Tris(2-methoxyethoxy)vinylsilane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial reverse mutation assay:  for mutagenicity in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (OECD Test Guideline 471) (BioReliance, 1999).

In vitro cytogenicity in mammalian cells: tris(2-methoxyethoxy)vinylsilane was negative for clastogenicity in Chinese hamster ovary cells (OECD Test Guideline 473) (BioReliance, 2004).

In vitro gene mutation in mammalian cells: tris(2-methoxyethoxy)vinylsilane was negative for mutagenicity in L5178 mouse lymphoma cells (OECD Test Guideline 476) (Harlan Cytotest Cell Research GmbH, 2010).

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:

1999-07-09 to 1999-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

100, 333, 1000, 3333, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Stability in solvent

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without activation

Details on test system and experimental conditions:

METABOLIC ACTIVATION
0.5 ml of S9 mix containing 10% Aroclor induced rat liver S9 with NADP as cofactor. The S9 was assayed for ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to S. typhimurium TA100

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

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 hours
- Expression time (cells in growth medium): 48-72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 hours

SELECTION AGENT (mutation assays): not specified

NUMBER OF REPLICATIONS: concentrations were plated in triplicate, experiment was repeated

NUMBER OF CELLS EVALUATED: 0.3 x 10E09 cells per ml

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:

OTHER: The study was performed in two phases, using the preincubation and plate incorporation methodology. The first phase, the preliminary toxicity study, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article.

Evaluation criteria:

A substance is considered positive if it causes a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concnetrations of test substance, of greater than two times (TA 98, 100 and E. coli) or 3 times (TA 1535 and 1537) the vehicle control value.

Statistics:

No statistical evaluation carried out.

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Preliminary Toxicity Test: Ten dose levels, ranging from 6.7 to 5000 ug/plate were tested with the four Salmonella strains and WP2 uvrA in the presence or absence of S9.  Neither precipitate nor appreciable toxicity was observed. 

No positive responses were observed in the mutagenicity assays. Neither precipitate nor appreciable toxicity was observed. All bacterial strains exhibited mutagenic responses to the positive control substances, indicating that the tests were sensitive and valid.

Table 1: Preincubation experiment 1: Number of revertants per plate (mean of 3 plates)  

Concentration µg/plate	Strain    TA 98		Strain    TA 100		Strain    TA 1535		Strain    TA 1537		E. coli WP2 uvrA
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0*	16	21	137	134	10	9	8	8	12	17
100	12	18	146	122	11	14	5	6	15	16
333	11	16	141	154	9	10	7	7	15	15
1000	13	17	141	161	9	8	6	6	16	15
3333	12	11	134	159	12	7	6	6	16	16
5000	12	15	135	147	9	7	7	5	19	17
Positive control	745	416	700	611	420	100	229	78	371	173

* Solvent control with DMSO

Table 2: Preincubation experiment 2: Number of revertants per plate (mean of 3 plates)

Concentration µg/plate	Strain    TA 98		Strain    TA 100		Strain    TA 1535		Strain    TA 1537		E. coli WP2 uvrA
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0*	14	16	104	131	13	12	9	9	12	15
100	14	15	112	136	10	13	10	9	15	12
333	13	17	127	143	13	12	9	9	16	13
1000	14	16	108	147	13	11	6	8	13	14
3333	11	14	111	123	11	12	6	9	15	14
5000	12	17	109	153	13	13	5	7	12	14
Positive control	480	400	620	617	294	68	51	68	282	91

* Solvent control with DMSO

Conclusions:

In an in vitro gene mutation study in bacteria, conducted accordig to OECD test guideline 471 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane has been tested using S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA. No increase in the number of revertants was observed in any strain with or without metabolic activation, neither in the preliminary plate incorporation range-finding study nor in the initial and repeat preincubation assays. The substance was tested to limit concentrations and appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-10-08 to 2003-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

350 (toxicity test only), 700, 1400, 2801 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (dried before use)
- Justification for choice of solvent/vehicle: based on informatin from sponsor and compatibility with target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (with and without activation); 20 hours (without activation)
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid added 2 hours before harvest.
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 200 metaphase spreads were examined and scored for chromatid and chromosome-type aberrations


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell count and percent viability

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Test substances considered positive if it produced a statistically significant, dose-dependent increase relative to control in the percentage of cells with aberrrations, in excess of histroic solvent controls.

