Registration Dossier

Diss Factsheets

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 activation in all strains tested (OECD TG 471)
Cytogenicity in mammalian cells: Negative without activation, positive structural, negative numerical with activation (OECD TG 473)

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 homogenate 
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: the solvent was chosen based on 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:
2-nitrofluorene
Remarks:
TA 98 (without 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, 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:
WP2, WP2uvrA (without activation)
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:
other: Sterigmatocystin
Remarks:
All strains (with activation)
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 to 72 hours

NUMBER OF REPLICATIONS: 2 plates per test concentration

DETERMINATION OF CYTOTOXICITY

- Method: relative total growth; background lawn assessment
Evaluation criteria:
The test article is evaluated as positive when it causes a dose-related increase in mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article.
Statistics:
Positive controls and tester strains with revertant counts greater than 100 were counted with a colony counter (Mini Count) and those less than 100 were counted manually.
Key result
Species / strain:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537; Escherichia coli WP2 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
> 5000 ug/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none recorded
- Effects of osmolality: none recorded
- Evaporation from medium: not reported
- Precipitation: Slight precipitation at higher concentrations did not affect assay
- Other confounding effect: In first two tests, a variation in the precipitation pattern was observed, so the tests were repeated. The findings from the iinitial two experiments are not recorded.

RANGE-FINDING/SCREENING STUDIES:
No toxicity observed

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the controls lie within the range of the historical control data.


ADDITIONAL INFORMATION ON CYTOTOXICITY:

 

Table 1 Preliminary toxicity test

 

TA 100

WP2 uvrA (pKM101)

Concentration (μg/Plate)

Plate 1

+ MA

Plate 2

- MA

Cytotoxic (Yes/No)

Plate 1

+ MA

Plate 2

- MA

Cytotoxic (Yes/No)

0

199

174

no

250

208

no

6.7

180

173

no

234

187

no

10

165

157

no

241

210

no

33

174

163

no

200

198

no

67

179

197

no

228

185

no

100

196

152

no

195

217

no

333

177

170

no

186

184

no

667

175

175

no SP

230

214

no SP

1000

169

156

no SP

138

168

no SP

3333

199

174

no SP

221

169

no SP

5000

174

158

no SP

247

180

no SP

SP Slight precipitate

 

Table 2: Experiment 1 Plate incorporation assayNumber of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

-MA

+ MA

Cytotoxic
(yes/no)

-

 MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

0*

13

17

no

95

144

no

10

11

no

100

13

17

no

89

127

no

9

11

no

333

13

18

no

86

135

no

8

11

no

1000

14

16

no

85

116

no

9

11

no

3333

17

21

no

97

116

no

6

14

no

5000

13

21

no

95

111

no

7

13

no

Positive Control

1304

1050

-

581

997

-

497

124

-

*solvent control with DMSO

 

Table 2: Experiment 1 Plate incorporation assayNumber of revertants per plate (mean of 3 plates)

 

TA1537

WP2 uvrA (pKM101)

WP2 (pKM101)

Conc.
(
µg/plate)

-MA

+ MA

Cytotoxic
(yes/no)

-

 MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

0*

4

6

no

226

263

no

65

64

no

100

4

10

no

236

295

no

54

64

no

333

3

5

no

239

258

no

87

60

no

1000

6

8

no

210

294

no

62

53

no

3333

5

8

no

213

273

no

67

64

no

5000

5

6

no

197

247

no

67

61

no

Positive Control

215

130

-

1554

1262

-

1476

499

-

 

 

*solvent control withDMSO

 

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

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

 MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

15

24

no

102

129

no

11

10

no

50

26

24

no

112

122

no

10

11

no

160

21

20

no

105

116

no

11

9

no

500

24

14

no

101

120

no

12

10

no

1600

23

17

no

110

128

no

9

12

no

5000

19

18

no

100

123

no

10

11

no

Positive Control

764

909

-

527

902

-

433

117

-

*solvent control with DMSO

 

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

 

TA1537

WP2 uvrA (pKM101)

WP2 (pKM101)

Conc.
(
µg/plate)

-MA

+ MA

Cytotoxic
(yes/no)

-

 MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

0*

5

7

no

204

334

no

23

21

no

100

5

5

no

274

278

no

25

21

no

333

5

5

no

173

316

no

26

25

no

1000

5

6

no

208

296

no

26

22

no

3333

5

4

no

206

285

no

31

28

no

5000

5

6

no

214

283

no

28

24

no

Positive Control

250

119

-

2225

1304

-

358

1222

-

 

*solvent control withDMSO

 

Conclusions:
Trimethoxysilane has been tested according to OECD 471 and in compliance with GLP. No increase in the number of revertants was observed with or without metabolic activation in either the initial or the repeat assay using Salmonella typhimurium strains and E. coli WP2 uvrA, using the preincubation method. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of this study.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-08-16 to 2006-09-17
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:
other: CHO cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
152.5 to 1220 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: solubility of the test article 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: 16 - 24 hours

- Exposure duration: 4 - 20 hours (-MA), 4 hours (+MA)

NUMBER OF REPLICATIONS: 2 flasks per concentration

DETERMINATION OF CYTOTOXICITY

- Method: Cell growth inhibition relative to the solvent control

Evaluation criteria:
Toxicity based on cell growth inhibition relative to solvent control.

The number and types of aberrations found, % aberrant cells in the total population of cells examined, and mean aberrations per cell were calculated and reported for each treatment group.

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).
Statistics:
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 Fisher's Exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
other: positive structural, negative numerical
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1220 µg/ml (4-hour group)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1220 µg/ml (4-hour group)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Preliminary toxicity test using Trimethoxysilane in CHO cells with and without S9 metabolic activation (4 hour treatment / 16 hour recovery) 

 

 

- MA

+ MA

- MA

 

4 hour treatment / 16 hour recovery

20 hour continuous treatment)

Treatment

μg/ml

Cell Viability (%)

Cell Growth Index (%) *

Cell Growth Inhibition (%) **

Cell Viability

(%)

Cell Growth Index (%) *

Cell Growth Inhibition (%) **

Cell Viability (%)

Cell Growth Index (%) *

Cell Growth Inhibition (%) **

DMSO

98

100

-

98

100

-

100

100

-

0.122

99

95

5

100

89

11

99

86

14

0.366

99

79

21

100

96

4

99

92

8

1.22

100

82

18

97

97

3

100

85

15

3.66

97

76

24

99

80

20

97

87

13

12.2

99

78

22

97

81

19

95

86

14

36.6

98

67

33

98

86

14

96

99

1

122

98

63

37

100

98

2

97

94

6

366

99

68

32

99

78

22

98

87

13

1220

93

41

59

95

42

58

91

67

33

* Cell Growth Index = (cells per flask treated group/cells per flask control group), expressed as a percentage

** Cell Growth Inhibition = 100 % - % cell growth index; not calculated for negative controls 

Table 2: Cytogenetic analysis of CHO cells in the absence of metabolic activation (4 hour treatment, 16 hour recovery period)

 

 

Control*

152.5 μg/ml

305 μg/ml

610 μg/ml

Positive control

Flask

A

B

A

B

A

B

A

B

A

B

Cytotoxicity

no

no

no

no

no

no

yes

yes

no

no

Chromatid aberrations***

Breaks

0

0

0

0

0

0

1

0

10

7

Interchanges

0

0

0

0

0

0

0

0

0

1

Chromosome aberrations***

Gaps**

0

0

1

0

0

0

0

0

1

0

Breaks

0

0

0

0

0

0

0

0

0

0

Dicentric

0

0

0

0

0

0

0

0

0

0

Rings

0

0

0

0

0

0

0

0

0

0

% aberrant cells

Numerical

2

3

5

5

4

5

3

3

1

2

Structural

0

0

0

0

0

0

1

0

28

24

Mitotic index

 

11.0

11.4

10.8

10.4

10.0

10.4

9.6

8.8

7.4

6.8

* Solvent control with DMSO

** Total gaps

*** Total number of aberrations

Mitotic Index = number of mitotic figures x 100/500 cells counted

% Aberrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps

 

Table 3: Cytogenetic analysis of CHO cells in the presence of metabolic activation (4 hour treatment, 16 hour recovery period)

 

 

Control*

152.5 μg/ml

305 μg/ml

1220 μg/ml

Positive control

Flask

A

B

A

B

A

B

A

B

A

B

Cytotoxicity

no

no

no

no

no

no

yes

yes

yes

yes

Chromatid aberrations***

Breaks

0

0

0

1

0

2

1

8

2

7

Interchanges

0

0

1

1

3

1

7

4

8

6

Chromosome aberrations***

Gaps**

1

0

1

0

2

0

2

3

0

1

Breaks

0

0

0

0

0

0

0

0

0

0

Dicentric

0

1

0

0

0

0

1

1

0

0

Rings

0

0

0

0

0

0

0

0

2

1

% aberrant cells

Numerical

5

4

5

5

5

4

3

4

5

4

Structural

0

1

1

2

3

3

8

9

16

18

Mitotic index

 

11.6

11.6

11.2

11.4

10.6

11.2

5.4

6.4

4.0

3.8

* Solvent control with DMSO

** Total gaps

*** Total number of aberrations

Mitotic Index = number of mitotic figures x 100/500 cells counted

% Aberrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps

 

Table 4: Cytogenetic analysis of CHO cells in the absence of metabolic activation (20 hour continuous treatment)

 

 

Control*

152.5 μg/ml

305 μg/ml

610 μg/ml

Positive control

Flask

A

B

A

B

A

B

A

B

A

B

Cytotoxicity

no

no

no

no

no

no

yes

yes

no

no

Chromatid aberrations***

Breaks

0

0

0

0

0

1

0

0

4

3

Interchanges

0

0

0

0

0

0

0

0

2

3

Chromosome aberrations***

Gaps**

0

0

0

0

0

0

0

1

1

0

Breaks

0

0

0

0

0

0

0

0

0

0

Dicentric

0

0

0

0

0

0

0

0

0

0

Rings

0

0

0

0

0

0

0

0

0

0

% aberrant cells

 

 

Numerical

3

4

4

4

4

3

4

4

4

3

Structural

0

0

0

0

0

1

0

0

24

20

Mitotic index

 

10.4

10.0

10.2

10.4

9.4

10.0

5.2

4.8

6.6

6.0

* Solvent control with DMSO

** Total gaps

*** Total number of aberrations

Mitotic Index = number of mitotic figures x 100/500 cells counted

% Aberrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps

 

 

Conclusions:
In a reliable study, conducted in accordance with OECD 473, in compliance with GLP, trimethoxysilane was concluded to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in CHO cells in the S9-activated test system at the highest dose tested.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981-03-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The restrictions were that only four dose concentrations were tested with single replicates and the range of strains does not comply with current guidelines. The study was considered to be reliability 2. Read-across is considered to be scientifically valid and also reliability 2.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 doses no duplicates; the range of strains does not comply with current guidelines.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0.5, 5, 100 and 500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: none given
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without activation strain TA 1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without activation strains TA 98 and TA 1538
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
all strains with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without activation TA 1535 and TA 100
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- 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


NUMBER OF REPLICATIONS: One


DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in number of revertants (reviewers assessment)
Evaluation criteria:
A reproducible dose-responsive increase in the number of revertants over 3 concentrations to at least twice the solvent control (TA 1535, 1537, 1538) or an increase over 3 concentrations with the highest increase twice the solvent control is considered posiitve.
Statistics:
The number of colonies was counted using a Model C111 Automated colony counter. Each count is listed as the average of 10 replicate counts on each plate.
Species / strain:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: hydrolysis of test compound may have occurred. This is not considered by the reviewer to affect the relevance of the result.


RANGE-FINDING/SCREENING STUDIES: No information


COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data not presented in study report


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

TA98

TA100

TA1535

Conc.
(
µg/plate)

-

MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

0*

16

30

no

325

287

no

14

14

no

0.5

15

26

no

281

262

no

17

17

no

5

14

32

no

226

291

no

14

16

no

100

11

18

no

245

268

no

10

20

no

500

11

28

no

281

233

no

5

14

yes

Positive control

>1000

>1000

no data

>1000

>1000

no data

1000

62

no data

*solvent control withethanol

 

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

TA1537

TA1538

Conc.
(
µg/plate)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

11

6

no

4

24

no

0.5

8

4

no

3

24

no

5

12

7

no

1

23

no

100

12

5

no

2

10

yes

500

2

7

yes

4

7

yes

Positive control

44

22

no data

>1000

316

no data

*solvent control with ethanol

Conclusions:
Trichlorosilane was tested according to a protocol that is similar to OECD 471 and in compliance with GLP. No test substance related increase in the number of reversions was observed when tested to limit concentration in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA 1538, with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. The substance is considered to be non-mutagenic under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus assay inhalation study in rat: Negative (similar to OECD TG 474)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1982-04-21 to 1982-05-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The deviations are that only 1000 cells were scored compared with 2000 in current guideline. The study was therefore considered to be reliability 2. Read-across is considered to be scientifically valid and also reliability 2.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1000 cells scored
Principles of method if other than guideline:
Micronucleus test in vivo: Matter and Schmid, 1971, Mut. Res. 12: 417-425.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Spartan Research Laboratories, Inc. Harlet, MI

- Weight at study initiation: 100 to 175 grams

- Housing: Animals housed individually

- Diet (e.g. ad libitum): PURINA Rodent Laboratory Chow (ad libitum)

- Water (e.g. ad libitum): ad libitum
Route of administration:
inhalation
Vehicle:
- Vehicle(s)/solvent(s) used: air

- Concentration of test material in vehicle: 100 ppm
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: specially constructed glass chamber

- System of generating particulates/aerosols: vapours were generated by bubbling clean, dry air through the liquid test material
Duration of treatment / exposure:
4 hour(s)
Frequency of treatment:
Single 4 hour exposure
Post exposure period:
30 hours
Dose / conc.:
100 ppm (nominal)
No. of animals per sex per dose:
5 animals per dose level
Control animals:
yes, concurrent no treatment
Positive control(s):
triethylenemelamine

- Route of administration: split-dose intraperitoneal injection (0 and 24 hours)

- Doses / concentrations: 0.5 mg/kg/dose
Tissues and cell types examined:
Animals were exposed to the test article by acute inhalation. They were sacrificed, the bone marrow is extracted and smear preparations made and stained. Polychromatic erythrocytes are scored for micronuclei under the microscope. Both positive and negative (solvent) controls are used in each experiment.
Details of tissue and slide preparation:
At sacrifice the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was aspirated from the bone and transferred to centrifuge tubes containing 5 ml fetal calf serum (one tube for each animal). Following centrifugation to pellet the tissue, the supernatant was drawn off and portions of the pellet was spread on slides and air-dried. The slides were then stained in May-Gruenwald Solution and Giemsa. A thousand polychromatic erythrocytes (PCEs) per animal were scored. The frequency of micronucleated was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field.
Evaluation criteria:
In the normal animal, the normocytes/PCEs is approximately 2. If an agent inhibits the proliferation of erythroblasts the proportion of PCEs is generally reduced. If the agent promotes chromosome breakage or acts as a spindle poison, generally the proportion of red normocytes increases.
Statistics:
Mean normocyte and polychromatic erythrocytes calculated as well as ratio of normocytes to PCEs
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY

- Dose range: 100 ppm

- Clinical signs of toxicity in test animals: To ensure that the Micronucleus Assay was perfomed on animals exposed to a lethal concentration of the test material (Group A), a second group of animals (Group B) was simultaneously exposed to the chemical via inhalation as a positive control for lethality. The data demonstrates that both groups (A&B) was exposed to a lethal concentration of the test material. All five animals in Group B experienced weight loss and died within the 14 day observation period. At autopsy all five animals showed extensive lung damage with hemorrhage and atelectasis. Animals exposed via inhalation to the test material all showed slight to moderate evidence of lung damage in the form of petechial hemorrhage and focal altelectasis. Some evidence of kidney congestion was noted in several animals. No abnormal pathology was evident in either the positive control (C) or negative control (D) groups.

- Evidence of cytotoxicity in tissue analyzed: The ratio of of normocytes to PCE's obtained with both the test material treatment (Group A = 10.64) and the negative control (Group D = 10.28) closely approximate the normal expected ratio and were fairly consistent. The mean normocyte count from the positive control group was elevated (Group C = 19.4) indicating that the animals responded to a known clastogen (chromosome breaking agent). This same trend verified by the increase in percentage of micronucleated PCEs in the positive control group.

