Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tungsten zirconium (hydroxide) oxide tested negative in a bacterial reverse mutation test with and without metabolic activation (Orovecz, 2018; Klimisch 1; OECD 471).

No data are available on in vitro cytogenicity and gene mutation in mammalian cells for tungsten zirconium oxide. However, since the comparison of basic toxicological (Annex VII) data for tungsten zirconium (hydroxide) oxide and zirconium dioxide indicated that the addition of tungsten to the crystal lattice of zirconium dioxide does not alter the unhazardous character of zirconium dioxide, it was considered acceptable to cover higher toxicological endpoints using data for zirconium dioxide alone.

The endpoints on in vitro cytogenicity and gene mutation in mammalian cells are therefore covered using studies performed with zirconium dioxide. In an OECD 473 and an OECD 476 study (NOTOX, 2010a,b; Klimisch 1), zirconium dioxide tested negative in respectively cultured peripheral human lymphocytes and mouse lymphoma L5178Y cells with and without metabolic activation. Similar results would be expected for tungsten zirconium oxide.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-04-19 to 2010-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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 did not 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 did not 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.
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:
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:
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. certificate)
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
 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.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-30 to 2017-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Based on the solubility test, a 100 mg/mL stock solution was prepared in DMSO. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10.
Target gene:
Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537); tryptophan locus (E. coli strain WP2uvrA)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital-/β-naphthoflavone-induced rat liver post-mitochondrial S9 fraction
Test concentrations with justification for top dose:
Preliminary concentration range finding test: 10, 31.6, 100, 316, 1000, 2500, 5000 μg/plate with and without S9-mix (TA98 and TA100, plate incorporation);
Initial mutation test: 15.81, 50, 158.1, 500, 1581, 5000 μg/plate with and without S9-mix (all strains, plate incorporation);
Confirmatory mutation test: 5, 15.81, 50, 158.1, 500, 1581, 5000 μg/plate with and without S9-mix (all strains, pre-incubation).

The test item was found soluble in DMSO at 100 mg/mL (= 5000 μg/plate). Therefore, 5000 μg/plate was selected as top dose for the preliminary concentration range finding test. Based on the results of the range finding test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO), acetone and N,N-dimethylformamide (DMF). At a 100 mg/mL concentration suspension with quick sedimentation was observed using acetone. At this concentration suspension with slower sedimentation was observed using distilled water, DMSO and DMF. After 2 minutes ultrasonic water bath there was no change using distilled water, DMF and acetone but a homogeneous suspension was observed using DMSO. DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.
The solubility of the test item in DMSO is presented in the section "Any other information on materials and methods including tables".
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylenediamine
Remarks:
without S9; 4 μg/plate (TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; 2 μg/plate (TA100, TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9; 50 μg/plate (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; 2 μL/plate (WP2uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9; 2 μg/plate (TA98, TA100, TA1535, TA1537); 50 μg/plate (WP2uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION
- Preliminary concentration range finding test/Initial mutation test: in agar (plate incorporation)
Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C). The content of the tubes was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

- Confirmatory mutation test: pre-incubation
For the pre-incubation method, bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C. Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

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

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains); tryptophan (E. coli strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background inhibition; decrease in the number of revertant colonies
Evaluation criteria:
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

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

According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow-up experiment.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft Excel TM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537
Remarks:
Preliminary range finding test, initial mutation test, and confirmatory mutation test.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Preliminary range finding test and confirmatory mutation test.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: slow sedimentation in suspension at 100 mg/mL
- Precipitation:
Preliminary Range Finding Test: no precipitate detected
Initial Mutation Test: precipitate at 5000 μg/plate with and without S9 (all strains)
Confirmatory Mutation Test: precipitate at 1581 and 5000 μg/plate with and without S9 (all strains)
Note: In these concentrations, the background lawn development could not be assessed due to the strong precipitate, but the colony counting was not affected.

PRELIMINARY RANGE FINDING TEST
The observed number of revertant colonies was in the normal range. Slight decreases of the revertant counts were observed compared to the solvent control sporadically. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
No precipitate of the test item was detected in the Preliminary Range Finding Test.
Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%))
- Positive historical control data: The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
- Untreated, negative (solvent/vehicle) and positive control plates were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Inhibitory, cytotoxic effects of the test item were not detected in the Initial Mutation Test and Confirmatory Mutation Test.

Initial Mutation Test/Confirmatory Mutation Test

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 500 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.30). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 50 μg/plate concentrations without metabolic activation (the observed mutation factor value was: MF: 1.68). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro

Bacterial Reverse Mutation Test

Orovecz (2018) performed a bacterial reverse mutation study with tungsten zirconium (hydroxide) oxide according 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 5 to 5000 µg/plate with and without metabolic activation in two independent experiments. Vehicle and positive controls included in the study were valid. Tungsten zirconium (hydroxide) oxide did not induce mutations 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 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 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.

Justification for classification or non-classification

Tungsten 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 tungsten zirconium oxide) did not induce chromosome aberrations or mutations during in vitro testing in mammalian cells. Since tungsten oxide is, according to the read across justification, not expected to affect the toxicological properties of zirconium dioxide, tungsten zirconium oxide should not be classified for genetic toxicity based on the available information.