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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
3 November 2005 to 17 May 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Cited as Directive 2000/32/EC, B.17
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid
- Storage condition of test material: at room temperature

Method

Target gene:
HPRT
Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
- Type and identity of media: minimal essential medium (MEM) supplemented with 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction of rats induced with Phenobarbital/beta-Naphthoflavone
Test concentrations with justification for top dose:
- Experiment I: 143.8; 287.5; 575; 1150; and 2300 µg/mL with and without S9-mix
- Experiment II: 143.8; 287.5; 575; 1150; and 2300 µg/mL without S9-mix, 287.5; 575; 1150; 1725; and 2300 µg/mL with S9-mix
See also table 1 below.
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: data not available
Controlsopen allclose all
Negative controls:
yes
Remarks:
culture medium
Solvent controls:
yes
Remarks:
medium with solvent or vehicle alone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: (EMS): 0.3 mg/mL without metabolic activation
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Migrated to IUCLID6: (DMBA): 2.0 µg/mL with metabolic activation
Details on test system and conditions:
- METHOD OF APPLICATION: in suspension

- DURATION
- Exposure duration: 4h in experiment I with or without metabolic activation, 24h in the absence and 4h in the presence of metabolic activation in experiment II
- Expression time: 7 days
- Selection time: 8 days in 6-TG
- Fixation time: Day 15 or day 16

- SELECTION AGENT: thioguanine (6-TG)
- STAIN: in 10 % methylene blue in 0.01 % KOH solution
- NUMBER OF REPLICATES: two

- DETERMINATION OF CYTOTOXICITY: cloning efficiency
- OTHER: SCORING METHOD: The stained colonies with more than 50 cells are counted. In doubt the colony size was checked with a preparation microscope
Evaluation criteria:
A test material producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test material is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test material is classified as mutagenic if there is 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:
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect observed
- Effects of osmolality: no effect observed
- Precipitation: Precipitation was observed at 1150 µg/mL and above in the first main experiment with and without metabolic activation (4 h treatment). In the second experiment without metabolic activation (24 h treatment), precipitation occurred at 575 µg/mL and above. In the second experiment with metabolic activation, precipitation was noted at 1150 µg/mL and above (4 h treatment).

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments.
No relevant toxic effect (relative cloning efficiency at or below 50 %) occurred up to the highest concentration following 4 h treatment in the presence of metabolic activation. In the absence of metabolic activation relevant toxicity was noted at the maximum concentration of 4600 µg/mL.
Following 24 h treatment, performed solely without metabolic activation, no relevant toxic effect was observed up to 143.8 µg/mL. At the concentration of 575 µg/mL the cloning efficiency was reduced to 40.2 % of solvent control. No toxicity data were generated at 287.5 µg/mL and at 1150 µg/mL and above due to microbial contamination of the cells.
The test medium was checked for precipitation with the unaided eye at the end of each treatment period (4 or 24 hours) just prior to removal of the test material. Precipitation was observed at 575.0 µg/mL and above in the absence and presence of metabolic activation at both treatment intervals.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutant frequencies generally remained within the historical range of negative and solvent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects solely occurred at the maximal concentration of 2300 µg/mL in the second experiment without metabolic activation.

See detailed results in table 2 (attached document)

Applicant's summary and conclusion

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

In conclusion it can be stated that under the experimental conditions reported the test material did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus) (Wollny, 2006), Chinese hamster V79 cells cultured in vitro were exposed to Cerium carbonate, in deionised water, at concentrations of 143.8 to 2300 µg/mL in the presence and absence of mammalian metabolic activation. Cerium carbonate was tested up to precipitating concentrations.

The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

Therefore, Cerium carbonate is considered to be non-mutagenic in this HPRT assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline EU Method B.17.