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Protocol no. 1 
BOVINE ISOLATED CORNEA TEST 

This test is designed to detect damage to the eye after application to the conjunctiva. 

CONTACT

Dr. B. Northover Department of Pharmacology School of Pharmacy Leicester Polytechnic Leicester, LE1 9BH UK Tel: England - 533 551551 ext 2271 

RATIONALE

This method permits the precise measurement of opacity development in isolated corneal preparations. The occurrence of opacity in response to a toxic or irritant compound is the basis for employing this technique as an indication of the likelihood that a chemical would damage the eye in vivo after application to the conjunctiva. 

BASIC PROCEDURE

Corneas are removed from bovine eyes (epithelium and endothelium layers can be removed if necessary). The thickness of the preparation can then be determined. The opacity of the cornea may be measured by quantifying the ability of light to pass through it. Comparisons of the thickness of and, more importantly, the opacity of the cornea before and after exposure to chemical compounds provides an indication of the likely irritant/toxic effect of the chemical. 

CRITICAL ASSESSMENT

At present, this technique has the limitation that only water soluble compounds can easily be tested. Interference with opacity measurement may occur when using highly coloured compounds due to staining of the cornea, it may be possible to overcome this by using suitable coloured filters in the opacitometer. A further limitation of this technique is that it cannot be truly representative of the eye in situ as it does not incorporate a drainage system. In some parts of the world it may not be possible to obtain fresh bovine eyes. General advantages of this technique include, an accurate quantifiable endpoint, relatively simple technique to implement once necessary equipment has been obtained, animal material is obtained from the slaughter house and the technique is inexpensive. Comparisons to other tests: The in vivo Draize test involves a more subjective measurement of damage and can not be as readily quantified. The same argument applies to the fertilised egg chorio-allantoic membrane test. Cell culture cytotoxicity tests, e.g. cultured corneal cells, fail to imitate the multilayered structure of the cornea. This system maintains the integral structure and, therefore, the measured toxicity of a compound will reflect both its ability to penetrate the tissue and its direct cytotoxic effect, thus making it more representative of the in vivo situation. Other more complex tests such as the rabbit ileum require a higher degree of operator proficiency, and intact eye preparations may not provide as precise, or sensitive, a measurement of damage as this technique. Modification of the system: Although the basic procedure for determining opacity is the same, it is possible to remove the epithelium and/or endothelium. In some cases this may be useful to determine whether a toxic response is related to a particular layer of the cornea, e.g. it may be endothelium- or epithelium-dependent. It is also possible to place the test chemical in contact with either the epithelial or endothelial sides of the cornea or both; the benefits of such a procedure and which method provides the best correlation to the in vivo situation are still being assessed. The authors are currently assessing whether prediction of in vivo irritancy is best obtained by application of substances to one or both sides of the cornea, and whether the epithelium and/or endothelium should be removed. Their personal viewpoint at this time and until further evidence is accumulated is that the intact cornea, exposed to the test chemical on the epithelial surface alone, is probably the better model for correlation to the in vivo situation. 

TEST STATUS

Undergoing in-house development. 

CHEMICALS TESTED

Anionic surfactants: triethanolamine decyl sulphate triethanolamine lauryl sulphate triethanolamine myristyl sulphate sodium decyl sulphate sodium lauryl sulphate Cationic surfactants: cetyltrimethylammonium bromide myristyltrimethylammonium bromide lauryltrimethylammonium bromide Non-ionic surfactants: Tween 20 Tween 80 Miscellaneous and industrial chemicals: Allyl alcohol Dioxane Ethanol Carbitol Propylene glycol 

ORGANISATIONS USING THE TEST 

None at present 

REFERENCES

  1. Muir, C.K. (1984) A simple method to assess surfactant-induced bovine corneal opacity in vitro: preliminary findings. Toxicology Letters, 22, 199-203 
  2. Muir, C.K. (1985) Opacity of bovine cornea in vitro induced by surfactants and industrial chemicals compared with ocular irritancy in vivo. Toxicology Letters, 24, 157-162. 
  3. Igarashi, H. (1986) With which component of the bovine isolated cornea does sodium lauryl sulphate react to produce opacity? Toxicology Letters, 32, 249-253. 
  4. Igarashi, H. (1987) The opacification of the bovine isolated cornea by surfactants and other chemicals - a process of protein denaturation? ATLA, 15, 8-19. 
  5. Igarashi, H. & Northover, A.M. (1987) Increases in opacity and thickness induced by surfactants and other chemicals in the bovine isolated cornea. Toxicology Letters, 39, 249-254. 
  6. Muir, C.K. (1987) Surfactant-induced opacity of bovine isolated cornea: an epithelial phenomenon? Toxicology Letters, 38, 51-54. 
IP-1 June 1989