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EC number: 200-663-8 | CAS number: 67-66-3
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- Aquatic toxicity
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Carcinogenicity
Administrative data
Description of key information
A combined chronic toxicity/carcinogenicity inhalation study was carried out with chloroform in female and male BDF1 mice (Yamamoto et al. 2002) receiving repeated dose inhalation exposure to chloroform vapours at concentrations of 0, 5, 30 or 90 ppm (0, 24.5, 147 or 440 mg/m3) for 104 weeks. Adverse effects were mainly observed in the liver and kidneys of exposed animals. Inhalation exposure to chloroform of mice over a period of 2 years led to a significantly increased incidence of renal adenomas and carcinomas at exposure concentrations of 30 and 90 ppm (147 and 440 mg/m3). For the exposed mice, the histopathological changes in the kidneys including the cytoplasmic basophilia which occurred at 30 ppm (147 mg/m3) or greater, were employed as the biologically significant endpoint to deduce a NOAEC of 5 ppm (24.5 mg/m3). In the same study, F 344 rats exposed to concentrations of 0, 10, 30 or 90 ppm (0, 49.5, 147 or 440 mg/m3) did not show a statistically significant, exposure-related increase in the incidence of liver and kidney tumours. Exposure to chloroform at 30 or 90 ppm (147 or 440 mg/m3) induced nuclear enlargement of the proximal tubules and dilatation of the tubular lumen without development of renal tumours. No significantly increased incidence of histopathological lesions in the liver was found. Slight but significant increases in the serum levels of GPT and gamma-GTP occurred in exposed male rats from 10 ppm (49.5 mg/m3) exposure concentration.
A chronic exposure study was carried out with chloroform using the following strains of mice: C57BL, CBA, CF/1, ICI (Roe et al. 1979). The studies were carried out according to principles similar to those of the EU method B.32 with restrictions. Chloroform administered via the oral route induced kidney tumours in male mice. The lowest oral NOAEL for these effects was 17 mg/kg bw/day. In a carcinogenicity study in male Osborne-Mendel rats receiving chloroform via drinking water for a period of 104 weeks, an increased incidence of renal cell tumours was observed which was clearly dose-related. The NOAEL for renal tumours was 200 mg/L (19 mg/kg bw/day; Jorgensen et al., 1985)). Several other studies are available in rats or mice which were either positive or negative; in the positive studies kidney and/or liver tumors were seen. A 7 -year oral study in Beagle dogs did not show an exposure-related increase in the incidence of tumors; the NOAEL was at least 30 mg/kg bw/day.
According to the US EPA, (2001) studies in animals reveal that chloroform can cause an increased incidence of kidney tumours in male rats or mice and an increased incidence of liver tumours in mice of either sex. These induced tumours responses are postulated to be secondary to sustained or repeated cytotoxicity and secondary regenerative hyperplasia, according to the dose levels tested. “The weight of the evidence indicates that a mutagenic mode of action via DNA reactivity is not a significant component of the chloroform carcinogenic process. The persistent cell proliferation presumably would lead to higher probabilities of spontaneous cell mutation and subsequent cancer".
Key value for chemical safety assessment
Carcinogenicity: via oral route
Link to relevant study records
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.32 (Carcinogenicity Test)
- Deviations:
- yes
- Remarks:
- The study was not carried out with the pure substance but with a formulation containing only a small fraction of the test substance. Only two dose groups were tested.
- GLP compliance:
- no
- Species:
- mouse
- Strain:
- other: C57BL, CBA, CF/1, ICI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The long-term study included female and male mice of the ICI strain (Imperial Chemical Industries, Ltd.), the CBA strain (Laboratory Animal Centre, Carshalton), C57BL strain (Laboratory Animal Centre, Carshalton), and CF/1 (Carworth Farms, USA). Mice were aged not more than 10 weeks and were acclimated for 1 week, weighed and allocated at random four to a cage to different exposure groups or to a reserve group. Mice had free access to tap water and to autoclaved Spratts Laboratory Animals Diet. Autoclaved sawdust was provided as cage litter. Room temperature and relative humidity were controlled at 21 +/- 2 °C and 50 +/- 5 % respectively.
