Replace, Reduce, Refine – Recent Developments in Alternative Testing Methods in Pharmaceutical Toxicology and their Regulatory Acceptability in the EU Wissenschaftliche Prüfungsarbeit zur Erlangung des Titels „ Master of Drug Regulatory Affairs “ der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Dr. rer. nat. Sandra Mahr aus Berlin Bonn 2018 Betreuer und 1. Referent PD Dr. Gerd Bode 2. Referent PD Dr. Elke Röhrdanz 2 Table of Contents Table of Contents ......................................................................................................... 3 Table of Figures ........................................................................................................... 5 Table of Tables ............................................................................................................ 5 List of Abbreviations ..................................................................................................... 6 List of Abbreviations (continued) .................................................................................. 7 1 Introduction ........................................................................................................... 8 1.1 Aim of the present thesis ............................................................................... 8 2 Regulatory requirements ...................................................................................... 8 2.1 European legislation ...................................................................................... 8 2.1.1 Directive 2001/83/EC ................................................................................ 9 2.1.2 Directive 2001/20/EC and Regulation (EU) 536/2014 ...............................10 2.1.3 European Pharmacopoeia (Ph. Eur.) ........................................................11 2.1.4 Further requirements ................................................................................11 2.1.5 Regulatory requirements for medical devices, chemicals and other products ...................................................................................................11 2.1.5.1 Certification of medical devices ...........................................................11 2.1.5.2 Registration, evaluation, authorisation of chemicals (REACH) ............11 2.1.5.3 Regulation on the classification, labelling and packaging of chemicals ............................................................................................11 2.1.5.4 Biocidal Products Regulation (BPR) ....................................................12 2.1.5.5 Cosmetics Regulation No 1223/2009 ..................................................12 2.2 Toxicology guidelines from the International Council for Harmonisation (ICH) ............................................................................................................12 2.3 Current data on the use of animals for scientific purposes ...........................14 2.4 Principle of 3Rs: Replace, Reduce, Refine ...................................................16 2.4.1 Background ..............................................................................................16 2.4.2 EU regulatory activities fostering the principle of the 3Rs .........................16 2.5 International initiatives promoting alternative testing strategies ....................17 2.5.1 EMA Joint CVMP/CHMP Working Group on the Application of the 3Rs in Regulatory Testing of Medicinal Products (J3RsWG) ....................17 2.5.2 European Union Reference Laboratory for alternatives to animal testing (EURL-ECVAM) ............................................................................17 2.5.3 European Partnership for Alternative Approaches to Animal Testing (EPAA) .....................................................................................................18 2.5.4 EU-ToxRisk ..............................................................................................19 2.5.5 International programs outside the EU .....................................................19 2.5.6 Implementation of the 3Rs by the ICH ......................................................20 3 Regulatory acceptance of alternative testing approaches ....................................21 3 3.1 Guideline on the principles of regulatory acceptance of 3Rs (replacement, reduction, refinement) testing approaches .............................21 3.1.1 Regulatory acceptance criteria according to the EMA ..............................21 3.1.2 Proof of scientific validity ..........................................................................21 3.1.3 Method validation .....................................................................................21 3.1.4 Data submission .......................................................................................22 3.1.5 Scientific advice and qualification of alternative testing approaches by the Scientific Advice Working Party (SAWP) ........................................22 3.2 Validation process by the EURL-ECVAM .....................................................23 3.3 Validation status of alternative testing methods at EURL-ECVAM ................23 4 Recent developments in alternative testing strategies and their status of regulatory acceptance ......................................................................................28 4.1 Challenges in the development of alternative testing approaches ................28 4.2 Organoid systems – Organs in a dish ..........................................................28 4.2.1 What are organoids? ................................................................................28 4.2.2 Use of organoids in regulatory toxicity testing ..........................................30 4.3 Microphysiological systems (MPS) – Organs on a chip ................................31 4.3.1 What are MPSs? ......................................................................................31 4.3.2 Current MPS developments and application in regulatory toxicity testing ......................................................................................................32 4.3.2.1 Liver-islet-chip .....................................................................................32 4.3.2.2 Skin-tumour model ..............................................................................33 4.3.2.3 Lung-on-a-chip ....................................................................................33 4.3.2.4 Female-reproductive-tract-on-a-chip ...................................................34 4.3.2.5 Four-organ-ADMET-chip .....................................................................35 4.3.2.6 Human-on-a-chip ................................................................................35 4.3.3 Use of MPSs in regulatory toxicity testing .................................................36 4.4 “Omics” ........................................................................................................38 4.4.1 What is “omics” technology? ....................................................................38 4.4.2 Application of “omics” technology in regulatory toxicity testing: toxicogenomics ........................................................................................39 4.5 In-silico tools ................................................................................................41 4.5.1 What are in-silico methods? .....................................................................41 4.5.2 EU development projects on in-silico tools ...............................................42 4.5.3 Application of in-silico methods in regulatory toxicity testing .....................42 4.5.4 Further promising developments in alternative testing strategies .............44 4.6 Information on regulatory acceptability of alternative testing methods from EMA assessment reports .....................................................................45 5 Conclusion and Outlook .......................................................................................46 6 Summary .............................................................................................................49 4 7 References ..........................................................................................................50 8 Annex ..................................................................................................................67 Table of Figures Figure 1: Purposes of animal experiments reported 2011, Source: [18].......................15 Figure 2: Illustration of the stepwise EURL-ECVAM validation process .......................23 Figure 3: Schematic overview of organoid generation from pluripotent stem cells, Source: [83] ...............................................................................................29 Figure 4: Generation of different organoids, Source: [84] ............................................30 Figure 5: Multi-organ-chip with PDMS layer, Source: [86] ...........................................31 Figure 6: Two-organ-MPS (liver-islet-chip). Structure of liver-islet-chip device (left), tissue-loading scheme of organ co-culture (right), Source: [87] ........32 Figure 7: Skin-tumour model for cancer drug candidate “safficacy” testing, Source: [89] ...............................................................................................33 Figure 8: Lung-on-a-chip, Source: [91] ........................................................................34