United States Environmental Protection Agency Health Effects Research Laboratory Research Triangle Park NC 27711 Research and Development EPA-600/S1 -85-001 Feb. 1985 Project Summary Investigation of the Metabolism of Chlorinated Hydrocarbons in Subhuman Species Carl C. Smith, Steven T. Cragg, Geraldine F. Wolfe, and Walter W. Weigel The metabolic fate and pharmacoki- netics of four common contaminants of chlorinated drinking water were studied in rats and rhesus monkeys. Rats ex- creted single oral or intravenous (i.v.) doses of 1,2,4-trichlorobenzene (TCB) primarily in the urine and feces; tissue levels were highest in the fat, kidneys, and liver. In monkeys, excretion of TCB following single oral or i.v. doses was slower than in rats, and fecal excretion was less important. In a subchronic toxicity study with monkeys, TCB was toxic at daily doses of 90 mg/kg body weight or more and was lethal at 174 mg/kg. TCB at 90-174 mg/kg in- creased hepatic enzyme activity in monkeys, and these increases correlated well with changes in urinary chlorgu- anide metabolite profiles. In rats, the toxicity of single oral doses of TCB was decreased by pretreatment with pheno- barbital and increased by pretreatment. with 3-methylcholanthrene. In rats, bromodichloromethane(BDC) tended to accumulate in fat, especially after i.v. administration. BDC given to monkeys both i.v. and orally was ex- creted primarily via the lung, with metabolism probably accounting for about 5% of the dose. In monkeys receiving single i.v. or oral doses of bis(2-chloroisopropyl) ether (BCIE), no more than one-third of the dose was recovered in the urine and feces. Repeated 30-mg/kg oral doses of BCIE to monkeys had toxic effects; again, the lungs appeared to be an important route of elimination. Monkeys excreted single oral doses of bis(2- chloroethyl) ether (BCEE) primarily in the urine. This Project Summary was developed by EPA's Health Effects Research Labo- ratory, Research Triangle Park, NC, to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction Rats and monkeys were used to study the metabolic fate and pharmacokinetics of four common halogenated organic contaminants of chlorinated drinking water: 1,2,4-trichlorobenzene (TCB); bro- modichloromethane (BDC); bis(2-chloro- isopropyl) ether (BCIE); and bis(2-chloro- ethyl) ether (BCEE). Emphasis was on the use of rhesus monkeys, because there is evidence that, in general, monkeys are more similar to man in their metabolism of compounds than are other common laboratory animals. The purpose of these studies was to provide data useful in judging the potential toxicity of these contaminants to man. Procedure Studies with 1,2,4-Trichlorobenzene Excretion and tissue distribution of TCB following single oral or intravenous (i.v.) doses were measured in young male Charles River albino rats. The treatment ------- solution contained 2.0 mg (0.45 fjC\ 14C)1,2,4-TCB per ml of waterethanol: Emulphor® (8:1:1, v/v/v). Rats (four per treatment) were given this solution in an oral dose of 10 mg/kg. Treatment groups were sacrificed at 3,6,12,24,48,72, and 96 h post-treatment. Urine and feces were collected at 24-h intervals or at necropsy. The following tissues were analyzed for total 14C activity by liquid scintillation counting: whole blood, plas- ma, liver, spleen, kidneys, lungs, heart, testes, brain, fat, muscle, and (together with their contents) stomach, small intes- tine, cecum, and large intestine. Excretion and blood levels of TCB following single oral or i.v. doses (10 mg/kg b.w.) were measured in young adult female rhesus monkeys (two per treatment). Blood samples were taken before treatment and at 1, 2, 4, 6, 8, 12, 24, 48, 72, and 96 h post-treatment. Urine samples were collected at 6,8,12, 24,48, 72, and 96 h post-treatment, and feces samples were collected at 24-h intervals. Subchronic oral TCB toxicity was studied in rhesus monkeys. Initial dose levels were 1, 5, and 25 mg/kg b.w. (five monkeys per treatment). When after one month these doses had not caused weight loss, changes in clinical chemistry anal- yses, or changes in urinary chlorguanide metabolite profiles, an additional dose level of 125 mg/kg was added. Of five monkeys receiving this dose for a month, one died after 15 days, one showed temporary weight loss, and the remaining three showed no signs or symptoms of TCB toxicity. Therefore, after