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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-06 to 2011-12-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment: 0.020, 0.039, 0.078, 0.16, 0.31, 0.63, 1.25, 2.5, 5 and 10 mM (+/-MA); Expt I: 0.02, 0.04 and 0.06 mM (-MA); 0.100, 0.150 and 0.175 mM (+MA); Expt II: 0.03, 0.04 and 0.05 mM (-MA); 0.11, 0.14 and 0.16 mM (+MA)

Vehicle / solvent:
-Vehicle (s)/solvent(s) used: Tetrahydrofurane (final concentration of 1% v/v solvent)
-Justification for choice of solvent/vehicle: Due to the nature of the test item Octyltrichlorosilane could be dissolved in THF and diluted in cell culture medium (MEM) at a concentration of 10 mM. The solvent was compatible with the survival of the cells and the S9 activity.

Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation 400 and 900 µg/mL
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation 0.83 µg/mL
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density
METABOLIC ACTIVATION: S9 mix included glucose-6-phospate and NADP as cofactors. S9 added so that the final protein concentration in cultures was 0.75 mg/ml.
Evaluation criteria:
There are several 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 (0.0% - 4.0% aberrant cells (with and without metabolic activation)).


Statistics:
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.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No biologically relevant increase in the frequency of polyploid cells was observed.

Any other information on results incl. tables

Results of chromosome analysis
without metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I                              
negative control 200 - 2 1 0 1 1 0 0 0 91 101 1 2.5 1.0
solvent control 200 - 5 2 1 0 0 0 0 0 100 100 1 3.5 1.5
0.0025 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 89 n.d. n.d. n.d. n.d.
0.005 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 93 n.d. n.d. n.d. n.d.
0.01 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 95 n.d. n.d. n.d. n.d.
0.02 mM 200 no 1 3 0 0 2 0 0 0 97 101 1 2.5 1.0
0.04 mM 200 no 3 1 0 1 0 0 0 0 76 97 1 2.5 1.0
0.06 mM 200 yes   2 0 0 0 0 0 0 0 36 68 0 1.0 0.0
0.08 mM - yes   n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.10 mM - yes   n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.12 mM - yes   n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
EMS 900 µg/mL 200 - 7 13 8 1 1 0 1 1 84 95 1 15.0 11.0
Experiment II                                  
negative control 200 - 4 1 0 0 1 0 2 3 90 101 2 4.5 2.0
solvent control 200 - 1 0 0 0 0 0 1 0 100 100 0 1.0 0.5
0.0025 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 93 n.d. n.d. n.d. n.d.
0.005 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 94 n.d. n.d. n.d. n.d.
0.01 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 102 n.d. n.d. n.d. n.d.
0.02 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 99 n.d. n.d. n.d. n.d.
0.03 mM 200 no 3 1 0 0 0 0 0 0 85 100 6 2.0 0.5
0.04 mM 200 no 2 2 0 0 0 0 1 0 76 99 4 2.0 1.5
0.05 mM 200 yes 3 2 0 0 0 0 0 0 38 49 0 2.5 1.0
0.06 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 17 n.d. n.d. n.d. n.d.
0.07 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 8 n.d. n.d. n.d. n.d.
EMS 400 µg/mL 200 - 4 14 6 0 0 0 2 0 85 101 0 12.0 10.5

Results of chromosome analysis
with metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I                              
negative control 200 - 0 1 1 0 0 0 0 0 94 100 0 1.0 1.0
solvent control 200 - 1 2 1 0 0 0 0 0 100 100 0 1.5 1.0
0.025 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 106 n.d. n.d. n.d. n.d.
0.050 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 91 n.d. n.d. n.d. n.d.
0.100 mM 200 no 3 0 0 1 0 0 0 0 80 100 1 2.0 0.5
0.125 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 62 n.d. n.d. n.d. n.d.
0.150 mM 200 yes 1 0 0 0 1 0 0 0 63 93 1 1.0 0.0
0.175 mM 200 yes 2 1 0 0 0 0 0 0 43 73 1 1.5 0.5
0.200 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 16 n.d. n.d. n.d. n.d.
0.225 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 9 n.d. n.d. n.d. n.d.
0.250 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0 n.d. n.d. n.d. n.d.
0.275 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0 n.d. n.d. n.d. n.d.
0.300 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 21 n.d. n.d. n.d. n.d.
CPA 0.83 µg/mL 200 - 3 12 3 1 0 0 3 0 73 94 0 9.5 8.0
Experiment II                                  
negative control 200 - 6 3 1 2 0 0 0 0 99 100 1 4.5 2.0
solvent control 200 - 4 3 0 0 0 0 0 0 100 100 1 3.5 1.5
0.03 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 85 n.d. n.d. n.d. n.d.
0.06 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 114 n.d. n.d. n.d. n.d.
0.09 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 86 n.d. n.d. n.d. n.d.
0.11 mM 200 no 0 1 0 0 0 0 0 0 79 100 1 0.5 0.5
0.13 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 61 n.d. n.d. n.d. n.d.
0.14 mM 200 yes 1 1 0 0 0 0 0 0 62 71 0 1.0 0.5
0.16 mM 200 yes 1 1 0 0 0 0 0 0 50 59 1 1.0 0.5
0.18 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 24 n.d. n.d. n.d. n.d.
0.20 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 15 n.d. n.d. n.d. n.d.
0.22 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 5 n.d. n.d. n.d. n.d.
CPA 0.83 µg/mL 200 - 7 10 4 0 1 0 2 1 85 100 1 11.0 8.0

n.d. not determined

Applicant's summary and conclusion

Conclusions:
Trichloro(octyl)silane has been tested in a cytogenicity study conducted according to OECD TG 473 and in compliance with GLP. No evidence of the induction of chromosome aberrations or polyploidy was observed with or without metabolic activation when tested to cytotoxic concentrations in Chinese hamster V79 cells. It is concluded that octyltrichlorosilane is non-clastogenic (does not induce chromosome aberrations) under the conditions of the test.
Executive summary:

To investigate the potential of octyltrichlorosilane to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in Experiment I. In Experiment II the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

The test item Octyltrichlorosilane could be dissolved in THF and diluted in cell culture medium (MEM) at a concentration of 10 mM.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 0.02, 0.04 and 0.06 mM

with metabolic activation: 0.100, 0.150 and 0.175 mM

Experiment II:

without metabolic activation: 0.03, 0.04 and 0.05 mM

with metabolic activation: 0.11, 0.14 and 0.16 mM

In the Experiments I and II no precipitation of the test item was seen with and without metabolic activation at all concentrations evaluated.

Toxic effects of the test item were noted in Experiment I without metabolic activation at a concentration of 0.06 mM, with metabolic activation at concentrations of 0.15 mM and higher.

In Experiment II without metabolic activation toxic effects of the test item were observed at a concentration of 0.05 mM, with metabolic activation toxic effects of the test item were noted at concentrations of 0.14 mM and higher.

In both experiments 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.

In both experiments with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

The positive controls induced the appropriate responses.

There was no evidence of test item Octyltrichlorosilane induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data.