Voronin et al. ( 1987) assessed the carcinogenic potential of bromodichloromethane in male and female CBA x C57Bl/ 6 mice. Groups of mice ( 50 55/ sex/ concentration) were exposed to bromodichloromethane provided in drinking water at concentrations of 0.04, 4.0, or 400 mg/ L for 104 weeks. Untreated control groups of 75 male and 50 female mice were also included in the study design. No significant differences were observed in total tumor incidence when evaluated by Chi square analysis. The study authors concluded that, under the conditions of this bioassay, bromodichloromethane was not carcinogenic in mice. Draft  Do Not Cite or Quote February 20, 2003 V  91 Theiss et al. ( 1977) examined the carcinogenic potential of bromodichloromethane in Strain A mice ( 6 to 8 weeks old). Male animals ( 20 mice/ group) were injected intraperitoneally up to three times weekly over a period of 8 weeks. Three dose levels ( 20, 40, or 100 mg/ kg bromodichloromethane) were used with concurrent positive and negative control groups that contained 20 animals each. Mice were sacrificed 24 weeks after the first injection, and the frequency of lung tumors in each test group was compared with vehicle treated controls. No statistically significant increase in the incidence of lung tumors/ mouse was reported.

Melnick et al. ( 1998) investigated the mechanistic relationship between liver toxicity and tumorigenicity of bromodichloromethane. Female B6C3F 1 mice ( 10 animals per group) were treated with bromodichloromethane in corn oil via gavage for 3 weeks ( 5 days/ week). Doses of bromodichloromethane used in this study were 0 ( vehicle only), 75, 150, or 326 mg/ kg day. A significant dose related increase in absolute liver weight and liver weight/ body weight ratio was noted for the 150 and 326 mg/ kg day dose groups. Serum ALT activity was significantly increased in the two highest dose groups and serum SDH activity was elevated at all doses tested. At necropsy, there was clear evidence of hepatocyte hydropic degeneration in animals treated with 150 and 326 mg/ kg day. BrdU was administered to the animals during the last 6 days of the study, and hepatocyte labeling index ( LI) analysis was conducted. The two highest ( 150 and 326 mg/ kg day) doses resulted in significantly elevated hepatocyte proliferation as measured by the LI. Using the Hill equation model, these authors compared the dose response for liver toxicity ( enzyme and LI data) and tumorigenicity ( data from previously published NTP sponsored bioassays) for bromodichloromethane. This analysis indicated that the shape of the dose response as well as the Hill exponents were different for liver toxicity and tumorigenicity. It was concluded that the results of this comparison do not support a causal relationship between liver toxicity/ reparative hyperplasia and tumor development.

George et al. ( 2002) evaluated the carcinogenicity of bromodichloromethane in male F344/ N rats ( 78 animals/ dose) exposed to the compound via drinking water for 104 weeks. Nominal concentrations of 0.07, 0.35, or 0.70 g/ L were administered in drinking water containing 0.25% Emulphor ® . The vehicle control solution consisted of 0.25% Emulphor ® . The study authors indicated that testing of higher concentrations was prevented by refusal of the test animals to drink solutions containing more than 0.7 g/ L. Six animals per exposure concentration were sacrificed at 13, 26, 52, and 78 weeks for gross observation and histopathological examination of the thyroid, liver, stomach, duodenum, jejunum, ileum, colon, rectum, spleen, kidneys, urinary bladder, and testes. A complete rodent necropsy was performed at terminal sacrifice and representative samples of the tissues listed above were examined microscopically. A complete pathological examination was performed on five rats from the high dose group. Serum profiles of LDH, ALT, ALP, AST, SDH, BUN, total protein, creatine, and total antioxidant activities were determined at 26, 52, and 104 weeks. Hepatocyte and renal tubular cell proliferation were measured at each sacrifice by bromodeoxyuridine labeling.

The measured drinking water concentrations of bromodichloromethane were 0.06, 0.38, and 0.76 g/ L. When corrected for loss of bromodichloromethane as a result of volatility, instability, or adsorption to glass surfaces during treatment, the corresponding administered Draft  Do Not Cite or Quote February 20, 2003 V  92 concentrations were 0.06, 0.33, and 0.62 g/ L. Based on measured water consumption, these levels correspond to mean daily doses for the entire study of 3.9, 20.6, and 36.3 mg/ kg day as calculated by the study authors. Mean daily doses of 6.4, 32.6, and 58.9 mg/ kg day were calculated for the first 13 weeks of the study when the growth rate of the test animals was highest. No significant differences were observed among groups for feed consumption or survival. Twenty one to 22 unscheduled deaths were observed in each treatment group. Mononuclear cell leukemia was seen in all dose groups and was reported to be the primary cause of morbidity and mortality prior to 104 weeks. Exposure to bromodichloromethane did not affect the growth rate of test animals when compared to the control. Kidney weight was significantly depressed at the high dose and a significant negative trend was observed for relative kidney weight. No significant changes were observed in clinical chemistry parameters. Observed nonneoplastic changes in the liver ( e. g., biliary fibrosis, bile duct inflammation, and chronic inflammation) were considered to be age related background changes, since neither the incidence nor severity of the lesions differed from the control values. Bromodichloromethane had no effect on hepatocyte proliferation as measured by bromodeoxyuridine labeling. Renal tubular cell hyperplasia was significantly decreased in the 3.9 mg/ kg day group and significantly increased in the 36.3 mg/ kg day group ( 15.8%) relative to the control value ( 8.7%).

The absence of effect on body weight and other examined endpoints suggests that a maximum toxic dose may not have been achieved in this study. However, the dosing regimen used by George et al. ( 2002) was sufficient to increase the incidence of hepatocellular neoplasia ( Table V 14). The data for hepatic tumors indicate a biphasic pattern of dose response. The prevalence and multiplicity of hepatocellular adenoma and combined hepatocellular adenoma and carcinoma were significantly increased at 3.9 mg/ kg day, nonsignificantly increased at 20.6 mg/ kg day, and comparable to the control values at 36.3 mg/ kg day. The prevalence and multiplicity of hepatocellular carcinoma were increased at 20.6 mg/ kg day when compared to control values, but the response did not reach statistical significance. The underlying basis for the biphasic response is unknown, but the study authors noted that the observed pattern of response could be explained by inhibition of the hepatic metabolism of bromodichloromethane by the compound itself. Exposure to bromodichloromethane decreased the prevalence of basophilic ( control, 67%; 3.6 mg/ kg day, 62.2%; 20.6 mg/ kg day, 46%; 36.6 mg/ kg day, 34.7%) and clear cell ( 17.8%, 2.2%, 2.1%, 4.1%) altered foci of cells ( AFC) in a dose dependent manner, but had no significant effect on the prevalence of eosinophilic AFCs when compared to the controls. The decreases in prevalence were statistically significant at the mid and high doses for basophilic AFCs and at all doses for clear cell AFCs. Exposure to bromodichloromethane had no significant effect on the prevalence of renal tubular adenomas or carcinomas ( Table V 14). One renal tubular adenoma was observed in the 3.6 mg/ kg day group and two tumors were observe in the 36.3 mg/ kg day. The historical incidence of renal tubular adenomas in male F344/ N rats is very low ( 2/ 327 or 0.6%), as determined from control groups in NTP drinking water studies ( NTP, 2002; no data were reported from the study laboratory). Therefore, the occurrence of these tumors in the present study may be of biological significance. No increased incidences of neoplasia were evident in the five high dose animals selected for a histopathological examination of all organs. On the basis of the increased prevalence and multiplicity of hepatocellular neoplasms in the 3.9 Draft  Do Not Cite or Quote February 20, 2003 V  93 and 20.6 mg/ kg day groups, the study authors concluded that bromodichloromethane was carcinogenic in male F344/ N rats under the Table V 14 Hepatic and Renal Tumors in Male F344/ N Rats Administered Bromodichloromethane in the Drinking Water for Two Years Tumor Type Mean Daily Dose of Bromodichloromethane ( mg/ kg day)

Vehicle Control 3.9 20.6 36.3 Liver Hepatocellular adenoma 1/ 45 ( 2.2%) a 0.02 ± 0.02b, c 7/ 45 ( 15.5%)* 0.16 ± 0.04* 3/ 48 ( 6.2%) 0.06 ± 0.02 2/ 49 ( 4.1%) 0.04 ± 0.02 Hepatocellular carcinoma 1/ 45 ( 2.2%) 0.02 ± 0.02 1/ 45 ( 2.2%) 0.02 ± 0.01 4/ 48 ( 8.3%) 0.10 ± 0.03 2/ 49 ( 4.1%) 0.04 ± 0.02 Hepatocellular adenoma and carcinoma ( combined) 2/ 45 ( 4.4%) 0.04 ± 0.02 8/ 45 ( 17.8%)* 0.19 ± 0.00* 7/ 48 ( 14.6%) 0.17 ± 0.04 4/ 49 ( 8.2%) 0.08 ± 0.28 Kidney Tubular cell adenoma 0/ 46 ( 0%) 1/ 45 ( 2.2%) 0/ 51 ( 0%) 2/ 44 ( 4.5%)

Tubular cell carcinoma 0/ 46 ( 0%) 0/ 45 ( 0%) 0/ 51 ( 0%) 0/ 44 ( 0%)

Tubular cell adenoma or carcinoma ( combined) 0/ 46 ( 0%) 1/ 45 ( 2.2%) 0/ 51 ( 0%) 2/ 44 ( 4.5%)

Source: George et al. ( 2002) * Statistically significant when compared to the control value, p

0.05 a Prevalence ( percentage of animals with tumor) b Multiplicity, number of tumors per animal c Mean ± standard deviation conditions of the bioassay. A source of uncertainty in this conclusion is lack of knowledge on the biological mechanism underlying the biphasic dose response observed for hepatic tumors.

George et al. ( 2002) also evaluated the carcinogenicity of bromodichloromethane in male B6C3F 1 mice ( 78 animals/ dose) exposed via drinking water for 100 weeks. Nominal concentrations of 0.05, 0.25, or 0.50 g/ L were administered in drinking water containing 0.25% Emulphor ® . The vehicle control solution consisted of 0.25% Emulphor ® . Seven animals per exposure concentration were sacrificed at 13, 26, 52, and 78 weeks for gross observation and histopathological examination of the liver, stomach, duodenum, jejunum, ileum, colon, rectum, spleen, kidneys, urinary bladder, and testes. A complete rodent necropsy was performed at Draft  Do Not Cite or Quote February 20, 2003 V  94 terminal sacrifice and representative samples of the tissues listed above were examined microscopically. A complete pathological examination was performed on five rats from the high dose group. Serum profiles of LDH, ALT, ALP, AST, SDH, BUN, total protein, creatine, and total antioxidant activities were determined at 26, 52, and 100 weeks. Hepatocyte and renal tubular cell proliferation were measured by bromodeoxyuridine labeling at each sacrifice.

The measured drinking water concentrations of bromodichloromethane were 0.06, 0.30, and 0.55 g/ L. When corrected for loss of bromodichloromethane as a result of volatility, instability, or adsorption to glass surfaces during treatment, the corresponding administered concentrations were 0.06, 0.28, and 0.49 g/ L. Based on measured water consumption, these levels correspond to mean daily doses of 8.1, 27.2, and 43.4 mg/ kg day as calculated by the study authors. Water consumption was significantly reduced at the mid and high doses; the study authors attributed the reduced intake to taste aversion. No significant differences were observed among groups for feed consumption or survival. Exposure to bromodichloromethane did not affect the growth rate of test animals when compared to the control. Kidney weight was significantly depressed at 27.2 and 43.4 mg/ kg day when compared to the control values. No significant changes were observed in clinical chemistry parameters. Mild, treatment related nonneoplastic hepatic lesions were observed in the 27.2 and 43.4 mg/ kg day dose groups ( identity and prevalence not reported). Increased incidences of hepatocellular karyomegaly and necrosis with inflammation ( prevalence and severity not reported) were not dose related. The prevalence of renal tubular hyperplasia was 3%, 0%, 6% and 0% for the vehicle control, 8.1, 27.2, and 43.4 mg/ kg day groups, respectively. Other observed preneoplastic and neoplastic lesions ( identity and prevalence not reported) were considered background events for the male B6C3F 1 mouse. BrdU labeling index in hepatocytes and renal tubular cells was not altered at any time point. Hepatocellular adenomas and carcinomas were observed in all treatment groups. Neither the prevalence nor multiplicity of these tumors was significantly increased by exposure to bromodichloromethane. Renal tubular cell neoplasia was not observed in any treatment group. No increased incidences of neoplasia were evident in the five high dose animals subject to a full histopathological examination. On the basis of these data, the study authors concluded that bromodichloromethane was not carcinogenic to male mice under the conditions employed in this study. However, in the absence of compound related effects on body weight or other toxicologic endpoints, it is not evident that an adequately high dose was tested in this study.

De Angelo et al., ( 2002) evaluated the ability of bromodichloromethane administered in drinking water to induce aberrant crypt foci ( ACF), putative early preneoplastic lesions, in the colons of male F344/ N rats. Groups of weanling rats ( 6 animals/ group) were exposed to distilled water, 0.25% Alkamuls EL 620 ® , or 0.7 g/ L bromodichloromethane in 0.25% Alkamuls EL 620 for 13 weeks. A single intraperitoneal injection of 30 mg/ kg azoxymethane ( AOM) served as the positive control. Body weight and water consumption were measured twice during the first week of the study and once per week thereafter. Colons were collected at study termination, fixed, stained with 0.2% methylene blue, divided into three equal segments, and scanned for ACF. The measured concentration of bromodichloromethane averaged 0.64 ± 0.06 mg/ L ( mean and standard error) over the course of the study. When adjusted for volatilization and adherence to glass, the corrected concentration was 0.51 mg/ L. Water consumption was significantly reduced Draft  Do Not Cite or Quote February 20, 2003 V  95 ( 38%) in the bromodichloromethane exposure group when compared to the deionized water control. The average daily dose was 45 mg/ kg day as calculated by the study authors. Average terminal body weight of the rats exposed to bromodichloromethane was within 10% of the control values. No ACF were observed in colons from control animals. ACF were observed in five of six colons from bromodichloromethane exposed animals. The total number of ACF ( 30) and number of aberrant crypts per focus ( 3.33 ± 0.47) were significantly increased relative to the combined deionized water and vehicle controls. All observed ACF were located in the distal ( rectal) segment of the colon. For comparison, 807 ACF and 4.95 ± 0.25 crypts per focus ( mean and standard error) were observed in the AOM positive control group. Eight percent, 42% and 50% of the ACF induced by AOM were located in the proximal, middle, and distal segment of the colon, respectively. The study authors noted that ACF induced by bromodichloromethane do not to progress to neoplasia, as judged by the absence of colon neoplasms in the two year cancer study conducted by George et al. ( 2002).

