tert-butyl-dimethyl-piperazin-1-ylsilane

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:

2011-12-20 to 2012-01-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

duplicate plates were used instead of triplicate

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Reverse Mutagenicity test on Bacteria" of "Mutagenicity Test" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011; No. 0331-7, Pharmaceutical and Food Safety Bureau, the Ministry of Health, Labour and Welfare

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-[(1,1-Dimethylethyl)dimethylsilyl]piperazine

Cas Number:

138938-64-4

Molecular formula:

C10H24N2Si

IUPAC Name:

1-[(1,1-Dimethylethyl)dimethylsilyl]piperazine

Test material form:

liquid

Details on test material:

- State of aggregation: colourless transparent liquid
- Stabilisation: stable is storage conditions

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:

39.1, 78.1, 156, 313, 625, 1250 µg/plate
Bacterial growth inhibition was observed at greater than or equal to 1250 µg/plate, therefore 1250 µg/plate was selected to be the highest dose.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was soluble and stable in dehydrated DMSO. The test substance solution of 50.0 mg/mL prepared with dehydrated DMSO was considered to be stable, because there were no change in colour, exothermic reaction nor gas generation at room temperature within 2 hours after preparation.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

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 and WP2uvrA without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

TA1537 without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA100, TA1535, TA98, TA1537 and WP2uvrA with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): 2.3 x 10⁹ cells/mL S. typhimurium and 3.8x10⁹ cells/mL in E. coli

ACTIVATION SYSTEM: 1mL of S9 mix consisted of 8 µmol MgCl2, 33 µmol KCl, 5 µmol Glucose-6-phosphate, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.

DURATION
- Preincubation period: 0.1 ml of test substance solution and 0.5 mL of S9 mix were added to 0.1 ml of the bacterial culture and the mixture was shaken for 20 min at 37 +/- 0.5°C
- Exposure duration: The mixture was incubated for 48 hours at 37 +/- 0.5°C

SELECTION AGENT (mutation assays): histidine and biotin deficient agar for S. typhimurium strains, and tryptophan deficient agar for E.coli.

NUMBER OF REPLICATIONS: duplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies

Rationale for test conditions:

The highest selected concentration for the main experiment was 1250 µg/plate, at which bacterial growth inhibition was observed during the range-finding test.

Evaluation criteria:

The test substance was concluded to be positive when the number of relevant colonies increased to twice or more than that in the negative control and when the responses were dose related and/or reproducible. Five lower doses diluted with a geometric progression of 2 were selected.

Statistics:

No statistical methods were used.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: TA1535, TA1537, TA100, TA98 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1250 µg/plate for test strains with and without S9 mix

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was not observed with or without S9 mix

RANGE-FINDING/SCREENING STUDIES:

HISTORICAL CONTROL DATA
- Positive historical control data: the positive control results were within the historical control data
- Negative (solvent/vehicle) historical control data: the negative control results were within the historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: the bacterial growth inhibition was observed by a stereomicroscope
- Other observations when applicable: colony counting was performed manually for plates with bacterial growth inhibition; a colony analyser was used for the rest of the colonies.

Any other information on results incl. tables

Table 1: Mean number of revertants with and without metabolic activation

Concentration μg/plate	TA100		TA1535		WP2uvrA		TA98		TA1537
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
Negative control	106	94	10	6	34	27	27	16	17	10
39.1	115	108	6	9	33	32	39	22	16	12
78.1	107	111	9	9	33	36	32	18	16	9
156	109	101	12	8	26	28	28	19	18	9
313	101	99	8	8	31	35	25	17	19	9
625	116	100	11	8	36	29	25	20	20	12
1250	87	74	11	9	29	19	23	11	12	8
Positive control substance	2-AA	AF-2	2-AA	NaN3	2-AA	AF-2	2-AA	AF-2	2-AA	ICR-191
Positive control dose (µg/plate)	1	0.01	2	0.5	10	0.01	0.5	0.1	2	0.5
Number of revertants (positive control)	875	840	227	396	696	377	363	505	191	1436

AF-2: 2 -(2 -Furyl)-3 -(5 -nitro-2 -furyl)acrylamide

NaN3: Sodium azide

2AA: 2 -Aminoantracene

Applicant's summary and conclusion

Conclusions:

1-(Tert-butyldimethylsilyl)piperazine has been tested in a valid Bacterial reverse mutation assay, conducted according to a protocol similar to OECD TG 471, and in compliance with GLP, using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli WP2uvrA. 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 positive and negative (solvent) 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.