Trimethoxy(2,4,4-trimethylpentyl)silane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April - 01 July 2002

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

FREIE UND HANSESTADT HAMBURG, BEHÖRDE FÜR ARBEIT, GESUNDHEIT UND SOZIALES, Hamburg (Germany)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxy(2,4,4-trimethylpentyl)silane

EC Number:

251-995-5

EC Name:

Trimethoxy(2,4,4-trimethylpentyl)silane

Cas Number:

34396-03-7

Molecular formula:

C11H26O3Si

IUPAC Name:

trimethoxy(2,4,4-trimethylpentyl)silane

Details on test material:

- Name of test material (as cited in study report): BS 1316 (Test substance No. 60006325)
- Physical state: colourless liqiud at 20 °C
- Lot/batch No.: KE 01282
- Expiration date of the lot/batch: 01 Jan 2004
- Stability under test conditions: at least 1 week at +4°C in the dark (in solvent)
- Storage condition of test material: at room temperature, dark
- Density: 0.90 g/cm³
- Melting point: < -78 °C
- pH value: approx. 7
- Receipt No.: 24388
- Date of receipt: 21 Feb 2002

Method

Target gene:

his-operon

Species / strain

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:

Type and composition of metabolic activation system:
- source of S9 : Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was prepared according to Maron and Ames (Mutation Res. 113, 173-215; 1983). S9 was collected from 20-30 rats. The protein content of the pooled S9 fraction was 32.92 mg/mL and the P-450 content 0.32 nmol/mg. The S9 fraction was stored in liquid nitrogen.

- method of preparation of S9 mix: The S9 mix was freshly prepared on the day of the test according to Maron and Ames (Mutation Res. 113, 173-215; 1983), containing 5% S9 and the following components per 100 mL:
- 5.0 mL rat liver S9 (Aroclor 1254-induced)
- 2.0 mL 0.4 M MgCl2 + 1.65 M KCl-salt solution (sterile stock solution)
- 141.0 mg glucose-6-phosphate
- 306.5 mg NADP
- 50 mL 0.2 M phosphate buffer, pH 7.4 (sterile stock solution)
- sterile aqua ad iniectabilia ad 100 mL
Afterwards, the S9 mix was filter-sterilised by using a 0.45 µm filter and the kept on ice.

- volume of S9 mix in the final culture medium (plate incorporation test): 0.5 mL S9 mix was added to 2 mL top agar, 0.1 mL of cell suspension and 0.1 mL test material (or solvent or positive control), giving a final concentration of approximately 1% S9

Test concentrations with justification for top dose:

- Test 1: 100, 316, 1000, 3160 and 5000 µg/plate (plate incorporation test)
- Test 2: 3.16, 10, 31.6, 100 and 316 µg/plate (preincubation test)

Vehicle / solvent:

- Vehicle/solvent used: ethylene glycol dimethylether
- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

methylmethanesulfonate

other: +S9: 2-anthracene amide (TA 98, TA 102, TA 1537; 2µg/plate)

Details on test system and experimental conditions:

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 in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of colonies by more than 50% compared with the solvent control and/or sparse background lawn

Evaluation criteria:

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.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking on histidine-free agar plates.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.

Statistics:

MANN and WHITNEY and Spearman’s rank.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preincubation: at 316 µg/plate ±S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 5000 µg/plate ±S9; preincubation: at 316 µg/plate ±S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preincubation: at 316 µg/plate ±S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preincubation: at 316 µg/plate ±S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 5000 µg/plate ±S9; preincubation: at 100 and 316 µg/plate +S9 and at 316 µg/plate -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDY:
The test substance was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100. Ten concentration of the test substance ranging from 0.316 to 5000 µg/plate were examined. Slight cytotoxicity (scarce background lawn) was noted at the concentration of 5000 µg/plate. Hence, 5000 µg/plate was chosen as the top concentration for the main study.

SIGNS OF TOXICITY:
In the plate incorporation test without and with metabolic activation slight cytotoxicity (scarce background lawn) was noted at the top concentration of 5000 µg/plate in test strain TA 100 and TA 102.
In the preincubation test without metabolic activation slight cytotoxicity (scarce background lawn) was noted at the top concentration of 316 µg/plate in test strains TA 98, TA 100, TA 1535 and TA 1537. Reduction in the number of revertants by more than 50% and scarce background lawn was noted in test strain TA 102 at concentrations of 100 and 316 µg/plate.
In the preincubation test with metabolic activation slight cytotoxicity (scarce background lawn) was noted in all test strains at the top concentration of 316 µg/plate. Reduction in the number of revertants by more than 50% was observed in strain TA 102, TA 1535 and TA 1537 at the concentrations of 316 µg/plate.

