Triethoxy(2,4,4-trimethylpentyl)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): negative with and without metabolic activation in all strains tested (OECD Test Guideline 471 and in compliance with GLP) (RCC Cytotest Cell Research, 1998).
Cytogenicity in mammalian cells: negative with and without metabolic in Chinese lung fibroblasts (V79) (OECD Test Guideline 473 and in compliance with GLP) (BSL Bioservice, 2001e).
Mutagenicity in mammalian cells: read across from structural analogue triethoxy(octyl)silane (CAS 2943-75-1): negative with and without metabolic activation in mouse lymphoma L5178Y cells (OECD Test Guideline 476 and in compliance with GLP) (BSL Bioservice, 2012).

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:

1998-03-24 to 1998-04-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and Beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

33-5000 µg/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100 (without activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 1537, TA 98 (without activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 102 (without activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains (with activation)

Details on test system and experimental conditions:

ACTIVATION: Phenobarbital and beta-naphthoflavone induced rat liver S9 mix containing NADP as cofactor: Mix was 15% S9, 500 µl were mixed into a total volume of 2,700 µl prior to pouring giving a final concentration of approximately 1.2% S9.

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

DURATION

- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration. The initial plate incorporation experiment was repeated using the preincubation method.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn evaluation, revertant colony counts

Evaluation criteria:

The test substance is considered positive if there is a reproducible dose related increase in revertants, or reproducible increase in one test concentration.

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 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both experiments.

Cytotoxicity is defined as a reduction in the number of colonies compared with the solvent control and/or a sparse background lawn.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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

Additional information on results:

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

Table 2: Dose range-finding study Number of revertants per plate

	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)
Negative control*	41	21	No	148	137	No
0**	26	23	No	137	117	No
3	30	20	No	124	107	No
10	33	23	No	106	94	No
33	36	19	No	126	108	No
100	30	23	No	113	114	No
333	30	20	No	116	111	No
1000	36	28	No	153	117	No
2500	24	23	No	147	117	No
5000	34	16	No	156	118	No
Positive control	286	609	No	405	982	No

*negative control with water

**solvent control with DMSO

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA102
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
Negative control*	21	41	No	137	148	No	192	249	No
0**	23	26	No	117	137	No	178	273	No
33	19	36	No	108	126	No	169	209	No
100	23	30	No	114	113	No	163	206	No
333	20	30	No	111	116	No	158	227	No
1000	28	36	No	117	153	No	169	234	No
2500	23	24	No	117	147	No	161	202	No
5000	16	34	No	118	156	No	196	280	No
Positive control	609	286	No	982	405	No	983	1153	No

*negative control with water

**solvent control with DMSO

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

	TA1535			TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
Negative control*	12	9	No	18	14	No
0**	15	10	No	14	14	No
33	20	14	No	15	17	No
100	19	11	No	12	12	No
333	23	11	No	13	13	No
1000	16	18	No	13	15	No
2500	17	14	No	12	13	No
5000	14	15	No	13	15	No
Positive control	1024	147	No	153	112	No

*negative control with water

**solvent control with DMSO

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA102
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
Negative control*	29	52	No	159	156	No	279	245	No
0**	25	49	No	147	166	No	285	251	No
33	31	54	No	139	169	No	237	231	No
100	30	51	No	137	165	No	275	254	No
333	29	49	No	122	160	No	227	258	No
1000	30	49	No	139	166	No	222	252	No
2500	32	43	No	128	175	No	236	276	No
5000	28	51	No	134	157	No	231	243	No
Positive control	630	267	No	1236	688	No	1496	1049	No

*negative control with water

**solvent control with DMSO

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

	TA1535			TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
Negative control*	26	16	No	11	25	No
0**	29	13	No	11	22	No
33	22	18	No	10	24	No
100	21	16	No	9	30	No
333	22	16	No	7	23	No
1000	20	18	No	9	18	No
2500	21	11	No	9	16	No
5000	17	17	No	6	18	No
Positive control	847	121	No	128	126	No

*negative control with water

**solvent control with DMSO

Conclusions:

Triethoxy(2,4,4-trimethylpentyl)silane has been tested for mutagenicity to bacteria in a valid and reliable study conducted according to OECD Test Guideline 471 and in compliance with GLP. No mutagenic effect was observed for the test substance tested up to limit concentration in any of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in a plate incorporation experiment without and with metabolic activation. The result was confirmed in an independent pre-incubation assay. Appropriate positive, negative 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

Study period:

2000-11-02 to 2001-02-06

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)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - 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 lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Beta-Napthoflavone and Phenobarbital induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1, -S9: 500, 2000, 5000 µg/ml
Experiment 1, +S9: 250, 200, 5000 µg/ml
Experiment 2, -S9: 50, 200, 500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Cell culture medium. The test substance was prepared and diluted in cell culture medium. Freshly prepared solutions (applied within 1 h after preparation) were used.

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:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

ACTIVATION: S9 mix contained 0.75 mg/ml protein and NADP as cofactor. 50 µl were added to test organisms, test or control substance and medium in each chamber of Quadriperm dishes.

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours with and without S9 (exp 1); 20 hours without S9 (exp 2)

- Expression time (cells in growth medium): 16 hours (exp 1); 20 hours (exp 2)

- Selection time (if incubation with a selection agent): 2 days

- Fixation time (start of exposure up to fixation or harvest of cells): 17.5 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemide

STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: At least 200 per concentration

DETERMINATION OF CYTOTOXICITY

- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:

- Determination of polyploidy: determined in 100 cells per culture of each test group

Evaluation criteria:

A positive result is determined by a dose related increase in the number of cells with aberration, and a biologically relevant positive response for at least one of the test points.

The number of aberrations found in the negative control should be between 0 % and 4.5 %. The positive control should produce biologically relevant increases in the number of cells with structural chromosome aberrations.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500 μg/ml (exp 2, 20 h treatment without activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: noted in experiment 2 at 5000 μg/ml, 500 μg/ml selected as top concentration for evaluation in that experiment.

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

Table 2: Results of chromosome analysis Experiment 1, 4h treatment without activation (total count from 2 cultures)

		Solvent* Control	Positive Control	Low dose 500 μg/ml	Medium dose 2000 μg/ml	High dose 5000 μg/ml
Cytotoxicity		No	No	No	No	No
		Mean
Chromatid aberrations	gaps	3	4	0	1	2
	deletions	0	1	1	0	1
	interchanges	0	21	0	1	2
Chromosome	gaps	-	-	-	-	-
	deletions	0	0	0	0	0
	interchanges	0	4	0	0	0
Mitotic index		100 %	103 %	140 %	116 %	86 %
Polyploidy		6	7	1.5	1	4
Endo reduplication		ND	ND	ND	ND	ND

 *Solvent control with culture medium

ND not determined

Table 3: Results of chromosome analysis Experiment 1, 4h treatment with activation (total count from 2 cultures)

		Solvent* Control	Positive Control	Low dose 250 μg/ml	Medium dose 2000 μg/ml	High dose 5000 μg/ml
Cytotoxicity		No	No	No	No	No
		Mean
Chromatid aberrations	gaps	2	4	4	2	0
	deletions	0	1	0	0	0
	interchanges	2	17	1	1	1
Chromosome	gaps	-	-	-	-	-
	deletions	0	0	0	0	0
	interchanges	0	0	0	0	0
Mitotic index		100 %	126 %	93 %	95 %	83 %
Polyploidy		3.5	5	3.5	3.5	2
Endo reduplication		ND	ND	ND	ND	ND

 *Solvent control with culture medium

ND not determined

Table 4: Results of chromosome analysis Experiment 2, 20h treatment without activation (total count from 2 cultures)

		Solvent* Control	Positive Control	Low dose 50 μg/ml	Medium dose 200 μg/ml	High dose 500 μg/ml
Cytotoxicity		No	Yes	No	No	Yes
		Mean
Chromatid aberrations	gaps	1	11	3	1	0
	deletions	0	2	1	0	0
	interchanges	0	35	0	0	0
Chromosome	gaps	-	-	-	-	-
	deletions	0	0	0	0	0
	interchanges	0	2	0	0	0
Mitotic index		100 %	51 %	91 %	85 %	29 %
Polyploidy		3.5	2.5	4.5	1.5	3
Endo reduplication		ND	ND	ND	ND	ND

 *Solvent control with culture medium

ND not determined

Conclusions:

Triethoxy(2,4,4-trimethylpentyl)silane has been tested for clastogenicity in a valid and reliable test performed according to OECD Test Guideline 473 and in compliance with GLP. The test substance did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line with and without metabolic activation when tested up to limit concentrations. Appropriate solvent and postive controls were included and gave expected results. It is concluded 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

Study period:

2011-12-21 to 2012-03-20

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IWGT Recommendations

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-expt I: 0.2, 0.5, 2.5, 5.0, 7.5 and 10.0 mM (+/-MA); Pre-expt II: 0.01, 0.1, 1.0, 2.0, 5.0, 10.0 mM (-MA, 24 h exp); Expt I: 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 mM (+/-MA); Expt II: 0.15, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM (+MA); 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.10 and 0.20 mM (-MA)

Vehicle / solvent:

THF was used as solvent (0.35% THF v/v in the samples). To reach a final concentration of 0.35% THF v/v in the samples the test item stock solution was diluted in RPMI + 5% HS for short-term exposure or RPMI + 7.5% HS for long-term exposure. After adding the THF stock solution to cell culture medium precipitate formed.
The solvent was compatible with the survival of the cells and the activity of the S9 mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation: 3.5 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation: 200 µg/mL and 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in THF (0.35% THF v/v in the samples)
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/ml trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

ACTIVATION: S9 mix contained glucose-6-phosphate and NADP as co-factors, and sufficient S9 to give a final protein concentration in the cultures of 0.75 mg/ml

Evaluation criteria:

The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

RTG of 14.6% and 9.7% without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Pre-experiment for toxicity with and without metabolic activation

Concentration (mM)	Number of cells 4 h after treatment		Number of cells 24 h after treatment		Number of cells 48 h after treatment		Suspension growth		Relative suspension growth
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
Negative control	271000	278000	946000	948000	1600000	1570000	15.1	14.9	89.8	103.6
	307000	356000	1050000	1150000	1510000	1540000	15.9	17.7	94.1	123.3
Solvent control	307000	256000	104000	807000	1560000	1570000	16.2	12.7	100.0	100.0
	339000	313000	112000	101000	1560000	1590000	17.5	16.1
0.2	284000	271000	77000	593000	1540000	1480000	11.9	8.8	70.4	61.1
0.5	307000	252000	876000	377000	1490000	1190000	13.1	4.5	77.5	31.2
2.5	289000	220000	728000	186000	1620000	637000	11.8	1.9	70.0	13.3
5.0	316000	225000	798000	137000	1550000	286000	12.4	0.9	73.4	6.0
7.5 (P)	303000	271000	650000	239000	1590000	850000	10.3	2.6	61.3	17.8
10.0 (P)	305000	244000	728000	183000	1580000	481000	11.5	1.4	68.3	10.0

Summary: Experiment 1 and 2 with metabolic activation

	Treatment (mM)	RTG (%)	MF (mutants/10^6cells)	IMF (mutants/10^6cells)	Precipitate
	Negative control	103.5	125.6	/	-
		98.1		/	-
	Solvent control	100.0	135.2	/	-
				/	-
	0.1	72.3	142.3	7.2	-
Exp 1	0.2	73.5	143.3	8.1	-
	0.5	84.9	97.1	-38.1	-
	1.0	63.7	182.8	47.6	-
	2.5	67.1	144.4	9.2	-
	5.0	87.2	117.6	-17.6	-
	7.5	78.1	154.8	19.6	-
	10.0	74.3	130.3	-4.8	+
	Positive control	38.4	918.4	783.2	-
	Treatment (mM)	RTG (%)	MF (mutants/10^6cells)	IMF (mutants/10^6cells)	Precipitate
	Negative control	98.9	116.5	/	-
		111.8		/	-
	Solvent control	100.0	116.7	/	-
				/	-
	0.15	111.4	105.1	-11.6	-
Exp 2	0.3	96.2	131.1	14.4	-
	0.7	75.7	128.5	11.8	-
	2.0	42.3	220.5	103.8	-
	4.0	72.7	138.8	22.2	-
	6.0	58.9	170.6	53.9	-
	8.0	74.1	134.6	17.9	+
	10.0	76.4	154.9	38.2	+
	Positive control	50.5	1141.0	1024.3	-

MF - mutant frequency

IMF = induced mutant frequency

RTG = relative total growth

Summary: Experiment 1 and 2 without metabolic activation

	Treatment (mM)	RTG (%)	MF (mutants/10^6cells)	IMF (mutants/10^6cells)	Precipitate
	Negative control	122.0	142.0	/	-
		129.6		/	-
	Solvent control	100.0	144.3	/	-
				/	-
	0.1	100.3	177.2	32.9	-
Exp 1	0.2	87.3	157.4	13.0	-
	0.5	98.4	186.0	41.6	-
	1.0	49.2	190.1	45.7	-
	2.5	37.8*	179.4	35.1	-
	5.0	37.2	179.3	35.0	-
	7.5	17.1	224.1	79.8	+
	10.0	14.6	264.7	120.4	+
	Positive control 1	75.1	817.6	673.2	-
	Positive control 2	85.2	564.1	419.7	-
	Treatment (mM)	RTG (%)	MF (mutants/10^6cells)	IMF (mutants/10^6cells)	Precipitate
	Negative control	145.4	110.6	/	-
		122.8		/	-
	Solvent control	100.0	109.3	/	-
				/	-
	0.001	114.6	108.6	-0.8	-
Exp 2	0.002	99.3	94.7	-14.6	-
	0.005	103.4	120.0	10.6	-
	0.01	62.1	121.8	12.5	-
	0.02	82.5	160.0	50.6	-
	0.05	98.6	108.4	-0.9	-
	0.10	16.8	173.0	63.6	-
	0.20	0.9	119.1	9.7	-
	Positive control 1	21.3	2373.6	2264.2	-
	Positive control 2	14.7	1826.6	1717.3	-

Conclusions:

Triethoxy(octyl)silane has been tested for mutagenicity to mammalian cells in a reliable study conducted according to OECD Test Guideline 476 and in compliance with GLP. No biologically relevant increase in mutation rate was found in mouse lymphoma L5178Y cells with or without metabolic activation in either the initial or the repeat experiment, up to limit and cytotoxic concentrations. The global evaluation factor was not exceeded at any concentration. In addition, colony sizing showed no clastogenic effects in either experiment. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay oral study in mouse: Negative (OECD Test Guideline 474 and in compliance with GLP) (BSL Bioservice, 2001)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-11-27 to 2001-01-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: Harlan Winkelman (D-33178, Borchen)

- Weight at study initiation: 24 - 42 g

- Assigned to test groups randomly: yes, under following basis: assigned and tail tagged by chance

- Housing: Macrolon Type III (Hereto, D-79302 Emmendingen)

- Diet (e.g. ad libitum): ad libitum

- Water (e.g. ad libitum): ad libitum

- Acclimation period: minimum of 5 days


ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19 - 25 ˚c

- Humidity (%): 49.5 - 61 %

- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: To: 15 January 2001

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)

- Justification for choice of solvent/vehicle: The vehicle was chosen because it is relatively non toxic to the test animals

- Lot/batch no. (if required): 36H0738

Details on exposure:

Route of exposure: Oral

Duration of treatment / exposure:

24 - 48 hours

Frequency of treatment:

The animals received the test item once orally.

Post exposure period:

Animals were sacrificed 24 - 48 h after treatment.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

five

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide

- Justification for choice of positive control(s): standard guideline positive control

- Route of administration: oral

- Doses / concentrations: 10 ml/kg bw of 0.9 % solution in 0.9 % NaCl

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: limit dose

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): no further information

DETAILS OF SLIDE PREPARATION: centrifuged cells were suspended in a thin layer of FCS and smeared on a slide. The smears were air dried and stained with May-Grunwald (Merck, D-64293 Darmstadt) / Giemsa (Merck, D-64293 Darmstadt).

METHOD OF ANALYSIS: microscopic examination. 2000 PCE scored for incidence of polychromatic erythrocytes with micronuclei. Ratio of PCE / NCE was scored based on 1000 erythrocytes (PCE+NCE)

Evaluation criteria:

A test item is classified as mutagenic if it induces either a statistically significant dose related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.

Statistics:

Micronuclei in 2000 PCE per animal were counted for each sex. NCE per 1000 PCE, Sum and Mean calculated for each sex.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See table 3

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): negative

- Ratio of PCE/NCE (for Micronucleus assay): see table 3. The PCE/NCE ratio was unchanged in treated animals, so there is no evidence that the test substance reached the target tissue.

- Appropriateness of dose levels and route: appropriate dose and route

- Statistical evaluation: no statistically significant induction of micronuclei occurred.

Table 3: Results of in vivo micronucleus test

		Vehicle Control	Positive Control	200 mg/kg bw	1000 mg/kg bw	2000 mg/kg bw	2000 mg/kg bw
Number of cells evaluated		2000	2000	2000	2000	2000	2000
Sampling time (h)		24	24	24	24	24	48
Number of erythrocytes	normo­chromatic	NR	NR	NR	NR	NR	NR
	poly­chromatic	2000	2000	2000	2000	2000	2000
	polychromatic with micronuclei	7 Male 5.2 Female	70.2 Male 42.4 Female	7.6 Male 9.2 Female	8.4 Male 4.8 Female	9.4 Male 5.0 Female	7.4 Male 4.8 Female
Ratio of erythrocytes	polychromatic / normochromatic	Male 1000/679.4 Female 1000/766.6	Male 1000/608 Female 1000/657.6	Male 1000/712.4 Female 1000/655.8	Male 1000/656.4 Female 1000/611.6	Male 1000/657 Female 1000/641.2	Male 1000/1064.6 Female 1000/511
	polychromatic with micro­nuclei / normochromatic	NR	NR	NR	NR	NR	NR

 

Conclusions:

Triethoxy(2,4,4-trimethylpentyl)silane has been tested in a reliable in vivo mouse micronucleus assay according to OECD Test Guideline 474 and in compliance with GLP. No statistically significant increase in the number of cells with micronuclei was observed after oral administration of the limit dose of 2000 mg/kg bw. Appropriate positive and vehicle controls were included and gave expected results. The PCE / NCE ratio was slightly affected in treated males, indicating that the test item was of low toxicity to the target tissue. It is concluded that the test substance is negative for the induction in micronuclei under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Information is available for the registered substance, triethoxy(2,4,4-trimethylpentyl)silane, from reliable studies for in vitro mutagenicity to bacteria and cytogenicity, and from an in vivo micronucleus assay. No information is available for the registered substance for in vitro mutagencicity to mammalian cells, however, data are available for the structural analogue, triethoxy(octyl)silane. See attachment to Section 13 for justification of read-across.

Triethoxy(2,4,4-trimethylpentyl)silane has been tested for mutagenicity to bacteria in a valid and reliable study conducted according to OECD Test Guideline 471 and in compliance with GLP. No mutagenic effect was observed for the test substance tested up to limit concentration in any of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in a plate incorporation experiment without and with metabolic activation. The result was confirmed in an independent pre-incubation assay. Appropriate positive, negative 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 (RCC Cytotest Cell Research, 1998).

Triethoxy(2,4,4-trimethylpentyl)silane has been tested for clastogenicity in a valid and reliable test performed according to OECD Test Guideline 473 and in compliance with GLP. The test substance did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line with and without metabolic activation when tested up to limit concentrations. Appropriate solvent and postive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test (BSL Bioservice, 2001e).

An additional study for in vitro cytogenicity is available (Chemicals Evaluation and Research Institute, 2001b). This study reported a positive result in Chinese hamster fibrobalsts (V79 cells) in the absence of metabolic activation. The study report available for this result is an extended summary, and does not include tables of results, so there is not enough information to for a full independent assessment, but it is noted by the reviewer that the test substance was much more cytotoxic in the absence of activation, and the increase in abnormalities recorded was most marked at cytotoxic concentrations. In addition, the test substance is reported to be a mixture of 2,4,4-trimethylpentyltriethoxysilane and 5,5-dimethylhexyltriethoxysilane. In view of these factors, the availability of a reliable negative result in a similar study and the negative result in vivo, the study with the negative result was chosen as key.

The structural analogue, triethoxy(octyl)silane has been tested for mutagenicity to mammalian cells in a reliable study conducted according to OECD Test Guideline 476 and in compliance with GLP. No biologically relevant increase in mutation rate was found in mouse lymphoma L5178Y cells with or without metabolic activation in either the initial or the repeat experiment, up to limit and cytotoxic concentrations. The global evaluation factor was not exceeded at any concentration. In addition, colony sizing showed no clastogenic effects in either experiment. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test (BSL Bioservice, 2012).

Triethoxy(2,4,4-trimethylpentyl)silane has been tested in a reliable in vivo mouse micronucleus assay according to OECD Test Guideline 474 and in compliance with GLP.  No statistically significant increase in the number of cells with micronuclei was observed after oral administration of the limit dose of 2000 mg/kg bw. Appropriate positive and vehicle controls were included and gave expected results. The PCE / NCE ratio was slightly affected in treated males, indicating that the test item was of low toxicity to the target tissue. It is concluded that the test substance is negative for the induction in micronuclei under the conditions of the test (Bioservice, 2001).

Justification for classification or non-classification

Based on the available in vitro data and in vivo data on the registered substance and its structural analogue, triethoxy(2,4,4-trimethylpentyl)silane is not classified for mutagenicity according to Regulation (EC) No 1272/2008.