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Eye irritation

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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: Well-conducted and document study performed under GLP to current guideline.

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)
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
EC Number:
EC Name:
[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
Cas Number:
Molecular formula:
Test material form:
solid: flakes

Test animals / tissue source

other: cow
Details on test animals or tissues and environmental conditions:
Not applicable

Test system

other: mineral oil
other: see above
Amount / concentration applied:
0.75 ml of a 20% w/v solution of test material in mineral oil
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
Number of animals or in vitro replicates:
Twelve corneas were used; three for the test material, three for the negative control, three for the positive control and three for the vehicle control.
Details on study design:
Source of Bovine Eyes
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 and placed in Hanks' Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 hours of receipt. All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Preparation of Corneas
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 minimum essential medium (MEM) and plugged. The holders were incubated at 32 ±1°C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

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 was calculated. Three corneas were numerically allocated to the test item formulation. Three corneas were also numerically allocated to the negative control item, three corneas to the positive control item and three corneas to the mineral oil control.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item at a concentration of 20% w/v in mineral oil or control items were applied to the cornea. 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°C for 240 minutes.

At the end of the exposure period the test item formulation 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. Due to the limited solubility in the MEM rinsing solutions, the corneas treated with the mineral oil and the corneas treated with the test item in mineral oil were initially swabbed with 1 % Tween 80 before rinsing with MEM.

The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein and Permeability Determinations
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 (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ±1°C for 90 minutes.
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 492nm (00492) was measured. If values greater than 1.500 00492 were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.

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

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
Treatment In Vitro Irritancy Score
Test Item 20% w/v in Mineral Oil 0.7
Negative Control 6.1
Positive Control 103.6
Mineral Oil 3.3

Any other information on results incl. tables

Corneal Epithelium Condition Post Treatment

Treatment  Cornea Number  Observation Post Treatment
 Negative Control  1







 Positive Control









Mineral Oil









 Test Item 20% w/v in Mineral Oil









Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
The test item at a concentration of 20% w/v in mineral oil was considered not to be an ocular corrosive or severe irritant.
Executive summary:

A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 437 (2009) "Bovine Corneal Opacity and Permeability Assay" Method.

The test item was applied at a concentration of 20% w/v in mineral oil for 240 minutes. Negative, positive and mineral oil control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The in vitro Irritancy scores are summarised as follows:


 In Vitro Irritancy Score

 Test Item 20% w/v in Mineral Oil


 Negative Control


 Positive Control


 Mineral Oil Control


The test item at a concentration of 20% w/v in mineral oil was considered not to be an ocular corrosive or severe irritant.