Statistics:

Fisher's exact test was used for pairwise comparison of treatment with control. If positive, Cochran-Armitage used to measure dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

RANGE-FINDING/SCREENING STUDIES: in the preliminary toxicity assay, the maximum dose tested was 2801 µg/mL. The test substance was soluble in treatment medium at all dose levels tested. No substantial toxicity (more than or equal to 50% reduction in cell growth relative to the solvent control) was observed up to the highest test substance dose level in any treatment condition. Based on these findings, the dose levels for the chromosome aberration assay ranged from 350 to 2801 µg/mL for all three exposure groups. 

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

Due to a lack of substantial toxicity, the highest dose level selected for microscopic analysis was the
maximum dose selected for chromosome aberration. The percentage of cells with structural or numerical aberrations
in the test substance groups was not significantly different than the solvent control.

Table 1 Results of chromosome aberrations analysis, 4 hour treatment without activation

Treatment (µg/ml)	S9 Activation	Treatment time	Mean Mitotic Index	Cells With Aberrations
				Numerical (%)	Structural (%)
0 (DMSO)	-	4	10.1	2.5	2.5
700	-	4	9.4	2	0
1400	-	4	9.6	2	2
2801	-	4	8.5	1.5	1
Positive control 0.2	-	4	10.7	2.5	10.0**

 

Table 2 Results of chromosome aberrations analysis, 4 hour treatment with activation

Treatment (µg/ml)	S9 Activation	Treatment time	Mean Mitotic Index	Cells With Aberrations
0 (DMSO)	+	4	9.9	3.5	10.0**
700	+	4	10.7	4	1.5
1400	+	4	9.2	3	0
2801	+	4	10.5	5	1.5
Positive control 10	+	4	8.8	3	33.0**

 

Table 3 Results of chromosome aberrations analysis, 20 hour treatment without activation

Treatment (µg/ml)	S9 Activation	Treatment time	Mean Mitotic Index	Cells With Aberrations
0 (DMSO)	-	20	7.8	3	0
700	-	20	7.6	2	0.5
1400	-	20	9	6.5	0.5
2801	-	20	9	6.5	0.5
Positive control 0.1	-	20	9.9	2.5	9.5**

** 

Conclusions:

In an in vitro chromosome aberration study, conducted according to OECD test guideline 473 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane has been tested in a valid study. No increase in the number of cells with aberrations was observed at any concentration with and without metabolic activation when tested in Chinese hamster ovary cells up to limit concentrations. The expected results were obtained with solvent and positive controls. It is concluded that the test substance is negative for the induction of chromosomal aberrations under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital, beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

187.5-3000 µg/mL with and without activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.

Untreated negative controls:

no

Remarks:

13.0 - 19.5 µg/ml

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without activation

Untreated negative controls:

no

Remarks:

3.0 – 6.0 µg/ml

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

ACTIVATION: phenobarbital, beta-naphthoflavone induced rat liver S9, NADP and glucose-6-phosphate as cofactor. Concentration in final test medium: 5%
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (with and without activation), 24 hours (without activation)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT: selective medium with TFT

NUMBER OF REPLICATIONS: duplicate controls and test concentrations, duplicate microtiter plates for mutant selection and viability determination

NUMBER OF CELLS EVALUATED: 1 x 10E06 cells/flask treated; 4 x 10E03 cells per well of microtiter plates for mutant selection; 2 cells per well for viability assessment.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency is reproducibly dose dependant and exceeds a threshold of 126 colonies per 10E06 cells above the corresponding solvent control. Results rejected if the relative total growth is less than 10% of the vehicle control unless unless the exception criteria specified by the IWGT recommendations are fulfilled.

Statistics:

A linear regression (least squares) comparison of number of mutant colonies from treated and solvent control groups was used to assess dose dependence. Biological relevance was considered alongside statistical significance.

Key result

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:

not applicable

Positive controls validity:

valid

Table 1 - Experiment I / 4 h treatment

	Concentration µg/ml	S9 mix	Relative total growth	Mutant colonies/10x6 cells	Threshold	Relative total growth	Mutant colonies/10x6 cells	Threshold
Column	1	2	3	4	5	6	7	8
			Culture I			Culture II
Solvent control with DMSO		-	100.0	170	296	100	206	332
Positive control with MMS	19.5	-	42.6	477	296	70.9	379	332
Test item	93.8	-	culture was not continued#			culture was not continued#
Test item	187.5	-	208.9	167	296	108.6	220	332
Test item	375.0	-	160.4	213	296	104.9	215	332
Test item	750.0	-	101.9	174	296	83.9	267	332
Test item	1500.00	-	112.8	205	296	125.4	188	332
Test item	3000.0	-	173.5	167	296	142.0	155	332
Solvent control with DMSO		+	100.0	144	270	100.0	198	324
Positive control with CPA	3.0	+	59.6	374	270	84.0	229	324
Positive control with CPA	4.5	+	45.4	453	270	10.8	739	324
Test item	93.8	+	culture was not continued#			culture was not continued#
Test item	187.5	+	91.1	278	270	124.8	175	324
Test item	375.0	+	94.9	240	270	122.8	170	324
Test item	750.0	+	95.4	245	270	113.8	164	324
Test item	1500.0	+	118.4	212	270	98.4	186	324
Test item	3000.0	+	111.1	221	270	93.6	167	324

