2,2,4-trimethyl-1-oxa-4-aza-2-silacyclohexane

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:

Experimental start date: 31 May 2017, Experimental completion date: 08 August 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,4-trimethyl-1-oxa-4-aza-2-silacyclohexane

EC Number:

233-487-5

EC Name:

2,2,4-trimethyl-1-oxa-4-aza-2-silacyclohexane

Cas Number:

10196-49-3

Molecular formula:

C6H15NOSi

IUPAC Name:

2,2,4-trimethyl-1,4,2-oxazasilinane

Test material form:

liquid

Details on test material:

Batch number: 20140202

Specific details on test material used for the study:

Identification: 2,2,4-Trimethyl-1-oxa-4-aza-2-silacyclohexane
CAS No: 10196-49-3
CAS name: 1-Oxa-4-aza-2-silacyclohexane, 2,2,4-trimethyl-
EC name: 2,2,4-trimethyl-1-oxa-4-aza-2-silacyclohexane
EC number: 233-487-5
Empirical Formula: C6H15NOSi
Molecular Mass: 145.3 g/mol
Physical state/Appearance: Clear colourless liquid
Expiry Date: 09 December 2030
Storage Conditions: Room temperature in the dark

Method

Target gene:

Histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthaflavone induced rat liver was used at the metabolic activation system

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The maximum concentration was 5000 µg/plate as this is the maximum recommended dose level.

Experiment 2: 15, 50, 150, 500, 1500, 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test

Vehicle / solvent:

The test item was fully miscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL in solubility checks performed in–house. Sterile distilled water was selected as the vehicle for the study. The test item was found to be stable at 4 and 24 hours when stored at room temperature in the light, the formulations were also found to be homogeneously prepared.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

80 µg/plate for TA1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/plate for TA98

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Test Item Preparation and Analysis
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 5 minutes at 40 °C Formulated concentrations were adjusted to allow for the stated impurity content (1.4%) of the test item. All formulations were used within four hours of preparation. Analysis for concentration of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, 5000 µg/plate.
Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to
pre-incubation.
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Due to a hardware failure, Ames study manager and sorcerer system suffered an extended downtime, resulting in manual counts being performed on all of the plates produced for Experiment 2.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation fell within prescribed historical control ranges. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in Experiment 1 at 5000 µg/plate (TA1535 dosed in the absence of S9) and 150 µg/plate (TA1537 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.9 times the concurrent vehicle controls.

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
104		17		25		16		15
84	(87)	22	(17)	11	(24)	16	(16)	11	(12)
72		12		36		15		9

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
63		14		18		20		12
65	(67)	14	(14)	25	(19)	19	(17)	6	(9)
72		13		13		13		9

 

 

Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 10 July 2017					To: 13 July 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	71 77 82	(77) 5.5#	12 9 10	(10) 1.5	21 27 23		(24) 3.1	13 27 20	(20) 7.0	18 18 22	(19) 2.3
	1.5 µg	93 74 87	(85) 9.7	10 10 13	(11) 1.7	20 23 20		(21) 1.7	16 13 19	(16) 3.0	12 10 8	(10) 2.0
	5 µg	82 79 95	(85) 8.5	12 13 18	(14) 3.2	23 20 17		(20) 3.0	16 33 13	(21) 10.8	24 15 14	(18) 5.5
	15 µg	90 91 79	(87) 6.7	15 15 14	(15) 0.6	24 19 17		(20) 3.6	20 15 17	(17) 2.5	14 23 13	(17) 5.5
	50 µg	85 81 81	(82) 2.3	17 10 8	(12) 4.7	17 18 28		(21) 6.1	13 19 25	(19) 6.0	14 13 30	(19) 9.5
	150 µg	83 85 83	(84) 1.2	18 14 20	(17) 3.1	23 26 20		(23) 3.0	25 21 24	(23) 2.1	9 21 16	(15) 6.0
	500 µg	92 98 90	(93) 4.2	24 19 7	(17) 8.7	25 22 30		(26) 4.0	25 25 23	(24) 1.2	15 6 10	(10) 4.5
	1500 µg	90 74 109	(91) 17.5	18 20 12	(17) 4.2	17 20 19		(19) 1.5	17 21 21	(20) 2.3	10 17 20	(16) 5.1
	5000 µg	77 88 80	(82) 5.7	22 24 14	* (20) 5.3	19 35 31		(28) 8.3	19 27 28	(25) 4.9	16 20 15	(17) 2.6
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		300 481 425	(402) 92.7	77 72 100	(83) 14.9	402 299 279		(327) 66.0	249 284 288	(274) 21.5	103 77 148	(109) 35.9

