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Genetic toxicity in vitro

Description of key information

In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 (OECD Test Guideline 471, 1997) (LPT, 2002).

Chromosome aberration in mammalian cells: a statistically significant but not dose-dependent increase in the number of aberrant cells was observed without metabolic activation in Chinese hamster V79 cells (OECD Test Guideline 473). With metabolic activation, a dose-dependent increase was observed which was not statistically significant (Eurofins, 2017).

In vitro micronucleus assay: negative with and without metabolic activation in Chinese hamster V79 cells (OECD Test Guideline 487) (Eurofins Biopharma, 2018).

Mammalian mutagenicity: negative with and without metabolic activation in mouse lymphoma L5178Y cells (OECD Test Guideline 490) (Eurofins, 2018).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-04-18 to 2002-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC directive 92/69/, L383 A part B, 1992
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 3160 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethylene glycol dimethylether

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 (without activation) 10µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 (without activation) 10 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without activation) 100 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102 (without activation) 1300 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthracene amide
Remarks:
TA 98, TA 102, TA 1537 (with activation) 2 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
TA 100, TA 1535 (with activation) 1500 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION

- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration. The experiment was repeated using the pre-incubation method

DETERMINATION OF CYTOTOXICITY

- Method: other: background lawn assessment

METABOLIC ACTIVATION
S9 was prepared from Aroclor 1254 induce rats, and checked for protein content and P-450 content. S9 mix contained 5% S9; 2% 0.4 M MgCl2/1.65 M KCl solution; glucose-6-phosphate and NADP as co-factors. 0.5 ml of S9 mix was added to 2 ml of top agar, 0.1 ml test material (or control) and 0.1 ml cell suspension, giving a final concentration of approximately 1% S9.
Evaluation criteria:
A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both experiments.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking on histidine-free agar plates.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.
Statistics:
MANN and WHITNEY and Spearman’s rank.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed.


Table 2: Dose range-finding study Number of revertants per plate (TA 100 - MA)

 

TA100

Conc.
(µg/plate)

Plate 1

Plate 2

Cytotoxic
(yes/no)

0*

160

134

No

0.316

152

196

No

1

152

172

No

3.16

141

150

No

10

143

162

No

31.6

157

135

No

100

143

151

No

316

155

162

No

1000

181

144

No

3160

0

0

Yes

5000

0

0

Yes

*solvent control with Ethylene glycol dimethylether

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA102

Conc.
µg/plate

MA

+

 MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

0*

36

36.7

No

151

134.3

No

268.7

278.7

No

31.6

35

35

No

143

159

No

268.3

274.7

No

100

32

36

No

134.7

158.7

No

286.3

282

No

316

37.3

37.3

No

140.7

169.3

No

290

283

No

1000

30.7

37.3

No

149

136.3

No

288

270

No

3160

0

0

Yes

0

0

Yes

284.7

265.3

No

Positive control

1291

845.7

No

1243

1266.3

No

1310

1228

No

*solvent control with Ethylene glycol dimethylether

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

 

