Lithium fluoride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic potential was observed in a bacterial reverse mutation assay conducted with the lithium fluoride. A mouse lymphoma assay with the source substance LiOH*H2O was also negative.

Based on the several in vitro studies with the two source substances LiOH and NaF, further investigations have to be made with regards to the clastogenic activity of the source substance sodium fluoride.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21st July 1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(30th May 2008)

Deviations:

no

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

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

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 / 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.

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)

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)

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)

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)

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-04-06 to 2000-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 1999

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: 27.05.97
- Expiration date of the lot/batch: 19 November 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; in the dark
- Stability under test conditions: Stability of DMSO solution: at least 96 hrs

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Dose range finding test:
10, 33, 100, 133, 1000 µg/mL with and without S9 mix

Chromosome aberrations:
Without S9 mix: 275, 300, and 530 µg lithium hydroxide/mL culture medium (24 h treatment, 24 h fixation time);
350, 375 and 400 µg lithium hydroxide/mL culture medium (48 h treatment, 48 h fixation time),
With S9 mix: 400, 425 and 450 µg lithium hydroxide/mL culture medium (3 h treatment, 48 h fixation time)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

MMC-C

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

CP

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

Cytogenetic assay:
Lithium hydroxide was tested in the absence and presence of 1.8 % (v/v) S9-fraction in duplicate in two independent experiments.

Experiment 1:
Lymphocyte cultures (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation, respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed in duplicate to selected doses of lithium hydroxide for 3 h in the absence and presence of S9-mix.
After 3 h treatment, the cells exposed to lithium hydroxide were rinsed once with 5 mL of HBSS and incubated in 5 mL of culture medium for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay, appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modification of experimental conditions.

Experiment 2:
Lymphocyte cultures (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation, respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed in duplicate to selected doses of lithium hydroxide for 3 h in the absence and presence of S9-mix.
After 3 h treatment, the cells exposed to lithium hydroxide in the presence of S9-mix were rinsed once with 5 mL of HBSS and incubated in 5 mL of culture medium for another 44-46 h (48 h fixation time).
The cells which were treated for 24 and 48 h in the absence of S9-mix were not rinsed after treatment but were worked up immediately after 24 h and 48 h (24 h and 48 h fixation time).

Chromosome preparation:
During the last 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol:acetic acid fixative (3:1 v/v).

Preparation of slides:
Fixed cells were dropped onto cleaned slides which were immersed for 24 h in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the study identification number and group number. Two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5 % (v/v) Giemsa solution in tap water.
Thereafter the slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring the cytogenetic assay:
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50 % or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations:
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was >= 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 chromosomes were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test, if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics:

X*2 = (N-1)x(ad-bc)*2/(a+b)(c+d)(a+c)(b+d)

where b = the total number of aberrant cells in the control cultures,
d = the total number of non aberrant cells in the control cultures,
n0 = the total number of cells scored in the control cultures,
a = the total number of aberrant cells in treated cultures to be compared with the control,
c = the total number of non aberrant cells in treated cultures to be compared with the control,
n1 = the total number of cells scored in the treated cultures,
N = sum of n0 and n1

If P [ X*2 > (N-)x(ad-bc)*2/(a+b)(c+d)(a+c)(b+d)] (two-tailed) is small (P < 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding test:
Lithium hydroxide precipitated in the culture medium at a concentration of 1000 µg/mL, therefore a concentration of 1000 µg/mL was used as the highest concentration of lithium hydroxide.
In the dose range finding test, blood cultures were treated with 10, 33, 100, 333 and 1000 µg lithium hydroxide per mL culture medium with and without S9 mix.
The pH of a concentration of 1000 mg lithium hydroxide/mL was 11.83 (compared to 8.15 in the solvent control).

Cytogenetic assay:
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Experiment 1A:
Without S9 mix: 100, 180, 333, 420 and 560 µg lithium hydroxide/mL culture medium (3 h treatment time, 24 h fixation time)
With S9 mix: 100, 333, 420 and 560 µg lithium hydroxide/mL culture medium (3h treatment, 24 h fixation time)
Lithium hydroxide precipitated in the culture medium at a concentration of 560 µg/mL, therefore a concentration of 560 µg/mL was used as the highest concentration of lithium hydroxide in the first cytogenetic assay.
Since the highest dose level of 560 µg lithium hydroxide/mL was too cytotoxic to the cells (mitotic index of 21 % both in the absence and in the presence of S9 mix) and no dose level resulting in a mitotic index of 50 % could be selected in both the absence and presence of S9 mix, an additional experiment was performed with the following dose levels:

Experiment 1B:
With and without S9 mix: 300, 350, 400, 450, 500 and 550 µg lithium hydroxide/mL culture medium (3 h treatment, 24 h fixation time)
Because of the high cytotoxicity in cultures treated with 350 µg/mL lithium hydroxide and upwards in the presence and absence of S9 mix, the test was not used for evaluation but a third experiment was performed with the following dose levels: see Experiment C

Experiment 1C:
With and without S9 mix: 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg lithium hydroxide/mL culture medium (3 h treatment, 24 h fixation time).

Despite the narrow concentration range used, the mitotic index of cultures treated with 375 and 400 µg/mL lithium hydroxide (without S9 mix) drastically decreased from 128 % to 0 %. In the presence of S9 mix, cytotoxicity was observed at a concentration of 375 µg/mL lithium hydroxide and upwards.
The pH of the concentrations 275, 300, 325, 350, 375, 400 and 425 µg/mL was 9.61, 9.69, 9.66, 9.68, 9.66, 9.80 and 10.19, respectively. Possibly these high pH values also play a role in the cytotoxicity of lithium hydroxide.

