Document ZmeyGbGDjRkEkyJpqqKmMK7O

The Science of the Total Environment, 47 (1985) 385--413 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands 335 CARCINOGENIC ANO MUTAGENIC PROPERTIES OF CHEMICALS IN DRINKING WATER R.J. BULL College of Pharmacy, Washington State University, Pullman, Washington 99164 ABSTRACT Isolated cases of careless handling of industrial and domestic waste has lead to a wide variety of dangerous chemicals being inadvertently introduced into drinking water. However, chemicals with established carcinogenic and mutagenic properties that occur with a high frequency and in multiple locations are limited in number. To date, the chief offenders have been chemicals of relatively low carcinogenic potency. Some of the more conmon chemicals are formed as by-products of disinfection. The latter process is generally regarded as essential to the production of a "microbiologically safe" drinking water. Consequently, any reductions in what may be a relatively small carcinogenic risk must be balanced against a potential for a higher frequency of waterborne infectious disease. The results of recent toxicological investigations will be reviewed to place the potential carcinogenic and mutagenic hazards frequently associated with drinking water into perspective. First, evidence for the carcinogenicity of certain volatile organic compounds such as trichloroethylene, tetrachloroethylene and carbon tetrachloride is considered. Second, the carcinogenic activity that can be ascribed to various by-products of chlorination is reviewed in some detail. Finally, recent evidence that other chemicals derived from the treatment and distribution of drinking water is highlighted as an area requiring move systematic attention. INTRODUCTION A wide variety of chemicals have been found in drinking waters around the world. Modern analytical techniques have identified such a diversity of chemicals that it renders comprehensive review of toxic and potentially carcinogenic properties of each substance virtually impossible. However the sources of toxic chemicals in finished drinking water are derived from three general sources: 1. Contamination of source water. 2. By-products of treatment processes. 3. Leaching from the distribution system. Examples of contaminants from each source will be discussed in turn. CONTAMINANTS OF SOURCE A great variety of chemicals can contaminate sources of drinking water. By far the most frequent contaminants in this category are trichloroethylene 0048-9697/85/503.30 1985 Elsevier Science Publishers B.V. SL 034437 and tetrachloroethylene because of their large production volumes and widespread use in commerce. The carcinogenicity of trichloroethylene, tetrachloroethylene and to a lesser degree carbon tetrachloride are somewhat more controversial. Consequently, this section will provide an updated review of the data bearing on the carcinogenic properties of these chemicals. Consideration of other contaminants will be for comparative purposes only. A summary of pertinent carcinogenicity and mutagenicity data associated with contaminants of source waters Is provided in Tables 1 and 2. Carbon Tetrachloride Studies of carbon tetrachloride (Cameron and Karunaratne, 1936; Reuber and Glover 1967a; Reuber and Glover 1967b) have documented the ability of carbon tetrachloride to Induce preneoplastic changes in the liver of rats (e.g., hyperplastic nodules and cholangiofibrosis). Subsequent studies (Reuber and Glover, 1970) demonstrated that subcutaneous administration of carbon tetrachloride in corn oil at a dose of 2080 mg/kg twice weekly gave rise to hepatocellular carcinomas in male Japanese rats, Osborne-Mendel rats, and Wistar rats. Black rats and Sprague-Dawley rats failed to survive long enough to develop carcinomas following the same treatment. The authors' called attention to the fact that carcinoma development appeared to be Inversely related to the degree of liver cirrhosis across the strains tested. However, this relationship may be fortuitous since those strains developing severe cirrhosis failed to survive. The single level of treatment utilized in this study precludes a clear resolution of this issue. A more recent study (NCI, 1976) of the carcinogenicity of carbon tetrachloride in Osborne-Mendel rats failed to confirm its carcinogenic effects at doses of up to 160 mg CCl^/kg in com oil administered S times weekly for 78 weeks (47 and 94 mg/kg for males, 80 and 159 mg/kg for females). However, it should be noted that the doses utilized were much lower than used in previous studies. Eschenbrenner and Miller (1943) demonstrated that carbon tetrachloride was able to induce hepatomas in Strain A/J mice. Tumor yield was generally dose-related, but the effect was enhanced as the spacing between doses was Increased from 1 to S day intervals to the same total dose of carbon tetrachloride. In subsequent experiments Eschenbrenner & Miller (1946) examined the relationship between liver necrosis and tumor development and found that the liver became resistant to necrotizing doses of carbon tetrachloride with chronic treatment. This observation was most probably related to the Inactivation of cytochrome P-450 by carbon tetrachloride observed in modern metabolic studies (Glende et aK, 1975). Overall, there 387 appeared to be a relationship between obvious necrosis and the development of hepatomas. However, hepatomas could be Induced by doses of carbon tetrachloride that did not induce obvious necrosis in the liver. The authors pointed out that they could not rule out the possibility of a slightly greater rate of liver cell replacement at these lower doses. They did find evidence of cirrhosis at doses not producing obvious necrosis, indicating some degree of liver injury. ICiplinger and Kensler (1963) also observed the induction of hepatomas following oral administration of carbon tetrachloride in C3H mice relative to historical controls, however, this study did not include concurrent controls. The National Cancer Institute (NCI, 1976) documented a high incidence of hepatocellular carcinomas in the B6C3F1 mice administered at doses of 1250 and 2500 mg/kg by gavage in a corn oil vehicle. Despite the fact of its recognized carcinogenic effects in rodents there is little convincing evidence that carbon tetrachloride possesses genotoxic properties (IARC, 1979b; USEPA, 1984). On the other hand, the ability of hepatonecrotic doses of carbon tetrachloride to promote the yield of liver tumors in mice is well documented (Pound and McGuire, 1975). Therefore, the bulk of the evidence indicates that the cancer producing activity of carbon tetrachloride are secondary to Its hepatoxic effects and carbon tetrachloride might be considered as a carcinogen that does not act by a "genotoxic" mechanism. Trichloroethylene Trichloroethylene (TCE) was first shown to be carcinogenic in a study sponsored by the National Cancer Institute (NCI, 1976). In this study, male B6C3F1 mice were exposed to time-weighted average doses of 1169 and 2339 mg/kg body weight, whereas, females received 869 and 1739 mg/kg of TCE dissolved in corn oil by gavage for a period of 78 weeks. In the same study both male and female Osborne-Mendel rats were administered doses of 549 and 1097 mg/kg TCE by the same means and for the same duration of exposure. Dose-related increases in the Incidences of hepatocellular carcinomas were observed in both male and female mice, although the response was much more marked in the males. There were no statistically significant Increases in tumor yield in either male or female Osborne-Mendel rats. Inhalation studies conducted by Henschler et al_. (1980) at concentrations of TCE in air of 100 and 500 ppm for 6 h/day, 5 days/week for a period of 18 months demonstrated an Increased incidence of malignant lymphoma in female NMRI mice but was without apparent effect in male mice, Uistar rats or Syrian SL 034439 388 TABLE 1. Evidence for Carcinogenicity of Carbon Tetrachloride, Trichloroethylene and Tetrachoroethylene Species ft Strain Chemical (Sexl Carbon Tetrachloride C3H Mice (MSF) Hiqhest Dose 0.04 ml 2 to 3 times weekly for 8-16 weeks Route Oral Vehicle Olive Oil Result Hepatoma Reference Edwards, et al., 1941 Strain L 0.04 ml 46x over a 4 month period (no M M Hepatoma Edwards, et al.. 1941 m Strain A Mice (M&F) 2.5g/kg to a total Oral Olive Oil Hepatoma Eschenbrenner of 30 doses 1 Miller, 1943 H B4C3F1 Mice (HSF) 2500/mg/kg 5 X weekly Oral 2-5S Hepatocellular NCI, 1976 for 78 wks solution carcinoma in corn oil II C3H Mice 1.6 g/kg 3 X weekly Oral Corn Oil Hepatoma Kiplinger 6 for 10 wks Kensler, 1963 U C3H Mice 0.04 ml 2 times Rectal Olive Oil Liver Tumors Confer & weekly for 20-26 Stenger, weeks 1965 N Os borne-Mendel 160 mg/kg 5 X weekly Oral Corn Oil Negative NCI, 1976 Rat (M&F) 78 weeks N Syrian golden 12.5 ul/hamster weekly Oral Corn Oil Liver cell Della Porta hamsters no controls carcinomas et.al., 1976 M Japanese rat (M) 2g/kg bw 2 X weekly s.c. Corn Oil Hepatocellular Reuber & carcinoma Glover, 1970 M Osborne-Mendel (M) U MM M s.c. WH Hepatocellular Reuber & carcinoma Glover, 1970 K Wlstar - (M) M MM M s.c. MN Hepatocellular Reuber & Carcinoma Glover, 1970 Species & Strain Chemical . (Sex) Carbon Tetrachloride Black rat (M) TABLE 1. (Continued) Hjqhest Dose 2g/kg 6w 2 X weekly Route Vehicle s.c. Corn Oil 11 Sprague-Dawley (M) ft ft ft ft ft ft II Trichloroethylene N ft ft ft B6C3F1 Mice (M&F) Osborne-Mendel (M&F) ICR Mice (F) Sprague-Dawley rats (F) Mice (M&F) 2339 mg/kg TWA for 78 weeks 1097 mg/kg TWA for 78 weeks 0, ISO & 450 ppm in air 7 h 0, 150 & 450 ppm in air 7 h per day, S days a week 500 ppm for 18 months Oral Oral Inh. Inh. Inh. ft ft Tetrachloroethylene ft ft Rats (M&F) SOO ppm for 18 months Hamsters (M&F) 500 ppm for 18 months B6C3F1 Mice (M&F) 1072 mg/kg TWA for 78 weeks Osborne-Nendel (M&F) Sprague-Dawley (M&F) 600 ppm in air for 12 months Inh. Inh. Oral Oral Inh. Corn Oil Corn Oil Air Air Air Air Air Corn Oil Corn Oil - Result (Died early) ft Reference Rebuer & Clover, 1970 Reuber & Glover, 1970 Hepatocellular NCI, (1976) carcinoma Negative NCI, (1976) Pulmonary Fukuda, et adenocarcinoma al., 1983 Negative ft ft Malignant 1 ymphoma Negative Negative Henschler et al., 1980 ft ft ft II Hepatocellular NCI, (1977) carcinoma Negative NCI, (1977) Negative Rampy et al. 1976 0\3> Trichloroethylene N 0 * It M TABLE 2. (Continued) Test System Covalent binding to protein in rat liver microsomes Snecial Conditions In vitro Hiqhest Dose Tested Unlike vinyl chloride binding is not specific to SH containing protein. Result Reference Bolt and Filser 1977 5. TvoMmurium ------" -- TA100 +/- Aroclor 1254 induced rat liver S-9 fraction 100 ug/ml in top agar neg. with and without S-9 Kenschler et al., 1977 Saccharomyces cerevisiae kVl85-14C /- mouse liver S-9 20 ul/ml fraction Pos. with S-9 only Shahin S Von Borstel 1977 Fischer Rat F1706 embryo cell system Covalent binding to hepatic proteins In the S.D. Rat Saccharomyces cerevisiae 07 1.1 x 104 M In vivo and In vitro Suspension test, 40 mM +/- mouse liver 10,000 x g supernatant Pos. Price et al., 1977 Pos. Allemand, et al., 1976 Pos. point mut. and give conversion required S-10 to Bronzettl, et al., 1978 SL 034443 Chemical Trichloroethylene H M It It Tetrachloroethylene Test System Saccharomyces cerevlsiae til " 04 D4 Schizosaccharomyces pombe Schizosaccharomyces pombe OKA binding in in vitro & in vTvo E. Col 1 K12 TABLE 2. (Continued) Special Conditions Host mediated HM Host mediated Highest Dose Tested 40o mg/kg 400 mg/kg 3700 mg/kg (22 repeated doses of ISO mg/kg + 400 mg/kg day of sacrifice). Result pos. ilv reversion + trp conversion Pos. at ade and trp loc. pos. trp and ade loci Reference Bronzetti et al., 1978 MH 4 M PI +/- mouse and rat 22 mM liver S-9 fraction pretreated phenobarbital or B-naphthoflavone neg. at ade loci 1.3.4. 5&9 Rossi, et al. 1983 PI Host mediated 2 9A9 neg. Rossi, et al. 1983 Calf thymus, in vitro mouse, in vivo gal + arg + nad * KTR +/- House microsomes, 0.9 mM in media phenobarbital pre treated pos. in vitro neg. in vivo neg. Bergman, 1983 Greim et al. 1975 SL 034444 Chemical Tetrachloroethylene Test S>yystem S. typnimurium S. typhimurium Hacroraolecular binding in rat and liver tested in vivo Saccharomyces cerevisiae TABLE 2. (Continued) SSppeecial Conditions TMT Spot test-no S-9 used TA19S1 In vitro TA1952 TA1535 TA1538 TA100 TA98 TA1950 Host mediated TA1951 Female ICR mice TA1952 LD5Q and 1/2 LD,60 500 mg/kg (oral dose) D7 Suspension assay /- 85 mM mouse liver S-9 11 g/kg (single Host mediated assay dose) 2g/kg k 12 + A 9/kg Result neg. neg. neg. neg. neg. pos. neg. Reference Cerna 1 Kypenova, 1977 Pos. no dose response evident Cerna & Kypenova, 1977 Binding to Schumann et macro al. 1980 molecules but no binding detected in purified ONA Neg. Heg. Neg. Bronzettt et al. 1983 394 hamsters. More recently, Fukuda et aK (1983) were able to show a small Increase In the incidence of pulmonary adenocarcinomas in female ICR Swiss mice exposed to 150 and 450 ppm TCE (16 and 15S, respectively, versus a control Incidence of ZS) in air 7 h per day, 5 days a week for 104 weeks. No evidence of carcinogenic activity was observed in female Sprague-Oawley rats subjected to the same treatment. Consequently, evidence that TCE is carcinogenic is limited to mice, although three different tumors have been identified in each of the mouse strains that have been studied. As a result of the initial observation that TCE was carcinogenic in mice and because it comes from the