Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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:

April 1, 1992 to April 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

Deviations:

no

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

276-374-6

EC Name:

Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

72139-17-4

Molecular formula:

C25H16ClF2N5O8S2.2Na

IUPAC Name:

disodium 1-amino-4-({3-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-methyl-5-sulfophenyl}amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

No further details specified in the study report.

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:

Dose: 0, 8, 40, 200, 1000 & 5000 μg/plate
The doses for the first trial were routinely determined on the basis of a standard protocol: 5000 μg per plate were used as the highest dose, if not limited by solubility. At least four 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. In case of a positive response, however, 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.

Vehicle / solvent:

Deionized water.
Justification for choice of solvent/vehicle: solvent for the test substance was selected from the following list in the order water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dirnethylether (EGDE) according to information provided 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

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

Details on test system and experimental conditions:

For the mutant count, four plates were used, both with and without S9 mix, for each strain and dose. The same 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.
In the first assay, tubes were plated immediately after addition of the last component. In the repeat tests, all tubes were preincubated at 37°C for 30 minutes before plating.

The toxicity of the substance was assessed in three ways. The first 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", "H" 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 the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure when 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 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, plates were temporarily stored in a refrigerator.

The following doses per plate were evaluated in the first test and in the repeat tests:
μg per plate
1. Negative control 0
2. Levafix Br. Blau E-BRA FW 5000
3. Levafix Br. Blau E-BRA FW 1000
4. Levafix Br. Blau E-BRA FW 200
5. Levafix Br. Blau E-BRA FW 40
6. Levafix Br. Blau E-BRA FW 8
7. Positive control, sodium azide 10 (only TA 1535)
8. Positive control, nitrofurantoin 0.2 (only TA 100)
9. Positive control, 4-nitro-1,2-phenylene diamine 10 (only TA 1537)
10. Positive control, 4-nitro-1,2-phenylene diamine 0.5 (only TA 98)
11. Positive control, 2-aminoanthracene 3

The solvent employed for Levafix Br. Blau E-BRA FW was deionized water and for the positive controls DMSO.
The solvent for the test substance was selected from the following list in the order water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether (EGDE) according to information provided by the internal sponsor.
No "untreated" negative control was set up for deionized water, since sufficient evidence was available in the literature (i.e. Maron and Ames, 1983) and from our own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the bacterial strains used.

Rationale for test conditions:

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

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 (i.e. Maron and Ames, 1983) and 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 not met, an assay was accepted if it showed mutagenic activity of the test compound.

Statistics:

Not specified.

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:

Higher doses had a weak strain-specific bacteriotoxic effect (>1000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

Higher doses had a weak strain-specific bacteriotoxic effect (>1000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

Higher doses had a weak strain-specific bacteriotoxic effect (>1000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

Higher doses had a weak strain-specific bacteriotoxic effect (>1000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The Salmonella/microsome test, employing doses up to 5000 μg per plate, showed Levafix Br . Blau E-BRA FW to only produce bacteriotoxic effects at 5000 μg per plate yet all doses could be used for assessment purposes.
Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility), revealed no biologically relevant variations from the respective negative controls.
In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity. The positive control for the repeat test on TA 98 (without S9 mix) did not fulfill the criteria for acceptance. An additional repeat assay on TA 98 without S9 mix was therefore performed which confirmed the result of the first test.
Despite this sensitivity, no indications of mutagenic effects of Levafix Br. Blau E-BRA FW could be found at assessable doses up to 5000 μg per plate in any of the Salmonella typhimurium strains used.

