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The Isolated Chicken Eye (ICE) test can be used to evaluate the potential of chemicals/substances to cause eye irritancy.

Specifically, it is used to assess irritancy potential on the basis of changes in corneal thickness, corneal opacity and permeability. While the method has some limitations, it is recommended for use as part of a wider testing strategy to classify chemicals and substances as ocular corrosives or severe irritants.

The method was assessed as part of a retrospective validation study that was conducted by ICCVAM-NICEATM in collaboration with EURL ECVAM.

Read more about ICE test method on ICCVAM-NICEATM website here.

Eye irritation

[collapsed]The end-points of the ICE test are changes in corneal thickness, corneal opacity and permeability. Individually, each of these end-points are considered indicative of eye irritancy potential.

Increases in corneal thickness indicate swelling and excessive fluid retention (oedema) between the different layers of the cornea. As the cornea is responsible for a considerable amount of refraction of light that is required for proper focussing of the eye, increases in corneal thickness can result in blurry sight.

Corneal opacity gives an indication of the amount of light that can pass into the eye. It is the result of proteins in the cornea undergoing denaturation or precipitation and basically means they change from being clear to opaque. Any opacity changes suggest improper functioning of the eye.

Corneal permeability results from damage to corneal epithelium cells that normally serve as a barrier for the interior of the eye. Any permeability means foreign objects, substances or chemicals can pass into the eye resulting in damage and loss of function.[/collapse]

The Isolated Chicken Eye (ICE) test

[collapsed]The ICE test is based on isolated chicken eyes which are a by-product of slaughter houses. After application of the test substance, the assessment of the end-points of the test (see above) is then carried out using a slit-lamp microscope and a previously published scoring system.

Corneal permeability is assessed by application of fluorescein (a dye). A standard operating procedure is available (see below).[/collapse]

Animal testing replacement

[collapsed]The ICE test was developed as an alternative in vitro method to the Draize rabbit eye test. While the method still involves animals (i.e. chickens), it is based on material obtained from slaughterhouses that would otherwise be disposed of.

In comparison to other similar methods (based on rabbit, bovine or porcine eyes), the ICE test is considered to be more consistent in terms of eye quality since the material can be obtained from chickens that are the same strain and age.

In terms of regulations, a positive test outcome is considered to be indicative of a substance being an ocular corrosive or severe irritant and in such an event, no further animal-based testing is needed.

However, a negative outcome in the test would require testing with other in vitro methods and finally in tests on rabbits as the ICE test cannot accurately identify substances that are not irritants or only mild irritants.[/collapse]

Validation study

[collapsed]The retrospective validation study involved a re-analysis of pre-existing data relating to four organotypic assays: the bovine corneal opacity and permeability assay (BCOP), the isolated chicken eye test (ICE), the isolated rabbit eye test and the hen's egg test.

The study was conducted by ICCVAM-NICEATM between 2003 and 2006. The study was subsequently peer reviewed in the US. ESAC then peer reviewed the same data for the purposes of recommending the methods in the EU.

The study assessed whether the assays could detect severe eye irritants and ocular corrosives. Subsequently the same methods were assessed for whether they could identify mild or non-irritants.[/collapse]

Validation study outcomes

[collapsed]According to the ESAC review, the essential components of modular validation of the ICE method were satisfied in the ICCVAM retrospective validation study. Specifically this means high quality protocols are available and end-points of the assay are clearly defined.

A wide variety of test substances have previously been evaluated, which means there is a wide domain of applicability but with the caveat that certain classes of materials carry a risk of either false-positive or false-negative outcomes.

Within- and between-laboratory, transferability, and predictive capacity were all reportedly satisfactory and that with some limitations, the ICE test method should be considered validated.[/collapse]

EURL ECVAM recommendations

[collapsed]Following a shadow peer review by ESAC, it was agreed that the ICCVAM recommendations should be endorsed by EURL ECVAM.

Subsequently an OECD test guideline was formulated and adopted 7 September 2009.[/collapse]