Cellular viability and death biomarkers enables the evaluation of ocular irritation using the bovine corneal opacity and permeability assay

Highlights

• We investigated fluorescence staining on cell-based biomarkers of death and viability in the BCOP assay.

• A new prediction model that combines the depth of injure (DOI) to the bovine cornea with BCOP assay was developed.

• Basing on the endpoint of the DOI, the BCOP assay can differentiate all regulatory eye irritation classifications, especially to distinguish between GHS 2A and 2B.

Abstract

The BCOP assay is used in the identification of chemicals that cause no ocular irritation or serious damage. However, this method has not been found to adequately discriminate between mild from moderate ocular irritation (category 2A/2B), based upon the animal data. In this study, we aimed to establish methods for discerning ocular irritation by chemicals. We used the BCOP assay and the fluorescence staining methods based on biomarkers for cellular viability and death. The potential for ocular irritation by 12 chemicals from different UN GHS categories was assessed by the BCOP assay. Cryosections of bovine corneas were obtained. The necrotic nucleus was TUNEL labeled, cytoplasmic f-actin was stained by phalloidin while the nucleus was stained by DAPI. The depth of injury (DOI) was then measured. According to BCOP assay, in vivo data of Draize eye test and DOI, the results showed that category NC irritants caused ≤ 10 % epithelial DOI, irritants of category 2B caused >10 % epithelial DOI and showed no stromal damage, while category 2A showed damage to the stroma. Based on these results, the GHS prediction model could distinguish between GHS 2A and 2B. Authenticating the viability of BCOP by DOI measurements can provide a more reliable basis for classifying ocular irritants.

Introduction

The BCOP assay is a valid in vitro alternative method for evaluating the potential ocular irritancy of chemicals and products using the corneal tissue. Injuries attributed to the test chemical are quantitatively determined by opacity changes and permeability to fluorescein (Alépée et al., 2019). The BCOP assay has the ability to identify the categories at the extremes of the eye irritation spectrum. These extreme categories are classified as ‘no categories’ and ‘category 1’ according to the GHS classification system. However, this assay has not been sufficiently proven to differentiate between moderately irritating and mildly irritating chemicals (e.g. GHS category 2A/2B) (Verstraelen et al., 2013; Hayashi et al., 2012). Based on their ability to irritate the eyes, most chemicals belong to both ends of the irritation spectrum (Scott et al., 2010). There exist a lot of chemicals that fall in the middle region of ocular irritation (e.g. GHS category 2) for which no alternative methods to the Draize test have been approved. The reversibility of damage caused by GHS category 2 substances in in vivo experiments cannot be demonstrated or verified by in vitro methods (OECD TG 437, 438, 460, 491 and 492) (Lotz et al., 2018). Therefore, more effective methods are needed to determine the ocular irritation abilities of GHS category 2 substances and to predict reversibility.

The ability for ocular irritation by chemicals is directly associated with the depth of corneal damage (Jester, 2006). As such, ocular irritation can be classified according to the depth of the damage (Andreda et al., 2019). Histopathologic tests have been used to determine the depth of damage in both in vivo and in vitro experimental designs (Cater and Harbell et al., 2006; Goswami et al., 2019; Maurer et al., 1999). Even though histopathological evaluation of the corneal tissue has not been incorporated in the OECD Test Guideline number 437, the OECD has published a guidance document for this method (OECD, 2018). Studies have shown that exposure to irritants enhances inflammatory cell inflow that helps to mark the zone of injury in in vivo experiments (Cater and Harbell et al., 2013). In in vitro experimental designs, the isolated cornea tissue cannot undergo secondary inflammatory cell infiltration. Determination of cellular viability by assessing the associations between the nucleus or cytoplasmic structures and apoptosis, injury, or even changes in staining require an experienced pathologist. F-actin can be stained with rhodamine-labeled phalloidin to identify live cells (Jester et al., 1994). This approach is more objective in evaluating the depth of injury. Research in this field has primarily been centered on isolated rabbit eyes. Other methods such as DOI measurements based on biomarkers would be viable for isolated corneal models such as BCOP models. This method has, however, not been scientifically validated. The aim of this study was, therefore, to quantify DOI using fluorescent labeling based on live/dead cell biomarkers. To determine the feasibility of the DOI approach, we combined the DOI measurements of bovine cornea with the conventional BCOP method and in vivo data. Objective DOI measurements were done by processing for cryosectioning and using TUNEL labeling to detect necrotic nucleus and phalloidin staining to detect intracellular f-actin. The major challenge associated with DOI measurements is the expensive reagents and equipment. Compared to histopathological analysis, the edge of the damage area can be clearly distinguished by TUNEL or phalloidin staining. The DOI measurements can then be easily done by any experimenters. This is a notable advantage of this method. In addition, the results of histopathologic are still descriptive. The DOI approach can be used to compensate for this deficiency. In this study, we aimed at establishing methods for evaluating the ocular irritation potential of chemicals using BCOP assay with fluorescence staining on cell-based biomarkers for death and viability.