Zirconium dichloride oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro mutagenicity in bacteria

A single reliable (Klimisch 2) key study is published (Mortelmans, 1986), performed according to OECD guideline 471. In this study, zirconium dichloride oxide did not show mutagenic activity in the applied bacterium tester strains in the absence or presence of metabolic activation under the conditions of the test system.

In vitro mammalian chromosome aberration test

A single reliable (Klimisch 1) key study is available (Ciliutti, 2013), performed according to OECD guideline 473 and conform GLP requirements. In this study, the test substance did not induce structural chromosome aberrations in chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions.

In vitro gene mutation study in mammalian cells

No in vitro gene mutation study in mammalian cells with zirconium dichloride oxide is available. Data generated with zirconium acetate is used for endpoint coverage. Justification of this read-across approach is included in section 13. It was concluded from the study that zirconium acetate 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. The same is assumed for zirconium dichloride oxide.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well documented, scientifically sound study that is similar to OECD Guideline 471 "Bacterial Reverse Mutation Test", however, only four strains were evaluated and there was no strain present to detect cross linking mutagens. Study was run on 270 coded chemical samples multiple laboratories.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains were evaluated and there was no strain used to detect cross linking mutagens.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA98, and TA100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat and hamster metabolic activation system

Test concentrations with justification for top dose:

10, 33, 100, 333, 1000, 3333, 6666 µg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (ET 95%)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

Potassium chloride

Positive controls:

yes

Positive control substance:

other: Sodium azide (TA1535 and TA100); 4-nitro-o-phenylenediamine (TA98); 9-aminoacridine (TA1537); 2-aminoanthracene (all strains with hamster and rats liver metabolic activation systems). 9-aminoacridine hydrochloride H2O; 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: The test substance was assayed for mutagenicity in a preincubation assay. The following was added to each test tube: 0.5 mL of S-9 mix or 0.1 M PO4 buffer (pH7.4), 0.05 mL of the overnight culture, and 0.05 mL of solvent. The mixture was mixed and allowed to incubate without shaking at 37 degrees C for 20 min, at which time 2.0 mL of molten top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 mL of minimal glucose bottom agar in 15 x 100 mm plastic petri dishes. When the top agar had solidified, the plates were inverted and incubated at 37 degrees C for 48 hr.

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): L-histidine and D-biotin

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: no data

Evaluation criteria:

The criteria used for data evaluation are summarized as follows: (1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; (2) nonmutagenic response: when no increase in the number of revertants was elicited by the test substance; (3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A preliminary dose-setting test was initiated with strain TA100, in the presence and the absence of the metabolica activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was acknowledged by one or more of the following parameters: appearance of his- pinpoint colonies, reduced number of revertant colonies per plate or thinning or absence of the bacterial lawn.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the test conditions and based on the results the test substance is non-mutagenic in the absence and presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

The endpoint was covered using an in vitro gene mutation study in mammalian cells performed with zirconium acetate, another 'water soluble' zirconium compound with similar behaviour as zirconium dichloride oxide. The read across justification is attached to IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not predictable (read across)

Remarks on result:

other:

Remarks:

Based on the results of an in vitro gene mutation study in mammalian cells performed with zirconium acetate, another 'water soluble' zirconium compound with similar behaviour as zirconium dichloride oxide, negative results (both in the absence and presence of metabolic activation) would also be expected under the conditions of the test when a similar study would be performed with zirconium dichloride oxide.

Conclusions:

No in vitro gene mutation study in mammalian cells with zirconium dichloride oxide is available. Data generated with zirconium acetate is used for endpoint coverage. Justification of this read-across approach is included in section 13. It was concluded from the study that zirconium acetate 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. The same is assumed for zirconium dichloride oxide.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Oct 2012- 03 Jan 2013

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase locus

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.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

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.

