Magnesium fluoride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): non-mutagenic with or without metabolic activation

In vivo cytogenicity assay (OECD 473): negative with or without metabolic activation

In vitro gene mutation assay (OECD 476): negative with or without metabolic activation

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:

10 January 2017 - 21 February 2017

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:

In vitro gene mutation study in bacteria

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 662599-1
- Expiration date of the lot/batch: 31 July 2017
- Purity test date: 18 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature, protected from moisture
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Test item is poorly soluble in water according to the literature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Because of the poor solubility of the test item, a preliminary experiment was conducted to determine the insolubility. Three solvent (deionised water, dimethyl sulfoxide and ethanol) were tested. The test item did not show cytotoxicity when used any of the test solvents. DMSO was selcted as a solvent for the actual test. To increase the solubility the grinding in glass mortar and 3 hour- period of solubilisation were applied. The same procedure was applied in experiments when deionised water was used as vehicle.

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: .

Remarks:

The Salmonella strains used in the study are histidine-dependent. The strain has rfa mutation which changes the properties of the bacterial cell wall, increasing the permeability of cells to certain types of chemicals.

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

other:

Remarks:

The Salmonella strains used in the study are histidine-dependent. The strain has rfa mutation which changes the properties of the bacterial cell wall, increasing the permeability of cells to certain types of chemicals.

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other:

Remarks:

The Salmonella strains used in the study are histidine-dependent. The strain has rfa mutation which changes the properties of the bacterial cell wall, increasing the permeability of cells to certain types of chemicals.

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other:

Remarks:

The Salmonella strains used in the study are histidine-dependent. The strain has rfa mutation which changes the properties of the bacterial cell wall, increasing the permeability of cells to certain types of chemicals.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other:

Remarks:

The E.coli WP2 uvrA strain is tryptophan-dependent. Strain has a DNA deletion that affects repair of ultraviolet light damage which greatly increases sensitivity in detecting mutagens.

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

concentrations studied 50, 150, 500, 1500 and 5000 mg/plate.

Vehicle / solvent:

DMSO. In the range finding study also deionised water and ethanol were used as solvents.

Untreated negative controls:

yes

Remarks:

DMSO/sterile water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO/sterile water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene - with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): app. 10^9 cells per mL

DURATION
- Preincubation period: 20 min (without metabolic activation)
- Selection time (if incubation with a selection agent): 48 - 72 h

NUMBER OF REPLICATIONS: triplaicate cultures were set up
After the incubation period, the number of revertant colonies per plate was counted by hand

Evaluation criteria:

Considering biological relevance the test item is considered positive if the assay is valid and the following conditions are met:
- concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- Mutation factor >2.

The positive result indicates that the test item induces mutations in Salmonella typhimurium or E.coli cells.
The test item for which results do not meet the above criteria is considered non-mutagenic in this test.
Negative results indicate that under the test conditions, the test item does not produce mutations in Salmonella typhimurium/E.coli cells.

Statistics:

The mutation frequency at each dose concentration level of the test item was compared to the one observed in negative and positive controls.
The statistical analysis was carried out using unpaired T-test.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The results indicate that test item Magnesium Fluoride did not induce a statistically significant dose-related increase in the number of revertants when tested in Salmonella typhimurium TA100, TA1535, TA97, TA98 and E.coli uvrA, both with and without metabolic activation.
According to the results obtained in this test system the test item is considered non mutagenic.

Executive summary:

Magnesium Fluoridewas tested for the mutagenic potential using in vitro Bacterial Reverse Mutation Test (Ames test) (corresponding to the OECD TG No. 471). A preliminary range-finding assay was performed using three strains of Salmonella typhimurium (TA97, TA100, TA1535) up to a maximum dose of 5.0 mg/plate to determine cytotoxicity and the optimal non-toxic test dose.

