Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

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

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

EC Number:

252-305-5

EC Name:

Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

Cas Number:

34977-63-4

Molecular formula:

C27H19N5O7S.2Na

IUPAC Name:

tetrasodium 3-[(1E)-2-{4-[(1E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]naphthalen-1-yl}diazen-1-yl]-2-hydroxybenzoate 5-[(1E)-2-{4-[(1E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]naphthalen-1-yl}diazen-1-yl]-2-hydroxybenzoate

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

Appearance: black powderTrade name: Saturn Black LNBatch No: 9013CAS No: 34977-63-4

Method

Target gene:

his (trp) [gene for synthesis histidine or tryptophan]

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injection, Ardeapharma, Lot. No.: 1501210041

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Sodium azide, CAS 26647-22-8 4-nitro-1,2-phenylenediamine, CAS 99-56-9 2-aminofluorene, CAS 153-78-6 2-aminoanthracene, CAS 613-13-8 N-methyl-N´-nitro-N-nitrosoguanidine, CAS 70-25-7 9-aminoacridine hydrochloride monohydrate, CAS 52417-22-8

Details on test system and experimental conditions:

TEST PROCEDURE100 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 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. After incubation 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. Each experiment was repeated. As there was no cytotoxicity, no precipitation or dose-responsiveness, doses in the second experiments remained the same as in the first experiment.SELECTION OF DOSES/TOXICITY2 mL of water for injection was added to 100 mg of the test substance to reach the maximum dose recommended in guidelines - 5000 µg per plate (per 0.1 mL). Some part of the test substance remained undiluted. 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 100 without metabolic activation. No toxicity of the test substance (but particles of the test substance occurred on Petri dishes from 50 to 5000 µg per plate) was observed at evaluation so the dose of 5000 µg per plate was used as maximum for the first mutagenicity experiments as well. The maximum concentration was diluted according to the rules given in guidelines (five different analysable concentrations with approximately half log, i.e. approximately √10, intervals between test points). The doses used were 50, 150, 500, 1500 and 5000 µg per plate. The same maximum dose was used in the second mutagenicity experiments, because no toxicity and increased number of revertants was observed up (and including) to the highest dose. To obtain a dose-effect dependence, non-mutagenic, the lowest concentration, was omitted and concentrations were generally increased (100-5000 µg per plate).Fresh suspensions of the test substance were prepared before each experiment. All concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate. Suspensions, used for diluting of concentration row, were shaken before withdrawal of an aliquot for dilution. All application forms were shaken before dosing to top agar.PREPARATION AND USING OF METABOLIC ACTIVATIONThe 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, 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 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 µL S9 (the concentration of S9 in the S9mix was 5.7 %). In experiments without metabolic activation only buffer was added to the top agar.CONTROLSEach experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of water for injection. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers. GENOTYPES OF STRAINSGenotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached. An increase is considered as „biologically relevant“: - if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10; - if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10; A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

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:

E. coli WP2 uvr A

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 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

See attached file

Applicant's summary and conclusion

Conclusions:

Under the experimental design, the test substance, Direct Black 51, was mutagenic for Salmonella typhimurium TA 98 and TA 1537 in experiments with as well as without metabolic activation. In the other bacterial strains Salmonella typhimurium TA 100, TA 1535 and E. coli WP2uvrA signs of mutagenicity were also observed. These increasing of number of revertants were not sufficiently confirmed, so the test substance is considered to be non-mutagenic for these bacterial strains.

Executive summary:

The test substance Direct Black 51 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in water for injection and assayed in doses of 50-5000 µg per plate, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the experimental design, the test substance, Direct Black 51, was mutagenic for Salmonella typhimurium TA 98 and TA 1537 in experiments with as well as without metabolic activation. In the other bacterial strains Salmonella typhimurium TA 100, TA 1535 and E. coli WP2uvrA signs of mutagenicity were also observed. These increasing of number of revertants were not sufficiently confirmed, so the test substance is considered to be non-mutagenic for these bacterial strains.