Since it was not possible to determine a concentration which caused the appropriate 50 % inhibition of the mitotic index, the following doses were selected for scoring of chromosome aberrations:
From experiment 1A:
With and without S9 mix: 333, 420 and 560 µg lithium hydroxide/mL (3 h treatment, 24 h fixation time)

From experiment 1C:
Without S9 mix: 325, 350, and 375 µg lithium hydroxide/mL culture medium (3 h treatment time, 24 h fixation time)
With S9 mix: 325, 350, 375, and 400 µg lithium hydroxide/mL culture medium (3 h treatment, 24 h fixation time)
For cultures with S9 mix four doses were selected, since only one of the duplicate cultures contained scorable metaphases at concentrations of 375 and 400 µg/mL lithium hydroxide.

Based on the results of the dose range finding test and experiments 1A, 1B and 1C the following dose levels were selected to perform an independent repeat:

Experiment 2:
Without S9 mix: 275, 300, 325, 350, 375, 400 and 425 µg lithium hydroxide/mL culture medium (24 and 48 h treatment time, 24 and 48 h fixation time);
With S9 mix: 350, 375, 400, 425, 450, 475, 500 and 525 µg lithium hydroxide/mL culture medium (3 h treatment time, 48 h fixation time).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9 mix: 275, 300 and 350 µg lithium hydroxide/mL culture medium (24 h treatment time, 24 h fixation time); 350, 375 and 400 µg lithium hydroxide/mL culture medium (48 h treatment time, 48 h fixation time),
With S9 mix: 400, 425 and 450 µg lithium hydroxide/mL culture medium (3 h treatment time, 48 h fixation time).

Evaluation of the results:
The ability of lithium hydroxide to introduce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in three independent experiments.
The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range [min = 0, max = 5 (mean = 0.8, standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9 mix; gaps excluded and min = 0 max = 5 (mean = 0.8, standard deviation = 0.9) aberrant cells per 100 metaphases in the presence of S9 mix, gaps excluded].
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly.

Experiments 1A and 1C:
Due to the steepness of the dose response curve for cytotoxicity of lithium hydroxide it was not possible to determine the number of chromosomal aberrations at a mitotic index of 50 %. Therefore, chromosome aberrations were scored from two independent experiments (experiment 1A and 1C) at different concentrations. As a result of extreme cytotoxicity, only 102 and 103 metaphases could be scored in the absence and presence of S9 mix, respectively, in experiment 1A at a concentration of 560 µg/mL lithium hydroxide. At the other concentrations tested, 200 metaphases were scored per concentration. In experiment 1C in the presence of S9 mix at the highest concentrations of 375 and 400 µg/mL only one of the two duplicate cultures could be scored due to extreme cytotoxicity.
Both in the absence and presence of S9 mix lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in both experiments 1A and 1C.

Experiment 2:
In the absence of S9 mix, at the 24 hours continuous treatment time, lithium hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 µg/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 µg/mL lithium hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations.
Since the increase of chromosome aberrations at 275 µg/mL was observed only when gaps were included and furthermore the increase was within the historical control data range, it was not considered biologically relevant.

Scoring of the additional 200 metaphases at the concentration of 350 µg/mL lithium hydroxide verified the statistically significant increase. However, the observed increase was within or just on the border of our historical control data range (min = 0, max = 5 aberrant cells per 100 metaphases, gaps excluded), and was observed at a very toxic concentration. In addition, higher concentrations tested at the prolonged treatment time of 48 hours in the absence of metabolic activation did not induce significant increases in the number of cells with chromosome aberrations. Furthermore, the irregular toxicity profile and the non-physiological test conditions (pH > 9) may be considered as confounding factors. Therefore, the observed increase in the number of aberrant cells at the concentration of 350 µg/mL is considered not biologically relevant.

At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 µg/mL lithium hydroxide did not induce a significant increase in the number of cells with chromosome aberrations.

In the presence of S9 mix, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.

Conclusion:
Finally, it is concluded that this test is to be considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test.

Conclusions:

The effect of lithium hydroxide on the induction of chromosome aberrations in culture peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9 mix) was investigated.
It was concluded that this test should be considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test.

Executive summary:

The effect of lithium hydroxide on the induction of chromosome aberrations in culture peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9 mix) was investigated according to OECD Guideline 473 and EU method B.10. In the absence of S9 mix lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 375 µg/mL in experiment 1C. In the second experiment lithium hydroxide was tested up to 350 µg/mL for a 24 hours continuous treatment time and up to 400 µg/mL for a 48 hours continuous treatment time. 

In the presence of 1.8 % (v/v) S9-fraction lithium hydroxide was tested up to 560 μg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 400 µg/mL in experiment 1C. In the second experiment lithium hydroxide was tested up to 450 μg/mL for a 3 h treatment time with a 48 h fixation time. 

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. 

Experiment 1A and 1C:

Both in the absence and presence of S9 mix lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in both experiments 1A and 1C. 

Experiment 2:

In the absence of S9 mix, at the 24 hours continuous treatment time, lithium hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 μg/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 µg/mL lithium hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations.

Since the increase of chromosome aberrations at 275 µg/mL was observed only when gaps were included and furthermore the increase was within the historical control data range and revealed no dose-response-relationship, it was not considered biologically relevant. 

Scoring of the additional 200 metaphases at the concentration of 350 µg/mL lithium hydroxide verified the statistically significant increase. However, the observed increase was within or just on the border of the historical control data range (min = 0, max = 5 aberrant cells per 100 metaphases, gaps excluded), and was observed at a very toxic concentration. In addition, higher concentrations tested at the prolonged treatment time of 48 hours in the absence of metabolic activation did not induce significant increases in the number of cells with chromosome aberrations. Furthermore, the irregular toxicity profile and the non-physiological test conditions (pH > 9) may be considered as confounding factors. Therefore, the observed increase in the number of aberrant cells at the concentration of 350 µg/mL is considered not biologically relevant.

At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 µg/mL lithium hydroxide did not induce a significant increase in the number of cells with chromosome aberrations. 