- Harvest times: Group A (Treatment group): 30 hours after exposure. Group B (Toxicity Control): 14 days. Group C (Positive Control): 30 hours. Group D (Negative Control): 30 hours.

- High dose with and without activation: 100 ppm

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): Within normal range for all groups

- Ratio of PCE/NCE (for Micronucleus assay): Within normal range for all groups

- Statistical evaluation: Comparison of the study groups by Student T-test using a SAS computer program confirms their is no difference between the mean PCE micronucleus count for study groups A and D (test article and negative control) (<.0001) while the positive control is definitely positive (p>0.5) compared to the former groups.

 The following table indicates that the amimals responded to the positivecontrol substance. Both Normocytes/PCE ** ratio and % of micronucleated PCEs were significantly increased in the positive control group when compared to the test material-treated group.

Table 3 : Mean Results of in vivo micronucleus test with mouse bone marrow

 

Test Group

(A)

Positive Control (C)

Negative Control (D)

Number of cells evaluated

1000

1000

1000

Sampling time (h)

30h

30h

30h

Number of erythro-cytes

Normocytes / Field

10.64

19.4

10.28

PCE / Field

4.36

4.56

4.36

Micronuclei / 1000 PCE

6.8

39.2

5.6

Ratio of erythro­cytes

Normochromatic / polychromatic

2.52

4.38

2.39

% Micronucleated PCE

0.68

3.92

0.56

 

Conclusions:
Under test conditions, trimethoxysilane did not induce chromosome breakage or act as a spindle poison in the rodent micronucleus assay even when animals were exposed to lethal concentrations. There is no evidence from this study that the mechanism of kill at toxic levels of exposure involves genotoxicity. The test substance is non-mutagenic in Sprague-Dawley rats in this mammalian micronucleus assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The read-across substance trimethoxysilane (CAS 2487-90-3) has been tested for bacterial mutagenicity according to OECD 471 (Microbiological Associates, 1995) and no increase in the number of revertants was observed with or without metabolic activation in either the initial or the repeat assay using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA using the preincubation method. It was therefore concluded that trimethoxysilane was negative for mutagenicity to bacteria under the conditions of the study. A second read-across substance trichlorosilane (CAS 10025-78-2) was tested according to a protocol that is similar to OECD 471 and no test substance-related increase in the number of reversions was observed when tested to limit concentrations in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA1538, with and without metabolic activation, the substance was considered to be non-mutagenic under the conditions of the test.

Trimethoxysilane, when tested in a mammalian cytogenicity study according to OECD 473 (BioReliance, 2007), was found to be positive for the induction of structural, but negative for the induction of numerical, chromosome aberrations in CHO cells with metabolic activation (S9-activated) at the highest dose tested. However, the evidence for the clastogenicity of trimethoxysilane was not confirmed when it was testedin an in vivo micronucleus assay according to a protocol similar to OECD 474 (Dow Corning, 1982). Trimethoxysilane did not induce chromosome breakage or act as a spindle poison in the rodent micronucleus assay even when the animals were exposed to lethal concentrations and was therefore considered to be negative for clastogenicity in Sprague-Dawley rats under the conditions of the test.

No data are available for mutagenicity to mammalian cells. However, as the substance tested was positive in the in vitro cytogenicity assay, in vitro testing for mutagenicity to mammalian cells is not required.

Read-across justification

Non-testing methods including read-across from surrogate substances are able to provide information on mutagenic toxicity (REACH Guidance part 07a, R.7.7.3). In the case of genetic toxicity, the presence or absence of functional groups that are known to be related to genetic toxicity is considered important, as the presence or absence of reactive groups and molecular substructures is associated with mutagenic and carcinogenic properties of chemicals (Benigni and Bossa, 2006). Consideration is therefore given to the structural similarity, particularly presence or absence of structural alerts for genetic toxicity, when selecting surrogate substances for genetic toxicity endpoints.

Read-across hypothesis

Trichlorosilane is a close structural analogue of dichlorosilane; trichlorosilane has one hydrogen and three chlorine atoms bound to silicon, dichlorosilane has two hydrogen and two chlorine atoms. Trimethoxysilane is a trialkoxysilane. All three substances hydrolyse in contact with water generating silanediol or silanetriol and then monosilicic acid (which will condense to form amorphous polysilicic acid at concentrations above approximately 100 -150 mg/l). Hydrolysis is likely to occur under the conditions of the studies and also in vivo. The non-silanol products of hydrolysis are methanol and hydrogen chloride, which are not expected to contribute to genetic toxicity, as explained below.

