Disodium 4-hydroxy-3-[[2-methoxy-5-methyl-4-[(4-sulphonatophenyl)azo]phenyl]azo]-7-(phenylamino)naphthalene-2-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-hydroxy-3-[[2-methoxy-5-methyl-4-[(4-sulphonatophenyl)azo]phenyl]azo]-7-(phenylamino)naphthalene-2-sulphonate

EC Number:

228-326-0

EC Name:

Disodium 4-hydroxy-3-[[2-methoxy-5-methyl-4-[(4-sulphonatophenyl)azo]phenyl]azo]-7-(phenylamino)naphthalene-2-sulphonate

Cas Number:

6227-14-1

Molecular formula:

C30H25N5O8S2.2Na

IUPAC Name:

sodium 4-hydroxy-3-((E)-(2-methoxy-5-methyl-4-((E)-(4-sulfonatophenyl)diazenyl)phenyl)diazenyl)-7-(phenylamino)naphthalene-2-sulfonate

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732)were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.

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 were: 30, 100, 300, 1000 and 3000 µg/plate.
Selection of doses/toxicity: 2.0 mL of water for injection were added to 100 mg of the test substance in a test tube to reach the maximum dose recommended in guidelines - 5000 µg per plate (per 0.1 mL).

Media:
Nutrient Broth for microbiology, Merck, Lot. VM648943433, exp. 25/07/2019
Nutrient agar, Merck,VM664850449, exp. 05/11/2019
Agar-agar, Merck, Lot. K47292515 604, minimum shelf life 12/2019
Procedures of media preparation are described in detail in internal SOP M/12.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2aminofluorene

Remarks:

TA100 / TA98 with metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535/TA1537/Escherichia coli with metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

TA98 without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100/TA1535 without metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without metabolic activation

Controls 6

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Remarks:

Escerichia coli without metabolic activation

Details on test system and experimental conditions:

Test procedure:
The first experiments and toxicity test: 100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 108-109 CFU/mL, 0.5 mL relevant buffer and 30 (100) µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45 +- 3 °C. After shaking the mixture was poured into a minimal glucose agar plate.
The second experiments: 0.5 mL of relevant buffer, 100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 108-109 CFU/mL and 30 (100) µL of S9 post mitochondrial fraction were mixed and shaken at 37 +- 1 °C for 30 minutes. Then, 2 mL of molten top agar (with trace of histidine or tryptophan) was added and the mixture was poured into a minimal glucose agar plate.
Petri dishes prepared one or the other way were incubated of 48 - 72 h at 37 +- 1 °C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.
For an adequate estimate of variation, triplicate plating was used at each dose level except in the toxicity test with strain TA 98, where test substance was tested in duplo.

For toxicity experiment, the starting suspension (5000 µg/0.1 mL) was diluted to concentration series 10 - 5000 µg per plate. The concentration row was tested for toxicity
in strain TA 98 without metabolic activation (see Table B). The test substance was dissolved in the two lowest concentrations only (100 and 10 µg/0.1 mL), in the other concentrations the test substance was tested in suspension.
Particles of the test substance were seen on Petri dishes from the concentration of 500 µg per plate. The highest concentration (5000 µg/0.1 mL) was hardly evaluable due to dark coloured agar and presence of particles of the test substance in background. No toxicity was observed in any dose.
Due to difficult evaluation of the highest concentration, the concentration of 3000 µg/0.1 mL was used as maximum in the first mutagenicity experiments.
The first experiments showed troubles with evaluation also in the concentration of 3000 µg/0.1 mL, therefore, the concentration of 1000 µg/0.1 mL was used as maximum in the second mutagenicity experiments. The second mutagenicity experiments were performed with pre-incubation for increase of sensibility of the assay.
Fresh solutions of the test substance were prepared before each experiment. Concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate.

Preparation and using of S9: The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg·mL-1, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL.g-1 wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 C. 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 30 or 100 L S9 (the concentration of S9 in the S9mix was 5.7 or 19%). In experiments without metabolic activation only buffer was added to the top agar.

Results and discussion

Test results

Species / strain:

other: TA98/TA100/TA1535/TA1537/Escherichia coli

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Under the above-described experimental design, the test substance was non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation

Applicant's summary and conclusion

Conclusions:

Not mutagenic

Executive summary:

The test substance was non‑mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.