Tetrakis(2-butoxyethyl) orthosilicate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-06 to 2015-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014-09-26

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(2-butoxyethyl) orthosilicate

EC Number:

242-560-0

EC Name:

Tetrakis(2-butoxyethyl) orthosilicate

Cas Number:

18765-38-3

Molecular formula:

C24H52O8Si

IUPAC Name:

tetrakis(2-butoxyethyl) orthosilicate

Constituent 2

Reference substance name:

tetrakis(2-butoxyethoxy)silane

IUPAC Name:

tetrakis(2-butoxyethoxy)silane

Test material form:

other: liquid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimum essential medium) with 10 % FBS, 100 U/100 μg/mL penicillin/streptomycin solution, 2 mM L-glutamine, 2.5 μg/mL amphotericin, 25 mM HEPES
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9

Test concentrations with justification for top dose:

Experiment I
With metabolic activator (S9 mix) - 0.1 μg/mL , 0.2 μg/mL , 0.5 μg/mL
Without metabolic activator (S9 mix) - 5 μg/mL, 10 μg/mL, 20 μg/mL

Experiment II
Without metabolic activator (S9 mix) - 0.02 μg/mL, 0.05 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.5 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofurane (THF)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

ACTIVATIOR: Phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9. An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:8 mM MgCl2, 33mM KCl, 5mM Glucose-6-phosphate, 5mM NADP in 100 mM sodium-phosphate-buffer pH 7.4

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Experiment I: 4 hours, with and without metabolic activation
Experiment II: 21 hours, without metabolic activation
- Expression time (cells in growth medium):
Experiment I: 21 hours
Experiment II: The complete medium containing the different concentrations of the test material is not changed until preparation of the cells.
- Fixation time (start of exposure up to fixation or harvest of cells): The cells are fixed at least two times with 3 + 1 methanol + glacial acetic acid and spread onto the slides.
Experiment I: 21 hours
Experiment II: 21 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 150 metaphases per culture were scored for chromosomal aberrations (300)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and relative increase in cell count (RICC)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A result is considered positive when there is a clear and dose-related increase in the number of cells with aberrations, and a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment I, precipitation of the test item was noted without metabolic activation at a concentration of 1 μg/mL and with metabolic activation at a concentration of 20 μg/mL.
In experiment II, precipitation of the test item was seen without metabolic activation at a concentration of 5 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive controls were within the range of historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Summary of Experimental Results

	Dose Group	Concentration µg/mL	Relative Mitotic Index  (%)	RICC (%)	Mean % Aberrant Cells			Historical Laboratory Negative Control Range	Precipitation
					Including Gaps		Excluding Gaps
Experiment I and II, without metabolic activation
Experiment I 4 hour treatment, 21 hour preparation interval	C	0	111	114	2.3	1.3		0.0% - 4.0 % aberrant cells	-
	S	0	100	100	2.7	2.0			-
	2	0.2	105	93	5.0	3.3			-
	3	0.5	103	79	6.0	2.7			-
	4 (P)	1	94	79	8.0	4.0			+
	EMS	900	122	108	12.0	9.3			-
Experiment II 21 hour treatment, 21 hour preparation interval	C	0	108	119	4.0	2.7		0.0 % - 4.0 % aberrant cells	-
	S	0	100	100	4.7	2.3			-
	6	1	104	95	4.3	1.7			-
	7	2	144	98	3.7	1.3			-
	8	5	120	94	3.0	1.3			+
	EMS	400	81	84	10.7	9.3			-
Experiment I, with metabolic activation
Experiment I 4 hour treatment, 21 hour preparation interval	C	0	86	121	2.0	1.3		0.0 % - 4.3 % aberrant Cells	-
	S	0	100	100	2.7	2.0			-
	1	5	89	113	1.7	1.3			-
	2	10	101	90	1.7	1.3			-
	3 (P)	20	94	87	2.0	1.0			+
	CPA	1.5	103	100	8.3	7.7			-

The mitotic index was determined in 1000 cells per culture of each test group.

C: Negative Control (Culture Medium)

S: Solvent Control (THF)

EMS: Ethylmethanesulfonate

CPA: Cyclophosphamide

Relative Mitotic Index: the relative mitotic index is related to the negative controls; the mitotic index is determined in 1000 cells per culture of each test group.

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the control groups. The cell count was determined by a cell counter per culture for each test group

- without precipitation, + with precipitation

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation

Tetrakis(2-butoxyethyl)silicate has been tested in a valid study according to OECD 473 and under GLP, up to precipitation concentrations 1 μg/mL and 20 μg/mL. No increase in the number of cells with aberrations was observed either with or without metabolic activation in Chinese hamster lung fibroblasts (V79). Appropriate solvent and positive 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 this study.