1. QSAR in CANCER ASSESSMENT PURPOSE and AGENDA Gilman Veith Duluth MN May 19-21, 2010

2. McKim Workshop Goals  Summarize regulatory contexts for hazard identification and risk estimation of carcinogenesis  Review progress on alternative methods for estimating endpoints needed in cancer assessment  Discuss the need for a mechanistic framework for improving the transparency of alternative models  Identify examples of chemical categories for which alternatives reliably predict outcomes of rodent assays

3. REGULATORY CONTEXTS Precautionary Perspective “What additional other test results would I want to see before I am willing to conclude a chemical is safe?” Holistic Risk Perspective “How can I identify greatest carcinogenic risks among all chemicals regardless of their testing status?”

4. HAZARD CLASSIFICATION CARCINOGENICITY IARCGHSEPAACGIHDescription/Status of Available Data 11A1A1 Human carcinogen, definite, human studies 2A1B2A2 Probably human, animal data multiple species, no epi data 2B23A3 Possibly human, suspected, some animal data 34A4 Not classifiable, inadequate data to assess 45A5 Probably not human, not suspected, no tumors in at least two species

5. HAZARD CLASSIFICATION CARCINOGENICITY  Carcinogenic to Humans (epi data required)  Likely to be Carcinogenic to Humans (tumors in two species)  Suggestive Evidence of Carcinogenic Potential  Inadequate Information to Assess Carcinogenic Potential  Not Likely to be Carcinogenic to Humans (no tumors in two species)

6. DATA DRIVEN HAZARD ASSESSMENT CARCINOGENIC POTENTIAL

7. New Product Development Testing/ Assessment Requirements Number Of Chemicals Safety Assessment Process

8. INITIAL HAZARD ASSESSMENTS  Screening Information Datasets – SIDS  Globally Harmonised System of C&L – GHS  REACH –Dossier Testing Plan Reviews  TSCA –PMNs (predictive hazard identification)

9.  QSAR in chemical engineering has always been used to focus attention/resources on specific activities  QSAR orders chemicals according to intrinsic attributes and generates initial hypotheses for more strategic use of testing and assessment resources  Structural alerts are a convenient and transparent approach to identifying hazard-specific priorities STREAMLINING DATA REQUIREMENTS

10. Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical Conceptual Framework Schultz et al. 2006. SAR QSAR in Environ.Res. 17(4) 1-16.

11. Molecular Initiating Events Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical Conceptual Framework QSAR ResponsePathways Chemistry/Biochemistry

12. 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 Center Targets -Receptor Targets

13. 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 Assessment Question is: “What are the known biological effects from this altered target…. organelles, cells, organs, species ”

14. Library Of Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical From the Library of Initiating Events ChemicalProfilers Available in A Variety of Commercial & Public Software Targets Interactions Structural Requirements Conformations Metabolic Simulators Inventories

15. Library Of Molecular Initiating Events Chemical Speciation and Metabolism Measurable Biological Effects Adverse Outcomes Parent Chemical From the Library of Initiating Events DNA Binding Altered Genes/Proteins Genetic Instability/ Cell Immortality Acquisition of Tumorigenicity Premature Death

16. GROUPING BY CANCER PATHWAYS  Genotoxic Carcinogenesis  Direct DNA damage through abiotic chemical “binding”  Most electrophiles bind to many DNA/protein sites  Metabolic differences impact cell, organ, species sensitivity  Epigenetic Carcinogenesis  Cytoxicity-induced cell proliferation  Receptor-mediated pathways  Disturbance of homeostatic control  Loss of immune surveillance  Oxidative Stress- Indirect DNA damage  Loss of intercellular communication

17. SCREENING LEVEL HAZARD ID Direct DNA Binding Nongenotoxic Mechanisms Activated Metabolites Indirect DNA Damage Parent Chemical Individual Initiating Events/ Structural Alerts Grouping Data for Interaction Categories Structural Evidence of Cancer Potential Category with Data for Cancer Potential No Evidence Of Cancer Potential

