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EC number: 939-960-9 | CAS number: 39318-32-6
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
1. Information on zirconium dioxide
Genetic toxicity in vitro:
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 magnesium oxide
Because magnesium oxide transforms to magnesium hydroxide when in contact with moisture (e.g. physiological liquids), data for magnesium hydroxide were added to the weight of evidence approach to cover the endpoint of gene mutation in bacterial cells. The study was performed according to OECD guideline 471 and EU method B13/14. Magnesium hydroxide tested negative with and without metabolic activation in Salmonella typhimurium strains and E. coli WP2 uvr A.
3. Conclusion on magnesium zirconium oxide
Based on the available data on the individual components, magnesium
zirconium oxide is not expected to be genotoxic.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2010-04-19 to 2010-05-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- In the dose range finding study/first cytogenetic assay during incubation period, temperature was outside the range of 37.0±1.0°C as specified in the protocol with a minimum of 31.3°C for approx 1.5 hour. This deviation had no effects on the results
- GLP compliance:
- yes
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- See section 'Any other information on materials and methods incl. tables'
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany) (S9 fraction)
- Test concentrations with justification for top dose:
- Dose range finding test/first cytogenetic assay: at 3 h exposure time: 10, 33 and 100 µg zirconium dioxide/mL culture medium with and without S9-mix; at 24 and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg zirconium dioxide/mL culture medium without S9-mix
Second cytogenicity test: without S9-mix: 10, 33 and 100 µg/mL culture medium (24 and 48 h exposure time, 24 h and 48 h fixation time); with S9-mix: 10, 33 and 100 µg/mL culture medium (3 h exposure time, 48 h fixation time) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Without metabolic activation (-S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation (+S9-mix); solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 24 and 48 h in the absence of S9-mix or for 3 h in the presence of S9 mix (second cytogenetic assay)
- Expression time (cells in growth medium): after 3 h exposure, the cells exposed to zirconium dioxide in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 44-46 h; the cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): see above
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium) (Acros Organics, Belgium) - during the last 2.5-3 h of the culture period
STAIN (for cytogenetic assays): Cell cultures were centrifuged for 5 min at 1300 rpm (365 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa (Merck) solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.
NUMBER OF REPLICATIONS: duplicate cultures
NUMBER OF CELLS EVALUATED: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was placed over the marked slide. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. in case the number of aberrant cells, gaps excluded, was > or = 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. The highest concentration analysed was based on the solubility of zirconium dioxide in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: no
OTHER: Test substance preparation: Zirconium dioxide was suspended in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany) at concentrations of 0.3 mg/mL and above. the stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dioxide was dissolved in dimethyl sulfoxide at concentrations of 0.1 mg/mL and below. Zirconium dioxide concentrations were used within 2.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v) - Evaluation criteria:
- A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-side, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision. - Statistics:
- The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:
X²=[(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]
where b = the total number of aberrant cells in the control cultures, d = the total number of non aberrant cells in the control cultures, n0 = the total number of cells scored in the control cultures, a = the total number of aberrant cells in treated cultures to be compared with the control, c = the total number of non aberrant cells in treated cultures to be compared with the control, n1 = the total number of cells scored in the treated cultures, N = sum of n0 and n1
If P [X² > [(N-1) (ad-bc)²]/[(a+b) (c+d) (a+c) (b+d)]] (one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence interval. - Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Remarks:
- all strains/cell types tested
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Remarks:
- all strains/cell types tested
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- The mitotic index of the test substance didn't reach 50% of the control value for all tested concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: yes
RANGE-FINDING/SCREENING STUDIES: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Zirconium dioxide was tested in the absence and presence of 1.8% (v/v) S9-fraction. Lymphocytes (0.4 mL blood of a healthy male donor + 5 mL or 4.8 mL culture medium + (+ or - S9) + 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of zirconium dioxide for 3h, 24h, and 48h in the absence of S9-mix or for 3 h in the presence of S9-mix. The highest tested concentration was determined by the solubility of zirconium dioxide in the culture medium at the 3h exposure time. At a concentration of 100 µg/mL zirconium dioxide precipitated in the culture medium. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate vehicle and positive controls were included. At the 24h and 48h exposure time, zirconium dioxide was tested beyond the limit of solubility to obtain adequate toxicity data. After 3 h exposure to zirconium dioxide in the absence or presence fo S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 and 48h fixation time).Cytotoxicity of zirconium dioxide in the lymphocyte cultures cultures was determined using the mitotic index. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The first cytogenetic assay was ommited. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility.
COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the mutation frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
ADDITIONAL INFORMATION ON CYTOTOXICITY: - 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
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- Deviations of temperature and humidity caused by adjustment after opening of the incubator door. However the study integrity was not adversely affected by the deviations
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Food and Consumer Product Safety Authority (VWA), Prinses Beatrixlaan 2, 2595 AL Den Haag, Postbus 19508, 2500,CM Den Haag, The Netherlands
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- thymidine-kinase (TK) locus L5178Y
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9-mix induced by a combination of phenobarbital and beta-naphtoflavone
- Test concentrations with justification for top dose:
- 0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Without metabolic activation; MMS was dissolved in dimethyl sulfoxide. The stock solutions of MMS were prepared immediately before use.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation; CP was dissolved in Hanks' balanced salt solution (HBSS) without calcium and magnesium. The stock solutions of CP were stored in aliquots at < or = -15°C in the dark and one sample was thawed immediately before use.
- Details on test system and experimental conditions:
- In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11 or 12 days (TFT selection)
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours (MTT staining)
SELECTION AGENT (mutation assays): TFT
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS EVALUATED: for the determination of mutation frequency a total number of 9.6 x 1E05 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium, with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 1E05 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (trifluorothymidine-selection).
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHER:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Type and identity of media: horse serum was inactivated by incubiation at 56°C for at least 30 minutes. Basic medium: RPMI 1640 Hepes buffered medium (Dutch modificiation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: for 3 hour exposure: cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT) (Sigma). Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- State of the suspension/solution according to the concentration: at a concentration of 0.12 mg/mL and higher zirconium dioxide was suspended in dimethyl sulfoxide (DMSO, SeccoSolv, Merck Darmdstadt, Germany). At a concentration of 0.04 mg/mL and lower the test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Zirconium dixoide concentrations were used within 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.8% (v/v). - Evaluation criteria:
- The global evaluation factor (GEF) has been defined as the mean of the negative/solvent mutation frequency distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more then mutation frequency (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: a) non of the tested concentrations reaches a mutation frequency of mutation frequency (controls) + 126; b) the results are confirmed in an independent repeated test.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- all strains/cell types tested
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- first and second experiment
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Zirconium dioxide precipitated in the exposure medium at concentration of 100 µg/mL and above. Zirconium dioxide was tested beyond the limit of solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period. After 3 hours of treatment: both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control. After 24 hours of treatment with various concentrations of Zirconium dioxide, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control.
COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. - 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:
- weight of evidence
- Study period:
- 25-02-2010 to 11-03-2010
- 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
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver microsomal enzymes were routinely prepared from adult male Wistar rats
- Test concentrations with justification for top dose:
- In the dose range finding test, magnesium hydroxide was tested up to concentrations of 5000 µg/plate. Based on the results, magnesium hydroxide was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate. In an independent repeat of the assay, magnesium hydroxide was tested at the same concentration range as in the first assay.
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- dimethyl sulfoxide
- Positive controls:
- yes
- Positive control substance:
- not specified
- Details on test system and experimental conditions:
- Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria.
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C). - Evaluation criteria:
- No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. - Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- 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
- Conclusions:
- All bacteria strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two repeated experiments.
The negative strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that magnesium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. - Executive summary:
Evaluation of the mutagenic activity of magnesium hydroxide in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).
Magnesium hydroxide was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone).
The study procedures described in the report were based on the most recent OECD and EC guidelines.
In the dose range finding test, magnesium hydroxide was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Magnesium hydroxide did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.
Based on the results of the dose range finding test, magnesium hydroxide was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, magnesium hydroxide was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Magnesium hydroxide did not induce a significant dose-related increase in the number of relevant (His+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. The results were confirmed in an independently repeated experiment.
In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that magnesium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- 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
- Conclusions:
- Interpretation of results: negative
CC10 zirconium oxide is considered as "not mutagenic under the conditions of the test." - Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- in vitro gene mutation in bacteria, in vitro cytogenicity and gene mutation in mammalian cells
- 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 studies performed with zirconium dioxide (in vitro gene mutation in bacteria, in vitro gene mutation in mammalian cells, in vitro cytogenicity in mammalian cells) and magnesium oxide (in vitro gene mutation in bacteria). 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
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Remarks on result:
- other: Magnesium zirconium oxide is concluded not to be genotoxic.
