicity of hepatocellular tumors in both male and female mice increased significantly in a dose-related manner (males, 2/50, 11/49, and 16/46; females, 0/50, 3/48, and 28/48). There were 27/57 (47%) males and 31/56 (55%) females with multiple tumors. The increased incidence of hepatocellular tumors is consistent with the results of the previous NTP (1982, unpublished) gavage study, the Maltoni (1984) study, and the National Coffee Association (1983) drinking water study. The National Coffee Association study produced a borderline significant increase in liver tumors at a dose significantly less than the maximum tolerated dose. There was also an increase in hemangiosarcoma (1/50, 2/50, and 5/50) or hemangioma and hemangiosarcoma (2/50, 2/50, and 6/50) in male mice (Table 7), which occurred with a positive trend by life table analysis. 3.1.3. Summary The results of the NTP inhalation bioassay (1985, draft) using F344/N rats showed an increased incidence of benign mammary gland neoplasms, primarily 32 ------- TABLE 8. MULTIPLICITY OF LIVER TUMORS IN MICE EXPOSED TO DICHLOROMETHANE Exposure groups (ppm) Diagnoses 2,000 4,000 Male One adenoma and one carcinoma Multiple adenomas Multiple carcinomas Multiple adenomas and multiple carcinomas One adenoma and multiple carcinomas Multiple adenomas and one carcinoma Incidence of mice with multiple tumors No. of mice with multiple tumors/no, of mice with pulmonary tumors Female One adenoma and one carcinoma Multiple adenomas Multiple carcinomas Multiple adenomas and multiple carcinomas One adenoma and multiple carcinomas Multiple adenomas and one carcinoma Incidence of mice with multiple tumors No. of mice with multiple tumors/no, of mice with pulmonary tumors 1/50 0/50 1/50 0/50 0/50 0/50 2/50(4%) 2/22(9%) 0/50 0/50 0/50 0/50 0/50 0/50 0/5(0%) 0/3(0%) 2/49 3/49 3/49 0/49 0/49 3/49 11/49(22%) 11/24(46%) 1/48 0/48 2/48 0/48 0/48 0/48 3/48(6%) 3/16(19%) 3/49 3/49 6/49 1/49 2/49 1/49 16/49(33%) 16/33(48%) 6/48 4/48 10/48 3/48 • 1/48 4/48 28/48(58%) 28/40(70%) SOURCE: NTP, 1985. 33 ------- fibroadenomas, in both male and female rats. In female rats, there was also a significant increase in hepatocellular neoplastic nodules and hepatocellular carcinomas (combined) by the trend test only and a statistically significant increase of mononuclear cell leukemias by age adjustment. In male rats, there was a significant increase in mesotheliomas, primarily from the tunica vagina- lis. Lastly, there was a marginally significant increase in adrenal pheochro- mocytomas and interstitial cell tumors in males and pituitary gland adenomas and carcinomas (combined) in male and female rats by the trend test only. In the NTP inhalation bioassay using B6C3F1 mice, a highly significant increase in alveolar/bronchiolar adenoma and/or carcinoma was observed in both sexes. The incidence of hepatocellular adenoma and hepatocellular carcinoma (combined) was increased in the high-dose males and in both dosed groups of females. There was also a dose-related increase in the number of mice bearing multiple lung and liver tumors. Only one lung tumor per mouse was found, whereas 38% of all dosed male mice and 42% of all dosed female mice had mul- tiple lung tumors. The incidence of multiple hepatocellular tumors in the exposed groups increased in both sexes in a dose-related manner. Multiple hepatocellular tumors were found in only 4% of the male controls, but none were found in the female controls; in contrast, 28% of the exposed males and 32% of the exposed females exhibited multiple liver tumors. The NTP concluded that, under the conditions of this bioassay, there was: some evidence of DCM carcinogenicity for male F344/N rats as shown by an increased incidence of benign neoplasms of the mammary gland, sufficient or clear evidence of DCM carcinogenicity for female F344/N rats as shown by an increased incidence of benign neoplasms of the mammary gland, and sufficient or clear evidence of DCM carcinogenicity in male and female B6C3F1 mice as shown by increased incidences of lung and liver tumors. 34 ------- 3.2. PHARMACOKINETICS/METABOLISM The purpose of this analysis is to determine if there are available pharmacokinetic/metabolism data that can be useful in the assessment of the carcinogenic risk arising from exposure to methyl ene chloride (DCM). Most of the data used in this analysis have previously been reviewed (U.S. EPA, 1985) and therefore will not be discussed in detail. Data on routes of exposure, rates of ingestion, metabolism, and administered versus effective dose in the NTP bioassay have been reviewed in order to determine if there are qualitative or quantitative differences or similarities between species which may alter the assumptions used in estimating risks. Relevant new data have been incorporated as appropriate. 3.2.1. In Vitro Metabolism/Pathways The preponderance of data obtained from both in vivo and in vitro experiments indicate that DCM and .other dihalomethanes are biotransformed to both carbon monoxide and carbon dioxide. Carbon monoxide is the end product of microsomal oxidation, and carbon dioxide is an end product of cytosolic metabolism. A number of investigators (Kubic and Anders, 1975, 1978; Hogan et al., 1976; Stevens and Anders, 1978, 1979; and Ahmed and Anders, 1978) have studied the metabolism of DCM and other dihalomethanes in experiments using rat liver microsomes. These studies have resulted in the following observations: o NADPH and molecular oxygen are required for maximal activity; « Anaerobic conditions reduce the rate of DCM conversion to carbon monoxide by 80 percent; o There is a high correlation betw°en the in vitro production of carbon monoxide and microsomal Pg content; o Pretreatment of test animals with P inducers resulted in 35 ------- increased conversion of DCM to carbon monoxide by rat liver microsomes; • Pretreatment of test animals with P^Q inhibitors resulted in decreased conversion of DCM to carbon monoxide by rat liver microsomes; and • Cleavage of the carbon-hydrogen bond is the rate-limiting step in dihalomethane metabolism. Based on these observations, Anders et al. (1977) postulated that DCM is biotransformed to carbon monoxide by the microsomal mixed-function oxidases via formation of a formyl halide intermediate. The proposed mechanism for the metabolism of dihalomethanes by liver microsomes is summarized in Figure 5. A number of investigators (Heppel and Porterfield, 1948; Kubic and Anders, 1975; Ahmed and Anders, 1976, 1978) have studied the metabolism of DCM and other dihalomethanes to carbon dioxide, formaldehyde, and formic acid using a rat liver cytosolic fraction. They have made the following observa- tions: • The reaction does not require molecular oxygen; • The reaction is glutathione-dependeht; • Chemicals known to complex with glutathione inhibit the reaction; and • Removal of formaldehyde dehydrogenase by ammonium hydroxide fractionation results in the formation of formaldehyde only, and not formic acid. Based on these observations, Ahmed et al. (1980) postulated that DCM is bio- transformed to carbon dioxide by the liver cytosolic fraction via formation of a reactive formyl intermediate. The proposed mechanism for the metabolism 36 ------- Microsomal Pathway MFO X«CH~ X/. NADPH -H -X > ,c \ OH Nonenzymatic rearrangement f Covalent ^ H Spontaneous lipid 0 Decomposition protein "H, -X Cytosolic Pathway formyl halide CO X - CH GSH transferase NAD GS - CH2X S-halomethylglutathione HOH Nonenzymatic hydrolysis GS - CH2OH S-hydroxymethyl glutathione GS - C 0 S-formyl glutathione HOH S-formyl glutathione hydrolase HC " •»• GSH ^ OH formic acid formaldehyde dehydrogenase metabolic incorporation H2C=0 + GSH formaldehyde v co2 Figure 5. Proposed reaction mechanisms for the metabolism of dihalomethanes to carbon monoxide, carbon dioxide, formaldehyde, formic acid, and inorganic halide. SOURCE: Ahmed et al., 1980. 37 ------- of dihalomethanes by liver cytosol is summarized in Figure 5. 3.2.2. Tissue Distribution There have been a number of studies which have compared DCM metabolism by means of microsomes and/or cytosol prepared from various tissues. Kubic and Anders (1975) compared the biotransformation of DCM to carbon monoxide by liver, kidney, and lung microsomes. They found liver microsomes to be five times more active than lung microsomes, and 30 times more active than kidney microsomes. Ahmed and Anders (1976) compared the biotransformation of DCM to carbon dioxide by cytosolic fractions prepared from various tissues of the rat. The-highest activities were found in liver, lung, and kidney cytosol. Liver cytosol was found to be 15 times more active than lung cytosol and 12.5 times more active than kidney cytosol. Ahmed and Anders (1978) measured the metabolism of dibromomethane to bromide, formaldehyde, and formic acid using rat liver cytosol, and observed that free bromide was formed at the rate of 27.7 * 3.8 nmoles/mg protein/minute, and that the amount of formaldehyde plus formic acid formed was 12.6 * 0.8 nmoles/mg protein/minute. Kubic and Anders (1975), using rat liver microsomes or cytosol from the same liver preparation, measured dibromomethane metabolism and found that the microsomes yielded 3.9 nmoles carbon monoxide/mg protein/minute and 14.0 nmoles free bromide/mg protein/minute. The cytosol fraction from the same liver preparation con- verted dibromomethane to bromide at a rate of 9.1 nmoles/mg protein/minute. Based on these data, it appears that similar amounts of DCM/mg protein/unit time are converted to carbon monoxide or carbon dioxide by microsomes and by cytosol. Since microsomes comprise 2% to 5% of liver and cytosol comprises about 10% of liver protein (Estabrook et al., 1971; Hogeboom et al., 1953), it would be anticipated, on a mass basis, that the cytosol would metabolize more DCM than would microsomes. It has been postulated that each pathway 38 ------- involves the formation of an active intermediate, and each intermediate can be a potential alkylating agent. The available data also indicate that most metabolism of DCM occurs in the liver, although small amounts of activity have been detected in the lung and kidney. Analysis of rat tissues 48 hours post-exposure to ^C-DCM showed that liver, kidney, and lung contain the most radioactivity (McKenna et al., 1982). At the present time, the implications of these observations in assessing the carcinogenic potency of DCM are unclear. 