title: Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.
abstract: In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (μM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results.
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Hess
authors.affiliation: Department of Pre-clinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany; German Center for Lung Research, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Germany.
authors.forename: L
authors.initials: L
authors.lastname: Wang-Lauenstein
authors.affiliation: Department of Pre-clinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany; German Center for Lung Research, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Germany.
authors.forename: A
authors.initials: A
authors.lastname: Braun
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany.
authors.forename: S N
authors.initials: SN
authors.lastname: Kolle
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Landsiedel
authors.affiliation: German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Liebsch
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Ma-Hock
authors.affiliation: German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Pirow
authors.affiliation: Institute of Pharmacology and Toxicology, Medical Faculty of RWTH Aachen University, Wendlingweg 2, Aachen, Germany.
authors.forename: X
authors.initials: X
authors.lastname: Schneider
authors.affiliation: German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Steinfath
authors.affiliation: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Vogel
authors.affiliation: Institute of Pharmacology and Toxicology, Medical Faculty of RWTH Aachen University, Wendlingweg 2, Aachen, Germany. Electronic address: chmartin@ukaachen.de.
authors.forename: C
authors.initials: C
authors.lastname: Martin
authors.affiliation: Department of Pre-clinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany; German Center for Lung Research, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Germany. Electronic address: katherina.sewald@item.fraunhofer.de.
authors.forename: K
authors.initials: K
authors.lastname: Sewald
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D002470:Cell Survival; D005260:Female; D066298:In Vitro Techniques; D053582:Interleukin-1alpha / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007753:Laboratories; D008168:Lung / Q000378:metabolism; D008954:Models, Biological; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013778:Tetrazolium Salts / Q000378:metabolism; D018675:Toxicity Tests
chemical_list: C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D053582:Interleukin-1alpha; D013778:Tetrazolium Salts; D007770:L-Lactate Dehydrogenase
keywords: Acute inhalation toxicity; Industrial chemicals; Precision-cut lung slices; Prevalidation; Reproducibility; Respiratory toxicity; Submersed culture conditions; Transferability; Two-group classification model