2-[2-(2,3-dihydro-2-methyl-1H-indol-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium chloride

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:

November 21, 1995 to November 30, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69/EEC B.14. Salmonella typhimurium Reverse Mutation Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals "Genetic Toxicology: Salmonella typhimurium Reverse Mutation Assay" Adopted: 26 May 83, No. 471

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA: HG - Gene Muta - S. typhimurium, October 1984

Version / remarks:

New and Revised Health Effects Test Guidelines October 1984 (U.S.) Environmental Protection Agency Washington, DC (PB 84-233295) .
HG - Gene Muta - S. typhimurium, October 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(2,3-dihydro-2-methyl-1H-indol-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium chloride

EC Number:

228-800-7

EC Name:

2-[2-(2,3-dihydro-2-methyl-1H-indol-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium chloride

Cas Number:

6359-50-8

Molecular formula:

C22H25N2.Cl

IUPAC Name:

2-[2-(2,3-dihydro-2-methyl-1H-indol-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium chloride

Test material form:

solid: particulate/powder

Details on test material:

Basic Yellow 21

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The doses of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as μg/tube for better separation of plate incorporation and preincubation trials, despite the fact that μg/plate and μg/tube could be used synonymously.

0, 16, 50, 158, 500, 1581 and 5000 μg/plate – with and without S9 mix
0, 45, 90, 180, 360, 720 and 1440 μg/tube – with and without S9 mix

The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 µg or 5 µI per plate were used as the highest dose. At least five additional doses were routinely used. The doses used in the first trial (plate incorporation test) were: 0, 16, 50, 158, 500, 1581 and 5000 μg/plate.
Doses up to and including 158 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 1581 µg per plate for assessment purposes.
Due to the substance's toxicity, doses ranging from 45 µg to 1440 µg per tube were chosen for the repeat tests. The doses used in the second trial (preincubation test) were: 0, 45, 90, 180, 360, 720 and 1440 μg/tube (equivalent to 0, 16, 50, 158, 500, 1581 and 5000 μg/plate).

Vehicle / solvent:

The test substance was formulated in deionized water and formed a light-yellow suspension (5000 μg per plate) and light yellow clear solutions (1581 μg per plate and below) respectively. The positive controls were dissolved in DMSO.
The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether, and DMF according to information given by the internal sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

other: Nitrofurantoin (NF); 4-nitro-1,2-phenylene diamine (4-NPDA); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
For the mutant count, three plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained three plates per strain.
The amount of solvent for the test substance and for the controls was 0.1 ml/plate.
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 μg or 5 μl per plate were used as the highest dose. At least five additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
Because the first assessable trial showed no mutagenic effects of the test substance, the repeat was performed as preincubation in a water bath at 37 °C for 20 minutes. At the end of the preincubation period 2 ml of molten soft agar were added to the tubes, the content mixed and plated.
For the mutant count, three plates were used for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained three plates per strain. In experiments without S9 mix buffer was used as replacement.
The doses of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as μg/tube for better separation of plate incorporation and preincubation trials, despite the fact that μg/plate and μg/tube could be used synonymously.
The toxicity of the substance was assessed in three ways. The first method was a gross appraisal of background growth on the plates for mutant determination. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37 °C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per millilitre, since the chosen method of incubation normally produces the designed density. However, the numbers of viable cells were established in a parallel procedure by determining the titers
The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased fivefold to per -it the complete growth of bacteria.
The tests were performed both with and without S9 mix.
The count was made after the plates had been incubated for 48 hours at 37 °C. If no immediate count was possible, plates were temporarily stored in a refrigerator.

The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/or the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.
The following doses per plate were evaluated in the first test:
μg per plate
1. Negative control 0
2. ASTRAZON Gelb 7GLL 5000
3. ASTRAZON Gelb 7GLL 1581
4. ASTRAZON Gelb 7GLL 500
5. ASTRAZON Gelb 7GLL 158
6. ASTRAZON Gelb 7GLL 50
7. ASTRAZON Gelb 7GLL 16
8. Positive control, sodium azide 10 (only TA 1535)
9. Positive control, nitrofurantoin 0.2 (only TA 100)
10. Positive control, 4-nitro-1,2-phenylene diamine 10 (only TA 1537)
11. Positive control, 4-nitro-1,2-phenylene diamine 0.5 (only TA 98)
12. Positive control, Cumene hydroperoxide 50 (only TA 102)
13. Positive control, 2-aminoanthracene 3

Due to the substance's toxicity, doses ranging from 45 μg to 1440 μg per tube were chosen for the repeat tests.
ASTRAZON Gelb 7GLL was formulated in deionized water and formed a light-yellow suspension (5000 μg per plate) and light yellow clear solutions (1581 μg per plate and below) respectively. The positive controls were dissolved in DMSO.
No ''untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature (e.g. Maron and Ames, 1983) and from the laboratories own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.