Methanol has been tested in vitro in bacterial and mammalian mutagenicity assays and in micronucleus and chromosome aberration assays. The majority of the results were negative (OECD, 2004). In the ECHA disseminated dossier for methanol, the conclusions of all the key in vitro studies and the weight of evidence of the in vivo assays are negative.

Hydrogen chloride gave negative results in the most reliable of the bacterial mutagenicity studies. Positive results were obtained in mutagenicity and cytogenicity assays using mammalian cells (OECD, 2002; ECHA disseminated dossier for hydrogen chloride). The positive results were associated with decrease in pH, and it is considered that the positive results were likely to have been caused by reduced pH. Positive results caused by high or low pH effects are considered not to be relevant for in vivo situations (ECHA 2012), and testing should be carried out at neutral pH.

Dichlorosilane hydrolyses very rapidly with a hydrolysis half-life <0.3 min at 25°C and pH 4, 7 and 9 (analogue read-across). Trichlorosilane hydrolyses very rapidly, with a hydrolysis half-life < 0.3 minutes at 25°C and pH 4, 7 and 9 (read-across from methyltrichlorosilane) and trimethoxysilane also hydrolyses very rapidly, with a hydrolysis half-life of < 0.3 minutes at pH 4, 7 and 9 and 2°C (measured).

Analogue approach justification

(a) Structural similarity

Trichlorosilane is a close structural analogue of dichlorosilane; trichlorosilane has one hydrogen and three chlorine atoms bound to silicon, dichlorosilane has two hydrogen and two chlorine atoms. Trimethoxysilane is a trialkoxysilane. All three substances hydrolyse in contact with water generating silanetriol and then monosilicic acid (which will condense to form amorphous polysilicic acid at concentrations above approximately 100 -150 mg/l).

(b) Structural alerts for genotoxicity

Dichlorosilane, trichlorosilane and trimethoxysilane do not include structural alerts for genotoxicity.

(c) Lack of genetic toxicity (other than pH effects) of non-silanol products of hydrolysis.

Trimethoxysilane and trichlorosilane were chosen as read-across substances as they form a similar hydrolysis product as that formed by the registered substance, and none of these substances has any functional groups that are associated with genetic toxicity. The genetic toxicity data available for these and other structural analogue substances are summarised in the table below. Amorphous polysilicic acid is among the substances included in the table, as supporting evidence that production of inorganic silica does not increase the potential for genotoxicity of the registered substance. Additional information is given in a supporting report (PFA, 2013aa) attached in Section 13 of the IUCLID dossier.

Name

Bacterial Mutagenicity

In Vitro Mammalian Cytogenicity

In Vitro Mammalian Mutagenicity

In Vivo Genotox

10025-78-2

Trichlorosilane

Negative

(Dow Corning Corporation, 1981)

-

-

-

2487-90-3

Trimethoxysilane

Negative

(Microbiological Associates, 1995)

Positive (+MA)

(BioReliance, 2007)

 

Negative in micronucleus

(Dow Corning Corporation, 1982)

998-30-1

Triethoxysilane

 

 

Negative

(Dow Corning Corporation, 1995)

 

681-84-5

Tetramethyl orthosilicate

[tetramethoxysilane]

Negative

(Safepharm Laboratories, 2001)

-

-

Negative in micronucleus

(Dow Corning Corporation, 1987)

78-10-4

Tetraethyl orthosilicate

[tetraethoxysilane]

Negative

(Hüls AG, 1993)

Negative

(BioReliance, 2002)

Negative

(Bushy Run Research Centre, 1981)

-

682-01-9

Tetrapropyl orthosilicate

[tetrapropoxysilane]

Negative

(LPT, 2002)

-

-

-

 N/a

 Synthetic amorphous silica (SAS) [polysilicic acid]

 Negative (OECD, 2004)

 NegativeOECD (2004)

 Negative

(OECD, 2004)

Negative in chromosome aberration assay and rodent dominant lethal assay (OECD, 2004) 

References

Benigni and Bossa (2006). Current Computer-Aided Drug Design 2, (2), 169-176.