- Route of administration:
- oral: gavage
- Vehicle:
- other: toothpaste of the above-mentioned composition and/or arachis oil
- Details on exposure:
- Fresh batches of toothpaste were prepared monthly. The toothpaste (assaying 3.33-3.67 % CHCl3) had a specific gravity such that a chloroform dose level was achieved by 1 mL of toothpaste/kg body weight. For the dose level of 17 mg CHCl3/kg body weight, toothpaste to the above formula but containing only 1.0 % CHCl3 was given in the same dose volume.
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 80 weeks
- Frequency of treatment:
- 6 days per week
- Post exposure period:
- 16 to 24 weeks
- Dose / conc.:
- 17 mg/kg bw/day (nominal)
- Remarks:
- only ICI strain
- Dose / conc.:
- 60 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- see Table 1, Table 2, Table 3
- Control animals:
- yes
- Details on study design:
- In high-dose male ICI mice receiving 60 mg chloroform/kg body weight/day, a slight increase in moderate to severe fatty degeneration of the liver was seen and kidney tumours (adenomas and carcinomas) were statistically higher than in the controls. Treatment with chloroform was associated with increased incidence of moderate to severe kidney lesions in CBA and CF/1 mice.
- Observations and examinations performed and frequency:
- Examination for debility or death twice daily; individual weighing at weekly intervals for the first 6 months, then fortnightly for the next 6 months, and thereafter monthly; food consumption was not recorded in the first study but was recorded on a cage basis for the whole second study and from weeks 9-12 only in the third study
- Sacrifice and pathology:
- The following organs were weighed after the mice were killed: adrenals, kidneys, lungs, liver, and spleen. All macroscopically-observed tumours and abnormal growths were processed for histopathological examination along with a wide range of available tissues such as brain, lungs, liver and kidneys
- Other examinations:
- Haematological records were made in the first study during weeks 21 and 56
- Statistics:
- Statistical analysis was performed using a one-tailed test (no further specification)
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Details on results:
- A slight increase in moderate to severe fatty degeneration of the liver was seen and kidney tumours (adenomas and carcinomas) were statistically higher in high-dose male ICI mice receiving 60 mg chloroform/kg body weight/day, than in the controls. Treatment with chloroform was associated with increased incidence of moderate to severe kidney lesions in CBA and CF/1 mice.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 17 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: hepatic changes and kidney tumors at the next higher dose of 60 mg/kg bw/day
- Conclusions:
- An increased incidence of kidney tumours (adenomas and carcinomas) was seen in male ICI mice receiving 60 mg CHCl3/kg body weight/day. The overall NOAEC was established as 17 mg/kg bw/day.
- Executive summary:
Three chronic exposure studies were carried out with chloroform using four different strains of mice (C57BL, CBA, CF/1, ICI). The studies were carried out according to principles similar to those of the EU method B.32 with restrictions. Chloroform was administered orally by gavage in toothpaste or in arachis oil. Male ICI mice receiving 60 mg CHCl3/kg body weight/day in arachis oil exhibited a slight increase in moderate to severe fatty degeneration of the liver and a statistically significant increase in the incidence of kidney tumours (adenomas and carcinomas). Treatment with chloroform was associated with increased incidence of moderate to severe kidney lesions in CBA and CF/1 mice. The experimental NOAEL value established in the study was 17 mg/kg body weight/day.
Reference
Table 1: Incidence of renal tubule adenomas and carcinomas in ICI mice exposed orally to chloroform over a period of 80 weeks.
Treatment |
Sex |
Incidence of renal tumours |
First study |
||
Vehicle control (toothpaste) |
Male |
0/72 |
17 mg/kg bodyweight/day CHCl3 |
0/37 |
|
60 mg/kg bodyweight/day CHCl3 |
8/38 |
|
Vehicle control (toothpaste) |
Female |
0/59 |
17 mg/kg bodyweight/day CHCl3 |
0/35 |
|
60 mg/kg bodyweight/day CHCl3 |
0/38 |
|
Second study |
||
Control |
Male |
1/48 |
Vehicle control (toothpaste) |
6/237 |
|
60 mg/kg bodyweight/day CHCl3 |
9/49 |
|
Third study |
||
Control |
Male |
0/3 |
Vehicle control (toothpaste) |
1/49 |
|
Vehicle control (arachis oil) |
1/50 |
|
60 mg/kg bodyweight/day (toothpaste) CHCl3 |
5/47 |
|
60 mg/kg bodyweight/day (arachis oil) CHCl3 |
12/48 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 17 mg/kg bw/day
- Study duration:
- chronic
- Species:
- mouse
- Quality of whole database:
- Several supporting oral toxicity studies available
- Organ:
- kidney
- liver
Carcinogenicity: via inhalation route
Link to relevant study records
- Endpoint:
- carcinogenicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- Fischer 344/DuCrj
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- F-344/DuCrj rats of both sexes were obtained at the age of 4 weeks from Charles River Japan Inc., Kanagawa, Japan; animals were quarantined and acclimated for 2 weeks and were housed individually in stainless steel wire hanging cages in stainless steel chambers maintained at 23 +/- 2 °C, 55 +/- 10 % relative humidity, 12 air changes per hour; fluorescent lightning gave a 12 hours light/12 hours dark cycle; animals had free access to a pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo) and sterilised water
- Route of administration:
- inhalation: vapour
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- air
- Details on exposure:
- Chloroform vapour-air mixture was produced by bubbling clean air through the liquid chloroform, further diluted with clean air, and supplied to the inhalation exposure chambers by the method and apparatus described by Kano et al. (2002, Journal of Occupational Health 44, 119-124).
