Triethoxyhexadecylsilane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Dec 2011 - 20 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

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:

other: mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Triethoxyoctylsilane

EC Number:

220-941-2

EC Name:

Triethoxyoctylsilane

Cas Number:

2943-75-1

Molecular formula:

C14H32O3Si

IUPAC Name:

Triethoxy(octyl)silane

Test material form:

liquid

Method

Target gene:

Thymidine kinase

Species / strain

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:

Preliminary experiment I:
- 0.2, 0.5, 2.5, 5.0, 7.5 and 10.0 mM (with and without metabolic activation)
Preliminary experiment II:
- 0.01, 0.1, 1.0, 2.0, 5.0, 10.0 mM without metabolic activation)
Experiment I:
- 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 mM (with and without metabolic activation)
Experiment II:
- 0.15, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM (with metabolic activation)
- 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.10 and 0.20 mM (without metabolic activation)

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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: in medium

DURATION:
- Exposure duration: 4 h (short-term exposure), 24 h (long-term exposure)
- Expression time (cells in growth medium): 48 h
- Selection time: about 14 days

SELECTION AGENT: 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 10E6 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.

Results and discussion

Test results

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was noted in preliminary experiment I with and without metabolic activation, preliminary experiment II without metabolic activation, Experiment I with and without metabolic activation and in Experiment II with metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In Experiment I with metabolic activation the relative total growth (RTG) was 74.3% for the highest concentration (10.0 mM) evaluated. At two concentrations (1.0 mM and 2.5 mM) growth inhibition was observed with a RTG of 63.7% and 67.1%, respectively. However, considering all evaluated concentrations this effect showed no concentration relationship. The highest concentration evaluated without metabolic activation was 10.0 mM with a RTG of 14.6%. The Experiment without metabolic activation displayed a concentration related decrease in RTG starting at 1.0 mM with a RTG of 49.2%. In Experiment II with metabolic activation the relative total growth (RTG) was 76.4% for the highest concentration (10.0 mM) evaluated. At two concentrations (2.0 mM and 6.0 mM) growth inhibition was observed with a RTG of 42.3% and 58.9%, respectively. However, considering all evaluated concentrations this effect showed no concentration relationship. The highest concentration evaluated without metabolic activation was 0.20 mM with a RTG of 0.9%. Due to high cytotoxicity the highest concentration was not considered for evaluation of mutagenicity. Experiment II without metabolic activation displayed a concentration related decrease in RTG starting at 0.1 mM with a RTG of 16.8%. In Experiments I and II no biologically relevant increase of mutants were found after treatment with the test item (with and without metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed in Experiment I with metabolic activation and in Experiment II with and without metabolic activation. In Experiment I without metabolic activation a dose-response relationship was observed in the higher concentrations. Additionally, in Experiments I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

Any other information on results incl. tables

Table 1: 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

Table 2: Summary of 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

Table 3: Summary of 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	-

MF - mutant frequency

IMF = induced mutant frequency

RTG = relative total growth

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Triethoxyoctylsilane has been tested for mutagenicity to mammalian cells in a reliable study conducted according to OECD TG 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.