the second month of the study, the 5- and 25-mg/kg groups were divided in half and assigned to two new dose levels, 90 and 174 mg/kg; the original control group was continued. Weights and blood samples were taken weekly. Urinary profiles of chlorguanide metabolites were measured monthly. After 12 weeks or at death, animals were necropsied, and liver slices were frozen for enzyme assays (described below). The chlorguanide metabolite profile test, developed in this laboratory, is based on the principle that the^activity of mixed function oxidases (MFO) in the liver (the P-450 system) can be estimated from the profile of urinary metabolites of a com- pound metabolized primarily by this system. Chlorguanide (CG) is oxidized by P-448 to chlorguanide triazine (CGT) and by P-450 to p-chlorophenyl biguanide (PBG). Parent CG is also excreted. Thus, the urinary concentrations of CGT and PBG can be used to estimate the relative activities of P-448 and P-450 in the liver. The relative activity of each monoxyge- nase component can be measured by comparing urinary levels of CGT and PBG metabolites with levels of CG. We have developed colorimetric and microbiologi- cal methods for measuring CG, CGT, PBG (after conversion to CGT), and total CG metabolites. Through the use of radio- labelled CG, a method for separating CG and its metabolites using thin-layer chromatography (TLC) was developed. In the TCB subchronic toxicity study, CG, CGT, and PBG were extracted and sepa- rated from a two-day urine pool obtained following a single oral dose of chlorgua- nide hydrochloride(10 mg/kg b.w.), and the radioactivity for each of the CG metabolites was determined by liquid scintillation counting. Microsomal suspensions of monkey liver samples were assayed for rates of MFO (P-448 and P-450) activity, via N- demethylation of aminopyrine and benz- phetamine and hydroxylation of zoxazol- amine and aniline. In a study of the effects of MFO inducers on the acute toxicity of TCB, groups of four rats were pretreated with phenobar- bital (in water, 75 mg/kg for 4 days), Aroclor®* 1254 (in corn oil, 50 mg/kg.for 5 days), 3-methylcholanthrene(3-MC)(in corn oil, 20 mg/kg for 2 days), water (2 ml/kg for 4 days), or corn oil (2 ml/kg for 5 days). TCB was administered 24 h after the last pretreatment, in a single oral dose of either 800 or 1000 mg/kg. Rats were weighed daily until sacrifice 6 days after dosing with TCB. Studies with Bromodichloromethane In a pilot study of bromodichlorome- thane tissue distribution, male Charles River albino rats (two per treatment) received 14C-BDC i.v. at 10 mg/kg or orally at 20 mg/kg, with a specific activity of 448 dpm//ug of BDC. Rats were sacri- ficed at 3 and 6 h post-treatment. Urine was collected until the animals were sacrificed, and tissues were analyzed as described above. In a "crossover" study of BDC blood levels, excretion, and tissue distribution, two female rhesus monkeys received oral or i.v. doses of 14C-BDC(10 mg/kg). One animal received an i.v. dose initially and •Mention of trade names or commercial products does not constitute endorsement or recommenda- tion for use. oral doses 9 and 82 days later; the oth< received an oral dose initially and i. doses 9 and 82 days later. Blood sample were taken before treatment and at 2, < 6,8,12, 24, and 48 h after the two initii doses. Urine and feces samples were als collected at 4,12,24, and 48 h. Followin the final dose, blood samples were take at 1, 2, 4, 6, 8, 12, and 24 h. Urine an feces samples were collected over th 24-h period. After 24 h, the monkey were necropsied, and tissue levels of14 were determined. Studies with Bis(2-chloroisopropyl) Ether Blood levels, excretion, and tissu distribution of 14C-BCIE were determine in female rhesus monkeys followin single oral or i.v. doses (30 mg/kg) with specific activity of 88.68 dpm///g. On animal in each treatment group wa sacrificed after 4 h, and one additions orally dosed animal was sacrificed after i h. Blood samples were collected at 1,2,4 and 6 h, urine samples at 4 and 6 h, an feces samples when available. The re maining two animals in each treatmen group were sacrificed after 168 h. Blooi samples were taken before treatment, a 2, 4, 6, 8, 12, and 24 h post-treatment and daily thereafter. Urine samples wen collected at 4,6,8,12, and 24 h and dail< thereafter. Feces samples were collectei daily. To