Exploration of alternative methods for toxicity assessment of pesticide metabolites Alternative in vitro methods to characterize the role of endocrine active substances (EAS) in hormone-targeted tissues, Istituto Superiore di Sanita` , Rome, 17 th December 2012
Background Regulation (EC) No 1107/2009 concerning the placing of PPPs on the market requires: identification of metabolites • determination of their “toxicological relevance” • for in/exclusion in the residue definition
Background OECD G uidance Document on the Definition of Residue Proposes residue definition for risk assessment, reflecting • the actual toxicological burden consisting of active substance and relevant metabolites Requires quantitative and qualitative assessment of • metabolites
Pesticide metabolites as residues • In contrast to active substances for which a full toxicological dossier needs to be provided usually very limited or no toxicological data are available although their toxic potency and toxicological profile might differ substantially from the parent compound • Reference values of parent compounds are applied for metabolites present in food commodities
Assessment tools Conventional testing of metabolites? • Number of metabolites • Difficulties in synthesising sufficient amounts • Costs • Research capacities • Animal welfare concerns
Assessment tools Preparatory Work for the Toxicology Part of the Opinion • Project: Impact of Metabolism on Toxicity (AGES, Austria) • Project on Threshold of Toxicological Concern (TTC) Concept (CRD, UK) • 2 projects on (Quantitative) Structure-Activity Relationships (Q)SARs (JRC, EC)
Assessment tools – Impact of Metabolism Impact of metabolic and degradation processes on the toxicological properties of residues of pesticides in food commodities (published 6 May 2010) http://www.efsa.europa.eu/en/supporting/pub/49e.htm
Assessment tools – Impact of Metabolism Review of transformation pathways (11 pesticide classes) and comparison • with toxicity data (DARs, public literature) Based on lack of other information analyses largely based on acute tox • studies In total more than 140 chemical changes identified and analysed • Conclusions: Toxification/detoxification cannot be attributed to certain metabolic steps • (Conjugates not necessary less toxic due to cleavage) Metabolite > 10% to be considered as contributing significantly to • toxicity Improvement of ADME studies (more accuracy, more information) • necessary
Assessment tools - TTC Applicability of thresholds of toxicological concern in the dietary risk assessment of metabolites, degradation and reaction products of pesticides (published 24 March 2010) http://www.efsa.europa.eu/en/supporting/pub/44e.htm
Assessment tools – TTC Threshold of Toxicological Concern (TTC) Concept • Level of exposure below which no appreciable risk is expected • Based on comparison of chemical structures and chronic data (CPDB data base - Gold et al. 1984, 1989) So far only applicable for chronic exposures • • Already in use for assessment of food flavourings
Assessment tools - TTC Threshold of Toxicological Concern (TTC) Concept Classification TTC TTC Structural features threshold threshold (µg/person/d) (µg/kg bw/d) Cramer class I 1800 30 Simple structure + Metabolism Cramer class II 540 9 Less innocuous than class I Cramer Class III 90 1.5 Suggestive of significant toxicity – Functional groups Cramer class III 18 0.3 Parent compound neurotoxic – + Neurotoxicity OP + Carbamate structure Cramer class III 0.15 0.0025 Parent compound genotoxic + Genotoxicity Identification with QSAR
Assessment tools - TTC • Review on the TTC concept and current use • Validation of TTC for pesticides: Comparison of 100 pesticide ADIs with TTC classification • Case study with 79 metabolites of 15 selected pesticides Conclusions: • TTC appropriate for assessment of metabolites • Exposure to metabolites considered to be covered by TTC thresholds allocated – critical is the genotoxicity threshold • QSARs predictions for genotoxicity considered reliable
Assessment tools – QSAR I Applicability of QSAR analysis to the evaluation of the toxicological relevance of metabolites and degradates of pesticide active substances for dietary risk assessment (published 7 May 2010) http://www.efsa.europa.eu/en/supporting/pub/50e.htm
