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Other Ocular Assays

Ocular Irritection Assay

The Ocular Irritection Assay is an in chemico eye irritation assay that detects, ranks, and predicts the corneal irritation potential of a test material. The assay assesses changes to the reagent solution (containing proteins, glycoproteins, lipids), which mimics the denaturation and disruption that occurs in corneal proteins in vivo.

The test article is applied to a membrane disc that controls the delivery to the reagent solution. The changes in the protein structure are measured by optical density readings in the reagent solution by a photo-spectrometer.

IIVS participated in the validation and preparation of the resulting manuscript published in Toxicology In Vitro. The guidance document is currently under evaluation by EURL/ ECVAM for identifying ocular hazards according to UN GHS.

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Chorioallantoic Membrane Vascular Assay (CAMVA) – Ocular Irritation Potential and Efficacy Assessment

The CAMVA is an in vitro assay that measures the presence and degree of vascular effect to the chorioallantoic membrane (CAM) of 10-day fertilized eggs following exposure to test article.

After exposure, the CAM is examined visually for vascular hemorrhaging, capillary injection and/or ghost vessels at the test material application site. The concentration that elicits a positive response in 50% of the treated CAM specimens (RC50) indicates ocular irritation potential of a test material. Exposed CAM can also be evaluated for efficacy endpoints, such as vascular constriction.

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Trans-Epithelial Permeability (TEP) Assay

The TransEpithelial Permeability (TEP) Assay, is a cell-based assay used to evaluate the potential ocular irritancy of a test article by measuring the permeability of sodium fluorescein across a confluent monolayer of MDCK cells.

At confluency MDCK cells form tight junctions similar to those formed in the outermost epithelial layer of the eye. Disruptions of the tight junctions by a test article allows for fluorescein permeability through the membrane, which can be quantified.

The assay can be used in product development or internal safety assessments by evaluating the concentration that causes a 50% leakage of fluorescein (EC50)., The assay can also be used in the identification of ocular corrosives and severe Irritants as outlined in OECD Test Guideline 460: Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants (adopted 2 October 2012).

TEP Image 1
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Short Time Exposure Assay (STE)

The Short Time Exposure (STE) assay, developed by Kao Corporation (Japan), is an in vitro assay used to assess acute eye irritation potential as an alternative to the traditional in vivo Draize test. The test method evaluates the cytotoxicity induced by a series of test chemical dilutions in a monolayer of rabbit corneal fibroblasts (Statens Seruminstitut Rabbit Cornea – SIRC) after a single five-minute exposure.

Two prediction models were initially developed for the STE assay – one categorizes the test material as either an ocular irritant or non-irritant; the second provides a rank classification of ocular irritation (mild, moderate or severe). The prediction models are based on the mean % viability of a 5% and 0.05% dilution of the test material relative to solvent controls.

• A test material is categorized as Non-Irritant if the mean % viability of the 5% dilution was > 70%; and categorized as Irritant if the mean % viability was ≤ 70%.

Figure 1: Categorization of Eye Irritation Potential Using Mean Viability of 5% dilution

• A rank irritancy classification of a test material may be further determined based upon the evaluation of cytotoxicity at both dilutions. For the 5% dilution, a score of 0 is assigned if the cell viability is > 70%, and a score of 1 is assigned if the cell viability is ≤ 70%. For the 0.05% dilution, a score of 1 is assigned if the cell viability is > 70%, and a score of 2 is assigned if the cell viability is ≤ 70%. The sum of the scores, ranging from 1 to 3, determines the eye irritation rank classification as a minimal, moderate, or severe eye irritant.

Figure 2. Eye Irritation Potential Using STE Rank Score

These two approaches may be taken to support test material screening or product development and characterization goals.

For regulatory classification and labeling, the assay may be used to identify test substances and mixtures that induce serious eye damage as well as those that do not require classification for either serious eye damage or eye irritation, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labeling of Chemicals (GHS), as described in OECD Test Guideline 491.

As stated in OECD TG 491: “The STE test method cannot be used for the identification of test chemicals as UN GHS Category 2, Category 2A (eye irritation) or UN GHS Category 2B (mild eye irritation), due to the considerable number of UN GHS Category 1 chemicals under-predicted as UN GHS Category 2, 2A, or 2B and UN GHS No Category chemicals over-predicted as UN GHS Category 2, 2A, or 2B. For this purpose, further testing with another suitable method may be required.”

Figure 3. Prediction Model for GHS Classification and Labelling using the STE Test Method

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