Calcium 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:

16 to 25 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary GLP guideline-compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Calcium fluoride

EC Number:

232-188-7

EC Name:

Calcium fluoride

Cas Number:

7789-75-5

Molecular formula:

CaF2

IUPAC Name:

calcium difluoride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Calcium fluoride, batch no. F1510740 834, test substance no. 10/0041-1, described as a white solid and stored at room temperature; hygroscopic. The substance had a calculated composition of >98% CaF2. The stability of the test substance under storage conditions was guaranteed until 31 Dec 2014 (by the sponsor).

Method

Target gene:

The study determined reverse gene mutations resulting in reversion to histidine prototrophy (S. typhimurium) or tryptophan independence (E. coli).

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers on phenobarital and ß-naphthoflavone induced male Wistar rats

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

The test substnace was dissolved in dimethylformamide (DMF). The vehicle was chosen due to the limited solubility of test substance in water. The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

sterility control

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; with S9 mix 2.5 µg/plate for Salmonella strains and 60 ↨0g/plate for E. coli.

Controls 2

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); without S9, TA1535 and TA100, 5 µg/plate

Controls 3

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD), without S9, TA98, 10 µg/plate

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, TA1537, 100 µg/plate

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9, E. coli, 5 µg/plate

Details on test system and experimental conditions:

Deep-frozen bacterial cultures were thawed at room temperature prior to use. 0.1 ml of the bacterial suspension was inoculated in nutrient broth soltuion (8 g/l Difco nutrient broth +5 g/l NaCl) and incubated in a shaking water bath at 37°C for 12-16 hours. Generally a germ density of ≥10E8. The overnight cultres were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

Standard plate test (plate incorporation method): S. typhimurium; test tubes containing 2 ml portions of soft agar consisiting of 100 ml agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42-45°C, and the remaining components were added in the following order: 0.1 ml test solution or vehicle, 0.1 ml fresh bacterial culture, 0.5 ml S9 mix or 0.5 ml phosphate buffer. After mixing the samples were poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds . After incubation at 37°C for 48-72 hours in the dark, the revertant bacterial colonies were counted.
E. coli; test tubes containing 2 ml portions of soft agar and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42-45°C, and the remaining components added in the following order: 0.1 test solution or vehicle, 0.1 ml fresh bacterial culture, 0.5 ml S9 mix or phosphate buffer. After mixing the samples were poured onto minimal agar plates within approx 30 seconds. After incubation at 37°C for 48-72 hours in the dark, the revertant bacterial colonies were counted.

Preincubation test: 0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S9 mix or phosphate buffer were incubated at 37°C for approx 20 minutes using a shaker. Then 2 ml of soft agar was added and after mixing, the samples were immediately poured onto agar plates. After incubation at 37°C for 48-72 hours in the dark, the revertant bacterial colonies were counted.

Titer determination: the titer was determined in the experiments with S9 mix both for the negative controls and for the two highest doses. In the standard plate test, 0.1 ml of the overnight cultures was diluted to 10E-6. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42-45°C and the remaining components were added in the following order: 0.1 ml vehicle with and without test substance, 0.1 ml fresh bacterial culture, 0.5 ml S9 mix. After mixing, the samples were immediately poured onto agar plates. After incubation at 37°C for 48-72 hours in the dark, the bacterial colonies were counted.

All test concentrations and controls were plated in triplicate.

Evaluation criteria:

Toxicity was detected by a decrease in the number of revertants, a clearing or reduction of the background lawn, and/or a reduction in the titer.
The test substance was considered to demonstrate a positive mutagenic response if a dose-related and reproducible increase in the number of revertant colonies (i.e. about a doubling of the spontaneous mutation rate in at least one tester strain) was observed.

Statistics:

Not required in this study.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight, see below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight, see below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test up to the highest required concentration. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants, reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2 500 μg/plate onward. In addition, in the presence of metabolic activation in the preincubation assay reduced his- or trp- background growth was observed depending on the strain from about 33 μg/plate onward.
No test substance precipitation was found with and without S9 mix. The negative and positive controls all gave the expected results and fulfilled the acceptance criteria of the study. Negative control values were within the range of historical negative control data.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

The test substance did not induce a relevant increase in the number of revertant colonies, in either of two independent experiments (standard plate test and preincubation test) with and without S9 mix.

Applicant's summary and conclusion

Conclusions:

Calcium fluoride was not mutagenic in the bacterial reverse mutation assay with and without metabolic activation.

Executive summary:

Calcium fluoride was evaluated for its mutagenic potential in Salmonella typhimurium strains TA1535, TA100, TA98, TA1537 and E. coli WP2 uvrA. Two independent experiments were performed in the presence and absence of metabolic activation (S9 mix); the standard plate test and the preincubation method. Appropriate negative, vehicle and positive controls were also included.

A bacteriotoxic effect, indicated byt the decreased numbers of revertants, was occasionally observed at concentrations of 2500 µg/plate and above. The test substance did not induce a relevant increase in the number of revertant colonies, in either of the two independent experiments with and without S9 mix.

Under the conditions of the study, it was concluded that Calcium fluoride is not mutagenic in the bacterial reverse mutation test in the presence and absence of metabolic activation.