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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
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- 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: inhalation
Administrative data
- Endpoint:
- sub-chronic toxicity: inhalation
- 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:
- A 90-day chloroform inhalation study in female and male B6C3F1 mice: implications for cancer risk assessment
- Author:
- Larson JL, Templin MV, Wolf DC, Jamison KC, Leininger JR, Méry S, Morgan KT, Wong BA, Conolly RB, Butterworth BE
- Year:
- 1 996
- Bibliographic source:
- Fund. Appl. Toxicol 30, 118-137
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
- GLP compliance:
- not specified
- 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 % purity and stabilised with 0.006 % amylenes obtained from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin, USA was stored in 10-gallon stainless steel pressure vessels
Constituent 1
Test animals
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Seven-week old male and female B6C3F1 mice obtained from Charles River Breeding Laboratories, Inc., Raleigh, North Carolina, USA; housed one per cage in 8-m3 stainless steel and glass inhalation chambers; separate chambers for each exposure concentration; acclimation for 2 weeks; chambers maintained on a 12-hours light/12-hours dark cycle, NIH-07 rodent chow (Ziegler Bros., Gardener; Pennsylvania), and deionised filtered tap water available ad libitum; temperature at 22.2 +/- 2 °C and 50 +/- 10 % relative humidity, continuous flow of HEPA and charcoal-filtered air at flow rates of 2000 litre/min
Administration / exposure
- Route of administration:
- inhalation
- Type of inhalation exposure:
- whole body
- Vehicle:
- air
- Details on inhalation exposure:
- Exposure atmosphere was generated by a vaporisation technique; Nitrogen, metered by a mass flow controller, was admitted into the chloroform storage vessel through a dip tube; the chloroform-containing nitrogen gas flowed into the supply air duct for the exposure chamber; in the 2 and 10 ppm chambers, nitrogen was used to pressurise the vessel to 5 psi and carry the chloroform into the chamber through a mass flow controller
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Chamber concentrations of chloroform were monitored using a Miran 1A infrared gas analyser (see Table 1)
- Duration of treatment / exposure:
- see Table 2
- Frequency of treatment:
- 6 hours on 5 days/week or 7 days/week
Doses / concentrationsopen allclose all
- Dose / conc.:
- 1.47 mg/m³ air
- Remarks:
- 0.3 ppm (target concentration)
- Dose / conc.:
- 9.8 mg/m³ air
- Remarks:
- 2 ppm (target concentration)
- Dose / conc.:
- 49 mg/m³ air
- Remarks:
- 10 ppm (target concentration)
- Dose / conc.:
- 147 mg/m³ air
- Remarks:
- 30 ppm (target concentration)
- Dose / conc.:
- 441 mg/m³ air
- Remarks:
- 90 ppm (target concentration)
- No. of animals per sex per dose:
- ranging from 10 to 60, see Table 2
- Control animals:
- yes
Examinations
- Sacrifice and pathology:
- Mice in unlabelled group were weighed and killed subsequently; livers and kidneys were removed, weighed and examined macroscopically, then slides were prepared from the tissues; in rats exposed for 3 or 13 weeks, a complete tissue screen was collected including adrenals, brain, cecum, cervix, colon, duodenum, ear canal, esophagus, eye with haderian gland, femoral-tibial joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mesenteric lymph nodes, ovaries, pancreas, parathyroid gland, ribs, prostate, salivary gland, skin with mammary gland, sciatic nerve, seminal vesicles, spinal cord, sternum, stomach, spleen, testes, thigh muscle, thymus, thyroid, trachea, urinary bladder, uterus, vagina and vertebrae.
- Other examinations:
- Nasal passages were examined as well
- Statistics:
- The Williams test was used to determine significant differences in organ weights, body weights and labelling index between treatment and control groups; the student's t-test was used to determine statistical differences in body weight and organ weights and labelling index between the exposure groups of 7 and 5 days/week and between exposure groups of 13-week continuous chloroform exposure and 6-week stop.
Results and discussion
Results of examinations
- 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):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- effects observed, treatment-related
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Details on results:
- Animal health: No clinical signs of toxicity were noted at any time throughout the exposure study in females, and few of the male mice had rough hair coats and slight hair loss at the latter time points. No statistically significant differences between control and exposure groups were observed in body weight gain of females or males exposed for 13 weeks.
