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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 activation (OECD TG 471)
Cytogenicity in mammalian cells: negative in CHO cells (OECD TG 473)
Mutagenicity in mammalian cells: negative in CHO-K1 cells (OECD TG 476)

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:
2002-05-06 to 2002-08-08
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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535, TA1537
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
100-5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given in report
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 metabolic activation
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 metabolic activation
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 metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102 without metabolic activation
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, TA102, TA 1537 with metabolic activation
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 metabolic activation
Details on test system and experimental conditions:
ACTIVATION: Aroclor induced rat liver S9; NADP and glucose-6-phosphate as cofactors; S9 mix contained 5% S9; 0.5 ml S9 mix added to 2.2 ml agar/cell suspension/test material

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: 3 plates per concentration; 2 independent experiments, the first plate incorporation, the second pre-incubation

NUMBER OF CELLS EVALUATED: 10 E07 to 10 E08 cells

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn
Evaluation criteria:
A chemical is considered positive if there is a reproducible dose-related statistically significant increase in the number of revertants compared with solvent control to at least 2 fold (strains TA 98, TA100 and TA 102) or 3-fold (strains TA 1535 and TA 1537). In addition, the histidine independence of teh revertants has to be confirmed.
Statistics:
Statistically significant: p
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
3160 µg/plate (TA 98, preincubation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential

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

Concentration µg/plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

5000

35.7

41.0

132.3

128.3

269.3

310.3

11.7

12.0

8.0

7.0

3160

31.3

38.0

128.7

136.7

259.3

302.7

12.3

11.3

6.3

7.3

1000

34.7

36.0

117.0

125.0

258.7

300.0

11.7

11.0

5.7

7.7

316

31.7

37.3

116.7

126.0

310.7

307.3

11.0

11.7

5.3

6.3

100

25.0

39.7

134.3

144.0

276.7

280.7

11.3

12.7

4.0

5.0

0**

36.0

42.0

127.0

122.7

265.0

298.3

12.3

11.3

5.7

6.3

Positive control

986.3

990.3

1206.3

1196.7

1005.0

1135.0

213.3

207.0

207.7

208.7

** Solvent control with DMSO

Table 2 Experiment 2 Pre-incubation: Number of revertants per plate (mean of 3 plates)

Concentration µg/plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

5000

33.7*

0.0*

122.3*

118.0*

261.0*

281.3*

12.3*

15.0*

3.3*

0.0*

3160

38.3*

27.7*

143.3

122.0

274.7

278.7

11.7

14.7

3.0

4.0

1000

39.7

36.3

148.0

151.7

250.3

265.0

10.7

15.3

2.7

2.7

316

50.7

30.0

141.0

154.3

282.0

287.3

12.0

13.3

3.0

3.3

100

38.0

31.0

145.7

159.0

284.7

285.3

13.0

16.7

3.3

3.0

0**

29.0

40.0

121.7

137.7

290.0

287.0

13.3

15.7

3.0

3.7

Positive control

1164.0

1139.3

1181.3

1189.0

1179.7

1188.0

674.0

671.7

696.0

700.3

* Scarce background lawn

** Solvent control with DMSO

Conclusions:
negative with and without metabolic activation

Tetramethylsilane was tested in valid study according to OECD 471 and under GLP. No increase in the number of revertants was observed in the plate incorporation assay with and without metabolic activation. The results was confirmed in the repeat pre-incubation experiment, also with and without metabolic activation. Vehicle and positive controls gave expected results. 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:
1998-07-30 to 1998-11-16
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:
1998
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenoarbitol and beta-naphtoflavone induced rat liver S9
Test concentrations with justification for top dose:
Test 1: 23,102, 187 µg/ml(-S9), 59, 260, ~500 µg/ml (+S9); Test 2: 25, 116, ~100 (-S9), 69, 260, 511 (+S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: none given in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: S9 mix contained NADP as cofactor

METHOD OF APPLICATION: other: the gaseous test substance was injected into the tightly closed cell culture flasks through a septum. At the end of the incubation period samples of the gaseous phase were withdrawn and concentration in medium determined from gas chromatography of the sample and the known air/medium distribution coefficient.

DURATION
- Preincubation period: none
- Exposure duration: 3 hours. In with metabolic activation assay the medium was exchanged at the end of this period by medium without S9.
- Expression time (cells in growth medium): 17 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid added 2 hours before cell preparation
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures, experiment repeated

NUMBER OF CELLS EVALUATED: 2000 cells were evaluated for determination of mitotic index; 100 cells per culture were analysed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A substance was considered positive if it induced a statistically significant increase in one or more concentrations at levels exceeding the normal range of the test system (>>5%).
Statistics:
Chi-square: statistically significant. if p < 0.05
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ca 500 µl/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential

Table 1 Experiment 1: Exposure 3 hours Harvest 20 hours, with and without activation

Concentration µg/ml

Metabolic activation

No. of cells examined

Mitotic index % mean *

Cells with aberrations % mean including gaps

Cells with aberrations % mean excluding gaps

0*

-

200

7

2

0

23

-

200

6.5

2

1

102

-

200

7.6

1.5

0

187

-

200

6.3

3.5

1

MMC 0.003

-

200

2.8

30.5

22

0*

+

200

4.5

2

0.5

59

+

200

4

3.5

1.5

260

+

200

5.6

2.5

1

500

+

211

3.2

4.7

0

CP 3

+

200

2.2

19.5

12.5

Positive controls: MMC Mitomycin C; CP Cyclophosphamide

* Negative control with culture medium

 

Table 2 Experiment 2: Exposure 3 hours Harvest 20 hours, with and without activation

Concentration µg/ml

Metabolic activation

No. of cells examined

Mitotic index % mean

Cells with aberrations % mean including gaps

Cells with aberrations % mean excluding gaps

0*

-

200

8.4

4.5

0.5

25

-

200

6.4

3.1

1.4

116

-

200

6.4

1

0

200

-

200

6.7

1.5

0

MMC 0.003

-

200

4.5

28

21.5

0*

+

200

5.1

1.5

0.5

59

+

200

4.9

4.5

2

260

+

200

3.6

4

1.5

511

+

211

3.6

2.3

0.9

CP 3

+

200

2.3

18.1

15.5

Positive controls: MMC Mitomycin C; CP Cyclophosphamide

* Negative control with culture medium

Conclusions:
negative with and without metabolic activation

Tetramethylsilane has been tested in a valid and reliable study according to OECD 473 and under GLP. The volatility of the substance was taken into account in the study: exposure was in sealed flasks and an analytical determination of the concentrations by head space analysis was carried out at the end of the incubation period. No increase in the number of cells with aberrations was observed. Positive and vehicle controls produced expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitol/beta-naphthoflavone activated rat liver S9
Test concentrations with justification for top dose:
Test 1: 9-215 µg/ml (-S9), 28-543 µg/ml (+S9); Test 2 12-2125 µg/ml (-S9), 28-534 µg/ml (+S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: Phenobarbitol/beta-naphthoflavone activated rat liver S9, NADP as cofactor

METHOD OF APPLICATION: other: the gaseous test substance was injected into the tightly closed cell culture flasks through a septum. At the end of the incubation period samples of the gaseous phase were withdrawn and concentration in medium determined from gas chromatography of the sample and the known air/medium distribution coefficient.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: none
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-26 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 flasks per concentration were treated, then pooled and 3 dishes per culture were plated for determination of cloning efficiency, 5 cultures per treatment group

NUMBER OF CELLS EVALUATED: 5 x 10 E05 (mutant frequency)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A substance is mutagenic if it causes a statistically significant dose related increase in mutant frequency at concentrations of the test substance resulting in > 20% cell survival. The increase is only considered biologically significant if it exceeds the spontaneous mutant frequency.
Statistics:
T-test ("Two sample analysis")
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential

In trials 1 and 2, treatment induced statistically significant increases in mutant frequency relative to the control, but these were within the range of the historical negative control, and not dose related, so were considered to be the result of normal assay variation, and a consequence of low negative control value.

Table 1 Test 1 Mutant frequency - average of 5 flasks

Without S9 mix

With S9 mix

Concentration µg/ml

Absolute C.E.

Mutant frequency

Significance

Concentration µg/ml

Absolute C.E.

Mutant frequency

Significance

0

88

0

0

91

2

9

96

5

**

29

93

2

NS

24

91

3

***

60

87

3

NS

57

98

0

NS

143

89

0

NS

98

87

2

*

228

97

0

NS

200

97

2

**

543

91

3

NS

Positive control 300

92

279

###

Positive control 10

106

108

###

 

Table 2 Test 2 Mutant frequency - average of 5 flasks

Without S9 mix

With S9 mix

Concentration µg/ml

Absolute C.E.

Mutant frequency

Significance

Concentration µg/ml

Absolute C.E.

Mutant frequency

Significance

0

83

0

0

88

2

12

90

1

NS

28

92

5

*

23

80

2

NS

60

87

3

NS

56

85

5

**

138

96

1

NS

110

82

0

NS

Positive control 10

121

135

###

215

85

4

**

Positive control 300

83

378

###

 

Table 3 Test 2c Mutant frequency - average of 5 flasks

With S9 mix 

Concentration µg/ml

Absolute C.E.

Mutant frequency

Significance

0

91

1

 

233

97

3

*

534

89

5

*

Positive control 10

93

275

###

NS not significant

* statistically significant

** statistically highly significant

*** statistically very highly significant

### highly significant, no statistics performed

Conclusions:
negative with and without activation

Tetramethylsilane has been tested in a valid and reliable study according to OECD 476 and under GLP. The volatility of the substance was taken into account in the study: exposure was in sealed flasks and an analytical determination of the concentrations by head space analysis was carried out at the end of the incubation period. The statistically significant increase in the number of revertants was neither dose related nor exceeding historical negative control valueS, and the increase in the presence of S9 was not reproducible. It is concluded that the test substance is negative for mutagenicity to mammalian cells 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

Information is available from reliable studies for all the required in vitro endpoints. There were reliable positive and negative bacterial mutagenicity results: the negative study was selected as key, as it was obtained in a more recent study to the current guideline, and used test substance with a higher degree of purity. In addition, tests in mammalian cells gave negative results.


Justification for classification or non-classification

The available information for the substance indicates that Tetramethylsilane (CAS 75 -76 -3) does not induces mutations in bacteria, as the positive result observed in one strain was not observed when test substance with a higher degree of purity was used in a more recent study. In addition, no potential for genetic toxicity was observed when Tetramethylsilane was tested in mammalian cells: no evidence of mutagenicity or cytogenicity was seen in mutagenicity and cytogenicity studies. In vivo testing is not considered necessary as the in vitro result are negative overall. It is concluded that classification for mutagenicity is not required according to Regulation (EC) No 1272/2008.