Diethoxy(methyl)silane

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:

2007-02-01 to 2007-02-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

2-AA was the sole positive control for S9 mix and no information was provided, if the S9 batch used was tested before with an additional positive control substance.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, Munich, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethoxy(methyl)silane

EC Number:

217-982-3

EC Name:

Diethoxy(methyl)silane

Cas Number:

2031-62-1

Molecular formula:

C5H14O2Si

IUPAC Name:

diethoxy(methyl)silane

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: all strains except TA102 carry a mutation of the uvrB gene coding for the DNA excision repair system (uvrB-), all strains carry the deep rough mutation (rfa), TA102, TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated withPhenobarbital and ß-Naphthoflavone

Test concentrations with justification for top dose:

- Cytotoxicity pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
- Experiment I: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (with and without metabolic activation - plate incorporation)
- Experiment II: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (with and without metabolic activation - pre-incubation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility properties of the test article, compatibility with the S9 activity, and relative non-toxicity to bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

aqua dest.

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 1 h
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- A cytotoxicity pre-experiment was carried out with the tester strains TA 98 and A 100 to determine the non-toxic concentrations for the main genotoxicity experiments.
- Method: Background lawn assessment / revertant colony counts

Evaluation criteria:

A test system is considered as mutagenic if
- there is a clear and dose-related increase in the number of revertants 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 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 and TA 102 and 3-fold of the solvent control for TA 98, TA 1535 and TA 1537.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Any other information on results incl. tables

Table 1: Dose range-finding study. Number of revertants per plate (2 plates per strain).

	TA 98			TA 100
Concentration (μg/Plate)	Plate 1 - MA	Plate 2 + MA	Cytotoxic (Yes/No)	Plate 1 - MA	Plate 2 + MA	Cytotoxic (Yes/No)
DMSO	1	1	No	1	1	No
3.16	0.8	1.3	No	1	0.9	No
10	0.9	1.3	No	0.9	0.9	No
31.6	1.1	1.3	No	1	0.9	No
100	1	1.2	No	1	0.9	No
316	0.7	1.3	No	0.9	0.8	No
1000	0.7	1.1	No	0.9	1	No
2500	0.7	1.3	No	0.9	0.9	No
5000	0.9	1.3	No	0.9	0.9	No
Positive control	21.5	113.3	No	8	20.1	No

table2: Test results of Experiment I (plate incorporation)

with or without S9 -Mix	Test substance	Mean number of revertant colonies per plate (average of 3 plates ± SD)
	concentration	Base-pair substitution type			Frameshift type
	[µg/plate]	TA100	TA1535	TA102	TA98	TA1537
-	DMSO	111 ± 3.6	10 ± 2.6	150 ± 4.0	22 ± 2.3	7 ± 0.6
-	31.6	107 ± 6.2	7 ± 3.0	128 ± 25.0	24 ± 5.2	15 ± 3.5
-	100	109 ± 11.7	11 ± 3.6	153 ± 16.5	22 ± 6.1	16 ± 5.1
-	316	100 ± 7.1	8 ± 1.7	138 ± 12.5	15 ± 3.5	11 ± 1.0
-	1000	98 ± 8.9	7 ± 1.5	148 ± 7.8	16 ± 4.6	8 ± 2.5
-	2500	99 ± 7.5	9 ± 2.0	127 ± 21.9	16 ± 2.9	11 ± 2.6
-	5000	96 ± 4.6	9 ± 1.5	135 ± 19.5	20 ± 2.5	15 ± 6.8
Negative control	Aqua dest.	108 ± 8.2	10 ±2.1	188 ± 8.1	19 ± 2.6	13 ± 2.1
Positive controls	Name	NaN3	NaN3	MMS	4 -NOPD	4 -NOPD
	concentrations [µg/plate]	10	10	1µL	10	40
	Mean No. of colonies/plate (average of 3 ± SD)	893 ± 47.4	828 ± 38.9	1611 ± 208.4	481 ± 13.5	129 ± 10.6
+	DMSO	127 ± 5.3	9 ± 4.5	192 ± 14.4	26 ± 2.6	10 ± 4.0
+	31.6	114 ± 15.8	9 ± 2.1	149 ± 19.9	35 ± 1.0	11 ± 3.2
+	100	119 ± 6.8	6 ± 3.1	182 ± 10.3	31 ± 6.0	12 ± 0.6
+	316	103 ± 6.2	10 ± 0.6	188 ± 11.5	34 ± 3.6	11 ± 2.6
+	1000	128 ± 15.7	8 ± 1.2	212 ± 19.3	28 ± 5.0	12 ± 0.6
+	2500	120 ± 10.2	12 ± 1.0	201 ± 24.8	35 ± 6.0	10 ± 3.5
+	5000	112 ± 6.4	9 ± 3.1	167 ± 23.5	34 ± 2.5	14 ± 1.5
Negative control	Aqua dest.	124 ± 11.0	7 ± 2.0	211 ± 9.7	35 ± 3.6	13 ± 4.7
Positive controls	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	concentrations [µg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	2547 ± 100.3	102 ± 6.9	1156 ± 49.0	2945 ± 191.1	358 ± 12.0