TA1535

TA1537

Conc.
µg/plate

MA

+ MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

0*

18.3

16.7

No

6

4.3

No

31.6

19.3

16

No

3.7

5.3

No

100

12.3

15.3

No

4

4.3

No

316

20.3

15.3

No

5.3

4.3

No

1000

18

13

No

4.7

5

No

3160

0

0

Yes

0

0

Yes

Positive control

964.3

970

No

360.7

383.7

No

*solvent control with Ethylene glycol dimethylether

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA102

Conc.
µg/plate

— MA

+

 MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

0*

34.7

29

No

131

132.3

No

269.7

266.3

No

31.6

31.3

27.3

No

130.7

133.3

No

258.7

267

No

100

31.7

29

No

128

131.7

No

283.3

282.3

No

316

38.3

27.3

No

122.7

118.7

No

259.7

278.3

No

1000

0

0

Yes

0

0

Yes

0

0

Yes

3160

0

0

Yes

0

0

Yes

0

0

Yes

Positive control

1238

1238.7

No

1349.3

1351

No

1317

1216.3

No

*solvent control with Ethylene glycol dimethylether

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA1535

TA1537

Conc.
µg/plate

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

0*

16

14.3

No

13

14

No

31.6

12.3

15.3

No

12.7

14

No

100

13.3

15.3

No

9.3

13.3

No

316

12

11.3

No

7

11

No

1000

0

0

Yes

0

0

Yes

3160

0

0

Yes

0

0

Yes

Positive control

1354.7

1341.7

No

1337.7

1330.7

No

*solvent control with Ethylene glycol dimethylether

Conclusions:
tert-Butyl(chloro)dimethylsilane has been tested in a reliable test conducted in accordance with OECD 471 (1997) and in compliance with GLP, using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. No test-substance induced increase in the number of revertants was observed with or without metabolic activation up to cytotoxic concentrations in either the initial plate incorporation assay or the independent repeat experiment using the pre-incubation method. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 February 2017 to 20 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: chromosome aberration in mammalian cells
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The sample was re-covered with nitrogen after opening and sealed with parafilm
- Stability under test conditions: The solvent was compatible with the survival of the cells and the S9 activity.
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed with different solvents up to the maximum recommended concentration of 10 mM. According to the results of the solubility test, the test item was dissolved in tetrahydrofuran (THF). The test material was stable and soluble in THF.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: From the highest test item stock solution (2 M) separate dosing solutions of the test item were prepared for each of the concentrations by serial dilution with THF and added to cell culture medium (0.5%; v/v; final concentration).
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich V79 in stock cultures
- Suitability of cells: V79 cells in vitro are widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens.
- Cell cycle length, doubling time or proliferation index: 12 - 14 h
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments.
- Modal number of chromosomes: diploid number, 2n = 22
- Normal (negative control) cell cycle time: not specified

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 5% carbon dioxide atmosphere (95% air)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S9 mix: 0.02, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5, 7.5, 10 mM. On the basis of the data and the observations from the pre experiment and taking into account the recommendations of the guidelines, the following concentrations were selected for the main experiments I and II. Experiment I, 4-hour treatment without metabolic activation: 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 mM. Experiment II, 4-hour treatment with metabolic activation: 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 mM.
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): About 1 x 104 cells/mL were seeded into cell culture flasks with complete culture medium.

ACTIVATION: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.

DURATION
- Exposure duration: 4 hours exposure with and without metabolic activation
- Expression time (cells in growth medium): 17 hours after exposure
- Fixation time (start of exposure up to fixation or harvest of cells): at 21 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid for 2.5 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: single cultures

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): At least 300 well spread metaphases per concentration and validity controls were scored for cytogenetic damage.

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxic effect the cell count (RICC)
- Any supplementary information relevant to cytotoxicity: not applicable

OTHER EXAMINATIONS:
- Determination of polyploidy: number of polyploid cells is scored
- Determination of endoreplication: not determined
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

Rationale for test conditions:
The test conditions were determined on the basis of the data and the observations from the pre experiment and taking into account the recommendations of the guidelines
Evaluation criteria:
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data
Statistics:
Fisher´s exact test was performed to verify the results in the experiment. x² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 mM and higher with S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2, 3, 4 and 5 mM without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: Precipitate of the test item was noted first at 3 mM (without metabolic activation) and 9 mM (with metabolic activation) in experiment I with the unaided eye.
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: No precipitation of the test item was noted. The highest dose group evaluated in the pre-experiment was 10 mM. The relative increase in cell count (RICC) was used as parameter for toxicity.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: EMS (600 µg/mL) and CPA (1.11 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.
- Negative (solvent/vehicle) historical control data: Historical laboratory negative control range -0.28% - 3.7% aberrant cells, excl. gaps 4-hour treatment without metabolic activation and -0.23% - 3.95% aberrant cells, excl. gaps 4-hour treatment with metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC
- Other observations when applicable: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
Remarks on result:
other: the increase in chromosome aberrations with S9 was dose-dependent when evaluated by the x² trend test but was not statistically significant when evaluated using Fisher’s exact test, therefore was considered by the reviewer to be negative.

Table 1: Experiment I, without and with metabolic activation

Dose Group

Concentration [mM]

RICC [%]

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

-S9

4 h treatment

21 h preparation interval

 

 

 

 

 

C

0

123

4.3

3.3

-0.28% - 3.7% aberrant cells, excl. gaps

-

-

S

0

100

4.0

2.7

-

/

2

1.5

73

8.0

7.7

-

+

3

2

45

6.0

4.7

-

-

4

3

41

8.0

6.3

 

+

+

EMS

600 µg/mL

54

11.4

8.4

-

+

+S9

4 h treatment

21 h preparation interval

 

 

 

 

 

