2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium 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:

August 16th, 1988 to September 15th, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

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 471 Genetic Toxicology : Salmonella typhimurium, Reverse Mutation Assay, Adopted : May 26th, 1983

Deviations:

no

Test guideline 2

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).

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

EC Number:

288-950-4

EC Name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

Cas Number:

85940-63-2

Molecular formula:

C22H17N4Na3O13S4

IUPAC Name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

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:

0, 4, 20, 100, 500, 2500, 5000 & 10000 μg/plate
The test compound was tested at doses and proved to be not toxic to most of the bacterial strains. For mutagenicity testing 10 000 microgram/plate was chosen as the highest dose.

Vehicle / solvent:

At the day of the experiment the test substance was dissolved in DMSO at appropriate concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

other: 2-Aminoanthracene (2AA); Benzidine

Details on test system and experimental conditions:

Preparation and storage of a liver homogenate fraction ("S-9")
Liver preparations were performed from liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrien hamsters. Male Sprague Dawley rats (200 - 300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5 - 6 Sprague Dawley rats or from 5 - 6 male Syrien golden hamsters (7 - 8 weeks old) are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KCl . The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and stored at -80 °C for not longer than three months.

Preparation of S-9 Mix
Sufficient S-9 fraction is thawed immediately before each test at room temperature. Three volumes of S-9 fraction is mixed with 7 volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix of the rat liver are:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7.4
According to the modification proposed by Prival using 30 minutes preincubation in the presence of 30% hamster S-9 (Syrien golden hamster), the S-9 Mix consisted of:
8 Mm MgCl2
33 mM KCl
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4 mM NADP+
2 mM NADH
2 mM FMN (Riboflavin-5'-phosphate -Na-salz)
100 mM phosphate buffer pH 7.4

Bacteria
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2/liter) at 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537. Identification of the different bacterial strains is performed periodically.

Toxicity experiments and dose range finding
Preliminary toxicity tests were performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occurring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically. In combination with the main experiment, toxicity testing was performed as follows:
0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (=surviving fraction).

Mutagenicity test
Two independent experiments which each of the two protocols (Ames, Prival) were performed.
a) Ames Test
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0 .6% agar, 0.6 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top agar at 45 °C:
0.1 ml test compound solution
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.5 ml S-9 Mix (if required) or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2% glucose). After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.

b) Prival modification
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incubated at 30 °C for the duration of 30 minutes. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.6% agar+ 0.6% NaCl) and 10 ml amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 Mm histidine + 0.5 mM biotin) is added . After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Rationale for test conditions:

The test was performed according to the methods described. No unforeseen circumstances were observed which have affected the quality and integrity of this study.

Evaluation criteria:

Not specified in the study report.

Statistics:

Not specified in the study report.

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Sterility of S-9 Mix and the test compound was indicated by the absence of contamination on the test material and S-9 Mix sterility check plates . Control plates (background control and positive controls) gave the expected number of colonies.

Any other information on results incl. tables

Preliminary test with 30% rat liver S-9 Mix

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	170	155	164	192
4	-	180	168	175	197
20	-	175	184	161	181
100	-	185	167	197	191
500	-	152	153	145	158
2500	-	197	173	204	214
10000	-	216	229	223	195
0	+	171	181	147	184
4	+	185	189	181	186
20	+	180	192	162	186
100	+	183	196	161	191
500	+	184	183	191	177
2500	+	202	181	215	210
10000	+	197	187	199	204

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	18	19	18	16
4	-	14	16	12	14
20	-	16	17	12	19
100	-	17	17	16	19
500	-	14	15	12	16
2500	-	18	15	20	18
10000	-	19	17	15	25
0	+	16	13	20	14
4	+	16	19	13	17
20	+	17	13	22	16
100	+	16	16	17	16
500	+	11	15	11	8
2500	+	19	18	22	16
10000	+	21	24	21	19

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	8	7	12	6
4	-	10	13	8	8
20	-	8	9	7	7
100	-	8	7	12	4
500	-	8	7	8	10
2500	-	11	9	12	11
10000	-	9	4	9	14
0	+	6	6	6	7
4	+	7	8	7	7
20	+	7	4	6	10
100	+	8	6	8	9
500	+	8	8	8	8
2500	+	10	11	8	10
10000	+	6	8	4	7

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	21	21	26	17
4	-	18	15	19	19
20	-	24	27	30	15
100	-	23	21	20	28
500	-	20	17	20	22
2500	-	21	22	20	20
10000	-	19	17	20	20
0	+	30	37	28	26
4	+	29	30	32	26
20	+	28	16	35	34
100	+	23	16	26	26
500	+	31	22	36	35
2500	+	27	21	31	29
10000	+	30	27	34	29

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Preliminary test with 30% syrien golden hamster liver S-9 Mix and preincubation