same chemical class as vinyl chloride (chloroethylene), an established human carcinogen, there have been many studies of the compound's genotoxic properties. Simmon et al_. (1977) reported that TCE increased the reversion rate of S. typhimurium strain TA100 only in the presence of a 9000 X g supernatant of homogenates made of mouse or rat liver. The experiments in this study were conducted in a desiccator and avoided the volatilization of TCE that is encountered in the standard plate assay of the Ames test when such precautions are not taken. Cerna and Kypenova (1977) were unable to demonstrate mutagenic activity of TCE in the spot test utilizing a variety of strains of S. typhimurium in the absence of a metabolic activation system, but were able to demonstrate positive activity in host-mediated assays in female ICR mice using strains TA1950, TA1951 and TA195Z. Greim et al_. (1975) demonstrated that TCE was capable of more than doubling the spontaneous mutation rate at the arg+ locus of E. coli strain K12 after incubation with a liver microsomal preparation taken from mice that had been pretreated with phenobarbital. No activity was attributable to TCE in the absence of metabolic activation. Henschler et al_. (1977) failed to confirm the mutagenic activity of TCE in S. typhimurium strain TA10O both in the presence and absence of a metabolic activation system derived from Aroclor 1Z54 pretreated rats. In addition to studies in bacterial systems, TCE has been shown to Induce mutations in one yeast test system by two different research groups (Shahin & von Borstel, 1977; Bronzetti et ah, 1978) either in the presence of a metabolic activation system or in host-mediated assays, but negative under similar circumstances in another (Rossi et al_., 1983). Price et al_. (1978) were able to demonstrate that TCE was capable of transforming Fischer rat embryo cells. Transformed cells were capable of growth in semisolid agar and produced undifferentiated fibrosacromas when inoculated into newborn Fischer rats. Henschler et al_. (1977) suggested that the mutagenic activity and the carcinogenic activity of TCE might be accounted for by the presence of SL 034445 395 eplchlorohydrin and 1,2-epoxibutane. However, these chemicals produced a mutagenic effect in the absence of a metabolic activation system, an observation that was not observed in prior positive tests of TCE by other authors. It has been clearly shown that TCE is metabolized to a form capable of covalently interacting with tissue proteins at a level comparable to that observed with vinyl chloride (Van Ouuren & Banerjee, 1976; Bolt S Filser, 1977; All emend et aK 1978). Only very low levels of interaction of TCE with DNA jn vivo have been demonstrated (Stott et a1_., 1982; Bergman, 1983). Under in vitro conditions and in the presence of a metabolic activation system, a higher degree of binding of a TCE metabolite to calf thymus DNA has been observed (Bergman, 1983). The evidence that TCE Is carcinogenic was considered to be limited by IARC (1979d). At that time the evidence that TCE has genotoxic properties was obscured by reports of impurities present in the technical grade solvent (Henschler et aU, 1977). However, the mutagenic activity associated with impurities have always shown activity in the absence of metabolic activation, whereas samples of TCE have been routinely negative in the absence of metabolic activation. As indicated above, positive results have been specifically observed under circumstances where metabolic activation was possible in E. coli, S. typhimurlum, and Saccharomyces cerevisiae. Moreover demonstration of transformation in Fischer rat embryo cells supports the notion that TCE is able to act as a carcinogen. Negative results using bacterial and yeast systems |n vitro appear to involve failure to consider the volatility of TCE or employed lower concentrations than utilized in positive studies. The reasons for the discrepancy in results using two yeast strains in host-mediated assays is not as clear. The lack of strong evidence of TCE interaction with DNA in vivo (Stott et a1_., 1982; Bergman, 1983) presents a problem in invoking a genotoxic event as being responsible for the carcinogenic response. The difficulty in demonstrating such an interaction is in sharp contrast to that observed with vinyl chloride, a closely related chemical (Green and Hathway, 1978). On the other hand, it is difficult to completely rule out the possibility that the minor interaction observed (Stott et al., 1982) could be responsible for some of the apparent genotoxic effects of TCE. The observations of pulmonary adenocarcinomas (Fukuda et al_., 1983) and malignant lymphoma (Henschler et a1_., 1980) in addition to hepatocellular carcinomas previously reported (NCI, 1976), indicate that TCE has some carcinogenic activity in mice. It should be pointed out, however, that these SL 034446 396 tumors have an appreciable background incidence in the strains of mice utilized. In the liver of mice, doses that produced liver tumors produced definite histological evidence of hepatocellular damage and a dose-related hypertrophic response to trichloroethylene (Stott et al_., 1982). These data suggest that liver tumors in long-term studies may have resulted from repeated tissue damage and hypertrophy. Tetrachloroethylene To date, the evidence that tetrachloroethylene (perchloroethylere, PCE) is carcinogenic is limited to a study conducted by the National Cancer Institute (1977). The results of this study indicated that time weighted average doses of 536 and 1072 mg/kg to male and 386 and 772 mg/kg given to female B6C3F1 mice by gavage using a corn oil vehicle for 78 weeks resulted in the development of hepatocellular carcinomas. No evidence of increased tumor incidence was observed in Osborne-Mendel rats given time weighted average doses of 471 and 941 mg/kg in males and 474 and 949 mg/kg in females administered under the similar conditions in the same study. Rampy et aK (1978) also failed to observe increased tumor incidences In Sprague-Dawley rats exposed to 600 ppm TCE in air for a period of 12 months. Evidence that PCE has genotoxic properties is also limited. PCE failed to increase the mutation frequency Of E. coll K12 strain (Greim et al_. 