Diacetoxydi-tert-butoxysilane

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:

From February 5 to February 27, 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according to OECD Guideline 476. GLP 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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Propyltriacetoxysilane

EC Number:

241-816-9

EC Name:

Propyltriacetoxysilane

Cas Number:

17865-07-5

IUPAC Name:

propylsilanetriyl triacetate

Test material form:

liquid

Method

Target gene:

Thyamidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

Experiment I:
With metabolic activation: 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 9.0 and 10.0 mM
without metabolic activation: 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 5.5 mM.
Experiment II:
With metabolic activation: 0.1, 0.3, 0.6, 1.2, 2.5, 5.0, 7.5 and 8.5 mM
without metabolic activation: 0.5, 1.0, 2.0, 3.0, 3.5, 4.0, 4.5 and 5.0 mM.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI cell culture was used as solvent (RPMI + 5% HS). The pH of cell culture medium as adjusted to physiological range with NaOH. The solvent was compatible with the survival of the cells and the activity of the S9 mix.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(treatment medium)

Controls 2

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Short-term exposure (4h): 1x10E+07 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence of absence of metabolic activation. After exposure, test item was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period. The cell density was determined each day and adjusted to 3x10E+05 cells/mL in a total culture volume of 20 mL, if necessary.
Long-term exposure (24h): 5x10E+06 cells were suspended in 25 mL RPMI medium with 7.5% horse serum (75 cm2 flasks) and exposed to designated concentrations of the test item in the absence of metabolic activation. After exposure, test item was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with PBS. Subsequently 3x10E+05 cells were suspended in 14 mL complete culture medium and incubated for an expression and growth period. The cell density was determined each day and adjusted to 3x10E+05 cells/mL in a total culture volume of 20 mL, if necessary.
After the expression period cloning efficiency and mutant frequency were analysed (see below).

DURATION
- Exposure duration:
Experiment 1: 4 hours (with and without metabolic activation)
Experiment II: 4 hours (with metabolic acitvation) and 24 hours (without metabolic activation)
- Expression time (cells in growth medium): 2 days, at 37 ºC in 5% Co2/95% humidified air.
- Selection time (if incubation with a selection agent): ~ 14 days at 37 ºC in 5% CO2/95% humidified air

SELECTION AGENT (mutation assays): TFT.

NUMBER OF REPLICATIONS: 1 replicate per dose (negative control, 2 replicates).

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY:
Method: Retalive total growth and relative cloning efficiency:
- Relative total growth (RTG): The RTG is the product of the relative suspension growth and the relative cloning efficiency for each culture.
- Suspention growht (SG): The SG reflects the number of times the cell number increases from the starting cell density.
- Cloning efficiency (CE): After the expression period the CE of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plated. The cells were incubated for at least 6 days at 37 ºC in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells) and those without cell growth (negative wells) compared to the total number of cultures seeded.
- Mutant frequency: Cultures were seed in selective medium. Cells from each experimental grow were seeded in four 96-well plates and were scored after incubation period. The mutant frequency was calculated dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. The mutant frequencies were compared with the Global Evaluation Factor (GEF= 126).

OTHER EXAMINATIONS:
Clastogenicity: Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. Small colonies were defined by slow growth and/or morphological alteration of the cell close.

Evaluation criteria:

The test item is considered mutagenic if following criteria are met: the inducing mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10E+06 cells, and a dose-dependent increase in mutant frequency is detected.
Combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (>=40% of total colonies) is an indicative for potential clastogenic effects and/or chromosomal aberrations.

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 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

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH detected with the test item was within the physiological range.
- Water solubility: a solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 10 mM. Based on the results of the solubility test RPMI cell culture was used as solvent (RPMI + 5% HS). The solvent was compatible with the survival of the cells and the activity of the S9 mix.
- Precipitation: No precipitation of the test item was noted in pre-experiment I and II and experiment I and II with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment for toxicity: The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 10.0 mM. In pre-experiment I six concentrations [0.1, 0.5, 2.5, 5.0, 7.5 and 10.0 mM] were tested with and without metabolic activation for 4h exposure. In the pre-experiment II (24h long-term exposure without metabolic activation) six concentrations [0.1, 0.5, 1.0, 2.0, 3.0 and 5.0 mM] were tested. The experimental conditions were the same as described below in the experimental performance. In experiment I 10.0 mM (with metabolic activation) and 5.5 mM (without metabolic activation) were selected as the highest concentrations. In experiment II 8.5 mM (with metabolic activation) and 5.0 mM (without metabolic activation were selected as the highest concentrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed in the pre-experiment I. However, this was not reproducible in the main experiments. In experiment I and II with and without metabolic activation growht inhibition was observed. In experiment I with metabolic activation to relative total growth (RTG) as 23.1% for the highest concentration (10.0 mM) evaluated. The highest concentration evaluated without metabolic activation was 5.5 mM with RTG of 10.1%. In experiment II with metabolic activation the relative total growth (RTG) was 10.5% for the highest concentration (8.5 mM) evaluated. The highest concentration evaluated without metabolic activation was 5.0 with a RTG of 10.3%.

Any other information on results incl. tables

MUTAGENICITY: All dose groups with and without metabolic activation were considered non-mutagenic under test conditions.

Experiment I and II with metabolic activation:

Test Group	Conc. [mM]	RCE [%]	RTG [%]	MF [mutants /10^6cells]	IMF [mutants /10^6cells]	GEF exceeded	Statistical significance	Precipitate
C1	0	100.0	100.0	82.0	/	/	/	-
C2					/	/	/	-
2	0.5	95.0	91.4	87.9	5.9	-	-	-
3	1.0	102.1	100.5	91.7	9.7	-	-	-
4	2.0	113.2	114.9	91.0	9.1	-	-	-
5	4.0	91.0	80.4	96.7	14.7	-	-	-
6	6.0	96.4	94.6	104.9	23.0	-	-	-
8	8.0	116.7	77.9	83.7	1.7	-	-	-
9	9.0	106.7	36.6	99.0	17.0	-	-	-
10	10.0	103.6	23.1	153.8	71.8	-	+	-
B[a]P	2.5	77.9	42.1	949.9	867.9	+	+	-

 

Test Group	Conc. [mM]	RCE [%]	RTG [%]	MF [mutants /10^6cells]	IMF [mutants /10^6cells]	GEF exceeded	Statistical significance	Precipitate
C1	0	100.0	100.0	82.0	/	/	/	-
C2					/	/	/	-
1	0.1	101.5	89.9	77.1	-4.4	-	-	-
2	0.3	114.5	106.7	79.5	-1.9	-	-	-
3	0.6	97.1	77.7	73.4	-8.1	-	-	-
4	1.2	122.0	107.4	69.0	-12.5	-	-	-
5	2.5	94.3	83.6	73.7	-7.7	-	-	-
6	5.0	106.1	96.0	112.7	31.2	-	-	-
7	7.5	87.8	23.5	90.0	8.5	-	-	-
8	8.5	91.6	10.5	91.7	10.3	-	-	-
B[a]P	3.5	77.2	46.8	601.3	519.9	+	+	-

 

Experiment I and II without metabolic activation:

Test Group	Conc. [mM]	RCE [%]	RTG [%]	MF [mutants /10^6cells]	IMF [mutants /10^6cells]	GEF exceeded	Statistical significance	Precipitate
C1	0	100.0	100.0	82.0	/	/	/	-
C2					/	/	/	-
1	0.1	93.4	95.4	97.4	13.2	-	-	-
2	0.5	102.1	108.3	105.5	21.3	-	-	-
3	1.0	93.4	97.7	105.2	21.0	-	-	-
4	2.0	96.2	90.6	115.6	31.4	-	+	-
5	3.0	88.2	60.2	134.9	50.7	-	+	-
6	4.0	92.1	35.1	187.0	102.8	-	+	-
7	5.0	93.4	16.2	177.5	93.3	-	+	-
8	5.5	90.8	10.1	167.1	82.9	-	+	-
EMS	300	70.2	48.8	664.8	580.7	+	+	-
MMS	10	64.5	43.6	531.3	447.2	+	+	-

 

