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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial reverse mutation assay:  for mutagenicity in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (OECD Test Guideline 471) (BioReliance, 1999).

In vitro cytogenicity in mammalian cells: tris(2-methoxyethoxy)vinylsilane was negative for clastogenicity in Chinese hamster ovary cells (OECD Test Guideline 473) (BioReliance, 2004).

In vitro gene mutation in mammalian cells: tris(2-methoxyethoxy)vinylsilane was negative for mutagenicity in L5178 mouse lymphoma cells (OECD Test Guideline 476) (Harlan Cytotest Cell Research GmbH, 2010).

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:
1999-07-09 to 1999-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
100, 333, 1000, 3333, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Stability in solvent
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535 without 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 activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E. coli WP2 uvrA without 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 activation
Details on test system and experimental conditions:
METABOLIC ACTIVATION
0.5 ml of S9 mix containing 10% Aroclor induced rat liver S9 with NADP as cofactor. The S9 was assayed for ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to S. typhimurium TA100

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

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 hours
- Expression time (cells in growth medium): 48-72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 hours

SELECTION AGENT (mutation assays): not specified

NUMBER OF REPLICATIONS: concentrations were plated in triplicate, experiment was repeated

NUMBER OF CELLS EVALUATED: 0.3 x 10E09 cells per ml

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:

OTHER: The study was performed in two phases, using the preincubation and plate incorporation methodology. The first phase, the preliminary toxicity study, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article.
Evaluation criteria:
A substance is considered positive if it causes a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concnetrations of test substance, of greater than two times (TA 98, 100 and E. coli) or 3 times (TA 1535 and 1537) the vehicle control value.
Statistics:
No statistical evaluation carried out.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid

Preliminary Toxicity Test: Ten dose levels, ranging from 6.7 to 5000 ug/plate were tested with the four Salmonella strains and WP2 uvrA in the presence or absence of S9.  Neither precipitate nor appreciable toxicity was observed. 

No positive responses were observed in the mutagenicity assays. Neither precipitate nor appreciable toxicity was observed. All bacterial strains exhibited mutagenic responses to the positive control substances, indicating that the tests were sensitive and valid.

Table 1: Preincubation experiment 1: Number of revertants per plate (mean of 3 plates)  

Concentration

µg/plate

Strain    TA 98

Strain    TA 100

Strain    TA 1535

Strain    TA 1537

E. coli WP2 uvrA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

0*

16

21

137

134

10

9

8

8

12

17

100

12

18

146

122

11

14

5

6

15

16

333

11

16

141

154

9

10

7

7

15

15

1000

13

17

141

161

9

8

6

6

16

15

3333

12

11

134

159

12

7

6

6

16

16

5000

12

15

135

147

9

7

7

5

19

17

Positive control

745

416

700

611

420

100

229

78

371

173

* Solvent control with DMSO

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

Concentration

µg/plate

 

Strain    TA 98

Strain    TA 100

Strain    TA 1535

Strain    TA 1537

E. coli WP2 uvrA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

0*

14

16

104

131

13

12

9

9

12

15

100

14

15

112

136

10

13

10

9

15

12

333

13

17

127

143

13

12

9

9

16

13

1000

14

16

108

147

13

11

6

8

13

14

3333

11

14

111

123

11

12

6

9

15

14

5000

12

17

109

153

13

13

5

7

12

14

Positive control

480

400

620

617

294

68

51

68

282

91

* Solvent control with DMSO

Conclusions:
In an in vitro gene mutation study in bacteria, conducted accordig to OECD test guideline 471 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane has been tested using S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA. No increase in the number of revertants was observed in any strain with or without metabolic activation, neither in the preliminary plate incorporation range-finding study nor in the initial and repeat preincubation assays. The substance was tested to limit concentrations and appropriate solvent and positive 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.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-10-08 to 2003-11-18
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)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
350 (toxicity test only), 700, 1400, 2801 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dried before use)
- Justification for choice of solvent/vehicle: based on informatin from sponsor and compatibility with target cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (with and without activation); 20 hours (without activation)
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid added 2 hours before harvest.
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 200 metaphase spreads were examined and scored for chromatid and chromosome-type aberrations


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell count and percent viability

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Test substances considered positive if it produced a statistically significant, dose-dependent increase relative to control in the percentage of cells with aberrrations, in excess of histroic solvent controls.
Statistics:
Fisher's exact test was used for pairwise comparison of treatment with control. If positive, Cochran-Armitage used to measure dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: in the preliminary toxicity assay, the maximum dose tested was 2801 µg/mL. The test substance was soluble in treatment medium at all dose levels tested. No substantial toxicity (more than or equal to 50% reduction in cell growth relative to the solvent control) was observed up to the highest test substance dose level in any treatment condition. Based on these findings, the dose levels for the chromosome aberration assay ranged from 350 to 2801 µg/mL for all three exposure groups. 

COMPARISON WITH HISTORICAL CONTROL DATA: results were within the range of historical control data

Due to a lack of substantial toxicity, the highest dose level selected for microscopic analysis was the
maximum dose selected for chromosome aberration. The percentage of cells with structural or numerical aberrations
in the test substance groups was not significantly different than the solvent control.


Table 1 Results of chromosome aberrations analysis, 4 hour treatment without activation

Treatment (µg/ml)

S9 Activation

Treatment time

Mean Mitotic Index

Cells With Aberrations

Numerical (%)

Structural (%)

0 (DMSO)

-

4

10.1

2.5

2.5

700

-

4

9.4

2

0

1400

-

4

9.6

2

2

2801

-

4

8.5

1.5

1

Positive control 0.2

-

4

10.7

2.5

10.0**

 

Table 2 Results of chromosome aberrations analysis, 4 hour treatment with activation

Treatment (µg/ml)

S9 Activation

Treatment time

Mean Mitotic Index

Cells With Aberrations

0 (DMSO)

+

4

9.9

3.5

10.0**

700

+

4

10.7

4

1.5

1400

+

4

9.2

3

0

2801

+

4

10.5

5

1.5

Positive control 10

+

4

8.8

3

33.0**

 

Table 3 Results of chromosome aberrations analysis, 20 hour treatment without activation

Treatment (µg/ml)

S9 Activation

Treatment time

Mean Mitotic Index

Cells With Aberrations

0 (DMSO)

-

20

7.8

3

0

700

-

20

7.6

2

0.5

1400

-

20

9

6.5

0.5

2801

-

20

9

6.5

0.5

Positive control 0.1

-

20

9.9

2.5

9.5**

** 

Conclusions:
In an in vitro chromosome aberration study, conducted according to OECD test guideline 473 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane has been tested in a valid study. No increase in the number of cells with aberrations was observed at any concentration with and without metabolic activation when tested in Chinese hamster ovary cells up to limit concentrations. The expected results were obtained with solvent and positive controls. It is concluded that the test substance is negative for the induction of chromosomal 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
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital, beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
187.5-3000 µg/mL with and without activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Untreated negative controls:
no
Remarks:
13.0 - 19.5 µg/ml
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without activation
Untreated negative controls:
no
Remarks:
3.0 – 6.0 µg/ml
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with activation
Details on test system and experimental conditions:
ACTIVATION: phenobarbital, beta-naphthoflavone induced rat liver S9, NADP and glucose-6-phosphate as cofactor. Concentration in final test medium: 5%
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (with and without activation), 24 hours (without activation)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT: selective medium with TFT

NUMBER OF REPLICATIONS: duplicate controls and test concentrations, duplicate microtiter plates for mutant selection and viability determination

NUMBER OF CELLS EVALUATED: 1 x 10E06 cells/flask treated; 4 x 10E03 cells per well of microtiter plates for mutant selection; 2 cells per well for viability assessment.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency is reproducibly dose dependant and exceeds a threshold of 126 colonies per 10E06 cells above the corresponding solvent control. Results rejected if the relative total growth is less than 10% of the vehicle control unless unless the exception criteria specified by the IWGT recommendations are fulfilled.
Statistics:
A linear regression (least squares) comparison of number of mutant colonies from treated and solvent control groups was used to assess dose dependence. Biological relevance was considered alongside statistical significance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1 - Experiment I / 4 h treatment

 

Concentration µg/ml

S9 mix

Relative total growth

Mutant colonies/10x6 cells

Threshold

Relative total growth

Mutant colonies/10x6 cells

Threshold

Column

1

2

3

4

5

6

7

8

 

 

Culture I

Culture II

Solvent control with DMSO

 

-

100.0

170

296

100

206

332

Positive control with MMS

19.5

-

42.6

477

296

70.9

379

332

Test item

93.8

-

culture was not continued#

culture was not continued#

Test item

187.5

-

208.9

167

296

108.6

220

332

Test item

375.0

-

160.4

213

296

104.9

215

332

Test item

750.0

-

101.9

174

296

83.9

267

332

Test item

1500.00

-

112.8

205

296

125.4

188

332

Test item

3000.0

-

173.5

167

296

142.0

155

332

 

 

 

 

 

 

 

 

 

Solvent control with DMSO

 

