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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 27th to March 24th, 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Principles of method if other than guideline:
The potential ocular irritancy/toxicity of the test substance is measured by the test substance's ability to induce opacity and permeability to fluorescein in an isolated bovine cornea.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl 3-[4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl]propanoate
EC Number:
Cas Number:
Molecular formula:
ethyl 3-[4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl]propanoate

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
- Source: local abattoir as a by-product from freshly slaughtered animals (J.W. Treuth & Sons, Inc., Baltimore, MD)
- Indication of any existing defects or lesions in ocular tissue samples: the bovine eyes were grossly examined for damaged and those exhibiting defects were discarded.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied: 750 µl.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Five corneas treated with test substance
Three corneas each for positive and negative control
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: the bovine eyes was excised from the freshly slaughtered animals and then placed in Hanks' Balanced Salt Solution containing Penicillin/Streptomycin and transported on ice packs to the laboratory. Upon arrival, corneal preparation was immediately conducted. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised so that a 2-3 mm rim of sclera was left surrounding the cornea. The isolated corneas were stored in a petri dish containing HBSS until they were mounted in a corneal holder where the endothelial side was placed against the O-ring of the posterior chamber. The anterior chamber was placed on top of the cornea and the screws were tightened. Beginning with the posterior chamber, the two chambers were flled with Minimum Essential Medium (EMEM) without phenol red, containing 1 % foetal bovine serum and 2 mM L-glutamine (Complete MEM). The corneal holders were labelled and incubated at 32 ± 1 °C for a minimum of 1 hour.

BOVINE CORNEAL OPACITY AND PERMEABILITY ASSAY: after the incubation period, the corneas were removed from the incubator. The medium was removed from both chambers and replaced with fresh Complete MEM. The initial opacity was determined for each cornea using a Spectro Designs OP-KIT opatocimeter. The medium was then removed from the anterior chamber and replaced with the test article, positive control or negative control.

EXPOSURE TO THE TEST SUBSTANCE: 750 μl of the test aricle, positive control or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure unifrom distribution over the cornea. Each treated cornea was completely covered with the test article and were incubated at 32 ± 1 °C for 10 minutes.

NUMBER OF REPLICATES: five corneas for the test item and three corneas for the positive control and negative control.

NEGATIVE CONTROL: three corneas, whose initial opacity readings were close to the median opacity for all the corneas.

- Washing steps after exposure period: the epithelial side of the corneas was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test articles. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hours after which a final measure of opacity was obtained.

METHODS FOR MEASURED ENDPOINTS: after the final opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh Complete MEM and 1 ml of a 4 mg/ml fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1 °C. At the end of the incubation, the medium was removed from the posterior chambers and placed into tubes numbered with corresponding chamber numbers.
- Corneal permeability: optical density at 490 nm was determined using a Molecular Devices Vmax kinetic microplate reader. If the OD490 value of a control or test article sample was greater than 1500, a 1:5 dilution of the sample was prepared in Complete MEM. A 360 μl sample of each 1:5 dilution was transferred to its specified well and the plate was read again.

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

The mean OD490 for the blank wells was calculated and then subtracted from the raw OD490 of each well (corrected OD490). Any dilutions made were considered (multiplied). The final corrected OD490 values of the test articles and the positive control was calculated via subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each treated cornea.
Final corrected OD490 = (raw OD490- mean blank OD490) - average corrected negative control OD490.
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition. In vitro score = Mean opacity value + (15 x Mean OD490 Value).

The following classification was established by Sina (1995) based on studies with a wide range of test substances:
In Vitro Score:
from 0 to 25 = mild irritant
from 25.1 to 55 = moderate irritant
from 55.1 and above = severe irritant
Sima, J.F., Galer, D.M., Sussman, R.G., Gautheron, P.D., Sargent, E.V., Leong, B., Shah, P.V., Curren, R.D. and Miller, K. (1995). A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates. Fundamental and Applied Toxicology 26:20-31.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
other: Mild irritant
Average of 3 test systems
Other effects / acceptance of results:
BCOP assay was accepted when the positive control (ethanol) caused an in vitro score that fell within two standard deviations of the historical mean.
Ethanol was used as a positive control. The score was recorded to be 44.5 and is acceptable, according to the classification system established by Sima et al.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
a further study is necessary for the evaluation of the classification of the substance according to the OECD 437 criteria.
IVIS = 20.1
Executive summary:

The aim of the study was to assess the potential ocular irritancy of test article, to isolated bovine corneas, as measured by the test article’s ability to induce opacity and permeability to fluorescein (relative to the control corneas) that can ultimately be used to determine an in vitro score.

Bovine corneas obtained as a by-product from freshly slaughtered animals, were mounted in special holders and exposed to the test articles. The positive control used in the study was ethanol and the negative control used was sterile, deionised water. The corneas were incubated at 32 ± 1 °C for 10 minutes and the epithelial side was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of test article or control and then finally rinsed with Complete MEM (without phenol red). An opacity measurement was taken and then the corneas were returned to the anterior chambers refilled with fresh Complete MEM (without phenol red) and incubated for approximately 2 hours before a final opacity measurement was taken. The posterior chamber was filled with fresh Complete MEM and fluorescein solution was added to the anterior chamber and incubated at 32 ± 1 °C for 90 minutes and then 360 µl aliquots were taken from the posterior chambers and the OD490 value was measured. A 1:5 dilution with Complete MEM was carried for any aliquot whose OD490 value exceeded 1.500 and the plate was read again. The permeability measurement and in vitro score was then calculated. The BCOP assay was proved to be valid since the in vitro score for the positive control (ethanol) fell within two standard deviations of the historical mean (within a range of 39.4 to 64.3).

The test substance, produced an in vitro score of 20.1, as measured by changes in opacity and permeability (to fluorescein), which classifies it as a mild irritant to bovine corneas, based on the classification system established by Sina et al.