Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Jan 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
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those 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

POSITIVE SUBSTANCE
- Amount applied in the test: 0.75 mL
- Purity: 99%

NEGATIVE CONTROL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
triplicates for each treatment and control 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 cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity >7 was discarded.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) 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 the 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 position. The anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.

- POST-EXPOSURE INCUBATION: 2 h in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer, Electro Design, France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax Molecular Devices) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Results and discussion

Results of ex vivo / in vitro study
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
0.03
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
The positive control (2-ethoxyethanol) showed clearly increased opacity and distinctive permeability of the corneae fullfilling the criteria as severe irritating/corrosive.
Relative to the negative control, the test substance did not cause an increase of the corneal opacity or permeability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in an IVIS which was within two standard deviations of the current historical mean.
- Acceptance criteria met for positive control: The positive control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Any other information on results incl. tables

Table 2. Results after 10 min incubation time.

Test group

Opacity value =
Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

Mean

 

Mean

 

 

Negative

control

0

0.00

0.061

0.064

0.92

0.96

0

0.068

1.02

0

0.063

0.95

Positive

control

63*

1.420*

79.10

79.95

55*

1.376*

59.08

59*

1.393*

76.89

Test substance

0*

0.023*

0.35

0.03

-1*

0.026*

-0.61

0*

0.023*

0.35

*: corrected values

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the BCOP assay the test substance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.03.
Executive summary:

A Bovine Corneal Opacity and Permeability (BCOP) test was performed according to OECD 437.

The in vitro irritancy score (IVIS) was 0.03 (threshold for classification ≥55.1 or ≤ 3). The test item did not cause an increase of the corneal opacity or permeability and is therefore not an eye irritant.