EXITOX-II: Development of an animal free testing strategy for the risk assessment of inhalable compounds

In human risk assessment there is a paradigm shift toward mechanistic risk assessment with the aim to replace as far as possible in vivo animal testing by new approach methodologies (NAMs). These NAMs include different techniques based on in silico (QSAR, grouping and PBPK), in vitro, and ex vivo approaches. In this study we hypothesize that adverse outcomes of chronic respiratory diseases are shared between similar substances and change signaling pathways in single cells, tissues, and organisms. We aim to develop an integrated approach for testing and assessment (IATA) to replace animal studies with repeated inhalational exposure. EXITOX-II, explain inhalation toxicity II, is a public funded project and started in January 2017 as a follow up project of EXITOX. In this project we selected five groups of structurally similar compounds for testing findings/adverse outcomes in repeated dose toxicity because of (RDT)shared toxicological studies with inhalation exposure. The RDT studies were taken from the FhG database RepDose. Three groups induce inflammation, one hyperplasia and one pulmonary fibrosis. Based on their generic physico-chemical properties these compounds are tested in vitroin i) alveolar epithelial cells A549 and ii) in fresh human lung tissue. So far we exposed cells and tissues to chemicals at air-liquid interface or submerse. Cellular readouts such as cytotoxicity and chemokine release are currently measured. Based on these results, samples are collected for omic analyses with the TempoSeqTM technology (mRNA) and Affymetrix arrays (miRNA). By dose dependent testing, we will investigate the onset of gene changes and miRNA regulation. Further we aim to better differentiate between group-specific toxicological changes and general stress responses e.g. related to high dosing. Finally, we will confirm gene changes by RTqPCR testing. In this presentation we will show the current results from in vitro testing, QSAR models for ADME parameters such as ppb (plasma protein binding), and PBPK models for lung uptake. Furthermore, we will present our concept for biomarker discovery based on e.g. differentially expressed genes and the unique upstream analysis available from the geneXplain platform. A first indication how we see these building blocks in the context of an IATA will be provided