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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name Estragole
Appearance Clear liquid
Composition 4-allylanisole
Purity 99.53%
Storage Room Temperature: (20 ± 5 °C), keep away from light

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Vehicle / solvent:
DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene
Details on test system and experimental conditions:
All Salmonella typhimurium strains were stored as lyophilizates in the refrigerator at 2-8 °C.
The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.
Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the fridge to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Results and discussion

Test results
Key result
Species / strain:
other: Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.
The test item Estragole was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).
The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
The bacterial background lawn was not reduced at any of the concentrations and no rele-vant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.
The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Based on the first experiment, the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all bacteria strain using the pre-incubation method.
The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not visible at the highest concentration (5 µL/plate) and a decrease in the number of revertants was observed in the bacteria strain TA97a. The other four bacteria strains showed no growth in this concentration.
In the next lower concentration (2.5 µL/plate), the bacterial background lawn was observed in all bacteria strains, but the bacteria growth was clearly reduced.
The results of this experiments showed that the test item caused no increase in the num-ber of revertants in all bacteria strains compared to the solvent control, in both the ab-sence and presence of metabolic activation.
The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.