Zirconium oxide, erbium and yttrium doped

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria

Read-across to structurally similar substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8)

The test material did not show any mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

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:

25 May 2016 to 30 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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
- A 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps spaced by factors of 2, 2.5 and approximately √10.
- Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation.
- The formulations were stirred with magnetic stirrer during formulation and treatment.
- No purity conversion was applied in the study.

Target gene:

Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537); tryptophan locus (E. coli strain WP2 uvrA)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital-/β-naphthoflavone induced rat liver post-mitochondrial S9 fraction

Test concentrations with justification for top dose:

Preliminary concentration range finding test: 10, 31.6, 100, 316, 1 000, 2 500, 5 000 µg/plate (TA98 only, plate incorporation);
Initial mutation test (5 strains)/complementary initial mutation test (S. typhimurium strains TA100, TA1535 and TA1537): 15.81, 50, 158.1, 500, 1 581, 5 000 µg/plate (plate incorporation);
Confirmatory mutation test: 5, 15.81, 50, 158.1, 500, 1 581, 5 000 µg/plate (all strains, pre-incubation).

The test material was found to form a slowly settling formulation in DMSO at 100 mg/mL (= 5 000 µg/plate). Therefore, 5 000 µg/plate was selected as top dose for the preliminary concentration 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 5 000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test material was examined using distilled water, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone and ethanol. The test material was insoluble (immediately settling formulation in each case) in distilled water and acetone at 100 mg/mL and 50 mg/mL concentration. The test material was insoluble at these concentrations using DMF (quickly settling formulation). The test material was insoluble at these concentrations using ethanol and DMSO (slowly settling formulation in each case). Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (preliminary compatibility test).

The formulations were stirred with magnetic stirrer during formulation and treatment.

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

Remarks:

without S9; 4 µg/plate (TA98)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; 2 µg/plate (TA100, TA1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9; 50 µg/plate (TA1537)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 2 µL/plate (WP2uvrA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9; 2 µg/plate (TA98, TA100, TA1535, TA1537); 50 µg/plate (WP2uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Preliminary concentration range finding test/Initial mutation test/complementary initial mutation test: In agar (plate incorporation).
Bacteria (cultured in Nutrient Broth) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45 °C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).
The content of the tubes was:
2 000 mL top agar
50 µLvehicle (solvent) or test material solution (or reference control)
100 µL overnight culture of test strain
500 µL phosphate buffer (pH 7.4) or S9 mix
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 hours.
- Confirmatory mutation test: Pre-incubation.
For the pre-incubation method, bacteria (cultured in Nutrient Broth) were exposed to the test material 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, 50 μL of test material formulations or its vehicle (or 50 μL of reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test material. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 °C 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 non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

DURATION
- Pre-incubation period: 48 h (confirmatory mutation test)
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 48 h (simultaneously with exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT: Histidine (S. typhimurium strains); tryptophan (E. coli strains).

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Bacterial background inhibition; decrease in the number of revertant colonies.

Evaluation criteria:

Criteria for a Positive Response:
A test material was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions was 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 was 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, a 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:
The test material was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test material and for the controls using Microsoft Excel™ software.
* Mutation factor (MF): Mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Remarks:

Initial and Confirmatory Mutation test

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, other: TA100, TA1535 and TA1537

Remarks:

Complementary Initial Mutation Test

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

Remarks:

Initial Mutation Test/Confirmatory Mutation Test

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble (immediately settling formulation in each case)
- Precipitation:
Preliminary concentration range finding test: Slight precipitate at 2 500 and 5 000 µg/plate with and without S9.
Initial mutation test: (Slight) precipitate at 5 000 µg/plate with and without S9 in all strains.
Complementary initial mutation test: (Slight) precipitate at several concentrations with and without S9 in all strains.
Confirmatory mutation test: (Slight) precipitate at several concentrations with and without S9 in all strains.

RANGE-FINDING/SCREENING STUDIES:
The observed number of revertant colonies was mostly in the normal range. Sporadically, minor differences compared to the solvent control numbers were detected. However, they did not have biological significance and were considered as biological variability of the test system.
No inhibitory, cytotoxic effects of the test material were observed in the preliminary experiment.
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 5 000 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95 %)
- Positive historical control data: The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.
- Untreated, negative (vehicle/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 in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Inhibitory, cytotoxic effects of the test material were not detected in the Initial Mutation Test, in the Complementary Initial Mutation test* or in the Confirmatory Mutation Test.
* Note: Inhibitory, cytotoxic effects of the test material were detected in the Initial Mutation Test in case of the positive controls. However, in the performed Complementary Initial Mutation Test the study was fully valid.

