Page 12: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Trihalomethanes
10.0 Effects on experimental animals and in vitro
At acutely toxic doses, chloroform causes central nervous system depression and cardiac effects. In rats, the clinical signs of acute toxicity for all of the THMs are similar and include piloerection, sedation, flaccid muscle tone, ataxia, and prostration. LD50s for chloroform, BDCM, DBCM, and bromoform were 908, 916, 1186, and 1388 mg/kg bw, respectively, in male rats and 1117, 969, 848, and 1147 mg/kg bw, respectively, in female rats. In surviving animals, there were a variety of effects, including reduced food intake, growth retardation, increased liver and kidney weights, haematological and biochemical effects, and histological changes in the liver and kidney (Chu et al., 1980). Keegan et al. (1998) characterized the no-observed-adverse-effect level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) for acute hepatotoxicity in F344 rats for both chloroform and BDCM delivered in an aqueous vehicle. For both chloroform and BDCM, the oral NOAEL was 0.25 mmol/kg bw, and a LOAEL of 0.5 mmol/kg bw was determined. Assessment at later time points indicated that liver damage caused by BDCM is more persistent than that caused by chloroform.
Based on data on chloroform, and limited data on DBCM, BDCM and bromoform, the literature suggests that rats are more sensitive than mice to acute effects of THMs. The critical effects associated with acute oral exposure in animals, irrespective of the target organ, are cellular degeneration, damage, and/or necrosis (GlobalTox, 2002).
The liver and thyroid, rather than the liver and kidney, were the organs most affected following administration of each of the THMs in a subchronic study (Chu et al., 1982a,b). Groups of 20 male and female SD rats ingested drinking water containing chloroform, BDCM, DBCM, or bromoform at concentrations of 5, 50, 500, or 2500 mg/L for 90 days; estimated doses were 0.11-0.17, 1.2-1.6, 8.9-14, and 29-55 mg/day per rat, respectively. Ten animals in each group were killed at the end of exposure, and the remaining animals were sacrificed 90 days later.
The growth rate was suppressed in animals administered chloroform and BDCM at 2500 mg/L at the end of exposure but not following the 90-day recovery period. Food consumption was also depressed during both exposure and recovery periods in groups receiving chloroform, DBCM, or BDCM at 2500 mg/L. Food consumption in males was depressed during exposure to 2500 mg bromoform/L but was normal at the end of the recovery period. Lymphocyte counts were decreased at the end of the recovery period in groups receiving 500 mg chloroform/L, 2500 mg DBCM/L, or 2500 mg bromoform/L. Mild, reversible histological changes in the liver and thyroid of exposed groups were reported, with the hepatotoxicity being greatest for bromoform, followed by, in descending order, BDCM, DBCM, and chloroform; however, the incidence of the lesions was not dose-related, although the frequency of more severe changes was greater in higher dose groups (statistical significance not reported). As the histological effects were mild and reversible and the haematological effects observed in chloroform-exposed animals were not dose-related, the NOAEL for all of the THMs in this study is considered to be 500 mg/L; the LOAEL is considered to be 2500 mg/L.
In a 90-day study in which CD-1 male and female mice (7-12 animals of each sex per treatment group) received 50, 125, or 250 mg chloroform/kg bw per day by intubation in Emulphor deionized water, there was a dose-related increase in liver weights and a decrease in hepatic microsomal activities in high-dose males and in females at all dose levels (Munson et al., 1982). Hexobarbital sleeping times were also increased in mid- and high-dose females. Blood glucose was increased in the high-dose groups of both sexes, and humoral immunity was decreased in high-dose males and mid- and high-dose females. Cellular immunity was decreased in high-dose females. The authors also reported slight histopathological changes in the kidney and liver of both sexes but did not provide information on the prevalence, severity, or dose-response relationship. The LOAEL for female mice in this study is considered to be 50 mg/kg bw; for males, the LOAEL is 250 mg/kg bw and the NOAEL is 125 mg/kg bw. The absence in this investigation of an increase in serum glutamic-pyruvic transaminase and serum glutamic-oxaloacetic transaminase observed in the high-dose groups in a 14-day study with a similar dosing regimen by the same investigators led the authors to conclude that some tolerance to the hepatotoxic action of chloroform may develop following long-term exposure.
The importance of the vehicle of administration in the toxicity of chloroform was demonstrated in a study in which groups of 80 male and female B6C3F1 mice were exposed to 60, 130, or 270 mg/kg bw per day by gavage in corn oil or a 2% Emulphor suspension for 90 days. Chloroform caused more marked hepatotoxic effects when administered in corn oil than in aqueous suspension, as determined by body and organ weights, serum chemistry, and histopathological examination (Bull et al., 1986).
Chloroform was administered by corn oil gavage to five male B6C3F1 mice per dose group at doses of 0, 34, 90, 138, or 277 mg/kg bw for 4 days or 3 weeks (5 days per week). Mild degenerative changes in centrilobular hepatocytes were noted in mice given 34 or 90 mg/kg bw per day after 4 days of treatment, but these effects were absent at 3 weeks. At 138 and 277 mg/kg bw per day, centrilobular necrosis was observed at 4 days and with increased severity at 3 weeks. Hepatic cell proliferation was increased in a dose-dependent manner at all chloroform doses after 4 days, but only in the 277 mg/kg bw group at 3 weeks. Renal tubular necrosis was observed in all treated groups after 4 days, while 3 weeks of exposure produced severe nephropathy at the highest dose and regenerating tubules at the lower doses. The nuclear labelling index was increased in the proximal tubules at all doses after 4 days of treatment, but was elevated only in the two highest dose groups after 3 weeks (Larson et al., 1994a).
In a similar study, five female B6C3F1 mice per dose group were administered chloroform dissolved in corn oil by gavage at doses of 0, 3, 10, 34, 238, or 477 mg/kg bw per day for 4 days or 3 weeks (5 days per week). Dose-dependent changes included centrilobular hepatic necrosis and markedly elevated labelling index in mice given 238 or 477 mg/kg bw per day. The NOAEL for histopathological changes (cytolethality and regenerative hyperplasia) was 10 mg/kg bw per day, and for induced cell proliferation, 34 mg/kg bw per day. In the same study, 14 female B6C3F1 mice per dose group were continuously exposed to chloroform in the drinking water at concentrations of 0, 60, 200, 400, 900, or 1800 mg/L for 4 days or 3 weeks. There was no increase in the hepatic labelling index after either 4 days or 3 weeks in any of the dose groups, nor were any microscopic alterations observed in the liver, even though the cumulative daily amount of chloroform ingested in the high-dose group was 329 mg/kg bw per day. The authors suggested that mice provided with chloroform in the drinking water ad libitum received the dose over the entire day with much smaller peak tissue levels than when the compound was administered as a bolus dose (Larson et al., 1994b).
