Dihydro-2,2-dioctyl-6H-1,3,2-oxathiastannin-6-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 January 1999 to 01 June 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro gene mutation study in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Dihydro-2,2-dioctyl-6H-1,3,2-oxathiastannin-6-one

EC Number:

221-218-4

EC Name:

Dihydro-2,2-dioctyl-6H-1,3,2-oxathiastannin-6-one

Cas Number:

3033-29-2

Molecular formula:

C19H38O2SSn

IUPAC Name:

2,2-dioctyl-1,3,2-oxathiastanninan-6-one

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: white powder
- Storage conditions: ambient conditions

Method

Target gene:

6-thioguanine resistant mutants

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- The cell line, V79 4(H) can be traced back directly to the original V79 isolate prepared by Ford and Yerganian.
- The karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
- Permanent stocks of the V79 cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.

MEDIA USED
- The following culture media were used: EMEM Complete: Minimal medium 900 mL + Foetal Calf Serum 100 mL
- Minimal Medium: Eagle's Minimal Essential Medium (10X) 58.7 mL, L-glutamine (200 mM) 5.9 mL, Sodium bicarbonate (7.5 %) 15.7 mL, Non-essential amino acids (100X) 5.9 mL, Streptomycin sulphate 50 mg/mL and Penicillin G 50.000 IU/mL 1.2 mL, Sterile bidistilled water 500mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 Tissue Homogenate

Test concentrations with justification for top dose:

CYTOTOXICITY ASSAY
- 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/mL

MUTATION ASSAY
- First Assay:
With S9: 150, 75.0, 37.5, 18.8 and 9.38 µg/mL
Without S9: 14.1, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/mL
- Second Assay:
With S9: 150, 100, 66.7, 44.4 and 29.6 µg/mL
Without S9: 18.0, 15.0, 12.0, 8.00, 5.33 and 3.55 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetic acid : ethanol (1:1)
- Justification for choice of solvent/vehicle: The test material was found to be soluble in acetic acid : ethanol (1:1) at a maximum concentration of 100 mg/mL (after heating at approximately 45 °C). Due to the cytotoxicity of acetic acid, solutions of the test substance were furtherly diluted in sterile distilled water in the ratio 15 : 100 (v/v). An aliquot of the stock solution at a concentration of 15 mg/mL was added to EMEM minimal in the ratio 1:100 and generated a clear solution. On the basis of these results a maximum concentration of 150 µg/ml was selected as the highest dose-level to be used in the cytotoxicity test.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

CYTOTOXICITY ASSAY
- The toxicity test was undertaken in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose-levels of the test substance was used; cell cultures were treated using the same treatment conditions as the mutation assays, and the survival of the cells was subsequently determined.
- The test material was assayed at a maximum concentration of 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/mL in the cytotoxicity test
- Treatment was performed both in the absence and presence of S9 metabolism; a single culture was used at each test point and positive controls were not included. Following treatment, cell monolayers were washed with Phosphate Buffered Saline (PBS); EMEM complete was added to the flasks which were then returned to the incubator at 37 °C in a 5 % CO2 atmosphere (100 % nominal relative humidity). The following day the cultures were trypsinised, counted, diluted and plated. After incubation for six days the colonies were stained with Giemsa solution and counted.

MUTATION ASSAYS
- Three experiments were performed including negative and positive controls, in the absence and presence of S9 metabolising system.
- Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
- On the day before the experiment, sufficient numbers of 75 cm² flasks were inoculated with 2 million freshly trypsinised V79 cells from a common pool. The cells were allowed to attach overnight prior to treatment.
- The treatment media were prepared as follows:
Without S9 metabolism (10 mL total volume): Minimal medium 8.9 mL, Hepes buffer (200 mM) 1.0 mL and control or test material solution 0.1 mL
With S9 metabolism (10 mL total volume): Minimal medium 4.9 mL, S9 mix 5.0 mL and control or test material solution 0.1 mL
- The cultures were incubated at 37 °C for three hours. At the end of the incubation period, the treatment medium was removed and the cell monolayers were washed with Phosphate Buffered Saline (PBS). Fresh complete medium was added to the flasks which were then returned to the incubator at 37 °C in a 5 % CO2 atmosphere (100 % nominal relative humidity) to allow for expression of the mutant phenotype.
- The following day, the cultures were trypsinised and an aliquot was diluted and plated to estimate the viability of the cells. A number of cells were then re-plated in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give one million viable cells.

- On day 3 and day 6, the cell populations were sub-cultured in order to maintain them in exponential growth. The number of cells taken forward was adjusted according to the expected viability, to give at least one million viable cells seeded in F175 flasks.

- On day 6 and day 9, each culture was trypsinised, resuspended in complete medium and counted by microscopy. After dilution, an estimated 1 x 10^5 cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine (at 7.5 µg/mL). These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating Efficiency (P.E.).

