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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: GLP study performed according to a published procedure.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
method according to Clive, D. et al Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutation Res. 59, 61-108 (1979)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate
EC Number:
EC Name:
Bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate
Cas Number:
Molecular formula:
bis(oxiran-2-ylmethyl) cyclohexane-1,2-dicarboxylate
Test material form:


Target gene:
Thymidine kinase genes
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse Lymphoma cells L5178Y, heterocygous subline, TK +/- (Obtained from Dr. D. Clive with burroughs wellcome Company Research TRiangle Park, N. C., USA).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
without metabolic activation: 1.2, 2.5, 5, 7.5, 10, 11.5 and 12.5 nl/ml (first experiment)
without metabolic activation: 2, 4, 8, 12, 16, 18 and 20 nl/ml (second experiment)

with metabolic activation: 10, 20, 40, 60, 80, 90 and 100 nl/ml (first experiment)
with metabolic activation: 8, 16, 32, 48, 64, 72 and 80 nl/ml (second and third experiment)
Vehicle / solvent:
DMSO (1%)
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Ethylmethane sulfonate (without S9 activation) and dimethylnitrosamine (with S9 activation)
Details on test system and experimental conditions:
The test substance was dissolved in DMSO, and diluted into the culture medium.
In a preliminary toxicity test the concentration of TGIC causing a 85% reduction of the viability of cells was determined in suspension growth after 4-hour treatment and 72 hour suspension growth of the cells.
For this test cells were taken from exponential growth phase in Fischer’s medium containing 10% horse serum and anti-biotics; the final concentration of DMSO in the cell culture medium was 1%.
Seven concentrations were tested (51.563 – 3300 nl/ml) in the first and second (0.7183 - 50 nl/ml) test.
Suspension growth was compared to control cultures of the same initial cell density. The concentration calculated to cause 85% reduction of relative growth was used as highest dose in the mutagenicity experiment.
The mutagenicity test was carried out using exponentially growing cells (free of TK -/- cells) at 3 x 10E5 cells/ml, in round bottom flasks. After cleaning of spontaneous mutants and an additional three-days incubation period in THG, the cells were treated for four hours with the selected concentrations.
S9-activating mix was prepared from livers of male rats treated with Aroclor 1254.
After the 4-hour treatment, cells were washed and resuspended in growth medium and grown for 72 hours. Cell counts were regularly adjusted to 3 x 10E5 cells/ml. At the end of this expression period, cells were transferred to culture tubes containing 5 ml semi-solid agar cloning medium: for each concentration 8 tubes were seeded with 4 x 10E5 cells, in cloning medium containing BudR at 50 ug/ml, for viability counts 4 tubes of each concentration were seeded with 200 cells each in cloning medium without BudR.
Incubation time was 14 to 16 days at 37°C, and 12 days for the viability tubes.
Colonies were counted with a Colony counter (Fisher Count-All, Model 600), and with the aid of the viability counts the relative number of mutant colonies (TK-/-) was determined.
The results are expressed as induced TK -/- mutants /10E6 surviving cells. Mutagenicity criteria is an increase of 2.5 above control in any of the concentrations tested.
Positive control were ethylmethanesulfonate for the experiment without, and dimethylnitrosamine for the experiment with S9-activation
Evaluation criteria:
The results are expressed as induced TK -/- mutants /10E6 surviving cells. Mutagenicity criteria is an increase of 2.5 above control in any of the concentrations tested.
no data

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):
positive with and without metabolic activation

TK 12 608 caused mutagenic effects in the presence and absence of metabolic activation, and is therefore a direct-acting mutagen under the conditions of the assay.
Executive summary:

TK 12 608 was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 )ug/0.1 ml. In order to confirm the results, the experiments were repeated with the same concentrations.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone backmutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture(rat liver microsomes and co-factors).

In the experiments performed without microsomal activation treatment with TK 12 608 led to a marked increase in the number of back-mutant colonies of strains TA 100 and TA 1535. In the experiments carried out with microsomal activation this effect was more pronounced.

TK 12 608 and its metabolites formed as a result of microsomal activation thus displayed a clear-cut mutagenic effect in this test system.