Key result

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: Consistent with historical controls

Conclusions:

It is concluded that the 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.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Between 21 February 2013 and 21 March 2013

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: cultures were grown in Ham's F10 medium supplemented with 15% foetal bovine serum. All incubations are at 37°C in a 5% carbon dioxide atmosphere at 100% humidity (nominal).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolising system (S9) induced by a mixture of phenobarbital – 5,6-benzoflavone

Test concentrations with justification for top dose:

Experiment 1 (3 hour treatment): 7.50, 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 mM (corresponding to 1580, 1060, 528, 264, 132, 66.0, 33.0, 16.5, 8.24 and 4.12 μg/mL of zirconium dichloride oxide).
Experiment 2 (20 hour exposure): 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391, 0.0195 and 0.00977 mM (corresponding to 1060, 530, 265, 133, 66.3, 33.1, 16.6, 8.28, 4.14 and 2.07 μg/mL of zirconium dichloride oxide).

Vehicle / solvent:

Suspensions of the test item, as received, were prepared immediately before use in DMSO.
- Vehicle(s)/solvent(s) used: Sterile water of injectable grade.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
- Exposure duration: Experiment 1: 3 hours; experiment 2: 20 hours
- Expression time (cells in growth medium): No data
- Harvest of cells at 20 hours (approximately 1.5 cell cycle length)
- Fixation time (start of exposure up to fixation or harvest of cells): No data, two cultures were prepared at each test point. Air-dried slides were prepared from each culture and stained with 3% Giemsa.

STAIN: 3% Giemsa

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 100 metaphase spreads were scored for chromosomal aberrations from each culture with the exception of one replicate culture treated with Mitomycin and two replicate culture treated with cyclophosphamide from the first main experiment, where, due to the high incidence of aberrant cells (excluding gaps), scoring was terminated at 50 metaphases.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of population doubling

OTHER EXAMINATIONS:
Polyploidy and endoreplicated cells

Evaluation criteria:

The test material was considered to have clastogenic properties if statistically significant increases in the incidence of cells bearing aberrations were observed at any dose level over the concurrent control, the increases exceeded the historical control values and the increases were reproduced in both replicate cultures.

Statistics:

For the statistical analysis, Fisher's Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis was performed using sets of data either including or excluding gaps.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A statistically significant increase (p<0.05), excluding gaps, was observed at the highest dose level selected for analysis (2.50 mM), in the absence of S9 metabolic activation. However, this increase only occurred in one replicate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the 20-hr treatment at the highest dose level (5.00 mM) mild toxicity was observed in the second experiment. No cytotoxicity was observed in the first experiment at any dose level.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the first experiment, dose related reductions of pH values compared to the concurrent vehicle controls were observed at higher dose levels both in the absence and presence of S9 metabolism. The observed changes in pH were more than one unit at the highest concentration of 7.5 mM. No relevant variation of pH was observed in the second experiment.
- Effects of osmolality: Osmolality of media was within the range of physiological values for both experiments.
- Solubility: Solubility of the test item was evaluated in a preliminary trial using sterile distilled water. The test item was found to be soluble in this solvent at a concentration of 1.00 M (corresponding to 211 mg/mL of zirconium dichloride oxide and 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.
- Precipitation: During the first main experiment, at the beginning and by the end of treatment, dose related precipitation was observed at dose levels from 7.50 to 1.25 mM, both in the absence and presence of S9 metabolic activation. In the absence of S9 metabolism precipitation was observed at dose levels from 7.50 to 1.25 mM both in the absence and presence of S9 metabolism, opacity of the treatment media was observed at the next lower dose level of 0.625 mM. During the second experiment, dose related precipitation was observed at the beginning and at the end of treatment at the three highest dose levels of 5.00, 2.50 and 1.25 mM. Opacity of the medium was noted at 0.625 mM.

COMPARISON WITH HISTORICAL CONTROL DATA:
For the first experiment, values of population doubling observed in negative control cultures were within the range of the laboratory historical control data.
For the second experiment, the value of population doubling observed in the untreated control slightly exceeded the range of the laboratory control data (population doubling of 2.24; range 1.15 - 2.07).