 Threshold = number of mutant colonies per 10x6 cells of each solvent plus 126

# culture was not continued since a minimum of four concentrations is required by the guidelines

Table 2 - Experiment II / 24 h treatment

	Concentration µg/ml	S9 mix	Relative total growth	Mutant colonies/10x6 cells	Threshold	Relative total growth	Mutant colonies/10x6 cells		Threshold
Column	1	2	3	4	5	6	7		8
			Culture I			Culture II
Solent. control with DMSO		-	100.0	169	295	100.0		143	269
Positive control with MMS	13.0	-	10.1	333	295	6.7		303	269
Test item	93.8	-	culture was not continued#			culture was not continued#
Test item	187.5	-	126.7	168	295	83.5		136	269
Test item	375.0	-	130.5	156	295	66.2		144	269
Test item	750.0	-	108.1	124	295	80.5		161	269
Test item	1500.0	-	95.5	165	295	82.2		142	269
Test item	3000.0	-	95.9	108	295	60.9		136	269

Threshold = number of mutant colonies per 10x6 cells of each solvent plus 126

# culture was not continued since a minimum of four concentrations is required by the guidelines

Table 3 - Experiment II / 4 h treatment

	Concentration µg/ml	S9 mix	Relative total growth	Mutant colonies/10x6 cells	Threshold	Relative total growth	Mutant colonies/10x6 cells	Threshold
Positive control with CPA	3	+	45.2	289	268	44.1	197	230
Positive control with CPA	4.5	+	29.9	326	268	26	308	230
Test item	93.8	+	culture was not continued#			culture was not continued#
Test item	187.5	+	98.9	154	268	85.9	130	230
Test item	375.00	+	86.2	163	268	88.9	141	230
Test item	750.00	+	72.5	162	268	106.3	109	230
Test item	1500.00	+	84.3	196	268	69.1	201	230
Test item	3000.00	+	127.5	168	268	68.5	178	230

Threshold = number of mutant colonies per 10x6 cells of each solvent plus 126

# culture was not continued since a minimum of four concentrations is required by the guidelines

Conclusions:

In an in vitro mammalian mutagenicity study, conducted according to OECD test guideline 476 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane has been tested in a valid study. No increase in the number of mutants was detected either with or without metabolic activation in either of the cultures of mouse lymphoma L5178Y cells at any dose up to limit concentrations. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo testing is not required as the results of all the in vitro studies were negative.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 and in compliance with GLP, tris(2-methoxyethoxy)vinylsilane was tested using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. No increase in the number of revertants was observed in any strain with or without metabolic activation, neither in the preliminary plate incorporation range-finding study nor in the initial and repeat preincubation assays. The substance was tested to limit concentrations and appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Bioreliance, 1999).

In a supporting in vitro gene mutation study in bacteria, conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP, no increase in the number of revertants per plate was observed with or without activation. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Microtest Research, 1988).

In a supporting in vitro gene mutation study in bacteria, conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP, the test substance was negative for mutagenicity to bacteria under the conditions of the test (Hüls, 1993).

In an in vitro cytogenicity study in mammalian cells, conducted according to OECD Test Guideline 473 and in compliance with GLP, no increase in the number of cells with aberrations was observed at any concentration with and without metabolic activation when tested in Chinese hamster ovary cells up to limit concentrations. The expected results were obtained with solvent and positive controls. It is concluded that the test substance is negative for the induction of chromosomal aberrations under the conditions of the test (BioReliance, 2004).

In an in vitro gene mutation study in mammalian cells, conducted according to OECD Test Guideline 476 but not in compliance with GLP, no increase in the number of mutants was detected either with or without metabolic activation in either of the cultures of mouse lymphoma L5178Y cells at any dose up to limit concentrations. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of this test (Harlan Cytotest Cell Research GmbH, 2010).

Justification for classification or non-classification

Based on the available data tris(2-methoxyethoxy)vinylsilane does not require classification for genotoxicity according to Regulation (EC) No 1272/2008.