Lilly et al. ( 1994) examined the effect of vehicle on the toxicity of bromodichloromethane Male F344 rats ( number unknown) were administered a single dose of 0, 200, or 400 mg/ kg bromodichloromethane by gavage in corn oil or in an aqueous 10% Emulphor ® solution. Body weights were significantly decreased at 400 mg/ kg only in the animals receiving the aqueous gavage. Absolute and relative kidney weights were significantly increased at 400 mg/ kg in both vehicles, with a significantly greater increase observed in the animals gavaged with oil compared to those gavaged with 10% Emulphor ® solution. Serum markers of hepatotoxicity were significantly increased at 400 mg/ kg in both vehicles with one nonsignificant increase in the aqueous vehicle. The increases were significantly greater for two of these markers in animals receiving the oil vehicle compared to those receiving the aqueous vehicle. Clinical observations were supported by histopathology findings. Hepatocellular degeneration and necrosis were observed at 400 mg/ kg in both vehicles. The difference in vehicles was reflected in more severe hepatocellular degeneration and a higher incidence of centrilobular necrosis in animals receiving the oil gavage compared to those receiving the aqueous gavage. Numerous increases in urinary markers of renal toxicity were observed 24 hours after dosing. Based on the differences observed, renal toxicity at 200 mg/ kg was similar or greater in the aqueous vehicle. Renal toxicity at 400 mg/ kg, however, was greater in the oil vehicle. The time to peak toxicity was both doseand vehicle dependent. At 200 mg/ kg, peak damage was observed at 24 hours in both vehicles. At 400 mg/ kg, peak damage was observed at 48 hours following oil gavage and at 24 36 hours following aqueous gavage. Histopathology revealed both renal tubule degeneration and necrosis at both dose levels. The incidence of renal tubule degeneration was greater in animals receiving the aqueous gavage at the low dose; however, the severity of renal degeneration and necrosis was greater in the animals receiving the oil gavage at the high dose. The authors attributed the vehicle differences to slower gastrointestinal uptake of bromodichloromethane from the oil vehicle compared to the aqueous vehicle. At the high dose, more bromodichloromethane would be converted to a reactive metabolite following oil dosing, while saturation would occur following aqueous dosing. At the low dose, the difference in uptake would have less of an effect. Overall, this study found that the kidney was more sensitive than the liver to a single dose of bromodichloromethane. A LOAEL of 200 mg/ kg was identified for each vehicle based on minimal renal tubule degeneration and changes in markers of renal function.

Lilly et al. ( 1996) investigated the effect of subchronic pretreatment with corn oil on the toxicity of bromodichloromethane. Prior to initiation of dosing with bromodichloromethane, male Draft  Do Not Cite or Quote February 20, 2003 V  5 Fischer 344 rats ( 6 animals/ group) were gavaged with oral doses of corn oil or water for six weeks ( 5 days/ week) at a constant volume of 5 mL/ kg. Following pretreatment, the animals were gavaged with a single dose of 0, 200, or 400 mg bromodichloromethane/ kg in 10% Emulphor ® . Urine was collected at 24, 36, and 48 hours following bromodichloromethane administration. The rats were sacrificed at 48 hours and necropsies were performed. Activities of the hepatotoxicity indicators alanine aminotransferase ( ALT), aspartate aminotransferase ( AST), lactate dehydrogenase ( LDH), and sorbitol dehydrogenase ( SDH) were measured in the serum, and the renal toxicity indicators alkaline phosphatase ( ALK), AST, and LDH, were measured in the urine. Additional analyses included determination of serum levels of bile acids, triglycerides, cholesterol and albumin, and urine levels of N acetylglucosaminidase and gamma glutamyl transpeptidase activity. Enzymatic activity of cytochrome P450 isoforms CYP2E1 and CYP2B1/ B2 was measured in the microsomal fraction of the liver to investigate whether corn oil was an inducer of bromodichloromethane metabolizing enzymes.

Liver weight was significantly reduced only in the water pretreatment group at the high dose. Kidney weight was reduced in both pretreatment groups at the high dose. Activities of serum AST and LDH were significantly elevated in both pretreatment groups at 400 mg/ kg. ALT levels increased in a dose dependent manner in the water pretreatment group, but significant elevations were noted only at the 400 mg/ kg dose in animals pretreated with corn oil. Activities of urinary AST and LDH were greater than controls in both pretreatment groups after 24, 36, and 48 hours. ALK levels were significantly increased in both pretreatment groups at 24 hours. At 36 and 48 hours, ALK levels were elevated only in water pretreated animals. High incidences of renal tubular necrosis occurred at 200 and 400 mg/ kg in both pretreatment groups. There were no significant differences in the histopathological lesion scores between the pretreatment groups. No significant differences were noted in the hepatic activity of CYP2E1 or CYP2B1/ B2 in the corn oil pretreated animals compared to the water controls. Although a number of differences between the pretreatment groups were noted in results for specific endpoints, the overall results from this study indicate that 6 weeks of pretreatment with corn oil did not significantly enhance the acute hepato or nephrotoxicity of bromodichloromethane. In addition, the reported data suggest that vehicle related differences in toxicity observed in other bromodichloromethane studies are most likely due to pharmacokinetic differences in absorption rather than altered enzyme activity induced by corn oil. This study confirms the acute LOAEL of 200 mg/ kg day previously identified by Lilly et al. ( 1994) for renal toxicity.

Lilly et al. ( 1997) administered single doses of bromodichloromethane by gavage in aqueous 10% Emulphor ® solution to male F344 rats at dose levels of 0, 123, 164, 246, 328, or 492 mg/ kg. Groups of 5 animals/ dose were sacrificed at 24 and 48 hours post dosing. Body weights were significantly decreased at or above 246 mg/ kg after 48 hours. At 24 hours, absolute and relative kidney weights were significantly increased at or above 328 mg/ kg and 246 mg/ kg, respectively. At 48 hours, only relative kidney weight at the high dose was significantly increased. At 24 hours, serum markers of liver damage ( ALT and AST) were significantly increased at or above 246 mg/ kg with one marker ( SDH) increased at all dose levels. Although smaller statistically significant increases were observed at the low doses at 24 hours for ALT ( 123 and 164 mg/ kg) and AST ( 164 mg/ kg), the biological significance of these increases is unclear. After Draft  Do Not Cite or Quote February 20, 2003 V  6 48 hours, serum levels of these markers were decreased from 24 hour levels with statistically significant changes noted only at the higher doses. No effects in urinary markers of kidney damage were found at either 123 or 164 mg/ kg. These markers, however, were significantly elevated after 24 hours for doses at or above 246 mg/ kg with few exceptions. No histopathological examination was conducted. These results were generally consistent with earlier results ( Lilly et al., 1994), although the present study was conducted at doses low enough to identify a NOAEL. In contrast to the earlier results of Lilly et al. ( 1994), this study did not find that the kidney was more sensitive than the liver to the toxic effects of bromodichloromethane. Based on hepatotoxicity and nephrotoxicity, this study identified a NOAEL of 164 mg/ kg and a LOAEL of 246 mg/ kg.

Keegan et al. ( 1998) investigated the acute toxicity of bromodichloromethane administered orally in an aqueous vehicle. Male Fischer 344 rats ( 6 animals/ group) were gavaged with a single dose of 0, 0.125, 0.1875, 0.250, 0.5, 0.75, 1.0 or 1.5 mmol/ kg dissolved in a 10% aqueous solution of Alkamuls EL 620. These doses of bromodichloromethane are equivalent to 0, 20.5, 30.7, 41.0, 81.9, 122.9, 163.8, and 245.7 mg/ kg, respectively. Control animals were dosed with vehicle only ( 10% Alkamuls EL 620). Gavage volumes were kept constant at 5 ml/ kg body weight. Animals were sacrificed 24 hours after dose administration and the liver, kidneys, and serum were harvested. Significant decreases in body weight were observed in the 0.75, 1.0, and 1.5 mmol bromodichloromethane/ kg treated animals. Decreases in absolute liver weights were observed in the 0.5, 0.75, 1.0, and 1.5 mmol bromodichloromethane/ kg treated animals. No change was noted in relative liver weights. Absolute kidney weights were not effected by bromodichloromethane treatment, but relative kidney weights were significantly increased in the two highest dose groups ( 1.0 and 1.5 mmol bromodichloromethane/ kg). Serum levels of ALT, SDH, and AST were assessed as an indication of liver toxicity. Dose dependent elevations in ALT ( 45% to 239% increase), AST ( 25% to 130% increase) and SDH ( 74% to 378% increase) were observed in the 0.5, 0.75, 1.0, and 1.5 mmol dose groups. Based on these findings, 0.25 mmol/ kg ( 41.0 mg/ kg) represents the NOAEL and 0.5 mmol/ kg ( 81.9 mg/ kg) represents the LOAEL for orally administered bromodichloromethane in an aqueous vehicle. These authors used the NOAEL of 41.0 mg/ kg to calculate One Day Health Advisories for drinking water of 4 mg/ L for a 10 kg child and 14 mg/ L for a 70 kg adult.

2. Dibromochloromethane The acute oral lethality of dibromochloromethane has been assessed in rats and mice of both sexes. Chu et al. ( 1980) reported LD 50 values in male and female Sprague Dawley rats of 1,186 and 848 mg/ kg day for males and females, respectively. Bowman et al. ( 1978) reported LD 50 values in mice of 800 and 1,200 mg/ kg day, respectively.

Bowman et al. ( 1978) investigated the acute oral toxicity of dibromochloromethane in ICR Swiss mice ( 10/ sex/ group). Doses of 500 to 4000 mg/ kg ( individual doses not reported) were administered by gavage in Emulphor ® : alcohol: saline ( 1: 1: 8) to fasted animals. Sedation and anesthesia occurred at 500 mg/ kg. Males were more sensitive than females to the acute lethal effects of dibromochloromethane. Draft  Do Not Cite or Quote February 20, 2003 V  7 NTP ( 1985) evaluated the acute toxicity of dibromochloromethane in male and female F344/ N rats. The rats ( 5 animals/ sex/ dose) received single doses of 160, 310, 630, 1,250, or 2,500 mg/ kg dibromochloromethane by gavage in corn oil. The observation period following treatment was 14 days. Mortality in high dose rats was 100% by day 3. At the 1,250 mg/ kg dose, four male rats and one female rat died. One female rat died in the 630 mg/ kg group. Doses of 310 mg/ kg or greater produced lethargy in all animals for 3 hours after dosing. A gross necropsy was conducted on one or two animals from each group. No treatment related effects were observed in rats selected for gross necropsy.

In a concurrent study, NTP ( 1985) evaluated the acute toxicity of dibromochloromethane in male and female B6C3F 1 mice ( 5/ sex/ dose). The mice received single doses of 160, 310, 630, 1,250, or 2,500 mg/ kg dibromochloromethane by gavage in corn oil. The observation period following treatment was 14 days. All male mice receiving the 2,500 mg/ kg and 1,250 mg/ kg doses died. Three male mice receiving the 630 mg/ kg dose died, while a single male mouse died at the 310 mg/ kg dose. All female mice receiving the 2500 mg/ kg dose died. Four of the female mice administered the 1,250 mg/ kg dose died between days 2 and 8 post treatment. No female mice died at doses of 630 mg/ kg or lower. A gross necropsy was conducted on one or two animals from each group. At necropsy, aberrations of the kidney ( dark red or pale medullae) and liver ( discolored foci) were reported to be more frequently observed in treated animals than in control animals ( raw data were not presented in the study).

Müller et al. ( 1997) investigated the cardiotoxic effects of acute dibromochloromethane exposure. Male Wistar rats were administered a single dose of dibromochloromethane by gavage in olive oil at dose levels of 0, 83, 167, 333, or 667 mg/ kg. Telemetric measurements of cardiovascular parameters ( heart rate, blood pressure, body temperature, and physical activity) were recorded in conscious rats ( 6/ group) 24 hours prior to administration to 72 hours following administration. Heart rate and blood pressure were also measured in urethane anesthetized rats ( 10/ group) 25 minutes following administration. For these rats, contractility parameters, such as the Krayenbühl index, were also calculated. Treatment related arrhythmias were not observed in conscious rats dosed with 83 to 333 mg/ kg of dibromochloromethane while rats in the high dose group exhibited premature ventricular contractions one minute following administration. Heart rate and body temperature were initially decreased in all treatment groups following administration, but returned to control values 24 hours post exposure in rats administered 83 to 333 mg/ kg. In the high dose rats, heart rate remained depressed up to 48 hours post exposure, and body temperature decreased 4.5  C below control values by 72 hours post exposure. Blood pressure was initially increased in all treatment groups following administration, but began to return to control values within 48 hours post exposure in rats administered 83 to 333 mg/ kg. Blood pressure in the high dose group, however, decreased below control values 72 hours postexposure Physical activity was decreased in conscious rats administered 333 and 667 mg/ kg during the entire observation period. In urethane anesthetized rats, negative effects on muscle contractility were observed at dose levels of 333 and 667 mg/ kg, negative chronotropic ( rate of contraction) effects were observed at the 333 mg/ kg dose level, and negative dromotropic ( defined as influencing the velocity of conduction of excitation, as in nerve or cardiac muscle Draft  Do Not Cite or Quote February 20, 2003 V  8 fibers) effects were observed at dose levels of 167 to 667 mg/ kg. Heart rate, blood pressure, and several contractility parameters, however, did not exhibit dose related trends.

3. Bromoform Bowman et al. ( 1978) assessed the acute oral toxicity of bromoform in ICR Swiss mice. Groups of ten male ( 30 to 35 g) and ten female ( 25 to 30 g) mice were treated with single doses ranging from 500 to 4,000 mg/ kg. Compounds were solubilized in Emulphor ® : alcohol: saline ( 1: 1: 8) and administered by gavage to fasted animals. The period of observation following treatment was 14 days. LD 50 values were 1400 and 1550 mg/ kg for males and females, respectively. Ataxia, sedation, and anesthesia occurred within 60 minutes of treatment at doses of 1000 mg/ kg and above. Sedation lasted approximately 4 hours.