Magnesium Fluoride was tested for the mutagenic potential in strains Salmonella typhimurium TA100, TA 98, TA 97, TA1535 and E.coli uvrA in the Main assay and in the assay with Pre incubation up to a maximal dose of 5.0 mg/plate in the absence external metabolic activation and in presence of external metabolic activation with S9 fractions prepared from Sprague-Dawley rat after induction Aroclor.

Magnesium Fluoride did not produce any significant increases of mutation frequency in strains up to the maximum dose of 5.0 mg/plate in strains Salmonella typhimurium TA100, TA 98, TA 97, TA1535 and E.coli uvrA both in the absence and presence of metabolic activation.

In accordance with these results Magnesium Fluoride is considered to be non mutagenic in bacterial gene mutation test system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1. 2017 - April 3, 2017

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 662599-1
- Expiration date of the lot/batch: 31 July 2017
- Purity test date: 18 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature, protected from moisture
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Test item is poorly soluble in water according to the literature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item is poorly soluble in water according to the literature on solubility of Magnesium Fluoride (0.0076 g/100 g water). Based on these data the stock solution was prepared by dissolving the test item in Dulbecco’s Modified Eagle’s medium and shaking overnight.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures), UK
- Number of passages if applicable: The cell cultures obtained from the stock ampules will be
maintained by subculturing for a maximum of 8 passages and used to initiate cultures for the assay
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco’s Modified Eagle’s medium (DMEM) with 4.5 g/L of glucose supplemented with L-glutamine + 10 % FBS + PS
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

76 µg/mL, the top concentration that was able to dissolve into vehicle.

Vehicle / solvent:

Dulbecco’s Modified Eagle’s medium (DMEM)

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- Cell density at seeding (if applicable): 3.5 x 105 V79 cells were seeded in duplicate into Petri dishes (diameter 60 mm)

DURATION
- Preincubation period: 1 day
- Exposure duration: In the non -activated tests (direct method) the cultures were treated for 3 or 24
hours. In the activated tests the cultures were treated for 3 hours.
- Expression time (cells in growth medium): 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 2h

SPINDLE INHIBITOR (cytogenetic assays): demecolcine

STAIN (for cytogenetic assays): Giemsa stain (5% in phosphate buffer, pH = 6.8) for 4-6 min.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the Demecolcine exposure, the medium with dividing cells were transferred into labelled centrifuge tubes, the monolayer of cells was dissociated with 0.25% trypsin and resuspended in the collected medium. An aliquot of this cell suspension were counted using Bürker`s chamber for determination of RCG. The cells were centrifuged (1000 rpm for 5 min). The culture medium was removed and approximately 8 ml of hypotonic potassium chloride was added to swell the cells. The suspension was incubated for 8 min at 37°C until the hypotonic solution was replaced with a fixative (cooled methanol mixed with glacial acetic acid in a ratio of 3:1). The cells were centrifuged and the fixation process was repeated twice. A slightly turbid solution of suspended cells was prepared and two drops of fixed cells were dropped onto clean microscope slides which had been dipped in cold distilled water. The slides were air dried and stained.

NUMBER OF CELLS EVALUATED: A total of 1000 cells were scored from each concentration (500
from each duplicate dish)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well-spread metaphases containing 22 ± 2 centromeres were analysed per each concentration, solvent and positive controls equally divided among the duplicates by microscopic examination

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test item was assessed by determining the Relative Cell Growth
(RCG) and/or the Relative Mitotic Index (RMI) of the treated cells.

Evaluation criteria:

The metaphases were analysed for the following structural aberrations: chromatid gaps and breaks, isochromatid gaps and breaks, and exchange (dicentrics, double minutes, quadriradials, triradials and rings). Since, the genetic significance of gaps is not clearly understood; they were not included in the assessment of chromosomal damage and thus were not evaluated statistically.

Criteria for a valid assay
- In the solvent control, the percentage of cells with aberrations should not exceeded 4%.
- The positive controls induce a statistically significant increase (p < 0.05) over the solvent control group in percentage of aberrant cells.
- At least one of the test concentrations scored should show approximately 50 % reduction in the RCG and/or RMI. This requirement should not be applied to test articles where no apparent toxicity could be achieved at the maximum soluble concentration or highest allowable concentration.

