3-methyl-2-[(1-methyl-2-phenyl-1H-indol-3-yl)azo]thiazolium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Start of sperimental phase: 25 May 2017; End of experimental phase: 09 June 2017; Study completion: 02 November 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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

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:

3-methyl-2-[(1-methyl-2-phenyl-1H-indol-3-yl)azo]thiazolium chloride

EC Number:

255-785-4

EC Name:

3-methyl-2-[(1-methyl-2-phenyl-1H-indol-3-yl)azo]thiazolium chloride

Cas Number:

42373-04-6

Molecular formula:

C19H17N4S.Cl

IUPAC Name:

3-methyl-2-[(1-methyl-2-phenyl-1H-indol-3-yl)azo]thiazolium chloride

Test material form:

solid: particulate/powder

Method

Target gene:

the test item for the ability to induce gene mutations in Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these
stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures,
which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement No Growth onMinimal plates+Biotin.Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement No Growth onMinimal agar plates.Growth onMinimal plates+Tryptophan.
-uvrA, uvrB : Sensitivity to UV irradiation.
-rfa : Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

Preliminary Toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate

Main Assay I:
TA98 without S9: 2000, 1000, 500, 250, 125 µg/plate; with S9: 2000, 1000, 500, 250, 125, 62.5 µg/plate
TA1535 with and without S9: 1000, 500, 250, 125, 62.5, 31.3 µg/plate
TA1537 and TA100 with and without S9: 500, 250, 125, 62.5, 31.3 µg/plate
WP2 uvrA with and without S9: 250, 125, 62.5, 31.3, 15.6 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water for injection.
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Details on test system and experimental conditions:

The preliminary toxicity test and the first experiment were perfomed using a plate-incorporation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate ( Chu et al. 1981); Regression line

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium (TA1537 tester strain)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

the test item induces reverse mutation by frameshifts in Salmonella typhimurium (TA1537 tester strain) in the absence and presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item Basic Red 29 induces reverse mutation by frameshifts in Salmonella typhimurium (TA1537 tester strain) in the absence and presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Basic Red 29 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The test item was used as a solution in sterile water for injection.

Toxicity test

The test item Basic Red 29 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Dose-related toxicity was noted with TA1535, TA1537, TA98 and TA100 at the two or three highest dose levels, in the absence and presence of S9 metabolism. A more pronunced toxic effect was noted with WP2 uvrA, where thinning of the background lawn was observed starting from 158 µg/plate, both in the absence and presence of metabolic activation. Large increases in the number of revertant colonies were observed with TA1537 tester strain at slight or not toxic concentrations, both in the absence and presence of S9 metabolism.

Main Assay

On the basis of toxicity test results, in the Main Assay, using the plate incorporation method, the test item was assayed at the following dose levels:

TA98 without S9: 2000, 1000, 500, 250, 125 µg/plate; with S9: 2000, 1000, 500, 250, 125, 62.5 µg/plate

TA1535 with and without S9: 1000, 500, 250, 125, 62.5, 31.3 µg/plate

TA1537 and TA100 with and without S9: 500, 250, 125, 62.5, 31.3 µg/plate

WP2 uvrA with and without S9: 250, 125, 62.5, 31.3, 15.6 µg/plate

Toxicity was observed with all tester strains at higher dose levels in the absence and presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period at any concentration tested. The test item induced large increases in the number of revertant colonies in TA1537 tester strain, both in the absence and presence of S9 metabolism. Since a clear positive response was observed, no further experiment was undertaken.

Conclusion

It is concluded that the test item Basic Red 29 induces reverse mutation by frameshifts in Salmonella typhimurium (TA1537 tester strain) in the absence and presence of S9 metabolism, under the reported experimental conditions.