C

0

101

5.7

3.3

-0.23% - 3.95% aberrant cells, excl. gaps

-

-

S

0

100

5.0

3.3

-

/

1

2

75

6.0

3.0

-

-

2

4

63

9.7

6.3

-

-

3

5

41

8.3

6.7

 

-

-

CPA

1.11 µg/mL

79

20.7

18.3

-

+

C: Negative Control (Culture Medium)

S:  Solvent Control (THF)

EMS: Ethylmethanesulfonate

CPA:  Cyclophosphamide

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the(solvent) control groups. The cell count was determined by a cell counter per culture for each test group.

a: - without precipitation, + with precipitation

b: statistical significant increase compared to solvent controls (Fishers exact test, p< 0.05),
+: significant; -not significant

Conclusions:
tert-Butyl(chloro)dimethylsilane has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD TG 473 and in compliance with GLP (Eurofins, 2017). In the absence of metabolic activation, the increase was observed at all concentrations, and was statistically significant (Fisher’s exact text) at the mid and high doses of 1.5 mM and 3 mM. The increases were not dose-dependent but were considered biologically relevant as all test item concentrations were outside the historical control values. With metabolic activation, a dose-dependent increase was observed at the mid and high doses of 4 mM and 5 mM. This increase was not statistically significant. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations under the conditions of this study.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-05 to 2018-01-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: gene mutation in mammalian cells
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The sample was re-covered with argon after opening and sealed with parafilm
- Stability under test conditions: The substance is hydrolytically unstable; a non-aqueous solvent
- Solubility and stability of the test substance in the solvent/vehicle: Based on the pre-experiment for solubility test, performed for the Chromosome Aberration Test, the test item was dissolved in tetrahydrofuran (THF). The test material was stable and soluble in THF.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 10 mM solution in THF was prepared.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
clone TK+/- -3.7.2C
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich stock culture
- Suitability of cells: Applicable to the assay
- Cell cycle length, doubling time or proliferation index: 10-12 h doubling time
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable



MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced rat liver S9
Test concentrations with justification for top dose:
without metabolic activation: 0.5, 1.0, 3.0, 5.0, 5.5, 6.0 and 6.5 mM
and with metabolic activation: 0.5, 1.0, 2.5, 3.0, 4.0, 5.0 and 6.0 mM

The top dose was based on observations in the pre-experiment for toxicity. In the experiment with metabolic activation the selection of concentration was based on the occurrence of precipitation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): trifluorothymidine [TFT]

NUMBER OF REPLICATIONS: single cultures were used

NUMBER OF CELLS EVALUATED: Approximately 2000 cells per well were incubated with TFT

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: no relevant information

- OTHER: large and small colonies were counted
Rationale for test conditions:
The test conditions were determined on the basis of the data and the observations from the pre-experiment for toxicity and taking into account the recommendations of the guidelines
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- A dose-dependent increase in mutant frequency is detected

A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
- The cloning efficiency of the negative and/or solvent controls is in the range 65% -120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 106 cells
- The cell number of the negative/solvent controls should undergo 8-32-fold increase during a 2-day growth period (short-term treatment)
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 106 cells.
- The RTG must be greater than 10%.
Statistics:
Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The relative total growth (RTG) was 19.7% (without metabolic activation) and 61.0% (with metabolic activation) at the highest dose tested
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no pH confounding effects - pH of buffer was 7.4, pH of test item was within the physiological range.
- Effects of osmolality: osmolality was within the physiological range.
- Evaporation from medium: no
- Water solubility: test item was not soluble in water
- Precipitation: Precipitation was observed in the pre-experiment for toxicity
- Definition of acceptable cells for analysis: not applicable
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Toxicity was observed at 5 mM, the highest concentration tested; the relative suspension growth was 29.0% without metabolic activation and 9.1% with metabolic activation

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
EMS: mean 726.5, range 318.7-2919.0, SD 203.5
MMS: mean 763.4, range 376.4-2416.1, SD 421.6
B[a]P: mean 535.5, range 312.4-1108.9, SD 152.5
- Negative (solvent/vehicle) historical control data:
THF: mean 105.9, range 56.9-153.0, SD 30.0

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RTD
- Other observations when applicable: no increase was observed in the frequency of small colonies

Main Experiment, without metabolic activation

 

Test Group

Conc.