De Angelo et al. ( 2002) also evaluated the ability of bromodichloromethane administered in drinking water to induce ACF in the colons of male B6C3F 1 ( 6 animals/ group) and A/ J mice ( 9 animals/ group; sex not specified). Mice of the A/ J strain are sensitive to chemical induction of ACF. Test animals were exposed to distilled water, 0.25% Alkamuls EL 620 ® , or 0.5 g/ L bromodichloromethane in 0.25% Alkamuls EL 620 for 13 weeks ( both strains) or 30 weeks ( A/ J mice only). A single intraperitoneal injection of 50 mg/ kg 4 aminobiphenyl or 10 mg/ kg azoxymethane ( AOM) served as the positive controls for the B6C3F1 and A/ J strains, respectively. Body weight and water consumption were measured twice during the first week of the study and once per week thereafter. Colons were collected at study termination, fixed, stained with 0.2% methylene blue, divided into three equal segments, and scanned for ACF. The study report did not provide results for measured concentration of bromodichloromethane in drinking water solutions or an estimated dose. No differences were observed in between the control and any treatment group for body weight or water and feed consumption. ACF development was not observed in the colons of B6C3F 1 mice treated with bromodichloromethane in the drinking water or injected with 4 aminobiphenyl. Bromodichloromethane did not induce ACF in A/ J mice. Injection of A/ J mice with the positive control compound AOM induced 47.4 ± 4.9 ACF/ cm2 ( mean and standard error) and 7.2 ± 1.1 tumors/ cm2 after 13 weeks and 17.8 ± 2.6 tumors/ cm2 after 30 weeks of treatment. In comparison, 807 ACF and 4.95 ± 0.25 crypts per focus were observed in the AOM positive control group.

2. Dibromochloromethane NTP ( 1985) administered dibromochloromethane at doses of 0, 40, or 80 mg/ kg day ( in corn oil) to groups of 50 male and 50 female F344/ N rats via gavage 5 times/ week for 104 to 105 weeks. Survival of dosed male and female rats was comparable to that of the vehicle control groups. High dose males had lower body weights when compared with the vehicle control. Compound related nonneoplastic lesions ( fatty metamorphosis and ground glass cytoplasmic changes) were found in the livers of both sexes ( See section V. D. 1). Nephrosis was observed in female rats. No statistically significant increase in the incidence of any neoplastic lesion was Draft  Do Not Cite or Quote February 20, 2003 V  96 observed. Based on the results of this study, the authors concluded that there was no evidence of carcinogenicity in rats administered dibromochloromethane.

NTP ( 1985) administered dibromochloromethane ( in corn oil) to groups of 50 male and 50 female B6C3F 1 mice via gavage 5 times/ week for 104 to 105 weeks. The administered doses were 0, 50, or 100 mg/ kg day. Survival of female mice was comparable to that of the vehiclecontrol group. High dose male mice, however, had lower survival rates than the vehicle controls. At week 82, nine high dose male mice died of an unknown cause. An inadvertent overdose of dibromochloromethane given to low dose male and female mice at week 58 killed 35 male mice, but apparently did not affect the females. The low dose male mouse group was, therefore, considered to be unsuitable for analysis of neoplasms. Compound related nonneoplastic lesions were found primarily in the livers of male mice ( hepatocytomegaly, necrosis, fatty metamorphosis) and female mice ( calcification and fatty metamorphosis). Nephrosis was observed in male mice. In females, a statistically significant increase in the incidence of hepatocellular adenomas and adenomas and carcinomas combined was observed in the high dose group. In male mice, a statistically significant increase in the incidence of hepatocellular carcinomas and adenomas and carcinomas combined was observed in the high dose group. A summary of the incidence of these tumors is presented in Table V 15. A negative trend in the incidence of malignant lymphomas was evident in dibromochloromethane exposed male mice when compared to the vehicle control. The study authors concluded that the results of this study provided equivocal evidence of dibromochloromethane carcinogenicity in male B6C3F 1 mice and some evidence of carcinogenicity in female B6C3F 1 mice.

In other bioassays, Voronin et al. ( 1987) observed no significant tumor increases in CBAxC57B1/ 6 mice ( 50/ sex/ dose) treated with dibromochloromethane in the drinking water at concentrations of 0, 0.04, 4.0, or 400 mg/ L ( approximately 0, 0.008, 0.76, or 76 mg/ kg day) for 104 weeks. In an unpublished report of a two year dietary study, Tobe et al. ( 1982) reported no increase in gross tumors in male rats dosed with up to 210 mg/ kg day or female rats treated with up to 350 mg/ kg day.

Melnick et al. ( 1998) exposed female B6C3F 1 mice ( 10/ dose) to dibromochloromethane in corn oil via gavage for 3 weeks ( 5 days/ week). The doses of dibromochloromethane in this study were 0 ( vehicle only), 50, 100, 192, or 417 mg/ kg day. The corresponding time weighted doses are 0, 37, 71, 137, and 298 mg/ kg day. No treatment related signs of overt toxicity were observed during the study. Body weight and water intake were not significantly altered at any dose tested. However, a statistically significant and dose related increase in liver weight/ body weight ratio was seen in the 100, 192 and 417 mg/ kg day dose groups. Serum ALT activity was significantly increased in the two highest dose groups. The activity of serum SDH was significantly elevated at all doses tested except 50 mg/ kg day. However, the increase in activity ( shown graphically) was very small relative to the control at the 100 and 192 mg/ kg day doses. At necropsy, there was clear evidence of hepatocyte hydropic degeneration in the192 and 417 mg/ kg day dose groups. BrdU was administered to the animals during the last 6 days of the study, and hepatocyte labeling index ( LI) analysis was conducted. Only the highest dose tested ( 417 mg/ kg day) resulted in significantly elevated hepatocyte proliferation as measured by the LI. Draft  Do Not Cite or Quote February 20, 2003 V  97 Using the Hill equation model, these authors compared the dose response for liver toxicity ( enzyme and LI data) and tumorigenicity ( data from previously conducted NTP bioassays) for dibromochloromethane. This analysis indicated that the shape of the dose response as well as the Hill exponents were different for liver toxicity and tumorigenicity. It was concluded that the results of this comparison do not support a causal relationship between liver toxicity/ reparative hyperplasia and tumor development.

Table V 15 Frequencies of Liver Tumors in B6C3F1 Mice Administered Dibromochloromethane in Corn Oil for 105 Weeks  Adapted from NTP ( 1985)

Treatment ( mg/ kg day) Sex Adenoma Carcinoma Adenoma or Carcinoma ( combined)

Vehicle Control M F 14/ 50 2/ 50 10/ 50 4/ 50 23/ 50 6/ 50 50 M F  a 4/ 49  6/ 49  10/ 49 100 M F 10/ 50 11/ 50b 19/ 50b 8/ 50 27/ 50c 19/ 50d a Male low dose group was inadequate for statistical analysis. b p < 0.05 relative to controls. c p < 0.01 ( life table analysis); p = 0.065 ( incidental tumor test) relative to controls. d p < 0.01 relative to controls.

De Angelo et al. ( 2002) evaluated the ability of dibromochloromethane administered in drinking water to induce aberrant crypt foci ( ACF), putative early preneoplastic lesions, in the colons of male F344/ N rats. Groups of weanling rats ( 6 animals/ group) were exposed to distilled water, 0.25% Alkamuls EL 620 ® , or 0.9 g/ L dibromochloromethane in 0.25% Alkamuls EL 620 for 13 weeks. A single intraperitoneal injection of 30 mg/ kg azoxymethane ( AOM) served as the positive control. Body weight and water consumption were measured twice during the first week of the study and once per week thereafter. Colons were collected at study termination, fixed, stained with 0.2% methylene blue, divided into three equal segments, and examined for ACF. The measured concentration of dibromochloromethane averaged 0.80 ± 0.05 mg/ L ( mean and standard error) over the course of the study. When adjusted for volatilization and adherence to glass, the corrected concentration was 0.63 mg/ L. Water consumption was significantly reduced ( 32%) in the dibromochloromethane exposure group when compared to the deionized water control. The average daily dose of dibromochloromethane was 60 mg/ kg day as calculated by the study authors. Average terminal body weight of the rats exposed to dibromochloromethane was within 10% of the control values. No ACF were observed in colons from control animals. ACF were observed in three of six colons from dibromochloromethane exposed animals. The total number of ACF ( 17) and number of aberrant crypts per focus ( 2.43 ± 0.61) were significantly increased relative to the combined deionized water and vehicle controls. Fourteen percent of the Draft  Do Not Cite or Quote February 20, 2003 V  98 observed ACF were located in the middle segment of the colon and 86% were located in the distal ( rectal) segment. In comparison, 807 ACF and 4.95 ± 0.25 crypts per focus were observed in the AOM positive control group. Eight percent, 42% and 50% of the ACF induced by AOM were located in the proximal, middle, and distal segment of the colon, respectively. Draft  Do Not Cite or Quote February 20, 2003 V  99 3. Bromoform Groups of 50 male B6C3F 1 mice were exposed to bromoform via gavage ( corn oil) at doses of 0, 50, or 100 mg/ kg day of bromoform for 103 weeks ( 5 days/ week) ( NTP, 1989a). Groups of 50 female mice received doses of 0, 100, or 200 mg/ kg day bromoform by the same protocol. At termination, all animals underwent gross necropsy and thorough histological examinations of tissues. Survival in both treated female groups was reduced; however, the authors attributed this reduction in survival partly to utero ovarian infection. A statistically significant increase in the incidence of thyroid follicular cell hyperplasia was noted in high dose females; however, there were no statistically significant increases in the incidence of any neoplastic lesion in any dose group compared to controls. Based on the results of this study, the NTP ( 1989a) concluded there was no evidence of carcinogenic activity of bromoform in male or female mice.

In a similar experiment, groups of 50 male and 50 female F344/ N rats were administered bromoform via gavage in oil at doses of 0, 100, or 200 mg/ kg day for 5 days/ week for 103 weeks ( NTP 1989a). At termination, all animals were necropsied, and a thorough histological examination of tissues was performed. Adenomatous polyps or adenocarcinomas of the large intestine were noted in three high dose male rats, eight high dose female rats, and one low dose female rat ( Table V 16). Although the number of tumors found was small, the incidence was considered to be significant because these intestinal tumors are very rare in the rat. The NTP concluded that there was some evidence for carcinogenic activity in male rats and clear evidence in female rats.

Table V 16 Tumor Frequencies in the Large Intestine of F344/ N Rats Exposed to Bromoform in Corn Oil for 2 Years  Adapted from NTP ( 1989a)

Tumor Tumor Frequency Male rat Control 100 mg/ kg 200 mg/ kg Adenocarcinoma 0/ 50 0/ 50 1/ 50 Polyp ( adenomatous) 0/ 50 0/ 50 2/ 50 Female rat Control 100 mg/ kg 200 mg/ kg Adenocarcinoma 0/ 48 0/ 50 2/ 50 Polyp ( adenomatous) 0/ 48 1/ 50 6/ 50 Theiss et al. ( 1977) examined the carcinogenic activity of bromoform in Strain A mice. Male animals ( 6 to 8 weeks old, 20 mice/ group) were injected intraperitoneally up to three times Draft  Do Not Cite or Quote February 20, 2003 V  100 weekly over a period of 8 weeks. The dose levels utilized were 4, 48, or 100 mg/ kg bromoform. A positive and a negative control group were included in the study design and each contained 20 animals. Mice were sacrificed 24 weeks after the first injection and the frequency of lung tumors in each test group was compared with vehicle treated controls. Bromoform produced a significant increase ( p = 0.041) in tumor frequency only at the intermediate dose. U. S. EPA ( 1980) concluded that these results were suggestive of carcinogenic activity but were not an adequate basis for the assessment of cancer risk.

Kurokawa ( 1987) observed no evidence of carcinogenicity in male or female rats exposed to microencapsulated bromoform at concentrations of 400, 1600, or 6500 ppm in the diet for 24 months.

Melnick et al. ( 1998) exposed female B6C3F 1 mice ( 10 animals/ group) to bromoform in corn oil via gavage for 3 weeks ( 5 days/ week). Doses of bromoform used in this study were 0 ( vehicle only), 200, or 500 mg/ kg day. A dose related increase in absolute liver weight and liver weight/ body weight ratio was noted in both tested doses. Neither serum ALT nor serum SDH activity was significantly elevated at either dose of bromoform. At necropsy, there was no evidence of hepatocyte hydropic degeneration in animals treated with either dose. BrdU was administered to the animals during the last 6 days of the study, and hepatocyte labeling index ( LI) analysis was conducted. Only the 500 mg/ kg day dose resulted in marginally significant increase in hepatocyte proliferation as measured by the LI. Using the Hill equation model, these authors compared the dose response for liver toxicity ( enzyme and LI data) and tumorigenicity ( using data from previously published NTP bioassays) for bromoform. This analysis indicated that the shape of the dose response as well as the Hill exponents were different for liver toxicity and tumorigenicity. The authors concluded that these data do not support a causal relationship between liver toxicity/ reparative hyperplasia and tumor development.

