7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

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:

March 11, 1992 to March 30, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 84/449/EEC B.14. Other Effects – Mutagenicity Salmonella typhimurium Reverse Mutation Test

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: M. J. Prival, V. D; Mitchell

Version / remarks:

M. J. Prival, V. D; Mitchell: Analysis of a method for testing azo dyes for mutagenicity in Sallmonella typhimurium in the presence of flavin mononucleo­ tide and hamster liver S-9. Mut. Res.,103- 116 (1982).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

EC Number:

286-839-5

EC Name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

Cas Number:

85391-83-9

Molecular formula:

C21H14ClF2N5O8S2.xNa

IUPAC Name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

Test material form:

solid: particulate/powder

Details on test material:

Reactive Red 123

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 μg /plate was used as the highest dose.
The following doses per pate were evaluated in the first test:
0, 8, 40, 200, 1000, 5000 μg/plate
Due to the substance's weak toxicity, doses ranging from 8 μg to 5000 μg per tube were chosen for the repeat tests.
The following doses per plate were evaluated in repeat tests:
0, 8, 40, 200, 1000, 5000 μg/plate

Vehicle / solvent:

Levafix Scharlach E-2GA was dissolved in deionized water.
The solvent used was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone &, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

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

Details on test system and experimental conditions:

Test Design
For the mutant count, four 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 four 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 /plate was used as the highest dose. At least four additional doses were routinely used as progressive dilutions of the top dose. 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 obtain in the first experiment.

Because the first assessable trial showed no mutagenic effects of the test substance, the repeat was performed according to Prival and Mitchell (1982) due to the chemical structure of the compound. Preincubation was performed in a water bath at 30 °C for 30 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, four 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 four 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.

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. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with reduced background growth. (The same applies to the signs "c", "v", "p", "n" or "%", which may also be used in the tables.) 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 S the bacterial suspensions at a defined density of viable cells per millilitre, since the chosen method of incubation normally produces the desired 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 from 0.5 mM to 2.5 mM to permit 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, plate were temporarily stored in a refrigerator.

Rationale for test conditions:

The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
Because the first assessable trial showed no mutagenic effects of the test substance, the repeat was performed according to Prival and Mitchell (1982) due to the chemical structure of the compound.

Evaluation criteria:

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.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were it met, an assay was accepted if it showed mutagenic activity of the test compound.

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. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.

In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

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:

bacteriotoxic effects at 5000 μg/plate

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:

bacteriotoxic effects at 5000 μg/plate

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:

bacteriotoxic effects at 5000 μg/plate

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:

bacteriotoxic effects at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Plate Incorporation Method
There was no indication of a bacteriotoxic effect of Levafix Scharlach E-2GA at doses of up to and including 1000 μg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. 5000 μg per plate had a weak, strain-specific bacteriotoxic effect, but could nevertheless be used for assessment.
None of the four 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.

Prival Assay
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine 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.
There was no indication of a bacteriotoxic effect of Levafix Scharlach E-2GA at doses of up to and including 1000 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. 5000 μg per plate, a weak, strain-specific bacteriotoxic effect, but could nevertheless be used for assessment.
None of the four 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, benzidine, Congo red 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 Levafix Scharlach E-2GA in the Salmonella/Microsome Test

Plate Incorporation Method

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

-ve = negative

 

Summary of the Results with Levafix Scharlach E-2GA in the Salmonella/Microsome Test

Prival Assay

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

-ve = negative

 

Summary of Mean Values Without S9 Mix

Group	Strain
	TA 1535	TA 100	TA 1537	TA 98
μg/plate 0 8 40 200 1000 5000 Na-azide NF 4-NPDA	19 23 18 21 17 17 875	107 124 124 105 106 82   388	5 7 6 6 4 4     62	19 17 23 17 20 10     82
μg/tube 0 8 40 200 1000 5000 Na-azide NF 4-NPDA	11 12 10 11 10 10 883	82 103 109 115 94 86   316	13 13 13 14 13 11     44	37 32 30 30 27 28     75

 

Summary of Mean Values With S9 Mix

Group	Strain
	TA 1535	TA 100	TA 1537	TA 98
μg/plate 0 8 40 200 1000 5000 2-AA	26 20 28 27 24 17 140	150 140 156 155 140 102 865	6 5 6 9 7 5 47	28 27 27 33 22 15 245
μg/tube 0 8 40 200 1000 5000 Benzidine Congo red 2-AA	17 22 21 23 20 15     166	150 168 176 180 164 137     1392	24 21 22 19 23 17     251	55 56 47 53 53 42 119 85

 

