2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)prop-1-enyl]-1,3,3-trimethyl-3H-indolium acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested in an Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation (Eurofins 2018). The maximum concentration tested was limited mainly by a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 of solvent control. In two independent experiments no biologically relevant increase in revertant colony numbers of any of the five tester strains was observed. Therefore the substance is considered non-mutagenic.

In an additional Ames test (plate incorporation) with Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537, no mutagenic effects were found in presence and absence of metabolic activations (Bayer 1981). The number of concentrations evaluated was limited and positive controls were in some cases only marginally positive. It can be concluded that under the conditions of the test the substance did not induce mutagenic effects in bacteria.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2017 to 12 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: TA 98, TA 1535 and TA 102 MOLTOX, INC., NC 28607, USA; TA 100 and TA 1537 Xenometrix AG, Switzerland.
- Methods for maintenance in cell culture if applicable: stock cultures stored with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction from rats treated with phenobarbital / β-naphthoflavone

Test concentrations with justification for top dose:

exp 1 :
0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 nL/plate or TA 100, TA 1535 and TA 1537 (with and without metabolic activation),for TA 98 and 102 (without metabolic activation)
3.16, 10.0, 31.6, 100, 316, 100, 316 and 1000 nL/plate for TA98 and TA 102 with metabolic activation

exp 2
0.050, 0.158, 0.50, 1.58, 5.0, 15.8, 50.0, 158 and 500 nL/plate for all strains with and without metabolic activation

Top doses limited by cytotoxicity (reduction of bacterial background lawn or reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 of solvent controls)

Vehicle / solvent:

Aqua destillata (purified water)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Due to severe toxicity of the test item, the confirmatory experiment is carried out according to the plate incorporation method with a different spacing between dose levels)

DURATION
- Preincubation period: NA
- Incubation time: at 37 °C for at least 48 h in the dark

NUMBER OF REPLICATIONS: 3/concentration

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

Evaluation criteria:

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:

NA

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 nL/plate and 15.8 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 31.6 and 50 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 and 15.8 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 and 158 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 316 and 15.8 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 nL/plate and 500 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 and 158 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 and 50 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 and 158 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 15.8 and 500 nL/plate in exp 1 and 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The substance is considered non-mutagenic under the conditions of the test.

Executive summary:

The substance was tested in an Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation. The maximum concentration tested was limited mainly by a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 of solvent control. In two independent experiments no biologically relevant increase in revertant colony numbers of any of the five tester strains was observed. Therefore the substance is considered non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the information available the substance does not need to be classified for mutagenicity according to REGULATION (EC) No 1272/2008.