N,N-bis(trimethylsilyl)aminopropylmethyldiethoxysilane

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:

1 February 2002 to 26 March 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restriction was that duplicate cell cultures were used.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Notification No. 77 of JMOL on September 1, 1988

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Notification No. 287 of the Planning and Coordination Bureau, JEA, No. 127 of the Pharmaceutical Affairs Bureau, JMHW, No. 2 of the Basic Industries Bureau, JMITI on October 31, 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-890-5

EC Name:

-

Cas Number:

201290-01-9

Molecular formula:

C14H37NO2Si3

IUPAC Name:

7-ethoxy-2,2,7-trimethyl-3-(trimethylsilyl)-8-oxa-3-aza-2,7-disiladecane

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in room temperature, shield from light
- Stability under test conditions: stable in ordinary temperature
- Solubility and stability of the test substance in the solvent/vehicle: unstable in water (reactivity), stable in dimethylsulphoxide and acetone
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The highest concentration of the test substance solution used in this test was prepared using dehydrated acetone just before use.
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: the solutions of the lower concentration were prepared by the same acetone by serial dilution from the solution of the highest concentration

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 5, 20, 78, 313, 1250, 5000 µg/plate
Main test: 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: A solubility test was performed to determine the solvent of the test substance, where two different organic solvents of dimethylsulfoxide and acetone were used, because the test substance was unstable in water. In the solubility test the test substance dissolved in acetone. No exothermic reaction was seen, thus the test substance was thought to be stable in acetone.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

organic solvent of acetone

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

TA100, TA98, WPuvrA, without S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

organic solvent of acetone

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535, without S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

organic solvent of acetone

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

organic solvent of acetone

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535, WP2uvrA, with S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

organic solvent of acetone

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA100, TA98, TA1537, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 2.3x10⁹ (TA100); 3.5x10⁹ (TA1535); 6.1x10⁹ (WP2uvrA); 3.5x10⁹ (TA98); 2.9x10⁹ (TA1537)

ACTIVATION: Co-factor solution was added to the thawed S9 fraction at a concentration of 10 v/v% and then stored until use. The composition of S9 mix per ml was: S9 fraction 10 v/v%, MgCl2 8 µmol, KCl 33 µmol, G-6-P 5 µmol, NADPH 4 µmol, NADH 4 µmol, sodium phosphate buffer 100 µmol.

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS: duplicate cultures


DETERMINATION OF CYTOTOXICITY
- Method: number of revertants

Rationale for test conditions:

Neither toxic effect of the test substance nor two-fold or more increase in the number of revertant colonies with dose-relativity compared with the negative control was observed in any bacterial strains at any dose levels in the preliminary test regardless of the presence or absence of metabolic activation. Based on these results the highest dose selected for the main test was 5000 µg/plate, with and without metabolic activation.

Evaluation criteria:

The test substance was considered positive for mutagenic activity when clear dose-related increase in the number of revertant colonies and two-fold or more increase in the number of the revertant colonies compared with the negative control were observed with reappearance.

Statistics:

No statistical analysis was used for evaluation of the result.

Results and discussion

Test results

Key result

Species / strain:

other: TA100, TA98, TA1535, TA1537 and WPuvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed at any of the test concentrations, with or without metabolic activation

RANGE-FINDING/SCREENING STUDIES: Neither toxic effect of the test substance nor two-fold or more increase in the number of revertant colonies with dose-relativity compared with the negative control was observed in any bacterial strains at any dose levels in the preliminary test regardless of the presence or absence of metabolic activation.

HISTORICAL CONTROL DATA
- Positive historical control data: within the ranges of historical control data
- Negative (solvent/vehicle) historical control data: within the ranges of historical control data

Any other information on results incl. tables

Table 1: Mean number of revertants in the presence or absence of metabolic activation

Concentration μg/plate	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
Acetone	33	25	139	135	9	10	7	6	27	32
313	34	24	137	136	11	9	6	5	28	26
625	33	25	144	140	10	9	7	5	32	29
1250	33	22	133	130	8	7	7	7	33	31
2500	39	29	135	133	12	10	8	6	35	28
5000	36	28	131	129	11	9	8	5	34	28
AF-2 0.01				641						198
AF-2 0.1		578
NaN3						391
9AA								798
B[a]P	264		1256				68
2AA 2.0					252
2AA 10.0									636

Applicant's summary and conclusion

Conclusions:

N,N-bis(trimethylsilyl)aminopropylmethyldiethoxysilane has been tested in a valid bacterial reverse mutation assay, according to a protocol similar to OECD TG 471 and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WPuvrA. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to a limit concentration. Appropriate solvent and positive controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.