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- not specified, but reference is given to Kano et al. (2002; Journal of Occupational Health 44, 119-124).
- Duration of treatment / exposure:
- 104 weeks
- Frequency of treatment:
- 6 hours per day, 5 days per week
- Dose / conc.:
- 10 ppm
- Remarks:
- target concentration (49.5 mg/m3)
- Dose / conc.:
- 30 ppm
- Remarks:
- target concentration (147 mg/m3)
- Dose / conc.:
- 90 ppm
- Remarks:
- target concentration (440 mg/m3)
- No. of animals per sex per dose:
- 50 males and 50 females
- Control animals:
- yes
- Observations and examinations performed and frequency:
- Daily observations for clinical signs and mortality; animals were weighed and their food and water consumptions were measured weekly for the first 13 weeks and every 4 weeks thereafter
- Sacrifice and pathology:
- Animals found dead, in a moribund state or surviving to the end of the 2 year exposure received complete necropsy; urinary, haematological and blood biochemical parameters of all surviving animals were obtained from urine collected at the end of the 2-year exposure period and from blood samples taken at the end of the 2-year exposure period after overnight fasting; The urinary, haematological and blood biochemical parameters examined here were given in the OECD 453 guidelines. All organs were removed, weighed at necropsy and examined for macroscopically visible lesions. The histopathologically examined tissues were described in detail in Katagiri et al, 2001. The tissues for microscopic examination were fixed in 10% neutral buffered formalin, embedded in paraffin, and sections of all tissues and tumours were 5 micrometre thick, and stained with haematoxylin and eosin.
- Statistics:
- The incidence of non-neoplastic lesions and urinary data were analysed by chi-square test; the incidence of neoplastic lesions was analysed by Peto's test and Fisher's exact test; body weight, food consumption, and haematological and blood biochemical parameters were analysed by Dunnett test
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- effects observed, treatment-related
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Urinalysis findings:
- effects observed, treatment-related
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Details on results:
- There was no statistical difference in the 2-year survival rate between the exposed female groups and the control group, but the survival rate of the control male group significantly decreased as compared to the exposed male groups. The growth rate of the 90 ppm group of both sexes was significantly suppressed over the controls throughout the entire exposure period. Absolute and relative kidney weights of the 90 ppm female group were statistically higher than those of the controls. No statistically increase in the incidence of kidney tumours was seen in the exposed males and females, but a case of renal cell adenoma occurring in the 90 ppm female group was very rare as compared to the historical laboratory control data. Dose-dependent increases in the occurrence of nuclear enlargement of the proximal tubule and dilatation of the tubular lumen in the kidney were observed with exposure concentrations of 30 ppm and 90 ppm. The severity of chronic progressive nephropathy significantly decreased with increasing exposure concentrations in animals of both sexes. Hepatocellular adenomas were observed in the female rats, but the incidence was not exposure-related. There was a significantly increased incidence of vacuolated cell foci in the 90 ppm female group.