test the effect of multiple oral doses 14C-BCIE (30 mg/kg) was given daily foi three consecutive days to three femaU rhesus monkeys. One animal receivec only two doses, because of the severity o toxic effects. One of the other two animal: vomited an undetermined portion of tht second dose. Blood samples were taker before treatment and at 1,2,4,8, and 24 h post-treatment on days 1,2, and 3, daily on days 4-10, and for one monkey also or days 13,15, and 17. Urine samples were collected at 12 and 24 h on day 1 and daily thereafter. Feces were collected when- ever available during days 1 -3, and 12 and 24 h on day 4, and daily thereafter. Study with Bis(2-chloroethyl) Ether Two female rhesus monkeys received single 10-mg/kg oral doses of 14C-BCEE with a specific activity of 106.27 dpm/A/g of BCEE. Urine samples were collected at 6, 12, 24, 36, 48, 72, and 96 h post- treatment, and feces samples daily for 4 days. ------- Results and Discussion Excretion and Tissue Distribution of TCB in Rats Although single oral and i.v. doses of "C-TCB to rats produced similar meta- bolic patterns, tissue levels were higher after i.v. administration. Following single oral doses to rats, excretion of TCB and its metabolites was approximately 95% complete at 24 h post-treatment and complete at 48 h. Mean urinary excretion at 96 h was 85.1 %, and mean excretion in the f eces was 14.9%. Approximately 96% of the administered UC was accounted for in the urine, feces, and analyzed tissues after 12 h, and all of the dose was accounted for after 24 h. Following single i.v. doses, excretion was approximately 85% complete at 24 h and 95% complete at 48 h. At 96 h, mean urinary excretion was 83.2% of the dose, and mean excre- tion in the feces was 12.4%. In orally dosed rats, the highest tissue levels were observed at 3 h. Outside of the gastrointestinal (Gl) tract, 14C levels were highest in the kidney, liver, plasma, and fat. At 24 h, very little 14C was in the stomach; about 3.5% of the dose was in the rest of the Gl tract, and all other tissues accounted for about 1% of the dose. Some radioactivity persisted in the fat, kidneys, and liver for at least 96 h after single oral doses. Following i.v. administration, maximum tissue levels occurred at 3 or 6 h post-treatment; levels were much higher in the fat than in any other tissue analyzed (51.6 /jg/g at 3 h). Prominent radioactivity in the intestines at 3 h indicated biliary excretion of TCB or its metabolites and probable enterohe- patic circulation. At 24 h, 14C levels exceeded 1 yug/g in the fat, liver, and kidneys; at 96 h, radioactivity was still appreciable in these tissues. Pharmacokinetic Study of TCB in Monkeys Following a single i.v. dose of 14C-TCB at 10 mg/kg, 14C levels in blood declined very rapidly, from an estimated zero-time level of about 112//g/ml to 2.0-2.9//g/ml at 1 h post-treatment. In the one monkey observed for 96 h, blood levels declined gradually to 0.2 //g/ml. Peak blood levels for orally treated monkeys were 3.0/*g/ml at 1 h and 2.3 //g/ml at 2 h. For both routes, plasma levels were slightly higher than blood levels through about 2 h post- treatment. Urinary excretion following i.v. admin- istration accounted for about 22% of the dose at 24 h and 38% at 96 h. Following oral administration, urinary excretion levels were 36.7% and 39.8% at 24 h and 56.7% and 73.1 % at 96 h. It appears that following i.v. dosing, TCB was rapidly deposited in the fat, then slowly mobilized and metabolized by the liver, whereas following oral dosing, it was transported first to the liver, where it was more rapidly metabolized before entering the systemic circulation. Fecal excretion was insignifi- cant for both routes, accounting for less than 4% of the dose. The low level of fecal radioactivity following i.v. treatment pro- vides indirect evidence that enterohepatic circulation of TCB and its metabolites was less in the monkey than in the rat. Subchronic Toxicity of TCB in Monkeys Toxic effects of TCB were observed in monkeys at all three dose levels. At 174 mg/kg, all four monkeys lost weight rapidly, and two died after three weeks of treatment. Treatment of the two surviving monkeys was discontinued on day 25, at which time the monkeys showed weak- ness and tremors. After treatment was discontinued, both monkeys began to gain weight; however, one died during the sixth week, apparently