Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

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:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

EC Number:

233-311-7

EC Name:

Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

Cas Number:

10114-47-3

Molecular formula:

C28H20N4O6S4.2Na

IUPAC Name:

disodium 2,2'-(diazene-1,2-diyldi-4,1-phenylene)bis(6-methyl-1,3-benzothiazole-7-sulfonate)

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: orange powder- Batch No.: 31/94- Prevailing particle size: 2 - 4 µm

Method

Target gene:

gene for histidine synthesis

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 supernatant of rat liver and a mixture of cofactors

Test concentrations with justification for top dose:

10, 100, 500, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-1,2-phenylenediamine (CAS: 99-56-9); 2-aminofluorene (CAS: 153-78-6); 2-aminoanthracene (CAS: 613-13-8)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)NUMBER OF REPLICATIONS: 3PREPARATION OF S9 METABOLIC ACTIVATORThe metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats, previously induced with Delor 106 (mixture of PCBs). Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer. Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 20 µL S9. In experiments without metabolic activation only buffer was added to the top agar.DETERMINATION OF CYTOTOXICITY- Any supplementary information relevant to cytotoxicity: no cytotoxicity observed

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table No. 1: Bacteria reverse mutation test results

Dose (µg/plate)	S9 (μL)	Rt/Rc		S9 (μL)	Rt/Rc
Salmonella typhimurium TA 97
water	0	1.0	1.0	20	1.0	1.0
10	0	1.1	1.0	20	1.1	1.0
100	0	1.0	1.0	20	0.9	1.0
500	0	-	0.9	20	1.0	-
1000	0	1.1	0.9	20	1.0	1.0
2500	0	1.1	0.9	20	0.9	1.0
5000	0	0.9	-	20	-	0.9
NPD	0	7.7	6.0	20	16.3	12.8
2-AF	0	-	-	20	-	-
Salmonella typhimurium TA 98
water	0	1.0	1.0	20	1.0	1.0
10	0	0.8	1.0	20	1.1	1.2
100	0	0.9	1.1	20	0.9	1.1
500	0	-	1.0	20	-	1.3
1000	0	1.1	1.0	20	0.8	1.5
2500	0	0.9	1.0	20	0.9	1.1
5000	0	0.7	-	20	0.7	-
NPD	0	56.2	106.6	-	-	-
2-AF	-	-	-	20	101.2	92.9
Salmonella typhimurium TA 100
water	0	1.0	1.0	20	1.0	1.0
10	0	1.0	0.9	20	1.0	0.9
100	0	1.1	0.9	20	0.9	1.0
1000	0	1.0	1.0	20	1.0	1.1
2500	0	1.1	1.0	20	1.0	1.1
5000	0	1.1	1.0	20	1.0	1.0
AS	0	8.2	6.5	-	-	-
2-AF	-	-	-	20	14.4	16.1
Salmonella typhimurium TA 1535
water	0	1.0	1.0	20	1.0	1.0
10	0	1.0	1.3	20	1.1	1.0
100	0	1.2	1.0	20	1.0	0.9
500	0	-	1.2	-	-	-
1000	0	0.7	0.9	20	0.9	0.8
2500	0	0.6	0.8	20	1.0	0.7
5000	0	0.8	0.5	20	0.9	0.6
AS	0	51.8	52.0	-	-	-
2-AA	-	-	-	20	17.0	13.2

AS         sodium azide

NPD      4-nitro-1,2-phenylenediamine

2-AF     2-aminofluorene

2-AA     2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Based on results of this test, it is obvious that test substance, Reactive Orange 13, shouldn´t be considered as a mutagen for all the Salmonella typhimurium with as well as without metabolic activation.