1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212570_28446780.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Assessment of toxic effects Ludek Blaha et al. 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Outline • •Why toxicity assessment • •Who wants the assessment • •When •Where the assessment is needed • •What to assess for toxicity • •How to assess toxicity • • •What if not do we need tests? alternatives … 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •1996 - Chemicals in the environment •Do you believe that chemicals in products sold to consumers have been proven safe? • •Think again • • most chemicals in modern use have simply not been tested for their impacts on •human, even very basic effects. • •… what about the effects in nature, then ? OSF%20Hardback%20Large •How we stand 20 years later? 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif http://i.dailymail.co.uk/i/pix/2012/12/05/article-2243155-165C6117000005DC-311_634x522.jpg 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Impacts on biota à global effects •Mixing oceans •à cooling the atmosphere •[Nature 447, p.522, May 31, 2007] •Marine life supplies up to 50% of the mechanical energy required worldwide to mix waters from the surface to deeper cool layers •[Dewar, Marine Res 64:541 (2006)] •[Katija a Dabiri, Nature 460:624 (2009)] 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Who wants the toxicity assessment? Výsledek obrázku pro government icon 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Who wants the toxicity assessment? Výsledek obrázku pro government icon Researcher Government Goal To understand with joy! To survive (law or jail? $$ or hunger?) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Who wants the toxicity assessment? Výsledek obrázku pro government icon Researcher Government Goal To understand with joy! To survive (law or jail? $$ or hunger?) Approach Why rules? Strict and tough rules 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Who wants the toxicity assessment? Výsledek obrázku pro government icon Researcher Government Goal To understand with joy! To survive (law or jail? $$ or hunger?) Approach Why rules? Strict and tough rules Stakeholders Any? (… other scientists?) Many! •Businesses … providing jobs •People … wanting jobs but also health 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •Ethinylestradiol (EE2) •Estrogen receptor? in fish? snails? rabbits? •More proteins? yolk? vitellogenin? her2neu? •cyp19a? - •Adverse effects? Death? •Infertility? •Hyperactivity? Scientific approach – EE2 (part 1/2) http://www.leggilo.net/wp-content/uploads/2012/07/yasmin-drospirenone-ethinylestradiol__1982_big.jp g http://www.sfreporter.com/santafe/imgs/media.images/2761/08.25.10-FemFish-cover.widea.jpg 2. Hypotheses! 1. Problem definition •More cell proliferation? cancer? gut? •stem cell? - Výsledek obrázku pro cyp19 Výsledek obrázku pro caco2 https://www-pls.llnl.gov/data/assets/images/science_and_technology/life_sciences/cancer_diet/kulp1. jpg 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •Kidd, K.A. et al. 2007. Collapse of a fish population following exposure to a synthetic estrogen. Proceedings of the National Academy of Sciences 104(21):8897-8901 •Controls +Ethinylestradiol •5 ng/L (!) •7 years Scientific approach – EE2 (part 2/2) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Regulatory approach: risk assessment and management • • • • Hazard Exposure assessment Dose (or PEC) Effect assessment TDI (or PNEC) Risk characterisation RQ = Dose / TDI (or PEC / PNEC) •RQ < 1 RQ > 1 Risk management 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif • • • • Hazard Exposure assessment Dose (or PEC) Effect assessment TDI (or PNEC) Risk characterisation RQ = Dose / TDI (or PEC / PNEC) § EU Directive 98/83/EC (in addition to others) pesticide in drinking water DW in city of Bruno … atrazine 0.15 mg/L No pesticide in DW >0.1 mg/L •RQ < 1 RQ > 1 Risk management RQ = 0.15/0.1 = 1.5 DWTP company $$ for penalty $$ for DWTP improvement $$ lobbying to affect legislation Regulatory approach: risk assessment and management 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Summary on Who? •Toxicity assessment depends on goal –approaches, standardization, demands on quality etc •Science vs Regulatory/business reality are different worlds with specific requirements • •(Eco)Toxicological research is exciting and important: –if relevant (and if accepted by the society / polititans) the results continuously improve quality of life – •Risk assessment concept integrates is central to decision making –Integrates eco/toxicity testing (= „effect“ assessment or dose-response assessment) Výsledek obrázku pro in vitro toxicity 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif When & where the toxicity assessment is needed? Výsledek obrázku pro government icon Anytime! … depending on researcher’s budget As the law says! … what are the law(s)? 