In the presence of S9 mix, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations. 

It was concluded that this test is considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test. (Notox, 2000)

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-20 to 2010-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The indicator cell used for this study was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay is isolated by Dr. Donald Clive (Burroughs Wellcome Company, Research Triangle Park, NC).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

12.5, 25, 50 100 and 200 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua ad iniectabilia

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

positive control for non-activation mutation studies.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

positive control for assays performed with S9 metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

ASSAY WITHOUT METABOLIC ACTIVATION
The cells for the experiments were obtained from logarithmically growing laboratory stock cultures and were seeded into a series of tubes at 1 x 107 cells per tube. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were resuspended in a final volume of 20.0 mL of treatment medium that contained 5 % heat inactivated fetal bovine serum. The dosed tubes were closed, vortexed and placed on a roller drum at approx. 37 °C at 10 - 15 rpm for an exposure period of 3 hours. The cells were washed and resuspended in growth medium.
Cell densities were adjusted to 2 x 105/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (192 wells, averaging 1.6 cells/well) and incubated for 1 week at 37 ± 0.4 °C whereas the rest of the cells was incubated for 2 days at 37 ± 0.4 °C for the expression period.
The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells in the expression period were maintained below 106 cells per mL and a minimum of 4 concentration levels plus positive and negative control was selected for 5-trifluoro-thymidine (TFT) resistance.
At the end of the expression period, the selected cultures were diluted to 1 x 104 cells/mL and plated for survival and TFT resistance in parallel (plating efficiency step 2). The plating for survival was similar to the above described method. For the plating for TFT resistance, 3 μg/mL TFT (final concentration) were added to the cultures and 0.2 mL of each suspension was placed into four 96-well microtiter plates (384 wells, averaging 2 x 103 cells/well). The plates were incubated for 12 days at 37 ± 0.4 °C and wells containing clones were identified microscopically and counted.
In addition, the number of large and small colonies was recorded with an automated colony counter that can detect colony diameters equal or greater than 0.2 to 0.3 mm. Large colonies are defined as >= 1/3 and small colonies < 1/3 of the well diameter of 6 mm.

ASSAY WITH METABOLIC ACTIVATION
The activation assay is often run concurrently with the non-activation assay; however, it was an independent assay performed with its own set of solvent and positive controls. In this assay, the above-described activation system was added to the cells together with test item.

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment was a mutant frequency that was >= 2 times the concurrent background mutant frequency. The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations was not sufficient evidence to evaluate the test item as a mutagen.
A concentration-related or toxicity-related increase in mutant frequency should be observed.
The ratio of small : large colonies will be calculated from the results of the determination of small to large colonies.
If the test item is positive, the ratio of small to large colonies for the test item will be compared with the corresponding ratios of the positive and negative controls. Based on this comparison, the type of the mutagenic properties (i.e. basepair substitutions, deletions or large genetic changes frequently visible as chromosomal aberrations) of the test item will be discussed.
A test item is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or a range of applied concentrations extending to at least twice the solubility limit in culture media.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200 µg/mL.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50 % compared with the negative control. Exposure to the test item at the concentration of 200 µg/mL in the absence of metabolic activation resulted in relative survival of 28 % and 34 % (plating efficiency step 1) and 20 % and 33 % (plating efficiency step 2), and in the presence of metabolic activation of 28 % and 26 % (plating efficiency step 1) and 23 % and 17 % (plating efficiency step 2). Therefore, the test item was considered cytotoxic at the top concentration of 200 µg/mL.

No relevant change in pH and osmolality was noted.

Conclusions:

Under the present test conditions, lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration range investigated.
According to the evaluation criteria for this assay, these findings indicate that lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK +/- cells to test the potential of lithium hydroxide to cause gene mutation and/or chromosome damage according to OECD Guideline 476 and the EU method B.17. Lithium hydroxide monohydrate was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out twice. The test item was dissolved in aqua ad iniectabilia. A correction factor of 1.73 was used. The dose levels and concentrations given in the text and tables refer to lithium hydroxide monohydrate. The limit of solubility was about 34 mg/mL. In the preliminary experiment without and with metabolic activation, concentrations tested were 0.25, 1, 2.5, 10, 25, 100 and 200 µg/mL. Cytotoxicity (decreased survival) was noted at the top concentration of 200 μg/mL. Hence, in the experiments without or with metabolic activation the concentrations of 12.5, 25, 50 100 and 200 µg/mL were used. In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200 μg/mL. Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3 -methylcholanthrene in the presence of exogenous metabolic activation. The mean values of mutation frequencies of the negative controls ranged from 61.61 to 98.34 per 106 clonable cells in the experiments without metabolic activation, and from 68.23 to 82.61 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data range. The mutation frequencies of the cultures treated with lithium hydroxide monohydrate ranged from 64.74 to 92.63 per 106 clonable cells (3 hours exposure) and 50.42 to 92.34 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and 75.88 to 105.59 per 106 clonable cells (3 hours exposure, first assay) and 45.04 to 99.10 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Under the present test conditions, lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration range investigated. According to the evaluation criteria for this assay, these findings indicate that lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Based on a read-across approach, results of this study are applied to lithium fluoride and it can therefore be concluded that lithium fluoride is negative with regard to mutant frequency in the absence and presence of metabolic activation. (LPT, 2010)

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(Only two analysable concentrations were tested. Only single cultures were used for each experimental endpoint)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human blood lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium (supplemented with 15 % fetal calf serum, 1 % penicillin & streptomycin and 1 % phytohaemagglutinin)
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor-induced rat liver)

Test concentrations with justification for top dose:

0, 20, 40 µg/mL with and without metabolic activation for short-term (2 hours) and continuous treatment (28 hours)

Vehicle / solvent:

- Vehicle/solvent used: medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

CP

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

MMS

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 hours; 24 hours
- Fixation time: 72 hours after culture initiation

SPINDLE INHIBITOR (cytogenetic assays): 0.1 mL of vinblastine sulphate (100 µg/mL)
STAIN (for cytogenetic assays): 4 % Giemsa

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of endoreplication: Yes

Statistics:

The Fisher exact test for 2 x 2 tables and 2-sided significance levels was used to compare the incidence of chromosome aberrations between the two blood donors within each group. Further the data was pooled within each group before comparison with the vehicle control (Fisher's
exact test for 2-sided tables and 1-sided significance level).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

, the incidence of chromosome aberrations in cultures exposed to NaF was only significantly increased after a 28-hour exposure to 20 and 40 ug/mL without S9, and after a 2-hour exposure to 40 µg/mL with S9.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No confounding factors were observed during the test.