of acute gastric dilatation due to overeating. At the two lower doses, weight loss was variable. At 125 mg/kg, three mon- keys lost weight rapidly during the first two weeks of treatment; one of these animals died on day 16, and the other two stabilized and then gained weight rapidly during the remainder of the treatment period. The fourth monkey showed a gradual weight gain over the treatment period, with only a slight weight loss during weeks 7-8. At 90 mg/kg, one monkey rapidly lost 2.5 kg during the first 4 weeks, then stabilized and gained weight. The weight of a second monkey remained relatively stable throughout the period, though it declined slightly over the last 4 weeks of treatment. The two remaining monkeys at 90 mg/kg showed moderate weight loss throughout the first 6-7 weeks. One of these monkeys then gained weight rapidly during weeks 8-10. The other died in'the seventh week; however, the absence of definitive toxic manifestations before death or at necrop- sy suggests that the death may not have been drug related. A possible explanation for stabilization and weight gain following initial weight loss is that the animals responded by developing hepatic MFO induction, thus reducing the effective dose through increased metabolism of TCB. Hematocrit tended to decline gradually in all TCB treatment groups, with subse- quent increases after 5-7 weeks in the monkeys receiving 90 and 125 mg/kg. In the one surviving high-dose monkey withdrawn from treatment, hematocrit returned to the pretreatment value by the end of the study. Ratios of liver weight to body weight (at sacrifice) were significantly higher in the monkeys receiving TCB at 125 and 174 mg/kg than in the untreated controls. The effect was less in the high-dose group, all of whose members either died or were withdrawn from treatment after 3 weeks. This ratio was also elevated in the 90-mg/kg monkey that died after 7 weeks, though not in the other animals at that dose. Comparison of chlorguanide metabolite profiles between treatment groups and the control group appeared to indicate a significant decrease in percent CG in the urine, a significant increase in percent PBG, and no change in percent CGT following treatment with TCB for 30 days. However, large individual variation in baseline (pretreatment) profiles gave rise to large baseline differences among the groups, especially in percent CG and percent CGT. Comparison of the mean change in the parameters (the post- treatment value minus the baseline value for each group) indicated that percent CG decreased significantly in the 125 mg/kg group and percent PBG increased signifi- cantly in all three treatment groups. CGT tended to decrease in all groups; this decrease was statistically significant when post-treatment values for the 90- and 174-mg/kg groups were compared with their own baseline values, but not when change in CGT was compared between each treatment group and the control group. After 60 days' treatment, the 90-mg/kg and 125-mg/kg groups showed stabilization or partial reversal of all changes in CG metabolite profile. In Vitro Enzyme Assays of Monkey Livers Enzyme activity (as nanomoles per minute per gram liver) was significantly increased against all four substrates. Mean increases over control values for the 90- and 125-mg/kg groups ranged from about 185% to 260%. The 174- mg/kg dose also increased activity against all four substrates, but interpretation of the results is complicated by the general toxicity of the dose and the cessation of treatment after 3 weeks. MFO activity was similarly increased in the 90- and ------- 125-mg/kg groups. Enzyme activities were positively correlated with urinary levels of PBG and were not significantly correlated with urinary levels of CGT. Inhibition and Potentiation of the Hepatotoxicity of TCB in Rats Following pretreatment with Aroclor, two rats receiving TCB at 1000 mg/kg died within 24 h after TCB administration, and one receiving TCB at 800 mg/kg died after 4 days. One rat induced with 3-MC and given TCB at 1000 mg/kg died after 3 days. All other rats survived the observa- tion period. Rats pretreated with Aroclor or 3-MC lost more weight than did the vehicle control rats, but rats pretreated with phenobarbital lost less weight than the controls. Weight loss reached a maximum on days 3-4; all surviving rats then began to gain weight. The most