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A variety of animal and human studies give evidence that chloroform is well absorbed, metabolised and eliminated by mammals (including humans) after oral, inhalation or dermal exposure. Due to the available overview documents (WHO 1994, ATDSR 1997, Health Council of the Netherlands 2000, WHO 2004, European Commission 2008) no single studies were entered in this section, but an overview of available information is given.

Inhalation exposure in animals and humans:

B6C3F1 mice and Osborne-Mendel rats were exposed by inhalation to different vapour concentrations of [14C]-labelled chloroform for 6 days (Corley et al. 1990). The radioactivity was measured in exhaled air, urine, faeces, carcass, skin and as cage washes for 48 hours following exposure. In mice, the greatest portion of chloroform was absorbed, metabolised and then exhaled as [14C]-carbon dioxide (60 to 80 %). Minor portions were absorbed and then exhaled as [14C]-chloroform, excreted in urine or faeces, or found as residue in carcass, skin or cage wash at the end of the post-exposure collection period. For rats it seemed that less of the chloroform was metabolised to CO2 and exhaled and that more unchanged chloroform was exhaled or excreted via urine and faeces.

Morgan et al. (1970) exposed human volunteers to a total amount of approximately 5 mg of [38Cl]-labelled chloroform as vapour. The volunteers were asked to inhale the radiolabelled chloroform in a single breath and hold breath for approximately 20 seconds. It was estimated that under the conditions of the experiment, approximately 95 % of the applied dose was absorbed into the human body. The study also estimated the amount excreted 1 hour after exposure: 10 % of the applied dose was exhaled at that time, but that less than 1 % was excreted via urine. The radiological investigation showed that radiolabelled chloroform was transferred rapidly to the blood stream and that dissolved chloroform was removed either by excretion into alveolar air or by transfer to other compartments such as body lipids.

Dermal exposure in animals and humans:

There is evidence that considerable absorption of chloroform through the skin of test animals occurs. Morgan et al. (1991) measured blood concentrations in male F-344 rats during 24 -hour dermal exposure to pure chloroform or aqueous solutions of chloroform. The blood chloroform level peaked at 51 mg/L after exposure to the pure substance for 4 to 8 hours and remained about constant for the duration of the exposure period. The absorption was more rapid with the chloroform applied in aqueous solution and peak blood concentrations of chloroform were reached after approximately 2 hours.

The absorption of chloroform from bathing water through the human skin was investigated by Gordon et al. (1998) who studied concentrations of chloroform in exhaled air of volunteers breathing solely fresh air during the bathing period. The experiments showed that the absorption rate was temperature-dependent and the rate at 30 °C was by a factor of 30 lower than that at 40 °C. The results of the study were used to determine effective skin permeability coefficients by using a physiologically based pharmacokinetic model (Corley et al. 2000). The calculations for dermal absorption at 40 °C showed that in males the dermal dose resulting from the bathing would be between 13.9 and 22.9 % of an oral dose resulting of the consumption of two litres of drinking water at the same concentration as the bathing water, whereas in females this percentage was 9.4 to 27.8 %.

Oral exposure in animals and humans:

Chloroform is well absorbed after oral administration. After intragastric administration of 75 mg/kg chloroform in water or vegetable oil to male Wistar rats, peak blood concentrations were observed within approximately 6 minutes. The blood concentrations were higher in experiments using water as the vehicle (Withey et al. 1983). Mice, rats and squirrel monkeys were administered a dose of 60 mg/kg [14C]-radiolabelled chloroform by the oral route (Brown et al. 1974). The radioactivity in breath, faeces, urine and carcass was surveyed for 48 hours following exposure. In mice and rats, the greatest portion of the administered dose was recovered as exhaled [14C]-carbon dioxide (about 85 % in mice and 67 % in rats), followed by unchanged chloroform in exhaled air (about 6 % in mice and 20 % in rats). In the monkey, the excretion pattern was different and 18 % was excreted as CO2 in exhaled air and 79 % as unchanged chloroform in exhaled air. In humans given a single oral dose of 0.5 g chloroform, about 50-52% of the dose was absorbed, and virtually all the absorbed dose was metabolised to carbon dioxide. Blood levels peaked after 1.5 h and declined in line with a two-compartment model with half-lives of 13 and 90 min, respectively (Fry et al., 1972).

Metabolism in animals and humans:

The following text is copied from the Concise International Chemical Assessment Document 58 on chloroform published by the World Health Organisation (WHO 2004):

"Both oxidative and reductive pathways of chloroform metabolism have been identified, although data in vivo are limited. Carbon dioxide is the major metabolite of chloroform generated by the oxidative pathway in vivo. The oxidative pathway also generates reactive metabolites, including phosgene (Pohl et al., 1977; Pohl & Krishna, 1978) (determined in vitro, with phenobarbital induction), while the reductive pathway generates the dichloromethylcarbene free radical (Wolf et al., 1977; Tomasi et al., 1985; Testai & Vittozzi, 1986) (determined in vitro and in vivo, both with and without phenobarbital induction). Oxidative and reductive metabolism both proceed through a cytochrome P450 (CYP)-dependent enzymatic activation step. The balance between oxidative and reductive pathways depends on species, tissue, dose, and oxygen tension. In intact mammals, oxidative tension probably precludes any significant metabolism by the reductive pathway (Testai & Vittozzi, 1986; Ammann et al., 1998). Phosgene is produced by oxidative dechlorination of chloroform to trichloromethanol, which spontaneously dehydrochlorinates (Mansuy et al., 1977; Pohl et al., 1977). Dehydrochlorination of trichloromethanol produces one molecule of hydrochloric acid, and hydrolysis of phosgene produces another two molecules, so that three molecules of hydrochloric acid are produced in the conversion of chloroform to carbon dioxide.