COMPARISON WITH HISTORICAL CONTROL DATA:
The percentage of abberrations observed exceeded the historical control data (0 - 1.5 % for gaps, 0 - 0.5 % for breaks, 0 - 1 % for fragments)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The mitotic index of treated cells was comparable to the controls.

- The types of aberrations observed were predominantly gaps, breaks and fragments, there were no exchange-type aberrations

- The incidence of aberrations in the sodium fluoride-exposed cells were outside the historical control range for this laboratory (i.e., 0 -1.5 % for gaps, 0 -0.5% for breaks and 0 -1% for fragments)

-The positive control substances, methyl methane sulfonate and cyclophosphamide, induced statistically significant increases in the incidence of chromosome aberrations, indicating the responsiveness of the test system and metabolic competence of the S9 mix.

Conclusions:

Sodium fluoride, at concentrations of 20 and 40 µg/mL without metabolic activation, induced a statistically significant increase (3 and 9 %, respectively) in the incidence of chromosomal aberrations in human blood lymphocyte cultures when compared to the vehicle control cultures (0.5 %).

Executive summary:

An in vitro chromosome aberration test was performed similarly to OECD guideline 473 in human blood lymphocytes, with sodium fluoride at concentrations of 0, 20, 40 µg/mL with and without metabolic activation. Sodium fluoride was tested up to cytotoxic concentrations. A maximum mutation frequency of 0.5 % was observed in the control cultures. The incidence of chromosome aberrations in treatment cultures was low but significantly increased after a 28-hour exposure to 20 and 40 µg/mL without S9 mix (3 and 9 %, respectively). After a 2-hour exposure to 40 µg/mL with S9 a significant mutation frequency of 2.5 % was observed. Induced abberrations were mainly gap- and break-like. No exchange-type of aberrations were observed. Although the frequency of aberrations is rather low, a mutagenic effect cannot be totally excluded.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: rat/Sprague-Dawley bone marrow cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Medium consisted of Dulbecco's modification of Eagle's medium buffered at pH7.2 with 20 mM HEPES buffer supplemented with 2 0% heat-inactivated fetal bovine serum, 10% uterine extract, 100 IU/mL penicillin, and 100 ug/mL streptomycine.
- Properly maintained: yes

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 0.1, 1, 10 and 100 µM of sodium fluoride.

Untreated negative controls:

yes

Remarks:

, sodium chloride, used at a concentration of 1000 uM

Negative solvent / vehicle controls:

yes

Remarks:

deionized distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 1342 N-methyl-N'-nitrosoguanidine at a concentration of 1 uM

Key result

Species / strain:

primary culture, other: rat bone marrow cells (Sprague-Dawley)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

with one exception (0.1 µM sodium fluoride at 12 hours), all sodium fluoride treatments induced statistically significant increases in the percentage of aberrant cells and average breaks/cell as compared to the deionized distilled water controls.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

other: the negative control substance, sodium chloride at 1000 uM, caused cytoxicity so no metaphases were available for analysis.

Positive controls validity:

valid

Conclusions:

Sodium fluoride, at concentrations of 0.1, 1, 10 or 100 uM, induced statistically significant increases in chromosome aberrations in rat bone marrow cells at different exposure times (12, 24 and 36 hours) when compared to control cells exposed to deionized distilled water; an exception was no significant increase in aberrant cells treated with 0.1 µM sodium fluoride for 12 hours.

Executive summary:

An in vitro chromosome aberration test was performed similarly to OECD Guideline 473, in rat/Sprague-Dawley bone marrow cells with sodium fluoride in concentrations of 0.1, 1, 10, 100 µM without metabolic activation. Results showed that sodium fluoride, at concentrations of 0.1, 1, 10 or 100 µM, induced statistically significant increases in chromosome aberrations in rat bone marrow cells at different exposure times (12, 24 and 36 hours) when compared to control cells exposed to deionized distilled water; an exception was no significant increase in aberrant cells treated with 0.1 µM sodium fluoride for 12 hours.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(Only two analysable concentrations without metabolic activation were tested.)

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human foreskin fibroblasts (JHU-1 cells)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum in a humidified atmosphere with 5 % CO2 in air at 37 °C.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

Cytotoxicity testing: 50, 100, 150 µg/mL
12-h exposure: 0, 25, 50, 75 µg sodium fluoride/mL
24-hour exposure: 20, 40 µg sodium fluoride/mL

Vehicle / solvent:

- Vehicle used: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

no

Positive control substance:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 12 and 24 hours
- Expression time (cells in growth medium): overnight
- Fixation time: treatment with 0.8 % sodium citrate at room temperature for 20 min and fixed in Carnoys solution (methanol/acetic acid 3:1)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.05 µg/mL) added 3 hours before end of treatment time
STAIN (for cytogenetic assays): with Giemsa (3 % in 0.07 M phosphate buffer)

NUMBER OF REPLICATIONS: Not given.

