Registration Dossier

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:
2018

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
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
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 deep freezer 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 test strains TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
two exceptions in five experiments

Applicant's summary and conclusion

Conclusions:
Five valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.
The test item (R)-3,7-dimethyl-1,6-octadien-3-ol 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 five 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.

Experiment 1a:
In this 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.
In the highest concentration (5 µL/plate), the bacterial background lawn was not reduced, but a decrease in the number of revertants was observed for all five bacteria strains.
In the next lower concentrations, a relevant decrease in the number of revertants was ob-served for the bacteria strains TA98, TA100 and TA1535.
The test item showed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in the highest concentrations (5 µL/plate) and towards the bacteria strains TA98, TA100 and TA1535 (with and without metabolic activation) in the next lower concentration (1.5 µL/plate).
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.


Experiment 1b:
In this experiment, the test item was tested up to concentrations of 5 µL/plate in the ab-sence and presence of S9-mix in the strains TA97a and TA102 using the plate incorpora-tion method, the bacteria strains TA98, TA100 and TA1535 was tested up to concentra-tions of 1.5 µL/plate under the same conditions.
The test item showed no precipitates on the plates at any of the concentrations.
The bacterial background was observed at all tested concentrations.
In the highest concentration 5 µL/plate for the bacteria strain TA102, resp. in the highest concentration 1.5 µL/plate for the bacteria strain TA1535, a decrease in the number of re-vertants was observed.
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.
Due to a contamination on the agar plates, no evaluation of the bacteria strains TA97a, TA98 and TA100 was possible. Therefore the experiment was repeated with the bacteria strains TA97a, TA98 and TA100 (= experiment 1c).

Experiment 1c:
In this experiment, the test item was tested up to concentrations of 5 µL/plate in the ab-sence and presence of S9-mix in the strain TA97a using the plate incorporation method, the bacteria strains TA98 and TA100 was tested up to concentrations of 1.5 µL/plate under the same conditions.
The test item showed no precipitates on the plates at any of the concentrations.
The bacterial background was observed at all tested concentrations.
In the highest concentration for the bacteria strain TA97a (5 µL/plate), a decrease in the number of revertants was observed with and without metabolic activation.
In the concentration 1.5 µL/plate, a decrease in the number of revertants was observed with and without metabolic activation in the bacteria strains TA98 and TA100.
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.

Experiment 2a:
Based on the results of the experiments with the plate incorporation method, the test item was tested up to concentrations of 5 µL/plate for the bacteria strain TA102 resp. 1.5 µL/plate for the bacteria strain TA1535 in the absence and presence of S9-mix 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 reduced at all tested concentrations.
For the bacteria strain TA102 no bacteria growth and no bacterial background lawn was visible at the two highest concentrations (5 and 1.5 µL/plate).
For the bacteria strain TA1535 no bacteria growth and no bacterial background lawn was visible at the highest concentration (1.5 µL/plate).
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.

Experiment 2b:
Based on the results of the experiments with the plate incorporation method, the test item was tested up to concentrations of 1.5 µL/plate in the absence and presence of S9-mix in the strains TA98 and TA100 using the pre- incorporation method and up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a using the pre- incorporation method, too.
The test item showed no precipitates on the plates at any of the concentrations.
The bacterial background lawn was not reduced at all tested concentrations.
Signs of toxicity were observed as follows:
Bactria strain TA97a: 5 µL/plate (no bacteria growth and no bacterial background lawn), 2.5 and 1.25 µL/plate.
Bactria strain TA98: 1.5 µL/plate (no bacteria growth and no bacterial background lawn), 0.75 and 0.38 µL/plate.
Bacteria strain TA100: 1.5 µL/plate, 0.75 and 0.38 µL/plate.

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.

Based on the results of this study it is concluded that (R)-3,7-dimethyl-1,6-octadien-3-ol is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.