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif http://navier.engr.colostate.edu/whatische/images/ChEL02t01f01big.jpg • Industrial chemicals • • Cosmetics • PPP (pesticides) • Biocides • • Human pharmaceuticals • • Veterinary pharmaceuticals • Chemical laws („bulk“) REACH (ECHA) PPP (EFSA) MPs (EMA) §§ §§ WFD – surface w. GWD – ground w. Air quality Food and feed Soil & Sediments Wastes SOIL AIR WATER Two §§ approaches: ØProspective (chemicals…) Ø ØRetrospective (mixtures …) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif What to assess for toxicity? Výsledek obrázku pro government icon Current research topics As required by law Individual chemicals Engineered nanomaterials/particles Ecological effects (e.g. of pharmaceuticals) Endocrine disruption & chronic diseases Industry & biocides (REACH) PPPs = pesticides Pharmaceuticals Cosmetics Mixtures Multistressors +T°C, salinity, pathogens, irradiation, food Exposome Contaminated samples Can analyzed chemicals explain observed effects ? Chemical analyses & limits Effect testing rare: Remediation, dredged sediments (CZ), effluents (DE) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif What to assess for toxicity? Výsledek obrázku pro government icon Current research topics As required by law Individual chemicals Engineered nanomaterials/particles Ecological effects (e.g. of pharmaceuticals) Endocrine disruption & chronic diseases Industry & biocides (REACH) PPPs = pesticides Pharmaceuticals Cosmetics Mixtures Multistressors +T°C, salinity, pathogens, irradiation, food Exposome Contaminated samples Can analyzed chemicals explain observed effects ? Chemical analyses & limits Effect testing rare: Remediation, dredged sediments (CZ), effluents (DE) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif What to assess for toxicity? Výsledek obrázku pro government icon Current research topics As required by law Individual chemicals Engineered nanomaterials/particles Ecological effects (e.g. of pharmaceuticals) Endocrine disruption & chronic diseases Industry & biocides (REACH) PPPs = pesticides Pharmaceuticals Cosmetics Mixtures Multistressors +T°C, salinity, pathogens, irradiation, food Exposome Contaminated samples Can analyzed chemicals explain observed effects ? Chemical analyses & limits Effect testing rare: Remediation, dredged sediments (CZ), effluents (DE) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif What to assess for toxicity? Výsledek obrázku pro government icon Current research topics As required by law Individual chemicals Engineered nanomaterials/particles Ecological effects (e.g. of pharmaceuticals) Endocrine disruption & chronic diseases Industry & biocides (REACH) PPPs = pesticides Pharmaceuticals Cosmetics Mixtures Multistressors +T°C, salinity, pathogens, irradiation, food Exposome Contaminated samples Can analyzed chemicals explain observed effects ? Chemical analyses & limits Effect testing rare: Remediation, dredged sediments (CZ), effluents (DE) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Nanoparticles - examples https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcR52Y7PdqKN5Z97ZhIFGfyoY41Qv9l41niNcuE3T-F9sWy IjSKMpA http://nano.anl.gov/images/bifunctional_nanoparticle.jpg https://encrypted-tbn1.gstatic.com/images?q=tbn:ANd9GcR9LiHxdv4f1xLBnJm7j0ey82mAIkrqpx6dVbyiFdDTwcR n5G35 https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcTRyUwtGdjXOTAe8emSb0Ngkn0R-l8krCeEyFVPeK8-f5Y rg322ug https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS1YFciG79F0BpdYj74qJmGI-zgvfT7pAazIR4bDRuhmll hzbqG https://encrypted-tbn2.gstatic.com/images?q=tbn:ANd9GcQzw3I5Xb7Vq_3twyU5AzgXl6TZPl1WvVm-RGpTGUQSGNu BdlDu 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Toxicity