1975) in the presence or absence of mouse liver microsomes. It was reported (in abstract form) to produce an increase In the reversion rate of S. typhimurium strain TA100 in the absence of metabolic activation in the spot test, but was Inactive in all other strains employed (Cerna & Kypenova, 1977). On the other hand, these same authors indicated that PCE was positive in a host-mediated assay utilizing S. typhimurium strains TA1950, TA1951 and TA1952 in female ICR mice at the LD50 and 1/2 of the LD50 in the same abstract. There was no evidence of dose-response. Bronzettl et at. (1983) were unable to demonstrate an effect of PCE using Saccharomyces cerevisiae Strain 07 in a suspension assay in'the presence or absence of mouse liver S-9 fraction or in a host-mediated assay. It Is notable that these same authors had previously demonstrated positive results with TCE under both of these conditions (Bronzettl et aK 1978). The IARC (1979e) evaluated the evidence that PCE was carcinogenic and found only limited evidence that It was active in mice. There is no substantive basis on which to modify that opinion at the present time. SL 034447 397 BY-PRODUCTS OF TREATMENT PROCESSES The trihalomethanes were the first chemicals that were recognized to be by-products of the chlorination of drinking water (Bellar et aK, 1974; Rook, 1974). Trichloromethane (chloroform), dichlorobromomethane, dibromoehloromethane, and tribromomethane (bromoform) are the most commonly encountered trihalomethanes found in drinking water. lodinated derivatives are less commonly observed. A recent, comprehensive review of its carcinogenic, mutagenic, and teratogenic effects has been published by Davidson et ajk, (1982). The present section will primarily concern itself with data that has become available since this earlier review. More recently, it has become quite apparent that the trihalomethanes (THM) are only one class of by-products produced in the chlorination of drinking water. Many of the non-THM by-products have been shown to be mutagenic in bacterial' systems, but of these only the haloacetonitriles and chlorinated phenols have been shown carcinogenic in experimental animals. Thus, only these two additional classes will be covered in the present review. However, chlorine is known to increase mutagenic activity in drinking water (e.g. Zoeteman et aU 1982; Kool et aK 1982; Meier and Bull, 1985) and in reactions with humic acid, (Meier et al_. 1983; Coleman et <0., 1984; Meier et al_., 1985). On the other hand, it should be remembered that chlorination is the only practice extensively studied. Many by-products remain to be identified with chlorine as well as other alternative modes of disinfection and even those which have been identified have been poorly characterized toxicologically. In addition to by-products of drinking water disinfection, other treatment processes have the potential for the introduction of carcinogens into drinking water. In this light the carcinogenicity of acrylamide is discussed for illustrative purposes. Chloroform A study sponsored by the National Cancer Institute (NCI, 1976) demonstrated that B6C3F1 mice of both sexes developed hepatocellular carcinomas while male Osborne-Mendel rats had Increased yields of renal tumors following chronic exposure to chloroform (Table 3). Chloroform was administered to both mice and rats in a corn oil vehicle by stomach tube. Because of the primary source of chloroform in drinking water is side reactions of chlorination and the necessity for producing microbiologically safe drinking water a more thorough examination of the dose response relationships involved in chloroform-induced cancer was undertaken (Jorgenson et al. 1985). The doses used in this study was extended to a lower range and SL 034448 399 the group sizes were substantially expanded. Chloroform was administered in the drinking water. This design was to avoid problems that may have been associated with gavage dosing and the corn oil vehicle. The expanded group size at low doses would provide a better basis for extrapolation of the results to estimate human risks. Serum and tissue biochemical measurements were made in separate groups of animals on parallel treatments. Additional control groups were utilized for both species in which were restricted in their water consumption to the amount consumed by the high dose groups for each species. This provided a check on the effects that might be secondary to decreased water consumption. Only female 86C3F1 mice and male Osborne-Mendel rats were included in the experiment as these were the sexes which gave the greatest response in the NCI (1976) study. In the male Osborne-Mendel rat very similar results were observed in the Jorgenson et al_. study (1985) as had been obtained in the prior NCI study (1976). In particular, there was a dose-related increase in renal tubular adenomas and adenocarcinomas. The results obtained with the female B6C3F1 mice were, however, substantially different than observed in the original NCI study. There was a complete absence of any dose-related increases in the incidence of hepatocellular carcinomas in mice. This result was particularly significant, since the daily doses of chloroform in the two high exposure groups bracket the low dose which gave rise to an 80S incidence of hepatocellular carcinomas in the earlier NCI study. These results are interesting in the context of evidence of chloroform carcinogenicity that was obtained prior to the NCI study (Table 3). Eschenbrenner and Miller (1945) noted that hepatocarcinogenic effects of chloroform were confined to doses which produced frank liver necrosis. Studies quoted in Table 3 that failed to produce increases in tumor Incidence involved dose levels lower than those used in either the NCI (1976) or Jorgenson et a_l_. (1985) studies. Roe et al_. (1979), however, did find Increased Incidence of renal tumors in ICI mice when chloroform was administered in a toothpaste base at a dose of 60 mg/kg per day. Renal tumors were not produced In the C57BL, CBA, and CF/1 strains at the same dose in the same study. No liver tumors were not observed in any of these mouse strains. It has been argued that the carcinogenicity was secondary to tissue necrosis followed by regenerative hyperplasia largely because of the early observations of Eschenbrenner and Miller (1945). Chloroform at doses found capable of Inducing tumors produces substantial increases of tritiated thymidine incorporation into ONA of both the liver and kidney of the B6C3F1 mouse (Reitz et al., 198Z). This result provides evidence of a regenerative SL 034449 Species and Strain A Mice TABLE 3. Evidence for Carcinogenicity of Chloroform In Experimental Animals Vehicle Olive Oil Highest Dose Tested 2350 mg/kg Route oral Tumor Liver CS7BL Klee Toothpaste 60 mg/kg/d - oral none C8A Klee cm ICI Klee Sprague-Dawley Rat Beagle Dog M Osborne-Kendel Rat M M M B6C3F1 Klee H Osborne-Kendel Rat 86C3F1 Nice Toothpaste Toothpaste Toothpaste Toothpaste Toothpaste M Corn Oil M H M Corn Oil M Drinking Water Drinking Water 60 mg/kg/d 60 mg/kg/d 60 mg/kg/d 60 mg/kg/d 20 mg/kg/d M 1B0 mg/kg/d M m u 477 mg/kg/d II 130 mg/kg/d 400 mg/kg/d oral oral oral oral oral II oral M U H oral N oral oral none none Kidney none none Total Neoplasms Kidney Thyroid Cholangiofibromas Cholangiocarcinomas Liver Lymphoma Kidney Hone References Fschenbrenner & Killer, 1945 Roe et al., 1979 H M M M U Reuber, 1979 NCI, 1976 Reuber, 1979 HM MN HC1, 1976 Reuber, 1979 EPA-HCI Study. EPA-HCI Study. CD SL 034450 h',pW;04T`J*a .`f'l 400 response and Is consistent with this hypothesis. Along the same line, Moore et al_. (1982) provided evidence that doses of 60 mg/kg of chloroform in corn oil resulted in damage to the kidney and evidence of tubular regeneration as measured by tritiated thymidine uptake. These same doses administered in a toothpaste base were without effect. At 240 mg/kg similar effects were seen with both vehicles. This type of response was not strongly apparent in the liver or kidney of Osborne-Mendel rats. In animals receiving parallel treatments to those used in the Jorgenson et aH . study (1985) there was a dose-related increase in the liver fat content apparent in chloroform-treated mice (Jorgenson et al_., 1982). This effect was observed only at much higher doses in the rat. Before the changes in liver fat occurred in the rat they were preceded by substantial a depression of serum triglycerides. Data on serum triglycerides of the mice under these conditions are not presently available. Based on the rat data one can speculate that the use of corn oil may have contributed to the development of hepatocellular carcinomas in the first NCI study through some type of interaction with the hepatoxic effects of chloroform. The difficulty that has been encountered trying to demonstrate that chloroform possesses genotoxic properties (Table 4) indirectly supports the thesis that chloroform induced tumors are the result of overt tissue necrosis. A number of independent workers have failed to show a direct interaction of chloroform or a metabolite with DNA in the mouse or rat liver or kidney (Diaz-Gomez and Castro, 1980; Reitz, et aK 1982; Pereira et aK 1982). On the other hand, chloroform has been shown capable of Inducing chromosome breakage and Increased sister chromatid exhange frequencies in human lymphocytes in vitro and increased sister chromatid exchange in the bone marrow of mice treated with chloroform in vivo (Morimoto and Koizumi, 1983). It has also been found to enhance the transformation of Syrian Hamster Embryo (SHE) cells by S7 adenovirus (Hatch et aJ., 1983). It is notable that the concentrations of chloroform tested in these positive experiments tend to be higher than those utilized in similar experiments that had negative results (Kirkland et at., 1981). It must be said, however, that the exact mechanism by which chloroform induces cancer remains elusive. Attempts to directly demonstrate an alternative mechanism for chloroform-induced cancer have produced equivocal data. For example, Pereira et al_. (1982) failed to clearly demonstrate either tumor initiating or tumor promoting activity in the rat liver. In summary, the extent to which chloroform in drinking water represents a carcinogenic hazard to man is still uncertain. There is no doubt that SL 034451 chloroform Is capable of producing renal tumors in at least one strain of mice and rats. Chloroform can also induce liver tumors in mice, but this effect seems dependent on the vehicle used. In view of the apparent dependence of the carcinogenic effects of chloroform in the mouse liver on the vehicle in which it was administered, it is suggested that chloroform's carcinogenic activity in the rat may be more appropriate for estimating carcinogenic risk in humans. Haloacetonitriles The dihalogenated chlorine and bromine derivatives of acetonitrile have been the principal species identified in chlorinated drinking water. (Trehy & Bieber, 1981; Oliver, 1983). Investigations into the carcinogenicity of these chemicals have been confined to the three members of the chlorinated series, bromochloroacetonitrile and dibromoacetonitrile. Sitimon et al_. (1977) first documented that dichloroacetonitrile (DCAN) was mutagenic in Salmonella typhimurium. Bull et al_. (198S) confirmed the mutagenic properties of DCAN in Salmonella and demonstrated that bromochloroacetonitrile (BCAN) is mutagenic under similar circumstances. Chloroacetonitrile (CAN), trichloroacetonitrile (TCAN) and dibromoacetonitrile (DBAN) were found to be inactive in the presence or absence of a 9000 X g supernatant fraction (S-9) of a liver homogenate taken from rats previously treated with Arochlor 1254. All five haloacetonitriles induced sister chromatid exchange (SCE) in Chinese hamster ovary cells, vitro, in the absence of rat liver S-9 fraction. None of these same five haloacetonitriles, however, were found capable of inducing mutagenic activity in the mouse micronucleus assay in vivo (5 male and 5 female CD-I mice in each group) at doses of 0, 12.5, 25 and 50 mg/kg bw for 5 consecutive days in this same study. Direct evidence of the carcinogenicity of the haloacetonitriles is limited to initiation/promotion experiments in the mouse skin. Experiments employing the oral route of administration (a total dose of 150 and 300 mg/kg bw split between 6 Individual doses) followed by a 20 week promotion schedule with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) failed to demonstate a significantly elevated incidence of either benign or malignant skin tumors within a 1 year observation period (Bull et al_., 1984). On the other hand, higher doses applied topically (1200, 2400 and 4800 mg/kg, also split between 6 applications), yielded significantly higher tumor Incidences with CAN, BCAN and OBAN. DCAN and TCAN produced small increases in cumulative tumor yields, but the changes were not significantly different from controls. SL 034452 TABLE 4. Evidence for Genotoxic Activity of Chloroform Test System Special Conditions Highest Dose Tested Result S. Tvpimurium TA1535 II TA1537 TAI53B H TA9B N TA100 Syrian Hamster Embryo CellsEnhanced viral transformation V79 cells - 8-azaguanine +/- House A Rat Liver and kidney S-9 N H at M Applied in sealed chamber Applied in flow through system, then sealed 10 mg/plate M M M N 0.5 ml/chamber 31 in air neg. 41 M U M pos. neg. Human lymphocyte - Sister Chromatid Exchange Induction Human Lymphocyte Chromosome Damage E. Coll NP2p and WP2uvr A'p Human Lymphocyte - Sister Chromatid Exchange. in vitro House Bone Harrow - Sister Chromatid Exchange, in vivo Rat Liver, DKA - binding in vivo +/ - Rat Liver S-9 */ - Rat Liver S-9 +/ - Rat liver S-9 400 ug/plate 400 ug/plate 10 mg/plate 6 mg/ml 4 x 200 mg/kg/1 neg. neg. neg. pos. pos. neg. Reference Van Abbe et al. 1982 1 N II N Hatch et al.. 19B3 Sturrock, 1977 Kirkland et al., 1981 M M Horimoto A Koisumi, 19B3 H Diaz-Gomez A Castro, 1980 402 M TABLE 4. (Continued) Test System Specie! Conditions