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Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538, E. coli W3110/polA+, E. coli P3478/polA- and Saccharomyces cerevisiae: negative with and without metabolic activation (OECD 471)
Mammalian cytogenicity (Chromosome Aberration): negative with and without metabolic activation (OECD 473)
Mammalian mutagenicity (mouse lymphoma assay): negative with and without metabolic activation (OECD 476)

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977 - 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was conducted according to a Litton Bionetics Inc. method but full details are not available
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no analytical purity stated; only single cultures tested, tester strains TA 102 or E. coli WP2 were not used in the study as required in the appropriate OECD test guideline
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli, other: W3110/polA+
Species / strain / cell type:
E. coli, other: P3478/polA-
Species / strain / cell type:
yeast, other: Saccharomyces cerevisiae (D4)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9-mix
Test concentrations with justification for top dose:
Main experiment:
- 0.001, 0.01, 0.1, 1, 5 µL/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9-mix: MNNG: 10 µg/plate (TA 1535, TA 100, D4), QM: 10 µg/plate (TA 1537), NF: 100 µg/plate (TA 1538, TA 98); +S9-mix: ANTH: 100 µg/plate (TA 1535, TA 100), AMQ: 100 µg/plate (TA 1537), AAF: 100 µg/plate (TA 1538, TA 98), DMNA: 100 µM/plate (D4)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 h (Ames test), 24 h (DNA repair test)

NUMBER OF REPLICATIONS: single culture/test concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 100, TA 1535, TA 1537 and TA 1538, and 2-3 times the solvent control for TA 98 and D4.
Species / strain:
S. typhimurium, other: TA 1535, TA1537, TA98, and TA100
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
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥1 µl/plate -S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
yeast, other: Saccharomyces cerevisiae (D4)
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
Species / strain:
E. coli, other: W3110/po1A+
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
Species / strain:
E. coli, other: P3478/po1A-
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:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Marked cytotoxicity was observed in tester strain TA 1538 in the absence of metabolic activation at concentration ≥1 µl/plate. No relevant cytotoxicity was observed for the other tester strains at any concentration either in the presence or in the absence of metabolic activation.

Table 1: Experiment 1 Plate incorporation Number of revertants per plate

 

TA98

TA100

TA1535

Conc.
µg/plate

MA

+

 MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

 MA

+ MA

Cytotoxic
(yes/no)

0*

36

44

No

92

134

No

28

14

No

0.001

30

55

No

76

117

No

17

18

No

0.01

26

52

No

88

155

No

16

21

No

0.1

25

43

No

81

142

No

15

15

No

1

34

53

No

84

126

No

16

13

No

5

26

58

No

75

125

No

12

12

No

Positive control

>1000

>1000

No

610

811

No

>1000

299

No

*solvent control with DMSO

Table 2: Experiment 1 Plate incorporation Number of revertants per plate

 

TA 1537

TA 1538

D4

Conc.
µg/plate

MA

+ MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

 MA

+ MA

Cytotoxic
(yes/no)

0*

14

15

No

28

31

No

44

154

No

0.001

30

14

No

15

31

No

48

222

No

0.01

19

26

No

26

31

No

42

64

No

0.1

22

19

No

23

29

No

46

202

No

1

14

18

No

14

30

No

43

234

No

5

10

13

No

15

35

No

50

228

No

Positive control

>1000

392

No

701

>1000

No

746

242

No

*solvent control with DMSO

Conclusions:
No mutagenic effect was observed for the test substance tested up to cytotoxic concentration in any of the test strains in two independent experiments without and with metabolic activation. The test substance is non-mutagenic in test strains used.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977 - 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
only 50 cells scored - guideline requires 200. no duplicates. Number cells with 2 or more aberrations presented (rather than % with aberrations
GLP compliance:
no
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)
Target gene:
Not applicable
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
mouse liver S9-mix
Test concentrations with justification for top dose:
Main experiment:
- 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.4, 3.2, 4.8 µLl/mL(without metabolic activation)
- 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.4, 3.2 µL/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: It is assumed by the reviewer that solvent was chosen based on solubility properties and relative non toxicity to mouse lymphoma cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 24 h; 24 h treatment: 24 h