table2:Test results of Experiment II (pre-incubation)

with or without S9 -Mix	Test substance	Mean number of revertant colonies per plate (average of 3 plates ± SD)
	concentration	Base-pair substitution type			Frameshift type
	[µg/plate]	TA100	TA1535	TA102	TA98	TA1537
-	DMSO	95 ± 6.8	10 ± 3.1	178 ± 9.2	22 ± 3.5	11 ± 3.0
-	31.6	89 ± 22.7	7 ± 4.5	104 ± 21.6	19 ± 5.6	12 ± 2.0
-	100	101 ± 8.2	9 ± 1.0	146 ± 11.1	21 ± 1.7	10 ± 3.0
-	316	97 ± 10.5	6 ± 1.2	157 ± 7.2	21 ± 1.0	7 ± 2.1
-	1000	74 ± 13.2	6 ± 1.0	178 ± 14.8	18 ± 1.5	7 ± 2.5
-	2500	58 ± 10.6	4 ± 1.5	158 ± 1.5	25 ± 4.7	9 ± 2.5
-	5000	70 ± 7.5	7 ± 2.6	130 ± 39.5	23 ± 0.6	12 ± 2.0
Negative control	Aqua dest.	103 ± 14.4	7 ± 1.0	186 ± 12.1	27 ± 3.1	9 ± 1.2
Positive controls	Name	NaN3	NaN3	MMS	4 -NOPD	4 -NOPD
	concentrations [µg/plate]	10	10	1µL	10	40
	Mean No. of colonies/plate (average of 3 ± SD)	701 ± 14.5	842 ± 30.0	1716 ± 117.7	472 ± 53.1	141 ± 9.3
+	DMSO	77 ± 4.5	9 ± 3.0	269 ± 14.4	36 ± 3.2	10 ± 3.1
+	31.6	84 ± 2.5	7 ± 2.0	143 ± 38.9	30 ± 2.5	15 ± 2.3
+	100	88 ± 14.6	6 ± 1.2	208 ± 26.0	42 ± 13.3	16 ± 3.1
+	316	92 ± 11.0	7 ± 4.0	226 ± 10.6	41 ± 6.9	13 ± 2.3
+	1000	99 ± 6.0	7 ± 1.2	240 ± 22.9	39 ± 7.6	9 ± 1.5
+	2500	75 ± 0.6	9 ± 0.6	206 ± 32.4	33 ± 4.2	12 ± 1.5
+	5000	86 ± 3.2	7 ± 2.1	191 ± 44.5	43 ± 8.0	11 ± 1.5
Negative control	Aqua dest.	100 ± 5.8	8 ± 3.1	307 ± 13.8	37 ± 5.5	13 ± 1.7
Positive controls	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	concentrations [µg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	872 ± 112.1	39 ± 7.5	1386 ± 138.1	1762 ± 40.7	120 ± 3.5

Applicant's summary and conclusion

Conclusions:

The test item was investigated for mutagenicicty to bacteria according to the OECD TG 471, and in compliance with GLP. In two independent experiments (plate incorporation and preincubation) the test material was tested in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 102 up to limit concentrations with and without a metabolic activation system. No significant increase in the number of revertants was observered in any of the tester strain with and without metabolic activation. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not mutagenic to bacteria under the conditions of the test.