IIVS | BCOP (OECD 437) & Histology
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BCOP (OECD 437) & Histology

The BCOP (Bovine Corneal Opacity and Permeability) assay is an in vitro eye irritation test method developed by Gautheron et al. (1992), which uses living bovine corneal tissue, obtained as a by-product from abattoirs, to evaluate the potential ocular irritancy of a test article. Types of injury caused by exposure to the test article are quantitatively measured by changes in opacity and permeability to fluorescein.

The BCOP assay allows for the investigation of the mechanism of the damage caused. Corneal opacity can be caused by protein denaturation or the induction of stromal swelling, while corneal permeability reflects a loss in corneal barrier function and cell-to-cell membrane junctions of the corneal epithelium.

An additional histological endpoint can be added to assess the corneal swelling, hydration, or morphological alterations in the cornea. This assessment evaluates the type of observed lesions and the depth of injury into the corneas. For specific assay procedures, please see Step-by-Step or view our recent video.

Quick Facts
BCOP Bovine Eye

BCOP Bovine Eye

Assay Model: organotypic, bovine corneas (obtained as a by-product from abattoirs)

Endpoints:

  1. Opacity (determined by measuring the amount of light transmitted through the cornea).
  2. Permeability (determined by measuring the amount of fluorescein dye that penetrates through the cornea).

 

 

Depth of injury model in a Rabbit Cornea (Maurer and Jester, 2002)

Depth of injury model in a Rabbit Cornea (Maurer and Jester, 2002)

Each assay includes a positive and negative control. For more information about testing your materials using this assay, please see Applications. Specialized protocols may be prepared as requested through consultation with an IIVS Study Director.

Applications

Sensitivity

Ocular Irritation Assays – Continuum of Sensitivity

Ocular Irritation Assays – Continuum of Sensitivity

BCOP is primarily used to evaluate test materials in the moderate to extreme irritancy/corrosive range. However, exposure protocol modifications can be made to increase the sensitivity of the assay system to resolve among milder ingredients and formulations.

Test Material Compatibility

The assay is compatible with both water soluble and insoluble formulations. Both solid and liquid test materials can be tested. Since test materials are topically applied, the test system is suitable for creams, pastes, highly viscous materials, and powders.

Alternate Endpoints

BCOP’s multiple endpoints allow the investigations of mechanism of action. An additional histological endpoint can be added to assess the types of observed lesions and the depth and degree of injury. Based upon the work of Maurer and Jester (2002), the depth and degree of injury may be used to predict the severity and outcome of the corneal injury.

Test article concentrations and exposure times may be adjusted as necessary to accommodate specific physical characteristics or client needs. Specialized protocols may be prepared as requested through consultation with the Study Director.

Validation

  • In the US and in Europe, the BCOP assay has been approved for identification of corrosive substances and those chemicals not requiring classification according to UN GHS.
  • OECD Test Guideline was adopted (TG437) in 2009 and revised in 2013.
  • The US EPA accepts the BCOP assay as part of a tiered strategy for the labeling of cleaning products with anti-microbial claims.
Histology

The results of a histological evaluation may be used in several ways depending on the study goals. It may be used to confirm the level of corneal irritation predicted by the opacity and permeability values. In other cases, it is helpful to understand the types of lesions and the depth of injury that a material might induce in the cornea.

Water
Ethanol
SLS

Step-by-Step

  1. Isolation and Mounting of Corneas
  2. Initial Opacity
  3. Dosing and Rinsing
  4. Permeability Determination
  5. Preparation for Histology (optional)
  6. Calculate In Vitro Score
Step 1: Isolation and Mounting of Corneas

Upon receipt in the lab, the corneas are immediately prepared. The bovine corneas are excised and mounted in specially designed chambers. The chambers are then filled with media in a way that allows the corneas to retain their natural shape.

The mounted corneas are incubated to allow them to equilibrate for 1 hour. This is followed by a visual inspection to examine for imperfections (such as cuts, scratches, small opaque areas). Any corneas exhibiting such imperfections are removed from any further testing and discarded.

Bovine Cornea Excision
Mounting of a Bovine Cornea
Chamber Filling and Observation
Step 2: Initial Opacity
Opacity reading

Opacity reading

After the corneas have been examined, an initial baseline opacity measurement is performed for all the corneas. The initial opacity reading is representative of a normal, untreated cornea. Using a calibrated opacitometer, each cornea is compared against an air-filled chamber. After the initial opacity reading, each cornea is labeled with the appropriate test article or control treatment group and prepared for the dosing and rinsing procedure.

Step 3: Dosing and Rinsing

The medium is removed from the anterior chamber of the holder. The test article or control is dosed on the epithelial surface of the cornea for its designated exposure period. Test articles may be applied neat or in dilution. For particularly viscous materials or slurries, the formulation may be applied directly to the cornea by removing the anterior chamber window.

After each designated exposure time, the corneas are thoroughly rinsed and the chambers are refilled. A post exposure opacity is taken (for liquid protocols) and the corneas are returned to the incubator for a specified post-exposure “expression” incubation. A second opacity measurement may be taken after the post-exposure “expression” incubation.

BCOP Dosing
Rinsing of Corneas
Step 4: Permeability Determination

After the post-exposure opacity measurement, both chambers of the holder are aspirated, the posterior chamber is refilled with fresh media, and fluorescein is added to the anterior chamber. The chambers are then incubated vertically for 90 minutes. After 90 minutes, all of the medium in the posterior chamber is removed and then placed into a prelabeled glass tube.

The medium from each numbered tube is transferred to its corresponding well on a 96-well plate. The optical density at 490 nm is determined using a spectrophotometer.

Step 5: Preparation for Histology (optional)

Corneas may be saved for histology for analysis of the degree and depth of corneal damage. Each cornea is carefully placed into a plastic cassette, fixed in formalin and processed for hematoxylin and eosin staining.
Histological analysis can be performed at IIVS or through a subcontracted board certified pathologist.

If histology is elected, an additional histology report is prepared complete with photographic representations of the corneas and a full histological evaluation for each test article and control treatment group. Please refer to Histology section for more information.

Step 6: Calculate In Vitro Score

The opacity and permeability measurements are then evaluated in the calculation of the In Vitro Score. For example, a very mild ocular irritant may result in Mean Opacity Value of 1.4 and a Mean OD490 value of 0.158, resulting in an In Vitro Score of 3.77.

The following classification system (established by Sina et al.) provides a good initial guide to interpretation of these in vitro data. Please note that these specific ranges may not be applicable to all classes of materials.

In Vitro Score: * from 0 to 25 = mild irritant * from 25.1 to 55 = moderate irritant * from 55.1 and above = severe irritant

Cited
Sina, 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

Positive and Negative Control Treated Corneas
The Positive and Negative Control Treated Corneas
Histology Preparation
Histology Preparation
Placement of Corneas into Formalin
Placement of Corneas into Formalin