Registration Dossier

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Administrative data

Description of key information

Skin irritation/corrosion: 
Four studies were used to cover the endpoint with a weight of evidence approach: one study performed to determine the pH and the acidic reserve of zirconium dinitrate oxide (Klimisch 1, Solvay, 2014, see IUCLID Section 4.20), two in vitro tests using the EPISKIN Model to assess both irritation (OECD guideline 439) and corrosion potential (OECD guideline 431) of the substance (Klimisch 1, Hargitai, 2014a,b) and one in vivo skin irritation test performed according to OECD guideline 404 (Klimisch 1, Matting, 2015b). Those studies interpreted together conclude that it is unlikely that zirconium dinitrate oxide has a potential for skin irritation (solid test item).
Eye irritation/corrosion:
An isolated chicken eye test was performed according to OECD guideline 438 to assess the eye corrosion/irritation potential of the substance (Klimisch 1, Váliczkó, 2014). Given the results, zirconium dinitrate oxide is considered severely irritating (Eye Damage Category 1 under the CLP Regulation).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25/11/2014-28/11/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
Relative humidity of min. 26% with no impact on outcome of the study and interpretation of the results
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
S&K-LAP Kft.
2173 Kartal, Császár út 135, HUNGARY
- Age at study initiation: 11 weeks old
- Weight at study initiation: 2832 – 2944 g
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): municipal tap water, as for human consumption, ad libitum, from an automatic system
- Acclimation period: 6/8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 – 21.5 °C
- Humidity (%): 26 – 68 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
other: the untreated skin of each animal
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
The test item was moistened with water to ensure good skin contact. The untreated skin of each animal served as a control.
Duration of treatment / exposure:
4 hours
Observation period:
To assess skin irritation, the animals were examined 1, 24, 48 and 72 hours after patch removal.
Number of animals:
For ethical reasons, an initial test was performed using a single animal. One hour after application of the test item to the skin of the sentinel animal, the application site was examined and evaluated. After one hour in one rabbit no corrosive effect, no significant systemic toxicity and no other severe local effects were observed in the initial test, therefore the bandage was replaced and the exposure continued for a further 3 hours (a total 4 hours of exposure).
At 24 hour observation time the substance was found not to be corrosive. There was no significant systemic toxicity and no other severe local effects have been observed in the initial tested animal. Therefore the 2 other rabbits were exposed.
Details on study design:
TEST SITE
- Area of exposure:
The test item was applied to an approximately 6 cm² area of intact skin.
A single layer of a fine medical gauze (open-weave with large holes) of approximately 5x5 cm was placed over the application area.
The appropriate amount of test item was carefully spread over the application area (the gauze helped maintain the test item in place).
Three more layers of gauze were placed over the test item.
- Type of wrap if used:
These gauze patches were kept in contact with the skin by a patch of clear plastic with a surrounding adhesive hypoallergenic plaster to ensure continued good contact between the moistened test item and the shaved skin.
The entire trunks of the animals were wrapped with plastic wrap for 4 hours.
Medical elastic tubing was placed over the plastic to keep it in place.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the treatment period, the test item was removed with water at body temperature.
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable, score 0 from first reading
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable, score 0 from first reading
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable, score 0 from first reading
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable, score 0 from first reading
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable, score 0 from first reading
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable, score 0 from first reading
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
other: sum of all edema scores and erythema scores for each animal at each time point (18 values) divided by 9 (3 test animals and 3 time intervals)
Time point:
24/48/72 h
Score:
0
Max. score:
8
Reversibility:
other: not applicable, score 0 from first reading
Irritant / corrosive response data:
At observation time 1, 24, 48 and 72 hours after patch removal, there were no observed local and clinical signs noted on the skin of the treated animals. As no clinical signs were observed at 72 hours after patch removal, the study was terminated after the 72 hours observation period.
Other effects:
No mortality observed during the study
No test item related clinical signs noted
No test item related effect on body weight
Interpretation of results:
GHS criteria not met
Conclusions:
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals and the CLP regulation 1272/2008, zirconium dinitrate oxide does not require classification as a skin irritant.
According to the classification system based on the scheme devised by Draize (1959), zirconium dinitrate oxide is a "non-irritant".
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-09-18 to 2014-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified (adult)
Source strain:
other: not applicable
Justification for test system used:
The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD Guideline 431); therefore, it was considered to be suitable for evaluation of this endpoint.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (Manufacturer: SkinEthic, France)
- Tissue batch number(s): 14-EKIN-035
- Expiry date: 22 September 2014
- Date of initiation of testing: 18 September 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.6-24.7°C
- All incubations with MTT dye were performed protected from light in a > 95% humidified atmosphere containing 5% CO2 in air at 37°C.
- Overnight incubations for formazan extraction were performed at room termperature protected from light with gentle agitation (150 rpm).

