Yttrium zirconium oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro
Bacterial Reverse Mutation Test: One reliable bacterial reverse mutation study was available (Klimisch 1). The study (Chemical Inspection and Testing Institute, 1997) was performed according to a method equivalent or similar to OECD guideline 471. Yttrium zirconium oxide tested negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 strains and E. coli WP2 uvr A strain.

In vitro cytogenicity in mammalian cells

One reliable test (Klimisch 1) was performed according to OECD guideline 473 (chromosome aberration) with the read across substance zirconium dioxide, the main component in the crystal lattice of yttrium zirconium oxide. The substance tested negative in cultured peripheral human lymphocytes with and without metabolic activation (Notox, 2010a).

In vitro gene mutation in mammalian cells

One reliable mouse lymphoma test (Klimisch 1) was performed according to OECD guideline 476 with the read across substance zirconium dioxide, the main component in the crystal lattice of yttrium zirconium oxide. The substance tested negative in mouse lymphoma L5178Y cells with and without metabolic activation (Notox, 2010b).

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:

1997-10-20 to 1997-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus (Salmonella typhimurium)
tryptophan locus (Escherichia coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 induced rat liver

Test concentrations with justification for top dose:

Dose-setting test: 5, 10, 50, 500, 1000, and 5000 µg/plate
Final test: 0.156, 0.313, 0.625, 1.25, 2.5 and 5 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)

Remarks:

For strains: TA 98, TA 100 and WP2 uvrA without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strains: TA 1535 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-methoxy-6-Chloro-9-[3-(2-chloroethyl)-aminopropylamino] acridine ¿ 2HCl(ICR-191)

Remarks:

For strains: TA 1537 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

For strains: TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubation

DURATION
- Pre-incubation period: no data
- Exposure duration: at least 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): at least 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): histidine and tryptophan
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable

OTHER: NaN3 was dissolved in distilled water; AF-2 ICR-191 and 2AA were dissolved in DMSO.

Evaluation criteria:

It is determined positive if the number of revertant colonies of the test agent treatment group depends on the dosage and increased twice or more as the negative control and reproducibility is acknowledged. Other cases are determined negative.

Unpredictable situations that may affect the credibility of the test, and not following the test plan.

For not following the test plan, the minimum glucose agar plating medium Lot No. AN550JM (manufactured on Oct. 3, 1997) was used in addition, while only AN510IM was to be used according to the plan. It was determined, however, that it would have no bad effect to the test.
There was no other unpredictable situation that may affect the credibility of the test.

No other situation that may adversely affect the credibility of the test, or no other incident of not following the test plan was acknowledged.

Statistics:

Statistical analyses were not done.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: insoluble in water or oil
- Precipitation: Precipitation thought to be the test agent of this test was seen in 5 µg/plate regardless of presence/absence of S9 mix
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
5000 µg/plate as the maximum, the seven doses; 1000, 500, 100, 50, 10 and 5 µg/plate, were set. As a result, depositions which seem to be the test agent were seen in 5 µg/plate, regardless of the presence/absence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
It was confirmed that the positive control agent induces mutation in the test strains, and that the numbers of revertant colonies as well as the negative control value were within the range of the historical data of this institute, thus it was confirmed that the test was conducted properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Conclusions:

It is determined that yttrium zirconium oxide does not have reverse mutation inducing capacity under the conditions of this test.