Criterion for positive response
The test item was considered to have caused a positive response in this assay if the test item showed a statistically significant increase (p < 0.05) over that of the solvent control group in the percentage of aberrant cells at one or more concentrations.

Negative response
The test item was considered to have caused a negative response if none of the test concentrations showed a statistically significant increase in the percentage of aberrant cells.

Statistics:

The percentage of all aberrant cells were taken as the statistical variable to analyze. Each treatment group (including the positive control group) was compared individually to the solvent control group. The data in each outcoming contingency table were evaluated statistically applying. Significance level of 0.05 were adopted among statistical analyses to make appropriate conclusions. The component treatment group was considered statistically different from the solvent one at P<0.05. Statgraphics™ Centurion, version XV software was applied for the purpose of statistical evaluation.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes were observed
- Effects of osmolality: The osmolality values were within the physiological ranges for these cells
- Water solubility: Poorly soluble in water.
- Precipitation: A visible precipitate in the culture medium was observed at the test concentration 76 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
Concentrations selected for the chromosome aberration test were based on the results of the cytotoxicity Range-Finder Experiment from the Study No. 6003567130. This study is reported at IUCLID section 7.6.1. Genetic toxicity in vitro.003. Key_mutagenicity.

Conclusions:

The results from the In Vitro Mammalian Chromosome Aberration Test indicate that the test item, Magnesium Fluoride did not induce a statistically significant increase in the percentage of cells with aberrations both with and without metabolic activation when compared to the solvent controls, at the concentration of 76 µg/mL tested.
The maximum concentration tested was limited by solubility (poorly soluble test item) in the experiments with and without metabolic activation.
Therefore, under the conditions of this test and according to the criteria set for evaluating the test results, Magnesium Fluoride was negative both with and without metabolic activation in the V 79 Chromosome Aberration Test.

Executive summary:

Clastogenic effect ofMagnesium Fluorideagainst V79 cells was assessed in independent experiments with and without metabolic activation at 3-h exposure. Because these tests gave negative results, an additional experiment without metabolic activation at 24-h exposure was carried out. Only one concentration of test item, solvent control and positive control were tested in each experiment.

Because the test item Magnesium Fluoride is poorly soluble in the water only one concentration was selected for the test.

No marked changes of pH of treatment media were observed in the experiments at concentration 76 µg/mL tested as compared to the concurrent solvent controls.

The osmolality values were within the physiological ranges for these cells. The negative control cultures showed optimal growth under these conditions.

One concentration was evaluated for RCG and/or RMI in the Mammalian Chromosome Aberration Test.

In the non-activated system with 3h and 24h treatment the RCGs for the test item concentration of 76 µg/mL were 119.8 and 104.1 respectively. The RMIs were 95 and 100% respectively.

In the activated system with 3h treatment the RCG for the test item concentration of 76 µg/mL was 95.8% and RMI was 86.1%.

Based on the toxicity results (RCGs and/or RMIs), the chromosome aberrations were scored at the concentration of 76 µg/mL in both the non-activated and the activated systems. In addition, the corresponding solvent and positive (MMC at 0.1, 0.7 µg/mL and CP at 40 µg/mL) controls were also scored. One hundred and fifty (150) metaphases were scored from each of the two replicate cultures for test concentration and the controls.

In the short – term treatment (3h), the percentages of aberrant cells excluding gaps of Magnesium Fluoride treated groups bothin the non-activated system (-S9) and in the activated system (+S9) were similar to that of the solvent control.

In the case of continuous treatment (24h), the percentages of aberrant cells excluding gaps of Magnesium Fluoride treated group were 1.3%.

The percentages of aberrant cells excluding gaps of solvent control treated group ranged from 0.3 to 1.3%.

All solvent control cultures had percentages of aberrant cells within the expected range.

