Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in 2014 study at recognised contract research organisation.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
other: Liquid
Details on test material:
- Lot/batch No.: SC00008199
- Expiry Date: 19 August 2016
- Purity 99.5%

Test animals / tissue source

other: Bovine corneas
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival. All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Test system

unchanged (no vehicle)
Amount / concentration applied:
The test substance was used as supplied (100% concentration)
Duration of treatment / exposure:
The test substance was applied for 10 minutes followed by an incubation period of 120 minutes.
Observation period (in vivo):
Number of animals or in vitro replicates:
Details on study design:
Preparation of Corneas
Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they
were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’sminimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea
using a calibrated opacitometer. The average opacity for all corneas waere calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate
corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed. The
holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior
chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was
applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Evaluation of Results
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro
Irritancy Score.

Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity
reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean
opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea.
The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score: In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value).
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only
one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

Results and discussion

In vivo

Irritation parameter:
other: In Vitro Irritancy Score
Time point:
other: 120 minutes
Irritant / corrosive response data:
The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤4.7 and permeability ≤0.080. The negative control acceptance criteria were therefore satisfied.
The test substance gave an IVIS of 1.2

Applicant's summary and conclusion

Interpretation of results:
not classified
Migrated information Criteria used for interpretation of results: other: GHS EU
The test item was not irritating.