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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is considered to be non-mutagenic in vitro in the Ames test (according to OECD 471) with and without metabolic activation tested up to the highest guideline defined concentration.

A micronucleus test in vitro (according to OECD 487) was performed with and without metabolic activation. The test substance is considered to be able to induce chromosome breaks and/or gain or loss to human lymphocytes.

Link to relevant study records

Reference

Reference 1

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

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

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

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 / 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

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

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-13 until 2018-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

extended exposure, further details are given in the section "Principles of method if other than guideline"

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 611220160318
- Storage stability: 30 June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (approximately 4°C)
- Stability under test conditions: guaranteed by the sponsor under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: The test item was used as supplied by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- no prior treatment

Species / strain / cell type:

lymphocytes: Human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human volunteers
- Suitability of cells: as recognized in the OECD 487 guidelines
- Normal cell cycle time (negative control): approximately 16 hours

For lymphocytes:
- Sex, age and number of blood donors:
- preliminary experiment: non-smoking volunteer female, 26 years old
- main experiment: non-smoking volunteer male, 31 years old
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: only blood from one donor used
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
- Eagle's minimal essential medium with HEPES buffer (MEM) and supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), approximately 37 ºC with 5% CO2 in humidified air

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

At the end of the exposure period, the washed cell cultures were incubated for a further 24 hours in the presence of Cytochalasin B at 4.5 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : in-house batches PB/βNF S9 14/12/17 and 29/03/18; phenobarbital induced Sprague Dawley rats
- method of preparation of S9 mix : pre-prepared standardized in-house procedures
- concentration or volume of S9 mix and S9 in the final culture medium: 2% as dosed at a 10% volume of S9-mix into culture media
- quality controls of S9: enzymatic activity to diagnostic mutagens, sterility tested

Test concentrations with justification for top dose:

- preliminary experiment: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL; top dose based on maximum dose level

- main experiment:
- 4-h exposure without S9: 0, 15.63, 31.25, 62.5, 100, 125, 200, 250, 300 µg/mL
- 4-h exposure with S9 (2%): 0, 15.63, 31.25, 62.5, 100, 125, 200, 250, 300 µg/mL
- 24-h exposure without S9: 0, 31.25, 62.5, 125, 250, 300, 350, 400, 500 µg/mL
- top doses based on toxicity: 300 μg/mL for the 4-hour exposure groups and 500 μg/mL for the 24-hour exposure group

Vehicle / solvent:

- Vehicle used: dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2 (preliminary and main experiment) with three different expsure conditions

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
- 4-h exposure without S9, 4-h exposure with S9, 24-h exposure without S9

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- cytokinesis block described above

- Methods of slide preparation and staining technique used including the stain used:
- lymphocytes re-suspended in several mL of fresh fixative (methanol/glacial acetic acid (19:1 v/v)) before centrifugation and re-suspension in a small amount of fixative
- several drops of this suspension dropped onto clean, wet microscope slides and left to air dry with gentle warming
- each slide permanently labeled
- stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- minimum of approximately 500 cells per culture scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value
- 2000 binucleated cells analyzed for micronucleus frequency per concentration (1000 binucleated cells per culture, two cultures per concentration)

- Criteria for scoring micronucleated cells:
- cells with 1, 2 or more micronuclei were recorded as such
- primary analysis was on the combined data
- criteria for identifying micronuclei: round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei
- binucleate cells selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary
- two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
- cytokinesis block proliferation index (CBPI)
- microscopical checks to determine the quality of the binucleate cells and also the toxicity and extent of precipitation
- hemolysis

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- detection of micronuclei (MN) in the cytoplasm of interphase cells

ACCEPTABILITY CRITERIA
- criteria for the validity of the assay:
- concurrent negative control (vehicle control) within the laboratory historical control data range
- positive control chemicals induced a positive response (p≤0.01), demonstrated validity of the experiment and the integrity of the S9-mix
- cell proliferation criteria in the solvent control considered to be acceptable
- study performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
- required number of cells and concentrations analyzed

Evaluation criteria:

If acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase
- no dose-related increase
- results in all evaluated dose groups within the range of the laboratory historical control data (refer to table 1)

If acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- increase which can be considered to be dose-related
- results are substantially outside the range of the laboratory historical negative control data
- if all criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system

If the response is neither clearly negative/positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

Statistics:

- control of frequency of binucleate cells with micronuclei with the concurrent vehicle control: Chi-squared Test on observed numbers of cells with micronuclei, (Hoffman et al., 2003)
- toxicologically significant response: p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

near optimum toxicity was achieved at 200 μg/mL with 47% and 51% cytostasis in the absence and presence of S9 respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: near optimum toxicity was achieved at 200 μg/mL with 47% and 51% cytostasis in the absence and presence of S9 respectively

Remarks:

