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Description of key information

Repeated dose toxicity studies in laboratory animals identified the liver, kidneys and nasal cavity as the target organs for the toxic potential of chloroform. The lowest LOAEL for hepatotoxicity following repeated oral exposure to chloroform was 15 mg/kg bw/day reported from a 7-year study in dogs. A NOEL for the mentioned target organs of 34 mg/kg bw/day was determined in female F-344 rats receiving chloroform by oral gavage for three weeks.
The NOAEC for renal lesions, induction of cell proliferation and tumour formation in the kidneys following repeated inhalation exposure of male mice to chloroform vapours for 90 days was 25 mg/m3 (exposure for 5 days a week) or 50 mg/m3 (exposure for 7 days a week). Distinct nasal lesions in the form of a generalised atrophy of ethmoid turbinates were observed in male and female F-344 rats exposed to 50 mg/m3 for 90 days.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
LOAEL
15 mg/kg bw/day

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEC
25 mg/m³

Additional information

Oral exposure

A combined repeated dose and carcinogenicity study was carried out with chloroform orally administered to male and female Beagle dogs over a period of more than 7 years (Heywood et al. 1979). The study was performed according to principles comparable to those set out in the testing guideline for method B.33 suggested by the European Commission with minor restrictions. The chloroform was contained in toothpaste and administered in form of a gelatine capsule. Chronic effects related to chloroform exposure included a significantly increased level of alanine aminotransferase level in the high dose group throughout the study and in the lower-dose group during the later phase of the study and increased incidence of aggregations of vacuolated histiocytes forming "fatty cysts" in the liver of treated dogs. A NOAEL value could not be established and the LOAEL value found in this study was 15 mg/kg body weight/day established for female and male dogs together.

A study on the sub-acute toxicity of chloroform was carried out using female F-344 rats exposed by oral gavage to graded doses of chloroform dissolved in corn oil for 5 days per week for 3 consecutive weeks (Larson et al. 1995a). The study was comparable to a guideline study according to method B.7 of the European Commission with acceptable restrictions. Liver, kidneys and nasal passages were identified as the target organs for chloroform administered by oral gavage. Mild degenerative centrilobular changes and a dose-dependent increase in the hepatocyte labelling index (% of cells in S-phase), a dose-dependent increase in the labelling index in the kidneys, lesions and cell proliferation in the olfactory epithelium and changes in the nasal passages were observed at 100 mg/kg body weight/day of chloroform after 3 weeks of exposure. The NOAEL for systemic effects was 34 mg/kg body weight/day based on the finding of lesions and increased cell replication in the liver and kidneys at higher dose levels. Local effects in the nasal passage were seen after 4 days of exposure at a dose of 34 mg/kg/day. Male F-344 rats exposed by oral gavage to chloroform in a similar manner exhibited comparable adverse effects in the liver, kidneys and nasal cavity (Larson et al. 1995). However, male rats were less sensitive to chloroform exposure compared to female rats and the NOAEL established for male F-344 rats was 90 mg/kg body weight/day. It should be noted that the signification of nasal damages is not so clear, it is difficult to say if it is due to evaporation of chloroform from stomach or if it is a specific effect due to toxicity of chloroform. Moreover, this kind of effects are frequent and may not be related to chloroform only, but to vapors in general.

A less clear picture was obtained from repeated dose toxicity studies on oral exposure to mice. Hepatotoxicity was found in a 90-day repeated dose oral toxicity study on male and female B6C3F1 mice at a dose of 270 mg/kg and the NOAEL was 60 mg/kg/day (Bull et al. 1986). The liver was also the primary target organ for chloroform toxicity in a sub-acute study carried out with female B6C3F1 mice being exposed to chloroform in corn oil for 5 days per week for 3 weeks (Larson et al. 1994a, Larson et al. 1994b). The NOEL for hepatic degenerative changes indicated by liver histology and serum enzyme levels was 10 mg/kg/day and the NOEL for hepatic cell proliferation was 34 mg/kg/day. In male B6C3F1 mice administered chloroform in corn oil by oral gavage for 5 days per week for 3 weeks, the liver and kidneys were affected (Larson et al. 1994c). The LOEL for renal toxicity was 34 mg/kg/day in this study. The NOEL for hepatic toxicity and cell replication in the liver and the kidneys was 34 mg/kg/day. A 90-day repeated dose oral toxicity study with chloroform dissolved in 10 % Emulphor in deionised water was performed in male and female CD-1 mice. The study found a NOAEL of 50 mg/kg/day based on renal and liver toxicity (Munson et al. 1982). A sub-acute repeated dose oral toxicity study in male CD-1 mice receiving chloroform in corn oil for 14 days observed a LOEL of 37 mg/kg/day based on renal and liver toxicity (Condie et al. 1983).