Nasal lesions were observed at 10 ppm and above including thickening of the bone and atrophy and respiratory metaplasia of the olfactory epithelium. Lowered incidences of pituitary gland adenoma in the 90 ppm females and myocardial fibrosis in the 90 ppm males were observed. The exposed males exhibited significantly decreased serum levels of triglyceride, phospholipids and creatinine at 30 ppm and above and total cholesterol at 90 ppm. Serum levels of BUN significantly decreased in the three exposed male groups. GOT, GPT and gamma-GTP significantly increased in the exposed males. The exposed females exhibited a significant decrease in triglyceride and an increase in gamma-GTP at 90 ppm. Positive urinary glucose was observed in the 90 ppm male group and in the 10 ppm and above exposed females. - Dose descriptor:
- NOAEC
- Effect level:
- 10 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: changes in the kidneys at the next higher level tested of 30 ppm
- Dose descriptor:
- NOAEC
- Effect level:
- >= 90 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: absence of exposure-related increase in tumors
- Conclusions:
- Inhalation exposure of male and female F-344 rats to 10, 30 and 90 ppm chloroform vapour did not result in any statistically significant, exposure-related increase in the incidence of liver and kidney tumours. Exposure to chloroform at 30 and 90 ppm induced nuclear enlargement of the proximal tubules and dilatation of the tubular lumen without development of renal tumours. No significantly increased incidence of histopathological lesions in the liver was found. Slight but significant increases in the serum levels of GPT and gamma-GTP occurred in exposed male rats from 10 ppm (49.5 mg/m3) exposure concentration. Although these two biomarkers are biologically significant, (indicative of liver cell necrosis and regeneration), they were not considered to determine a LOAEL since no dose-relationship was observed.
- Executive summary:
A combined chronic toxicity/carcinogenicity study was carried out with chloroform using female and male F-344 rats receiving inhalation exposure to chloroform vapours of 0, 10, 30 or 90 ppm (0, 49.5, 147 or 440 mg/m3) during a period of 2 years. The study was in accordance with the method B.33 suggested by the European Commission with minor restrictions. The growth rate of female and male rats exposed to 90 ppm chloroform vapour was suppressed over the controls. Nasal lesions were observed at exposure concentrations of 10 ppm and above. The 2 -year inhalation exposure to 10, 30 or 90 ppm did not result in any significant increase in the incidence of liver and kidney tumours in both sexes. Exposure to chloroform at 30 or 90 ppm induced nuclear enlargement of the proximal tubules and dilatation of the tubular lumen without development of renal tumours. No significantly increased incidence of histopathological lesions in the liver was found. The NOAEL value for the histopathological changes in the kidneys was found to be 10 ppm (49.5 mg/m3).
- Endpoint:
- carcinogenicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Species:
- mouse
- Strain:
- other: BDF1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Crj:BDF1 mice of both sexes were obtained at the age of 4 weeks from Charles River Japan Inc., Kanagawa, Japan; animals were quarantined and acclimated for 2 weeks and were housed individually in stainless steel wire hanging cages in stainless steel chambers maintained at 23 +/- 2 °C, 55 +/- 10 % relative humidity, 12 air changes per hour; fluorescent lightning gave a 12 hours light/12 hours dark cycle; animals had free access to a pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo) and sterilised water
- Route of administration:
- inhalation: vapour
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- air
- Details on exposure:
- Chloroform vapour-air mixture was produced by bubbling clean air through the liquid chloroform, further diluted with clean air, and supplied to the inhalation exposure chambers by the method and apparatus described by Kano et al. (2002, Journal of Occupational Health 44, 119-124).
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- not specified in the publication, but reference to Kano et al. (2002, Journal of Occupational Health 44, 119-124)
- Duration of treatment / exposure:
- 104 weeks
- Frequency of treatment:
- 6 hours per day, 5 days per week
- Dose / conc.:
- 5 ppm
- Remarks:
- target concentration (24.5 mg/m3)
- Dose / conc.:
- 30 ppm
- Remarks:
- target concentration (147 mg/m3)
- Dose / conc.:
- 90 ppm
- Remarks:
- target concentration (440 mg/m3)
- No. of animals per sex per dose:
- 50 males and 50 females
- Control animals:
- yes
- Details on study design:
- Mice exposed to 30 and 90 ppm were found to die of acute poisoning in the first week of exposure in a preliminary experiment. Therefore, exposure concentrations of the 30 and 90 ppm exposure concentration groups were increased stepwise: mice exposed to 30 ppm were exposed to 5 ppm for the first 2 weeks, 10 ppm for another 2 weeks and 30 ppm for the subsequent 100 weeks. Mice exposed to 90 ppm were exposed to 5 ppm for the first 2 weeks, 10 ppm for the following 2 weeks, 30 ppm for the next 2 weeks and 90 ppm for the subsequent 98 weeks.