severe liver damage occurred in the groups that had lost the most weight; rats pretreated with phenobarbital showed less liver damage than did the controls. Thus, phenobarbital decreased and 3-MC increased the toxicity of single oral doses of TCB in rats, probably through changes in hepatotoxicity. Pharmacokinetic Studies on BDC in Rats When 14C-BDC was given to rats i.v., accumulation in fat accounted for more than 50% of the dose recovered in the tissues. Elevated levels were also ob- served in the liver, blood, plasma, kidneys, and lungs. Small amounts of 14C in the Gl tract suggested that the compound was excreted in the bile. The distribution pattern was very different following oral administration: the compound was slow to leave the stomach, levels in fat were lower than observed following i.v. admin- istration (6.9 vs. 24.4 fjg/g at 3 h), and levels in the liver were higher (9.0 vs. 3.7 fjg/g at 3 h). The tissues accounted for much less of the dose at 3 h when BDC was given orally (8.6% vs. 25.5% after i.v. administration); recovery in the urine was higher. Pharmacokinetics of' BDC in Monkeys Following i.v. administration of UC- BDC to monkeys, blood levels fell rapidly for the first 8 h and more gradually for the next 40 h (from about 2 fjg/g at 1 h to 0.2-0.4 fjg/Q at 48 h). Blood levels were more variable following oral dosing, peak- ing at 4 h and declining in a pattern similar to that seen following i.v. admin- istration. Urinary excretion of 14C account- ed for no more than about 6% of the dose and was generally between 2 and 3%, regardless of the route of administration. Fecal excretion was 0.2-1.65% of the dose. Little BDC appeared to be sequester- ed in any tissue; levels were highest in the bile, cecum, and liver by both routes, and were slightly higher following oral administration. BDC appeared to be ex- creted primarily via the lung. Metabolism of BDC probably accounted for no more than about 5% of the dose. Pharmacokinetics of BCIE in Monkeys Following a single i.v. dose of 1*C-BCIE, blood levels dropped rapidly, from an estimated zero-time level of about 330 jug/gto2-hvaluesof24.3-32.7//g/gand 12-h values of 8.4 fjg/g. Over the next 6 days, the levels declined gradually to about 2.5 fjg/g. During the first 5 days, plasma UC levels were slightly higher than values for whole blood. Monkeys receiving BCIE orally had peak values 2-4 h after administration, ranging from 21.8-26.1 A/g/g. The pattern of decline in blood levels was similar to that observed following i.v. administration; levels after 7 days were 1.2 and 1.9 fjg/g. Plasma and blood levels were similar. Monkeys re- ceiving BCIE i.v. excreted 28.6% and 34.8% of the dose in the urine; about half of this amount was eliminated in 4 h. After oral administration, urinary excre- tion was 24% and 30.1% of the dose; about half was eliminated by 8 h. Fecal excretion was 1.2% and 6% of the i.v. dose and 1.6% and 1.9% of the oral dose. Thus, no more than one-third of the dose administered by either route was recover- ed in the urine and feces. The only organs showing significant levels of 14C from BCIE after 7 days were the liver and kidneys. Fat also showed some sequester- ing activity, and there appeared to be some biliary excretion of BCIE or its metabolites. Multiple Dose Study of BCIE in Monkeys Two monkeys receiving three daily oral doses of 14C-BCIE developed edema and subdural hemorrhage in the tissues around the eyes, while the third monkey showed no ocular reaction to treatment. All three monkeys lost weight. Weight loss was greatest in the animals with ocular symptoms, and these animals also had decreased urine flow. One of the monkeys exhibiting toxic symptoms also had exhibited glucosuria and hypergly- cemia before treatment began, and was not given a third dose of BCIE. The other monkey with toxic symptoms began men- struation on the fifth day of the study, 14 days earlier than expected. Peak blood levels of BCIE generally occurred at 2 h post-treatment; 2-h values ranged from 17.2-34 /Lig/ml. Levels drop- ped rapidly during the next 6 h and more slowly through the remainder of the 24-h period. Residual radioactivity increased with repeated doses; 24-h values ranged from 5.0-8.9 //g/ml after the first dose, 7.8-10.5 //g/ml after the second, and 8.3- 14.9 fjg/m\ after the third. Plasma levels of BCIE were consistently higher than blood