Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro

In vitro mutagenicity in bacteria

A single reliable (Klimisch 1) key study is available (Váliczkó, 2017), performed according to OECD guideline 471 and conform GLP requirements. In this study, ytterbium oxide did not show mutagenic activity in the applied bacterium tester strains in the absence or presence of metabolic activation under the conditions of the test system.

In vitro chromosome aberration study

A single reliable (Klimisch 1) key study is available (Hargitai, 2018b), performed according to OECD guideline 473 and conform GLP requirements. In this study, ytterbium oxide induced a significant level of chromosome aberrations in Chinese hamster V79 cells with and/or without metabolic activation. Ytterbium oxide was considered as clastogenic in this test system.

In vitro gene mutation study in mammalian cells (HPRT assay)

A single reliable (Klimisch 1) key study is available (Hargitai, 2018a), performed according to OECD guideline 476 and conform GLP requirements. In this study, ytterbium oxide did not show mutagenic activity under the conditions of the HPRT assay in CHO K1 Chinese hamster ovary cells in the absence or presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

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
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
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.
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).
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.
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'.

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

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).
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-03-29 to 2018-09-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
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: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and short period of the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was formulated in the selected vehicle to provide a suitably concentrated stock formulation. The necessary amount of test item was weighed in calibrated centrifuge tube (no correction for purity of the test item was applied), 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. 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.

CORRECTION FACTOR
No correction for purity of the test item was applied.
Target gene:
hprt (hypoxanthine-guanine phosphoribosyl transferase) enzyme locus located on the X chromosome of Chinese Hamster Ovary (CHO) cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Sub-line K1
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection (Manassas, Virginia, United States)
ATCC No.: CCL-61
Prior to use in this test, the culture was cleansed of pre-exisitng mutant cells by culturing in HAT medium on 22 April 2016. Cells were stored as frozen stocks in a liquid nitrogen tank.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Four types of Ham's medium were prepared:
F12-1: 1% v/v foetal bovine serum (FBS, heat inactivated), 0.01 mL/mL L-glutamine, 0.01 mL/mL antibiotic-antimycotic solution (10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)
F12-5: 5% v/v FBS, L-glutamine and antibiotic-antimycotic solution see F12-1
F12-10: 10% v/v FBS, L-glutamine and antibiotic-antimycotic solution see F12-1
F12-SEL*: 10% v/v FBS, L-glutamine and antibiotic-antimycotic solution see F12-1
*: Hypoxanthine-free Ham's F-12 medium was used for preparation of the selection culture medium.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, checks carried out for each batch of frozen stock; the cell line was tested negative
- Periodically checked for karyotype stability: no data
- Periodically 'cleansed' against high spontaneous background: yes, prior to use in the test, the culture was cleansed of pre-exisitng mutant cells by culturing in HAT medium on 22 April 2016. Cells were stored as frozen stocks in a liquid nitrogen tank. For each experiment, one or more vials were thawed rapidly.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9, induced by treatment with a mixture of phenobarbital (PB) and β-naphthoflavone (BNF)
Test concentrations with justification for top dose:
Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. In the preliminary experiment, a 5-h treatment in the presence and absence of S9-mix and a 24-h treatment in the absence of S9-mix were performed with a range of test concentrations to determine toxicity immediately after the treatments.
The concentrations in the mutation assays were as follows:
Assay 1 and 2:
+S9: 2.74, 8.23, 24.69, 74.07, 222.2, 666.6, 2000 µg/mL
-S9: 31.25, 62.5, 125, 250, 500, 750, 1000, 1500, 2000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Based on the available solubility information of another study (Citoxlab 16/198-020C), the test item was soluble at a 200 mg/mL concentration in dimethyl sulfoxide (DMSO), and the physical appearance of the formulation was suitable for the test. Other recommended solvents were not appropriate for this test item (Citoxlab 16-198-007M). Therefore, DMSO was selected as vehicle for the study.
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
vehicle control, DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
DMSO was used as a vehicle of the positive control
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- in medium in plates
- at least 2E+06 cells were placed in each of a series of sterile dishes and incubated for about approximately 24 h before treatment at 37°C in a humidified atmosphere