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	149	156	132	160
4	+	153	161	148	149
20	+	156	161	148	159
100	+	160	169	162	150
500	+	148	145	156	142
2500	+	169	161	154	192
10000	+	206	195	197	226 (ibl)

Compound dissolved in 100 microliter DMSO

+ : presence

ibl : incomplete bacterial lawn

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	11	12	12	8
4	+	11	8	14	10
20	+	11	13	11	9
100	+	13	16	13	11
500	+	11	8	15	11
2500	+	9	12	9	6
10000	+	14	17	15	9 (ibl)

Compound dissolved in 100 microliter DMSO

+ : presence

ibl : incomplete bacterial lawn

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	9	7	10	10
4	+	13	9	17	13
20	+	10	11	7	13
100	+	6	5	9	5
500	+	9	6	12	9
2500	+	10	8	13	10
10000	+	6	4	9	4 (ibl)

Compound dissolved in 100 microliter DMSO

+ : presence

ibl : incomplete bacterial lawn

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	26	31	28	20
4	+	31	26	29	39
20	+	30	28	33	30
100	+	26	26	30	23
500	+	30	29	33	28
2500	+	36	38	34	37
10000	+	25	29	26	21 (ibl)

Compound dissolved in 100 microliter DMSO

+ : presence

ibl : incomplete bacterial lawn

 

Main experiment with 30% rate liver S-9 Mix

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate			Surviving fraction
0	-	184	185	184	184	1.0
20	-	176	177	182	170	0.9
100	-	171	191	166	157	0.9
500	-	173	189	172	159	0.9
2500	-	181	171	20	171	0.8
5000	-	216	225	187	235	0.6
10000	-	220	228	227	205	1.0
0	+	199	193	187	217	1.0
20	+	183	170	194	185	1.0
100	+	205	194	196	225	1.1
500	+	214	194	219	228	0.9
2500	+	218	185	217	251	1.0
5000	+	232	206	230	260	1.1
10000	+	225	227	223	226	1.0

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	15	21	14	11
20	-	17	17	17	16
100	-	15	13	14	17
500	-	13	10	15	14
2500	-	13	13	13	12
5000	-	14	18	13	11
10000	-	12	7	17	12
0	+	22	20	23	23
20	+	17	13	19	19
100	+	16	14	18	15
500	+	15	17	15	13
2500	+	16	18	18	12
5000	+	24	19	24	29
10000	+	21	26	20	18

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	8	9	8	6
20	-	8	6	7	10
100	-	9	13	10	5
500	-	8	6	5	14
2500	-	8	8	10	7
5000	-	7	4	10	6
10000	-	7	9	3	9
0	+	11	10	15	8
20	+	8	7	7	9
100	+	11	9	11	12
500	+	10	8	17	5
2500	+	9	7	9	11
5000	+	9	9	10	8
10000	+	13	9	14	15

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FWTR with and without metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	28	20	35	28
20	-	28	24	29	30
100	-	27	34	27	20
500	-	33	33	29	37
2500	-	28	31	26	26
5000	-	34	34	35	32
10000	-	24	26	23	22
0	+	35	36	34	34
20	+	40	43	42	36
100	+	42	40	44	42
500	+	38	41	39	35
2500	+	38	41	39	35
5000	+	42	39	53	35
10000	+	41	45	33	44

Compound dissolved in 100 microliter DMSO

- : absence

+ : presence

 

Main experiment with 30% syrien golden hamster liver S-9 Mix and preincubation

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate			Surviving fraction
0	+	153	175	128	155	1.0
20	+	169	215	144	148	1.0
100	+	169	190	157	160	0.9
500	+	164	161	143	188	1.0
2500	+	172	154	151	201	1.0
5000	+	193	185	173	221	1.0
10000	+	204	192	200	219	1.0

Compound dissolved in 100 microliter DMSO

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	10	6	12	12
20	+	15	12	18	14
100	+	11	11	9	13
500	+	12	19	8	10
2500	+	14	12	15	15
5000	+	16	22	12	15
10000	+	15	19	10	15

Compound dissolved in 100 microliter DMSO

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	11	13	8	11
20	+	10	14	7	8
100	+	9	7	12	8
500	+	14	13	12	16
2500	+	11	8	13	12
5000	+	13	16	9	13
10000	+	11	10	13	11

Compound dissolved in 100 microliter DMSO

+ : presence

 

Mutagenicity experiment with Remazol-Brillantgelb GL FTWR with metabolic activation

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose μg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	38	25	42	46
20	+	37	36	36	40
100	+	42	39	33	55
500	+	41	38	33	52
2500	+	44	44	43	46
5000	+	38	36	39	39
10000	+	37	45	36	29

Compound dissolved in 100 microliter DMSO

+ : presence

 

Positive control

 

Preliminary test

 

Mutability (positive controls) and sterility test of the experiment with Remazol-Brillantgelb GL FWTR

