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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and appropriate guidelines
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
US OPPTS 870.5375 and CFR 799.9537
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibromomethane
EC Number:
200-824-2
EC Name:
Dibromomethane
Cas Number:
74-95-3
Molecular formula:
CH2Br2
IUPAC Name:
dibromomethane
Details on test material:
Clear colourless liquid, 99.4% purity, DBM

Method

Species / strain
Species / strain / cell type:
other: human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
with and without S-9 from liver homogenate from rates induced with phenobarbitone and B-naphthofavone
Test concentrations with justification for top dose:
Experiment 1: 27.19, 54.38, 108.75, 217.5, 435, 870 ug/ml (with and without S9)
Experiment 2: without S9: 54.38, 108.75, 217.5, 652.5, 870 ug/ml
with S9: 13.60, 27.19, 54.38, 108.75, 217.5, 435 ug/ml
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: In the absence of S9, mitomycin C (MMC) at 0.4 ug/ml for culture in Experiment 1. It was dissolved in Minimal Essential Medium. In th presence of S9, cyclophosphamide (CP) at 5ug/ml. It was dissolved in dimethyl sulphoxide.
Details on test system and experimental conditions:
Two treatment conditions were used for the study.
Four hours exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration followed by cell harvest after a 20-hour expression period; and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period.
The test material (99.4% purity, dibromomethane) was dissolved in dimethyl sulphoxide and dilutions prepared. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls.
Positive control materials were Mitomycin C at 0.4 µg/ml in the absence of S-9, and dissolved in the media whereas, in the presence of S-9, cyclophosphamide at 5 µg/ml was used.
The dose levels were selected following a preliminary toxicity test using doses levels ranged from 27.19 to 870 µg/ml. The dose levels selected for metaphase analysis were 54.38, 108.75 and 217.5 µg/ml in both the absence and presence of S9.
Following mitosis arrest, cells were harvested, placed on slide and stained with 5% Gurrs Giemsa. Slides were checked microscopically to determine quality of the metaphase and also toxicity and presence of precipitation.
Evaluation criteria:
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation. A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value. Where possible the first 100 consecutive well-spread metaphases from each culture were counted except where there were approximately 50% cells with aberrations then slide evaluation was terminated at approximately 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix I). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
other:
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Evidence of toxicity and dose related mitotic inhibition in all three exposure groups (see mitotic index attached). Based on the toxicity, the maximum dose level tested was 870 ug/l for both 4(20) hour exposure groups.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: human lymphcytes taken from whole blood was drawn from vloenteers
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test material was considered to be clastogenic to human lymphocytes in vitro because of a statistically significant dose-related
increase in the frequency of cells with chromosome aberrations both in the presence and absence of a liver enzyme metabolizing
system.
Executive summary:

In vitro mammalian chromosome aberration test in human lymphocytes was conducted.

Two treatment conditions were used for the study. Four hours exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration followed by cell harvest after a 20-hour expression period; and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period.

The test material (99.4% purity, dibromomethane) was dissolved in dimethyl sulphoxide and dilutions prepared. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Positive control materials were Mitomycin C at 0.4 µg/ml in the absence of S-9, and dissolved in the media whereas, in the presence of S-9, cyclophosphamide at 5 µg/ml was used.

The dose levels were selected following a preliminary toxicity test using doses levels ranged from 27.19 to 870 µg/ml. The dose levels selected for metaphase analysis were 54.38, 108.75 and 217.5 µg/ml in both the absence and presence of S9.

Following mitosis arrest, cells were harvested, placed on slide and stained with 5% Gurrs Giemsa. Slides were checked microscopically to determine quality of the metaphase and also toxicity and presence of precipitation.

Results: The test material induced a statistically significant increase in the frequency of cells with aberrations at all dose levels analysed, in both exposure groups, using a dose range that included a dose level that induced greater than 50% mitotic inhibition. No increase in the incidence of polyploid cells was observed in either the absence or presence of S9.

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Therefore, the test material was considered clastogenic to human lymphocytes in vitro.