3.2.3. In Vivo Metabolism/Effect of Dose There have been a number of in vivo studies in which investigators have given animals l^C-DCM an(j tnen measured the amount of exhaled l^C-carbon monox- ide and l^C-carbon dioxide. Yesair et al. (1977) and Angelo (1985) assessed the metabolism of DCM by mice. Yesair et al. (1977) gave groups of mice 1 mg/ kg or 100 mg/kg l^C-DCM in corn oil by intraperitoneal injection, and then collected metabolic by-products for 96 hours. (The authors stated, without giving data, that most of the observed metabolism took place during the first 12 hours post-exposure.) Angelo (1985) gave groups of mice 10 mg/kg or 50 mg/ kg l^C-DCM in 25% polyethylene glycol by tail vein injection, and then collec- ted metabolic by-products for 4 hours. The data collected by these investiga- tors are summarized in Table 9. The data show that the mouse is able to metabolize DCM to carbon monoxide and carbon dioxide, and that over a large dose range, equal amounts of DCM are converted to carbon monoxide and carbon dioxide. However, because of different experimental designs used by Yesair et al. (1977) and Angelo (1985), it is not possible to assess whether there is a dose-response relationship. Indeed, the greater conversion of DCM to carbon monoxide and carbon dioxide in mice given 100 mg/kg compared to those given smaller doses is probably an artifact of experimental design, namely the use of corn oil carrier in the intraperitoneal injection of DCM. 39 ------- TABLE 9. IN VIVO METABOLISM OF DCM BY MICE Dose 1 mg/kgb 10 mg/kgc 50 mg/kgc 100 mg/kgb (11.76 umoles/kg) (117.6 umoles/kg) (588 umoles/kg) (1,176 umoles/kg) DCM exhaled3 ' 56.9 382.2 470.0 % of dose exhaled 48.4 65.0 40.0 C02a 5.9 23.8 80.6 294.0 C0a 5.3 17.5 29.4 235.0 aValues are umoles/kg. bYesair et al. (1977). cAngelo (1985). There has been one animal study reported on the.metabolism of inhaled 14C-DCM. McKenna et al. (1982) gave groups of rats a single 6-hour inhalation exposure to 50, 500, or 1500 ppm l^C-DCM. At the end of the exposure period, exhaled DCM, carbon dioxide, carbon monoxide, and urine were collected for 48 hours. At the end .of the 48-hour collection period, the rats were sacri- ficed and tissue levels of radioactivity were determined. The data obtained from this study are summarized in Tables 10 and 11 and in Figures 6 and 7. The authors interpreted these data to indicate saturability of metabolism because of the following observations: • The percent of administered DCM metabolized to carbon dioxide and carbon monoxide declined with increasing dose; and • There was less than a proportional increase in tissue levels of radioactivity. The key assumption made by McKenna et al. (1982) was that the disproportion- ate exhalation of DCM indicated saturated metabolism. However, a review of 40 ------- TABLE 10. BODY BURDENS AND METABOLIZED ^C-DCM IN RATS AFTER INHALATION EXPOSURE TO 11+C-DCM Exposure concentration 50 ppm 500 ppm 1,500 ppm Total body burden ,a mgEq ll*DCM/kg 5.53 ± 48.41 ± 109.14 ± 0.18 4.33 3.15 Metabolized 1£*DCMa mgEq 11+DCM/kg 5.23 ± 33.49 ± 49.08 ± 0.32 0.33 1.37 Metabol ized 94.6 69.2 45.0 aValues are mean ± standard deviation; number of animals in each group = 3. SOURCE: McKenna et al., 1982. TABLE 11. FATE OF i^C-DCM IN RATS AFTER A SINGLE 6-HOUR INHALATION EXPOSURE % body burden (x S.D., n = 3) Parameter measured Expi red Expired Expi red Urine Feces Carcass Skin CH2C12 C02 CO Cage wash 5 26 26 8 1 23 6 0 50 ppm .42 .20 .67 .90 .94 .26 .85 .75 0.73 1.21 3.00 0.39 0.19 1.62 1.62 0.33 30 22 18 8 1 11 6 0 500 ppm .40 .53 .09 .41 .85 .65 .72 .24 7 4 0 0 0 1 0 0 .10 .57 .81 .90 .68 .87 .13 .23 1500 ppm 55 13 10 7 2 7 3 0 .00 .61 .23 .20 .33 .24 .97 .43 1.92 1.20 1.68 0.74 0.05 0.65 0.15 0.15 SOURCE: McKenna et al., 1982. 41 ------- 10.0 r- "& •V U X U 5 U) 0.01 0.001 50 ppm 500 ppm 1500 ppm EXPOSURE I I I 4 5 TIME, hours 6/0 Figure 6. Plasma levels of DCM in rats during and after DCM exposure for 6 hours. Data points represent mean ± standard deviation for two to four rats. SOURCE: McKenna et al., 1982. 42 ------- 100.0 10.0 0 o o a O O XI X 1.0 0.1 i ' i ' i ' M i y EXPOSURE- I I 50 ppm 500 ppm 1500 ppm I I I I I I I I I I I I I I I 4 5 6/0 TIME, hours Figure 7. Blood COHb concentrations in rats during and after a 6-hour inhala- tion exposure to DCM. Each data point is the mean ± standard error for two to four rats. SOURCE: McKenna et al., 1982. 43 ------- the study indicates that the data do not support this assumption since the measurements were made when the DCM concentration within the animal was rapidly changing. For the purpose of analysis we selected a three-compartment model (lungs, blood, and tissues). In this model system the DCM concentration within the animal is dependent upon three components: 1) uptake by the lungs via passive diffusion, 2) diffusion into the blood, and 3) uptake by the tissues, metabolism, and exhalation. First, given the volatility of DCM, it is reason- able to expect that exhalation would be the major contributing component in modulating the post-exposure concentration within the animal. Indeed, the very rapid decline in the blood concentration of DCM (10 pg/mL to 0.1 pg/mL in less than 30 minutes) supports this assumption. Thus, the amount of DCM available for metabolism becomes a function of the amount remaining in the tissues post-exposure, and therefore is governed by the air/blood partition coefficient of DCM. While the data reported by McKenna et al. (1982) were not obtained under the steady-state conditions necessary for the comparison of different doses, the study showed that during the exposure period the carboxy- hemoglobin (COHb) levels in rats exposed to 500 and 1500 ppm DCM were the same (Figure 7). These data would support the concept that the carbon monox- ide pathway was saturated during the period of exposure. However, since similar data on the carbon dioxide pathway were not obtained, it is not possible to conclude that saturated metabolism occurred during the exposure period. One laboratory has reported results from similar experiments using mice and rats. Angelo (1985) gave 10 mg/kg or 50 mg/kg 14C-DCM intravenously to groups of mice or rats and then measured the amount of exhaled DCM, carbon dioxide, and carbon monoxide for 4 hours post-exposure. The data from these experiments are summarized in Tables 12 and 13. Rats given 10 mg/kg or 50 44 ------- TABLE 12. METABOLISM OF DCM FOLLOWING INTRAVENOUS ADMINISTRATION OF 10 mg/kg OR 50 mg/kg Mice Rats Exhaled 10 mg/kg 50 mg/kg 10 mg/kg 50 mg/kg DCM 48.4 ±6.1 65.0+4.2 44.5 ± 3.7 57.6+6.1 Carbon dioxide 20.2* 2.0 13.71 1.5 16.0 ± 1.0 9.9 ± 0.7 Carbon monoxide 14.9 ± 4.0 8.4 ± 2.9 12.7 ± 2.2 8.7 ± 2.6 aValues are percent of administered dose + standard deviation. SOURCE: Angelo, 1985. TABLE 13. METABOLISM OF DCM FOLLOWING INTRAVENOUS ADMINISTRATION TO CARBON MONOXIDE AND CARBON DIOXIDE Time post- exposure (min.) 0-60 60-240 Total 0-60 60-240 Total Mice Carbon Dose monoxide 10 mg/kg 0.7a 0.7 1.4 50 mg/kg 2.1 2.2 4.3 Rats Carbon dioxide 1.7 0.3 2.0 5.7 1.2 6.9 Carbon monoxide 0.2 1.1 1.3 0.7 3.7 4.4 Carbon dioxide 0.8 0.7 1.5 1.9 3.1 5.0 aValues are mg/kg. SOURCE: Angelo, 1985. 45 ------- mg/kg immediately (less than 20 minutes) exhaled about 35% of the administered dose, while mice immediately exhaled 45% of the low dose and 60% of the high dose (Table 14). The data, as shown in Table 14, indicate that in both species the amount of DCM metabolized to carbon monoxide and carbon dioxide is not proportional to the increase in administered dose. However, if the administered dose is adjusted by subtracting the amount of DCM immediately exhaled, there is a proportional relationship between dose and metabolites formed in the mouse and the rat. The adjusted data indicate that on a mg/kg basis the rat and mouse appear to be capable of metabolizing the same amount of DCM over a 4-hour period. This conclusion is supported by the findings of Yesair et al. (1977) and McKenna and Zempel (1981), who assessed the metabolism of small amounts of DCM in mice or rats. Both of the latter studies showed that most of the administered dose was metabolized to carbon monoxide and carbon dioxide. TABLE 14. METABOLISM OF DCM BY RATS AND MICE EFFECT OF DOSE CORRECTION FOR EXHALED SUBSTRATE Species Mouse Rat Dose 10 50 10 50 Amount exhaleda»b 4.6 29.8 3.2 17.6 Adjusted dose3 5.4 20.2 6.8 32.4 Amount of C0+C02 formed3 3.5 11.1 2.9 9.4 aValues are mg/kg. ^Amount exhaled in first 20 minutes. SOURCE: Adapted from Angelo, 1985. 46 ------- Rodkey and Collison (1977a) exposed a group of 4 rats to 154 ymoles of DCM (1255 ppm) in a chamber having a closed rebreathing system and determined the amount of carbon monoxide exhaled as a function of time. After a short lag period, the rats exhaled carbon monoxide at a rate of 30 ymoles/kg/hour. The authors stated that they obtained similar results after giving animals the same dose of DCM intraperitoneally. In a second experiment, a group of 4 rats exposed to 793 ymoles of DCM (6462 ppm) exhaled 40 ymoles carbon monoxide/kg/hour. These data indicate that in the rat the microsomal pathway is almost saturated at DCM exposures as low as 1255 ppm and that, therefore, the Vmax equals about 30 to 40 ymoles/kg/hour. Rodkey and Collison (1977b) also measured DCM biotransformation to carbon monoxide and carbon dioxide. Rats were exposed to 200 ymoles of DCM in a chamber with a closed rebreath- ing system, and the amount of carbon monoxide and carbon dioxide produced were determined at 7 to 9 hours post-exposure. The amount of carbon monoxide formed was about 90 ymoles, whereas the amount of excess carbon dioxide formed was only 56 ymoles. These data appear to differ significantly from those reported by McKenna et al. (1982) and DiVincenzo and Hamilton (1975), who found that more carbon dioxide than carbon monoxide was exhaled. However, since Rodkey and Collison (1977b) accounted for only about 75% of the admin- istered dose, these studies are not directly comparable. It has been estimated that.mice metabolize DCM to carbon monoxide at a rate of about 19 ymoles/kg/hr (EPA, 1985). This value was determined by assuming that the carbon monoxide formation was constant over the 12-hour post-exposure period in the experiments performed by Yesair et al. (1977). Given the volatility of DCM, it is unlikely that the substrate concentra- tion was saturating for 12 hours. Thus the rate constant for carbon monoxide formation in the mouse could well exceed 19 ymoles/kg/hr. 47 ------- The in vivo metabolic studies confirm the data obtained in vitro that DCM is biotransformed to carbon monoxide and carbon dioxide. There are some data which indicate that formation of carbon monoxide in the rat reached Vmax at exposures of less than 1200 ppm. There are no data on the exposure concen- tration required to saturate the carbon dioxide pathway. Comparative data on species differences and similarities are limited. At low doses both rats and mice metabolize similar amounts of DCM to carbon monoxide and carbon dioxide. 