P28-08 Evaluating the potential of ReproTracker as an alternative assay for regulatory use following the ICH S5 (R3) guideline

ICH S5 (3R) is paving the way for the acceptance and validation of alternative models for developmental and reproductive toxicity (DART) testing by establishing the requirements and promoting regulatory confidence in new approach methodologies. In vitro models, validated based on regulatory guidelines, can be used as powerful tools within weight-of-evidence approaches in multiple frameworks for DART testing. The ReproTracker assay is based on human induced pluripotent stem cells (hiPSCs) which are differentiated towards three germ layer-specific cell types: cardiomyocytes (mesoderm), hepatocytes (endoderm), and neural rosettes (ectoderm). This model enables the assessment of the teratogenic potential by tracking alterations in biomarker expression, as well as morphological disruptions in the differentiated cells upon compound exposure. To comprehensively validate the assay, we tested the 32 compounds listed in the ICH S5 (3R) guidelines. Following identification of human developmental toxicants in ReproTracker, we assessed the clinical relevance of the concentrations detected as potentially teratogenic by investigating their correlation with their corresponding human therapeutic Cmax. In this study, the ReproTracker assay was able to correctly classify 26 of the 29 positive reference compounds, indicating the potential of the assay for DART testing. Two of the missed compounds were identified as DNA modifying agents, namely 5-fluorouracil and methotrexate. These compounds exhibited highly cytotoxicy profiles leading to very low top concentrations (less than 1 and 0.01 µM, respectively) being tested in the assay. For the majority of compounds, the assay predicted teratogenicity at concentrations either below or similar to the clinically relevant concentrations (human Cmax). When the predicted lowest adverse effect levels were higher than the respective Cmax, they did not exceed a 10–40 fold difference. While two of the three negative controls were correctly classified as negatives, vildagliptin was falsely classified as a teratogen but at concentrations more than 400-fold higher than its human relevant concentration (0.3 µM). In this study, the ReproTracker assay demonstrated a high predictability 96% with a sensitivity of 90%, highligting the potential of the assay in teratogenicity assessment. This study showcases the predicitive potential of ReproTracker to detect teratogenicity of chemicals and positions the assay at the forefront of in vitro DART testing. Moreover, validation following the ICH S5 guidelines provides a solid foundation for further validation and integration of the assay into safety assessment frameworks.