- Remarks:
- Conclusion based on the results of a series of read across studies: Ames tests with zirconium dioxide (LAUS, 2008) and magnesium hydroxide (Verspeek-Rip, 2010b), an in vitro cytogenicity study in mammalian cells with zirconium dioxide (NOTOX, 2010a) and an in vitro gene mutation study in mammalian cells with zirconium dioxide (NOTOX, 2010b).
Referenceopen allclose all
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.
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
Dose range finding test
Magnesium hydroxide was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate in the absence and presence of S9-mix.
Table 1. Strain TA100 - Without S9 -mix
Plate Dose (micrograms/plate) |
1 |
2 |
3 |
Mean |
SD |
Positive control |
1039 |
990 |
983 |
1004 ± |
31 |
Solvent control |
99 |
94 |
112 |
102 ± |
9 |
3 |
95 |
87 |
85 |
89 ± |
5 |
10 |
84 |
101 |
86 |
90 ± |
9 |
33 |
123 |
139 |
107 |
123 ± |
16 |
100 |
106 |
100 |
94 |
100 ± |
6 |
333 |
111 |
110 |
108 |
110 ± |
2 |
1000 |
87 |
95 |
109 |
97 ± |
11 |
3330 |
84 |
106 |
84 |
91 ± |
13 |
5000 |
175 |
111 |
101 |
129 ± |
40 |
Table 2. Strain TA100 With S9 -mix
Plate Dose (micrograms/plate) |
1 |
2 |
3 |
Mean |
SD |
Positive control |
1283 |
1347 |
1240 |
1290± |
54 |
Solvent control |
156 |
134 |
126 |
139± |
16 |
3 |
96 |
65 |
66 |
76± |
18 |
10 |
71 |
83 |
95 |
83± |
12 |
33 |
84 |
80 |
112 |
92± |
17 |
100 |
95 |
107 |
99 |
100± |
6 |
333 |
107 |
99 |
93 |
100± |
7 |
1000 |
102 |
86 |
94 |
94± |
8 |
3330 |
108 |
100 |
97 |
102± |
6 |
5000 | 95 | 139 | 105 | 113± | 23 |
No precipitation of magnesium hydroxide was observed on the plates at start or end of incubation period.
No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed.
No increase in the number of revertants was observed upon treatment with magnesium hydroxide under all conditions tested.
Mutation assay
Magnesium hydroxide was tested in the absense and presence of S9-mix in two mutation assays. The first experiment was performed with the strains TA1535, TA1537 and TA98 and the second experiment was performed with strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Results are in the tables below.
Table 3. Experiment 1: Mutagenic response of magnesium hydroxide in Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Dose (µg/plate) |
Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain |
||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|
Without S9-mix |
|||||
positive control |
857 ± 14 |
343 ± 11 |
982 ± 89 |
1004 ± 31 |
513 ± 1 |
solvent control |
6 ± 2 |
3 ± 1 |
12 ± 1 |
102 ± 9 |
21 ± 4 |
|
|
|
|
|
|
3 |
|
|
|
89 ± 5 |
19 ± 5 |
10 |
|
|
|
90 ± 9 |
18 ± 3 |
33 |
|
|
|
123 ± 16 |
23 ± 2 |
100 |
7 ± 2 |
3 ± 1 |
16 ± 3 |
100 ± 6 |
16 ± 6 |
333 |
6 ± 1 |
3 ± 1 |
12 ± 1 |
110 ± 2 |
20 ± 2 |
1000 |
5 ± 1 |
4 ± 1 |
14 ± 5 |
97 ± 11 |
18 ± 4 |
3330 |
7 ± 1 |
4 ± 2 |
14 ± 1 |
91 ± 13 |
24 ± 7 |
5000 |
8 ± 1 |
3 ± 0 |
16 ± 3 |
129 ± 40 |
25 ± 3 |
With S9-mix |
|||||
positive control |
155 ± 11 |
300 ± 31 |
940 ± 21 |
1290 ± 54 |
282 ± 20 |
solvent control |
6 ± 1 |
3 ± 0 |
17 ± 3 |
139 ± 16 |
17 ± 5 |
|
|
|
|
|
|
3 |
|
|
|
76 ± 18 |
18 ± 2 |
10 |
|
|
|
83 ± 12 |
18 ± 5 |
33 |
|
|
|
92 ± 17 |
17 ± 3 |
100 |
8 ± 1 |
3 ± 0 |
21 ± 7 |
100 ± 6 |
16 ± 4 |
333 |
6 ± 3 |
4 ± 1 |
17 ± 1 |
100 ± 7 |
23 ± 2 |
1000 |
6 ± 2 |
3 ± 1 |
14 ± 4 |
94 ± 8 |
19 ± 5 |
3330 |
7 ± 1 |
3 ± 0 |
17 ± 3 |
102 ± 6 |
23 ± 3 |
5000 |
8 ± 2 |
5 ± 1 |
13 ± 1 |
113 ± 23 |
23 ± 3 |
