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Toxicological information

Genetic toxicity: in vitro

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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:
12 - 28 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
Principles of method if other than guideline:
The temperature was above the protocolled range of 37.0 ± 1.0°C for approximately
90 minutes in the second mutation experiment (with a maximum of 39.5°C).
Evaluation: This short term deviation was observed within three hours after initiation of the test and was caused by adjustment of the temperature in the incubator after placing an amount of selective agar plates in the incubator. The negative control data (number of spontaneous revertants per plate) were within the laboratory historical range for each tester strain, therefore this short deviation of the temperature has no effect on the results of the study.

The study integrity was not adversely affected by the deviations.

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LiPF6
- Substance type: inorganic
- Physical state: white powder
- Analytical purity: > 99.9%
- Lot/batch No.: P07102202
- Expiration date of the lot/batch: 31 December 2008
- Stability under storage conditions: stable
- Storage condition of test material: At room temperature protected from light under nitrogen

Method

Target gene:
histidine gene in S. typhimurium
tryptophan gene in E. Coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Mutation assay: 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
pos. control for TA1535 (-S9)

Migrated to IUCLID6: diluted in saline conc 5 µg/plate
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
pos. control for TA1537 (-S9)

Migrated to IUCLID6: diluted in water Conc. 60 µg/plate
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
pos. control for TA98 (-S9)

Migrated to IUCLID6: diluted in DMSO Conc. 10 µg/plate
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
pos. control for TA100 (-S9)

Migrated to IUCLID6: diluted in DMSO Conc. 650 µg/plate
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
pos. control for WP2uvrA (-S9)

Migrated to IUCLID6: diluted in DMSO Conc. 10 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene diluted in DMSO
Remarks:
Conc. 1 µg/plate for strains TA1535 (5 & 10% S9), TA98 (5 & 10% S9) and TA100 (5% S9); Conc. 2.5 µg/plate for strains TA1537 (5% S9) and TA100 (10% S9); Conc. 5 µg/plate for strain TA1537 (10% S9) and Conc. 10 µg/plate for strain WP2uvrA (5 and 10% S9).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.


DETERMINATION OF CYTOTOXICITY
- Method: other: 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:
- Other: precipitation

Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of LiPF6 on the plates was not observed at the start or at the end of the incubation period.


RANGE-FINDING/SCREENING STUDIES:
Precipitate: Precipitation of LiPF6 on the plates was not observed at the start or at the end of the incubation period in both tester strains.
Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with LiPF6 under all conditions tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our 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 LiPF6 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.