Evaluation criteria:

For a test substance to be considered mutagenic in this assay, it is required that:
- There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test material. If only the highest practicable dose-level (or the highest dose-level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
- There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).
The "five-fold increase" in mutant frequency above the concurrent negative control is used as arbitrary criteria for positive response and it was established in our laboratory based on analysis of variation of negative control data. If spontaneous mutation frequency is in the upper part of the historical range, significance of mutation increase is evaluated on the basis of historical control data.

Statistics:

- The results of experiments were subjected to an Analysis of Variance in which the effect of replicate culture, expression time and dose-level in explaining the observed variation was examined. For each experiment, the individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = (x + a)^b where a = 0 and b = 0.275. A three way analysis of variance was performed (without interaction) fitting to three factors:
1) Replicate culture: to identify differences between the replicate cultures treated.
2) Expression time: to identify differences in response at the expression times used.
3) Dose-level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
- The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism. The analysis was achieved using the GLIM package. The F values obtained for each fitted factor and the corresponding probability values.
- Replicate culture, expression time and dose-level were not significant factors in explaining the observed variation in the data, both in the absence and presence of S9 metabolism, in Experiment 1 and 2.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY TEST
- Following treatment in the absence of S9 metabolism a dose related decline in survival was observed reaching 8% of the control value at 18.8 µg/mL, while at higher concentrations survival was reduced to the limit of detection. In the presence of S9 metabolism, no signs of toxicity were observed up to the highest dose-level tested.
- On the basis of these results, maximum concentrations of 14.1 and 150 µg/mL were selected as the maximum dose-levels to be used in the first mutation assay in the absence and presence of S9 metabolism, respectively.

MUTATION ASSAYS
- Two independent assays for mutation to 6-thioguanine resistance were performed. The results presented in the first experiment were obtained in a repeat assay. In the original experiment unacceptably low plating efficiency values were obtained; the results were otherwise consistent with those presented.
- Survival after treatment:
In Main assay 1, following treatment in the absence of S9 metabolism, a dose-related decline in survival was observed reaching approximately 35% of the control value at the two higher concentrations. In Main assay 2, marked toxicity was observed at higher dose levels and survival was reduced to approximately 6% of the negative control value at 18.0 and 15.0 µg/mL.
- In both experiments, following treatment in the presence of S9 metabolism, slight reduction in survival was observed at the second last dose-level.
- Mutation results:
No five-fold increases over the spontaneous mutation frequency were observed at any treatment-level, either in the absence or presence of S9 metabolic activation.
The mutant frequencies in the negative control cultures fell within the normal range. Treatment with the positive control substances gave marked responses in all experiments indicating the correct functioning of the test system.
- Osmolality and pH:
The pH values and osmolality of the post-treatment media were determined in Experiment 1. The addition of the test substance solution did not have any obvious effect on the osmolality or pH of the treatment medium.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the test material does not induce mutation in Chinese hamster V79 cells after in vitro treatment, either in the absence or presence of S9 metabolic activation.

Executive summary:

The mutagenic activity of the test material was investigated in accordance with the standardised guidelines OECD 476 and EEC Council Directive 87/302 Part B, under GLP conditions, by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment.

Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test material solutions were prepared using acetic acid:ethanol (1 : 1) in sterile distilled water at a concentration of 15 % v/v.

A preliminary cytotoxicity assay was performed. The test material was assayed at a maximum dose-level of 150 µg/mL and a wide range of lower dose-levels: 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/mL. Treatment with the test material in the absence of S9 metabolic activation resulted in severe toxicity at higher dose-level tested reaching 8 % of the negative control value at 18.8 µg/mL. In the presence of S9 metabolic activation no toxicity was observed at any dose-level. On the basis of the survival data obtained, the maximum dose-levels for the first mutation assay were selected as 14.1 µg/mL for treatment in the absence of S9 metabolism, and 150 µg/mL in its presence.

Two assays for mutation to 6-thioguanine resistance were performed. In the absence of S9 metabolism the first experiment was performed using dose-levels of 14.1, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/mL. In the second experiment the dose range was modified to take account of toxicity results obtained in the first experiment and the following dose-levels were used: 18.0, 15.0, 12.0, 8.00, 5.33 and 3.55 µg/mL. In the presence of S9 metabolism the first experiment was performed using dose-levels of 150, 75.0, 37.5, 18.8 and 9.38 µg/mL. In the second experiment the dose-range was modified to focus on the highest concentration that could be tested (150, 100, 66.7, 44.4 and 29.6 µg/mL).

No significant increases in mutant numbers or mutant frequency were observed following treatment with the test substance at any dose-level, in the absence or presence of S9 metabolism. In both mutation assays dose-related toxicity was observed only in the absence of S9 metabolism.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S 9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

Under the conditions of this study the test material does not induce gene mutations in Chinese hamster V79 cells after in vitro treatment, either in the absence or presence of S9 metabolic activation.