STAIN: Giesma

NUMBER OF REPLICATIONS: single cultures/test concentration

NUMBER OF CELLS EVALUATED: 50 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


Statistics:
The average numbers of chromosome aberrations per cell was calculated and compared with values for the negative control compound to determine significance (Students t-test (p<0.05 and p<0.01)).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4.8 µL/mL -S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cytotoxicity was only recorded at a concentration of 4.8 µL/plate in the absence of metabolic activation.

Table 1: Summary of chromosome aberrations

Test item

Concentration in µl/ml

Mitotix index (%)

Aberrant cells (%)

Exposure period 4 h, fixation time 24 h, without S9-mix

Negative control

-

8.8

3.3

Solvent control

-

8.5

1.7

Positive control

0.5

5.8

14.3

 

 

 

 Test substance

0.1

8.3

0.0

0.2

11.3

4.0

0.4

10.2

2.0

0.8

4.2

0.0

1.2

10.6

4.0

1.6

9.6

6.0

2.4

6.6

0.0

3.2

7.2

2.0

4.8

-

-

Exposure period 4 h, fixation time 24 h, with S9-mix

Negative control

-

8.9

2.8

Solvent control

-

8.0

1.1

Positive control

0.3

5.4

14.8

 

 

Test substance

0.1

9.6

0.0

0.2

10.8

0.0

0.4

11.4

0.0

0.8

9.7

0.0

1.2

6.0

2.0

1.6

10.0

4.0

2.4

8.3

6.0

3.2

8.1

2.0

Conclusions:
Under test conditions, there was some evidence for increased levels of chromosome aberrations, but the levels of increase were minimal. No properly stained spreads were used for the tests. The test substance is considered non clastogenic in mouse lymphoma cells.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977 - 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
no
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
mouse liver S9-mix
Test concentrations with justification for top dose:
Experiment I:
- 0.1, 0.2, 0.4, 0.8, 1.6 µL/mL (without metabolic activation)
- 0.2, 0.4, 0.8, 1.6, 3.2 µL/mL (with metabolic activation)

Experiment II:
- 1.6, 2.4, 3.2, 4.8 µL/mL (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 3 days
- Selection time: 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 days

NUMBER OF REPLICATIONS: two independent experiments with single cultures/test concentration each

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
A compound is considered mutagenic in the mouse lymphoma assay if a dose response relationship is observed over three of the four dose levels employed; the minimum increase at the high level of the dose response curve is at least 2.5 times greater than the solvent control value; the solvent control data are within the normal range of the spontaneous background of the TK locus.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
0.4 and 3.2 µL/mL with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Evident toxicity was only recorded at concentrations of 0.4 and 3.2 µg/mL in the presence of metabolic activation.

Table 1: Summary of mutagenicity assay (experiment I)

Test item

Dose level (µl/ml)

Relative suspension growth (% of control)

Total mutant clones

Total viable clones

Relative cloning efficiency

(% of control)

Percent relative growth*

Mutant frequency

(x 10^-6)**

Exposure period 4 h, without S9-mix

Solvent control

-

100.0

9.0

216.0

100.0

100.0

4.2

Negative control

-

120.3

13.0

208.0

96.3

115.8

6.3

Positive control

5

44.1

276.0

82.0

38.0

16.8

336.6

 

 

Test substance

0.1

39.2

30.0

182.0

84.3

33.1

16.5

0.2

46.2

23.0

174.0

80.6

37.2

13.2

0.4

60.3

18.0

175.0

81.0

48.9

10.3

0.8

62.9

20.0

229.0

106.0

66.7

8.7

1.6

39.0

36.0

182.0

84.3

33.5

19.8

Exposure period 4 h, with S9-mix

Solvent control

-

100.0

24.5

252.0

100.0

100.0

9.7

Negative control

-

112.0

24.0

151.0

59.9

67.1

15.9

Positive control

5

25.6

117.0

39.0

15.5

4.0

300.0

 