De Angelo et al. ( 2002) evaluated the ability of bromoform administered in drinking water to induce aberrant crypt foci ( ACF), putative early preneoplastic lesions, in the colons of male F344/ N rats. Groups of weanling rats ( 6 animals/ group) were exposed to distilled water, 0.25% Alkamuls EL 620 ® , or 1.1 g/ L bromoform in 0.25% Alkamuls EL 620 for 13 weeks. A single intraperitoneal injection of 30 mg/ kg azoxymethane ( AOM) served as the positive control. Body weight and water consumption were measured twice during the first week of the study and once per week thereafter. Colons were collected at study termination, fixed, stained with 0.2% methylene blue, divided into three equal segments, and examined for ACF. The measured concentration of bromoform averaged 0.98 ± 0.08 mg/ L over the course of the study. When adjusted for volatilization and adherence to glass, the corrected concentration was 0.77 mg/ L. Water consumption was significantly reduced ( 30%) in the bromoform exposure group when compared to the deionized water control. The average daily dose of bromoform was 76 mg/ kgday as calculated by the study authors. The average terminal body weight of the rats exposed to bromoform was within 10% of the control values. No ACF were observed in colons from control animals. ACF were observed in four of six colons from bromoform exposed animals. The total number of ACF ( 26) and number of aberrant crypts per focus ( 3.71 ± 0.36) were significantly increased relative to the combined deionized water and vehicle controls. Fourteen percent of the Draft  Do Not Cite or Quote February 20, 2003 V  101 observed ACF were located in the middle segment of the colon and 86% were located in the distal ( rectal) segment. In comparison, 807 ACF and 4.95 ± 0.25 crypts per focus ( mean and standard error) were observed in the AOM positive control group. Eight percent, 42% and 50% of the ACF induced by AOM were located in the proximal, middle, and distal segment of the colon, respectively.

H. Other Key Health Effects 1. Immunotoxicity a. Bromodichloromethane Munson et al. ( 1982) administered bromodichloromethane by gavage to CD 1 male and female mice ( 8 12/ sex/ dose) for 14 days at levels of 0, 50, 125, or 250 mg/ kg day. Bromodichloromethane appeared to affect the humoral immune system, as judged by decreased antibody forming ( ABF) cells in serum and by decreased hemagglutination titers. These changes were clearly significant ( p < 0.05) at the high dose in both males and females, and decreased ABF cells were also noted at the mid dose ( 125 mg/ kg day) in females. This study identified a NOAEL of 50 mg/ kg day and a LOAEL of 125 mg/ kg day for bromodichloromethane on the basis of decreased immune function in females Additional information on other endpoints measured in this study is provided in Section V. B. 1.

French et al. ( 1999) investigated the immunotoxicity of bromodichloromethane in a series of four experiments conducted in mice and rats. Immunotoxicity in mice was examined following exposure via ingestion of drinking water or by gavage. The immunological parameters examined were antibody response to injected sheep red blood cells and T and B lymphocyte proliferation. Mitogens used in the proliferation assay were concanavalin A ( Con A) or phyto hemagglutinin p ( PHA) for T cells and lipopolysaccharide ( LPS) for B cells. Female C57BL/ 6 mice ( 6 animals per group) were treated for 14 or 28 days with drinking water containing 0, 0.05, 0.25 or 0.5 g/ L bromodichloromethane. All drinking water ( including controls) contained 0.25% Emulphor ® to reduce volatilization of bromodichloromethane. Based on measured water consumption, these concentrations were estimated by the authors to be equivalent to 0, 10, 37 or 62 mg/ kg day. There were no significant differences in the number of antibody forming cells, antibody production, or spleen weights in any treatment group. Likewise, splenic and mesenteric lymph node cell proliferative responses to T and B cell mitogens were similar in all groups. Continuation of this study for an additional 2 weeks did not affect any measured parameter. These data identify a NOAEL of 62 mg/ kg day for short term exposure.

French et al. ( 1999) conducted a second experiment in which female C57BL/ 6 mice were dosed by gavage with bromodichloromethane in 10% Emulphor ® once a day for 16 days. Treatment groups ( 6 animals per group) included controls ( deionized water or10% Emulphor ® ) , 50, 125 or 250 mg/ kg day bromodichloromethane. As in the previous experiment, there were no differences in ABF cells, antibody titers or mitogen induced proliferation in any treatment groups. A decrease in spleen weight and spleen to weight ratio was observed in the 125 mg/ kg day group Draft  Do Not Cite or Quote February 20, 2003 V  102 when compared to the Emulphor ® control. However, spleen weights in the Emulphor ® control were significantly higher than those in the deionized water control group, making this finding difficult to interpret.

French et al. ( 1999) investigated the immunotoxicity of bromodichloromethane in male Fisher 344 rats following two different in vivo exposure regimens: ingestion of drinking water containing bromodichloromethane and gavage. The immunological parameters examined were antibody response to injected sheep red blood cells and T and B lymphocyte proliferation. The mitogens used in the proliferation assay were concanavalin A ( Con A) or phyto hemagglutin p ( PHA) for T cells and S. typhimurium mitogen ( STM) for B cells. Six rats per treatment group were exposed for 26 weeks to drinking water containing 0, 0.07 or 0.7 g/ L bromodichloromethane and 0.25% Emulphor ® . Based on water consumption measurements, these concentrations were estimated by the authors to be equivalent to average daily doses of 0, 5 or 49 mg/ kg day. There was a significant suppression of Con A stimulated proliferation of spleen cells observed in the 49 mg/ kg day dose group. No effect on other immunological parameters was reported. These data suggest NOAEL and LOAEL values of 5 and 49 mg/ kg day, respectively, for immunotoxic effects.

French et al. ( 1999) also examined the effect of short term exposure to relatively large doses of bromodichloromethane on immune function. Female F344 rats ( 6 animals/ group) received gavage doses of deionized water, 10% Emulphor ® , or 75, 150, or 300 mg bromodichloromethane/ kg in 10% Emulphor ® for 5 days. Surviving high dose animals had decreased body, spleen, and thymus weights. Con A and PHA responses were depressed in spleen cells isolated from high dose animals. Two of the six rats in the 300 mg/ kg day group died during the exposure period. The remaining high dose animals had significantly decreased body, spleen and thymus weights compared to both control groups. Thymus weight, but not spleen or body weight, was also decreased in the 150 mg/ kg day group. Con A responses were significantly depressed in both spleen and mesenteric lymph node ( MLN) cells in the 300 mg/ kgday treatment group. All three ( 75, 150 and 300 mg/ kg day) dose groups exhibited suppression of PHA stimulated MLN cells when compared to the vehicle ( but not the water) controls. This discrepancy was due to the fact that Emulphor ® alone significantly elevated the proliferative response to PHA in MLN cells relative to the deionized water group. In contrast to the T cell responses, there was a significant increase in antibody production and proliferative responses to STM ( B cells) from spleen cells at the highest dose tested ( 300 mg/ kg day dose group). These data suggest a marginal NOAEL of 150 mg/ kg day and a LOAEL of 300 mg/ kg day for acute exposure based on depression of immune response.

b. Dibromochloromethane Munson et al. ( 1982) administered dibromochloromethane by gavage to CD 1 male and female mice ( 8 to 12/ sex/ dose) for 14 days at levels of 0, 50, 125, or 250 mg/ kg day and evaluated humoral and cell mediated immune system functions. Dibromochloromethane appeared to affect the humoral immune system, as judged by decreased antibody forming ( ABF) cells in serum and by decreased hemagglutination titers. These changes were significant Draft  Do Not Cite or Quote February 20, 2003 V  103 ( p < 0.05) at the high dose in both males and females. Decreased ABF cells were also noted at the mid dose ( 125 mg/ kg day) in females. This study identified a NOAEL of 50 mg/ kg day and a LOAEL of 125 mg/ kg day for dibromochloromethane on the basis of decreased immune function in females. Additional information on this study is provided in Section V. B. 2.

c. Bromoform Munson et al. ( 1982) administered bromoform ( aqueous) by gavage to CD 1 male and female mice ( 6 to 12/ sex/ dose) for 14 days at levels of 0, 50, 125, or 250 mg/ kg day. Endpoints evaluated included humoral immune system function. The authors judged that the humoral immune system was not significantly affected by bromoform, although a decrease in antibody forming ( ABF) cells was reported for high dose males. These data suggest a NOAEL of 250 mg/ kg day for effects of bromoform on the immune system. Additional information on this study is provided in Section V. B. 3.

2. Hormonal disruption No studies or case reports were identified that described hormonal disruption by dibromochloromethane or bromoform.

Bielmeier et al. ( 2001) examined the effect of bromodichloromethane on serum progesterone and luteinizing hormone levels in two experiments conducted as part of an investigation on full litter resorption ( FLR) in F344 rats ( see Section V. E. 1 for additional details). In the first experiment, rats ( 7 to 10/ treatment group) were administered a single 100 mg/ kg dose by aqueous gavage on gestation day 8 or 9. Hormone levels in samples of tail blood were determined on GD 9 through 12. FLR was observed in 0, 60 and 100% of the control, GD 8 dosed, and GD 9 dosed animals, respectively. A marked reduction in progesterone levels was noted 24 hours after dosing in all rats that resorbed their litters when compared to controls and to bromodichloromethane treated animals that retained their litters. The mean progesterone levels in animals dosed on GD 9 decreased from 137.94 ng/ mL ± 11.44 ng/ mL to 48.45 ± 23.57 ng/ mL within 24 hours ( n = 9). For animals treated on GD 8, the mean progesterone level 24 hours after bromodichloromethane treatment was 67.01 ± 16.22 ng/ mL in animals that resorbed litters ( n = 6) and 127.19 ± 14.89 in controls ( n= 7). The resorbed groups had reduced progesterone levels comparable to the progesterone levels in non pregnant animals ( n = 2) when assayed three days after compound administration. In contrast to the effect noted on progesterone levels, administration of bromodichloromethane had no apparent effect on LH when measured 24 hours after dosing. However, elevated LH concentrations were observed on GD 11 to 12 in animals experiencing resorption. LH levels in these groups increased from approximately 0.20 ng/ mL on GD 10 to approximately 0.80 ng/ mL on GD 11 and remained elevated through GD 12. In contrast, LH levels in the controls decreased from 0.31 to 0.14 ng/ mL over the same time period.

Bielmeier et al. ( 2001) performed a second experiment to further characterize the effect of bromodichloromethane treatment on progesterone and LH levels in pregnant F344 rats. The rats ( 8 11/ treatment group) were dosed with 0, 75, or 100 mg/ kg by aqueous gavage on GD 9. Blood Draft  Do Not Cite or Quote February 20, 2003 V  104 samples were collected at 0, 6, 12, and 24 hours after dosing. The incidence of FLR was 0, 64%, and 90% in the 0, 75, and 100 mg/ kg dose groups, respectively. The progesterone levels peaked in all dose groups ( including controls) at 6 hours. At 12 and 24 hours, the progesterone levels in bromodichloromethane treated animals that resorbed their litters were progressively reduced. Progesterone levels in bromodichloromethane treated animals that retained their litters remained comparable to levels observed in the control group. No significant differences in LH concentration were noted among dose groups at any time point.

3. Structure Activity Relationships Although the mechanism of brominated trihalomethane toxicity is not known with certainty, there is abundant evidence to indicate that adverse effects are secondary to metabolism. Bromine is generally a better leaving group than chlorine, suggesting that bromine substitution could potentially influence the pathway and rate of trihalomethane metabolism. Multiple studies ( described in Section III. C) indicate that metabolism of chloroform and the brominated trihalomethanes can occur through one or both of two cytochrome P450 mediated pathways: reductive metabolism to free radical intermediates or oxidative metabolism to dihalocarbonyls ( Figure 4 1). Although comparative data are limited, there is some evidence to indicate that chloroform and the brominated trihalomethanes are metabolized to a different extent by these pathways. Tomasi et al. ( 1985) examined the reductive metabolism of chloroform, bromodichloromethane, and bromoform in rats and obtained the following rank order for free radical formation: bromoform> bromodichloromethane> chloroform. Wolf et al. ( 1977) reported that bromoform was more extensively metabolized under anaerobic conditions in vitro than was chloroform. Gao and Pegram ( 1992) observed that binding of reactive intermediates to rat hepatic microsomal lipids and proteins was more than twice as high for bromodichloromethane as for chloroform when assayed under anaerobic conditions. These results collectively suggest that reductive metabolism may be a more important metabolic pathway for brominated trihalomethanes than for chloroform. At present, this apparent difference in metabolism has not been linked to specific differences in toxicity.

Two mutagenicity studies provide additional information on structure activity relationships among the trihalomethanes. Additional details of these studies are presented in Section V. F. Examination of mutagenicity in a strain of Salmonella typhimurium engineered to express rat theta class glutathione S transferase ( GST) indicated the following order for mutagenic potency ( number of revertants/ ppm) of the brominated trihalomethanes: bromoform

dibromochloromethane> bromodichloromethane ( DeMarini et al., 1997). The potency of the first two compounds was several times greater than that observed for bromodichloromethane. Analysis of the mutational spectra of the brominated trihalomethanes indicated that all three compounds have similar mutational spectra ( predominately GC

AT transitions) and site specificity ( middle C of a CCC sequence in target DNA). These observations suggest that a common reactive intermediate or class of intermediates is likely to mediate the mutagenicity of these compounds. Draft  Do Not Cite or Quote February 20, 2003 V  105 In the second study, Pegram et al.( 1997) compared the glutathione S transferase mediated mutagenicity of bromodichloromethane and chloroform in a GST+ strain of S. typhimurium ( See section V. F. 1). Revertants were produced in a dose related manner in the presence of low as well as high concentrations of bromodichloromethane. In contrast, chloroform induced a doubling of the number of revertants only at high concentrations. This result provides evidence that bromine substitution of trihalomethanes confers the capability for GST catalyzed transformation to mutagenic intermediates at low substrate concentrations. These data further suggest that chloroform and the brominated trihalomethanes may induce adverse effects via different modes of action, and indicate the need for care in extrapolating the characteristics of chloroform metabolism and toxicity to brominated trihalomethanes.