Five female F344 rats per dose group were given chloroform by corn oil gavage at doses of 0, 34, 100, 200, or 400 mg/kg bw per day for 4 consecutive days or 5 days per week for 3 weeks (Larson et al., 1995b). In the liver, mild degenerative centrilobular changes and dose-dependent increases in hepatocyte proliferation were noted at doses of 100, 200, and 400 mg/kg bw per day. At 200 and 400 mg/kg bw per day, degeneration and necrosis of the renal cortical proximal tubules were observed. Increased regenerative proliferation of epithelial cells lining proximal tubules was seen at doses of 100 mg/kg bw per day or more. Lesions of the olfactory mucosa lining the ethmoid region of the nose (new bone formation, periosteal hypercellularity, and increased cell replication) were seen at all doses, including the lowest dose of 34 mg/kg bw per day.
Larson et al. (1995a) also administered chloroform to 12 male F344 rats per dose group by corn oil gavage (0, 10, 34, 90, or 180 mg/kg bw per day) or in the drinking water (0, 60, 200, 400, 900, or 1800 mg/L) for 4 days or 3 weeks. Gavage of 90 or 180 mg/kg bw per day for 4 days induced mild to moderate degeneration of renal proximal tubules and centrilobular hepatocyte changes that were no longer present after 3 weeks. Increased cell proliferation in the kidney was noted only at the highest gavage dose after 4 days. The labelling index was elevated in the livers of the high-dose group at both time points. With drinking water administration, rats consuming the water containing 1800 mg/L were dosed at a rate of 106 mg/kg bw per day, but no increase in renal or hepatic cell proliferation was observed at this or any lower dose.
The cardiotoxicity of chloroform was examined in male Wistar rats given daily doses of 37 mg/kg bw (0.31 mmol/kg) by gavage in olive oil for 4 weeks. Chloroform caused arrhythmogenic and negative chronotropic and dromotropic effects as well as extension of the atrioventricular conduction time and depressed myocardial contractility (Muller et al., 1997).
In an inhalation study, Templin et al. (1996b) exposed BDF1 mice to chloroform vapour at concentrations of 0, 149, or 446 mg/m3 (0, 30, or 90 ppm) 6 hours per day for 4 days or 2 weeks (5 days per week). In the kidneys of male mice exposed to 149 or 446 mg/m3, degenerative lesions and 7- to 10-fold increases in cell proliferation were observed. Liver damage and an increased hepatic labelling index were noted in male mice exposed to 149 and 446 mg/m3 and in female mice exposed to 446 mg/m3. Both doses were lethal in groups exposed for 2 weeks (40% and 80% mortality at 149 and 446 mg/m3, respectively).
A 90-day chloroform inhalation study was conducted using male and female B6C3F1 mice and exposure concentrations of 0, 1.5, 10, 50, 149, and 446 mg/m3 (0, 0.3, 2, 10, 30, and 90 ppm) for 6 hours per day, 7 days per week. Large, sustained increases in hepatocyte proliferation were seen in the 446 mg/m3 groups at all time points (4 days and 3, 6, and 13 weeks). In the more sensitive female mice, a NOAEL of 50 mg/m3 for this effect was established. Renal histopathology and regenerative hyperplasia were noted in male mice at 50, 149, and 446 mg/m3 (Larson et al., 1996). In another 90-day inhalation study, F344 rats were exposed to chloroform as concentrations of 0, 10, 50, 149, 446, or 1490 mg/m3 (0, 2, 10, 30, 90, or 300 ppm) for 6 hours per day, 7 days per week. The 1490 mg/m3 level was extremely toxic and deemed by the authors to be inappropriate for chronic studies. Increases in renal epithelial cell proliferation in cortical proximal tubules were observed at concentrations of 149 mg/m3 and above. Hepatic lesions and increased proliferation were noted only at the highest exposure level. In the ethmoid turbinates of the nose, enhanced bone growth and hypercellularity in the lamina propria were observed at concentrations of 50 mg/m3 and above, and a generalized atrophy of the turbinates was seen at all exposure levels after 90 days (Templin et al., 1996c).
Jamison et al. (1996) reported that F344 rats exposed to a high concentration of chloroform vapour (1490 mg/m3 [300 ppm]) for 90 days developed atypical glandular structures lined by intestinal-like epithelium and surrounded by dense connective tissue in their livers. These lesions appeared to arise from a population of cells remote from the bile ducts. The authors also observed a treatment-related increase in transforming growth factor-alpha (TGF-α) immunoreactivity in hepatocytes, bile duct epithelium bile canaliculi, and oval cells and an increase in transforming growth factor-beta (TGF-β) immunoreactivity in hepatocytes, bile duct epithelium, and intestinal crypt-like ducts. The lesions occurred only in conjunction with significant hepatocyte necrosis, regenerative cell proliferation, and increased growth factor expression or uptake.
Palmer et al. (1979) exposed 10 male and 10 female SPF Sprague-Dawley rats to chloroform by intragastric gavage (in toothpaste) daily for 13 weeks. Dose levels were 0, 15, 30, 150, or 410 mg/kg bw per day. At 150 mg/kg bw per day, there was "distinct influence on relative liver and kidney weight" (significance not specified). At the highest dose, there was increased liver weight with fatty change and necrosis, gonadal atrophy in both sexes, and increased cellular proliferation in bone marrow.
Thornton-Manning et al. (1994) administered five consecutive daily BDCM doses to female F344 rats and female C57BL/6J mice by aqueous gavage and found that BDCM is both hepatotoxic and nepthrotoxic to female rats (150-300 mg/kg bw per day) but only hepatotoxic to female mice (75-150 mg/kg bw per day). Munson et al. (1982) administered BDCM (50, 125, or 250 mg/kg bw per day) to male and female CD-1 mice by aqueous gavage for 14 days and reported evidence for hepatic and renal toxicity as well as effects on the humoral immune system (decreases in both antibody-forming cells and haemagglutination titres)A subsequent study by French et al. (1999) found no effects of BDCM on immune function. Based on the degree of aspartate aminotransferase and alanine aminotransferase elevations in this study, BDCM was found to be a more potent hepatotoxicant than chloroform, DBCM, and bromoform.
F344/N rats and B6C3F1 mice were given BDCM by gavage in corn oil 5 days per week for 13 weeks. Rats (10 per sex per dose) were given 0, 19, 38, 75, 150, or 300 mg/kg bw per day. Male mice (10 per dose) were given 0, 6.25, 12.5, 50, or 100 mg/kg bw per day, and female mice were given 0, 25, 50, 100, 200, or 400 mg/kg bw per day. Of the male and female rats that received the highest dose, 50% and 20%, respectively, died before the end of the study. None of the mice died. Body weights decreased significantly in male and female rats given BDCM at 150 or 300 mg/kg bw per day. Centrilobular degeneration of the liver was observed at 300 mg/kg bw per day in male and female rats and at 200 and 400 mg/kg bw per day in female mice. Degeneration and necrosis of the kidney were observed at 300 mg/kg bw per day in male rats and at 100 mg/kg bw per day in male mice. The NOAELs in rats were 75 and 150 mg/kg bw per day for body weight reduction and for hepatic and renal lesions, respectively. The NOAEL for renal lesions in mice was 50 mg/kg bw per day (NTP, 1987).
DBCM-induced cardiotoxicity was reported in male Wistar rats after short-term exposure (4 weeks of daily dosing with 0.4 mmol/kg bw). Arrhythmogenic and negative chronotropic and dromotropic effects were observed, as well as extension of atrioventricular conduction times. Inhibitory actions of DBCM on calcium ion dynamics in isolated cardiac myocytes were also noted (IPCS, 2000).
