Trimethoxy(3,3,3-trifluoropropyl)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:

Experimental start date 29 May 2019, Experimental completion date 18 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries, 24 November 2000

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxy(3,3,3-trifluoropropyl)silane

EC Number:

207-059-3

EC Name:

Trimethoxy(3,3,3-trifluoropropyl)silane

Cas Number:

429-60-7

Molecular formula:

C6H13F3O3Si

IUPAC Name:

trimethoxy(3,3,3-trifluoropropyl)silane

Test material form:

liquid

Specific details on test material used for the study:

Identification: ST2092NM
Chemical Name: Trimethoxy(3,3,3-trifluoropropyl)silane
CAS Number: 429-60-7
Physical state/Appearance: Clear colourless liquid
Batch Number: 802496
Purity: 100%
Expiry Date: 13 March 2020
Storage Conditions: Room temperature in the dark

Method

Target gene:

Histidine and tryptophan

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-naphtha flavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation method:
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed.

Experiment 2 - Pre-Incubation method:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:
In solubility checks performed in house, the test item was initially noted as miscible in dimethyl sulphoxide at 50 mg/mL and acetone at 100 mg/mL. However, over time (approximately 45 minutes) the test item dropped out of solution when formulated in dimethyl sulphoxide, therefore acetone was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

negative (untreated) controls were performed to assess the spontaneous revertant colony rate.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Used in the absence of S9-mix

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

negative (untreated) controls were performed to assess the spontaneous revertant colony rate.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Used in the absence of S9-mix

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

negative (untreated) controls were performed to assess the spontaneous revertant colony rate.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Used in the absence of S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

negative (untreated) controls were performed to assess the spontaneous revertant colony rate.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Used in the presence of S9-mix

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

negative (untreated) controls were performed to assess the spontaneous revertant colony rate.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Used in the presence of S9-mix

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Study Controls:
The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.
The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (tested in singular prior to Experiment 1).

Microsomal Enzyme Fraction:
The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized in-house procedures.

S9-Mix and Agar:
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9: 5.0 mL
1.65 M KCl/0.4 M MgCl2 : 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media:
Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.

Test Item Preparation and Analysis:
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in extra dry acetone by mixing on a vortex mixer. No correction for purity was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots (Maron et al., 1981).

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined.

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 OECD TG 471 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:
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. 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 previouslyexcept 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 between 48 and 72 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:
Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

Dose selection:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.
Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation:
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the appropriate concentration of test item formulation, 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 between 48 and 72 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).

Rationale for test conditions:

The purpose of the study was to evaluate the test substance for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2 (pre-incubation) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2 (pre-incubation) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2 (pre-incubation) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2 (pre-incubation) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2 (pre-incubation) at 5000 µg/plate

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 (acetone) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation):
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 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).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (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). A number of statistically significant values were noted (TA100 at 150 µg/plate and TA98 at 1.5 and 500 µg/plate in the absence of metabolic activation (S9-mix) and TA100 at 5, 15, 150 and 5000 µg/plate in the presence of metabolic activation (S9-mix);, however, these responses were within the in-house historical vehicle/untreated control range for the strains and were, therefore considered of no biological relevance.

Experimental 2 (pre-incubation):
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).

Results from the second mutation test showed that the test item induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns noted to all of the tester strains at 5000 µg/plate in both the absence and presence of metabolic activation (S9-mix).

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

No biologically relevant 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). Two statistically significant values were noted (TA1535 at 50 and 500 µg/plate in the absence of metabolic activation (S9-mix), however, these responses were within the in-house historical vehicle/untreated control range for the strains and were, therefore considered of no biological relevance.

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
114		25		28		21		19
142	(128)	23	(23)	25	(27)	25	(24)	17	(17)
129		20		27		26		15

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
143		21		22		14		13
158	(146)	18	(21)	32	(29)	19	(17)	12	(14)
137		23		34		19		18

Test Results: Experiment 1 - Without Metabolic Activation (Plate Incorporation)

Test Period		From: 30 May 2019					To: 02 June 2019
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	107 123 125	(118) 9.9#	15 17 15	(16) 1.2	30 19 29		(26) 6.1	17 19 14	(17) 2.5	13 6 10	(10) 3.5
	1.5 µg	125 122 129	(125) 3.5	13 19 19	(17) 3.5	36 25 23		(28) 7.0	24 24 25	(24) 0.6	10 13 14	(12) 2.1
	5 µg	142 123 131	(132) 9.5	12 12 14	(13) 1.2	32 25 22		(26) 5.1	15 23 15	(18) 4.6	13 9 15	(12) 3.1
	15 µg	139 120 145	(135) 13.1	12 14 10	(12) 2.0	26 21 18		(22) 4.0	20 23 17	(20) 3.0	12 8 10	(10) 2.0
	50 µg	132 136 133	(134) 2.1	16 21 27	(21) 5.5	24 19 19		(21) 2.9	17 18 22	(19) 2.6	7 8 11	(9) 2.1
	150 µg	125 147 149	(140) 13.3	26 12 15	(18) 7.4	21 22 28		(24) 3.8	18 18 17	(18) 0.6	7 12 13	(11) 3.2
	500 µg	125 153 126	(135) 15.9	24 15 19	(19) 4.5	18 31 23		(24) 6.6	22 22 30	(25) 4.6	16 12 14	(14) 2.0
	1500 µg	137 143 135	(138) 4.2	17 26 18	(20) 4.9	20 22 25		(22) 2.5	22 20 17	(20) 2.5	13 15 15	(14) 1.2
	5000 µg	122 116 129	(122) 6.5	15 14 15	(15) 0.6	20 36 29		(28) 8.0	23 23 20	(22) 1.7	14 13 13	(13) 0.6
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
		549 533 698	(593) 91.0	285 352 399	(345) 57.3	709 729 575		(671) 83.7	121 158 166	(148) 24.0	124 339 129	(197) 122.7

Test Results: Experiment 1 - With Metabolic Activation (Plate Incorporation)

Test Period		From: 30 May 2019					To: 02 June 2019
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	91 120 107	(106) 14.5#	16 14 17	(16) 1.5	37 37 28		(34) 5.2	23 28 27	(26) 2.6	13 11 13	(12) 1.2
	1.5 µg	131 122 132	(128) 5.5	18 9 10	(12) 4.9	39 32 31		(34) 4.4	14 21 27	(21) 6.5	16 10 11	(12) 3.2
	5 µg	123 144 122	(130) 12.4	12 13 14	(13) 1.0	44 33 26		(34) 9.1	26 21 28	(25) 3.6	15 19 12	(15) 3.5
	15 µg	125 143 124	(131) 10.7	13 16 16	(15) 1.7	40 35 31		(35) 4.5	28 34 29	(30) 3.2	16 12 8	(12) 4.0
	50 µg	117 110 126	(118) 8.0	12 17 16	(15) 2.6	25 31 31		(29) 3.5	23 23 30	(25) 4.0	21 10 19	(17) 5.9
	150 µg	131 127 136	(131) 4.5	17 12 12	(14) 2.9	32 30 35		(32) 2.5	25 25 34	(28) 5.2	9 16 15	(13) 3.8
	500 µg	137 106 134	(126) 17.1	11 17 17	(15) 3.5	30 37 24		(30) 6.5	15 22 28	(22) 6.5	8 12 12	(11) 2.3
	1500 µg	140 117 129	(129) 11.5	10 15 14	(13) 2.6	31 31 34		(32) 1.7	25 26 27	(26) 1.0	18 10 17	(15) 4.4
	5000 µg	149 148 129	(142) 11.3	13 11 14	(13) 1.5	41 36 38		(38) 2.5	22 23 15	(20) 4.4	12 19 8	(13) 5.6
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
		1230 1918 1337	(1495) 370.2	311 308 271	(297) 22.3	171 176 231		(193) 33.3	197 170 160	(176) 19.1	229 264 228	(240) 20.5

Test Results: Experiment 2 - Without Metabolic Activation (Pre-Incubation)

