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Current Status of Reactivity Models in Aquatic Toxicity

Current Status of Reactivity Models in Aquatic Toxicity . Mark Cronin Liverpool John Moores University England. Aquatic Toxicology. Aquatic Toxicity Prediction. Acute toxicity Chronic toxicity Endocrine disruption. Modes / Mechanisms of Action. Non-reactive Narcosis – baseline effect

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Current Status of Reactivity Models in Aquatic Toxicity

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  1. Current Status of Reactivity Models in Aquatic Toxicity Mark Cronin Liverpool John Moores University England

  2. Aquatic Toxicology

  3. Aquatic Toxicity Prediction • Acute toxicity • Chronic toxicity • Endocrine disruption

  4. Modes / Mechanisms of Action • Non-reactive • Narcosis – baseline effect • Uncoupling • Reactive • Toxicity elevated above narcosis • Receptor Mediated

  5. Role of Mechanism of Action in Predicting Aquatic Toxicity • Mechanism based QSARs • Acute – Chronic Ratio (ACR) • Category formation Vonk et al (2009) MOA Workshop Report - ATLA

  6. Role of Reactivity in Predicting Aquatic Toxicity • 40-70% of industrial chemicals are non-reactive • Toxicity can potentially be predicted accurately • Acute-chronic ratios may be consistent • For reactive chemicals • Grouping may be of assistance

  7. Narcosis and QSARs • Narcosis is reversible; a baseline effect • There are a number of narcotic mechanisms • Well accepted and understood, if not defined at the molecular level • Consistent ACR • QSARs need to be robust models • Log P models preferred • Domain needs to be defined • Ellison et al for Tetrahymena

  8. Electrophilic Reactivity and QSARs • Mechanisms – see next slide • Acute toxicity greater than narcosis • ACR elevated • Traditionally difficult to model toxicity except within closely defined classes or mechanisms

  9. Generic Acute Fish Mortality Pathway for “Respiratory Irritation” • a direct-acting electrophile or can be abiotically or biotically transformed to an electrophile • molecular sites of action are specific nucleophiles, either thiol or amino-moieties, reactions are non-selective • molecular initiating event is covalent perturbation of proteins; • biochemical pathways affected are varied and result in general inhibition of cellular functions • cellular- and organ-level consequences are irreversible • target organ(s) or tissue(s) are the gill • key physiological response is general hypoxia • key target organ-response is sloughing of the gill epithelium • key organism response is a sharp reduction in blood oxygen level, asphyxiation, quickly leads to death Schultz (2010); McKim – FATS publications

  10. Applications of Reactivity in Predicting Aquatic Toxicity • Development of QSARs • Identification of narcotic / reactive / other mechanistic domains • Definition of reactive domains • Grouping to allow for read across

  11. QSARs Using Reactivity • Acute toxicity to Tetrahymena pyriformis of Michael Acceptors • Log (IGC50-1) = 1.05 (log RC50-1) + 1.53 • n = 20, s = 0.39, r2 = 0.975, q2 = 0.973 • F= 699, relationship covers 9 log units Information from Schultz et al

  12. Calculated Descriptors of Reactivity • LUMO • HOMO • Electrophilicity index (w) • Superdelocalisability • Atomic charges • Limited to categories or do not capture protein reactivity

  13. Is a Compound Narcotic? • If it is: • We can predict toxicity • We can extrapolate ACR • If it isn’t • More information / testing may be required • How do we determine if a compound is narcotic?

  14. Methods to Determine if a Compound is Narcotic • Mode of action assignment • Verhaar • Russom (ASTER) • OASIS • Domain definition • Excess acute toxicity • Cytotoxicity • Reactivity

  15. Narcosis: Killer Questions • Are physico-chemical properties consistent with narcosis? • Solubility, volatility • Is your compound in a narcotic domain? • Classes / analogues • Verhaar / Russom / OASIS • MOA definitions • Is the compound “unlikely” to be activated through metabolism? • Is your compound reactive?