F344/N rats and B6C3F1 mice (10 per sex per dose) were given DBCM by gavage in corn oil at dose levels of 0, 15, 30, 60, 125, or 250 mg/kg bw per day, 5 days per week for 13 weeks. The final body weights of rats that received 250 mg/kg bw were depressed. A dose-dependent increase in hepatic vacuolation was observed in male rats. Based on this hepatic effect, the NOAEL in rats was 30 mg/kg bw per day. Kidney and liver toxicity were observed in male and female rats and male mice at 250 mg/kg bw per day. Survival rates for treated animals and corresponding controls were comparable except in high-dose rats. Clinical signs in the treated animals and controls were comparable. Based on the renal and hepatic lesions, a NOAEL of 125 mg/kg bw per day was identified in mice (NTP, 1985).
A 90-day corn oil gavage study was conducted using Sprague-Dawley rats and doses of 0, 50, 100, or 200 mg/kg bw per day. Body weight gain was significantly depressed in the high-dose groups to less than 50% and 70% of the controls in males and females, respectively. Observations of liver damage included elevated alanine aminotransferase in mid- and high-dose males, centrilobular lipidosis (vacuolization) in males at all doses and in high-dose females, and centrilobular hepatic necrosis in high-dose males and females. Kidney proximal tubule cell degeneration was induced by DBCM in all high-dose rats and to a lesser extent at 100 mg/kg bw per day in males and at both 50 and 100 mg/kg bw per day in females (Daniel et al., 1990).
Young adult rats (10 per sex per dose) were given bromoform by gavage in corn oil at doses of 0, 12, 25, 50, 100, or 200 mg/kg bw per day, 5 days per week for 13 weeks. Male and female mice were given doses of 0, 25, 50, 100, 200, or 400 mg/kg bw per day. Growth was not affected except at the highest dose in male mice, in which it was slightly suppressed. Male mice at the two highest dose levels showed "minimal to moderate" hepatocellular vacuolation in a few cells. Male rats showed a dose-related increase in hepatocellular vacuolation, which became statistically significant at 50 mg/kg bw per day. The NOAELs for hepatocellular vacuolation were 25 and 100 mg/kg bw per day in male rats and male mice, respectively (NTP, 1989).
All four THMs have induced sister chromatid exchanges (SCE) in human lymphocytes in vitro (bromoform > DBCM > BDCM > chloroform) and in mouse bone marrow cells in vivo (Morimoto and Koizumi, 1983).
In contrast to the predominantly non-genotoxic and non-mutagenic finding for chloroform, the weight of evidence favours a finding of mutagenicity and genotoxicity for the brominated THMs. Pegram et al. (1997) provided evidence that the mutagenic metabolic pathway for brominated THMs is mediated by GSTT1-1 conjugation and that mutagenic effects were not nearly as common with chloroform as with brominated THMs. The ability of GSTT1-1 to mediate the mutagenicity of various brominated THMs and induce almost exclusively GC→AT transitions suggests that it is likely that these THMs are activated by similar pathways (DeMarini et al., 1997).
The current weight of evidence suggests that chloroform is only slightly mutagenic and unlikely to be genotoxic. Varma et al. (1988) reported that chloroform was mutagenic in Salmonella typhimurium without metabolic activation, although a mixture of chloroform (85%) and bromoform (15%) was not mutagenic in the same assay with or without metabolic activation. LeCurieux et al. (1995) and Roldan-Arjona and Pueyo (1993) found that chloroform was not mutagenic with or without metabolic activation using several strains in an S. typhimurium assay. Shelby and Witt (1995) reported that chloroform was genotoxic in a mouse micronucleus assay in B6C3F1 mice but negative in an in vivo chromosomal aberration assay. Pegram et al. (1997) reported chloroform to be mutagenic in S. typhimurium TA1535, although not to the same extent as brominated THMs. Chloroform was not genotoxic in a number of unscheduled DNA synthesis (UDS) and/or repair, micronuclei, chromosomal aberration, and SCE assays (GlobalTox, 2002).
Although BDCM has given mixed results in bacterial assays for genotoxicity, the results have tended to be positive in tests employing closed systems to overcome the problem of the compound's volatility (IARC, 1991, 1999; Pegram et al., 1997). LeCurieux et al. (1995) found that BDCM was negative both with and without metabolic activation in the Ames assay. BDCM tested positive in several independent chromosomal aberration assays with and without metabolic activation but was negative in UDS and a mouse micronucleus assay. Fujie et al. (1993) reported that BDCM induced SCE. In addition, Pegram et al. (1997) provided evidence that a mutagenic metabolic pathway for brominated THMs is mediated by GSTT1-1 conjugation.
DBCM is mostly positive in genotoxicity tests employing closed systems to overcome the problem of volatility (IARC, 1991, 1999; Pegram et al., 1997). DBCM has given mostly positive results in eukaryotic test systems (Loveday et al., 1990; IARC, 1991, 1999; McGregor et al., 1991; Fujie et al., 1993), although there is less consistency in results between the different assays when considered with or without an exogenous metabolic system (WHO, 2005). DBCM was positive in the Ames test with S. typhimurium strain TA100 without activation (Simmon et al., 1977; Ishidate et al., 1982) but negative in strains TA98, TA1535, and TA1537 with or without activation (Borzelleca and Carchman, 1982). It gave positive results for chromosomal aberration in Chinese hamster ovary cells with activation (Ishidate et al., 1982) and for SCE in human lymphocytes and mouse bone marrow cells in vivo (Morimoto and Koizumi, 1983); it was negative in the micronucleus assay (Ishidate et al., 1982) and UDS in the liver of rats (IPCS, 2000).
There is some evidence to suggest that bromoform may be weakly mutagenic (GlobalTox, 2002). Bromoform, in common with the other brominated THMs, is largely positive in bacterial assays of mutagenicity conducted in closed systems (Zeiger, 1990; IARC, 1991, 1999). Bromoform was positive in the Ames test in S. typhimurium strain TA100 without activation (Simmon et al., 1977; Ishidate et al., 1982), positive with and without activation in TA98, and negative or equivocal in strains TA1535 or TA1937 with and without activation (NTP, 1989).
Bromoform gave increased SCE and chromosomal aberrations in mouse and rat bone marrow cells (Morimoto and Koizumi, 1983; Fujie et al., 1990). It gave negative results in mouse bone marrow (Hayashi et al., 1988; Stocker et al., 1997), in the rat liver UDS assay (Pereira et al., 1982; Stocker et al., 1997), and in the dominant lethal assay (Ishidate et al., 1982). In studies carried out by the National Toxicology Program (NTP, 1989), it was positive for micronuclei and SCE, but negative for chromosomal aberrations in mouse bone marrow. Potter et al. (1996) found that bromoform did not induce DNA strand breaks in the kidneys of male F344 rats following seven daily doses of 1.5 mmol/kg bw. As with bacterial assays, bromoform appeared more potent than the other brominated THMs (Morimoto and Koizumi, 1983; Banerji and Fernandes, 1996).