3.2.4. Human Studies There have been three studies in which volunteers have been exposed to DCM. DiVincenzo and Kaplan (1981a, b) evaluated the conversion of DCM to carbon monoxide in sedentary, non-smoking individuals and in individuals engaged in physical activity. Exposure, in a chamber, was to 50, 100, 150, or 200 ppm DCM for 7.5 hours or for 7.5 hours/day for 5 consecutive days. The metabolism of DCM was also studied in men engaged in physical activity. DiVincenzo and Kaplan (1981a) found that in sedentary individuals the pul- monary uptake of DCM was linear over the range studied (50-200 ppm), and that excretion of carbon monoxide was proportional to the pulmonary uptake of DCM. The authors noted that only 25% to 34% of the DCM taken up was converted to carbon monoxide, and therefore hypothesized, based on data from animal studies, that up to 70% was metabolized to carbon dioxide. Sedentary volunteers exposed once for 7.5 hours to 50, 100, 150, or 200 ppm of DCM had peak COHb concentrations of 1.9%, 3.4%, 5.3%, and 6.8%, respec- tively. When sedentary volunteers were exposed to 100, 150, or 200 ppm DCM for 7.5 hours/day for 5 consecutive days, the COHb of those exposed to 150 ppm or 200 ppm increased to levels above that of the single exposure. The volunteer exposed to 200 ppm DCM had a COHb of about 5% on day 1 and a COHb of about 6.5% on day 4. The carbon monoxide concentrations in the breath of 48 ------- the volunteers also increased each day during exposure to high concentrations (150-200 ppm) of DCM. Conversely, the peak level of expired DCM in the breath of the volunteers remained constant (peak level did not change) throughout the exposure period. DiVincenzo and Kaplan (1981b) also measured DCM uptake and metabolism during exercise. The data show (see Table 15) that uptake of DCM is directly related to work intensity, and that the amount of DCM metabolized to carbon monoxide is directly proportional to pulmonary uptake. This is illustrated by the finding that a sedentary volunteer exposed to 200 ppm DCM exhaled 6.1 mmoles of carbon monoxide (with a pulmonary uptake 21.1 mmoles DCM), while an exercising volunteer exposed to just 100 ppm DCM exhaled 11.8 mmoles of carbon monoxide (with a pulmonary uptake of 41.9 mmoles DCM). The latter observation suggests that the metabolic capacity of people exposed to 400 ppm DCM would not be exceeded. McKenna et al. (1980) exposed volunteers to 100 or 350 ppm DCM for 6 hours and measured various parameters, including blood and exhaled air levels of DCM, COHb, and exhaled carbon monoxide. The data showed that the blood level of DCM for both concentrations reached a steady-state in about 2 hours. At the end of the 6-hour exposure, the COHb concentration of the group exposed to 350 ppm DCM was 1.4-fold higher than that of the group exposed to 100 ppm. Likewise, the concentration of exhaled carbon monoxide in the group exposed to 350 ppm DCM was 2.1-fold higher than that of the group exposed to 100 ppm. The authors assumed that once steady-state is achieved, further uptake of DCM would be proportional to the rate of metabolism of DCM. Consistent with this steady-state assumption, McKenna et al. (1980) interpreted these findings to mean that the COHb and carbon monoxide levels between the high and low groups were less than proportional as metabolism became saturated. 49 ------- TABLE 15. EFFECT OF EXERCISE ON THE PULMONARY UPTAKE AND METABOLISM OF METHYLENE CHLORIDE DURING EXPERIMENTAL EXPOSURES TO METHYLENE CHLORIDE VAPOR Work intensity Volunteer (mL Q£ min~l kg"*) 1 4 2 14 3 15 19 4 16 28 Pulmonary uptake of DCM (mmoles) 10.7 19.4 30.7 36.4 28.8 41.9 Total pulmonary excretion of CO (mmoles) 2.7 5.1 10.2 14.3 10.4 11.8 SOURCE: DiVincenzo and Kaplan, 1981b. A review of the McKenna et al. (1980) study indicates that the data do not support the assumption that a steady-state blood level of DCM is a reflec- tion of saturated metabolism. The DCM concentration in the blood in the sim- plest model is a function of three components: uptake by the tissues, metabo- lism, and exhalation. First, given the volatility of DCM, it is reasonable to expect that exhalation is the major contributing component that modulates the blood level. Indeed, the data from the study support this assumption, since the amount of DCM exhaled increased disproportionately with dose. In addition, the very rapid decline in the blood level of DCM post-exposure argues very strongly that exhalation is the major component governing the observed blood level. Furthermore, the data reported by DiVincenzo and Kaplan (1981a) on volunteers exposed to 50 to 200 ppm DCM clearly show a slight but distinct disproportionate exhalation of DCM when the body mechanism to metabolize DCM 50 ------- has not been saturated. The finding by McKenna et al. (1980) that the COHb and carbon monoxide levels in the group exposed to 350 ppm DCM were less than 3.5-fold higher than in the group exposed to 100 ppm, and the subsequent interpretation that this indicates saturated metabolism, ignores the limitations of the design of the experiment. The initial amount of product formed using the same amount of enzyme and two different concentrations of substrate appears to be the same, and if at least one concentration of substrate is less than saturating, the ratio of product formed will continue to change until a steady-state or con- stant rate of carbon monoxide formation is achieved. The McKenna et al. (1980a) data clearly showed that both the COHb and the concentration of ex- haled carbon monoxide concentration were increasing during the exposure portion of the study (Figures 8 and 9), and thus, it is not possible to predict steady-state concentrations of either COHb or exhaled carbon monoxide. Lastly, DCM is metabolized via two pathways. Data from animal studies indicate that both pathways are of equal importance, and that perhaps the carbon dioxide pathway is more important. The authors' conclusion, based on data from only one pathway, that metabolic saturation in humans is achieved at less than 350 ppm DCM is therefore premature. 3.2.5. Summary The results of both in vitro and in vivo studies indicate that DCM is metabolized via two pathways. One pathway yields carbon monoxide as an end product, and the other yields carbon dioxide as an end product with formalde- hyde and formic acid as metabolic intermediates. Each pathway involves formation of a metabolically active intermediate which is theoretically capable of irreversibly binding to cellular macromolecules. A comparative analysis of the capability of various tissues to metabolize DCM indicates 51 ------- 100—i 10—J •o o _ O I O O 1 - Exposure 0246 350 ppm 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours Figure 8. COHb level in volunteers exposed to 100 ppm or 350 ppm DCM. SOURCE: McKenna et al., 1980. 52 ------- 100 —, CO IN EXPIRED AIR 10 — c. O •3 X tu 1 __ Exposure I i I 0246 0 2 4 6.8 10 12 14 16 18 20 22 24 Hours Figure 9. Exhaled carbon monoxide by volunteers exposed to 100 ppm or 350 ppm OCM. SOURCE: McKenna et al., 1980. 53 ------- that the liver is the primary site of metabolism, with some metabolism taking place in the lung and kidney. An analysis of the available in vitro data suggests that the carbon dioxide pathway may metabolize significantly more DCM than the carbon monoxide pathway. Consistent with this observation is j[n_ vivo data which suggest that when rats or mice are exposed to high concentra- tions of DCM they exhale more carbon dioxide and excrete more formic acid than carbon monoxide. At exposure to low concentrations of DCM, both pathways are utilized about equally. The data from rat studies also suggest that the route of exposure, at low doses, results in similar metabolic profiles. A comparative analysis of the data from in vivo studies in mice, rats, and humans indicates that all three species metabolize DCM to carbon monoxide (Table 16). Both mice and rats metabolize DCM to carbon dioxide. There are no human data on the metabolism of DCM to carbon dioxide. However, based on uptake data, some investigators have speculated that this pathway is func- tional in humans. Groups of rats exposed to 500 or 1500 ppm DCM have the same COHb, sug- gesting that the carbon monoxide (microsomal) pathway has been saturated. There are no similar data on the cytosolic pathway. One group of investiga- tors has suggested that the microsomal pathway is saturated in humans at less than 350 ppm DCM. However, an analysis of the study indicates that the data do not support this conclusion. At present, the available data are insufficient for the purpose of esti- mating doses of DCM at which metabolism is saturated. The available data indicate that at low doses little unmetabolized DCM is exhaled, and that at high doses there is a significant exhalation of DCM immediately post-exposure. The data suggest that at high doses more DCM is taken up into the body. Currently, there are insufficient data to determine the relationship between 54 ------- TABLE 16. METABOLISM OF DCM TO CARBON MONOXIDE, CARBON DIOXIDE, AND FORMIC ACID IN MICE, RATS, AND HUMANS Species Exposure/uptake Carbon monoxide (ymoles/kg) Carbon dioxide (ymoles/kg) Mice3 Miceb Miceb Mice3 Ratsc Ratsb Ratsc Ratsb Ratsd Rats6 Rats6 Rats6 Humans^ 11.8 ymoles/kg 117.6 ymoles/kg 588.0 ymoles/kg 1176.0 ymoles/kg 11.8 ymoles/kg 117.6 ymoles/kg 588.0 ymoles/kg 588.0 ymoles/kg 6009.4 ymoles/kg 50 ppm (65.0 ymoles/kg) 500 ppm (569.3 ymoles/kg) 1500 ppm (1,283.5 ymoles/kg) 50 ppm (79.1 ymoles/kg) 100 ppm (152.9 ymoles/kg) 150 ppm (219.7 ymoles/kg) 200 ppm (301.0 ymoles/kg) 5.3 16.5 50.6 235 3.6 15.2 70 51.7 129.2 17 103 131 18.6 28.6 71.4- • 87.4 5.9 22.3 81.1 294 4.1 (4.7)9 17.6 37 (48.8) 58.8 182.9 (242.9) 17.3 (23.1) 128 (175.8) 174 (266.4) 55.8 107.1 154.3 210 aYesair et al. (1977). DCM given i.p. in corn oil. According to the authors, the metabolism of methylene was essentially complete 12 hours post-exposure. bAngelo (1985). DCM given i.v. in 25% polyethylene glycol. Metabolism was monitored for 4 hours post-exposure. GMcKenna and Zempel (1981). DCM given by gavage. Metabolism was monitored for 48 hours post-exposure. dDiVincenzo and Hamilton (1975). DCM given i.p. in corn oil. Metabolism was monitored for 48 hours post-exposure. 6McKenna et al. (1982). DCM given in a single inhalation exposure for 6 hours, Metabolism was monitored for 48 hours post-exposure. DDiVincenzo and Kaplan (1981). DCM given in a single inhalation exposure for 7.5 hours. The amount of post-exposure carbon monoxide exhaled was measured for 24 hours. The amount of carbon dioxide exhaled was based on assumptions made by the authors. For the purpose of comparison, it was assumed that the weight of each volunteer was 70 kg. 9Carbon dioxide + radioactivity in urine (assumed to be formic acid). 55 ------- exposure concentration and uptake. It is of interest to note that the obser- vation has been made that fat people take up more DCM than thin people, and and that high levels of DCM are found in fat tissues post-exposure (U.S. EPA, 1985). There is a paucity of data on the genotoxicity of DCM. Commercially available DCM gives weak but positive results in Salmonella, yeast, and Drosophila without metabolic activation. It has been shown to induce chro- mosomal aberrations in some cultured mammalian cell systems but not in others. DCM also causes a weak increase in sister chromatid exchange, but it has not been shown to cause unscheduled DNA synthesis or to inhibit DNA synthesis (U.S. EPA, 1985). At the present time, based on positive mutagenic studies and the likelihood that reactive intermediates are formed during biotransformation to carbon monoxide and carbon dioxide, it seems reasonable to assume that DCM exerts its carcinogenic effect via a genotoxic mechanism. Based on this analysis, it is concluded that the available pharmacokine- tic/metabolism data do not offer useful parameters for making assumptions in the calculation of quantitative carcinogenic risk assessments for DCM. 56 ------- 4. QUANTITATIVE ESTIMATION (USING THE NTP INHALATION BIOASSAY) The EPA Office of Health and Environmental Assessment has recently pub- lished a comprehensive document on the health effects of dichloromethane (methylene chloride, DCM) (U.S. EPA, 1985). After the completion of this report, the NTP released the findings of an inhalation toxicology and carcino- genesis study of DCM in F344/N rats and B6C3F1 mice (NTP, 1985, draft). The qualitative findings of this study are discussed in a preceding section of this document; here, the NTP findings are used to develop estimates of unit incremental cancer risks for humans exposed to DCM. Variations in extrapo- lated risks using different cancer end points from the NTP study are discussed, as well as the influence of the dose-response model selected. The quantita- tive findings are also compared with earlier experimental carcinogenesis studies of DCM and with the limited information available from epidemiologic studies. 4.1. SUMMARY OF NTP FINDINGS USED FOR QUANTITATIVE ANALYSIS In an earlier section of this document, the end points of mammary and subcutaneous tumors in rats and lung and liver tumors in mice were determined to be the sites where the NTP study produced the strongest findings of car- cinogenicity for DCM. Tables 17 and 18 present the NTP tumor incidence find- ings for these sites. The denominator for each data point is the number of animals that were examined at the specific tumor site. The statistical significance of the NTP findings has been discussed earlier in this report, and the findings are only summarized in Tables 17 and 18. The data in these two tables will serve as the basis for the quantitative risk estimates to be developed. The EPA draft guidelines for carcinogen risk 57 ------- TABLE 17. SUMMARY OF NTP INHALATION STUDY OF DCM: FINDINGS FOR MAMMARY AND SUBCUTANEOUS TUMORS IN RATS 01 00 . Dose Site/tumor Control Males Mammary gland: adenoma or fibroadenoma3 0/50 Subcutaneous tissue: fibroma0 1/50 Mammary gland or subcutaneous tissue: 1/50 adenoma, fibroadenoma or fibroma3 Females Mammary gland: fibroadenoma3 5/50 Mammary gland: adenoma, 7/50 fibroadenoma, adenocarcinoma, or mixed tumor, malignant3 1,000 ppm 0/50 1/50 ; 1/50 ll/50b 13/50° 2,000 ppm 2/50 2/50 4/50 13/50b 14/50b 4,000 ppm 5/50b 4/50 9/50d 22/50d 23/50d 3A11 trend tests for tumor incidence positive at p < 0.01 nominal level. D0ne or more positive pairwise comparison(s) with control group p < 0.05 nominal level. C0ne or more positive trend test(s) for tumor incidence p < 0.05 nominal level. dAll pairwise comparisons with control group positive p < 0.01 nominal level. 1 ' Note: Tumor trend tests reported by NTP: life table, incidental tumor, and Cochran-Armitage tests; pairwise tests: life table, incidental tumor, and Fisher Exact tests. SOURCE: NTP, 1985. ------- TABLE 18. SUMMARY OF NTP INHALATION STUDY OF DCM FINDINGS FOR LUNG AND LIVER TUMORS IN MICE Dose Tumor Control 2,000 ppm 4,000 ppm Males Alveolar/bronchiolar adenoma3 3/50 19/50b 24/50b Alveolar/bronchiolar carcinoma8 2/50 10/50C 28/50b Alveolar/bronchiolar adenoma or carcinoma3 . 5/50 27/50b 40/50b Hepatocellular adenoma^ 10/50 14/49C 14/49C Hepatocellular carcinomad 13/50 15/49 . 26/49c Hepatocellular adenoma or carcinoma3 22/50 24/49 33/49c Alveolar/bronchiolar or hepatocellular carcinoma6^ 15/50 21/49 39/49b Alveolar/bronchiolar or hepatocellular adenoma or carcinoma6'^ 27/50 34/49d . 45/49b 01 10 Females Alveolar/bronchiolar adenoma3 2/50 23/48b 28/48b Alveolar/bronchiolar carcinoma3 . 1/50 13/48b 29/48h Alveolar/bronchiolar adenoma or carcinoma3 3/50 30/48b 41/48b Hepatocellular adenoma3 . 2/50 6/48 22/48b Hepatocellular carcinoma3 1/50 ll/48b 32/48b Hepatocellular adenoma or carcinoma3 3/50 16/48b 40/48b Alveolar/bronchiolar or hepatocellular carcinoma6'' 1/50 21/48b 43/47b Alveolar/bronchiolar or hepatocellular adenoma or carcinoma6*' 5/50 36/48b 46/47b 3A11 trend tests for tumor incidence positive at p < 0.01 nominal level. bAll pairwise comparisons with control group positive p < 0.01 nominal level. C0ne or more positive pairwise comparison(s) with control group p < 0.05 nominal level. done or more positive trend test(s) for tumor incidence at p < 0-05 nominal level. eDenominators are number of animals examined for tumors at both luny and liver sites. 'Tumor grouping not presented in NTP report; significance determined using Fisher Exact Test. Note: Tumor trend tests reported by NTP: life table, incidental tumor, and Cochran-Armitage tests; pairwise tests: life table, incidental tumor, and Fisher Exact tests. SOURCE: NTP, 1985. ------- assessment (U.S. EPA, 1984) call for risk estimation using the combined inci- dence of statistically elevated tumors. The combined incidence of lung and liver tumors in mice is given for quantitative analysis, and does not imply that the tumors are biologically related. 4.2. DOSE-RESPONSE MODEL SELECTION The EPA draft guidelines for carcinogen risk assessment (U.S. EPA, 1984) express the fact that there is no rigorously established scientific basis for the selection of a dose-response model to predict carcinogen risks at low doses. In the typical situation where there is limited information on which to base the selection of a model, the draft EPA guidelines express a preference for the multistage dose-response model. The guidelines place emphasis on the upper confidence limit (UCL) risk estimates derived from this model. The bases for the preference include the following: 1) The multistage model incorporates the current scientific opinion that multiple steps are involved in the process of cancer develop- ment, and that a chemical carcinogen can contribute to one or more of these steps. 2) The UCL of the multistage model produces a risk estimate that is linear at low dose (LDL). An LDL dose-response is expected when a carcinogen accelerates stages of the carcinogenic process that lead to the background occurrence of cancer in unexposed members of the population. 3) The UCL of the multistage model produces a "plausible upper bound" estimate of risk, i.e., an estimate that is reasonable but is usu- ally as high or higher than estimates derived from other models; models that are not linear at low dose will generally lead to sub- 60 ------- stantially lower risk estimates. 4) The multistage UCL is stable under small changes in the input values for tumor incidence; in contrast, the maximum likelihood estimate (MLE) can be unstable if the results in one or a few animals are changed. Because of the role of genetic and mutational factors in the development of many cancers, a supportive biological argument for LDL can be made strongly for chemicals that are known to cause genetic damage. The EPA Health Assess- ment Document for Dichloromethane (U.S. EPA, 1985) concluded that the weight of evidence shows that DCM is capable of causing gene mutations and has the potential to cause such effects in exposed human cells. Further testing to determine the strength of mammalian evidence was recommended. These findings provide additional support for the application of an LDL dose-reponse model in estimating DCM cancer risks. Considering both EPA's policy for carcinogen evaluation and the biological information available specifically for DCM, the multistage model was selected as the primary model to be applied in this risk assessment. Versions of the multistage model which incorporate time-to-tumor information have been devel- oped. While time-to-tumor models generally do not produce risk estimates that differ greatly from similar dichotomous models, the results are presented for comparison. Several other models are also presented for comparison with the multistage model. 