Use of the Cultex Ò Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility Dirk Steinritz a,b,⇑ , Niklas Möhle c , Christine Pohl d , Mirko Papritz a , Bernhard Stenger d , Annette Schmidt a , Charles James Kirkpatrick d , Horst Thiermann a , Richard Vogel f , Sebastian Hoffmann e , Michaela Aufderheide c a Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany b Walther Straub Institute of Pharmacology and Toxicology, University of Munich, Goethestraße 33, 80336 Munich, Germany c Cultex Ò Laboratories, Feodor-Lynen-Straße 21, 30625 Hannover, Germany d Institute of Pathology, University Medical Center, Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55101 Mainz, Germany e Seh Consulting + Services, Rathausplatz 9, 33098 Paderborn, Germany f Berlin Toxicology Office, Leubnitzer Weg 3c, 13593 Berlin, Germany article info Article history: Available online 11 May 2013 Keywords: REACH Lung toxicity Aerosols Cultex Ò RFS In vitro toxicity Air–liquid-interface abstract Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is con- sidered as a serious health hazard. For a wide range of substances basic knowledge about the toxic prop- erties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicolog- ical in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general (‘‘3R’’ principle), pro- found in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex Ò RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials. Using the Cultex Ò RFS, human lung epithelial cells (A549 cells) were exposed to different concentra- tions of airborne metal dusts (nano- and microscale particles) at the air–liquid-interface (ALI). Cell viabil- ity (WST-1 assay) as a parameter of toxicity was assessed 24 h after exposure with special focus on the intra- and inter-laboratory (three independent laboratories) reproducibility. Our results show the general applicability of the Cultex Ò RFS with regard to the requirements of the ECVAM (European Centre for the Validation of Alternative Methods) principles on test validity underlin- ing its robustness and stability. Intra- and inter-laboratory reproducibility can be considered as sufficient if predefined quality criteria are respected. Special attention must be paid to the pure air controls that turned out to be a critical parameter for a rational interpretation of the results. Our results are encouraging and future work is planned to improve the inter-laboratory reproducibility, to consolidate the results so far and to develop a valid prediction model. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Since the beginning of industrialization, the respiratory system is challenged by an increasing number of harmful substances of natural or industrial origin. Especially the ongoing industrial devel- opment (most notably in emerging industrialized countries) has contributed to air pollution that is responsible for a multitude of health risks. In addition new everyday products (e.g. nanoparticle containing sprays) that have found wide applications in electron- ics, chemicals, environmental protection and biological medicine exhibit toxic characteristics [1]. It is therefore not surprising, that 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.05.001 ⇑ Corresponding author at: Bundeswehr Institute of Pharmacology and Toxicol- ogy, Neuherbergstraße 11, 80937 Munich, Germany. Tel.: +49 89 3168 2304; fax: +49 89 3168 2333. E-mail addresses: dirk.steinritz@lrz.uni-muenchen.de, dirksteinritz@bundes- wehr.org (D. Steinritz), N.Moehle@Cultex-laboratories.com (N. Möhle), pohlc@uni- mainz.de (C. Pohl), mirko_papritz@yahoo.de (M. Papritz), stengeb@uni-mainz.de (B. Stenger), annette2schmidt@bundeswehr.org (A. Schmidt), kirkpatrick@ukmainz.de (C.J. Kirkpatrick), horstthiermann@bundeswehr.org (H. Thiermann), drrvogel@aol.- com (R. Vogel), sebastian.hoffmann@seh-cs.com (S. Hoffmann), M.Aufderheide@- Cultex-laboratories.com (M. Aufderheide). Chemico-Biological Interactions 206 (2013) 479–490 Contents lists available at SciVerse ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint