Gadolinium zirconium oxide (Gd2Zr2O7)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-09 to 2016-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Gadolinium zirconium oxide

Cas Number:

11073-79-3

Molecular formula:

Gd2Zr2O7

IUPAC Name:

Gadolinium zirconium oxide

Test material form:

solid

Details on test material:

- Batch No.: 150273V2
- Colour: white
- Density: 1.76 g/cm³
- Active Components: > 98 %
- Purity: > 98 %
- Expiry Date: 5 years after production
- Storage Conditions: room temperature, dry in closed container

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL:
- 0.75 g of the test item was applicated directly onto the cornea and moistened with one drop of physiological saline 0.9 % NaCl (open-chamber-method). 750 µL of the control substance was introduced into the anterior chamber (closed-chamber-method)

VEHICLE
- Concentration (if solution): physiological saline (0.9 % NaCl)

Duration of treatment / exposure:

4 h ± 5 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 120 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (imidazole 20% in physiological saline 0.9% NaCl).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The eyes were carefully examined for defects and defectives eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C.

TREATMENT METHOD:
- Closed chamber for controls
- Open chamber for the test item

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Washing with MEM containing phenol red until the medium was free of test substance (at least 3 times), then completly rinsed with RPMI without phenol red.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. After the 4 hours incubation period and subsequent washing the anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490):
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 120 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in Table 1 (see "Any other information on materials and methods").
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made. For this purpose further testing with another suitable method is required.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (triplicates)

Value:

0.32

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Table 2: In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.33	0.05	1.02
2		0.33	0.035
3		0.41	0.048
MV		0.36	0.044
4	Positive Control	99.37	3.311	142.3
5		94.74	3.451
6		83.13	3.216
MV		92.41	3.326
7	Test Item	-0.21	0.034	0.32
8		0.71	-0.019
9		0.75	-0.033
MV		0.42	-0.0006

MV = mean value

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the mean in vitro irritation score of 0.32 obtained in the bovine corneal opacity and permeability assay (OECD 437), the test item is considered to be not irritating under the test conditions reported.

Executive summary:

The eye irritation potential of the test item (> 98% purity) was investigated in the bovine corneal opacity and permeability assay in accordance with OECD 437. The test item was applied directly to the cornea and moistened with one drop of 0.9% physiological saline. The corneal opacity was measured before and after treatment (4 hours). The mean in vitro irritation score was determined with 0.32. The positive control induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, Gadolinium zirconium oxide can be considered as not irritating to the eye (UN GHS No Category).