DURATION
- Pre-incubation period: Before treatment: 24 h (all treatments). After treatment and before start of expression period: 19 h (only for the 5-h treatments)
- Exposure duration: 5 h in the presence and absence of S9 in Assay 1; 5 h in the presence of S9 in Assay 2; 24 h in the absence of S9 in Assay 2
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): At the end of the expression period (Day 8), the cell number in the samples was adjusted to 4E+05 cells/mL. 1 mL of the adjusted cell suspension and 4 mL of F12-SEL medium were added into Petri dishes (diameter approximately 100 mm, 5 parallels per sample) for each sample. An additional 5 mL of F12-SEL medium containing 20 μg/mL 6-thioguanine (abbreviation: 6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 μg/mL) to determine mutation frequency. Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5 ± 0.3% CO2 in air) for 7 days for colony growing.
- Fixation time (start of exposure up to fixation or harvest of cells): After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.

SELECTION AGENT (mutation assays): 6-thioguanine, to determine mutation frequency

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.

NUMBER OF CELLS EVALUATED: The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2E+06 cells: 5 plates at 4E+05 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 1E+06 clonable cells.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency: Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
Rationale for test conditions:
no data, per OECD guideline 476
Evaluation criteria:
The test item was considered to be mutagenic in this assay if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p < 0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
According to the relevant OECD guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.

The assay was considered valid if all the following criteria are met:
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the historical control data.
2. The positive control chemicals induced a clear increase in mutant frequency.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8.
4. At least four test item concentrations in duplicate cultures were presented.
Statistics:
The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data were also checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency).
In the statistical analysis, negative trends were not considered significant.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Sub-line K1
Metabolic activation:
with and without
Genotoxicity:
negative
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 (PRELIMINARY EXPERIMENT)
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 µg/mL (the recommended maximum concentration).
Insolubility was detected in the preliminary experiment. The concentrations selected for the main experiments were based on results of the preliminary test according to the OECD No. 476 guideline instructions. Lower test concentrations were generally seperated by a factor of two, more closely spaced concentrations were selected in the expected cytotoxic range. At least seven concentrations were selected for the main experiments.