 

Test substance

0.2

81.3

33.0

200.0

79.4

64.5

16.5

0.4

135.7

30.0

133.0

52.8

71.6

22.6

0.8

92.2

41.0

172.0

68.3

62.9

23.8

1.6

76.7

44.0

152.0

60.3

46.2

28.9

3.2

15.9

27.0

188.0

74.6

11.8

14.4

* = Relative (Suspension Growth x Relative Cloning Efficiency) / 100

** = (Mutant Clones / Viable Clones) x 10^-4

Table 2: Summary of mutagenicity assay (experiment II)

Test item

Dose level (µl/ml)

Relative suspension growth (% of control)

Total mutant clones

Total viable clones

Relative cloning efficiency

(% of control)

Percent relative growth

Mutant frequency

(x 10^6)

Exposure period 4 h, without S9-mix

Solvent control

-

100.0

48.0

370.0

100.0

100.0

13.0

Negative control

-

136.2

34.0

325.0

87.8

119.7

10.5

Positive control

5

50.2

778.0

133.0

35.9

18.0

585.0

 

Test substance

1.6

106.2

52.0

250.0

67.6

71.8

20.8

2.4

83.8

18.0

308.0

83.2

69.7

5.8

3.2

59.5

57.0

303.0

81.9

48.7

18.8

4.8

24.9

50.0

270.0

73.0

18.2

18.5

Exposure period 4 h, with S9-mix

Solvent control

-

100.0

80.0

384.0

100.0

100.0

20.0

Negative control

-

205.1

45.0

299.0

77.9

159.7

15.1

Positive control

5

71.1

376.0

132.0

34.4

24.4

284.8

 

Test substance

1.6

128.0

60.0

309.0

80.5

103.0

19.4

2.4

104.1

68.0

375.0

97.7

101.6

18.1

3.2

159.5

55.0

208.0

54.2

86.4

26.4

4.8

35.3

72.0

267.0

69.5

24.5

27.0

* = Relative (Suspension Growth x Relative Cloning Efficiency) / 100

** = (Mutant Clones / Viable Clones) x 10^-4

Conclusions:
The substance was tested in an in vitro mutagenicity assay in mouse lymphoma L5178Y cells. The test substance is non-mutagenic in mouse lymphoma 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