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 2010-04-19 to 2010-05-18

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)

Deviations:

yes

Remarks:

In the dose range finding study/first cytogenetic assay during incubation period, temperature was outside the range of 37.0±1.0°C as specified in the protocol with a minimum of 31.3°C for approx 1.5 hour. This deviation had no effects on the results

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

See section 'Any other information on materials and methods incl. tables'

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany) (S9 fraction)

Test concentrations with justification for top dose:

Dose range finding test/first cytogenetic assay: at 3 h exposure time: 10, 33 and 100 µg zirconium dioxide/mL culture medium with and without S9-mix; at 24 and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg zirconium dioxide/mL culture medium without S9-mix
Second cytogenicity test: without S9-mix: 10, 33 and 100 µg/mL culture medium (24 and 48 h exposure time, 24 h and 48 h fixation time); with S9-mix: 10, 33 and 100 µg/mL culture medium (3 h exposure time, 48 h fixation time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 24 and 48 h in the absence of S9-mix or for 3 h in the presence of S9 mix (second cytogenetic assay)
- Expression time (cells in growth medium): after 3 h exposure, the cells exposed to zirconium dioxide in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 44-46 h; the cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): see above

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium) (Acros Organics, Belgium) - during the last 2.5-3 h of the culture period
STAIN (for cytogenetic assays): Cell cultures were centrifuged for 5 min at 1300 rpm (365 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa (Merck) solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was placed over the marked slide. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. in case the number of aberrant cells, gaps excluded, was > or = 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. The highest concentration analysed was based on the solubility of zirconium dioxide in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: no

OTHER: Test substance preparation: Zirconium dioxide was suspended in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany) at concentrations of 0.3 mg/mL and above. the stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dioxide was dissolved in dimethyl sulfoxide at concentrations of 0.1 mg/mL and below. Zirconium dioxide concentrations were used within 2.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v)

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-side, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:
X²=[(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]
where b = the total number of aberrant cells in the control cultures, d = the total number of non aberrant cells in the control cultures, n0 = the total number of cells scored in the control cultures, a = the total number of aberrant cells in treated cultures to be compared with the control, c = the total number of non aberrant cells in treated cultures to be compared with the control, n1 = the total number of cells scored in the treated cultures, N = sum of n0 and n1
If P [X² > [(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]] (one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence interval.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Remarks:

all strains/cell types tested

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Remarks:

all strains/cell types tested

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Zirconium dioxide was tested in the absence and presence of 1.8% (v/v) S9-fraction. Lymphocytes (0.4 mL blood of a healthy male donor + 5 mL or 4.8 mL culture medium + (+ or - S9) + 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of zirconium dioxide for 3h, 24h, and 48h in the absence of S9-mix or for 3 h in the presence of S9-mix. The highest tested concentration was determined by the solubility of zirconium dioxide in the culture medium at the 3h exposure time. At a concentration of 100 µg/mL zirconium dioxide precipitated in the culture medium. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate vehicle and positive controls were included. At the 24h and 48h exposure time, zirconium dioxide was tested beyond the limit of solubility to obtain adequate toxicity data. After 3 h exposure to zirconium dioxide in the absence or presence fo S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 and 48h fixation time).Cytotoxicity of zirconium dioxide in the lymphocyte cultures cultures was determined using the mitotic index. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The first cytogenetic assay was ommited. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the mutation frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Results:

Both in the absence and presence of S9-mix zirconium dioxide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of zirconium dioxide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that zirconium dioxide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions of this test.

Table 1: Mitotic index of human lymphocyte cultures treated with zirconium dioxide at the 24 h and 48 h continuous exposure time in the dose range finding test.

Zirconium dioxide concentration (µg/mL)	Number of metaphases per 1000 cells
	Absolute	Percentage of control
Without metabolic activation (-S9 -mix)
24 h exposure time, 24 h fixation time
Control a)	36	100
1	33	92
3	32	89
10	31	86
33	36	100
100 b)	34	94
333 c)	38	106
1000 c)	38	106
48 h exposure time, 48 h fixation time
Control a)	42	100
1	44	105
3	44	105
10	42	100
33	39	93
100 b)	42	100
333 c)	44	105
1000 c)	44	105

a) Dimethyl sulfoxide

b) Zirconium dioxide precipitated in the culture medium

c) Zirconium dioxide precipitated heavily in the culture medium which would interfere with the scoring of chromosome aberrations