Test Group	Conc. [mM]	RCE [%]	RTG [%]	MF [mutants /10^6cells]	IMF [mutants /10^6cells]	GEF exceeded	Statistical significance	Precipitate
C1	0	100.0	100.0	82.0	/	/	/	-
C2					/	/	/	-
5	0.5	109.6	143.6	85.7	11.7	-	-	-
6	1.0	114.6	145.7	98.6	24.6	-	-	-
7	2.0	116.3	148.5	81.3	7.3	-	-	-
8	3.0	109.6	109.6	65.6	-8.3	-	+	-
9	3.5	111.2	85.7	119.9	46.0	-	-	-
10	4.0	112.9	57.0	79.8	5.9	-	-	-
11	4.5	118.1	27.8	66.0	-8.0	-	-	-
12	5.0	97.6	10.3	71.8	-2.1	-	-	-
EMS	200	52.3	33.6	2628.9	2554.9	+	+	-
MMS	10	49.1	35.4	1088.4	1014.5	+	+	-

 

C: Negative controls

RCE: Relative cloning efficiency = [(mean value positive cultures/mean value positive cultures of corresponding controls)*100]

RTG: Relative total growth = (RSG*RCE/100)

MF: Mutant frequency = {-ln [negative cultures/total wells (selected medium)] / -ln [negative cultures/total wells (non selective medium)]}*800

IMF: Induced mutatn frequency = mutant frequency sample – mean value mutant frequency corresponding controls

GEF: Global evaluation factor (126); +: GEF exceeded, -: GEF not exceeded

Statistical significance difference in mutant frequency compared to negative controls (Mann Whitney test, p<0.05). +: signficant; -: not significant.

B[a]P: Benzo[a]pyrene (µg/mL)

EMS: Ethylmethanesulfonate (µg/mL)

MMS: Methylmethanesulfonate (µg/mL)

CLASTOGENICITY: All dose groups with and without metabolic activation were considered as not clastogenic under test conditions.

Main Experiment I and II – Colony sizing, with metabolic activation

 

Test Group	Conc. [mM]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	45	40	5	11.1
C2	0	45	43	2	4.4
8	8.0	53	48	5	9.8
9	9.0	57	44	13	22.8
10	10.0	83	67	16	19.3
B[a]P	2.5	259	149	110	42.5

 

Test Group	Conc. [mM]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	49	45	4	8.2
C2	0	46	37	9	19.6
6	5.0	68	59	9	13.2
7	7.5	46	39	7	15.2
8	8.5	49	37	12	24.5
B[a]P	3.5	204	106	98	48.0

 

 

Main Experiment I and II – Colony sizing, without metabolic activation

 

Test Group	Conc. [mM]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	51	42	9	17.6
C2	0	48	39	6	18.8
6	4.0	96	77	19	19.8
7	5.0	93	86	7	7.5
9	5.5	86	69	17	19.8
MMS	10	166	92	74	44.6

 

Test Group	Conc. [mM]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	38	30	8	21.1
C2	0	41	34	4	17.1
10	4.0	48	40	8	16.7
11	4.5	42	34	8	19.0
12	5.0	38	33	5	13.2
MMS	10	210	93	117	55.7

Applicant's summary and conclusion

Conclusions:

Propyl Triacetoxy Silane (PTA) was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells under test conditions.

Executive summary:

The test item Propyl Triacetoxy Silane PTA was assessed for its potential to induce mutations at the mouse lymphoma thyamidine kinase locus using the cell line L5178Y according to OECD Guideline 476.

The selection of the concentrations used in the main experiments was based on data from the toxicity pre-test. The Experiment I was performed as a 4h short-term exposure assay up to 10.0 mM with metabolic activation and up to 5.5 mM without metabolic activation test substance concentration. The Experiment II was performed as a 4h short-term exposure with metabolic activation up to 8.5 mM and as a 24h long-term exposure without metabolic activation up to 5.0 mM test concentration. No precipitation of the test item was noted in pre-experiment I and II and experiment I and II with and without metabolic activation. No toxicity was observed in pre-experiment I. However, this was not confirmed in the main test. In experiment I and II with and without metabolic activation growth inhibition was noted at highest doses. In experiment I and II no biologically relevant increase of mutants was 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) was not exceeded by the induced mutant frequency at any concentration. In experiment I without metabolic activation a dose dependent effect was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus provind the efficiency of the test system to indicate potential clastogenic effects.

In the described mutagenicity test under the experimental conditions reported, the test item Propyl Triacetoxy Silane PTA is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.