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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 476 and EC Guideline B17 with minor deviations of temperature and humidity that didn't influence the study integrity.

Data source

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

Materials and methods

Test guidelineopen allclose all
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:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Zirconium dioxide
Cas Number:
1314-23-4
Molecular formula:
ZrO2
IUPAC Name:
Zirconium dioxide
Constituent 2
Reference substance name:
Zirconium dioxide
EC Number:
215-227-2
EC Name:
Zirconium dioxide
IUPAC Name:
215-227-2
Details on test material:
- Name of test material (as cited in study report): Zirconium dioxide
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: off-white powder
- Analytical purity: 98.87%
- Impurities (identity and concentrations): SiO2 (0.5%), Na2O (0.16%), CaO (0.02%), MgO (< 0.01%), Fe2O3 (0.03%), TiO2 (0.07%), Al2O3 (0.13%), perte au feu(0.13%), H2O (0.08%), U + Th (< 500 ppm)
- Composition of test material, percentage of components: no data
- Isomers composition: not applicable
- Purity test date: no data
- Lot/batch No.: 10 01 002
- Expiration date of the lot/batch: 01 March 2015
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark
- Other: no data

Method

Target gene:
thymidine-kinase (TK) locus L5178Y
Species / strain
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
Controlsopen allclose all
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. Migrated to IUCLID6: at concentration of 15 and 5 µg/mL for a 3 and 24 hours treatment period, respectively
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 Migrated to
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 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.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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