Historical Controls of Plate Incorporation Method

 

Summary of historical negative and positive controls of experiments performed from January to June 1988 using mean values presented as 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 EGDME^2	- - - - - -	14 13 12 15 10 18	2 2 2 3 2	97 94 87 69 85 117	9 15 11 7 10	8 8 8 7 7 10	1 1 1 1 1	17 17 19 22 18 21	2 2 3 3 2
Na-azide NF 4-NPDA	- - -	839	115	382	46	90	13	109	20
30% Water DMSO DMF Ethanol Acetone EGDME^2	+ + + + + +	14 15 14 20 14 18	3 3 3 2 1	134 124 113 105 134 159	10 14 9 6 25	8 9 9 6 11 9	2 2 2 1 2	29 29 31 30 34 35	3 3 5 3 3
2-AA	+	282	63	601	164	66	17	532	160
10% Water DMSO DMF Ethanol Acetone	+ + + + +	147 14 -- 23 13	4 2	123 111 72 87 85	4 13   6	9 8 9 8 7	1	33 33 27 38 29	5
2-AA	+	357	67	1422	428	298	65	1323	323

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1988 using mean values presented as 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	- - - - -	14 14 12 10 15	3 2 2 2 2	97 93 70 71 138	9 25 4 2 10	8 8 7 7 8	2 1 1 1 3	20 19 13 21 39	5 10 1 3 6
Na-azide NF 4-NPDA	- - -	822	137	412	42	88	19	124	25
30% Water DMSO DMF Ethanol Acetone	+ + + + +	12 16 14 17 13	2 2 3 3 4	144 124 117 90 177	15 15 14 4 35	10 10 9 8 9	2 2 2 1 2	35 32 31 39 43	6 5 6 2 8
2-AA	+	261	69	755	196	93	21	583	171
10% DMSO DMF	+ +	7 11	1	110 121	12	9 6	1	32 26	5
2-AA	+	348	70	1544	572	416	75	1499	423

 

Summary of historical negative and positive controls of experiments performed from January to June 1989 using mean values presented as 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	- - - - - -	10 9 7 10 9 8	3 3 1 2 - 2	91 84 60 73 100 69	11 16 4 12 -- 16	7 7 6 7 7 6	1 2 1 2 - 2	18 16 14 18 18 17	3 2 2 4 - 4
Na-azide NF 4-NPDA	- - -	721	110	359	61	75	13	119	35
30% Water DMSO DMF Ethanol Acetone EGDE^2	+ + + + + +	14 14 14 17 15 14	2 3 2 3 - 2	133 114 100 118 138 115	12 18 9 12 -- 25	9 9 8 10 13 11	2 1 2 2 - 2	32 28 25 37 32 27	8 4 4 8 - 8
2-AA	+	195	33	633	127	63	28	392	133
10% DMSO DMF	+ +	12 --	2 -	105 --	28 --	7 7	2 -	25 31	4 -
2-AA	+	267	27	1455	348	283	64	1547	289

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1989 using mean values presented as 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
DMSO DMF Ethanol Acetone EGDE^2	- - - - -	10 9 8 15 8	4 4 3 - -	72 57 57 96 63	6 15 12 -- --	7 8 6 6 6	4 2 1 - -	16 17 14 13 21	5 5 6 - -
Na-azide NF 4-NPDA	- - -	853	147	326	47	91	26	87	25
30% DMSO DMF Ethanol Acetone EGDE^2	+ + + + +	14 15 11 21 13	2 3 6 - -	89 87 79 96 87	7 6 13 -- --	11 11	2 4 2 - -	23 26 23 20 26	2 4 5 - -
2-AA	+	157	42	500	83	73	22	498	101
10% DMSO Ethanol	+ +	14 11	5 -	91 53	7 -	10 4	1 -	24 18	4 -
2-AA	+	158	54	1464	152	289	117	1294	113

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to June 1990 using mean values presented as 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	- - - - - - -	15 12 10 17 13 10 14	3 2 4   3 1 4	74 72 65 87 77 69 95	10 13 10   11 4 14	7 8 7 7 8 6 8	1 2 2   2 1 1	22 17 10 19 19 11 18	5 3 6   2 2 5
Na-azide NF 4-NPDA	- - -	799	108	268	48	52	12	81	14
30% Water DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + +	18 18 13 22 19 13 15	2 3 3   3 1 2	108 86 97 121 98 104 97	17 11 17   15 8 9	9 9 7 11 8 7 9	2 2 3   2 3 3	27 27 20 28 29 22 28	5 3 5   4 3 8
2-AA	+	161	39	509	130	48	15	379	54
10% DMSO Ethanol Acetone	+ + +	18 16	2	89 85 107	20	11 8	4	30 29 17	6
2-AA	+	214	49	1196	181	235	38	1140	284