Chu et al. ( 1980) evaluated the acute toxicity of bromoform in male and female Sprague Dawley rats. Fasted adult rats ( 10/ sex/ dose) received doses of 546, 765, 1071, 1500, or 2100 mg/ kg bromoform dissolved in corn oil by gavage. Clinical observations were made for 14 days after treatment. The LD 50 values for male and female rats were 1388 and 1147 mg/ kg, respectively. Clinical signs observed in treated rats included sedation, flaccid muscle tone, ataxia, piloerection, and hypothermia. Gross pathological examination revealed liver and kidney congestion in treated animals. Chu et al. ( 1982a) reported results for growth, food intake, organ weight, histopathology, hematological indices, liver microsome aniline hydroxylase activity and serum chemistry in surviving rats. Bromoform treatment increased liver protein concentration in the serum of male rats at doses of 765 and 1071 mg/ kg. Lymphocyte counts were decreased in male ( 765 and 1071 mg/ kg doses) and female ( 765 mg/ kg) rats but the effect was not dosedependent Female rats at the 765 mg/ kg dose had elevated aniline hydroxylase levels.

NTP ( 1989a) investigated the acute oral toxicity of bromoform in male and female F344/ N rats. The rats ( 5/ sex/ group) were administered a single oral dose of bromoform ( by gavage, in corn oil) at dose levels of 125, 250, 500, 1,000, or 2,000 mg/ kg. Control groups were not included in the study design. Mortality was 10/ 10 at 2,000 mg/ kg, 6/ 10 at 1,000 mg/ kg, and 0/ 10 at 500 mg/ kg or lower. Shallow breathing was observed in rats that received the 1000 or 2000 mg/ kg doses. No other clinical signs were reported.

NTP ( 1989a) investigated the acute oral toxicity of bromoform in male and female B6C3F 1 mice. The mice received a single oral dose of bromoform ( by gavage, in corn oil) at dose levels of 125, 250, 500, 1,000, or 2,000 mg/ kg. There were no controls. Mortality was 0/ 10 at 2,000 mg/ kg, 6/ 10 at 1,000 mg/ kg, 1/ 10 at 500 mg/ kg, and 0/ 10 at 250 mg/ kg or lower. The final mean body weight of mice that survived to the end of the study period was unaffected by bromoform exposure. Male mice that received doses of 500, 1,000, or 2,000 mg/ kg and females that received 1,000 or 2,000 mg/ kg were lethargic. Shallow breathing was noted in male mice administered the 1,000 or 2,000 mg/ kg dose.

B. Short Term Exposures Draft  Do Not Cite or Quote February 20, 2003 V  9 This section summarizes short term studies ( less than approximately 90 days) on the health effects of brominated trihalomethanes in animals. Details of these studies are summarized in Table V 3.

Table V 3 Summary of Short Term Toxicity Studies for Brominated Trihalomethanes Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Bromodichloromethane Oral Exposure Chu et al. ( 1982a) Rat SD* Drinking water M 10 28 days 0 0.8 8 68 ( NOAEL) No signs of toxicity observed.

Munson et al. ( 1982) Mouse CD 1 Gavage ( aqueous) M, F 8 12 14 days 0 50 ( NOAEL) 125 ( LOAEL) 250 Decreased immune function; increased liver weight, decreased absolute and relative spleen wt. ( females)

Condie et al. ( 1983) Mouse CD 1 Gavage ( corn oil) M 8 16 14 days 0 37 74 ( NOAEL) 148 ( LOAEL) Liver and kidney histopathology NTP ( 1987) Rat F344/ N Gavage ( corn oil) M, F 5 14 days 0 38 75 150 ( NOAEL) 300 ( LOAEL) 600 Decreased body weight gain; renal pathology NTP ( 1987) Mouse B6C3F1 Gavage ( corn oil) M, F 5 14 days 0 19 38 75 ( NOAEL) 150 ( LOAEL) 300 Mortality, renal histopathology Aida et al. ( 1992a) Rat Wistar Diet M 7 1 month 0 21 62 ( NOAEL) 189 ( LOAEL) Liver histopathology Aida et al. ( 1992a) Rat Wistar Diet F 7 1 month 0 21 66 ( NOAEL) 204 ( LOAEL) Liver histopathology Table V 3 ( cont.)

Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Draft  Do Not Cite or Quote February 20, 2003 V  10 Thornton Manning et al. ( 1994) Rat F344 Gavage ( aqueous) F 6 5 days 0 75 ( NOAEL) 150 ( LOAEL) 300 Liver histopathology, renal histopathology; increased liver and kidney wt.; elevated markers of hepatotoxicity Thornton Manning et al. ( 1994) Mouse C57BL/ 6J Gavage ( aqueous) F 6 5 days 0 75 ( NOAEL) 150 ( LOAEL) Increased serum markers of hepatotoxicity Potter et al. ( 1996) Rat F344 Gavage ( aqueous) M 1, 3, or 7 days 123 246 ( NOAEL) No effect in hyaline droplet formation or cell proliferation Melnick et al. ( 1998) Mouse B6C3F1 Gavage ( corn oil) F 10 3 weeks ( 5 d/ wk) 0 75 ( NOAEL) 150 ( LOAEL) 326 Increased abs. and relative liver weight; increased serum markers of hepatotoxicity; hepatocyte degeneration; increased labeling index NTP ( 1998) Rat SD Drinking water M, F 6 2 weeks 0 11 45 ( NOAEL) 91 ( LOAEL) 124 Transient reduction in weight gain NTP ( 1998) Rat SD Drinking water M, F 5 13 35 days Group A males 0 9 ( NOAEL) 38 ( LOAEL) 67 Single cell hepatic necrosis in Group A males Coffin et al. ( 2000) Mouse B6C3F1 Gavage ( Corn oil)

Drinking water F 10 11 days 11 days 0 150 ( LOAEL) 300 0 138 ( LOAEL) Hydropic degeneration in liver ( corn oil gavage and drinking water); increased relative liver weight ( gavage); increased proliferating cell nuclear antigen labeling index ( gavage)

Inhalation Exposure Torti et al. ( 2001) Mouse C57BL/ 6 FVB/ N ( wildtype Vapor M 6 1 week ( 6 hr/ day) 0 ppm 1 ppm ( NOAEL) 10 ppm ( LOAEL) 30 ppm 100 ppm 150 ppm Dose dependent marginal increase in renal tubular degeneration in C57BL/ 6 mice; mild increase in renal tubular degeneration and marginal increase in hepatic degeneration in FVB/ N mice; sign. increased labeling index Table V 3 ( cont.)

Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Draft  Do Not Cite or Quote February 20, 2003 V  11 Torti et al. ( 2001) Mouse C57BL/ 6 ( p53 heterozygous Vapor M 6 1 week ( 6 hr/ day) 0 1 ppm ( NOAEL) 10 ppm ( LOAEL) 30 ppm 100 ppm 150 ppm Dose dependent marginal to mild increase in renal tubular degeneration; sign. increased labeling index Torti et al. ( 2001) FVB/ N ( p53 heterozygous Vapor M 6 1 week ( 6 hr/ day) 0 ppm 0.3 ppm 1 ppm 3 ppm ( NOAEL) 10 ppm ( LOAEL) 30 ppm Dose dependent mild increase in renal tubular degeneration and marginal increase in nephrosis; marginal increase in hepatic degeneration; sign. increased relative kidney wt. and labeling index.

Torti et al. ( 2001) Mouse C57BL/ 6 FVB/ N ( wildtype Vapor M 6 3 weeks ( 6 hr/ day) 0 ppm 0.3 ppm 1 ppm 3 ppm ( NOAEL) 10 ppm ( LOAEL) 30 ppm Marginal increase in renal tubular degeneration Torti et al. ( 2001) Mouse C57BL/ 6 FVB/ N ( p53 heterozygous Vapor M 6 3 weeks ( 6 hr/ day) 0 ppm 0.3 ppm 1 ppm 3 ppm ( NOAEL) 10 ppm ( LOAEL) 30 ppm Marginal or mild increase in renal tubular degeneration in both strains; marginal increase in hepatic degeneration in FVB/ N heterozygous strain Dibromochloromethane Munson et al. ( 1982) Mouse CD 1 Gavage ( aqueous) M, F 8 12 14 days 0 50 ( NOAEL) 125 ( LOAEL) 250 Decreased immune function Chu et al. ( 1982a) Rat SD Drinking water M 10 28 days 0 0.7 8.5 68 ( NOAEL) No effect on growth, clinical signs, biochemical or histopathological endpoints Condie et al. ( 1983) Mouse CD 1 Gavage ( corn oil) M 8 16 14 days 0 37 74 ( NOAEL) 147 ( LOAEL) Decreased PAH uptake, moderate liver and kidney histopathology Table V 3 ( cont.)

Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Draft  Do Not Cite or Quote February 20, 2003 V  12 NTP ( 1985) Rat F344/ N Gavage ( corn oil) M, F 5 14 days 0 60 125 250 ( NOAEL) 500 ( LOAEL) 1,000 Mortality; liver and renal gross pathology NTP ( 1985) Mouse B6C3F1 Gavage ( corn oil) M, F 5 14 days 0 30 60 ( NOAEL) 125 ( LOAEL) 250 500 Liver and kidney gross pathology Aida et al. ( 1992a) Rat Wistar Diet M 7 1 month 0 18 ( NOAEL) 56 ( LOAEL) 173 Liver histopathology Aida et al. ( 1992a) Rat Wistar Diet F 7 1 month 0 34 ( NOAEL) 101 ( LOAEL) 333 Liver histopathology; increased relative liver weight Potter et al. ( 1996) Rat F344 Gavage ( aqueous) M 1, 3, or 7 days 156 312 ( NOAEL) No effect on hyaline droplet formation or cell proliferation Melnick et al. ( 1998) Mouse B6C3F1 Gavage ( corn oil) F 10 3 weeks ( 5 d/ wk) 0 50 100 ( NOAEL) 192 ( LOAEL) 417 Liver histopathology; increased serum enzymes and liver weight Coffin et al. ( 2000) Mouse B6C3F1 Gavage ( Corn oil)

Drinking water F 10 11 days 11 days 0 100 ( LOAEL) 300 0 171 Increased proliferating cell nuclear antigen labeling index ( gavage); increased relative liver wt.

Bromoform Munson et al. ( 1982) Mouse CD 1 Gavage ( aqueous) M, F 6 12 14 days 0 50 125 ( NOAEL) 250 ( LOAEL) Increased serum enzyme activity ( AST); decrease in antibody forming cells and delayed type hypersensitivity response Chu et al. ( 1982a) Rat SD Drinking water M 10 28 days 0.7 8.5 80 ( NOAEL) No effect on growth, clinical signs, biochemical or histopathological endpoints Table V 3 ( cont.)

Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Draft  Do Not Cite or Quote February 20, 2003 V  13 Condie et al. ( 1983) Mouse CD 1 Gavage ( corn oil) M 8 16 14 days 0 72 145 ( NOAEL) 289 ( LOAEL) Decreased PAH uptake, moderate histopathological changes NTP ( 1989a) Rat F344/ N Gavage ( corn oil) M, F 5 14 days 0 100 200 ( NOAEL) 400 ( LOAEL) 600 800 Decreased body weight gain; 1/ 5 died at 400 mg/ kgday 100% mortality at two highest doses NTP ( 1989a) Mouse B6C3F1 Gavage ( corn oil) M 5 14 days 0 50 100 200 ( NOAEL) 400 ( LOAEL) 600 Stomach nodules; ataxia, lethargy; 1/ 5 died at high dose Aida et al. ( 1992a) Rat Wistar Diet M 7 1 month 0 62 ( NOAEL) 187 ( LOAEL) 618 Hepatic vacuolization, serum chemistry/ biochemistry Aida et al. ( 1992a) Rat Wistar Diet F 7 1 month 0 56 ( NOAEL) 208 ( LOAEL) 728 Hepatic vacuolization, serum chemistry/ biochemistry Potter et al. ( 1996) Rat F344 Gavage ( aqueous) M 1, 3, or 7 days 190 379 ( NOAEL) No effect on hyaline droplet formation or cell proliferation Melnick et al. ( 1998) Mouse B6C3F1 Gavage ( corn oil) F 10 3 weeks ( 5 d/ wk) 0 200 ( NOAEL) 500 ( LOAEL) Increase in absolute and relative liver wt.; marginally significant increase in LI at highest dose Coffin et al. ( 2000) Mouse B6C3F1 Gavage ( corn oil)

Drinking Water F 10 11 days 11 days 0 200 ( LOAEL) 500 0 301 Liver histopathology; increased proliferating cell nuclear antigen labeling index; increased relative liver wt.

* SD, Sprague Dawley Draft  Do Not Cite or Quote February 20, 2003 V  14 1. Bromodichloromethane Munson et al. ( 1982) administered bromodichloromethane by aqueous gavage to male and female CD 1 mice ( 8 to 12/ sex/ group) for 14 days at levels of 0, 50, 125, or 250 mg/ kg day. Endpoints evaluated included body and organ weights, hematology, serum enzyme levels ( SGOT, SGPT), and humoral and cell mediated immune system functions. At 250 mg/ kg day, body weights were significantly decreased. Significant organ weight changes included increased relative liver weight ( mid and high dose groups), decreased absolute spleen weight ( high dose males and mid and high dose females), and decreased relative spleen weight ( mid and high dose females).

Among the hematology endpoints, only fibrinogen levels were significantly decreased in high dose males and in mid and high dose females. Significant clinical chemistry findings included decreased glucose levels ( high dose males), increased ALT and AST activities ( highdose groups), and increased blood urea nitrogen ( BUN) levels ( high dose groups). Bromodichloromethane appeared to affect the humoral immune system, as judged by significantly decreased antibody forming cells ( high dose males and mid and high dose females) and hemagglutination titers ( mid and high dose males and high dose 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.

Chu et al. ( 1982a) administered bromodichloromethane to male Sprague Dawley rats ( 10/ group) in drinking water for 28 days at dose levels of 0, 5, 50, or 500 ppm. These levels corresponded to doses of 0, 0.8, 8.0, or 68 mg/ kg day, as calculated by the authors based on recorded fluid intake. The authors observed no effects on growth rate or food consumption and no signs of toxicity throughout the exposure. No dose related biochemical or histologic changes were detected ( no data were provided). This study identified a NOAEL of 68 mg/ kg day, but the reported data were too limited to allow an independent verification.

