Page 10: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Trihalomethanes

8.0 Kinetics and metabolism

8.1 Absorption

THMs are generally well absorbed, metabolized, and rapidly eliminated by mammals after oral or inhalation exposure (IPCS, 2000).

8.1.1 Chloroform

The absorption kinetics of chloroform following intragastric intubation are dependent upon the vehicle of delivery. Based on the calculated area under blood concentration-time curves (5 hours), uptake of chloroform following administration of 75 mg/kg bw by intragastric intubation in aqueous solution was 8.7 times greater than that for a similar dose administered in corn oil in paired Wistar rats (Withey et al., 1983).

Chloroform is readily absorbed through the skin of humans and animals, and significant dermal absorption of chloroform from water while showering has been demonstrated. Hydration of the skin appears to accelerate absorption of chloroform (Jo et al., 1990).

8.2 Distribution

8.2.1 Chloroform

Chloroform is distributed throughout the whole body, with levels being highest in the fat, blood, liver, kidneys, lungs, and nervous system. Distribution is dependent on exposure route; extrahepatic tissues receive a higher dose from inhaled or dermally absorbed chloroform than from ingested chloroform. Placental transfer of chloroform has been demonstrated in several animal species and humans. Unmetabolized chloroform is retained longer in fat than in any other tissue (WHO, 2005).

8.2.2 Brominated trihalomethanes

Brominated substitution would be expected to confer greater lipophilicity on the brominated THMs compared with chloroform, which would affect tissue solubility. Mink et al. (1986) found that the liver, stomach, and kidneys were the organs containing the highest BDCM levels. Mathews et al. (1990) found that repeated doses had no effect on the tissue distribution of BDCM in rats. Lilly et al. (1998) found slightly higher maximum concentrations of BDCM in the liver and kidneys after aqueous administration compared with corn oil delivery in male rats.

8.3 Metabolism

THMs are metabolized primarily to carbon dioxide and/or carbon monoxide.

8.3.1 Chloroform

Available data indicate that the toxicity of chloroform is attributable to its metabolites. Both oxidative and reductive pathways of chloroform metabolism have been identified, although in vivo data are limited. The metabolism of chloroform proceeds through a cytochrome P450-dependent activation step, regardless of whether oxidative or reductive reactions are occurring. The balance between oxidative and reductive pathways depends on species, tissue, dose, and oxygen tension. Tissues with chloroform-metabolizing ability include liver, kidney cortex, and tracheal, bronchial, olfactory, oesophageal, laryngeal, tongue, gingival, cheek, nasopharyngeal, pharyngeal, and soft palate mucosa. Of these, the liver is the most active, followed by the nose and kidney. The rate of biotransformation to carbon dioxide is higher in rodent (hamster, mouse, rat) hepatic and renal microsomes than in human hepatic and renal microsomes. Strain- and sex-related differences in sensitivity of mice to nephrotoxicity are correlated with the ability of the kidney to metabolize chloroform. Chloroform is biotransformed more rapidly in mouse than in rat renal microsomes (Environment Canada and Health Canada, 2001).

The oxidative biotransformation of chloroform is catalysed by cytochrome P450 to produce trichloromethanol. Loss of hydrogen chloride from trichloromethanol produces phosgene as a reactive intermediate. Phosgene may be detoxified by reaction with water to produce carbon dioxide or by reaction with thiols, including glutathione and cysteine, to produce adducts. Carbon dioxide is the major metabolite of chloroform generated by the oxidative pathway in vivo. Both products of oxidative activation, phosgene and hydrochloric acid, can cause tissue damage. Phosgene reacting with tissue proteins is associated with cell damage and death. Increased covalent binding of chloroform metabolites in the liver occurs when glutathione is depleted (Environment Canada and Health Canada, 2001). Phosgene can bind covalently to cellular nucleophiles, but little binding of chloroform metabolites to DNA is observed. Chloroform also undergoes cytochrome P450-catalysed reductive biotransformation to produce the dichloromethyl radical (with and without phenobarbital induction), which becomes covalently bound to tissue lipids.

Secondary metabolic pathways are reductive dehalogenation via CYP2B1/2/2E1 (leading to free radical generation) and glutathione conjugation via theta-class glutathione-S-transferase T1-1 (GSTT1-1), which generates mutagenic intermediates. Glutathione-S-transferase-mediated conjugation of chloroform to glutathione can occur only at extremely high chloroform concentrations or doses (IPCS, 2000). Reduced glutathione is capable of scavenging essentially all chloroform metabolites produced in incubations with mouse liver microsomes when chloroform concentrations are not too high (Environment Canada and Health Canada, 2001). Although the findings should be interpreted with caution, Delic et al. (2000) used PBPK modelling to estimate that humans would need to be exposed to 645 mg/m3 (130 ppm) by inhalation in order to attain levels of active metabolites associated with a concentration of 50 mg/m3 (10 ppm) in mice. Based on comparison of the formation of reactive metabolites as measured by binding of radioactivity from [14C]CHCl3 (0-10 mmol) in rat and human liver microsomes, it was concluded that the metabolism in these species is similar, although less efficient in humans (Cresteil et al., 1979).

In eight human volunteers ingesting gelatin capsules containing chloroform (500 mg in olive oil), a maximum of 68.3% and 50.6% of the dose was found in the expired air as chloroform and carbon dioxide, respectively, 8 hours post-administration (Fry et al., 1972; NAS, 1987). There was an inverse relationship between the adipose tissue content of the body and pulmonary elimination of chloroform (Fry et al., 1972).

8.3.2 Brominated trihalomethanes

BDCM is metabolized to phosgene, while DBCM and bromoform are metabolized to brominated analogues of phosgene. The rate of metabolism of these compounds to carbon monoxide both in vivo and in vitro generally follows the halide order, namely, bromoform >> DBCM > BDCM >> chloroform. The International Programme on Chemical Safety (IPCS, 2000) postulated that the brominated THMs may be more rapidly and more extensively metabolized than their chlorinated counterparts. Although this may be true for BDCM, support for this statement, as it pertains to DBCM or bromoform, is difficult to determine from the limited currently available literature. The majority of the comparative metabolism studies conducted to date are limited to chloroform or BDCM. Nonetheless, it would appear that the toxicity of BDCM and likely other brominated THMs is mediated through a bioactivation pathway (IPCS, 2000).

Thornton-Manning et al. (1994) concluded that there were clear interspecies differences in metabolism of BDCM, which may explain the greater sensitivity of rats, relative to mice, to the hepatotoxicity of orally administered BDCM. Within 8 hours following intragastric administration of 150 mg/kg bw (rats) or 100 mg/kg bw (mice) in corn oil, 4-18% and 40-81% of total radiolabelled THMs were eliminated as carbon dioxide through the lungs in expired air in rats and mice, respectively. In the same experiment, 41-67% and 5-26% of the parent compound were eliminated unchanged in rats and mice, respectively. Less than 10% of the total radiolabel for each of the chemicals was detected in the urine of both species 36-48 hours post-exposure; the proportion excreted in the urine for both species was greatest for chloroform, followed by, in descending order, bromoform, BDCM, and DBCM. The authors considered the metabolism of these compounds in the mouse to be 4- to 9-fold greater than that in the rat; however, it should be noted that the administered doses were high and that metabolism in both species is more complete following administration of lower, more relevant doses.

Pegram et al. (1997) provided evidence that the mutagenic metabolic pathway for brominated THMs is mediated by GSTT1-1 conjugation and that the mutagenic pathway of chloroform is not. These findings suggest that chlorinated and brominated THMs may be activated by different mechanisms. DeMarini et al. (1997) examined the ability of GSTT1-1 to mediate the mutagenicity of various THMs, reported nucleotide transitions (GC→AT) mediated by glutathione-S-transferase in Salmonella, and ranked the THMs according to relative mutagenic potency as follows: bromoform = DBCM > BDCM. GSTT1-1 conjugation of BDCM was confirmed by Ross and Pegram (2003), who characterized the reaction kinetics of the conjugation of BDCM with glutathione in mouse, rat, and human hepatic cytosols. Reactive glutathione conjugates produced may result in the formation of DNA adducts. Furthermore, these reactive intermediates produced by glutathione conjugation of BDCM are more mutagenic/genotoxic than intermediates produced from dichloromethane.