INITIAL AND CONFIRMATORY MUTATION TEST
- In the Initial Mutation Test and the Complementary Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 50 μg/plate concentration with metabolic activation (the observed mutation factor value was 1.35). 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 1581 μg/plate concentration without metabolic activation (the calculated mutation factor value was 1.77). There was no dose-response relationship, the calculated mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
Sporadically, higher numbers of revertant colonies were detected compared to the vehicle (solvent) control in the main tests. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The number of revertant colonies was within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
- Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Conclusions:

Under the experimental conditions applied, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial strains used.
In conclusion, the test material did not show any mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and US EPA OPPTS 870.5100, under GLP conditions, following the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Complementary Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of a solubility test, the test material was formulated in DMSO at a concentration of 100 mg/mL. Concentrations of 5 000; 2 500; 1 000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test in tester strains Salmonella typhimurium TA98 and TA100 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test (5 strains) and Complementary Initial Mutation Test (Salmonella typhimurium TA100, TA1535 and TA1537 strains) were 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate. Concentrations examined in the Confirmatory Mutation Test (5 strains) were 5 000, 1 581, 500, 158.1, 50, 15.81 and 5 μg/plate.

In the Initial Mutation Test, Complementary 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 dose-related trends and no indication of any treatment-related effect.

Precipitate and/or slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test, in the Complementary Initial Mutation Test and in the Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation at several concentrations. Precipitate or slight precipitate was detected on the plates in the Initial Mutation Test in all examined strains with and without metabolic activation at 5 000 μg/plate concentrations.

Inhibitory, cytotoxic effects of the test material were not detected in the Preliminary Concentration Range Finding Test, in the Complementary Initial Mutation Test* and in the Confirmatory Mutation Tests.

*Note: Inhibitory, cytotoxic effects of the test material were detected in the Initial Mutation Test in case of the positive controls. However, the performed Complementary Initial Mutation Test the study was fully valid.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, and the reference mutagens showed the expected increase in the number of revertant colonies, indicating that the test conditions were adequate. In addition, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test material had no mutagenic activity in the applied bacterial strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8). The only difference between the substance being registered and the read-across substance is that the substance to be registered is missing the gadolinium oxide content (about 1.4 %). The absence of this compound at such a low % is considered highly unlikely to affect its properties.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Remarks:

Initial and Confirmatory Mutation test

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

Species / strain:

S. typhimurium, other: TA100, TA1535 and TA1537

Remarks:

Complementary Initial Mutation Test

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

Species / strain:

E. coli WP2 uvr A

Remarks:

Initial Mutation Test/Confirmatory Mutation Test

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

> In vitro gene mutation study in bacteria

The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and US EPA OPPTS 870.5100, under GLP conditions, following the Bacterial Reverse Mutation Assay. The study was conducted on the read-across substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8). The only difference between the substance being registered and the read-across substance is that the substance to be registered is missing the gadolinium oxide content (about 1.4%). The absence of this compound at such a low % is considered highly unlikely to affect its properties.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Complementary Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of a solubility test, the test material was formulated in DMSO at a concentration of 100 mg/mL. Concentrations of 5 000; 2 500; 1 000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test in tester strains Salmonella typhimurium TA98 and TA100 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test (5 strains) and Complementary Initial Mutation Test (Salmonella typhimurium TA100, TA1535 and TA1537 strains) were 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate. Concentrations examined in the Confirmatory Mutation Test (5 strains) were 5 000, 1 581, 500, 158.1, 50, 15.81 and 5 μg/plate.

In the Initial Mutation Test, Complementary 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 dose-related trends and no indication of any treatment-related effect.

Precipitate and/or slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test, in the Complementary Initial Mutation Test and in the Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation at several concentrations. Precipitate or slight precipitate was detected on the plates in the Initial Mutation Test in all examined strains with and without metabolic activation at 5 000 μg/plate concentrations.

Inhibitory, cytotoxic effects of the test material were not detected in the Preliminary Concentration Range Finding Test, in the Complementary Initial Mutation Test* and in the Confirmatory Mutation Tests.

*Note: Inhibitory, cytotoxic effects of the test material were detected in the Initial Mutation Test in case of the positive controls. However, the performed Complementary Initial Mutation Test the study was fully valid.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, and the reference mutagens showed the expected increase in the number of revertant colonies, indicating that the test conditions were adequate. In addition, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test material had no mutagenic activity in the applied bacterial strains under the test conditions used in this study.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.