N,N'-methylenedistearamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +S9 negative, -S9 negative, S. typhimurium: TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA, OECD 471, Thompson 2018

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2018 to 25 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

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

Version / remarks:

24 November 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park ,Shardlow, Derbyshire, DE72 2GD, UK

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine and tryptophan locus

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fractions (CD Sprague-Dawley)

Test concentrations with justification for top dose:

Mutagenicity: Experiment 1 (with and without metabolic activation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).
Mutagenicity: Experiment 2 (with and without metabolic activation): 15, 50, 150, 500, 1500 and 5000 μg/plate. The dose range used for Experiment 2 was determined by the results of Experiment 1.

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl formamide (purity: 99.98%)
- Justification for choice of solvent/vehicle: The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl formamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Exposure duration: approximately 48 hours at 37°C
- Expression time: none

SELECTION METHOD:
The strains used will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium.

NUMBER OF REPLICATIONS: triplicates

METHOD:
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of 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).

Experiment 1 – plate incorporation
The dose range for Experiment 1 was based on OECD TG 471 and was 1.5 to 5000 μg/plate.
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 (or 0,5 mL of S9-mix for the metabolic activation) 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.
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). Manual counts were performed at 5000 μg/plate because of test item precipitation. Further sporadic manual counts were also performed due to spreading colonies which prevented an accurate automated count.

Experiment 2 – pre-incubation method
The experiment was repeated on a separate day 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.
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer (or 0,5 mL of S9-mix for the metabolic activation) and 0.1 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.
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). Manual counts were performed at 5000 μg/plate because of test item precipitation. Further sporadic manual counts were also performed due to spreading colonies which prevented an accurate automated count.

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.

Acceptability criteria are listed in 'Any other information on materials incl. methods'.

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. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not reported in the tables section.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98

Remarks:

Experiment 1 (plate incorporation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 1 (plate incorporation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MAIN TEST: Test results for experiment 1 can be found in Table 2,3 and experiment 2 in Table 3,4 in 'Any other information on results incl. tables'.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: tested up to levels of precipitation (above 500 μg/plate)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see Table 1 in ‘Any other information on results incl. tables’.
- Negative (solvent/vehicle) historical control data: see Table 1 in ‘Any other information on results incl. tables’.

Table 1. Historical positive control and vehicle/negative control values 2017

	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Combined vehicle and untreated control values
Values	472	463	950	477	862	431	935	462	918	449
Mean	92	92	18	17	25	31	22	26	13	14
SD	14.4	15.4	6.5	7.1	7.0	7.4	6.9	6.8	3.4	3.4
Positive control values
Values	472	463	475	472	431	431	468	462	459	448
Mean	769	1415	773	260	682	257	224	191	305	380
SD	353.2	553.3	5.66	71.1	231.3	132.5	62.9	83.9	132.9	113.8

Table 2. Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 17 May 2018					To: 20 May 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	150 125 118	(131) 16.8#	13 18 29	(20) 8.2	30 24 20	(25) 5.0	31 15 19	(22) 8.3	15 6 8	(10) 4.7
	1.5 µg	116 127 133	(125) 8.6	24 34 14	(24) 10.0	29 23 22	(25) 3.8	25 24 17	(22) 4.4	10 12 7	(10) 2.5
	5 µg	132 96 130	(119) 20.2	18 21 13	(17) 4.0	45 25 30	(33) 10.4	22 21 18	(20) 2.1	4 11 7	(7) 3.5
	15 µg	109 132 119	(120) 11.5	13 14 25	(17) 6.7	19 20 27	(22) 4.4	15 22 27	(21) 6.0	4 6 16	(9) 6.4
	50 µg	128 108 126	(121) 11.0	16 10 9	(12) 3.8	20 20 28	(23) 4.6	16 25 26	(22) 5.5	8 8 11	(9) 1.7
	150 µg	105 116 150	(124) 23.5	13 10 9	(11) 2.1	28 26 32	(29) 3.1	25 32 31	(29) 3.8	5 13 24	(14) 9.5
	500 µg	102 P 138 P 128 P	(123) 18.6	13 P 11 P 18 P	(14) 3.6	22 P 39 P 34 P	(32) 8.7	19 P 13 P 15 P	(16) 3.1	11 P 7 P 14 P	(11) 3.5
	1500 µg	149 P 127 P 98 P	(125) 25.6	11 P 18 P 12 P	(14) 3.8	30 P 21 P 23 P	(25) 4.7	16 P 23 P 19 P	(19) 3.5	15P 16P 25 P	(19) 5.5
	5000 µg	103 P 99 P 118 P	(107) 10.0	22 P 12 P 15 P	(16) 5.1	27 P 28 P 22 P	(26) 3.2	28 P 23 P 17 P	(23) 5.5	12 P 14 P 13 P	(13) 1.0
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
		404 549 541	(498) 81.5	866 905 708	(826) 104.3	341 389 421	(384) 40.3	206 224 253	(228) 23.7	272 439 425	(379) 92.6