Conclusions:

Zirconium dichloride oxide tested as solution (containing 29.2% of zirconium dichloride oxide anhydrous) does not induce structural chromosome aberrations in Chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

no data

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Insufficient information provided on methods and results to accurately evaluate the study. Study was not conducted according to GLP and by comparison to OECD guideline 473, there was an insufficient amount of doses, number of cells scored, no metabolic activation, positive controls or cytotoxicity examined.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: Only 1 test concentration was used rather than 3; only 100 cells were scored rather than 200; no positive control; no metabolic activation; no measure of cytotoxicity.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Test evaluated human peripheral venous blood from normal human healthy donors of both sexes of various age groups without addiction to tobacco and alcohol.

Metabolic activation:

without

Test concentrations with justification for top dose:

For each sample one set was treated with aqueous zirconium oxychloride solution (20 µg/mL of culture medium).

Vehicle / solvent:

culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Positive control substance:

no

Details on test system and experimental conditions:

RPMI 1640 (Gibco) supplemented with human (AB+) serum (20%) and phytohaemaglutinin (0.4 mL) was used as culture medium.
In choosing this dose a corresponding dilution of the LD50 dose was used for in vitro samples. Each set consisted of 2 culture tubes for the study of chromosomal aberrations (CAs).

Cells were harvested after 72 hour of incubation following the usual colchicine hypotonic-acetic/methanol (1:3) fixation and air drying-Giemsa schedule.

All cultures were performed in replicate for each parameter. Endpoints screened were chromosome and chromatid breaks, dicentrics and rearrangements. The frequencies of chromosomal aberrations were compared between the samples of different age groups. Observations were taken from both culture tubes and pooled. Slides were coded and scored blind. One hundred well scattered, evenly stained metaphase plates were scored to study different types of chromosomal aberrations.

Evaluation criteria:

For calculation of total breaks, a chromatid break was taken as one break, and chromosome breaks, dicentrics and translocations, as two breaks.

Statistics:

The frequencies of CAs were analysed statistically following Student's t-test to compare treatment and respective control sets. In addition, analysis of variance test, two-way ANOVA with replication was used to study age-related effects of the chemical.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

Treatment with zirconium oxychloride increased the frequencies of CAs in all cultures obtained from individuals of all age groups. Lymphocytes of the oldest age group (51-60) from both male and female samples showed a statistically significant increase in aberrations when compared with their respective untreated sets. Types of aberrations recorded were mostly, of chromatid breaks and gaps with a few chromosome breaks, dicentrics and translocations.
Increased frequencies of dicentrics and other rearrangements were mostly observed in age groups of 21-30, 31-40, 41-50, 51-60 years, but not to a significant level. Samples from females of the oldest age group showed significantly higher breaks/cell after treatment (P < 0.1) when compared with those of other age groups. Critical analysis by the two-way ANOVA model showed that the effect of the dose alone was significant (< 0.001) while the effects of age and interactions between age and dose were not significant.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Critical analysis by the two-way ANOVA model showed that the effect of the dose alone was significant while the effects of age and interactions between age and dose were not significant.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

no data

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Insufficient information provided on methods and results to accurately evaluate the study. Study was not conducted according to GLP and by comparison to OECD guideline 479, there was an insufficient amount of doses, no metabolic activation, positive controls or cytotoxicity examined.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

: Only 2 test concentrations were used rather than 3; no positive control; no metabolic activation; no measure of cytotoxicity.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Test evaluated human peripheral venous blood from normal human healthy donors of both sexes of various age groups without addiction to tobacco and alcohol (separate from the chromosomal aberration and micronuclei test group).

Metabolic activation:

without

Test concentrations with justification for top dose:

For each sample one set was treated with aqueous zirconium oxychloride solution (10 and 20 µg/mL of culture medium).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Positive control substance:

no

Details on test system and experimental conditions:

RPMI 1640 (Gibco) supplemented with human (AB+) serum (20%) and 5-bromodeoxyuridine (Brdu, Sigma) at a concentration of 8 µg/mL was used as culture medium.

Replicate cultures were maintained for all control and treatment sets, giving a total of 6 cultures for each sample. The treatment period was 72 h at 37 degrees C in darkness.

