Dibutyltin maleate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

4 August - 9 December 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Internal Method No. 515.0 Main Dept. Exp. Toxicology

Deviations:

not specified

Principles of method if other than guideline:

Internal Method No. 515.0 Main Dept. Exp. Toxicology

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyltin dichloride

EC Number:

211-670-0

EC Name:

Dibutyltin dichloride

Cas Number:

683-18-1

IUPAC Name:

dibutyltin dichloride

Details on test material:

- Name of test material (as cited in study report): ZK 22.663 (DBTCl)

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
Stocks of the V79 cell line are stored in liquid nitrogen allowing the repeated use of the same cell culture batch in experiments.
- Periodically checked for karyotype stability: Yes, the cells have a stable karyotype with a modal chromosome number of 22.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Without metabolic activation: 0.000001, 0.000003, 0.000010, 0.000030, 0.000060 µl/ml
With metabolic activation: 0.00020, 0.00030, 0.00040, 0.00045, 0.00050 µl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The solid test material was heated in a 50°C waterbath and diluted as a liquid in DMSO to give final
concentrations of 0.000001-0.00050 µl/ml.
Stability in DMSO is unknown, therefore, the solutions were prepared immediately before the start of the test.
In aqueous solution the test material hydrolizes.

The test compound was strongly toxic at 0.0005 µl/ml with metabolic activation therefore 0.0005 µl/ml was chosen as the highest final concentration in the activation assay.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate and 9,10-dimethyl-1,2-benzanthracene

Remarks:

The positive control used without metabolic activation was ethylmethane sulfonate dissolved in MEM medium in a final concentration of 1 µl/ml; with S9 mix 9,10-dimethyl-1,2-benzanthracene was used dissolved in DMSO to yield a final concentration of 15 µg

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Thawed stock cultures were propagated at 37°C and c. 5% COˇ2 in 25 cm2 plastic-flasks (Nunc, Roskilde, Denmark). Seeding was done with about 5 x 10ˆ4 cells per flask in 5 ml of MEM-medium (Minimal Essential Medium, Gibco-Europe, Karlsruhe, FRG) supplemented with 10% fetal calf serum = FCS (Gibco-Europe, Karlsruhe, FRG) and neomycin sulfate (0.01%; Byk Gulden, Konstanz, FRG). The cells were subcultured twice a week.

For the selection of mutants the medium was supplemented with 11 µg/ml 6-thioguanine (EGA (Aldrich), Steinheim, FRG).

DURATION
- Preincubation period: After 24 h incubation at 37°C in a humidified atmosphere with c. 5% COˇ2 the medium was replaced by serum free medium containing the test substance and in the test with metabolic activation additionally 40 µl S9 mix/ml. After 4 h in the COˇ2 incubator this medium was replaced by medium containing FCS.
- Exposure duration: 7 days
- Expression time (cells in growth medium): For mutation expression the cells were grown for seven days with one subculturing (c. 10ˆ6 cells/175 cmˆ2 flask). For subsequent mutant selection five 80 cmˆ2 plastic flasks per concentration (c. 5 x 10ˆ5 cells/flask) containing selective medium were established.
- Selection time (if incubation with a selection agent): 7 days

STAIN (for cytogenetic assays): After a selection period of c. 7 days the colonies were fixated and stained with 10 % methylene blue in 0.01% KOH solution.

Exponentially growing cultures more than 50% confluent were trypsinised (trypsin concentration: 0.2 % in Ca-Mg-free salt solution) and for the mutagenicity experiment a single cell suspension was prepared with about 10ˆ6 cells in 31 ml medium per 175 cmˆ2 flasks (1 flask per concentration) and for the plating efficiency test with about 500 cells in 5 ml medium per 25 cmˆ2 flask (in duplicate).

DETERMINATION OF CYTOTOXICITY
- Method: number of cells per flasks

OTHER:
Mutagenicity of test material measured as number of mutant colonies

Evaluation criteria:

Experience has shown that the following predetermined descriptive criteria are the most useful for interpretation of the results:
- the test substance is classified as mutagenic if it induces with one of the test substance concentrations reproducibly a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- the test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently from the enhancement factor for induced mutants.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range found in this laboratory a seemingly concentration-related increase of the mutations or a factor of three or even more within this range may be regarded as to be irrelevant.

Statistics:

So far no satisfactory mathematical methods are available for statistical analysis of mammalian cell mutagenicity experiments such as those performed here. An evaluation is made only after a repeat experiment has been carried out.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Not reported

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Without metabolic activation by S9 mix a marked toxicity was seen with the test material at 0.00006 µl/ml, with metabolic activation a clear toxic effect could be observed at 0.0003 µl/ml in the first experiment and at 0.0005 µl/ml in the second assay of the second experiment. The positive control EMS revealed also a high cytotoxicity. The plating efficiencies of untreated cells were in the expected high range of > 50%.

In the test without S9 mix the mutation rates of the negative controls ranged from 1 to 12 mutant colonies per 10^6 cells; the cultures treated with ZK 22.663 (0.000001-0.00006 µl/ml) showed comparable results. The positive control substance EMS was clearly mutagenic.

In the assay with S9 mix the mutation rate of the negative controls was 6 to 27 mutant colonies per 10^6 cells. The cells treated with ZK 22.663 at various concentrations (0.0001 to 0.0005 µl/ml) showed mutation rates which were comparable to those of the negative controls. The positive control DMBA was clearly mutagenic.

The test material did not show a mutagenic potential in the HGPRT/V79 mammalian cell gene mutation test neither in the absence nor in the presence of rat liver S9 mix in two independently performed experiments.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Without metabolic activation by S9 mix a marked toxicity was seen with the test material at 0.00006 µl/ml, with metabolic activation a clear toxic effect could be observed at 0.0003 µl/ml in the first experiment and at 0.0005 µl/ml in the second assay of the second experiment. The positive control EMS revealed also a high cytotoxicity.
The test material did not show a mutagenic potential in the HGPRT/V79 mammalian cell gene mutation test neither in the absence nor in the presence of rat liver S9 mix in two independently performed experiments.

Executive summary:

In an evaluation of gene mutations in mammalian cells in culture study: HGPRT-test with V79 cells (Schering report number: IC16 -89) the test material was found to have cytotoxic effects without metabolic activation by S9 mix at 0.00006 µl/ml and with metabolic activation a clear toxic effect could be observed at 0.0003 µl/ml in the first experiment and at 0.0005 µl/ml in the second assay of the second experiment. The test material did not show a mutagenic potential in the HGPRT/V79 mammalian cell gene mutation test neither in the absence nor in the presence of rat liver S9 mix in two independently performed experiments.