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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, performed on a similar substance

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:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
refer to confidential details on test material
refer to confidential details on test material
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
appearance: olive green powder
Storage: 4 °C in the dark
Stable in DMSO at room temperature for 60h
Stable in water for 24h


Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 0.05, 0.1, 0.25, 0.5, 1 µg/ml
Vehicle / solvent:
Positive controls:
Positive control substance:
7,12-dimethylbenzanthracene mutagenic with S9 mix
Details on test system and experimental conditions:
On day 1 of the experiment, 1.500.000 cells were seeded per I50 rnn Ø dish. On the following day, the cells were exposed to the test compound with the exception that 6-fold larger volumes of media and test solution were used. After removal of the test compound and washing of the plates with PBS, the cultures were maintained untíl day 8 with 30 ml normal DME-FCS with one subculture on day 5. This period is required for expression of the new genotype i.e., for sufficient dllution and catabolism of the previously formed hypoxanthíne guanine phosphoribosyl transferase. Afterwards the cells were harvested by trypsinisation and replated at a density of 1.000.000 per 150 mm Ø dish in DME-FCS containing 6-thioguanine (7 µg/ml) for selection of mutants (6 replicate plates), or at 100 cells per 60 mm Ø dish in medium without 6-thioguanine for the estimation of cloning efficiencies (3 replicate plates). The cultures were fixed and stained after 8 days (cloning efficiency plates) or 10 to 11 days (6-thioguanine plates).
Evaluation criteria:
Negative: if solvent and positive controls show results within the norm and if the test compound does not increase the mutation frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 10 x 10-6 and if at least 1.000.000 cells per condition have
been evaluated, the compound is considered as negative in the test and no further experlments are performed.

Positive: In case of a dose-dependent increase of the mutation frequency to at least 5-fold solvent control and at least 40 x 10-6 both in the presence and
absence of S-9 Mix, the compound is considered as positlve in the test without additional experiments.

In all other cases, the test at the suspected optimal concentration range is repeated. Then, the result' is considered as positive if in both experiments
(at similar concentrations) the mutation frequency is at least 2-fold above the solvent control and at least 10 x 10-6. Otherwise the result is considered
as negative.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative without metabolic activation

The substance was tested for genetic toxicity in mammalian cells (in vitro) following OECD 476. Under the experimental conditions the substance does not show mutagenic potential to V79 cells.

Executive summary:

The substance was tested for genetic toxicity to mammalian cells (in vitro) following OECD 476. At various concentrations up to the limits given by toxícity and solubility and it was negative in the V79 mammalian cell mutagenicity test under conditions where the positve controls exerted potent mutageníc effects. However, the acute toxicity of the substance to the V79 cells (in the direct test) is noteworthy.

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