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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 - 13 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine operon
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate (expressed as 2,2-di-(tert-amylperoxy) butane)
Vehicle:
- Vehicle used: ethanol
- Justification for choice: the test item was diluted in the vehicle at a concentration of 200 mg/mL for the preliminary toxicity test and for both mutagenicity experiments.
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: The preliminary toxicity test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method.
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.
DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
Evaluation criteriaIn all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship is evidenced.
The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship is noted.

Results and discussion

Test results
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Applicant's summary and conclusion

Conclusions:
LUPEROX® 520M50 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

The potential of LUPEROX® 520M50 to induce reverse mutations was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) 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, diluted in ethanol, to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both 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. Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, 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. Since the test item was found poorly soluble in the final treatment medium but non-toxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines. 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. The selected dose-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate (expressed as 2,2-di-(tert-amylperoxy) butane) for the five strains in both mutagenicity experimentswith and without S9 mix. A moderate to strong emulsion, which did not prevent any scoring, was observed in the Petri plates from the lowest selected dose-levels. No noteworthy toxicity was noted at any of the tested dose-levels, towards the five strains used, either with or without S9 mix.

LUPEROX® 520M50 did not induce any biologically relevant increase in the number of revertants, in any strains or test conditions. Consequently, the results met the criteria of a negative response.