Dilutetium oxide silicate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro mutagenicity in bacteria

A single reliable (Klimisch 1) key study is available (Váliczkó, 2017a), performed according to OECD guideline 471 and conform GLP requirements. In this study, dilutetium oxide silicate 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 mammalian chromosome aberration test

One key study is available (Klimisch 1, Kovács, 2018b). This GLP study was performed according to OECD guideline 473 and Commission Regulation (EU) 2017/735 of 14 February 2017 B.10. In this study, dilutetium oxide silicate induced a significant level of chromosome aberrations in Chinese hamster V79 cells with and/or without metabolic activation. Dilutetium oxide silicate was considered as clastogenic in this test system.

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

One key study is available (Klimisch 1, Kovács, 2018a). This GLP study was performed according to OECD guideline 476 and Commission Regulation (EC) No. 440/2008, B.17. In this study, no mutagenic effect of dilutetium oxide silicate 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2016-11-15 to 2017-02-16

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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 test, a 100 mg/mL stock formulation was prepared in distilled water, which was diluted by serial dilutions in seven steps to obtain eight dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
- Distilled water was used as solvent to prepare the stock solution 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.
- Correction for purity of the test item was applied with a correction factor of 1.05 considering the composition. Concentration was calculated to anhydrous form as requested by the Sponsor. The correction factor was used at dose formulation preparation.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension in distilled water

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 concentration of 5000 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: distilled water
- 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) and N,N-dimethylformamide (DMF). The formulations at 100 mg/mL concentration using distilled water, DMSO or DMF as vehicles were suspensions with fast sedimentation. Due to the better biocompatibility distilled water 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: plate incorporation method
- 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 positive effect 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.

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.

Criteria for a Negative Response:
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.

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 concentration of 5000 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 Confirmatory Mutation Test.
- In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain without metabolic activation at the concentration of 158.1 μg/plate. The mutation factor value was 1.76. 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 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration without metabolic activation. The calculated mutation factor value at this dose level was 1.52. 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.
- 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.
- Slight decreases of the revertant counts were observed compared to the solvent control sporadically in the Confirmatory Mutation Test. However, the mean number of revertant colonies was within the historical control range, thus they were considered as biological variability of the test system.
- Slight increases in the numbers of revertant colonies were detected compared to the solvent control during the study in several 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 (lutetium oxide silicate) had no mutagenic activity in the applied bacterium tester strains under the conditions used in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-02-17 to 2018-06-15

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:

Commission Regulation (EU) 2017/735 of 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%)
- 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
Stock formulations of the test item (200 mg/mL) were prepared as follows. The necessary amount of grounded test item was weighed into a calibrated volumetric flask. The required volume of the vehicle (distilled water) was added and the formulation was mixed until homogeneity was reached. From the stock formulation several dilutions were prepared using the selected vehicle to prepare dosing formulations. Sterile vehicle was used for formulation. The dosing formulations were prepared immediately before the treatment of the cells in a sterile hood.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and short period of study.

CORRECTION FACTOR
Correction for the composition of the test item was performed applying a correction factor of 1.05 to achieve the target concentration of the formulation expressed as anhydrous form.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cells Cultures)
- Sex: male
- Suitability of cells: 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 12-14 h).
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 β-naphthoflavone (BNF)

Test concentrations with justification for top dose:

The study included two Concentration Selection Cytotoxicity Assays and three Chromosome Aberration Assays.
-S9 and +S9: 3.70, 11.11, 33.33, 62.5, 100 µg/mL (Assay 1, 2 and 3)
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test. 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).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Due to the better biocompatilibity distilled water was selected as the vehicle of the study.

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

yes

Remarks:

10 µL/mL 1% (v/v) distilled water

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 a 5 x 1E+05 cells/dish concentration.