Organ weights: Relative liver weights were increased at each necropsy time point in female and male mice exposed to 90 ppm. No differences in relative kidney weights were observed between control and exposure groups at any time point.
Liver and kidney histopathology and cell proliferation: After 13 weeks of 7 days/week exposure, microscopic liver alterations (mild degenerative changes of slight centrilobular hepatocyte swelling and vacuolation) were present in 4/14 female mice exposed to 10 ppm and 10/15 mice exposed to 30 ppm. All female mice exposed to 90 ppm chloroform for 13 weeks at 5 days/week had mild changes of hepatocyte vacuolation and scattered enlarged nuclei. Increased cell proliferation was not observed at any time point with doses of 10 ppm or lower, but occurred at 3 and 6 weeks with doses of 30 ppm and at 13 weeks with doses of 90 ppm. Lesion scores for male mice exposed for 13 weeks at 7 days/week were increased above background at 30 and 90 ppm. Swollen centrilobular hepatocytes with pale eosinophilic cytoplasm, enlarged nuclei and centrilobular to midzonal hepatocyte vacuolation were observed. Significant increases in hepatocyte labelling index were found only in male mice exposed to 90 ppm.
Kidneys from female mice exposed to chloroform were not different histologically from controls at any time point. There was no statistically significant increase in the labelling index over controls in any of the treatment groups in the kidneys. Kidney lesions in male mice were confined to the epithelial cells of the proximal convoluted tubules of the cortex. Male mice exposed to 30 or 90 ppm had single or multiple foci of regeneration within the cortex. Mineralisation within the cortex was present in male mice exposed to 90 ppm. Enlarged nuclei were noted in PCT epithelial cells in male mice exposed to 10, 30 and 90 ppm. An increase in the labelling index was found in the epithelial cells of the PCT in the cortex and in the outer stripe of the outer medulla with 30 and 90 ppm, but not with 10 ppm or lower.
Nasal histopathology and cell proliferation: Nasal tissue had mild changes confined to the posterior ventral areas of the nose in and adjacent to the sites of attachment of the ethmoid turbinates to the lateral wall with doses of 10, 30 or 90 ppm. Nasal lesions were characterised by mild proliferative responses in the periosteum of the frontal and maxillary bones adjacent to the attachment of the osseous cores of the ethmoid turbinates. The response was characterised by a thickening of the normally delicate and regularly structured bone in this site through hypertrophy and hyperplasia of the periosteum and irregular formation of new, immature bone. The adjacent lamina propria exhibited variable loss of acini of Bowman's glands with mild vascular congestion and oedema.
Effects on other tissues: No microscopic changes were seen in the other tissues examined.
Effect levels
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 9.8 mg/m³ air
- Sex:
- male
- Basis for effect level:
- other: based on renal and nasal changes at the next higher concentration of 49 mg/m3
Target system / organ toxicity
- Key result
- Critical effects observed:
- not specified
Any other information on results incl. tables
Table 3: Relative liver and kidney weight of female and male B6C3F1 mice exposed to chloroform vapours
13 weeks of exposure |
||
Concentration (ppm) |
Liver |
Kidney |
Female mice |
||
0 |
5.3 ± 0.3 b) |
1.7 ± 0.1 b) |
0.3 |
5.1 ± 0.3 b) |
1.6 ± 0.2 c) |
2 |
5.0 ± 0.2 b) |
1.5 ± 0.1 b) |
10 |
5.1 ± 0.2 b) |
1.5 ± 0.2 b) |
30 |
5.6 ± 0.7 b) |
1.7 ± 0.1 b) |
90 |
6.7 ± 0.3 b)* |
