Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June-July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
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
Test material form:
liquid
Details on test material:
clear, colourless liquid
Production date: 14. Jan. 2018
Expiry date: 14. Jan. 2021
Storage: room temperature (20 ± 5 °C)
Batch no.: 701146_43343114

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA98: 5136D)

Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA100: 5141D)
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA102: 5145D)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA1535: 5138D)
Species / strain / cell type:
S. typhimurium, other: 97a
Details on mammalian cell type (if applicable):
Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 5033D)
Metabolic activation:
with and without
Metabolic activation system:
S9 was obtained by Trinova Biochem GmbH, Gießen. Batch nos. 3913, 3833, 3837,
Specification produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraper-itoneally.
Test concentrations with justification for top dose:
plate incorporation method: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
pre-incubation method: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate
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:
Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutri-ent broth. After incubation 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.

Experimental Parameters
First Experiment
Concentrations tested 5 / 1.5 / 0.5 / 0.15 / 0.05 μL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method

Second Experiment
Concentrations tested 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 μL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method

Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous re-vertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: 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:
The test item showed no increase in the number of revertants in all bacteria strains in both experiments.
Nearly all negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

Confirmation of the Criteria and Validity

All strains met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity

In the two experiment, the test item showed no precipitates on the plates in all tested concentrations.

No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity

No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.