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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Feb 2015 to 20 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

Method

Target gene:
histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
pre-experiment: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μl/plate
main experiments: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μl/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: aqueous solvent was in appropriate due to the hydrolytic instability of the substance. The solvent was compatible with the survival of the bacteria and the S9 activity
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535; 10 μg/plate; -MA
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
TA 98 (10 μg/plate), TA 1537 (40 μg/plate); -MA
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102; 1 μl/plate; -MA
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
all strains; 2.5 μg/plate (10 μg/plate for TA 102); +MA
Details on test system and experimental conditions:
ACTIVATION:
Phenobarbital and β-naphthoflavone induced rat liver S9
Protein concentration: 38.2 mg/ml adjusted to 25 mg/ml
S9 mix:
- 100 mM sodium-ortho-phosphate-buffer, pH7.4
- 8 mM MgCl2
- 22 mM KCl
- 5 mM glucose-6-phosphate
- 4 mM NADP

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

DURATION
- Preincubation period: 60 min at 37°C
- Selection time (if incubation with a selection agent): at least 48h at 37°C in the dark

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: 3 plates per concentration; the experiment was repeated. The initial assay was conducted using the plate incorporation method, the repeat experiment used pre-incubation.

DETERMINATION OF CYTOTOXICITY
- Method: other: clearing or diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control

OTHER:
Evaluation criteria:
A test item is considered mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is:
- if in strains TA 98, TA 100 & TA 102, the number of reversions is at least twice as high
- if in strains TA 1535 & TA 1537, the number of reversions is at least three times as high
Statistics:
A statistical evaluation is not regarded as necessary.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Plate incorporation - mean number of revertants (3 plates)

Dose (µl/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Negative control

16

25

69

96

9

9

6

6

246

287

Solvent control

13

18

67

79

8

9

5

4

210

284

0.0316

17

22

65

85

13

11

7

7

193

275

0.100

18

19

61

74

11

13

6

6

201

281

0.316

16

22

57

80

11

9

7

4

206

270

1.0

14

26

53

87

10

11

5

3

204

292

2.5

20

27

64

88

9

8

7

4

204

291

5.0

20

26

70

93

14

8

5

6

220

286

Positive control

326

2361

285

2424

846

182

66

211

2714

762

Table 2: Preincubation - mean number of revertants (3 plates)

Dose (µl/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Negative control

24

23

83

106

12

14

5

5

237

383

Solvent control

19

22

65

88

12

13

4

4

162

277

0.0316

17

32

68

100

17

12

4

9

214

369

0.100

20

22

75

87

15

10

8

7

174

337

0.316

15

23

66

95

15

14

4

5

161

329

1.0

19

23

73

95

19

12

4

3

130

319

2.5

23

28

64

90

15

12

3

6

105

307

5.0

18

24

87

94

12

12

2

7

253

386

Positive control

856

868

527

1598

1158

210

69

155

2094

725

Applicant's summary and conclusion

Conclusions:
Tris(isopropenyloxy)(vinyl)silane has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 102 in the initial plate incorporation assay or the repeat experiment using the pre-incubation method, up to limit concentrations. Appropriate positive, solvent and negative (water) controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.