Benzoic acid, 5-[(4-aminophenyl)azo]-2-hydroxy-, reaction products with 3-[(4-amino-3-methoxyphenyl)azo]benzenesulfonic acid and carbonic dichloride, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the above-described experimental design, the test substance, Direct Yellow 44, was non mutagenic for the Salmonella typhimurium TA 100 and E. coli without and Salmonella typhimurium TA 1535 with as well as without metabolic activation.

The test substance was mutagenic for Salmonella typhimurium TA 98 and TA 1537 with as well as without metabolic activation, and Escherichia coli with metabolic activation. Signs of mutagenicity occurred repeatedly in experiments in Salmonella typhimurium TA 100 with metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.08.2017 – 29.09.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No.440/2008. Published in O.J. L 142, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

gene for histidine or tryptophan synthesis

Species / strain / cell type:

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

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 μgSelection of doses/toxicity:The test substance was soluble in dimethyl sufoxide - after heating up to 90° it was dissolved up to the highest recommended concentration 5000 µg per 0.1 mL. For toxicity experiment the highest concentration 5 mg per mL (recommended in OECD TG 471) was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 98 without metabolic activation (see Table B). Precipitation occurred in top agar in the highest concentration 5000 µg per plate. The test substance in background occurred in Petri dishes also in the highest concentration. No toxicity was observed in any dose. Although, precipitation in top agar occurred in the highest concentration, the concentration of 5000 µg per plate was used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.No cytotoxicity occurred in the first experiments.Signs of the mutagenicity were observed in some bacterial strains especially in experiments with metabolic activation, so doses for the second mutagenicity experiments were changed for purchase of better dose-response. Doses used for the second mutagenicity experiments were 500-2500 µg per plate. Amount of S9 was increased to 50 µL per plate.In the first mutagenicity experiments in TA 98 with as well as without metabolic activation dose-response as well as doubling of revertants were obtained so the second mutagenicity experiments were not performed.

Vehicle / solvent:

dimethyl sulfoxide (DMSO), Merck Lot. No. K48069252 644, exp. 10/2018- Justification for choice of solvent/vehicle: solubility of the substance

Untreated negative controls:

yes

Remarks:

negative controls - 0.1 mL of DMSO

Remarks:

Untreated controls - no solvent

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(AS)

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

(NPD)

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Remarks:

(2-AF)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(2-AA)

Positive controls:

yes

Positive control substance:

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

Remarks:

(MNNG)

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine hydrochloride monohydrate

Remarks:

(9-AAc)

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 detection of base-pair substitution mutations, and strain E.coli WP2uvrA detects cross-linking mutagensMETHOD OF APPLICATION: in medium; in agar (plate incorporation)NUMBER OF REPLICATIONS: two seriesDETERMINATION OF CYTOTOXICITY- Method: decrease of number of revertants or diminution of bacterial background

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (see below). After this rule the result is positive, if a reproducible doseresponse effect 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 the application of statistical methods:Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays in The 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 for bacterial mutagenicity. Mutat. Res. 189. 83 - 91

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

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

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

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 DATA: Each 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:The test substance was soluble in dimethyl sufoxide - after heating up to 90° it was dissolved up to the highest recommended concentration 5000 µg per 0.1 mL. For toxicity experiment the highest concentration 5 mg per mL (recommended in OECD TG 471) was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 98 without metabolic activation. Precipitation occurred in top agar in the highest concentration 5000 µg per plate. The test substance in background occurred in Petri dishes also in the highest concentration. No toxicity was observed in any dose. Although, precipitation in top agar occurred in the highest concentration, the concentration of 5000 µg per plate was used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.No cytotoxicity occurred in the first experiments.

Conclusions:

Under the above-described experimental design, the test substance, Direct Yellow 44, was non mutagenic for the Salmonella typhimurium TA 100 and E. coli without and Salmonella typhimurium TA 1535 with as well as without metabolic activation. The test substance was mutagenic for Salmonella typhimurium TA 98 and TA 1537 with as well as without metabolic activation, and Escherichia coli with metabolic activation. Signs of mutagenicity occurred repeatedly in experiments in Salmonella typhimurium TA 100 with metabolic activation.

Executive summary:

The test substance, Direct Yellow 44, 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 dispersed in dimethylsulfoxide and assayed in doses of 50 - 5000 μg per plate, which were applied to plates in volume of 0.1 mL.

The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50 – 5000 μg per plate.

In the second mutagenicity experiments concentrations were modified because of expectancy of mutagenic effect and to reach prospective dose-response to 500-2500 μg per plate. At experiments with metabolic activation volume of S9 was increased to 50 μL.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

In the arrangement given above, the test substance, Direct Yellow 44, was non mutagenic for the Salmonella typhimurium TA 100 and E. coli without metabolic activation and Salmonella typhimurium TA 1535 with as well as without metabolic activation.

The test substance was mutagenic for Salmonella typhimurium TA 98 and TA 1537 with as well as without metabolic activation, and Escherichia coli with metabolic activation. Signs of mutagenicity occurred repeatedly in experiments in Salmonella typhimurium TA 100 with metabolic activation.