The positive control items, cyclophosphamide(40 µg/L) and mitomycin C (0.1µg/mL, 0.7 µg/mL) induced statistically significant increases (p< 0.05) in the incidence of aberrant cells, indicating that the experimental system employed was functioning correctly.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 09, 2016 - March 9, 2017

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)

GLP compliance:

yes

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 662599-1
- Expiration date of the lot/batch: 31 July 2017
- Purity test date: 18 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 20±5 °C
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Test item is poorly soluble in water according to literature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item is poorly soluble in water according to the literature on solubility of Magnesium Fluoride (0.0076 g/100 g water). Based on these data the stock solution was prepared by dissolving the test item in Dulbecco’s Modified Eagle’s medium and shaking overnight.

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures), UK (Lot No. 05F013)
- Suitability of cells:The V79 cell line is well established; the stability of the karyotype and morphology makes this cell line suitable for gene mutation studies, with a low background spontaneous mutation rate.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco’s Modified Eagle’s medium (DMEM) with 10% FBS and 4.5 g/L of glucose supplemented with L-glutamine. For the mutation test, the medium was removed and replaced with 5 mL of serum free DMEM.

Metabolic activation:

with and without

Metabolic activation system:

20-methylcholanthrene induced rat liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

As the test substance was poorly soluble to water, a visible precipitate in the culture medium was observed at this concentration. In the initial cytotoxicity Range-Finder Experiment two concentrations of 38 µg/mL and 76 µg/mL were tested in the absence of S9. The reductions in RPE values (%RPE reduced to 10 to 20%) were not achieved at any of concentrations tested. Therefore, maximum soluble concentration of 76 µg/mL was chosen to be used in the study.

In Experiment 1 (A, B), only one concentration of 76 µg/mL was tested in the absence of S9.
In Experiment 2 (A, B), only one concentration of 76 µg/mL was tested in the presence of S9.

Vehicle / solvent:

Dulbecco’s Modified Eagle’s medium (DMEM) with 4.5 g/L of glucose supplemented with L-glutamine.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): At least 1 000 000 cells in a volume of 5mL per Petri dish

DURATION
- Preincubation period: 24 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF CELLS EVALUATED: approximately 300 cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: relative plating efficiency
- Any supplementary information relevant to cytotoxicity: In the initial cytotoxicity Range-Finder Experiment two concentrations of 38 µg/mL and 76 µg/mL were tested in the absence of S9. The reductions in RPE values (%RPE reduced to 10 to 20%) were not achieved at any of concentrations tested. Therefore, maximum soluble concentration of 76 µg/mL was chosen to be used in the study.

In Experiment 1 with duplicate cultures, only one concentration of 76 µg/mL was tested in the absence of S9. The precipitate was observed upon addition of the test item to the cultures at the beginning and at the end of the 3- hour incubation period.
Seven days after treatment, one concentration (76 µg/mL) in the absence of S9 was selected to determine viability and 6TG resistance, which gave ~89% and ~84.0% RPE, respectively.

In Experiment 2 with duplicate culture, only one concentration of 76 µg/mL was tested in the presence of S9. The precipitate was observed upon addition of the test item to the cultures at the beginning and at the end of the 3-hour incubation period.
Seven days after treatment concentration 76 µg/mL in the presence of S9 was selected to determine viability and 6TG resistance, which gave ~86% and ~96% RPE, respectively.

Evaluation criteria:

For valid data, the test item is considered to induce forward mutation at the hprt locus in Chinese hamster lung V79 cells if:
• At least one of the test concentrations exhibits a statistically significant increase in mutant frequency compared with concurrent negative control. The mutant frequency at one or more concentrations is at least 3-fold greater than negative control.
• Any of the results are outside the distribution of the historical negative control data.

