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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:
2017-04-05 to 2018-09-xx
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
30 May 2008, amended 14 February 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Ytterbium (III) oxide
EC Number:
215-234-0
EC Name:
Ytterbium (III) oxide
Cas Number:
1314-37-0
Molecular formula:
O3Yb2
IUPAC Name:
ytterbium (III) oxide
Test material form:
solid: particulate/powder
Details on test material:
- Name: diytterbium trioxide
- Appearance: white solid
- Further information on test material confidential.
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%), protected from light and humidity
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was formulated in the selected vehicle to provide a suitably concentrated stock formulation (200 mg/mL in the preliminary experiment, 100 mg/mL in the main tests) as follows. The necessary amount of test item was weighed in a calibrated centrifuge tube and an appropriate amount of DMSO was used to prepare the stock formulation, and it was thoroughly mixed by a vortex (until it appeared as a homogenous suspension by visual assessment). From the stock formulation, several dilutions were prepared using the selected vehicle to prepare dosing formulations for lower doses. Before use, the vehicle was filtered sterile using a 0.22 µm syringe filter. The stock formulation as well as all dilutions (dosing formulations) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood. Formulations (suspensions) were mixed by a vortex thoroughly before the treatments, and were protected from light.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

CORRECTION FACTOR
No correction for purity of the test item was applied.

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection for Cell Cultures)
- Sex: male
- Suitability of cells: Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14h). The cell stocks were kept in a freezer at -80 +/- 10°C (for short-term storage) or in liquid nitrogen (long-term storage).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5°C in a humidified atmosphere containing approximately 5% CO2 in air.
- The V79 cells for this study 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).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
colchicine (0.2 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix, induced by mixture of phenobarbital (PB) and beta-naphthoflavone (BNF)
Test concentrations with justification for top dose:
The study included two Concentration Selection Cytotoxicity Assays and two Chromosome Aberration Assays.

A total of ten test concentrations between 2000 and 3.906 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 the performed Concentration Selection Cytotoxicity Assays according to the OECD No. 473 guideline instructions (up to the cytotoxicity limit).

Chromosome Aberration Assays:
Assay 1 +S9: 31.25, 62.5, 125, 250, 500, 750, 1000 µg/mL
Assay 1 -S9: 15.625, 31.25, 62.5, 125, 250, 500 µg/mL
Assay 2 +S9 and -S9: 6.25, 12.5, 25, 50, 75, 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s): dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Based on the available information (solubility of test item was examined in distilled water, DMSO, ethanol, acetone, and N,N-dimethylformamide in study 16/198-007M), DMSO was selected as vehicle for the study (solubility and sedimentation rate was taken into account for the decision).
Controls
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
10 µL/mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
For the cytogenetic experiments, 1-3 day old cultures (more than 50% confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5E+05 cells/dish concentration.

DURATION
- Exposure duration: 3-h (Assay 1: +S9, -S9; Assay 2: +S9), 20-h (Assay 2: -S9)
- Fixation time (start of exposure up to fixation or harvest of cells): Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles, Assay 1) or 28 hours (approximately 2 normal cell cycles, Assay 2) from the beginning of treatment.

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

STAIN (for cytogenetic assays): The slides were stained with 5% Giemsa solution

NUMBER OF REPLICATIONS: 2

METHOD OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- 2-2.5 hours prior to harvesting, cell cultures were treated with colchicine (0.2 µg/mL)
- The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, and were then 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
- The slides were stained with 5% Giemsa solution, air-dried and cover slips 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 (the examination of slides from a culture was halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture).
- Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

DETERMINATION OF CYTOTOXICITY:
- At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed as reduction in relative increase in cell count (RICC) of the treated cells as compared to the negative control.

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. 3n, 4n, etc).
- Determination of endoreplication: Yes, endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.
Rationale for test conditions:
no data, 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
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.
Statistics:
For statistical analysis, Fisher's exact test was used. 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:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING / SCREENING STUDIES:
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 3-hour treatment with metabolic activation and 20-hour treatment without metabolic activation, 28-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays.
A total of ten test concentrations between 2000 and 3.906 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 the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions (up to the cytotoxicity limit).

