Ecotoxicology – part 4 New topics and future issues Ludek Blaha + ecotox colleagues Current approaches (black box of apical endoints) vs Future (mechanistic understanding & AOPs) OrganismChemical Adverse Effects Death Altered Reproduction Inhibition of Growth Tumorigenicity Skin irritation … + Traditionally – Evaluation of adverse effects using the whole organism models REGULATORY FOCUS (APICAL ENDPOINTS) Hazard assessment OrganismChemical Adverse Effects Death Inhibition of Growth Altered Reproduction Tumor Skin irritation … + Traditionally – Evaluation of adverse effects using the whole organism models +10^4 Chemicals HTS High-Throughput-Screening Chemical-biological interactions, Mechanistic Toxicological Data Hazard assessment New – Ex vivo / in vitro / In chemico / In silico Methods Key task/question: How to link MECHANISTIC INFORMATION with APICAL ENDPOINTS ? MoA and omics are supported by strategic documents Toxicity Testing in the 21st Century: A Vision and a Strategy US National Academies of Sciences http://www.nap.edu/catalog/11970.html Adverse Outcome Pathways The EXISTING KNOWLEDGE is used to link the two anchor points: Molecular Initiating Event (MIE) and Adverse Outcome (AO) via a series of intermediate steps: Key Events Chemical Macro- Molecular Interaction Cellular Response Organ Response Organism Response Population Response Molecular Initiating Event Key Event 1 Key Event 2 Adverse Outcome Tissue Effect Key Event 3 Chemical Property Receptor/Ligand Interactions DNA Binding Protein Oxidation Gene Activation Protein Production Altered Signaling Altered Physiology Disrupted Homeostasis Altered Development / Function Lethality Impaired Development Impaired Reproduction Altered Sex Ratio Extinction Adverse Outcome Pathway Toxicity Pathway Mode of Action In silico, In chemico, In Vitro, Ex vivo In vivo Ankley, G. T., R. S. Bennett, et al. (2010). "Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment." Environmental Toxicology and Chemistry 29(3): 730-741. AOP = Global strategy with support from OECD, EU, USA http://www.oecd.org/chemicalsafety/testing/projects-adverse-outcome-pathways.htm http://aopkb.org/ Key documents OECD Guidance document and a template for developing and assessing adverse outcome pathways (Series No. 184, Series on Testing and Assessment) Handbook for AOP developers AOP Wiki • https://aopkb.org/aopwiki/index.php/Main_Page • Wiki-based platform for development of AOPs • Only members of an OECD AOP development project can create / edit AOPs What AOPs are now in AOP Wiki (May 2016?) OECD Endorsed (WNT and TFHA) 1 Covalent Protein binding leading to Skin Sensitisation EAGMST Approved 6 1x ecotoxicology: Aromatase inhibition leading to reproductive dysfunction (in fish) EAGMST Under Review 12 EAGMST Under Development 84 SAAOP AOP Under Development 15 • OECD Extended Advisory Group on Molecular Screening and Toxicogenomics (EAG MST) • The Working Group of the National Coordinators of the Test Guidelines Programme (WNT) https://aopwiki.org/aops https://aopwiki.org/aops AOP Example: MIE aromatase inhibition Environmental Toxicology and Chemistry, Vol. 30, No. 1, pp. 64–76, 2011 Aromatase inhibition leading to reproductive dysfunction (in fish) https://aopwiki.org/wiki/index.php/Aop:25 AOP Example from RECETOX: Modulation of RAR/RXR  developmental toxicity in fish Jonáš et al. 2014 Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2014.06.022 Activation of RAR/RXR in P19/A15 cells by atRA and cyanobacterial metabolites ZF exposed to ATRA and cyanobacterial (120 hpf) - Control (A), exudates of C. raciborskii 3.3× (B) and 10× (C), M. aeruginosa 10× (D) and D. quadricaudatus 17× (E). ATRA 4 μg/L (13.3 nM) (F), 12 μg/L (40 nM) ((G) and (H)), 36 μg/L (I) and 108 μg/L (J). atRA other RAs in cyanos Summary • Toxicology is about doses – The goal is LD(LC)50 or NOAEL/NOEC • Legislation defines … what assays and how to do them – About 30 assays – The most widely used standard - OECD Guidelines for Testing of Chemicals • Replacing „black box“ in traditional testing – Synthesis of mechanistic and omics data – Adverse Outcome Pathways – Strategically supported by OECD, EU, USA Do we need testing with animals? Are there alternatives 3Rs REDUCTION REFINEMENTREPLACEMENT „Alternatives“ to toxicity testing … 3R rules Why doing replacement, reduction, refinement? •Because activists put pressure to do so? •Because animal welfare is a concern for EU citizen? •Because animal testing is “bad” and “alternatives” are good? •Because I will get “better” results? •Because it is cutting edge technologies? •Because I have to? E.g. EU law directive 2010/63/eu, ban on animal testing for cosmetics 3Rs are driven by EU laws, little by Member States. Scientific agenda is not driven enough by scientists itself… Academia is in general more reactive than proactive e.g. stop vivisection’s ECI European Policies on 3Rs Use of animals in EU (2011) •VALIDATION •MoA •Reliable •Relevant Substance Tested e.g. endocrine disrupters receptor binding Validation Prevalidation Development Research Independent Review Implementation Regulatory Acceptance Alternative Methods – R&D to Implementation ESAC Industry Regulators Academia PARERE ESTAF 7-8 YEARS • >60 3Rs Tests submitted to ECVAM since 2008 (update 01/2015) • 10 validated or ongoing validation => Prioritisation! COMPUTATIONAL (ECO)TOXICOLOGY PBPK models PBPK (PBTK) Physiologically based pharmacokinetic (toxicokinetic) models Fragmentation of a complex systém to „boxes“  All Processes described by arrows (mathematical equations) Example – computational toxicology for EDCs Li (2011) BMC Systems Biology Conceptual model EE2 – ethinylestradiol ER, AR atd. – receptors VTG – vitellogenin (marker of toxicity) Arrows – differential equations Li (2011) BMC Systems Biology Results: MODELLED (white) Vs MEASURED (grey) …good comparable Closing remarks • Ecotoxicology is exciting science! • Interface: science and society • Many opportunities • Sometimes hard work 10% inspiration and 90% „perspiration“ • Be creative: move frontiers • Keep the purpose in mind • Be critical: do not accept perceptions as facts • Speak up: you have something to say!