Table 2: Mitotic index of human lymphocyte cultures treated with zirconium dioxide at the 3 h exposure time in the dose range finding test (first cytogenetic assay)

Zirconium dioxide concentration (µg/mL)	Number of metaphases per 1000 cells
Without metabolic activation (-S9 -mix)	Absolute	Percentage of control
3 h exposure time, 24 h fixation time
Control b)	46 - 51	100
10	48 - 50	101
33	47 - 49	99
100	51 - 53	107
MMC-C; 0.5 µg/mL	38 - 33	73
With metabolic activation (+ S9 -mix)
Control b)	54 -54	100
10	50 - 49	92
33	55 - 54	101
100 c)	50 - 53	95
CP; 10 µg/mL	21 - 28	45

a) Duplicate cultures

b) Dimethyl sulfoxide

c) Zirconium dioxide precipitated in the culture medium

Table 3: Mitotic index of human lymphocyte cultures treated with zirconium dioxide in the second cytogenetic assay

Zirconium dioxide concentration (µg/mL)	Number of metaphases per 1000 cells
	Absolute	Percentage of control
Without metabolic activation (-S9 -mix)
24 h exposure time, 24 h fixation time
Control b)	65 -68	100
10	63 - 69	99
33	60 65	94
100 c)	58 -61	89
MMC-C; 0.2 µg/mL	31 - 35	50
48 h exposure time, 48 h fixation time
Control b)	71 - 68	100
10	65 - 69	96
33	68 - 66	96
100 c)	62 - 60	88
MMC-C; 0.1 µg/mL	53 - 55	78
With metabolic activation (+S9 -mix)
3 h exposure time, 48 h fixation time
Control b)	75 - 77	100
10	72 - 76	97
33	79 - 79	104
100	78 - 75	101
CP; 10 µg/mL	28 - 25	d)

a) Duplicate cultures

b) Dimethyl sulfoxide

d) Zirconium dioxide precipitated in the culture medium

e) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Conclusions:

Interpretation of results: negative with and without metabolic activation

Finally, it is concluded that this test is valid and that zirconium dioxide is not clastogenic in human lymphocytes under the experimental conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

yes

Remarks:

Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority (VWA), Prinses Beatrixlaan 2, 2595 AL Den Haag, Postbus 19508, 2500,CM Den Haag, The Netherlands

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

thymidine-kinase (TK) locus L5178Y

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by a combination of phenobarbital and beta-naphtoflavone

Test concentrations with justification for top dose:

0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; MMS was dissolved in dimethyl sulfoxide. The stock solutions of MMS were prepared immediately before use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation; CP was dissolved in Hanks' balanced salt solution (HBSS) without calcium and magnesium. The stock solutions of CP were stored in aliquots at < or = -15°C in the dark and one sample was thawed immediately before use.

Details on test system and experimental conditions:

In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11 or 12 days (TFT selection)
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours (MTT staining)

SELECTION AGENT (mutation assays): TFT
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: for the determination of mutation frequency a total number of 9.6 x 1E05 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium, with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 1E05 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (trifluorothymidine-selection).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Type and identity of media: horse serum was inactivated by incubiation at 56°C for at least 30 minutes. Basic medium: RPMI 1640 Hepes buffered medium (Dutch modificiation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: for 3 hour exposure: cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT) (Sigma). Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- State of the suspension/solution according to the concentration: at a concentration of 0.12 mg/mL and higher zirconium dioxide was suspended in dimethyl sulfoxide (DMSO, SeccoSolv, Merck Darmdstadt, Germany). At a concentration of 0.04 mg/mL and lower the test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dixoide concentrations were used within 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.8% (v/v).