NUMBER OF CELLS EVALUATED: 112 - 500 metaphases (12 h treatment); 100 metaphases (24 h treatment)

DETERMINATION OF CYTOTOXICITY
- Method: other: colony forming ability
200 to 500 cells were plated on petri dishes and incubated overnight. After incubation cells were treated with NaF at concentrations of 50, 100 and 150 µg/mL for 1, 2, 6, 12 or 24 hours. After treatment cells were washed twice with fresh medium and incubated for an additional period of 7 days to form colonies. After staining with Giemsa solution the number of colonies was counted and the % cell survival was determined.

Evaluation criteria:

No details given.

Key result

Species / strain:

other: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No confounding factors were observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A linear decrease of cell survival was observed with increasing concentration of NaF or exposure time. After 24 hour treatment with 50 µg/mL a cell survival of about 30 % was observed. Similar cell survival was also seen at 100 µg/mL for 12 hours.

Conclusions:

Sodium fluoride did show a statistically significant increase of chromosome aberrations when treated with sodium fluoride at concentrations of 25, 50 and 75 µg/mL (Tsutsui et al. 1984).

Executive summary:

In a mammalian cell gene mutation assay, human foreskin fibroblasts (JHU-1 cells) cultured in vitro were exposed to sodium fluoride at concentrations of 25, 50 and 75 µg/mL for 12 hours. 20 and 40 µg/mL were examined for 24 hours. An additional test for cytotoxicity was conducted at concentrations of 50, 100 and 150 µg/mL. All three treatments were done without metabolic activation. Sister chromatide exchanges and chromosome aberrations were examined.

A linear decrease of cell survival was observed with increasing concentration of NaF or exposure time. After 24 hour treatment with 50 µg/mL a cell survival of about 30 % was observed. Similar cell survival was also seen at 100 µg/mL for 12 hours.

In the negative control group the percentage of aberrant metaphases was 0.8. At an exposure time of 12 hours significant increase was observed at 50 µg/mL with aberrant metaphases of 17.9 %. 75 µg/mL could not be analysed due to the high cytotoxicity observed at that concentration and exposure time. At the 24 hour treatment only 1 % of the negative control metaphases showed aberrations. 7 % aberrant metaphases were already observed at 20 µg/mL. Nearly half of the examined metaphases showed chromosome aberrations at the highest tested concentration (40 µg/mL).

The above results indicate a concentration related positive response of induced mutant colonies over background. The substance was therefore regarded as positive by the authors. But cytotoxicity was pretty high and only two doses were able to be scored for both exposure times.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The available in vivo studies with the source substance sodium fluoride show no mutagenic potential with regards to chromosome aberrations in humans, rats and mice. It is therefore concluded that the target substance is not mutagenic in vivo as well.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Only two dose levels and one treatment were performed.

GLP compliance:

not specified

Type of assay:

other: mammalian micronucleus test

Species:

rat

Strain:

Wistar

Remarks:

Alpk:APfSD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 90 - 140 g
- Assigned to test groups randomly: yes
- Fasting period before study: at least 7 days
- Housing: groupwise
- Diet: ad libitum, pelleted NDD diet (Special Diet Services)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

Route of administration:

oral: drinking water

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Concentration of test material in vehicle: 500 and 1000 mg/kg
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5/group/sample time

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP)
- Justification for choice of positive control(s): Substance known to cause chromosome aberrations in bone marrow and micronucleated peripheral blood erythrocytes.
- Route of administration: gavage

Tissues and cell types examined:

Bone marrow and peripheral blood

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Slides were prepared of bone marrow cells using the paint brush technique.

METHOD OF ANALYSIS:
The number of normochromatic erythrocytes in 1000 polychromatic erythrocytes (PE) and the incidence of micronucleated polychromatic erythrocytes (MPE) in 2000 PE was assessed on each slide.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

4 out of 5 rats of the high dose group were found dead prior to 48 h sampling.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Table 1 Results

Sample time (hours after dosing)	Treatment and dose	MPE/1000 PE (based on 2000 PE assessed per animal)
		Individual animal results	Mean ± SD
24	Distilled water 10 mL/kg (vehicle control	3, 1, 0, 1, 1	1.2 ± 1.0
	NaF 1000 mg/kg	1, 1, 1, 0, 1	0.8 ± 0.4
	NaF 500 mg/kg	1, 0, 2, 1, 1	1.0 ± 0.7
	Cyclophosphamide 20 mg/kg	19, 11, 20, 32, 25	21.4 ± 7.7
48	Distilled water 10 mL/kg (vehicle control	1, 1, 1, 1, 0	0.8 ± 0.4
	NaF 1000 mg/kg	1 (a)	-
	NaF 500 mg/kg	2, 0, 0, 2, 2	1.2 ± 1.0
	Cyclophosphamide 20 mg/kg	9, 15, 13, 8, 8	10.6 ± 3.2

(a) 4 out of 5 dosed animals were found dead before 48 hour sampling

Conclusions:

No significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow was observed after a single oral treatment of male rats with 500 and 1000 mg/kg bw.

Executive summary:

In a Wistar rat bone marrow micronucleus assay equivalent to OECD guideline 474, 5 male rats /group/sampling time were treated orally with sodium fluoride at doses of 0, 500 and 1000 mg/kg bw (Albanese, 1987). Bone marrow cells were harvested at 24 and 48 hours post treatment. The vehicle was distilled water. The test substance was applied by gavage as a single dose.

4 out of 5 animals died before the second harvest point (48 hours). No signs of abnormal behaviour were observed in the remaining animals. As the ratio between normochromatic and polychromatic erythrocytes was nearly 1:1 in the control and treated animals, cytotoxicity for sodium fluoride was excluded. There was no a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

On two occasions less than 200 metaphases were examined.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

yes

Remarks:

On two occasions less than 200 metaphases were examined. Colchecin treatment was not performed.

