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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in Salmonella typimurium strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 (according to OECD TG 471)

Cytogenicity in mammalian cells: negative with and without metabolic activation in CHO cells (according to OECD TG 473)

Mutagenicity in mammalian cells: negative without metabolic activation, positive with metabolic activation in L5178Y cells (according to OECD TG 490)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-05-2002 To 02-12-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: rfa- and pkM101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa-, uvrB- and pkM101 (TA 98 & TA100)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
100, 316, 1000, 3160 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthracene amide
Remarks:
+S9; TA 98, TA102 and TA1537: 2 µg/plate dissolved in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
-S9: TA1537: 100 µg/plate dissolved in ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
-S9: TA98: 10 µg/plate in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
-S9: TA1535 and TA100: 10 µg/plate in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
-S9: TA102: 1300 µg/plate dissolved in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
+S9; TA100 and TA1535: 1500 µg/plate dissolved in aqua ad iniectabilia.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation.

DURATION
- Preincubation period: 60 minutes (at 37°C)
- Exposure duration: 48 hours (at 37°C)


NUMBER OF REPLICATIONS: 3 plates per concentration in 2 independent experiments


DETERMINATION OF CYTOTOXICITY
- Method: background lawn evaluation, reduction of the number of revertants by more than 50%

METABOLIC ACTIVATION SYSTEM: Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was prepared according to Maron and Ames (1983). S9 was collected from 20-30 rats.
The S9 mix was freshly prepared on the day of the test according to Maron and Ames (1983): containing 5% S9 and the following components (per 100 mL):
-5.0 ml rat liver S9 (Aroclor 1254-induced)
-2.0 ml 0.4 M MgCl2 + 1.65 M KCl-salt solution
-141.0 mg glucose-6-phosphate
- 306.5 mg NADP
- 50.0 ml 0.2 M phosphate buffer, pH 7.4
- sterile aqua ad iniectabilia ad 100 ml
Evaluation criteria:
A test chemical is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤0.05, U-test according to MANN and WHITNEY) 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 independent experiments;
- in addition, a significant (p ≤0.05) concentration (log value)-related effect (Spearman’s rank) correlation coefficient) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate without and with activation, 3160 µg/plate without activation (pre-incubation); 3160 and 1000 µg/plate with activation (plate incorporation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate without and with activation, 3160 µg/plate without activation (pre-incubation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate without and with activation (pre-incubation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate without activation (plate incorporation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test substance was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Cytotoxicity (scarce background lawn) was noted at the top concentration of 5000 µg/plate. Hence, 5000 µg/plate was chosen as the top concentration for the main study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the plate incorporation test without metabolic activation cytotoxicity (scarce background lawn) was noted at the top concentration of 5000 µg/plate of the test substance in test strain TA 100. In the experiment with metabolic activation cytotoxicity (reduction of the number of revertants by more than 50%) was noted at concentration of 1000 and 3160 µg of the test substance/ plate in test strain TA 1535.
In the preincubation test without metabolic activation pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 or 3160 µg/plate onwards in test strains TA 1535 and TA 1537, respectively. Cytotoxicity (scarce background lawn) was noted at the top concentration of 5000 µg/plate of test substance in test strain TA 98. In the experiment with metabolic activation pronounced cytotoxicity ((scarce background lawn and reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 µg/plate in test strain TA 1537, and cytotoxicity (scarce background lawn) in test strain TA 98 and TA 1535.

Table 1. Plate incorporation test without metabolic activation:

Substance (µg/plate)

Plate incorporation test without metabolic activation. Number of reverted colonies.

 

TA 98

TA 100

TA 102

TA 1535

TA1537

Silane M2 Dimethoxy

Mean values ± SD

5000

M

32.3

110.7#

291.7

10.0

3.3

±SD

6.4

19.5

5.5

1.7

1.5

3160

M

30.3

101.0

288.7

8.0

4.3

±SD

5.9

5.6

5.5

1.0

1.2

1000

M

24.7

118.7

317.3

10.0

3.7

±SD

2.5

1.5

25.0

3.5

1.5

316

M

29.0

100.0

255.0

12.3

3.3

±SD

4.4

3.5

10.6

2.9

1.2

100

M

26.3

107.0

265.3

11.0

5.7

±SD

3.5

18.2

7.4

2.0

0.6

Negative control 100 µg/plate

M

25.7

102.7

270.7

10.0

3.3

±SD

2.5

4.7

22.7

1.7

2.1

Positive control substance

 

2-Nitrofluorene

Sodium azide

Methylmethane sulfonate

Sodium azide

Aminoacridine

Concentration µg/plate

 

10

10

1300

10

100

 

M

726.0

1129.0

1348.0

540.7

142.7

 

±SD

59.6

46.0

78.3

15.3

32.1

# = scarce background lawn.

Table 2. Plate incorporation test with metabolic activation

Substance (µg/plate)

Plate incorporation test with metabolic activation. Number of reverted colonies.