Statistics:

For each test concentration summary statistics (average, standard deviation, coefficient of variation, minimum, maximum) was calculated. The same was done for negative and positive control data.
Two sample comparison was carried out to identify the test concentration for which median of the observed frequency data is significantly different from the median in the negative control group. Mann Whitney (Wilcoxon) W test was applied for this purpose. Significance level of 0.01 was adopted among statistical analyses to make appropriate conclusions.
Statgraphics TM Centurion software (version XV) was employed for statistical data evaluation.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Acceptable survival was detected in the test item concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes of pH of treatment media were observed in the Range-Finder experiment at concentrations up to 76 µg/mL tested as compared to the concurrent vehicle controls
- Effects of osmolality: The osmolality values were within the physiological ranges for these cells
- Water solubility: The test item is poorly soluble in water. Therefore, a stock solution was prepared by dissolving the test item in Dulbecco’s Modified Eagle’s medium and shaking overnight
- Precipitation: Precipitate was observed upon addition of the test item to the cultures at the beginning and at the end of the 3- hour incubation period

RANGE-FINDING/SCREENING STUDIES:
In the initial cytotoxicity Range-Finder Experiment two concentrations of 38 μg/mL and 76 μg/mL were tested in the absence of S9. The reductions in RPE values (%RPE reduced to 10 to 20%) were not achieved at any of concentrations tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The induced number of mutants of positive controls are within the expected range. Historical data not shown in the report.
- Negative (solvent/vehicle) historical control data:
Summary Statistics for Negative Control
Experiment No Count Average Standard deviation Range
Exp1 10 2.06 1.11 3.45
Exp2 10 2.14 0.52 1.55
Exp3 10 1.75 0.78 2.30
Exp4 10 0.47 0.46 1.15
Exp5 10 1.08 0.74 2.17
Exp6 10 1.70 1.01 3.50
Exp7 10 0.63 0.51 1.46
Exp8 10 1.95 1.13 3.99
Exp9 10 0.69 0.49 1.38
Exp10 10 1.91 0.49 1.55
Exp11 10 2.17 1.08 3.29
Exp12 10 0.64 0.34 1.07
Exp13 10 0.92 0.40 1.02
Exp14 10 0.73 0.44 1.04
Exp15 10 2.09 0.90 2.38
Exp16 10 1.32 0.50 1.37
Exp17 10 1.84 0.62 2.11
Exp18 10 1.76 0.53 1.47
Exp19 10 1.95 0.78 2.30
Exp20 10 1.88 0.89 2.41
Total 200 1.48 0.91 4.43

Summary of mutation data Experiment 1 (A, B)^c without S9

Treatment (µg/mL)	3 hour-S9 % RPE	Mf^a	Treatment (µg/mL)	3 hour-S9 % RPE	Mf^a
Vehicle^bA	100.0	20.23	Vehicle^bB	100.0	19.52
TI(76)         A	88.5	19.66	TI(76)         B	84.3	16.42
EMS(600)  A	91.0	273.98^*	EMS(600)  B	102.4	227.49^*

^a6TG resistant mutants/10⁶viable cells 7 days after treatment

^bculture medium (DMEM); TI -Test Item

^cA or B - codes for two replicate cultures

* Statistically significant (p<0.01) (Mann-Whitney W test)

Summary of mutation data Experiment 2 (A, B)^cwith S9

Treatment (µg/mL)	3 hour+S9 % RPE	MF^a	Treatment (µg/mL)	3 hour+S9 % RPE	MF^a
Vehicle^bA	100.0	19.06	Vehicle^bB	100.0	18.76
TI(76)              A	85.6	18.17	TI(76)              B	95.7	19.67
DMBA (600)  A	58.3	254.88	DMBA (600)  B	58.0	269.64

^a 6TG resistant mutants/10⁶viable cells 7 days after treatment

^b culture medium (DMEM); TI- Test Item

^c A or B - codes for two replicate cultures

* Statistically significant (p<0.01) (Mann-Whitney W test)

Conclusions:

Magnesium fluoride did not induce mutation at the hprt locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study.

Executive summary:

In experiment 1 with duplicate cultures (A, B) in the absence of S-9, no statistically significant increases in mean mutant frequency were observed following treatment with Magnesium fluoride. The test item did not induce significant cytotoxicity (expressed in terms of % RPE at the end of treatment) in the concentration tested.