Dermal exposure

Studies on dermal repeated exposure to chloroform are unavailable. The repeated inhalation NOAEC of 25 mg/m3 (Templin et al. 1998) will be used to derive the dermal threshold value and route-to-route extrapolation is performed. The absorption efficiencies for chloroform are given in the EU risk assessment (France 2007): 80 % via inhalation route and 10 % via dermal route. The inhalation volume of the testing animals (mouse: 0.41 m3/kg for the six hours, France 2007) is taken into account. There is no extrapolation from subchronic to chronic effects because comparable effect levels were seen at both exposure durations. The inhalation NOAEC can thus be corrected to produce a dermal NOAEL: 25 * 0.41 * 0.8 / 0.1 = 82 mg/kg bw/day (the NOAEL derived from the oral LOAEL is 150 mg/kg bw/day).

Inhalation exposure

A reliable combined chronic repeated dose/carcinogenicity study by the inhalation route was performed with male and female BDF1 mice (Yamamoto et al. 2002). Animals received whole-body exposure to chloroform vapours for 6 hours/day for 5 days per week over a period of 104 weeks. Animals exposed to 25 mg/m3 chloroform vapours showed reduced body weight gain, nasal lesions and changes in serum enzyme levels. The NOEC for renal cell carcinoma in male mice as the most sensitive carcinogenic endpoint was 25 mg/m3.

A 90-day repeated dose inhalation study was performed with male and female BDF1 mice according to procedures similar to those of the EU Method B.29 with restrictions (Templin et al. 1998). Mice were exposed for 6 hours per day, for 5 days per week to vapour concentrations of chloroform of 0, 5, 25, 147, 441 mg/m3. The study focussed on histopathological changes in the kidneys and the livers of the test animals as these endpoints were the most sensitive toxicological endpoints identified in preceding studies. Male mice were more susceptible with regard to chloroform exposure showing histopathologic changes in the kidneys at exposure concentrations of 147 and 441 mg/m3. The NOAEC for cell proliferation and tumour formation in the male mouse kidneys was 25 mg/m3, which covers the most sensitive toxicological endpoints. Female mice were less susceptible with regard to chloroform exposure than male BDF1 mice.

A 90-day repeated dose inhalation toxicity study was carried out with chloroform using male and female B6C3F1 mice exposed to chloroform vapours at concentrations of 0, 1.5, 10, 50, 147 or 441 mg/m3 for 6 hours/day for 5 or 7 days per week (Larson et al. 1996). The liver of male and female animals was affected by exposure in a dose-dependent manner. The kidney of male mice was also affected by exposure. The NOEC for liver lesions in male and female mice was 50 mg/m3, the NOEC for regenerative cell proliferation in the liver of female and male mice was 50 mg/m3 and 441 mg/m3, respectively. The NOEC for lesions in the kidneys of male mice was 10 mg/m3. At 13 weeks, increased regenerative cell proliferation in the kidneys occurred at 147 mg/m3 when male mice were exposed continuously for 7 days a week and at 50 mg/m3 when male mice were exposed for 5 days a week. Local effects in the respiratory tract consisting of nasal lesions and induced cell proliferation essentially were confined to male and female mice exposed to concentrations of 50 mg/m3 or greater. These effects were observed only at the early time point after 4 days and were transient during the remainder of the study. In conclusion, male mice tended to be more susceptible to inhalation exposure to chloroform vapours than female mice. The NOEC for lesions in kidney cells and increased regenerative cell proliferation in the kidneys of male mice was 10 mg/m3.

A 90-day subchronic toxicity study was carried out in male and female F-344 rats according to principles similar to those of the relevant OECD guideline (Templin et al. 1996b). Different groups of rats were exposed by inhalation to chloroform vapours at concentrations of 0, 10, 50, 147, 441 or 1470 mg/m3 for 6 hours per day for 5 or 7 days per week for 13 weeks. Systemic effects were found in the kidneys and the liver of rats. The NOEC for renal toxicity and increased cell replication in the kidneys of male and female rats was 50 mg/m3. The NOEC for liver toxicity in male and female rats was 147 mg/m3 and the NOEC for increased liver cell replication was 441 mg/m3. Minimal local effects in the respiratory tract of male and female rats were found at 10 mg/m3 after 13 weeks consisting of atrophy in the lamina propria and the olfactory epithelium. The local effects were more distinct at 50 mg/m3.


Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; respiratory: nose; urogenital: kidneys

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver; respiratory: nose; urogenital: kidneys

Justification for classification or non-classification

Based on the available data on repeated dose toxicity the proposed GHS classification for chloroform should be STOT Category 2, "Harmful to human health", with the hazard statement: may cause damage to organs (liver, kidneys, nasal passages) through prolonged or repeated exposure via oral or inhalation routes. According to EU directive 67/548/EEC the substance is classified R48/20/22 (danger of serious domages to health by prolonged exposure throught inhalation and if swallowed however during the revision of classification of chloroform of Technical Committee and Labelling in September 2007, it was agreed to keep only the classification R48/20 and to delete the R48/22 since this effect was only seen at high doses. Thus, only the inhalation route will be considered for the STOT RE2.