Transcript Fertile Egg – Based Toxicology Models

BCOP
Bovine Cornea Opacity
& Permeability Test
Acute and Subchronic Toxicology
Alternative / In Vitro Studies
Custom Research and Method Development
© 2009 MB Research Laboratories. All Rights Reserved.
BCOP: Bovine Cornea Opacity/Permeability Test
Alternative Eye Irritation Model
•
Uses excised Bovine Corneas normally discarded as waste
•
Measures two endpoints:
1)
Changes in light transmission
2)
Trans-corneal Fluorescein permeability
Normal
Opacity
© 2009 MB Research Laboratories. All Rights Reserved.
Background
Since the cornea is of primary interest in assessing ocular
irritation, the BCOP provides a useful parallel for possible
human exposure.
Bovine corneas have been used since the early 1950s as
an alternative ocular model.
Current BCOP evaluations were developed by Pierre
Gautheron and Joseph Sina in 1992* as an alternative to
the Draize Rabbit Eye Test .
Reviewed by ICCVAM as a regulatory alternative to the
Draize Rabbit Eye Test.
Regulatory agencies accept BCOP for the purpose of
classification for labeling under the Federal Hazardous
Substance Act
* “Bovine Corneal Opacity and Permeability Test: An In Vitro Assay of Ocular Irritancy”, Gautheron P.,
Dukic, M., Alix, D. and Sina, J. Fundamental and Applied Toxicology 18, 442-449.
© 2009 MB Research Laboratories. All Rights Reserved.
Assay Procedures
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The bovine eyes are harvested and shipped in cold HBSS.
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The corneas are carefully checked for any damage (opacity, cuts,
vascularization, etc.) before being excised and mounted in the special BCOP
chambers with Complete Minimal Essential Media in contact with the epithelium
and endothelium.
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After a 1 hour incubation a pretest opacity reading is taken using an
opacitometer, an instrument which measures light penetration through the
cornea
-
The corneas are divided into two groups – test article and control.
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Assay Procedures (cont.)
- Cornea is exposed to the test material. The material can be tested at
any concentration and over a range of exposure times to differentiate
potential mild to severe eye irritants.
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Assay Procedures (cont.)
- The test article is rinsed from the cornea, opacities scored and recorded before an
additional incubation.
- Incubation times may be varied to enhance post exposure expression of irritancy.
-A post incubation opacity reading is taken on each cornea and recorded.
-Opacity readings are subtracted from the pretest opacity readings to calculate the
Corrected Opacity Score.
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BCOP - Opacity
Normal
Opacity Caused by
swelling of
basal epithelial
cells.
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BCOP – Permeability
Different Optical Density Scores are measured by the amount of
fluroscein that permeates through the cornea into the posterior
chamber filled with Minimal Essential Media (MEM). Intensity of
response increases with the amount of fluorescein permeability.
- A permeability test is performed to measure the passage of fluorescein stain
through the cornea. A sample of the media is removed from the posterior end of
the chamber and measured spectrophotometrically (490nm) to determine the
amount of fluorescein leakage.
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Interpretation
In Vitro Score
Classification
0 to 25
Mild Irritant
25.1 to 55
Moderate Irritant
55.1 & above
Severe Irritant
Based upon our experience, test articles scoring 0 –4 are not a Draize
Irritant, test articles scoring 4 – 10 are borderline, and test articles scoring
10 – 25 are mild to moderate Draize Irritants.
Corneas may also be fixed, sectioned, and examined by a board certified
pathologist.
© 2009 MB Research Laboratories. All Rights Reserved.
Alternative Ocular Toxicology Models
Chorioallantoic Membrane Vascular Assay
EpiOcular - 3D Human Tissue Models – Air/Liquid Interface
Tissue Culture Well
Culture Insert
Membrane ALI Tissue
Medium
MB Research has pioneered the use and development of alternative
and in vitro ocular toxicity models used mainly for cosmetics and
consumer product safety evaluations. We have performed over
10,000 alternative ocular irritancy tests, which include approx. 2500
BCOPs to date.
BCOP can be used in combination with other alternatives such as the
CAMVA or HET-CAM to further determined ocular irritancy
classification.
© 2009 MB Research Laboratories. All Rights Reserved.
Thank you
MB Research Labs
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1765 Wentz Road
Spinnerstown, PA 18968
Tel: 215-536-4110
Fax: 215-536-1816
[email protected]
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© 2009 MB Research Laboratories. All Rights Reserved.