24-hour exposure

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

statistically significant increases in the frequency of micronuclei at all test item dose levels scored

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

400 μg/mL with 63% cytostasis, dose level below (350 μg/mL) achieved 15% cytostasis

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: please refer to table 8
- Precipitation and time of the determination:
- preliminary toxicity test: a precipitate of the test item observed in the parallel blood-free cultures at the end of the exposure, at 2000 μg/mL in the 24-hour exposure group only
- main test: no precipitate of test item or hemolysis observed

RANGE-FINDING/SCREENING STUDIES:
The dose range for the preliminary toxicity test was 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum dose was the maximum recommended dose level.
Hemolysis was observed following exposure to the test item at and above 250 μg/mL in the 4-hour exposure group in the presence of S9. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 250 μg/mL in all three exposure groups. The CBPI data are presented in Table 1. The test item induced evidence of toxicity in all three exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 300 μg/mL for the 4-hour exposure groups and was 500 μg/mL for the 24-hour exposure group.

Table 3: CBPI results from the 4-h experiments, with and without S9 mix

4-Hour exposure without S9								4-hour exposure with S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis	Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		Mono	Bi	Multi						Mono	Bi	Multi
0	A	306	180	14	1.42	1.45	0	0	A	345	146	9	1.33	1.34	0
	B	282	201	17	1.47				B	339	150	11	1.34
15.63	A	-	-	-	-	-	-	15.63	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
31.25	A	-	-	-	-	-	-	31.25	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
62.5	A	328	151	21	1.39	1.37	18	62.5	A	366	128	6	1.28	1.27	21
	B	335	159	6	1.34				B	382	113	5	1.25
100	A	340	150	10	1.34	1.37	18	100	A	384	111	5	1.24	1.27	19
	B	320	164	16	1.39				B	355	139	6	1.30
125	A	344	150	6	1.32	1.31	30	125	A	365	130	5	1.28	1.32	6
	B	359	131	10	1.30				B	330	164	6	1.35
200	A	386	111	3	1.23	1.24	47	200	A	445	53	2	1.11	1.17	51
	B	385	110	5	1.24				B	392	106	2	1.22
250	A	NB	NB	NB	NB	NB	NB	250	A	NB	NB	NB	NB	NB	NB
	B	NB	NB	NB	NB				B	NB	NB	NB	NB
300	A	NB	NB	NB	NB	NB	NB	300	A	NB	NB	NB	NB	NB	NB
	B	NB	NB	NB	NB				B	NB	NB	NB	NB
MMC 0.2	A	386	112	2	1.23	1.24	46	CP 5	A	458	42	0	1.08	1.09	75
	B	378	121	1	1.25				B	454	45	1	1.09

MMC = Mitomycin C

CP = Cyclophosphamide

- = Not selected for scoring

NB = No binucleate cells or insufficient binucleate cells for scoring

Table 4: CBPI results from the 24-h experiment, without S9 mix

24-Hour exposure without S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		Mono	Bi	Multi
0	A	342	149	9	1.33	1.36	0
	B	321	168	10	1.38
31.25	A	-	-	-	-	-	-
	B	-	-	-	-
62.5	A	-	-	-	-	-	-
	B	-	-	-	-
125	A	345	148	7	1.32	1.39	0 +
	B	282	210	8	1.45
250	A	310	174	16	1.41	1.40	0 +
	B	317	175	8	1.38
300	A	334	154	12	1.36	1.37	0 +
	B	322	168	10	1.38
350	A	346	138	16	1.34	1.30	15
	B	376	117	7	1.26
400	A	430	62	8	1.16	1.13	63
	B	452	45	3	1.10
500	A	NB	NB	NB	NB	NB	NB
	B	NB	NB	NB	NB
DC 0.075	A	388	107	5	1.23	1.23	35
	B	389	108	3	1.23

DC = Demecolcine

- = Not selected for scoring

NB = No binucleate cells or insufficient binucleate cells for scoring

+ = Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control 

Table 5: CBPI and micronucleus data from the 4-h experiment without metabolic activation

ExposureTime+/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi				1 MN	2 MN	>2 MN
4Hr-S9	0	A	306	180	14	1.42	1.45	0	3	0	0	0.30	0.20
		B	282	201	17	1.47			1	0	0	0.10
	62.5	A	328	151	21	1.39	1.37	18	11	0	0	1.10	0.70
		B	335	159	6	1.34			3	0	0	0.30
	100	A	340	150	10	1.34	1.37	18	4	0	0	0.40	0.45
		B	320	164	16	1.39			5	0	0	0.50
	125	A	344	150	6	1.32	1.31	30	13	0	0	1.30	1.05
		B	359	131	10	1.30			6	1	1	0.80
	200	A	386	111	3	1.23	1.24	47	6	2	1	0.90	0.75
		B	385	110	5	1.24			4	1	1	0.60
	MMC 0.2	A	386	112	2	1.23	1.24	46	31	0	2	3.30	3.65***
		B	378	121	1	1.25			38	2	0	4.00