Chloroform has been carcinogenic in two animal species in extensive bioassays. In an early study conducted by the National Cancer Institute (NCI), chloroform was administered by gavage in corn oil to groups of 50 male and 50 female Osborne-Mendel rats and B6C3F1 mice. Male rats received 0, 90, or 180 mg/kg bw 5 times per week for 78 weeks; female rats received 0, 125, or 250 mg/kg bw 5 times per week for the first 22 weeks and the same doses as the males thereafter. In the first 18 weeks, doses of 0, 100, or 200 mg/kg bw were administered to male mice, and 0, 200, or 400 mg/kg bw were administered to female mice. After 18 weeks, the doses were changed to 0, 150, and 300 mg/kg bw for male mice and 0, 250, and 500 mg/kg bw for female mice for the remainder of the exposure period (NCI, 1976a).
In male rats, there was a statistically significant dose-related increase in the incidence of carcinomas of the kidney (0/99, 4/50, and 12/50 for control, low doses, and high doses, respectively). These tumours were not observed in female rats, although there was a non-significant increase in tumours of the thyroid (adenocarcinomas and carcinomas) in this sex.
Highly significant increases in hepatocellular carcinomas were observed in both sexes of mice (males: 1/18, 18/50, 44/45; females: 0/20, 36/45, 39/41 for control, low doses, and high doses, respectively). Nodular hyperplasia was also observed in low-dose males. It should be noted, however, that the weight loss in exposed animals was greater than 10%.
Upon re-examination of tissue samples from the NCI carcinogenesis bioassay, Reuber (1979) also reported increases in the incidence of several types of benign and malignant tumours of the liver in female rats and malignant lymphomas in both sexes of mice.
In a more recent and larger study, 0, 200, 400, 900, or 1800 mg chloroform/L was administered in drinking water (a more appropriate vehicle than that used in the NCI bioassay described above) to male Osborne-Mendel rats (50-330 animals per group) and female B6C3F1 mice (50-430 animals per group) for 104 weeks; the time-weighted average doses on a body weight basis ranged from 19 to 160 mg/kg bw per day for the rats and from 34 to 263 mg/kg bw per day for the mice (Jorgenson et al., 1985). To increase the sensitivity for detecting low response rates, group sizes were larger for the lower doses; there were two control groups (n = 330 and n = 50), one of which (n = 50) was matched for water intake with the high-dose groups.
In rats, there were dose-related decreases in water consumption and body weight gain that persisted in the two highest dose groups; survival increased with dose, probably as a result of leaner body composition in the higher dose groups (e.g., after 104 weeks, only 12% of controls had survived, whereas 66% of the animals in the high-dose group were still alive; this is a common occurrence in such studies). Consistent with the results of the NCI bioassay described above, there was also a dose-related increase in the incidence of kidney tumours. The incidence of tubular cell adenomas and adenocarcinomas combined was slightly lower than that in the NCI bioassay: 1/50, 4/313, 4/148, 3/48, and 7/50 in the matched control and increasing dose groups, respectively. Although there were increases in other neoplastic lesions in rats, including neurofibromas, leukaemias, lymphomas, and circulatory system tumours, they were not considered to be treatment-related because of a lack of a clear dose-response relationship or statistical significance or because they appeared to be attributable to the longer survival of the chloroform-treated animals.
With respect to the non-neoplastic histopathological changes in the kidney in this study, the authors commented only that "nontumour pathology of the kidney was high in all animals regardless of treatment." As a result, "it was not possible to relate tumour pathology with other tissue damage on either an individual animal or across-group basis." The incidence of nephropathy was 91% in the control group, 90% in the matched control, and 95%, 95%, 100%, and 92% in the increasing dose groups, respectively. Kidney tissue from this investigation (Jorgenson et al., 1985) has recently been microscopically re-evaluated for evidence of cytotoxicity and regeneration. Toxic injury in proximal tubular epithelial cells was observed in all high-dose males (1800 mg/L, the dose at which there was a statistically significant increase in tumour incidence) at all time points and approximately half of animals receiving the second highest dose (900 mg/L) for 18 or 24 months. None of the other treatment groups or controls had these characteristic changes. Although a systematic evaluation was not possible due to degradation of the slides and frequent autolytic change, the authors confirmed that such changes were also present in males of the same strain in the 1976 NCI bioassay in which exposure was by corn oil gavage (Hard and Wolf, 1999).
In mice, drinking water consumption was markedly depressed, leading to the death of about 25% of the two highest dose groups and 6% of the next highest dose group in the first week; after this initial period, survival did not differ significantly among groups. In contrast to the NCI bioassay described above, in which hepatic tumours in both sexes of mice were observed, there were no treatment-related increases in the incidence of any tumours in female mice in this study. Jorgenson et al. (1985) suggested that the hepatic tumours in mice in the NCI study may have been attributable to the interaction of chloroform with the corn oil vehicle.
In different studies in which four strains of mice (C57Bl, CBA, CF/1, and ICI) were administered chloroform for 80 weeks by gavage in toothpaste (0, 17, or 60 mg/kg bw per day in ICI male and female mice) or in toothpaste or arachis oil (0 or 60 mg/kg bw per day in males of all four strains), there were no treatment-related effects on the incidence of any type of tumour in males of three of the four strains (C57Bl, CBA, and CF/1 mice). There was, however, an increase in the incidence of epithelial tumours of the kidney at 60 mg/kg bw per day in male ICI mice, which was greater when chloroform was administered in arachis oil than in toothpaste (Roe et al., 1979).
Several other studies on the potential carcinogenicity of chloroform have been conducted. In B6C3F1 male mice (35 animals per group) ingesting chloroform in drinking water (0, 600, or 1800 mg/L) for periods up to 52 weeks, there were no increases in tumour incidence (Klaunig et al., 1986). However, these results may have been a function of the short observation period or small group sizes. The potential of chloroform to promote tumours induced by known initiators was also investigated in this study. Mice of the same strain (35 animals per group) ingested drinking water containing diethylnitrosamine (DENA) at 10 mg/L for 4 weeks followed by 600 or 1800 mg chloroform/L for up to 52 weeks. There were two control groups: after DENA treatment, the positive control group ingested drinking water containing phenobarbital (500 mg/L), while the vehicle control group received untreated drinking water. The induction of liver tumours was enhanced by exposure to phenobarbital but not by exposure to chloroform after DENA treatment. In contrast, in a study conducted by Deml and Oesterle (1985), chloroform administered in corn oil (100, 200, and 400 mg/kg bw, twice weekly for 11 weeks, 1 week after administration of a single dose of 8 mg DENA) promoted the development of DENA-initiated preneoplastic foci liver tumours in Sprague-Dawley rats.
In a study designed to assess the safety of chloroform in toothpaste, beagle dogs (eight per sex per dose) were given chloroform in a toothpaste base in gelatin capsules, 6 days per week for 7.5 years, at doses of 0, 15, or 30 mg/kg bw per day (Heywood et al., 1979). After 6 weeks of treatment, there were significant increases in serum glutamate-pyruvate transaminase levels in dogs given the high dose. At the low dose level, significant increases were observed at 34 weeks and after. Similar effects were not observed in the vehicle control (16 dogs of each sex) or untreated control (eight dogs of each sex) groups. "Fatty cysts" characterized by aggregations of vacuolated hepatocytes and minimal hepatic fibrosis were observed in animals within each group (including controls). These findings were more frequent and of greater magnitude in animals of either gender treated with chloroform at either dose level than in controls. The LOAEL in this study was 15 mg/kg bw per day.
