EPAA 3Rs Science Award 2012

Transcription

1 EPAA 3Rs Science Award 2012 Systematic approach to investigate outliers of the fish embryo test to increase its predictive capacity and applicability domain for acute fish toxicity and beyond Nils Klüver, PhD 8th EPAA ANNUAL CONFERENCE Brussels, EPAA industrial partner:

2 Purpose and aim of the study improve the fish embryo toxicity (FET) test to foster international acceptance as alternative for the fish acute toxicity test Replacement Systematic Identification of FET outliers Reduce uncertainties Define or extend the applicability domain Improve predictive capacity Page 2

3 Usage of fish for toxicity and safety evaluation ~ fish per annum in the EU (2008) most frequently used vertebrates in regulatory ecotoxicology REACH (>10t/a) Plant Protection Products Biocides Veterinary Medicinal Products Effluent Testing Amount of fish test needed within REACH is expected to increase (~1.2 million fish per year) On a global level, about fish per year are used in the USA for effluent testing Page 3

4 Acute Fish Toxicity Test OECD TG 203 most widely used vertebrate toxicity test in environmental risk assessment! 7-10 fish per aquarium control increasing concentrations ( 5x) 96 h screen for mortality takes 42 animals and overall 5 working days Alternatives? Page 4

5 Fish embryos as alternatives? fish embryos up to the stage of independent feeding are not considered as protected stages (DIRECTIVE 2010/63/EU) similar as in-vitro methods and can be considered as alternative test system Fish embryos are already widely used in screening approaches FET has emerged as a potential replacement method for acute fish toxicity testing Page 5

6 The fish embryo toxicity (FET) test 24 h 48 h 72 h 96 h modified after Lammer et al., 2009 FET is easy to perform and has a low cost! Page 6

7 Zebrafish embryo toxicity test (ZFET) is a promising alternative for acute fish test Various studies have indicated a high correlation of fish embryo and acute fish toxicity similar sensitivity The FET is a mandatory component in routine whole effluent testing in Germany since 2005 OECD draft guideline (2006) ZFET validation study coordinated by ECVAM (Phase 2 completed) transferability and very good intraand inter-laboratory reproducibility Page 7 Limitations? Lammer et al., 2009

8 ZFET outliers compounds with higher LC 50 in fish embryos compounds that do not provoke any embryo toxicity Page 8

9 Reasons for ZFET outliers Physico-chemical properties (e.g. lipophilicity, volatilisation, water solubility, molecular size ) Toxicokinetic (uptake, metabolism) Toxicodynamic (mode of action) Page 9

10 Project outline ZFET Database update Data mining for acute fish toxicity Outlier identification / understanding Implementation and communication Embryo toxicity data for >590 compounds is already available ( UFZ collaboration) Page 10

11 Outlier understanding ZFET outlier testing Including potential modifications of exposure scenarios (e.g. closed glass vials, exchange of media, shaking, window, duration) Chemical analytics and Metabolisation Determination of exposure concentration (24h intervals) Optional: Internal concentrations (time to reach equilibrium) Metabolic activation (include potential metabolites) MoA related endpoints Neurotoxicity (behaviour) heart beat measurements Others? Kühnert et al. submitted Page 11

12 3Rs beyond acute fish toxicity testing Page 12 FET has a chance to be accepted in order to replace the acute fish toxicity test big impact on the use of fish within REACH! Fish early life stage (FELS) OECD TG 210 Bioaccumulation in fish OECD TG 305 Endocrine disruption Developmental Toxicity (human risk) Acute systemic toxicity (human risk) Impact for various industrial sectors including chemical, cosmetic and pharmaceutical industry

13 Page 13 Thank you for your attention!

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15 Summary A ZEFT assay OECD draft guideline is available OECD validation was established similar sensitivities as acute fish toxicity test ZFET database Correlation analysis Priority list of outliers Adapting and improving the test system ZFET decision tree Suggestion to amend draft test guideline Page 15

16 Advantage of zebrafish embryos complexity of all physiological and morphological interactions (closer to adult compared to cell lines) Small size, high fecundity, optical transparent, easy to maintain, low costs one cell stage 24 hpf 48 hpf Page hpf

17 Generation of ZFET database and outlier identification ZFET Database update Embryo toxicity data for 590 compounds is already available (L Oréal UFZ collaboration) include data compilation of S. Belanger (P&G) and colleagues Page 17

18 Generation of ZFET database and outlier identification Acute fish toxicity data search in publically available databases (Duluth database, US- EPA ECOTOX, ECHA,EFSA, echemportal) Page 18

19 Generation of ZFET database and outlier identification Outlier identification Correlation and statistical analysis Definition of an outlier: arbitrary 10-fold difference in zebrafish embryo vs. acute fish toxicity and statistical analysis Priority list for outlier characterisation Page 19

20 Database and outlier identification Correlation analysis Critical evaluation of original data for outliers List of ZFET outliers Hypothesis generation based on existing knowledge for outliers (e.g. toxicokinetic, MoA) Priority list for outlier analysis (reflecting potential main reasons) Page 20

21 Implementation and Communication Defining applicability domain and suggestions to amend test guideline Communication of results to national members of the ZFET validation management group, ECETOC, ILSI HESI EUROECOTOX consortium Animal Alternatives in Environmental Risk Assessment Project Committee Page 21

22 Collaboration with L Oréal Dr. Marc Léonard Hypothesis for outliers Priority list of outliers Chemical analytics Discussion of results Feasibility ZFET decision tree Page 22