levels. The slopes of the dieaway curves for both blood and plasma levels appeared flatter on day 3 than previously. The monkey exhibiting the most severe toxic symptoms also had (after the second dose) the highest peak blood concentra- tion observed in the study (34 /yg/ml vs. 16.5 and 23.0 ;ug/ml in the other two monkeys, one of which had vomited part of this dose). Urinary excretion of 14C was variable. Only 30-45% of the dose was accounted for in the urine and feces (no more than 4% in the feces), suggesting that the lungs were an important route of elimina- tion of this compound. Pharmacokinetic Study of BCEE in Monkeys Two monkeys given 14C-BCEE orally excreted about 43% of the dose in the urine in 24 h post-treatment. One monkey had excreted about 17% of the dose by 6 h and about 30% by 12 h, while the other monkey had excreted only 26.5 ml of urine by 12 h, accounting for only 0.003% of the administered dose. At 72 h, re- coveries were 53.3% and 63.4%. Very little radioactivity was recovered in the feces (1.10% and 1.63% of the dose through 96 h). The monkey with reduced urine flow also showed hemorrhagic darkening below the eyes, reduced food consumption, and ketones in the urine, and began menstruation at 24 h after dosing. Conclusions and Recommendations In rats, single oral doses of TCB were excreted in the urine (85%) and feces (15%). Tissue levels were highest in the fat, kidneys, and liver. After i.v. admin- istration, fat TCB levels were higher, and excretion was slower. In monkeys, excre- tion of TCB was slower than in rats, and fecal excretion was less important. In 4 ------- subchronic studies with monkeys, TCB had toxic effects at daily doses of 90 mg/kg body weight or more and was lethal in about 25 days at 174 mg/kg. TCB at 90-174 mg/kg increased liver enzyme activity in monkeys. It is sug- gested that the effect of TCB on hepatic enzymes be tested in humans. Hepatic enzyme induction results correlated well with effects on chlorguanide metabolite profiles, suggesting that the GC metabo- lite profile test merits further trial as an in vivo assay for hepatic drug-metabolizing enzymes in humans. The toxicity of single oral doses of TCB in rats was decreased by pretreatment with phenobarbital and increased by pretreatment with 3-MC; these results were unexpected, and it is suggested that the experiment be re- peated. BDC given to rats i.v. accumulated in the fat; oral administration resulted in lower levels in fat and higher revels in the liver. In monkeys, BDC was excreted primarily via the lung, with metabolism probably accounting for no more than about 5% of the dose. Additional studies are needed to measure the chronic toxicity of BDC in rats and monkeys and to char- acterize the urinary metabolites. When BCIE was given to monkeys either i.v. or orally, no more than one- third of the dose was recovered in the urine and feces. BCIE persisted longest in the liver, kidneys, and fat. Repeated 30- mg/kg doses of BCIE to monkeys caused toxic effects, including periocular irrita- tion. Only 30-45% of the dose was accounted for in the urine and feces, suggesting that the lungs were an im- portant route of elimination. Single oral doses of BCEE were excreted primarily in the urine (53-63% of the dose at 72 h); toxic signs were seen at 10 mg/kg in one of the two monkeys tested. The present studies suggest that BCIE and BCEE may affect the menstrual cycle in monkeys; this possible effect should be tested. More extensive metabolic and chronic toxicity studies are needed to determine whether renal and periorbital toxic effects are due to these compounds or to metabolites. Car 1C. Smith. Steven T. Cragg, GeraldineF. Wolfe, and Walter W. We/gel are with University of Cincinnati, Cincinnati, OH 45267. Robert Lingg is the EPA Project Officer (see below). The complete report, entitled "Investigation of the Metabolism of Chlorinated Hydrocarbons in Subhuman Species," (Order No. PB 85-152 387/AS; Cost: $14.50, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield. VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Health Effects Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, NC 27711 *USGPO: 1985-559-111/10787 ------- ------- United States Center for Environmental Research Environmental Protection Information Agency Cincinnati OH 45268 Official Business Penalty for Private Use $300 OC00329 PS ------- |