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The objective of the study of Sarlang (2010d) was to evaluate the potential of the test item to induce reverse mutation inSalmonella typhimurium.The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

  

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes,).

 Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.The test item was diluted in ethanol.

 The dose-levels of the positive controls were as follows:

 without S9 mix

.           1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

.           50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

.           0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

.           0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

 with S9 mix

.           2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537 and TA 98 strains,

.           5 µg/plate of Benzo(a)pyrene (BAP): TA 100 strain,

.           10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

 

Results:

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid.

 Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines

Experiments without S9 mix

The selected treatment-levels were:

.           0.39, 0.78, 1.56, 3.13, 6.25 and 12.5 µg/plate for the first experiment,

.           0.63, 1.25, 2.5, 5, 10 and 20 µg/plate for the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

 

In the first experiment, a moderate toxicity (thinning of the bacterial lawn) was noted at 12.5 µg/plate towards all the tested strains.

In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >= 5 µg/plate in the TA 1537 strain, >= 10 µg/plate in the TA 98 and TA 102 strains and at 20 µg/plate in the TA 1535 and TA 100 strains.

 

An increase in the number of revertants was noted in the TA 102 strain in the second experiment. This increase reached the threshold of 2‑fold the vehicle control value, however it was neither dose-related nor reproducible (not observed in the first experiment performed under the same experimental conditions). Therefore, this increase was considered not to be biologically significant.

The test item did not induce any other noteworthy increase in the number of revertants, in any of the other strains.

Experiments with S9 mix

The selected treatment-levels were:

.           3.13, 6.25, 12.5, 25, 50 and 100 µg/plate for the first experiment,

.           10, 20, 40, 80, 160 and 320 µg/plate for the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

 

In the first experiment performed using the direct plate incorporation method, no noteworthy toxicity was noted towards any of the five strains tested up to 100 µg/plate.

In the second experiment performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels >= 80 µg/plate in the TA 100 strain, >= 160 µg/plate in the TA 1537 and TA 98 strains and at 320 µg/plate in the TA 1535 and TA 102 strains.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Under these experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium, either in the presence or the absence of a rat metabolizing system.


Short description of key information:
The potential of the substance to induce gene mutation in bacteria was investigated using a gene mutation assay on Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 according to OECD guideline 471 and Good laboratory Practice (Sarlang, 2010d).
The test item was found to be not mutagenic in this assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance is not mutagenic in the vitro gene mutation test in bacteria. However and according to the criteria laid down in EU regulation (EC) n° 1272/2008 (CLP) and the EU directive 67/548/EEC, the substance cannot be classified for genetic toxicity on a basis of a sole gene mutation test in bacteria.