Tetravinylsilane

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Japan MHLW Testing Methods for New Substance

Version / remarks:

March 29 2011, Revised April 2 2012

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.