of nanoparticles … http://ars.els-cdn.com/content/image/1-s2.0-S0165993611003748-gr1.jpg •(Mostly unknown) •Parameters may •Affect ecotoxicity • •Composition (chemical) •Surface (size, area) •Charge •Reactivity •Interactions with ions, other chemicals… • •è Effects on environmental Fate and toxicity 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Ecotoxicity of nanoparticles – RECETOX example •Comparison of toxicity - 4 „appeared to be the same“ particles (one producer – 4 different lots) •(zerovalent iron – ZVI – Fe0) • ?? Why is H16 so toxic ?? … despite of detailed investigation never revealed 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •PHARMACEUTICALS 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Example 1 - DICLOFENAC •Unexpected effects at NON-TARGET species • - nephrotoxicity at vultures • - Relevant also in EU • (ESP, EL,CY) • • • • • • • • • http://img2.allvoices.com/thumbs/image/609/480/103574038-diclofenac-drug.jpg http://www.painstopanswers.com/images/diclofenac.jpg 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Example 2 – AVERMEKTIN-like antiparasitics Ivermectin – antiparasitics in large herds nUsed 2-times per season per sheep/cow nKills 100% parasites in sheep nReleased in dung - kills 80-90% larvae of dung flies nHigh concentrations in dung (released 2 days post application) nPersistent in the soil (half-life 30 days) nCan be washed into adjacent streams (highly toxic to water insects) https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcQDHBMH2T3nCNNs0D0dL3i9DAZFsRcqXDrax6t3C410Rx3E OLYBWg • • http://www.aphotofauna.com/images/flies/fly_scathophagidae_scathophaga_stercoraria_yellow_dung_15-0 4-06.jpg •Moxidectin – used e.g. in home „spot on” products https://encrypted-tbn0.google.com/images?q=tbn:ANd9GcQYuav5FXXhPNb_O-2Bpl0rfQjaDBUqZl867IJ47-6yMdCb b36UkA •? 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •Boyles et al. (2011) Science 332 (60251) 41-42 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Stress à multigeneration effects Epigenetics à DNA methylations 2x difference Výsledek obrázku pro stickleback http://helicase.pbworks.com/f/DNAmeth.jpg 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif International ring test (2012-13) Testing comparability of existing and innovative bioassays for water quality assessment Main questions: Are current limits (for individual compounds) safe? Relevance of “Something from Nothing” phenomenon ? 3 samples à 12 European laboratories – different bioassays à ČR – RECETOX: 11 bioassays Carvalho, R. et al. (2014) Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they? Toxicol Sci 141(1): 218-233 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif EU WFD priority substances Different concentrations EQS = limit (Environmental Quality Standard) International ring test (2012-13) Testing comparability of existing and innovative bioassays for water quality assessment 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif ISO-A-3C ISPRA-A-C-6c ISPRA-A-a-1c Example: Effects of mixtures on D. rerio fish embryos Control Effects of RM 3 (i.e. safe) mixtures International ring test (2012-13) Testing comparability of existing and innovative bioassays for water quality assessment Carvalho, R. et al. (2014) Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they? Toxicol Sci 141(1): 218-233 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Example: Effects of mixtures on X. laevis frog embryos ISPRA kontrola s popisky (2).JPG ISPRA roztok C s popisky.JPG Controls International ring test (2012-13) Testing comparability of existing and innovative bioassays for water quality assessment Effects of RM 3 (i.e. safe) mixtures Carvalho, R. et al. (2014) Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they? Toxicol Sci 141(1): 218-233 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •Active sampling particles vs gaseous phase • •Reference locality – agriculture (Košetice observatory) •Region A – industrial (historically OCPs production) •Region B – combined: industry, agriculture, traffic • Novák et al. (2009) Environment International 200470718-001 63632 600-00171325 CB035867 Contaminated samples? Case study “air“ 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Chemical analyses 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Dioxin-like