Zirconium dinitrate oxide

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Zirconium dinitrate oxide

EC Number:

237-529-3

EC Name:

Zirconium dinitrate oxide

Cas Number:

13826-66-9

Molecular formula:

N2O7Zr

IUPAC Name:

nitric acid; oxozirconium

Test material form:

solid

Details on test material:

Name of test material (as cited in study report): zirconium dinitrate oxide

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.