Cells were harvested in darkness following the standard protocol. Fluorescence plus Giemsa staining was employed with modification. All slides were coded. For each set of experiments 50 well scattered second division metaphase plates were observed to study SCEs.

Statistics:

The frequencies of SCEs were analysed statistically following Student's t-test to compare treatment and respective control sets. In addition, analysis of variance test, two-way ANOVA with replication was used to study age-related effects of the chemical.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

The frequencies of SCEs were increased after treatment (10 µg and 20 µg of ZrOCI2) in male samples of all age groups. Samples of the 21-30, 31-40, and 41-50 years age groups showed a significant increase in SCEs after treatment with low dose. At the group level (between individuals), however, mean SCE frequencies were found to be independent of donor age as observed from the range of SCEs among the age groups and by analysis of variance.
In female donors, mean SCEs were slightly higher than normal male donors and range of SCE frequencies was increased after treatment. Increase in SCEs was also linearly age dependent and older age groups (41-50 and 51-60) showed signficant increase (P<0.1 to P<0.05). Within each age group, the difference between the effects of the two doses was not significant.
Critical analysis of the data in a two-way ANOVA model, taking age and dose as factors, revealed that the increase in mean SCEs of female samples was dependent on donor's age and on the treatment doses at the group level (P<0.001). But the interaction of the two factors was not significant.

Conclusions:

The frequencies of SCEs were increased after treatment (10 µg and 20 µg of ZrOCI2) in male samples of all age groups. Samples of the 21-30, 31-40, and 41-50 years age groups showed a significant increase in SCEs after treatment with low dose. At the group level (between individuals), however, mean SCE frequencies were found to be independent of donor age as observed from the range of SCEs among the age groups and by analysis of variance.
In female donors, mean SCEs were slightly higher than normal male donors and range of SCE frequencies was increased after treatment. Increase in SCEs was also linearly age dependent and older age groups (41-50 and 51-60) showed signficant increase (P<0.1 to P<0.05). Within each age group, the difference between the effects of the two doses was not significant.
Critical analysis of the data in a two-way ANOVA model, taking age and dose as factors, revealed that the increase in mean SCEs of female samples was dependent on donor's age and on the treatment doses at the group level (P<0.001). But the interaction of the two factors was not significant.

Reference 7

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Insufficient information provided on methods and results to accurately evaluate the study. Study was not conducted according to GLP and by comparison to OECD draft guideline 487, there was an insufficient amount of doses, number of cells scored, no metabolic activation, positive controls or cytotoxicity examined.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Draft Guideline 487

Deviations:

yes

Remarks:

: Only 1 test concentration was used rather than 3; 1000 metaphases were scored rather than 2000; no positive control; no metabolic activation; no measure of cytotoxicity.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Test evaluated human peripheral venous blood from normal human healthy donors of both sexes of various age groups without addiction to tobacco and alcohol.

Metabolic activation:

without

Test concentrations with justification for top dose:

For each sample one set was treated with aqueous zirconium oxychloride solution (20 µg/mL of culture medium).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Positive control substance:

no

Details on test system and experimental conditions:

RPMI 1640 (Gibco) supplemented with human (AB+) serum (20%) and phytohaemaglutinin (0.4 mL) was used as culture medium.
In choosing this dose a corresponding dilution of the LD50 dose was used for in vitro samples. Each set consisted of 2 culture tubes used for the micronucleus analysis.

Cells were harvested after 72 hour of incubation following the usual colchicine hypotonic-acetic/methanol (1:3) fixation and air drying-Giemsa schedule. The hypotonic used was NaCl (0.09%) 9 parts and KCl (0.56%) 1 part for 5 minutes.

All cultures were performed in replicate for each parameter. One thousand cells were observed for the micronuclei count.

Statistics:

The frequencies of micronuclei were analysed statistically following Student's t-test to compare treatment and respective control sets. In addition, analysis of variance test, two-way ANOVA with replication was used to study age-related effects of the chemical.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

The frequency of micronuclei formation was enhanced in samples from 21-30 to 51-60 years age groups of both sexes after treatment with the chemical but not to a statistically significant level. Age of the sample did not affect the frequencies of micronuclei in control subjects.