4.3. APPLICATION OF THE MULTISTAGE MODEL TO NTP BIOASSAY DATA The multistage dose-response model, as incorporated in the GLOBAL83 computer program developed by Howe (1983), has been applied to the NTP bio- assay data given in Section 4.2. The model is applied to the experimentally 61 ------- rodents exposed to DCM following the same time pattern as the NTP bioassay. A separate section of this report reviews the available pharmacokinetic and metabolic data on DCM and concludes that these data do not provide an adequate basis for modifications to the experimental doses for use in quantitative risk assessment. The GLOBAL83 program enables the user to select the highest degree of polynomial that the program will allow in the model. GLOBAL83 runs were made for a polynomial degree equal to the number of doses (counting the control) minus one. Tables 19, 20, 21, and 22 present the results of these computa- tions. Several conclusions can be drawn from these data. 1) The multistage model provides an adequate fit for all tumor groupings analyzed in both rats and mice. 2) In rats, the highest value for the UCL of the linear multistage term was obtained for mammary tumors in female rats. The highest value in males was lower by a factor of three. 3) In mice, the highest value for the UCL of the linear term was obtained in females having either adenomas or carcinomas of the lung and/or liver. The corresponding value for males was lower by a factor of two. 4) In mice, the male and female high-dose groups had a high percen- tage of tumors in both the lung and liver. As discussed in Section 4.4., the high-dose mice also showed elevated mortality in comparison to controls. In these circumstances, competing risks can lead to underestimates of risk attributable to indivi- dual tumor types. 5) The NTP (1985) noted that all male rat groups (including controls) 62 ------- TABLE 19. GLOBAL83 MODEL PARAMETERS FOR NTP (1985) RAT DCM DATA OJ 3-stage model Site q0 q^lO"3 (ppnr1) Males Mammary gland3 0.0 0.0' Subcutaneous13 0.0191 0.00093 Mammary gland or 0.0197 0.00075 subcutaneous0 Females Mammary glandd O.ill 0.107 Mammary gland6 0.161 0.0985 q2xlO'6 (ppnr2) 0.00696 0.00389 0.0117 0.0 0.0 q3xlO"10 q^xlO"3 (ppm-3) (ppm-1) 0.0 0.0311 0.0 0.0306 0.0 0.0540 0.00544 0.164 0.00846 0.164 aAdenoma or fibroadenoma. ^Fibroma. cAdenoma, fibroma, or fibroadenoma. ^Fibroadenoma. eAdenoma, fibroadenoma, adenocarcinoma, or mixed tumor, malignant. q^ = The itn power coefficient in the multistage model. q^ = The 95% upper confidence limit estimate of the linear coefficient. Values for the q^ apply for rats exposed under the NTP protocol dose time schedule. Calculations are based on an extra risk analysis. ------- TABLE 20. COMPARISON OF THREE-STAGE GLOBAL83 ESTIMATES WITH OBSERVED TUMOR RESPONSE - RATS Tumor Males Mammary gland . adenoma or fibroadenoma Subcutaneous Mammary gland or subcutaneous Females Mammary gland fibroadenoma Mammary gland, Observed 1,000 Control ppm 0.0 0.0 2.0 2.0 2.0 2.0 10.0 22.0 14.0 26.0 response (%) 2,000 4,000 ppm ppm 4.0 10.0 4.0 8.0 8.0 18.0 26.0 44.0 28.0 46.0 Predicted 2,000 Control ppm ' 0.0 0.7 1.9 2.4 1.8 3.0 10.5 19.7 14.9 22.9 response (%) 4,000 4,000 ppm ppm 2.7 10.5 3.6 8.2 6.4 18.8 28.1 43.7 30.6 45.6 Chi squared 0.66 0.06 0.42 0.30 0.46 all tumors ------- TABLE 21. GLOBAL83 MODEL PARAMETERS FOR NTP (1985) MOUSE DCM DATA Two-stage model Tumor Males Lung3 adenoma Lung carcinoma Lung adenoma or carcinoma Li verb adenoma Liver carcinoma Liver adenoma or carcinoma Lung or liver carcinoma Lung or liver adenoma or carcinoma Females . Lung adenoma Lung carcinoma Lung adenoma or carcinoma Liver adenoma Liver carcinoma Liver adenoma or carcinoma Lung or liver carcinoma Lung or liver adenoma or carcinoma <0 0.0672 0.0398 0.105 0.237 0.285 0.565 0.333 0.771 0.0445 0.0202 0.0619 0.0356 0.0193 0.0576 0.0197 0.105 qi*io-3 (ppnr1) 0.166 0.0 0.295 0.0306 0.0 0.0 0.0 0.0 0.242 0.0690 0.453 0.0 0.0 0.0 0.0 0.345 q2xlO"6 (ppnr2) 0.0 0.0481 0.0202 0.0 0.0283 0.0338 0.0739 0.107 0.0 0.0394 0.0032 0.0336 0.0655 0.101 0.147 0.148 (ppnr1) 0.223 0.141 0.452 0.0810 0.142 0.195 0.190 0.429 0.310 0.233 0.579 0.0872 0.145 0.160 0.244 0.870 aLung = alveolar/bronchiolar. ^Liver = hepatocellular. C0nly the linear parameters are given for the 4-stage model. q.j = The ith power coefficient in the multistage model. q-^ = The 95% upper confidence limit estimate of the linear coefficient. Values for the q-j apply for mice exposed under the NTP protocol dose time schedule. Calculations are based on an extra risk analysis. 65 ------- TABLE 22. COMPARISON OF TWO-STAGE GLOBAL83 ESTIMATES WITH OBSERVED TUMOR RESPONSE - MICE CTi Observed response (%) Tumor Females Lung adenoma Lung carcinoma Lung carcinoma or adenoma Liver adenoma Liver carcinoma Liver carcinoma or adenoma Liver/lung carci- noma Liver/'lung carci- noma or adenoma Males Lung adenoma Lung carcinoma Lung carcinoma or adenoma Liver adenoma Liver carcinoma Liver carcinoma or adenoma Lung/liver carci- noma Lung/liver carci- noma or adenoma Control 4.0 2.0 6.0 4.0 2.0 6.0 2.0 10.0 6.0 4.0 10.0 20.0 26.0 44.0 30.0 54.0 2,000 ppm 47.9 27.1 ' 62.5 12.5 22.9 33.3 43.8 75.0 38.0 20.0 54.0 28.6 30.6 49.0 42.9 69.4 4,000 ppm 58.3 60.4 85.4 45.8 66.7 83.3 91.5 97.9 48.0 56.0 80.0 28.6 53.1 67.3 79.6 91.8 Two-stage predicti Control 4.3 2.0 6.0 3.5 1.9 5.6 2.0 10.0 6.5 3.9 10.0 21.1 24.8 43.1 28.3 53.7 2,000 ppm 41.0 27.1 62.5 15.6 24.5 37.0 45.5 75.0 33.0 20.7 54.0 25.8 32.8 50.3 46.6 69.9 on (%) 4,000 ppm 63.7 60.4 85.4 43.6 65.6 81.3 90.6 97.9 52.0 55.5 80.0 30.2 52.2 66.9 78.0 91.7 Chi square 1.54 <.01 <.01 0.49 0.93 0.43 0.10 <.01 0.90 0.02 <.01 0.30 0.16 0.06 0.42 0.08 ------- experienced higher than usual mortality before final sacrifice. In female rats, there was increased mortality in the high-dose group relative to controls. Under these conditions, competing risks may lead to underestimation of the risk attributable to the tumors observed in the rats. The NTP indicated that the decreased rat survival is likely to be due to the frequent occurrence of leukemia in all groups. The draft EPA carcinogen evaluation guidelines (U.S. EPA, 1984) indicate that weight should be placed on the analysis of risks using the animals that have tumors in any one of the sites found to be statistically elevated. The draft guidelines also indicate that weight should be placed on the experimental species and sex group showing the highest risks. On these grounds, combined carcinomas and adenomas of the lung and/or liver in female mice is the end point of most weight. Thus, additional analyses were conducted, with emphasis on the data for mice having lung and/or liver tumors. The following sections describe these analyses. 4.4. RISK ANALYSIS CONSIDERING TIME-TO-TUMOR INFORMATION In the NTP study, there was a small number of deaths in mice before the first lung or liver tumors were found. The first lung or liver tumor was a liver tumor in a high-dose male mouse at week 61. Table 23 shows the numbers of male and female mice alive at 61 weeks that were subsequently examined at the lung and liver sites. While the number of animals lost to early mortality was small, because of the very high incidence of tumor responses seen in the high-dose groups, analyses were conducted to determine if the early deaths might have an effect on estimated risks. Table 24 shows the results of applying the two-stage multistage model to the mouse data using the denominators from Table 23. 67 ------- TABLE 23. MICE SURVIVING TO 61 WEEKS AND RECEIVING EXAMINATION OF THE LUNG AND LIVER Response Males Females Control 50 46 Dose 2,000 pptn 45 46 4,000 ppm 47 46 Note: In all, 13 mice were excluded from counting due to early death. The times of these deaths were: 0-3 months, 5 deaths; 3-6 months, 1 death; 6-9 months, 4 deaths; 9-12 months, 1 death; 12 months-61 weeks, 2 deaths. SOURCE: NTP, 1985. TABLE 24. GLOBAL83 MODEL PARAMETERS FOR MOUSE LUNG AND LIVER TUMORS COMBINED—NTP (1985) DCM DATA ON ANIMALS SURVIVING TO 61 WEEKS (WHEN FIRST TUMOR OCCURRED) Two-stage model parameters Tumor o c qQ q-^xlO'0 q2x!0"° (ppm"1) (ppm"2) (ppm-1) Males Lung or liver carcinoma Lung or liver carci- noma or adenoma Females Lung or liver carci- noma Lung or liver carci- noma or adenoma 0.341 0.777 0.0211 0.114 0.0 0.0371 0.0 0.0 0.0854 0.139 0.159 0.363 0.235 0.582 0.245 0.785 Values of the q-j apply for the NTP protocol dose schedule. 68 ------- It should be noted that this simplified analysis has the weakness that animals dying before the first tumor was observed may in reality (as would be seen in a large population) have experienced some risk of cancer, and secondly, that animals dying after the first tumor was observed are treated as having been at full lifetime risk of cancer independent of their time of death. The data in Table 24, in comparison with the data in Table 21, show that the GLOBAL q^ parameter estimates are consistent with the earlier analysis and are not strongly influenced by the adjustment of the denominators. In contrast, for some of the analyses, the MLE q^ estimates are unstable: for male mice the qj estimate for combined adenomas or carcinomas of the lung or liver in Table 21 is zero, whereas in Table 24 it is positive. For female mice, for the same combination of tumors and sites, this situation is reversed. The variability of the MLE linear term is consistent with the CAG's experience that this parameter estimate is often unstable in response to small changes in the input data. To provide a comparison with the dichotomous multistage model risk esti- mates developed in the preceding section, a time-to-tumor formulation of the multistage model, the WEIBULL82 time-to-tumor program, also developed by Crump (19 ), was applied to NTP cancer results in mice. The WEIBULL82 pro- gram is based on the following equation: Prob [effect] = 1 - exp C-Q (dose) x (time - T0)K] where Q is a fitted polynomial of the same form utilized in GLOBAL83, and T0 and K are fitted parameters for the time-dependence of the tumor response. Risk calculations using this model can be made for two end points: either tumors are assumed to be "incidental" and unrelated to the cause of an animal's 69 ------- death, or the tumors are "fatal" and are assumed to produce death directly. NTP studies do not attempt to identify the cause of death for animals in cancer bioassays; therefore, a time-to-tumor analysis must hypothesize as to the causes of deaths in study animals dying before final sacrifice. The NTP (1985) DCM bioassay report provides statistical analyses showing that in mice both the male and female high-dose groups experienced elevated mortality in the latter part of the 2-year study. A simple comparison demonstrates that the observed tumors may reasonably have produced this mortality. Table 25 shows the study mortality divided into three time periods, deaths before 61 weeks (when the first lung or liver tumor was observed), deaths between 61 and 103 weeks, and deaths at the final sacrifice at 104 weeks. The table also indicates the numbers of animals, in each death category, which were observed to have carcinomas of either the lung or liver; these are the tumor types that can be most strongly expected to contribute to mortality. Table 26 shows that mortality before the occurrence of the first lung or liver tumor was small and comparable in all groups. In the 61-103 week period, where elevated treatment-associated mortality is seen, the number of animals dying without carcinomas was relatively stable among the male dose groups. In high-dose females, few animals died without carcinomas. These data are consistent with DCM-induced tumors leading to the increased mortality observed in the NTP mice. In the following analysis, using the WEIBULL82 program, the effect of the alternate assumptions of incidental or fatal tumors will be compared. For completeness, both "fatal" and "incidental" analyses are presented for adenoma response as well as for the other tumor groupings. However, it is noted that a "fatal" tumor analysis may be less appropriate for adenomas. The data from these analyses are given in Table 26. 70 ------- TABLE 25. MORTALITY IN NTP MOUSE DCM BIOASSAY Dose Males Control 2,000 ppm ' 4,000 ppm Females Control 2,000 ppm 4,000 ppm Deaths before 61 weeks 0(0)a 4(0) 3(0) 4(0) 4(0) 4(0) Deaths 61-103 weeks 11(5) 22(10) 36(29) 21(0) 21(8) 38(35) Final sacrifice deaths 39(10) 24(11) 11(10) 25(1) 25(13) 8(8) aNumbers in parentheses indicate the number of animals in each group found to have lung or liver carcinomas. SOURCE: NTP, 1985. 71 ------- TABLE 26. COMPARISON OF HEIBULL82 AND GLOBAL83 PREDICTIONS FOR RISK AT 104 WEEKS-- MICE LUNG AND LIVER TUMORS8 "Fatal" tumor WEIBULL82 analysis Tumor Males Lung adenoma Lung carcinoma Liver adenoma Liver carcinoma Lung or liver carcinoma Lung or liver carcinoma or adenoma Females Lung adenoma Lung carcinoma Liver adenoma Liver carcinoma Lung or liver carcinoma Lung or liver carcinoma or adenoma q xlO-3 (ppm-1) 0.125 -- 0.0344 0.0417 0.0573 0.106 0.0 <.001 0.0 0.002 0.0153 0.0769 q*xlO-3 (ppm-1) 0.173 — 0.0787 0.1502 0.199 0.252 0.148 0.0651 0.0440 0.0975 0.137 0.210 nb 5 - 5 5 4 4 2 4 4 4 4 4 "Incidental" tumor WEIBULL82 analysis (ppm-1) 0.232 0.0561 0.0687 0.0369 0.0526 0.178 0.352 0.109 0.0084 0.0526 0.119 0.0 q*xlO-3 (ppm-1) 0.299 0.199 0.131 0.159 0.304 0.644 0.447 0.248 0.105 0.198 0.392 0.822 n 5 5 5 5 4 4 4 4 4 4 4 4 GLOBAL83 Two-stage (ppm-1) 0.166 0.0 0.0306 0.0 0.0 0.0 0.242 0.690 0.0 0.0918 0.0 0.345 q*xlO-3 (ppm-1) 0.223 0.141 0.0810 0.142 0.190 0.429 0.310 0.233 0.0872 0.145 0.244 0.870 aFor the WEIBULL82 model, q^ is defined aj the first-degree polynomial coefficient multiplied by the time function evaluated at week 104. q, is derived from model risk predictions at low dose. bn indicates the degree of the dose polynomial used in the WEIBULL82 analysis. Different degrees were used in this exploratory analysis. Values of the qi apply for the NTP protocol dose schedule. 72 ------- The following observations can be drawn from these data: 1) The q1 and qj values for the "incidental" tumor analysis from the WEIBULL83 program were generally higher than the values from the "fatal" tumor analysis. However, the size of this difference was moderate, constituting approximately a factor of two, with a maximum difference of a factor of four for q^ Such differences are expected because the "fatal" tumor analysis excludes tumors found at final sacrifice, and these tumors provide important quantitative contributions to the NTP findings (see Table 25 for examples). While the qj estimates generally followed this same pattern, the deviations were greater in some cases. 2) q^ and q^ estimates from the two-stage GLOBAL83 analysis agreed overall with the range of "incidental" and "fatal" tumor WEIBULL82 analyses. In all cases, the GLOBAL83 q^ values fell either within or very close to the range of the two WEIBULL q^ values. From this exploratory analysis, it can be seen that use of the WEIBULL82 time-to-tumor analysis does not lead to risk estimates strongly different from those derived from GLOBAL83. The WEIBULL82 program has been less widely utilized than the GLOBAL83 program, and requires assumptions as to the cause of animal death. For these reasons, WEIBULL82 is less well suited than GLOBAL83 for formal use in the present risk assessment. 4.5. COMPARISON OF RISKS ESTIMATED WITH OTHER DOSE-RESPONSE MODELS In order to provide comparison with the two-stage (restricted) multi- stage estimates presented in Table 5, calculations for combined lung and liver tumors were also made with the one-stage (one-hit) and four-stage (dichotomous) formulations of the multistage model. The one-stage or linear model has been one of the most widely used models in carcinogen risk assess- 73 ------- ment, and provides a simple formulation of the hypothesis that many fundamental carcinogenic processes are linear in nature. The four-stage model shares the multistage rationale of the two-stage model, but allows a sharper upward cur- vature in risk estimates. Table 27 shows parameter estimates for one-stage and four-stage versions of the GLOBAL83 program for tumor response data from Table 18. The one-stage model fits the experimental data acceptably for the combined carcinomas and adenomas in both the male and female groups. For the females, the one-stage model response estimates are within 4% of the observed response for the three experimental doses; for males, the estimates are within 6% of the observed response. For carcinomas alone, the one-hit model does not provide a good fit to the data in either sex (p < 0.05 males, p < 0.01 females, by the chi square test). As can be seen from Tables 21 and 22, the two-stage model provides an acceptable fit to the data for all four tumor end points. The four-stage model provides an exact fit for all four data sets. These analyses demonstrate that for combined adenomas and carcinomas of the lung and/or liver in both male and female mice, the data are compatible with a linear dose-response, and that the 95% UCL linear term estimates from the three models are consistent within 20% in males and within 10% in females. For the carcinoma response, where the one-stage model did not fit well, the four-stage model yielded positive MLE linear term estimates for both sexes, while the two-stage model did not. The UCL four-stage risk estimates were approximately 50% and 20% higher than the UCL two-stage estimates for combined lung and liver carcinomas in the males and females, respectively. To provide comparisons with multistage risk model estimates, two models with different theoretical formulations, the Weibull (dichotomous) and probit models, were used to analyze the combined lung and liver tumor data in both the male and female mice. These models were fitted to the data using the RISK81 74 ------- TABLE 27. ONE-STAGE AND MULTISTAGE MODEL PARAMETERS FOR TUMORS IN MICE3 Tumor One-stage Chi square Two-stage Cni square Four-stage Chi square in Male mice Lung or liver carcinoma Lung or liver carcinoma or adenoma 0.238 0.329 4.44 0.348 0.505 1.94 0.0 0.190 0.42 0.0 0.424 <.01 0.058 0.223 <.01. 0.125 0.429 <.01 Female mice Lung or liver carcinoma Lung or liver carcinoma or adenoma 0.418 0.522 6.77 0.736 0.936 1.25 0.0 0.244 <.01 0.345 0.870 <.01 0.120 0.368 <.01 0.472 0.870 <.01 aUnits: ppm~l ID"3. ------- computer program developed by Kovar and Krewski (1981). The RISK81 program provides two formulations of both models, one which is based on the assumption that the observed tumor incidence is independent of the background tumor rates observed in the controls, and a second formulation which assumes that the carcinogen contributes a dose that is additive to the background effects seen in the controls. In the additive case, the probit and Weibull models produce risk estimates that are linear at low dose. Tables 28 through 31 provide the results from these models in comparison to the two-stage GLOBAL83 results. The tables show the DCM doses that are estimated to produce four given levels of risk under the different models, as well as lower confidence limits (LCLs) on the dose that produces the specified effect. LCL dose estimates correspond to UCL risk estimates. In all cases, the doses estimated to produce a given risk by the back- ground-independent probit model are markedly higher than those predicted by the multistage model (four orders of magnitude difference in the female mice com- bined tumor group). While the independent Weibull MLE estimates of dose are substantially below those obtained with the independent probit model, they are substantially higher than those obtained with the two-stage multistage model in all four analyses. The independent Weibull MLE estimates are broadly com- parable to the multistage MLE estimates for cases where the MLE multistage linear term is zero. The LCL dose estimates of the independent Weibull model are notably lower than the MLE estimates. The additive background formulation of both the Weibull and probit models converged to provide acceptable parameter estimates only for the female mouse data sets. In these two cases, both models produced estimates of dose for a fixed risk that were linear at low dose and were markedly higher than the corresponding independent background models. The MLE and LCL dose estimates 76 ------- TABLE 28. MALE MICE CARCINOMAS OF LUNG OR LIVER: DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE) Risk level 10-2 10-4 10-6 10-8 GLOBAL83 two-stage MLE LCL 369 52.4 36.8 0.524 3.68 5.24 x 10-3 0.368 5.24 x 10-5 Independent probit MLE 1,080 543 329 217 LCL 531 185 84.7 44.4 Independent Wei bull MLE 723 123 20.9 3.57 LCL 217 11.0 0.556 0.0281 Calculations are based on excess risk over background. Probit and Weibull model estimates calculated using RISK81 computer program. LCL estimates are the 95% confidence lower bound on dose (variance based on log dose for RISK81). Note: The additive probit and additive Weibull models failed to converge for this data set. ------- TABLE 29. MALE MICE CARCINOMA OR ADENOMA OF LUNG OR LIVER: DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE) —I 00 Risk level 10-2 io-4 ID'6 10-8 GLOBAL83 two-stage MLE LCL 306 23.4 30.6 0.233 3.06 2.33 x IO-3 0.306 2.33 x 10-5 Independent probit MLE 885 410 236 150 LCL 353 101 41.0 19.5 Independent Weibull MLE . 