18. Simulated 2-Acetylaminofluorene Metabolism Models for the Metabolism Gap

19. PATHWAYS IN REACTIVE CHEMICALS Michael Addition Schiff base Formation S N 2 Acylation Michael Addition Schiff base Formation S N 2 Acylation Atom Centered Irreversible (Covalent) Binding Atom Centered Irreversible (Covalent) Binding Interaction Mechanisms Molecular Initiating Events In vivo Endpoints Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts In vitro Endpoints Death Impaired Growth Impaired Development Impaired Reproduction Cancer Membrane Alteration _ Oxidative Stress _ Genotoxicity

20. OXIDATIVE STRESS from GSH DEPLETION Pr-S Adducts GSH Oxidation GSH Depletion NH2 Adducts RN Adducts DNA Adducts Oxidative Stress Cell toxicity Other Effects Direct GSH Reactions Altered Synthesis Oxidation How Many Ways to Deplete GSH?How Many Downstream Effects?

21. Mode of Action Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY

22. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY DNA Mechanism #1 DNA Mechanism #2 DNA Mechanism #3 DNA Mechanism #n …………………………… Prot Mechanism #1 Prot Mechanism #2 Prot Mechanism #3 …………………………… Prot Mechanism #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/AlertMode of action

23. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY Mechanism/Alert DNA #1 DNA #2 DNA #3 DNA #n …………. Prot #1 Prot #2 Prot #3 ………….. Prot #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/Metabolites DNA Mechanism #1-Met 1…m DNA Mechanism #2-Met 1…m DNA Mechanism #3-Met 1…m …………………………… DNA Mechanism #3-Met 1…m Prot Mechanism #1 - Met 1..m Prot Mechanism #2 - Met 1..m Prot Mechanism #3 - Met 1..m …………………………… Prot Mechanism #n - Met 1..m Receptor-mediated pathways -Met 1…m Disturbance of homeostatic control -Met 1…m Loss of immune surveillance-Met 1…m Oxidative Stress- Indirect DNA damage -Met 1…m Loss of intercellular comm.-Met 1…m Cytotoxicity-induced cell proliferation-Met 1…m Mode of action

24. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY Mechanism/Alert DNA #1 DNA #2 DNA #3 DNA #n …………. Prot #1 Prot #2 Prot #3 ………….. Prot #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/Metabolites Receptor-mediated pathways -Met 1…m Disturbance of homeostatic control -Met 1…m Loss of immune surveillance-Met 1…m Oxidative Stress- Indirect DNA damage -Met 1…m Loss of intercellular comm.-Met 1…m Cytotoxicity-induced cell proliferation-Met 1…m DNA #1-Met 1…m DNA #2-Met 1…m …………………………… DNA #3-Met 1…m Prot #1 - Met 1..m Prot #2 - Met 1..m Prot #3 - Met 1..m …………………………… Prot #n - Met 1..m DNA #3-Met 1…m In vitro data AmesAmes+S9CA CA+S9 MLAMLA+S9CTA Mode of action

25. Receptor-mediated pathways Disturbance of homeostaticcontrol Loss of immune surveillance Oxidative Stress- Indirect DNA damage Loss of intercellular communication Cytotoxicity-induced cell proliferation GENOTOXICITY Direct DNA Damage Indirect DNA Damage NON-GENOTOXICITY Mechanism/Alert DNA #1 DNA #2 DNA #3 DNA #n …………. Prot #1 Prot #2 Prot #3 ………….. Prot #n AR Binding ER Binding Aromatase Inh. Thyroid Disturbance Mechanism/Metabolites Receptor-mediated pathways -Met 1…m Disturbance of homeostatic control -Met 1…m Loss of immune surveillance-Met 1…m Oxidative Stress- Indirect DNA damage -Met 1…m Loss of intercellular comm.-Met 1…m Cytotoxicity-induced cell proliferation-Met 1…m DNA #1-Met 1…m DNA #2-Met 1…m …………………………… DNA #3-Met 1…m Prot #1 - Met 1..m Prot #2 - Met 1..m Prot #3 - Met 1..m …………………………… Prot #n - Met 1..m DNA #3-Met 1…m In vitro data Ames Ames+S9 CA CA+S9 MLA MLA+S9 CTA In vivo data COMETUDSCAMN(BN)CTARCA Mode of action