1. McKim Conference on Predictive Toxicology The Inn of Lake Superior Duluth, Minnesota September 16-18, 2008 Toxicity Pathways as an Organizing Concept Gilman Veith

2. Summary of Past Discussions Need for a Paradigm Shift – Kees, Bradbury Need to Integrate Toxicology – Veith Soft Reactive Toxicity – Schultz, Comber Need for Organizing Chemistry – Mekenyan ReproTox Pathways – Schmieder, Ankley … NeuroTox Pathways – Fowle, Bushnell,

3. Purpose of McKim 2008 Status on the new mindset in Europe Progress on skin sensitisation models Progress on the QSAR Application Toolbox Explore organizing methods for targets Expand the delineation of toxicity pathways

4. QSAR QSAR is the study of how to organize experiments of chemical behavior to find order Order is found when similarity is well-grounded in chemistry…”small” changes are often huge. The hypothesis-driven paradigm will require integration of QSAR with more diagnostic assays

5. Why Build QSAR Methods? Example: estimated ER Binding Affinity for 6,518 High Production Volume Chemicals RBA

6. Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical Our Conceptual Framework

7. Molecular Initiating Events Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical Our Conceptual Framework 1. Identify Plausible Molecular Initiating Events 2. Design Database for Abiotic Binding Affinity/Rates 3. Develop QSARs to Predict Initiating Event from Structure 4. Quantify Response Pathways to Downstream Effects QSAR ResponsePathways Chemistry/Biochemistry

8. Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical Conceptual Framework Mortality -systemic toxicity -disease -cancer Impaired Development -terata -prenatal deficits Reproductive Fitness -fertility -viable offspring Chemical Inventories and Categories (~200,000) Interaction Mechanisms -Nonspecific Targets -Atom Centers Targets -Receptor Targets

9. Major Pathway for Reactive Toxicants To Fish Michael Addition Schiff base Formation S N 2 Acylation Atom Centered Irreversible (Covalent) Protein Binding Interaction Mechanisms Molecular Initiating Events “Any Exposed Surface” Changes Necrosis of the Gill Epithelium In vivo Endpoints Pathogenesis Vulnerable Organ Pathology Death from Suffocation Complexes Membranes, etc

10. Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical At the Molecular Initiating Event The QSAR Question is: “How many other chemicals can interact at this target?” While the Toxicology Question is: “What are the known biological effects from this altered target?”

11. Library Of Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical From the Library of Initiating Events OECD Toolbox ChemicalProfiler Handles the Chemistry for QSAR Models Targets Interactions Structural Requirements Conformations Metabolic Simulators Inventories

12. Simulated 2-Acetylaminofluorene Metabolism Which Metabolite should we use in modeling interactions?

13. Which Conformation should we use to model interactions?

14. Library Of Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical From the Library of Initiating Events OECD Toolbox Libraries Will Link Targets to Adverse Effects ER Binding Gene Activation Protein Production Altered Gonad Development Impaired Reproduction

15. Pathways for Reactive Toxicity from Soft Electrophiles Systemic Responses Skin Liver Lung Michael Addition Schiff base Formation S N 2 Acylation Atom Centered Irreversible (Covalent) Protein Binding Immunogenic Mechanisms Molecular Initiating Events Exposed Surface Irritation Systemic Immune Responses Necrosis Skin Lung/Gills GI Tract In vivo Endpoints Yes No

16. Major Pathways for Reactive Toxicity from Moderate Electrophiles Systemic Responses Skin Liver Lung Michael Addition Schiff base Formation S N 2 Acylation Atom Centered Irreversible (Covalent) Binding Interaction Mechanisms Molecular Initiating Events Exposed Surface Irritation Systemic Immune Responses Necrosis Which Tissues? In vivo Endpoints Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Oxidative Stress Dose-Dependent Effects

17. Chemical Activity (or comparable metric) Observable Response Level NOAEL—Long Term Nonspecific Reactive Nonpolar Narcosis 1.0 0.10.01 Receptor Off-Target Therapeutic Effects

18. Pathways for Reactive Toxicity Michael Addition Schiff base Formation S N 2 Acylation Atom Centered Irreversible (Covalent) Binding Interaction Mechanisms Molecular Initiating Events Membrane Alteration _ Oxidative Stress _ Genotoxicity Death Impaired Growth Impaired Development Impaired Reproduction In vivo Endpoints Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Dose-Dependent Pathways Species/Sex/Life-Stage In vitro Endpoints

19. Two Questions for Building Pathways Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Effect #1 Effect #2 Effect #3 Direct Reaction Altered Synthesis Oxidation How Many Ways to Deplete GSH?How Many Downstream Effects?

20. Delineation of Toxicity Pathways Linkages Across Levels of Biological Organization Chemical Reactivity Profiles Reversible Nonspecific Binding Reversible Specific Binding Covalent Binding Lethality Growth Development Reproduction Molecular/ Subcellular CellOrganIndividual In Silico Methods In vitro Methods In vivo Methods Electronic Molecular Initiating Events Membranes Energy Charge Nuclear Receptors Protein Synthesis DNA Integrity Chemical Inventories Response Pathways Regulatory Endpoints Exposure/ Metabolism Penetration Routes Detoxification Pathways Activation Pathways More Relevant Endpoints Better Defined Endpoints Intrinsic Chemical Attributes Tissue