I. Summary 1. Health Effects of Acute and Short Term Exposure of Animals Large oral doses of brominated trihalomethanes are lethal to laboratory animals. Reported acute LD 50 values range from 450 to 969 mg/ kg for bromodichloromethane, 800 to 1,200 mg/ kg for dibromochloromethane, and 1,388 to 1,550 mg/ kg for bromoform. Acute lethality values are summarized in Table V 1.

Acute oral exposure to sublethal doses of brominated trihalomethanes can also produce effects on the central nervous system, liver, kidney, and heart. Acute duration studies investigating endpoints other than death are summarized in Table V 2. Ataxia, anaesthesia, and/ or sedation were noted in mice receiving 500 mg/ kg bromodichloromethane, 500 mg/ kg dibromochloromethane, or 1,000 mg/ kg bromoform. Renal tubule degeneration, necrosis, and elevated levels of urinary markers of renal toxicity have been observed in rats receiving 200 to 400 mg/ kg bromodichloromethane. Elevated levels of serum markers for hepatotoxicity and have been observed in rats at doses of bromodichloromethane ranging from approximately 82 to 400 mg/ kg day, and hepatocellular degeneration and necrosis were observed at 400 mg/ kg. Effects on heart contractility were reported in male rats at doses of 333 and 667 mg/ kg dibromochloromethane.

Short term studies of brominated trihalomethanes are summarized in Table V 3. Short term exposure of laboratory animals to brominated trihalomethanes has been observed to cause effects on the liver and kidney. Hepatic effects, including organ weight changes, elevated serum enzyme levels, and histopathological changes, became evident in mice and/ or rats administered 38 to 250 mg/ kg day bromodichloromethane, 147 to 500 mg/ kg day dibromochloromethane, or 187 to 289 mg/ kg day bromoform for 14 to 30 days. Kidney effects, characterized by decreased p aminohippurate uptake, histopathological changes, and organ weight changes, became evident in mice and/ or rats administered 148 to 300 mg/ kg day bromodichloromethane, 147 to 500 mg/ kg day dibromochloromethane, or 289 mg/ kg day bromoform for 14 days. Evidence for decreased immune function was noted at bromodichloromethane or dibromochloromethane doses of 125 mg/ kg day. Studies examining Draft  Do Not Cite or Quote February 20, 2003 V  106 strain differences in response to short term brominated trihalomethane exposure have not been reported.

2. Health Effects of Longer term Exposure of Animals Subchronic studies of brominated trihalomethanes are summarized in Table V 4. The predominant effects of subchronic oral exposure occur in the liver and kidney. The effects produced in these two organs are similar in nature to those described for short term exposures, with liver appearing to be the most sensitive target organ for dibromochloromethane and bromoform exposure. Histopathological changes in the liver were reported in mice and/ or rats administered 200 mg/ kg day bromodichloromethane, 50 to 250 mg/ kg day dibromochloromethane or 50 to 283 mg/ kg day bromoform. Histopathological changes in the kidney were reported in mice and/ or rats administered 100 mg/ kg day bromodichloromethane, or 250 mg/ kgday dibromochloromethane. Studies examining strain differences in response to subchronic brominated trihalomethane exposure have not been reported.

Chronic toxicity studies of brominated trihalomethanes are summarized in Table V 5. As observed for exposure for shorter durations, the predominant effects of chronic oral exposure are observed in the liver and kidney. Histopathological signs of hepatic toxicity in mice and/ or rats became evident at doses of 6 to 50 mg/ kg day for bromodichloromethane, 40 to 50 mg/ kg day for dibromochloromethane, and 90 to 152 mg/ kg day for bromoform. Signs of bromodichloromethane induced renal toxicity became evident in mice and rats treated with doses of 25 and 50 mg/ kg day, respectively. Studies examining strain differences in response to chronic brominated trihalomethane exposure have not been reported.

3. Reproductive and Developmental Effects Reproductive and developmental studies of brominated trihalomethanes are summarized in Table V 9. Data on the developmental effects of brominated trihalomethanes suggest that these chemicals are toxic to the fetus in most cases only at doses that result in maternal toxicity. Signs of maternal toxicity ( decreased body weight, body weight gain and/ or changes in organ weight) were reported in rats administered bromodichloromethane at 25 to 200 mg/ kg day and in rabbits administered 4.9 to 35.6 mg/ kg day. Signs of maternal toxicity were observed in rats or mice administered 17 ( marginal) to 200 mg/ kg day dibromochloromethane and in mice administered 100 mg/ kg day bromoform. Maternal toxicity was not observed in female rats dosed with up to 200 mg/ kg day of bromoform. Several well conducted studies on the developmental toxicity of bromodichloromethane gave negative results at doses up to 116 mg/ kg day in rats and 76 mg/ kgday in rabbits when administered in drinking water. However, in other studies, slightly decreased numbers of ossification sites in the hindlimb and forelimb were observed in fetuses of rats administered 45 mg/ kg day in the drinking water on gestation days 6 to 21and sternebral aberrations were observed in the offspring of rats administered 200 mg/ kg day by gavage in corn oil. Reductions in mean pup weight gain and pup weight were observed when the pups were administered bromodichloromethane in the drinking water at concentrations of 150 ppm and above ( biologically meaningful estimates of intake on a mg/ kg day basis could not be calculated Draft  Do Not Cite or Quote February 20, 2003 V  107 for this study). Full litter resorption has been noted in F344 rats, but not Sprague Dawley rats, treated with bromodichloromethane at doses of 50 to 100 mg/ kg day during gestation days 6 to 10. Chronic oral exposure to bromodichloromethane resulted in reduced sperm velocities at doses of 39 mg/ kg day. This response was not accompanied by histopathological changes in any reproductive tissue examined. Adverse clinical signs and reduced body weight and body weight gain were observed in parental generation female rats and F 1 male and female rats at 150 ppm ( approximately 11.6 to 40.2 mg/ kg day) in a two generation study of bromodichloromethane administered in drinking water. In the same study, small but statistically significant delays in sexual maturation occurred in F 1 males at 50 ppm ( approximately 11.6 to 40.2 mg/ kg day) and F1 females at 450 ppm ( approximately 29.5 to 109 mg/ kg day). These delays may have been secondary to dehydration caused by taste aversion to bromodichloromethane in the drinking water.

Four of five studies on the reproductive or developmental toxicity of dibromochloromethane gave negative results when tested at doses of up to 200 mg/ kg day. In the fifth study, dibromochloromethane administered at 17 mg/ kg day in a multigenerational study resulted in reduced day 14 postnatal in one of two F2 generation litters. At 171 mg/ kg day, the mid dose in the study, decreased litter size, viability index, lactation index, and postnatal body weight were observed in some F1 and/ or F2 generation. The developmental and reproductive toxicity of bromoform was examined in two studies.

Bromoform administered to rats at 100 mg/ kg day in corn oil by gavage resulted in a significant increase in sternebral aberrations in the apparent absence of maternal toxicity. In a continuous breeding toxicity protocol, gavage doses of 200 mg/ kg day in corn oil resulted decreased postnatal survival, organ weight changes, and liver histopathology in F1 mice of both sexes. No effects on fertility or other reproductive endpoints were noted.

4. Mutagenicity and Genotoxicity In vitro and in vivo studies of the mutagenic and genotoxic potential of bromodichloromethane, dibromochloromethane, and bromoform have yielded mixed results. Synthesis of the overall weight of evidence from these studies is complicated by the use of a variety of testing protocols, different strains of test organisms, different activating systems, different dose levels, different exposure methods ( gas versus liquid), and in some cases, problems due to evaporation of the test chemical. Overall, a majority of studies yielded more positive results for bromoform and bromodichloromethane. The genotoxicity and mutagenicity data for dibromochloromethane are variable. Recent studies in strains of Salmonella engineered to contain rat theta class glutathione S transferase suggest that mutagenicity of the brominated trihalomethanes may be mediated by glutathione conjugation.

5. Carcinogenicity Studies in Animals The carcinogenic potential of individual brominated trihalomethanes administered in oil has been investigated in chronic oral exposure studies in mice and rats. Ingestion of Draft  Do Not Cite or Quote February 20, 2003 V  108 bromodichloromethane caused liver tumors in female mice, renal tumors in male mice and in male and female rats, and tumors of the large intestine in male and female rats. Ingestion of dibromochloromethane caused liver tumors in male and female mice, and ingestion of bromoform caused intestinal tumors in male and female rats. Comparison of dose response data for hepatotoxicity, cell proliferation, and tumorigenesis in female mice suggests that the hepatic carcinogenicity of brominated trihalomethanes is not a simple consequence of cytotoxicity and regenerative cell proliferation Draft  Do Not Cite or Quote February 20, 2003 V  109 6. Other Key effects The immunotoxicity of brominated trihalomethanes has been investigated in mice and rats. Short term bromodichloromethane exposure resulted in decreased antibody forming cells in serum, decreased hemagglutinin titers, and/ or suppression of Con A stimulated proliferation of spleen cells at doses of 125 to 250 mg/ kg day.

No evidence has been reported for hormonal effects following exposure to dibromochloromethane or bromoform. Studies in pregnant F344 rats detected decreased levels of progesterone in animals administered 75 or 100 mg/ kg bromodichloromethane by aqueous gavage on gestation day 8 or 9. Increased levels of luteinizing hormone were observed two to three days after dose administration. Disruption of luteal responsiveness to luteinizing hormone has been proposed as a possible mode of action by which bromodichloromethane elicits full litter resorption in F344 rats.

Limited structure activity data for brominated trihalomethanes and chloroform suggest that bromination may influence the proportion of compound metabolized via the oxidative and reductive pathways, with brominated compounds being more extensively metabolized by the reductive pathway. Additional evidence suggests that a GSH mediated pathway may play an important role in metabolism of brominated trihalomethanes, but not chloroform. These data raise the possibility that brominated trihalomethanes may induce some adverse effects via different modes of action than chloroform. Draft  Do Not Cite or Quote February 20, 2003 VI  1 VI. HEALTH EFFECTS IN HUMANS A. Clinical Case Studies 1. Bromodichloromethane No clinical reports or short term studies were located on the effects in humans from ingestion of bromodichloromethane.

2. Dibromochloromethane No clinical case reports or short term studies were located on the effects in humans from ingestion of dibromochloromethane.

3. Bromoform In the past, bromoform was used as a sedative for children with whooping cough. Typical doses were approximately one drop ( about 180 mg), given three to six times/ day ( Burton Fanning, 1901). This dosing usually resulted in mild sedation in children, although a few rare instances of death or near death were reported ( e. g., Dwelle, 1903; Benson, 1907). These cases were believed to be due to accidental overdoses. Based on these clinical observations, the estimated lethal dose for a 10 to 20 kg child is approximately 300 mg/ kg, and the LOAEL for mild sedation is approximately 54 mg/ kg day.

B. Epidemiological Studies Multiple epidemiological studies have investigated the relationship between exposure to disinfection by products in chlorinated drinking water and adverse health effects. These studies fall into two basic categories: studies of association with cancer ( Table VI 1) and studies of association with adverse pregnancy or birth outcomes or alteration of menstrual function ( Table VI 2). Because the purpose of this document is to isolate the health effects of individual brominated trihalomethanes, a detailed examination of all available studies on disinfection byproducts is beyond the scope of this report. Epidemiologic studies published prior to 1994 are discussed in greater detail in the Drinking Water Criteria on Chlorine ( U. S. EPA, 1994a). A number of recent publications have reviewed the association between chlorination disinfection byproducts and cancer and adverse reproductive or developmental outcomes ( e. g., Reif et al., 1996; Mills et al., 1998; Nieuwenhuijsen et al., 2000; Bove et al., 2002).

A subset of epidemiologic studies has examined possible associations between exposure to bromodichloromethane and adverse reproductive outcomes. The relationship between exposure to brominated trihalomethanes and alterations in menstrual function has also been investigated. These studies are described in greater detail in the sections below. Draft  Do Not Cite or Quote February 20, 2003 VI  2 Table VI 1 Epidemiological Studies Investigating an Association Between Chlorinated Drinking Water and Cancer Reference Study Description Observation Alavanja et al. ( 1978) Case control study in seven New York State counties. Greater risk of gastrointestinal and urinary tract cancer mortality, both sexes, in chlorinated water areas of the counties.

Cantor et al. ( 1978) Ecological study using age standardized cancer mortality rates, 1968 1971; and halomethane levels from U. S. EPA surveys. Strongest correlation between brominecontaining trihalomethanes and bladder cancer.

Struba ( 1979) Case control study of mortality in North Carolina, 1975 1978. Small but significant odds ratios for rectum, colon and bladder cancers in rural areas but not in urban areas.

Brenniman et al. ( 1980) Case control study in 70 Illinois communities, 1973 1976. Questionnaires sent to water treatment plants to verify 1963 inventory data on chlorine levels. Statistically significant relative risks of cancer of gall bladder, large intestine, and total gastrointestinal and urinary tract in females served by systems with chlorinated versus nonchlorinated ground water. Due to many uncontrolled confounding factors, authors concluded that chlorination was not a major factor in the etiology of gastrointestinal and urinary tract cancers.

Gottlieb et al. ( 1981) Case control study using mortality data in Louisiana and estimations of exposure. Rectal cancer significantly elevated with respect to surface or Mississippi River water consumption.

Young et al. ( 1981) Case control study in Wisconsin, 1972 1977. Questionnaires sent to waterworks superintendents on chlorine content. Colon cancer showed significant ( p< 0.05) association with chlorine intake in all three dosage categories.

