Hydrogen [4-[4-(p-ethoxyanilino)-4'-[ethyl(m-sulphonatobenzyl)amino]-2'-methylbenzhydrylene]-3-methylcyclohexa-2,5-dien-1-ylidene](ethyl)(m-sulphonatobenzyl)ammonium, monosodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 20085/B TE did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, FAT 20085/B TE is considered to be non-mutagenic in this bacterial reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-08-19 to 2014-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: A.dest.

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 140808

Negative solvent / vehicle controls:

yes

Remarks:

A. dest., BSL Lot No. 140808

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

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

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.



NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 20085/B TE is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to FAT 20085/B TE at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I and II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

FAT 20085/B TE was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increased induced mutant colonies over background with any of the test substance concentrations. Based on the findings of the study, it was clear that FAT 20085/B TE did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Hence, FAT 20085/B TE is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

ACcid Blue 090 was screened for mutagenic potential in the bacterial reverse mutation assay. In this assay, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to FAT 20085/B TE at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I and II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II). FAT 20085/B TE was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increased induced mutant colonies over background with any of the test substance concentrations. Based on the findings of the study, it was clear that FAT 20085/B TE did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Hence, FAT 20085/B TE is considered to be non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

FAT 20085/B TE is considered to be non-mutagenic in this bacterial reverse mutation assay. Hence, it does not warrant classification according to EU CLP (Regulation EC No. 1272/2008).