REMOVAL OF TEST MATERIAL AND CONTROLS
After the incubation time, all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with approximately 25 mL PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: plate reader, not further specified
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 2

COLOUR CONTROL
- As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if both disks have mean viability of < 35%.
- The test substance is considered to be non-corrosive to skin if both disks have mean viability of >=35%.
- Justification for the selection of the cut-off point(s): The prediction model corresponds to the methods agreed by EU regulatory agencies in line with OECD guideline 431. The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
Duration of treatment / exposure:
4 h
Duration of post-treatment incubation (if applicable):
Not applicable (except for 3 h with MTT dye and overnight incubation for formazan extraction before optical density measurement).
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
relative tissue viability (compared to the negative control)
Run / experiment:
mean of two replicates
Value:
68.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosiveness as mean relative viability of the two replicates was > 35%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: no
- Colour interference with MTT: NSC (non-specific colour) was determined to be 2.3%, which was below the threshold of 5%, consequently, no correction due to colouring potential was needed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Validity of the test

After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper condition.

The mean OD value of the two negative control tissues was in the recommended range (0.900).

The positive control treated tissues showed 1.1% viability demonstrating the proper performance of the assay.

The difference of viability between the two test item-treated tissue samples in the MTT assay was 1.5%.

All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the in vitro EPISKIN Model test, zirconium dinitrate oxide was found to be not corrosive to skin.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-09-25 to 2014-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult, not further specified
Source strain:
other: not applicable
Justification for test system used:
The EPISKIN (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439). Therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN (SM), SkinEthic, France
- Tissue batch number(s): 14-EKIN-036
- Expiry Date: 29 September 2014
- Date of initiation of testing: 25 September 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.4-23.8°C
- Temperature of post-treatment incubation (if applicable): 37°C
- All post-treatment incubations were carried out in a > 95% humidified atmosphere containing 5% CO2 in air, protected from light, and at 37°C (except for incubation for formazan extraction after the 3-h incubation with MTT-dye - the 2-h incubation for formazan extraction was at room temperature).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After 15 min incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with phosphate buffered saline (PBS) to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: yes, plate reader, not further specified
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 3

COLOUR CONTROL
- As the test item was coloured, one additional test item-treated tissue was used for the non-specific OD evaluation.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritant to skin if the relative mean viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than 50% of the mean viability of the negative control treatments.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
As the test item was solid, first an appropriate amount (at least 10 µL) of distilled water was applied to the epidermal surface (in order to improve further contact between powder and epidermis) and then 20 mg of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a curved flat spatula (or other appropriate tool) in order to cover evenly all the epidermal surface (without damaging the epidermis). The amount was sufficient to cover the epidermal surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
relative viability (compared to negative control)
Run / experiment:
mean of 3 replicates
Value:
83.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
no indication of irritation as the mean relative tissue viability in the 3 treated replicates was > 50%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: no
- Colour interference with MTT: NSC (non-specific colour) was determined to be 3.6%, which is lower than the cut-off value of 5%, consequently, correction for colouring potential was not necessary

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kits were checked in each case. Based on the observed colours, the epidermis units were in proper condition.