GLP compliance:

not specified

Type of assay:

other: chromosome aberration

Species:

mouse

Strain:

other: Swiss Webster and male BALB/c

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Swiss Webster: 10 - 12 weeks; BALB/c: 10 weeks
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: drinking water

Vehicle:

- Vehicle used: water

Duration of treatment / exposure:

-Swiss Webster mice, 10-12 weeks of age, from colonies maintained on a low fluoride (0.5 ppm F) diet for 2 years plus 50 ppm fluoride applied via drinking water
- BALB/c male mice were fed a low fluoride (0.5 ppm) diet for six weeks plus application of 1, 5, 10, 50 and 100 ppm fluoride in drinking water

Frequency of treatment:

continuous treatment via drinking water

Dose / conc.:

1 ppm (nominal)

Remarks:

in water (BALB/c mice)

Dose / conc.:

5 ppm (nominal)

Remarks:

in water (BALB/c mice)

Dose / conc.:

10 ppm (nominal)

Remarks:

in water (BALB/c mice)

Dose / conc.:

50 ppm (nominal)

Remarks:

in water (Swiss Webster mice; BALB/c mice)

Dose / conc.:

100 ppm (nominal)

Remarks:

in water (BALB/c mice)

No. of animals per sex per dose:

For Swiss Webster mice: 9 animals per control dose and 6 animals per 50 ppm dose.
For BALB/c mice: 5 animals per negative control; 4 animals per positive control; 10 animals per 1, 5, 10 and 100 ppm NaF doses, and 5 animals per 50 ppm NaF dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine was injected in the positive control BALB/c mice (1 mg/kg) one day before sacrifice.

Tissues and cell types examined:

Bone marrow cells and testicular cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Fixed suspensions of bone marrow and testes were made. From each suspension of bone marrow and testis, 4 slides were prepared and stained.

Statistics:

For each animal the percentage of mitotic cells in bone marrow containing aberrations and the percentage of examined testis cells with aberrations was calculated. Subsequently, mean and standard error of the percentage for each group of animals and for each type of tissue were calculated. The mean and standard error of the aberration rate for each tissue of each group were related to their bone-ash fluoride percent by plotting. The regression on the fluoride dose in drinking water and on the mean fluoride content in bone were computed using both linear and logarithmic models with the 6 week and life-time groups considered separately and together. Comparison of the means of different groups was done by using the t-statistic.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Swiss Webster mice

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Lifetime treatment

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

BALB/c mice

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: 6-week treatment

Conclusions:

Under the study conditions, no chromosomal aberrations were detected in bone marrow and testicular cells of male Swiss Webster and Balb/c mice treated with sodium fluoride.

Executive summary:

In the available publication two different cell types (bone marrow and testicular cells) were analysed for chromosome aberrations (CA) equivalent or similar to OECD guideline 475 and 483 (Martin et al., 1979). Two testing regimens were applied. Male Swiss Webster mice were taken from colonies that have been treatment over a period of 2 years. These animals were maintained under a low fluoride diet (0.5 ppm) and treatment groups additionally received 50 ppm of fluoride in drinking water. Control animals only received distilled water. 6 to 9 animals were examined per concentration group.

Male BALB/c mice received several concentrations of sodium fluoride via drinking water while being held on a low fluoride diet (0.5 ppm) for a period of 6 weeks. Animals received 0, 1, 5, 10, 50 and 100 ppm of sodium fluoride in drinking water. 4 to 10 animals were used per treatment group.

The animals were sacrificed and fixed cell suspensions of both cell types were prepared. A minimum of 50 chromosome spreads (per cell type) were analysed for each animal. The number of cells including CAs as well as the type of CA was documented.

No animal died due to treatment under both testing regimens. The treatment with 50 ppm sodium fluoride over a period of 2 years led to less CAs compared to the control group. This was observed in both bone marrow and testis cells but was not considered as statistically significant or biologically relevant.

The chromosome spreads of the bone marrow cells in the 6-week consumption experiment showed similar CA rates compared to the examined control cells. With regards to the testis cells the result show some heterogeneity in aberration rate due to one animal with an increased number of aberrations at a dose of 10 ppm compared to the other tested animals. No dose response could be observed and the other animals of the same dose group did also not show an increased aberration rate. Therefore the effect was not regarded as treatment related or biologically relevant. Positive controls induced the appropriate response on bone marrow cells.

Based on the above results there was no evidence or a concentration related positive response of chromosome aberrations induced over background.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro testing

Bacterial reverse mutation assay (Ames test)

Lithium fluoride was tested in the bacterial reverse mutation assay according to OECD guideline 471 (Toxicoop, 2015). 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 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 is considered non-mutagenic in this bacterial reverse mutation assay.

No other data on mutagenicity are available for lithium fluoride. Consequently, read-across was applied using study results obtained from sodium fluoride and lithium hydroxide as they are characteristically similar compounds to lithium fluoride or at least covers the toxicity potential of both fluoride and lithium ion.

Chromosome Aberration assays

The effect of lithium hydroxide on the induction of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated according to OECD Guideline 473 and EU method B.10 (Notox, 2000).

In the absence of S9-mix lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 375 µg/mL in experiment 1C. In the second experiment lithium hydroxide was tested up to 350 µg/mL for a 24 hours continuous treatment time and up to 400 µg/mL for a 48 hours continuous treatment time. 

In the presence of 1.8 % (v/v) S9-fraction lithium hydroxide was tested up to 560 μg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 400 µg/mL in experiment 1C. In the second experiment lithium hydroxide was tested up to 450 μg/mL for a 3 h treatment time with a 48 h fixation time. 

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. 

Experiment 1A and 1C:

Both in the absence and presence of S9-mix lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in both experiments 1A and 1C. 

Experiment 2:

In the absence of S9-mix, at the 24 hours continuous treatment time, lithium hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 μg/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 µg/mL lithium hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations.

Since the increase of chromosome aberrations at 275 µg/mL was observed only when gaps were included and furthermore the increase was within the historical control data range and revealed no dose response-relationship, the increase was not considered biologically relevant. 