- Observations and examinations performed and frequency:
- Daily observations for clinical signs and mortality; animals were weighed and their food and water consumptions were measured weekly for the first 13 weeks and every 4 weeks thereafter.
- Sacrifice and pathology:
- Animals found dead, in a moribund state or surviving to the end of the 2 year exposure received complete necropsy; urinary, haematological and blood biochemical parameters of all surviving animals were obtained from urine collected at the end of the 2-year exposure period and from blood samples taken at the end of the 2-year exposure period after overnight fasting . The urinary, haematological and blood biochemical parameters examined here were given in the OECD 453 guidelines. All organs were removed, weighed at necropsy and examined for macroscopically visible lesions. The histopathologically examined tissues were described in detail in Katagiri et al, 2001. The tissues for microscopic examination were fixed in 10% neutral buffered formalin, embedded in paraffin, and sections of all tissues and tumours were 5 micrometre thick, and stained with haematoxylin and eosin.
- Statistics:
- The incidence of non-neoplastic lesions and urinary data were analysed by chi-square test; the incidence of neoplastic lesions was analysed by Peto's test and Fisher's exact test; body weight, food consumption, and haematological and blood biochemical parameters were analysed by Dunnett test
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Urinalysis findings:
- effects observed, treatment-related
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not specified
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Details on results:
- The survival rate in the 2-year exposure study was not different between exposure and control groups. The body weight in male mice exposed to chloroform was significantly reduced compared to the control group. Body weight in all female mice exposed to chloroform was significantly reduced compared to the control group, however, it was recovered in the 5 ppm and 30 ppm groups at the timepoints of weeks 52 and 94. The incidence of renal nodules significantly increased in the 30 ppm and 90 ppm exposure groups. The incidence of renal cell carcinoma in the exposed males increased significantly in a dose-dependent way. The exposed males exhibited significantly increased incidences of cytoplasmic basophilia, nuclear enlargement and atypical tubule hyperplasia at 30 ppm and 90 ppm. The incidence of hepatocellular carcinomas occurring in the exposed females showed a significant positive trend, which appeared in the combined incidence of hepatocellular adenomas and carcinomas. A significantly increased incidence of total altered cell foci was found in the female 90 ppm group. Statistically significant exposure-related increases in the incidences of fatty change in the 90-ppm exposed males and females and of total altered cell foci in the 90 ppm exposed females were noted. The exposed males showed significantly increased serum levels of ALP at 5 ppm and above, BUN at 30 ppm and above, GOT and GPT at 90 ppm and total protein at 90 ppm. Significantly increased levels of triglyceride, BUN, GOT and GPT were observed in the 90 ppm exposed females.
- Relevance of carcinogenic effects / potential:
- 2 years inhalation exposure to chloroform significantly increased the tumour incidence in mice. The determined NOAEC of 5 ppm can be interpreted as a threshold level above which the cytoplasmic basophilia in the kidneys occurs as a regenerative, proliferative response to necrosis, leading to the development of renal cell adenomas and carcinomas through the pre-neoplastic atypical tubule hyperplasia based on the hypothesis of a non-genotoxic-cytotoxic mode of action of chloroform.
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 5 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no hepatic and renal changes and no increase in exposure-related tumors
- Conclusions:
- Inhalation exposure to chloroform of mice over a period of 2 years led to an significantly increased incidence of renal adenomas and carcinomas at exposure concentrations of 30 and 90 ppm.
- Executive summary:
A combined chronic toxicity/carcinogenicity study was carried out with chloroform in female and male BDF-1 mice receiving repeated dose inhalation exposure to chloroform vapours at target concentrations of 0, 5, 30 or 90 ppm (0, 24.5, 147, or 440 mg/m3) for 104 weeks according to the method B.33 suggested by the European Commission. Adverse effects were mainly observed in the liver and kidneys of exposed animals. Inhalation exposure to chloroform of mice over a period of 2 years led to a significantly increased incidence of renal adenomas and carcinomas at exposure concentrations of 30 and 90 ppm. The incidence of hepatocellular carcinomas occurring in the exposed females showed a significant positive trend, which appeared in the combined incidence of hepatocellular adenomas and carcinomas. For the exposed mice, the histopathological changes in the kidneys including the cytoplasmic basophilia which occurred at 30 ppm and above, were employed as the biologically significant endpoint to deduce a NOAEL value, which was 5 ppm (24.5 mg/m3).
