Dimethoxydimethylsilane

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:

17-05-2002 To 02-12-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethoxydimethylsilane

EC Number:

214-189-4

EC Name:

Dimethoxydimethylsilane

Cas Number:

1112-39-6

Molecular formula:

C4H12O2Si

IUPAC Name:

dimethoxydimethylsilane

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa- and pkM101

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa-, uvrB- and pkM101 (TA 98 & TA100)

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

100, 316, 1000, 3160 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthracene amide

Remarks:

+S9; TA 98, TA102 and TA1537: 2 µg/plate dissolved in DMSO

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-S9: TA1537: 100 µg/plate dissolved in ethanol

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

-S9: TA98: 10 µg/plate in DMSO

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-S9: TA1535 and TA100: 10 µg/plate in water

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

-S9: TA102: 1300 µg/plate dissolved in DMSO.

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9; TA100 and TA1535: 1500 µg/plate dissolved in aqua ad iniectabilia.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation.

DURATION
- Preincubation period: 60 minutes (at 37°C)
- Exposure duration: 48 hours (at 37°C)


NUMBER OF REPLICATIONS: 3 plates per concentration in 2 independent experiments


DETERMINATION OF CYTOTOXICITY
- Method: background lawn evaluation, reduction of the number of revertants by more than 50%

METABOLIC ACTIVATION SYSTEM: Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was prepared according to Maron and Ames (1983). S9 was collected from 20-30 rats.
The S9 mix was freshly prepared on the day of the test according to Maron and Ames (1983): containing 5% S9 and the following components (per 100 mL):
-5.0 ml rat liver S9 (Aroclor 1254-induced)
-2.0 ml 0.4 M MgCl2 + 1.65 M KCl-salt solution
-141.0 mg glucose-6-phosphate
- 306.5 mg NADP
- 50.0 ml 0.2 M phosphate buffer, pH 7.4
- sterile aqua ad iniectabilia ad 100 ml

Evaluation criteria:

A test chemical is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤0.05, U-test according to MANN and WHITNEY) compared with the solvent control to at least 2-fold of the solvent control for TA-98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent experiments;
- in addition, a significant (p ≤0.05) concentration (log value)-related effect (Spearman’s rank) correlation coefficient) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate without and with activation, 3160 µg/plate without activation (pre-incubation); 3160 and 1000 µg/plate with activation (plate incorporation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate without and with activation, 3160 µg/plate without activation (pre-incubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate without and with activation (pre-incubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate without activation (plate incorporation)

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 examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Cytotoxicity (scarce background lawn) was noted at the top concentration of 5000 µg/plate. Hence, 5000 µg/plate was chosen as the top concentration for the main study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the plate incorporation test without metabolic activation cytotoxicity (scarce background lawn) was noted at the top concentration of 5000 µg/plate of the test substance in test strain TA 100. In the experiment with metabolic activation cytotoxicity (reduction of the number of revertants by more than 50%) was noted at concentration of 1000 and 3160 µg of the test substance/ plate in test strain TA 1535.
In the preincubation test without metabolic activation pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 or 3160 µg/plate onwards in test strains TA 1535 and TA 1537, respectively. Cytotoxicity (scarce background lawn) was noted at the top concentration of 5000 µg/plate of test substance in test strain TA 98. In the experiment with metabolic activation pronounced cytotoxicity ((scarce background lawn and reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 µg/plate in test strain TA 1537, and cytotoxicity (scarce background lawn) in test strain TA 98 and TA 1535.

Any other information on results incl. tables

Table 1. Plate incorporation test without metabolic activation:

Substance (µg/plate)	Plate incorporation test without metabolic activation. Number of reverted colonies.
		TA 98	TA 100	TA 102	TA 1535	TA1537
Silane M2 Dimethoxy	Mean values ± SD
5000	M	32.3	110.7#	291.7	10.0	3.3
	±SD	6.4	19.5	5.5	1.7	1.5
3160	M	30.3	101.0	288.7	8.0	4.3
	±SD	5.9	5.6	5.5	1.0	1.2
1000	M	24.7	118.7	317.3	10.0	3.7
	±SD	2.5	1.5	25.0	3.5	1.5
316	M	29.0	100.0	255.0	12.3	3.3
	±SD	4.4	3.5	10.6	2.9	1.2
100	M	26.3	107.0	265.3	11.0	5.7
	±SD	3.5	18.2	7.4	2.0	0.6
Negative control 100 µg/plate	M	25.7	102.7	270.7	10.0	3.3
	±SD	2.5	4.7	22.7	1.7	2.1
Positive control substance		2-Nitrofluorene	Sodium azide	Methylmethane sulfonate	Sodium azide	Aminoacridine
Concentration µg/plate		10	10	1300	10	100
	M	726.0	1129.0	1348.0	540.7	142.7
	±SD	59.6	46.0	78.3	15.3	32.1

# = scarce background lawn.

Table 2. Plate incorporation test with metabolic activation

Substance (µg/plate)	Plate incorporation test with metabolic activation. Number of reverted colonies.
		TA 98	TA 100	TA 102	TA 1535	TA1537
Silane M2 Dimethoxy	Mean values ± SD
5000	M	32.3	113.7	241.7	9.3	7.7
	±SD	0.6	15.9	17.8	0.6	2.1
3160	M	38.0	113.3	277.7	7.7	4.3
	±SD	7.0	11.0	22.5	0.6	1.5
1000	M	34.7	117.0	256.7	7.0	5.7
	±SD	7.6	23.6	36.4	1.0	3.2
316	M	31.3	114.7	371.7	17.7	5.7
	±SD	7.1	10.6	46.2	3.5	0.6
100	M	49.0	114.3	284.3	13.3	6.3
	±SD	7.0	6.8	11.9	3.5	2.3
Negative control 100 µg/plate	M	38.7	114.7	296.0	16.0	5.0
	±SD	1.5	5.0	8.5	3.6	1.7
Positive control substance		2-Anthracene amide	Cyclophosphamide	2-Anthracene amide	Cyclophosphamide	2-Anthracene amide
Concentration µg/plate		10	10	1300	10	100
	M	724.0	1118.3	937.0	288.7	138.3
	±SD	43.3	45.8	14.0	25.9	26.1

Table 3. Preincubation test without metabolic activation

Substance (µg/plate)	Preincubation test without metabolic activation. Number of reverted colonies.
		TA 98	TA 100	TA 102	TA 1535	TA1537
Silane M2 Dimethoxy	Mean values ± SD
5000	M	18.3#	94.0#	298.7	7.3#	1.7#
	±SD	5.5	43.5	12.7	6.4	1.2
3160	M	23.0	134.3	306.7	17.3	2.0#
	±SD	5.0	10.6	17.9	0.6	3.5
1000	M	18.3	137.7	294.7	21.3	6.7
	±SD	4.5	3.2	14.2	1.5	2.5
316	M	23.7	136.0	309.7	16.0	6.7
	±SD	4.5	9.2	8.0	6.9	1.2
100	M	23.0	130.0	318.0	17.0	8.0
	±SD	2.0	7.5	8.2	2.0	2.6
Negative control 100 µg/plate	M	22.3	114.0	293.7	18.0	7.7
	±SD	5.0	10.8	21.1	6.0	0.6
Positive control substance		2-Nitrofluorene	Sodium azide	Methylmethane sulfonate	Sodium azide	Aminoacridine
Concentration µg/plate		10	10	1300	10	100
	M	514.0	1131.3	1102.3	234.0	292.0
	±SD	56.2	101.2	17.9	33.4	56.1

Table 4. Preincubation test with metabolic activation

Substance (µg/plate)	Preincubation test with metabolic activation. Number of reverted colonies.
		TA 98	TA 100	TA 102	TA 1535	TA1537
Silane M2 Dimethoxy	Mean values ± SD
5000	M	22.0#	144.7	387.0	17.7#	4.0#
	±SD	7.0	20.3	60.9	1.5	4.0
3160	M	31.0	135.7	443.0	17.0	5.7
	±SD	5.3	15.5	23.6	2.0	3.5
1000	M	26.7	131.7	446.0	16.7	9.7
	±SD	2.1	18.2	16.5	1.2	3.5
316	M	34.3	129.0	407.3	18.3	7.7
	±SD	4.2	5.3.6	16.5	1.2	1.2
100	M	32.0	132.0	412.3	17.7	9.3
	±SD	12.1	14.9	30.4	1.5	2.1
Negative control 100 µg/plate	M	31.0	128.0	375.7	15.3	8.7
	±SD	2.6	15.1	22.1	2.5	2.1
Positive control substance		2-Anthracene amide	Cyclophosphamide	2-Anthracene amide	Cyclophosphamide	2-Anthracene amide
Concentration µg/plate		2	1500	2	1500	2
	M	524.3	1190.7	1233.7	432.3	285.3
	±SD	39.8	73.5	92.2	20.0	52.3

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

In a bacterial mutagenicity assay according to OECD 471 and GLP, no mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for the test substance tested up to a concentration of 1000 µg/plate in any of the five test strains in two independent experiments without and with metabolic activation (plate incorporation or preincubation test).