Bovine corneas are used in the Bovine Corneal Opacity and Permeability (BCOP) assay, which is based on the quantification of opacity and permeability as damage parameters after exposure to substances applied to the corneal epithelium surface (OECD, 2020b). Corneal opacity is measured as the amount of light transmitted through the cornea using an opacitometer, while permeability is assessed by the amount of dye (sodium fluorescein) that penetrates the corneal thickness, measured with a visible light spectrophotometer.

The BCOP test was originally developed by Muir, 1985, Muir, 1987 and later refined by Gautheron et al., 1992, Gautheron et al., 1994 to assess the ocular irritation potential of chemicals. The method was then evaluated by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM) and the Japanese Center for the Validation of Alternative Methods (JaCVAM), in 2006 and 2010 (ICCVAM, 2010; ICCVAM, 2006). In the first stage, the BCOP test method was evaluated for its usefulness to identify chemicals inducing serious eye damage and in the second stage was evaluated for its usefulness to identify chemicals not classified for eye irritation or serious eye damage (OECD, 2020b).

To classify substances according to their ocular irritation potential under the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) and United States Environmental Protection Agency (US EPA) systems, it is necessary not only to evaluate the severity of the lesions but also to determine their reversibility. This aspect represents a major limitation since current approved in vitro methodologies do not directly model reversibility (Kolle et al., 2015, Kolle et al., 2017). One example of a non-validated method specifically developed to assess the reversibility of corneal epithelial damage is the PorCORA assay, an ex vivo system employing whole cultured porcine corneas (Piehl et al., 2011). The development of new alternative methods to the Draize test requires a better understanding of the different corneal layers, as well as the cellular and molecular changes involved in both ocular injury and repair.

A significant contribution to this knowledge was provided by Maurer and Parker (1996), who analyzed, via in vivo confocal microscopy, the eyes of rats and rabbits exposed to different types of surfactants. They identified quantifiable differences in the corneal epithelium and stroma. For instance, all surfactants caused erosion, denudation, and/or necrosis of the corneal epithelium. Cationic surfactants also induced keratocyte necrosis and endothelial changes. Subsequently, using a similar methodology, Jester et al., 1996, Jester et al., 1998 studied ocular irritation phenomena through optical microscopy, in vivo confocal microscopy, and laser scanning and demonstrated that the final response to ocular irritation induced by surfactants is directly related to the area and depth of corneal injury post-exposure. Their results led to a refinement of the previously proposed damage model, suggesting that, regardless of the mechanisms leading to the lesion, slight irritants damage the corneal epithelium, mild irritants affect the corneal epithelium and superficial stroma, and moderate/severe irritants damage the epithelium, deep stroma, and, in some cases, the corneal endothelium. Additionally, they proposed that the Depth of Initial Injury (DOI) should be accepted as a biomarker of ocular irritation response, correlating with lesion severity (Jester et al., 2010; Lebrun et al., 2019).

When evaluating antimicrobial products using alternative methods to the Draize test, Redden et al. (2009) established histopathological decision criteria by correlating histological changes in the epithelium, stroma, and endothelium with US EPA and UN GHS classification categories. Following a similar approach, Kolle et al., 2015, Kolle et al., 2017 evaluated agrochemical formulations and developed a histopathological irritation scoring system with different severity grades (ranging from 0 to IV) based on the depth of injury.

Using an isolated rabbit eye model, researchers investigated whether DOI could be objectively measured through fluorescent staining with cell death and viability biomarkers. The results showed that slight irritants damage less than 40 % of the epithelium, mild and moderate irritants damage more than 50 % of the epithelium—sometimes extending into the anterior stroma (<20 %)—and severe irritants damage more than 50 % of the stroma (Jester et al., 2010).

Furthermore, Maurer et al. (2001) demonstrated that oxidative agents induce delayed toxicity in vivo, affecting stromal keratocytes. Peroxide-containing formulations require histopathological evaluation to determine the full depth of the lesion, as observed cellular changes predict subsequent corneal degeneration (OECD, 2018).

The BCOP assay is widely used to categorize products falling within the extreme ends of the ocular irritation scale under the GHS system—i.e., Category 1 (severe irritants/corrosives) or No Category (non-irritants). However, substances with an IVIS (In Vitro Irritation Score) between 3 and 55 are classified as “No stand-alone prediction can be made” meaning they could potentially cause reversible damage in the in vivo model. To date, only one validated method exists for predicting Category 2 substances, based on a three-dimensional human corneal epithelium (HCE) model, the OECD TG 492B (OECD, 2022). However, no in vitro test has yet proven capable of distinguishing between Categories 2A and 2B, though combinations of tests have been proposed as a possible solution (Andrade et al., 2020; Lenze et al., 2024; Scott et al., 2010; van der Zalm et al., 2023).

Several reviews summarize the available in vivo, in vitro, and ex vivo test methods and define the scope of each (Clippinger et al., 2021). Three-dimensional models and those incorporating the entire cornea provide valuable insights into lesion severity (Lebrun et al., 2019; van der Zalm et al., 2023). Mechanistic injury data indicates that these methods replicate human eye pathophysiology as well as, or better than, the rabbit assay.

As an additional parameter in the BCOP assay, microscopic analysis of corneal structure has been recommended (Andrade et al., 2019; Jeong and Kim, 2022). In fact, the OECD's, 2018 guidance (OECD, 2018) endorses histopathological evaluation to assess damage caused by substances or formulations not well characterized by BCOP, determine lesion depth, characterize damage severity or extent, and differentiate between categories in borderline cases. Conducting a histopathological study following BCOP can help elucidate cellular and structural changes, provide evidence of corneal injury not revealed by BCOP endpoints, or even offer insights into the mechanisms by which a compound induces damage.

Corneal thickness, particularly central corneal thickness, is a clinically relevant parameter in the evaluation of various ocular pathologies (Gundreddy et al., 2023; Ondas and Keles, 2014; Palamar et al., 2017; Pazos González et al., 2004). Thickness changes due to damage primarily result from stromal edema. Measuring corneal thickness is crucial in assessing corneal injuries, as they compromise corneal integrity. Therefore, stromal thickness measurement could serve as an indicator of lesion severity.

Based on these considerations, the objective of this study was to identify histopathological parameters observed in bovine corneas treated with reference substances from different irritation categories that correlate with their potential reversibility. To achieve this, we used the DOI model, quantified all histopathological parameters observed in each corneal layer, and assessed corneal stromal thickness following exposure.