Methylsilanetriyl triacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data are available from reliable in vitro studies of bacterial mutagenicity and cytogenicity to mammalian cells for methylsilanetriyl triacetate. No further information is available for the registered substance, however, data are available for the closely related substances triethoxy(methyl)silane (CAS number 2031-67-6) and trichloro(methyl)silane (CAS number 75-79-6) from in vitro assays on mutagenicity to mammalian cells.
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA (OECD TG 471) (BioReliance, 2002a).
Cytogenicity in mammalian cells: negative in CHO cells (OECD TG 473) (BioReliance, 2002b).
Mutagenicity in mammalian cells: read-across from analogous substance trichloro(methyl)silane (CAS 75-79-6): negative in mouse lymphoma L5178Y cells (similar to OECD 476) (Litton Bionetics, 1978b).
Mutagenicity in mammalian cells: read-across from analogous substance triethoxy(methyl)silane (CAS 2031-67-6): negative in mouse lymphoma L5178Y cells (similar to OECD 476) (Litton Bionetics, 1978a).

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:

2001-10-24 - 2001-11-28

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 1254 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: Sponsor's request due to compatibility with the target cells. A fresh bottle, containing less that 0.1% water, to be opened and used once for each phase of the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 10 µg/plate

Remarks:

WP2 uvrA with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 1.0 µg/plate

Remarks:

TA98, TA100, TA1535, TA1537 with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without metabolic activation 1.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 without metabolic activation 1.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without metabolic activation 75 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without metabolic activation 1000 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

ACTIVATION: S9 mix contained glucose-6 phosphate and NADP as co-factors, and 10% S9. 0.5 ml of S9 was added to 2 ml top agar, 100 µl tester strain and 50 µl of test solution giving a final concentration of 1% S9.

DURATION

- Exposure duration: 48 - 72 hours at 37±2°C


SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated


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

Evaluation criteria:

The mean of each positive control must exhibit at least a 3 fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn. Data sets will be judged positive if the increase in mean revertants at the peak dose response is equal to or greater than 2 times the mean negative control value.

Statistics:

None shown in report

Key result

Species / strain:

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

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

 Experiment 1 Plate incorporation - Number of revertants per plate (mean of three plates)

Treatment µg/plate	TA98		TA100		TA1535		TA1537		WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0*	15	18	154	181	14	12	4	7	11	11
100	11	21	170	161	13	10	3	5	10	8
333	11	16	155	196	9	13	4	6	9	6
1000	11	17	140	178	13	11	3	5	9	8
3333	15	18	166	152	21	8	6	4	10	12
5000	20	21	149	159	20	14	5	5	10	8
Positive control	92	365	479	584	215	102	633	58	71	296

*solvent control with DMSO

 Experiment 2 Plate incorporation - Number of revertants per plate (mean of three plates)

Treatment µg/plate	TA98		TA100		TA1535		TA1537		WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0 *	16	20	134	154	19	12	8	7	12	12
100	10	19	138	165	21	15	5	10	14	13
333	15	23	152	163	17	19	5	10	12	17
1000	15	28	137	181	19	14	13	9	12	16
3333	15	25	136	145	21	16	9	6	10	14
5000	13	22	124	151	18	16	7	4	10	13
Positive control	67	495	441	690	203	124	352	137	84	291

 *solvent control with DMSO

Conclusions:

Methylsilanetriyl triacetate has been tested for mutagenicity to bacteria in a study which was conducted according to the OECD TG 471, and in compliance with GLP. No evidence of a test substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments. Appropriate positive and solvent 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

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

275, 550, 1100, 2200 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: information from the sponsor and compatibility with the 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 metabolic activation 10 and 20 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation 1 and 2 mg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (with and without metabolic activation) and 20 hours (without metabolic 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
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 200 cells were examined for chromosome aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: cell growth inhibition

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

Evaluation criteria:

A result was considered positive when there was a dose-dependent and statistically significant increase in the number of cells with aberrations.