[mM]

RCEa[%]

RTGb[%]

MFc[mutants/ 106cells]

IMFd[mutants/ 106cells]

GEFeexceeded

Statistical

Significant Increasef

Precipitate

C1

0

110.9

125.1

79.3

/

/

/

-

C2

 0

81.6

90.7

 see above

/

/

/

-

S1

0

100.0

100.0

75.4

/

/

/

-

S2

 0

 see above

/

/

/

-

1

0.5

95.2

104.8

62.7

-12.7

-

-

-

2

1.0

107.0

109.9

70.7

-4.6

-

-

-

4

3.0

112.9

87.6

63.1

-12.3

-

-

-

5

5.0

82.8

57.2

98.3

22.9

-

-

-

7

5.5

101.7

38.7

88.8

13.4

-

-

-

9

6.0

117.2

27.3

74.6

-0.8

-

-

-

10

6.5

107.0

19.7

80.3

4.9

-

-

-

EMS

300 µg/mL

95.2

105.1

674.9

599.5

+

+

-

MMS

10 µg/mL

69.5

74.3

668.2

592.8

+

+

-

Main Experiment, with metabolic activation

 

Test Group

Conc.

[mM]

RCE [%]

RTG [%]

MF [mutants/ 106 cells]

IMF [mutants/ 106 cells]

GEF exceeded

Statistical

Significant Increase

Precipitate

C1

0

113.0

117.6

76.9

/

/

/

-

C2

0

119.2

120.4

 see above

/

/

/

-

S1

0

100.0

100.0

77.2

/

/

/

-

S2

 see above

/

/

/

-

1

0.5

87.5

92.9

94.5

17.4

-

-

-

2

1.0

97.4

101.6

75.8

-1.3

-

-

-

3

2.5

111.1

96.2

68.9

-8.2

-

-

-

4

3.0

105.6

97.1

72.5

-4.6

-

-

-

8

4.0

95.9

74.5

94.7

17.5

-

-

-

12

5.0

111.1

67.3

67.5

-9.6

-

-

-

13

6.0

107.4

61.0

88.9

11.8

-

-

+

B[a]P

1.5 µg/mL

87.5

76.3

505.7

428.5

+

+

-

C:       Negative Controls

S:       Solvent Controls (THF 0.25%)

a:        Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]

          Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b:        Relative Total Growth, RTG = (RSG x RCE)/100

c:        Mutant Frequency,

          MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d:        Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e:        Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f:         statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test, p<0.05).
       +: significant; -not significant

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

B[a]P:  Benzo[a]pyrene

Conclusions:
tert-Butyl(chloro)dimethylsilane has been tested for mutagenicity to mammalian cells in mouse lymphoma L5178Y cells according to OECD 490 and in compliance with GLP (Eurofins, 2018). No statistically significant increase in the mutation frequency was observed, and there was no test substance-related effect on the proportion of small colonies. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2017 to 10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The sample was re-covered with argon after opening and sealed with parafilm
- Stability under test conditions: The substance is hydrolytically unstable; a non-aqueous solvent
- Solubility and stability of the test substance in the solvent/vehicle: Based on the pre-experiment for solubility test, performed for the Chromosome Aberration Test, the test item was dissolved in tetrahydrofuran (THF). The test material was stable and soluble in THF.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 10 mM solution in THF was prepared.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: not specified
- Suitability of cells: recommended in guideline
- Cell cycle length, doubling time or proliferation index: not specified
- Sex, age and number of blood donors if applicable: not applicable
- Methods for maintenance in cell culture if applicable: stored over liquid nitrogen
- Modal number of chromosomes: not specified
- Normal (negative control) cell cycle time: not specified

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced rat liver S9
Test concentrations with justification for top dose:
A pre-experiment was conducted under identical conditions as described for the main experiment I and the concentrations in the main experiment were based on pre-experiment findings. The maximum concentration was 10 mM, as recommended in the OECD TG 487.
Experiment I:
- without and with metabolic activation: 0.25, 0.50, 1.0, 2.5, 5.0, 6.0, 7.0, 8.0, 9.0 and 10 mM
Experiment II:
- without metabolic activation: 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 6.0, 7.0 ,8.0 and 10 mM
- with metabolic activation: 0.20, 0.40, 0.75, 1.5, 2.0, 3.0, 4.0 and 5.0 mM
The following concentrations were selected for the microscopic analyses of micronuclei frequencies:
Experiment I (short-term exposure 4 h):
- without metabolic activation: 0.50, 1.0 and 2.5 mM
- with metabolic activation: 0.50, 1.0, 2.5 and 5.0 mM
Experiment II (short-term exposure 4 h with, long-term exposure 24 h without metabolic activation):
- without metabolic activation: 0.25, 0.50 and 1.0 mM
- with metabolic activation: 0.40, 0.75 and 1.5 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: Based on the pre-experiment for solubility test, performed for the Chromosome Aberration Test, the test item was dissolved in tetrahydrofuran (THF). The test material was stable and soluble in THF.
Untreated negative controls:
yes
Remarks:
cell culture medium
Negative solvent / vehicle controls:
yes
Remarks:
cell culture medium with 0.5% THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Approx. 50 000 cells per cell culture flask.

