Hexasodium 4-amino-3,6-bis[[5-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulphonatophenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: read across

Adequacy of study:

key study

Study period:

August 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

EC Number:

269-284-3

EC Name:

Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Cas Number:

68214-04-0

Molecular formula:

C44H24Cl2N14Na6O20S6

IUPAC Name:

hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Constituent 2

Reference substance name:

Reactibe Red 120

IUPAC Name:

Reactibe Red 120

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Reaktiv Rot 120
- Appearance, consistency: red powder
- Storage: Room temperature (protected from light)

Method

Target gene:

His+ (Salmonella), Trp (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Plate incorporation assay S9 from Aroclor 1254 induced rat livers; Prival pre-incubation assay S9 from uninduced Syrian Hamster livers (additionally supplemented with FMN and NADH)

Test concentrations with justification for top dose:

both experiments: 0; 25 ; 125 ; 625; 3,125 and 6,250 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: high water solubility
- Homogeneity (pre-formulation) and stability in water was assessed

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

sterility control

Positive controls:

yes

Remarks:

without S9: N-methyl-N'-nitro-N-nitrosoguanidine (MINING) (FLUKA, 68051) - 5 pg/plate, dissolved in DMSO - - strains : TA 1535, TA 10 0 • 4-nitro-o-phenylendiamine (NOPID) (SIGMA, N-9504) - 10 pg/plate, dissolved in DIVIS O - strain : TA 98 • with rat S9

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

congo red

other: aminoanthrocene; benzidine; nitrophenylenediamine

Details on test system and experimental conditions:

POSITIVE CONTROLS:
without S9:
- N-methyl-N'-nitro-N-nitrosoguanidine - 5 ug/plate, dissolved in DMSO - TA 1535, TA 10 0
- 4-nitro-o-phenylendiamine - 10 ug/plate, dissolved in DMSO - TA 98
- 9-aminoacridine - 100 ug/plate, dissolved in DMSO - TA 1537
- 4-nitroquinoline-N-oxide - 5 ug/plate, dissolved in DMSO - E . coli WP2 uvrA
with rat S9:
- 2-aminoanthracene - 2.5 ug/plate, dissolved in DMSO- TA 1535, TA 100, TA 1537, TA 98
- 60 ug/plate, dissolved in DMSO - Escherichia coli WP2 uvrA
with hamster S9:
- 2-aminoanthracene - 10 ug/plate, dissolved in DMSO - TA 1535, TA 100, TA102, TA 1537, TA 98
- Congo red - 0.3 umol/plate, dissolved in DMSO - TA 98
- Benzidine - 0.3 umol/plate, dissolved in DMSO - TA 98

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: at 37° C for 48 - 72 hours in the dark

NUMBER OF REPLICATIONS: 3/concentration

CYTOTOXICITY:
-decrease in the number of revertants
- clearing or diminution of the background lawn reduced his- or trp- background growth)
- reduction in the titer

Evaluation criteria:

Positive:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system

Negative:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other

Statistics:

NA

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3125 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3125 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Positive controls Congo Red and benzidine showed high numbers of revertants (positive result)

Applicant's summary and conclusion

Conclusions:

negative without metabolic activation
negative with metabolic activation

The substance is not mutagenic in the Ames test

Executive summary:

The substance was tested in a plate incorporation assay (with and without rat S9) and a pre-incubation assay (with and without hamster S9 with FMN) in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A. Limited cytotoxicity was observed and the number of revertants in both assays was within control ranges. Therefore it is concluded that the substance does not induce mutations in bacterial cells.