Rationale for test conditions:

The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).

Evaluation criteria:

A reproducible and dose- related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

None

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:

cytotoxicity

Remarks:

In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Plate Incorporation Method
As may be seen, there was no indication of a bacteriatoxic effect of ASTRAZON Gelb 7GLL at doses of up to and including 158 μg per plate. The total bacteria count's consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only partly be used for assessment purposes up to and including 1581 μg per plate.
None of the five strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Preincubation Method
As may be seen, there was no indication of a bacterioxic effect of ASTRAZON Gelb 7GLL at doses of up to and including 90 μg per tube. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. Higher doses had a strong, strain-specific bacterio-toxic effect. Therefore they could only partly be used for assessment purposes up to and including 720 μg per tube.
None of the five strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls and thus confirmed the results of the plate incorporation method.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Any other information on results incl. tables

Summary of the Results with ASTRAZON Gelb 7GLL in the

Salmonella/Microsome Test Plate Incorporation Method

S9 mix	TA 1535	TA 100	TA 1537	TA 98	TA 102
Without	-ve	-ve	-ve	-ve	-ve
With	-ve	-ve	-ve	-ve	-ve

-ve = Negative

 

Summary of the Results with ASTRAZON Gelb 7GLL in the

Salmonella/Microsome Test Preincubation Method

S9 mix	TA 1535	TA 100	TA 1537	TA 98	TA 102
Without	-ve	-ve	-ve	-ve	-ve
With	-ve	-ve	-ve	-ve	-ve

-ve = Negative

 

Summary of Mean Values Without S9 Mix

group	Strain
	TA 1535	TA 100	TA 1537	TA 98	TA 102
μg/plate 0 16 50 158 500 1581 5000 Na-azide NF 4-NPDA Cumene	8 7 9 10 6 3 -- 822	100 94 92 89 81 19 0   288	11 10 8 12 12 10 --     182	22 27 34 29 33 17 --     210	278 309 351 330 305 241 ---       685
μg/tube 0 45 90 180 360 720 1440 Na-azide NF 4-NPDA Cumene	10 9 9 9 7 6 -- 711	91 91 81 84 77 88 40   493	13 11 2 11 9 6 5     168	31 28 28 33 28 22 20     201	330 446 458 421 298 248 209       708

 

Summary of Mean Values With S9 Mix

Group	Strain
	TA 1535	TA 100	TA 1537	TA 98	TA 102
μg/plate 0 16 50 158 500 1581 5000 2-AA	11 10 9 13 8 3 -- 224	103 112 100 113 94 49 -- 1583	12 9 11 12 8 6 -- 472	37 29 37 37 38 37 -- 1509	361 370 380 407 349 324 --- 1179
μg/tube 0 45 90 180 360 720 1440 2-AA	14 12 13 15 10 7 -- 264	122 131 22 118 116 80 18 1602	13 14 13 13 14 12 7 320	38 44 41 47 41 33 30 1261	403 440 479 463 432 403 241 749

 

Historical Controls

Plate Incorporation Method

 

Summary of historical negative and positive controls of experiments performed from January to June 1993 using mean values present medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water DMSO DMF Ethanol Acetone EGDE^2	- - - - - -	13 11 10 10 14 14	2 2 - 1 1 2	96 80 70 77 88 95	13 9 - - 11 14	10 8 11 10 9	3 1 - - 1	25 22 16 26 25	4 3 - - 3
Na-azid NF 4-NPDA	- - -	683	86	337	52	57	9	82	15
30% Water DMSO Ethanol EGDE^2	+ + + +	17 15 28 36	- 2 - -	116 111 135 137	- 7 - -	11 9 10 14	- 2 - -	36 30 38 45	- 5 - -
2-AA	+	157	35	571	190	82	258	494	76
10% Water DMSO DMF Ethanol Acetone EGDE^2	+ + + + + +	18 14 13 29 16 16	4 2 - - 3 -	118 101 87 113 114 118	9 13 - - 5 9	10 9 10 13 11 10	3 1 - - 1 -	39 31 23 45 43 36	5 3 - - 7 -
2-AA	+	154	34	1177	246	225	100	1236	195

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1993 using mean values present medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water DMSO Ethanol Acetone EGDE^2	- - - - -	11 11 17 16 17	2 2 - - 3	82 86 121 92 95	21 15 - - 20	7 8 8 9 9	3 2 - - 2	21 19 27 20 18	2 2 - - 5
Na-azid NF 4-NPDA	- - -	643	145	284	74	64	11	58	9
30% Water DMSO EGDE^2	+ + +	16 18 15	- 4 -	127 105 140	- 16 -	6 10 10	- 3 -	26 37 27	- 9 -
2-AA	+	220	23	448	203	88	21	478	86
10% Water DMSO Ethanol Acetone EGDE^2	+ + + + +	12 14 25 15 18	4 3 - - -	100 98 125 113 118	19 21 - - 11	8 10 13 10 12	2 2 - - 1	27 29 47 32 29	7 6 - - 3
2-AA	+	145	39	812	342	164	40	1127	223

