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IN VITRO METHODS FOR PREDICTING SERIOUS EYE DAMAGE AND IRRITATION
| METHOD | PRINCIPLE OF THE TEST | APPLICABILITY DOMAIN | GHS CATEGORISATION |
|---|---|---|---|
| Defined Approaches | |||
| OECD TG 467: Defined Approaches for Serious Eye Damage and Eye Irritation, comprising two defined approaches for liquids (DAL1 and DAL2) | DAL1 combines information from three sources: (1) physiochemical properties of the test substance, (2) results from testing in OECD TG 437 BCOP assay using the laser light based opacitometer, and (3) results from testing in OECD TG 492 (either the EpiOcular Eye Irritation Test or the SkinEthic Human Corneal Epithelium Eye Irritation Test. DAL2 combines information from two sources: (1) results from testing in OECD TG 437 BCOP assay using the laser light– based opacitometer and (2) OECD TG 491 Short Time Exposure assay. | DAL1 is applicable to neat non-surfactant liquids, and DAL2 is applicable to neat and diluted non-surfactant liquids. For both approaches, the applicability domain of each test guideline used within the defined approach should also be considered. | Both approaches can be used for the identification of substances causing serious eye damage (GHS Cat 1), causing eye irritation (GHS Cat 2), and not requiring classification for eye irritation or serious eye damage (GHS No Cat). Each approach can be used as a full replacement for the Draize rabbit eye irritation test. |
| Reconstructed Three-Dimensional Reconstructed Tissue Assays | |||
| OECD TG 492: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage (e.g. EpiOcular™, SkinEthic™, LabCyte, MCTT HCE™) | Test substance is applied to reconstructed tissue from human cells, which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in the human cornea. Cell viability (MTT or WST-8 assay) is used to predict toxicity. | Applicable to substances and mixtures, and to solids, liquids, semi-solids, and waxes. | For the identification of substances not requiring classification for eye irritation or serious eye damage (GHS No Cat). |
| OECD TG 492B: Reconstructed Human Cornea-like Epithelium Test Method for Eye Hazard Identification (SkinEthic™) | The test substance is applied to reconstructed tissue from human cells (as in OECD TG 492). Depending on whether the test substance is a solid or a liquid, cell viability is assessed at two or three exposure times, respectively. | This assay is applicable to substances and mixtures and to solids, liquids, semi-solids, and waxes. | This assay can be used for the identification of substances causing serious eye damage (GHS Cat 1), causing eye irritation (GHS Cat 2), and not requiring classification for eye irritation or serious eye damage (GHS No Cat). This method can be used as a full replacement for the Draize rabbit eye irritation test. |
| OECD TG 494: Vitrigel-Eye Irritancy Test (EIT) Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage | Human corneal epithelium (hCE) models fabricated in a collagen vitrigel membrane are exposed to a test substance. Damage to the barrier function of the models is assessed by analysing time-dependent changes in transepithelial electrical resistance (TEER) values. | Applicable to substances and mixtures with a pH > 5 (based on 2.5% weight/volume preparation). Not applicable to solids. | For the identification of substances not requiring classification for eye irritation or serious eye damage (GHS No Cat) |
| Organotypic Ex Vivo Assays | |||
| OECD TG 437: Bovine Corneal Opacity and Permeability (BCOP) Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage | Test substance is directly applied to cow eyes obtained as by-products from abattoirs. Corneal opacity (measured quantitatively as the amount of light transmission through the cornea) and permeability (measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea) are measured. Optional histopathology can be conducted for additional information. | Applicable to solids, liquids (including semi-solids, creams, and waxes), and mixtures | For the identification of substances causing serious eye damage (GHS Cat. I) and substances not requiring classification for eye irritation or serious eye damage (GHS No Cat). OECD TG 437 Training Video available at: https://www.youtube.com/watch?v=TiZbp5KDHI8. |
| OECD TG 438: Isolated Chicken Eye (ICE) Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage | Test substance is directly applied to chicken eyes obtained as by-products from abattoirs. Corneal swelling, opacity and fluorescein retention are assessed. | Applicable to solids (may be soluble or insoluble in water), liquids, emulsions, and gels. | For the identification of substances causing serious eye damage (GHS Cat. I) and substances not requiring classification for eye irritation or serious eye damage (GHS No Cat). |
| Cytotoxicity and Cell Function Based In Vitro Assays | |||
| OECD TG 460: Fluorescein Leakage (FL) Test Method for Identifying Ocular Corrosives and Severe Irritants | Epithelial monolayer Madin-Darby Canine Kidney (MDCK) cells are cultured on permeable inserts. The test chemical is applied for 1 minute and then removed; next, the non toxic highly fluorescent sodium-fluorescein dye is added, and the amount of dye that passes through the cell layer is measured spectrofluormetrically and used to predict toxicity. | Applicable to water-soluble chemicals or mixtures. Limitations for coloured or highly viscous substances (predictivity is improved by increasing the number of wash steps). Not applicable to strong acids and bases, cell fixatives, or highly volatile substances. | For the identification of substances causing serious eye damage (GHS Cat. I). |
| OECD TG 491: Short Time Exposure (STE) in vitro method | Measures cell viability (MTT assay) of corneal epithelial cells (Statens Seruminstitut Rabbit Cornea [SIRC] cells) in 96 well plates. As compounds are generally cleared from human or rabbit eye in 1-2 or 3-4 minutes, respectively, this test requires a 5-minute exposure. | Applicable to test chemicals that are soluble in saline, DMSO, or mineral oil. | For the identification of substances causing serious eye damage (GHS Cat. I) and substances not requiring classification for eye irritation or serious eye damage (GHS No Cat). |
| Macromolecular Matrix Assays | |||
| OECD TG 496: In Vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage | Test substance is directly applied to an in chemico macromolecular matrix model composed of lipids, proteins, glycoproteins, carbohydrates, and low molecular weight substances that model the cellular structure of the human corneal epithelium. An increase in optical density is used to predict the ocular hazard effects of chemicals. | Applicable to solids (may be soluble or insoluble in water) and liquids (may be viscous or non-viscous) whose 10% solution/dispersion has a pH in the range 4 ≤ pH ≤ 9. Some limitations for intensely coloured chemicals, chemicals that cause salting-out precipitation, high concentrations of some surfactants, and highly volatile chemicals. Also applicable to mixtures. | For the identification of substances causing serious eye damage (GHS Cat I) and substances not requiring classification for eye irritation or serious eye damage (GHS No Cat) |
For more information on these methods, please see our publication, Clippinger AJ et al. Human-relevant approaches to assess eye corrosion/irritation potential of agrochemical formulations. Cutan Ocul Toxicol. 2021;40(2):145-167.
For more information on tiered testing strategies for serious eye damage and eye irritation, please see the following publications:
- Organisation for Economic Co-operation and Development. 2023. Guidance document on integrated approaches to testing and assessment (IATA) for serious eye damage and eye irritation. No 263. Series on Testing and Assessment.
- US Environmental Protection Agency. 2015. Use of an alternative framework for classification of eye irritation potential of EPA pesticide products.
- European Chemicals Agency. 2017. Guidance on information requirements and chemical safety assessment. Chapter R.7a: Endpoint specific guidance Version 6.0. See R.7.2.8-R.7.2.11.