CHROMOSOME ABERRATION ASSAYS:
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of test item were 500, 250, 125, 62.5, 31.25 and 15.625 µg/mL (experiment without metabolic activation), and 1000, 750, 500, 250, 125, 62.5 and 31.25 µg/mL (experiment with metabolic activation).

In Assay 1, there were no large changes in the pH and osmolality. Insolubility (precipitate or minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 500-125 µg/mL concentration range without metabolic activation, and in the 1000-125 µg/mL concentration range with metabolic activation. Excessive cytotoxicity was observed at the 500 µg/mL concentration without metabolic activation (cytotoxicity value of 69%) and at 1000 and 500 µg/mL concentrations with metabolic activation (cytotoxicity values of 85% and 63%, respectively). Marked cytotoxicity was also observed at 250, 125 and 62.5 µg/mL concentrations without metabolic activation (cytotoxicity values of 58%, 49% and 42%, respectively), and at 500 and 250 µg/mL concentrations with metabolic activation (cytotoxicity values of 53% and 47%, respectively). Concentrations of 250, 125, 62.5, 31.25 and 15.625 µg/mL were selected for evaluation in the experiment without metabolic activation, and concentrations of 500, 250, 125 and 62.5 µg/mL were selected for evaluation in the experiment with metabolic activation.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 100, 75, 50, 25, 12.5 and 6.25 µg/mL (experiment with and without metabolic activation).

In Assay 2, similarly to the first experiment, there were no large changes in the pH and osmolality. Insolubility (precipitate or minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 100-50 µg/mL concentration range with and without metabolic activation. Marked cytotoxicity was observed at 100 and 75 µg/mL concentrations without metabolic activation (cytotoxicity values of 55% and 42%, respectively), and at 100 and 75 µg/mL concentrations with metabolic activation (cytotoxicity values of 56% and 50%, respectively). Concentrations of 100, 75, 50 and 25 µg/mL were selected for evaluation in the experiment without metabolic activation and concentrations of 100, 75, 50, 25 and 12.5 µg/mL were selected for evaluation in the experiment with metabolic activation.

In both assays, with and without metabolic activation, significant increases in chromosome aberrations were observed in at least two concentrations. Since all criteria for a positive response were met, the test item was concluded to be clastogenic under the conditions of this study.

The positive control treatments in each assay all caused a highly statistically significant increase, demonstrating the sensitivity of the test system.

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, but their incidence was not related to the test item treatment. No endoreduplicated metaphases were detected in the performed experiments.

Summary tables of the main experiments are given under 'Any other information on results incl. tables'.

Any other information on results incl. tables

Validity of the study

The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility and cytotoxicity was detected in all experiments with and/or without metabolic activation. The evaluated concentration ranges of Assay 1 and Assay 2 were considered to be adequate, as they covered the range from toxicity to no or little toxicity*, meanwhile the range from insolubility to no insolubility was also covered. (*Note: The degree of cytotoxicity of the highest evaluated concentration (expected to be in the range of 50-60%) was considered to be acceptable: it was 53% and 58% in Assay 1 with and without metabolic activation, respectively, and 56% and 55% in Assay 2 with and without metabolic activation, respectively.)

At least four test item concentrations were evaluated in each experiment.

Due to the marked cytotoxicity and/or test item precipitate on the slides, less than 300 cells (or 25 aberrant metaphases) were scored in some cases. However, at least one code was fully scored for each sample, and additional concentrations were also scored for all those cases to ensure the scientific validity of the observed results. Based on the overall outcome of the study (positive result in at least one fully scored experiment), this fact was having no impact on the results or integrity of the study.

The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were in line with the general historical control range* of the testing laboratory. (*Note: In the Assay 1 using the short treatment with metabolic activation (harvesting period of 20 hours), the aberration frequency of the negative (vehicle) control were slightly higher (6.3%) than required (0-5% aberration frequency). However, the strong results of the positive control showed the appropriate responsiveness of cells. Based on the overall outcome of the study, this fact was considered as having no impact on the results or integrity of the study.)