Allis et al. (2001) and Lilly et al. (1997) investigated the metabolism of BDCM following inhalation exposure in male rats. The findings suggest that CYP2E1 is the dominant enzyme involved in the metabolism of inhaled BDCM in rats (GlobalTox, 2002). Lilly et al. (1998) also found that more of the parent BDCM compound was eliminated unmetabolized via exhaled breath after aqueous dosing than after corn oil gavage.

8.3.3 Mixtures of THMs

A PBPK model was developed by DaSilva et al. (2000), who found that exposures to binary mixtures of chloroform and BDCM, DBCM, or bromoform would likely result in significant increases in the levels of unmetabolized chloroform in the blood, relative to chloroform administered alone. This study also demonstrated that clearance of THMs may be impacted by toxicokinetic interactions between THMs. Bromoform and DBCM appear to persist in blood and tissues for longer periods of time when co-administered with chloroform than when given alone (GlobalTox, 2002).

8.4 Excretion

8.4.1 Chloroform

In animals and humans exposed to chloroform, carbon dioxide and unchanged chloroform are rapidly eliminated in the expired air. The fraction of the dose eliminated as carbon dioxide varies with the dose and the species (IPCS, 2000).

8.4.2 Bromodichloromethane

Mink et al. (1986) estimated BDCM half-lives at 1.5 and 2.5 hours in the rat and mouse, respectively. Mathews et al. (1990) found that urinary and faecal elimination were low at all dose levels in male rats. Elimination kinetics of BDCM have been studied in humans who had been swimming in chlorinated pools; BDCM half-lives of 0.45-0.63 minutes for blood were estimated using breath elimination data (Lindstrom et al., 1997; Pleil and Lindstrom, 1997).

8.5 PBPK models

PBPK modelling is a technique that may inform and improve toxicological assessments, through a better assessment of the magnitude of the uncertainty factors applied in current risk assessment by informing on issues relating to extrapolation between and within species (Delic et al., 2000).

8.5.1 Chloroform

The 2001 CEPA assessment report on chloroform (Environment Canada and Health Canada, 2001) indicated that the exposure-response relationship for exposure to chloroform associated with cancer and rates of formation of reactive metabolites in the target tissue is upheld by evidence supporting the following assumptions inherent in the PBPK modelling:

  1. In both experimental animals and humans, metabolism of chloroform by CYP2E1 is responsible for production of the critical reactive metabolite, phosgene.
  2. The ability to generate phosgene and phosgene hydrolysis products determines which tissue regions in the liver and kidney are sensitive to the cytotoxicity of chloroform.
  3. This dose-effect relationship is consistent within a tissue, across gender, and across route of administration, and it may also be consistent across species.

The CEPA report presented a PBPK model that was a "hybrid" animal model of the International Life Sciences Institute Expert Panel, which was revised for their assessment and developed to permit its extension to humans (ILSI, 1997; ICF Kaiser, 1999). For this assessment, maximum rate of metabolism per unit kidney cortex volume (VRAMCOR) and mean rate of metabolism per unit kidney cortex volume during each dose interval (VMRATEK) were considered (Environment Canada and Health Canada, 2001).

a) Neoplastic assessment: The results of the exposure-response neoplastic assessment presented were for the combined incidence of renal adenomas and adenocarcinomas in Jorgenson et al. (1985). The VMRATEK associated with a 5% increase in tumour risk (TC05) in humans estimated on the basis of the PBPK model is 3.9 mg/L per hour (95% confidence limit = 2.5, chi-square = 0.04, degrees of freedom = 1, P-value = 0.84). This dose would result from continuous lifetime exposure to chloroform at 3247 mg/L in water or 149 mg/m3 (30 ppm) in air. Respective lower 95% confidence limits for these values are 2363 mg/L and 74 mg/m3 (15 ppm).

Although the data on dose-response were less robust than those for the cancer bioassay, for comparison, a benchmark dose was developed for histological lesions in the kidney in the re-analysis of a subset of the slides from the Jorgenson et al. (1985) biassay. The VMRATEK in humans associated with a 5% increase in histological lesions characteristic of cytotoxicity is 1.7 mg/L per hour (95% lower confidence limit = 1.4, chi-square = 3.9, degrees of freedom = 2, P-value = 0.14). This dose rate would result from continuous lifetime exposure to 1477 mg/L in water or 33.8 mg/m3 (6.8 ppm) in air (Environment Canada and Health Canada, 2001).

b) Non-neoplastic assessment: Short-term exposure by inhalation resulted in cellular proliferation in nasal passages in rats and mice at concentrations as low as 9.9 mg/m3 (2 ppm), with ossifications being observed at slightly higher concentrations following long-term exposure. Moderate hepatic changes were observed in short-term studies in mice at 50 mg/m3 (10 ppm); following both short- and long-term exposure to 124-149 mg/m3 (25-30 ppm), there were multiple adverse effects in the kidney and liver in both rats and mice in several studies. Following ingestion in drinking water, regenerative proliferation after short-term exposure of mice to doses as low as 17 mg/kg bw has been observed. Following bolus dosing, increases in proliferation in the liver of rats have been observed after short-term exposure of rats at 10 mg/kg bw per day, and fatty cysts have been observed in the liver of dogs given 15 mg/kg bw per day. As one of the lowest oral dose levels at which effects on liver and kidney have been observed was in dogs in a study by Heywood et al. (1979), a PBPK model in dogs was developed, keeping in mind that effects on the liver of rodents have also been observed in a similar dose range. Two dose metrics were investigated in exposure-response: the mean rate of metabolism per unit centrilobular region of the liver and the average concentration of chloroform in the non-metabolizing centrilobular region of the liver. The two dose metrics were selected in order to evaluate the possibility of the fatty cyst formation in the dogs being the result of the solvent effects of chloroform or effects of a reactive metabolite. Results of a model fitting supported the assumption that a metabolite, rather than chloroform itself, was responsible for the observed effects. This means that the effect of chloroform on the liver will vary depending on the rate of metabolism. The mean rate of metabolism per unit centrilobular region of the liver in humans associated with a 5% increase in fatty cysts estimated on the basis of the PBPK model is 3.8 mg/L per hour (95% lower confidence limit = 1.3, chi-square = 0.00, degrees of freedom = 1, P-value = 1.00). This dose rate would come from continuous lifetime exposure to 37 mg/L in water or 9.9 mg/m3 (2 ppm) in air. Both levels should be interpreted with caution, because they are not derived using a complete risk assessment approach. They represent only an estimate of levels of exposure in drinking water and/or in air to which humans would need to be exposed over a lifetime in order to attain the selected effect level (5% increase of fatty cysts in the liver).

The 2001 CEPA assessment report concluded, based on the above PBPK models, that the exposure of the general population is considerably less than the level to which it is believed a person may be exposed daily over a lifetime without deleterious effect. Underestimates in exposure due to use of hot rather than cold water and increased chloroform levels in the distribution system compared with the water treatment plant were noted (Environment Canada and Health Canada, 2001).

8.5.2 Bromodichloromethane

A PBPK model has been developed to describe the absorption, distribution, tissue uptake and dosimetry, metabolism, and elimination of BDCM in rats. The metabolism model, derived from inhalation exposure data, was subsequently linked to a multicompartment gastrointestinal tract submodel. This model accurately predicted tissue dosimetry and plasma bromide ion concentrations following oral exposure to BDCM and can be utilized in estimating rates of formation of reactive intermediates in target tissues (Lilly et al., 1997, 1998)

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