Since the previous Canadian drinking water guideline was drafted for total THMs (based on chloroform), significant effort has been made to characterize the mechanism of carcinogenicity and to understand the variability in effects from different routes and vehicles of administration. The current weight of evidence suggests that chloroform is a threshold carcinogen in rodents. There is strong evidence that the carcinogenic activity of chloroform in both rats and mice is mediated by a non-genotoxic mechanism of action that is secondary to cytotoxicity and cellular proliferation. There is strong evidence that the tumorigenicity of chloroform depends on the rate of its delivery to the target organ, and this suggests that detoxification mechanisms must be saturated before the full carcinogenic potential of chloroform is realized (GlobalTox, 2002). The weight of available evidence also indicates that chloroform has little, if any, capability of inducing gene mutation or other types of direct damage to DNA (IPCS, 2000).
IPCS (2000) summarized the pattern of chloroform-induced carcinogenicity in rodent bioassays conducted up to that time as follows: Chloroform induced hepatic tumours in B6C3F1 mice (males and females) when administered by gavage in corn oil at doses in the range of 138-477 mg/kg bw per day (NCI, 1976a,b). However, when similar doses were administered to the same strain in drinking water, hepatic tumours were not increased (Jorgenson et al., 1985). Liver tumours are observed, therefore, only in mice following administration by gavage in corn oil. This observation is consistent with those in initiation/promotion assays in which chloroform has promoted development of liver tumours, particularly when administered by gavage in a corn oil vehicle.
Chloroform also induces renal tumours, but at lower rates than liver tumours in mice. Chloroform induced kidney tumours in male Osborne-Mendel rats at doses of 90-200 mg/kg bw per day in corn oil by gavage (NCI, 1976a,b). However, in this strain, results were similar when the chemical was administered in drinking water, indicating that the response is not entirely dependent on the vehicle used (Jorgenson et al., 1985). It should be noted, however, that at the higher doses in this study, there were significant reductions in body weight. In an early, more limited investigation, kidney tumours were increased in ICI mice but not in CBA, C57BL, or CF1 mice administered chloroform by gavage in toothpaste (Roe et al., 1979). Therefore, the tumorigenic response in the kidney, although observed in both rats and mice (males), is highly strain-specific.
To investigate the possible role of replicative proliferative effects in the carcinogenicity of chloroform, a wide range of studies have been conducted in which replicative proliferative effects have been examined in similar strains of rats and mice exposed to similar doses or concentrations of chloroform, although for shorter periods, as in the principal carcinogenesis bioassays (Larson et al., 1993, 1994a,b,c, 1995a,b, 1996; Lipsky et al., 1993; Pereira, 1994; Templin et al., 1996a,b,c). Most of these studies involved evaluation of histopathological changes and cell proliferation in the kidney and liver, the latter determined as a BrdU labelling index in histological tissue sections. Results of available studies also indicate that the proliferative response is less when exposure is not continuous (e.g., inhalation for 5 days per week versus 7 days per week) (Larson et al., 1996; Templin et al., 1996c) and returns to baseline following a recovery period.
Based on studies conducted primarily in the F344 rat, available data are consistent with a mode of action for carcinogenicity in the kidney based on tubular cell regeneration. Studies in this strain indicate that chloroform causes damage and increases cell replication in the kidney at doses similar to those that induce tumours in Osborne-Mendel rats following gavage in corn oil for periods up to 3 weeks (Larson et al., 1995a,b). However, there has been no clear dose-response for renal damage or proliferation in F344 rats exposed to concentrations in drinking water that were similar to those that induced tumours in Osborne-Mendel rats in the carcinogenesis bioassay of Jorgensen et al. (1985) (Larson et al., 1995b). In a single study in which the proliferative response was compared in F344 and Osborne-Mendel rats at 2 days following a single gavage administration, it was concluded that these strains were about equally susceptible to chloroform-induced renal injury, although a statistically significant increase in labelling index was observed at a much lower dose in the Osborne-Mendel rat (10 mg/kg bw) than in the F344 rat (90 mg/kg bw); this latter observation may have been a function of the low value in controls for the Osborne-Mendel rats.
Data on the proliferative response in the strain in which renal tumours have been observed (Osborne-Mendel rats) are limited to examination at 2 days following a single administration by gavage in corn oil (Templin et al., 1996b); studies in which the proliferative response was examined in Osborne-Mendel rats following administration in drinking water have not been identified. Although the results of this study are not inconsistent with a mode of action of induction of tumours based on tubular cell regeneration, they are considered inadequate in themselves to quantitatively characterize the dose-response relationship for an intermediate endpoint for cancer induction (IPCS, 2000).
Environment Canada and Health Canada (2001) also discussed the weight of evidence for the mechanism of carcinogenicity for chloroform. This report stated that for Osborne-Mendel rats, the results of re-analyses of the original renal tissues (Hard and Wolf, 1999; Hard et al., 2000), from both the drinking water bioassay (Jorgenson et al., 1985) and the gavage study (NCI, 1976a), have been critical. They provide strong support for the argument that the mode of induction of these tumours is consistent with the hypothesis that sustained proximal tubular cell damage is a requisite precursor lesion for chloroform-induced tumours.
When comparing short-term studies in rats and mice using similar chloroform exposure regimens, the experimental conditions employed in studies that led to cellular proliferation and cytotoxicity led to tumour formation when employed in cancer bioassays. However, the converse is not always true.
The hypothesized mode of carcinogenesis for chloroform is in keeping with the growing body of evidence supporting the biological plausibility that prolonged regenerative cell proliferation can be a causal mechanism in chemical carcinogenesis. This has been addressed in numerous articles, including Ames and Gold (1990, 1996), Cohen and Ellwein (1990, 1991, 1996), Preston-Martin et al. (1990), Ames et al. (1993), Tomatis (1993), Cohen (1995), Cunningham and Matthews (1995), Butterworth (1996), Farber (1996), and Stemmermann et al. (1996).
In summary, chloroform has induced liver tumours in mice and renal tumours in mice and rats. The weight of evidence of genotoxicity, sex and strain specificity, and concordance of cytotoxicity, regenerative proliferation, and tumours is consistent with the hypothesis that cytotoxicity with a period of sustained cell proliferation likely represent a secondary mechanism for the induction of tumours following exposure to chloroform. This is consistent with a non-linear dose-response relationship for induction of tumours. This cytotoxicity is primarily related to rates of oxidation of chloroform to reactive intermediates, principally phosgene and hydrochloric acid. The weight of evidence for this mode of action is strongest for hepatic and renal tumours in mice and more limited for renal tumours in rats (Environment Canada and Health Canada, 2001).
There has been little evidence to support other mechanisms of carcinogenicity, especially at low doses where cytotoxicity and cellular proliferation are not expected. Chloroform toxicity is clearly enhanced in rodents when administered in corn oil, compared with when it is received in drinking water, supporting the hypothesis that tumorigenicity of chloroform depends on the rate of its delivery to the target tissue and further suggesting that detoxification mechanisms must be saturated before the full carcinogenic potential of chloroform is realized (GlobalTox, 2002).
