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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-06-20 to 2017-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: wet powder
Details on test material:
- Name of test material (as cited in study report): digadolinium oxalate
- CAS nr: 867-64-1
- Physical state: solid
- Appearance: wet white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature (15-25°C, below 70 RH %)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: insoluble, suspension only

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Stock formulations of 200 mg/mL anhydrous gadolinium oxalate in vehicle were prepared.

CORRECTION FACTOR
- A correction factor of 2.05 was applied during formulation to achieve the target concentration of the formulation expressed in anhydrous form.

Method

Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells:
ECACC (European Collection of Cells Cultures)
- Sex: male
MEDIA USED
- Laboratory cultures were maintained in 150 cm2 plastic flasks at 37 +/- 0.5°C in a humidified atmosphere containing approximately 5% CO2 in air.
- V79 cells were grown in Dulbecco's Modified Eagle's Medium supplemented with 2 mM L-glutamine, 1% v/v antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and 10% v/v heat-inactivated fetal bovine serum (DMEM-10, culture medium).
- During the treatments, the serum content of the medium was reduced to 5% v/v (DMEM-5).
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, induced by a mixture of phenobarbital and beta-naphthoflavone
Test concentrations with justification for top dose:
Treatment concentrations for the mutation assays were selected based on the results of a short preliminary test. Two concentration selection cytotoxicity assays were performed to establish an appropriate concentration range for the chromosome aberration assays (assay A: 3-h treatment with and without metabolic activation, 20-h harvesting time; assay B: 3-h treatment with metabolic activation or 20-h treatment without metabolic activation, 28-h harvesting time). A total of 10 concentrations between 2000 and 3906 µg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay. Treatment concentrations for the chromosome aberration assays were selected on the basis of results of these assays according to the OECD guideline instructions.

Chromosome Aberration Assay 1
3-hour treatment in the presence of S9-mix:
2000, 666.7, 222.2, 74.08 and 24.69 μg/mL
3-hour treatment in the absence of S9-mix:
2000, 666.7, 222.2, 74.08 and 24.69 μg/mL

Chromosome Aberration Assay 2
3-hour treatment in the presence of S9-mix:
2000, 666.7, 222.2, 74.08 and 24.69 μg/mL
20-hour treatment in the absence of S9-mix:
1500, 1000, 500, 250, 125 and 62.5 μg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: 1% w/v methyl cellulose aqueous solution
- Justification for choice of solvent/vehicle: Based on available data (CiToxLAB study code 16/093-007M) and due to the chemical nature of the test item. The test item was insoluble in generally used vehicles (distilled water, DMSO, DMF, acetone), only a suspension could be achieved up to 100 mg/mL. To ensure suspension homogeneity (lower sedimentation speed), 1% w/v methyl cellulose aqueous solution was selected as vehicle of the study.
Controls
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
1% methyl cellulose aqueous solution (negative (vehicle) control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and conditions:
METHOD OF APPLICATION
- Cell density at seeding (if applicable): 5 x 10^5 cells/dish

DURATION
- Exposure duration: 3 h (assay 1, with and without metabolic activation), 3 h (assay 2, with metabolic activation), 20 h (assay 2, without metabolic activation)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h (assay 1), 28 h (assay 2)

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.2 µg/mL)

STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
- 2 to 2.5 h prior to harvesting, cell cultures were treated with colchicine (0.2 µg/mL).
- Cells were swollen with 0.075 M KCl hypotonic solution for 8 min, then were washed in fixative (methanol:acetic acid 3:1 v/v mixture) until the preparation became plasma free (4 washes).
- A suspension of the fixed cells was dropped onto clean microscope slides and air-dried.
- Slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted.
- At least three slides were prepared for each culture.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells)
- At least 150 metaphases with 22 +/- 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible.
- Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

DETERMINATION OF CYTOTOXICITY
- Method: At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

OTHER EXAMINATIONS
- Determination of polyploidy: Yes. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc.).
- Determination of endoreplication: Yes. Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids.
Rationale for test conditions:
Per Guideline
Evaluation criteria:
The assay is considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.
Statistics:
Fisher's exact test. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Cytotoxicity was only observed at the highest concentration (1500 µg/mL) in assay 2 without metabolic activation (RICC = 51%). In both assays, the test item was tested up to precipitating concentrations.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
In assay 1, insolubility (precipitation / minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 2000-74.08 µg/mL concentration range with and without metabolic activation. There were no large changes in pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 666.7, 222.2, 74.08 and 24.69 µg/mL were chosen for evaluation in the experiment with and without metabolic activation.

In assay 2, insolubility was detected at the end of the treatment period in the final treatment medium in the 1500-62.5 µg/mL concentration range without metabolic activation and in the 2000-74.08 µg/mL concentration range with metabolic activation. There were no large changes in pH and osmolality. No cytotoxicity was observed in the experiment with metabolic activation, while cytotoxicity was detected in the experiment without metabolic activation (RICC value of the highest evluated concentration was 51%). Concentrations of 1500, 1000, 500, 250 and 125 µg/mL were evaluated in the experiment without metabolic activation, and concentrations of 666.7, 222.2, 74.08 and 24.69 µg/mL were evaluated in the experiment with metabolic activation.

The test item did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments without metabolic activation. In the experiments with metabolic activation, a marginal increase was seen at one concentration (222.2 µg/mL) in assay 1, however there was no dose response (no other increases were observed including the higher evaluated concentration in the same experiment), and the observed effect was not reproducible between replicates (increase was found only in one replicate of assay 1) nor between experiments using the same experimental conditions (no similar increase was seen in assay 2 with metabolic activation). Based on these facts, these data did not meet the criteria of a positive response, therefore, the test item was considered to have a negative response.

Polyploid metaphases (1-4) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. No endoreduplicated metaphases were detected in the performed experiments.

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency and the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation, demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least four test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

Applicant's summary and conclusion

Conclusions:
Gadolinium oxalate was considered not to be clastogenic in the absence and presence of metabolic activation under the conditions of the test system.