  16. Killer Question: Is Your Compound Reactive

  17. Mechanistic Basis for Needing to Understand Reactivity • Need to understand target nucleophile • Possible covalent interactions with nucleophile • How to capture the possibility of interactions

  18. Mapping Toxicity onto the Spectrum of Soft-Hard Nucleophiles • Nucleophilic sites in amino acids primary amino-groups of lysine and arginine secondary amino-group in histidine thiol-group of cysteine S-atoms of methionine

  19. Mapping Toxicity onto the Spectrum of Soft-Hard Nucleophiles • Nucleophilic sites in amino acids primary amino-groups of lysine and arginine secondary amino-group in histidine thiol-group of cysteine S-atoms of methionine Increasing Hardness

  20. Mapping Toxicity onto the Spectrum of Soft-Hard Nucleophiles • Nucleophilic sites in amino acids Aquatic Tox primary amino-groups of lysine and arginine secondary amino-group in histidine thiol-group of cysteine S-atoms of methionine Increasing Hardness

  21. Mapping Toxicity onto the Spectrum of Soft-Hard Nucleophiles • Nucleophilic sites in amino acids Aquatic Tox primary amino-groups of lysine and arginine secondary amino-group in histidine thiol-group of cysteine S-atoms of methionine Increasing Hardness

  22. Excess Toxicity: If Seen in Vitro – Extrapolate to in Vivo:Pre-Michael Acceptors: Oxidised in the Air or Medium of the Assay 2- or 4-substituted 3-substituted

  23. Information from Other Species • Is a skin sensitiser a reactive acute toxicant in fish? • Is a non-sensitiser a narcotic? • Need to map toxicity onto electrophilic spectrum

  24. Information from Other Species • Is a skin sensitiser a reactive acute toxicant in fish? • Is a non-sensitiser a narcotic • Need to map toxicity onto electrophilic spectrum Aquatic Toxicity Skin Sensitisation primary amino-groups of lysine and arginine secondary amino-group in histidine thiol-group of cysteine S-atoms of methionine

  25. Reactive Groupings and Categories • Groups reactive (and hence non-reactive) chemicals together • Allows for (Q)SAR formation and read-across • Groupings can be formed on the basis of mechanistic knowledge • QSARs can be developed using in chemico data

  26. Decreasing electrophilicity  decreasing reactivity Increasing steric hindrance decreasing reactivity Transition state effect decreasing reactivity

  27. What About Chemicals with More Than a Single Mechanism? Michael addition Schiff base formation Aromatic nucleophilic substitution Schiff base formation

  28. Current Status • 2-D methods • Verhaar / Russom – type rules are accepted, have potential to be developed further • ECOSAR classes are accepted, relate to mechanism indirectly • Molecular Orbital • Little practical use • Reactivity Measurement • Great potential; little acceptance

  29. What is Needed In the European Union ... • Methods that work • Methods that are simple • Methods that can be justified • Methods that will be accepted by EChA, national regulatory agencies

  30. What is Needed In the European Union ... • Methods that work • Methods that are simple • Methods that can be justified • Methods that will be accepted by EChA, national regulatory agencies • ... science is less important?

  31. Non-Testing Workflow

  32. Where Does the MOA Fit Within an ITS? • To select relevant QSARs • To select relevant analogues for chemical category development or read-across purposes • To rationalise/resolve disagreements in experimental data • Chemical similarity is context dependent i.e. dependent on the relevant parameters driving the toxicity • Mechanism of Action provides the frame of reference

  33. Conceptual ITS Chemical MODE/MECHANISM (Q)SAR, TTC In vitro Existing data In vivo Exposure information Read-across/ Chemical Categories Hazard information Risk Assessment MODE/MECHANISM

  34. Fish Acute Toxicity Workflow 1 Compound • Is this a single organic compound of known structure Domain of the assay • Inside of the solubility, volatility, stability domain of the assay? Existing Data • Do satisfactory toxicity data already exist for this compound? Other QSAR Predictions • Can reliable predictions of toxicity be made with an ad hoc QSAR or an expert system such as ECOSAR, TOPKAT, MultiCASE, TerraQSAR etc.? Metabolism • Does the compound have significant and / or relevant metabolites?

  35. Fish Acute Toxicity Workflow 2 • Mechanistic Profiler

  36. Fish Acute Toxicity Workflow 3 • Category Formation

  37. Fish Acute Toxicity Workflow 3 • In vitro information

  38. Fish Acute Toxicity Workflow 4 • In chemico information

  39. Conclusions: Current Status of Reactivity in Fish Acute Toxicity • Predicting narcotic vs non-narcotic mechanisms • Rationale grouping of chemicals • Descriptors for read-across / QSARs • Technology / models are here? • Needs • Expansion of domains • Practical workflows (to enter into ITS) • Case studies • Acceptance and implementation by industry, regulatory, wider scientific community

  40. Acknowledgements • This project was sponsored by Defra through the Sustainable Arable Link Programme • European Union 6th Framework OSIRIS Integrated Project (GOCE-037017-OSIRIS) • CAESAR Specific Targeted Project (SSPI-022674-CAESAR) • European Chemicals Agency (EChA) Service Contract No. ECHA/2008/20/ECA.203 • InSilicoTox Marie Curie Project (MTKD-CT-2006-42328) www.inchemicotox.org

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