Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories

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.

In vitro eye irritation testing using the open source reconstructed hemicornea – a ring trial

“…1 chemical methyl pentynol). The predictive capacities were comparable for both assays and thus confirmed results from previous studies conducted with the hemicornea (Engelke et al, 2013;Bartok et al, 2015;Tandon et al, 2015;Zorn-Kruppa et al, 2014), whereas the selectivity of both assays must be optimized.…”

“…The results of this part of the ring trial confirmed a tendency that was already observed during the developmental phase and protocol transfer of this method (Zorn-Kruppa et al, 2014). The DOI data for the Cat.…”

“…In order to overcome this limitation in predictivity, two test methods, both based on a bioartificially produced 3D human corneal equivalent (hemicornea), were developed (Zorn-Kruppa et al, 2014;Bartok et al, 2015). Both methods predict the eye-irritation potential of chemicals for all 3 GHS categories within one test.…”

In vitro eye irritation testing using the open source reconstructed hemicornea – a ring trial_suppl

“…The protocols for the production of the hemicornea models and the performance of the eye irritation test have been previously published by Zorn-Kruppa et al (2014) and Bartok et al (2015). More details, including the list of test items used in this ring trial, can be found in the supplementary file (doi: 10.14573/ altex.1610311s).…”

“…Both methods predict the eye-irritation potential of chemicals for all 3 GHS categories within one test. Assay performance, reliability and predictivity of both methods have been demonstrated in the developers' laboratories with sets of reference chemicals representing all GHS categories and different physicochemical properties (Zorn-Kruppa et al, 2014;Bartok et al, 2015;Tandon et al, 2015).…”