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

# Standard deviation

 

Table 3. Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 17 May 2018					To: 20 May 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	147 125 141	(138) 11.4#	26 20 18	(21) 4.2	29 29 19	(26) 5.8	19 27 25	(24) 4.2	5 8 9	(7) 2.1
	1.5 µg	112 95 99	(102) 8.9	22 24 23	(23) 1.0	26 14 30	(23) 8.3	26 18 21	(22) 4.0	3 8 8	(6) 2.9
	5 µg	150 106 114	(123) 23.4	16 26 23	(22) 5.1	23 22 36	(27) 7.8	21 28 21	(23) 4.0	3 8 6	(6) 2.5
	15 µg	114 138 140	(131) 14.5	12 21 17	(17) 4.5	28 22 35	(28) 6.5	24 14 35	(24) 10.5	7 10 5	(7) 2.5
	50 µg	153 128 134	(138) 13.1	14 10 14	(13) 2.3	27 37 34	(33) 5.1	29 15 33	(26) 9.5	8 12 11	(10) 2.1
	150 µg	147 139 101	(129) 24.6	10 10 23	(14) 7.5	27 23 27	(26) 2.3	19 20 32	(24) 7.2	13 10 16	(13) 3.0
	500 µg	83 P 93 P 102 P	(93) 9.5	12 P 8 P 17 P	(12) 4.5	32 P 27 P 40 P	(33) 6.6	26 P 28 P 36 P	(30) 5.3	7 P 15 P 19 P	(14) 6.1
	1500 µg	124 P 17P 18P	(109) 13.0	15 P 21 P 7 P	(14) 7.0	33 P 29 P 30 P	(27) 5.5	17 P 36 P 21 P	(25) 10.0	9 P 9 P 11 P	(10) 1.2
	5000 µg	100 P 102 P 110 P	(104) 5.3	13 P 17 P 16 P	(15) 2.1	30 P 30 P 24 P	(28) 3.5	24 P 20 P 22 P	(22) 2.0	12 P 12 P 11 P	(12) 0.6
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
		2441 1766 2256	(2154) 348.8	386 360 356	(367) 16.3	217 221 183	(207) 20.9	133 130 133	(132) 1.7	371 290 333	(331) 40.5

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item precipitate

# Standard deviation

 

Table 4. Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 22 May 2018					To: 25 May 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	104 103 107	(105) 2.1#	9 8 17	(11) 4.9	21 19 26	(22) 3.6	14 18 13	(15) 2.6	13 7 5	(8) 4.2
	15 µg	120 116 121	(119) 2.6	12 14 19	(15) 3.6	29 25 19	(24) 5.0	15 13 15	(14) 1.2	4 6 3	(4) 1.5
	50 µg	105 101 92	(99) 6.7	12 9 14	(12) 2.5	25 9 15	(16) 8.1	18 13 7	(13) 5.5	12 5 3	(7) 4.7
	150 µg	103 111 110	(108) 4.4	12 7 18	(12) 5.5	20 21 28	(23) 4.4	17 18 16	(17) 1.0	7 8 3	(6) 2.6
	500 µg	105 P 109 P 97 P	(104) 6.1	18 P 10 P 9 P	(12) 4.9	31 P 29 P 15 P	(25) 8.7	16 P 21 P 19 P	(19) 2.5	4 P 10 P 16 P	(10) 6.0
	1500 µg	86 P 98 P 97 P	(94) 6.7	14 P 9 P 11 P	(11) 2.5	20 P 18 P 18 P	(19) 1.2	15 P 23 P 13 P	(17) 5.3	6 P 5 P 8 P	(6) 1.5
	5000 µg	89 P 88 P 91 P	(89) 1.5	16 P 13 P 9 P	(13) 3.5	24 P 27 P 18 P	(23) 4.6	11 P 17 P 14 P	(14) 3.0	8 P 10 P 12 P	(10) 2.0
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
		947 811 852	(870) 69.8	687 535 665	(629) 82.1	554 543 412	(503) 79.0	160 150 130	(147) 15.3	412 356 266	(345) 73.7

