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EC number: 200-663-8 | CAS number: 67-66-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: 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
Data source
Reference
- Reference Type:
- publication
- Title:
- Toxicity and cell proliferation in the liver, kidneys and nasal passages of female F-344 rats induced by chloroform administered by gavage
- Author:
- Larson JL, Wolf DC, Méry S, Morgan KT, Butterworth BE
- Year:
- 1 995
- Bibliographic source:
- Fd Chem. Toxicol. 33(6), 443-456
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
- Deviations:
- yes
- Remarks:
- animals were exposed 5 d/wk for 3 wks
- GLP compliance:
- no
- Limit test:
- no
Test material
- Reference substance name:
- Chloroform
- EC Number:
- 200-663-8
- EC Name:
- Chloroform
- Cas Number:
- 67-66-3
- Molecular formula:
- CHCl3
- IUPAC Name:
- trichloromethane
- Details on test material:
- chloroform, > 99.5 % and stabilised with 0.006 % amylenes, purchased from Fluka Chemika (Buchs, Switzerland)
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, North Carolina, USA)
- Age at study initiation: 9 weeks
- Weight at study initiation: 139.5 +/- 5 g
- Fasting period before study: no data
- Housing: rats were housed two or three per cage in polystyrene shoe-box cages with cellulose bedding (ALPHA-dri, Shepherd Specialty Papers, Kalamazoo, USA), and filter top lids
- Diet (e.g. ad libitum): NIH-07 rodent chow (Ziegler Bros, Gardener, Pennsylvania, USA) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- Rats received chloroform dissolved in corn oil at doses of 0, 34, 100, 200 and 400 mg/kg/day in a constant dosing volume of 2 mL/kg administered by oral gavage. Each dose concentration of chloroform was prepared fresh on the morning that it was dispensed. All doses were administered between 8.00 and 10.00 hr to minimise diurnal variation.
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 4 consecutive days or 3 weeks
- Frequency of treatment:
- Daily in the 4-day study and one administration per day on five days per week in the 3-week study
Doses / concentrationsopen allclose all
- Dose / conc.:
- 34 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Dose / conc.:
- 400 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 rats per dose group
- Control animals:
- yes, concurrent vehicle
Examinations
- Observations and examinations performed and frequency:
- no data, but regular observations were performed during the testing period
- Sacrifice and pathology:
- At autopsy, rats were anaesthetised with sodium pentobarbital and the kidneys perfused in situ. Following a 3-min perfusion, the entire livers and kidneys were removed. Livers were weighed, examined macroscopically, and longitudinal 3-4 mm mid-sections of the left, median and right anterior hepatic lobes taken. The right and left kidneys were examined grossly and a 3-4 mm mid-saggital section of the left kidney and a 3-4 mm midtransverse section of the right kidney were taken. A 3-4 mm section of the duodenum was also included to confirm the systemic distribution and nuclear incorporation of bromodeoxyuridine (BrdU). Nasal cavities were flushed with neutral buffered 10 % formalin through the trachea; excess soft tissue was removed from the head, the lower jaw was removed, and the head was subsequently immersed in fresh fixative for no less than 48 hours and then decalcified in 5 % formic acid with ion exchange resin. Uniform blocks were cut at six levels of the nose to include all major epithelial types and blocks processed to paraffin. Tissue sections were cut at 4-5 micrometre thick. Serial sections were stained with haematoxylin and eosin or left unstained for BrdU immunohistochemistry.