Cragle et al. ( 1985) Case control study using colon cancer cases from seven hospitals in North Carolina. Consumption of chlorinated water strongly associated with colon cancer, above age 60.

Young et al. ( 1987) Case control study of colon cancer cases in Wisconsin. Water consumption was determined by interview, and chloroform levels by historical records and measurement. No association found between trihalomethane exposure and colon cancer incidence. Table VI 1 ( cont.)

Reference Study Description Observation Draft  Do Not Cite or Quote February 20, 2003 VI  3 Morris et al. ( 1992) Meta analysis of nine case control studies and one cohort study analyzing cancer and consumption of chlorinated water or water containing high chloroform levels. Statistically significant relative risk of rectal cancer and bladder cancer in exposed groups. No colon cancer.

McGheehin et al. ( 1993) Population based case control study Association between bladder cancer risk and exposure to chlorinated water and trihalomethanes.

King and Marret ( 1996) Case control study conducted by Health Canada Increased risk of bladder cancer associated with total trihalomethane exposure.

Hildesheim et al. ( 1997) Population based case control study of colon and rectal cancer risk. Iowa, 1986 1989. Rectal cancer risk associated with duration of chlorinated water use. No association of colon cancer risk with duration of chlorinated water use or trihalomethane estimates.

Cantor et al. ( 1998) Population based case control study of bladder cancer risk. Iowa, 1986 1989. Positive findings for risk restricted to men and to current or former smokers. In men, smoking and exposure to chlorinated water enhanced the risk of bladder cancer.

Marcus et al. ( 1998) Ecologic study of association between TTHM in 71 North Carolina public water supplies and incidence of histologically confirmed female invasive breast cancer obtained from cancer registry data. TTHM levels not associated with breast cancer risk when adjusted for potential confounding factors. Data were consistent with TTHMs being unrelated or weakly related to breast cancer risk.

Table VI 2 Epidemiological Studies Investigating an Association Between Chlorinated Drinking Water and Adverse Pregnancy Outcomes or Altered Menstrual Function Table VI 2 ( cont.)

Draft  Do Not Cite or Quote February 20, 2003 VI  4 Reference Study Description Observation Aschengrau et al. 1989 Hospital based case control study of spontaneous abortion and multiple water quality parameters in Boston, MA area. After adjustment for potential confounders and chemical constituents, frequency of spontaneous abortion was increased for consumption of surface water when compared to use of mixed surface and ground water ( OR 2.2, 95% C. I. 1.3  3.6) The association between surfance water and increased risk of spontaneous abortion was not confirmed by a comparison of chlorinated vs. chloraminated surface water. Chloraminated water was used as a surrogate for low exposure to disinfection by products.

Aschengrau et al. 1993 Case control study of drinking water quality and occurrence of late adverse effects among women who delivered infants during August 1977  March 1980 in Massachusetts After adjustment for confounding, frequency of stillbirths was increased for women exposed to chlorinated surface water ( OR 2.6, 95% CI 0.9 7.5).

Nuckols et al. ( 1995) Cross sectional study in Colorado of populations drinking chlorinated and chloraminated water No statistically significant effects of exposure, although odds ratio was elevated for risk of low birth weight infants.

Bove et al. ( 1995) Cross sectional study in New Jersey Relationship between total trihalomethane levels and " small for gestational age."

Savitz et al. ( 1995) Population based case control study in North Carolina Statistically significant association of miscarriage with increasing concentration of TTHM and with the highest sextile of exposure( OR= 2.8, 95% C. I. 1.1, 2.7), but no relationship with ingested dose or water source. Small increase in risk of low birth rate.

Gallagher et al. ( 1998) Retrospective cohort study of relationship between THM exposure during third trimester of pregnancy and low birthweight, low term birth weight, and preterm delivery. Colorado birth certificate data matched to historical water data based on census block groups. 1990 1993. Possible association of trihalomethane concentration in tap water at maternal residence during third trimester and risk of term low birth weight deliveries. Little association with preterm delivery. Weak association with low birth weight. Table VI 2 ( cont.)

Reference Study Description Observation Draft  Do Not Cite or Quote February 20, 2003 VI  5 Waller et al. ( 1998) Prospective study of association between total and individual THM exposure and spontaneous abortion. Concurrent THM data obtained from public water supplies. Women who drank

5 glasses/ day of cold tap water containing

75 µ g/ L TTHMs had an adjusted odds ratio of 1.8 for spontaneous abortion. Of individual THMs, only consumption of

5 glasses of water containing

18 µ g/ L bromodichloromethane ( or a compound co occurring with bromodichloromethane) was associated with spontaneous abortion.

Klotz and Pyrch ( 1998; 1999) Case control study of association between drinking water contaminants ( including disinfection byproducts) and neural tube defects. Births with neural tube defects reported to New Jersey's Birth Defects Registry in 1993 and1994 were matched against control births chosen randomly from across the State. Elevated odds ratios, generally between 1.5 and 2.1, for the association of neural tube defects with total THMs ( TTHMs). The only statistically significant results were seen when the analysis was isolated to those subjects with the highest THM exposures ( greater than 40 ppb) and was limited to those subjects with neural tube defects in which there were no other malformations ( OR = 2.1, 95% CI = 1.1   4.0).

Dodds et al. ( 1999) Retrospective cohort study in Nova Scotia women with singleton births, 1988 1995. Little association between TTHM level and fetal weight or gestational agerelated outcomes. Elevated relative risk for stillbirths for exposure to > 100 µ g/ L TTHM levels during pregnancy. Little evidence for increased prevalence or dose response for congenital abnormalities with possible exception of chromosome aberrations for exposure > 100 µ g/ L.

Magnus et al. ( 1999) Ecologic study in Norway of chlorinated water consumption and birth defects observed in births during period 1993 1995. 1994 data on water quality and disinfection practice. Water color used as an indicator for natural organic matter content. Among 141,077 births, 1.8% had birth defects. Adjusted odds ratios ( high color, chlorination vs. low color, no chlorination) of 1.14 ( 0.99 1.31) for any malformation; 1.26 ( 0.61 2.62) for neural tube defects; and 1.9 ( 1.10 3.57) for urinary tract defects.

Yang et al. ( 2000) Study in Taiwan of association between chlorination of drinking water and low birth weight. Examination of 18,025 births showed no association between consumption of chlorinated drinking water and low birth weight. Table VI 2 ( cont.)

Reference Study Description Observation Draft  Do Not Cite or Quote February 20, 2003 VI  6 King et al. ( 2000) Population based retrospective cohort study in Nova Scotia, Canada to examine the relationship between TTHM or individual THMs and risk for stillbirth of fetuses greater than 500 grams. Study cohort assembled from a perinatal database and consisted of 49,756 singleton births that occurred between 1988 and 1995. Risk doubled for women exposed to a bromodichloromethane level

20 µ g/ L when compared to women consuming concentrations of less than 5 µ g/ L ( relative risk = 1.98, 95% confidence interval of 1.23  3.49). When categories of stillbirth ( unexplained deaths and asphyxia related deaths) were examined, relative risk estimates for asphyxia related deaths increased by 32% for each 10 µ g/ L increase in bromodichloromethane concentration.

Dodds and King ( 2001) Retrospective cohort study conducted using data from a population based perinatal database in Nova Scotia, Canada and routine water monitoring data. The cohort consisted of women who had a singleton birth in Nova Scotia between 1988 and 1995 and who lived in an area with a municipal water supply. Exposure to bromodichloromethane at concentrations of 20 µ g/ L and over was associated with increased risk of neural tube defects ( adjusted relative risk = 2.5; 95% confidence interval 1.2 to 5.1) and decreased risk of cardiovascular anomalies ( adjusted relative risk = 0.3; 95% confidence interval 0.2 to 0.7). No association observed for bromodichloromethane and cleft defects.

Waller et al. ( 2001) Reanalysis of total trihalomethane exposure data reported in Waller et al. ( 1998). The study authors reported no apparent advantage in using a closest site ( vs. utility wide) measurement approach for estimation of exposure to total trihalomethanes.

Windham et al. ( 2003) Prospective study of association between total and individual THM exposure and menstrual cycle function. Concurrent THM data obtained from public water supplies. Exposure to dibromochloromethane and sum of brominated trihalomethanes was associated with a reductions in length of the menstrual cycle and follicular phase of the menstrual cycle, suggesting possible effects on ovarian function. Concentrations of

20 µ g/ L for dibromochloromethane and

45 µ g/ L for total brominated trihalomethanes were associated with reductions in cycle and follicular phase lengths of approximately one day. No effect was noted on length of luteal phase or duration of menses. Draft  Do Not Cite or Quote February 20, 2003 VI  7 In their assessment of available data on the available data for reproductive and developmental effects of disinfection byproducts, Reif et al. ( 1996) stress that interpretation of epidemiologic findings for these contaminants are potentially complicated by unmeasured confounding variables and misclassification errors. Smoking, socioeconomic status, alcohol consumption, other environmental exposures, and reproductive history are examples of confounding variables that have the potential to bias estimates of risk in studies of disinfection byproducts if not measured. Misclassification errors can arise from failure to account for spatial and temporal variability in contaminant measurement s, migration of study participants, incorrect assumptions related to water source or use, and use of water treatment data as a surrogate for tap water concentrations. These factors may result in under or over classification of health risks associated with the consumption of disinfected water. Of greatest concern are variables or errors which might lead to underestimation of the true public health risks associated with exposure to tap water containing brominated trihalomethanes. The positive findings in studies of brominated trihalomethanes thus form a foundation for further studies, but should be interpreted cautiously.

1. Bromodichloromethane Kramer et al. ( 1992) conducted a population based case control analysis to determine if exposure to trihalomethanes in drinking water is associated with low birthweight, prematurity, or intrauterine growth retardation ( lower than the 5th percentile of weight for gestational age). A separate analysis was conducted for each endpoint, using five randomly selected controls for each affected newborn. Data were collected from Iowa birth certificates from January 1, 1989, to June 30, 1990; the study population was restricted to residents of small towns where all of the drinking water was derived from a single source ( surface water, shallow wells, or deep wells). Exposure data were based on a 1987 municipal water survey; birth certificate data from 1987 were not used because data on maternal smoking status first became available in 1989. The study authors adjusted for maternal age, number of previous children, marital status, education, adequacy of prenatal care, and maternal smoking. An association was observed between exposure to at least 10 µ g/ L bromodichloromethane and intrauterine growth retardation ( odds ratio = 1.7). However, the confidence interval in these cases included 1, indicating that the increases were not statistically significant.

Waller et al. ( 1998) conducted a prospective study in pregnant women to examine the association between trihalomethanes in drinking water and spontaneous abortion ( pregnancy loss at 20 or less completed weeks of gestation). The study participants were recruited from three facilities of a large managed health care organization which were located in regions of California that primarily received either mixed, surface, or groundwater. Recruitment occurred when the women scheduled their first prenatal exam after confirmation of pregnancy. A group of 5,342 subjects completed a telephone interview that obtained information on demographics, previous pregnancy history, employment status, consumption of tap and bottled water, substance use ( alcohol, tobacco, and caffeine), and other factors. At the time of enrollment in the study, each woman was at least 18 years of age, at 13 or less weeks of gestation, spoke English or Spanish, and could provide with certainty the date of her last menstrual period. Following adjustment for Draft  Do Not Cite or Quote February 20, 2003 VI  8 elective termination of pregnancy, ectopic or molar pregnancies, and multiple gestations, a total of 5,144 pregnancies remained for analysis.

Waller et al. ( 1998) quantified exposure to trihalomethanes by estimating the subject's daily tap water intake at 8 weeks gestation. Concentration of total trihalomethanes and any available data on individual trihalomethanes were obtained directly from the utility supplying drinking water to a subject's address or zip code. Total trihalomethane levels were calculated by averaging all measurements taken by the utility supplying a participant's home. Each participant was assigned a personal exposure classification ( high or low) to total trihalomethanes ( TTHM) and individual trihalomethanes ( THM) based on the following criteria. A high personal exposure to TTHM was defined as drinking 5 or more glasses of cold tap water per day and having a TTHM level of 75 µ g/ L or higher. Low personal exposure to TTHM was defined as either 1) drinking less than 5 glasses of cold tap water per day, 2) having a TTHM level of less than 75 µ g/ L, or 3) receiving water from a utility that provided 95% or greater groundwater. Personal exposures to the individual THMs ( bromoform, bromodichloromethane and dibromochloromethane) were defined in a similar manner, with a high personal exposure being defined as drinking 5 or more glasses of cold tap water per day with an individual brominated THM level of 16 µ g/ L or higher for bromoform, 18 µ g/ L or higher for bromodichloromethane, or 31 µ g/ L or higher for dibromochloromethane. Low personal exposures to the individual THMs were defined as either 1) drinking less than 5 glasses of cold tap water per day, 2) having an individual THM level below the cutoff, or 3) having a TTHM level less than 72 µ g/ L if individual THM levels were not reported.

The authors found a modest association between consumption of trihalomethanecontaining water and incidence of spontaneous abortion. Increased risk of spontaneous abortion was noted starting at approximately 75 µ g/ L. The adjusted odds ratio ( OR) for women who drank 5 or more glasses of cold tap water per day containing an average TTHM level of 75 µ g/ L or higher during their first trimester was 1.8 ( 95% confidence interval ( C. I.) 1.1, 3.0). An estimated 18.4% of the study participants were exposed at or above this level. Of the four individual THMs, only high bromodichloromethane exposure ( or exposure to another compound closely associated with bromodichloromethane) was associated with spontaneous abortion alone ( adjusted OR = 2.0, 95% C. I. 1.2, 3.5) and after adjustment for other THMs ( adjusted OR = 3.0, C. I. 1.4, 6.6). Waller et al. ( 1998) noted that there was no additive or other effect from showering or swimming. Therefore, no adjustment was required for these variables. Misclassification of exposure was identified as the primary limitation of this study. Concentration levels for most subjects were based on test results for a single day, and thus do not reflect potential variation in trihalomethane levels over time. In addition, the exposure to THMs from sources other than ingestion could not be fully characterized.

