2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

12 February to 15 March 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.14 (Mutagenicity - Salmonella Typhimurium Reverse Mutation Test)

Principles of method if other than guideline:

According to Ames et al. 1973 and 1975; Maron and Ames 1983

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

EC Number:

258-946-7

EC Name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

Cas Number:

54060-92-3

Molecular formula:

C20H24N3O.CH3O4S C21H27N3O5S

IUPAC Name:

2-{[2-(4-methoxyphenyl)-2-methylhydrazin-1-ylidene]methyl}-1,3,3-trimethyl-3H-indol-1-ium methyl sulfate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): C. I. Basic Yellow 28 (Astrazon Goldgelb GLN)

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

First test: 8, 40, 200, 1000, 5000 µg/plate
Repeat tests: 25, 50, 100, 200, 400, 800 µg/plate
10, 20, 30, 40, 50, 60, 70 µg/plate TA 100 with 10 %, 30 %, 50 % S9

Vehicle / solvent:

methanol

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

historical for methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 w/o S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

historical for methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantion

Remarks:

TA 100 w/o S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

historical for methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Remarks:

TA 1537, TA 98 w/o S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

historical for methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation: 0.1 mL TS+0.1 mL bacteria+0.5 mL S9+2 mL soft agar: 30 sec at 45 °C
- Incubation period: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates/strain/concentration

DETERMINATION OF CYTOTOXICITY
- Method: - background growth
- marked and dose-dependent reduction in mutant count compared to negative controls
- titer determination

Acceptance criteria:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.

Statistics:

NA

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

TA 100: 1.6-fold increase at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect of C.I. Basic Yellow 28 at 8 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used to a limited extent up to 1000 µg per plate for assessment purposes.
In the first trial, none of the four strains concerned showed a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix.
The negative findings for Salmonella typhimurium TA 1535, TA 1537 and TA 98 were confirmed by further repeat tests. In the TA 100 strain, a dose-related minimal increase in mutant counts to 1.6-fold of negative controls was found in the second test. The findings of the second trial for Salmonella typhimurium TA 100 were confirmed by further repeat tests using 30 % and 50 % S9 mix. No relevant increase was found using 10 % S9 mix.
The lowest dose at which this finding was reproducible was approximately 30 µg per plate for Salmonella typhimurium TA 100. Minimally increased mutation rates - below the theshold for positive effects - were obtained only with S9 mix containing 30 % and 50 % S9 fraction in S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Basic Yellow 28 methyl sulfate showed a minimal increase (1.6-fold) in revertant mutations in TA 100 at cytotoxic concentrations with an increased amount of the metabolic activation system.

Executive summary:

C.I. Basic Yellow 28 methyl sulfate was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

8 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to 1000 µg per plate for assessment purposes.

On Salmonella typhimurium TA 100, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. A minimal increase in reverse mutations (1.6 -fold) was found only with S9 mix depending on S9 mix composition. The lowest reproducible effective dose was 30 µg per plate for Salmonella typhimurium TA 100.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2‑aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.