 

TA 98

TA 100

TA 102

TA 1535

TA1537

Silane M2 Dimethoxy

Mean values ± SD

5000

M

32.3

113.7

241.7

9.3

7.7

±SD

0.6

15.9

17.8

0.6

2.1

3160

M

38.0

113.3

277.7

7.7

4.3

±SD

7.0

11.0

22.5

0.6

1.5

1000

M

34.7

117.0

256.7

7.0

5.7

±SD

7.6

23.6

36.4

1.0

      3.2

316

M

31.3

114.7

371.7

17.7

5.7

±SD

7.1

10.6

46.2

3.5

0.6

100

M

49.0

114.3

284.3

13.3

6.3

±SD

7.0

6.8

11.9

3.5

2.3

Negative control 100 µg/plate

M

38.7

114.7

296.0

16.0

5.0

±SD

1.5

5.0

8.5

3.6

1.7

Positive control substance

 

2-Anthracene amide

Cyclophosphamide

2-Anthracene amide

Cyclophosphamide

2-Anthracene amide

Concentration µg/plate

 

10

10

1300

10

100

 

M

724.0

1118.3

937.0

288.7

138.3

 

±SD

43.3

45.8

14.0

25.9

26.1

Table 3. Preincubation test without metabolic activation

Substance (µg/plate)

Preincubation test without metabolic activation. Number of reverted colonies.

 

TA 98

TA 100

TA 102

TA 1535

TA1537

Silane M2 Dimethoxy

Mean values ± SD

5000

M

18.3#

94.0#

298.7

7.3#

1.7#

±SD

5.5

43.5

12.7

6.4

1.2

3160

M

23.0

134.3

306.7

17.3

2.0#

±SD

5.0

10.6

17.9

0.6

3.5

1000

M

18.3

137.7

294.7

21.3

6.7

±SD

4.5

3.2

14.2

1.5

2.5

316

M

23.7

136.0

309.7

16.0

6.7

±SD

4.5

9.2

8.0

6.9

1.2

100

M

23.0

130.0

318.0

17.0

8.0

±SD

2.0

7.5

8.2

2.0

2.6

Negative control 100 µg/plate

M

22.3

114.0

293.7

18.0

7.7

±SD

5.0

10.8

21.1

6.0

0.6

Positive control substance

 

2-Nitrofluorene

Sodium azide

Methylmethane sulfonate

Sodium azide

Aminoacridine

Concentration µg/plate

 

10

10

1300

10

100

 

M

514.0

1131.3

1102.3

234.0

292.0

 

±SD

56.2

101.2

17.9

33.4

56.1

Table 4. Preincubation test with metabolic activation

Substance (µg/plate)

Preincubation test with metabolic activation. Number of reverted colonies.

 

TA 98

TA 100

TA 102

TA 1535

TA1537

Silane M2 Dimethoxy

Mean values ± SD

5000

M

22.0#

144.7

387.0

17.7#

4.0#

±SD

7.0

20.3

60.9

1.5

4.0

3160

M

31.0

135.7

443.0

17.0

5.7

±SD

5.3

15.5

23.6

2.0

3.5

1000

M

26.7

131.7

446.0

16.7

9.7

±SD

2.1

18.2

16.5

1.2

      3.5

316

M

34.3

129.0

407.3

18.3

7.7

±SD

4.2

5.3.6

16.5

1.2

1.2

100

M

32.0

132.0

412.3

17.7

9.3

±SD

12.1

14.9

30.4

1.5

2.1

Negative control 100 µg/plate

M

31.0

128.0

375.7

15.3

8.7

±SD

2.6

15.1

22.1

2.5

2.1

Positive control substance

 

2-Anthracene amide

Cyclophosphamide

2-Anthracene amide

Cyclophosphamide

2-Anthracene amide

Concentration µg/plate

 

2

1500

2

1500

2

 

M

524.3

1190.7

1233.7

432.3

285.3

 