In the performed experiments, the positive control substances (cyclophosphamide (CP) in the experiments with metabolic activation and ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations demonstrating the sensitivity of the test system in each assay.

In conclusion, the study was considered to be valid.

Summary tables of the main experiments:

Summary table of Chromosome Aberration Assay 1 without metabolic activation

Concentration (µg/mL)

[number of analysed cells]

Time of treatment/sampling RICC# (%) Insolubility## Mean % aberrant cells###
Untreated control 3h/20h 103 - NE
Negative (vehicle) control [300] 3h/20h 100  - 3.3
500 µg/mL 3h/20h 31 +b NE
250 µg/mL [162] 3h/20h 42 +b 10.5**
125 µg/mL [156] 3h/20h 51 +a,b 7.1
62.5 µg/mL [181] 3h/20h 58 - 10.5**
31.25 µg/mL [300] 3h/20h 73 - 13.7***
15.625 µg/mL [300] 3h/20h 78 - 8.0*
Positive control [228] 3h/20h 67 - 18.9***

Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration (µg/mL)

[number of analysed cells]

Time of treatment/sampling RICC# (%)  Insolubility##  Mean % aberrant cells### 
Untreated control 3h/20h  105 - NE
Negative (vehicle) control [300] 3h/20h  100 - 6.3
1000 µg/mL 3h/20h  15 +b NE
750 µg/mL 3h/20h  37 +b NE
500 µg/mL [174] 3h/20h  47 +b 13.8**
250 µg/mL [244] 3h/20h  53 +b 14.8**
125 µg/mL [300] 3h/20h  67 +a,b 11.7***
62.5 µg/mL [300] 3h/20h  82 - 10.7***
31.25 µg/mL 3h/20h  79 - NE
Positive control [61] 3h/20h 60 - 82.0***

Summary table of Chromosome Aberration Assay 2 without metabolic activation

Concentration (µg/mL)

[number of analysed cells]

Time of treatment/sampling

RICC#

(%)

Insolubility## Mean % aberrant cells###
Untreated control 20h/28h 97 - NE
Negative (vehicle) control [300] 20h/28h 100 - 2.3
100 µg/mL [180] 20h/28h 45 + 14.4***
75 µg/mL [161] 20h/28h 58 + 9.3**
50 µg/mL [159] 20h/28h 65 +a 1.9
25 µg/mL [182] 20h/28h 85 - 3.3
12.5 µg/mL 20h/28h 94 - NE
6.25 µg/mL 20h/28h 99 - NE
Positive control [129] 20h/28h 65 - 19.4***

Summary table of Chromosome Aberration Assay 2 with metabolic activation

Concentration (µg/mL)

[number of analysed cells]

Time of treatment/sampling RICC# (%)  Insolubility## Mean % aberrant cells###
Untreated control 3h/28h 109 - NE
Negative (vehicle) control [300] 3h/28h 100 - 2.0
100 µg/mL [238] 3h/28h 44 + 21.0***
75 µg/mL [261] 3h/28h 50 + 15.3***
50 µg/mL [170] 3h/28h 69 +a 29.4***
25 µg/mL [300] 3h/28h 72 - 2.3
12.5 µg/mL [300] 3h/28h 88 - 2.0
6.25 µg/mL   3h/28h 99 - NE
Positive control [243] 3h/28h 66 - 14.8***

Negative (vehicle) control (DMSO)

Positive control

NE: not evaluated

RICC: relative increase in cell counts

# compared to the negative (vehicle) control

## in the final treatment medium at the end of the treatment

### excluding gaps

a: minimal amount of precipitate

b: discoloured medium/minimally discoloured medium

*p<0.05 comparing numbers of aberrant cells excluding gaps with corresponding negative control

**p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

***p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Applicant's summary and conclusion

Conclusions:
Diytterbium trioxide induced a significant level of chromosome aberrations in the performed experiments with and without metabolic activation. Therefore, diytterbium trioxide was considered as clastogenic in this test system (Chimese hamster V79 cells).