493 54.0 5.93 0.652 LCL 100 2.09 0.0439 9.16 x IO-4 Calculations are based on excess risk over background. Probit and Weibull model estimates calculated using RISK81 computer program. LCL estimates are the 95% lower confidence limits on dose (variance based on log dose for RISK81), Note: The additive probit and additive Weibull models failed to converge for this data set. ------- TABLE 30. FEMALE MICE CARCINOMAS OF LUNG OR LIVER: DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE) Risk level 10-2 10-^ ID'6 10-8 GLOBAL83 two-stage MLE 262 26.1 2.61 0.261 LCL 40.7 0.410 4.10xlO-3 4*10x10-5 Independent probit MLE 769 412 260 176 LCL 503 218 117 70.1 MLE 163 1 0 1 Additive probit LCL 81.4 .92 0.78 .0192 0.0078 .92xlO-4 7.8x10-5 Independent Additive Weibull Weibull MLE 309 35.8 4.15 0.482 LCL MLE 142 134 8.03 1.52 0.452 0.0152 0.0255 1.52xlO-4 LCL 68.8 0.686 0.00686 6.86x10-5 Calculations are based on excess risk over background. Probit and Weibull model estimates calculated using RISK81 computer program. LCL estimates are the 95% lower bound on the dose (variance based on log dose for RISK81). ------- TABLE 31. FEMALE MICE CARCINOMA OR ADENOMA OF LUNG OR LIVER: DCM DOSES ESTIMATED TO PRODUCE GIVEN RISK LEVELS (DOSES IN PPM UNDER NTP PROTOCOL SCHEDULE) CO o GLOBAL83 two-stage Risk level 10-2 10-4 ID'6 10-8 MLE 28.8 0.290 2.90x10-3 2.90x10-5 LCL 11.6 0.115 1.15x10-3 1.15x10-5 Independent probit MLE 478 238 141 92.5 LCL 198 67.4 30.2 15.6 Additive probit MLE 45.1 0.464 4.64x10-3 4.64x10-5 LCL 24.6 0.249 2.49x10-3 2.49x10-5 Independent Wei bull MLE 93.4 4.75 0.242 0.0124 LCL 17.2 0.196 2.23x10-3 2.53x10-5 Additive Wei bull MLE 36.3 0.369 3.69x10-3 3.69x10-5 LCL 15.7 0.156 1.56x10-3 1.56x10-5 Calculations are based on excess risk over background. Probit and Weibull model estimates calculated using RISK81 computer program. LCL estimates are the 95% lower bound on dose (variance based on log dose for RISK81). ------- were quite comparable between the two models (maximum differences under 50% at low dose). For female mouse carcinomas, the MLE estimates from these models lead to dose estimates that are markedly lower than the (non-linear) MLE multi- stage estimates. For the combined carcinomas and adenomas, the MLE estimates of additive Weibull, additive probit, and multistage models agreed within approximately 50%. For both groupings of tumors in female mice, the LCL dose estimates of the additi.ve probit, additive Weibull, and multistage models were within a factor of two of each other, with the two-stage multistage model leading to the lower estimates of dose for a fixed risk. 4.6. COMPARISON OF NTP (1985) RESULTS WITH OTHER BIOASSAYS There are several recent long-term animal studies of DCM in addition to the NTP (1985) bioassay. These studies are reviewed in detail in the EPA Health Assessment Document for DCM (U.S. EPA, 1985). While there were signi- ficant limitations in these studies, which often included maximum doses well below what the animals could tolerate, some positive and suggestively positive carcinogenic responses were obtained. This section develops GLOBAL83 parameter estimates (for a multistage model with the maximum polynomial degree equal to the number of dose groups minus one) for the responses found in rats and mice, using data taken from EPA (1985). 1) The Dow Chemical Company (1980) 2-year inhalation study in Sprague- Dawley rats found some statistically positive elevations in mammary tumors in this strain, which has a normally high spontaneous mammary tumor rate, making positive results more difficult to obtain statistically. The strongest find- ing was the elevation of the total number of mammary tumors seen in female rats (165/99 in controls vs. 287/97 in the high-dose groups). However, the GLOBAL83 model cannot accommodate data of this form. The percentages of mammary tumors in the different female groups was not statistically elevated, but can be used 81 ------- in GLOBAL83 to calculate an upper-bound risk q^ on the hypothesis of a real biological effect. The male rat data, which indicated an elevated mammary tumor response at the high-dose point (not significant) are also used in this manner (Table 32). For comparison, the combined mammary tumor data from the NTP (1985) study in F344/N rats (using the same dosing schedule) yielded values of q^ = 0.0311 x 10"^ for males and q^ = 0.164 x 10"^ for females. Thus, while the mammary tumor incidences were not statistically elevated in the Dow (1980) study, the upper bounds on q^ that can be derived from the Dow results are in close agreement with the NTP (1985) study. At a second site, sarcomas in or around the salivary gland in male rats, the Dow (1980) study found statistically positive results (Table 33). The NTP study did not find an elevated sarcoma incidence in or around the salivary gland. 2) The Dow Chemical Company (1982) inhalation study found evidence of an elevated mammary tumor incidence in female rats (the tumor percentage was sta- tistically elevated at 200 ppm compared with controls). The total number of mammary tumors found also showed an increase with dose, as in the Dow (1980) study. GLOBAL83 is applied to the incidence data to determine the maximum linear dose-response component that is compatible with these data (Table 34). The estimate of q^ derived from this study is high compared with that for mammary tumor incidence in female F344/N rats in the NTP (1985) study (q^ = 0.164 x 10-3 ppm-1). 3) The National Coffee Association (NCA)(1982a, b) study found evidence of an increased occurrence of liver tumors in female F344 rats given DCM in drinking water. The GLOBAL83 value for q^ is shown in Table 35, calculated on the basis of the experimentally applied dose units and also calculated using the same dose units as in the NTP (1985) study. (This second estimate 82 ------- TABLE 32. RAT MAMMARY TUMORS Dose3 Response Females Males Control 179/96 7/95 500 ppm 81/96 3/95 1,500 ppm 80/95 7/95 3,500 ppm 83/97 14/95 GL.OBAL83 ql ppm-1 0.201 x 10-3 0.040 x 10~3 aDose administered 6 hrs/day, 5 days/wk, for 2 years. Value for q^ based on administered doses and dose time schedule. SOURCE: Dow Chemical Co., 1980. TABLE 33. MALE RAT SALIVARY GLAND TUMORS Dose3 500 1,500 3,500 ^ GLOBAL83 Control ppm ppm ppm q (x 10"3 ppm"1) 1/93 0/94 5/91 11/88 0.043 3Dose administered 6 hrs/day, 5 days/wk, for.2 years. Value for q^ based on administered dose and dose time schedule. Note: These data show a statistically significant (p < 0.001) test for a linear trend, and the high-dose group response is elevated compared to the controls (p = 0.002). SOURCE: Dow Chemical Co., 1980. 83 ------- TABLE 34. FEMALE RAT MAMMARY TUMORS Dose3 Control 50 ppm 200 ppm 500 ppm 52/70 58/70 61/70 55/70 1.22 x lO-3 aDose administered 6 hrs/day, 5 days/wk, for 2 years. Value for q^ based on administered dose and dose time schedule. SOURCE: Dow Chemical Co., 1982. ' TABLE 35. NEOPLASTIC NODULES OR HEPATOCELLULAR CARCINOMA IN FEMALE F344 RATS Control Dose (mg/kg/day)a * * ql ^1 50 125 250 exp. dose units NTP dose units 0/134 1/85 4/83 1/85 6/85 0.470 x ID'3 0.22 x 10-3 aDose delivered in drinking water. Note: The tumor responses in the 50 and 250 mg/kg/day groups were elevated in comparison with controls at the p < 0.05 level. SOURCE: NCA, 1982a, b. 84 ------- was derived using the ppm to mg/kg/day conversion factors presented in sec- tion 4.7. below.) The NTP (1985) report noted that a positive, but marginal, increase in female rat hepatocellular neoplastic nodules or hepatocellular carcinomas was observed in that study (q^ approximately 0.03 x 10~3 ppm"1). 4) The NCA (1983) study in mice found evidence of an increased incidence of liver tumors in males. As with rats, a value of q^ using the dose units of the NTP (1985) study was derived, and is shown in Table 36. For comparison, the NTP study, which found elevated rates for the same tumors, yields a qt = 0.195 x 10-3 estimate. In conclusion, the studies discussed in this section provide some evi- dence for DCM-induced tumors at sites where the NTP (1985) study found tumors; in addition, the Dow (1980) study showed an increase in salivary gland region tumors in male rats. Estimates of q£ have been derived for these sites to indicate the maximum linear component of a tumor dose-response that is consis- tent with study findings. These upper-bound risk estimates are comparable to, or in some cases, larger than, corresponding estimates derived from the NTP (1985) study for the same tumor sites. 4.7. DERIVATION OF HUMAN UNIT RISK ESTIMATES FOR INHALATION OF DCM The EPA Health Assessment Document for Dichloromethane (U.S. EPA, 1985) developed estimates of unit risks to humans on the basis of the Dow (1980) findings of salivary gland tumors in rats. The same standard CAG assumptions that were used in that document to convert between animal and human doses are also applied here. It is noted that in this assessment, the extrapolation between high and low doses in humans and animals is done in a different order than in the earlier CAG assessment; however, this does not affect the results. Table 37 summarizes the values of q^ derived for tumors found in both sexes of rats and mice in the NTP study (data taken from Tables 19 and 20). 