In experiment 2 with duplicate cultures (A, B) in the presence of S-9, no statistically significant increases in mean mutant frequency were observed following treatment with Magnesium fluoride. Survival (expressed in terms of % RPE at the end of treatment) was not reduced at the concentration tested.

Mutant frequencies in vehicle controls were within the acceptable range and clear increases in mutant frequency were observed by the positive controls, ethyl methanesulphonate (without S‑9) and 7,12 -dimethyl-benz(a)anthracene (with S‑9). The assay system was therefore considered to be both sensitive and valid.

The study was considered reliable without restrictions since the study was conducted in accordance with current international guideline and in compliance with GLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro genetic toxicity of the target substance has been evaluated in one study with bacterial cells and in two studies with mammalian cells. These studies are performed according to the current guidelines and in accordance with GLP, and evaluated to be reliable without restrictions. Thus, it is justified to select all three studies as key studies.

Mutagenicity in bacterial test systems

A bacterial mutagenicity study by Múčková, M. (2017) (OECD guideline 471) was conducted by using four strains of Salmonella typhimurium bacteria (TA97, TA98, TA100, and TA1535) and one strain of E.coli (WP2 uvr A) with and without metabolic activation. The target substance was tested for its ability to induce mutations in the main assay at the concentration range of 0.050 – 5.0 mg/plate. The test substance in concentration range 0.050 – 5.0 mg/plate was evaluated for mutagenic potential in test with preincubation in the absence and presence of metabolic activation. The test substance did not produce any significant increases of mutation frequency in strains up to the maximum dose of 5.0 mg/plate in strains Salmonella typhimurium TA97, TA98, TA100, and TA1535 and E.coli uvrA both in the absence and presence of metabolic activation.

Cytogenicity in mammalian cells

The potential of the test substance to induce chromosome aberration (OECD guideline 473) was assessed in Chinese hamster lung fibroblasts (V79) by Lazovâ (2017). The test substance was tested at a concentration of 76 μg/ml for a 3 h exposure time in the absence and presence of metabolic activation (S9 - fraction). The test substance was also tested at a concentration of 76 μg/ml for a 24 h exposure time in the absence of metabolic activation. The target substance did not induce a statistically significant increase in the percentage of cells with aberrations both with and without metabolic activation when compared to the solvent controls, at the concentration tested.

Mutagenicity in mammalian cells

The potential gene mutagenic activity of the target substance was evaluated in Chinese hamster lung fibroblasts (V79) by Lazovâ (2017) according to the OECD guideline 476. Chinese hamster lung fibroblasts (V79) were exposed to the test substance, in the presence and absence of rat liver S-9 for three hours. In the inactive system (no S-9) and in the active system (with S-9) the cells were exposed to test material at concentration of 76 μg/mL. In addition to treated cells, a solvent control group and positive controls consisting of ethylmethane sulfonate for inactive studies (no S-9) and 7,12 -dimethyl-benz[a]anthracene (DMBA) for active studies (with S-9) were also used to test the efficacy of the mutagenicity assay. This assay was performed in two sets to ensure reproduction of experimental results.

In the presence and absence of metabolic activation (S9-mix), no statistically significant increases in mean mutant frequency were observed following treatment with test sample at the concentration tested in the two independent repeat experiments. Survival (expressed in terms of %RPE at the end of treatment) was not reduced at any concentration tested. Mutant frequencies in vehicle controls were within the acceptable range and clear increases in mutant frequency were observed by the positive controls, ethyl methanesulphonate (without S‑9) and 7,12 -dimethyl-benz(a)anthracene (with S‑9). The assay system was therefore considered to be both sensitive and valid.

Based on the above facts, the test substance is not considered to be mutagenic in humans.

Justification for classification or non-classification

Based on the results of in vitro bacterial gene mutation study, in vitro mammalian chromosomal aberration and gene mutation studies no classification is proposed for genotoxicity according to the criteria of CLP regulation 1272/2008.