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1990 using mean values presented as 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	- - - - - - -	13 14 13 13 12 12 13	2 2 2 1 3 2 2	105 105 82 105 93 116 112	16 7 16 16 14 2 15	9 8 6 8 9 6 8	1 1 2 1 1 1 2	21 21 12 21 22 23 18	4 3 4 4 3 1 3
Na-azide NF 4-NPDA	- - -	882	114	380	60	48	9	71	15
30% Water DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + +	18 17 15 22 19 13 18	3 2 3 2 3 1 3	143 137 109 144 118 131 135	15 5 14 16 18 4 14	11 10 10 11 10 9 11	2 2 1 2 1 1 2	29 28 23 33 39 26 32	3 4 3 3 7 1 5
2-AA	+	175	41	800	243	84	17	485	93
10% DMSO Acetone EGDE^2	+ + +	16 12	2	127 124 140	19	9 10	3	32 26	5
2-AA	+	179	69	1321	148	298	39	1206	168

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to June 1990 using mean values presented as 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	- - - - - - -	12 13 9 11 12 10 11	3 2 - 2 1 - 3	111 113 80 105 96 55 108	10 14 -- 14 15 -- 5	9 10 7 8 9 5 8	2 2 - 2 2 - 1	28 30 23 29 31 21 23	5 3 - 5 5 - 8
Na-azide NF 4-NPDA	- - -	623	102	398	56	49	10	89	20
30% Water DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + +	16 18 11 23 19 14 15	3 3 - 5 3 - 4	152 154 84 152 127 84 132	15 11 -- 7 17 -- 6	12 12 9 10 10 14 8	2 2 - 3 3 - 1	38 40 29 48 43 18 40	7 7 - 10 6 - 9
2-AA	+	182	33	800	163	86	24	472	105
10% Water DMSO Methanol	+ + +	15 16 --	- 3 -	102 132 150	- 5 -	5 10 --	- 1 -	46 39 --	- 4 -
2-AA	+	208	48	1408	216	314	147	754	369

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1991 using mean values presented as 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 Buffer DMSO DMF Methanol Ethanol Acetone EGDE^2	- - - - - - - -	12 13 12 7 10 12 12 14	3 2 3   1 4 2 3	89 97 92 75 84 80 87 108	10 10 15   11 8 6 22	9 8 9 7 8 8 8 8	3 1 1   1 3 1 1	27 25 24 17 25 23 26 26	4 2 4   3 4 4 5
Na-azide NF 4-NPDA	- - -	605	122	339	52	53	9	71	17
30% Water Buffer DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + + +	19 17 19 11 25 18 18 22	4   3     5 2 4	138 159 130 142 134 119 111 144	21   11     19 9 11	13 13 10 9 12 11 13 13	2   2     2   3	33 38 33 32 37 37 28 32	4   4     2 11 3
2-AA	+	164	38	727	139	91	32	520	161
10% Water Buffer DMSO DMF Methanol Ethanol Acetone EGDE^2	+ + + + + + + +	16 14 16 15 16 19 17 20	4   2     3   2	113 94 118 114 111 94 112 153	18   14 6   6   11	10 10 10 11 9 12 11 11	3   3     2   1	33 34 31 21 29 32 32 34	5   3     2   5
2-AA	+	197	50	1431	260	304	116	1097	207

²) Ethylene glycol dimethylether

Applicant's summary and conclusion

Conclusions:

Due to the results, the test item has to be regarded as non-mutagenic. The substance is not classifiable according to CLP criteria.

Executive summary:

Levafix Scharlach E-2GA was initially investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to 5000 μg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

 

In the plate incorporation assay doses of up to and including 1000 μg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At 5000 μg per plate, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes.

 

In the plate incorporation assay evidence of mutagenic activity of Levafix Scharlach E-2GA 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 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.

 

Levafix Scharlach E-2GA was investigated in an independent repeat using the Salmonella/microsome test, modified with S9 mix according to Prival and Mitchell, for point mutagenic effects in doses of up to 5000 μg per tube on the same strains. Without S9 mix preincubation was used.

 

Doses of up to and including 1000 μg per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At 5000 μg per plate, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes.

 

Evidence of mutagenic activity of Levafix Scharlach E-2CA 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, benzidine, Congo red and 2-aminoanthracene had a marked mutagenic effect, as was see by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

Therefore, Levafix Scharlach E-2GA was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the Prival modification of the Salmonella/microsome test.