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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:
weight of evidence
Study period:
From 2015-07-28 to 2015-09-08
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
Guideline:
other: Japan MHLW Testing Methods for New Substance
Version / remarks:
March 29 2011, Revised April 2 2012
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
Duplicate cultures were used for the test material, whereas triplicate plates are specified by OECD 471. This deviation does not affect the integrity of the study given the clear negative responses seen in three separate assays.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetravinylsilane
EC Number:
214-192-0
EC Name:
Tetravinylsilane
Cas Number:
1112-55-6
Molecular formula:
C8H12Si
IUPAC Name:
tetraethenylsilane
Test material form:
liquid
Specific details on test material used for the study:
Lot No.: 150715
Purity: 99.1%

Method

Target gene:
Reversion to histidine / tryptophan independence
Species / strain
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:
Phenobarbital / 5,6-benzoflavone-induced male SD rat liver S9 fraction
Composition of S9 mix:
S9 mix was prepared just before use. One milliliter of S9 mix consisted of 8 μmol MgCl2, 33 μmol KCl, 5 μmol Glucose-6-phophate, 4 μmol NADPH, 4 μmol NADH, 100 μmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
Test concentrations with justification for top dose:
Test concentrations were based on the results of a preliminary cytotoxicity assay performed at concentrations of up to 5000 µg/plate and were limited by toxicity. There was no evidence of precipitation.
In the main assay, the highest concentrations (-S9) were 19.5 µg/plate (TA100, TA1535, TA98, TA1537) and 313 µg/plate (WP2uvrA); (+S9) 78.1 µg/plate (TA1535) and 313 µg/plate (TA100, WP2uvrA, TA98, TA1537). Cytotoxicity (reduced numbers of revertant colonies and/or reduced background lawn) was seen at the highest concentrations used for each strain in the absence and presence of cytotoxicity.
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: AF-2 (TA100, WP2uvrA, TA98; -S9); ICR-191 (TA1537, -S9). 2-aminoanthracene (all strains; +S9)
Remarks:
Activity of the S9 batch had also been demonstrated using 2-AA, B(a)P and dimethylanthracene
Details on test system and experimental conditions:
The study was performed using the pre-incubation method using duplicate plates (triplicate plates were used for the solvent controls). Cultures were exposed to the test material or positive controls with or without S9 mix for 20 minutes at 37°C with shaking. Following the addition of soft agar, revertant colonies were counted after incubation for 48 hours.

- Confirmation of Sterility:
The highest concentration of the test item solution (0.05 mL) and S9 mix (0.5 mL) were individually mixed with 2 mL of the soft agar and were poured onto each minimal glucose agar plate in order to examine bacterial contamination. Bacterial contamination was judged with those plates after incubation at 37±0.5°C for 48 hours.

- Colony counting:
For the plates at which the bacterial growth inhibition was observed, the number of colonies was counted manually, and for the other plates with a colony analyzer (CA-11D, SYSTEM SCIENCE). Square correction and miscounting correction were conducted when counting with the colony analyzer.
Rationale for test conditions:
A preliminary range-finding assay was performed using concentrations of up to 5000 µg/plate in the absence and presence of S9. A second range-finding assay using lower concentrations was performed due to the level of toxicity seen in the initial assay. There was no evidence of precipitation at any concentration
Evaluation criteria:
A positive response was concluded where the number of revertant colonies in any strain exceeded twice that seen in the relevant control, and where the response was concentration-related and/or reproducible.
Statistics:
Not required.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There was no evidence of precipitation at any concentration of the test material.
The bacterial growth inhibition was observed at 19.5 μg/plate for TA100, TA98 and TA1537 without S9 mix, at 9.77 μg/plate or more for TA1535 without S9 mix, at 156 μg/plate or more for WP2urA without S9 mix. The bacterial growth inhibition was observed at 156 μg/plate or more for TA100, TA98 and TA1537 with S9 mix, at 78.1 μg/plate for TA1535 with S9 mix, at 313 μg/plate for WP2uvrA with S9 mix.
Exposure to the test material did not induce any increase in the number of revertant colonies at any concentration either in the absence or presence of metabolic activation.

Any other information on results incl. tables

Results of Range-finding Study 1

µg/plate

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

107

98

12

10

30

29

24

31

13

15

4.88

110

108

11

6

33

40

21

35

13

18

19.5

88*

108

6*

9

40

35

18*

37

9*

16

78.1

91*

88

9*

8*

35

25

14*

22

8*

12

313

100*

80*

5*

9*

33*

19*

16*

16*

10*

13*

1250

88*

87*

4*

4*

31*

26*

11*

20*

8*

12*

5000

0*

29*

0*

2*

26*

21*

0*

17*

3*

2*

AF-2

934

 

 

 

143

 

360

 

 

 

NaN3

 

 

267

 

 

 

 

 

 

 

ICR-191

 

 

 

 

 

 

 

 

532

 

2AA

 

1159

 

273

 

519

 

276

 

147

Mean numbers of revertant colonies

*growth inhibition observed

Results of Range-finding Study 2

µg/plate

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

118

100

11

13

28

21

23

26

9

10

0.610

129

 

11

 

 

 

19

 

9

 

1.22

110

 

7

 

 

 

22

 

10

 

2.44

107

 

10

14

 

 

23

 

9

 

4.88

103

 

12

7

 

 

26

 

14

 

9.77

84

112

12*

9

36

29

17

24

9

11

19.5

76*

114

6*

14

43

30

17*

29

12*

7

39.1

 

103

 

12

22

27

 

35

 

9

78.1

 

89*

 

5*

27

28

 

25

 

13

156

 

86*

 

 

24*

35

 

19*

 

11

313

 

78*

 

 

23*

26*

 

18*

 

10

AF-2

1358

 

 

 

145

 

377

 

 

 

NaN3

 

 

295

 

 

 

 

 

 

 

ICR-191

 

 

 

 

 

 

 

 

639

 

2AA

 

1023

 

294

 

396

 

318

 

176

Mean numbers of revertant colonies

*growth inhibition observed

 

Results of Main Study

µg/plate

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

98

109

11

9

34

36

20

24

14

12

0.610

112

 

6

 

 

 

27

 

15

 

1.22

109

 

11

 

 

 

18

 

12

 

2.44

94

 

9

12

 

 

23

 

10

 

4.88

106

 

13

8

 

 

21

 

7

 

9.77

106

97

9*

8

41

23

16

20

11

17

19.5

96*

103

9*

7

128

34

16*

17

7

14

39.1

 

104

 

8

25

46

 

27

 

9

78.1

 

114

 

8*

43

36

 

25

 

9

156

 

71*

 

 

23*

42

 

23*

 

12*

313

 

92*

 

 

26*

35*

 

23*

 

10*

AF-2

1113

 

 

 

173

 

461

 

 

 

NaN3

 

 

337

 

 

 

 

 

 

 

ICR-191

 

 

 

 

 

 

 

 

527

 

2AA

 

1075

 

325

 

397

 

329

 

184

Mean numbers of revertant colonies

*growth inhibition observed

Applicant's summary and conclusion

Conclusions:
No evidence of mutagenicity was seen under the conditions of this study.
Executive summary:

The mutagenicity of the test material was investigated in an Ames test (pre-incubation method) similar to OECD 471. Duplicate cultures of strains S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA (triplicate cultures for the negative control) were exposed to concentrations of the test material (dissolved in acetone), negative (solvent) or positive controls in the absence or presence of an exogenous metabolic activation system. Test concentrations were based on the results of a preliminary cytotoxicity assay performed at concentrations of up to 5000 µg/plate and were limited by toxicity. There was no evidence of precipitation. In the main assay, the highest concentrations (-S9) were 19.5 µg/plate (TA100, TA1535, TA98, TA1537) and 313 µg/plate (WP2uvrA); (+S9) 78.1 µg/plate (TA1535) and 313 µg/plate (TA100, WP2uvrA, TA98, TA1537). Cytotoxicity (reduced numbers of revertant colonies and/or reduced background lawn) was seen at the highest concentrations used for each strain in the absence and presence of cytotoxicity. Exposure to the test material did not induce any biologically relevant increase in the number of revertant colonies of any strain at any concentration either in the absence or presence of metabolic activation. The laboratory's criteria for a positive response was not seen for any strain. Appropriate positive control substances confirmed the sensitivity of the assay and the metabolic activation system. It was concluded that the test item was not mutagenic under the conditions of this Ames test.

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