Any other information on results incl. tables

Summary of the Results with Levafix Br. Blau E-BRA FW in the Salmonella/Microsome Test

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

Table and group μg/plate	Strain
	TA 1535	TA 100	TA 1537	TA 98
1-4 0 8 40 200 1000 5000 Na-azid NF 4-NPDA	25 26 25 24 24 22 702	93 88 97 92 95 91   321	9 10 9 10 9 5     38	18 23 23 33 25 15     56
5-8 0 8 40 200 1000 5000 Na-azid NF 4-NPDA	10 11 11 11 12 6 541	106 105 99 103 83 75   372	13 15 15 16 14 15     39	36 36 37 32 30 20     64
9 0 8 40 200 1000 5000 4-NPDA				31 23 29 25 29 19 77

 

Summary of Mean Values With S9 Mix

Table and group μg/plate	Strain
	TA 1535	TA 100	TA 1537	TA 98
1-4 10% S9 0 8 40 200 1000 5000 2-AA	28 30 28 29 26 22 158	120 104 118 125 122 107 1416	13 10 12 13 10 7 273	30 34 31 32 27 15 1187
5-8 10% S9 0 8 40 200 1000 5000 2-AA	14 17 12 14 14 12 121	146 135 166 163 141 94 1061	13 17 15 16 12 11 158	46 45 42 49 39 23 1248

 

Summary of historical negative and positive control of experiments performed from January to June 1987 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	- -	14 14	3 1	91 98	13 9	6 6	1 1	15 15	2 2
Na-azid NF 4-NPDA	- - -	974	99	319	83	75	9	91	18
30% DMSO DMF	+ +	15 14	2 2	136 138	14 4	8 11	2 0	30 35	3 6
2-AA	+	407	84	1292	298	59	18	870	205
10% DMSO DMF	+ +	13 17	2	113 90	13	8 6	1	29 32	3
2-AA	+	504	65	2381	289	298	35	1799	344

 

Summary of historical negative and positive control of experiments performed from July to December 1987 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	- - - -	12 12 9 3	2 2 2 2	80 80 65 71	15 12 12 11	8 7 8 8	2 1 2 1	16 15 16 18	2 2 8 5
Na-azid NF 4-NPDA	- - -	808	119	295	43	92	24	89	15
30% Water DMSO DMF Ethanol	+ + + +	15 15 14 19	3 1 2 6	122 109 91 100	15 15 16 22	9 9 10 8	3 1 3 2	31 29 24 32	5 5 10 5
2-AA	+	218	58	585	154	60	24	596	165
10% Water DMSO DMF Ethanol	+ + + +	13 13   18	2   11	137 102   112	6   15	8 7 11 7	3   2	28 25 30 39	3
2-AA	+	252	63	1270	586	294	84	1005	366

 

Summary of historical negative and positive control 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 EGDE^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-azid NF 4-NPDA	- - -	839	115	382	46	90	13	109	20
30% Water DMSO DMF Ethanol Acetone EGDE^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	+ + + + +	14 14 -- 23 13	4 2	123 111 72 87 85	4 13   6	9 8 9 8 7		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 control 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-azid 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 control 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-azid 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 control 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-azid 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 8 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 DMF	+ +	14 11	5 -	91 53	7 -	1- 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 control 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-azid 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 control 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-azid 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 control of experiments performed from January to June 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 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-azid 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	14	754	369

²) Ethylene glycol dimethylether

 

Summary of historical negative and positive control 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 87 107	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-azid NF 4-NPDA	- - -	605	122	339	52	53	9	79	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:

Evidence of mutagenic activity of Levafix Br. Blau E-BRA FW was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Therefore, Levafix Br. Blau E-BRA FW was considered to be non-mutagenic with and without S9 mix in the salmonella/microsome test.
The substance is not classifiable according to CLP criteria.

Executive summary:

Levafix Br. Blau E-BRA FW was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 μg per plate on four histidine- auxotrophic Salmonella typhimurium LT2 mutant strains (TA 153 5, TA 100, TA 98, and TA 1537).

 

Doses 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 the dose of 5000 μg per plate, the substance had a weak, strain-specific bacteriotoxic effect, yet this range could nevertheless be used for assessment purposes.

 

Evidence of mutagenic activity of Levafix Br. Blau E-BRA FW was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Therefore, Levafix Br. Blau E-BRA FW was considered to be non-mutagenic with and without S9 mix in the salmonella/microsome test.

 

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.