Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 09, 2017 to August 17, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: WD0066442; Purity: 100 %; Appearance: homogeneous amber liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The following nominal concentrations were prepared for the first experiment:
5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
The following nominal concentrations were prepared for the second experiment:
5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.
Vehicle / solvent:
DMSO was chosen as vehicle, because the test substance was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontane-ous revertants in the tested concentrations.
Controls
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:
Origin and Culture:
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and 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.

Test solutions preparation:
On the day of the start of the first and the second experiment, a stock solution containing 50 g/L of the test substance in DMSO was prepared. The test substance solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested.

Description of the Method:

Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
The test substance solutions were prepared. For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidinebiotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
- 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 μL S9 mix phosphate buffer (for test without metabolic activation).
- 100 μL bacteria suspension
- 2000 μL overlay agar (top agar)

The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method:
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
- 100 μL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
- 500 μL S9 mix or phosphate buffer (for test without metabolic activation).
- 100 μL bacteria suspension

After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
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 substance solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) 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 resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Confirmation of the Criteria and Validity:
All strains met the criterion of at least 10+E9 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 nearly all were within the historical control data ranges.

Solubility and Toxicity:
The test substance showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not re-duced.

Mutagenicity:
No significant 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.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method 13/14, in compliance with GLP. Five strains of Salmonella typhimurium were used, namely TA97a, TA98, TA100, TA102 and TA1535. The test was performed in two experiments in the presence and absence of metabolic activation (S9-mix). In the first experiment, the substance (dissolved in DMSO) was tested up to concentrations of 5000 μg/plate in all strains using the plate incorporation method. As the first experiment did not show mutagenic effects, a confirmatory second experiment was conducted with concentrations up to 5000 μg/plate, in the absence and presence of S9-mix with all bacteria strains using the pre-incubation method. The substance showed no precipitates on the plates at any of the concentrations and no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. None of the concentrations showed a significant increase in the number of revertants in the tested strains, in the presence and the absence of metabolic activation. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation (Andres, 2017).