Condie et al. ( 1983) investigated the renal and hepatic toxicity of bromodichloromethane in male CD 1 mice ( 8 to 16/ group). Bromodichloromethane was administered by gavage in corn oil for 14 days at dose levels of 0, 37, 74 or 148 mg/ kg day. Biochemical evidence of liver damage ( significantly elevated ALT) was observed at the high dose, while biochemical evidence of kidney damage ( significantly decreased p aminohippurate ( PAH) uptake by kidney slices) was observed at the mid and high dose. Significantly decreased BUN levels were observed in the lowand mid dose groups, but not in the high dose group. Histopathology revealed no consistent or important changes at the low or mid level doses, with minimal to moderate liver and kidney injury observed in the majority of animals at the high dose. Liver lesions included centrilobular pallor and focal inflammation. Kidney lesions included intratubular mineralization, epithelial hyperplasia, and cytomegaly. Although the severity of these lesions was primarily minimal to slight, a few animals in the high dose group exhibited moderate to moderately severe intratubular mineralization and/ or epithelial hyperplasia. This study identified a NOAEL value of 74 mg/ kgday and a LOAEL value of 148 mg/ kg day for bromodichloromethane, based on histopathology. Draft  Do Not Cite or Quote February 20, 2003 V  15 NTP ( 1987) administered doses of 0, 38, 75, 150, 300, or 600 mg/ kg day of bromodichloromethane in corn oil by gavage to male and female F344/ N rats ( 5/ sex/ dose) for 14 days. One low dose and one high dose female died before study termination. All high dose animals were hyperactive after dosing and either lost weight or gained no weight during the study. Final mean body weights were not significantly affected in groups given 38, 75, or 150 mg/ kgday At 300 mg/ kg, body weights of males and females were decreased by 21% and 7%, respectively, relative to vehicle controls. At 600 mg/ kg day, body weights of males and females were decreased by 44% and 22%, respectively, relative to vehicle controls. Necropsy was performed on all animals. Renal medullae were reddened in all high dose males and in one female in each of the control, low dose, and high dose groups. This study identified a NOAEL of 150 mg/ kg day and a LOAEL of 300 mg/ kg day in rats, based on decreased body weight gain. In a parallel experiment, NTP ( 1987) administered doses of 0, 19, 38, 75, 150, or 300 mg/ kg day bromodichloromethane in corn oil by gavage to male and female B6C3F 1 mice ( 5/ sex/ dose) for 14 days. All male mice that received 150 or 300 mg/ kg day bromodichloromethane died before study termination. Clinical signs included lethargy, dehydration, and hunched posture. The final mean body weights of the mice that survived were not significantly different from the controls. The renal medullae were reddened in four males in the 150 mg/ kg day group, all males in the 300 mg/ kg day group, and one female in the 150 mg/ kg day group. Based on behavior, appearance, gross necropsy, and mortality, this study identified a NOAEL of 75 mg/ kg day and a frank effect level ( FEL) of 150 mg/ kg day in male mice. An interesting point to note is that this study and the study by Condie et al. ( 1983) were conducted under similar conditions ( mice administered bromodichloromethane by gavage in corn oil for 14 days), but with dramatically different results. In contrast to the 100% mortality observed in this study for male mice, Condie et al. ( 1983) found only moderate histopathology in male CD 1mice at 148 mg/ kg day with no deaths occurring. The reason for this difference is unclear, but may be related to strain specific differences in sensitivity.

Aida et al. ( 1992a) administered bromodichloromethane to Slc: Wistar rats ( 7/ sex/ group) for one month at dietary levels of 0%, 0.024%, 0.072%, or 0.215% for males and 0%, 0.024%, 0.076%, or 0.227% for females. The test material was microencapsulated and mixed with powdered feed; placebo granules were used for the control groups. Based on the mean food intakes, the study authors reported the mean compound intakes for the one month period as 0, 20.6, 61.7, or 189.0 mg/ kg day for males and 0, 21.1, 65.8, or 203.8 mg/ kg day for females. Clinical effects, body weight, food consumption, hematology parameters, serum chemistry, and histopathology of all major organs were determined. Body weights were significantly decreased in the high dose groups relative to the controls. The high dose animals also exhibited slight piloerection and emaciation. Relative liver weight was increased only in high dose females. Significant, dose related biochemical findings at the low dose were limited to decreased LDH levels in males, but the biological significance of this effect is unclear. Serum LDH levels were also significantly decreased at the low and high dose in females. Other statistically significant, dose related changes included decreased glucose ( high dose males), decreased serum triglycerides ( high dose groups), decreased serum cholinesterase activity ( high dose males and mid and highdose females), and increased total cholesterol ( mid and high dose males). The changes in cholinesterase activity and cholesterol levels in males were not dose related. The cholesterol Draft  Do Not Cite or Quote February 20, 2003 V  16 levels were within normal ranges at all doses. Treatment related histopathological lesions were limited to the liver and were rated as very slight or slight. The lesions were mostly confined to the high dose groups. Vacuolization observed in mid dose females and in a single low dose male was not considered an adverse effect. Other observed effects included swelling of hepatocytes, single cell necrosis, hepatic cord irregularity, and bile duct proliferation. These lesions were observed only in high dose males and females with the exception of very slight to slight changes in individual low dose males. No effect was observed on any hematology parameter. Based on the histopathology observed in high dose males and females, the LOAELs identified in this study for bromodichloromethane in rats were 189.0 mg/ kg day in males and 203.8 mg/ kg day in females; the NOAELs were 61.7 mg/ kg day in males and 65.8 mg/ kg day in females.

Thornton Manning et al. ( 1994) administered bromodichloromethane at dose levels of 0, 75, 150, or 300 mg/ kg day by gavage to female F344 rats ( 6 animals/ dose) for five consecutive days. The dosing vehicle consisted of an aqueous 10% Emulphor ® solution. Animals were sacrificed on day 6. Two animals in the high dose group died on day 5. Final body weights of the high dose group were significantly decreased compared to the controls. Absolute and relative kidney and liver weights were significantly increased at 150 and 300 mg/ kg day with the exception of a nonsignificant increase in absolute liver weight at 150 mg/ kg day. Toxic effects on the kidney and liver were reflected in significantly increased LDH, AST, SDH, creatinine, and BUN at 300 mg/ kg day. These results were supported by the histopathology findings. In the liver, centrilobular vacuolar degeneration was observed at both 150 and 300 mg/ kg day with the severity of the effect increased with increasing dose. Centrilobular hepatocellular necrosis was also observed in one high dose animal. In the kidney, renal tubular vacuolar degeneration and renal tubule regeneration were observed at 150 and 300 mg/ kg day with the incidence and severity increased with increasing dose. While minimal renal tubule necrosis was observed in only one animal at the mid dose, all animals at the high dose exhibited mild to moderate renal tubule necrosis. Significant decreases in the hepatic activity of the CYP1A and CYP2B markers ethoxyresorufin O dealkylase ( EROD) and pentoxyresorufin O dealkylase ( PROD), were observed at all doses. The effect, however, was not dose related. No effect on the CYP2E1 marker pNP hydroxylase, was observed. Based on kidney and liver lesions observed at the mid dose, this study identified a NOAEL of 75 mg/ kg day and a LOAEL of 150 mg/ kg day.

Thornton Manning et al. ( 1994) conducted an analogous experiment with female C57BL/ 6J mice. Six mice per group were administered an aqueous solution ( 10% Emulphor ® ) of bromodichloromethane by gavage for five consecutive days at dose levels of 0, 75 and 150 mg/ kg day. Animals were sacrificed on day 6. All mice survived to the termination of the experiment. No effect on body, kidney, or liver weight was observed with the exception of a significant increase in absolute liver weight at 150 mg/ kg day. No change in cytochrome P450 activity was observed, although a nonsignificant dose related decrease in total P450 content was observed. ALT was significantly increased at 150 mg/ kg day, and a significant dose related increase in SDH activity was observed. Creatinine and BUN were not significantly increased. No kidney or liver lesions were observed at either dose. Based on increases in serum enzyme activity, a LOAEL of 150 mg/ kg day and a NOAEL of 75 mg/ kg day were identified for this study. Draft  Do Not Cite or Quote February 20, 2003 V  17 Potter et al. ( 1996) investigated hyaline droplet formation and cell proliferation in the kidney of male F344 rats. Test animals ( 4/ dose) 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. No exposure related increase in hyaline droplet formation was observed at either dose. Binding of bromodichloromethane to  2u globulin was not measured. 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.

Melnick et al. ( 1998) exposed female B6C3F 1 mice ( 10 animals/ group) to 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. There were no treatment related signs of overt toxicity observed during the study. Body weight and water intake were not significantly altered at any dose tested. However, a significant doserelated 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/ kgday doses resulted in significantly elevated hepatocyte proliferation as measured by the LI. NOAEL and LOAEL values of 75 and 150 mg/ kg day were identified on the basis of elevated serum enzyme activity, increased liver weight, and histological findings.

NTP ( 1998) evaluated the effect of bromodichloromethane on food and water consumption by Sprague Dawley rats in the course of a range finding experiment for a study of developmental and reproductive effects. This study was conducted in compliance with the Good Laboratory Practice Regulations as described in 21 CFR 58. Bromodichloromethane was administered to test animals ( 6 animals/ sex/ dose) at nominal concentrations of 0, 100, 500, 1000, and 1500 ppm in the drinking water for 2 weeks. The average doses of bromodichloromethane estimated based on water consumption were 11, 45, 91 and 124 mg/ kg day for the 100, 500, 1000 and 1500 ppm dose groups, respectively. All animals were observed twice daily for signs of toxicity. Body weight data were obtained twice weekly and at termination of the experiment. Feed and water consumption were measured twice weekly. Animals were euthanized at termination of the experiment without necropsy. No mortality or treatment related clinical signs were observed in any dose group. Body weights and weight gains were comparable among all dose groups, except for body weight gains on Study Day 5 ( the first day of compound administration) in the 1000 and 1500 ppm dose groups which were decreased 127.5% and 118.5%, respectively. Feed consumption was also comparable across dose groups, with the exception of male rats dosed with 1000 and 1500 ppm. Male rats in these dose groups showed decreases in consumption of 31% and 41% , respectively, on Study Days 1 to 5. Water consumption was reduced in the 500, 1000, and 1500 ppm dose groups, suggesting that bromodichloromethane is unpalatable at higher concentrations. The greatest reduction in water Draft  Do Not Cite or Quote February 20, 2003 V  18 intake was noted on Study Days 1 to 5 ( 61% and 62% for males in the 1000 ppm and 1500 ppm dose groups, respectively, and 38%, 40% and 52% for females in the 500, 1000 and 1500 ppm dose groups, respectively).

NTP ( 1998) conducted a short term reproductive and developmental toxicity screen in Sprague Dawley rats to evaluate the potential toxicity of bromodichloromethane administered in drinking water for 35 days. This study was conducted in compliance with the Good Laboratory Practice Regulations as described in 21 CFR 58. Groups of male and female rats ( 5 13/ sex/ dose) 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. E. 1). Feed and water consumption, body weight, hematology, clinical chemistry, cell proliferation, and pathology were evaluated in addition to developmental and reproductive endpoints. Based on water consumption and analytical measurements of bromodichloromethane in the provided drinking water, the calculated average daily doses were 0, 9, 38, and 67 mg/ kg day for Group A males; 0, 7, 43, and 69 mg/ kg day for Group B males; and 0, 14, 69, or 126 mg/ kg day for Group C females.

The results for reproductive and developmental effects are reported in Section V. E. 1. Alterations in hematological endpoints or clinical chemistry were not observed following bromodichloromethane exposure, with the exception of a 14% drop in creatinine in the 100 ppm Group A males and a 43% increase in 5 nucleotidase in the 1300 ppm Group A males when compared to controls. An increase in 5 nucleotidase is an indication of hepatobiliary dysfunction in which there is interference with the secretion of bile, and should be accompanied by a parallel change in alkaline phosphatase activity. Since alkaline phophatase activity was unaltered in this study, the toxicological significance of the observed increase in 5 nucleotidase was considered uncertain. Organ weight and organ/ body weight ratios reported by NTP ( 1998) were comparable in all treatment groups for both males and females. Histopathological examination identified three tissue changes that were potentially treatment related. Cytoplasmic vacuolization of hepatocytes and mild liver necrosis were observed in Group A males ( see Table V 6 for details of group assignment) treated with 700 and 1300 ppm bromodichloromethane and in Group B males treated with 1300 ppm bromodichloromethane. Hepatic necrosis was dose dependent, with incidences of 0/ 10, 0/ 10, 4/ 9, and 10/ 10 observed at 0, 100, 700, and 1300 ppm, respectively. These changes were not accompanied by an increase in alkaline phosphatase activity. Hematopoietic cell proliferation in the spleen was observed in Group A males at all doses of bromodichloromethane. However, the biological significance of this finding with respect to bromodichloromethane treatment was unclear, since cell proliferation in the spleen may occur as a response to general stress. Evidence of mild kidney necrosis was evident in Group A males in the 1300 ppm dose group, but may have resulted from decreased water intake. BrdU labeling index ( LI), a measurement of cell proliferation, was unchanged in the livers and kidneys of Group B males in all dose groups. 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.

As discussed in Sections V. E. 1, results from this study indicate that bromodichloromethane did not result in reproductive or developmental toxicity at drinking water concentrations Draft  Do Not Cite or Quote February 20, 2003 V  19 up to 1300 ppm. However, exposure to concentrations of 700 ppm and 1300 ppm produced changes in liver histopathology in male rats and resulted in decreases in body weight and food and water consumption in both sexes. On the basis of these results, NTP ( 1998) concluded that bromodichloromethane is unpalatable at these concentrations and is a possible general toxicant in male and female rats at concentrations of 700 ppm and above. Although not accompanied by changes in alkaline phosphatase activity, the occurrence of individual hepatocyte cell necrosis was clearly dose related and thus considered appropriate for identification of NOAEL and LOAEL values. Based on calculated average daily doses for Group A males at the 100 and 700 ppm concentrations ( Table 6A in NTP, 1998), these data identify NOAEL and LOAEL values of 9 mg/ kg day and 38 mg/ kg day, respectively, for occurrence of hepatic cell necrosis.

Coffin et al. ( 2000) examined the effect of bromodichloromethane administered by gavage in corn oil or in drinking water on cell proliferation and DNA methylation in the liver of female B6C3F1 mice. Gavage doses of 0, 0.92, or 1.83 mmol/ kg ( 0, 150, or 300 mg/ kg, respectively) were administered to test animals ( 7 8 weeks old; 10/ group) daily for five days, off for two days, and then again daily for four days. The high dose was selected on the basis that it had previously been shown to be carcinogenic in female mice. In a separate experiment, bromodichloromethane was administered in drinking water for 11 days at approximately 75% of the saturation level, resulting in an average daily dose of 0.85 mmol/ kg ( 138 mg/ kg). The mice were sacrificed 24 hours after the last gavage dose and the livers were removed, weighed, and processed for histopathological examination, proliferating cell nuclear antigen  labeling index ( PCNA LI) analysis, and determination of c myc methylation status. A significant, dose dependent increase in relative liver weight was observed in animals dosed by gavage; however, relative liver weight was unaffected in animals administered the compound in drinking water, when compared to controls. Histopathological findings in gavage dosed animals consisted of hydropic degeneration at the low dose and necrosis, fibrosis, and giant cell reaction at the high dose. No severity or incidence data were provided. The histopathology findings for animals receiving bromodichloromethane in the drinking water were similar to those observed in the low dose gavage group. Bromodichloromethane administered by gavage caused a dose dependent increase in the PCNA LI which was significant at each dose tested when compared to the control. There was no significant effect when the compound was administered in drinking water. Administration of bromodichloromethane by gavage or in drinking water decreased methylation of the c myc gene. A LOAEL of 150 mg/ kg, the lowest dose tested, was identified on the basis of liver toxicity ( hydropic degeneration) and increased cell proliferation in animals administered bromodichloromethane by corn oil gavage. A NOAEL was not identified. The results of the single dose drinking water experiment suggest a slightly lower LOAEL of 138 mg/ kg day, based on hydropic degeneration of the liver.

Torti et al. ( 2001) conducted a one week inhalation exposure study of bromodichloromethane in male wild type ( p53+/+) and genetically engineered p53 heterozygous ( p53+/) mice. C57BL/ 6, FVB/ N, and C57BL/ 6 p53+/ mice ( 6 mice/ type/ concentration) were exposed to target exposure concentrations of 0, 1, 10, 30, 100, or 150 ppm for six hours per day, seven days per week. FVB/ N p53+/ mice were exposed to concentrations of 0, 0.3, 1, 10, or 30 ppm for six hours per day, seven days per week. The test animals were evaluated for clinical and Draft  Do Not Cite or Quote February 20, 2003 V  20 pathological changes and induced regenerative cell proliferation in kidney and liver. Osmotic pumps for delivery of bromodeoxyuridine for determination of labeling index were implanted at 3.5 days prior to scheduled termination. Test animals were euthanized approximately 18 hours after the last scheduled exposure. Average measured concentrations of bromodichloromethane in the middle dose range were 102 to 104% of the target concentration ( coefficients of variation 2.6 to 10.6%). The lowest dose average concentration was 114% ( coefficient of variation 38.6%). The average high dose concentration was 78.8% of the target concentration as a result of technical problems with the metering system. Deaths were observed at concentrations of 30 ppm and greater, with 100% mortality observed for C57BL/ 6 heterozygous and FVB/ N wild type mice at 150 ppm. Clinical signs in mice surviving exposure at 100 and 150 ppm included lethargy and labored breathing. Reddened skin and eyes were also observed, primarily at concentrations of 30 ppm and above. Relative body weight was decreased at exposure concentrations of 30 ppm and above. Body weight loss was greater in the heterozygous p53+/ than in the corresponding wild type strain. Significantly increased relative kidney weight was evident in all mice exposed to concentrations of 30 ppm and above and in FVB/ N mice exposed to 10 ppm. Significantly increased relative liver weight was observed in wild type and heterozygous FVB/ N mice exposed to concentrations of 10 ppm and above. Reduced body weight gains were observed in surviving mice exposed to 30 ppm and above, with greater reductions observed in heterozygous mice when compared to wild type mice. Histopathologic evaluation revealed severe renal damage consisting of nephrosis, tubular degeneration, and associated regeneration. The averaged severity scores indicated greater damage in heterozygous compared to wild type mice and in FVB/ N compared to the C57BL/ N mice. Centrilobular degeneration and necrosis were observed in the livers of moribund mice sacrificed before study termination. These lesions were also observed in surviving animals at study termination. Hydropic degeneration occurred at concentrations of 30 ppm and above in wild type and heterozygous C57BL/ N mice and at concentrations of 10 ppm and above in FVB/ N mice. Necrosis was evident at concentrations of 100 and 150 ppm. No histopathologic lesions were observed in the bladder. Regenerative cell proliferation in the kidney cortex was significantly increased at exposure concentrations of 10 ppm and above. Renal labeling indices were approximately 20 to 30 % at 10 ppm and 45 to 60% at the high dose. Regenerative cell proliferation in the liver was less pronounced than in the kidney. Minimal increases in the labeling index ( approximately 10% or less) were observed in wild type C57BL/ N and FVB/ N mice. Labeling index was significantly increased only in C57BL/ N wild type mice exposed at the 100 ppm level. A modest increase in labeling index was observed in heterozygous FVB/ N mice exposed to 10 or 30 ppm; the response reached statistical significance at 30 ppm. Relatively large increases in labeling index ( up to 40%) were observed in heterozygous C57BL/ N mice at doses of 10 ppm and above. The response was statistically significant at the 30 and 100 ppm exposure levels. Bromodichloromethane did not induce cellular proliferation in the transitional epithelium of the bladder. These data identify NOAEL and LOAEL values of 1 and 10 ppm, respectively, based on histopathological changes in the kidney of male p53 wild type and heterozygous C57BL/ 6 and FVB/ N mice.

Torti et al. ( 2001) also conducted a three week inhalation exposure study of bromodichloromethane in wild type ( p53+/+) and genetically engineered p53 heterozygous ( p53+/) male mice. C57BL/ 6, FVB/ N, C57BL/ 6 p53+/, and FVB/ N p53+/ mice ( 6 Draft  Do Not Cite or Quote February 20, 2003 V  21 mice/ type/ concentration) were exposed to target exposure concentrations of 0.3, 1, 3, 10, or 30 ppm for six hours per day, seven days per week. The test protocol and endpoints measured were the same as those used for the one week study described above. Test animals were euthanized approximately 18 hours after the last scheduled exposure. Average measured concentrations were 92 to 97% of the target concentrations, with coefficients of variation ranging from 3.1 to 6.9%. Mortality was observed in all 30 ppm dose groups with the exception of wild type C57BL/ 6 mice. No clinical signs of toxicity were reported. Body weight gain was significantly reduced only in C57BL/ 6 wild type mice exposed at 30 ppm. Relative kidney weights in exposed groups did not differ significantly from the control values. Significantly increased relative liver weight was observed in only in heterozygous C57BL/ 6 and wild type FVB/ N mice exposed at 30 ppm. Histopathologic evaluation revealed near normal kidney architecture. Minimal to moderate degenerative tubular change and regenerative tubules were observed in the 10 and 30 ppm groups, but the acute tubular nephrosis observed in the one week study was not evident. Minimal hepatocyte degeneration was observed in heterozygous C57BL/ 6 mice exposed at 30 ppm and in heterozygous FVB/ N mice exposed at 10 or 30 ppm. These observations suggest that the liver and severe renal toxicity observed in the one week experiment conducted by Torti et al. ( 2001) are transient and were resolving by three weeks. No histopathologic lesions were observed in the bladder. Regenerative cell proliferation in the kidney cortex was near baseline levels, with only the 30 ppm groups showing small elevations. These elevations were statistically significant in all 30 ppm groups except C57BL/ N wild type mice. No increases in regenerative cell proliferation were evident in the liver or bladder. The NOAEL and LOAEL values in this study are 3 and 10 ppm, respectively, based on histopathologic changes in the liver and kidney of male p53 wild type and heterozygous C57BL/ 6 and FVB/ N mice.

2. Dibromochloromethane Chu et al. ( 1982a) administered dibromochloromethane to male Sprague Dawley rats ( 10/ group) in drinking water for 28 days at dose levels of 0, 5, 50, or 500 ppm. Based on recorded fluid intake, these levels corresponded to doses of 0, 0.7, 8.5, or 68 mg/ kg day, as calculated by the authors. The authors observed no effects on growth rate or food consumption and no signs of toxicity throughout the exposure. No dose related biochemical or histologic changes were detected ( no data provided). This study identified a NOAEL of 68 mg/ kg day, but the reported data were too limited to allow an independent verification.

Munson et al. ( 1982) administered dibromochloromethane by aqueous gavage to male and female CD 1 mice ( 8 to 12/ sex/ group) for 14 days at dose levels of 0, 50, 125, or 250 mg/ kg day. Endpoints measured included body and organ weights, hematology, clinical chemistry, and humoral and cell mediated immune system function. At 250 mg/ kg day, body weights were significantly decreased only in high dose males. Significant organ weight changes included increased absolute liver weight ( high dose females), increased relative liver weight ( mid and highdose groups), and decreased absolute and relative spleen weight ( high dose males). The only hematology parameter significantly affected by treatment was fibrinogen concentration, which was decreased in high dose males and females. Significant clinical chemistry findings were limited to the high dose groups. Specifically, glucose levels were significantly decreased in both males and Draft  Do Not Cite or Quote February 20, 2003 V  22 females, and ALT and AST activities were significantly increased in both males and females. Dibromochloromethane appeared to affect the humoral immune system, as judged by significantly decreased antibody forming cells ( mid and high dose groups) and hemagglutination titers ( highdose groups). The cell mediated immune system also appeared to be affected in male animals, as judged by a significant decrease in the popliteal lymph node stimulation index at the high dose. This study identified a NOAEL of 50 mg/ kg day and a LOAEL of 125 mg/ kg day for dibromochloromethane, based on decreased immune function.

Condie et al. ( 1983) investigated the renal and hepatic toxicity of dibromochloromethane. Male CD 1 mice ( 8 to 16/ group) were administered 0, 37, 74, or 147 mg/ kg day of dibromochloromethane by gavage in corn oil for 14 days. Biochemical evidence of liver damage ( elevated ALT) and kidney damage ( decreased PAH uptake by kidney slices) was observed at the high dose, but not at the mid level or low doses. Similarly, histopathology revealed no consistent or important changes at the low or mid doses with minimal to moderate liver and kidney injury at the high dose. Liver lesions included mitotic figures, focal inflammation, and cytoplasmic vacuolation, while kidney lesions included epithelial hyperplasia and mesangial nephrosis. On this basis, this study identified a NOAEL value of 74 mg/ kg day and LOAEL value of 147 mg/ kg day for dibromochloromethane.

In a 14 day study by NTP ( 1985), groups of male and female F344/ N rats ( 5/ sex/ dose) were administered 0, 60, 125, 250, 500, or 1,000 mg/ kg day of dibromochloromethane by gavage in corn oil. Animals were observed twice daily for mortality and were weighed once per week. Necropsies were performed on all animals. All high dose rats and all females that received 500 mg/ kg day died by day 6. Three males at 500 mg/ kg day died between days 5 and 8. No deaths occurred at or below 250 mg/ kg day. At 500 or 1000 mg/ kg day, clinical observations included lethargy, ataxia, and labored breathing. Treatment related macroscopic findings included mottled livers and darkened renal medullae in animals administered 500 or 1,000 mg/ kg day. Based on behavior, gross pathology, and mortality, this study identified a NOAEL of 250 mg/ kg day and a LOAEL of 500 mg/ kg day.

In a parallel study ( NTP, 1985), male and female B6C3F 1 mice ( 5/ sex/ dose) were administered 0, 30, 60, 125, 250, or 500 mg/ kg day of dibromochloromethane in corn oil by gavage for 14 days. Treatment related deaths occurred in 80% of the males and in 60% of the females at the high dose. Clinical signs at this dose included lethargy, ataxia, and labored breathing. Treatment related macroscopic findings included mottled livers and darkened renal medullae in high dose males and females. White papillomatous nodules in the stomach were also observed in males at 125, 250, or 500 mg/ kg day and in female mice at 250 or 500 mg/ kg day. Based on gross lesions, this study identified a NOAEL of 60 mg/ kg day and a LOAEL of 125 mg/ kg day in mice.

Aida et al. ( 1992a) investigated the effects of administering dibromochloromethane to Slc: Wistar rats ( 7/ sex/ group) for one month at dietary levels of 0%, 0.020%, 0.062%, or 0.185% for males, and 0%, 0.038%, 0.113%, or 0.338% for females. The test material was microencapsulated and mixed with powdered feed; placebo granules were used for the control Draft  Do Not Cite or Quote February 20, 2003 V  23 groups. Based on the mean food intakes, the study authors reported calculated doses of 0, 18.3, 56.2, or 173.3 mg/ kg day for males and 0, 34.0, 101.1, or 332.5 mg/ kg day for females. Clinical effects, body weight, food consumption, hematology parameters, serum chemistry, and histopathology of all major organs were determined. Body weights were significantly reduced in high dose females relative to the controls. High dose females also exhibited slight piloerection and emaciation. Dose related increases in both absolute and relative liver weights were observed in males ( significant at the high dose) and females ( significant at all dose levels with the exception of a nonsignificant increase in absolute liver weight at the low dose). Relative kidney weights were also significantly increased in the high dose females. Significant decreases in alkaline phosphatase ( mid and high dose males and all female dose groups) and LDH ( all female dose groups) were observed, but the biological significance of these changes is unclear. Significant, dose related changes in serum biochemistry included reduced nonesterified fatty acids in highdose males, reduced T GLY in high dose groups, and increased cholesterol in mid and high dose males and in females at all dose levels. The cholesterol levels, however, were within normal ranges at all dose levels. Serum cholinesterase activity was also significantly decreased in highdose males and mid and high dose females with the trend clearly dose related in females. Liver cell vacuolization was generally noted at a similar incidence in the controls and all dosing groups, but dose related increases in severity were observed in mid and high dose males and females. The incidence and very slight severity of the effects at the low dose were similar to those observed in the control groups and were not considered adverse. The severity of the liver cell vacuolization at the mid dose was rated as very slight to slight, while the severity at the high dose was rated as moderate to remarkable. Swelling and single cell necrosis were also observed, primarily in the high dose groups. No effect was observed on any hematology parameter. Based on the histopathology findings, NOAELs of 18.3 ( males) and 34.0 ( females) mg/ kg day and LOAELs of 56.2 ( males) and 101.1 ( females) mg/ kg day were identified for dibromochloromethane in rats.

