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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 June 2019 to 27 June 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

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

Test material

Test material form:


Target gene:
Histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
15, 50, 150, 500, 1500, 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains
Vehicle / solvent:
Demineralised water, prepared by LAUS GmbH, from an ion-exchanger, batch: T20190510
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
sodium azide
other: 2-Amino-anthracene, 4-Nitro-1,2-phenylene diamine
Details on test system and experimental conditions:
Test System:
Species: Salmonella typhimurium LT2, Escherichia coli
Strains: Salmonella typhimurium LT2: TA97a, TA98, TA100, TA1535; E. coli: WP2

Experimental Parameters:
Concentrations tested - 5000 / 1500 / 500 / 150 / 50 / 15 μg/plate
Incubation time - 48 h Incubation temperature 37 ±1°C
Tested strains- TA97a, TA98, TA100, TA1535, Escherichia coli WP2
Method - pre-incubation method
Incubation time- 20 min
No. of replicates - 3

Description of the Method:

General preparation:
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM resp. 10 mL of the tryptophan-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±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 , for test with metabolic activation) 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.

Sterility Control:
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37 ±1°C, four replicates.

Plates were checked for precipitation of test item at the end of the incubation by visual in-spection.

Positive Controls:
Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.

Evaluation criteria:
Six different analysable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test substance.
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 three fold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spon-taneous revertants) of the test substance solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants less mean spontaneous revertants) was given.
A substance is considered to be mutagenic, if a reproducible increase with or without meta-bolic activation of revertant colonies per plate exceeding an increasefactor of 2 for the bac-teria strains TA97a, TA98, TA100, TA1535 and E.coli compared to vehicle controls in at least one strain can be observed and there is a concentration-related increase. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
A substance is not mutagenic if it does not meet these criteria. If the criteria listed above are not clearly met, the results are assessed as equivocal and are discussed.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
True negative controls validity:
Positive controls validity:
Additional information on results:
All of the means of all replicates of the spontaneous revertants (in vehicle and negative controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.

Any other information on results incl. tables

In the experiment, no precipitation of the test substance was observed at any of the tested concentrations up to 5000 μg/plate.

In the experiment using the pre-incubation method, the test item caused no cytotoxicity towards all bacteria strains. The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 10E-9 bacteria/mL.The test item substance showed no increase in the number of revertants in all bacteria strains in the experiment with the pre- incubation method. No concentration-related increase over the tested range was found.

Applicant's summary and conclusion

Under the study conditions, the test substance was not found to be mutagenic.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA in the Ames plate incorporation and pre-incubation methods up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range study was performed in range of 15 to 5000 μg/plate. The test substance 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 relevant decrease in the number of revertants was observed in all bacteria strains. The test substance 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. Under the study conditions, the test substance was not found to be mutagenic (Andres, 2019).