Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 - 19 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 2008

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

EC Number:

300-346-5

EC Name:

Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

Cas Number:

93925-43-0

Molecular formula:

C4H8O2, C20H44O4SiSn, C24H52O6SiSn, C40H84O8SiSn2, C60H128O12Si2Sn3, C80H172O16Si3Sn4, C100H216O20Si4Sn5

IUPAC Name:

Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

Method

Target gene:

his operon; trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial factor (S9 mix), prepared from the livers of male Sprague Dawley rats treated with Aroclor (500 mg/kg bw).

Test concentrations with justification for top dose:

Dose range finding test (TA100): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (5% (v/v) S9 mix) and without metabolic activation
First experiment (TA98, TA 1535, TA1537, WP2uvrA): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (5% (v/v) S9 mix) and without metabolic activation
Second experiment (all strains): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (10% (v/v) S9 mix) and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (extra pure)
- Justification for choice of solvent/vehicle: Due to the reactivity to moisture of the test substance, ethanol was selected as the vehicle. At concentrations of 10 mg/mL and higher the test substance was suspended in ethanol. At concentrations of 3.33 mg/mL and lower the test substance was dissolved in ethanol.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191: 2.5 µg/plate for TA1537 (-S9); 2-aminoanthracene: 1, 2.5, 5 or 10 µg/plate for all strains (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

OTHER
Justification for using S9-mix prepared from rats treated with different substances:
Since rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone and by a combination of Aroclor are recommended by the OECD guideline and the positive control data were within the laboratory historical range for each tester strain, this deviation of the S9 homogenate had no effect on the results of the study.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduction of the revertant colonies at concentrations of 3333 and 5000 µg/plate without metabolic activation; tested up to the limit dose concentration of 5000 μg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate

Vehicle controls validity:

valid

Untreated 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:

other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate with and without metabolic activation. This dose range finding test is reported as a part of the first experiment of the mutation test. Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 1000 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period. To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA100 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance. In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The slight precipitate does not influence automated counting of the plate, the moderate precipitate required the plate to be hand counted.
Experiment I: Precipitation on the plates was observed at the start of the incubation period at concentrations of 1000 μg/plate and above. Precipitation on the plates was observed at the end of the incubation period at concentration of 333 μg/plate and above, except in tester strain TA98 where precipitation was observed at 1000 μg/plate and above. The precipitation of the test material at the dose level of 333 μg/plate contained a few particles only (please refer to Table 1 under "Any other information on results incl. tables").
Experiment II: Precipitation on the plates was observed at the start of the incubation period at the concentration of 1000 μg/plate and above. Precipitation on the plates was observed at the end of the incubation period at concentrations of 1000 μg/plate and above. Except in tester strain TA1535 (with and without metabolic activation) and TA98 (with metabolic activation) where a few particles of the test material were also observed at the dose level of 333 μg/ml (please refer to Table 2 under "Any other information on results incl. tables").

HISTORICAL CONTROL DATA
- Positive historical control data: The obtained data falls within the historical control data range (please refer to Table 4 under "Any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The obtained data falls within the historical control data range (please refer to Table 3 under "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Test results of Experiment I

With or without S9 Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 1535	TA100	WP2uvrA	TA98	TA1537
–	Solvent control	13 ± 5	110 ± 26	23 ± 9	20 ± 6	10 ± 1
–	3	-	116 ± 19	-	-	-
–	10	-	129 ± 22	-	-	-
–	33	13 ± 2	99 ± 14	22 ± 5	22 ± 2	5 ± 1
–	100	8 ± 3	94 ± 3	22 ± 6	28 ± 14	3 ± 1
–	333	4 ± 2 SP	92 ± 27	23 ± 3 SP	12 ± 4	3 ± 2 SP
–	1000	4 ± 1 SP	77 ± 8	19 ± 4 SP	11 ± 2 SP	4 ± 1 SP
–	3333	3 ± 1 SP	85 ± 11	22 ± 3 SP	8 ± 4 SP	5 ± 1 SP
–	5000	3 ± 2 SP	93 ± 14	26 ± 1 SP	9 ± 2 SP	4 ± 1 SP
Positive controls, -S9	Name	SA	MMS	4-NQO	NF	ICR-191
	Concentrations (μg/plate)	5	650	10	10	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	728 ± 18	885 ± 27	925 ± 347	1067 ± 29	886 ± 10
+	Solvent control	16 ± 5	129 ± 17	25 ± 7	20 ± 5	9 ± 3
+	3	-	127 ± 9	-	-	-
+	10	-	146 ± 43	-	-	-
+	33	12 ± 3	118 ± 32	22 ± 6	26 ± 8	5 ± 1
+	100	8 ± 1	89 ± 10	26 ± 3	17 ± 2	4 ± 1
+	333	5 ± 2 SP	83 ± 12	25 ± 7 SP	24 ± 5	3 ± 1 SP
+	1000	6 ± 1 SP	54 ± 9	25 ± 2 SP	18 ± 3 SP	4 ± 3 SP
+	3333	5 ± 2 SP	57 ± 7	27 ± 4 SP	18 ± 2 SP	3 ± 1 SP
+	5000	5 ± 2 SP	58 ± 3	24 ± 5 SP	19 ± 2 SP	2 ± 1 SP
Positive controls, +S9 (5% (v/v))	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	1	10	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	197 ± 6	1009 ± 16	925 ± 347	1009 ± 16	415 ± 36