Scoring of the additional 200 metaphases at the concentration of 350 µg/mL lithium hydroxide verified the statistically significant increase. However, the observed increase within or just on the border of the historical control data range (min = 0, max = 5 aberrant cells per 100 metaphases, gaps excluded), and is observed at a very toxic concentration. In addition, higher concentrations tested at the prolonged treatment time of 48 hours in the absence of metabolic activation did not induce significant increases in the number of cells with chromosome aberrations. Furthermore, the irregular toxicity profile and the non-physiological test conditions (pH > 9) may be considered as confounding factors. Therefore, the observed increase in the number of aberrant cells at the concentration of 350 µg/mL is considered not biologically relevant.

At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 µg/mL lithium hydroxide did not induce a significant increase in the number of cells with chromosome aberrations. 

In the presence of S9 mix, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations. 

Finally, it is concluded that this test is considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test.

Supporting literature regarding chromosome aberration assays

In a chromosome aberration assay, human peripheral blood lymphocytes cultured in vitro were exposed to sodium fluoride at concentrations of 0.0001, 0.0003, 0.001, 0.003, 0.01 M equivalent to 4, 13, 42, 126, 420 µg/mL without metabolic activation (Thomson et al, 1985). Sister chromatide exchanges and chromosome aberrations were examined. Sodium fluoride was tested at concentrations up to 60 times the level normally used in drinking water for prevention of tooth decay.

The highest concentration of sodium fluoride lead to pulverised nuclei in all three different lymphocyte cultures. The positive control induced the appropriate response. There was no evidence or a concentration related positive response or increase of chromosome aberrations or sister chromatide exchanges over background.

In a chromosome aberration assay, human foreskin fibroblasts (JHU-1 cells) cultured in vitro were exposed to sodium fluoride at concentrations of 0, 1, 2, 4, 8, 10 µg/mL without metabolic activation (Tsutsui et al., 1995). The cells were exposed to the low concentrations for prolonged time periods of 1, 2 and 3 weeks with medium renewal twice weekly. Positive controls induced the appropriate response with aberrant metaphases of 15.5 % at the highest tested concentration and a concentration related positive response. No significant differences were observed between control and treated cells with regard to decrease of cells (mitotic index). The percentage of aberrant metaphases ranged from 0 - 1.3 % in the treated cells. 0.3 % aberrant metaphases were observed on the control. Conclusively, there was no evidence or a concentration related positive response of induced mutant colonies over background.

An in vitro chromosome aberration test was performed similarly to OECD guideline 473 in human blood lymphocytes, with sodium fluoride at concentrations of 0, 20, 40 µg/mL with and without metabolic activation (Albanese et al. 1987). Sodium fluoride was tested up to cytotoxic concentrations. A maximum mutation frequency of 0.5 % was observed in the control cultures. The incidence of chromosome aberrations in treatment cultures was low but significantly increased after a 28-hour exposure to 20 and 40 µg/mL without S9 mix (3 and 9 %, respectively). After a 2-hour exposure to 40 µg/mL with S9 a significant mutation frequency of 2.5 % was observed. Induced aberrations were mainly gap- and break-like. No exchange-types of aberrations were observed. Although the frequency of aberrations is rather low, a mutagenic effect cannot be totally excluded.

In a chromosome aberration assay, human foreskin fibroblasts (JHU-1 cells) cultured in vitro were exposed to sodium fluoride at concentrations of 25, 50 and 75 µg/mL for 12 hours (Tsutsui et al., 1984). 20 and 40 µg/mL were examined for 24 hours. An additional test for cytotoxicity was conducted at concentrations of 50, 100 and 150 µg/mL. All three treatments were done without metabolic activation. Sister chromatide exchanges and chromosome aberrations were examined. A linear decrease of cell survival was observed with increasing concentration of NaF or exposure time. After 24 hour treatment with 50 µg/mL a cell survival of about 30 % was observed. Similar cell survival was also seen at 100 µg/mL for 12 hours. In the negative control group the percentage of aberrant metaphases was 0.8. At an exposure time of 12 hours significant increase was observed at 50 µg/mL with aberrant metaphases of 17.9 %. 75 µg/mL could not be analysed due to the high cytotoxicity observed at that concentration and exposure time. At the 24 hour treatment only 1 % of the negative control metaphases showed aberrations. 7 % aberrant metaphases were already observed at 20 µg/mL. Nearly half of the examined metaphases showed chromosome aberrations at the highest tested concentration (40 µg/mL). The above results indicate a concentration related positive response of induced mutant colonies over background. The substance was therefore regarded as positive by the authors. But cytotoxicity was pretty high and only two doses were able to be scored for both exposure times.

An in vitro chromosome aberration test was performed similarly to OECD Guideline 473, in rat/Sprague-Dawley bone marrow cells with sodium fluoride in concentrations of 0.1, 1, 10, 100 µM without metabolic activation (Khalil, 1995). Results showed that sodium fluoride, at concentrations of 0.1, 1, 10 or 100 µM, induced statistically significant increases in chromosome aberrations in rat bone marrow cells at different exposure times (12, 24 and 36 hours) when compared to control cells exposed to deionized distilled water; an exception was no significant increase in aberrant cells treated with 0.1 µM sodium fluoride for 12 hours.