Assessment tools – QSAR I • Review on the use of QSAR for regulatory purposes • Extensive evaluation of potentially useful QSARs (e.g. Toxtree, Lazar, Derek, Hazard Expert , Caesar, Topkat) using different data sets (Pesticide metabolites, EU classified substances, CPDB) • Case studies focused on genotoxicity based on the outcome of the TTC project Conclusions: • Currently limited use of QSARs for regulatory purposes • Recommendations for the application of different models covering all relevant toxicological endpoints - i.e. combination of models recommended to optimise sensitivity and specificity in regard to genotoxicity
Assessment tools – QSAR II Applicability of QSAR analysis in the evaluation of developmental and neurotoxicity effects for the assessment of the toxicological relevance of metabolites and degradates of pesticide active substances for dietary risk assessment (published 16 June 2011) http://www.efsa.europa.eu/en/supporting/pub/169e.htm
Assessment tools – QSAR II Evaluation of QSAR models (Derek, Caesar, Topkat, • Leadscope, Hazard Expert, PASS, ADME predictor, Accord) and read across (OECD toolbox) for assessment of developmental effects and neurotoxicity Conclusions: No appropriate QSAR models for neurotoxicity identified • Stepwise approach for developmental effects combining • read across and QSAR might be promising
Extension of the mandate including assessment of isomers (September 2011) In order to: • investigate if the approaches proposed for assessment of metabolites are applicable for isomers • identify specific issues, suitable methodologies, research needs for isomers
Scientific Opinion on Evaluation of the Toxicological Relevance of Pesticide Metabolites for Dietary Risk Assessment (published 26 July 2012) Available from: http://www.efsa.europa.eu/en/efsajournal/p ub/2799.htm
Scientific Opinion – Toxicology Part Chapters: • Presentation and evaluation of projects on Impact of Metabolism, TTC and QSARs • Development of a single TTC value for acute exposures (based on short term NOAELs) • Proposal for toxicity assessment and for acute and chronic exposures (decision trees) • Evaluation of the assessment tools for isomer assessment • Critical Issues/Uncertainties
Is an ARfD assigned to parent substance NO NO Has the parent compound a QSAR analysis for YES developmental toxicity neurotoxic mechanism of action See chapter 7.2 YES Acute exposure estimate of metabolite See chapter 8 Compounds predicted Compounds predicted No positive negative/inconclusive further testing No or Exposure > 0.3 µg/kg bw/d See chapter 5.3.1 ** The YES metabol Read-across OP,carbamate Compounds predicted ite is See chapter 6.3 positive /inconclusive toxicophore not See chapter 5 YES relevant No Compounds predicted Is the metabolite in Cramer negative Class II or III? Non TTC approach No YES No see chronic Exposure > 5 µg/kg bw/d (modified acute value) No acute See chapter 5.3.1 RA Is the metabolite in Cramer necessary Class I? YES No Exposure > 30 µg/kg bw/d YES (modified acute value) See chapter 5.3.1
Conclusions/Recommendations Toxification/detoxification not attributable to specific metabolic steps • Use of PBPK models recommended for assessment of ADME • TTC concept is an appropriate and sufficiently conservative evaluation • tool for metabolites , but should not be used for parent compounds For acute exposures an acute TTC threshold of 5 µg/kg bw/d can be • applied Where exposure exceeds TTC values, targeted testing can be applied • TTC can in principle used for evaluation of endocrine active substances • (except steroids) but should preferably be based on testing if data already indicate that a compound has endocrine mediated adverse effects
Conclusions/Recommendations • Once an EU agreed approach for defining and assessing of endocrine active substances is available, the use of the TTC approach for EAS needs to be revisited In principle the TTC concept and QSARs could be used for assessment of • stereoisomers present in mixtures, however, in practice information on stereochemistry is not provided in the data sets underlying these tools QSARs should not be used as a stand alone tool for assessments but can • together with the application of read-across complement the TTC approach • The usability of QSARs for the assessment of endocrine active active substance has not been investigated based on lack of a definition and appropriate data bases A guidance should be developed based on the scientific opinion •
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