Eye Irritation Test ET50



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.


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

The test described here is a non-regulatory method for the potency assessment of mild and ultra-mild chemicals in terms of human eye irritation potential. It provides discrimination between Mild, Milder and Mildest formulations.

The test is based on the depth of injury model or Maurer and Jester, using 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.

The method utilises reconstructed human cornea-like epithelium (RhCE), which in its overall design mimics the biochemical and physiological properties of the corneal epithelium of the human eye. The test item is applied directly to the cornea surface, providing a good model of “real life” exposure. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from the corneal tissues. 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 the irritancy classification based on the proven prediction model and, where relevant, to place a series of items into a rank order of irritation potential.


Reconstructed human cornea-like epithelium is a corneal model composed of living human cells which have been cultured to form a multi-layered, differentiated corneal epithelium. The levels of differentiation obtained are at the cutting edge of in vitro tissue technology. The model consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal human in vivo corneal epithelium. The profiles of key differentiation markers also 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. Reconstructed human cornea-like epithelium is 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.


  • Corneal models are pre-warmed in a cell culture incubator (37°C / 5% CO2) for 60 minutes or overnight. The culture medium is replaced prior to applying treatment.
  • The test item is applied neat or as a 1:4 dilution in ultra-pure water, according to the most appropriate protocol.
  • The test item is applied to the surface of the corneal models: triplicate models are dosed at the apical surface with 100μl.
  • Controls consist of ultra-pure water (negative control) and 0.3% Triton X-100 (positive control).
  • The dosed corneal models are placed into a cell culture incubator for 16 minutes.
  • Test items and control substances are removed from the corneal models surface by washing.
  • Following a post-exposure soak in culture medium, the viability of the corneal models is assessed by MTT conversion. MTT solution is applied to the surface of the models and placed into a cell culture incubator for 3 hours. The blue formazan metabolite produced by viable cells is then extracted into isopropanol by incubation at roomtemperature for 2 hours.
  • Triplicate samples of the extracted formazan solution are transferred to a microplate and the formazan product is quantified by absorbance spectrophotometry (wavelength 570nm).
  • Absorbance readings of the formazan product from corneal models incubated with test items are compared with those of negative controls to calculate percentage viability.
  • The value obtained from this initial 16 minute incubation with testsubstance determines the time points for 2 further incubations, which are
    then carried out using a repeat of the above process. Time points are determined as follows. If the viability is >90% after the initial 16 minute exposure, subsequent time points are 64 and 256 minutes. If the viability is <90% but >30% after the initial 16 minute exposure, subsequent time points are 4 and 64 minutes. If the viability is <30% after the initial 16 minute exposure, the subsequent time points are 1 and 4 minutes. Triplicate positive controls (0.3% Triton X-100) are incubated for 4, 15 and 45 minutes.
  • Absorbance values from all 3 time points are used to calculate ET50 (the time, in minutes, taken to reduce the viability of the corneal model to 50% of the negative control value). The ET50 for the positive control should fall between 12.2 and 37.5 minutes.
  • A statistical model is then used to convert the ET50 value to an equivalent in vivo Draize score.
  • Draize scores are converted to eye irritation potential according to accepted classification criteria as follows: Draize score 0-15: Minimal / Non-Irritant. Draize score 15.1- 25: Mild Irritant. Draize score 25.1-50: Moderate Irritant. Draize score 50.1-110: Severe / Extreme Irritant.
  • A range of acceptance criteria must be satisfied in order for the experimental run to be valid.

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Eye irritation test

At a glance

What it measuresViability of corneal cells
Turnaround time5-8 weeks
MethodReconstructed human cornea-like epithelium
Sample required10ml (liquids) / 10g (solids)
Test codeCT-005

Always animal and animal product-free

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