Evaluation criteria:

The global evaluation factor (GEF) has been defined as the mean of the negative/solvent mutation frequency distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more then mutation frequency (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: a) non of the tested concentrations reaches a mutation frequency of mutation frequency (controls) + 126; b) the results are confirmed in an independent repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

all strains/cell types tested

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

first and second experiment

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Zirconium dioxide precipitated in the exposure medium at concentration of 100 µg/mL and above. Zirconium dioxide was tested beyond the limit of solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period. After 3 hours of treatment: both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control. After 24 hours of treatment with various concentrations of Zirconium dioxide, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

The growth rate over the two-day expression period for cultured treated with DMSO was between 20 and 28 (3 hours treatment) and 40 and 50 (24 hours treatment).

Mutation frequencies in cultures treated with positive control chemicals were increased by 26- and 14-fold for MMS in the absence of S9-mix, and by 19-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Experiment 1: Cytotoxic and mutagenic response of zirconium dioxide in the mouse lymphoma L5178Y test system (3 hours treatment)

Without metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	118	100	100	53	31	20
SC2	100	113	100	100	51	31	19
0.03	112	101	87	98	50	23	25
0.1	105	110	95	100	54	29	23
0.3	110	94	81	90	54	26	26
1	117	111	96	113	50	21	28
3	112	101	87	97	49	25	22
10	106	98	85	90	58	34	23
33	102	97	84	85	58	30	27
100 (1)	103	105	91	94	52	29	22
MMS	66	57	49	32	1334	804	318

With 8% (v/v) metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	88	100	100	54	32	21
SC2	100	89	100	100	53	29	23
0.03	100	102	116	116	53	34	18
0.1	99	83	94	93	54	38	15
0.3	99	79	90	89	59	32	26
1	100	81	92	93	67	33	33
3	92	74	83	77	78	47	29
10	99	86	98	97	60	31	27
33	92	90	102	94	56	33	21
100 (1)	100	77	87	87	61	32	28
CP	53	72	82	44	1000	674	191

 

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = DMSO; MMS = Methylmethanesulfonate; CP = cyclophosphamide

(1) zirconium dioxide precipitated in the exposure medium

Experiment 2: Cytotoxic and mutagenic response of zirconium dioxide in the mouse lymphoma L5178Y test system (24 hours)

Without metabolic activation

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	118	100	100	57	32	23
SC2	100	104	100	100	63	36	25
0.03	120	88	79	95	72	43	27
0.1	127	107	96	122	66	34	29
0.3	137	120	108	148	50	29	20
1	127	111	100	128	54	34	18
3	139	110	99	138	55	37	17
10	140	91	82	115	80	48	29
33	138	115	103	143	69	41	25
100 (1)	153	97	87	133	54	38	15
MMS	119	77	69	83	815	564	157

With 12% (v/v) metabolic activation:

dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency x 1E-06
					total	(small	large)
SC1	100	111	100	100	67	40	25
SC2	100	80	100	100	85	44	37
0.03	107	77	80	86	85	57	26
0.1	97	86	90	87	86	45	37
0.3	99	102	107	105	64	34	28
1	97	107	111	108	69	43	24
3	99	97	101	100	75	53	20
10	90	99	104	93	77	54	20
33	89	107	111	99	94	49	40
100 (1)	91	102	107	97	71	45	24
CP	42	54	56	24	1422	832	355

(1) = Zirconium dioxide precipitated in the exposure medium

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamid (1) = Zirconium dioxide precipitated in the exposure medium

Conclusions:

Interpretation of results: negative with and without metabolic activation

In conclusion, zirconium dioxide is not mutagenic in the TK mutation test system under the specified experimental conditions.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

The endpoint was covered using a chromosome aberration study performed with zirconium dioxide. The read across justification is attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: read across conclusion

Remarks on result:

other: Yttrium zirconium oxide is concluded not to cause chromosome aberrations in mammalian cells in vitro.

Remarks:

Conclusion based on an in vitro chromosome aberration study in mammalian cells performed with zirconium dioxide (NOTOX, 2010a).