DURATION
- Exposure duration: Cells were incubated for approximately 24 hours at 37°C in 10 mL of culture medium (DMEM-10).
- Expression time (cells in growth medium): 17.5 to 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) 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

METHODS 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.
- Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
*Note: The examination of slides from a culture was halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

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. ca. 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 osmolarity 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:
Based on the available solubility information (which was performed in the previous study of Citoxlab study code 16/251-007M), the test item formed suspensions with fast sedimentation at a 100 mg/mL concentration in distilled water, in dimethyl sulfoxide (DMSO) and in N,N-dimethylformamide (DMF). Due to the better biocompatibility, distilled water was selected as the vehicle for the study. The highest examined concentration in the preliminary test was 2000 μg/mL.
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-h treatment with and without metabolic activation, 20-h harvesting time; and Assay B: 20-h treatment without metabolic activation, 20-h harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system.
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-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 100, 62.5, 33.33, 11.11 and 3.70 μg/mL (experiment with and without metabolic activation).
In Assay 1, no insolubility was detected in the experiment with and without metabolic activation. There were no large changes in the pH and osmolality with and without metabolic activation. Cytotoxicity was observed in the experiment with and without metabolic activation (RICC value of the highest evaluated concentration with metabolic activation was 46% and without metabolic activation was 42%). Concentrations of 100, 33.33 and 11.11 μg/mL (a total of three) were evaluated in the experiment with and without metabolic activation.
In case of the experiment without metabolic activation, no statistically significant change in the aberrant cells as compared to the negative control were observed at the three concentrations evaluated. In case of the experiment with metabolic activation, at a concentration of 100 μg/mL, statistically significant increases were observed in the number of aberrant cells (p < 0.001) when compared with the appropriate negative (vehicle) control values.
The biological significance of the observed increase in frequency of aberrant methaphases depends on the level of toxicity caused by the test item at those test points. Since the statistically significant increase has been observed at one concentration (100 µg/mL) with metabolic activation, showing less than 60% toxicity, these data meet the criteria of a positive response. However, since this was only observed at one concentration with metabolic activation and at none of the concentrations tested without metabolic activation, the results are deemed inconclusive. This resulted in further investigations.

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 20 h after the beginning of the treatment in both cases. The examined concentrations of the test item were 100, 62.5, 33.33, 11.11 and 3.70 μg/mL (experiment with and without metabolic activation).
In Assay 2, no insolubility was detected in the experiment with and without metabolic activation. There were no large changes in the pH and osmolality with and without metabolic activation. Cytotoxicity was observed in the experiment with and without metabolic activation (RICC value of the highest evaluated concentration with metabolic activation was 50% and without metabolic activation was 44%). Concentrations of 100, 33.33 and 11.11 μg/mL (a total of three) were evaluated in the experiment with and without metabolic activation.
In case of the experiments with and without metabolic activation, at 100 and 33.33 μg/mL concentrations statistically significant increases were observed in the number of aberrant cells (0.05 > p > 0.01 or 0.01 > p > 0.001) when compared with the appropriate negative (vehicle) control values.
Since the statistically significant increases have been observed at concentrations with and without metabolic activation showing less than 60% toxicity, these data meet the criteria of a positive response. Therefore, the test item was considered to have given a positive response in this experiment.

Due to equivocal results in the study (increases were not completely reproducible between replicate cultures and between tests, when treatment conditions were the same) an additional experiment (Assay 3) was performed for validity of the study using the same experimental conditions as in Assay 2 to give more information about the test item effect.

In Chromosome Aberration Assay 3, 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 20 h after the beginning of the treatment in both cases. The examined concentrations of the test item were 100, 62.5, 33.33, 11.11 and 3.70 μg/mL (experiment with and without metabolic activation).
In Assay 3, no insolubility was detected in the experiment with and without metabolic activation. There were no large changes in the pH and osmolality with and without metabolic activation. Cytotoxicity was observed in the experiment with and without metabolic activation (RICC value of the highest evaluated concentration with metabolic activation was 49% and without metabolic activation was 43%). Concentrations of 100, 33.33 and 11.11 μg/mL (a total of three) were evaluated in the experiment with and without metabolic activation.
In case of the experiments without metabolic activation, at 100, 33.33 and 11.11 µg/mL, statistically significant increases were observed in the number of aberrant cells (p < 0.001, 0.05 > p > 0.01, 0.01 > p > 0.001, respectively) when compared to the appropriate negative (vehicle) control values. In the case of the experiments with metabolic activation, at 100 and 33.33 µg/mL, statistically significant increases were observed in the number of aberrant cells (p < 0.001, 0.05 > p > 0.01, respectively) when compared with the appropriate negative (vehicle) control values. No statistically significant increase in aberrant cells was observed at 11.11 µg/mL with metabolic activation, when compared with the negative control values.
Since the statistically significant increases have been observed at concentrations with and without metabolic activation showing less than 60% toxicity, these data meet the criteria of a positive response. Therefore, the test item was considered to have given a positive response in this experiment.