Highest Dose ________________________________________________________ _______________ Tested Sprague Dawley Rat, DNA binding in vivo Liver R.A. B6C3F1 mice, DMA binding, B6C3F1 mice, DNA binding in vivo Kidney Liver Liver N.A. N.A. N.A. Result neg. neg. neg. neg. Reference Pereira et al., 1982 N H Reitz et al., 1982 i'W1'1 U1 r4 403 A preliminary investigation of the haloacetonitriles ability to increase the incidence of lung tumors in strain A/J mice was reported by Bull and Robinson (1985). Doses of 10 mg/kg administered three times weekly for 8 weeks significantly increased lung tumor yields at 9 months of age with CAN, TCAN, and SCAN (Bull and Robinson, 1985). However, the tumor incidence was increased to only 32, 28 and 31" respectively. Control incidence of this spontaneous tumor was 105, Although statistically significant, this small increase in lung tumors is difficult to interpret because of the variable background rate of this tumor in A/J mice. These data indicate that certain members of the haloacetonitrile class do possess weak carcinogenic properties. This evidence has been confined to the topical route of administration and coupled to the subsequent application of a potent tumor promoting agent, TPA. There is no evidence available concerning the ability of these compounds to induce cancer by a systemic route of administration. Because of their relatively comnon occurrence as a result of the chlorination of drinking water it is critical that studies examining this issue be initiated. Chlorinated Phenols Chlorination of drinking water often gives rise to small quantities of chlorinated phenol derivatives. The principle products are 2-chlorophenol, 2,4-dichlorophenol and 2,4,6-trichlorophenol. There is substantive evidence of carcinogenic properties for only 2,4,6-trichlorophenol. The National Cancer Institute (NCI, 1979) conducted a feeding study of the effects of 5,000 and 10,000 ppm of 2,4,6-trichlorophenol in the diet of F344 rats and 5,214 and 10428 ppm in the diet of B6C3F1 mice. The incidence of lymphomas or leukemias was increased in male rats in a dose-related manner. Hepatocellular carcinomas were increased in both male and female mice and the incidences were dose-dependent. Chlorinated Aldehyde and Ketone Derivatives A number of chlorinated aldehydes and ketones have been identified in drinking water or related matrices following chlorination. A number of these by-products have been shown to be mutagenic (Bull and Robinson, 1985). Preliminary results from our studies, Indicate that at least two of these chemicals, 2-chloropropenal and 1,3-dichloroacetone are capable of initiating tumors in mouse skin (unpublished results). SL 034455 405 Other Treatment Chemicals . A wide variety of chemicals are necessary in the production of a safe and wholesome drinking water. For the most part these chemicals are generally regarded as safe and pose no substantive hazard to the health of the consuming public. However, if the synthesis of some products is improperly controlled they can contain contaminants that are of potential concern. An example of such products is the group of polymeric chemicals that are commonly used clarifying water, the coagulant aids. The coagulant aids are high molecular weight polymers that are essentially non-toxic and are removed from the finished water by precipitation and filtration. However, the monomeric components used in the synthesis of these polymers are often reactive chemicals that are highly toxic and if sufficiently stable in water could be expected to be in the finished drinking water. One example of such a monomer is acrylamide. Acrylamide has been recently shown capable of initiating papillomas and carcinomas in the skin of mice whether administered by the oral, intraperitoneal or topical routes of administration (Bull et aK, 1984a). It was also shown to be capable of increasing the yield of lung adenomas in strain A/J mice in the same study. More recently, very similar results have been reported with ICR-Swiss mice (Bull 1984b). Although acrylamide is Inactive in the standard plate assay of the Ames' test (Bull et aK, 1984a) it does increase the frequency of sister chromatid exchange in Chinese hamster ovary cells, in vitro (Bull et al_., 1983). Shiraishi and Yamamoto (1978) and Shiraishi (1978) reported that acrylamide produces aneuploid and polyploid cells in both bone marrow cells and spermatogonia of mice treated with acrylamide by oral or intraperitoneal administration. Bull et al_. (1983) observed dose-related increases in Spermhead abnormalities in mice with acrylamide. More recently, Vanhorick and Moens (1983) found that acrylamide was a weak inducer of SV40 DNA amplification in SV40-transformed Chinese hamster cells as measured by in situ hybridization techniques. However, acrylamide synergistically enhanced the Induction of SV40 DNA synthesis by a number of other carcinogens. DISTRIBUTION SYSTEM Contribution of toxic chemicals from the distribution system to finished drinking water can be considerable under certain circumstances. It Is beyond the scope of this paper to deal comprehensively with this subject. However, it should be pointed out that certain products used in drinking water distribution are made of monomeric components that are recognized carcinogens. The most obvious example is polyvinyl chloride pipe which is a polymer of the SL 034456 406 well established human carcinogen vinyl chloride (IARC 1979). Modern manufacturing techniques have reduced the monomer content of these products to negligible concentrations, but users of such products should carefully avoid substandard materials. Another product that was widely used in lining of certain water mains, storage tanks and pipes are coal tar and asphaltic-based paints. The high polyaromatic hydrocarbon content of coal tar paints is partially, but not completely responsible for the high carcinogenic activity of these products in the mouse skin (Robinson et aK, 1984). Although still containing carcinogenic components, the asphaltic based paints used in contact with potable water are approximately 3 orders of magnitude less potent than their coal tar analogs. Because of the extremely low water solubility of the polyaromatic hydrocarbons that are partially responsible for the carcinogenic activity of coal tar these chemicals would not be expected to be rapidly leached from coatings. However, the extent to which these coatings provide a relatively constant reservoir for the leaching of carcinogen from the surface of the distribution system has not been well studied. However, some information exists to suggest that as these coatings age they can begin to shed fine particulates containing high concentrations of polyaromatic hydrocarbons into the