Solvent control: 0.1 mL ethanol

SA = sodium azide

MMS = methyl methane sulfa

4NQO = 4-nitroquinoline-N-oxide

NF = 2-nitrofluorene

2AA = 2-aminoanthracene

SP = slight precipitate

Table 2: Test results of Experiment II

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 1535	TA100	WP2uvrA	TA98	TA1537
–	Solvent control	9 ± 1	120 ± 30	17 ± 4	16 ± 4	6 ± 1
–	100	4 ± 1	84 ± 5	20 ± 1	16 ± 3	8 ± 4
–	333	3 ± 1 SP	92 ± 12	18 ± 4	12 ± 4	5 ± 5
–	1000	3 ± 1 SP	89 ± 7 SP	24 ± 3 SP	16 ± 1 SP	2 ± 1 SP
–	3333	3 ± 2 SP	94 ± 2 SP	20 ± 4 SP	15 ± 3 SP	2 ± 1 SP
–	5000	3 ± 1 SP	89 ± 9 SP	19 ± 1 SP	13 ± 1 SP	2 ± 1 SP
Positive controls, –S9	Name	SA	MMS	4-NQO	NF	ICR-191
	Concentrations (μg/plate)	5	650	10	10	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	844 ± 14	838 ± 15	1232 ± 41	1081 ± 28	797 ± 35
+	Solvent control	11 ± 4	110 ± 2	22 ± 3	15 ± 3	7 ± 4
+	100	8 ± 5	79 ± 15	18 ± 2	26 ± 8	1 ± 1
+	333	4 ± 1 SP	93 ± 13	20 ± 2	19 ± 6 SP	5 ± 4
+	1000	3 ± 2 SP	92 ± 12 SP	16 ± 2 SP	19 ± 7 SP	3 ± 1 SP
+	3333	4 ± 1 SP	89 ± 10 SP	15 ± 4 SP	17 ± 3 SP	5 ± 1 SP
+	5000	4 ± 2 SP	69 ± 6 SP	16 ± 6 SP	14 ± 6 MP	1 ± 2 SP
Positive controls, +S9 (10% (v/v))	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	10	1	5
	Mean No. of colonies/plate (average of 3 ± SD)	189 ± 18	1341 ± 68	387 ± 21	780 ± 54	267 ± 11

Solvent control: 0.1 mL ethanol

SA = sodium azide

MMS = methyl methane sulfa

4NQO = 4-nitroquinoline-N-oxide

NF = 2-nitrofluorene

2AA = 2-aminoanthracene

SP = slight precipitate

Table 3: Historical control data: Negative control

	TA 1535		TA1537		TA98		TA100		WP2uvrA
S9 mix	-	+	-	+	-	+	-	+	-	+
Range	3 - 25	3 - 31	2 - 19	2 - 16	11 - 49	12 - 59	61 - 195	58 - 179	8 - 45	6 - 46
Mean	10	8	5	5	19	24	106	88	22	23
SD	4	3	2	2	5	7	22	21	7	7
n	1208	1216	1073	1098	1191	1208	1224	1221	1090	1105

SD = Standard deviation

n = Number of observations

Table 4: Historical control data: Positive control

	TA 1535		TA1537		TA98		TA100		WP2uvrA
S9 mix	-	+	-	+	-	+	-	+	-	+
Range	24 - 1270	60 - 943	89 - 1086	82 - 677	401 - 1342	225 - 1656	348 - 1417	229 - 1752	138 - 1479	122 - 1248
Mean	887	265	314	374	1016	792	950	1103	1074	372
SD	112	111	146	104	133	286	117	261	212	137
n	1123	1150	1011	1039	1136	1157	1153	1170	1023	1047

SD = Standard deviation

n = Number of observations

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative