Elizabeth Julien (Consultant) Mary Alice Smith (University of Georgia) Steve Gendel (FDA/CFSAN)

1. Key Events Dose-Response Analysis.Part 2: Application to Nutrients, Pathogenic Microorganisms, and Food AllergensSOT RASS TeleconferenceFebruary 10, 2010 Elizabeth Julien (Consultant) Mary Alice Smith (University of Georgia) Steve Gendel (FDA/CFSAN) Steve Olin (ILSI Research Foundation)

2. Dose-Response and Thresholds • Recognition of the centrality of dose-response concept in life sciences • QUESTION: Can our increasing understanding of modes of action provide insights for characterizing dose-response relationships at low doses (including thresholds) ? • Not only for chemicals but also for other bioactive agents.

3. ILSI RF Threshold Working Group • Characterizing fundamental biology of human health effects for chemicals, pathogens, allergens, nutrients • Implications for dose-response, practical thresholds, public health standards • Fostering cross-disciplinary discussion • → Key Events Dose-Response Framework

4. ILSI RF Threshold Working Group • Chemical Group: Alan Boobis (Imperial College London), George Daston (Procter & Gamble), and Julian Preston (EPA). • Nutrient Group: Sanford Miller (U Maryland), Joseph Rodricks (ENVIRON), Ian Munro (CANTOX), A. Catharine Ross (Pennsylvania State), Robert Russell (Tufts), 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. Work Products and Next Steps • 5 papers – Crit Rev Food SciNutr, 49 (8), Sept 2009 (Overview, Chemicals, Nutrients, Pathogens, Allergens) – open access. • 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 • CONTACT: Steve Olin (solin@ilsi.org)

6. The Key Events Analytical Framework: A case study with retinol (Vitamin A)Beth Julien, Ph.D.SOT RASS Telecon, Feb 10 2010

7. Acknowledgements This presentation describes the work of the ILSI Threshold Project “Nutrient Group”:Catharine Ross and Robert RussellSanfordMiller, Ian Munro, Joseph Rodricks, Elizabeth Yetley …. and incorporates ideas developed by the entire Threshold Project Working Group. See Crit Rev Food Sci Nutr, 49 (8), Sept 2009

8. Increasing refinement in approach INITIAL DOSE (Exposure or Intake) INITIAL DOSE (Exposure or Intake) INITIAL DOSE (Exposure or Intake) Target Tissue Dose, Adjustment Factors, etc. Multiple events, Multiple dose levels; Multiple d-r relationships Ultimate Effect of Concern Ultimate Effect of Concern Ultimate Effect of Concern

9. Overall KEDRF Concept INITIAL DOSE (exposure or intake) key event(s) (e.g., absorption, inhalation) key event(s) (e.g., transport to target tissue) key event(s) (interaction in target tissue) At various events, homeostatic mechanisms may affect progression along pathway ultimate effect of concern

10. Looking at the whole pathway of events … • Which events may be “control points” where mechanisms exist to maintain 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. Examining an individual key event Factors that combine to determine outcome of individual events: • Dose (level and frequency) • Homeostatic mechanisms (e.g., repair, immune, response, compensatory pathways) • Host factors (life-stage, disease state, genetic makeup, nutritional status, co-exposure)

12. Dose Host factors Homeostatic mechanisms Event (Process or Interaction) ↓ likelihood of effect of concern Progression toward effect of concern

13. Looking at an individual event (esp. “control points” or “determining” events) • Can we characterize dose-response at this event? • If not, what data are needed? • Is there evidence for threshold? • What homeostatic mechanisms exist? • What host factors come into play? • How can this information be used for practical purposestoward informing public health standards? • Human relevance? • Identifying susceptible sub-populations? • Quantifying variability?

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

15. For certain nutrients, two types of thresholds exist • An intake level that must be exceeded (usually on a regular basis) to provoke a toxic effect • A minimum intake level required on a regular basis to support health and prevent deficiency A range of safe and sufficient intake levels is situated between these two thresholds

16. 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, ↓ immune response, ↓bone growth Teratogenicity – Severe toxicity/lethality Organ damage, affecting metabolism Focus of case study: upper levels of intake

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

18. 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.

19. Analysis of Events II Liver Metabolism and Storage: R → RE by LRAT for storage. 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” .

20. Analysis of Events III • Release of retinol from Liver Storage • Possible feedback loop: Circulating retinoid metabolites (?) • may signal liver to release stored RE, and convert it to R. • R binds RBP and is released into circulation, where it forms a • trimolecular complex with transthyretin (R-RBP-T). • Plasma retinol concentration is nearly constant in a given individual. Only when liver storage goes below or above a wide normal range (~ < 20 µg or >300 µg), do circulating levels change. • Plasma retinol levels are not a good indicator of VA status. • Conclusion: Not a mechanism for control of circulating retinol when there is high intake.

21. Analysis of Events IV Uptake into Extrahepatic Tissues: Mechanism unknown. R may dissociate from R-RBP-T, diffuse into cell. • In cell nucleus, RA binding proteins bind specific isomers of RA and regulate activity of retinoid responsive genes. • Expression of a subset of binding proteins can be induced in some tissues by the metabolite all-trans retinoic acid. Conclusion: Binding activity appears to be only a partially regulated event; not a control point for regulating RA levels. 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.

22. Dietary Vitamin A oxidative inactivation LRAT RE Retinol RA polar metabolites of RA storage and release oxidative activation conjugation and excretion Excess dietary Vitamin A LRAT RE Retinol RA CYPs polar metabolites of RA

23. Case Study Conclusions • Overwhelmed LRAT capacity, leading to excessive RA levels is likely a “determining event” Research question: how high must RA (or its metabolites) rise in order to cause effect? How long must it remain high? Research need: study induction of Cyp26 and accumulation of polar metabolites of RA in blood and urine as potential early signals of toxicity .

24. General lessons • 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 supports a practical use: strengthens connection between regulatory standards for a population (RfD, ULs) and the underlying biology

25. Application of the Key Events Dose Response Framework to:Pathogenic Microorganisms Working Group: Robert L. Buchanan Arie H. Havelaar Mary Alice Smith Richard C. Whiting Elizabeth Julien

26. Current Approaches and Practice • “Infectious Dose” or “Minimum Infectious Dose” - traditionally used to describe the ability of a pathogenic microorganism to cause illness and disease • Concept presumes a threshold dose • Microbiological equivalent to NOAEL in toxicology

27. Microbial Pathogen Categories • Toxigenic bacteria – Threshold assumed • Toxins are preformed in food • Clostridium botulinum, Staphylococcus aureus • Toxicoinfectious bacteria – No threshold assumed • Colonize GI tract, not invasive • Toxins act locally (Vibrio parahemoliticus) and/or in distant tissues (Escherichia coli O157:H7) • Invasive bacteria – No threshold assumed • Colonize GI tract and disseminate in host • Intercellular spread • in mucosa (Salmonella enterica), • to lymphoid system (Yersinia enterocolitica) • to bloodstream (Salmonella Typhi) • Intracellular spread • to fetus (Listeria monocytogenes)

28. Data Sources for Current Understanding of Microbial Dose-Response • Expert elicitation (experience) • In vitro studies • Cell, tissue or organ cultures • Non-living experimental systems (fermenters, model intestinal systems, test tubes); predictive microbiology: mechanistic models • Animal studies • Human studies • Volunteer feeding studies • Outbreak investigations • Surveillance and annual health statistics • Biomarkers

29. Current Basis of Microbial Dose-Response Modeling • Conditional chain of events Exposure  Infection  Illness • Single hit—One microorganism has a probability • Independent action by each microorganism • No threshold • All single hit models are approximately linear at low doses (a mathematical property) Haas, 1983; Teunis et al., 1996; FAO/WHO 2003

30. Dose -Response Models 1 0.8 0.6 Data Prob. Illness 0.4 0.2 0 10 1 0 1 2 3 4 5 6 7 8 9 0.1 0.01 Prob. Illness 0.001 Public health and regulatory concern 0.0001 0.00001 0.000001 0.0000001 0 2 4 6 8 10 log Dose

31. Pathogen-Host Interactions • The interactions between the pathogen and the host can be very complex • Immune or adaptive response of host • Homeostatic mechanisms of host • Pathogens can evolve mechanisms to use host resources to help with survival and growth • Host characteristics such as age, health status, immune status can also effect interactions

32. Key Events Dose-Response Framework: Listeriosis Exposure to L. monocytogenes via ready- to-eat foods (soft cheeses, deli meats, smoked fish, pates) Invasive, infects spleen, liver and CNS Risk groups: fetus and neonate, elderly, immunocompromised Rare but high case-fatality ratio (~20%) Pregnant women at greatly increased risk: spontaneous abortion, stillbirth, neonatal meningitis