MUTATION ASSAYS
In Assay 1, insolubility (precipitate / minimal amount of precipitate) was detected in the final treatment medium at the end of the treatment in the 2000 – 74.07 µg/mL concentration range in the experiment with metabolic activation and in the 2000 – 62.5 µg/mL concentration range in the experiment without metabolic activation. The precipitation did not interfere with the cell counting after treatment. There were no large changes in pH and osmolality after treatment in any cases.
In the presence of S9-mix (5-hour treatment), marked but not extreme cytotoxicity of the test item was observed (the highest evaluated concentration of 2000 µg/mL showed 25% adjusted relative survival). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data. No dose response relationship was observed (a trend analysis showed no effect of treatment).
In the absence of S9-mix (5-hour treatment), excessive cytotoxicity of the test item was observed: cells of the 2000 and 1500 µg/mL concentrations died after treatment or by the end of the expression period. The first two surviving concentrations of 1000 and 750 µg/mL showed extreme cytotoxicity (adjusted relative survival values of 1% and 3% on the plates, respectively), thus they were excluded from evaluation. An evaluation was made using data of the five concentrations with acceptable levels of cytotoxicity in the concentration range of 500-31.25 µg/mL (the highest evaluated concentration showed 15% adjusted relative survival). No statistically significant increases in the mutation frequency were observed at any examined concentration when compared to the negative (vehicle) control data. No dose response relationship was observed (a trend analysis showed no effect of treatment).
In Assay 2, insolubility (precipitate / minimal amount of precipitate, oily film / minimal amount of oily film) was detected in the final treatment medium at the end of the treatment in the 2000 – 74.07 µg/mL concentration range in the experiment with metabolic activation and in the 2000 – 62.5 µg/mL concentration range in the experiment without metabolic activation. The precipitation did not interfere with the cells counting. There were no large changes in pH and osmolality after treatment in any case.
In the presence of S9-mix (5-hour treatment), marked but not extreme cytotoxicity of the test item was observed (the highest concentration of 2000 µg/mL showed 10% adjusted relative survival). Thus an evaluation was made using data of all seven concentrations. A statistically significant increase (at p<0.05) in the mutation frequency was observed at the 2.74 µg/mL concentration when compared to the negative (vehicle) control results. However, the observed data (11.3 x 10-6) were still within the expected range (between 5-20 x 10-6 as suggested by the OECD No. 476 guideline) and within the historical control range, thus indicating no biological relevance. Furthermore, no dose response relationship was observed (a trend analysis showed no effect of treatment). Therefore, the observed results were considered as not being test item related.
In the absence of S9-mix (24-hour treatment), similarly to the first test, excessive cytotoxicity of the test item was observed: cells of the 2000 and 1500 µg/mL concentrations died after treatment or by the end of the expression period. The first two surviving concentrations of 1000 and 750 µg/mL showed extreme cytotoxicity (adjusted relative survival values were 3% and 9%, respectively), thus they were excluded from evaluation. An evaluation was made using data of the five concentrations with acceptable levels of cytotoxicity in the concentration range of 500-31.25 µg/mL (the highest evaluated concentration showed 18% adjusted relative survival). No statistically significant increases in the mutation frequency were observed at any examined concentration when compared to the negative (vehicle) control data. No dose response relationship was observed (a trend analysis showed no effect of treatment).
Any other sporadic, statistically non-significant differences were examined for consistency; none of them were repeatable when comparing Assay 1 and Assay 2. Furthermore, all the observed mutation frequency values were comparable with the general historical control range and in line with the OECD No. 476 guideline. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between treated samples and negative (vehicle) controls.

Validity of the mutation assays:

The spontaneous mutation frequency of the negative (vehicle) control samples was within the historical control range, and the observed values were in harmony with the expected range (generally between 5-20 x 10-6) as shown in the OECD No. 476 guideline.

The positive controls (DMBA in the presence of metabolic activation and EMS in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays.

The cloning efficiencies for the negative (vehicle) controls on Days 1 and 8 were within the target range of 60-140% and 70-130% in all assays .

The tested concentration range in the study was considered to be adequate as they covered the range from cytotoxicity to no cytotoxicity (in each case the highest evaluated concentration was in the proper range (showing approx. 10-20% of adjusted relative survival) according to the requirement of the OECD No. 476 guideline). The examined range also covered the range from insolubility to no insolubility.

At least five test item concentrations (in duplicate) were evaluated in each experiment. 

The overall study was considered valid.

Conclusions:
The HPRT Assay with diytterbium trioxide performed on CHO K1 Chinese hamster ovarian cells was considered to be valid and reflect the real potential of the test item to cause mutations in the cultured mammalian cells used in this study. 
Treatment with the test item did not result in a statistically and biologically significant dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9) in this study. 
In conclusion, no mutagenic effect of diytterbium trioxide was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-07-05 to 2017-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: Based on the results of the preliminary tests, a 100 mg/mL stock formulation was prepared in dimethyl sulfoxide (DMSO). Seven test concentrations were prepared by successive dilutions of the stock formulation, to obtain lower doses. The maximum test concentration was 5000 μg test item/plate.
- Dimethyl sulfoxide (DMSO) was used as solvent to prepare the stock formulation of the test material. Test suspensions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock formulation using the selected solvent and were used within 4 hours after preparation.
- No correction for purity of the test item was applied.

