Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 2017 - 15 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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:
July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: isolated bovine cornea
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 months old
- Storage temperature: ambient temperature
- Transport conditions of ocular tissue: The isolated eyes were transported to the laboratory in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin).
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3 corneas per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. 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. Care was taken to assure no air bubbles were present within the compartments. The corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. At the end of the incubation period, the basal opacity was determined (t0). Each corneae with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 min

POST-INCUBATION PERIOD: yes (2 hours)

REMOVAL OF TEST SUBSTANCE
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: OP_KIT opacitometer (changes in the light transmission passing through the cornae)
- Corneal permeability: Microtiter plate reader (OD490, passage of sodium fluorescein dye)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3 No Category (UN GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Category 1 (UN GHS)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
87.07
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
96.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
75.48
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
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:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the results of the OECD 437 study, the test item is serious eye damaging (CLP/EPA/GHS (Cat 1)).
Executive summary:

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was performed.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive control (2-Ethoxyethanol), and the negative control (saline) were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

The test item was tested undiluted. Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 86.42 (threshold for serious eye damage: IVIS ≥ 55).

The required acceptability criteria were met.

The test item is considered to be classified as serious eye damaging.