Lithium fluoride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-01 to 2015-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21st July 1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(30th May 2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TOXI-COOP Toxikological Research Zenter Zrt., Hungary

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lithium fluoride

EC Number:

232-152-0

EC Name:

Lithium fluoride

Cas Number:

7789-24-4

Molecular formula:

FLi

IUPAC Name:

lithium(1+) fluoride

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: Lot No.: 1812
- Expiration date of the lot/batch: May 2025
- Purity test date: May 12, 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; Avoid contact with acid
- Stability under test conditions: stable under recommended test conditions

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix ( Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver)

Test concentrations with justification for top dose:

Rangefinding experiment: 5, 16, 50, 160, 500, 1600, 5000 µg/plate with and without S9 mix
Main experiments: 16, 50, 160, 500, 1600, 5000 µg/plate with and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Test substance was suspendable in ultrapure water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

without S9 mix (TA 98)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix (TA 100, TA 1535)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix (TA 1537)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix (WP2 uvrA)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with S9 mix (TA 98, TA 100, TA 1535, TA 1537, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: 20 min at 27 °C (only Experiment II)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduced background lawn

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

Not applicable.

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
The obtained revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA100, at the examined concentration of 5000 μg/plate, without and with addition of exogenous metabolic activation (±S9 Mix); furthermore at 500 μg/plate, without metabolic activation (-S9 Mix). In these cases the revertant colony numbers were below the corresponding historical control data ranges; however were evaluated as being within the biological variability range of the applied test system.
Slightly higher revertant colony counts were obtained in S. typhimurium TA98 at 160 μg/plate without metabolic activation (-S9 Mix) and at 16 μg/plate, with addition of metabolic activation (+S9 Mix). These slightly higher revertant counts remained in the corresponding historical control data and biological variability range of the applied test system.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, it is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Lithium fluoride was tested in the bacterial reverse mutation assay according to OECD guideline 471. Five bacterial strains were used to investigate the mutagenic potential of lithium fluoride in two independent experiments, in a plate incorporation test (Experiment I, Initial Mutation Test) and in a pre-incubation test (Experiment II, Confirmatory Mutation Test). The test item was dissolved (suspended) in ultrapure water resulting in concentrations of 16, 50, 160, 500, 1600 and 5000 µg/plate. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. All of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Lithium fluoride, technical at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges, were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, lithium fluoride, technical, is considered non-mutagenic in this bacterial reverse mutation assay.