In one carcinogenesis bioassay conducted for BDCM, groups of 50 male and 50 female F344/N rats and B6C3F1 mice were administered the compound by gavage in corn oil, 5 days per week for 102 weeks. Rats received 0, 50, or 100 mg/kg bw per day; male mice received 0, 25, or 50 mg/kg bw per day, while female mice received 0, 75, or 150 mg/kg bw per day (NTP, 1987).
In rats, there was some decrease in body weight gain in the high-dose groups of both sexes (statistical significance not specified), increased incidence of cytomegaly of the renal tubular epithelial cells in males (both doses), nephrosis in the high-dose group of females, and hepatic changes, including necrosis, clear cell change, eosinophilic cytoplasmic change, focal cellular change, and fatty metamorphosis, in both sexes, but predominantly in the high-dose group of females. There was clear evidence of carcinogenicity in male and female rats, with increases in the incidence of renal tubular cell adenomas and adenocarcinomas (combined incidence in control, low-dose, and high-dose groups: males, 0/50, 1/50, and 13/50; females, 0/50, 1/50, and 15/50) and rare tumours (adenomatous polyps and adenocarcinomas) of the large intestine (combined incidence: males, 0/50, 13/50, and 45/50; females, 0/46, 0/50, and 12/47). Increased incidence of skin neoplasms in low- but not high-dose male rats was also observed but was not considered to be compound-related. The neoplasms of the kidney in rats in this bioassay were not similar to those observed for other compounds, such as 1,4-dichlorobenzene, for which tumours occurred principally in males and were associated with severe nephropathy and increased incidence of calcification and hyaline droplet formation, associated with reabsorption of alpha-2-microglobulin (Charbonneau et al., 1989).
There was a decrease in body weight gain of female mice, and survival was significantly lower than that of controls, due partly to ovarian abscesses not considered to be treatment-related. The incidence of renal cytomegaly and hepatic fatty metamorphosis in male mice was also increased. Pathological changes in the thyroid gland and testis were also observed but were not considered to be treatment-related. There was also clear evidence of carcinogenicity in male and female B6C3F1 mice, based on increased incidence of adenomas and adenocarcinomas (combined) of the kidney in males (incidence in control, low-dose, and high-dose groups, 1/49, 2/50, and 9/50, respectively) and of hepatocellular adenomas and carcinomas (combined) in female mice (incidence 3/50, 18/48, and 29/50, respectively).
Moore et al. (1994) administered BDCM in drinking water (containing 0.25% Emulphor) to male F344 rats and B6C3F1 mice for 1 year and evaluated clinical indicators of kidney toxicity. Water containing BDCM concentrations of 0.08, 0.4, and 0.8 g/L for rats and 0.06, 0.3, and 0.6 g/L for mice resulted in average daily doses of 4.4, 21, and 39 mg/kg bw for rats and 5.6, 24, and 49 mg/kg bw for mice. A urinary marker for renal proximal tubule damage, N-acetyl-β-glucosaminidase, was elevated above controls in each dose group in rats and at the highest treatment level in mice. Significant increases in urinary protein, indicative of glomerular damage, were also noted in low- and mid-dose rats as well as high-dose mice.
While cytotoxic effects of BDCM may potentiate tumorigenicity in certain rodent tissues at high dose levels, direct induction of mutations by BDCM metabolites may also play a carcinogenic role. The extent to which each of these processes contributes to the induction of tumours observed in chronic animal studies is, however, questionable (IPCS, 2000).
DeAngelo et al. (2002) examined the ability of THMs administered in drinking water to induce aberrant crypt foci in the colons of B6C3F1 mice and F344/N rats. Preneoplastic aberrant crypt foci were induced in the colon of rats following the administration of some brominated THMs. However, unlike DBCM and bromoform, colon neoplasms were not found upon chronic administration of BDCM to rats via drinking water. BDCM did, however, induce colon cancer in male rats when administered in corn oil gavage.
In a recent draft study from NTP (2004), male F344/N rats and female B6C3F1 mice were exposed to BDCM in drinking water for 2 years. Groups of 50 male F344/N rats were exposed to target concentrations equivalent to average daily doses of 0, 6, 12 or 25 mg/kg bw BDCM. Survival and mean body weights of all exposed groups were generally similar to those of the controls throughout the study. There were no increased incidences of neoplasms that were attributed to BDCM. The incidences of chronic inflammation in the liver of the two higher dose groups were significantly greater than that in the controls; however, the biological significance of these increases is uncertain. Groups of 50 female B6C3F1 mice were exposed to target concentrations equivalent to average daily doses of 0, 9, 18 or 36 mg/kg bw BDCM. Survival of exposed groups was similar to that of the controls, but mean body weights of all exposed groups were generally less than those of the controls from week 4 through the end of the study. The incidences of hepatocellular adenoma or carcinoma occurred with a negative trend, and the incidence in the higher dose group was significantly decreased relative to the control group. The incidence of haemangiosarcoma in all organs was significantly decreased in the 18 mg/kg bw group.
The authors of the study concluded that under the conditions of this 2-year drinking water study, there was no evidence of carcinogenic activity of BDCM in male F344/N rats exposed to target concentrations of 6, 12 or 25 mg/kg bw and in female B6C3F1 mice exposed to target concentrations of 9, 18 or 36 mg/kg bw (NTP, 2004). However, this report has not yet been peer-reviewed and as such is not final, and cannot be used in the risk assessment at this time.
In a National Toxicology Program (NTP) carcinogenesis bioassay, DBCM was administered in doses of 0, 40, or 80 mg/kg bw by gavage in corn oil 5 times per week for 104 weeks to groups of 50 male and female F344/N rats. In addition, 0, 50, or 100 mg/kg bw per day was administered in similar fashion to groups of 50 male and female B6C3F1 mice 5 days per week for 105 weeks. Body weight gain in the high-dose group of male rats was decreased, and there was a dose-related increase in lesions (fatty metamorphosis and ground-glass cytoplasmic changes) of the liver in both sexes and nephrosis of the kidney (dose-related) in females. There was, however, no evidence of carcinogenicity in rats (NTP, 1985).
In male mice, survival was significantly lower in both dose groups, and 35 animals in the low-dose group were accidentally killed during weeks 58-59. In both sexes, the incidences of hepatic lesions were increased, including fatty metamorphosis (both sexes), hepatocellular necrosis (dosed males), hepatocytomegaly (high-dose males), and calcification of the liver (high-dose females). Nephrosis (high dose) and renal calcification in males and follicular cell hyperplasia of the thyroid gland (possibly related to a bacterial infection) in females were also increased. There was equivocal evidence of carcinogenicity in male B6C3F1 mice based on an increased incidence of hepatocellular carcinomas, but only a marginal increase in hepatocellular adenomas or carcinomas (combined) (incidence of hepatocellular carcinomas in control and high-dose groups, 10/50 and 19/50, respectively; incidence of hepatocellular adenomas and carcinomas combined, 23/50 and 27/50, respectively). The number of surviving animals in the low-dose group of male mice, however, was inadequate for analysis of tumour incidence, owing to a dosing error. There was also some evidence of carcinogenicity in female mice, based on an increased incidence of hepatocellular adenomas and hepatocellular adenomas or carcinomas (combined). The incidence of hepatic adenomas and carcinomas (combined) in the control, low-dose, and high-dose groups was 6/50, 10/49, and 19/50, respectively.