Because exposure misclassification appeared to be a limitation of the Waller et al. ( 1998) study, Waller et al. ( 2001) reported a reanalysis of exposure data from that study. The objective of the new analysis was to examine how use of alternative methods for estimation of exposure would affect associations between TTHM exposure and risk of spontaneous abortion. This reanalysis did not address dose response relationships between individual brominated Draft  Do Not Cite or Quote February 20, 2003 VI  9 trihalomethanes and occurrence of spontaneous abortion. Two exposure analyses were tested. The first method used the utility wide average concentration ( the metric used in Waller et al., 1998). The second method used THM measurements taken from the water system sampling site nearest the subject's home. For each method, the authors performed 1) an unweighted analysis; 2) an analysis weighted by a factor intended to reduce exposure misclassification; and 3) an analysis within a subset of the cohort that possibly had less exposure misclassification than the entire cohort. The weighted and subset analyses were performed in an effort to reduce exposure misclassification. The utility wide average method estimated the concentration of total trihalomethanes by averaging all distribution measurements taken by the subject's utility during the first trimester of pregnancy. In contrast to the method used in Waller et al. ( 1998) the time interval was not expanded in order to capture a measurement and thus reduce missing data in cases where no measurements were available in the first trimester. The closest site method took the average of all measurements taken during the first trimester of pregnancy at the water distribution site nearest to the subject's home. For the weighted analyses, the utility wide approach used the variance of the utility wide average as a proxy for accuracy. The closest site approach used TTHM measurements taken from the water system sampling site nearest the subject's home and adjusted for distance between the subject's home and the sampling site. For the subset approach, analyses were restricted to groups of women for whom the exposure assessment was likely to be more accurate. Subset analyses using the utility wide average TTHM concentration included women whose utility measurements were all within 20 µ g/ L of each other and women served by groundwater utilities. Subset analyses using the closest site average concentrations used women who lived within 0.5 miles of the utility sampling site and all women served by groundwater utilities. An ingestion metric was calculated using individual daily cold tap water intake at eight weeks gestation as determined in Waller et al. ( 1998). A categorical ingestion exposure metric was created using the first trimester THM concentration dichotomized at 75 µ g/ L and cold tap water ingestion dichotomized at 5 glasses per day. Ingestion was also estimated by multiplying the TTHM concentration by cold tap water consumption. A metric to capture exposure to trihalomethanes during showering was created by multiplying THM concentration by typical shower duration and the frequency of showering.

Use of the utility wide approach generally resulted in odds ratios equivalent to or slightly higher than the closest site approach. Odds ratios obtained using the utility wide average method for estimating TTHM ( but not the closest site method) became progressively stronger in the weighted and subset analyses. The study authors reported a positive, monotonic dose response relationship between spontaneous abortion rate and an exposure metric incorporating TTHM and personal ingestion. Rates of spontaneous abortion obtained using this approach ranged from 8.3% to 13.7% ( unweighted); 7.9% to 16.6 % ( variance weighted); and 6.6% to 16.6% ( lowvariance subset). The study authors noted that a major limitation of this reanalysis is the lack of a " gold standard" with which to compare the estimated TTHM ingestion of subjects in the study. In the absence of such a standard, it is not possible to determine whether the reanalysis actually reduced exposure misclassification. The conclusions reached by the study authors were 1) there was no advantage in using the closest site method over the utility wide method for exposure analysis; 2) use of variance based weighting factors and subset analyses is defensible and resulted in some increases of odds ratio, but resulting loss of sample size may limit the utility of these techniques; and 3) use of a variety of exposure assessment techniques may lessen the impact of Draft  Do Not Cite or Quote February 20, 2003 VI  10 bias resulting from utility specific factors such as inconsistencies in sampling density or unrecognized contamination problems.

The reanalysis conducted by Waller et al. ( 2001) identified evidence for differential misclassification in the prior analysis of a ground water predominate area (" Zone A") reported in Waller et al. ( 1998). The effect of this misclassification was to bias the original estimate of the relationship between TTHM ingestion and spontaneous abortion away from the null. Over 400 of the women in the study cohort resided in Zone A, an area within a large mixed source utility that received predominately groundwater. Zone A was not sampled for THMs during the study period. Because other areas within the utility frequently had high TTHM concentrations, use of a utility wide approach for estimating TTHM concentration probably resulted in an overestimation of exposure for Zone A residents. An investigation by the study authors revealed that although the spontaneous abortion rate of women in Zone A was low overall, women who drank at least 5 glasses of water per day had a spontaneous abortion rate of 14.6%. The reason for the high spontaneous abortion rate among women consuming large amounts of Zone A water was unclear, but was reported to be consistent with other epidemiological studies that found high rates of spontaneous abortion among women ingesting large amounts of unchlorinated water in Region 1 of the original study. Exclusion of Zone A residents or recoding them to a level determined by later testing within the zone decreased the adjusted OR for high exposure to TTHM ( TTHM  75 µ g/ L and intake  5 glasses per day) to 1.5 ( 95% C. I. 0.8, 2.8) as compared to the adjusted OR of 1.8 ( 95% C. I. 1.1, 3.0) identified in the original analysis ( Waller et al., 1998). The impact of this finding on the adjusted OR calculated for individual brominated THMs is currently unknown, but is expected to be addressed in a future publication by Waller et al.

King et al. ( 2000) conducted a population based retrospective cohort study to examine the relationship between TTHM or individual THMs and risk for stillbirth of fetuses greater than 500 grams. The study cohort was assembled from a perinatal database in Nova Scotia, Canada and consisted of 49,756 singleton births that occurred between 1988 and 1995. Exposure was assigned by relating the mother's residence at the time of delivery to the levels of total and individual THMs measured in public water supplies. Maternal age, parity, smoking during pregnancy, infant's sex, and neighborhood family income were evaluated as potential cofounders. Relative risks were adjusted for smoking and maternal age. Exposure to TTHMs, chloroform, and bromodichloromethane were associated with increased risk of stillbirth. Analysis of the results suggested that exposure to bromodichloromethane was a stronger predictor of risk than exposure to chloroform. Risk doubled for women exposed to a bromodichloromethane level of greater than or equal to 20 µ g/ L when compared to women consuming concentrations of less than 5 µ g/ L ( relative risk = 1.98, 95% C. I. 1.23, 3.49). When categories of stillbirth ( unexplained deaths and asphyxia related deaths) were examined, relative risk estimates for asphyxia related deaths increased by 32% for each 10 µ g/ L increase in concentration of bromodichloromethane. As noted by the authors, a potential limitation of this study was misclassification of exposure as a result of mobility within the study population ( estimated to affect less than 10% of study subjects). This study did not examine early fetal death ( e. g. spontaneous abortion) because the perinatal database employed in this investigation contained information only on fetuses that weighed 500 grams or more. Draft  Do Not Cite or Quote February 20, 2003 VI  11 Dodds and King ( 2001) conducted a retrospective cohort study of singleton births among 49,842 residents of Nova Scotia, Canada between 1988 and 1995 to assess the relationship between exposure to THMs and birth defects. Of the brominated THMs, only bromodichloromethane was examined. Information on exposure concentrations consisted of routine water monitoring data obtained from the Nova Scotia Department of the Environment and reflected samples collected from within the water distribution system. The birth defects examined had previously been reported in other epidemiological studies, and included neural tube defects, cardiovascular defects, cleft defects, and chromosomal abnormalities. The perinatal information used in the study was obtained from the Nova Scotia Atlee perinatal database. This database contains information abstracted from medical records and includes infant diagnoses among stillborn and live born infants up to the time of discharge from the hospital after birth. In addition, information on prenatally diagnosed congential anomalies was obtained from pregnancy terminations. Inclusion of this data was deemed important because, in Nova Scotia, approximately 80% of neural tube defects are detected antenatally and the pregnancy is electively terminated. Exposure windows were selected to target the period before or during gestation when exposure to a potential developmental toxicant or mutagen might have the most profound effect on a particular developmental or genotoxic endpoint. The selected windows were as follows: average bromoform concentrations from one month and one month after were used for analysis of neural tube defects; concentrations during the first two months of pregnancy were used for analysis of cardiac defects and cleft defects; and the average concentrations three months before pregnancy were used for the analysis of chromosomal abnormalities. Estimates of relative risks and 95% confidence intervals were obtained from Poisson regression models. Maternal age, parity, maternal smoking, and neighborhood family income were assessed as potential confounders. The categories used for bromodichloromethane concentration were < 5 µ g/ L; 5  9 µ g/ L; 10  19 µ g/ L; and  20 µ g/ L.

Exposure to bromodichloromethane at concentrations  20 µ g/ L was associated with increased risk of neural tube defects ( adjusted relative risk 2.5; 95% C. I. 1.2, 5.1) when adjusted for maternal age and income level. However, there was no evidence of a dose response trend with increasing concentration of bromodichloromethane. In addition, the study authors noted that this point estimate was " fairly unstable" as a result of the low number of cases ( n= 10) in the  20 µ g/ L exposure category. For cardiac defects, a significant reduction in risk was associated with exposure to concentrations of  20 µ g/ L ( relative risk 0.3; 95% C. I. 0.2, 0.7) and there was a trend of decreasing risk with increasing exposure. The study authors considered it unlikely that exposure above  20 µ g/ L was actually protective. They suggested that this may be a chance finding or a reflection of a negative association of bromodichloromethane with other disinfection by products in this region which may increase cardiac risks. There was no apparent trend or significant association for exposure to bromodichloromethane and occurrence of cleft defects or chromosomal aberrations.

2. Dibromochloromethane Windham et al. ( 2003) examined menstrual cycle characteristics in relation to the presence of brominated trihalomethanes in tap water in a prospective study of women living in Northern California. Data were also reported for chloroform and TTHM. The relationships examined Draft  Do Not Cite or Quote February 20, 2003 VI  12 included: 1) cycle characteristics and concentration of individual THMs, TTHM, and total brominated THMs in tap water; 2) cycle characteristics and estimated water consumption ( TTHM); and 3) cycle characteristics and duration of showering ( TTHM). The target population was married women of reproductive age ( 18 39 years old) who were members of the Kaiser Permanente Medical Care Program. Participants in the study were enlisted between May 1990 and June 1991. Participants were selected from among nearly 6500 women using a short screening interview to identify women who were more likely to become pregnant ( i. e., those who reported a menstrual period within the last six weeks, were not surgically sterilized, did not use birth control pills or IUDs, and were non contracepting for less than 3 months). Out of 1092 eligible women, a total of 403 women collected first morning urine samples daily for 2 9 menstrual cycles ( average 5.6 cycles) for measurement of steroid metabolites. These measurements were used to estimate menstrual parameters such as cycle and phase length. Cycle length was calculated from the first day of menses to the day before onset of the next menses. When the available data permitted, the cycle was divided into the follicular phase ( first day of menses through estimated day of ovulation) and the subsequent luteal phase. Between 1424 and 1714 cycles were available for evaluation of each parameter. Information on water consumption ( as unheated tap water or drinks made from unheated tap water, drinks made from heated tap water, and bottled water) and other variables ( age, race, education, employment, income, pregnancy history, exercise type and frequency, smoking, alcohol and caffeine consumption) was collected in a baseline telephone interview prior to urine collection. Information on the number of showers taken at home per week and their duration was also collected. Showering was examined as minutes per week and by combining the duration and cycle specific THM level to create combinations of high and low exposure. The participants filled out a daily diary during the urine collection phase and recorded vaginal bleeding as number of pads or tampons. Exposure to THMs was estimated from quarterly utility monitoring data and information on drinking water and other tap water use collected during the baseline interview. A 90 day exposure time period was assigned for each cycle because THM monitoring was conducted by the utilities on a quarterly ( i. e., about 90 days) basis. A period of 60 days before and 30 days after each cycle start date was selected for the 90 day window. Cycle specific exposures to TTHM and individual THMs were calculated by averaging all THM measurements taken by a participant's utility at various points in the distribution system ( i. e., the " utility wide average" described by Waller et al., 1998, 2001) during the that 90 day period. Because the brominated THMs were highly correlated and thus difficult to examine independently, the study authors also examined the sum of the levels of the three brominated compounds. Exposure levels for the brominated THMs were examined as categorical variables. For TTHM, ingestion metrics were calculated for unheated tap water and the sum of heated and unheated tap water. Statistical analyses were conducted using the menstrual cycle as the unit of observation. Menstrual parameters were analyzed as continuous or categorical variables in relation to categorical exposure indices and the methods used accounted for repeated measures. Numerous covariates reflecting demographic, reproductive history, and lifestyle factors were examined in relation to categorical trihalomethane levels and ingestion to identify potential confounders. Age, pregnancy history, body mass index, caffeine consumption, and alcohol consumption, as well as race and smoking, were included as variables in all adjusted models. Draft  Do Not Cite or Quote February 20, 2003 VI  13 Increasing levels of individual brominated compounds or total brominated THMs were associated with significantly shorter cycles when examined by quartile ( Table VII 3). Similar decrements were observed in follicular, but not luteal, phase length. Dose response patterns were evident for both individual and total brominated THMs. The strongest association for an individual compound was observed for dibromochloromethane, with adjusted decrements of 1.2 Draft  Do Not Cite or Quote February 20, 2003 VI  14 Table VI 3 Means and Adjusted Differences in Menstrual Cycle and Follicular Phase Length by Quartile of Individual and Summed Brominated Trihalomethanes Compound Quartile of Exposurea 1b 2 3 4 Mean in days ( SE) Adjusted Differencec ( 95% CI) Adjusted Difference ( 95% CI)

Cycle Length Bromodichloromethane 29.8 ( 0.30)  0.59 ( 1.2,  0.02)  0.74 ( 1.5,  0.02)

