Sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate

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:

20.01.-20.02.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

A deviation from SOP occurred in the first experiments in E. coli - number of revertants in solvent control was above historical control limits. As number of revertants was within historical control limits for spontaneous reversion we are convinced.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate

EC Number:

228-810-1

EC Name:

Sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate

Cas Number:

6359-85-9

Molecular formula:

C23H21N5O6S2.Na

IUPAC Name:

sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate

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:

- Name of test material (as cited in study report): Acid Yellow 25- Physical state: yellow solid, powder- Analytical purity: 95 % (w/w)- Impurities (identity and concentrations): 5%(w/w) NaCl (CAS No. 7647-14-5 - Lot/batch No.: 7003/2007- Expiration date of the lot/batch: 2 years/June 2018- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

Method

Target gene:

gene for histidine or tryptophan synthesis

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:

supernatant of rat liver and a mixture of cofactors.

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 μg30, 100, 300, 1000, 3000 μgSelection of doses/toxicity: : At first, the test substance was tried to dissolve in water for injection. The test substance was insoluble in the highest concentration recommended in guidelines (5000 μg.0.1mL-1). Afterwards, the test substance was dissolved in dimethyl sulfoxide and the maximum recommended concentration was reached.

Vehicle / solvent:

Water for injection, Ardeapharma, Lot. No.: 1501210041, exp. 01/2017- Justification for choice of solvent/vehicle: solubility of the substance

Controls

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxid

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, N-methyl-N´-nitro-N-nitrosoguanidine, 9-aminoacridine hydrochloride monohydrate

Details on test system and experimental conditions:

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) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - 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 detectionof base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagensMETHOD OF APPLICATION: in agar (plate incorporation)NUMBER OF REPLICATIONS: two seriesDETERMINATION OF CYTOTOXICITY- Method: relative total growth

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible withthe application of statistical methods (2, 3). After this rule the result is positive, if a reproducible doseresponseeffect occurs and/or a doubling of the ratio Rt/Rc is reached.

Statistics:

For the evaluation of results, the modified two-fold increase rule was used, which is compatible with theapplication of statistical methods:Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays. inThe Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation. Elsevier North-Holland Biomedical Press. 231 - 417Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests forbacterial mutagenicity. Mutat. Res. 189. 83 - 91

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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 4

Key result

Species / strain:

S. typhimurium TA 1537

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 5

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

Additional information on results:

HISTORICAL CONTROL DATAEach 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 DMSO. 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. ADDITIONAL INFORMATION ON CYTOTOXICITY:No toxicity was observed in any dose. Precipitation in the top agar occurred from 2500 μg.0.1mL-1. The occurrence of precipitation in top agar was then investigated in the test substance concentration of 1500 μg.0.1mL-1. This concentration of the test substance did not cause precipitation in top agar so concentrations for the first mutagenicity experiments were chosen as 5000, 1500, 500, 150 and 50 μg per plate.

Applicant's summary and conclusion

Conclusions:

Under the above-described experimental design, the test substance, Acid Yellow 25, was non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.

Executive summary:

The test substance Acid Yellow 25 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 diluted in dimethyl sulfoxide and assayed in doses of 30 - 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.

In the arrangement given above, the test substance, Acid Yellow 25, was non-mutagenic for all the used tester strains without as well as with metabolic activation.