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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Section 13: see the attached document containing the justification for the read-across.

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

Constituent 1
Reference substance name:
Analogue Substance #2
Analogue Substance #2
Test material form:
solid: particulate/powder


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 test: range with metabolic activation: 0.24 to 500.0 µg/ml; range without metabolic activation: 0.24 to 500.0 µg/ml.
Mutagenicity test original experiment: range with metabolic activation: 18.52 to 500.0 µg/ml; range without metabolic activation: 14.81 to 400.0 µg/ml.
Confirmatory experiment: range with metabolic activation: 18.52 to 500.0 µg/ml; range without metabolic activation: 12.96 to 350.0 µg/ml.
Vehicle / solvent:
Dimethylsulfoxide (suspension): since the test substance was insoluble in all common solvents, it had to be prepared as a suspension in DMSO. The highest suitable concentration of test substance in DMSO was determined in a preliminary solubilisation test to be 50.0 mg/ml. Lower concentrations of the test substance were obtained by 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, 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 and the effects described above are reproducible.
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
not examined
Untreated negative controls validity:
True negative controls validity:
not examined
Positive controls validity:

Applicant's summary and conclusion

Negative. The test substance was tested for in vitro gene mutation in mammalian cells following OECD 476. Under the experimental conditions the test substance did not show any mutagenic potential.
Executive summary:

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