Referenceopen allclose all
Table 1: Incidence of neoplastic lesions in the rats exposed to chloroform vapour for 104 weeks
Male |
Female |
|||||||
Group |
Control |
10 ppm |
30 ppm |
90 ppm |
Control |
10 ppm |
30 ppm |
90 ppm |
Number of animals examined |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
49 |
Liver |
||||||||
Hepatocellular adenoma |
0 |
0 |
0 |
0 |
1 |
0 |
2 |
1 |
Kidneys |
||||||||
Renal cell adenoma |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
Pituitary gland |
||||||||
Adenoma |
22 |
23 |
21 |
17 |
24 |
20 |
18 |
11* |
*: significant difference at p 0.05 by Fisher exact test
Table 2: Incidence of selected non-neoplastic lesions in the liver and kidneys of rats exposed to chloroform vapour for 104 weeks
Male |
Female |
|||||||
Group |
Control |
10 ppm |
30 ppm |
90 ppm |
Control |
10 ppm |
30 ppm |
90 ppm |
Number of animals examined |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
49 |
Liver |
||||||||
Total altered cell foci |
11 |
16 |
16 |
18 |
15 |
9 |
20 |
26 |
Clear cell foci |
4 |
4 |
5 |
6 |
4 |
1 |
2 |
7 |
Acidophilic cell foci |
2 |
5 |
2 |
3 |
0 |
1 |
0 |
1 |
Basophilic cell foci |
4 |
6 |
8 |
8 |
7 |
5 |
10 |
4 |
Mixed cell foci |
1 |
1 |
1 |
1 |
4 |
2 |
6 |
9 |
Vacuolated cell foci |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
5* |
Kidneys |
||||||||
Nuclear enlargement: proximal tubules |
0 |
0 |
5* |
32** |
0 |
0 |
6* |
34** |
Dilatation: tubular lumen |
0 |
0 |
9* |
27** |
0 |
0 |
5* |
38** |
Chronic progressive nephropathy b) + |
3 |
11* |
10* |
17** |
8 |
19** |
27** |
15** |
2 + |
6 |
10 |
24 |
14 |
15 |
7 |
5 |
3 |
3 + |
19 |
15 |
8 |
2 |
14 |
3 |
3 |
1 |
4 + |
19 |
8 |
2 |
1 |
4 |
2 |
0 |
2 |
Significant different at p 0.05 (*) and p 0.01 (**) by Chi square test; b) the severity of chronic progressive nephropathy was classfied into four different grades according to the criteria described by Kawai (1980)
Table 3: Serum levels of blood biochemical parameters and urinalysis in rats exposed to chloroform vapour for 104 weeks
Male |
Female |
|||||||
Group |
Control |
10 ppm |
30 ppm |
90 ppm |
Control |
10 ppm |
30 ppm |
90 ppm |
Number of animals examined |
27 |
39 |
36 |
38 |
37 |
35 |
40 |
34 |
Biochemistry |
||||||||
Total protein (g/dL) |
6.7 |
7.1 |
7.0 |
6.9 |
7.0 |
7.4* |
7.3 |
7.2 |
Glucose (mg/dL) |
162 |
169 |
165 |
154 |
170 |
168 |
164 |
160 |
Total cholesterol (mg/dL) |
173 |
164 |
153 |
125** |
142 |
131 |
135 |
149 |
Triglyceride (mg/dL) |
222 |
167 |
146* |
87** |
191 |
126 |
109 |
94** |
Phospholipids (mg/dL) |
289 |
268 |
241* |
196** |
280 |
252 |
255 |
271 |
Creatinine (mg/dL) |
0.9 |
0.6 |
0.6** |
0.7** |
0.5 |
0.5 |
0.5 |
0.5 |
BUN (mg/dL) |
28.6 |
20.6** |
18.3** |
23.2** |
17.9 |
18.5 |
18.5 |
18.6 |
GOT (IU/L) |
67 |
79 |
81 |
98* |
128 |
113 |
124 |
144 |
GPT (IU/L) |
21 |
25* |
24 |
26* |
37 |
40 |
39 |
48 |
gamma-GTP (IU/L) |
4 |
8* |
10** |
7* |
4 |
4 |
5 |
6** |
LDH (IU/L) |
164 |
239 |
179 |
311 |
297 |
245 |
239* |
344 |
ALP (IU/L) |
215 |
265 |
283 |
243 |
152 |
133 |
157 |
174 |
Urinalysis |
||||||||
Glucose |
0/27 |
0/39 |
2/37 |
20/39** |
2/41 |
8/37* |
29/41** |
24/34** |
Occult blood |
3/27 |
6/39 |
4/37 |
10/39 |
2/41 |
1/37 |
4/41 |
2/34 |
