Regulatory Ocular Irritection® Test

OVERVIEW

Corneal opacity is an important driver for the classification of eye hazards. Changes to corneal opacity (a precursor of irritation and serious eye damage) can be caused by disruption of the highly organised structure of the corneal proteins and carbohydrates.

The test system used for this method contains a macromolecular reagent composed of a mixture of proteins, glycoproteins, carbohydrates, lipids, and low molecular weight components. When rehydrated, this mixture forms a complex macromolecular matrix which mimics the highly ordered structure of the transparent cornea. Test items that are hazardous will produce turbidity of the macromolecular reagent through promoting protein denaturation, unfolding, and changes in conformation (as well as disruption and disaggregation of the macromolecular matrix components).

Before proceeding to the main test, the item to be tested is characterised through determining the pH of a 10% dilution in water and assessing whether the product is a surfactant. Samples with a pH ≥4 or ≤9 are suitable to be tested by this method. Certain items are not suitable to be tested by this method. These include intensely coloured chemicals, substances which cause salting out precipitation, high concentrations of surfactants, and highly volatile substances.

During the main test, based on their physico-chemical properties, items are applied directly to the macromolecular matrix at room temperature, or onto a membrane disc above the matrix. Items without surfactant-like properties are tested with a series of five doses (125, 100, 75, 50, 25 µl (liquids)/mg (solids)) applied neat to the membrane disc. Items with surfactant-like properties are diluted in sterile water and 125 µl applied directly to the macromolecular matrix at concentrations of 5%, 2.5%, 1.25%, 0.625%, 0.3125%, then covered with the membrane. Waxy solids are applied directly to the matrix then covered with the membrane disc.

The matrix is exposed for 24hr ± 0.5hr at 25°C±1°C, and visually inspected for damage to the membrane disc. The contents are transferred to a 96 well plate, and their optical density (OD) recorded at 405nm using a spectrophotometer.

The OD readings recorded are used to calculate an Irritection® Draize Equivalent Score (IDE) for each item tested. A Maximal Qualified Score (MQS) is determined and represents the highest IDE score obtained from various tested concentrations of an item. The MQS can range from 0 to 51, and is used to predict the UN GHS classification. According to the OECD Test Guideline, the method is validated to predict No Category, or Category 1. Where the MQS would indicate a classification between these, a result of No Prediction is obtained, and further testing will be required to determine a category.