1. Perspective on the current state-of-knowledge of mode of action as it relates to the dose response assessment of cancer and noncancer toxicity Jennifer Seed, PhD Office of Pollution Prevention & Toxics US EPA This presentation represents the view of presenter & does not necessarily represent the decisions or stated policies of the EPA.

2. 2 Evolution of Risk Assessment RA/RM Paradigm Guidelines/Methods Dosimetry/PbPK RA/RM Paradigm Guidelines/Methods Dosimetry/PbPK 1980s Mode of Action Susceptible Populations Mixtures Mode of Action Susceptible Populations Mixtures Toxicity Pathways Integrated Approaches CompTox Toxicity Pathways Integrated Approaches CompTox 1990s 2000s 21 st Century 1983 1994 2007 2009

3. 3 Characterize Uncertainty Promote Harmonized approach for all endpoints Mode of Action Toxicity Pathways Identify Key Events Understand Human Relevance Susceptibility Dose Extrapolation Mode of Action information is core to the risk assessment paradigm and its evolution into the 21 st century Inform Multiple Chemical Risks

4. 4 How do you determine the Weight of Evidence for establishing MoA? Needs to be based around specific hypothesis judged against data International Framework Introduced by USEPA & IPCS (1999, 2001) ILSI builds on EPA/IPCS work (2003, 2005) IPCS continues work (2006, 2008) Based on Bradford Hill Criteria for Causality Distinguishes MoA vs Mechanism Human Relevance (Concordance) Analysis

5. 5 Mode of Action/Human Relevance (MoA/HR) Framework Why is it important? –Increases predictive capacity and utility of risk assessment –Provides rigor and structure to analysis and consistency of documentation – Provides transparency –Guides and focuses research/testing 5

6. Figure 5.8 New unified process for selecting approach and methods for dose-response assessment for cancer and noncancer end points.

7. 7 Mode of Action Human Relevance (MoA/HR) Framework (v.IPCS) Compare “Key Events” & relevant biology between animals & humans Dose Response & Species Extrapolations Exposure Assessment

8. 8 Question 1: Sufficient Weight of Evidence to establish MoA in animals? Postulated MoA (hypothesis) –Describe sequence of Key Events (measurable) –Consider other possible MoAs Experimental support for key events –Concordance of dose-response relationships –Temporal association –Strength, consistency and specificity of association of toxicological effect with key events –Biological plausibility and coherence Uncertainties, inconsistencies, missing data

9. 9 Cacodylic Acid: Sequence of “Key Events” in MOA DMA III Metabolite Hyperplasia Urothelial Toxicity Regenerative Proliferation Urinary Bladder Tumors Sustained Urinary bladder from a female F344 rat treated with 100 ppm DMA V BrdU Labeling Measurable Key Events in Target Tissue Other MoAs: DNA damage via ROS?

10. 10 Q1: WoE Sufficient to Establish MoA? Postulated mode of action –Sequence of key events Experimental support –Relationship of key events & adverse effects –Dose-response –Temporal response Biological plausibility & coherence Strength, consistency & specificity Identify uncertainties Dose (mg/kg bw/day) Metabolism DMA V  DMA III Urothelial Toxicity Regenerative Proliferation Urothelial Hyperplasia Transitional Cell Carcinoma 0.2 (2 ppm) + (wk 3-0.03 ± 0.01 uM) + (wk 10-6/10, grade 3 or 4) --- 1 (10 ppm) + (wk 3-0.12 ± 0.02 uM) + (wk 3-2/7, grade 3) (wk- 10; 8/10, grade 3 or 4) slight (wk 10-1.5X inc) -- 4 (40 ppm) + (wk 3-0.28 ± 0.09 uM) + (wk 3-7/7, grade 3) (wk 10-5/10, grade 3 or 4) + (wk 10-4.3X inc) + (wk 10- 4/10) - 9.4 (100 ppm) + (wk 3-0.55 ± 0.15 uM) + (6 hrs-6/7, grade 3) (24 hrs-4/7, grade 3 or 4) (wk 2 6/10, grade 5)(wk 10-0/10, grade 4 or 5) + (wk 1- 2.2X inc) (wk 2- 3.9X inc) (wk 10-4.2X inc) + (wk 8-7/10) (wk 10-9/10) + (papilloma first obs at wk 107; carcinoma first obs at wk 87) Temporal Dose Response 6 hrs 1 wk 8 wk 104 wk

11. 11 Q1. Weight of evidence sufficient to establish MoA in animals? Q2. Fundamental qualitative differences in key events? Q3. Fundamental quantitative differences in key events? 11 Q.2 & 3 Evaluation of Human Relevance Compare “Key Events” & relevant biology between animals & humans * Use all existing knowledge: chemical specific & generic (e.g., genetic/disease models, related cpds)

12. 12 Q. 2 & 3 The Concordance Analysis Key Event Qualitative Concordance Animals Humans Strength Quantitative Concordance Humans Presence of DMA III in urine Yes, Data DMA III present following exposure to iAs; Direct exposure to DMA V ? Considerable In animals; but limited data in humans PbPK Model--based on use of DMA III dosimetry at the target tissue because it represents the rate-limiting event leading to proliferation Sustained urothelial cell damage and regenerative proliferation Yes, Data Unknown: Potential if sufficient DMA III and cytotoxicity is produced Considerable in animals, possible in humans but no data No data Bladder tumours Yes, Data PossibleConsiderable in animals; plausible in humans Limited evidence indicates significantly less DMA III produced 12

13. 13 Q1. Weight of evidence sufficient to establish MoA in animals? Q2. Fundamental qualitative differences in key events? Q3. Fundamental quantitative differences in key events? 13 Carrying MoA information forward into the dose-response analysis Dose extrapolation based on MoA understanding Key events - shape of the dose-response curve & points of departure MoA-Based vs Default

14. 14 Urothelial cytotoxicity (3 wk; 10 wk) Proliferation (10 wks) Hyperplasia (10 wks) Tumors (104 wks) Point of Departure for Cacodylic Acid: Benchmark dose modeling using key events 0.7; 0.02 0.9 2.0 7.7 0.2; 0.0008 0.4 1.5 6.0 BMD 10 (mg/kg/day) BMDL 10 (mg/kg/day) Key Events. PoD that provides a level of exposure that will not trigger regenerative proliferation

15. 15 Carrying MoA information forward - Implications for inter- and intra- species extrapolation Interspecies Kinetics (4) Human Variability in Disposition (3) Human Variability in Sensitivity (3) Interspecies Dynamics (2.5) Default = 10X Data Derived Extrapolation Factors

16. 16 IPCS Harmonization Guidance Relevant to MoA-Based Risk Assessment Conceptual Framework for Evaluating a Mode of Action for Chemical Carcinogenesis Human Relevance Framework for Cancer Non-cancer Human Relevance Framework Principles of Characterizing and Applying PBPK Models in Risk Assessment Chemical Specific Adjustment Factors Combined Exposures to Multiple Chemicals information can be found at: http://www.who.int/ipcs/methods/harmonization/en/index.htmlhttp://www.who.int/ipcs/methods/harmonization/en/index.html

17. 17 2007 NRC Toxicity Testing in the 21 st Century Compounds Metabolite(s) Assess Biological Perturbation Affected Pathway Measures of dose in vitro Dose Response Analysis for Perturbations of Toxicity Pathways Calibrating in vitro and human Dosimetry Human Exposure Data Population Based Studies Exposure Guideline Mode of Action Chemical Characterization Dose Response Assessment Hazard Characterization Risk Characterization Exposure Assessment Fig 3-7 Risk Assessment Components