Genetic toxicity (mutagenicity) in bacteria in vitro

A reliable bacterial gene mutation study (Ames test) performed equivalent or similar to OECD TG 471 with Diethoxy(dimethyl)silane (CAS 78-62-6) is available (SEHSC, 1978). The strains Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100, E. coli W3110/polA+, E. coli P3478/polA- and Saccharomyces cerevisiae were tested according to the plate incorporation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted with single cultures at concentrations from 0.001, 0.01, 0.1, 1, and 5 µL/plate. Marked cytotoxicity was observed in tester strain TA 1538 in the absence of metabolic activation at concentration ≥1 µL/plate. No relevant cytotoxicity was observed for the other tester strains at any concentration either in the presence or in the absence of metabolic activation. Appropriate solvent (DMSO) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and Saccharomyces cerevisiae with and without metabolic activation. In addition, no zones of inhibition were recorded for the tester trains E. coli W3110/polA+ and E. coli P3478/polA- in the presence or absence of metabolic activation in the DNA repair test. Taken together and based on the results of the study, the test material was considered to be non-mutagenic under the conditions of the test.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro chromosome aberration test in mouse lymphoma L5178Y cells was performed with Diethoxy(dimethyl)silane (CAS 78-62-6) equivalent or similar to OECD TG 473 (SCHSC, 1978). Mouse lymphoma cells were treated with Diethoxy(dimethyl)silane or vehicle (ethanol) in the absence or presence of a metabolic activation system (non-induced mice liver S9-mix) at concentrations of 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.4, 3.2, 4.8 µL/mL (without metabolic activation) and 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.4, 3.2 µL/mL (with metabolic activation) for 4 h. After the 4 h exposure period with the test material cells were incubated with BUdR for additional 20 h and fixed and stained with Giemsa and either analysed for chromosome aberrations or sister chromatid exchanges (SCE). Appropriate negative, solvent and positive controls were included in the test and gave the expected results. Cytotoxicity was only recorded at a concentration of 4.8 µL/plate in the absence of metabolic activation. However, only single cultures for each test concentration were performed and only 50 cells per concentration were evaluated which is a deviation of the current OECD TG. Evaluation of sister chromatid exchanges revealed no statistically significant, dose-related increase of SCEs in the absence of metabolic activation. In contrast, a statistically significant, dose-related increase of SCEs was observed in the presence of metabolic activation. Nevertheless, since only single cultures were performed and only 10 cells were evaluated for SCEs for each test concentration, the statistically significant increase of SCEs in the presence of metabolic activation is considered not to be reliable. Taken together and based on the results of the chromosome aberration test the test substance did not cause a statistically significant, dose-related increase in chromosome aberrations and is therefore considered to be non-clastogenic in mouse lymphoma L5178Y cells under the tested conditions.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro Mammalian Cell Gene Mutation Test was performed with Diethoxy(dimethyl)silane (CAS 78-62-6) in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) equivalent or similar to OECD TG 476 (SCHSC, 1978). Mouse lymphoma cells were treated with Diethoxy(dimethyl)silane or vehicle (ethanol) in two independent experiments in the absence or presence of a metabolic activation system (non-induced mice liver S9-mix) at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, 4.8 µL/mL (without metabolic activation, experiment I) and 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, 4.8 µL/mL (with metabolic activation, experiment I) and 1.6, 2.4, 3.2 and 4.8 µL/mL (with and without metabolic activation, experiment II) for 4 h. After the 4 h exposure period with the test material mouse lymphoma cells were cultured in growth medium for 3 days followed by incubation in selective medium for additional 10 days. Fixation of the cells was performed 13 days after start of exposure with the test material. Evident toxicity was only recorded at concentrations of 0.4 and 3.2 µg/mL in the presence of metabolic activation. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5l78Y cell line at the TK locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not cause a statistically significant, dose-related increase in mutant frequency either in the absence or in the presence of metabolic activation. Therefore, the test substance is considered not to be mutagenic in the mouse lymphoma L5178Y test system under the conditions of the test.

 

A further gene mutation test as part of the previously described in vitro Mammalian Cell Gene Mutation Test was performed with Diethoxy(dimethyl)silane (CAS 78-62-6) investigating the primary DNA damage in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) (SCHSC, 1978). Prior to treatment, mouse lymphoma L5178Y cells were exposed to 0.5 µCi/mL 14C-thymidine for at least 20 h to label the cellular DNA followed by further incubation with non-radiolabeled medium for 24 h. Duplicate cultures are exposed to Diethoxy(dimethyl)silane at concentrations of 0.6, 1.2 and 2.4 µL/mL for 4 or 2 h in the absence or presence of metabolic activation, respectively followed by primary DNA damage evaluation via alkaline elution. No marked toxicity was recorded at any test material concentration either in the presence or absence of metabolic activation. Appropriate solvent (ethanol) and positive controls were included and gave the expected results. The test material did not induce a statistically significant, dose-related response in the alkaline elution assay either in the presence or absence of metabolic concentration. Therefore, the test substance is considered not to induce primary DNA damage in the mouse lymphoma L5178Y test system under the conditions of the test.

 

Based on the available data on in vitro genetic toxicity with Diethoxy(dimethyl)silane (CAS 78-62-6) sufficient evidence is available to conclude that the registration is neither mutagenic in bacterial and mammalian cells nor clastogenic in mammalian cells.


Justification for classification or non-classification

The available data on genetic toxicity of Diethoxy(dimethyl)silane do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.