Trihalomethanes

10.0 Effects on experimental animals and in vitro

10.1 Acute toxicity

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).

10.2 Subchronic toxicity

10.2.1 Trihalomethanes

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.

10.2.2 Chloroform

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.

10.2.3 Bromodichloromethane

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).

10.2.4 Dibromochloromethane

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).

10.2.5 Bromoform

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).

10.3 Genotoxicity

10.3.1 Trihalomethanes

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).

10.3.2 Chloroform

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).

10.3.3 Bromodichloromethane

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.

10.3.4 Dibromochloromethane

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).

10.3.5 Bromoform

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).