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

Any other information on results incl. tables

Table 1: Dose range-finding study: number of revertants per plate (2 plates)

	TA 100
Conc. (µg/plate)	Plate 1	Plate 2	Cytotoxic (yes/no)
0*	132	175	No
0.316	150	195	No
1	127	125	No
3.16	169	152	No
10	138	135	No
31.6	164	149	No
100	128	133	No
316	154	126	No
1000	133	174	No
3160	151	140	No
5000	163	167	Yes

*solvent control with Ethylene glycol dimethylether

Table 2: Experiment 1 Plate incorporation: number of revertants per plate (mean of 3 plates)

	TA 98			TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	26.7±7.6	30.3±4.5	No	123.3±10.1	167.7±7.4	No	302.0±8.7	287.3±14.8	No
100	24.0±2.6	47.7±5.5	No	151.7±19.7	147.3±8.0	No	240.3±17.6	245.7±9.9	No
316	30.7±8.5	34.7±2.1	No	150.7±9.3	151.0±2.6	No	234.7±6.4	225.0±19.1	No
1000	20.3±3.8	34.0±3.0	No	159.3±1.2	144.7±3.2	No	226.3±29.9	245.7±17.9	No
3160	26.0±7.0	35.3±4.7	No	204.0±7.2	158.7±4.5	No	248.7±35.6	224.0±22.3	No
5000	24.0±6.2	28.7±3.2	No	192.7±15.9	173.3±10.4	Yes	259.3±23.9	206.3±20.6	Yes
Positive control	331.3±23.7	343.7±2.5	No	1091.3±37.8	1061.3±14.4	No	1087.7±12.0	1159.3±70.9	No

*solvent control with ethylene glycol dimethylether

Table 2: Experiment 1 Plate incorporation: number of revertants per plate (mean of 3 plates)

	TA 1535			TA 1537
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	19.0±4.4	16.3±1.5	No	9.3±1.5	11.7±0.6	No
100	17.7±5.9	14.3±4.0	No	9.7±2.1	11.7±3.8	No
316	19.0±7.0	15.0±4.6	No	7.7±3.1	12.0±2.0	No
1000	17.3±3.2	17.3±2.3	No	11.0±1.0	13.7±1.5	No
3160	17.7±0.6	19.3±5.7	No	9.0±2.0	15.0±2.6	No
5000	19.0±1.7	15.3±6.7	No	11.3±2.5	14.0±3.6	No
Positive control	387.0±7.2	393.7±1.2	No	1000.0±5.2	986.0±3.5	No

*solvent control with ethylene glycol dimethylether

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

	TA 98			TA 100			TA 102
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	26.7±9.0	34.7±2.5	No	125.0±14.8	119.3±8.7	No	291.0±7.5	280.3±13.3	No
3.16	31.3±2.1	31.7±11.4	No	133.0±12.1	117.3±15.9	No	291.3±1.5	267.7±27.4	No
10	28.7±2.5	30.7±5.5	No	186.7±3.1	127.3±8.5	No	263.7±11.2	260.7±7.4	No
31.6	39.3±16.2	31.7±4.5	No	155.7±8.1	106.0±9.6	No	265.3±0.6	278.0±28.6	No
100	28.3±5.0	31.0±4.0	No	144.3±15.5	124.0±2.6	No	0.0±0.0	270.3±16.3	Yes
316	33.7±3.5	30.0±1.0	Yes	113.7±7.5	112.7±8.4	Yes	0.0±0.0	0.0±0.0	Yes
Positive control	855.7±22.7	924.0±54.7	No	1003.0±7.5	926.0±119.7	No	1165.3±6.1	1200.7±77.6	No

*solvent control with ethylene glycol dimethylether

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

	TA 1535			TA 1537
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	12.0±1.0	14.7±2.1	No	5.7±3.8	10.0±1.0	No
3.16	14.0±3.6	15.0±1.7	No	6.7±2.5	6.7±1.5	No
10	14.7±0.6	12.3±4.2	No	7.3±3.2	10.3±1.5	No
31.6	14.0±4.0	15.0±2.6	No	5.3±1.2	9.3±2.1	No
100	12.3±1.5	12.7±2.1	No	6.3±1.5	7.7±1.5	No
316	16.0±1.0	0.0±0.0	Yes	10.3±1.5	0.0±0.0	Yes
Positive control	632.0±6.1	581.7±46.3	No	617.0±4.6	423.3±86.4	No

*solvent control with ethylene glycol dimethylether

MA: metabolic activation

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation

The test item has been tested in a reliable study conducted according to the OECD TG 471 and in compliance with GLP. No test-substance related increase in the number of revertants was observed when tested with and without metabolic activation up to cytotoxic concentration in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in either the initial plate incorporation assay or the independent repeat experiment using the pre-incubation method. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.