Test Period		From: 14 June 2019					To: 17 June 2019
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	156 122 127	(135) 18.4#	15 15 13	(14) 1.2	28 22 34		(28) 6.0	21 13 39	(24) 13.3	7 11 15	(11) 4.0
	15 µg	141 155 157	(151) 8.7	19 15 15	(16) 2.3	25 34 28		(29) 4.6	16 15 17	(16) 1.0	8 10 13	(10) 2.5
	50 µg	137 130 156	(141) 13.5	16 24 21	(20) 4.0	29 37 41		(36) 6.1	17 28 17	(21) 6.4	7 9 11	(9) 2.0
	150 µg	127 150 128	(135) 13.0	18 15 15	(16) 1.7	23 32 24		(26) 4.9	18 18 10	(15) 4.6	16 16 17	(16) 0.6
	500 µg	146 140 160	(149) 10.3	21 21 16	(19) 2.9	31 27 22		(27) 4.5	25 24 17	(22) 4.4	5 13 10	(9) 4.0
	1500 µg	116 122 141	(126) 13.1	16 13 12	(14) 2.1	26 24 22		(24) 2.0	15 18 23	(19) 4.0	6 16 10	(11) 5.0
	5000 µg	0 V 0 V 0 V	(0) 0.0	9 S 9 S 9 S	(9) 0.0	41 S 31 S 30 S		(34) 6.1	19 S 17 S 14 S	(17) 2.5	19 S 12 S 6 S	(12) 6.5
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
		819 764 812	(798) 29.9	1497 1307 1298	(1367) 112.4	784 781 781		(782) 1.7	191 176 186	(184) 7.6	333 290 225	(283) 54.4

S: Sparse bacterial background lawn

V: Very weak bacterial background lawn

Test Results: Experiment 2 - With Metabolic Activation (Pre-Incubation)

Test Period		From: 14 June 2019					To: 17 June 2019
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	153 153 149	(152) 2.3#	14 18 20	(17) 3.1	31 53 38		(41) 11.2	49 17 28	(31) 16.3	20 17 7	(15) 6.8
	15 µg	139 139 149	(142) 5.8	17 12 12	(14) 2.9	42 37 41		(40) 2.6	32 24 37	(31) 6.6	16 21 14	(17) 3.6
	50 µg	159 149 155	(154) 5.0	10 13 15	(13) 2.5	40 42 52		(45) 6.4	31 31 32	(31) 0.6	15 10 13	(13) 2.5
	150 µg	136 144 142	(141) 4.2	17 11 8	(12) 4.6	41 27 39		(36) 7.6	22 20 26	(23) 3.1	16 15 9	(13) 3.8
	500 µg	152 153 144	(150) 4.9	12 17 12	(14) 2.9	42 48 41		(44) 3.8	32 20 23	(25) 6.2	13 12 13	(13) 0.6
	1500 µg	152 162 140	(151) 11.0	14 11 13	(13) 1.5	45 38 43		(42) 3.6	18 23 29	(23) 5.5	7 11 13	(10) 3.1
	5000 µg	128 S 126 S 128 S	(127) 1.2	14 S 16 S 7 S	(12) 4.7	38 S 32 S 35 S		(35) 3.0	25 S 20 S 27 S	(24) 3.6	16 S 15 S 13 S	(15) 1.5
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
		1351 1274 1272	(1299) 45.0	249 220 230	(233) 14.7	218 180 195		(198) 19.1	103 212 201	(172) 60.0	246 213 309	(256) 48.8

S: Sparse bacterial background lawn

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test the test item was considered to be non-mutagenic.

Executive summary:

Introduction:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1)guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using 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) was based on OECD TG 471 and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxicity of the test item following the change in test methodology.

Results:

The vehicle (acetone) 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 and 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 OECD TG 471 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).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method).Results from the second mutation test showed that the test item induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns noted to all of the tester strains at 5000 µg/plate in both the absence and presence of metabolic activation (S9-mix). 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

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). A number of statistically significant values were noted (TA100 at 150 µg/plate and TA98 at 1.5 and 500 µg/plate in the absence of metabolic activation (S9-mix) and TA100 at 5, 15, 150 and 5000 µg/plate in the presence of metabolic activation (S9-mix); however,these responses were within the in-house historical vehicle/untreated control range for the strains and were, therefore considered of no biological relevance.

Similarly, no biologically relevant 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). Two statistically significant values were noted (TA1535 at 50 and 500 µg/plate in the absence of metabolic activation (S9-mix); however,these responses were within the in-house historical vehicle/untreated control range for the strains and were, therefore considered of no biological relevance.

Conclusion:

The test item was considered to be non-mutagenic under the conditions of this test.