Remarks on result:

other: all strains/cell types tested

Conclusions:

No statistically significant increases in micronuclei were found following exposure to the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

no data

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Bone marrow was only collected once at 24 hours rather than at 12-18 hours and 24 h thereafter, colchicine was given 2 hours before sampling rather than 3-5 hours before sampling; a positive control was not used; results were not presented per animal and no listing of the different types of structural chromosome aberrations was presented. Oral administration was unspecified.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

See rationale for reliability scoring.

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-9 weeks of age
- Weight at study initiation: 28-30 g
- Diet (e.g. ad libitum): ad libitum Gold Mohur mice feed; manufactured by Lipton India Limited
- Water (e.g. ad libitum): ad libitum
- Houding: Temperature controlled chambers

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water

Details on exposure:

no data

Duration of treatment / exposure:

single oral administration

Frequency of treatment:

single dose

Post exposure period:

not applicable

Dose / conc.:

225 other: mg/kg actual ingested - males

Dose / conc.:

750 other: mg/kg actual ingested - males

Dose / conc.:

2 250 other: mg/kg actual ingested - males

Dose / conc.:

220 other: mg/kg actual ingested - females

Dose / conc.:

734 other: mg/kg actual ingested - females

Dose / conc.:

2 200 other: mg/kg actual ingested - females

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

Bone marrow was flushed out and prepared for analysis of chromosomal aberration.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses were calculated as fractions of the LD50 dose determined for the test substance.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): A single oral administration of the test substance and animals were sacrificed 24 hours after adminstration.

DETAILS OF SLIDE PREPARATION: 2 h before sacrifice, all mice were intraperitoneally injected with colchicine, 4 mg/kg. Bone marrow was flushed out and prepared for analysis of chromosomal aberrations, following the usual hypotonic acetic acid-ethanol fixation and Giemsa staining schedule. Slides were coded and scored blind for total chromosomal abnormalities.

Evaluation criteria:

50 well-scattered metaphase plates per animal and a total of 250 metaphase plates per sex per treatment group were observed for chromosomal aberrations. 5000 cells per sex per treatment were scored to estimate divisional frequency.

Statistics:

The results obtained for total chromosomal abnormalities were statistically analysed for dose response following trend test according to Margolin et al., 1986. The level of significance was established at p

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

Observations made 24 h after the administration of zirconium oxychloride in concentrations of 2250 and 750 mg/kg body weight in males and 2200 and 734 mg/kg body weight in females increased the divisional frequency in comparison to control. However, mitotic divisional frequency was not enhanced appreciably by the lowest dose used (225 mg/kg for males and 220 mg/kg for females).
Control: 2.74% mitotic cells in males and 2.90% in females
Low dose: 3.10% mitotic cells in males and 3.96% in females
Mid dose: 5.50% mitotic cells in males and 5.60% in females
High dose: 4.90% mitotic cells in males and 5.80% in females

Animals exposed to the test substance reported significant enhancement of the frequency of aberrant metaphases as compared with the control. The percentages of total abnormalities were increased considerably in both sexes with all concentrations used. The degree of increase was directly proportional to the concentration and was significant between the control and the next dose used and also between the consecutive doses.
In general, the abnormalities were relatively higher in the female mice than in the male ones, but the difference was not statistically significant. Induced abnormalities included both chromosomal aberrations and spindle disturbances. The former were mainly chromatid and chromosome breaks with a few cases of centric fusion. The action of zirconium appears to be on different phases of the cell cycle.
Total aberrant metaphases (mean):
control: 5.6% in males and 5.2% in females
Low dose: 10.8% in males and females
Mid dose: 18.0% in males and 19.0% in females
High dose: 22.5% in males and 24.8% in females

Conclusions:

Under the test conditions the frequencies of aberrations were directly proportionate to the concentration used. Female mice were found to be more susceptible than male mice, though not to a significantly higher level. The results of this study should be treated with great care for the following reasons:
1) Only total chromosomal abnormalities are presented, which include structural chromosome aberrations and spindle disturbances, the latter being
polyploidy. Polyploidy is a disturbance in mitotic processes and cell cycle progression. An increase in polypoloidy may indicate that a chemical has the potential to induce numerical aberrations. However, polyploidy is not related to clastogenicity, and should therefore be included separately from the structural chromosome aberrations. As this study does not distinguish between the two, a conclusion on clastogenicity cannot be drawn.
2) An aqueous solution of zirconium oxychloride is given to the mice. It is known from water solubility/hydrolysis experiments that the substance in water will cause a drop in pH by the release of H+ ions. Apart from the fact that the mice have probably been dosed corrosive solutions, there is some evidence that H+ ions can cause chromosomal damage (refer to IARC monograph 54-8 (1992; p. 203) on hydrochloric acid). It can thus not be ruled out that the observed effects in the bone marrow have been caused by the low pH after dissolving the test substance in water.
3) No data was presented on whether the substance can actually reach the bone marrow and whether this test is thus biologically relevant. Some evidence exists that zirconium oxychloride is poorly absorbed by mice after oral administration of an aqueous solution of 1500 mg/kg (Delongeas et al., 1983, J. Pharmacol. 14(4) 437-447). It reaches the blood and after 6 h a maximal blood concentration of 2.9 mg Zr/L (i.e. about 10 mg zirconium oxychloride per liter) was reached. In a similar but more extensive study in rats given aqueous solutions of 3000 mg zirconium oxychloride/kg, the maximal blood concentration was reached after 6 hours being about 0.3 mg Zr/L (i.e. about 1 mg zirconium oxychloride per liter). Using metabolic cages it was shown that within 24 hours 90-98% of the given dose was excreted via feces and minimal amounts via urine. This shows that the substance is hardly absorbed as it is excreted via feces.
In the same study of Delongeas et al. (1983), tissue distribution reveals that the small absorbed fraction is distributed and fixed in the ovaries, liver and lung, and to a lesser degree in bone and CNS. All together this raises the question whether the substance in the current study has reached the bone marrow to a sufficient extent in order to induce effects.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Data waiving:

other justification

Justification for data waiving:

other:

Additional information

Genetic toxicity in vitro:

In vitro gene mutation study in bacteria

An Ames test with zirconium dichloride oxide is performed by Mortelmans et al. (1986). The test has been performed with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. Both with and without Aroclor 1254-induced rat and hamster liver S9 mix, zirconium dichloride oxide tested negative in this study. This study was considered as key study for this endpoint.

In vitro chromosome aberration test in mammalian cells

Ciliutti (2013) performed an in vitro Chromosome Aberration test in Chinese hamster ovary cells (OECD 473) with zirconium dichloride oxide. Two experiments were performed using different test concentrations with and without S9 activation. Both negative and positive controls were considered to be valid. On the basis of the results obtained, it was concluded that zirconium dichloride oxide did not induce structural chromosome aberrations after in vitro treatment and under the reported conditions. This study was selected as key study for this endpoint.

In vitro mammalian cell gene mutation test

No reliable data are available for zirconium dichloride oxide for this endpoint. Therefore, read across data from zirconium acetate, another 'water soluble' zirconium compound with similar behaviour as zirconium dichloride oxide, were used to cover the endpoint. In this read across study, Bisini (2013) performed an in vitro mammalian gene mutation assay in L5178Y TK+/- lymphoma cells using the fluctuation method according to OECD Guideline 476. Two experiments were performed using different concentrations with and without metabolic S9 activation (experiment I) and without metabolic S9 activation (experiment II). Plates were tested in duplicate. Plates were exposed for 3 h (experiment I) and 24 h (experiment II). The positive and negative (vehicle) controls were considered to be valid. It was concluded that the test substance did 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 experimental conditions.

Other studies

Next to the abovementioned key studies, some other in vitro data on genetic toxicity of zirconium dichloride oxide have been identified as well, more specifically from a publication of Ghosh et al. (1992), presenting the results of three in vitro tests conducted using human lymphocytes collected from several age groups. The three tests include a chromosome aberration test, a micronucleus test and a sister chromatid exchange assay. These studies were however not considered reliable (Klimisch 3).

