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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 September 2013 (Study Plan completion) to 05 November 2013 (Quality Assurance statement)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material : Lithium cryolite
- Molecular formula : Li3AlF6
- Molecular weight : 162
- Physical state: White powder
- Storage condition of test material: At room temperature in the dark

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany
Test concentrations with justification for top dose:
Dose range finding test with tester strain WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Mutation assay: 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
Preparation of test solutions started with solutions of 50 mg/ml dilutions in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany) applying treatment with ultrasonic waves resulting in a homogeneous white suspension. The lower test concentrations were prepared by subsequent dilutions in DMSO. At concentrations of 0.33 mg/ml and higher Lithium cryolite formed a suspension in DMSO.
At concentrations of 0.1 mg/ml and lower the test substance was fully soluble.
Test substance concentrations were used within 2 hours after preparation.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test substance, which was DMSO.
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48h +/-4h in the dark at 37 °C +/- 1°C
- Expression time (cells in growth medium): cells were counted just after the exposure

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: 0.1 mL of fresh bacterial culture (10E9 cells/mL)

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of Lithium cryolite, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

OTHER EXAMINATIONS:
precipitation of the test substance
Evaluation criteria:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test article precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes for two strains, see details on results
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES and FIRST MUTATION EXPERIMENT:
Lithium cryolite was tested in tester strain WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate.

Precipitate: Precipitation of Lithium cryolite on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards but not at the end of the incubation period.
Toxicity: Cytotoxicity was only observed in the tester strains TA1535 in the presence S9-mix and TA1537 in absence of S9-mix, where an extreme reduction of the revertant colonies was observed at the test substance concentration of 5000 µg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.
Mutagenicity : In the first mutation experiment, no increase in the number of revertants was observed upon treatment with Lithium cryolite under all conditions tested.

MUTATION EXPERIMENT 2:
Based on the results of the dose range finding test, Lithium cryolite was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537, TA100 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitate: Precipitation of Lithium cryolite on the plates was observed at the start of the incubation period at the concentration of 1000 µg/plate and upwards and no precipitate was observed at the end of the incubation period.

Toxicity : Cytotoxicity was observed in tester strain TA1535 in the presence of S9-mix, TA98 in absence and presence of S9-mix and TA100 in the presence of S9-mix, where an extreme reduction of the revertant colonies or no revertant colonies were observed at the test substance concentration of 5000 µg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.
Mutagenicity : In the second mutation experiment, no increase in the number of revertants was observed upon treatment with Lithium cryolite under all conditions tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

List of deviations

Protocol deviations:

1. Selection of an adequate range of doses for the mutation experiments was based on a dose range finding test with tester strain WP2uvrA (in the absence and presence of S9-mix). Tester strain TA100 was tested in the first mutation experiment.

Evaluation: The performing of the dose range finding test with tester strain WP2uvrA and the testing of tester strain TA100 in the first mutation experiment had no effect on the results of the study.

2. In the second experiment, the positive control substances of the following tester strains: TA1535 (absence of S9-mix) and TA100 (presence of S9-mix) and in the first experiment WP2uvrA (absence and presence of S9-mix) showed responses (mean plate count) which were not within the laboratory historical range.

Evaluation: These values were more than 3 times greater than the concurrent solvent control values, therefore this deviation in the mean plate count of the positive controls had no effect on the results of the study.

The study integrity was not adversely affected by the deviations.

 

Standard operating procedures deviations:

Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that Lithium cryolite is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenic activity of Lithium cryolite was investigated in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay according to the OECD Testing Guideline 471 and under GLP.

 

Lithium cryolite was tested with four histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA98 and TA100) and with a tryptophan-requiring strain if E. coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

At concentrations of 0.33 mg/ml and higher Lithium cryolite formed a suspension in dimethyl sulfoxide whereas at 0.1 mg/ml and lower it was fully soluble.

 

In the dose range finding test, Lithium cryolite was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in tester strain WP2uvrA. Lithium cryolite did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

 

Based on the results of the dose range finding test, Lithium cryolite was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1537 in the absence of S9-mix and TA1535 in the presence of S9-mix at the highest tested concentration.

In an independent repeat of the assay with additional parameters, Lithium cryolite was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1535 and TA100 in the presence of S9-mix and TA98 in the absence and presence of S9-mix at the highest tested concentration.

Lithium cryolite did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

The negative control values were within the laboratory historical control data ranges.

 

The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate.

 

Based on the results of this study it is concluded that Lithium cryolite is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.