effects o Difference B>A o Difference B vs A – particles vs gas Výsledek obrázku pro toxicology exercise laboratory Labs on Wed + Thu 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Antiandrogenic effects o Quantitative – comparable o Clear differences in patterns … no effects on particles in „B“ (?) o 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Summary on When, Where, What •Regulatory world –Assessment of „chemicals“! – •Contaminated samples - effects rarely tested –Great value of bioassays in assessment of contaminated samples –Effects observed (!) –How to set the „limits“? • •Research issues and questions –Nanomaterials, Pharmaceuticals, EDCs –Mixtures! –Exposome – http://www.observatoiredescosmetiques.com/media/photo/1436012289.jpg http://www.theispot.com/images/source/Michael_Waraksa__Mapping_the_Exposome.jpg 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif • •Assessment of chemical hazards • •…to… • • Humans Other organisms • (TOXICOLOGY) (ECOtoxicology) • https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcTE7qm7tLqavKV1VUwQpKDwC6XUksMmHXLKvoBzqHIKaqjv q_mACQ https://encrypted-tbn2.google.com/images?q=tbn:ANd9GcRorg12adBHNbxSdT8AiWMQmO7b7vltAdBZohD_pqfDehNn re7fkg https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcQvHwZACb-TU516T7ZMDFe-QBU598rfeCvHryOWorunBunH oi4zpA https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD _trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif (Eco)Toxicology – science of „doses“ •‘What is there which is not a poison? • „Cause-effect paradigm“ •All things are poison and nothing without poison. •Solely the dose determines that a thing is not a poison. • 2 • •Paracelsus (1493 - 1541) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Toxicology – ultimate goal ? •To identify (or predict) •safe vs hazardous levels • • • 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif http://3.bp.blogspot.com/-dokK9_P3A4I/UZ38uN8AoUI/AAAAAAAAAJc/d1N8vDjsmAw/s1600/what+is+a+chemical+ reactions.jpg http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD _trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg Organism Chemical Adverse Effects Death Altered Reproduction Inhibition of Growth Tumorigenicity Skin irritation … + Traditionally – Evaluation of adverse effects using the whole organism models Hazard assessment REGULATORY FOCUS (APICAL ENDPOINTS) Výsledek obrázku pro cyp19 Výsledek obrázku pro caco2 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •Depends on legislation (… of course !) •… but current EU legislations tend to be harmonized (use similar approaches) • à example of REACH What assays and how exactly? EU%2520Flag 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •REACH Registration, Evaluation and Authorisation •of Chemicals –27-2-2001: White Paper on the Strategy for Future Chemicals Policy –23-10-2003: Commission’s proposal REACH –December 2008: Pre-registration mandatory (all chemicals in EU must be registered at ECHA •European Chemicals Agency (http://echa.europa.eu) • 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif –> 95,000,000 known chemicals (…and counting http://www.cas.org/) –100,000 substances in EINECS (i.e. commercial use) –30,000 relevant for REACH –cc 3000 HPVCs (High Production Volume Chemicals) Existing substances and REACH http://www.sigmaaldrich.com/content/dam/sigma-aldrich/safc/safc-reach-timeline.jpg 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif REACH legislation in EU Registration, Evaluation and Authorisation and Restriction of Chemicals 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif REACH: what data type must be registered? EU%2520Flag •Physico-chemical properties, e.g.: –Vapour pressure, boiling point, Kow,… • •Human toxicology, e.g.: –Acute and chronic toxicity, skin irritation, carcinogenity,… • •Environment/ Ecotoxicological information, e.g.: –Acute and/or chronic toxicity for aquatic organisms, biodegradation, … • • 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif REACH: testing EU%2520Flag •Total costs: 2,8 to 5,6 billion € (industry pays) •Testing costs (50-60% of total): 86% for Human, 14% Ecotox 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •Depends on legislation (… of course !) •… but current EU legislations tend to be harmonized (use similar approaches) • à example