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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
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:
Acute and subacute cytogenetic effects of the trihalomethanes on rat bone marrow cells in vivo
Author:
Fujie K, Aoki T and Wada M
Year:
1990
Bibliographic source:
Mutat Res 242, 111-119

Materials and methods

GLP compliance:
not specified
Type of assay:
other: Mammalian bone marrow chromosome aberration assy

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroform
EC Number:
200-663-8
EC Name:
Chloroform
Cas Number:
67-66-3
Molecular formula:
CHCl3
IUPAC Name:
chloroform
Test material form:
liquid

Test animals

Species:
rat
Strain:
Long-Evans
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Department of Pathology, Kobe University School of Medicine (Outbred from the colony of Ben May Laboratory for Cancer Research, University of Chicago)- Age at study initiation: 4 to 5 weeks- Weight at study initiation: 50 to 100 g- Housing: Commercial bedding (White Flakes, Charles River Japan Co.)- Diet (e.g. ad libitum): commercial solid food, Oriental Yeast Co., Tokyo- Water (e.g. ad libitum): ad libitum- Acclimation period:ENVIRONMENTAL CONDITIONS- Temperature (°C): 22 - Humidity (%): 60- Photoperiod (hrs dark / hrs light): natural light cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Physiological saline
Details on exposure:
0.01, 0.1, and 1 mmol/kg bodyweight (1.2, 11.9, and 119.4 mg/kg) dissolved in physiological saline
Duration of treatment / exposure:
five consecutive days
Frequency of treatment:
daily administration
Post exposure period:
Sampling 18 hours after the last sampling (1.2, 11.9, and 119.4 mg/kg)6, 12, 18 and 24 hours after the last sampling (119.4 mg/kg)
Doses / concentrations
Remarks:
Doses / Concentrations:1.2, 11.9, and 119.4 mg/kgBasis:
No. of animals per sex per dose:
three
Control animals:
yes, concurrent vehicle
Positive control(s):
1.5 mmol/kg (250.5 mg/kg) of potassium bromate (KBrO3)

Examinations

Tissues and cell types examined:
Metaphase cells in femoral bone marrow
Details of tissue and slide preparation:
Chromosome analysis: At various times after treatment with the chemicals, the animals were killed to study the frequency of aberrant metaphase cells in femoral bone marrow. The animals were injected with colchicine (0.3 mg dissolved in 0.3 ml of physiological saline) 1 h before death. Chromosome specimens were prepared from the femoral bone marrow by a conventional method, stained in 2% Giemsa solution (pH 6.8) for 15 min, and microscopically analyzed under code. Chromosome aberrations (CA) consisted of breaks and gaps: gaps were defined as complete interruptions in the continuity of one or both chromatids not exceeding the width of a chromatid, and breaks as discontinuities greater than the width of a chromatid, irrespective of whether or not the distal fragment was dislocated. Cells with gaps were not included in the category of damaged cells, based on the general opinion that gaps are not a good indicator of chromosome damage. About 100-200 metaphase cells were examined per rat, and the percentage of aberrant metaphase cells were calculated. Each group consisted of 3-6 rats.
Evaluation criteria:
Positive results were defined by significant differences revealed by the X2-test. A trend test was used for evidence of dose response.
Statistics:
Positive results were defined by significant differences revealed by the X2-test. A trend test was used for evidence of dose response.

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose-response relationships were studied in cells sampled 18 h after the fifth and last day of treatment. Compared to the values for the untreated control, significant increases in CA were noted with 1 mmole CHCl3/kg (119.4 mg/kg). The percent of aberrant metaphase cells over time was determined 6, 12, 18, and 24 h after the fifth and last day of oral treatment at1 mmole CHCl3/kg (119.4 mg/kg) Compared to the values for the untreated control, significant increases in CA were noted at 24h x5+12h,and 24hX5+18h with CHCl3. The incidence of aberrant cells increased gradually, and reached the maximum level at 18 h after the last treatment, decreasing to the control level at 24 h after the last treatment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positiveThe kinetic of the chromosomal aberrations at 1 mmol/kg is not traditional: positive 12 and 18 hours after five daily administration and negative 6 and 24 hours after the same treatment. Such a kinetic is not compatible with the kinetic of specific chromosomal aberrations but more with a cytotoxic activity. Unfortunately, no information about the cytotoxic activity in the bone marrow is available.In addition, considering the toxicokinetic data available in rats after oral or i.p. administrations of chlororoform (e.g. Wang et al., 1995)(1) , it is not plausible that the very low tissue levels of chloroform resulting from an oral or i.p. dose of 1.2 mg/kg/ could be at the origin of a clastogenic activity. Such positive results should then be considered as equivocal.(1): for example, Wang et al (1995) found that chloroform blood concentrations are around 0.1 mM (12 µg/mL) 60 minutes after an oral administration of 100 mg/kg. Wang P-Y, Kaneko T, Sato A, Charboneau M & Plaa GL (1995) Dose- and route-dependant alteration of metabolism and toxicity of chloroform in fed and fasting rats. Toxicol. appl. Pharmacol., 135, 119-126.
Executive summary:

In a chromosomal aberration assay, groups of 3 male Long-Evans rats were treated orally (gavage) with chloroform (dissolved in redistilled water and physiological saline) at doses of 1.2, 11.9, and 119.4 mg/kg bodyweight once daily for five consecutive days.

One group of 3 males, acting as control group, received the vehicle (physiological saline solution) and another group of 3 males received the positive control test item (potassium bromate) at the dose-level of 250.5 mg/kg, under the same experimental conditions than the chloroform treated groups.

The animals were killed 18 hours after receiving the last dose to study the frequency of aberrant metaphase cells in femoral bone marrow cells. Compared to control group, a dose-related increase in the incidence of aberrant cells and of the number of aberration per cell was observed. This difference was statistically significant at the high dose (6% versus 1% for the controls). Various times of sampling (6, 12, 18 and 24 hours after the last dosing) were also performed at the high dose and indicated that the incidence of aberrant cells increased gradually to reach a maximum level 18 hours after the last treatment. No more increase was observed 24 hours after the last treatment.