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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6th December 1988 - 21st February 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity is 96.3%

Method

Target gene:
The gene for the enzyme HGPRT (Hypoxanthine-Guanine Phos-phoribosyl Transferase) is located on the X-chromosome of the Chinese hamster.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
NDA
Additional strain / cell type characteristics:
other: HGPRT
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from liver of male Sprague-Dawley rats
Test concentrations with justification for top dose:
Without metabolic activation (µM): 1, 5, 10, 20, 30, 50, (first test); 1, 5, 7.5, 10, 15, 20, 30 (repetition).

With metabolic activation (µM): 1, 5, 10, 20, 30, 50, 75 (first test); 1, 5, 10, 20, 30, 40, 50 (repetition).
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
blank medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
blank medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 2 days
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 4 days
- Fixation time (start of exposure up to fixation or harvest of cells): 10 - 11 days


SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for mutagenic assays): 10 % methylene blue in 0.01 n KOH


NUMBER OF REPLICATIONS: Subcultured into 5 cells per flask per concentration.


NUMBER OF CELLS EVALUATED: 5 x 10^5 cells per flask


DETERMINATION OF CYTOTOXICITY
The toxicity of the test material was checked in a pilot test. 500 cells per 5 ml medium were seeded in 25cm^2 flasks; two flasks per concentration were used. After 24 hours the medium was replaced by medium without serum containing different doses of the test compound for 3 hours. After washing the cells, new nutrient medium was added. The cells were grown for 5 days, then the colonies were stained and counted. The toxicity test was performed without and with metabolic activation

Evaluation criteria:
The test substance is classified as mutagenic if there is a reproducible statistically significant dose-dependent increase in the mutation rate. This increase should be three times higher than the spontaneous mutation rate.

The mutation rate is enhanced by mutagenic substances with different mechanisms: base substitutions, frameshifts and chromosome rearrangements. The number of living cells in the selective medium, which contains purine analogues, is therefore a criterion for the mutagenic properties of a substance.
Statistics:
The statistical method to be used, if required is the U-test according to Mann-Whithney.

Because the results were obviously negative in this case, no statistical analysis was performed.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic to the cells at concentrations of 30 uM and above without metabolic activation and of 50 RM and above with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test material was checked in a pilot test. 500 cells per 5 ml medium were seeded in 25cm^2 flasks; two flasks per concentration were used. After 24 hours the medium was replaced by medium without serum containing different doses of the test compound for 3 hours. After washing the cells, new nutrient medium was added. The cells were grown for 5 days, then the colonies were stained and counted. The toxicity test was performed without and with metabolic activation

COMPARISON WITH HISTORICAL CONTROL DATA:
NDA

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The pre-test for toxicity of the substance showed a steep slope of the dose-response curve. Without metabolic activation the substance is not toxic for the cells at 20 µM/mL, but at 50 µM/mL the toxicity was nearly 100 %. With metabolic activation there was no toxicity at 20 µM/mL , at 50 µM/mL about 67 % of the cells were growing compared to the control and at 100 µM/mL no cells were growing.
Remarks on result:
other: strain/cell type: HGPRT
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The number of spontaneous mutations were 50 per 106 cells and 16 per 106, resp., without metabolic activation. With metabolic activation these values were 28 per 106 cells and 31 per 106 cells. The positive controls showed the expected results: Ethylmethanesulfonate: 253 and 271 mutant colonies per 106 cells; Dimethylbenz(a)anthracene: 547 and 388 mutant colonies per 106 cells. The test material ZK 21979 caused in the tests with metabolic activation no enhancement of the number of mutant cell colonies compared to the solvent control. In the test without metabolic activation there was with 10 µM ZK 21979 an enhancement up to 72 mutant colonies per 106 cells (spontaneous rate: 50, solvent control: 35). The other concentrations showed no enhancement. In the repetition test therefore additional concentrations (7.5 µM/mL and 15 µM/mL) were used. In this test no enhancement of the number of mutant colonies was observed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this assay the test material ZK 21979 had no mutagenic effect.
Executive summary:

An in vitro test was performed to assess the potential mutagenic activity of ZK 21979 (dioctyltin dichloride) in the HGPRT-test with the Chinese hamster cell line V79. The study was conducted as per OECD 476 to GLP standard.

Under the conditions of this assay the test material ZK 21979 had no mutagenic effect without or with metabolic activation.