of REACH • •Assays must be STANDARDIZED for REACH should follow OECD Guidelines • •Other standardization agencies (also include toxicity tests) e.g. ISO, ASTM • What assays and how exactly? Organisation for Economic Co-operation and Development EU%2520Flag 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif OECD guidelines for testing of chemicals •5 main sections –Section 1: Physical Chemical Properties –Section 2: Effects on Biotic Systems (i.e. Ecotoxicity) –Section 3: Degradation and Accumulation –Section 4: Health Effects (i.e. Toxicity) –Section 5: Other Test Guidelines Organisation for Economic Co-operation and Development Organization for Economic Cooperation Development 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif OECD guidelines (examples – selection) Organisation for Economic Co-operation and Development Test No. 201: Alga, Growth Inhibition Test 11 July 2006 Test No. 221: Lemna sp. Growth Inhabition Test 11 July 2006 Test No. 202: Daphnia sp. Acute Immobilisation Test 23 Nov 2004 Test No. 211: Daphnia magna Reproduction Test 16 Oct 2008 Test No. 203: Fish, Acute Toxicity Test 17 July 1992 Test No. 204: Fish, Prolonged Toxicity Test: 14-Day Study 04 Apr 1984 Test No. 210: Fish, Early-Life Stage Toxicity Test 17 July 1992 Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages 21 Sep 1998 Test No. 215: Fish, Juvenile Growth Test 21 Jan 2000 Test No. 229: Fish Short Term Reproduction Assay 08 Sep 2009 Test No. 230: 21-day Fish Assay 08 Sep 2009 Test No. 231: Amphibian Metamorphosis Assay 08 Sep 2009 SECTION 2 - Aquatic organisms 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif OECD guidelines (examples – selection) Organisation for Economic Co-operation and Development Test No. 401: Acute Oral Toxicity Test No. 402: Acute Dermal Toxicity Test No. 403: Acute Inhalation Toxicity Test No. 404: Acute Dermal Irritation/Corrosion Test No. 405: Acute Eye Irritation/Corrosion Test No. 406: Skin Sensitisation Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents Test No. 409: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents Test No. 410: Repeated Dose Dermal Toxicity: 21/28-day Study Test No. 411: Subchronic Dermal Toxicity: 90-day Study Test No. 412: Subacute Inhalation Toxicity: 28-Day Study SECTION 4 – Human health effects 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Try it! … download and study your guideline for free! 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif http://3.bp.blogspot.com/-dokK9_P3A4I/UZ38uN8AoUI/AAAAAAAAAJc/d1N8vDjsmAw/s1600/what+is+a+chemical+ reactions.jpg http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD _trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg Organism Chemical 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) Výsledek obrázku pro government icon Hazard assessment 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif http://3.bp.blogspot.com/-dokK9_P3A4I/UZ38uN8AoUI/AAAAAAAAAJc/d1N8vDjsmAw/s1600/what+is+a+chemical+ reactions.jpg http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD _trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg Organism Chemical Adverse Effects Death Inhibition of Growth Altered Reproduction Tumor Skin irritation … + Traditionally – Evaluation of adverse effects using the whole organism models http://www.tdi.ox.ac.uk/_asset/image/tdi-barcoded-plates.jpeg/fit/300/300/FF0000 http://wallpoper.com/images/00/27/10/25/rainbows-vials_00271025.jpg + http://ehp.niehs.nih.gov/wp-content/uploads/118/4/ehp.0901392.g001.png 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 ? 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 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 https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcTAlr7MmMMxBP1Gffwd_0Gk4X2faiMuSVai0zxTF1WtiV1 DANMq https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQyQs5sVs1TzKletUOC20iLNg93Liu4X5Q_XF_I6rQWsL4 04Y83 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 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. 