DESIGN AND DOSE SELECTION FOR CHRONIC RODENT STUDIES SOT RASS TELECON April 9, 2008

1. DESIGN AND DOSE SELECTION FOR CHRONIC RODENT STUDIESSOT RASS TELECONApril 9, 2008 Lorenz Rhomberg Gradient Corporation Dale Hattis Clark University Stephen Olin ILSI Research Foundation

2. Lorenz Rhomberg Gradient Corporation Karl Baetcke U.S. EPA/OPP Jerry Blancato U.S. EPA/ORD/NERL James Bus Dow Chemical Company Samuel Cohen U. of Nebraska Medical Center Rory Conolly U.S. EPA/ORD/NCCT Rakesh Dixit MedImmune, Inc. John Doe Syngenta CTL, UK Karen Ekelman U.S. FDA/CVM Penny Fenner-Crisp ILSI RF/RSI Paul Harvey NICNAS, Australia Dale Hattis Clark University Abigail Jacobs U.S. FDA/CDER David Jacobson-Kram U.S. FDA/CDER Tom Lewandowski Gradient Corporation Robert Liteplo Health Canada Olavi Pelkonen U. of Oulu, Finland Jerry Rice Georgetown University Diana Somers PMRA, Canada Angelo Turturro U.S. FDA/NCTR Webster West U. of South Carolina Stephen Olin ILSI RF/RSI Authors

3. Process • Convened working group: • International • Multi-disciplinary • Government, industry, academia • Building on ILSI 1997 “Principles for the Selection of Doses in Chronic Rodent Bioassays”, working group: • Examined issues that come into play in putting those principles into practice • Offered perspectives and insights in the context of increasing demands on the bioassay and the growing importance of mechanism/mode of action in the assessment of human health risks

4. Scope and Focus • Not a new testing paradigm, but an effort to facilitate continuing improvement in quality, consistency and utility of bioassay data • Recognizes the ‘yin and yang’ of design and interpretation • Broadly focused on dose selection but not just MTD • More weight on cancer bioassay but also applicable to chronic toxicity studies

5. Other Information • Peer consultation (2005) informed development of final document • OECD draft guidance on dose selection based on ILSI 1997 and 2007 • Financial support provided by EPA/OPP and Health Canada ********************************************************** • Citation: “Issues in the Design and Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: Approaches to Dose Selection” (2007) Critical Reviews in Toxicology, 37(9): 729-837.

6. Dose Selection Questions: • Top Dose? • Number of Dose Levels? • Placement of Dose Levels? • Numbers of Animals per Dose Level?

7. Cancer vs. Noncancer Endpoints Different dose-selection criteria • Cancer: • Power to Detect • Stochastic Endpoints • Noncancer: • Severity Spectrum • NOAEL / BMD Identification

8. Prospective vs. Retrospective? RETROSPECTIVE (evaluate the success of a design by interpreting outcomes) PROSPECTIVE (plan a design likely to succeed in providing needed information)

9. Bioassay Objectives • screening chemicals for identifying carcinogens or other toxic effects; • characterizing the dose-response curve in the observable range; • characterizing the dose-response curve to facilitate low-dose extrapolation; • defining a threshold or benchmark dose point of departure; • providing data on health effects at human exposure levels; • providing data to test hypotheses regarding mode of action.

10. Combining Objectives • Different objectives invariably conflict in demands on dose selection • Illuminate conflicts by considering ideal designs for different objectives • No single ideal design – compromises are necessary • Hedging against poor choices

11. A Systematic Approach to Selecting Doses

12. 3 CONTEXTS: • scientific • regulatory / risk mgmt • practical A Systematic Approach to Selecting Doses

13. A Systematic Approach to Selecting Doses • OBJECTIVES: • Haz Screening • D-R • Low-Dose Extrapol’n • ID Threshold • BMD • Human Safety Study • MoA

14. A Systematic Approach to Selecting Doses • PK • MoA

15. A Systematic Approach to Selecting Doses • Top Dose • Number of Doses • Location of Doses • Animal Allocation

16. A Systematic Approach to Selecting Doses • Imagine Possible Outcomes • & • Evaluate Design • Performance

17. A Systematic Approach to Selecting Doses • Iterate to Improve • Likelihood of Desired • Performance

18. Design / Interpretation Factors • mortality • clinical signs • site-of-administration effects • pharmacokinetics and altered metabolism • physiological effects • nutritional effects • hormonal effects • organ-weight changes • cell proliferation and apoptosis

19. Case Studies • Formaldehyde • Methylene Chloride • Drug X

20. Appendix: Criteria for MTD Attainment:Clinical Pathology and Pathology-based Endpoints • Liver • Kidney • Hematopoietic System • Reproductive System

21. Looking to the Future of the Chronic Bioassay--Potential Paradigm Shifts Decision-making questions to be addressed are different Experimental tools available are different Scientific issues that can be and need to be addressed are different

22. Decision-Making Questions--From Qualitative to Quantitative • How extensive is the cleanup that is needed for a superfund site? • How quantitatively “significant” are the risks posed by occupational exposures to specific (air levels x durations) of X? • How large are the health prevention benefits from defined reductions in exposures to X achievable by specific interventions?

23. New Experimental Tools as Sources of Paradigm Shifts • Carcinogenesis • From quantal observations of one+ tumor per animal after lifetime dosing to shorter term measurements of continuous parameters (somatic mutation rates for relevant mutations, rates of epigenetic changes and relative growth advantages for initiated clones) • More definitive mode of action characterizations with genetic “knockout” animals and related manipulations (e.g. RNAi, animals with specific genes over expressed) • “-omics” based assay systems applied to quantitative elucidation of dose/time/response relationships after identification/separation of specific relevant cell types

24. Newer Tools for Newer Questions for both Carcinogenesis and Other Health Endpoints • Accounting for more diversity in toxic susceptibility (by age, other interacting factors) • Chronic cumulative modes of harm (e.g. Parkinson’s, Alzheimer’s, obstructive lung disease conditions where there are long term losses of functional units). Need biomarkers of: • Cumulative total of past damage (e.g. FEV1) • Current rate of increase in damage (e.g. enzymes released into the blood that indicate death of key types of cells or structural damage) • Dysfunction indicators (e.g. heart rate variability changes)

25. Building a Deeper Understanding of Homeostatic Control Systems • How exactly are the numerous set points for different levels of biological organization set? • Coded in the genome? If so, how? • Developed by some sort of evolutionary learning process? How? • How do failures of homeostatic controls happen? • Barker observations--chronic cumulative loss of “wetware” to accomplish control • Other reasons for deterioration of homeostatic system controls with ageing

27. New Paradigms for Risk Analysis-- Tools for Probabilistic Analyses • From analysis of data sets to data bases • Quantitative structure activity relationships specific to different modes of action require standardized effect levels (e.g. ED50s) rather than no-effect levels or even BMDL’s that mix in effects of inter-animal variability and assay uncertainty/measurement errors

28. Conclusion • The current chronic animal bioassay is perhaps best seen as an aircraft carrier—a central anchor of a large naval battle group including many subsidiary vessels/experiments that both protect the carrier and enhance the capabilities to address increasingly diverse information needs. It remains a mainstay for current risk assessment, but will increasingly be supplemented, if not supplanted, with numerous other tools involving exposures during specific life-stages and measuring a variety of endpoints short of apical measures of adverse health endpoints.