3,3,6,6-tetramethoxy-2,7-dioxa-3,6-disilaoctane

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:

2018-02-05 until 2018-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3,6,6-tetramethoxy-2,7-dioxa-3,6-disilaoctane

EC Number:

242-285-6

EC Name:

3,3,6,6-tetramethoxy-2,7-dioxa-3,6-disilaoctane

Cas Number:

18406-41-2

Molecular formula:

C8H22O6Si2

IUPAC Name:

3,3,6,6-tetramethoxy-2,7-dioxa-3,6-disilaoctane

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 611220160318
- Expiration date of the batch: The stability of the test substance under storage conditions is guaranteed until Jun 2018.
- Purity test date: 27 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Stability under test conditions: see above
- Solubility and stability of the test substance in the solvent/vehicle: All test substance formulations were prepared immediately before administration.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No test substance precipitation was found with and without S9 mix.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO).

FORM AS APPLIED IN THE TEST
clear solution

Method

Target gene:

Salmonella typhimurium
TA 1535, TA 100: his G46; TA 1537: his C3076; TA 98: his D3052; TA 98, TA 100: R factor plasmid pKM101

Escherichia coli
WP2 uvrA: trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: Salmonella and E.coli: deficient excision repair system (uvrB) and reduced hydrophilic polysaccharide layer (rfa) TA 98 and TA 100: modified postreplication DNA

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2700 and 5400 μg/plate
Standard plate test with and without S9 mix (ratio 1:9 = 1 part of S9 fraction was mixed with 9 parts S9 supplement (cofactors))

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2700 and 5400 μg/plate μg/plate
Standard plate test with and without S9 mix (ratio 3:7)

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
In this study, due to the purity of the test substance 5.4 mg/plate was used as top dose in all experiments.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Untreated negative controls:

other: Not applicable

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: with S9 mix: 2-aminoanthracene (2-AA), 2.5 µg/plate; without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, 5 µg/plate; 4-nitro-o-phenylenediamine, 10 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72h
- Expression time (cells in growth medium): for about 12-16h

SELECTION AGENT (mutation assays): N/A

NUMBER OF REPLICATIONS: 2 independent experiments, each with 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6) and clearing or diminution of the background lawn (= reduced his- or trp- background growth)

OTHER EXAMINATIONS:
- Solubility in the final treatment mixture: observation of precipitation of the test material

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
- a dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling or tripling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system

A test substance was generally considered non-mutagenic in this test if:
- the number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other

Acceptance criteria:
- the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
- the positive control substances (with / without S9 mix) induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
- sterility controls revealed no indication of bacterial contamination
- fresh bacterial culture containing approximately 109 cells per mL used

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: No test substance precipitation was found with and without S9 mix.
- Sterility: No contamination was observed in the sterility controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test neighter with S9 mix ratio 1:9 nor with a ratio of 3:7 up to the highest test substance concentration.

Any other information on results incl. tables

Table 1: Results of the 1st standard plate test experiment without metabolic activation