Mechanistic issues for DBCM are similar to those addressed for BDCM.
In an NTP carcinogenesis bioassay, 0, 100, or 200 mg bromoform/kg bw was administered by gavage in corn oil 5 days per week for 103 weeks to groups of 50 F344/N rats of each sex and to female B6C3F1 mice (NTP, 1989). Male B6C3F1 mice were administered 0, 50, or 100 mg/kg bw on the same schedule. In rats, there was a reduction of body weight gain in low- and high-dose males and high-dose females; survival in the high-dose group of males was also significantly lower than that in controls. As well, dose-related, non-neoplastic effects in the salivary gland (squamous metaplasia and chronic active inflammation in both sexes), prostate (squamous metaplasia), forestomach (ulcers in the males), lung (chronic active inflammation -- males only), and spleen (pigmentation -- high-dose females) were also observed, although the lesions of the salivary gland and lung were characteristic of infection by rat corona virus, to which a positive serological reaction was observed early in the study. There was some evidence of carcinogenicity in male rats and clear evidence in female rats, based on increased incidences of uncommon neoplasms (adenomatous polyps and adenocarcinomas of the large intestine) in both sexes. The incidences of these tumours (combined) in the control, low-dose, and high-dose groups of females were 0/50, 1/50, and 8/50, respectively; in males, the comparable values were 0/50, 0/50, and 3/50. Although the incidence of these tumours in females was similar to that observed in the NTP bioassay for BDCM, the incidence in males was much less. Reduced survival in the high-dose group of male rats administered bromoform may, however, have lowered the sensitivity of the bioassay for detecting a carcinogenic response. The incidence of neoplastic nodules in low-dose female rats was also greater than that in controls, but it was not considered to be a chemically induced neoplastic effect, as the lesions did not fit the current NTP criteria for hepatocellular adenomas, nor was the incidence significantly increased in high-dose female rats or in dosed male rats.
In female mice, there was a decrease in body weight gain and survival (partially attributable to utero-ovarian infection) and increases in the incidence of follicular cell hyperplasia of the thyroid (high dose) and fatty change of the liver (both doses). There was no evidence of carcinogenicity in male or female mice (NTP, 1989).
Bromoform was administered in drinking water (containing 0.25% Emulphor) to male F344 rats and B6C3F1 mice for 1 year, and clinical indicators of kidney toxicity were examined (Moore et al., 1994). Water containing bromoform concentrations of 0.12, 0.6, and 1.2 g/L for rats and 0.08, 0.4, and 0.8 g/L for mice resulted in average daily doses of 6.2, 29, or 57 mg/kg bw for rats and 8.3, 39, or 73 mg/kg bw for mice. Several indicators of tubular and glomerular damage were elevated at each treatment level in mice, and mice appeared more susceptible to the nephrotoxic effects of bromoform than to those of BDCM. As in mice, urinary protein was increased in all rat dose groups, but little evidence of loss of tubule function was observed in rats.
Although bromoform seems to have a greater propensity for metabolism and is a more potent mutagen than BDCM, it appears to be a less potent toxicant and carcinogen based on the results of the NTP (1985, 1987) bioassays and numerous other in vivo studies of toxicity. As with DBCM, a possible explanation is less bioavailability resulting from the greater lipophilicity of this compound and the use of corn oil as the vehicle of administration. This concept may be supported by the occurrence of bromoform-induced tumours in the intestinal tract, but not in the liver or kidneys. Greater lipophilicity and reactivity of bromoform metabolites may also prevent it from reaching critical target sites. Moreover, when bromoform was injected intraperitoneally, its metabolism was greater than that of the other THMs (Anders et al., 1978; Tomasi et al., 1985). When administered by corn oil gavage, however, bromoform was the least metabolized THM (Mink et al., 1986).
The teratogenicity of THMs was investigated in one study in which BDCM, DBCM, or bromoform at doses of 50, 100, or 200 mg/kg bw per day or chloroform at doses of 100, 200, or 400 mg/kg bw per day was administered to groups of 15 pregnant Sprague-Dawley rats by oral intubation in corn oil on gestation days 6-15. Maternal weight gain was depressed in the high-dose groups (200 mg/kg bw per day) receiving BDCM and DBCM, but to a lesser extent than that in the high-dose group for chloroform (400 mg/kg bw per day). Maternal liver weight was also increased at the highest dose of BDCM (200 mg/kg bw per day). BDCM and bromoform were considered to be fetotoxic, based on the observation of interparietal anomalies, although the statistical significance of the observed increases was not reported. These compounds also appeared to increase the incidence of aberrations of the sternebrae. The LOAEL based on this fetotoxic effect was 50 mg/kg bw per day (Ruddick et al., 1983).
A survey of available toxicological literature on reproductive and developmental effects of DBPs including chloroform and BDCM was conducted for the U.S. EPA by Tyl (2000), who concluded that current published studies are not sufficient for quantitative assessment of reproductive or developmental risk but are sufficient for determination of hazard. The potential hazards identified for chloroform and BDCM were whole litter resorption and fetotoxicity, and for BDCM, male reproductive toxicity (Tyl, 2000).
Available data on the teratogenicity of the THMs are confined principally to chloroform. In studies conducted to date, chloroform has not been teratogenic in rats, rabbits, or mice at doses up to 400 mg/kg bw following administration by gavage in corn oil or emulphor:saline (Thompson et al., 1974; Burkhalter and Balster, 1979; Ruddick et al., 1983). Fetotoxic effects (e.g., decreased body weights and sternebral and interparietal malformations) were sometimes observed, but only at doses that were toxic to the mothers.
In a continuous breeding study, male and female CD-1 mice were administered chloroform in corn oil by gavage at actual doses of 0, 6.6, 15.9, or 41.2 mg/kg bw per day for 7 days prior to and throughout the 98-day cohabitation period. Control and high dose F1 pups were administered chloroform after weaning at postnatal day 21 according to the same dosing schedule as their F0 parents. There were no significant effects on fertility or reproduction in either gender over two generations. Histopathological changes indicative of hepatotoxicity were observed in the F1 females in all treatment dose levels (Gulati et al., 1988).
Hoechst (1991) examined the embryotoxicity and developmental toxicity of inhaled chloroform. Female Wistar rats were mated, then exposed by whole-body inhalation to chloroform at 0, 15, 50, or 149 mg/m3 (0, 3, 10, or 30 ppm) for 7 hours per day between gestation days 7 and 16. Slight reductions in food consumption and significant reductions in body weights were observed in dams exposed at 50 and 149 mg/m3. These findings were hypothesized to result in the slight stunting of fetuses produced in these animals. A NOAEL of 15 mg/m3 was established based on the lack of embryotoxicity or teratogenicity (GlobalTox, 2002).
