Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Between 30 August 2012 and 22 March 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented GLP study with cerium trinitrate, performed according to OECD Guideline 473 and EU Method B10. The read-across justification for extrapolation to cerium ammonium nitrate is added in section 13 of this IUCLID dataset. The study has been identified as K2 study as it was performed with a 'read across' substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37°C with 5% CO2 in humidified air. The lymhocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system (S9) induced by a mixture of phenobarbitone and beta-naphthoflavone
Test concentrations with justification for top dose:
Four hours exposure with 20-hour expression period without S9: 101.9, 203.75, 407.5, 815, 1222.5 and 1630 µg/mL
Four hours exposure with 20-hour expression period with S9 (2%): 203.75, 407.5, 815, 1630, 2445, 3260 µg/mL
24-hour exposure without S9: 101.9, 203.75, 407.5, 815, 1222.5 and 1630 µg/mL
Four hours exposure with 20-hour expression period with S9 (1%): 203.75, 407.5, 815, 1630, 2445, 3260 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous media at 32.6 mg/mL but was soluble in dimethyl sulphoxide at 326 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide at 326 mg/ml was, therefore, selected as the vehicle.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
0.4 and 0.2 µg/mL mitomycin C was used in experiment 1 and 2 respectively in the absence of S9. It was dissolved in Minimal Essential Medium. In the presence of S9, cyclophosphamide was used at 5 µg/mL in both experiments. It was dissolved in DMSO.
Details on test system and experimental conditions:
- Exposure duration: 4 and 24 hours
- Expression time: 20 hours

SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa

NUMBER OF CELLS EVALUATED: 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid.
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploidy cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Qualitative observations in that inhbition of mitotic index was observed, and 52% and 82% mitotic inhibition was achieved at 815 and 1222.5 µg/mL, respectively, in the absence of S9. In the presence of S9, 26% mitotic inhibition was achieved at 2445 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Above 2445 µg/mL metaphases could not be accurately assessed due to the presence of excessive precipitate on the slides.

The maximum dose level selected for metaphase analysis was therefore 815 µg/mL and 2445 µg/mL in the absence and presence of S9, respectively. The toxicity observed at 1222.5 µg/mL, in the absence of S9, was considered to be excessive and precluded the dose from chromosome analysis.

Cerium trinitrate did not induce any statistically significant increases in the frequency of cells with aberrations in either exposure group, which included a dose level that was generally within the optimal 50% mitotic inhibition. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes, determined by the in-house historical range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and without metabolic activation

Cerium trinitrate was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic with and without metabolic activation.