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

*               p ≤ 0.05

#         Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 10 July 2017					To: 13 July 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	72 81 85	(79) 6.7#	9 13 19	(14) 5.0	23 33 40		(32) 8.5	26 25 25	(25) 0.6	8 14 8	(10) 3.5
	1.5 µg	102 86 80	(89) 11.4	10 10 11	(10) 0.6	22 30 29		(27) 4.4	25 11 20	(19) 7.1	13 12 9	(11) 2.1
	5 µg	71 83 85	(80) 7.6	9 21 9	(13) 6.9	24 22 20		(22) 2.0	23 14 27	(21) 6.7	18 14 11	(14) 3.5
	15 µg	74 88 94	(85) 10.3	15 21 12	(16) 4.6	25 23 20		(23) 2.5	12 13 19	(15) 3.8	12 11 10	(11) 1.0
	50 µg	88 91 84	(88) 3.5	16 9 11	(12) 3.6	26 16 43		(28) 13.7	18 16 22	(19) 3.1	13 16 11	(13) 2.5
	150 µg	82 83 84	(83) 1.0	15 8 11	(11) 3.5	36 32 25		(31) 5.6	14 18 23	(18) 4.5	16 16 21	* (18) 2.9
	500 µg	71 90 107	(89) 18.0	9 17 9	(12) 4.6	25 28 32		(28) 3.5	19 21 14	(18) 3.6	11 13 7	(10) 3.1
	1500 µg	84 87 83	(85) 2.1	9 9 10	(9) 0.6	33 29 32		(31) 2.1	22 21 19	(21) 1.5	20 15 10	(15) 5.0
	5000 µg	86 92 113	(97) 14.2	10 7 9	(9) 1.5	22 26 28		(25) 3.1	20 12 17	(16) 4.0	13 15 15	(14) 1.2
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		732 1479 1516	(1242) 442.3	214 229 227	(223) 8.1	235 208 217		(220) 13.7	185 209 204	(199) 12.7	248 279 319	(282) 35.6

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

*               p ≤ 0.05

#            Standard deviation

 

 

Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 01 August 2017					To: 04 August 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	78 68 68	(71) 5.8#	12 16 13	(14) 2.1	24 21 18		(21) 3.0	24 22 27	(24) 2.5	9 8 16	(11) 4.4
	15 µg	69 65 65	(66) 2.3	10 9 14	(11) 2.6	20 25 12		(19) 6.6	18 22 22	(21) 2.3	11 9 9	(10) 1.2
	50 µg	65 67 69	(67) 2.0	18 16 14	(16) 2.0	14 14 18		(15) 2.3	24 27 16	(22) 5.7	9 9 10	(9) 0.6
	150 µg	81 79 61	(74) 11.0	16 21 12	(16) 4.5	24 15 25		(21) 5.5	17 22 14	(18) 4.0	12 11 9	(11) 1.5
	500 µg	67 69 64	(67) 2.5	11 11 13	(12) 1.2	24 12 24		(20) 6.9	16 18 21	(18) 2.5	9 14 13	(12) 2.6
	1500 µg	80 65 66	(70) 8.4	12 10 17	(13) 3.6	20 15 18		(18) 2.5	11 16 15	(14) 2.6	3 16 14	(11) 7.0
	5000 µg	81 70 69	(73) 6.7	18 7 14	(13) 5.6	16 19 14		(16) 2.5	21 17 21	(20) 2.3	19 9 7	(12) 6.4
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		799 833 798	(810) 19.9	1457 1692 1480	(1543) 129.5	553 527 459		(513) 48.5	295 306 302	(301) 5.6	359 246 317	(307) 57.1

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

#         Standard deviation

 

 

Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 01 August 2017					To: 04 August 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	86 68 62	(72) 12.5#	15 21 19	(18) 3.1	21 24 19		(21) 2.5	28 29 18	(25) 6.1	15 8 6	(10) 4.7
	15 µg	68 67 65	(67) 1.5	20 21 16	(19) 2.6	27 19 27		(24) 4.6	25 17 21	(21) 4.0	9 12 15	(12) 3.0
	50 µg	66 72 64	(67) 4.2	17 12 16	(15) 2.6	19 16 24		(20) 4.0	31 13 16	(20) 9.6	11 11 7	(10) 2.3
	150 µg	65 65 64	(65) 0.6	18 19 13	(17) 3.2	19 20 18		(19) 1.0	24 19 17	(20) 3.6	8 9 10	(9) 1.0
	500 µg	69 60 65	(65) 4.5	18 19 11	(16) 4.4	18 13 20		(17) 3.6	21 21 27	(23) 3.5	15 9 11	(12) 3.1
	1500 µg	61 64 63	(63) 1.5	12 13 23	(16) 6.1	16 14 21		(17) 3.6	28 23 31	(27) 4.0	11 10 9	(10) 1.0
	5000 µg	73 72 64	(70) 4.9	23 18 21	(21) 2.5	26 25 24		(25) 1.0	22 30 24	(25) 4.2	9 11 16	(12) 3.6
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		902 907 898	(902) 4.5	194 186 205	(195) 9.5	192 166 197		(185) 16.6	141 156 180	(159) 19.7	337 368 386	(364) 24.8

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

2,2,4-Trimethyl-1-oxa-4-aza-2-silacyclohexane (CAS no. 10196-49-3) was considered to be non-mutagenic under the conditions of this test.

Executive summary:

  Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

 Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with2,2,4-Trimethyl-1-oxa-4-aza-2-silacyclohexaneusing both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (Experiment 2, pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 2 was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). Small, statistically significant increases in revertant colony frequency were observed in Experiment 1 at 5000 µg/plate (TA1535 dosed in the absence of S9) and 150 µg/plate (TA1537 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.9 times the concurrent vehicle controls.

Conclusion

2,2,4-Trimethyl-1-oxa-4-aza-2-silacyclohexanewas considered to be non-mutagenic under the conditions of this test.