Tetraethyl 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:

1993-01-29 to 1993-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Tetraethyl orthosilicate

EC Number:

201-083-8

EC Name:

Tetraethyl orthosilicate

Cas Number:

78-10-4

Molecular formula:

C8H20O4Si

IUPAC Name:

tetraethyl orthosilicate

Test material form:

liquid

Method

Target gene:

Histidine operon (S. typhimurium strains); tryptophan operon (E. coli).

Species / strain

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:

125 to 1500 µg/mL (without activation, 4 and 20h exposure), and 500 to 2080 µg/mL (witn activation 4 hour exposure)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: based on infomation provided by the Sponsor and compatibility with the target cells.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(without activation)

Controls 2

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:

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4h / 20h (-S9), 4h (+S9)

- Expression time (cells in growth medium): 20h

- Fixation time (start of exposure up to fixation or harvest of cells): 20h

NUMBER OF REPLICATIONS: duplicate flasks

NUMBER OF CELLS EVALUATED: 200 metaphase spreads (100 per duplicate flask), per concentration.

DETERMINATION OF CYTOTOXICITY

- Method: percentage total growth

Evaluation criteria:

Cells examined for numerical and structural aberrations, and statistical significance calculated. For the result to be positive, the increase in aberrations must be statistically significant and dose related.

Toxicity is defined as a 50 % or greater reduction in cell growth.

Statistics:

The number and type of aberrations found, the percentage of structurally and numerically damaged cells (% aberrant cells) in the total population examined and mean aberrations per cell were calculated for each group. Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control.

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With metabolic activation: 1250 µg/ml; Without metabolic activation: 1250 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: at highest test concentration was 7.4
- Effects of osmolality: checked

RANGE-FINDING/SCREENING STUDIES: Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was observed at dose level 2080 pg/mL in the non-activated 4 and 20 hour exposure groups and in the S9 activated 4 hour exposure group.


COMPARISON WITH HISTORICAL CONTROL DATA: The percentage of cells with structural aberrations in the test article-treated groups was statistically increased above that of the solvent control at dose level 1250 pg/mL (p

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

For the chromosome aberration assay, CHO cells were seeded at approximately 5 x l0 5 cells/25 cm² flask and were incubated at 37+/- 1C  in a humidified atmosphere of 5+/-1% CO2 in air for 16-24 hours.

Treatment time: 4hrs, S9: none, Toxicity at highest dose scored: 52% at 1250 µg/ml; Mitotic index reduction: 4%
Treatment time: 20 hrs, S9: none, Toxicity at highest dose scored: 56% at 1250 µg/ml; Mitotic index reduction: none
Treatment time: 4 hrs, S9: yes, Toxicity at highest dose scored: 54% at 1250 µg/ml; Mitotic index reduction: 10%

The percentage of structurally damaged cells in the mitomycin (positive  control in non-activated system) treatment (15%) was statistically  significant.
The percentage of structurally damaged cells in the cyclophosphamide  (positive control in activated system) treatment (14%) was statistically  significant.

Table 1 Summary of results (200 cells scored)

Treatment µg/ml	Metabolic activation	Treatment time (h)	Mean mitotic index	Aberrations per cell (mean (±SD)	Cells with aberrations %
					numerical	Structural
0*	-	4	15.7	0.015 ± 0.122	3.0	1.5
250	-	4	16.0	0.005 ± 0.071	2.5	0.5
750	-	4	15.2	0.005 ± 0.071	3.0	0.5
1250	-	4	15.0	0.000 ± 0.000	2.5	1.0
Positive control	-	4	15.5	0.130 ± 0.484	4.0	10.0
0*	+	4	16.6	0.000 ± 0.000	2.0	0.0
250	+	4	15.1	0.005 ± 0.071	2.5	0.5
750	+	4	15.7	0.000 ± 0.000	1.5	0.0
1250	+	4	14.9	0.030 ± 0.198	3.5	2.5
Positive control	+	4	13.5	0.200 ± 0.576	3.0	14.0
0*	-	20	15.6	0.000 ± 0.000	2.0	1.0
250	-	20	14.8	0.000 ± 0.000	3.5	0.0
750	-	20	15.6	0.010 ± 0.141	2.0	0.5
1250	-	20	15.9	0.005 ± 0.071	2.5	0.5
Positive control	-	20	15.3	0.210 ± 0.598	3.5	15.0

* solvent control with DMSO

Applicant's summary and conclusion

Conclusions:

Tetraethyl orthosilicate has been tested in a reliable study conducted according to OECD Test Guideline 473 in compliance with GLP (reliability score 1). No increase in the number of structural and numerical chromosome aberrations in CHO cells was observed. Appropriate positive and solvent 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.