OECD TG 437 / 438 / 492: Comparative Analysis of In Vitro Alternatives for Eye Irritation

The assessment of eye irritation and corrosion is a critical component of the safety profile of chemicals, pesticides, cosmetics, and industrial products. Traditionally, this assessment was conducted using in vivo tests (OECD TG 405) in a rabbit model; however, in line with the 3R principles and scientific advancements, validated in vitro and ex vivo alternatives have come to the forefront. The most widely used of these alternatives are the OECD TG 437 (BCOP), OECD TG 438 (ICE), and OECD TG 492 (RhCE) guidelines.

Over the past three years, these three in vitro/ex vivo methods have accounted for more than 70 percent of the eye irritation studies we have conducted in our laboratory. In particular, following the complete ban on animal testing under the EU Cosmetics Regulation in 2023, we have observed a significant increase in requests from manufacturers of cosmetics and personal care products. In this article, we will share the scientific basis of each of these three guidelines, the differences in their practical application, and our laboratory’s field experience in this area.

OECD TG 437: Bovine Corneal Opacity and Permeability (BCOP) Test

OECD TG 437 is an ex vivo test that uses the corneas of bovine eyes obtained from slaughterhouses. The basic principle of the test involves placing isolated corneas in special holders and applying the test substance to the corneal surface. Toxic effects are evaluated by measuring corneal opacity (cloudiness) and permeability. The in vitro irritation score, derived by combining these two parameters, is used to determine the substance’s GHS classification.

The most common challenge we face when conducting the BCOP test in our laboratory is maintaining the freshness of the corneal samples. Eyes obtained from the slaughterhouse must be delivered to the laboratory within the first four hours after slaughter, and the corneas must be isolated and prepared for testing within this timeframe. Due to this logistical requirement, we schedule the BCOP test only on specific days and coordinate with the slaughterhouse in advance. Furthermore, although bovine corneas share a high degree of structural similarity with human corneas, differences in corneal thickness and the number of epithelial layers can lead to false-negative results with certain sensitive reagents. In such cases, we enhance sensitivity by incorporating histopathological examination.

The most significant advantage of the BCOP is that, since it is obtained as a byproduct of slaughterhouses, it does not pose any ethical concerns. It was adopted by the OECD in 2009 and updated in 2013 and 2025. However, the test measures only the opacity and permeability parameters of the cornea; it cannot model factors such as the tear film, immune response, and tissue healing. It may be limited in predicting the potential for serious eye damage from complex mixtures, such as pesticide formulations.

OECD TG 438: Isolated Chicken Eye (ICE) Test

OECD TG 438 is an ex vivo test that uses eyes obtained from chickens. Since the chickens are slaughtered for food consumption, the test does not require the use of animals. The principle of the test involves placing the isolated eye in a holder and applying the test substance to the corneal surface. Toxic effects are evaluated based on four parameters: corneal opacity, epithelial damage (fluorescein retention), increased corneal thickness (swelling), and macroscopic morphological damage.

In our laboratory’s application of the ICE test, we achieve high accuracy, particularly for surfactants and alcohol-based cleaning products. Last year, in an ICE study we conducted for an industrial cleaning product manufacturer, we successfully identified the product’s potential for “Serious Eye Damage” (Category 1) and facilitated the manufacturer’s revision of the formulation. This case is valuable in demonstrating just how effective the ICE test is, particularly with surfactants.

The ICE test was adopted by the OECD in 2009 and updated in 2013 and 2025. Its most significant advantages are its ability to measure physical parameters such as corneal swelling and the potential to enhance its sensitivity through histopathological examination. However, like the BCOP, the ICE test uses only ex vivo tissue and does not fully reflect human biology; it cannot model vascular and immune interactions.

OECD TG 492: Reconstituted Human Corneal-Like Epithelium (RhCE) Test

OECD TG 492 is an in vitro test that uses three-dimensional tissue models mimicking the human corneal epithelium. These models are tissue cultures derived from human corneal epithelial cells that preserve the multilayered structure, barrier function, and metabolic activity of the corneal epithelium. The principle of the test involves applying the test substance to the RhCE tissue for a specific period of time and then measuring cell viability using the MTT assay.