Narotsky et al. (1997) examined the effects of BDCM in F344 rats using doses of 0, 25, 50, or 75 mg/kg bw per day in aqueous or oil gavage vehicles. BDCM induced full-litter resorptions in the 50 and 75 mg/kg bw per day dose groups with either vehicle of administration. For dams receiving corn oil, full-litter resorptions (FLR) were noted in 8% and 83% of the litters at 50 and 75 mg/kg bw per day, respectively. All vehicle control litters and litters from the group given 25 mg/kg bw per day survived the experimental period. BDCM had been shown to cause maternal toxicity at these doses in a previous study (Narotsky et al., 1992).
In a developmental study conducted by Christian et al. (2001), Sprague-Dawley rats and New Zealand White rabbits were dosed with BDCM continuously in drinking water on gestation days 6-21 in rats and gestation days 6-29 in rabbits. Mean consumed doses were 0, 2.2, 18.4, 45.0, or 82.0 mg/kg bw per day for rats and 0, 1.4, 13.4, 35.6, or 55.3 mg/kg bw per day for rabbits. In rats, water consumption was reduced in all treatment doses, and body weight gain and feed consumption were reduced at ≥45.0 mg/kg bw per day. In rabbits, body weight gain and feed consumption were reduced at ≥35.6 mg/kg bw per day. The maternal NOAELs were 18.4 and 13.4 mg/kg bw per day for rats and rabbits, respectively. Minimal delays in the ossification of forepaw phalanges and hindpaw metatarsals and phalanges occurred in rat fetuses at 82.0 mg/kg bw per day and were considered marginal, reversible, and associated with severely reduced maternal weight gain. There were no treatment-related effects observed in rabbit fetuses. The developmental NOAELs were 45.0 and 55.3 mg/kg bw per day for rats and rabbits, respectively (Christian et al., 2001).
In a two-generation reproduction study conducted by Christian et al. (2002), Sprague-Dawley rats were treated with BDCM continuously via the drinking water at concentrations of 0, 50, 150, or 450 mg/L (equal to 0, 4.1-12.6, 11.6-40.2, or 29.5-109.0 mg/kg bw per day). In the two top dose groups, mortality and clinical signs associated with reduced water consumption, reduced body weights and weight gains, and reduced food consumption were observed. Reduced body weights were associated with reduced organ weights and increased organ weight ratios. Small delays in sexual maturation (preputial separation, vaginal patency) and more F1 rats with prolonged diestrus were also attributed to severely reduced body weights. The NOAEL for general toxicity and the NOAELs for reproductive and developmental toxicity were at least 4.1-12.6 mg/kg bw per day. If the delayed sexual maturation associated with severely reduced body weights is considered general toxicity, reproductive and developmental NOAELs for BDCM are greater than 29.5-109.0 mg/kg bw per day (Christian et al., 2002).
Bielmeier et al. (2001) investigated rat strain sensitivity between F344 and Sprague-Dawley rats as measured by FLR after dosing with BDCM. Following aqueous gavage with BDCM at 75 mg/kg bw per day on gestation days 6-10, F344 rats had a 62% incidence of FLR, whereas all SD rats maintained their litters. Additionally, rats treated with BDCM at 75 mg/kg bw per day on gestation days 6-10, the critical period encompassing the luteinizing hormone (LH)-dependent period of pregnancy, had a 75% incidence of FLR, but rats treated on gestation days 11-15 with BDCM at 75 or 100 mg/kg bw per day were unaffected. Twenty-four hours after a single dose, all dams with FLR had markedly reduced serum progesterone levels; however, LH levels were unaffected. The high FLR rate during the LH-dependent period, the lack of response thereafter, and the reduced progesterone levels without an associated reduction in LH levels suggest that BDCM disrupts luteal responsiveness to LH (GlobalTox, 2002).
Klinefelter et al. (1995) studied the potential of BDCM to alter male reproductive function in F344 rats. BDCM was consumed in the drinking water for 52 weeks, resulting in average dose rates of 22 and 39 mg/kg bw per day. No gross lesions in the reproductive organs were revealed by histological examination, but exposure to the high BDCM dose significantly decreased the mean straight-line, average path, and curvilinear velocities of sperm recovered from the cauda epididymis (IPCS, 2000).
Chen et al. (2003) examined the effect of BDCM on chronic gonadotrophin secretion by human placental trophoblast cultures. A BDCM dose-dependent reduction in the secretion of bioactive and immunoreactive chorionic gonadotrophin from human placental trophoblasts was observed, suggesting that BDCM targets these cells. A reduction in chorionic gonadotrophin could have adverse effects on pregnancies, since this hormone plays a vital role in maintaining pregnancy.
In a multigeneration reproduction study, groups of 10 male and 30 female ICR mice were treated with DBCM in Emulphor at 0, 0.1, 1.0, or 4.0 g/L (0, 17, 171, or 685 mg/kg bw per day) in drinking water for 35 days, then mated; subsequent re-matings occurred 2 weeks after weaning. The F1 mice were treated with the same test solution for 11 weeks after weaning and then mated; re-mating occurred 2 weeks after weaning. At 17 mg/kg bw per day, there was only a slight depression in the body weight of the newborn pups in the F2b generation. At 171 mg/kg bw per day, there was a significant decrease in female body weight and an increase in the occurrence of gross liver pathology of F0 and F1b mice; the lesions varied in severity from fat accumulation to distinct masses on the liver surface. Although not occurring in every generation, there were significant decreases in litter size, pup viability, postnatal body weight, and lactation index. At 685 mg/kg bw per day, the effects were of the same types but more severe. Body weight gain was significantly reduced in both males and females at the highest dose (685 mg/kg bw per day) and in females at the middle dose (171 mg/kg bw per day). Animals in both these groups exhibited enlarged livers with gross morphological changes. In addition, the gestation index, fertility, and survival of the F1 generation were significantly reduced. Only fertility was decreased (high dose) in the F2 generation (IPCS, 2000). Based on maternal toxicity and fetotoxicity, a NOAEL of 17 mg/kg bw per day was identified (Borzelleca and Carchman, 1982).
Bromoform was found to induce FLR in pregnant F344 rats when administered orally on gestation days 6-15, but at higher doses (150 and 200 mg/kg bw per day) than those required to produce the same effect for BDCM (Narotsky et al., 1993).
The effect of bromoform on fertility and reproduction was investigated in Swiss CD-1 mice (20 pairs per dose) dosed for 105 days at 0, 50, 100, or 200 mg/kg bw per day in corn oil by gavage. No apparent effect on fertility or reproduction (e.g., litters per pair, live pups per litter, sex of live pups, pup body weights) was reported in either the parental or the F1 generation, and a reproductive NOAEL of 200 mg/kg bw per day was identified (NTP, 1989).
Neurotoxicological findings reported for the THMs are observations of anaesthesia associated with acute high-dose exposures to brominated THMs (bromoform, BDCM, DBCM) and results from a behavioural study conducted by Balster and Borzelleca (1982) in adult male mice dosed by aqueous gavage for up to 90 days. Treatment with 1.2 or 11.6 mg/kg bw per day was without effect in various behavioural tests, and dosing for 30 days with 100 mg/kg bw per day did not affect passive avoidance learning. Animals dosed with either 100 or 400 mg/kg bw per day for 60 days exhibited decreased response rates in an operant behaviour test. These effects were greatest early in the regimen, with no evidence of progressive deterioration (IPCS, 2000).
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