2-Pyridinamine, 5-bromo-3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames study (Verspeek-Rip, 2010) is available which is key study. It is concluded that the test substance is not mutagenic in the 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:

From 28 June to 08 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene in S. typhimurium
Tryptophan gene in Escherichia coli

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate
Mutation assay: 10, 33, 100, 333, 1000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: The test substance was dissolved in dimethyl sulfoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix for strain TA1535.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix for strain TA1537.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix for strain TA98.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

In the absence of S9 mix for strain TA100.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

In the absence of S9 mix for strain WP2uvrA.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

In the presence of S9 mix for all strains.

Details on test system and experimental conditions:

Dose range finding test
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. In the first experiment the test substance was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537, and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethyl sulfoxide and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony counting
The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

None stated

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitate:
Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period.

Toxicity:
In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity:
In both mutation assays, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test substance is not mutagenic in the bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

An Ames study was conducted according to OECD 471 using S.typhimurium and E. coli strains (Verspeek-Rip, 2010). Key study.

It is concluded that the test substance is not mutagenic in the bacterial reverse mutation assay.

Justification for selection of genetic toxicity endpoint
Study run to a method comparable with current guidelines and to GLP

Justification for classification or non-classification

Genetic toxicity in vitro: Negative result (Not mutagenic to S. typhimurium and E. coli strains).

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1 the substance is not classified for the genetic toxicity endpoint.