The most significant advantage of the RhCE test is that, because it uses human cells, it allows for direct extrapolation to human biology. In our laboratory, we use this method as a primary screening test, particularly for cosmetic and personal care products. Since animal testing is prohibited under the EU Cosmetics Regulation, RhCE is the only mandatory option for this sector.

However, the RhCE test models only the superficial corneal epithelium; it cannot assess interactions with deeper eye structures, tear film dynamics, or tissue healing. Furthermore, TG 492 can only distinguish between “Not Requiring Classification” and “Irritant”; it is not sufficient on its own for a “Serious Eye Damage” (Category 1) classification. Therefore, we recommend conducting complementary testing with BCOP or ICE, particularly for potential Category 1 substances.

The table below provides a comparative summary of the key features of the OECD TG 437, TG 438, and TG 492 guidelines.

Defined Approaches and Integrated Testing Strategies

The OECD has developed Defined Approaches (DA) for cases where a single in vitro test is insufficient for eye irritation assessment. These approaches aim to reduce or completely eliminate the need for the in vivo Draize test by combining the results of different in vitro tests. There are five DAs accepted under OECD TG 467: DAs for Liquids (DAL-1 and DAL-2), DAs for Solids (DAS), and DAs for Surfactants (DASF).

In our laboratory, we apply these DAs, particularly for complex formulations. For example, last year we used the DAL-2 (BCOP + STE) approach for a cosmetic manufacturer’s new facial cleansing gel. While the RhCE test alone yielded a “non-irritating” result, the DAL-2 combination revealed the product’s “mildly irritating” potential, leading us to help the manufacturer make a minor revision to the formulation. This case demonstrates how integrated testing strategies provide more reliable results compared to single tests.

Conducting Eye Irritation Tests in Our Laboratory

In our GLP-certified laboratory, we conduct eye irritation tests covered by OECD TG 437, TG 438, and TG 492 in full compliance with international standards. We use validated test systems for all three methods; for ex vivo tests, we source fresh bovine corneas and chicken eyes, while for in vitro tests, we use validated RhCE models.

Our testing process begins with a comprehensive preliminary evaluation. The test substance’s physicochemical properties, pH value, solubility, and chemical structure are examined. Based on this information, the most appropriate test method or combination of test methods is determined. Test results are interpreted and reported according to GHS criteria.

One of our laboratory’s key advantages is that we offer various in vitro and ex vivo methods as part of an integrated strategy. For a given product, the most appropriate screening test is performed first; if the results are inconclusive or if the regulatory authority specifically requests it, supplementary tests or DA’s are used. This stepwise approach ensures efficiency in terms of both time and cost.

Frequently Asked Questions

One of the most common questions we receive regarding OECD TG 437, 438, and 492 is which test is suitable for which product. TG 437 (BCOP) is suitable for substances in liquid, solid, and paste forms, as well as water-insoluble substances. TG 438 (ICE) is particularly ideal for surfactants, alcohols, and water-soluble substances. TG 492 (RhCE), on the other hand, is preferred for cosmetics, personal care products, and water-soluble substances. Our laboratory evaluates the properties of the test substance to recommend the most appropriate method or DA combination.

In vitro test results are often asked whether they can replace in vivo tests. Yes, validated in vitro tests and DA combinations allow for classification without the need for in vivo testing. The OECD and EU REACH recognize these in vitro methods as primary tests. However, in vivo validation may be required if test results are inconclusive or if the regulatory authority specifically requests it.

Regarding the testing duration, TG 437 and TG 492 typically yield results within 1–2 weeks; TG 438, however, may take 2–3 weeks due to tissue procurement and swelling measurements. Including reporting and quality assurance review, the total duration is 3–5 weeks. Finally, the question arises as to which regulatory submissions these tests can be used for. TG 437, 438, and 492 reports are valid for EU REACH, the CLP Regulation, GHS classification, the EU Cosmetics Regulation, and Turkish KKDIK requirements.

In vitro and ex vivo methods in eye irritation assessment form the foundation of a modern approach that combines scientific accuracy with ethical responsibility. With our tests, which are fully compliant with OECD TG 437, TG 438, and TG 492 standards, we evaluate the ocular safety profile of your products to the highest scientific standards—without the need for animal testing. Our GLP-certified laboratory, equipped with validated tissue models, an experienced team, and integrated testing strategies, is your reliable partner for all your eye irritation testing needs. Contact us to demonstrate your product’s ocular safety.