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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 May to 30 July 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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
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
Details on test material:
Calcium fluoride, batch no. F1510740 834, BASF test item no. 10/0041-1, expiry date 14 December 2014. Purity >98% (provided in BASF Certificate of Analysis), dose calculation adjusted to purity. The test substance was described as a white solid, and has a molecular weight of 78.07 g/mol. The test substance was stored at room temperature protected from moisture.

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cells were stored in liquid nitrogen. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch of cells were screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from phenobarbital/ß-naphthoflavone induced livers of young male Wistar rats
Test concentrations with justification for top dose:
Preliminary test: 6.3 to 800 µg/mL
Main test: 25, 50, 100, 200, 400 and 800 µg/mL.
Vehicle / solvent:
Deionised water. The solvent was chosen based on its solubility properties and relative non-toxicity to cells. The final concentration of deionised water in culture medium was 10%.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: without S9, 0.15 mg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Migrated to IUCLID6: with S9, 1.1 µg/mL
Details on test system and experimental conditions:
The test material was suspended in deionised water immediately before treatment.
Thawed stock cultures were propagated at 37°C in 80 cm² plastic flasks. Approx. 5x10E5 cells were seeded into each flask with 15 mL of MEM (minimum essential medium) supplemented with 10% fetal calf serum (FCS) and 1% neomycin, and subcultured weekly. Cultures were incubated at 37°C in a 4.5% carbon dioxide atmosphere. For the selection of mutant cells the medium was supplemented with 11 µg/mL 6-thioguanine (6-TG).

Preliminary toxicity test: A pre-test was performed to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions were the same as for the mutagenicity experiment. In the pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item was indicated by a reduction of the cloning efficiency (CE).

Main test: two independent experiments were performed, both in the presence and absence of S9 mix.
Two day old (experiment I) and three day old (experiment II) exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37°C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2% in Ca-Mg-free salt solution. Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA. The cell suspension was seeded into plastic culture flasks. Approximately 1.5x10E6 cells (single culture) and 5x10E2 cells (in duplicate) were seeded in MEM with 10% FCS (complete medium) for the determination of mutation rate and toxicity, respectively.
After 24 h the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 h this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 h in complete medium in the absence of metabolic activation. The pH was adjusted to 7.2.
Four days (experiment I) and three days (experiment II) after treatment 1.5106 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO2 for about 7 days. The colo¬nies were stained with 10 % methylene blue in 0.01 % KOH solution (E. Merck, 64293 Darmstadt, Germany).
The stained colonies with more than 50 cells were counted. If in doubt the colony size was checked with a preparation microscope

Evaluation criteria:
A test item was classified as positive if it induced either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic.
A test item was classified as mutagenic if it reproducibly induced a mutation frequency three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item was classified as mutagenic if there was a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
Statistics:
Least squares linear regression.

Results and discussion

Test results
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
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: No relevant cytotoxic effect occurred up to the maximum concentration with and without metabolic activation. . Precipitation was observed at 200 µg/mL and above without metabolic activation and at 400 µg/mL and above with metabolic activation following 4 hours treatment. After 24 hours treatment precipitation occurred at 400 µg/mL and above. There was no relevant shift of pH and osmolarity of the medium even in the stock solution of the test item.

Main test: Precipitation of the test item was observed in the first experiment at 100 µg/mL and above with and without S9. In the second experiment precipitation was noted at 200 µg/mL and above with (4 hours treatment) and without (24 hours treatment) S9. No cytotoxic effects indicated by a relative cloning efficiency I below 50% occurred in both main experiments with and without S9. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor exceeded the threshold of 3.0 at 25, 50, and 400 µg/mL in the first culture of experiment I without metabolic activation and at 400 µg/mL in the first culture of the second experiment with metabolic activation. This effect however, was judged as irrelevant since it was neither dose dependent as indicated by the lacking statistical significance nor reproduced in the parallel culture under identical conditions.
A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture of the second experiment without S9. This trend however, was judged as irrelevant since the mutation frequency did not reach the threshold by far and all absolute values of the mutation frequency remained within the historical range of solvent controls.

The range of the solvent controls (with and without S9) was from 5.5 up to 34.4 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.6 up to 38.9 mutant colonies per 10E6 cells. The highest solvent control value (34.4 colonies per 10E6 cells) exceeded the range of historical solvent control data. The mean value of both parallel cultures however, (22.3 and 34.4 equal to 28.4) colonies per 10E6 cells) was fully acceptable.
EMS (0.15 mg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Remarks on result:
other: strain/cell type: V79
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test material did not induce gene mutations at the HPRT locus in V79 cells.       

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Calcium fluoride did not induce gene mutations at the HPRT locus in V79 cells, with and without metabolic activation.
Executive summary:

The potential of calcium fluoride to cause gene mutations at the HPRT locus in Chinese hamster V79 cells was evaluated in two independent experiments. Each experiment included parallel cultures, with and without metabolic activation (S9 mix). The first main experiment was performed with and without S9 mix and a treatment period of 4 hours. The second experiment was performed for 4 hours with S9 and for 24 hours without S9. The highest concentration tested was 800 µg/mL.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in either of the main experiments.

The test material did not induce gene mutations at the HPRT locus in V79 cells, under the conditions of the study. It was concluded, therefore, that calcium fluoride is not mutagenic in the HPRT assay.