The mean OD value of the three negative control tissues was in the recommended range (1.027). Standard deviation for negative control samples was 17.1.

The positive control treated tissues showed 9.3% viability demonstrating the proper performance of the assay. The standard deviation value for positive control samples was 2.5.

The standard deviation for viability values of the three test item-treated tissue samples in the MTT assay was 17.3.

All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure to zirconium dinitrate oxide, the mean relative viability was 83.8% compared to the negative control value. This is above the threshold of 50%, therefore the substance was considered as being non-irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
other: COBB 500
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129.
Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

EYES SELECTION AND PREPARATION
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, and placed on a layer of tissue with the cornea facing upwards. The eyes were held in horizontal position, while the test item was applied onto the cornea. The test item was applied in an amount of 30 mg onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 mg powdered imidazole. The negative control eye was treated with 30 µL of physiological saline (Salsol solution, NaCl 0.9% w/v).
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
Number of animals or in vitro replicates:
One eye was treated with physiological saline, three eyes with the test item and another three with imidazole (positive control).
Details on study design:
REMOVAL OF TEST SUBSTANCE
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all the residual test item if possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or control material remaining on the cornea was observed.

EVALUATION
Corneal swelling was calculated according to the following formulae:

CS at time t = (CT at time t – CT at t=0) / (CT at t=0) x 100

Mean CSmax at up to 75 min = (FECSmax(30min to 75min) + SECSmax(30min to 75min) + TECSmax(30min to 75min)) / 3

Mean CSmax at up to 240 min = (FECSmax(30min to 240min) + SECSmax(30min to 240min) + TECSmax(30min to 240min)) / 3

Remark:
CS = cornea swelling
CT = cornea thickness
FECS = first eye cornea swelling
SECS = second eye cornea swelling
TECS = third eye cornea swelling
max(30min to 75min) = maximum swelling of the individual eye at 30 to 75 minutes
max(30min to 240min) = maximum swelling of the individual eye at 30 to 240 minutes

For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (>12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.

Corneal opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t=0

Mean ΔCOmax = (FECOmax(30min to 240min) + SECOmax(30min to 240min) + TECOmax(30min to 240min)) / 3

Remark:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = base line cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity base line
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus base line cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t – FR at t=0

Mean ΔFR = (FEFR (30min) + SEFR(30min) + TEFR(30min)) / 3

Remark:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = base line fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention base line
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus base line fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus base line fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus base line fluorescein retention

STORAGE OF CORNEAS
At the end of the procedures, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin) for potential histopathology and stored.
Irritation parameter:
percent corneal swelling
Remarks:
maximum
Run / experiment:
mean / 3 corneas / 75 min
Value:
4.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
maximum
Run / experiment:
mean / 3 corneas / 240 min
Value:
-3.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
cornea opacity score
Remarks:
maximum
Run / experiment:
mean / 3 corneas / 30-240 min
Value:
2.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III
Irritation parameter:
fluorescein retention score
Run / experiment:
mean / 3 corneas / 30 min
Value:
1.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III
Other effects / acceptance of results:
The test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared 240 minutes after the post-treatment rinse. Loosening of epithelium was observed in two eyes (2/3) at 180 minutes after the post-treatment rinse.

In this in vitro eye irritation assay with isolated chicken eyes exposed to zirconium dinitrate oxide, very significant corneal opacity, fluorescein retention and loosening of the epithelium were observed. The weight of evidence indicates a classification in Category 1.

Negative control:

The negative control, physiological saline, was classified as non irritating.

Positive control:

The positive control, imidazole, was classed as severely irritating.

