Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 November 2017 - 23 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
2017
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:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Characteristics of donor animals: 12 - 60 months old
- Storage, temperature and transport conditions of ocular tissue: transported in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container
- indication of any existing defects or lesions in ocular tissue samples: corneas were examined and only corneas which were free from damages were used

Test system

Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL of the dissolved test item (150 mg/750 µL 20% HBSS)
- Concentration: 20 %

VEHICLE
- Amount applied: 750 µL
- Concentration: 20 %
- Lot/batch no.: 20171207
Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
3 replicates per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from damages were used.

NUMBER OF REPLICATES: 3 replicates per group

SOLVENT CONTROL USED: Balanced Salt Solution (HBSS)

POSITIVE CONTROL USED: Imidazole solution (20%)

APPLICATION DOSE AND EXPOSURE TIME:
- application dose: 750 µL of the dissolved test item (150 mg/750 µL 20% HBSS)
- exposure time: 4 h

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in OECD guideline 437

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Replicate 1
Value:
94.27
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Replicate 2
Value:
94.74
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Replicate 3
Value:
94.9
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle 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:
The test item was determined to be have an eye damaging potential (UN GHS Category 1).
Executive summary:

The eye damaging potential of the test item was assessed in a Bovine Corneal Opacity and Permeability (BCOP) test according to OECD guideline 437. Bovine corneas were collected from slaughtered cattle that were between 12 and 60 months old. The test item solution (20% in HBSS) was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) was 1.81. 20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS was 94.64. Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS was 99.44. According to OECD guideline 437, a substance with an IVIS > 55 induces serious eye damage and should be classified as UN GHS Category 1. Therefore, the test item was determined to have an eye damaging potential and is classified as UN GHS Category 1.