Strain		Test group	Dose (µg/plate)	Mean revertants per plate		Standard deviation	Factor	Individual revertant colony count
TA 1535		DMSO	-	10.3		1.5	-	12, 10, 9
		Test item	33	7.0		2.6	0.7	9, 8, 4
			100	5.7		2.5	0.5	6, 8, 3
			333	8.0		2.0	0.8	6, 10, 8
			1000	9.3		4.5	0.9	14, 5, 9
			2700	8.7		6.4	0.8	4, 16, 6
			5400	7.3		3.8	0.7	3, 10, 9
		MNNG	5.0	4284.0		244.9	414.6	4016, 4496, 4340
TA 100		DMSO	-	78.3		4.0	-	76, 83, 76
		Test item	33	77.3		2.9	1.0	79, 74, 79
			100	72.7		15.6	0.9	58, 89, 71
			333	85.3		3.2	1.1	89, 83, 84
			1000	77.7		14.2	1.0	93, 75, 65
			2700	79.0		6.2	1.0	81, 84, 72
			5400	66.3		5.0	0.8	67, 71, 61
		MNNG	5.0	2188.0		66.1	27.9	2156, 2144, 2264
TA 1537		DMSO	-	4.3		1.2	-	3, 5, 5
		Test item	33	4.0		2.0	0.9	2, 4, 6
			100	4.3		1.5	1.0	3, 4, 6
			333	3.0		1.0	0.7	3, 2, 4
			1000	2.3		0.6	0.5	2, 2, 3
			2700	4.7		2.1	1.1	7, 3, 4
			5400	6.3		3.8	1.5	8, 2, 9
		AAC	100	670.7		24.1	154.8	648, 668, 696
TA 98	DMSO		-	17.7	1.2		-	19, 17, 17
	Test item		33	16.7	1.5		0.9	17, 15, 18
			100	19.7	1.2		1.1	21, 19, 19
			333	18.0	1.7		1.0	17, 20, 17
			1000	15.0	1.0		0.8	16, 15, 14
			2700	18.0	1.0		1.0	17, 19, 18
			5400	13.3	1.2		0.8	14, 14, 12
	NOPD		10	778.7	32.1		44.1	812, 748, 776
E. coli	DMSO		-	20.7	3.5		-	17, 24, 21
	Test item		33	20.3	5.8		1.0	17, 27, 17
			100	21.0	2.6		1.0	22, 23, 18
			333	22.3	3.2		1.1	21, 26, 20
			1000	16.7	0.6		0.8	16, 17, 17
			2700	19.7	5.0		1.0	15, 19, 25
			5400	21.7	7.5		1.0	29, 14, 22
	4-NQO		5	797.3	39.7		38.6	784, 766, 842

DMSO: dimethyl sulfoxide

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine

AAC: 9-aminoacridine
NOPD: 4-nitro-o-phenylenediamine

4 -NQO: 4-nitroquinoline-N-oxide

Table 2: Results of the 1st standard plate test experiment with metabolic activation (ratio 1:9)

Strain		Test group	Dose (µg/plate)	Mean revertants per plate		Standard deviation	Factor	Individual revertant colony count
TA 1535		DMSO	-	6.3		2.5	-	6, 9, 4
		Test item	33	9.3		3.2	1.5	7, 13, 8
			100	8.7		4.0	1.4	4, 11, 11
			333	4.3		1.5	0.7	4, 6, 3
			1000	7.7		1.2	1.2	7, 9, 7
			2700	6.3		2.5	1.0	9, 6, 4
			5400	7.0		1.0	1.1	7, 8, 6
		2-AA	2.5	186.0		17.3	29.4	206, 176, 176
TA 100		DMSO	-	72.3		3.5	-	72, 69, 76
		Test item	33	70.0		6.1	1.0	73, 63, 74
			100	72.3		3.2	1.0	71, 70, 76
			333	76.3		3.8	1.1	72, 79, 78
			1000	81.0		8.7	1.1	76, 76, 91
			2700	74.3		4.5	1.0	70, 74, 79
			5400	72.7		3.2	1.0	69, 75, 74
		2-AA	2.5	2125.3		24.1	29.4	2148, 2100, 2128
TA 1537		DMSO	-	7.0		4.4	-	12, 4, 5
		Test item	33	4.7		3.5	0.7	8, 1, 5
			100	6.7		4.7	1.0	3, 12, 5
			333	8.7		4.0	1.2	5, 8, 13
			1000	9.0		2.0	1.3	9, 11, 7
			2700	7.0		1.7	1.0	8, 5, 8
			5400	4.7		3.5	0.7	5, 8, 1
		2-AA	2.5	107.3		1.2	15.3	106, 108, 108
TA 98	DMSO		-	21.7	1.2		-	21, 23, 21
	Test item		33	22.0	3.0		1.0	22, 25, 19
			100	19.7	0.6		0.9	20, 20, 19
			333	22.7	5.1		1.0	24, 17, 27
			1000	20.3	2.1		0.9	22, 21, 18
			2700	20.3	1.5		0.9	22, 19, 20
			5400	14.3	1.2		0.7	15, 15, 13
	2-AA		2.5	1098.7	23.4		50.7	1124, 1078, 1094
E. coli	DMSO		-	24.0	12.3		-	19, 38, 15
	Test item		33	25.3	5.8		1.1	22, 22, 32
			100	25.0	4.4		1.0	23, 22, 30
			333	20.7	3.5		0.9	24, 17, 21
			1000	31.0	13.9		1.3	47, 23, 23
			2700	21.7	2.1		0.9	21, 24, 20
			5400	31.7	7.1		1.3	38, 33, 24
	2-AA		60	96.0	4.6		4.0	95, 101, 92

DMSO: dimethyl sulfoxide

2 -AA: 2-aminoanthracene

Table 3: Results of the 2nd standard plate test experiment without metabolic activation

Strain		Test group	Dose (µg/plate)	Mean revertants per plate		Standard deviation	Factor	Individual revertant colony count
TA 1535		DMSO	-	10.3		2.5	-	10, 13, 8
		Test item	33	10.3		0.6	1.0	10, 10, 11
			100	10.0		1.0	1.0	11, 9, 10
			333	12.0		3.6	1.2	11, 16, 9
			1000	13.7		1.2	1.3	13, 15, 13
			2700	9.3		0.6	0.9	10, 9, 9
			5400	11.7		2.1	1.1	14, 10, 11
		MNNG	5.0	4162.3		64.5	402.8	4088, 4204, 4195
TA 100		DMSO	-	98.7		17.2	-	83, 117, 96
		Test item	33	98.0		1.7	1.0	99, 96, 99
			100	101.0		9.5	1.0	90, 106, 107
			333	96.0		3.6	1.0	97, 92, 99
			1000	103.7		8.7	1.1	111, 106, 94
			2700	95.7		9.9	1.0	91, 107, 89
			5400	94.0		7.5	1.0	101, 95, 86
		MNNG	5.0	4062.7		96.8	41.2	4170, 3982, 4036
TA 1537		DMSO	-	6.3		1.5	-	5, 6, 8
		Test item	33	6.7		2.3	1.1	8, 8, 4
			100	6.7		1.5	1.1	7, 8, 5
			333	5.7		1.5	0.9	4, 7, 6
			1000	5.3		1.5	0.8	5, 7, 4
			2700	7.3		1.2	1.2	8, 8, 6
			5400	7.3		4.9	1.2	5, 4, 13
		AAC	100	596.7		48.0	94.2	598, 548, 644
TA 98	DMSO		-	14.3	1.5		-	16, 13, 14
	Test item		33	16.7	1.5		1.2	18, 17, 15
			100	15.3	3.1		1.1	16, 18, 12
			333	12.3	1.2		0.9	13, 11, 13
			1000	16.3	1.5		1.1	18, 16, 15
			2700	18.3	1.2		1.3	19, 19, 17
			5400	15.0	1.0		1.0	16, 15, 14
	NOPD		10	598.7	12.2		41.8	596, 588, 612
E. coli	DMSO		-	21.3	2.1		-	23, 19, 22
	Test item		33	24.3	2.5		1.1	22, 24, 27
			100	16.7	3.1		0.8	14, 16, 20
			333	20.7	2.1		1.0	20, 23, 19
			1000	19.0	5.6		0.9	18, 14, 25
			2700	23.3	4.0		1.1	27, 24, 19
			5400	28.3	3.2		1.3	26, 32, 27
	4-NQO		5	573.3	43.7		26.9	526, 612, 582