The electrophilic metabolite phosgene binds covalently to nucleophilic components of tissue proteins (Pohl et al., 1980). It also interacts with other cellular nucleophiles (Uehleke & Werner, 1975) and binds to some extent to the polar heads of phospholipids (Vittozzi et al., 1991). Alternatively, phosgene reacts with water to release carbon dioxide and hydrochloric acid (Fry et al., 1972; B.R. Brown et al., 1974; D.M. Brown et al., 1974). The interaction of phosgene with glutathione results in the formation of S-chlorocarbonyl glutathione, which can either interact with an additional glutathione to form diglutathionyl dithiocarbonate (Pohl et al., 1981) or form glutathione disulfide and carbon monoxide (Ahmed et al., 1977; Anders et al., 1978). Incubation of mouse renal microsomes with glutathione increases production of these metabolites from chloroform and decreases irreversible binding to proteins and further metabolism to carbon dioxide (Smith & Hook, 1984). Reduced glutathione is capable of scavenging essentially all chloroform metabolites produced in incubations with mouse liver microsomes when chloroform concentrations are not too high (Vittozzi et al., 1991). The relative importance of the minor pathways of phosgene metabolism depends upon the availability of glutathione, other thiols, and other nucleophilic compounds, such as histidine and cysteine (see Figure 2).

Oxidative metabolism, with CYP2E1 (an ethanol-inducible mono-oxygenase isoenzyme system present in the liver of mammals, including humans) playing a key role, is probably the only significant in vivo pathway at low exposures, and available data indicate that oxidative metabolism has a major role in toxicity. The dominant role of CYP2E1 in metabolizing chloroform to toxic metabolites has been demonstrated in studies involving treatment of animals with enzyme inducers or inhibitors, as well as studies in mice lacking CYP2E1 (Brady et al., 1989; Guengerich et al., 1991; Nakajima et al., 1995a,b; Constan et al., 1999; see also section 8.8). Immunoinhibition studies with anti-CYP2E1 monoclonal protein have shown that CYP2E1 is responsible for 81% of the metabolism assayed at a low chloroform (0.5 mmol/litre) concentration in liver microsomes from acetone-induced rats (Brady et al., 1989). Toxicity to rat and mouse hepatocytes incubated in vitro with up to 5 mmol chloroform/litre was prevented by the addition of a CYP2E1 inhibitor or by reduced oxygen tension, underscoring the importance of oxidative metabolism in toxicity (Ammann et al., 1998). Regional distribution of liver lesions in rats and mice correlates well with the hepatic distribution of CYP2E1 and glutathione (Smith et al., 1979; Ingelman-Sundberg et al., 1988; Tsutsumi et al., 1989; Johansson et al., 1990; Dicker et al., 1991; Nakajima et al., 1995a,b).

CYP2B1 may also have a role in chloroform metabolism, although this is likely to be only minor at low tissue chloroform concentrations (studies reviewed in Environment Canada & Health Canada, 2001). However, at high tissue concentrations (e.g., resulting from an oral dose of 0.5 ml/kg body weight), chloroform hepatotoxicity was dramatically potentiated in Wistar rats treated with phenobarbital (a CYP2B1 inducer) but not in rats treated with n-hexane (a CYP2E1 inducer), compared with uninduced controls (Nakajima et al., 1995b).

A study in which rats were exposed to [14C]chloroform showed that metabolism was most active in the liver, followed by the nose and kidney. Metabolic activity was correlated with accumulation of metabolites (Löfberg & Tjälve, 1986)."

Additional information on the human metabolism of chloroform has been provided from in vitro tests in which it was shown that the substance can undergo both oxidative and reductive metabolism in the human liver, depending on oxygen and substrate concentration (Gemma et al. 2003). At low levels, chloroform is metabolised primarily to phosgene by CYP2E1. When the CYP2E1-mediated reaction is saturated, the predominant role in phosgene production is for CYP2A6, which is efficient even in highly hypoxic conditions. CYP2E1 is also able to catalyse dichloromethyl radical formation. This latter pathway seems to be scarcely relevant in human liver, since it is active only at high substrate concentrations and strictly anaerobic conditions (Gemma et al. 2003).

The relevance of CYP2E1 in chloroform metabolism in humans provides useful information to identify eventual differences in susceptibility to chloroform-induced adverse effects. Variations in the level of expression of CYP2E1 is not uncommon, being determined either by genetic features, pathophysiological conditions such as diabetes, or by environmental factors, such as alcoholic beverages consumption or exposure to other xenobiotics known to affect CYP2E1 expression. Also the human foetus may be affected, when the foetal brain CYP2E1 mRNA is expressed at relatively high amounts corresponding to a fairly constant level of enzymatic activity of ethanol metabolism (Gemma et al. 2003).

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