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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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:
solid: particulate/powder

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
supplied by Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rats liver induced with Aroclor 1254
Test concentrations with justification for top dose:
0, 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/plate
Vehicle / solvent:
Ethanol
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: product insoluble in all conventional solvents, used as a suspension in ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other:
Remarks:
2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

NUMBER OF REPLICATIONS: 2 (3 with T98 -S9)

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
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 number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.

Results and discussion

Test results
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix:
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at tested dose-levels = 55.6 µg/plate in the first experiment without S9 mix, in the second experiment in strains TA 1537, TA 98 and TA 102, and in the third experiment.
Furthermore, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at tested dose-levels = 18.5 µg/plate in TA 1535 and TA 100 strains during the second experiment.
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards any of the tested strains.
In the second experiment, an increase in the number of revertants was observed in the TA 98 strain at the highest tested dose-level of 500 µg/plate. This increase was above the threshold of 2-fold the vehicle control, and the mean and individual revertant colony counts were above the historical data (i.e. up to 57 revertants vs. [12-39] for the corresponding vehicle control).
As this increase was not observed in the first experiment performed under the same experimental conditions and was noted without any clear evidence of a dose-response relationship, a third assay was implemented in the TA 98 strain without S9 mix.
It is to be noted that manual scoring was used in the third experiment when precipitate was noted in Petri plates, in order to ensure that any increase in the number of revertants is not due to the presence of precipitate. In the third experiment, no increase in the number of revertants was noted, in any of the tested dose-levels.
Consequently, the increase observed in the second experiment was not considered to be biologically relevant, and the results meet thus the criteria of a negative response.
The test item did not induce any significant increase in the number of revertants in the other tested strains.

Experiments with S9 mix:
The selected treatment-levels were 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/plate for both mutagenicity experiments.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at tested dose-levels = 55.6 µg/plate in the five tested strains during the first experiment and in TA 1537, TA 98 and TA 102 strains during the second experiment.
Furthermore, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at tested dose-levels = 18.5 µg/plate in TA 1535 and TA 100 strains during the second experiment.
No toxicity was noted towards all the strains used, either with or without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

Applicant's summary and conclusion

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

The potential of EKKE, to induce reverse mutations in Salmonella typhimurium  was evaluated in a study s 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 EKKE to be used for the mutagenicity experiments. The test item was then tested in two or three independent experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Three experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteriaSalmonella typhimuriumwere used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. 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 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. The test item was suspended in ethanol. The selected treatment-levels were 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/plate, for the three mutagenicity experiments, with and without S9 mix.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at tested dose-levels = 55.6 µg/plate in the first experiment both with and without S9 mix, in the second experiment in TA 1537, TA 98 and TA 102 strains both with and without S9 mix, and in the third experiment. Furthermore, a moderate to strong precipitate was observed in the Petri plates when scoring the revertants at tested dose-levels = 18.5 µg/plate in the second experiment with S9 mix in TA 1535 and TA 100 strains. No toxicity was noted towards all the strains used, either with or without S9 mix. In the second experiment without S9 mix, an increase in the number of revertants was observed in the TA 98 strain at the highest tested dose-level of 500 µg/plate. This increase was above the threshold of 2-fold the vehicle control, and the mean and individual revertant colony counts were above the historical data. As this increase was not observed in the first experiment performed under the same experimental conditions and was noted without any clear evidence of a dose-response relationship, a third assay was implemented in the TA 98 strain without S9 mix. Manual scoring was used in the third experiment when precipitate was noted in Petri plates, in order to ensure that any increase in the number of revertants was not due to the presence of precipitate. In the third experiment, no increase in the number of revertants was noted, in any of the tested dose-levels. Consequently, the increase observed in the second experiment was not considered to be biologically relevant, and the results meet thus the criteria of a negative response. The test item did not induce any significant increase in the number of revertants in the other tested conditions. In conclusion, EKKE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.