ACTIVATION: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice. The final concentration of S9 mix in the cultures was 5%.

DURATION
- Exposure duration: experiment I, 4 hours with or without metabolic activation; experiment II, 24 hours without metabolic activation and 4 hours with metabolic activation.
- Expression time (cells in growth medium): 20 hours following 4-hour treatment or 23 hours following 24-hour treatment.
- Fixation time (start of exposure up to fixation or harvest of cells): at 24 hours

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B.

STAIN (for cytogenetic assays): acridine orange solution.

NUMBER OF REPLICATIONS: duplicate cultures were used. The 4-hour treatment with metabolic in Experiment I activation gave inconclusive results so was repeated in Experiment II.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 ml complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter. After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.

NUMBER OF CELLS EVALUATED: For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges.

DETERMINATION OF CYTOTOXICITY
- Method: relative cell growth; cytokinesis block proliferation index (CBPI); cytostasis.


Rationale for test conditions:
A pre-experiment was conducted under identical conditions as described for the main experiment I and the concentrations in the main experiment were based on pre-experiment findings.
Evaluation criteria:
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
Statistics:
Statistical significance was determined using the χ² test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment I cytostasis was 44% at 5.0 mM (-MA); 32% at 2.5 mM and 67% at 5 mM (+MA). In experiment II, cytostatis was 43% at 1.0 mM (-MA); 37% at 1.5 mM (+MA).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none observed.
- Effects of osmolality: none observed.
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: Precipitation of the test item was observed at 2.5 mM and higher with and without metabolic activation in experiment I and at 1 mM and 1.5 mM without and with metabolic activation, respectively, in experiment II in the cultures at the end of treatment.
- Definition of acceptable cells for analysis: not specified

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment no precipitate of the test item was noted which was attributed to sticking of the precipitate to the tube walls in which redilution was performed before transfer to culture flasks. The highest dose group evaluated in the pre-experiment was 10 mM without metabolic activation and 7.5 mM with metabolic activation.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: not specified

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: MMS (25 µg/mL) and CPA (2.5 and 5.0 µg/mL) were used as clastogenic controls and colchicine as aneugenic control (0.16 and 2.0 µg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay
- Negative (solvent/vehicle) historical control data: In experiment I without metabolic activation the micronucleated cell frequency of the negative control (1.00%) was within the historical control limits of the negative control (0.39% – 1.40%) and the micronucleated cell frequency of the solvent control (1.00%) was within the historical control limits of the solvent control (0.45% – 1.55%). In experiment I with metabolic activation the micronucleated cell frequency of the negative control (0.75%) was within the historical control limits of the negative control (0.37% – 1.68%) and the micronucleated cell frequency of the solvent control (0.80%) was within the historical control limits of the solvent control (0.23% – 1.88%). In experiment II without metabolic activation the micronucleated cell frequency of the negative control (1.25%) was within the historical control limits of the negative control (0.39% – 1.40%) and the micronucleated cell frequency of the solvent control (0.60%) was within the historical control limits of the solvent control (0.45% – 1.55%). In experiment II with metabolic activation the micronucleated cell frequency of the negative control (1.10%) was within the historical control limits of the negative control (0.37% – 1.68%) and the micronucleated cell frequency of the solvent control (1.45%) was within the historical control limits of the solvent control (0.23% – 1.88%).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula: CBPI= (c1 x 1) + (c2 x 2) + (cx x 3)/n. The CBPI can be used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures: % Cytostasis= 100 – 100 x ((CBPIt – 1) / (CBPIc – 1))
CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures

In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to 1.0 mM. At 2.5 mM a cytostasis of 44% was observed. In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to 1.0 mM. At 2.5 mM a cytostasis of 32% and at 5.0 mM a cytostasis of 67% was observed. In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 0.50 mM. At 1.0 mM a cytostasis of 43% was observed. In experiment II with metabolic activation no increase of the cytostasis above 30% was noted up to 0.75 mM. At 1.5 mM a cytostasis of 37% was observed.
Remarks on result:
other: see "Remarks"
Remarks:
In experiment I with metabolic activation a statistically significant enhancement of cells with micronuclei was noted at 5.0 mM. The observed increase in micronucleus frequency at this concentration can be attributed to the strong cytotoxicity and/or strong level of precipitation observed at this concentration that led to chromosomal damage as a secondary effect and is thus considered as not biologically relevant. In experiment II without metabolic activation a statistically significant enhancement of cells with micronuclei was noted at 1.0 mM. Since the value of the micronucleus frequency was within the historical control limits of the negative and solvent control and no dose-response relationship was observed the observed increase was considered as not biologically relevant.

Table1:  Summary: Experiment I and II, without metabolic activation

Dose Group

Concentration [mM]

Number of cells evaluated

Cytostasis

[%]

Relative Cell Growth
[%]

Micro-nucleated
Cells Frequency
[%]

Historical Control Limits Negative Control

P

Statistically Significant Increasea

 

Exp. I

4 h treatment

24 h fixation interval

 

 

 

0.39% - 1.40%

 

 

C

0

2000

 0*

116

1.00

/

/

S

0

2000

0

100

1.00

/

/

2

0.50

2000

6

94

0.75

-

-

3

1.0

2000

20

80

0.75

-

-

4

2.5

2000

44

56

0.45

+

(+)

MMS

25 µg/mL

2000

8

92

3.85

-

+

Colc

2.0 µg/mL

2000

0

100

4.25

 

-

+

 

 

 

 

Exp. II

24 h treatment

24 h fixation interval

 

 

 

0.39% - 1.40%

 

 

C

0

2000

 0*

225

1.25

/

/

S

0

2000

0

100

0.60

/

/

2

0.25

2000

 0*

115

1.10

-

-

3

0.50

2000

22

78

0.70

-

-

4

1.0

2000

43

57

1.25

 

+

+

MMS

25 µg/mL

2000

0

190

5.60

-

+

Colc

0.16 µg/mL

2000

0

162

5.95

 

-

+

Table2:   Summary: Experiment I and II, with metabolic activation

Dose Group

Concentration [mM]

Number
 of cells evaluated

Cytostasis

[%]

Relative Cell Growth
[%]

Micro-

Nucleated
Cells
Frequency
[%]

Historical Control Limits Negative Control

P

Statistically Significant Increasea

 

Exp. I

4 h treatment

24 h fixation interval

 

 

 

0.37% - 1.68%

 

 

C

0

2000

 0*

106

0.75

/

/

S

0

2000

0

100

0.80

/

/

2

0.50

2000

0

100

0.95

-

-

3

1.0

2000

26

74

1.10

-

-

4

2.5

2000

32

68

1.05

 

+

-

5

5.0

1779

67

33

4.65

 

+

+

CPA

5.0 µg/mL

2000

57

43

9.35

-

+

 

Exp. II

4 h treatment

24 h fixation interval

 

 

 

0.37% - 1.68%

 

 

C

0

2000

 0*

110

1.10

 

/

/

S

0

2000

0

100

1.45

 

/

/

2

0.40

2000

8

92

1.10

 

-

-

3

0.75

2000

18

82

1.05

 

-

-

4

1.5

2000

37

63

1.30

 

+

-

CPA

2.5 µg/mL

2000

41

59

5.35

 

-

+

C: Negative Control (Culture medium)

S: Solvent Control (THF 0.5% v/v in culture medium)

CPA: Cyclophosphamide, Positive Control (with metabolic activation) [2.5 and 5.0 µg/mL]

a:  statistical significant increase compared to solvent control (c² test , p<0.05).