In the publication from Ghosh et al. (1992) the effects of age on in vitro genetic toxicity were assessed. Human lymphocytes from various age groups and from both men and women were collected and used to assess chromosome aberrations, micronuclei and sister chromatid exchanges. No metabolic activation system was used. Zirconium dichloride oxide caused an increase in the number of chromosome aberrations in all cultures tested, but mostly not significant compared to the controls. The substance also caused an increase in the number of micronuclei in all cultures, but not to a significant extent. The number of sister chromatid exchanges were increased significantly in various age groups in both males and females, but not in all age groups. Taking into account the reliability of the study, it can be concluded that the positive results in some of the age groups are ambiguous and can be disregarded.

Conclusion based on in vitro testing

Based on the available information, zirconium dichloride oxide is considered negative for in vitro genetic toxicity.

Genetic toxicity in vivo:

According to REACH Annex IX section 8.4, column 2, no further in vivo testing is required as no positive results were obtained in any of the three in vitro studies performed according to REACH Annexes VII and VIII section 8.4.

An in vivo mouse bone marrow chromosome aberration test is available (Ghosh et al., 1990). This study was not considered reliable (Klimisch 3). Under the test conditions the frequencies of aberrations were directly proportionate to the concentration used. Female mice were found to be more susceptible than male mice, though not to a significantly higher level.

However, after a critical review of the study, it was concluded that the results are ambiguous and therefore the study is disregarded.

The results of the study should be treated with great care for the following reasons:

1) Only total chromosomal abnormalities are presented, which include structural chromosome aberrations and spindle disturbances, the latter being polyploidy. Polyploidy is a disturbance in mitotic processes and cell cycle progression. An increase in polypoloidy may indicate that a chemical has the potential to induce numerical aberrations. However, polyploidy is not related to clastogenicity, and should therefore be included separately from the structural chromosome aberrations. As this study does not distinguish between the two, a conclusion on clastogenicity cannot be drawn.

2) An aqueous solution of zirconium oxychloride is given to the mice. It is known from water solubility/hydrolysis experiments that the substance in water will cause a drop in pH by the release of H+ ions. Apart from the fact that the mice have probably been dosed corrosive solutions, there is some evidence that H+ ions can cause chromosomal damage (refer to IARC monograph 54-8 (1992; p. 203) on hydrochloric acid). It can thus not be ruled out that the observed effects in the bone marrow have been caused by the low pH after dissolving the test substance in water.

3) No data were presented on whether the substance has actually reached the bone marrow and whether this test is thus biologically relevant. Some evidence exists that zirconium oxychloride is poorly absorbed by mice after oral administration of an aqueous solution of 1500 mg/kg (Delongeas et al., 1983, J. Pharmacol. 14(4) 437-447). It reaches the blood and after 6 h a maximal blood concentration of 2.9 mg Zr/L (i.e. about 10 mg zirconium oxychloride per liter) was reached. In a similar but more extensive study in rats given aqueous solutions of 3000 mg zirconium oxychloride/kg, the maximal blood concentration was reached after 6 hours being about 0.3 mg Zr/L (i.e. about 1 mg zirconium oxychloride per liter). Using metabolic cages it was shown that within 24 hours 90-98% of the given dose was excreted via feces and minimal amounts via urine. This shows that the substance is hardly absorbed as it is excreted via feces. In the same study of Delongeas et al. (1983), tissue distribution reveals that the small absorbed fraction is distributed and fixed in the ovaries, liver and lung, and to a lesser degree in bone and CNS. All together this raises the question whether the substance in the current study has reached the bone marrow to a sufficient extent in order to induce effects.
In vivo:
No reliable studies are available for zirconium dichloride oxide. However, since the results of all in vitro tests were negative, no in vivo testing is needed according to the REACH Regulation.

Justification for classification or non-classification

Based on the results of three different types of in vitro tests, the substance is considered not to be genotoxic. Therefore the substance does not need to be classified for mutagenicity.