Potter et al. ( 1996) evaluated hyaline droplet formation and cell proliferation in the kidney of male F344 rats following exposure to dibromochloromethane. The rats ( 4/ dose) were dosed with 0.75 or 1.5 mmol/ kg ( 156 or 312 mg/ kg day, respectively) of dibromochloromethane in 4% Emulphor ® by gavage for 1, 3, or 7 days. No exposure related increase in hyaline droplets was observed in dosed rats. Binding to  2u globulin was not measured. Changes in kidney tubule cell proliferation were assessed by in vivo incorporation of [ 3H] thymidine. No statistically significant effect of dibromochloromethane exposure on this endpoint was noted following exposures of up to 7 days duration.

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 were 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 Draft  Do Not Cite or Quote February 20, 2003 V  24 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 the 192 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. Evaluation of the data in this study suggest a LOAEL of 192 mg/ kg day, based on a consistent pattern of positive results for indicators of hepatotoxicity at this dose.

Coffin et al. ( 2000) examined the effect of dibromochloromethane administered by gavage in corn oil or in drinking water on cell proliferation and DNA methylation in the liver of female B6C3F1 mice. Gavage doses of 0, 0.48, or 1.44 mmol/ kg ( 0, 100, or 300 mg/ kg, respectively) were administered to test animals ( 7 8 weeks old; 10/ group) daily for five days, off for two days, and then again daily for four days. The high dose was selected on the basis that it had previously been demonstrated to be carcinogenic in female mice. Dibromochloromethane was administered in drinking water at approximately 75% of the saturation level, resulting in an average daily dose of 0.82 mmol/ kg ( 171 mg/ kg). The mice were sacrificed 24 hours after the last gavage dose and the livers were removed, weighed, and processed for histopathological examination, proliferating cell nuclear antigen  labeling index ( PCNA LI) analysis, and determination of c myc methylation status. For histopathological analysis, stained liver sections were evaluated for toxicity using a semi quantitative procedure using the following severity scoring system: Grade 1 consisted of mid lobular ballooning hepatocytes; Grade 2 consisted of mid lobular ballooning hepatocytes extending to the central vein; Grade 3 consisted of centrilobular necrosis with ballooning hepatocytes; and Grade 4 consisted of necrosis extending from the central vein to the mid lobule zone. A significant, dose dependent increase in relative liver weight was observed in animals dosed by gavage; however, relative liver weight was unaffected in animals administered the compound in drinking water, when compared to controls. At the low gavage dose, liver toxicity consisted mainly of a Grade 1 response. At the high dose, liver toxicity consisted mainly of a Grade 2 response. No incidence data were provided in the study report, nor was a severity grade reported for the control group. The histopathology findings for animals receiving bromodichloromethane in the drinking water were similar to those observed in the low dose gavage group. Dibromochloromethane administered by gavage caused a dose dependent increase in the PCNA LI. The increases observes at each dose were significantly different from the control. There was no significant effect on PCNA LI when the compound was administered in drinking water. Administration of dibromochloromethane by gavage or in drinking water decreased methylation of the c myc gene. A LOAEL of 100 mg/ kg, the lowest dose tested, was identified on the basis of liver toxicity ( ballooning hepatocytes) and increased cell proliferation in gavaged animals.

3. Bromoform Chu et al. ( 1982a) administered bromoform to male Sprague Dawley rats ( 10/ group) in drinking water for 28 days at dose levels of 0, 5, 50, or 500 ppm. Based on recorded fluid intake, these levels corresponded to doses of 0, 0.7, 8.5, or 80 mg/ kg day, as calculated by the authors. Draft  Do Not Cite or Quote February 20, 2003 V  25 The authors observed no effects on growth rate or food consumption and no signs of toxicity throughout the exposure. No dose related biochemical or histologic changes were detected ( no data provided). This study identified a NOAEL of 80 mg/ kg day, but the reported data were too limited to allow an independent confirmation.

Munson et al. ( 1982) administered bromoform by aqueous gavage to male and female CD 1 mice ( 6 to 12/ sex/ group) for 14 days at levels of 0, 50, 125, or 250 mg/ kg day. Parameters observed included body and organ weights, hematology, clinical chemistry, and humoral and cellmediated immune system functions. Body weights were significantly decreased in high dose females, while body weights in males were significantly increased at the mid and high doses. Absolute and relative liver weights were significantly increased in males at the mid and high dose and in females at the high dose. Absolute spleen weight was also decreased in mid and high dose females. Hematologic effects included significantly decreased fibrinogen in males at the high dose and significantly decreased prothrombin time in all treated males. The changes in prothrombin time, however, were not dose related. Significant clinical chemistry findings included decreased glucose levels ( high dose males), increased AST activity ( high dose groups), and decreased BUN levels ( high dose males). Both the humoral and cell mediated immune systems appeared to be affected in males at the high dose with a significant decrease in antibody forming cells and a significant decrease in delayed type hypersensitivity response. The authors stated that no treatment related effects on the immune system in females were observed ( no data were reported). Based on changes in clinical chemistry parameters, this study identified a NOAEL of 125 mg/ kg day and a LOAEL of 250 mg/ kg day.

Condie et al. ( 1983) investigated the renal and hepatic toxicity of bromoform. Male CD 1 mice ( 8 to 16/ group) were administered 0, 72, 145, or 289 mg/ kg day of bromoform by gavage in corn oil for 14 days. Biochemical evidence of liver damage ( elevated ALT) and kidney damage ( decreased PAH uptake by kidney slices) was observed at the high dose, but not at the mid or low dose. Histopathological examination revealed no consistent or important changes at the low or mid doses, with minimal to moderate liver and kidney injury at the high dose. Specific microscopic changes included intratubular mineralization, epithelial hyperplasia, mesangial hypertrophy and mesangial nephrosis in the kidney, and centrilobular pallor, mitotic figures, focal inflammation, and cytoplasmic vacuolation in the liver. On this basis, this study identified a NOAEL value of 145 mg/ kg day and a LOAEL value of 289 mg/ kg day.

NTP ( 1989a) investigated the short term oral toxicity of bromoform in F344/ N rats and B6C3F 1 mice. Groups of male and female rats ( 5/ sex/ group) and female mice ( 5/ group) were administered doses of 0, 100, 200, 400, 600, or 800 mg/ kg day of bromoform in corn oil by gavage for 14 days. Male mice were administered 0, 50, 100, 200, 400, or 600 mg/ kg day. All rats that were dosed at 600 or 800 mg/ kg day died before the end of the study. At 400 mg/ kgday only one male rat died before study termination. These rats exhibited lethargy, labored breathing, and ataxia. At 400 mg/ kg day, final body weights were decreased by 14% in male rats and by 4% in female rats relative to controls. In mice, one male and one female administered the high dose died before study termination. At dose levels of 600 mg/ kg day or above, ataxia and lethargy were noted. Final body weights of mice were comparable to those of the controls. Draft  Do Not Cite or Quote February 20, 2003 V  26 Raised stomach nodules were observed in males at 400 and 600 mg/ kg day and in females at 600 and 800 mg/ kg day. Based on decreased body weight and mortality in rats and on stomach nodules in mice, this study identified a NOAEL of 200 mg/ kg day and a LOAEL of 400 mg/ kgday

Aida et al. ( 1992a) administered bromoform to Slc: Wistar rats ( 7/ sex/ group) for one month at dietary levels of 0%, 0.068%, 0.204%, or 0.612% for males and 0%, 0.072%, 0.217%, or 0.651% for females. The test material was microencapsulated and mixed with powdered feed; placebo granules were used for the control groups. Based on the mean food intakes, the study authors reported the mean compound intakes as 0, 61.9, 187.2, or 617.9 mg/ kg day for males and 0, 56.4, 207.5, or 728.3 mg/ kg day for females. Clinical effects, body weight, food consumption, hematology parameters, serum chemistry, and histopathology of all major organs were determined. Body weights were significantly reduced in high dose males relative to the controls. High dose animals of both sexes exhibited slight piloerection and emaciation. Relative liver weight was significantly increased in mid and high dose males and females. Significant changes in serum chemistry were primarily observed in the mid and high dose animals with the females more significantly affected. These changes included significant decreases in ( a) serum glucose in low and high dose males and in mid and high dose females, ( b) triglycerides in high dose males and in mid and high dose females, ( c) cholinesterase activity in high dose males and in all female treatment groups, ( d) LDH in mid and high dose females, and ( e) BUN in mid and high dose females. All of these changes in the groups noted exhibited strong dose related trends with the exception of serum glucose in males. Creatinine levels and alkaline phosphatase activity were also significantly decreased in all female treatment groups, but the changes were not dose related. Significant increases, although not dose related, were observed for phospholipids and cholesterol in mid and high dose animals with the exception of a nonsignificant increase in phospholipids in high dose females. The only change of clear biological significance at the low dose was a decrease in cholinesterase activity in females. No effect was observed on any hematology parameter. Microscopic and macroscopic findings were limited to the liver. Specifically, discoloration was observed in all males and females in the high dose group. The incidence and severity of liver cell vacuolization and swelling were dose related. Severe hepatic cell vacuolization was observed in 5/ 7 high dose males and in 6/ 7 females at the mid and high dose. Slight to moderate liver cell swelling was observed in three high dose males, while all high dose females displayed slight signs of liver cell swelling. Females appeared to be more sensitive for development of histopathological effects, but the changes observed in low dose females were not considered an adverse effect. Based on the histopathology and serum chemistry changes in the mid dose animals, this study identified NOAELs of 61.9 mg/ kg day for males and 56.4 mg/ kg day for females, and LOAELs of 187.2 mg/ kg day for males and 207.5 mg/ kg day for females.

Potter et al. ( 1996) evaluated the effect of bromoform on hyaline droplet formation and cell proliferation in the kidney of male F344 rats. Animals ( 4/ dose) received doses of 0.75 or 1.5 mmol/ kg of bromoform in 4% Emulphor ® by gavage for 1, 3, or 7 days. These doses correspond to 190 or 379 mg/ kg day, respectively. No exposure related increase in hyaline droplet formation was observed. Cell proliferation in the kidney following bromoform exposure was measured in vivo by [ 3H] thymidine incorporation. No statistically significant effects were noted Draft  Do Not Cite or Quote February 20, 2003 V  27 following exposures of up to 7 days duration. Binding of bromoform to  2u globulin was not measured.

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. There were no treatment related signs of overt toxicity observed during the study. Body weight and water intake were not significantly altered at any dose tested. However, 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 were 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. These data suggest a NOAEL of 200 mg/ kg day and a LOAEL of 500 mg/ kg day based on increased hepatocyte proliferation.

Coffin et al. ( 2000) examined the effect of bromoform administered by gavage in corn oil or in drinking water on liver toxicity, cell proliferation and DNA methylation in female B6C3F1 mice. Gavage doses of 0, 0.79, or 1.98 mmol/ kg ( 0, 200, or 500 mg/ kg, respectively) were administered to test animals ( 7 8 weeks old; 10/ group) daily for five days, off for two days, and then again daily for 5 days. The high dose was selected on the basis that it had previously been demonstrated to be carcinogenic in female mice. Bromoform was administered in drinking water at approximately 75% of the saturation level, resulting in an average daily dose of 1.19 mmol/ kg ( 301 mg/ kg). The mice were sacrificed 24 hours after the last gavage dose and the livers were removed, weighed, and processed for histopathological examination, proliferating cell nuclear antigen  labeling index ( PCNA LI) analysis, and determination of c myc methylation status. For histopathological analysis, stained liver sections were evaluated for toxicity using a semiquantitative procedure using the following severity scoring system: Grade 1 consisted of mid lobular ballooning hepatocytes; Grade 2 consisted of mid lobular ballooning hepatocytes extending to the central vein; Grade 3 consisted of centrilobular necrosis with ballooning hepatocytes; and Grade 4 consisted of necrosis extending from the central vein to the mid lobule zone. A significant, dose dependent increase in relative liver weight was observed in animals dosed by gavage; however, relative liver weight was unaffected in animals administered the compound in drinking water, when compared to controls. At the low gavage dose, liver toxicity consisted mainly of a Grade 1 response. At the high dose, liver toxicity consisted mainly of a Grade 2 response. No incidence data were provided in the study report, nor were severity data presented for the control group. The histopathology findings for animals receiving bromoform in the drinking water were similar to those observed in the low dose gavage group. Bromoform administered by gavage caused a significant, dose dependent increase in the PCNA LI. Bromoform also significantly enhanced cell proliferation when the compound was administered in drinking water. Administration of bromoform by gavage or in drinking water decreased methylation of the c myc gene. A LOAEL of 200 mg/ kg, the lowest dose tested, was identified on the basis of liver toxicity in gavaged animals. Draft  Do Not Cite or Quote February 20, 2003 V  28 C. Subchronic Exposure This section addresses studies of brominated trihalomethanes that are of approximately 90 days in duration. Table V 4 summarizes the details of these subchronic studies. Draft  Do Not Cite or Quote February 20, 2003 V  29 Table V 4 Summary of Subchronic Toxicity Studies for Brominated Trihalomethanes Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Bromodichloromethane Oral Exposure Chu et al. ( 1982b) Rat SD* Drinking water M 20 90 days 0 0.57 6.5 53 212 Non dose dependent hepatic and thyroid lesions Chu et al. ( 1982b) Rat SD Drinking water F 20 90 days 0 0.75 6.9 57 219 Non dose dependent hepatic and thyroid lesions NTP ( 1987) Rat F344/ N Gavage ( corn oil) M, F 10 13 weeks ( 5 d/ wk) 0 19 38 75 ( NOAEL) 150 ( LOAEL) 300 Reduced body weight gain NTP ( 1987) Mouse B6C3F1 Gavage ( corn oil) M 10 13 weeks ( 5 d/ wk) 0 6.3 13 25 50 ( NOAEL) 100 ( LOAEL) Focal necrosis of proximal renal tubular epithelium NTP ( 1987) Mouse B6C3F1 Gavage ( corn oil) F 10 13 weeks ( 5 d/ wk) 0 25 50 100 ( NOAEL) 200 ( LOAEL) 400 Hepatic microgranulomas Inhalation Exposure Torti et al. 2001 Mouse C57BL/ 6 FVB/ N p53 ( heterozygous Vapor M Not reported 13 weeks ( 6 h/ day) 0 ppm 0.5 ppm 3 ppm 10 ppm 15 ppm Text reported minimal cortical scarring and occasional regenerative tubules in C56BL/ 6 mice and mild renal cortical tubular karyocytomegaly. Concentrations at which these effects occurred were not reported. Table V 4 ( cont.)

Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Draft  Do Not Cite or Quote February 20, 2003 V  30 Dibromochloromethane Chu et al. ( 1982b) Rat SD Drinking water M 20 90 days 0 0.57 6.1 49 ( NOAEL) 224 ( LOAEL) Hepatic lesions Chu et al. ( 1982b) Rat SD Drinking water F 20 90 days 0 0.64 6.9 55 ( NOAEL) 236 ( LOAEL) Hepatic lesions NTP ( 1985) Rat F344/ N Gavage ( corn oil) M, F 10 13 weeks ( 5 d/ wk) 0 15 30 ( NOAEL) 60 ( LOAEL) 125 250 Hepatic vacuolization indicative of fatty metamorphosis ( males)

NTP ( 1985) Mouse B6C3F1 Gavage ( corn oil) M, F 10 13 weeks ( 5 d/ wk) 0 15 30 60 125 ( NOAEL) 250 ( LOAEL) Fatty liver and toxic nephropathy in males Daniel et al. ( 1990) Rat SD Gavage ( corn oil) M, F 10 90 days 0 50 ( LOAEL) 100 200 Hepatic vacuolization ( males); renal lesions ( females)

Bromoform Chu et al. ( 1982b) Rat SD Drinking water M 20 90 days 0 0.65 6.1 57 ( NOAEL) 218 ( LOAEL) Hepatic lesions and vacuolation Chu et al. ( 1982b) Rat SD Drinking water F 20 90 days 0 0.64 6.9 55 ( NOAEL) 283 ( LOAEL) Hepatic lesions and vacuolation Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Draft  Do Not Cite or Quote February 20, 2003 V  31 NTP ( 1989a) Rat F344/ N Gavage ( corn oil) M, F 10 13 weeks ( 5 d/ wk) 0 12 25 ( NOAEL) 50 ( LOAEL) 100 200 Hepatic vacuolation in males NTP ( 1989a) Mouse B6C3F1 Gavage ( corn oil) M, F 10 13 weeks ( 5 d/ wk) 0 25 50 100 ( NOAEL) 200 ( LOAEL) 400 Hepatic vacuolation in males * SD, Sprague Dawley 1. Bromodichloromethane Chu et al. ( 1982b) administered bromodichloromethane to male and female weanling Sprague Dawley rats ( 20/ sex/ dose) in drinking water at levels of 0, 5, 50, 500, or 2,500 ppm for 90 days. Half of each group ( 10/ sex/ dose) was sacrificed at the end of the exposure period, and the remaining animals were given tap water for another 90 days. As calculated by the authors ( using data on water consumption and the average initial and final body weights in the vehicle controls and the high dose groups), these levels corresponded to doses of approximately 0, 0.57, 6.5, 53, and 212 mg/ kg day for males and 0, 0.75, 6.9, 57, and 219 mg/ kg day for females. At 2,500 ppm, food consumption was significantly depressed and significant growth suppression occurred in both males and females. Mild histologic changes were observed in the liver and thyroid of the male animals. Neither incidence nor severity were clearly dose related. Specifically, the incidence of hepatic lesions was increased in males at concentrations equal to or greater than 50 ppm, with similar statistically significant increases in the severity of these lesions in these dose groups compared to the control. The author noted that the hepatic lesions were mild and similar to the control following the 90 day recovery period. Increased incidence of thyroid lesions was also observed in males at concentrations equal to or greater than 50 ppm. The severity of these lesions was similar to that observed in the control group. These lesions were also mild and similar in nature to those of the control after the 90 day recovery period. The incidence of hepatic lesions in the female treatment groups ( 3 5/ 10) was slightly increased compared to that of the control group ( 0/ 10) with the severity significantly increased in the 50 and 2,500 ppm treatment groups, but not in the 500 ppm group. No significant numbers of females were reported as having thyroid lesions. Lack of a clear dose response relationship for either incidence or severity of lesions prevented identification of reliable NOAEL or LOAEL values.

NTP ( 1987) administered doses of 0, 19, 38, 75, 150, or 300 mg/ kg day of bromodichloromethane to male and female F344/ N rats ( 10/ sex/ dose) by gavage in corn oil for 5 days/ week for 13 weeks. The low dose group was administered 1.9 mg/ kg day for the first Draft  Do Not Cite or Quote February 20, 2003 V  32 3 weeks of the study. A necropsy was performed on all animals. Before study termination, 50% of the males and 20% of the females in the high dose group died. Although food consumption was not recorded, animals in the high dose groups appeared to eat less food. These animals were also emaciated. At 300 mg/ kg day, final body weights of the males and females were decreased by 55% and 32%, respectively, relative to the controls. At 150 mg/ kg day, final body weights of the males and females were decreased by 30% and 12%, respectively, relative to the controls. Treatment related lesions were observed only at the high dose. At 300 mg/ kg day in males, centrilobular degeneration of the liver and occasional necrotic cells were observed in 4/ 9 animals. Mild bile duct hyperplasia was also observed in these animals. Kidney lesions in high dose males consisted of degeneration of renal proximal tubular epithelial cells ( 4/ 9) and definite foci of coagulative necrosis of the tubular epithelium ( 2/ 9). High dose males ( 4/ 9) also exhibited lymphoid degeneration of the thymus, spleen, and lymph nodes, and mild to moderate atrophy of the seminal vesicles and/ or prostate. Enlarged hepatocytes were observed in females ( 2/ 9) at 300 mg/ kg day. Although degeneration of the spleen, thymus, and lymph nodes was noted in high dose females, the extent of the atrophy was much less than that observed in males. This study identified a NOAEL of 75 mg/ kg day and a LOAEL of 150 mg/ kg day based on reduced body weight gain.

In a parallel experiment, NTP ( 1987) administered bromodichloromethane in corn oil by gavage to male and female B6C3F 1 mice ( 10/ sex/ dose) for 5 days/ week for 13 weeks. Doses were 0, 6.25, 12.5, 25, 50, or 100 mg/ kg day for males and 0, 25, 50, 100, 200, or 400 mg/ kgday for females. All animals survived to the end of the study. The final body weights of highdose males were decreased by 9% relative to the controls. The final body weights of females that received 200 and 400 mg/ kg day were decreased 5% and 6%, respectively, relative to the controls. No treatment related clinical signs were noted. Treatment related lesions were observed only at 100 mg/ kg day in males and at 200 and 400 mg/ kg day in females. Kidney lesions in high dose males included focal necrosis of the proximal renal tubular epithelium ( 6/ 10) and nephrosis of minimal severity ( 2/ 10). Microgranulomas were observed in the liver of 70% of the females that received the 200 mg/ kg day dose. NOAEL and LOAEL values for female mice were 100 and 200 mg/ kg day, respectively, based on occurrence of microgranulomas. This study identified a NOAEL of 50 mg/ kg day and a LOAEL of 100 mg/ kg day for male mice on the basis of liver histopathology.

Torti et al. ( 2001) reported results from a 13 week interim sacrifice conducted as part of an inhalation cancer bioassay in p53 heterozygous C57BL/ 6 and FVB/ N male mice. Test animals were exposed to vapor concentrations of 0, 0.5, 3, 10, or 15 ppm, 6 hours/ day for 13 weeks. No exposure related effects were noted for mortality, morbidity, relative body weight, relative kidney or liver weight, or cell proliferation in liver, kidney or bladder. Histopathologic lesions were limited to the kidney. The study authors reported minimal cortical scarring and occasional regenerative tubules in the C57BL/ 6 strain. The only lesion reported for the FVB/ N strain was limited to mild renal cortical tubular karyocytomegaly. No incidence data were presented for these lesions and the concentrations at which they occurred was not stated.

2. Dibromochloromethane Draft  Do Not Cite or Quote February 20, 2003 V  33 Chu et al. ( 1982b) administered dibromochloromethane to male and female weanling Sprague Dawley rats ( 20/ sex/ dose) in drinking water at levels of 0, 5, 50, 500, or 2,500 ppm for 90 days. Half of each group ( 10/ sex/ dose) was sacrificed at the end of the exposure period, and the remaining animals were given tap water for another 90 days. Based on calculations by the authors, these levels corresponded to doses of approximately 0, 0.57, 6.1, 49, and 224 mg/ kg day for males and 0, 0.64, 6.9, 55, and 236 mg/ kg day for females. At 2,500 ppm, food consumption was depressed in both males and females, with the decrease reaching statistical significance in the males. Body weight gain was also decreased at the high dose, but not significantly. Mild histologic changes occurred in the liver and thyroid in both males and females. Neither the incidence nor severity exhibited clear dose response trends with the possible exception of the incidence and severity of hepatic lesions in the males. The severity of hepatic lesions was significantly increased at 50 ppm in females and at 2,500 ppm in both males and females. Hepatic lesions included increased cytoplasmic volume and vacuolation due to fatty infiltration. Lesions of the thyroid included decreased follicular size and colloid density and occasional focal collapse of follicles. The severity of these lesions was not significantly different from that of the control. The authors noted that histological changes were mild and similar to controls when evaluated after the 90 day recovery period. These data identified a NOAEL of 49 mg/ kg day and a LOAEL of 224 mg/ kg day for males, and a NOAEL of 55 mg/ kg day and a LOAEL of 236 mg/ kg day for females.

NTP ( 1985) administered dibromochloromethane by gavage in corn oil to male and female F344/ N rats ( 10/ dose/ sex). Doses of 0, 15, 30, 60, 125, or 250 mg/ kg were given 5 days/ week for 13 weeks. Animals were weighed weekly. All animals were submitted for gross necropsy, while histopathology was conducted on animals in the control and high dose groups with the exception that the liver was examined in all males and in females at 125 mg/ kg day, and that the kidney and salivary glands were examined in males and females at 125 mg/ kg day. Only one male and one female in the high dose group survived, with most deaths occurring during weeks 8 to 10. At 125 mg/ kg day, final body weights of males were decreased 7% relative to controls. Histopathological examination revealed severe lesions and necrosis in kidney, liver, and salivary glands, primarily at the high dose. Males, however, exhibited a dose dependent increase in the frequency of clear cytoplasmic vacuoles indicative of fatty metamorphosis in the liver; this effect was statistically significant at doses of 60 mg/ kg day or higher. On this basis, this study identified a NOAEL of 30 mg/ kg day and a LOAEL of 60 mg/ kg day in rats for dibromochloromethane. BMD modeling was conducted on the incidence of fatty metamorphosis in male rats.

NTP ( 1985) performed a similar 13 week gavage study with dibromochloromethane in male and female B6C3F 1 mice ( 10/ sex/ dose). The doses and dosing schedule were the same as for the rat study. No treatment related effects on body weight or histopathology were observed at doses of 125 mg/ kg day or lower. At the high dose, final body weights of males and females were decreased by 6% relative to controls. Fatty metamorphosis of the liver and toxic nephropathy were observed in high dose males, but not in high dose females. This study identified a NOAEL of 125 mg/ kg day and a LOAEL of 250 mg/ kg day in mice. Draft  Do Not Cite or Quote February 20, 2003 V  34 Daniel et al. ( 1990) administered gavage doses ( in corn oil) of 0, 50, 100, or 200 mg/ kgday of dibromochloromethane to male and female Sprague Dawley rats ( 10/ sex/ dose) for 90 consecutive days. Individual dosages were adjusted weekly based on individual body weights. During the final week of the study, urinalysis was conducted following an overnight fast. Ophthalmoscopic examinations were performed prior to treatment and during the last week of the study. Hematology, serum clinical chemistry, and a thorough histopathologic examination were also conducted. No deaths, clinical signs of toxicity, or treatment related changes in the ophthalmoscopic examinations or hematology were observed. Final body weights were significantly reduced in the high dose groups by 32% in males and by 13% in females. Body weight decreases in the other groups were less than 10% of control weights. A dose related increase was observed in liver weight in females that reached statistical significance at the high dose. Clinical chemistry values indicative of hepatotoxicity and suggestive of nephrotoxicity included increased levels of alkaline phosphatase ( high dose males and females), ALT ( mid and high dose males), and creatinine ( mid and high dose males and high dose females), and decreased potassium levels ( high dose males). Centrilobular lipidosis ( vacuolization) was observed in the liver of almost all high dose males and females and all mid and low dose males ( with one exception at each level), but in only one mid dose female. The severity of the effect was dose related. Centrilobular necrosis was also observed in high dose males and females. Slight to moderate degeneration within the kidney proximal tubular cells occurred in all high dose males and females and to a lesser extent in mid dose males and low and mid dose females. Based on the liver histopathology in males and kidney histopathology in females, the LOAEL for dibromochloromethane in this study was 50 mg/ kg day.

3. Bromoform Chu et al. ( 1982b) administered bromoform to male and female weanling Sprague Dawley rats ( 20 rats/ sex/ group) for 90 days in drinking water at levels of 0, 5, 50, 500, or 2,500 ppm. Half of each group ( 10/ sex/ dose) was sacrificed at the end of the exposure period, and the remaining animals were given tap water for another 90 days. Based on calculations by the authors, these levels corresponded to doses of approximately 0, 0.65, 6.1, 57, and 218 mg/ kg day for males and 0, 0.64, 6.9, 55, and 283 mg/ kg day for females. At 2,500 ppm, food consumption was depressed in both males and females, with the decrease reaching statistical significance in males. Body weight gain was also decreased at the high dose, but not significantly. Lymphocyte counts were significantly decreased in high dose males and females when evaluated 90 days after cessation of treatment. The only change in serum biochemistry was a significant decrease in LDH in both males and females at the high dose. This effect was also noted 90 days after cessation of treatment. Mild histologic changes occurred in the liver and thyroid of male and female animals. Although neither incidence nor severity were clearly dose related, these parameters did tend to increase with dose. The severity of hepatic lesions was significantly increased in high dose males and in females at 500 and 2,500 ppm. Hepatic lesions included increased cytoplasmic volume and vacuolation due to fatty infiltration. Lesions of the thyroid included decreased follicular size and colloid density and occasional focal collapse of follicles. The severity of these lesions in the treated animals was not significantly different from that in the controls. Although the authors noted that histologic changes were mild and similar to controls when evaluated after the 90 day Draft  Do Not Cite or Quote February 20, 2003 V  35 recovery period, males in the high dose group continued to exhibit an increased incidence of hepatic lesions with greater severity relative to the control. These data identified a NOAEL of 57 mg/ kg day and a LOAEL of 218 mg/ kg day for males, and a NOAEL of 55 mg/ kg day and a LOAEL of 283 mg/ kg day for females.