85 ------- TABLE 36. HEPATOCELLULAR ADENOMAS OR CARCINOMAS IN MALE MICE Dose (mg/kg/day)a Control 60 125 185 250 exp. dose units NTP dose units 24/125 51/200 30/100 31/99 35/125 0.995 x 10'3 0.78 x 10-3 aDose administered in drinking water. Note: The response at the 125 and 185 mg/kg/day doses were elevated in com- parison with controls (p < 0.05). SOURCE: NCA, 1983. TABLE 37. VALUES OF qT FOR NTP (1985) BIOASSAY USED TO DERIVE HUMAN ql ESTIMATES Male rat mammary or subcutaneous tumors Female rat mammary tumors Male mouse lung or liver adenoma or carcinoma Female mouse lung or liver adenoma or carcinoma "Jt "31 :H = 0.0540 x 10" J (ppm"1 exp. protocol) = 0.164 x 10"^ (ppm"1 exp. protocol) •fc "51 ^1 = 0.429 x 10~J (ppm"1 exp. protocol) = 0.870 x 10~3 (ppm"1 exp. protocol) 86 ------- 1) These values are first converted to equivalent rodent values for con- tinuous exposure to DCM in units of mg/kg/day. To make this conversion, the / inhalation rates for the rodents must first be estimated. This is done using the following formulas (U.S. EPA, 1985): For mice: I = 0.0345 (wt/0.025)2/3 m3/day For rats: I = 0.105 (wt/0.113)2/3 m3/day Inhalation rates are calculated using the NTP (1985) average weights for male and female mice and rats at the midpoint of the bioassay (51-week data point; the NTP report does not give the average animal weight over the whole study period). The data are given in Table 38. Dose conversion factors can now be calculated between the NTP schedule and a continuous mg/kg/day exposure. For example, in male rats: 1 ppm NTP schedule = 10~6 x 3,478 g DCM/m3 x 1,000 mg/g x average exposure 4.29 hrs/day x 1 day/24 hrs x 0.268 m3/day /0.462 kg = .361 mg/kg/day These data are given in Table 39. 2) Equivalent human DCM doses and q^ values are now calculated using the CAG methodology for well-absorbed vapors (DCM in air is likely to be absorbed by the lungs to a high degree at low doses in both humans and rodents; the interspecies conversion is being applied for the risks estimated at low doses). The CAG assumes (U.S. EPA, 1985) that humans and animals exposed to equal doses of a carcinogen on a (mg/kg)2/3 basis over equivalent proportions 87 ------- TABLE 38. ESTIMATED INHALATION RATES FOR NTP (1985) TEST ANIMALS Weight at Estimated bioassay midpoint inhalation rate (kg) (m3/day) Male rat Female rat Male mouse Female mouse 0.462 0.278 0.037 0.032 0.268 0.191 0.0448 0.0407 TABLE 39. DOSE CONVERSION FACTORS AND EQUIVALENT q? VALUES FOR NTP (1985) STUDY - mg/kg/day equivalent (mg/kg/day)-1 of 1 ppm exposure, for continuous NTP protocol exposure3 Male rat Female rat . Male mouse Female mouse 0 0 0 0 .361 .467 .753 .791 0. 0. 0. 1. 149 383 570 10 X X X X lO-3 •ID'3 1.0-3 10-3 athese values of q^, which apply to the same^tumor types as are listed in Table 31, are obtained by multiplying the q^^ values in Table 31 by the reciprocals of the values in the first column of this table. 88 ------- of a lifetime will encounter the same degree of cancer risk. This implies that a rodent with weight WR, exposed to a dose of D mg/kg/ day and a human exposed to a dose of D^)"1/3 mg/kg/day encounter the same lifetime cancer risks. Table 40 contains human dose equivalents and values for q^. 3) To obtain an estimate of the unit risk for a human inhaling 1 yg/m3 of DCM over a lifetime, the standard CAG assumption of a human inhalation rate of 20 m3/day (U.S. EPA, 1985) is applied. A continuous exposure to 1 yg/m3 of DCM is equal to an exposure of 1 yg/m3 x 10~3 mg/yg x 20 m3/day x 1/70 kg = 2.86 x 10"4 mg/kg/day Using the value of q^ in female mice from Table 34, an upper-limit lifetime cancer risk estimate of 4.1 x 10~6 is estimated for this exposure. Alterna- tively expressed, q^ = 4.1 x 10~6 (yg/m3)"1. Using the relation that 1 yg/m3 DCM is equivalent to 2.88 x 10~4 ppm, qjj[ = 1.4 x 10~2 ppm"1 (continuous exposure). 4) The CAG's potency index is derived by multiplying q^ (mg/kg/day)"1 by the molecular weight of the compound (84.9 g/mole for DCM) to obtain q-^ (mmol/kg/ day) . For lung and liver tumors, combined, in female mice, q-^ (mmol/kg/ day)~l =1.1. This value is in the fourth quartile of the CAG histogram for the potency index distribution. 4.8. HUMAN UNIT RISK ESTIMATE FOR INGESTION OF DCM Data both from the NTP inhalation study and from the earlier NCA studies of DCM in drinking water will be considered in connection with a unit risk estimate for DCM ingestion. All of the existing studies have limitations for estimating the risks from ingestion of DCM. DCM is rapidly absorbed and sys- 89 ------- temically distributed following either inhalation or ingestion exposure; thus an inhalation study is relevant for assessing hazards from ingestion exposure. Nonetheless, exposures via the two routes are likely to lead to differing doses reaching individual organs; in particular, an inhalation study may result in a higher degree of exposure to lung tissue and a lesser exposure to tissues in the digestive system than an ingestion exposure. For this reason the NTP findings for liver tumors, but not lung tumors, in mice are used for quantitative estimation of risk from DCM ingestion. On the other hand, it should be noted that an analysis based on the NTP study may underestimate risks of liver tumors or other digestive system tumors. TABLE 40. DOSE CONVERSION FACTORS AND EQUIVALENT qt VALUES FOR RODENTS IN NTP (1985) STUDY AND FOR HUMANS Human mg/kg/day ^ equivalent for rodent q^a 1 mg/kg/day (mg/kg/day)-1 exposure human Male rat 0.188 0.793 x lO'3 Female rat 0.158 2.43 x 10"3 Male mouse 0.0809 7.05 x 10'3 Female mouse 0.0770 14.3 x 10"3 aThese values of q^, which apply to the same^tumor sites as those given in Table 31, are obtained by multiplying the q-^ values in Table 33 by the reciprocals of the values in the first column of this table. 90 ------- The NCA (1983) drinking water study in mice yielded suggestive but not conclusive evidence of a treatment-associated increase in hepatocellular carci- nomas and/or adenomas in males (U.S. EPA, 1985). This finding can be directly used to make an upper-bound risk estimate from ingestion exposure to DCM. However, the NCA study utilized doses that were well below the maximum tolera- ted dose (MTD) (U.S. EPA, 1985). Thus, it is possible that elevated tumor incidences would have been found in other tissues if the study had been conduc- ted nearer to the MTD. a) The unit risk from ingestion exposure is obtained using the q^ esti- mate for liver tumors in female mice (the sex with the higher risk estimate): q^ = 0.160 (ppm"1). The female mouse data are used for this calculation because the stronger qualitative and quantitative data for carcinogenicity in the liver were obtained for this sex in the NTP (1985) study. If the NTP findings for male mice were used instead, the unit risk estimate given below would be 20% higher. Using the above dose-conversion procedures, this value * O 1 leads to an equivalent human estimate of q^ = 2.6 x 10 (mg/kg/day)"1. If a 70-kg human drinks 2 L/day of water containing 1 ug/L DCM, the average daily exposure is 1 yg/L x ID'3 mg/ug x 2 L/day x 1/70 kg = 2.86 x 10~5 mg/kg/day Using the above value for q^, the lifetime incremental cancer risk is estimated at 7.5 x ID'8 (ug/L)'1. b) Using the NCA (1983) study, the value for q^ in male mice is 0.995 x 10~3 (mg/kg/day)-1, based on hepatocellular carcinomas and adenomas. The male mouse data from the NCA study are used for this calculation because only the male mice showed evidence for carcinogenicity in the liver in this study, which 91 ------- was conducted well below the MTD. Following the dose-conversion procedure given earlier in this section, the equivalent human value is 1.23 x 10~2 (mg/ kg/day)"1. The corresponding UCL unit risk estimate for drinking water is 3.5 x 10-7 (ug/L)-l. The unit risks calculated on the basis of the two mouse studies are com- parable; therefore, the mean value of 2.1 x 10~7 (yg/L)~l is used for the unit risk estimate for drinking water exposure to DCM. 4.9. COMPARISON OF ANIMAL AND HUMAN DATA RELEVANT TO CANCER RISK The risk prediction for human exposure to DCM can be compared with the observed cancer mortality in a cohort of Eastman Kodak employees exposed to DCM (Friedlander et al., 1978; Hearne and Friedlander, 1981).* Such an analysis was included in U.S. EPA (1984) with reference to the finding of salivary tumors in rats, and a parallel calculation will be shown here. The continuous lifetime equivalent DCM exposure of the Kodak employees was estimated to be between 1.88 and 7.52 ppm, based on a 20-year exposure for the 252 long-term workers. Based on the 95% upper-limit slope factor for the preceding section (q^ = 1.4 x 10"^ ppm~l for continuous human exposure), the upper bound on the lifetime cancer risk encountered by these workers is estimated to be between • 0.026 and 0.105. For the 252 workers, this would translate to a 95% upper limit of 6.6 to 26.5 excess cancer deaths over a lifetime. However, because the study follow-up period was 17 years and most workers were not observed until death, it is probable that only a fraction of the estimated excess cancer deaths were seen. In the absence of a more rigorous method, it is estimated *0tt et al. (1983) also reported an epidemiologic study of DCM workers; how- ever, the limited follow-up in this study prevents any adequate comparison with estimated lifetime cancer risks. In the Ott study, the overall mortality was less than 10% for all subgroups during the study period. See U.S. EPA (1985) for further discussion of this study. 92 ------- that the fraction of cancer cases that would be observed in the follow-up period is approximated by the overall mortality expected in the follow-up: 65.9/252 deaths, or 26%. Thus, a 95% upper limit of between 1.7 and 6.8 cancer deaths due to DCM exposure would have been predicted for the cohort. Using the statistical methods presented by Beaumont and Breslow (1981), the power of the Friedlander study, with 17.8 expected cancer deaths, to detect an excess of 1.7 deaths from total cancer (with 95% confidence) is 0.06; the power to detect 6.8 cancer deaths is 0.31. Tumors of several types were found to be elevated in the NTP mouse and rat bioassays. Additionally, it cannot be generally expected that humans and experimental animals will show a carcinogenic response at the same sites when exposed to a chemical which is carcinogenic to both. These factors pre- vent rigorous comparison of the DCM cancer risk estimated from the NTP study with findings of an epidemiologic study for particular cancer sites. However, as a tentative example of power comparisons that may be made, the ability of the Friedlander study to detect excess lung cancer deaths is calculated. For this example, an estimate of risk for lung cancer is obtained by ap- plying the observed excess of lung tumors in female mice (the sex with the stronger response) to estimate lung cancer deaths in the Friedlander cohort. Taking the q± value for female mouse lung carcinoma or adenoma from Table 5 (0.579 x 10~3) and applying the procedure of Section 4.7., the upper-bound unit risk estimate for humans is 9.5 x 10~3 (ppm~l) for continuous exposure. 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