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

# Standard deviation

 

Table 5. Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 22 May 2018					To: 25 May 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	118 117 125	(120) 4.4#	9 10 9	(9) 0.6	39 26 37	(34) 7.0	25 19 29	(24) 5.0	10 10 18	(13) 4.6
	15 µg	125 111 112	(116) 7.8	7 9 13	(10) 3.1	21 36 29	(29) 7.5	21 30 27	(26) 4.6	21 12 16	(16) 4.5
	50 µg	120 125 134	(126) 7.1	17 11 8	(12) 4.6	28 22 28	(26) 3.5	27 25 30	(27) 2.5	6 11 11	(9) 2.9
	150 µg	113 118 111	(114) 3.6	9 14 18	(14) 4.5	23 29 25	(26) 3.1	21 32 26	(26) 5.5	12 14 17	(14) 2.5
	500 µg	131 P 121 P 111 P	(121) 10.0	11 P 11 P 14 P	(12) 1.7	29 P 38 P 31 P	(33) 4.7	30 P 28 P 23 P	(27) 3.6	19P 20P 10 P	(16) 5.5
	1500 µg	100 P 124 P 84 P	(103) 20.1	15 P 17 P 13 P	(15) 2.0	19 P 16 P 14 P	(16) 2.5	21 P 32 P 19 P	(24) 7.0	8 P 17 P 14 P	(13) 4.6
	5000 µg	93 P 97 P 101 P	(97) 4.0	10 P 31 P 32 P	(12) 1.5	30 P 21 P 23 P	(25) 4.7	24 P 25 P 19 P	(23) 3.2	14 P 12 P 4 P	(10) 5.3
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
		1701 2056 1818	(1858) 180.9	262 207 233	(234) 27.5	156 161 163	(160) 3.6	160 175 148	(161) 13.5	310 256 262	(276) 29.6

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item precipitate

# Standard deviation

Conclusions:

In this GLP compliant study, performed according to OECD 471, the test substance and its metabolites did not induce gene mutations in the tested strains of S. typhimurium and E. coli

Executive summary:

In this GLP compliant study, performed according to OECD 471, the mutagenic potential of the test substance was evaluated in vitro inSalmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. These strains were treated with suspensions of 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, 5, 15, 50, 150, 500, 1500 and 5000 µg/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, 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. All of the plates were incubated at 37 ± 3^oC 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). Manual counts were performed at 5000 µg/plate because of test item precipitation. A single manual count was also performed due to spreading colonies which prevented an accurate automated count.

The vehicle (dimethyl formamide) 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.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). 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). Minor statistical values were noted in Experiment 2 (TA100 at 15 μg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. In conclusion, the test substance was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation in bacteria

In this GLP compliant study, performed according to OECD 471, the mutagenic potential of the test substance was evaluated in vitro in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. These strains were treated with suspensions of 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 metabolising 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, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. A second experiment was done 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, 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. All of the plates were incubated at 37 ± 3 ^oC 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). Manual counts were performed at 5000 µg/plate because of test item precipitation. A single manual count was also performed due to spreading colonies which prevented an accurate automated count.

The vehicle (dimethyl formamide) 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. 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). 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). Minor statistically significant differences were noted in Experiment 2 (TA100 at 15 μg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. In conclusion, the test substance was considered to be non-mutagenic under the conditions of this test.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.