+: significant increase; (+): significant decrease ; -: not significant

MMS: Methylmethanesulfonate, Positive Control (without metabolic activation) [25 µg/mL]

Colc:  Colchicine, Positive Control (without metabolic activation) [0.16 and 2.0 µg/mL]

CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1x 1) + (c2x 2) + (cxx 3))/n

Relative Cell Growth:  100 x ((CBPITest conc– 1) / (CBPIcontrol -1))

c1:  mononucleate cells

c2:  binucleate cells

cx:  multinucleate cells

n:  total number of cells

P:  Precipitation 

 

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

Conclusions:
tert-Butyl(chloro)dimethylsilane has been tested in a reliable in vitro micronucleus assay conducted in accordance with OECD TG 487 and in compliance with GLP, using Chinese hamster V79 cells (Eurofins, 2018). In the first experiment cells were exposed for 4 hours without metabolic activation, and no test-substance induced increase in the frequency of micronuclei was observed up to the first concentration with a visible precipitate and significant toxicity. A second experiment without metabolic activation was carried out in which cells were exposed for 48 hours. No increase in micronuclei was observed following continuous exposure to the test item at 1.0 mM, at which concentration a precipitate was observed and cytostasis was 43%. In the first experiment, 4-hour exposure with metabolic activation, no increase in the frequency of micronuclei was observed up to 2.5 mM, however, at 5 mM a statistically significant increase in micronuclei was observed. 5 mM was not the lowest concentration with a visible precipitate, and the level of cytotoxicity as measured by relative CBPI was 33% which is lower than that recommended by the OECD TG 487. The short-term exposure with metabolic activation was therefore repeated and no biologically significant increase in micronuclei was observed at 1.5 mM which was the first concentration at which precipitation was observed. The cytotoxicity at this concentration was within the limits recommended by the guideline. It was therefore considered that the increase of the micronuclei frequency in the first experiment with S9 at a concentration of 5 mM can be attributed to the strong cytotoxicity (exceeding the level recommended by OECD TG 487) and/or strong level of precipitation observed at this concentration that led to chromosomal damage as a secondary effect. It is concluded that the test substance is not clastogenic under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

tert-Butyl(chloro)dimethylsilane has been tested in a reliable test conducted in accordance with OECD Test Guideline 471 (1997) and in compliance with GLP, using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 (LPT, 2002). No test-substance induced increase in the number of revertants was observed with or without metabolic activation when tert-butyl(chloro)dimethylsilane was tested up to cytotoxic concentrations in either the initial plate incorporation assay or the independent repeat experiment using the pre-incubation method. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

tert-Butyl(chloro)dimethylsilane has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD Test Guideline 473 and in compliance with GLP (Eurofins, 2017). In the absence of metabolic activation, an increase in the number of aberrant cells was observed at all concentrations. This increase was statistically significant (Fisher’s exact text (p < 0.05) at the mid and high doses of 1.5 mM and 3 mM. The increases were not dose-dependent when evaluated by the x² trend test but were considered biologically relevant as all test item concentrations were outside the historical control values. With metabolic activation, a dose-dependent increase was observed at the mid and high doses of 4 mM and 5 mM. This increase was not statistically significant when evaluated by the Fisher’s exact test. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations under the conditions of this study.

tert-Butyl(chloro)dimethylsilane has been tested for mutagenicity to mammalian cells in mouse lymphoma L5178Y cells according to OECD Test Guideline 490 and in compliance with GLP (Eurofins, 2018). No statistically significant increase in the mutation frequency was observed, and there was no test substance-related effect on the proportion of small colonies. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.

tert-Butyl(chloro)dimethylsilane has been tested in a reliable in vitro micronucleus assay conducted in accordance with OECD Test Guideline 487 and in compliance with GLP, using Chinese hamster V79 cells (Eurofins Biopharma, 2018). In the first experiment cells were exposed for 4 hours without metabolic activation, and no test-substance induced increase in the frequency of micronuclei was observed up to the first concentration with a visible precipitate and significant toxicity. A second experiment without metabolic activation was carried out in which cells were exposed for 48 hours. No increase in micronuclei was observed following continuous exposure to the test item at 1.0 mM, at which concentration a precipitate was observed and cytostasis was 43%. In the first experiment, 4-hour exposure with metabolic activation, no increase in the frequency of micronuclei was observed up to 2.5 mM, however, at 5 mM a statistically significant increase in micronuclei was observed. 5 mM was not the lowest concentration with a visible precipitate, and the level of cytotoxicity as measured by relative CBPI was 33% which is lower than that recommended by the OECD Test Guideline 487. The short-term exposure with metabolic activation was therefore repeated and no biologically significant increase in micronuclei was observed at 1.5 mM which was the first concentration at which precipitation was observed. The cytotoxicity at this concentration was within the limits recommended by the guideline. It was therefore considered that the increase of the micronuclei frequency in the first experiment with S9 at a concentration of 5 mM can be attributed to the strong cytotoxicity (exceeding the level recommended by OECD Test Guideline 487) and/or strong level of precipitation observed at this concentration that led to chromosomal damage as a secondary effect. It is concluded that the test substance is not clastogenic under the conditions of the test.