NTP ( 1989a) exposed male and female F344/ N rats to bromoform by gavage for 5 days/ week for 13 weeks. Animals ( 10/ sex/ dose) received doses of 0, 12, 25, 50, 100, or 200 mg/ kg day. None of the rats died before the end of the study, and body weights were not significantly affected. All high dose animals, as well as males dosed with 100 mg/ kg day, were lethargic. At sacrifice, tissues were examined for gross and histologic changes. A dosedependent increase in the frequency of hepatocellular vacuolation was observed in male rats, which reached statistical significance at 50 mg/ kg day ( IRIS, 1993b). These hepatic effects were not observed in females. This study identified a NOAEL of 25 mg/ kg day and a LOAEL of 50 mg/ kg day, on the basis of the hepatic vacuolation seen in male rats.

In a parallel study, NTP ( 1989a) exposed male and female B6C3F 1 mice to bromoform by gavage for 5 days/ week for 13 weeks. Animals ( 10/ sex/ dose) received doses of 0, 25, 50, 100, 200, or 400 mg/ kg day. One female died at 100 mg/ kg day, but no other deaths at any other dose level occurred. At sacrifice, tissues were examined for gross and histologic changes. Body weights were not significantly affected, although males receiving 400 mg/ kg day had body weights about 8% less than controls. A dose related increase in the number of hepatocellular vacuoles was seen in male mice ( incidence of 5/ 10 at 200 mg/ kg and 8/ 10 at 400 mg/ kg reported in text; incidence in controls not explicitly stated), but not in females. Based on hepatocellular vacuolation, this study identified a NOAEL of 100 mg/ kg day and a LOAEL of 200 mg/ kg day in male mice.

D. Chronic Exposure This section addresses studies on the health effects of brominated trihalomethanes that are of one to two years in duration. These studies are summarized in Table V 5.

1. Bromodichloromethane Tobe et al. ( 1982) evaluated the chronic effects of bromodichloromethane administered in the diet to male and female Slc: Wistar SPF rats ( 40/ sex/ group) for 24 months. The histopathology data for the animals exposed to bromodichloromethane in this study were reported by Aida et al. ( 1992b). The animals were 5 weeks old at the start of the study and weighed approximately 100 g. Bromodichloromethane was microencapsulated, and an appropriate amount was mixed with powdered feed. The concentrations administered were 0.0. 0.014, 0.055, or 0.22%. Control groups ( 70 rats/ sex) received microcapsules without the test compound. Body weight and food consumption were monitored weekly for the first 6 months, every 2 weeks from 6 to 12 months, and every 4 weeks during the second year of the study. Interim sacrifices of at least 9 animals/ sex/ control group and 5 animals/ sex/ dose group were conducted at 6, 12, and 18 Draft  Do Not Cite or Quote February 20, 2003 V  36 months. All surviving animals were sacrificed at 2 years. At each time of sacrifice, necropsies, hematology, and serum biochemistry were conducted. Based on mean food intakes, the reported average doses were approximately 0, 6, 26, or 138 mg/ kg day for males and 0, 8, 32, or 168 mg/ kg day for females ( Aida et al., 1992b). Marked suppression of body weight gain was seen in males and females of the high dose group. Males and females of the high dose group exhibited mild piloerection and emaciation. Relative liver weight was significantly increased in the mid and high dose groups, while relative kidney weight was significantly increased only in the high dose groups. At 18 months, dose dependent reductions Draft  Do Not Cite or Quote February 20, 2003 V  37 Table V 5 Summary of Chronic Toxicity Studies for Brominated Trihalomethanes Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Bromodichloromethane NTP ( 1987) Rat F344/ N Gavage ( corn oil) M, F 50 2 years ( 5 d/ wk) 0 50 ( LOAEL) 100 Renal and hepatic histopathology NTP ( 1987) Mouse B6C3F1 Gavage ( corn oil) M 50 2 years ( 5 d/ wk) 0 25 ( LOAEL) 50 Renal and hepatic histopathology NTP ( 1987) Mouse B6C3F1 Gavage ( corn oil) F 50 2 years ( 5 d/ wk) 0 75 ( LOAEL) 150 Reduced body weight gain Aida et al. ( 1992b); Tobe et al. ( 1982) Rat Wistar Diet M 40 2 years 0 6 ( LOAEL) 26 138 Hepatic vacuolization, serum chemistry Aida et al. ( 1992b); Tobe et al. ( 1982) Rat Wistar Diet F 40 2 years 0 8 ( NOAEL) 32 ( LOAEL) 168 Hepatic vacuolization, serum chemistry Klinefelter et al. ( 1995) Rat F344 Drinking water M 7 1 year 0 22 39 ( NOAEL) No evidence of treatment related histopathological or organ weight effects ( see reproductive effects, section V. E for additional data from this study)

Dibromochloromethane Tobe et al. ( 1982) Rat Wistar SPF Diet M 40 2 years 0 12 ( NOAEL) 49 ( LOAEL) 196 Serum biochemistry, liver appearance at necropsy; decreased body weight gain Tobe et al. ( 1982) Rat Wistar SPF Diet F 40 2 years 0 17 ( NOAEL) 70 ( LOAEL) 278 Serum biochemistry, liver appearance at necropsy; decreased body weight gain NTP ( 1985) Rat F344 Gavage ( corn oil) M, F 50 2 years ( 5 d/ wk) 0 40 ( LOAEL) 80 Histologic changes in liver, including fat accumulation and ground glass appearance, and altered basophilic staining NTP ( 1985) Mouse B6C3F1 Gavage ( corn oil) M, F 50 105 weeks ( 5 d/ wk) 0 50 ( LOAEL) 100 Fatty metamorphosis in liver and follicular cell hyperplasia in thyroid Table V 5 ( cont.)

Reference Species Route Sex Number per dose group Duration Dose ( mg/ kg day) Results Draft  Do Not Cite or Quote February 20, 2003 V  38 Bromoform Tobe et al. ( 1982) Rat Wistar SPF Diet M 40 2 years 0 22 ( NOAEL) 90 ( LOAEL) 364 Enzyme changes and altered liver appearance at necropsy Tobe et al. ( 1982) Rat Wistar SPF Diet F 40 2 years 0 38 ( NOAEL) 152 ( LOAEL) 619 Enzyme changes and altered liver appearance at necropsy NTP ( 1989a) Rat F344/ N Gavage ( corn oil) M, F 50 103 weeks ( 5 day/ wk) 0 100 ( LOAEL) 200 Decreased body weight, lethargy, mild hepatotoxicity NTP ( 1989a) Mouse B6C3F1 Gavage ( corn oil) M 50 103 weeks ( 5 day/ wk) 0 50 100 ( NOAEL) No observed effects on body weight or hepatotoxicity NTP ( 1989a) Mouse B6C3F1 Gavage ( corn oil) F 50 103 weeks ( 5 day/ wk) 0 100 ( LOAEL) 200 Decreased body weight, minimal to mild fatty changes in liver * SD, Sprague Dawley Draft  Do Not Cite or Quote February 20, 2003 V  39 in serum cholinesterase activity and increases in   glutamyl transpeptidase (   GTP) activity ( indicative of bile duct proliferation) were observed in males, with the changes significant at the high dose. The mid and high dose males also displayed a 27% and 65% reduction, respectively, in total serum triglycerides ( T Gly) levels when compared to the control group. At 18 months, serum cholinesterase levels were significantly decreased, while total cholesterol levels were significantly increased, in all dose groups for the treated females. Serum T Gly levels ( decreased) and   GTP activity ( increased) were also reported to deviate significantly from control values in the mid and high dose females. The most sensitive markers at 24 months were T GLY and serum cholinesterase, with significant changes seen in all of the male treatment groups. Gross necropsy revealed dose related yellowing and roughening of the liver surface. Treatment related lesions were limited to the liver. At 24 months, fatty degeneration and granuloma were observed in all dose groups with the exception of granulomas in low dose females. Specifically, fatty degeneration and granulomas were observed in low dose males, but not control males, and fatty degeneration was observed in low dose females at a higher rate ( 8/ 19) than in control females ( 2/ 32). Cholangiofibrosis was also observed in the high dose groups. Bile duct proliferation was observed in most high dose animals at 6 months, and was prevalent in the controls and all dose groups by 24 months. Histopathology was observed in all dose groups as early as 6 months with the exception of low dose females. Based on the results of Tobe et al. ( 1982) alone, the NOAEL was 6 mg/ kg day in males and 8 mg/ kg day in females. The LOAEL was identified as 26 ( males) to 32 ( females) mg/ kg day, based on serum enzyme changes and altered liver appearance. Based on the histopathology data reported for this study by Aida et al. ( 1992b), however, the entire study identified a LOAEL of 6 mg/ kg day in male rats and 8 mg/ kg day in female rats.

NTP ( 1987) administered doses of 0, 50, or 100 mg/ kg day of bromodichloromethane in corn oil by gavage to male and female F344/ N rats ( 50/ sex/ dose), 5 days/ week for 102 weeks. The authors observed all animals for clinical signs and recorded body weights ( by cage) once per week for the first 12 weeks of the study and once per month thereafter. A necropsy was performed on all animals, including those found dead, unless they were excessively autolyzed or cannibalized. During necropsy, all organs and tissues were examined for grossly visible lesions. Complete histopathology was performed on all female rats and on high dose and vehicle control male rats. Male rats in the low dose group that died early in the study were also examined histologically. Survival of dosed rats was comparable to that of vehicle controls. Mean body weight of high dose male and female rats was decreased during the last 1.5 years of the study; body weight gain of high dose male and female rats was 86% and 70% of the corresponding vehicle control values. Body weight gain of low dose male and female rats was comparable to that of the vehicle control group. No treatment related clinical signs were observed. In males, treatment related nonneoplastic effects included renal cytomegaly, tubular cell hyperplasia, hepatic necrosis, and fatty metamorphosis. In females, changes included eosinophilic cytoplasmic change, clear cell change, focal cellular change, fatty metamorphosis of the liver, and tubular cell hyperplasia of the kidney. Based on these histologic findings, this study identified a LOAEL of 50 mg/ kg day in rats.

NTP ( 1987) administered bromodichloromethane in corn oil by gavage to male and female B6C3F 1 mice ( 50/ sex/ dose), 5 days/ week for 102 weeks. For males, doses were 0, 25, or Draft  Do Not Cite or Quote February 20, 2003 V  40 50 mg/ kg day. For females, doses were 0, 75, or 150 mg/ kg day. Final survival of treated male mice was comparable to that of vehicle controls. At week 84, survival of female mice was greater than 50% in all dose groups. After week 84, survival of dosed and vehicle control female mice was reduced ( final survival: 26/ 50, 13/ 50, 15/ 50 for the 0, 5, and 50 mg/ kg day groups, respectively), and this decreased survival was associated with ovarian abscesses ( 8/ 50, 19/ 47, 18/ 49). Body weight gain of high dose male mice was 87% of that of the vehicle control group; the body weight gain of low dose male mice was comparable to that of the vehicle control group. Mean body weight of high dose female mice was decreased during the last 1.5 years of the study. The body weight gain was reduced 55% compared to the controls at the high dose and by 25% among low dose females. In males, treatment related nonneoplastic changes included fatty metamorphosis of the liver, renal cytomegaly, and follicular cell hyperplasia of the thyroid gland. In females, hyperplasia of the thyroid gland was observed. This study identified a LOAEL of 25 mg/ kg day, based on histopathological findings in male mice.

Klinefelter et al. ( 1995) reported interim ( 52 week) necropsy data from a cancer bioassay in which male F344 rats were administered average concentrations of 0, 330, or 620 mg/ L bromodichloromethane in drinking water. Corresponding doses of 0, 22, and 39 mg/ kg day were calculated by the authors using water consumption and body weight data. For the interim sacrifice, 7 animals per dose group were killed, and the testis, epididymis, liver, spleen, kidney, thyroid, stomach, intestine, and bladder were evaluated histopathologically. Bromodichloromethane had no effect on body weight or on the kidney, liver, spleen, or thyroid weight. There was no histopathological evidence of bromodichloromethane related noncancer or cancer effects on any of the examined organs. High levels of nephropathy and interstitial cell hyperplasia were observed, but these lesions were not treatment related. The NOAEL and LOAEL for this study are based on reproductive endpoints. These reproductive effects are summarized in Section V. E. 1.

2. Dibromochloromethane Tobe et al. ( 1982) evaluated the chronic effects of dibromochloromethane administered in the diet to male and female Slc: Wistar SPF rats ( 40/ sex/ group) for 24 months. The animals were 5 weeks old at the start of the test and weighed approximately 100 g. Dibromochloromethane was microencapsulated, and an appropriate amount was mixed with powdered feed. Control groups ( 70 rats/ sex) received microcapsules without test compound. Body weight and food consumption were monitored weekly for the first 6 months, every 2 weeks from 6 to 12 months, and every 4 weeks during the second year of the study. Data were reported from the sacrifices of 9 animals/ sex/ control group and 5/ sex/ dose group at 18 months; all surviving animals were sacrificed at 24 months. Necropsies, hematology, and serum biochemistry were conducted at the time of sacrifice. No histopathology data for dibromochloromethane have been published from this study. Dibromochloromethane was administered at dietary levels of 0.0%, 0.022%, 0.088%, or 0.35%. Based on reported body weights ( 150 to 475 g for males and 100 to 215 g for females) and food consumption ( 15 to 20 g/ day for males and 10 to 15 g/ day for females), these levels corresponded to doses of approximately 0, 12, 49, and 196 mg/ kg day for males and 0, 17, 70, and 278 mg/ kg day for females. Marked suppression of body weight gain was seen in males Draft  Do Not Cite or Quote February 20, 2003 V  41 and females at the high dose, and mild suppression of body weight gain ( about 10%) was seen in males and females at the mid dose. Decreased T GLY and serum cholinesterase activity and increased   GTP were seen in the mid and high dose males and females. Yellowing of the liver surface was noted in the mid and high dose groups, and roughening of the liver surface was noted in high dose males. This study suggests NOAELs of 12 mg/ kg day ( males) and 17 mg/ kgday ( females), and LOAELs of 49 mg/ kg day ( males) and 70 mg/ kg day ( females), based on serum biochemistry data, decreased body weight, and gross necropsy findings.