Dibromochloromethane 30.0 ( 0.33)  0.69 ( 1.4,  0.02)  1.2 ( 2.0,  0.38)

Bromoform 29.7 ( 0.26)  0.42 ( 0.96, 0.13)  0.79 ( 1.4,  0.14)

Sum of Brominated Compounds 30.0 ( 0.34)  0.72 ( 1.4,  0.04)  1.2 ( 2.0,  0.40)

Follicular Phase Bromodichloromethane 17.0 ( 0.31)  0.54 ( 1.1, 0.01)  0.80 ( 1.5,  0.08)

Dibromochloromethane 17.1 ( 0.34)  0.62 ( 1.3, 0.05)  1.1 ( 1.9,  0.25)

Bromoform 16.9 ( 0.27)  0.30 ( 0.83, 0.23)  0.78 ( 1.4,  0.14)

Sum of Brominated Compounds 17.2 ( 0.35)  0.66 ( 1.3, 0.02)  1.1 ( 1.9,  0.29)

Source: Windham et al. ( 2003) a Top quartiles for bromodichloromethane, dibromochloromethane, bromoform, and the summed brominated compounds are

16,

20,

12, and

45 µ g/ L, respectively. b Reference group; the mean provided is unadjusted with standard error ( SE) c Adjusted for age, race, body mass index, income, pregnancy history, caffeine and alcohol consumption, and smoking.

days ( 95% C. I.  2.0,  0.38) for mean cycle length and 1.1 days ( 95% C. I.  1.9,  0.25) for mean follicular phase length at the highest quartile (  20 µ g/ L). In comparison, a clear association with reduced cycle length was not observed for chloroform ( difference  0.3 days; 95% C. I.  1.0, 0.40), even at the highest quartile (  17 µ g/ L). Menses length was slightly increased at the highest quartile for bromodichloromethane exposure. For categorical variables, the odds of having a long cycle ( Adjusted Odds Ratio, AOR 0.55; 95% C. I. 0.28, 1.08) or long follicular phase ( AOR 0.26; 95% C. I. 0.12, 0.59) were significantly reduced at the highest quartile for summed brominated THM concentration (  45 µ g/ L). These data suggest that brominated THMs or other disinfection by products that co occur with brominated THMs may affect ovarian function. Draft  Do Not Cite or Quote February 20, 2003 VI  15 Windham et al. ( 2003) also examined relationships between TTHM ( brominated compounds plus chloroform) exposure and menstrual parameters. A monotonic decrease in mean cycle length was observed with increasing TTHM exposure category. At concentrations greater than 60 µ g/ L, the adjusted decrement was 1.1 day ( 95% C. I.  1.8,  0.40) when compared to concentrations less than 40 µ g/ L. The decrease in follicular phase length was similar ( 0.94 day; 95% C. I.  1.6,  0.24). Cycles with TTHM concentrations above the current MCL of 80 µ g/ L were appeared to be shorter by about one day ( 0.99 days; 95% C. I.  2.2, 0.17). A unit decrement in mean cycle and follicular phase length of 0.18 days per 10 µ g/ L increase in total trihalomethane concentration ( 95% C. I.  0.29,  0.07) was determined when the cycle specific TTHM level was examined as a continuous variable. When ingestion patterns were examined, mean cycle and phase lengths showed little variation in relation to consumption of unheated tap water at home. In contrast, increased consumption of heated tap water was associated with significantly decreased cycle and follicular phase lengths. The observed decrements were greater than one day with daily consumption of three or more drinks made from heated tap water. These decrements were reduced by adjustment, particularly when caffeine was included in the model; the adjusted decrement in cycle length was 0.68 days ( 95% C. I.  2.1, 0.72). A non monotonic relationship was observed for mean cycle length and an ingestion metric combining TTHM concentration and consumption of unheated tap water, with the highest category (> 60 µ g/ L) showing a decrement of 0.4 days and the third category (> 40 60 µ g/ L) showing a decrement of one day. Use of an ingestion metric based on total home tap water consumption ( i. e., heated and unheated tap water) revealed a more consistent pattern of reduced cycle length, with adjusted decrements of greater than one day for cycle ( 1.1 day; 95% C. I.  2.2,  0.06) and follicular phase ( 1.1 day; 95% C. I.  2.2, 0.03) lengths. Examination of time spent showering did not reveal additional risks with longer showers. The unadjusted mean cycle length varied little by time spent showering. Following adjustment, there was a tendency toward decreased length with any category of showering above 35 minutes/ week. This relationship was stronger for follicular phase duration than for cycle length. For example, the adjusted mean decrements at the longest duration (  105 minutes) were  0.68 days ( 95% C. I.  2.0, 0.68) for cycle length and  1.2 days ( 95% C. I.  2.6, 0.26). However, the confidence intervals were wide for all duration categories and a clear dose response pattern ( i. e., shorter lengths at higher durations) was not evident.

The study authors noted several strengths and potential limitations of this study. Strengths include the use of a prospective study design, use of biologic measures to determine menstrual parameters, and consideration of many potential confounders. Potential limitations include exposure misclassification, use of a study sample that is not representative of the general population, lack of information on other sources of exposure such as washing, cooking and cleaning, as well as exposures outside the home. There are two observations in this study that might suggest involvement of compounds other than THMs in the reduction of cycle length. First, the more consistent association of decreased cycle length reported for heated compared to unheated tap water is unexpected if THMs alone are the causative agent. This is because THMs volatilize from heated water and exposure to these compounds should therefore be lower for heated tap water, unless the volatilized compound is inhaled. Second, examination of time spent showering did not reveal additional risks with longer showers. This is also counter to the expected trend, as elevated blood levels of THMs have been documented after showering ( Backer et al., 2000; Lynberg et al., 2001) as a result of dermal and inhalation exposure. However, Draft  Do Not Cite or Quote February 20, 2003 VI  16 information on shower duration was collected by interview and the reported lengths may not have accurately reflected actual shower duration.

Because the study by Windham et al. ( 2003) is the first to examine changes in menstrual cycle function in relation to tap water exposure, there are no supporting data on the association of disinfection by products other than the THMs with changes in menstrual cycle function. Although this study suggests that disinfection by products may have effects on ovarian function, no conclusions can be drawn regarding the identity of the compounds responsible for these effects. The relationships reported in this study underscore the need for additional research on the effects of drinking water disinfection by products on menstrual cycle function.

3. Bromoform Epidemiological studies identifying adverse health effects specifically associated with exposure to bromoform were not identified.

C. High Risk Populations High risk ( or susceptible) populations are those which experience more adverse effects at comparable levels of exposure, which experience adverse effects at lower exposure levels than the general population, or which experience a higher than average exposure because they live or work in settings with elevated environmental concentrations of the chemical of interest. The enhanced response of these susceptible subpopulations may result from intrinsic or extrinsic factors. Factors that may be important include, but are not limited to: impaired function of detoxification, excretory, or compensatory processes that protect against or reduce toxicity; differences in physiological protective mechanisms; genetic differences in metabolism; developmental stage; health status; gender; or age of the individual. For brominated trihalomethanes, high risk populations may potentially include those with elevated levels of CYP2E1 ( via exposure to inducing substances or because of altered physiological or health states) or elevated levels of glutathione S transferase theta. These factors are discussed in greater detail in Section VII. D. 3 of this document.

A growing body of scientific evidence indicates that children may suffer disproportionately from some environmental health risks. These risks may arise because the neurological, immunological, and digestive systems of children are still developing ( U. S. EPA, ( 1998a). In addition, children may incur greater exposure because they eat more food, consume more fluids, and breathe more air in proportion to their body weight when compared to adults ( U. S. EPA, 1998a). Factors contributing to potentially greater risk in children are discussed in Section VII. D. 2 of this document. Draft  Do Not Cite or Quote February 20, 2003 VI  17 D. Summary Limited human health data are available for the brominated trihalomethanes. In the past, bromoform was used as a sedative for children with whooping cough. Doses of 50 to 100 mg/ kgday usually produced sedation without apparent adverse effects. Some rare instances of death or near death were reported, although these cases were generally attributed to accidental overdoses. No human toxicological data were available for bromodichloromethane or dibromochloromethane.

Numerous epidemiological studies have examined the association between water chlorination and increased cancer mortality rates. None of these studies has examined the association between cancer and exposure to any individual brominated trihalomethane. Recent studies have examined the association of chlorinated water use with various pregnancy outcomes, including low birth weight, premature birth, intrauterine growth retardation, spontaneous abortion, stillbirth , and birth defects. An association has been reported for exposure to bromodichloromethane ( or a closely associated compound) and a moderately increased risk of spontaneous abortion during the first trimester. A confirmation of this finding is pending reanalysis of the original data to correct a differential misclassification error identified in a subsequent analysis of the study data. An association has also been reported for exposure to bromodichloromethane ( or a closely associated compound) 1) and stillbirth of fetuses weighing more than 500 g and 2) increased risk of neural tube defects in women exposed to  20 µ g/ L of bromodichloromethane prior to conception through the first month of pregnancy. An association has been reported for total brominated trihalomethanes and reduced menstrual cycle and follicular phase length in women of child bearing age. Among the individual brominated trihalomethanes, dibromochloromethane displayed the strongest association with altered menstrual function. It is not possible to directly conclude from these studies that bromodichloromethane and dibromochloromethane are developmental toxicants in humans, because chlorinated water contains many disinfection by products. Nevertheless, these studies raise significant concern for possible human health effects. The methodology used to estimate exposure to brominated trihalomethanes in tap water has been examined with the goal of refining estimates of intake of these compounds in epidemiological studies.

For the brominated trihalomethanes, populations at high risk may potentially include those with elevated levels of CYP2E1 ( via exposure to inducing substances or because of altered physiological or health states) or elevated levels of glutathione S transferase theta. In addition, users of hot tubs and swimming pools may experience additional exposure to brominated trihalomethanes. Draft  Do Not Cite or Quote February 20, 2003 VII  1 VII. MECHANISM OF TOXICITY A. Role of Metabolism The toxicity of the brominated trihalomethanes is related to their metabolism. This conclusion is based largely on the observation that liver and kidney, the chief target tissues for these compounds, are also the primary sites of their metabolism. In addition, treatments which increase or decrease metabolism also tend to increase or decrease trihalomethane induced toxicity in parallel. Pankow et al. ( 1997), for example, examined the relationship between metabolism of dibromochloromethane and hepatotoxicity. Serum leucine aminopeptidase ( LAP) activity ( an indicator of hepatotoxicity) increased in a dose dependant fashion with any treatment that increased the metabolism of dibromochloromethane ( e. g. pretreatment with isoniazid or mxylene

B. Biochemical Basis of Toxicity The precise biochemical mechanisms which link brominated trihalomethane metabolism to toxicity are not certain, but many researchers have proposed that toxicity results from the production of reactive intermediates. These reactive intermediates are believed to form covalent adducts with various cellular molecules and to impair the function of those molecules resulting cell injury. Reactive intermediates may arise from the oxidative ( dihalocarbonyls) or the reductive ( free radicals) pathways of metabolism as discussed in Section III. C. Support for this mode of action has been obtained from in vitro studies of bromodichloromethane. Under both aerobic and anoxic conditions, bromodichloromethane is metabolized to intermediates that covalently bind to rat microsome proteins and lipids. Direct evidence showing a relationship between the levels of covalent binding intermediates generated by the oxidative or reductive pathways and the extent of toxicity is not currently available for brominated trihalomethanes.

Free radical generation by the reductive pathway for brominated trihalomethane metabolism may result in lipid peroxidation. Although evidence demonstrating that lipid peroxidation actually accounts for the observed cellular toxicity associated with brominated trihalomethanes is lacking, at least one study has established that lipid peroxidation does occur in conjunction with brominated trihalomethane metabolism. De Groot and Noll ( 1989) reported that all three brominated trihalomethanes induced lipid peroxidation in rat liver microsomes in vitro, and that this was maximal at low oxygen levels ( between 1 and 10 mm Hg of O 2). The authors interpreted these data to support the concept that lipid peroxidation is caused by free radical metabolites generated by the reductive metabolism of trihalomethanes.

Glutathione has been implicated in both defense against toxicity induced by brominated trihalomethanes and in generation of mutagenic metabolites. Gao et al. ( 1996) examined the effect of glutathione on the toxicity of bromodichloromethane in vivo and in vitro. Depletion of glutathione by pretreatment of male F344 rats with the glutathione synthesis inhibitor buthionine sulfoximine increased the hepatotoxicity of a single gavage dose of 400 mg/ kg bromodichloromethane administered in 10% Emulphor ® . Biochemical indicators of hepatotoxicity ( e. g. AST, ALT, LDH) were increased approximately 11 fold and the severity of Draft  Do Not Cite or Quote February 20, 2003 VII  2 morphological changes ( centrilobular vacuolar degeneration and hepatocellular necrosis) was greater in the glutathione depleted animals. Serum and urinary markers of renal damage were also significantly increased by glutathione depletion. Renal tubule necrosis was observed only in the glutathione depleted group. Overall, glutathione depletion appeared to enhance hepatotoxicity more than nephrotoxicity, an effect that was attributed to organ specific differences in bromodichloromethane metabolism. The addition of glutathione to reaction mixtures of rat hepatic or renal microsomal fraction and radiolabeled bromodichloromethane resulted in 92% and 20% reductions in protein binding to bromodichloromethane, respectively. The difference in response to glutathione addition was interpreted as evidence for existence of different metabolic pathways in liver and kidney. Bromodichloromethane binding to lipid in liver microsomes under anaerobic conditions was decreased in the presence of glutathione, suggesting that glutathione can react with the dihalomethyl radical.