- Other examinations:
- The labelling index (percentage of nuclei in S-phase) was evaluated in rats by bromodeoxyuridine (BrdU) immunohistochemistry. On the Monday afternoon preceding a Friday autopsy, osmotic pumps containing 20 mg/mL BrdU were aseptically implanted sc over the thoracumbular area using isofluorane anaesthesia. Autopsies were conducted on Friday mornings; consequently, rats received BrdU for about 3.5 days. BrdU-labelled tissues were mounted on "ProbOn Plus" slides (Fisher Scientific, Pittsburg, Pennsylvania, USA) to ensure adhesion during processing. The immunohistochemical detection of BrdU-labelled cells was done as described by Elridge et al. (1990, Carcinogenesis 11, 2245-2251). Cells that had incorporated BrdU were identified by the red pigment within the nuclei. Computer-generated random fields were used for scoring BrdU-labelled nuclei in the liver and kidney by light microscopy. In the liver tissue sections, at least 1000 hepatocellular nuclei in the left lobe were counted. For kidney tissue sections, at least 1000 proximal tubular cells in the cortical region were scored, as well as 1000 epithelial cells in the outer stripe of the medulla, 1000 epithelial cells in the inner stripe of the outer medulla and 1000 epithelial cells in the inner medulla. The labelling index, representing the percentage of nuclei in S-phase, was calculated by dividing the number of labelled nuclei by the total number of nuclei counted (expressed in %). For the nasal passages, quantitative studies of cell replication were confined to the base of the dorsal scroll of the first endoturbinate. These studies used the unit length labelling index method as modified by Méry et al. (1994, Toxicology and Applied Pharmacology 125, 214-227) for investigation of nasal lesions.
- Statistics:
- The Williams test was used to determine significant differences in liver and body weights and labelling index between treatment and control means. The Williams test is designed to establish the lowest dose that produces a response that is significantly elevated over the control in a dose-response study.
Results and discussion
Results of examinations
- 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):
- not specified
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- effects observed, treatment-related
- 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:
- no effects observed
- Details on results:
- Mild degenerative centrilobular changes and dose-dependent increases in the hepatocyte labelling index (LI ) were observed after administration of 100 mg or more chloroform/kg/day. Rats given 200 or 400 mg/kg/day for 4 days or 3 wk had degeneration and necrosis of the proximal tubules of the renal cortex. Regenerating epithelium lining proximal tubules was seen histologically and as an increase in LI. Dose-dependent increases in LI were observed in the kidneys at doses of 100 mg or more chloroform/kg/day at both 4 days and 3 wk. Two distinct treatment-induced responses were observed in specific regions of the olfactory mucosa lining the ethmoid region of the nose. A peripheral lesion was seen at all doses used and included new bone formation, periosteal hypercellularity and increased cell replication. A central lesion was seen at doses of 100 mg or more chloroform/kg/day and was characterized by degeneration of the olfactory epithelium and superficial Bowman's glands. These observations define the dose-response relationships for the liver, kidneys and nasal passages as target organs for chloroform administered by gavage in the female F-344 rat.
Effect levels
- Dose descriptor:
- NOAEL
- Effect level:
- 34 mg/kg bw/day (nominal)
- Sex:
- female
- Basis for effect level:
- other: changes in liver and kidneys at the next higher level of 100 mg/kg bw/day
Target system / organ toxicity
- Critical effects observed:
- not specified
Any other information on results incl. tables
Table 1: Chloroform-induced proliferation in the liver of female F-344 rats
