# Non-invasive in vitro NAM for the detection of reversible and irreversible eye damage after chemical exposure for GHS classification purposes (ImAi)

## TL;DR

This study introduces a non-invasive in vitro method to detect eye damage from chemicals, offering a potential replacement for animal testing.

## Contribution

A non-invasive in vitro test (ImAi) is developed to distinguish reversible and irreversible eye damage, enabling full replacement of the Draize eye test.

## Key findings

- The ImAi method achieved 90.9% correct classification for Cat. 1 and 83.3% for Cat. 2 chemicals.
- The method uses impedance spectroscopy to monitor tissue recovery over 14 days.
- It overcomes limitations of existing in vitro methods in predicting tissue recovery.

## Abstract

The potential risk of chemicals to the human eye is assessed by adopted test guidelines (TGs) for regulatory purposes to ensure consumer safety. Over the past decade, the Organization for Economic Co-operation and Development (OECD) has approved new approach methodologies (NAMs) to predict chemical eye damage. However, existing NAMs remain associated with limitations: First, no full replacement of the in vivo Draize eye test due to limited predictability of severe/mild damage was reached. Second, the existing NAMs do not allow reliable differentiation between reversible and irreversible eye damage. Especially the prediction of tissue recovery remains challenging in vitro. Existing in vitro NAMs are based on destructive analysis with no consideration of tissue recovery. In this study, we developed a standalone eye-irritation test method based on non-invasive impedance spectroscopy (ImAi) to discriminate between damaging and irritating chemicals. Tissue effects were analyzed via transepithelial electrical resistance (TEER) measurements of human in vitro epithelial models over 14 days. The TEER was performed using a developed impedance spectrometer. For development of the EIT, a chemical reference list of 329 chemicals was compiled. The applicability of the ImAi-test was exemplified by the discrimination of Cat. 1 vs. Cat. 2 for 23 reference chemicals. Correct classification was achieved for 90.9% of Cat. 1 and 83.3% of Cat. 2 chemicals. Our non-invasive in vitro test overcomes the limitations of Cat. 2 classification of the existing in vitro methods and provides for the first time a non-animal test method that can fully replace the Draize eye test.

The online version contains supplementary material available at 10.1007/s00204-024-03940-x.