In contrast to the apparent role protective role of glutathione described above, studies in strains of S. typhimurium engineered to express rat theta class glutathione S transferase suggest that conjugation with glutathione leads to formation of mutagenic metabolites ( Pegram et al., 1997; DeMarini et al., 1997). These studies are described in greater detail in Section V. F. 1. Proposed pathways for generation of the mutagenic species are outlined in Figure V 2, also located in Section V. F. 1. Briefly, similar mutational specificity, site specificity, and mutation spectra for the three brominated trihalomethanes support the conclusion that they are activated by one or more common pathways. In contrast, the data do not support a glutathione S transferase mediated pathway for the structurally related trihalomethane chloroform. This finding suggests that chloroform and the brominated trihalomethanes may in some instances be metabolized by different pathways.

C. Mode of Action of Carcinogenesis Administration of individual brominated trihalomethanes has been associated with formation of liver tumors ( bromodichloromethane, dibromochloromethane), kidney tumors ( bromodichloromethane), and tumors of the large intestine ( bromodichloromethane, bromoform) in some experimental animals. The mode of action by which brominated trihalomethanes induce tumors in laboratory animals is not known. However, two general modes of action have been proposed: 1) formation of DNA adducts resulting from interaction with one or more classes of reactive metabolites and 2) production of cytotoxicity coupled with regenerative hyperplasia.

The production of reactive metabolites from trihalomethanes is well established. Classes of reactive metabolites produced include dihalocarbonyls produced by oxidative metabolism and and dihalomethyl radicals produced by reductive metabolism. Additional evidence suggests that reactive species can be also formed via glutathione conjugation ( DeMarini et al., 1997; Pegram et al., 1997). Detection of adduct formation and consistent evidence of DNA reactivity in standard assays are two lines of experimental evidence that would strongly support the adduct formation hypothesis. At present there are no in vivo data available on DNA adducts resulting from metabolism of brominated trihalomethanes. DNA reactivity can be inferred from test results of mutagenic and genotoxic potential. As noted previously ( U. S. EPA, 1994b), synthesis of the Draft  Do Not Cite or Quote February 20, 2003 VII  3 overall weight of evidence for genotoxicity of individual brominated trihalomethanes is complicated by the use of a variety of testing protocols, different strains of test organisms, different activating systems, different dose levels, different exposure methods ( gas versus liquid), and in some cases, problems due to evaporation of the test chemical. Overall, a majority of studies yielded more positive results for bromoform and bromodichloromethane, and evidence of mutagenicity is considered adequate for these chemicals. Study results for the mutagenicity of dibromochloromethane are mixed, and the overall evidence for mutagenicity of this chemical is judged to be inconclusive ( U. S. EPA, 1994b).

Alternatively, the induction of tumors by individual brominated trihalomethanes could involve an epigenetic mode of action. Induction of tumors in animal studies has been noted to occur primarily at sites where cytotoxicity was observed ( i. e., liver and kidney), and there appears to be a correlation between hepatotoxicity and liver tumorigenicity of brominated trihalomethanes in mice ( bromodichloromethane > dibromochloromethane > bromoform) ( U. S. EPA, 1994b). This raises the possibility that regenerative hyperplasia caused by the cytotoxic effects of brominated trihalomethanes may contribute to the tumorigenic potential of these chemicals. A brief review of studies that have evaluated regenerative hyperplasia following exposure to brominated trihalomethanes is provided below.

A number of studies have measured cell proliferation in liver and/ or kidney following exposure to brominated trihalomethanes. Miyagawa et al. ( 1995) observed evidence for the induction of hepatocyte proliferation in male B6C3F 1 mice following a single oral gavage dose of dibromochloromethane in corn oil at the maximum tolerated dose ( MTD) or at one half the MTD ( 200 or 400 mg/ kg). Proliferation was assessed by incorporation of [ 3H] thymidine using the in vivo in vitro replicative DNA synthesis assay at 24, 39, and 48 hours postexposure.

Potter et al. ( 1996) investigated cell proliferation in the kidney of male F344 rats. Test animals received 0.75 or 1.5 mmol/ kg of bromodichloromethane in 4% Emulphor ® by gavage for 1, 3, or 7 days. The administered doses corresponded to 123 or 246 mg/ kg day for bromodichloromethane, 156 or 312 mg/ kg day for dibromochloromethane, and 190 or 379 mg/ kg day for bromoform. Cell proliferation in the kidney was assessed in vivo by [ 3H] thymidine incorporation. No statistically significant effect of bromodichloromethane on tubular cell proliferation was observed following exposures of up to 7 days, although high labeling levels were observed in 3 of 4 rats at the 246 mg/ kg day dose of bromodichloromethane.

NTP ( 1998) evaluated cell proliferation in the kidney and liver of Sprague Dawley rats as part of a short term reproductive and developmental toxicity screen of bromodichloromethane. The compound was administered in drinking water for 35 days. Groups of male and female rats were exposed to drinking water concentrations of 0, 100, 700 and 1300 ppm bromodichloromethane using the study design described in Table V 6 ( Section V. D. 1). BrdU labeling index ( LI) was unchanged in the livers and kidneys of Group B males at doses up to 69 mg/ kg day. A small but statistically significant increase in the LI was noted in the livers and kidneys of Group C females in the 1300 ppm dose group ( 126 mg/ kg day). The study authors noted that the result in females was probably biologically insignificant. Draft  Do Not Cite or Quote February 20, 2003 VII  4 Melnick et al. ( 1998) exposed female B6C3F 1 mice ( 10/ dose) to bromodichloromethane, dibromochloromethane, or bromoform in corn oil via gavage for 3 weeks ( 5 days/ week). BrdU was administered to the animals during the last 6 days of the study, and hepatocyte labeling index ( LI) analysis was conducted. Time adjusted doses of 107, 336, and 357 mg/ kg of bromodichloromethane, dibromochloromethane, and bromoform, respectively, resulted in significantly elevated hepatocyte proliferation as measured by the LI. The authors compared the dose response for liver toxicity ( including hepatic enzyme activity and LI data) and tumorigenicity ( using data from previously published NTP bioassays) for the brominated trihalomethanes using the Hill equation model. This analysis indicated that the shape of the dose response as well as the Hill exponents were different for liver toxicity and tumorigenicity. The authors concluded that these data do not support a causal relationship between liver toxicity/ reparative hyperplasia and tumor development.

Torti et al. ( 2001) conducted 1 week and 3 week inhalation exposure studies of bromodichloromethane in wild type and transgenic mice. Bromodichloromethane toxicity was transient. Regenerative lesions and increased labeling index were evident in the kidney cortex of mice exposed to concentrations of 10 ppm and above for one week. After three weeks of bromodichloromethane exposure, damaged areas of kidney cortex were entirely regenerated ( residual scarring was present) and labeling index measurements had returned to near baseline levels. The study authors noted that these results are in contrast to those observed in similar experiments performed with chloroform, where treatment of F344 rats and B6C3F 1 mice resulted in continued cytotoxicity and elevated cell turnover for up to 90 days ( Larson et al., 1996; Templin et al., 1996). The mechanistic basis for these different responses to structurally similar compound is unclear, but may reflect an induced change in metabolism or emergence of a resistant cell population in animals treated with bromodichloromethane.

George et al. ( 2002) reported that exposure of male F344/ N rats to bromodichloromethane in drinking water for two years at a level that significantly enhanced the prevalence and multiplicity of hepatocellular adenomas and carcinomas had no effect on hepatocellular proliferation. In the same study, the prevalence of renal tubular hyperplasia, but not tumor incidence, was significantly increased at the high dose.

Based on an extensive evaluation of carcinogenicity data, cytotoxicity coupled with regenerative hyperplasia is considered the primary mode of action for tumor formation following exposure to high concentrations of chloroform, a structurally related trihalomethane which has low genotoxic potential ( U. S. EPA, 2000d). However, two lines of evidence suggest that chloroform is not a prototypical trihalomethane. First, the weight of evidence for at least two of the brominated trihalomethanes indicates that they are genotoxic. This contrasts with the negative weight of evidence evaluation for chloroform. Second, there is evidence that the brominated trihalomethanes are readily bioactivated to mutagenic products via a glutathione S transferase mediated pathway, while chloroform is bioactivated only at very high concentrations. Therefore, a common mode of action for carcinogenicity of chloroform and brominated trihalomethanes cannot be assumed on the basis of current experimental evidence. Data to support a nonlinear primary mode of action for tumor development in liver, kidney, and large intestine are currently Draft  Do Not Cite or Quote February 20, 2003 VII  5 lacking for the brominated trihalomethanes. In the absence of such information, combined with a positive weight of evidence evaluation for genotoxicity, the mode of action for tumor development is assumed to be a linear process.

D. Interactions and Susceptibilities 1. Potential Interactions The toxicity of the brominated trihalomethanes appears to result from cytochrome P450 mediated metabolism to reactive metabolites ( U. S. EPA, 1994b). Therefore, agents which increase or decrease the activity of enzymes responsible for metabolism of brominated trihalomethanes may modify toxicity. Pankow et al. ( 1997) observed that pretreatment with isoniazid or m xylene ( inducers of CYP2E1 and CYP2B1/ CYP2B2, respectively) increased the hepatotoxicity of dibromochloromethane in male rats, as measured by elevated serum leucine aminopeptidase activity. Hewitt et al. ( 1983) observed that pretreatment with acetone, a CYP2E1 inducer, potentiated the acute toxicity of bromodichloromethane and dibromochloromethane in male rats. Thornton Manning et al. ( 1993) also found that pretreatment with acetone potentiated the acute hepatotoxicity of bromodichloromethane in male rats. Conversely, the cytochrome P450 inhibitor 1 aminobenzotriazole prevented bromodichloromethane induced hepatotoxicity in rats ( Thornton Manning et al. 1993). Current findings regarding the existence of glutathionemediated pathways for brominated trihalomethane metabolism ( see sectionV. E. 1) suggest that treatments or agents which alter glutathione S transferase activity may potentially modify the toxicity of brominated trihalomethanes.

The severity of brominated trihalomethane toxicity is potentially affected by the vehicle of administration. Vehicle effects are well documented in the toxicity of chloroform ( e. g., Bull et al. 1986; Jorgenson et al. 1985) and there is some evidence that similar effects occur with brominated trihalomethanes. In a study of vehicle effects on the acute toxicity of bromodichloromethane, a high dose ( 400 mg/ kg) of the chemical was more hepato and nephrotoxic when given in corn oil compared to aqueous administration, but this difference was not evident at a lower dose ( 200 mg/ kg) ( Lilly et al. 1994). Draft  Do Not Cite or Quote February 20, 2003 VII  6 2. Greater Childhood Susceptibility A growing body of scientific evidence indicates that children may suffer disproportionately from some environmental health risks. These risks may arise because the neurological, immunological, and digestive systems of children are still developing ( U. S. EPA, ( 1998a). In addition, children may incur greater exposure because they eat more food, consume more fluids, and breathe more air in proportion to their body weight when compared to adults ( U. S. EPA, 1998a).

U. S. EPA ( 1998a) recently identified three key questions to consider when evaluating health risks in children from exposure to drinking water disinfection byproducts ( DBP) such as the brominated trihalomethanes:

° Is there information which shows that the disinfectant or DBP causes effects in the developing fetus or impairs ability to conceive and bear children? If it causes this type of problem will it occur at a lower dose than that which will cause other types of effects?

° If the disinfectant or DBP causes cancer, are children more likely to be affected by it than are adults?

° If the disinfectant or DBP causes some noncancer toxic effect, are children more likely to be affected by it than are adults?

The data available for evaluation of these issues as they relate to brominated trihalomethanes are addressed below.

a. Effects on the fetus and ability to conceive and bear children General Results from Animal Studies Studies on the reproductive and developmental effects of brominated trihalomethanes are summarized in Section V. E. At the present time, the available data suggest that dibromochloromethane or bromoform cause effects in the developing fetus only at doses which also produce signs of maternal toxicity. Furthermore, there is no evidence that indicates that either of these chemicals impairs ability to conceive and bear children. Studies of these chemicals in animals indicate that reproductive and developmental effects would likely occur only at doses higher than those observed to cause liver and renal effects.

Bromodichloromethane has the most extensive database for developmental and reproductive effect among the brominated trihalomethanes. Study results for the reproductive and developmental effects of bromodichloromethane are mixed. No reproductive or developmental effects were observed at doses up to approximately 116 mg/ kg day in females or 68 mg/ kg day in males in studies conducted in Sprague Dawley rats ( NTP, 1998). Adverse Draft  Do Not Cite or Quote February 20, 2003 VII  7 reproductive or developmental effect were not observed in rabbits exposed to doses as high as 55 or 76 mg/ kg day in drinking water on gestation days 6 to 29 ( CCC, 2000c, d). Increased incidences of sternebral aberrations ( Ruddick et. al., 1983) and decreased ossification sites in the forelimb and hindlimb ( CCC, 2000b) have been observed in Sprague Dawley rats administered bromodichloromethane in corn oil and drinking water, respectively, at doses which induced maternal toxicity.

Reproductive effects of bromodichloromethane have been noted in rodent assays. Klinefelter et al. ( 1995) observed effects on sperm motility in rats administered 39 mg/ kg day in drinking water for 52 weeks, but these effects were not accompanied by histopathological changes in male reproductive tissues. Narotsky et al. ( 1997) observed a significantly increased incidence of full litter resorption ( FLR) in F344 rats treated with 75 mg/ kg day bromodichloromethane by aqueous gavage throughout the period of organogenesis. This effect was described as an all or nothing phenomenon, in that the litter was either fully resorbed or appeared normal at parturition. This pattern was interpreted by the study authors as evidence for a maternally mediated mechanism, rather than a direct effect of bromodichloromethane on the developing embryo. Bielmeier et al. ( 2001) observed increased incidence of FLR in F344 rats treated with 75 or 100 mg/ kg day BDCM by aqueous gavage on one or more days during the interval from gestation day 6 to 10. This response was strain specific and FLR was not observed in Sprague Dawley rats treated with up to 100 mg/ kg day on gestation days 6 to10.