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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, following official guidelines, GLP compliant performed on a mixture containing the substance and other analogue substances

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference substance name:
not applicable being a mixture
not applicable being a mixture
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
Black powderwater solubility: 40g/lstorage: room temperatureexpiration date: March 2003


Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
clone 65/3Origin: Dr. D. Wild, Freiburg, Germany
Metabolic activation:
Metabolic activation system:
Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Cytotoxicity testRange with metabolic activation: 0.24 to 500.0 ug/mlRange without metabolic activation: 0.24 to 500.0 ug/mlMutagenicity testOriginal experiment:Range with metabolic activation: 18.52 to 500.0 ug/mlRange without metabolic activation: 14.81 to 400.0 ug/mlConfirmatory experiment:Range with metabolic activation:18.52 to 500.0 ug/mlRange without metabolic activation: 12.96 to 350.0 ug/ml
Vehicle / solvent:
dimethylsulfoxide (suspension)Since the test item was insoluble in all common solvents, it had to be prepared as a suspension in DMSO. The highest suitable concentration of test item in DMSO was determined in a preliminary solubilisation test to be 50.0 mg/ml. Lower concentrations of the test substance were obtainedby appropriate dilution of the stock suspension with DMSO. The respective suspensions were added 1:100 to the cell culture medium. The final concentration of DMSO in the culture medium was 1%. The highest concentration caused a homogenious turbidity after 100-fold dilution with culture medium as determined in the preliminary solubility test by microscopic evaluation. Due to the dense black staining by the test compound, observation of the cultures with the naked eye did not allow todecide whether the compound precipitated or not. It was therefore not determined which concentrations caused the formation of precipitates in the toxicity and mutagenicity tests. The test substance suspensions were prepared immediately before the start of the test.
Untreated negative controls:
Positive controls:
Positive control substance:
N-dimethylnitrosamine with metabolic activation, ethylmetanesulphonate without metabolic activation
Evaluation criteria:
All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of theresult. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.Criteria for a positive responseThe test substance will be considered to be mutagenic if:• The assay is valid (see assay acceptance criteria)• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.• There is a significant dose-relationship as indicated by the linear trend analysis.• The effects described above are reproducible.
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines [9]

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Untreated negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results (migrated information):negativeThe test item was tested for in vitro gene mutation in mammalian cells following OECD 476. Under the experimental conditions the test item did not show any mutagenic potential.
Executive summary:

The test item was tested for in vitro gene mutation in mammalian cells following OECD 476. Chinese Hamster lung fibroblasts V79 were exposed to the test item with and without metabolic activation at dose ranging up to 500 ug/l and 400 ug/l respectively. After the expression period, the mutant frequency was determined by 6 -thioguanine screening and the substance resulted not having mutagenic potential.