Skin corrosion testing plays a crucial role in assessing the potential harm caused by chemicals to human skin, influencing regulatory decisions for safety, transportation, and labeling (EU, 2008). Traditionally, animal testing, particularly using rabbits, was the norm based on the Organization for Economic Co-operation and Development (OECD) Test Guideline (TG) 404 (OECD, 2015a). However, ethical concerns and the evolving understanding of animal welfare have driven a shift towards alternative in vitro methods using human skin models (OECD, 2019; OECD, 2015b; OECD, 2015c; OECD, 2021).

Reconstructed human epidermis (RhE) models, such as EpiSkin™ (Alépée et al., 2014a), EpiDerm™ (Liebsch et al., 2000), SkinEthic™ RHE (Alépée et al., 2014b), epiCS (Hoffmann et al., 2005) and LabCyte EPI-MODEL 24 (LabCyte Validation Management Team, 2018) have emerged as key players in in vitro skin corrosion testing. These three-dimensional structures, composed of human-derived epidermal keratinocytes, closely replicate the multi-layer cell organization of the epidermis. Notably, RhE models are employed in various applications (Coquette et al., 2003; Kose et al., 2018; Sugibayashi, 2017), with a significant focus on skin corrosion testing for categorizing chemicals as per the United Nations (UN) Global Harmonized System of classification and labeling of chemicals (GHS) (Pistollato et al., 2021; Alépée et al., 2019; Desprez et al., 2015a; Kandárová et al., 2006).

The regulatory landscape, governed by the UN GHS and implemented in the European Union System of Classification of Chemicals, emphasizes the importance of sub-categorization within skin corrosion assessments (European Chemicals Agency, 2017). The classification into sub-categories 1A, 1B, and 1C carries distinct implications for transportation safety measures, with sub-category 1A chemicals demanding stringent precautions and incurring higher costs (European Chemicals Agency, 2017). Therefore, accurate sub-categorization is essential to avoid unnecessary in vivo testing, reduce costs, and align with regulatory requirements.

The OECD Test Guideline No. 431, a cornerstone in skin corrosion testing, has undergone revisions to enhance its sub-categorization capabilities with the most recent update in 2019 (OECD, 2019). Initially allowing binary categorization, the guideline was updated in 2012–2014 to address the limitations in discriminating between Cat1B and Cat1C corrosive chemicals. RhE test methods, including EpiSkin™, EpiDerm™, SkinEthic™ RHE, epiCS® and LabCyte EPI-MODEL24, were incorporated into the guideline, showcasing their ability to partially sub-categorize within the corrosive category (OECD, 2019). However, challenges persist, particularly in the over-prediction rates for certain methods, necessitating further testing for confirmation.

Furthermore, the assessment of skin corrosion goes beyond individual countries, with international cooperation reflected in the Integrated Testing Strategy (Alépée et al., 2015) and Integrated Approach on Testing and Assessment (IATA) (OECD, 2017). RhE test methods contribute significantly to these approaches, playing a key role in advancing the scientific and technical aspects of skin corrosion evaluation.

The present study focuses on analyzing the capability of a novel in vitro reconstructed epithelium method, specifically the QileX-RhE test system (Chacón et al., 2020), for the subcategorization of chemicals within the framework of OECD TG 431 criteria. This approach is based in the Minimum List of Chemicals for determining the reproducibility and predictive capacity of similar or modified RhE methods (OECD, 2015d) as a new me-too alternative method. The study not only focuses on traditional metrics like sensitivity and specificity, but also delves into nuanced parameters such as positive predictive value (PPV), negative predictive value (NPV), post-test odds and likelihood ratios. These additional metrics offer a comprehensive assessment of test's performance, bridging the gap between traditional validation studies and real-world applications (Desprez et al., 2021).

This study aims to provide valuable insights into the applicability and effectiveness of this new method, contributing to the continuous improvement of skin corrosion assessment practices and advancing alternative testing methods.