FORM AS APPLIED IN THE TEST (if different from that of starting material): formulation in dimethyl sulfoxide
Target gene:
histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial S9 fraction (rat liver)
Test concentrations with justification for top dose:
100 mg/mL (5000 μg/plate), 31.62 mg/mL (1581 μg/plate), 10 mg/mL (500 μg/plate), 3.162 mg/mL (158.1 μg/plate), 1 mg/mL (50 μg/plate), 0.3162 mg/mL (15.81 μg/plate) and 0.1 mg/mL (5 μg/plate)

The selection of the doses was based on the results of a range finding study, in which doses of 10, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate were used.
The observed number of revertant colonies was in the normal range. Minor differences compared to the solvent control numbers were observed.
However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
Slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate.
Inhibitory or toxic effects of the test item were not detected in the Preliminary Range Finding Test.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The appropriate vehicle (solvent) and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO), ethanol, acetone and N,N-dimethylformamide (DMF). The formulations at 100 mg/mL concentration using distilled water, ethanol or acetone as vehicle were suspensions with fast sedimentation. Slower sedimentation was observed in the formulations using DMSO or DMF as vehicle. Due to the better biocompatibility, DMSO was selected as vehicle for the test with continuous stirring of the formulations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
INITIAL MUTATION TEST
- Method of application: in agar (plate incorporation)
- Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes: top agar 2000 μL; vehicle or test item formulation (or reference controls) 50 μL; overnight culture of test strain 100 μL; phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

CONFIRMATORY MUTATION TEST (pre-incubation method)
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since no biologically relevant increase in the number of revertant colonies was observed in the Initial Mutation Test.
- Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
- Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of nonactivated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

EVALUATION OF EXPERIMENTAL DATA
- The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft Excel TM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

In the main tests each sample (including the controls) was tested in triplicate.
Evaluation criteria:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.
Statistics:
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
- The observed number of revertant colonies was in the normal range. Minor differences compared to the solvent control numbers were observed. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
- Slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate.
- Inhibitory or toxic effects of the test item were not detected in the Preliminary Range Finding Test.
- Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

ADDITIONAL INFORMATION
- Slight precipitate was observed at 5000 μg/plate with and without metabolic activation in all tester strains in the Initial Mutation Test and in all Salmonella typhimurium strains in the Confirmatory Mutation Test.
- Slight decreases of the revertant counts were observed compared to the solvent control sporadically in the study. However, the mean number of revertant colonies was within the historical control range, thus they were considered as biological variability of the test system.
- No signs of inhibitory, cytotoxic effect of the test item (such as reduced background lawn development and/or reduced number of revertant colonies) were observed in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any concentrations with or without metabolic activation.
- In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent
controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
- In the Initial Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain with metabolic activation at the concentration of 50 μg/plate. The mutation factor value was 1.52. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
- In the Confirmatory Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 500 μg/plate concentration with metabolic activation. The calculated mutation factor value at this dose level was 1.60. However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
- Slight increases in the numbers of revertant colonies were detected compared to the solvent control during the study in some sporadic cases. However, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and were within the historical control range. They were considered as reflecting the biological variability of the test.

Validity of the tests

Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range-finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event.

The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item (diytterbium trioxide) had no mutagenic activity in the applied bacterium tester strains under the conditions used in this study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo

No in vivo data are available at this point in time, but due to the observations made in the in vitro mammalian chromosome aberration test, it was decided to include a test proposal for an in vivo study according to OECD guideline 474 (in vivo mammalian erythrocyte micronucleus test).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Study period:
Awaiting feedback from ECHA
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : ytterbium trioxide

