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ExITox (Explain Inhalation Toxicology) aims to develop an integrated approach for the hazard characterization of inhalable compounds without in vivo animal testing. Different in vitro and in silico models are developed by assessing groups of compounds that share structural properties and induced a shared toxicological effect pattern in existing in vivo studies with repeated exposure via inhalation. The shared toxicological effects in these read-across groups might indicate a shared mode of action. We hypothesize that in vitro data can be used to strengthen the read-across hypothesis e.g. by showing shared adverse outcome pathways. We selected a group of four aliphatic acrylates, which induced inflammation and hyperplasia in the respiratory ract of the test animals at dose levels ranging from 15-25 ppm. These acrylates were tested submerse in A549 cells and human precision-cut lung slices (PCLS) dose-dependently. Exposure occured over three days for one hour daily. In A549 cells the cytotoxicity of acrylates was assessed with EC50 values of 2.15, 3.7, 1.12 and 1.57 mM for methyl acylate, ethyl acrylate, propyl acrylate and butyl acrylate, respectively. Comparable to cells, PCLS showed similar sensitivity with the EC50 values of 1.61, 3.02, 2.99 and 4.48 mM for methyl acylate, ethyl acrylate, propyl acrylate and butyl acrylate, respectively. The bioinformatic process chain includes analysis of whole transcriptome with RNA-seq technology of all compounds to detect the expression changes, focus on regulation via enhancers and underlying miRNA levels, involves network analysis to identify potential biomarker up to a postulated diagnostic profile for each specific read-across group. The results for DEG and miRNA will be presented. This work was supported by BMBF grant FKZ 031A269A-D.

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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.