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif AOP = Global strategy with support from OECD, EU, USA http://www.oecd.org/chemicalsafety/testing/projects-adverse-outcome-pathways.htm 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 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 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 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 • • https://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 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://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif https://aopwiki.org/aops 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif AOP Example: MIE aromatase inhibition Environmental Toxicology and Chemistry, Vol. 30, No. 1, pp. 64–76, 2011 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Aromatase inhibition leading to reproductive dysfunction (in fish) https://aopwiki.org/wiki/index.php/Aop:25 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif AOP Example from RECETOX: Modulation of RAR/RXR à developmental toxicity in fish Jonáš et al. 2014 Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2014.06.022 Concentration-response curves of the retinoid-like activity (expressed as % of ... Activation of RAR/RXR in P19/A15 cells by atRA and cyanobacterial metabolites Comparison of phenotypes of control and zebrafish embryos exposed to ... 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 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Summary on How •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 – Organisation for Economic Co-operation and Development EU%2520Flag 2 https://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •3Rs REDUCTION REFINEMENT REPLACEMENT „Alternatives“ to toxicity testing … 3R rules 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •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 Ø3Rs are driven by EU laws, little by Member States. ØScientific agenda is not driven enough by scientists itself… ØAcademia is in general more reactive than proactive e.g. stop vivisection’s ECI 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •European Policies on 3Rs 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Use of animals in EU (2011) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •VALIDATION •MoA •Reliable •Relevant •Substance Tested • • •e.g. endocrine disrupters receptor binding 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •Validation •Prevalidation •Development •Research •Independent Review •Implementation •Regulatory Acceptance •Alternative Methods – R&D to Implementation •ESAC Industry Regulators Academia •PARERE •ESTAF •7-8 YEARS 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif • >60 3Rs Tests submitted to ECVAM since 2008 (update 01/2015) • 10 validated or ongoing validation => Prioritisation! 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif •COMPUTATIONAL •(ECO)TOXICOLOGY 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif PBPK models •PBPK (PBTK) •Physiologically based pharmacokinetic (toxicokinetic) models http://upload.wikimedia.org/wikipedia/en/thumb/2/22/WholeBody_wiki.svg/220px-WholeBody_wiki.svg.png https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcS1Xr-zyM_wH8apSCpkALlZltgIBwQ4IcylkdluXxtRsQN mocsqJw •Fragmentation of •a complex systém •to „boxes“ • •à All Processes described by arrows (mathematical equations) 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Example – computational toxicology for EDCs 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Li (2011) BMC Systems Biology •Conceptual model •EE2 – ethinylestradiol •ER, AR atd. – receptors • •VTG – vitellogenin (marker of toxicity) • •Arrows – differential equations 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Li (2011) BMC Systems Biology •Results: • •MODELLED (white) •Vs •MEASURED (grey) • •…good comparable 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif Wrap-up •Eco/Toxicology matters •Relevant especially for „chemicals“ •… but also for „mixtures“ and contaminated samples •Effect based tools in monitoring •Important results improving lives •Exciting with many open questions • •Regulatory and Science worlds are different … but are getting closer and closer •Mechanistic knowledge and utilization of „omics“ data •Development of AOPs •In vitro (alternative) models •Quantitative computational toxicology Výsledek obrázku pro sweat 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif 1212569_21823227.jpg recetox-cerna-kulate-CZ.gif logo_mu_cerne.gif logo_sci-black.gif http://globe.setac.org/images/2010/mixtox.jpg Thank you for your attention blaha@recetox.muni.cz