Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine, sodium polysulfide and sodium metabisulfite

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:

24 January 2018 - 24 April 2018

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:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine, sodium polysulfide and sodium metabisulfite

EC Number:

600-520-3

Cas Number:

1040874-53-0

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine, sodium polysulfide and sodium metabisulfite

Test material form:

solid: particulate/powder

Details on test material:

Test item: Blue TBR
Appearance: black bluish powder
CAS No: 1040874-53-0

Method

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:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver

Test concentrations with justification for top dose:

5, 16, 50, 160, 500, 1600 and 5000 µg/plate
Justification of top dose: Selection of the concentration range was done on the basis of solubility test and concentration range finding test.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: based on solubility test

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes, 37 °C (preincubation method)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

Evaluation criteria:

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

Results and discussion

Test results

Test results 1

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:

not examined

Positive controls validity:

valid

Test results 2

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:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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:

not examined

Positive controls validity:

valid

Test results 5

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 μg/plate in the absence and presence (±S9 mix) of exogenous metabolic activation following the plate incorporation procedure, and at 5000 and 1600 μg/plate in the absence and at 5000 μg/plate in the presence of exogenous metabolic activation following the pre-incubation procedure.

RANGE-FINDING/SCREENING STUDIES: The concentration range investigated in the main tests was based on a preliminary range finding test.

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions a correction of the concentrations for the purity (71.99 %) of test item was made in the experiments. Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate. The results of the preliminary concentration range finding tests allowed the applying of the recommended maximum test concentration of 5000μg/plate.

When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 μg/plate in the absence and presence (±S9 mix) of exogenous metabolic activation following the plate incorporation procedure, and at 5000 and 1600 μg/plate in the absence and at 5000 μg/plate in the presence of exogenous metabolic activation following the pre-incubation procedure. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system. The revertant colony numbers of solvent control dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in the revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.