Several treatment concentrations caused statistically significant increases in the number of cells with structural chromosome aberrations in Assay 1, Assay 2 and Assay 3, with and/or without metabolic activation, when compared with the appropriate negative (vehicle) control values (for more details, please refer to the Table in the section "Any other information on results, incl. tables"). In conclusion, dilutetium oxide silicate induced overall a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and/or without metabolic activation. Thus dilutetium oxide silicate was considered as clastogenic in this 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 treatment with dilutetium oxide silicate. No endoreduplicated metaphases were detected in the performed experiments.

Validity of the study:

The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. No insolubility was detected in all experiments with and without metabolic activation, while cytotoxicity was detected in any experiment with and without metabolic activation. The evaluated concentration ranges of Assay 1, Assay 2 and Assay 3 were considered to be adequate, as they covered the range from toxicity to no or little toxicity.

Three test item concentrations were evaluated in each experiment.

The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the general historical control range of the testing laboratory. The cell line and methods are used routinely in this laboratory, normally with negative results, hence the system is not considered to be overly sensitive.

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.

The study was considered to be valid.

Summary table of number of aberrant metaphases:

The obtained RICC (%) data, compared to the negative (vehicle) control, are indicated between brackets.

Test group	No. aberrant metaphases #
	Assay 1		Assay 2		Assay 3
	3h-/20h	3h+/20h	20h-/20h	3h+/20h	20h-/20h	3h+/20h
negative (vehicle) control	1 (100)	4 (100)	7 (100)	1 (100)	4 (100)	4 (100)
	5	2	4	3	2	7
100 µg/mL	7 (42)	25*** (46)	19** (44)	11* (50)	17*** (43)	20*** (49)
	6	15	8	1	14	18
33.33 µg/mL	6 (80)	7 (70)	8* (57)	7* (73)	10* (84)	14* (71)
	4	7	16	5	7	13
11.11 µg/mL	4 (92)	5 (86)	3 (94)	3 (89)	10** (83)	5 (84)
	3	6	2	6	12	7
positive control	25*** (79)	25*** (71)	25*** (52)	25*** (63)	25*** (52)	25*** (51)
	23	25	25	25	25	25

3h-/20h: 3-h treatment without S9-mix, harvesting 20 h from the beginning of treatment

3h+/20h: 3-h treatment with S9 mix, harvesting 20 h from the beginning of treatment

20h-/20h: 20-h treatment without S9 mix, harvesting 20 h from the beginning of treatment

* 0.05 > p > 0.01 comparing number of aberrant cells excluding gaps with corresponding negative control

** 0.01 > p > 0.001 comparing number of aberrant cells excluding gaps with corresponding negative control

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

# excluding gaps

## compared to the negative (vehicle) control

Summary table of the results:

	without metabolic activation (S9 mix)	with metabolic activation (S9 mix)
Assay 1	negative	positive at 100 µg/mL negative at 33.33 and 11.11 µg/mL
Assay 2	positive at 100 and 33.33 µg/mL negative at 11.11 µg/mL	positive at 100 and 33.33 µg/mL negative at 11.11 µg/mL
Assay 3	positive at 100, 33.33 and 11.11 µg/mL	positive at 100 and 33.33 µg/mL negative at 11.11 µg/mL

Conclusions:

The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.
Despite inconsistent results between assays, dilutetium oxide silicate induced overall a significant level of chromosome aberrations in Chinese hamster V79 cells with and/or without metabolic activation. Thus, dilutetium oxide silicate was considered as clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2018-01-16 to 2018-06-15

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
Stock formulations of the test item (200 mg/mL) were prepared as follows. The necessary amount of grounded test item was weighed into a calibrated volumetric flask. Approximately 80% of the required volume of vehicle (solvent) was added and the formulation was stirred until homogeneity was reached, then the volume was adjusted to the required final level. From the stock solution, several dilutions were prepared using the selected vehicle (solvent) to prepare dosing solutions for lower doses. The vehicle (solvent) was filtered sterile using a 0.22 μm syringe filter before the preparation of the dosing formulations in each case. The stock solutions as well as all dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.

CORRECTION FACTOR
Correction for the composition of the test item was performed applying a correction factor of 1.05 to achieve the target concentration of the formulation expressed as anhydrous form.

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-existing 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 F12 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 , check 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-existing 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, 666.7, 2000 µg/mL
-S9: 2.74, 8.23, 24.69, 74.07, 222.2, 666.7, 1500, 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Based on the available data (Citoxlab study codes: 16/251-007M and 16/251-020C), distilled water was considered suitable as vehicle for the test.

