Magnesium fluoride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 January 2017 - 21 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium fluoride

EC Number:

231-995-1

EC Name:

Magnesium fluoride

Cas Number:

7783-40-6

Molecular formula:

F2Mg

IUPAC Name:

magnesium difluoride

Test material form:

solid: crystalline

Details on test material:

- median particle size 873 um

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.

Method

Species / strain

Species / strain 1

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 2

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 3

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 4

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 5

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Applicant's summary and conclusion

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.