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

The endpoint was covered using an in vitro gene mutation study in mammalian cells performed with zirconium dioxide. The read across justification is attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: read across conclusion

Remarks on result:

other: Yttrium zirconium oxide is concluded not to be mutagenic in mammalian cells in vitro.

Remarks:

Conclusion based on the results of an in vitro gene mutation study in mammalian cells (NOTOX, 2010b) performed with zirconium dioxide.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

Bacterial Reverse Mutation Test

The Chemicals Inspection and Testing Institute (1997) performed a bacterial reverse mutation study according to a method similar or equivalent to OECD guideline 471. Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvr A were exposed to concentrations from 0.156 to 5000 µg/plate with and without metabolic activation in two independent experiments. Vehicle and positive controls included in the study were valid. Yttrium stabilised zirconium oxide did not induce mutation with and without metabolic activation and no cytotoxicity was observed.

In vitro cytogenicity assay

NOTOX B.V. (2010a) performed a chromosome aberration test according to OECD guideline 473 with the read across substance zirconium dioxide. Cultured peripheral human lymphocytes were exposed for 3 hours to 10, 33 and 100 µg zirconium dioxide/mL culture medium with and without S9-mix (dose range finding test/first cytogenetic assay); at 24 and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg zirconium dioxide/mL culture medium without S9-mix. A second cytogenicity test was performed as follows: without S9-mix: 10, 33 and 100 µg/mL culture medium (24 and 48h exposure time, 24h and 48h fixation time); with S9-mix: 10, 33 and 100 µg/mL culture medium (3h exposure time, 48h fixation time). Vehicle and positive control substances were tested simultaneously and considered valid. Zirconium dioxide tested negative with and without metabolic activation. No cytotoxicity was observed.

In vitro gene mutation assay

NOTOX B.V. (2010b) performed a mouse lymphoma test according to OECD guideline 476 with the read across substance zirconium dioxide. Mouse lymphoma L5178Y cells were exposed to 0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL zirconium dioxide with and without metabolic activation. In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Zirconium dioxide tested negative in both experiments with and without metabolic activation. No cytotoxicity was observed and positive and vehicle controls were considered valid.

In conclusion, yttrium zirconium oxide tested negative in a bacterial reverse mutation test with and without metabolic activation. Two other in vitro tests are available with the read across substance zirconium dioxide, the main component in the crystal lattice of yttrium zirconium oxide. In these two studies, zirconium dioxide tested negative. As yttrium zirconium oxide is expected to have a similar toxicological profile as zirconium dioxide (i.e., yttrium oxide is not expected to affect the toxicological properties of zirconium dioxide), the substance is considered not genotoxic.

The read across justification is included in IUCLID Section 13 of the registration dossier.

Genetic toxicity in vivo

No reliable data were available on the genetic toxicity in vivo endpoint. Yttrium zirconium oxide tested negative in the in vitro gene mutation test in bacteria (Ames test), and an in vitro chromosome aberration test as well as an in vitro gene mutation test in mammalian cells yielded negative results for zirconium dioxide (i.e. the main component in the crystal lattice of yttrium zirconium oxide). As yttrium oxide, according to the read across justification, is not expected to change the toxicological properties of zirconium oxide, yttrium zirconium oxide can be concluded to be not genotoxic. Therefore, and according to column 2 of Annex VIII of the REACH Regulation, no in vivo genetic toxicity study should be performed.

Justification for classification or non-classification

Yttrium zirconium oxide did not induce any mutations in an Ames test in the absence and presence of metabolic activation. The read across substance zirconium dioxide (the main component in the crystal lattice of yttrium zirconium oxide) did not induce chromosome aberrations or mutations during in vitro testing in mammalian cells. Since yttrium oxide is, according to the read across justification, not expected to affect the toxicological properties of zirconium dioxide, yttrium zirconium oxide should not be classified for genetic toxicity based on the available information.