Octanethioic acid, S-[3-(triethoxysilyl)propyl] ester, reaction products with 2-methyl-1,3-propanediol and 3-(triethoxysilyl)-1-propanethiol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 December 2006 - 14 May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

Colour: Light yellow

Specific details on test material used for the study:

Storage: at room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Duplicate cultures were treated at each concentration. The selection of the concentrations was based on the results from the pre-test (see below).

Experiment I:
without metabolic activation: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36 µg/mL
with metabolic activation: 200, 400, 600, 750, 850 and 1000 µg/mL

Experiment II:
without metabolic activation: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 µg/mL
with metabolic activation: 150, 250, 350, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL

Vehicle / solvent:

Ethanol

The test item could not be dissolved in DMSO, but the test item was found to dissolve in ethanol.
Precipitation of the test item was noted at 400 µg/mL and higher.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Exposure period experiment I: 4 hours (with and without metabolic activation)
Exposure period experiment II: 4 hours (with metabolic activation, 20 hours (with metabolic activation)

Seeding of Cultures:
3 or 4 days old stock cultures with higher than 50% confluency were trypsinised at 37°C for 5 min. by adding a trypsin solution in Ca-Mg-free PBS solution. The enzymatic treatment was stopped with complete culture medium. A single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2%. The cells were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment.
The cells were seeded into Quadriperm dishes which contain microscopic slides (at least 2 chambers per dish and test group). Into each chamber 1 x 10^4 - 5x 10^5 cells were seeded with regard to preparation interval. The medium was minimum essential medium supplemented with 10% FCS.

Experiment II: Short time exposure (with metabolic activation), long time exposure (without metabolic activation)
The treatment with metabolic activation:
2 days after seeding of the cells, the culture medium was replaced with serum-free medium containing the test item and 50 µL/mL S9 mix (with metabolic activation). Additional negative and positive controls were performed with and without exogenic metabolic activation.
4 hours after treatment the cultures were washed twice with PBS and cultured in complete medium for the remaining culture time.
In the experiment without metabolic activation, 2 days after seeding the cells were incubated with the test item in complete medium (MEM with 10% FCS) for 20 hours. The cells were prepared at the end of the incubation. Additional negative and positive controls were tested.

All cultures were incubated at 37°C in a humidified atmosphere with 5.0% CO2 (95.0% air).

The cytotoxicity was determined by the mitotic index (% cells in mitosis) by counting the number of mitotic cells per 1000 cells. As an additional parameter the relative cell density was calculated as the mean of twenty cell counts per test group.

Rationale for test conditions:

A pre-test was conducted under identical conditions as described for the main experiment. The following concentrations were tested:
Without S9-mix: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 µg/mL
With S9-mix: 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL

Evaluation criteria:

Criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells).
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary (3)(10). [...]
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Statistics:

The non-parametric Fisher's exact test was used.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The following concentrations were selected for microscopic analyses:
Experiment I
without metabolic activation: 10, 12, 14 and 16 µg/mL
with metabolic activation: 400, 600 and 750 µg/mL

Experiment I
without metabolic activation: 26, 28 and 30 µg/mL
with metabolic activation: 750, 800, 850 and 900 µg/mL

Precipitation: Precipitate was noted in all dose groups with metabolic activation. No precipitate of the test item was observed in the dose groups without metabolic activation.

Observations:
No biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

No increase in the frequencies of polyploid cells was found.

The results are included below in tabular form (including historical control data).

Applicant's summary and conclusion

Conclusions:

Based on the results of an in vitro chromosome aberration test in Chinese Hamster V79 cells according to OECD/EC guidelines and GLP principles, the test item is considered to be non-clastogenic.