NTP ( 1985) investigated the chronic oral toxicity of dibromochloromethane in male and female F344/ N rats. Groups of 50 animals/ sex/ dose were administered doses of 0, 40, or 80 mg/ kg day by gavage in corn oil for 5 days/ week for 104 weeks. Survival was comparable in all dose groups. Body weight gain was decreased in high dose males after week 20; final weight gain was 88% of the control value. Females in both dose groups gained more weight than did the controls. Histologic lesions in the liver were observed in both males and females at both dose levels. Changes included fat accumulation, " ground glass" appearance of the cytoplasm, and altered basophilic staining. This study identified a LOAEL of 40 mg/ kg day for dibromochloromethane in rats.

NTP ( 1985) performed a similar chronic oral exposure study of dibromochloromethane toxicity in male and female B6C3F 1 mice. Groups of 50 animals/ sex/ dose were administered doses of 0, 50, or 100 mg/ kg day by gavage in corn oil for 5 days/ week for 105 weeks. Survival in females was not different from controls, while survival in high dose males was significantly decreased. An overdosing accident at week 58 killed 35/ 50 male mice in the low dose group, and this group was not considered further. Mean body weight was decreased in high dose males and females, but not in low dose females. Treatment related hepatocytomegaly and focal necrosis were observed in livers of high dose males. Females showed liver calcification at the high dose and fatty metamorphosis at both the low and high doses. An increased incidence of follicular cell hyperplasia in the thyroid was observed in low and high dose females relative to the control. Thyroid lesions were not observed in treated males. This study identified a LOAEL of 50 mg/ kgday for dibromochloromethane in mice.

3. Bromoform Tobe et al. ( 1982) evaluated the chronic effects of bromoform administered in the diet to male and female Slc: Wistar SPF rats ( 40/ sex/ group) for 24 months. The animals were 5 weeks old at the start of the test and weighed approximately 100 g. Bromoform was microencapsulated, and administered at dietary levels of 0.0%, 0.04%, 0.16%, or 0.65%. Control groups ( 70 rats/ sex) received microcapsules without test article. Body weights and food consumption were monitored weekly for the first 6 months, every 2 weeks from 6 to 12 months, and every 4 weeks during the second year of the study. Data were reported from the sacrifices of 9 animals/ sex in the control group and 5/ sex/ dose in the exposure groups at 18 months; all surviving animals were sacrificed at 24 months. At each time of sacrifice, necropsies, hematology, and serum biochemistry were conducted. No histopathology data for bromoform have been published from this study. Based on reported body weights ( 150 to 475 g for males Draft  Do Not Cite or Quote February 20, 2003 V  42 and 100 to 215 g for females) and food consumption ( 15 to 20 g/ day for males and 10 to 20 g/ day for females), these levels corresponded to doses of about 0, 22, 90, and 364 mg/ kg day for males and 0, 38, 152, and 619 mg/ kg day for females. Marked suppression of body weight gain was seen in males and females at the high dose, and mild suppression of body weight gain ( about 15%) was seen in males and females at the mid dose. Dose related decreases in non esterified fatty acids were observed in all treated males and in females at the mid and high dose. Females also exhibited a dose related increase in levels of   GTP with the increases significant at the mid and high dose. Other serum biochemistry changes in the high dose groups included decreased serum triglyceride ( T GLY) and increased AST and ALT activity. Specifically, T GLY levels significantly decreased by 86% and 80% in the male and female high dose groups, respectively, by study termination. AST and ALT activities at study termination were significantly increased 1.6 to 2.6 fold in animals at the high dose compared to controls, with the exception that the increase in AST activity in males was statistically nonsignificant. Yellowing and small white spots, and roughening of the surface were seen in the livers of the mid and high dose animals. Roughening of the liver surface was observed in the high dose groups. Based on the necropsy findings and the serum biochemistry data, this study indicated NOAELs of 22 mg/ kg day for males and 38 mg/ kgday for females, and LOAELs of 90 mg/ kg day for males and 152 mg/ kg day for females.

NTP ( 1989a) exposed male and female F344/ N rats ( 50/ sex/ group) to bromoform by gavage in oil for 103 weeks ( 5 days/ week) at doses of 0, 100, or 200 mg/ kg day. Animals were observed for clinical signs throughout the study ( 2 days/ week). At termination, necropsy and histopathological examination were performed on all animals. Body weight gain was decreased by 37% in high dose females and by 29% in high dose males relative to the respective controls. Survival of the high dose males was also decreased. Both males and females were lethargic. Hepatic fatty change and chronic inflammation were noted in both males and females at both doses, and minimal necrosis was increased in high dose males. Nonneoplastic changes were not reported in other tissues. This study identified a LOAEL of 100 mg/ kg day in both male and female rats.

NTP ( 1989a) exposed groups of 50 male B6C3F 1 mice by gavage in oil to doses of 0, 50, or 100 mg/ kg day of bromoform for 103 weeks ( 5 days/ week). Groups of 50 female mice were administered doses of 0, 100, or 200 mg/ kg day. Animals were observed for clinical signs 2 days/ week throughout the study. At termination, all animals were necropsied, and a thorough histological examination of tissues was performed. Decreased survival was observed in females, but not males. This was at least partly due to a utero ovarian infection. No clinical signs were noted. Body weight gains were 82% and 72% of the control values for low and high dose females, respectively, but body weight gain was not affected in males. Increased incidences of minimal to mild fatty changes were noted in the livers of both low and high dose females, but not males. Nonneoplastic changes were not detected in other tissues. This study identified a LOAEL of 100 mg/ kg day for female mice, based on decreased body weight and fatty changes of the liver. No NOAEL for females was identified. For males, a NOAEL of 100 mg/ kg day was identified. Draft  Do Not Cite or Quote February 20, 2003 V  43 E. Reproductive and Developmental Effects Studies that have examined the reproductive and developmental toxicity of the brominated trihalomethanes are summarized in Table V 9 at the end of this section.

1. Bromodichloromethane a. Studies in Rats Ruddick et al. ( 1983) investigated the teratogenicity and developmental toxicity of bromodichloromethane in Sprague Dawley rats. Pregnant dams ( 15/ dose group) were administered 0, 50, 100, or 200 mg/ kg day by gavage in corn oil on gestation days ( GD) 6 to 15. Body weights were measured on GD 1, on GD 1 through GD 15, and before and after fetuses were removed by caesarean section on GD 22. On GD 22, females were sacrificed and body tissues ( including the uterus) were removed for pathological examination. Females were evaluated for the number of resorption sites, and number of fetuses. Maternal blood samples were collected and evaluated for standard hematology and clinical chemistry parameters. The liver, heart, brain, spleen, and one kidney were weighed. Standard histopathology was conducted on control and high dose females ( 5/ group). All fetuses were individually weighed, and evaluated for viability and external malformations. Histopathologic examination was performed on two pups per litter. Of the remaining live fetuses, approximately two thirds were examined for skeletal alterations and one third for visceral abnormalities.

Although 15 inseminated females per dose group were exposed to bromodichloromethane not all females became pregnant and/ or delivered litters. Therefore, the number of litters per dose group ranged from 9 to 14. One animal died in the control group, but no deaths occurred in any of the exposed groups. In the high dose group, maternal weight gain was significantly depressed by 38% as compared with controls. Although maternal weight gains were also reduced in the low and mid dose groups ( 13% and 15%, respectively, as compared with controls), these differences were not reported as statistically significant. Relative maternal liver weight was significantly increased in all exposed groups ( 110%, 110%, and 117% for the low, mid, and high dose groups, respectively as compared with control values). Relative kidney and brain weights were also statistically increased in the high dose group only. These increases in relative organ weights may have been associated with the decreased body weight gains in treated females. No treatment related changes in hematology, clinical chemistry, histopathology, number of resorptions, and the number of fetuses per litter were noted. No differences between treated and control groups were reported for fetal weights, gross malformations ( terata), and visceral abnormalities. However, an increase in the incidence of sternebral anomalies was observed in all treated groups. The number of affected fetuses/ number of affected litters were 2/ 2, 8/ 4, 9/ 7, 10/ 6 for the control, low, mid, and high dose groups, respectively. Statistical significance of fetotoxic endpoints was not reported by the study authors. An independent statistical analysis ( using the Fisher Exact test) was conducted on the published data for development of this Criteria Document and demonstrated that none of these increases differed significantly from control values ( p> 0.05). A trend test showed a statistically significant dose related trend ( p= 0.03); stepwise Draft  Do Not Cite or Quote February 20, 2003 V  44 analysis indicated that the trend became nonsignificant if the high dose ( 200 mg/ kg day) was omitted from the analysis. These findings suggest that the LOAEL and NOAEL for developmental toxicity are 200 and 100 mg/ kg day, respectively. However, it should be noted that the small sample sizes ( the sampling unit is the litter) limited the statistical power of the experiment to detect possible significant differences at lower doses. Based on significantly decreased maternal body weight gain, the LOAEL and NOAEL for maternal toxicity are 200 and 100 mg/ kg day, respectively.

Klinefelter et al. ( 1995) evaluated the effects of bromodichloromethane exposure on male reproduction during a chronic cancer bioassay study in which F344 rats were administered bromodichloromethane in drinking water at concentrations of 0, 330, or 620 mg/ L. The authors estimated the doses to be 0, 22, and 39 mg/ kg day. At 52 weeks, the authors conducted an interim sacrifice, which included an evaluation of epididymal sperm motion parameters and histopathology of the testes and epididymides. No histologic alterations were observed in any reproductive tissue. Sperm velocities ( mean straight line, average path, and curvilinear), however, were significantly decreased at 39 mg/ kg day. No effect on sperm motility was observed at 22 mg/ kg day. The NOAEL and LOAEL for reproductive effects are thus 22 and 39 mg/ kg day, respectively.

Narotsky et al. ( 1997) examined both the developmental toxicity and the effect of dosing vehicle on the developmental toxicity of bromodichloromethane. F344 rats ( 12 to 14/ group) were administered bromodichloromethane by gavage, in either corn oil or an aqueous vehicle containing 10% Emulphor ® , at dose levels of 0, 25, 50, or 75 mg/ kg day on GD 6 to 15. Dams were allowed to deliver naturally, and pups were evaluated postnatally. Maternal body weights were assessed on GD 5, 6, 8, 10, 13, and 20, and all rats were observed for clinical signs of toxicity throughout the test period. Postnatal day ( PND) 1 was defined as GD 22 irrespective of the actual time of parturition. All pups were examined externally for gross malformations and weighed on PND 1 and 6. Skeletal and visceral anomalies in the pups were not evaluated. Following PND 6 examination, the dams were sacrificed and the number of uterine implantation sites per female was recorded. The uteri of females that did not deliver litters were stained and evaluated histopathologically to detect any cases of full litter resorption ( FLR). In order to compare the kinetics of dosing vehicles, a separate experiment was conducted in which pregnant females ( 3 to 4 animals per vehicle per time point) were administered a single dose of 75 mg/ kg on GD 6 and whole blood samples were collected at 30 minutes, 90 minutes, 4.5 hours, or 24 hours postdosing. Following blood collection, the animals were sacrificed, blood concentrations of bromodichloromethane were measured, and pregnancy status was confirmed at necropsy.

In the developmental toxicity study, one animal that received 75 mg/ kg day in corn oil died before study termination. In the mid and high dose groups, clinical signs of toxicity were evident among animals administered bromodichloromethane in either dosing vehicle. At 75 mg/ kg day, kyphosis ( humpback) was observed in animals receiving the oil vehicle, and piloerection was observed in animals receiving either vehicle. At 50 mg/ kg day, piloerection was observed in animals receiving the aqueous gavage, and chromodacryorrhea/ lacrimation was observed in animals receiving the oil gavage. Maternal weight gain was significantly decreased in Draft  Do Not Cite or Quote February 20, 2003 V  45 all dosed groups receiving the aqueous vehicle and in the 50 and 75 mg/ kg day groups in animals receiving the oil vehicle on GD 6 to 8 ( data not reported for other time periods). Although maternal weight gain was also reduced at 25 mg/ kg day in animals given the oil vehicle, this decrease was not statistically significant. However, a two way analysis of variance ( ANOVA) indicated that there was no interaction between vehicle and dose for this maternal endpoint. All control and 25 mg/ kg day litters survived the test period; however, FLR was observed at 50 and 75 mg/ kg day with both dosing vehicles. Statistical analysis ( ANOVA) of FLR incidence showed a significant vehicle dose interaction. For females receiving bromodichloromethane in corn oil, FLR was reported in 8 and 83% of the litters at 50 and 75 mg/ kg day, respectively; an additional high dose litter was carried to term but was delivered late ( GD 23), and all pups died by PND 6. For females receiving the aqueous vehicle, FLR was observed in 17 and 21% of the litters at 50 and 75 mg/ kg day, respectively. There were no effects on gestation length, pre or postnatal survival, or pup morphology in surviving litters, with the exception noted above in the 75 mg/ kgday oil vehicle group. Based on full litter resorption, the LOAEL for developmental toxicity is 50 mg/ kg day for both vehicles, and the corresponding NOAEL is 25 mg/ kg day. Based on significantly reduced body weight gain during GD 6 to 8 in dams receiving the aqueous vehicle, the LOAEL for maternal toxicity is the lowest dose tested, 25 mg/ kg day, and a NOAEL could not be determined.

Analysis of bromodichloromethane concentrations in blood indicated that circulating levels decreased over time with both vehicles, but tended to be higher following corn oil administration. Bromodichloromethane blood concentrations were thus vehicle dependent and differed statistically at both 4.5 and 24 hours postdosing ( mean of 3.1 ng/ mL versus 0.4 ng/ mL for oil and aqueous vehicles, respectively, at 24 hours). The elimination half life of bromodichloromethane was estimated to be 3.6 hours when administered in corn oil and 2.7 hours when given in the aqueous vehicle.