±SD

39.8

73.5

92.2

20.0

52.3

Conclusions:
Interpretation of results:
negative with and without metabolic activation

In a bacterial mutagenicity assay according to OECD 471 and GLP, no mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for the test substance tested up to a concentration of 1000 µg/plate in any of the five test strains in two independent experiments without and with metabolic activation (plate incorporation or preincubation test).
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-11-2007 To 22-12-2007
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)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Toxicity test: +S9 : 9.34, 18.75, 37.5, 75, 150, 300, 600 and 1203 µg/ml
Toxicity test: -S9: 75, 150, 300, 600 and 1203 µg/ml
Cytogenetic analysis:
4 and 20 hours treatment without S9 mix - 300, 600 and 1203 µg/ml
4 Hours treatment with S9 mix - 37.5, 75 and 150 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone.
- Justification for choice of solvent/vehicle: Acetone was used as the solvent based on the solubility of the test article and compatibility with the target cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
+ S9: at final concentrations of 10 and 20 µg/ml.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
-S9: at final concentrations of 0.1 and 0.2 µg/ml.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration: In the non-activated study, the cells were exposed to the test article for 4 hours or continuously for 20 hours. In the S9-activated study, the cells were exposed for 4 hours.
- Expression time (cells in growth medium): 16 hours for the 4 hours non-activated and activated cells.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Two hours prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/ml and the flasks returned to the incubator until cell collection.
STAIN (for cytogenetic assays): The dried slides were stained with 5% Giesma, air dried and permanently mounted.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures as quadriradials (symmetrical and asymmetrical interchange), triradicals, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatids or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverised chromosome(s), pulverised cells and severely damaged cells (≥aberrations) were also recorded. Chromatid gaps and isochromatids gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using the microscopic stage.

METABOLIC ACTIVATION SYSTEM: Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 mix was mixed with a cofactor pool containing:
- 2 mM magnesium chloride
- 6 mM potassium chloride
- 1 mM glucose-6-phosphate
- 1 mM nicotinamide adenine dinucleotide phosphate (NADP)
- 20 µL S9 per ml medium (McCoy's 5A serum-free medium supplemented with 100 units penicillin/ml, 100 µg stretomycin/ml, 2mM L- glutamine and 2.5 µg/ml amphotericin B).
Evaluation criteria:
The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control. The number and types of aberrations found the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each treatment group. Chromatid and isochromatid gaps do not include the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher’s Exact test. Fisher’s Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fishers’s Exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p≤0.05). However, values that are statistically significant but do not exceed the range of historical solvent may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test article in treatment medium was approximately 7.0.
- Effects of osmolality: In the preliminary toxicity assay the osmolality in treatment medium of the highest concentration tested, 1203 µg/ml, was 291 mmol/kg. The osmolality of the solvent (acetone) in treatment medium was 290 mmol/kg. The osmolality of the test article concentration in treatment medium is acceptable because it did not exceed the osmolality of the solvent by more than 20%.
- Water solubility: The test article was soluble in actone at a concentration of approximately 500 mg/ml, the maximum concentration tested for solubility.

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was not observed at any dose level in the non-activated 4 and 20-hour exposure groups. Substantial toxicity was observed at doses ≥360.9 µg/mL in the S9-activated 4-hour exposure group. Based upon the results of the toxicity study, the dose level for the chromosome aberration assay was selected.


COMPARISON WITH HISTORICAL CONTROL DATA:

The percent aberrant cells in the test article-treated groups were within the historical solvent control range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity of Dimethoxydimethylsilane (cell growth inhibition relative to the solvent) in CHO cells when treated for 4 hours in the absence of S9 activation was 14% at 1203 µg/ml, the highest test concentration evaluated for chromosome aberration. The mitotic index at the highest dose level evaluated for chromosome aberration, 1203 µg/ml, was 10% reduced relative to the solvent control. The percentage of cells with structural or numerical aberrations in the test article- treated group was not significantly increased relative to solvent control at any dose level (p≥0.05, Fisher Exact test). The percentage of structurally damaged cells in the MMC (positive control) treatment group (22%) was statistically significant.
Toxicity of Dimethoxydimethylsilane (cell growth inhibition relative to the solvent) in CHO cells when treated for 4 hours in the presence of S9 activation was 54% at 150 µg/ml, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 150 µg/mL, was 27% reduced relative to the solvent control. The percentages of cells selected for microscopic analysis were 37.5, 75 and 150 µg/ml. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased relative to solvent control at any dose level (p≥0.05, Fisher’s Exact test). The percentage of structurally damaged cells in the CP (positive control) treatment group (23.0%) was statistically significant.
Toxicity of Dimethoxydimethylsilane (cell growth inhibition relative to the solvent) in CHO cells when treated for 20 hours in the absence of S9 activation was 14% at 1203 µg/ml, the highest test concentration evaluated for chromosome aberrations in the non-activated 20-hour continuous exposure group. The mitotic index at the highest dose level evaluated for chromosome aberrations, 1203 µg/ml, was 29% reduced relative to the solvent control. The dose level selected for microscopic analysis was 300, 600 and 1203 µg/ml. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased relative to solvent control at any dose level (p≥0.05, Fisher’s Exact test).The percentage of structurally damaged cells in the MMC (positive control) treatment group (22.0%) was statistically significant.

Table 1: Summary

Treatment

µg/mL

S9 Activation

Treatment time

Mean Mitotic index

Cells scored

Aberrations per cells

Cells with Aberrations

Numerical

Structural

(Mean ± SD)

Numerical %

Structural %

Acetone

-S9

4

7.1

200

200

0.000

±0.00

1.5

0.0

Dimethoxydimethylsilane

300

-S9

4

8.6

200

200

0.000

±0.000

2.0

0.0

600

S9

4

7.3

200

200

0.015

±0.122

2.0

1.5

1203

S9

4

6.4

200

200

0.010

±0.100

2.5

1.0

MMC -0.2

S9

4

6.6

200

50

0.280

±0.573

2.0

22.0**

Acetone

+S9

4

9.4

200

200

0.025

±0.186

3.0

2.0

Dimethoxydimethylsilane

37.5

+S9

4

8.6

200

200

0.010

±0.100

4.5

1.0

75

+S9

4

8.6

200

200

0.020

±0.140

5.0

2.0

150

+S9

4

6.9

200

200

0.035

±0.232

4.5

2.5

CP- 10.0

+S9

4

4.8

200

100

0.350

±0.744

4.0

23.0**

Acetone

-S9

20

10.0

200

200

0.000

±0.000

1.5

0.0

Dimethoxydimethylsilane

300

-S9

20

8.2

200

200

0.000

±0.000

2.5

0.0

600

-S9

20

6.8

200

200

0.000

±0.000

3.5

0.0

1203

-S9

20

7.1

200

200

0.010

±0.100

3.0

1.0

MMC- 0.1

-S9

20

6.2

200

50

0.260

±0.565

1.5

22.0**

Treatment: Cells from all treatment conditions were harvested 20 hours after the initiation of the treatments.

Aberrations per cells: Severely damaged cells were counted as 10 aberrations.

Percent Aberration Cells: *, p0.05; **, p0.01; using Fishers’s Exact test.

Conclusions:
Interpretation of results:
negative with and without metabolic activation.

In an in vitro cytogenicity test according to OECD 473 and to GLP, the test substance, dimethoxydimethylsilane was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated systems.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Nov 2019 - 30 Jan 2020
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:
July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length, doubling time or proliferation index: 10-12 h
- Modal number of chromosomes: 40 ± 2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Complete culture medium: RPMI 1640 medium supplemented with 10% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B
Treatment medium: RPMI 1640 medium supplemented with 5% horse serum
100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B
Selective medium: RPMI 1640 medium supplemented with 20% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B and 5 µg/mL trifluorothymidine (TFT)
5%CO2
- 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:
Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix: Cofactors were added to the S9 mix in 100 mM sodium-phosphate-buffer pH 7.4 to reach the concentrations as follows: 8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; and 5 mM NADP.
- concentration or volume of S9 mix and S9 in the final culture medium: Final protein concentration: 0.75 mg/mL in the cultures
- quality controls of S9: The following quality control determinations were performed by Trinova Biochem GmbH: a) alkoxyresorufin-O-dealkylase activities; b) test for the presence of adventitious agents; c) promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
Test concentrations with justification for top dose:
0.5, 1, 2, 4, 6, 8 and 10 mM with and without metabolic activation (4 h exposure)
Justification for top dose: 10 mM is the recommended maximum test concentration in an OECD 490 study.
Vehicle / solvent:
- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: According to the Sponsor’s recommendations a solubility test was performed with DMSO. Based on the results DMSO (Supplier: AppliChem; Lot: 0001446873) was used as the best suitable solvent (1% DMSO in cell culture medium v/v) up to the maximum recommended concentration of 10 mM.

Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
1% DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates for negative and solvent control, single for test substance concentrations
- Number of independent experiments: 1

METHOD OF TREATMENT:
- Cell density at seeding: 1 x 10E7 cells/25 cm² flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h with and without metabolic activation
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time: 12 days
- Method used: microtiter plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 7 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells, PW) and those without cell growth (negative wells, NW) compared to the total number of cultures seeded (total wells, TW). Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT.
- Criteria for small (slow growing) and large (fast growing) colonies: Small colonies (produced predominantly by chromosomal rearrangements) approximately ≤ ¼ of well diameter. Large colonies (produced by point mutations) approximately > ¼ of well diameter

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth (SG; the number of times the cell number increased from the starting cell density); relative total growth (RTG; the product of the relative suspension growth [RSG] and the relative cloning efficiency [RCE] for each culture: RTG = RSG x RCE / 100)
Rationale for test conditions:
The assay was considered acceptable if the following criteria were met:

1. At least 3 out of 4 of the 96-well plates were scorable;
2. The cloning efficiency of the negative and/or solvent controls was between 65-120%;
3. The spontaneous mutant frequency in the negative and/or solvent controls was between 50 to 170 mutant per 10E6 cells;
4. The cell number of negative and/or solvent controls increased between 8 to 32 fold during the 2-day growth period; and
5. Positive controls responded appropriately and produced either at least 300 mutants per 10E6 cells with at least 40% of the colonies being small OR indcued a small colony mutant frequency of at least 150 mutants per 10E6 cells. The RTG for positive controls also must be greater than 10%.
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 1E06 cells and
- a concentration-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistical methods might be used as an aid in evaluation of the test result.

A test item is considered to be negative if the induced mutant frequency is below the GEF or the trend of the test is negative.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the solvent controls.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH-value detected with the test item was within the physiological range.
- Precipitation and time of the determination: No precipitation of the test item was noted in the experiment.


STUDY RESULTS
- Negative and positive controls responded appropriately. EMS, MMS and B[a]P showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies (≥40%), thus proving the efficiency of the test system to indicate potential clastogenic effects.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : Concentration-related increases were determined in the χ² test for trend (p < 0.05).
- Statistical analysis: The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the solvent controls (p< 0.05).
- Any other criteria: GEF for MLA is 126 mutants/10E6 cells

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: No growth inhibition was observed without and with metabolic activation.
- Genotoxicity results: For result tables please refer to attachment
In the main experiment without and with metabolic activation all validity criteria were met. The negative and solvent controls showed mutant frequencies within the acceptance range of 50 - 170 mutants/10E6 cells, according to the IWGT criteria.
In the experiment without metabolic activation the mutant frequencies induced by the test item did not show any biologically relevant increase. The GEF of 126 mutants/106 cells was not exceeded in any of the concentrations (Table 3). Some of the observed mutant frequencies were statistically significant increased over those of the solvent controls (Table 8). Since there was no significantly concentration-related increase determined in the χ² test for trend (Table 10), this effect was considered as not biologically relevant.
In the experiment with metabolic activation the mutant frequencies induced by the test item showed a distinct biologically relevant increase. The GEF of 126 mutants/106 cells was exceeded at concentrations of 4, 6, 8 and 10 mM (146.6, 199.4, 271.3 and 347.7 mutants/106 cells, Table 6). A statistical analysis displayed that the corresponding values of the mutant frequencies were significantly increased over those of the solvent controls (Table 9). Moreover, a statistically significant concentration-related increase was determined in the χ² test for trend (Table 10).
Clastogenicity:
In the main experiment without and with metabolic activation the percentage of small colonies in the negative controls and in the solvent controls, was found to be lower than 40%. Due to the increased number of small colonies in the experiment with metabolic activation and corresponding mutagenicity in the highest concentrations (4, 6, 8 and 10 mM), these concentrations of the test items were considered as potential clastogenic (Table 7).