Statistics:

Fischer's exact test was used to compare the percent aberrant cells of each treatment group with the solvent control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

15% and 2200 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none - pH was adjusted to approximately 7.0 by addition of 1N NaOH
- Effects of osmolality: none

Summary of results

Treatment time (hours)	Recovery time (hours)	Harvest time (hours)	Metabolic activation	Toxicity at highest dose scored (µg/ml)	Mitotic reduction	Structural abnormalities observed	Numerical abnormalities observed
4	16	20	-	None	None	None	None
20	0	20	-	None	None	None	None
4	16	20	+	None	13%	None	None

Conclusions:

Methylsilanetriyl triacetate has been tested according to OECD 473 and under GLP. No increase in the number of chromosome aberrations per cell or the number of cells with aberrations was detected at any concentration with and without metabolic activation in Chinese hamster ovary cells. Solvent and positive controls gave expected results. It is considered that the test substance is negative for the induction of chromosome 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:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

non-activated mouse liver S9 mix used for activation; positive control toxicity expt 2 +MA was lower than required by current guideline

Principles of method if other than guideline:

Method: Litton Bionetics standard procedure: Screening Program for the Identification of Potential Mutagens and Carcinogens. Protocol Number : DMT 100

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Mouse liver S9

Test concentrations with justification for top dose:

0.01, 0.02 0.04, 0.08, 0.16, and 0.32 µL/ml, equivalent to approximately 10, 20, 40, 80, 160 and 320 µg/ml
Vehicle

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: It is assumed by the reviewer that the solvent was chosen based on solubility properties and relative non-toxicity to L5178Y cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

(with activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours

- Expression time (cells in growth medium): 3 days

- Selection time (if incubation with a selection agent): 10 days

- Fixation time (start of exposure up to fixation or harvest of cells): 3 days 4 hours


SELECTION AGENT (mutation assays): THMG (thymidine, hypoxanthine, methoxotrexate, glycine)

NUMBER OF REPLICATIONS: 3 plates for each test concentration, assay repeated

DETERMINATION OF CYTOTOXICITY

- Method: relative total growth

Evaluation criteria:

A compound is considered mutagenic if:

- A dose response relationship is observed over three of the four dose levels employed

- The minimum increase at the high level of the dose response curve is at least 2.5 times greater than the solvent control value

- The solvent control data are within the normal range of spontaneous background for the TK locus.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 0.32 µl/ml, equivalent to 320 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance did not induce mutation at the thymidine kinase locus with or without activation. Because there was very little toxicity observed in the non activation tests, the study was repeated. The repeat study produced greater levels of toxicity but was still negative for inducing mutations.

Table 2 :Results of Mammalian Mutagenicity assay (Test 1) with L5178Y/TK^+/- Mouse Lymphoma cells

Concentration µg/ml	Mutant* Frequency	Mutant* Frequency	%Relative Growth.	%Relative Growth.	Cytotoxicity (yes/no)
	- MA	+ MA	- MA	+ MA	-
Solvent control	10.6	12.2	100	100	-
Negative control	12.2	10.3	123	64.1	-
Positive control	511.0	368.1	18.5	5.8	yes
0.1	8.0	-	134.2	-	no
0.02	10.8	23.1	140	72.5	no
0.04	7.3	10.5	134.1	99.1	no
0.08	11.3	14.1	118.1	101.2	no
0.16	18.1	17.6	112.4	94.1	no
0.32	-	12.9	-	45.7	yes

*Per 10⁶surviving cells

Solvent control with Ethanol

Table 3 : Results of Mammalian Mutagenicity assay (Test 2) with L5178Y/TK^+/-  Mouse Lymphoma cells

Concentration µg/ml	Mutant* Frequency	Mutant* Frequency	%Relative Growth.	%Relative Growth.	Cytotoxicity (yes/no)
	— MA	+ MA	— MA	+ MA	-
Solvent Control	14	17	100	100	No
Negative Control	25.3	15.8	83.1	89.2	No
Positive Control	385	514	27	2.8**	No
0.04	-	15.4	-	87.8	No
0.08	26.9	22.1	65.7	75.6	No
0.16	27.5	16.5	60.1	48	Yes
0.24	16.5	-	58.6	-	No
0.32	26.6	58	0.2	0.1	Yes

*Per 10⁶surviving cells

Solvent control with Ethanol

**It is noted that this level of toxicity is lower than acceptable under current guidelines.

Conclusions:

Trichloro(methyl)silane was tested for mutagenicity in mammalian cells in a reliable and reproducible assay according to a protocol that is similar to OECD 476. Appropriate concurrent negative and positive controls were included and the expected responses were observed, though the cytotoxicity observed with the positive controls in the with activation experiments was high (2.8% and 5.8%). No test-substance related increase in mutant frequency was observed. It is concluded that trichloro(methyl)silane is negative for the induction of mutation in mammalian cells under the conditions of the test.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The restrictions are that the experiment did not include replicate culture, and mouse liver S9 was used for metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Insufficient test concentrations / no duplicates

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase operon

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

mouse liver S9

Test concentrations with justification for top dose:

+/- MA 0.1, 0.2, 0.4, 0.8, 1.6 µl/ml

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethylnitrosamine

Remarks:

(with activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: four hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): medium containing thymidine kinase, hypoxanthine, methotrexate and glycine

NUMBER OF REPLICATIONS: single doses, triplicate counts

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A compound is considered mutagenic if there is an increase in the mutant frequency relative to the control, showing a dose response relationship over three of the doses employed, with a minimum increase at the highest dose at least 2.5 times greater than the solvent control value, which should be within the normal range for the TK locus.

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 Mutagenicity in L5178Y cells (mutant frequencies are the mean of three counts)

Concentration µl/ml	-MA		+MA
	Mutant frequency	% growth	Mutant frequency	% growth
Solvent control*	3.3	100	6.5	100
Negative control	4.2	80.3	3.0	84.4
0.1	10.9	103.7	12.1	75.0
0.2	15.5	71.1	8.3	64.7
0.4	6.4	58.5	13.6	68.1
0.8	3.9	80.2	14.4	89.6
1.6	4.9	51.5	7.5	50.7
Positive control	485.6	12.9	643.9	2.9

* Solvent control with ethanol

Conclusions:

Triethoxy(methyl)silane has been tested according to a protocol that is similar to OECD 476, without replication. No increase in mutant frequency was observed at any concentration with or without metabolic activation. The solvent and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity in mouse lymphoma L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data are available from reliable in vitro studies of bacterial mutagenicity and cytogenicity to mammalian cells for methylsilanetriyl triacetate. No further information is available for the registered substance, however, data are available for the closely related substances triethoxy(methyl)silane (CAS number 2031-67-6) and trichloro(methyl)silane (CAS number 75-79-6) from in vitro assays on mutagenicity to mammalian cells.

Methylsilanetriyl triacetate has been tested for mutagenicity to bacteria in a study which was conducted according to the OECD TG 471, and in compliance with GLP (BioReliance, 2002a). No evidence of a test substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments. Appropriate positive and solvent 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.

Methylsilanetriyl triacetate has been tested according to OECD 473 and under GLP (BioReliance, 2002b). No increase in the number of chromosome aberrations per cell or the number of cells with aberrations was detected at any concentration with and without metabolic activation in Chinese hamster ovary cells. Solvent and positive controls gave expected results. It is considered that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.

The structural analogue triethoxy(methyl)silane has been tested according to a protocol that is similar to OECD 476, but without replication (Litton Bionetics, 1978a). No increase in mutant frequency was observed at any concentration with or without metabolic activation. The solvent and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity in mouse lymphoma L5178Y cells under the conditions of the test.

A second structural analogue, trichloro(methyl)silane, was tested for mutagenicity in mammalian cells in a reliable and reproducible assay according to a protocol that is similar to OECD 476 (Litton Bionetics, 1978b). Appropriate concurrent negative and positive controls were included and the expected responses were observed, though the cytotoxicity observed with the positive controls in the experiments with activation was high (2.8% and 5.8%). It is noted by the reviewer that the toxicity of the positive control in the experiments with activation was greater than is acceptable under current guidelines. No test-substance related increase in mutant frequency was observed. It is concluded that trichloro(methyl)silane is negative for the induction of mutation in mammalian cells under the conditions of the test.

No evidence for genetic toxicity was observed in the in vitro studies, so it is considered that in vivo testing is not required.

Justification for classification or non-classification

Based on the available in vitro genotoxicity data, methylsilanetriyl triacetate is not classified for mutagenicity according to Regulation (EC) No 1272/2008.