2,2,4,4,6,6-hexamethylcyclotrisilazane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-07 to 2015-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to an appropriate OECD guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,4,4,6,6-hexamethylcyclotrisilazane

EC Number:

213-773-6

EC Name:

2,2,4,4,6,6-hexamethylcyclotrisilazane

Cas Number:

1009-93-4

Molecular formula:

C6H21N3Si3

IUPAC Name:

2,2,4,4,6,6-hexamethyl-1,3,5,2,4,6-triazatrisilinane

Test material form:

other: liquid

Method

Target gene:

Histidine operon (S. typhimurium), tryptophan operon (E. coli).

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/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: anhydrous Acetone (purity > 99 %)
- Justification for choice of solvent/vehicle: the vehicle was chosen based on the hydrolysis and solubility properties of the test material.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

anhydrous acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100, without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

anhydrous acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 1537, TA 98, without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

anhydrous acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA, without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

anhydrous acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, with metabolic activation

Details on test system and experimental conditions:

ACTIVATION: The protein concentration of the S9 preparation was 39.3 mg/mL. The S9 mix included 10 % v/v S9 and the following cofactors: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. 0.5 ml S9 mix were added to a total volume of 2.7, giving a final concentration of approximately 2% S9 in the top agar.

METHOD OF APPLICATION: in agar plates for plate incorporation and preincubation

DURATION
- Preincubation period: at 37 °C for 60 minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): histidine-deficient agar plates for Salmonella typhimurium and tryptophan-deficient agar plates for Escherichia coli

NUMBER OF REPLICATIONS: triplicatem plates, initial plate incorporation assay repeated using the preincubation method

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; condition of background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed

Statistics:

Relevant statistics (st.dev, mean) were calculated.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate during plate incorporation, and at 2500 and 5000 μg/plate during pre-incubation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION: No precipitation of the test item occurred up to the highest investigated dose

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA showed reduced background growth at concentration of 5000 μg/plate test material, during the plate incorporation experiment, with and without metabolic activation (Experiment I). Strains TA 1535, TA 1537 and WP2 uvrA showed reduced background growth, following incubation with the test material at 5000 μg/plate during the pre-incubation experiment, with metabolic activation.
Cytotoxicity was observed in all the strains, when treated with 2500 and 5000 μg/plate of the test substance during plate incorporation and pre-incubation experiments, with and without metabolic activation.

No significant increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1. Results from plate incorporation experiment, mean revertant colonies count, with and without metabolic activation

Concentration μg/plate	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
Acetone	14	18	20	12	36	23	137	158	49	38
Untreated	18	20	18	15	40	27	155	171	52	37
3 μg	12	21	19	12	40	22	140	167	49	41
10 μg	14	16	19	14	38	20	139	166	46	43
33 μg	13	23	22	10	35	22	141	163	45	37
100 μg	15	21	15	13	33	25	143	167	52	37
333 μg	15	21	20	13	40	24	139	166	46	41
1000 μg	15	19	16	12	39	18	141	138	59	44
2500 μg	12	18	19	13	38	23	75	77	53	41
5000 μg*	10^R	11^R	1^R*	2^R*	8^R*	7^R*	8^R*	28^R*	19^R*	29^R
NaN310 μg	-	1156	-	-	-	-	-	2144	-	-
4-NOPD 10 μg	-	-	-	-	-	3 96	-	-	-	-
4-NOPD 50 μg	-	-	-	89	-	-	-	-	-	-
MMS 0.2 μg	-	-	-	-	-	-	-	-	-	841
2-AA 2.5 μg	451	-	246	-	3052	-	3663	-		-
2 -AA 10.0 μL	-	-	-	-	-	-	-	-	278	-

Table 2. Results from pre-incubation experiment, mean revertant colonies count, with and without metabolic activation

Concentration µg/plate	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
Acetone	10	11	20	11	32	20	164	172	43	35
Untreated	10	9	23	9	41	29	188	187	47	39
10 µg	9	12	15	10	40	27	170	160	45	37
33 µg	10	9	17	11	34	26	181	159	47	36
100 µg	12	10	19	9	40	21	177	178	51	38
333 µg	12	10	20	10	32	27	135	170	51	33
1000 µg	11	12	19	7	36	20	137	108	47	35
2500 µg*	10	2*	18	1*	26	4*	62*	43*	39	28
5000 µg*	0ᴿ*	0*	0ᴿ*	1*	3*	0*	0*	0*	0ᴿ*	0*
NaN3 10 µg		1054						2128
4-NOPD 10 µg						329
4-NOPD 50 µg				97
MMS 2.0 µL										645
2-AA 2.5 µg	401		194		2828		3387
2-AA 10.0 µg									387

 

NaN3 sodium azide

2-AA 2-aminoanthracene

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

R Reduced background growth

* Cytotoxic effect

Applicant's summary and conclusion

Conclusions:

Hexamethylcyclotrisilazane has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD Technical Guideline 471, in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 in the initial plate incorporation study or the repeat experiment using the pre-incubation method, when tested up to limit concentrations. Appropriate positive, solvent and negative 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.