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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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.
Controlsopen allclose all
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.
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); without S9, TA1535 and TA100, 5 µg/plate
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD), without S9, TA98, 10 µg/plate
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: without S9, TA1537, 100 µg/plate
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: 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 resultsopen allclose all
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
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
Remarks:
Migrated from field 'Test system'.

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

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.