Values shown are the means for each group and the urinalysis data indicate the number of animals having positive glucose or occult blood/total number of animals examined. Significant differences at p 0.05 (*) and p 0.01 (**) by Dunnett's test for biochemistry and by Chi-square test for urinalysis. BUN: blood urea nitrogen; GOT: glutamate oxaloacetate transminase; GPT: glutamate pyruvate transaminase: ALP: alkaline phosphatase; gamma-GTP: gamma-glutamyl transpeptidase; LDH: lactate dehydrogenase
Table 1: incidences of neoplastic lesions in the mice exposed to chloroform vapour for 104 weeks
Male |
Female |
|||||||||
Group |
Control |
5 ppm |
30 ppm |
90 ppm |
Peto |
Control |
5 ppm |
30 ppm |
90 ppm |
Peto |
Number of animals examined |
50 |
50 |
50 |
48 |
-- |
50 |
49 |
50 |
48 |
-- |
Liver |
||||||||||
Hepatocellular adenoma |
5 |
7 |
6 |
8 |
-- |
1 |
1 |
4 |
3 |
-- |
Hepatocellular carcinoma |
10 |
0** |
7 |
10 |
↑ |
1 |
1 |
0 |
3 |
↑ |
Hepatocellular adenoma + carcinoma |
14 |
7 |
12 |
17 |
↑ |
2 |
2 |
5 |
6 |
↑↑ |
Hemangioma |
0 |
0 |
1 |
0 |
-- |
0 |
0 |
0 |
0 |
-- |
Hemangiosarcoma |
3 |
0 |
2 |
1 |
-- |
2 |
0 |
0 |
1 |
-- |
Histiocytic sarcoma |
2 |
0 |
0 |
0 |
-- |
0 |
0 |
1 |
0 |
-- |
Kidneys |
||||||||||
Renal cell adenoma |
0 |
0 |
3 |
1 |
-- |
1 |
0 |
2 |
1 |
-- |
Renal cell carcinoma |
0 |
1 |
4 |
11** |
↑↑ |
0 |
0 |
0 |
1 |
-- |
Renal cell adenoma + carcinoma |
0 |
1 |
7* |
12** |
↑↑ |
24 |
20 |
18 |
11* |
-- |
*: p 0.05; **: p 0.01 with Fisher exact test; ↑: p 0.05; ↑↑: p 0.01 with Peto's test Table 2: Incidences of selected non-neoplastic lesions in the liver and kidneys of mice exposed to chloroform vapour for 104 weeks
Male |
Female |
|||||||
Group |
Control |
5 ppm |
30 ppm |
90 ppm |
Control |
5 ppm |
30 ppm |
90 ppm |
Number of animals examined |
50 |
50 |
50 |
48 |
50 |
49 |
50 |
48 |
Liver |
||||||||
Necrosis: central |
0 |
0 |
0 |
3 |
1 |
0 |
1 |
2 |
Necrosis: focal |
1 |
2 |
6 |
2 |
0 |
0 |
2 |
3 |
Fatty change |
4 |
2 |
6 |
24** |
0 |
0 |
0 |
6* |
Total altered cell foci |
10 |
1** |
1** |
5 |
0 |
1 |
2 |
6* |
Clear cell foci |
6 |
0* |
0* |
3 |
0 |
1 |
0 |
3 |
Basophilic cell foci |
3 |
1 |
1 |
1 |
0 |
0 |
1 |
2 |
Mixed cell foci |
1 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
Kidneys |
||||||||
Nuclear enlargement: proximal tubules |
0 |
3 |
43** |
42** |
0 |
0 |
0 |
4 |
Cytoplasmic basophilia a) + |
33 |
40 |
8** |
9** |
0 |
4 |
3 |
5* |
2+ |
7 |
1 |
36 |
34 |
0 |
0 |
0 |
2 |
3+ |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
Atypical tubule hyperplasia |
0 |
0 |
11** |
14** |
0 |
0 |
0 |
0 |
Tubular necrosis: proximal tubules |
0 |
0 |
1 |
2 |
1 |
0 |
0 |
0 |
Significant differences at p 0.05 (*) and p 0.01 (**) by Chi square test; a) the severity of cytoplasmic basophilia was qualitatively scored as follws: +, a few lesions involving a single tubule in the whole histological section; 2 +, more than 4 lesions involving two or more tubules in the whole histological section; 3 +, numerous lesions throughout whole section; b) the severity of chronic progressive nephropathy was classified into four different grads according to the criteria described by Kawai (1980) Table 3: Serum level blood biochemical parameters and urinalysis in mice exposed to chloroform vapours for 104 weeks.
Male |
Female |
|||||||
Group |
Control |
5 ppm |
30 ppm |
90 ppm |
Control |
5 ppm |
30 ppm |
90 ppm |
Number of animals examined |
33 |
38 |
36 |
35 |
28 |
34 |
24 |
24 |
Biochemistry |
||||||||
Total protein (g/dL) |
5.6 |
5.8 |
5.9 |
6.1** |
5.5 |
5.5 |
5.6 |
6.0 |
Glucose (mg/dL) |
175 |
185 |
166 |
166 |
136 |
129 |
140 |
156 |
Total cholesterol (mg/dL) |
111 |
107 |
125 |
124 |
78 |
71 |
86 |
77 |
Triglyceride (mg/dL) |
83 |
82 |
101 |
80 |
82 |
72 |
81 |
58** |
BUN (mg/dL) |
25.8 |
22.5 |
26.3* |
30.8** |
17.2 |
21.4 |
19.3 |
21.4** |
GOT (IU/L) |
85 |
58 |
115 |
111* |
144 |
148 |
125 |
175* |
GPT (IU/L) |
29 |
16 |
40 |
44** |
28 |
47 |
39 |
50** |
LDH (IU/L) |
316 |
263 |
388 |
390 |
745 |
560 |
1437 |
748 |
ALP (IU/L) |
171 |
184* |
219** |
205** |
264 |
322 |
235 |
303 |
Urinalysis |
||||||||
Glucose |
0/33 |
0/39 |
0/38 |
0/35 |
0/29 |
0/36 |
1/27 |
1/24 |
Occult blood |
5/33 |
2/3 |
5/38 |
1/35 |
5/29 |
2/36 |
1/27 |
2/24 |
Values shown are the means for each group and the urinalysis data indicate the number of animals having positive glucose or occult blood/total number of animals examined. Significant difference at p 0.05 (*) by Dunnett's test for biochemistry and by Chi-square for urinalysis. BUN: blood urea nitrogen; GOT: glutamate oxaloacetate transminase; GPT: glutamate pyruvate transaminase; ALP: alkaline phosphatase
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 24.5 mg/m³
- Study duration:
- chronic
- Species:
- mouse
- Quality of whole database:
- Two inhalation studies available in rats and mice; in the mouse kidney and liver tumors were observed at 30 ppm and 90 ppm (147 mg/m3 and 440 mg/m3); in rats, however, no increased incidence of tumors was observed up to and including 90 ppm (440 mg/m3).
- Organ:
- kidney
- liver
Carcinogenicity: via dermal route
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
Renal tumors were observed in male BDFl mice exposed to chloroform via inhalation (Matsushima, 1994) and in male
ICI mice exposed to chloroform in a toothpaste or arachis oil vehicle (Roe et al., 1979), but not in male B6C3F1 mice administered chloroform by corn oil gavage (NCI, 1976) or in drinking water (Jorgenson et al., 1985). ILSI (1997) noted that males of the DBA strain, from which BDFl mice were derived, have higher tubular levels of the enzymes that bioactivate chloroform, and are much more susceptible to chloroform-induced renal damage, than male C57BL mice, the parental strain for B6C3F1 mice. Thus, the observed data for kidney tumors in mice are consistent with the hypothesis that the chloroform is metabolized to a cytotoxic compound, and that the resulting toxicity and cell proliferation can result in the development of chloroform-induced cancer.Formation of DNA adducts has not been shown with chloroform exposure. Although the formation of DNA adducts is the traditional hypothesis of tumor formation, the weight ofevidence favors the hypothesis that carcinogenesis may be related to increased cell proliferation following direct tissue injury.
Justification for classification or non-classification
According to Commission Regulation (EU) No 944/2013 of October 2013, amending Annex VI to the Regulation (EC) No 1272/2008 (CLP), chloroform is classified is classified as a Category 2 carcinogen, "Suspected human carcinogen", with the hazard statement H351: suspected of causing cancer.
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