Validity of the test:

The results from all eyes used met the quality control standards. The negative control and positive control results were in good correlation with the historical data. This experiment was considered to be valid.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on this in vitro eye irritation test with isolated chicken eyes exposed to zirconium dinitrate oxide, the test item was considered to be severely irritating and needs to be classified in Eye Damage Category 1 according to the CLP regulation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Determination of the pH value and the acidic/alkaline reserve (see IUCLID Section 4.20):

The pH of a 10% zirconium dinitrate oxide solution was determined electrochemically at 20°C with a calibrated pH meter. A pH value of 1.17 was determined. In addition, the acidic reserve of the solution was determined via titration of a 1 mol/L NaOH solution, which was required to achieve a pH of 4 of a 10% solution (or slurry) at 20°C. The acidic reserve value was determined to be 9.62. The calculation for the corrosivity, based on the pH value and on the acidic reserve value, was 0.368 (higher than -0.5) and the substance was considered not to be corrosive.

Skin irritation/corrosion:

Two in vitro studies and one in vivo study were used to cover the endpoint for skin irritation/corrosion potential.

In the in vitro skin corrosivity test, disks of EPISKIN (two units) were treated with the test item zirconium dinitrate oxide and incubated for 4 hours at room temperature. This study was performed according to OECD guideline 431 and in accordance with GLP and was thus scored as Klimisch 1. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis units were used as negative and positive controls. Two additional disks were used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to the skin.

Following exposure to zirconium dinitrate oxide, the mean viability was 68.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with zirconium dinitrate oxide, the results indicate that the test item is not corrosive to skin.

In the in vitro skin irritation test, disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. This study was performed according to OECD guideline 439 and in accordance with GLP and was thus scored as Klimisch 1. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) sodium dodecyl sulphate (SDS) solution treated epidermis units were used as negative and positive controls, respectively (three units/control). An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (<=) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure to zirconium dinitrate oxide, the mean cell viability was 83.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with zirconium dinitrate oxide, the results indicate that the test item is non irritant.

Finally, an acute skin irritation study was performed with zirconium dinitrate oxide in New Zealand White rabbits. This study was performed according to OECD guideline 404 and in accordance with GLP and was thus scored as Klimisch 1. Parameters monitored during this study included mortality, body weight measurements and clinical observations. The irritancy of the test item was evaluated according to the Draize method.

An amount of 0.5 g of the powdered test item was applied to the skin of three experimental animals. The test item was applied as a single dose. The test item was moistened with water to ensure good skin contact. An adhesive clear plastic patch was applied. The trunk was wrapped in clear plastic with medical tubing used to hold the patch in place. The untreated skin of each animal served as control. After 4 hours, the remaining test item was removed with water at body temperature.

To assess skin irritation, the animals were examined at 1, 24, 48 and 72 hours after the patch removal. Additional general examinations were performed daily for general clinical health. There was no mortality during the observation period. There was no test item related effect on body weight.

At observation time 1, 24, 48 and 72 hours after patch removal, no local and clinical signs have been noted on the skin of the treated animals. As no clinical signs were observed at 72 hours after patch removal, the study was terminated after the 72 hours observation period. According to the UN Globally Harmonised System of Classification and Labelling of Chemicals and the CLP Regulation 1272/2008, zirconium dinitrate oxide does not require classification as a skin irritant.

Eye irritation

An in vitro eye irritation study of the test item was performed in isolated chicken's eyes. The irritation effects of the test item were evaluated according to OECD guideline 438 and in accordance with GLP and was thus scored as Klimisch 1.

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 µL of physiological saline (Salsol solution, NaCl 0.9% w/v).

The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.

In this in vitro eye irritation assay with isolated chicken eyes exposed to zirconium dinitrate oxide, very significant corneal opacity, fluorescein retention and loosing of the epithelium were observed. The test item was then concluded to be severely irritating: Eye Damage Category 1 (H318).

Justification for classification or non-classification

Based on the available data on skin irritation and according to the criteria of the CLP Regulation, the substance does not have to be classified as a skin irritant.

Based on the available data from the in vitro eye irritation test and according to the criteria of the CLP Regulation, the substance is classified in Eye Damage Category 1 (H318).