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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): zirconium dioxide
- Substance type: off-white powder (8.40% < 1 micron)
- Physical state: solid
- Analytical purity: 98.87% ZrO2 + HfO2
- Impurities (identity and concentrations): 0.50% SiO2, 0.16% Na2O, 0.02% CaO, <0.01% MgO, 0.03% Fe2O3, 0.07% TiO2, 0.13% Al2O3, 0.08% H2O
- Composition of test material, percentage of components: no data
- Purity test date: 3 May 2010
- Lot/batch No.: 10 01 002
- Expiration date of the lot/batch: 1 March 2015
- Stability under test conditions: responsibility of the sponsor and on file at WIL Research Laboratories, LLC (WIL)
- Storage condition of test material: the test substance was stored at room temperature protected from light
- Other: a reserve sample of the test substance (approximately 3 g) was collected on 7 May 2010, and stored in the WIL Archives.

Method

Target gene:
Not applicable
Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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:

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.