Zirconium oxide sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

18 Oct 2012- 03 Jan 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study in accordance with OECD Guideline 476 and Method B.17. A maximal reliability score of 2 (reliable with restrictions) was assigned because the study is used for read across purposes in this dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Zirconium acetate

EC Number:

231-492-7

EC Name:

Zirconium acetate

Cas Number:

7585-20-8

IUPAC Name:

zirconium(2+) diacetate

Details on test material:

- Name of test material (as cited in study report): Zirconium acetate solution
- Substance type: Clear colourless solution
- Physical state: Liquid
- Analytical purity: Not indicated
- Composition of test material, percentage of components: aqueous solution containing 40.7% zirconium acetate anhydrous, dry content > 99% purity
- Lot/batch No.: 12/201
- Expiration date of the lot/batch: 31-Jul-2013
- Storage condition of test material: Room temperature

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Prior to use cells were cleansed of pre-existing mutants.
A cell suspension (1 x 1E06 cells/mL) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.
The cultures were incubated at 37°C in a water bath for three hours and transferred in an incubator (only 24 hours treated cultures) until the end of treatment. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).

Following culture media were used:
Minimal medium A:
RPMI 1640 (1 X): 516.1 mL
L-glutamine (200 mM): 5.4 mL
Sodium pyruvate (100 mM): 6.0 mL
Non-essential amino acids (100 X): 5.4 mL

Streptomycin sulphate 50.000 IU/mL + Peicillin G 50.000 units/mL: 1.1 mL
F68 Pluronic: 6.0 mL

Minimal medium B:
RPMI 1640 (1X): 522.1 mL
L-glutamine (200 mM): 5.4 mL
Sodium pyruvate (100 mM): 6.0 mL
Non-essential amino acids (100X): 5.4 mL
Streptomycin sulphate 50.000 units/mL + Penicillin G 50.000 units/mL: 1.1 mL

Complete medium (5%): 950 mL minimal medium A and 50 mL horse serum (heat-inactivated)
Complete medium (10%): 900 mL minimal medium A and 100 mL horse serum (heat-inactivated)
Complete medium A (20%): 800 mL miminmal medium A and 200 mL horse serum (heat-inactivated)
Complete medium B (20%): 800 mL minimal medium B and 200 mL horse serum (heat-inactivated)

The treatment media were prepared as follows:
Without S9 metabolism - 3-hour treatment time:
Cell suspension (1 x 1E06 cells/mL in complete medium 5%): 10.0 mL
Complete medium (5%): 9.8 mL
Control or test item solution: 0.2 mL
Total: 20.0 mL

Without S9 metabolism - 24-hour treatment time:
Cell suspension (1 x 1E06 cells/mL in complete medium 10%): 3.0 mL
Complete medium (10%): 16.8 mL
Control or test item solution: 0.2 mL
Total: 20.0 mL

With S9 metabolism - 3-hour treatment time:
Cell suspension (1 x 1E06 cells/mL) in complete medium 5%): 10.0 mL
S9 mix: 9.8 mL
Control of test item solution: 0.2 mL
Total: 20.0 mL

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Without metabolic activation:
Assay 1-0.500, 0.250, 0.125, 0.0625, 0.0313 and 0.0156 mM
Assay 2-2.50, 1.25, 0.625, 0.313, 0.156, 0.0781 and 0.0391 mM

With metabolic activation:
Assay 1-0.500, 0.250, 0.125, 0.0625, 0.0313 and 0.0156 mM
Assay 2-0.250, 0.147, 0.0865, 0.0509 and 0.0299 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test item was found to be soluble in this solvent at a concentration of 1.00 M (corresponding to 223 mg/mL of Zirconium acetate and to 91.2 mg/mL of zirconium). This concentration, when added to culture medium in the ratio of 1:100, gave a maximum dose level of 10 mM corresponding to the upper limit to be tested as indicated in the Study Protocol.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hr in test 1; 24 hr in test 2 without S9
- Expression time (cells in growth medium): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1.6 cells/well were plated in each well of two 96-well plates.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it was required that:

1. The induced mutant frequency (IMF) was higher than the global evaluation factor (GEF) suggested for the microwell method (126 x 1E-06) at one or more doses.

2. There was a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria were dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Any increase in mutant frequency lied outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines.
- Test for consistency between plates: chi-squared distribution (alpha = 0.001) with M-1 degrees of freedom (M = number of plates used)
- Heterogeneity factors for replicate cultures: For negative control and test item treatments, the consistency between replicate cultures was evaluated by calculation of the heterogeneity factor (H).
- Test for overall consistency: The overall consistency was evaluated by the calculation of the following ratio: H experiment / current heterogeneity factor. This ratio should not have exceed the one-sided 1% critical values from the F-distribution (the number of degrees of freedom at the numerator is equal to the number of pairs of cultures, whereas the number of degrees of freedom at the denominator is infinite).
- Updated heterogeneity factors: The estimated H experiment values were combined with the current heterogeneity factors to define the updated estimate factors as follows:
Updated heterogeneity = 1/20 H experiment + 19/20 current heterogeneity
- Comparison of each treatment with the control:
The control log mutant frequency was compared with the log mutant frequency from each treatment dose.
- Test for linear trend: The evaluation of a linear trend in mutant frequency with treatment dose was performed using weighted regression.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Main Assay II, slight toxicity was observed after 24 hours of treatment only at 0.625 mM.

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- In the first experiment at the end of the experimental period, dose related precipitation of the test item was noted at the two highest concentrations tested, both in the absence and presence of S9 metabolic activation.
In the second experiment, dose related precipitation of the test item, as indicated by the treatment medium opacity, was noted after 24 hours of treatment from the highest concentration tested down to 0.313 mM. No precipitation of the test iem was observed following the short treatment time.
Solvent and positive control cultures were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range (50‑200 x 1E-06 viable cells). The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65‑120%. The control growth factor over 2 days fell within the range of 8-32 in both experiments.
- Osmolality and pH:
The pH values and osmolality of the post-treatment media were dtermined. The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

Cytotoxicity test (Preliminary Test)
Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 5.00 mM and at a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 mM.
No relevant toxicity was noted using the 3 hour treatment time, both in the absence and presence of S9 metabolic activation, at any concentration tested.
Following 24 hour treatment, marked toxicity was observed at 5.00 mM (7% RS), moderate toxicity (27% RS) was noted at 2.50 mM, no relevant toxicity was observed over the remaining dose levels tested.
At the end of each treatment time, dose related precipitation of the test item was noted from the highest concentration down to 0.313 mM.

COMPARISON WITH HISTORICAL CONTROL DATA: Consistant with historical controls

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that zirconium acetate solution does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.