Eye Irritation Test ET50

WHY CONDUCT AN EYE IRRITATION TEST?

This is a non-regulatory eye irritation test which assesses the potency of an ingredient or formulation. The ET50 method provides a profile of damage to the cornea over time resulting in a classification of Severe, Moderate, Mild or Minimal / Non-Irritant. This can be used to rank test items in order of irritation potential or compare them against industry benchmarks.

OVERVIEW

Eye irritation is defined as changes in the eye following the application of a test chemical to the surface, which are fully reversible within 21 days of application

This test is a non-regulatory method for potency assessment of chemicals in terms of human eye irritation potential. It provides classification as Severe, Moderate, Mild, or Minimal/Non-Irritant, and can be useful to classify a series of products or ingredients in rank order of eye irritation potential.

The method has two variants depending on your formulation type:

Dilute Protocol: recommended for surfactant-based and water-soluble formulations. The test item is applied as 1:4 dilution (20%) in sterile water. It is suitable for shampoos, body washes, cleansers, and micellar waters where undiluted application would damage tissue viability too quickly to provide a useful potency profile.

Neat Protocol: recommended for non-surfactant formulations. The test item is applied undiluted. It is suitable for oils, creams, serums, balms, and solids, or any test item where direct potency assessment without dilution is required.

Both variants use the depth of injury model, based on the rationale that the degree of irritation caused by a test substance correlates with the degree of penetration into cell layers and subsequent impact on cell viability.

TEST SYSTEM: RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM

Reconstructed human cornea-like epithelium (RhCE) is a corneal model composed of living human cells cultured to form a multi-layered, differentiated corneal epithelium. The model consists of highly organized basal cells which progressively flatten as the apical surface is approached, analogous to normal human in vivo corneal epithelium. Key differentiation marker profiles mirror those seen in vivo.

The cells are both metabolically and mitotically active, and release many of the pro-inflammatory agents (cytokines) known to be important in eye irritation and inflammation. RhCE tissues are grown on special platforms at the air-liquid interface, allowing for direct application of test items in a way that accurately models real-life eye exposure.

SUMMARY OF TEST METHOD

Pre-test Assessment

Prior to both the dilute method and the neat methods being performed, the test item must undergo preliminary assessment to ensure the test item does not interfere with the endpoint MTT dye. This ensures that the MTT reaction is only measuring tissue viability and that the test item is not directly reducing the MTT dye.

A small volume (100 μl or 100 mg) of the neat test item is incubated with MTT solution in the dark at room temperature for 60 minutes. If the MTT solution colour turns blue/purple or significantly darkens, the test item directly reduces the MTT and parallel freeze-killed tissues should be dosed alongside the main test to account for any interference.

Additionally, the dilute protocol requires the items to undergo a density check. All items with a density of ≥ 0.95 need to be diluted to 20% in the assay.

Any items with a density <0.95 will be tested without dilution following the neat protocol. 

Test Application

Dilute Protocol: 100 μl of the test item is applied as a 20% dilution in sterile water to the surface of corneal models in triplicate.

Neat Protocol: 100 μl of liquid, or 200 mg of a 1:1 slurry of solid test items are applied neat to the surface of triplicate corneal models. These are dosed at the apical surface.

Controls

Sterile water (negative control) and 0.3% Triton X-100 (positive control). 

EXPOSURE

Dilute Protocol: Dosed corneal models are placed in a cell culture incubator for 16 minutes. The value obtained from the initial 16-minute incubation determines time points for two further incubations. If viability is >90%, subsequent time points are 64 and 256 minutes. If viability is <90% but >30%, subsequent time points are 4 and 64 minutes. If viability is <30%, subsequent time points are 1 and 4 minutes.

Neat Protocol: Fixed time points of 3, 30, and 60 minutes are used.

In both protocols the tissues are exposed to the positive control for 4, 15, and 45 minutes and the negative control for 60 minutes.

Viability Assessment

Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt and extracted from corneal tissues. Triplicate samples of the extracted formazan solution are transferred to a microplate and the formazan product is quantified by absorbance spectrophotometry (wavelength 570 nm).

Absorbance readings of the formazan product from corneal models incubated with test items are compared with those of negative controls to calculate percentage viability.

ET50 Determination

Irritation potential is calculated in terms of the “ET50” value: the time taken in minutes for the test item to reduce the viability of the corneal model to 50%. ET50 values are then used to assign irritancy classification based on the proven prediction model and, where relevant, to place a series of items into rank order of irritation potential.

Classification

Draize scores are converted to eye irritation potential according to accepted classification criteria:

A range of acceptance criteria must be satisfied in order for the experimental run to be valid.

Turnaround Time

5-8 weeks

Sample Required

10 ml (liquids) / 10 g (solids). Please enquire if sample availability is limited.

Price

Test prices are dependent on the quantity of test items. Please enquire for a quote.