+

100.0

144

270

100.0

198

324

Positive control with CPA

3.0

+

59.6

374

270

84.0

229

324

Positive control with CPA

4.5

+

45.4

453

270

10.8

739

324

Test item

93.8

+

culture was not continued#

culture was not continued#

Test item

187.5

+

91.1

278

270

124.8

175

324

Test item

375.0

+

94.9

240

270

122.8

170

324

Test item

750.0

+

95.4

245

270

113.8

164

324

Test item

1500.0

+

118.4

212

270

98.4

186

324

Test item

3000.0

+

111.1

221

270

93.6

167

324

 Threshold = number of mutant colonies per 10x6 cells of each solvent plus 126

# culture was not continued since a minimum of four concentrations is required by the guidelines

Table 2 - Experiment II / 24 h treatment

 

Concentration µg/ml

S9 mix

Relative total growth

Mutant colonies/10x6 cells

Threshold

Relative total growth

Mutant colonies/10x6 cells

Threshold

Column

1

2

3

4

5

6

7

8

 

 

              Culture I 

              Culture II

Solent. control with DMSO

 

 -

100.0

169

295

100.0

143

269

Positive control with MMS

13.0

 -

10.1

333

295

6.7

303

269

Test item

93.8

 -

culture was not continued#

culture was not continued#

Test item

187.5

 -

126.7

168

295

83.5

136

269

Test item

375.0

 -

130.5

156

295

66.2

144

269

Test item

750.0

 -

108.1

124

295

80.5

161

269

Test item

1500.0

 -

95.5

165

295

82.2

142

269

Test item

3000.0

 -

95.9

108

295

60.9

136

269

Threshold = number of mutant colonies per 10x6 cells of each solvent plus 126

# culture was not continued since a minimum of four concentrations is required by the guidelines

Table 3 - Experiment II / 4 h treatment

 

Concentration µg/ml

S9 mix

Relative total growth

Mutant colonies/10x6 cells

Threshold

Relative total growth

Mutant colonies/10x6 cells

Threshold

Positive control with CPA

3

 +

45.2

289

268

44.1

197

230

Positive control with CPA

4.5

 +

29.9

326

268

26

308

230

Test item

93.8

 +

culture was not continued#

culture was not continued#

Test item

187.5

 +

98.9

154

268

85.9

130

230

Test item

375.00

 +

86.2

163

268

88.9

141

230

Test item

750.00

 +

72.5

162

268

106.3

109

230

Test item

1500.00

 +

84.3

196

268

69.1

201

230

Test item

3000.00

 +

127.5

168

268

68.5

178

230

Threshold = number of mutant colonies per 10x6 cells of each solvent plus 126

# culture was not continued since a minimum of four concentrations is required by the guidelines

Conclusions:
In an in vitro mammalian mutagenicity study, conducted according to OECD test guideline 476 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane has been tested in a valid study. No increase in the number of mutants was detected either with or without metabolic activation in either of the cultures of mouse lymphoma L5178Y cells at any dose up to limit concentrations. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo testing is not required as the results of all the in vitro studies were negative.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane was tested using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. No increase in the number of revertants was observed in any strain with or without metabolic activation, neither in the preliminary plate incorporation range-finding study nor in the initial and repeat preincubation assays. The substance was tested to limit concentrations and appropriate solvent and positive 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 (Bioreliance, 1999).

In a supporting in vitro gene mutation study in bacteria, conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP, no increase in the number of revertants per plate was observed with or without activation. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Microtest Research, 1988).

In a supporting in vitro gene mutation study in bacteria, conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP, the test substance was negative for mutagenicity to bacteria under the conditions of the test (Hüls, 1993).

In an in vitro cytogenicity study in mammalian cells, conducted according to OECD Test Guideline 473 and in compliance with GLP, no increase in the number of cells with aberrations was observed at any concentration with and without metabolic activation when tested in Chinese hamster ovary cells up to limit concentrations. The expected results were obtained with solvent and positive controls. It is concluded that the test substance is negative for the induction of chromosomal aberrations under the conditions of the test (BioReliance, 2004).

In an in vitro gene mutation study in mammalian cells, conducted according to OECD Test Guideline 476 but not in compliance with GLP, no increase in the number of mutants was detected either with or without metabolic activation in either of the cultures of mouse lymphoma L5178Y cells at any dose up to limit concentrations. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of this test (Harlan Cytotest Cell Research GmbH, 2010).


Justification for classification or non-classification

Based on the available data tris(2-methoxyethoxy)vinylsilane does not require classification for genotoxicity according to Regulation (EC) No 1272/2008.