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains

Strain	Compound	Dose μg/plate	Metab. Activ.	Mean value	Colonies per plate
TA100	Sodium-azide	1	-	608	582	600	641
TA1535	Sodium-azide	1	-	475	477	471	476
TS1537	9-Aminoacridine	50	-	1258	118	136	120
TA98	2-Nitrofluorene	2.5	-	403	468	358	383
-	Remazol-Brillantgelb GL FWTR	10000	-	0	0	0	0

- : absence

 

Mutability (positive controls) and sterility test of the experiment with Remazol-Brillantgelb GL FWTR

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains

Strain	Compound	Dose μg/plate	Metab. Activ.	Mean value	Colonies per plate
30% rat liver S-9 Mix
TA100	2-Aminoanthracen	0.5	+	318	305	342	307
TA1535	2-Aminoanthracen	10	+	267	258	280	264
TS1537	2-Aminoanthracen	10	+	432	430	478	387
TA98	2-Aminoanthracen	0.5	+	205	223	172	218
TA100	Benz[a]pyrene	10	+	739	755	728	735
TA1535	Benzo[a]pyrene	10	+	45	50	47	39
TA1537	Benzo[a]pyrene	10	+	199	226	224	148
TA98	Benzo[a]pyrne	10	+	722	724	682	761
30% syrien golden hamster liver S-9 Mix and preincubation
TA100	2-Aminoanthracen	0.5	+	621	605	553	705
TA1535	2-Aminoanthracen	0.5	+	116	141	96	112
TA1537	2-Aminoanthracen	2.5	+	134	154	154	94
TA98	Congored	500	+	85	98	77	81
TA98	Benzidine	2.14	+	262	231	272	284
-	S-9 Mix	500 μl	+	0	0	0	0
-	Remazol Brillantgelb GL FWTR	10000 μg	+	0	0	0	0

+ : presence

 

Main experiment

 

Mutability (positive controls) and sterility test of the experiment with Remazol-Brillantgelb GL FWTR

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains

Strain	Compound	Dose μg/plate	Metab. Activ.	Mean value	Colonies per plate
TA100	Sodium-azide	1	-	681	655	727	662
TA1535	Sodium-azide	1	-	430	444	461	386
TS1537	9-Aminoacridine	50	-	132	123	152	122
TA98	2-Nitrofluorene	2.5	-	463	474	475	441
-	Remazol-Brillantgelb GL FWTR	2500	-	0	0	0	0

- : absence

 

Mutability (positive controls) and sterility test of the experiment with Remazol-Brillantgelb GL FWTR

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains

Strain	Compound	Dose μg/plate	Metab. Activ.	Mean value	Colonies per plate
30% rat liver S-9 Mix
TA100	2-Aminoanthracen	0.5	+	364	385	361	346
TA1535	2-Aminoanthracen	10	+	266	258	267	273
TS1537	2-Aminoanthracen	10	+	309	336	279	313
TA98	2-Aminoanthracen	0.5	+	224	202	216	254
TA100	Benz[a]pyrene	10	+	813	740	893	806
TA1535	Benzo[a]pyrene	10	+	50	54	51	45
TA1537	Benzo[a]pyrene	10	+	186	224	164	170
TA98	Benzo[a]pyrne	10	+	710	672	734	723
30% syrien golden hamster liver S-9 Mix and preincubation
TA100	2-Aminoanthracen	0.5	+	616	676	584	587
TA1535	2-Aminoanthracen	0.5	+	151	137	160	157
TA1537	2-Aminoanthracen	2.5	+	208	196	190	237
TA98	Congored	500	+	195	190	201	193
TA98	Benzidine	2.14	+	475	498	441	487
-	S-9 Mix	500 μl	+	0	0	0	0
-	Remazol Brillantgelb GL FWTR	2500 μg	+	0	0	0	0

+ : presence

Applicant's summary and conclusion

Conclusions:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S-9 Mix. No dose dependent effect was obtained.
In the presence of hamster liver S-9 using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described.
It is concluded that Remazol-Brillantgelb GL FWTR is not mutagenic in the absence and presence of rat S-9 Mix using the standard Ames Test procedure. Also in the presence of hamster liver S-9 Mix and preincubation the test compound did not induce a significant increase in the number of revertant colonies.
The substance is not classifiable according to CLP criteria

Executive summary:

Remazol-Brillantgelb GL FWTR was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium.

 

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 20 microgram/ plate to 10 000 microgram/plate was used.

 

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

 

Toxicity: The test compound proved to be not toxic to most of the bacterial strains. 10 000 microgram/plate was chosen as top dose level for the mutagenicity study.

 

a) Ames Test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Remazol-

Brillantgelb Gl FWTR did not result in relevant increases in the number of revertant colonies.

 

b) Prival Test:

In the presence of hamster liver S-9 using the preincubation method according to Prival Remazol-Brillantgelb GL FWTR did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

 

Summarizing, it can be stated that Remazol -Brillantgelb GL FWTR is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.