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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
Study period:
2007-08-30 till 2007-09-10
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no rescrictions, fully adequate for assessment.

Data source

Reference Type:
study report

Materials and methods

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

Test material

Details on test material:
Test substance No.: 07/0323-1
Batch identification: B 561 laufender Betrieb
Purity: 99.8 area-%
Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity.
Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Date of production: 08 Jun 2007
Molecular weight: 144.21 g/mol
Physical state, appearance: Liquid, colorless to yellow
Storage conditions: Room temperature


Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO (Chinese hamster ovary) cell line (substrain K3) (1, 2) is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254
Test concentrations with justification for top dose:
1 - 10 - 50 - 100 - 250 - 500 - 750 - 1000 - 1500 μg/mL
Vehicle / solvent:
Due to the limited solubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT test and for which historical data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
without metabolic activation: positive control substance; ethylmethanesulphonate. With metabolic activation: positive control substance: methylcholanthrene.
Details on test system and experimental conditions:
Day 1: Seeding of the cells pretreated with "HAT" medium: in 175 cm² flasks (1x106 cells in 20 mL) and in 25 cm² flasks (200 cells in 5 mL)
Day 2: Test substance incubation (20 – 24 hours after seeding); exposure period (4-hour and 24-hour); washing of the cultures (4-hour exposure);
1st cytotoxicity determination (cloning efficiency 1: survival)
Day 3: Washing of the cultures (24-hour exposure)
Day 5: 1st passage of the treated cells
Day 9: 2nd passage of the treated cells with seeding in the selection medium ("TG" medium); 2nd cytotoxicity determination (cloning efficiency 2: viability)
From day 16: Drying, fixation, staining and counting of the selected colonies
In this study all incubations were performed at 37°C with a relative humidity of > 90% in a 5% (v/v) CO2 atmosphere.
Evaluation criteria:
• The absolute cloning efficiencies of the negative controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative controls should fall within our historical negative control data range of 0 – 15 mutants per 106 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 10 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (MFcorr.: 91.24 – 553.18 per 106 cells) were clearly within our historical positive control data range (without S9 mix 4 hours treatment: MFcorr.: 48.83 –587.77 per 106 cells; without S9 mix 24 hours treatment: MFcorr.: 272.94 – 331.63 per 106 cells; with S9 mix: MFcorr.: 29.06 – 413.54 per 106 cells.

The osmolarity was not influenced by test substance treatment. The pH of the stock solutions were adjusted to physiological values using small amounts of 2 N NaOH.
In this study, in the absence and the presence of S9 mix no precipitation of the test substance in culture medium was observed up to the highest applied test substance concentration.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion