[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

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:

Start of experimental phase: 23 January 2018; End of experimental phase: 05 March 2018;

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

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:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

EC Number:

248-957-5

EC Name:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

Cas Number:

28313-51-1

Molecular formula:

C22H29N6S.CH3O4S

IUPAC Name:

{2-[ethyl({3-methyl-4-[(E)-2-(3-phenyl-1,2,4-thiadiazol-5-yl)diazen-1-yl]phenyl})amino]ethyl}trimethylazanium methyl sulfate

Test material form:

solid: particulate/powder

Details on test material:

Basic Red 23 MeSO4

Method

Target gene:

The test item Basic Red 23 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.

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:

liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone and a liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification method)

Test concentrations with justification for top dose:

Preliminary toxicity test: 5000,1580, 500, 158 and 50.0 µg/plate.
Main Assay I:
Tester strain S9 Dose level (µg/plate)
TA1535 ± 250, 125, 62.5, 31.3 and 15.6
TA1537 − 250, 125, 62.5, 31.3 and 15.6
TA1537 + 1000, 500, 250, 125, 62.5 and 31.3
TA98, WP2 uvrA ± 2000, 1000, 500, 250 and 125
TA100 − 62.5, 31.3, 15.6, 7.81, 3.91 and 1.95
TA100 + 125, 62.5, 31.3, 15.6, 7.81, 3.91 and 1.95

Main Assay II:
Tester strain S9 Dose level (µg/plate)
TA1535 ± 188, 93.8, 46.9, 23.4 and 11.7
TA1537 − 188, 93.8, 46.9, 23.4 and 11.7
TA1537 + 375, 188, 93.8, 46.9 and 23.4
TA98, WP2 uvrA ± 1500, 750, 375, 188 and 93.8
TA100 - 23.4, 11.7, 5.86, 2.93 and 1.46
+ 62.5, 31.3, 15.6, 7.81, 3.91 and 1.95


Main Assay III
Tester strain S9 Dose level (µg/plate)

TA1535, TA1537 − 11.3, 5.63, 2.81, 1.41 and 0.703
TA98, WP2 uvrA − 90.0, 45.0, 22.5, 11.3, 5.63 and 2.81
TA98 + 90.0, 45.0, 22.5, 11.3, 5.63 and 2.81

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:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

methylmethanesulfonate

other: Trypan blue

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 Main assay were performed using the plate-incorporation method and the standard metabolic activation system (Rat-Mixed Induction).
The second and third Main assays were performed using the pre-incubation method and the reductive metabolic activation system (Prival modification)

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 TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item Basic Red 23 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Basic Red 23 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 a liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation) in the preliminary toxicity test and in Main Assay I, and a liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification method), in Main Assay II and Main Assay III.

The test item was used as a solution in sterile water for injection.

Toxicity test

The test item Basic Red 23 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 observed with all tester strains at higher dose levels, both in the absence and presence of S9 metabolism. A less pronounced toxic effect was seen with TA98 and WP2 uvrA tester strains.

Main Assays

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

Tester strain        S9       Dose level (µg/plate)

TA1535                ±         250, 125, 62.5, 31.3 and 15.6

TA1537                −         250, 125, 62.5, 31.3 and 15.6

TA1537                +         1000, 500, 250, 125, 62.5 and 31.3

TA98, WP2 uvrA  ±         2000, 1000, 500, 250 and 125

TA100                   −         62.5, 31.3, 15.6, 7.81, 3.91 and 1.95

TA100                   +         125, 62.5, 31.3, 15.6, 7.81, 3.91 and 1.95

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

As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed. Based on the chemical structure of the test item (azo-dye), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The dose-range was slightly modified to take into account the toxicity results of Main Assay I. The test item was assayed at the following dose levels:

Tester strain           S9               Dose level (µg/plate)

TA1535                   ±                 188, 93.8, 46.9, 23.4 and 11.7

TA1537                   −                 188, 93.8, 46.9, 23.4 and 11.7

TA1537                  +                  375, 188, 93.8, 46.9 and 23.4

TA98, WP2 uvrA     ±                1500, 750, 375, 188 and 93.8

TA100                     -                  23.4, 11.7, 5.86, 2.93 and 1.46

                              +                  62.5, 31.3, 15.6, 7.81, 3.91 and 1.95

No increase in the revertant colonies was noted with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. However, dose related toxicity, mainly indicated by thinning of the background lawn, was observed at higher dose levels with all tester strains, in the absence and presence of S9 metabolic activation. Since mutagenic effects have to be evaluated at non-toxic concentrations, an additional experiment was performed in the absence of S9 metabolism with all tester strains with the exception of TA100 and in the presence of S9 metabolism with TA98 tester strain.

In Main Assay III, the test item was assayed at the following dose levels:

Tester strain           S9               Dose level (µg/plate)

TA1535, TA1537      −               11.3, 5.63, 2.81, 1.41 and 0.703

TA98, WP2 uvrA      −                90.0, 45.0, 22.5, 11.3, 5.63 and 2.81

TA98                        +                 90.0, 45.0, 22.5, 11.3, 5.63 and 2.81

A sufficient number of analysable dose levels were obtained and no increase in revertant colonies was noted at any concentration tested, with any tester strain/activation condition combinations.

No precipitation of the test item, evident to the unaided eye, was observed at the end of the incubation period, at any concentration, in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

Conclusion

It is concluded that the test item Basic Red 23 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.