33. Key Events Pathway: L. monocytogenes intake and potential fetal death Interplay of host and pathogen can influence progression at various events Intake of contaminated food 1 - P P Do not survive Pathogens survive in upper GI tract Do not establish, etc Establish; attach; taken up into epithelial cells Do not escape Escape from phagosomes; transfer to phagocytes Do not transfer Cross placenta Do not grow, no mortality Growth; results in fetal mortality 33

34. Key Event 1: Survival in Stomach • Microbial death rate affected by • digestive enzymes • the food matrix • quantity and composition/acidity of foods consumed • general level of acidity (may be reduced by advanced age, antacids consumption, achlorhydria) • Adaptation by L. monocytogenes to acid environment • Research--Measure whether probability of survival is proportional to number ingested, adaptation, strain & host differences

35. Key Event 2: Establish; attach; taken up into epithelial cells • Current knowledge - InlA on L. monocytogenes andE-cadherin receptors in host • Research • Does growth correspond to number internalized? • Role of host innate immune response • Gene control of InlA and E-cadherin (alleles, quantities)

36. Key Event 3: Escape from phagosomes; transfer to phagocytes • Current knowledge • L. monocytogenes synthesizes listeriolysin O (LLO) which forms small pores in phagosome and ultimately L. monocytogenes escapes from phagosome • Uses host actin to move to membrane-membrane interface with adjacent cells • Spread to other enterocytes and/or phagocytes which disseminate pathogen to other organs including placenta Research • Strain and host differences • Model responses (quantitative)

37. Key Event 4: Transfer of Pathogen across Placenta • Mechanism by which L. monocytogenes crosses placenta is not known, but 2 mechanisms hypothesized: • Invasion of endothelial cells via In1A and E-cadherin interaction (Lecuit et al 1999, 2004) • Actin-mediated cell-to-cell transfer from infected phagocytes to placental endothelial cells (Drevets et al, 1995). • Knowledge of this step could potentially provide method to prevent passage to the fetus.

38. Key Event 5: Pathogen Growth Leading to Fetal Morbidity and Mortality • Once across placenta, gain entry to fetal circulation and spread to fetal liver and brain • Immature fetal immune system puts fetus at great risk of infection • Asymptomatic maternal infection but may result in spontaneous abortion, delivery of stillborn infant or infected infant. • Unknowns: is fetal death a reaction of maternal system to fetal infection, loss of placental integrity, infection of fetus directly, or some combination?

39. Key Events Dose-Response Framework for Pathogens • For L. monocytogenes: • Some events appear probabilistic in nature (survival through GI, attachment to intestinal epithelium) • Other events engage host mechanisms and may have a finite capacity that can be overcome. These would likely be non-linear. • Other pathogens may be very different • pH tolerance, quorum sensing, toxin production, etc, may affect the dose response relationship.

40. Key Events Dose-Response Framework for Pathogens - Conclusions • Provides a structure for systematically considering complex factors influencing dose response • Highlights research needs • Generates new hypotheses and focused research • Ultimately provides new data to refine dose response • Basis for iterative improvement in microbial dose-response assessment

41. Potential Application of the Key Events Dose Response Framework to Food Allergens Allergen Working Group Steven Gendel Steve Taylor Geert Houben

42. Food Allergy – What is it? • An immunologic reaction to a food • Usually IgE mediated • IgE antibodies bind to one or more proteins in a food • Two step process • Sensitization • Elicitation

43. Gastrointestinal nausea vomiting abdominal pain diarrhea Cutaneous urticaria angioedema atopic dermatitis Respiratory rhinitis laryngeal edema asthma Systemic anaphylactic shock What Can Happen?

44. How Much of a Problem Is It? • 30,000 ER visits/ 2500 hospitalizations/ 150 deaths/yr • Up to 2-3% of adults & 6-8% of children have true food allergies • Over 150 foods implicated; 8-10 commonly allergenic foods • No cure, avoidance of allergenic food is critical

45. What is different about food allergens? • Allergic response is to a food component that is nutritious for most of the population • Sensitivity and severity (biological endpoints) have large range in the population • No animal models or in vitro tests for dose/response modeling

46. The Immunology of an Allergic Response Step 1 – Sensitization Step 2 – Elicitation

47. Food Allergic Responses – Application of The Key Event Approach

48. Sensitization • Very poorly understood • May be breakdown of oral tolerance • No data on thresholds for sensitization

49. Elicitation • More data on elicitation process • Clinical evidence for thresholds • Thresholds may change over time in an individual • Cross-reactivity and cross-sensitivity can lead to reactions to different foods

50. Major Steps in Elicitation Ingestion Digestion Uptake Cellular Events Signs and Symptoms