Aluminium cerium lutetium oxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 15, 2016 - January 15, 2016

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

Test guideline 1

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)

Version / remarks:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) No. 1152/2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

aluminium cerium lutetium oxide

EC Number:

942-299-9

Cas Number:

1279099-21-6

Molecular formula:

Lu2.8-2.99Ce0.01-0.2Al5O12

IUPAC Name:

aluminium cerium lutetium oxide

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Characteristics of donor animals: Age: 14 - 36 months; Corneal diameter: 25 - 27 mm
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL of the suspended test item (i.e. 150 mg/750 µL)
- Concentration: 20% (w/v) suspension in a 0.9% sodium chloride solution

VEHICLE
- Amount applied: 750 µL

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without Stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES: 3


NEGATIVE CONTROL USED: No


SOLVENT CONTROL USED: 0.9% sodium chloride solution


POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of the suspended test item (i.e. 150 mg/750 µL) and 240 min

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. The corneas were gently rinsed with wash medium using a syringe. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments. To remove the solvent and positive control, the corneal surface was washed three times.

METHODS FOR MEASURED ENDPOINTS:

- Corneal opacity: calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: Each cornea was observed visually and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: IVIS <= 3 (no UN GHS category), IVIS > 3; <= 55 (No prediction can be made), IVIS > 55 (UN GHS category 1)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 1/experiment 1

Value:

0.104

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 2/experiment 1

Value:

2.391

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 3/experiment 1

Value:

1.962

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The IVIS obtained after treatment with the test item was 1.5 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.

Executive summary:

This in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Cornea Opacity and Permeability Assay).

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used.

Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 μL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes.

After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro lrritancy Score (IVIS).

All validity criteria were fulfilled.

The IVIS obtained after treatment with the test was 1.5 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.