Abstract
The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.
Highlights
To date, the rabbit Draize eye irritation test [1] is still the only OECD-approved test for the prediction of all three GHS categories for eye irritation in one single test system [2]
We developed a method to analyze the depth of injury (DOI) by combining the MTT viability assay with cryosectioning techniques and computer-aided analysis with the aim to establish a prediction model to discriminate all three GHS classes for eye irritation within one model
In contrast to the ex vivo DOI measurements on isolated rabbit eyes (IRE-DOI) described by Jester [33], where classification is based on differential DOI values from both epithelium and stroma, our classification system is exclusively based on DOI in the entire tissue
Summary
The rabbit Draize eye irritation test [1] is still the only OECD-approved test for the prediction of all three GHS categories for eye irritation in one single test system [2]. Some methods have gained regulatory acceptance for selected GHS categories Both the BCOP and the ICE test method have been implemented at OECD level to screen for corrosives and severe eye irritants (Cat. 1) on the one hand and for non-classified chemicals on the other hand [23], [24]. Test methods which reliably distinguish the mild/moderate irritatants (Cat. 2) from Cat. 1 and the non-irritants (No Cat.) directly, are not yet available Many of these test methods are intended to be used only within the framework of an integrated testing strategy, either in a top-down or in a bottom-up approach [8], [27], [28]
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