MMC = Mitomycin C

*** = P<0.001

Table 6: CBPI and micronucleus data from the 4-h experiment with metabolic activation

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi				1 MN	2 MN	>2 MN
4Hr+S9	0	A	345	146	9	1.33	1.34	0	3	1	0	0.40	0.65
		B	339	150	11	1.34			8	1	0	0.90
	62.5	A	366	128	6	1.28	1.27	21	3	0	0	0.30	0.25
		B	382	113	5	1.25			2	0	0	0.20
	100	A	384	111	5	1.24	1.27	19	5	0	0	0.50	0.65
		B	355	139	6	1.30			7	1	0	0.80
	125	A	365	130	5	1.28	1.32	6	7	1	0	0.80	0.90
		B	330	164	6	1.35			8	2	0	1.00
	200	A	445	53	2	1.11	1.17	51	13	0	0	1.30	1.10
		B	392	106	2	1.22			9	0	0	0.90
	CP 5	A	458	42	0	1.08	1.09	75	34	2	0	3.60	3.20***
		B	454	45	1	1.09			27	1	0	2.80

CP = Cyclophosphamide

*** = P<0.001

Table 7: CBPI and micronucleus data from the 24-h experiment without metabolic activation

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean %Binucleate Cells with MN
			Mono	Bi	Multi				1 MN	2 MN	>2 MN
24Hr-S9	0	A	342	149	9	1.33	1.36	0	6	0	0	0.60	0.50
		B	321	168	10	1.38			4	0	0	0.40
	250	A	310	174	16	1.41	1.40	0 +	4	1	0	0.50	1.90***
		B	317	175	8	1.38			27	6	0	3.30
	300	A	334	154	12	1.36	1.37	0 +	13	0	0	1.30	1.35**
		B	322	168	10	1.38			11	2	1	1.40
	350	A	346	138	16	1.34	1.30	15	31	1	0	3.20	2.65***
		B	376	117	7	1.26			14	6	1	2.10
	400	A	430	62	8	1.16	1.13	63	Considered too toxic for scoring
		B	452	45	3	1.10			Considered too toxic for scoring
	DC 0.075	A	388	107	5	1.23	1.23	35	30	6	0	3.60	4.20***
		B	389	108	3	1.23			36	6	6	4.80

DC = Demecolcine

+ = Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control

** = P<0.01

*** = P<0.001

Table 8: PH and osmolality measurement of test item dosed into media.

Dose Concentration(µg/mL)	0	7.81	15.63	31.25	62.5	125	250	500	1000	2000
pH	7.14	7.28	-	7.32	-	-	7.31	-	-	7.32
Osmolality mOsm	453	448	-	451	-	-	448	-	-	433

Conclusions:

The test item was considered to be able to induce chromosome breaks and/or gain or loss to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reverse Bacterial Mutation:

The test material 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.

The incubation was performed with TA 1535, TA 100, TA 1537, TA 98 of Salmonella typhimurium and E. coli WP2 uvrA with and without metabolic activation. The dose range was 33 μg - 5400 μg/plate in the strains. No precipitation of the test substance was found with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. 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.

According to the results of the present study, 1,2- Bis(trimethoxysilyl)ethane is thus not mutagenic in the Ames test under the experimental conditions chosen here.

Micronucleus test in human lymphocytes for detection of the clastogenic and aneugenic potential:

A micronucleus test in human lymphocytes was performedin vitroto detect the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. The study was performed according to OECD 487.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The following exposure groups were tested with final concentrations of1,2- Bis(trimethoxysilyl)ethane (µg/mL) as follows:

- 4-hour without S9: 0, 15.63, 31.25, 62.5, 100, 125, 200, 250 and 300

-4-hour with S9 (2%): 0, 15.63, 31.25, 62.5, 100, 125, 200, 250, 300

-24-hour without S9: 0, 31.25, 62.5, 125, 250, 300, 350, 400, 500

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item demonstrated marked toxicity in all three exposure groups. The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure groups in the absence and presence of S9, using a dose range that included a dose level that achieved near optimum toxicity with approximately 50% cytostasis. The 24-hour exposure group induced statistically significant increases in the frequency of micronuclei at all test item dose levels scored. The numbers of micronuclei observed in the binucleate cells of the test item dose levels all exceeded the upper limit of the laboratory historical control range and at dose levels which all achieved acceptable toxicity.

The test item,1,2- Bis(trimethoxysilyl)ethane(MEOS) was considered to be able to induce chromosome breaks and/or gain or loss to human lymphocytesin vitro.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, classification as "suspected of causing genetic defects" (Mut. Cat. 2 (H341)) is warranted.