Dose (mg/kg/day) |
Labelling index in hepatocytes after: |
|
4 days |
3 weeks |
|
0 |
1.6 ± 1.1 |
0.6 ± 0.5 |
34 |
1.0 ± 0.6 |
0.8 ± 0.4 |
100 |
6.0 ± 2.5 * |
2.7 ± 1.5 |
200 |
11.7 ± 3.1 * |
14.0 ± 9.0 * |
400 |
14.0 ± 5.7 * |
11.8 ± 15.9 * |
Values are means ± SD (n = 5 rats); Asterisks indicate significant differences from the control (p < 0.05; William’s test)
Table 2: Chloroform-induced cell proliferation in the kidneys of female F-344 rats
Duration of administration |
Dose (mg/kg/day) |
Labelling index in: |
|||
Cortex |
Outer stripe, outer medulla |
Inner stripe, outer medulla |
Inner medulla |
||
4 days |
0 |
2.1 ± 0.6 |
2.9 ± 0.7 |
1.4 ± 0.7 |
0.4 ± 0.4 |
34 |
2.4 ± 0.8 |
2.1 ± 0.9 |
2.0 ± 1.3 |
0.8 ± 0.7 |
|
100 |
3.2 ± 0.7 |
1.7 ± 0.5 |
1.0 ± 0.4 |
0.6 ± 0.3 |
|
200 |
17.7 ± 12.2 * |
9.1 ± 16.0 |
6.6 ± 12.1 |
2.3 ± 4.3 |
|
400 |
41.0 ± 5.3 * |
31.1 ± 2.2 * |
27.3 ± 4.1 * |
3.2 ± 1.8 * |
|
3 weeks |
0 |
1.3 ± 1.0 |
1.2 ± 0.8 |
1.2 ± 0.7 |
0.4 ± 0.3 |
34 |
1.5 ± 0.3 |
1.0 ± 0.5 |
1.1 ± 0.6 |
0.2 ± 0.3 |
|
100 |
22.4 ± 20.9 * |
4.9 ± 6.8 |
3.3 ± 3.9 |
0.6 ± 0.6 |
|
200 |
33.8 ± 20.9 * |
10.4 ± 12.0 * |
6.1 ± 7.1 |
5.1 ± 7.5 |
|
400 |
13.5 ± 6.9 * |
5.7 ± 2.4 * |
2.0 ± 1.4 |
0.5 ± 0.6 |
Values are means ± SD (n = 5 rats); Asterisks indicate significant differences from the control (p < 0.05; William’s test)
Table 3: Severity of chloroform-induced nasal lesions (mean subjective score) in femal F-344 rats
Dose (mg/kg/day) |
Peripheral olfactory mucosal lesion a) |
Central olfactory mucosal lesion b) |
||
4 days |
3 weeks |
4 days |
3 weeks |
|
0 |
0 |
0 |
0 |
0 |
34 |
1.2 |
0.4 |
0.2 |
0 |
100 |
1.8 |
1.7 |
0.4 |
0.8 |
200 |
1.6 |
2.0 |
0.8 |
1.2 |
400 |
1.8 |
3.0 |
2.8 |
1.6 |
a) Periosteal hyperplasia, new bone formation, and loss of adjacent Bowman’s glands. Grading system for the turbinate bones: 0 = normal, 1 = uneven boundary of nasal bone, 2 = uneven boundary of nasal bone with slight new bone formation, 3 = moderate new bone formation, 4 = severe bone enlargement. Standard deviations are not presented owing to the subjective nature of the data and the consistent intragroup scores.
b) Degeneration of olfactory epithelium and superficial Bowman’s glands. Scoring system used for degeneration of the olfactory epithelium of the dorsal medial region of the nose: 0 = normal, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.
Table 4: Chloroform-induced cell proliferation in the nasal turbinates of female F-344 rats.
Dose (mg/kg/day) |
ULLI a) |
|
4 days |
3 weeks |
|
0 |
15 ± 4 |
16 ± 3 |
34 |
145 ± 97 * |
24 ± 9 |
100 |
306 ± 48 * |
61 ± 10 * |
200 |
321 ± 19 * |
63 ± 5 * |
400 |
377 ± 121 * |
63 ± 17 * |
a) Unit length labelling index of cells in the lamina propria of the proximal portion of the dorsal scroll of the first endoturbinate expressed as labelled nuclei per 0.25 mm bone. Values are means ± SD. Asterisks indicate significant differences from the control (p < 0.05, William’s test).
Applicant's summary and conclusion
- Conclusions:
- The LOAEL for oral exposure to chloroform dissolved in corn oil administered by gavage in a subacute study using female F-344 rats was 34 mg/kg body weight/day after exposure for 4 consecutive days; the NOAEL was 34 mg/kg body weight/day after exposure for three weeks. The LOAEL was based on observations of lesions and cell proliferation in the olfactory epithelium and changes in the nasal passages.
- Executive summary:
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 3 consecutive weeks on 5 days per week. 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 dose-dependent increase in the hepatocyte labelling index (% of cells in S-phase), dose-dependent increase in 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 observed after repeated oral doses administered for 3 weeks. Local effects in the nasal passage were seen after 4 days of exposure at a dose of 34 mg/kg/day.
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