Zirconium oxide (ZrO2), erbium-doped

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1. Information on zirconium dioxide
Gene mutation in bacteria:
One reliable bacterial reverse mutation study is available. The study was performed according to OECD guideline 471 and EU method B13/14. Zirconium dioxide tested negative with and without metabolic activation in Salmonella typhimurium strains.

In vitro cytogenicity in mammalian cells:
One reliable test was performed according to OECD guideline 473. Zirconium dioxide tested negative in cultured peripheral human lymphocytes with and without metabolic activation.

In vitro gene mutation in mammalian cells:
One reliable mouse lymphoma test was performed according to OECD guideline 476. Zirconium dioxide tested negative in mouse lymphoma L5178Y cells with and without metabolic activation.

 

2. Information on erbium oxide

Gene mutation in bacteria:

One reliable bacterial reverse mutation study is available. The study was performed according to OECD guideline 471. Erbium oxide tested negative with and without metabolic activation in Salmonella typhimurium strains.

 

3. Conclusion on erbium zirconium oxide

As erbium zirconium oxide is expected to have similar properties as the read across substances zirconium dioxide and erbium oxide, it should not be classified for genetic toxicity.

Link to relevant study records

Reference

Reference 1

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: Erbium 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.

Reference 2

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:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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 3

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 an in vitro cytogenicity 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: Erbium zirconium oxide is concluded not to be cytotoxic in mammalian cells in vitro based on the results of an in vitro cytogenicity study in mammalian cells (NOTOX, 2010a) performed with zirconium dioxide.

Reference 4

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:

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 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Read across based on Ames studies performed with zirconium dioxide and dierbium trioxide. The read across justification document is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: read across conclusion

Remarks on result:

other: Erbium zirconium oxide is concluded not to cause gene mutations in bacteria. Conclusion based on the results of Ames tests with zirconium dioxide (LAUS, 2008) and dierbium trioxide (Thompson, 2013).

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008-03-04 to 2008-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate provided by Rheinlandpfalz

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

4998, 1499, 500, and 50 µg/plate - Experiment one
4998, 2499, and 1250 µg/plate - Experiment two
As the test item was not soluble in any suitable solvent, a stock suspension containing 50 g/L was prepared and diluted as necessary.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine in DMSO (without at 80 µg for strains TA 97a, TA98 and TA102); Sodium azide in deionised water (without at 6 µg for strains TA100 and TA1535)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo-a-pyrene; 2-Amino-anthracene in DMSO (with at 40 µg for stain TA98); 2-Aminoanthracene in DMSO (with at 3 µg for strains TA97a, TA100, TA102 and TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION:

- In agar (plate incorporation) - Experiment one
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated (size of pores was 0.2 µm) into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mmol per 100 mL basis was added and the bottle was placed in the water bath at 45 degrees C.
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added. The mixture was gently vortexed, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 degrees C.

- Pre-incubation - Experiment two
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mmol per 100 mL basis was added and the bottle was placed in the water bath at 45 degrees C.
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37 degrees C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 mL top agar was added. The mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 degrees C.

DURATION
- Pre-incubation period: 20 minutes at 37 degrees C
- Exposure duration: 48 hours at 37 degrees C - Both experiments

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED: at least 10^9 cells/mL correlating to 100 colonies / plate

Evaluation criteria:

A test substance is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >/= 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

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:

- The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only).

- Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased.

- No toxicity was observed

Remarks on result:

other: all strains/cell types tested

Mean Revertants First Experiment:

Strain		97a		98		100		102		1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	139	109	13	11	152	185	212	192	15	16
	sd	58.2	11.6	1.7	3.8	25.1	31.1	22.0	58.2	3.6	5.7
DMSO	Mean	136	155	8	10	206	178	204	221	18	15
	sd	16.8	49.5	4.7	3.4	30.2	34.9	12.4	73.3	2.4	5.4
Pos Contr	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	7.36	6.46	125.1	100.1	6.59	5.62	4.91	4.53	66.73	66.73
4998 µg/pl.	Mean	171	100	10	9	129	183	198	209	19	14
	sd	28	8	3	1	11	17	18	68	6	2
	f(I)	1.23	0.92	0.77	0.82	0.85	0.99	0.93	1.09	1.27	0.88
1499 µg/pl.	Mean	156	146	12	9	170	160	198	178	15	18
	sd	17	29	3	3	19	30	32	54	4	3
	f(I)	1.12	1.36	0.92	0.82	1.12	0.86	0.93	0.93	1.00	1.13
500 µg/pl.	Mean	139	133	16	8	160	152	157	176	13	15
	sd	43	10	4	2	25	59	31	51	2	4
	f(I)	1.00	1.22	1.23	0.73	1.05	0.82	0.74	0.92	0.87	0.94
150 µg/pl.	Mean	134	113	10	9	152	178	209	168	15	12
	sd	41	7	4	2	17	37	50	45	4	4
	f(I)	0.96	1.04	0.77	0.82	1.00	0.96	0.99	0.88	1.00	0.75
50 µg/pl.	Mean	135	145	10	6	145	127	218	200	17	20
	sd	42	34	4	2	11	26	18	54	2	5
	f(I)	0.97	1.33	0.77	0.55	0.95	0.69	1.03	1.04	1.13	1.25

In this table ">1000" is represented by "1001"

Mean Revertants Second Experiment:

Strain		97a		98		100		102		1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	99	118	5	12	160	151	156	137	15	13
	sd	54.9	13.6	1.7	3.9	23.5	20.1	14.0	25.0	1.8	4.9
DMSO	Mean	152	115	7	12	142	133	167	164	8	11
	sd	9.6	6.1	0.8	3.4	17.0	1.9	8.6	31.0	2.1	2.2
Pos.Contr.	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	6.59	8.70	143.0	83.42	6.26	7.53	5.99	6.10	66.73	91.00
4998 µg/pl.	Mean	126	115	8	11	112	187	141	156	10	15
	sd	35	45	4	5	10	46	15	27	3	5
	f(I)	1.27	0.97	1.60	0.92	0.70	1.24	0.90	1.14	0.67	1.15
2499 µg/pl.	Mean	123	142	8	7	150	130	185	143	12	9
	sd	39	24	4	6	14	47	13	46	6	3
	f(I)	1.24	1.20	1.60	0.58	0.94	0.86	1.19	1.04	0.80	0.69
1250 µg/pl.	Mean	145	149	10	9	164	157	204	182	15	12
	sd	10	10	5	1	5	16	9	33	5	2
	f(I)	1.46	1.26	2.00	0.75	1.03	1.04	1.31	1.33	1.00	0.92

In this table "> 1000" is represented by "1001"

Conclusions:

Interpretation of results: negative

CC10 zirconium oxide is considered as "not mutagenic under the conditions of the test."

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 August 2012 - 22 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Additional strain / cell type characteristics:

other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (10 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test
Experiment 1: 0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
0.1 mL aliquots of the bacterial cultures were dispensed into sets of test tubes, followed by 2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli added to each 100 mL of top agar), 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation). The contents were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.
The positive and untreated controls were dosed using the standard plate incorporation method described above.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9 to 9 x 10⁹ bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1537 (with and without S9 mix) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA100 (without S9-mix only)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed with TA100 and WP2uvrA in both the absence and presence of S9-mix with ten different concentrations of the test material, ranging from 0.15 to 5000 µg/plate. The test material was not toxic at any concentration both in the absence and presence of S9-mix.


DEFINITIVE STUDY
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The mean number of revertant colonies for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiments 1 and 2, respectively.