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

DMSO was used as vehicle of the positive control materials

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.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples were taken into consideration if necessary). Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. In cases with survival lower than 10%, extreme caution is taken in the interpretation.
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:
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-h treatment in the presence and absence of S9-mix and 24-h 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 the observed cytotoxicity and/or insolubility in the preliminary experiments according to the OECD guideline 476 instructions (up to the maximum recommended concentration). Lower test concentrations were separated by a factor of three, but more closely spaced concentrations were selected in the expected cytotoxic concentration range in case of experiments without metabolic activation. At least seven concentrations were selected for the main experiments.

MUTATION ASSAYS:
In Assay 1, insolubility (precipitate or minimal amount of precipitate) was detected in the 2000–74.07 μg/mL or 2000-24.69 μg/mL concentration range in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation, respectively. There were no large changes in pH and osmolality after treatment in any case.
In the presence of S9-mix (5-h treatment), no marked cytotoxicity of the test item was observed (the highest concentration of 2000 μg/mL showed a relative survival of 107% on the survival plates). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any of the examined concentrations when compared to the negative (vehicle) control data and there was no dose response relationship with the treatment (a trend analysis showed no effect of treatment).
In the absence of S9-mix (5-h treatment), marked cytotoxicity of the test item was observed (the highest concentration of 2000 μg/mL and the following concentration of 1500 μg/mL showed a relative survival of 47% and 19%, respectively, on the survival plates). An evaluation was made using data of all eight concentrations. A statistically significant increase (at p < 0.01 level) was observed in this experiment at the 8.23 μg/mL concentration, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (12E-06) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5E-06 to 20E-06). No dose response relationship with the treatment was observed (a trend analysis showed no effect of treatment).
In Assay 2, insolubility (precipitate or minimal amount of precipitate) was detected in the 2000–74.07 μg/mL or 2000-24.69 μg/mL concentration range in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation, respectively. There were no large changes in pH and osmolality after treatment in any case.
In the presence of S9-mix (5-h treatment), similarly to the first test, no marked cytotoxicity of the test item was observed (the highest concentration of 2000 μg/mL showed a relative survival of 106% on the survival plates), thus an evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any of the examined concentrations when compared to the negative (vehicle) control data and there was no dose response relationship with the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results seen in Assay 1 with metabolic activation.
In the absence of S9-mix (24-h treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 2000 μg/mL and the following concentration of 1500 μg/mL showed a relative survival of 42% and 36%, respectively, on the survival plates). An evaluation was made using data of all eight concentrations. No statistically significant increases in the mutation frequency were observed at any of the examined concentrations when compared to the negative (vehicle) control data and there was no dose response relationship with the treatment (a trend analysis showed no effect of treatment). This result confirmed the negative results of Assay 1 without metabolic activation.
The 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 within the general historical control range*. 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.
*Note: The spontaneous mutant frequency is generally between 5E-06 and 20E-06 according to the guideline.

Validity of the mutation assays:

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays, and the observed values were in the expected range (5E-06 to 20E-06) as shown in OECD guideline 476.

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 the defined acceptance criteria of the relevant OECD guideline regarding cytotoxicity produced by the highest evaluated concentrations (approximately 80-90% toxicity, i.e. approximately 10-20% relative survival) or the recommended maximum concentration were considered to be fulfilled.

At least seven test item concentrations were evaluated in duplicate in each experiment.

The overall study was considered valid.

Conclusions:

The HPRT assay with dilutetium oxide silicate performed on CHO K1 Chinese hamster ovarian cells was considered to be valid and to 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 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 dilutetium oxide silicate was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Genetic toxicity in vivo

Description of key information

No in vivo data are available at this point in time. Although positive results were obtained in the in vitro mammalian chromosome aberration test, these results appear as an outlier considering altogether previous results observed in in vitro and in vivo genetic toxicity studies performed with various REACH registered rare earth compounds. Therefore it was decided not to include a test proposal for an in vivo study to investigate the in vivo clastogenic potential of lutetium oxide silicate.

The justification is attached to the section 13.2 (Genetic toxicity discussion) and in the attachment part.

 

 

Justification for classification or non-classification

Based on the available information, it is concluded that the test substance lutetium oxide silicate should not be classified for mutagenicity, as the results of the in vitro tests are considered conclusive but not sufficient for classification.