DMSO: dimethyl sulfoxide

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine

AAC: 9-aminoacridine
NOPD: 4-nitro-o-phenylenediamine

4 -NQO: 4-nitroquinoline-N-oxide

Table 4: Results of the 2nd standard plate test experiment with metabolic activation (ratio 3:7)

Strain		Test group	Dose (µg/plate)	Mean revertants per plate		Standard deviation	Factor	Individual revertant colony count
TA 1535		DMSO	-	11.3		2.1	-	9, 13, 12
		Test item	33	8.7		0.6	0.8	8, 9, 9
			100	8.3		2.9	0.7	5, 10, 10
			333	9.3		1.5	0.8	8, 9, 11
			1000	12.7		5.0	1.1	8, 18, 12
			2700	10.3		3.2	0.9	9, 14, 8
			5400	11.3		2.5	1.0	9, 11, 14
		Untreated	-	9.7		1.5	0.9	11, 8, 10
		2-AA	2.5	50.3		4.9	4.4	56 B, 48 B, 47 B
TA 100		DMSO	-	88.7		12.9	-	98, 74, 94
		Test item	33	91.0		6.9	1.0	87, 87, 99
			100	99.7		23.7	1.1	85, 87, 127
			333	84.0		13.0	0.9	71, 97, 84
			1000	84.3		2.5	1.0	87, 82, 84
			2700	77.0		3.6	0.9	74, 81, 76
			5400	87.0		14.8	1.0	104, 80, 77
		Untreated	-	85.7		5.7	1.0	92, 81, 84
		2-AA	2.5	413.3		10.1	4.7	404 B, 424 B, 412 B
TA 1535		DMSO	-	11.3		2.1	-	9, 13, 12
		Test item	33	8.7		0.6	0.8	8, 9, 9
			100	8.3		2.9	0.7	5, 10, 10
			333	9.3		1.5	0.8	8, 9, 11
			1000	12.7		5.0	1.1	8, 18, 12
			2700	10.3		3.2	0.9	9, 14, 8
			5400	11.3		2.5	1.0	9, 11, 14
		Untreated	-	9.7		1.5	0.9	11, 8, 10
		2-AA	2.5	50.3		4.9	4.4	56 B, 48 B, 47 B
TA 98	DMSO		-	21.3	1.5		-	21, 23, 20
	Test item		33	22.3	1.5		1.0	21, 24, 22
			100	20.7	3.8		1.0	18, 25, 19
			333	18.7	1.5		0.9	19, 17, 20
			1000	24.0	2.6		1.1	26, 21, 25
			2700	21.0	2.0		1.0	21, 19, 23
			5400	20.7	2.1		1.0	20, 19, 23
	Untreated		-	21.0	2.0		1.0	19, 23, 21
	2-AA		2.5	160.7	4.2		7.5	156 B, 162 B, 164 B
E. coli	DMSO		-	21.0	2.0		-	23, 21, 19
	Test item		33	19.3	3.2		0.9	17, 18, 23
			100	18.7	3.5		0.9	22, 19, 15
			333	18.3	8.5		0.9	15, 12, 28
			1000	26.7	6.8		1.3	29, 19, 32
			2700	20.0	8.0		1.0	28, 12, 20
			5400	21.7	2.9		1.0	20, 25, 20
	Untreated		-	20.0	1.0		1.0	20, 19, 21
	2-AA		60	90.0	7.9		4.3	81 B, 96 B, 93 B

DMSO: dimethyl sulfoxide

2 -AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

The test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate tests with different S9 fraction concentrations in the S9 mix (10 and 30% v/v)).

Executive summary:

The test substance 1,2-BIS(TRIMETHOXYSILYL)ETHANE was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5400 μg/plate (SPT)

TEST CONDITIONS: Standard plate test (SPT) with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: No bacteriotoxic effect was observed under all test conditions.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test without S9 mix or after the addition of a metabolizing system.

CONCLUSION:

Under the experimental conditions of this study, the test substance 1,2 - BIS(TRIMETHOXYSILYL)ETHANE is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.