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies : Three GLP-compliant, in vitro genetic toxicity studies are available. A key in vitro mutagenicity study in bacteria was performed according to OECD guideline 471. Ytterbium trioxide did not show mutagenic activity in the applied bacterium tester strains in the absence or presence of metabolic activation under the conditions of the test system. A key in vitro mammalian chromosome aberration test was performed according to OECD guideline 473. The test substance was considered as clastogenic in the test system (Chinese hamster V79 cells). A key in vitro gene mutation study in mammalian cells (HPRT assay) was performed according to OECD guideline 476. In this study, no mutagenic effect of ytterbium trioxide was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay in CHO K1 Chinese hamster ovary cells.
- Available non-GLP studies: No studies available
- Historical human data: No data available
- (Q)SAR: No data available
- In vitro methods: No other adequate in vitro method, other than the ones already conducted is available.
- Weight of evidence: No data is available which would allow a weight of evidence approach.
- Grouping and read-across: No chemical grouping or read-across approach was identified.
- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: Not applicable
- Other reasons [if applicable]: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- The test proposal is fully compliant with ECHA guidance document R.7a (Dec 2016). It is not possible to waive the study based on Column 2 adaptations of the REACH regulation.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- No additional information
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Genetic toxicity in vitro

In vitro mutagenicity in bacteria

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay (Váliczkó, 2017) according to OECD guideline 471 and conform GLP requirements. The experiments were carried out using histidine-requiring auxotrophic strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotrophic strain of Escherichia coli (WP2 uvrA) in the presence and absence of metabolic activation (cofactor-supplemented post-mitochondrial S9 fraction (rat liver)) prepared from the livers of phenobarbital/beta-naphthoflavone-induced rats. The study included a preliminary compatibility test, a preliminary concentration range finding test, an initial mutation test and a confirmatory mutation test. Based on the results of a solubility test, the test item was formulated in DMSO. Based on the results of the range finding test, the test item concentrations in the initial mutation test and in the confirmatory mutation test (5 strains) were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate. The tests were considered to be valid. The data demonstrate that the test item had no mutagenic activity in the applied bacterium tester strains in the absence or presence of metabolic activation under the test conditions used in this study. Untreated, negative (solvent) and positive controls were run concurrently. The results obtained in the control treatments were all within the historical control range. This study is considered reliable (Klimisch 1) and is considered as the key study for endpoint coverage.

In vitro chromosome aberration study

Ytterbium oxide was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells (Hargitai, 2018b).

The test item was formulated in DMSO and it was examined up to cytotoxic concentrations as well as solubility limit according to the OECD guideline recommendations.

In Chromosome Aberration Assay 1, a 3-h treatment with metabolic activation (in the presence of S9-mix) and a 3-h treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 h after the beginning of the treatment in both cases. The examined concentrations of the 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 Chromosome Aberration Assay 2, a 3-h treatment with metabolic activation (in the presence of S9-mix) and a 20-h treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 h 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 (experiments with and without 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, and the test was considered valid, the test item was considered as clastogenic under the conditions of the study.

In vitro gene mutation study in mammalian cells (HPRT assay)

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of ytterbium oxide to cause gene mutations (Hargitai, 2018a). Treatments were carried out for 5 hours with and without metabolic activation (+/-S9 mix) and for 24 hours without metabolic activation (-S9 mix) in 2 independent assays.

Dimethylsulfoxide (DMSO) was used as the vehicle for the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test.

At least seven test item concentrations were evaluated in duplicate in experiment 1 and 2.

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7-day expression period following the treatment) and mutagenicity (colony-forming ability at the end of the 7-day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

The overall study was considered to be valid.

Treatment with the test item did not result in a biologically significant dose-dependent increase in mutation frequencies in the presence or absence of a rat metabolic activation system (S9) in this study.

In conclusion, no mutagenic effect of ytterbium oxide was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Genetic toxicity - in vivo

No in vivo data are available at this point in time, but due to the observations made in the in vitro mammalian chromosome aberration test, it was decided to include a test proposal for an in vivo study according to OECD guideline 474 (in vivo mammalian erythrocyte micronucleus test).

Justification for classification or non-classification

Based on the available information, ytterbium oxide was considered to be clastogenic in an in vitro mammalian chromosome aberration test system, whereas negative results were obtained in an Ames study and an in vitro gene mutation test in mammalian cells (HPRT). Overall, the results on genetic toxicity are therefore considered to be inconclusive. A test proposal for an in vivo study according to OECD guideline 474 (in vivo mammalian erythrocyte micronucleus test) is therefore included in the dossier.