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to December 1994 using mean values present medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water DMSO DMF Methanol Ethanol Acetone EGDE^2	- - - - - - -	10 9 12 12 7 7 11	2 2 - - 2 3 3	89 82 94 95 56 79 79	14 9 - - 10 12 10	8 9 9 11 8 6 8	2 2 - - 2 2 2	22 21 25 15 24 18 16	3 4 - - 11 3 6
Na-azid NF 4-NPDA	- - -	706	98	263	33	105	35	138	23
30% Water DMSO EGDE^2	+ + +	13 11 10	- - -	98 96 115	- - -	7 5 7	- - -	29 22 22	- - -
2-AA	+	152	24	594	140	72	29	489	93
10% Water DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + +	13 11 13 20 10 10 14	2 2 - - 3 - 3	112 105 106 119 84 106 114	15 15 - - 11 11 18	8 8 9 11 4 6 8	2 2 - - 2 3 2	30 27 36 32 28 28 29	7 5 - - 4 2 10
2-AA	+	171	49	1111	330	103	73	1301	208

²) Ethylene glycol dimethylether

 

Preincubation Method

 

Summary of historical negative and positive controls of experiments performed from January to June 1993 using mean values present medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water DMSO Acetone	- - -	15 13 15	2 3 -	129 94 123	10 8 -	13 10 13	2 1 -	34 26 27	5 4 -
Na-azid NF 4-NPDA	- - -	689	106	475	67	60	10	101	22
10% Water DMSO Acetone	+ + +	20 16 16	4 3 -	140 114 141	10 11 -	12 10 14	2 1 -	45 34 33	4 5 -
2-AA	+	166	20	1176	149	255	114	1196	206

 

Summary of historical negative and positive controls of experiments performed from July to December 1993 using mean values present medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water DMSO Ethanol Acetone EGDE^2	- - - - -	13 15 10 15 14	2 4 - - 1	128 120 168 108 126	13 21 - - 20	10 9 7 1 1	1 2 - - -	22 24 26 24 20	1 3 - - 2
Na-azid NF 4-NPDA	- - -	754	162	443	46	65	13	73	8
10% Water DMSO Ethanol Acetone EGDE^2	+ + + + +	15 15 19 19 16	2 2 - - 2	153 129 131 147 152	25 20 - - 23	9 11 10 12 12	1 1 - - 3	26 34 28 36 30	3 5 - - 8
2-AA	+	167	25	1188	138	226	44	1100	198

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to December 1994 using mean values present medians (Z) and semi-Q range (QR)

Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
Water DMSO Methanol Ethanol Acetone EGDE^2	- - - - - -	11 10 12 9 11 13	2 2 - 2 - 2	131 99 139 103 113 105	18 10 - 10 - 22	8 8 6 10 8 10	2 2 - 4 - 2	19 22 18 20 23 20	6 5 - 4 - 5
Na-azid NF 4-NPDA	- - -	787	116	402	41	98	33	137	26
10% Water DMSO Methanol Ethanol Acetone EGDE^2	+ + + + + +	13 11 20 14 14 13	2 3 - 2 - 2	163 115 139 118 175 142	25 13 - 9 - 19	9 8 7 9 7 10	2 2 - 3 - 2	29 29 24 24 31 33	7 6 - 6 - 7
2-AA	+	185	35	1357	166	178	55	1268	208

²) Ethylene glycol dimethylether

Applicant's summary and conclusion

Conclusions:

Basic Yellow 21 was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.
The substance is not classified in accordance with CLP criteria.

Executive summary:

Basic Yellow 21 was initially investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 μg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.

 

Doses up to and including 158 μg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriatoxic effect, so that this range couldonly be used to a limited extent up to and including 158 μg per plate for assessment purposes.

 

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect , as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

The test substance was investigated in an independent repeat using the Salmonella/microsome test for point mutagenic effects in doses up to 1440 μg per tube after preincubation for 20 minutes at 37 °C on five Salmonella typhimurium LT2. mutants.

These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 und TA 102.

 

Doses up to and including 90 μg per tube did not cause any bacteriotoxic effects: Total bacteria counts, remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriatoxic effect, so that this range could only be used to a limited extent up to and including 720 μg per tube for assessment purposes.

 

Evidence of mutagenic activity of Basic Yellow 21 was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

The substance is not classified in accordance with CLP criteria.