The test material caused a visible reduction in the growth of the bacterial background lawns of Salmonella typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No toxicity was noted to any of the remaining bacterial strains at any test material dose level in both experiments in either the absence or presence of S9-mix. The test material was therefore tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix employing each exposure method.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

All of the positive control chemicals induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment	Mean number of colonies/plate
	Base-pair Substitution Type			Frameshift Type
	TA100	TA1535	WP2uvrA	TA98	TA1537
1	82	18	26	32	9
2	77	17	31	14	12

 

Table 2 Experiment 1

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - -	Solvent 50 150 500 1500 5000	91 82 85 85 80 70*	16 15 14 17 15 14	26 28 26 30 26 25	28 22 28 26 26 27	17 13 12 15 15 10*
+ + + + + +	Solvent 50 150 500 1500 5000	99 94 102 97 99 84	13 12 10 10 11 12	31 33 31 27 35 26	27 27 27 26 29 27	11 12 11 11 12 7*
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	749	541	267	159	113
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	1281	261	264	176	217

*Sparse bacterial background lawn

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

 

Table 3 Experiment 2

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - - - -	Solvent 5 15 50 150 500 1500 5000	79 77 75 74 71 70 70 70*	17 19 11 21 15 14 16 15	27 24 24 29 26 25 27 29	15 13 13 13 14 11 11 16	12 11 7 10 8 10 10 7*
+ + + + + + + +	Solvent 5 15 50 150 500 1500 5000	85 85 74 82 84 85 76 72	12 12 12 12 10 10 10 11	26 26 27 24 31 24 23 29	26 23 15 20 20 18 18 19	11 8 8 12 9 8 8 6*
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	514	590	484	141	714
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	1455	246	200	223	281

*Sparse bacterial background lawn

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Conclusions:

Interpretation of results: negative with and without metabolic activation.

Under the conditions of this study, the test material was considered to be non-mutagenic.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five and seven dose levels, respectively, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate using the plate incorporation method and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate using the pre-incubation method.

The test material caused a visible reduction in the growth of the bacterial background lawns of S. typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No toxicity was noted to any of the remaining bacterial strains at any test item dose level in both experiments in either the absence or presence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. 

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

1. Information on zirconium dioxide

Bacterial reverse mutation test:

LAUS (2008) performed a bacterial reverse mutation study according to OECD guideline 471 and EU method B13/14. Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were exposed to 50 to 4998 µg/plate with and without metabolic activation in two independent experiments. Vehicle and positive controls were valid. Zirconium dioxide did not induce mutation with and without metabolic activation and no cytotoxicity was observed.

 

In vitro cytogenicity in mammalian cells:

NOTOX (2010a) performed a chromosome aberration test according to OECD guideline 473. 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 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 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 in mammalian cells:

NOTOX B.V. (2010b) performed a mouse lymphoma test according to OECD guideline 476. 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.

 

2. Information on erbium oxide

Bacterial reverse mutation test:

Thompson (2013) perfomed a study on erbium oxide to assess the potential of the test material to cause mutagenic effects in bacteria in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

The test material caused a visible reduction in the growth of the bacterial background lawns of S. typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods (plate incorporation and pre-incubation). No toxicity was noted to any of the remaining bacterial strains at any test item dose level in both experiments in either the absence or presence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of this test.

 

3. Conclusion on erbium zirconium oxide

Based on the comparison of basic toxicological data (Annex VII endpoints), the read across assumption (i.e., addition of erbium (oxide) to the crystal lattice of zirconium dioxide does not alter the unhazardous character of zirconium dioxide) was considered valid. For gene mutation in bacterial cells (Ames test), data for both zirconium dioxide and erbium oxide were added to a weight of evidence approach. Both compounds tested negative both in the absence and presence of metabolic activation, under the conditions of the test. The higher endpoints (in vitro gene mutation in mammalian cells and cytogenicity in mammalian cells) were then covered by data for zirconium dioxide alone. Based on the available data and the read across approach, erbium zirconium oxide can safely be concluded not to be genotoxic.

Justification for classification or non-classification

1. Information on zirconium dioxide (CAS# 1314-23-4)

Zirconium dioxide is currently not classified for genetic toxicity. This is confirmed by the absence of effects in a bacterial reverse mutation assay as well as a cytogenicity study and a gene mutation study in mammalian cells.

 

2. Information on erbium oxide (CAS# 12061-16-4)

Erbium oxide is currently not classified for genetic toxicity. Only the Ames study has been included in this dossier.

 

3. Conclusion on erbium zirconium oxide

As erbium zirconium oxide is expected to have similar properties as the read across substances zirconium dioxide and erbium oxide, it should not be classified for genetic toxicity.