Supporting evidence that tert-butyl(chloro)dimethylsilane is not clastogenic

Chloro(trimethyl)silane (CAS 75-77-4) has been tested for the induction of chromosome aberrations in mouse lymphoma L5178Y cells in a reliable assay according to a protocol that is similar to OECD Test Guideline 473, but prior to GLP. Appropriate concurrent negative and positive controls were included and the expected responses were observed. The study reported that the test substance, chloro(trimethyl)silane, did show some clastogenic activity, but it was primarily of the simple type and was not dose dependent or of a sufficient magnitude to rate the test substance as a clastogen. The test substance was concluded to be not clastogenic (negative for induction of chromosome aberrations) in L1578Y mouse lymphoma cells under the conditions of the test (Litton Bionetics, 1978a).

Trichloro(methyl)silane (CAS 75-79-6) has been tested for the induction of chromosome aberrations in mouse lymphoma L5178Y cells in a reliable assay according to a protocol that is similar to OECD Test Guideline 473, but prior to GLP. Appropriate concurrent negative and positive controls were included and the expected responses were observed. The number of cells with 2 or more aberrations increased very slightly under activation conditions, and no increase was observed at the highest dose tested. Trichloro(methyl)silane was considered by the authors of the study to have weak clastogenic activity that may be related to the cytotoxic properties of the test substance. It is concluded by the reviewer that the study does not demonstrate a biologically relevant effect and that the test substance is negative for induction of chromosome aberrations under the conditions of the test (Litton Bionetics, 1978b).

Trichloro(octyl)silane (CAS 5283-66-9) has been tested in a cytogenicity study conducted according to OECD Test Guideline 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 (BSL Bioservice, 2012).

Chlorotri(isobutyl)silane (CAS 13154-25-1) has been tested in a cytogenicity study conducted according to OECD Test Guideline 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 peripheral human lymphocytes. Appropriate positive and solvent controls were included and gave expected results. It is concluded that chlorotri(isobutyl)silane does not induce chromosome aberrations under the condition of the test (SafePharm, 2002).

tert-Butyldimethylsilanol has been tested in a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471, and in compliance with GLP, using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli WP2uvrA. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to a limit concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (CERI Hita, 2015).

tert-Butyldimethylsilanol has been tested for ability to cause chromosome aberrations in Chinese hamster lung fibroblasts according to OECD Test Guideline 473 and in compliance with GLP. No increase in the number of cells with aberrations was observed either with or without metabolic activation when tested up to cytotoxic concentrations. Solvent (DMSO) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study (CERI Hita, 2016).

Chloro(trimethyl)silane (CAS 75-77-4) has been tested for the induction of chromosome aberrations in vivo in a reliable assay according to a protocol that is similar to OECD Test Guideline 475 and in compliance with GLP. Appropriate concurrent negative and positive controls were included, and the expected responses were observed. The test substance, chloro(trimethyl)silane, did not cause an increase in the  frequency of chromosomal breaks or aberrations in bone marrow cells of rats. Thus, there was no evidence in this study that it caused chromosomal damage and was concluded to be negative for induction of chromosome aberrations under the conditions of the test (Bioassay Systems Corporation, 1982).

Conclusion

tert-Butyl(chloro)dimethylsilane is not mutagenic to bacteria or to mammalian cells. The following have been taken into account in consideration of the potential for cytogenicity:

1. The negative result in the in vitro micronucleus assay (OECD Test Guideline 487) with the registration substance;

2. The lack of dose dependence in the increase in chromosome aberrations in Chinese hamster V79 cells in the absence of metabolic activation (OECD Test Guideline 473);

3. The lack of statistical significance in the increase in chromosome aberrations in Chinese hamster V79 cells in the presence of metabolic activation (OECD Test Guideline 473);

4. The weight of evidence for other alkylated chlorosilanes (all results negative);

5. The supporting data for the silanol hydrolysis product of the registration substance, tert-butyl(dimethyl)silanol (CAS 18173-64-3).

It is concluded that the test substance is not genotoxic, and further testing for genetic toxicity is not required.

Justification for classification or non-classification

Based on the available in vitro genotoxicity data, tert-butyl(chloro)dimethylsilane is not classified for mutagenicity according to Regulation (EC) No 1272/2008.