finished drinking water. DISCUSSION It is clear from the previous discussion that chemical carcinogens find their way into drinking water through a variety of means. Obviously, industrial contamination is only one and in most cases a minor source of chemicals in drinking water. Evidence that the chlorination by-products 2,4,6-trichlorophenol and the haloacetonitriles have carcinogenic properties indicate that we must look beyond the trihalomethanes as the only potential carcinogenic hazards associated with disinfection. However, whatever the carcinogenic risk these products might represent they must be balanced against the obvious reduction of disease from waterborne infectious disease that results from chlorination. Finally there is a clear need to carefully scrutinize products that are utilized in the treatment and distribution of drinking water since these products can be a major source of harmful chemicals presented at the tap for human consumption. Despite the need to accept the general notion that carcinogenicity in experimental animals signals cause for concern about human exposures to the same chemical, it must be recognized that considerable uncertainty underlies the extrapolation of that information to man. These uncertainties are both SL 034457 407 qualitative and quantitative and have been discussed in detail elsewhere (IARC, 1982c). For example, if a chemical induces malignant tumors in more than one species, particularly if it produces tumors that are rare in the particular strain or species its carcinogenic properties are widely accepted (IARC, 1982c). On the other hand, a chemical that increases the incidence of a tumor that has a high spontaneous incidence in the test animal employed are felt to provide only limited evidence of carcinogenicity. Certain of the low molecular weight chlorinated hydrocarbons fall into this category. Other chemicals from the same class, such as vinyl chloride, cause little controversy because they produce malignant tumors in multiple sites and different species. It should be recognized that this view does not suggest that increased incidence of spontaneous tumors is of less concern than induction of rare tumors. Rather it is a statement related to the wide variety of factors that can modify the yield of a spontaneous tumor, whereas induction of a rare tumor probably requires the properties of a complete carcinogen. It is ironic that most of the chemicals that occur frequently in drinking water fall into a class of carcinogens whose carcinogenic effects are controversial. In general, the controversy involves those chemicals which induce liver tumors, in particular those whose activity is limited to the Induction of liver tumors in mice. Part of this concern is illustrated by the fact that the yield of hepatocellular carcinomas in 86C3F1 mice can be increased substantially by simple performance of a partial hepatectomy (Newberne et aK, 1982). The chemicals most directly involved in this controversy are trichloroethylene and tetrachloroethylene, but also involve considerations of the carcinogenicity of carbon tetrachloride and chloroform. Carbon tetrachloride should in many ways be considered the prototype for this group, although its carcinogenicity seems somewhat more generally accepted than the other three compounds. As pointed out above, carbon tetrachloride Is capable of producing liver tumors in a variety of species. Despite many attempts (many of which have not been published), it has been virtually impossible to demonstrate any genotoxic activity with carbon tetrachloride (IARC, 1979b; USEPA, 1984). However, the studies that have demonstrated carbon tetrachloride Induced liver cancer has involved very high doses, from 1000 to more than 2000 mg/kg administered In an vegetable oil vehicle 2 to 5 times weekly for extended periods of time. The experimental results with trichloroethylene In many ways contrast sharply with that obtained with carbon tetrachloride. Trichloroethylene has been shown to only induce hepatocellular carcinomas in mice (NCI, 1976). SL 034458 Although of a rather low potency, the genotoxic properties of trichloroethylene have been demonstrated by a number of investigators. In this respect, evidence of its carcinogenic activity would have to be considered stronger than that of carbon tetrachloride. However, the fact remains that trichloroethylene has only been shown capable of increasing the yield of neoplasms only in strains of mice which have high background incidence of these same neoplasms (Henschler et al_., 1980; Fukuda et al_. 1983; NCI, 1976b). Examination of the livers of mice receiving chloroform in drinking water demonstrated a dose-related increase in liver fat content that extend to levels much lower than those utilized for the original NCI coreinogenes is study (Jorgenson et aK, 1982). Similar changes were not observed in the livers of Osborne-Mendel rats until much higher levels of chloroform were administered in drinking water. These results suggest that corn oil was in some way involved in the liver pathology that develops in mice treated with chloroform. If so, this would imply that tumors induced by other hepatoxic agents may depend heavily on the corn oil vehicle comnonly used in testing of non-polar chemicals. Such an interpretation would suggest that results that have been obtained with chemicals such as trichloroethylene and tetrachloroethylene may not be extrapolated to other species without appropriate qualification. In fact, these data also call into similar question the carcinogenicity of carbon tetrachloride since it has only been shown capable of producing liver tumors when administered in a vegetable oil vehicle. Tetrachloroethylene increases hepatic DNA synthesis in B6C3F1 mice but not in rats following doses of 1000 mg/kg per day for 11 days (Schumann, et a1_., 1980). Similarly, Reitz et al_. (1982) found substantial increases in DNA synthesis of the liver and kidney of B6C3F1 mice treated with single doses of 60 and 240 mg/kg of chloroform and much smaller increases in the liver and kidney of male Osborne-Mendel rats. These data indicate that the toxic effects of these chemicals in the mouse liver results in substantial tissue regeneration of dose levels utilized in the carcinogenesis bioassays of these chemicals. Consequently, the possibility exists that these chemicals are capable of increasing liver tumor development from spontaneously Initiated cells in the same way that carbon tetrachloride has been shown to increase hepatomas in the liver of diethylnitrosamines initiated mice (Pound and McGuire, 1978). These questions become extremely important when attempting to estimate the risk to human beings consuming drinking water. Presumably the damage that 409 Is