Table 4. Experiment 2: Mutagenic response of magnesium hydroxide in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Dose (µg/plate) |
Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain |
||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|
Without S9-mix |
|||||
positive control |
735 ± 16 |
312 ± 40 |
1109 ± 31 |
958 ± 24 |
952 ± 27 |
solvent control |
9 ± 2 |
3 ± 0 |
18 ± 3 |
104 ± 6 |
19 ± 4 |
|
|
|
|
|
|
100 |
9 ± 2 |
3 ± 1 |
15 ± 2 |
96 ± 6 |
21 ± 3 |
333 |
8 ± 1 |
3 ± 0 |
16 ± 5 |
106 ± 13 |
21 ± 3 |
1000 |
6 ± 1 |
6 ± 1 |
19 ± 4 |
98 ± 4 |
21 ± 3 |
3330 |
7 ± 1 |
3 ± 1 |
15 ± 4 |
101 ± 6 |
24 ± 3 |
5000 |
7 ± 3 |
3 ± 0 |
14 ± 3 |
103 ± 10 |
22 ± 4 |
With S9-mix |
|||||
positive control |
125 ± 18 |
368 ± 8 |
669 ± 33 |
745 ± 48 |
176 ± 18 |
solvent control |
4 ± 1 |
3 ± 0 |
19 ± 2 |
61 ± 2 |
20 ± 4 |
|
|
|
|
|
|
100 |
7 ± 4 |
3 ± 1 |
18 ± 1 |
66 ± 5 |
20 ± 4 |
333 |
7 ± 1 |
3 ± 0 |
22 ± 4 |
68 ± 3 |
21 ± 4 |
1000 |
6 ± 2 |
3 ± 1 |
21 ± 3 |
65 ± 7 |
18 ± 2 |
3330 |
7 ± 2 |
3 ± 0 |
19 ± 4 |
80 ± 6 |
30 ± 5 |
5000 |
7 ± 3 |
4 ± 2 |
19 ± 3 |
105 ± 6 |
28 ± 2 |
Precipitation of magnesium hydroxide on the plates was not observed at the start or at the end of the incubation period.
In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In both assays, no increase in the number of revertants was observed upon treatment with magnesium hydroxide under all conditions 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"
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
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 magnesium oxide
Bacterial reverse mutation test:
Verspeek-Rip (2010b) performed a bacterial reverse mutation test with magnesium hydroxide according to OECD guideline 471. This test is considered relevant for magnesium oxide because magnesium oxide is transformed to magnesium hydroxide when in contact with moisture, such as bodily fluids.
In this study, magnesium hydroxide was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone). The substance tested negative in all strains both in the absence and presence of metabolic activation, under the conditions of the test.
3. Conclusion on magnesium zirconium oxide
Based on the comparison of basic toxicological data (Annex VII endpoints), the read across assumption (i.e., addition of magnesium (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 magnesium (hydr)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, magnesium zirconium oxide can safely be concluded not to be genotoxic.
Justification for classification or non-classification
1. Information on zirconium dioxide
Zirconium dioxide did not induce any mutation or chromosome aberration in three different in vitro studies and therefore, the substance should not be classified for genetic toxicity.
2. Information on magnesium oxide
An Ames study with magnesium hydroxide (considered representative for magnesium oxide) yielded negative results in the absence and presence of metabolic activation, under the conditions of the test. Based on the read across approach it was not considered necessary to add further data on genetic toxicity for the substance to this dossier. Magnesium hydroxide and magnesium oxide are however not classified for genetic toxicity.
3. Conclusion on magnesium zirconium oxide
Based on the conclusions for zirconium dioxide and magnesium (hydr)oxide, no classification for genetic toxicity is required for magnesium zirconium oxide either.
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