Mammalian cell gene mutation assays

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK +/- cells to test the potential of lithium hydroxide to cause gene mutation and/or chromosome damage according to OECD Guideline 476 and the EU method B.17 (LPT, 2010). Lithium hydroxide monohydrate was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out twice. The test item was dissolved in aqua ad iniectabilia. A correction factor of 1.73 was used. The dose-levels and concentrations given in the text and tables refer to lithium hydroxide monohydrate. The limit of solubility was about 34 mg/mL. In the preliminary experiment without and with metabolic activation, concentrations tested were 0.25, 1, 2.5, 10, 25, 100 and 200 µg/mL. Cytotoxicity (decreased survival) was noted at the top concentration of 200 μg/mL. Hence, in the experiments without or with metabolic activation the concentrations of 12.5, 25, 50 100 and 200 µg/mL were used. In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200μg/mL. Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3 -methylcholanthrene in the presence of exogenous metabolic activation. The mean values of mutation frequencies of the negative controls ranged from 61.61 to 98.34 per 106 clonable cells in the experiments without metabolic activation, and from 68.23 to 82.61 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data range. The mutation frequencies of the cultures treated with lithium hydroxide monohydrate ranged from 64.74 to 92.63 per 10^6 clonable cells (3 hours exposure) and 50.42 to 92.34 per 10^6 clonable cells (24 hours exposure) in the experiments without metabolic activation and 75.88 to 105.59 per 10^6 clonable cells (3 hours exposure, first assay) and 45.04 to 99.10 per 10^6 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Under the present test conditions, lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration range investigated. According to the evaluation criteria for this assay, these findings indicate that lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Literature data regarding mammlian cell gene mutations

An in vitro mammalian cell assay was performed in Chinese hamster lung fibroblasts (V79) and human EUE fibroblasts to test the potential of sodium fluoride to cause gene mutation and/or chromosome damage (Slamenova et al. 1992). The test substance was applied in concentrations of 10, 50, 100, 150, 200, 250, 300, 350 or 400 µg NaF/mL and tested without metabolic activation. Concentrations of 10-150 µg NaF/mL in culture medium induced 10-75 % cytotoxicity in V79 cells. Cytotoxicity in EUE cells was observed at concentrations of 200 µg NaF/mL and above. Sodium fluoride did not show any mutagenic effects after 24 hours exposure of Chinese hamster V79 cells and after 24 and 48 hours exposure of EUE cells at the concentrations indicated above.

Conclusions for in vitro testing

Test data with lithium hydroxide did not show any treatment related effects either for chromosome aberrations or mammalian cell gene mutagenicity. Sodium fluoride did not show any effects in the mammalian cell gene mutation test either.

As can be seen above, the five different chromosome aberration tests in vitro partially indicated a mutagenic effect for sodium fluoride. Therefore three in vivo chromosome aberration assays (in humans, rats and mice) were also included in the chemical safety assessment of lithium fluoride.

In vivo testing

Three in vivo studies with humans, rats and mice are available in order to assess the induction of CAs in comparison to the results of the chromosome aberration studies of sodium fluoride in vitro.

In a Wistar rat bone marrow micronucleus assay equivalent to OECD guideline 474, 5 male rats/group/sampling time were treated orally with sodium fluoride at doses of 0, 500 and 1000 mg/kg bw (Albanese, 1987). Bone marrow cells were harvested at 24 and 48 hours post treatment. The vehicle was distilled water. The test substance was applied by gavage as a single dose.

4 out of 5 animals died before the second harvest point (48 hours). No signs of abnormal behaviour were observed in the remaining animals. As the ratio between normochromatic and polychromatic erythrocytes was nearly 1:1 in the control and treated animals, cytotoxicity for sodium fluoride was excluded. There was no a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

In the second available publication two different cell types (bone marrow and testicular cells) were analysed for chromosome aberrations (CA) equivalent or similar to OECD guideline 475 and 483 (Martin et al., 1979). Two testing regimens were applied. Male Swiss Webster mice were taken from colonies that have been treatment over a period of 2 years. These animals were maintained under a low fluoride diet (0.5 ppm) and treatment groups additionally received 50 ppm of fluoride in drinking water. Control animals only received distilled water. 6 to 9 animals were examined per concentration group.

Male BALB/c mice received several concentrations of sodium fluoride via drinking water while being held on a low fluoride diet (0.5 ppm) for a period of 6 weeks. Animals received 0, 1, 5, 10, 50 and 100 ppm of sodium fluoride in drinking water. 4 to 10 animals were used per treatment group.

The animals were sacrificed and fixed cell suspensions of both cell types were prepared. A minimum of 50 chromosome spreads (per cell type) were analysed for each animal. The number of cells including CAs as well as the type of CA was documented.

No animal died due to treatment under both testing regimens. The treatment with 50 ppm sodium fluoride over a period of 2 years led to less CAs compared to the control group. This was observed in both bone marrow and testis cells but was not considered as statistically significant or biologically relevant.

The chromosome spreads of the bone marrow cells in the 6-week consumption experiment showed similar CA rates compared to the examined control cells. With regards to the testis cells the result show some heterogeneity in aberration rate due to one animal with an increased number of aberrations at a dose of 10 ppm compared to the other tested animals. No dose response could be observed and the other animals of the same dose group did also not show an increased aberration rate. Therefore the effect was not regarded as treatment related or biologically relevant. Positive controls induced the appropriate response on bone marrow cells.

Based on the above results there was no evidence or a concentration related positive response of chromosome aberrations induced over background.

In the publication of van Asten et. al. (1998) the lymphocytes of 14 female human volunteers were investigated. 7 volunteers were included in the test group and exposed to fluoride containing formulations (i.e. sodium fluoride and disodium monofluorophosphate) for a period of 15 months up to 49 months. The peripheral blood lymphocytes of these patients were stimulated to divide in vitro. Biological endpoints included the frequencies of chromosomal aberrations and micronuclei in binucleated cells. 7 female volunteers of a comparable age range served as control group and remained untreated.

Chromosome aberrations as well as micronuclei were not induced in patients treated with fluoride and were comparable with the results of the untreated control group. Thus, fluoride ions did not show any clastogenic potential in vivo in humans.

Conclusions for in vivo testing

The above study results lead to the conclusion that a clastogenic potential of sodium fluoride can be excluded in vivo.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the target substance is not considered to be classified and labelled as mutagenic under Regulation (EC) No 1272/2008.