1.6 ± 0.2 b) |
Male mice |
||
0 |
4.9 ± 0.3 b) |
2.2 ± 0.1 b) |
0.3 |
4.9 ± 0.3 b) |
2.1 ± 0.2 b) |
2 |
5.1 ± 0.3 b) |
2.2 ± 0.1 b) |
10 |
5.3 ± 0.2 b) |
2.4 ± 0.2 b) |
30 |
5.5 ± 0.1 b)* |
1.9 ± 0.1 b) |
90 |
6.3 ± 0.8 b)* |
2.2 ± 0.2 b) |
a) values are presented as organ weights as % of body weight; means +/- SD; b) n=5; * statistically different from controls (Williams test, p 0.05) Table 4: Hepatic lesion scores and incidence in female and male maic exposed to chloroform vapours a)
Female mice |
Male mice |
|||||
Concentration (ppm) |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
0 |
0.1 (1/15, 1.0) |
0.1 (1/15, 1.0) b) |
0.1 (1/15, 1.0) b) |
0.3 (5/15, 1.0) |
0.3 (5/15, 1.0) b) |
0.3 (5/15, 1.0) b) |
0.3 |
0.3 (1/15, 1.0) |
c) |
c) |
0.4 (4/15, 1.5) |
c) |
c) |
2 |
0.5 (5/14, 1.4) |
c) |
c) |
0.3 (5/14, 1.0) |
c) |
c) |
10 |
0.6 (4/14, 2.0) |
0.2 (2/13, 1.0) |
c) |
0.3 (5/15, 1.0) |
0.5 (4/13, 1.5) |
c) |
30 |
0.8 (10/15, 1.2) |
c) |
0.3 (1/8, 2.0) |
1.2 (12/12, 1.2) |
c) |
0.6 (4/8, 1.3) |
90 |
2.9 (15/15, 2.9) |
1.8 (13/13, 1.8) |
0.8 (2/8, 3.0) |
1.9 (14/14, 1.9) |
0.8 (10/12, 1.3) |
0.8 (4/8, 1.5) |
a) chloroform-induced liver histopathological changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from lipid vacuolation to degenerative changes and necrosis. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with liver lesions; b) control animals are the same for all the 13 -week studies; c) animals were not examined at these time points. Table 5) Hepatocyte labelling indices in female and male mice exposed to chloroform vapours
Female mice |
Male mice |
|||||
Concentration (ppm) |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
0 |
1.3 ± 1.0 e) |
1.3 ± 1.0 g) |
1.3 ± 1.0 g) |
0.5 ± 0.4 e) |
0.5 ± 0.4 g) |
0.5 ± 0.4 g) |
0.3 |
1.4 ± 1.0 e) |
h) |
h) |
1.1 ± 0.8 e) |
h) |
h) |
2 |
0.8 ± 0.3 f) |
h) |
h) |
0.8 ± 0.6 f) |
h) |
h) |
10 |
2.4 ± 1.5 f) |
0.8 ± 0.3 c) |
h) |
1.0 ± 0.6 f) |
1.5 ± 0.9 c) |
h) |
30 |
1.8 ± 1.4 f) |
h) |
1.0 ± 0.6 d) |
0.9 ± 0.6 f) |
h) |
0.9 ± 0.7 d) |
90 |
18.1 ± 8.5 e)* |
7.1 ± 3.7 c)* |
0.8 ± 0.6 c) |
10.7 ± 4.9 e)* |
3.1 ± 2.4 c)* |
1.4 ± 0.9 c) |
c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05) Table 6) Severity of nasal lesions in female mice exposed to chloroform vapours a)
Concentration (ppm) |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
0 |
0.1 ± 0.3 e) |
0.1 ± 0.3 g) |
0.1 ± 0.3 g) |
0.3 |
0 e) |
h) |
h) |
2 |
0.1 ± 0.3 e) |
h) |
h) |
10 |
0.4 ± 0.5 f) |
0.4 ± 0.5 d) |
h) |
30 |
0.4 ± 0.5 e) |
h) |
0 d) |
90 |
0.1 ± 0.3 c) |
0.3 ± 0.5 d) |
0 d) |
a) chloroform-induced nasal histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of connective tissue proliferation in the lamina propria. The severity of the nasal lesion for each group is expressed as the mean score +/- SD; c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points Table 7: Nasal turbinate lamina propria labelling indices in female mice exposed to chloroform vapours a)
Concentration (ppm) |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
0 |
27 ± 9 e) |
27 ± 9 g) |
27 ± 9 g) |
0.3 |
29 ± 27 e) |
h) |
h) |
2 |
29 ± 17 d) |
h) |
h) |
10 |
67 ± 33 d) |
27 ± 18 b) |
h) |
30 |
43 ± 29 e) |
h) |
18 ± 8 b) |
90 |
27 ± 6 b) |
35 ± 8 e) |
14 ± 5 e)*** |
a) all values are expressed as means +/- SD, b) n=5; d) n=7; e) n=4; g) control animals are the same for all the 13 -week duration studies; h) animals were not examined at these time points; *** statistically significant decreased compared to controls (Williams test, p 0.05) Table 8: Renal lesion scores and incidence in male mice exposed to chloroform vapours a)
Concentration (ppm) |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
0 |
0.0 (0/15, 0.0) |
0.0 (0/15, 0.0) b) |
0.0 (0/15, 0.0) |
0.3 |
0.0 (0/15, 0.0) |
c) |
c) |
2 |
0.0 (0/14, 0.0) |
c) |
c) |
10 |
0.0 (0/15, 0.0) |
0.1 (1/13, 1.0) |
c) |
30 |
0.9 (11/12, 1.0) |
c) |
1.0 (8/8, 1.0) |
90 |
1.5 (14/14, 1.5) |
1.6 (12/12, 1.6) |
1.3 (8/8, 1.3) |
a) chloroform-induced kidney histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of nephropaty, which includes degenerative changes, foci of regeneration and mineralisation within the cortex. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a kidney lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with kidney lesions; b) control anaimals are the same for all the 13 -week studies; c) animals were not examined at these time points Table 9: Labelling indices in sections of the kidneys in male mice exposed to chloroform vapours a)
Concentration (ppm) |
13 weeks, 7 days/week |
13 weeks, 5 days/week |
13 weeks, exposure stop after 6 weeks |
Cortex |
|||
0 |
1.6 ± 0.6 f) |
1.6 ± 0.6 g) |
1.6 ± 0.6 g) |
0.3 |
1.9 ± 0.8 f) |
h) |
h) |
2 |
1.6 ± 0.6 e) |
h) |
h) |
10 |
2.0 ± 0.9 f) |
7.6 ± 3.0 c)* |
h) |
30 |
2.4 ± 1.2 f)* |
h) |
1.3 ± 0.4 c) |
90 |
3.0 ± 0.9 e)* |
5.7 ± 2.2 c)* |
1.8 ± 0.3 c) |
Outer stripe of outer medulla |
|||
0 |
0.8 ± 0.3 f) |
0.8 ± 0.3 g) |
0.8 ± 0.3 g) |
0.3 |
1.1 ± 0.3 f) |
h) |
h) |
2 |
0.8 ± 0.3 e) |
h) |
h) |
10 |
0.8 ± 0.3 f) |
1.6 ± 0.8 c)* |
h) |
30 |
0.6 ± 0.3 f) |
h) |
1.0 ± 0.5 c) |
90 |
1.0 ± 0.4 e) |
1.4 ± 0.6 c)* |
0.9 ± 0.2 c) |
a) values are expressed as means +/- SD; c) n=8; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05)
Applicant's summary and conclusion
- Conclusions:
- Inhalation exposure of B6C3F1 mice to chloroform caused mild nasal lesions of the ethmoid region, liver cell proliferation and kidneys cell proliferation in female and male mice; the NOAEC was 2 ppm (9.8 mg/m3).
- Executive summary:
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, 0.3, 2, 10, 30 or 90 ppm (0, 1.47, 9.8, 49, 147 and 441 mg/m3) for 6 hours/day for 5 or 7 days per week. 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 exposure setting, i.e. groups having regular exposure interruptions for two days/week and groups receiving continuous exposure for 7 days a week and the duration of exposure, had an influence on the observed systemic effects. Increases in the relative liver weight in male and female mice were seen only at exposure to 90 ppm (441 mg/m3) chloroform. Liver lesions in male and female mice occurred at exposure concentrations of 30 (147 mg/m3) ppm or greater. The NOEC for regenerative cell proliferation in the liver of female mice was 10 ppm (49 mg/m3) and in the liver of male mice was 90 ppm (441 mg/m3). Kidneys of female mice were not different histologically from controls at any exposure concentration or time point. In contrast, lesions in the kidneys of male mice (enlarged nuclei in PCT epithelial cells and scattered areas of regenerating foci) were observed at exposure concentrations of 10 ppm (49 mg/m3). At 13 weeks, increased regenerative cell proliferation in the kidneys occurred at 30 ppm (147 mg/m3) when male mice were exposed continuously for 7 days a week and at 10 ppm (49 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 10 ppm (49 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. Male mice exhibited lesions of the kidney cells and increased regenerative cell proliferation in the kidneys at exposure concentration of 10 ppm (49 mg/m3), which was the LOAEC in the study; the NOAEC was set at 2 ppm (9.8 mg/m3).
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