produced by initiators of cancer (i.e. those that act through damage to DNA) produce a certain degree of irreversible or non-repairable damage that is directly related to the dose of carcinogen. Theoretically, this would lend itself to a linear relationship to dose at low response rates and risks at low concentrations can be reliably estimated from experiments conducted at high doses. Increased tumor incidence due to non-genotoxic effects of chemicals or interactions with nutritional state presumably involve reversible processes. Consequently, the assumption of a linear relationship with dose at low response rates may not be justified. This may substantially affect any estimate of the health hazard associated with the low concentrations of these chemicals that occur in drinking water that is based upon the high doses used in animal experiments. The data available at present is not suited for projecting the risk to humans from the use of materials in the treatment and distribution of drinking water. Identification of carcinogenic properties of by-products of disinfection other than the trihalomethanes, acryamide and coal tar paints, however, does indicate that the use of such material in contact with potable water requires closer scrutiny. The industry should examine critically time-honored practices in the treatment and distribution of drinking water to gradually replace these materials with safer alternatives. In summary, evidence that a chemical induces cancer in experimental animals does suggest that there is a potential risk to man regardless of the > mechanism involved. Although it is certainly possible that a chemical that produces cancer in experimental animals would not do so in man, it is difficult to develop a prudent course of action on this assumption because the evidence which presently exists would suggest that the reverse is the general case. If tumorigenesis is dependent upon tissue damage. It should be remembered that these agents are also capable of inducing liver damage in humans. Consequently, it should be recognized that the argument presented regarding relationship of tissue damage to tumorigenicity is more of a quantitative consideration in the judgement of risks in man and less of a question of acrosS-species extrapolation than discussions of the issue would ordinarily imply. Therefore, the appearance of all the above chemicals in drinking water is cause for concern and to be avoided wherever possible and t practical. % ti- SL 034460 410 REFERENCES Allemand, H., Pessayre, 0., Descatoire, V., Degott, C., Feldmann, G., and Benhamou, J.P., (1978) Metabolic activation of trichloroethylene into a chemically reactive metabolite toxic to the liver. J. Pharmcol. Exp. Therapeut.. 204, 714-723. Bellar, T.A., L i cfTEenberg, J.J. and Kroner, R.C., (1974) The occurrence of organohalides in chlorinated drinking waters. J. Am. Mater Works Assn.. 66, 703-706 Bergman, K., (1983) Interactions of trichloroethylene with DNA in vitro and with RNA and ONA of various mouse tissues in vivo. Arch. ToTTcol.,54, 181-193. Bieber, T.I. and Trehy, M.L., (1983) Dihaloacetonitriles in chlorinated natural waters. In: Jolley, R.L.. et al. eds., Mater ChlorinaTion: Environmental Impact and Health Effects. 4, Ann Arbor Ml: Ann Arbor Science Pub 1 i., Inc., pp 85-96. Bronzetti, G., Zeiger, E., and Frezza, 0., (1978) Genetic activity of trichloroethylene in yeast. J. Toxicol. Environ. Health, 1, 411-418. Bronzetti, G., Bauer, C., Corsi, C., Del Carratore, R., Galli, ., Nieri, R., and Paolini, M., (1983) Genetic and biochemical studies on perchloroethylene in vitro and in vivo. Mutation Research 116, 323-331. Bolt, H.M. and Filser, TTG., (1977) "Irreversible binding of chlorinated ethylenes to macromolecules. Environ Health Persp. 21. 107-112. Bull, R.J., Meier, J.R. and Robinson, M. (lds3) Evidence of genotoxic effects with acrylamide. Pharmacologist 25, 640. Bull, R.J., Robinson, M., Laurie, R.5., Stover, G.D., Greisiger, E., Meier, J.R., and Stober, J., (1984a) Carcinogenic effects of acrylamide in sencar and A/J mice. Cancer Res. 44, 107-111. Bull, R.J., Robinson, M. and Stober, J.JC (1984b) Carcinogenic activity of acrylamide in the skin and lung of Swiss-ICR mice. Cancer Letters 24, 209-212. 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Environ. Hlth. Persp. 46, 163*168. Reuber, M.D. and Glover, E.L. (1970) Cirrhosis and carcinoma of the liver male rats given subcutaneous carbon tetrachloride. J. Natl. Cancer Inst., 44, 419-423. Reuber, M.D. and Glover, E.L., (1976a) Hyperplastic and early neoplastic lesions of the liver in buffalo strain rats of various ages given subcutaneous carbon tetrachloride. J. Natl. Cancer Insti,, 38, 891-895. Reuber, M.D. and Glover, E.L. (1976b) Cholangiofibrosis in the lTver of Buffalo strain rats injected with carbon tetrachloride. Exp. Pathol. 48, 319-322. Reuber, M.D. (1979) Carcinogenicity of chloroform. Environ. Hlth. Persp. 31, 171-182. Robinson, M., Bull, R.J., Munch, J. and Meier, J. (1984) Comparative carcinogenic and mutagenic activity of coal tarr and petroleum asphalt paints used in notable water supply systems. J. Appl. Toxicol., 4. 49-56. Roe, F.J.C., Palmer, A.R., Worden, A.N., Van Abbe, N.J., (1979) Safety evaluation of toothpaste containing chloroform. I. 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(1977) Mutagenic activity of chemicals identified in drinking water. Progress in Genetic Toxicology, pp 249. Stott, W.T., Quest, J.F and Watanabe, P.G. (1982) The pharmacokinetics and macromolecular intention of trichloroethylene in mice and rats. Toxicol. Appl. Pharmacol. 62, 137-151. Trehy, M.L. and Bieber, T.I., (1981) Detection, identification and Quantitative analysis of diha1oacetonitri1es~Tn chlorinated natural waters. In: Keith, L.H~., ed. Vol. 2. Advances in the Identification and Analysis of Organic Pollutants in Water.. (Ann Arbor, HI: Ann Arbor Science Publ., Inc.), pp, 941-975. IISEPA (1984) Research and Hea]th Assessment Document for Carbon Tetrachloride^ CPA"jTOo/8-82-001. Van Duuren, 8.L. and Banerjee, S., (1976) Covalent Interaction of metabolites of the carcinoqen trichloroethylene in rat hepatic microsomes. Cancer Research. 3, 2419-2422. Vanhovick, M. and Moens, W. (1983) Carcinogen-mediated induction of SV40 DNA amplification is enhanced by acrylamide in Chinese hamster CG60 cells. Carcinogenesis, 4, 1459-1463. Weisburger, J.H. and WilliamsT G.M. (1983) The distinct health risk analysis required for genotoxic carcinogens and promoting aqents. Environ. Hlth. Persp.. 50, 233-245. Withey, J.R.,"Collins, B.T. and Collins, P.G., (1983) Effect of vehicle on the pharmacokinetic and uptake of four halogenated hydrocarbons from the gastrointestinal tract of the rat. J, Appl. Toxicol. 3, 249-253. SL 034463