BioReliance (2002).In VitroMammalian Chromosome Aberration Test. Test article Tetraethyl orthosilicate CAS 87-10-4. Testing laboratory: BioReliance 9630 Medical Center.

BioReliance (2007). In Vitro Mammalian Chromosome Aberration test with Trimethoxysilane (CAS Number 2487-90-3). Testing laboratory: BioReliance 9630 Medical Center Drive, Rockville, Maryland 20850 USA. Owner company: SEHSC. Study number: AB34WR.331. BTL. Report date: 2007-01-17.

Bushy Run Research Centre (1981). Tetraethyl orthosilicate:in vitromutagenesis studies. Testing laboratory: Bushy Run Research Centre 4 Mellon Road Export Pennsylvania 15632 USA. Report no.: 44-68. Owner company: Momentive. Report date: 1981-06-28.

Dow Corning Corporation (1981). Mutagenicity Evaluation of Dow Corning Z-1210 Silane in the Ames bacterial assay. Testing laboratory: Dow Corning Toxicology Department. Report no.: I-0005-0847. Owner company: Dow Corning Corporation. Report date: 1981-04-06.

Dow Corning Corporation, (1982). Evaluation of Trimethoxysilane via acute vapour inhalation in the rodent micronucleus assay. Testing laboratory: Dow Corning toxicology department. Owner company: Dow Corning Corporation. Company study No.: 82-0241-FGM. Report date: 1982-06-29.

Dow Corning (1987). Acute Inhalation Toxicity. Testing laboratory: Dow Corning Corporation, Midland, MI. Report no.: Internal Report No. 1987-I0005-1665.

Dow Corning Corporation (1995). L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay. Testing laboratory: Dow Corning Corporation Health and Environmental Sciences, Dow Corning Corporation, 2200 West Salzburg Road, Auburn, MI 48611. Report no.: 1995-I0000-39993. Owner company: Dow Corning Corporation. Study number: 151-1193. Report date: 1995-01-05.

Hüls AG (1993a). Determination of mutations caused by DYNASIL A in Salmonella/microsome test based on Ames mutation test under Guideline 84/449/EEC B. 14. Certified translation from German. Testing laboratory: Hüls AG, Testing institute for Biology, building 9015 Postfach 13-20 D-4370 Marl Germany. Report no.: 93-0195-DGM, final report AM-93-3. Report date: 1993-03-26.

LPT(2002). Mutagenicity study of silan TPN in the Salmonella typhimurium reverse mutation assay (in vitro). Testing laboratory: LPT. Report no.: 15425/19/02. Owner company: Wacker. Report date: 2002-09-24.

Microbiological Associates (1995). Y-4398: Salmonella/Escherichia coli Preincubation Mutagenicity Assay with a Confirmatory Assay. Testing laboratory: Microbiological Associates, Inc. Rockwell MD 20850 USA. Owner company: Momentive. Study number: G95AD27.503001. Report date: 1995-04-17.

OECD (2002): SIDS Initial Assessment Report for SIAM 15, Boston, USA, 22-25 October 2002: hydrogen chloride, CAS 7647-01 -0.

OECD SIDS (2004). Soluble Silicates. CAS No. 1344-09-8, 6834-92-0, 10213-79-3, 13517-24-3 and 1312-76-1. SIDS Initial Assessment Report for SIAM 18 Paris, France 20-23 April, 2004.

OECD (2004a): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 18-20 October 2004, Methanol, CAS 67-56-1.

Safepharm Laboratories (2001). KBM-04: Reverse mutation assay 'Ames Test' using Salmonella typhimurium and Escherichia coli. Testing laboratory: Safepharm Laboratories Ltd, PO Box No.45, Derby, DE1 2BT, UK. Owner company: Shin Etsu Chemical Co Ltd. Study number: 763/189. Report date: 2001-03-26.



Justification for classification or non-classification

Based on the available in vitro and in vivo information from the read-across substances trimethoxysilane (CAS number 2487-90-3) and trichlorosilane (CAS 10025-78-2), the registered substance, dichlorosilane does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.