HISTORICAL CONTROL DATA
All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich (please refer to attachment).
Conclusions:
In an OECD 490 study, the test material is considered to be mutagenic in an in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells with metabolic activation. As the number of small colonies is clearly increased a clastogenic mechanism is assumed.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Due to positive results in an in vitro mammalian mutagenicity test a testing proposal for a comet assay (OECD TG 489) is inlcuded for further evaluation of the mutagenic potential of the test substance.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Study period:
The test will be conducted after a decision on the requirement to carry out the proposed test has been taken according to the procedure laid down in Regulation (EC) 1907/2006 and a deadline to submit the information required has been set by the Agency.
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : dimethoxy(dimethyl)silane, CAS 1112-39-6

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies : no GLP in vivo genotoxicity studies are available for dimethoxy(dimethyl)silane
- Available non-GLP studies : no non-GLP in vivo gentoxicity studies are available for dimethoxy(dimethyl)silane
- Historical human data : no historical human data is available
- (Q)SAR : Q)SAR tools sufficiently addressing the endpoint genetic toxicity in vivo are currently not available.
- In vitro methods : Three in vitro studies are available: bacterial mutagenicity, mammalian cytogenicity and mammalian mutagenicity. Bacterial mutagenicity and mammalian cytogenicity showed negative results for the test substance. However, in the in vitro mammalian mutagenicity study according to OECD 490 and GLP positive results were observed with metaoblic activation. Therefore, further evaluation of the mutagenic potential of dimethoxy(dimethyl)silane needs to be performed.
- Weight of evidence: There are no studies available on in vitro mammalian mutagenicity or in vivo mutagenicity with the registered substance which could be used in a weight of evidence approach.
- Grouping and read-across : No suitable read-across options are available on genetic toxicity.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- An in vitro mammalian mutagenicity assay according to OECD 490 indicated a mutagenic potential for the registered substance. In order to clarify this mutagenic potential an in vivo mutagenicity study is necessary according to standard information requirements according to Annex VIII, Column II, 8.4. No appropriate read-across substances are available.

Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Sex:
male
Route of administration:
oral: gavage
Tissues and cell types examined:
glandular stomach, liver, duodenum
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Studies were chosen as key when the available study was of relevance and of sufficient quality for classification, labelling and for risk assessment.

 

A key bacterial reverse mutation assay conducted in compliance with GLP and according to OECD TG 471 is available for dimethoxy(dimethyl)silane. No evidence for a test-substance related increase in the number of revertants was observed in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA102. The strains were treated with doses up to a concentration of 5000 µg/plate in two independent experiments without and with metabolic activation (plate incorporation or preincubation test). Appropriate positive and solvent controls were included and gave expected results. Under the conditions of this study, dimethoxy(dimethyl)silane was concluded to be non-mutagenic in the Salmonella typhimurium strains (Laboratory of Pharmacology and Toxicology KG, 2003).

 

The key in vitro cytogenicity study on dimethoxy(dimethyl)silane was conducted according to OECD TG 473 and to GLP. The test substance did not cause a statistically significant, dose related increase in chromosome aberrations in Chinese hamster ovary (CHO) cells with or without metabolic activation. Appropriate positive and solvent controls were included and gave expected results. The test substance was therefore considered non-clastogenic in Chinese hamster ovary CHO cells (Dow Corning Corporation, 2008b).

 

A key mammalian cell gene mutation study on dimethoxy(dimethyl)silane was conducted according to OECD TG 490 and to GLP. The test substance dimethoxy(dimethyl)silane was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The test item was investigated at the following concentrations: 0.5, 1, 2, 4, 6, 8 and 10 mM.

No precipitation of the test item was noted in the experiment. No growth inhibition was observed without and with metabolic activation. No biologically relevant increase of mutants was found after treatment with the test item in the experiment without metabolic activation.The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation) was not exceeded by the induced mutant frequency at any concentration. In the experiment with metabolic activation, a biologically relevant increase of mutants was found after treatment with the test item. The GEF was exceeded by the induced mutant frequency at concentrations of 4 mM and higher. A statistical analysis displayed that the corresponding values of the mutant frequencies were significantly increased over those of the solvent controls. Moreover, a statistically significantly concentration-related increase was determined in the x² test for trend.

Ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS) and benzo[a]pyrene (B[a]P) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test substance dimethoxy(dimethyl)silane is considered to be mutagenic in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells. As the number of small colonies is clearly increased a clastogenic mechanism is assumed.

 

Dimethoxy(dimethyl)silane is found to be mutagenic in an in vitro mammalian mutagenicity study. Therefore, a testing proposal for an in vivo comet assay (OECD TG 489) is included in order to further evaluate the mutagenic potential of the test substance.

Justification for classification or non-classification

The available in vitro data on mutagenicity of the test substance indicate a mutagenic potential. Therfore, further in vivo testing is proposed. For the time being no final conlcusion on the criteria for classification according to Regulation (EC) No 1272/2008 can be given.