Study Design of In Vitro Eye Irritation EpiOcular (OECD 492) Study Design
The EpiOcular™ Eye Irritation Test (OECD 492) is an in vitro test method used to assess the acute eye irritation potential of chemicals and mixtures. It utilizes a reconstructed human corneal epithelium (RhCE) model to measure cytotoxicity, allowing for the identification of substances not requiring classification and labeling for eye irritation or serious eye damage according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).
Test System:
- The EpiOcular™ test system consists of a multilayered, differentiated RhCE model cultured on a permeable membrane insert.
- The model mimics the stratified epithelium of the human cornea, including basal, wing, and squamous cell layers.
- It expresses key markers of corneal differentiation, such as cytokeratins 12 and 19.
Test Procedure:
- Test substance preparation: The test substance is prepared in appropriate vehicles at various concentrations.
- Tissue treatment: The RhCE tissues are pre-incubated in culture medium for equilibration.
- Exposure: The test substance solutions are applied topically to the RhCE inserts.
- Incubation: The tissues are incubated for a defined period (typically 24 hours) to allow for interaction with the test substance.
- Cell viability assessment: After incubation, cell viability is determined using a metabolic assay, such as MTT or Alamar Blue.
- Data analysis: The percentage of viable cells in the treated tissues is compared to control tissues. The concentration-response relationship is determined, and a cytotoxicity endpoint (IC50) is calculated.
Interpretation of Results:
Based on the IC50 value and predefined criteria, the test substance is classified into one of five categories:
- Category 1: Not requiring classification (IC50 > 50 μL/mL)
- Category 2: Reversible, mild irritation (IC50 ≤ 50 μL/mL but > 10 μL/mL)
- Category 3: Reversible, moderate irritation (IC50 ≤ 10 μL/mL but > 1 μL/mL)
- Category 4: Irreversible damage (IC50 ≤ 1 μL/mL)
- Category 5: Corrosive (IC50 ≤ 0.1 μL/mL)
This classification helps predict the potential eye irritation hazard of the test substance and informs regulatory decisions regarding labeling and safety classification.
Advantages of the EpiOcular™ Test:
- Human-relevant model: Uses a reconstructed human tissue, providing greater relevance to human eyes compared to animal models.
- Reduced animal use: Contributes to the 3Rs (replacement, reduction, and refinement) of animal testing.
- High throughput: Allows for testing of multiple substances simultaneously.
- Cost-effective: Relatively inexpensive compared to animal studies.
Limitations of the EpiOcular™ Test:
- Limited complexity: Does not fully replicate the complex structure and function of the human eye.
- Species differences: May not predict eye irritation potential in all species.
- Not suitable for all substances: Certain substances, such as those with volatile or corrosive properties, may not be accurately assessed.
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