Eye irritation (ultra-mild) test

POTENCY ASSESSMENT METHOD (ET50) FOR MILD AND ULTRA-MILD TEST ITEMS

WHY CONDUCT AN EYE IRRITATION TEST?

This test is a special extended protocol of our eye irritation ET50 method for mild and ultra-mild test items. It allows you to rank ingredients or formulations in order of irritation potential, in classifications of Severe, Moderate, Mild or Minimal / Non-Irritant, as well as comparing their potency 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

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 skin 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 skin model to 50%. ET50 values are then used to assign the irritancy classification based on the proven prediction model.

TEST SYSTEM: RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM

Reconstructed human cornea-like epithelium (EpiOcular™) 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 organised 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. EpiOcular™ 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.

SUMMARY OF THE TEST METHOD

  • EpiOcular™ 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 to the surface of the EpiOcular™ models: triplicate models are dosed at the apical surface with 100μl.
  • Controls consist of ultrapure water (negative control) and 0.3% Triton X-100 (positive control).
  • The dosed skin models are placed into a cell culture incubator for 1 hour, 5 hours and 24 hours (triplicate models per time point).
  • Test items and control substances are removed from the skin models surface by washing.
  • Following a post-exposure soak in culture medium, the viability of the EpiOcular™ 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 EpiOcular™ models incubated with test items are compared with those of negative controls to calculate percentage viability. Absorbance values from all 3 time points are used to calculate ET50 (the time, in minutes, taken to reduce the viability of the EpiOcular model to 50% of the negative control value).
  • ET50 values are placed into rank order and / or compared with those of known benchmarks to provide a prediction of the eye irritation potential of the test item.
  • 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
ComplianceNon-regulatory
Turnaround time5-7 weeks
MethodPotency assessment
Min. sample required10ml (liquids) / 10g (solids)
Test codeCT-027

Always animal and animal product-free

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