Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting date: 2015-04-07; experimental completion date: 2015-04-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
resp. EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
The syntax of the test item name was changed, this can be seen as uncritical
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Kaiser-Freidrich-Straße 7, D-55116 Mainz
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: viscous liquid
Details on test material:
Stabilized EMIM BF4:
Appearance: lightly yellow clear viscous liquid
Purity: 93.36% (EMIM+, HPLC); 86.68% (BF4-, titration); 4.89% (Stabilizer, HPLC)

Method

Species / strain
Species / strain / cell type:
other: 9005=S. typhimurium TA 1535, TA 97a, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: lipopolysaccharide side chain deficiency
Metabolic activation:
with and without
Test concentrations with justification for top dose:
1. Experiment:
5 concentrations of the test item ranging from 50 µg/plate to 5000 µg/plate using the plate incorporation method

2. Experiment
7 concentrations of the test item ranging from 78 µg/plate to 5000 µg/plate using the pre-incubation method
Vehicle / solvent:
dimethyl sulfoxide (DMSO) was used as vehicle
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Amino-anthracene; 4-Nitro-1,2-phenylene Diamine
Details on test system and experimental conditions:
Specification:
Species Salmonella typhimurium LT2
Strains TA97a, TA98, TA100, TA102 and TA1535

Origin and culture:
Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem (batch of the bacteria strains: TA97a: 4904D, TA98: 4903D, TA100: 4902D, TA102: 4872D, TA1535: 4908D) and were stored as lyophilisates in the fridge at 2-8 °C.
One day before the start of each experiment, one lyophilisat per strain to be used was taken from the fridge to inoculate a culture vessel containing nutrient broth. After incubation overnight 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.


Evaluation criteria:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor  2) in at least one strain can be observed. A concentration-related in-crease over the range tested can also be taken as a sign of mutagenic activity.
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

Results and discussion

Test results
Species / strain:
other: 4116=S. typhimurium TA 1535, TA 97a, TA 98 and TA 100
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 on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation and negative without metabolic activation

The test item stabilized EMIM BF4 is considered as “not mutagenic under the conditions of the test”.
Executive summary:

Two valid experiments were performed.

 

First Experiment:

Five concentrations of the test item, dissolved in dimethyl sulfoxide (ranging from 50 µg/plate to 5000 µg/plate) were used. 5 genetically changed strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 h at 37 ± 1°C, using the plate incorporation method.

None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item did not show any mutagenic effects in the first experiment.

The test item showed no precipitates on the plates in all tested concentrations

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using 7 concentrations of the test item (ranging from 78 µg/plate to 5000 µg/plate) and a modification in study performance (pre-incubation method at 37 ± 1°C).

The test item did not show mutagenic effects in the second experiment, either.

The test item showed no precipitates on the plates in all tested concentrations.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Under the conditions of the test, the test item did not show mutagenic effects towardsSalmonella typhimurium,strainsTA97a, TA98, TA100, TA102 and TA1535.

Therefore, no concentration-effect relationship could be determined.