Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

modified version for azo-dyes according to the method of Prival and Mitchell (1982)
Prival. M.J. and V.D. Mitchell (1982) Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9 Mutation Res. 97, 103-116

Deviations:

no

Test guideline 2

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:

Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

EC Number:

947-944-8

Cas Number:

70879-65-1

Molecular formula:

C23H17N4O

IUPAC Name:

Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

Method

Target gene:

His (S. typhimurium)
Trp (E. coli)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

non-induced hamster liver S9 mix

Test concentrations with justification for top dose:

Pre-Experiment (Experiment I): 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment Ia: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

DMSO (purity > 99 %) was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (without metabolic activation), 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

See p10-11 of the attached document

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at a concentration of 33 to 1000 μg/plate .

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

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 1537

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 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

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 5

Key result

Species / strain:

S. typhimurium TA 100

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 6

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment I, genotoxicity was observed in strain TA98 and TA100 with non-induced hamster liver S9 mix. For strain TA100, there was a slight and dose dependent increase in the number of revertants but the threshold of twice the number of the corresponding solvent control was not reached.
To clarify the results of experiment I a confirmatory experiment (experiment Ia) was performed as a pre-incubation assay with strains TA 98 and TA 100, both with non-induced hamster liver S9 mix. In the repeated experiment Ia an increase in revertant colony numbers exceeding the threshold of twice the number of the corresponding control was observed in strain TA 98 with S9 mix, which confirms the results of experiment I.
No increase was observed in strain TA 100 with S9 mix. Therefore it can be stated, that the erratic colony count in experiment I is probably caused by statistical fluctuations and can be judged as biologically irrelevant.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 in the presence of metabolic activation.