Steve Olin (ILSI Research Foundation) Elizabeth Julien (Consultant)

1. Key Events Dose-Response FrameworkTCEQ/ARA Workshop IBeyond Science and Decisions:From Issue Identification to Dose-Response AssessmentMarch 16-18, 2010Austin, Texas Steve Olin (ILSI Research Foundation) Elizabeth Julien (Consultant)

2. Origins • QUESTION: Can our increasing understanding of modes of action provide insights for characterizing dose-response relationships (including thresholds) and, if so, how? • Not only for chemicals but also for other bioactive agents: nutrients, pathogens, allergens. • → Key Events Dose-Response Framework

3. Key Events Dose-Response Framework ILSI Research Foundation established a tripartite, multidisciplinary expert group to • develop an integrated framework to incorporate advances in scientific knowledge, • based on mode of action concept, focus on understanding the fundamental biology and dose-response (including possible thresholds) at each key event, • inform hazard characterization and risk assessment, • foster cross-disciplinary discussion.

4. ILSI RF Threshold Working Group • Chemical Group: Alan Boobis (Imperial College London), George Daston (Procter & Gamble), and Julian Preston (EPA). • Nutrient Group: A. Catharine Ross (Pennsylvania State), Robert Russell (Tufts),Joseph Rodricks (ENVIRON), Sanford Miller (U Maryland), Ian Munro (CANTOX), and Elizabeth Yetley (retired NIH). • Pathogen Group: Bob Buchanan (U Maryland), Arie Havelaar (RIVM), Mary Alice Smith (U Georgia), and Richard Whiting (Exponent). • Allergen Group: Steven Gendel (FDA CFSAN), Geert Houben (TNO), and Steve Taylor (U Nebraska).

5. Products and Next Steps • 5 papers – Crit Rev Food SciNutr, 49 (8), Sept 2009 (Overview, Chemicals, Nutrients, Pathogens, Allergens) – open access: http://www.informaworld.com/smpp/title~db=all~content=g914018566 • Next Steps • Encourage the development of additional case studies illustrating and evaluating the utility of the Framework • Organize small meetings and workshops to work through specific examples • Explore application and integration of the Framework into MOA analysis for risk assessment

6. Mode of action and key events EXTERNAL DOSE (exposure) KEY EVENT (absorption) KEY EVENT (target tissue exposure to ultimate toxic species ) KEY EVENT[S] (biological perturbation[s]) KEY EVENT[S] (pathological change[s]) Adverse health effect 6

7. Examining an individual key event Factors that combine to determine outcome of individual events: • Dose (level, frequency and duration) • Physiological mechanisms (e.g., homeostasis, repair, immune response, compensatory pathways) • Host factors (life-stage, disease state, genetic makeup, nutritional status, co-exposure)

8. Factors operating at the level of key events Dose Host factors Physiologicalmechanisms Event (Process or Interaction) Probability of effect of concern modified Progression toward effect of concern

9. Address each key eventsystematically • Is a minimum dose level required in order for this key event to occur? What data would be needed to demonstrate this? • What response mechanisms (e.g. homeostasis, repair) are involved? At what dose would these be overwhelmed? • What modifying factors (e.g. lifestage, disease state, nutritional status) can potentially reduce the effectiveness of response mechanisms? What factors can increase the effectiveness of response mechanisms? • Do such modifying factors change the dose level at which response mechanisms become overwhelmed? What data would be needed to demonstrate this?

10. Looking at the whole pathway of events … • Which events may be “control points” where mechanisms exist to maintain the normal physiological environment (e.g., homeostasis)? Are any control points especially vulnerable (readily overwhelmed by dose? readily modified by host factors? ) • Is any particular key event a possible “determining event” – i.e., its outcome disproportionately affects the probability of seeing the outcome of interest? • Does any particular key event appear to drive the slope or shape of the overall dose-response relationship?

11. First Case Examples • Chemicals • Non-DNA-reactive carcinogen (chloroform) • DNA-reactive carcinogens • Endocrine disruptors (binding to estrogen receptor) • Nutrients • Vitamin A (retinol) toxicity • Pathogens • General discussion of toxigenic, toxicoinfectious, and invasive bacteria • Listeria monocytogenes • Food Allergens • General discussion of key events for elicitation

12. Postulated MOA for CHCl3 CYP2E1 Metabolism Chloroform Oxidative Cl Cl C H Cl Phosgene Sustained cytoxicity Regenerative cell proliferation Key events Tumor development

13. Key events for chloroform carcinogenicity

14. Critical determinant of threshold • There is evidence that several of the key events in chloroform carcinogenesis exhibit a threshold. • It is not possible to determine which of these is the critical determinant (if any), from available data. • For this, studies in which each event was studied in isolation would be necessary. • Potential contribution of toxicogenomics • Information from all biologically relevant models should be used in such analyses. This is of particular value for more distal key events, which will be less compound specific.

15. Individual vs. population thresholds • Thresholds will vary among individuals. • Once the determining key events are understood, research to study contributions to population variability (including identification of susceptible populations) can be targeted on those events. • The goal is to understand how various factors (age, gender, disease state, nutritional status, etc.) may quantitatively affect the doses at which those determining events occur.

16. Applying the KEDRF to nutrients • Typically, for a given nutrient there will be long-term intake, with a dose that varies day to day. • For some nutrients, homeostatic controls exist to regulate blood and tissue levels despite daily intake variation. Control via: • One or more kinetic events • One or more dynamic events • Various intake patterns may lead to adverse effects: acute excess intake, chronic excess intake, chronic deficiency.

17. Retinol (vitamin A) A range of clinically-evident effects, depending on dose level and dose frequency. Very High – Extremely High Acute Intake Moderately High Chronic Intake Chronic Inadequate Intake Visual abnormalities, impaired fertility, reduced immune response, decreased bone growth Teratogenicity – Severe toxicity/lethality Organ damage, affecting metabolism Focus of case study: upper levels of intake

18. Overview of Retinol Pathway Uptake from Lumen Intestinal Metabolism; Distribution, Elimination dose dose dose dose dose Uptake into Liver Liver Metabolism, Storage, Release Uptake into Extrahepatic Tissues Target Tissue Interactions

19. Analysis of Events I Uptake from Lumen Highly efficient hydrolysis RE → R; Carrier mediated passive absorption; ~ 70% R absorbed. Not down-regulated with ↑intake or with VA status. Not a control point.. dose dose Intestinal Metabolism; Distribution, excretion Almost all R is re-esterified, packaged into chylomicrons (CM) for transport. No evidence of regulation. Not a control point. Uptake into liver Most CM remnants rapidly taken into liver; Retinol is passively assimilated into hepatocytes. No evidence for homeostatic regulation. Not a control point.

20. Analysis of Events II Liver Metabolism and Storage: R → RE by LRAT for storage. With signal to release, RE→ R, R-RBP released Under normal intake,LRAT activity correlates with circulating VA levels. LRAT activity is reduced in states of low VA; increased with ↑ intake. With high intake levels, LRAT activity ↑ only slightly. • LRAT may become saturated. • Liver’s storage capacity is not inexhaustible; threshold may be signaled by accumulation of circulating retinoid products. • Conclusion: LRAT activity is a regulated event - a control point. Saturation of LRAT may be a “determining event” .

21. Analysis of Events III Uptake into Extrahepatic Tissues: Mechanism unknown. R may dissociate from R-RBP-T, diffuse into cell. Target Tissue Metabolism and Activity : R metabolite (RA) binds CRABPs, forms complex with RAR/RXR receptors. Binding to RARE (or RXRE) on DNA, affects transcription. Conclusion: Binding activity appears to be only a partially regulated event; not a control point for regulating RA levels.

23. Vitamin A Toxicity:Analysis and Research Needs • Saturation of LRAT, leading to excessive RA levels, is likely a “determining event”. • Question: how high must circulating RA (or its metabolites) be in order to cause effect? How long must it remain high? • Key research needs • Quantitative data on dose-response for LRAT saturation • Characterize sources of variability at control points • Explore the potential role of downstream events

24. Overall Observations • KEDRF is an analytical framework that facilitates a systematic evaluation of multiple elements that combine to determine overall dose-response. • It complements empirical, mechanistic and modeling approaches to dose-response. • It highlights research needs, generates new hypotheses and focused research, and ultimately provides new data to refine dose response. • It supports a practical use: strengthens connection between regulatory standards for a population ( e.g., RfD, ULs) and the underlying biology.

25. Another ILSI Project • “Risk Assessment in the 21st Century (Risk21)” • Joint ILSI HESI/ILSI Research Foundation project • Just getting underway; probably 3 years • Building on recent NAS reports, a Scoping Meeting, August 2009, proposed 4 broad focus areas: • Exposure Science • Dose Response • MOA and high-to-low dose extrapolation • In vitro → in vivo extrapolation in context of HTPS and ToxTesting21 • Tiered (Integrated) Testing • Cumulative Risk • Contact: Michelle Embry (HESI) – membry@ilsi.org