Diethoxy(methyl)silane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test, ): S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 102: negative with and without metabolic activation (according to OECD 471)
Mammalian cytogenicity (Chromosome Aberration, RA from CAS 2031-67-6): negative with and without metabolic activation (similar to OECD 473)
Gene Mutation (Mammalian Cells, RA from CAS 2031-67-6): negative with and without metabolic activation (similar to OECD 476)

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:

2007-02-01 to 2007-02-16

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:

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

his operon

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

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the attached justification below and the overall justification for grouping of substances attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

CAS 2031-67-6, Litton Bionetics, 1979

Conclusions:

In an in vitro cytogenicity test equivalent or similar to OECD 473 and not compliant with GLP, the source substance CAS 2031-67-6 did not cause a statistically significant, dose related increase in chromosome aberrations. Appropriate solvent and positive controls were included and gave expected results. The source substance was considered non-clastogenic in mouse lymphoma L5178Y cells. As explained in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity potential.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the attached justification below and the overall justification for grouping of substances attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50% growth inhibition at -S9 2.4 µL/mL; +S9: 3.2 µL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

CAS 2031-67-6, Litton Bionetics, 1978

Conclusions:

In an in vitro mouse lymphoma L5178Y cells gene mutation test equivalent or similar to OECD 476 and not compliant with GLP, the source substance CAS 2031-67-6 was found to be non-mutagenic. Appropriate solvent and positive controls were included and gave expected results. As explained in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Only limited data on the genetic toxicity of diethoxy(methyl)silane (CAS 2031-62-1) are available. Therefore, the risk assessment was performed based on the available data from the source substance triethoxy(methyl)silane (CAS 2031-67-6). In accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and in accordance with the Read across assessment framework (RAAF, ECHA 2017) read across from the analogue substance has been applied to support the human health hazard assessment of diethoxy(methyl)silane (CAS 2031-62-1).

 

Genetic toxicity (mutagenicity) in bacteria in vitro

Two genetic toxicity studies addressing mutagenicity in bacteria in vitro with diethoxy(methyl)silane (CAS 2031-62-1) are available and were performed according to OECD TG 471 and in compliance with GLP.

A key bacterial gene mutation study (Ames test) performed according to OECD TG 471 and in compliance with GLP with CAS 2031-62-1 is available (BSL, 2007).The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment 1) and pre-incubation (experiment 2) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Both experiments were conducted in three repetitions at each concentration from 31.6 to 5000 µg/plate (vehicle: DMSO). No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation.No cytotoxicity was observed at any of the tested concentrations. 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.

A supporting bacterial gene mutation study (Ames test) performed according to OECD TG 471 and in compliance with GLP with CAS 2031-62-1 is available (WIL, 1996).The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvr B were tested according to the pre-incubation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in three repetitions at each concentration from 100 to 5000 µg/plate (vehicle: ethanol). No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. No cytotoxicity was observed at any of the tested concentrations. Appropriate 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.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

No data on genetic toxicity (cytogenicity) in mammalian cells is available with diethoxy(methyl)silane (CAS 2031-62-1). Therefore, read across from the similar substance triethoxy(methyl)silane (CAS 2031-67-6) was applied (Litton Bionetics, 1979).

An in vitro chromosome aberration test in mouse lymphoma L5178Y cells was performed with CAS 2031-67-6 equivalent or similar to OECD TG 473 and in compliance with GLP (Litton Bionetics, 1979). Mouse lymphoma cells were treated with triethoxy(methyl)silane or vehicle (ethanol) in the absence or presence of a metabolic activation system (Aroclor-induced rat liver S9-mix) at concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL for 4 hours (with and without S9-mix). Fixation of the cells was performed 20 hours post-exposure with the test material (with and without S9-mix). Appropriate negative, solvent and positive controls were included in the test and gave the expected results. Cytotoxicity was not recorded under the tested conditions. Based on the results of the study the test substance did not cause a statistically significant, dose-related increase in chromosome aberrations and is therefore considered to be non-clastogenic in mouse lymphoma L5178Y cells under the tested conditions.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

No data on genetic toxicity (mutagenicity) in mammalian cells is available with diethoxy(methyl)silane (CAS 2031-62-1). Therefore, read across from the structural analogue substance triethoxy(methyl)silane (CAS 2031-67-6) was applied (Litton Bionetics, 1978).

An in vitro gene mutation test in Mouse lymphoma L5178Y cells was performed with CAS 2031-67-6 equivalent or similar to OECD TG 476 (Litton Bionetics, 1978). Mouse lymphoma cells were treated with triethoxy(methyl)silane or vehicle (ethanol) in two independent experiments in the absence or presence of a metabolic activation system (Aroclor-induced rat liver S9-mix) at concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 µL/mL (with and without S9-mix, experiment 1), 0.4, 0.8, 1.6 and 2.4 µL/mL (without S9-mix, experiment 2) and 0.4, 0.8, 1.6, 2.4 and 3.2 µl/ml (with S9-mix, experiment 2) for 4 hours. Fixation of the cells was performed 13 days post-exposure with the test material. Appropriate negative, solvent and positive controls were included in the test and gave the expected result. Cytotoxicity was observed at concentrations of 2.4 µL/mL (without S9-mix) and 3.2 µl/ml (with S9-mix). No increase of the mutation frequency in a dose-dependent manner in comparison with historical control range was recorded. Based on the results of the study test substance was found to be non-mutagenic with and without metabolic activation under the tested conditions.

 

Taking into consideration the above results generated from the structurally analogue substance the target substance is expected to show similar genetic toxicity in mammalian cells.

Justification for classification or non-classification

The available data on genetic toxicity in vitro and in vivo on the registered substance and structural related analogue substances do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.