McKim Workshop on Strategic Approaches for Reducing Data Redundancy in Cancer Assessment Duluth, MN, USA 19 May, 2010

 Goal & Problem  Categories  Mechanism, Modes, & Pathways  The Example of Skin Sensitization  Adverse Outcome Pathways  Critical Events & Integrated Testing Strategy  Summary

 To place every discreet organic chemical in to a category for every hazard endpoint of interest.  Easy for data-rich acute effects.  Hard for data-sparse chronic effects.  With this in mind the concept of outcome pathways is proposed.

 There is discontinuity between chemical and biological spaces (substances, which are “similar” in molecule structure, are often dissimilar in terms of their toxciity including the ability to elicit a particular hazard endpoint, as well as potency within that endpoint).  We often have difficulty in forming toxicologically meaningful groups, especially for elaborate hazard endpoints such as cancer.

 Provide a means of evaluating all members for common toxicological behavior or consistent trends among data for an endpoint (measured data on a few category members can be used to estimate the missing values for one or more untested member).  Identification of a consistent pattern of toxic effects within a category increases the confidence in the reliability of the results for all.  This is predicated on a priori binning the chemical in the correct category.  Category formation is a key topic of predictive toxicology.

 The goal is to develop a TMC, which enables a transparent, defensible assessment through mechanistic comparisons without further testing.  This shifts the emphasis to intrinsic chemical activity and critical biological events and away from statistical parameters, especially a fixation on fit and predictivity.  Data for different in vivo endpoints differs so several ways will be needed to form TMCs.

 Are formed as a result of a common 1) chemical reactivity mechanism, 2) biological mechanism, 3) mode of toxic action (based on receptor, enzyme or basic cellular processes), or 4) molecular similarity  When one moves from a common chemical reactivity-based category to a receptor or common cellular process-based category and even more so to molecular similarity-based category confidence in whether the chemical in question truly belongs to the category diminishes

 Toxicologically related to DNA- and protein- binding.  Directly applicable to a limited number of hazard endpoints where the Molecular Initiating Event (MIE) is the rate limiting factor in the in vivo effect.  Important but not the total answer to forming TMCs.

 In mammalian pharmacology and toxicology literature the MechTA denotes the sequence of events leading from the absorption of an effective dose of a chemical to the specific biological response in the target.  Understanding a chemical’s MechTA requires understanding the causality and temporal relationships between the steps to a particular toxic endpoint, as well as the steps that lead to an effective dose at the biological target(s).

 Meeting this definition of a MechTA requires an exceptionally large amount of high quality data, which only can be attained for a very limited number of compounds.  This is currently out of reach for the vast majority of industrial organic compounds.  One cannot impose the MechTA criteria to forming TMCs and expect to make progress in the near term.

 Foundation can be traced to the studies of McKim et al. (1987) and their fish acute toxicity syndromes, which are represented by selected biochemical and/or physiological effects of exposure selected as key responses measured in vivo from exposure to model chemicals.  Require less data than MechTA approach.  Successful in forming TMCs for acute aquatic toxicity.

 Designed to describe knowledge concerning the linkages between chemical structure of the target compound and the in vivo outcome of regulatory interest.  The term adverse outcome pathway has been selected so not to cause confusion with the term “Toxicity Pathway” (United States National Research Council in their document entitled “Toxicity Testing in the Twenty-first Century: A Vision and a Strategy”, 2007).

 Facilitates the use of in silico, in chemico, and in vitro (cellular, molecular, and biochemical) endpoints in forecasting in vivo effects.  Assimilates MIEs with measurements of key biological process.

Karlberg et al. Chem. Res. Toxicol. 2008, 21, Haptenation; 2. Epidermal inflammation & LC activation; 3. LC migration; 4. DC: T cell interaction; 5. T cell proliferation; 6. Increase in hapten-specific T cells; 7. Hapten re-exposure; 8. Acute inflammation; 9. T cell-mediated inflammation

 Events, which are:  Hypothesized in the pathway.  Essential to the induction of the adverse outcome.  Measurable

 A data generating and data gathering exercise.  Largely focused on in silico, in chemico, and in vitro endpoints.  Selected ITS endpoints must have biological relevance to the hazard endpoint in question, which is most transparent when linked to an AOP.

 Toxicants electrophile or chemicals converted to a reactive metabolite.  Molecular site(s) of action are nucleophilic sites (cysteine and lysine) in proteins.  MIE is covalent (irreversible) perturbation of dermal proteins.  Biochemical paths are incompletely known, but includes stimulation of selected cellular responses (e.g., antioxidant-response element).

EVENTMEASUREMENT Protein binding in silico predictions from structure Chemical reactivity w/ SH + for TH1, - for TH2 Chemical reactivity w/ NH2 - for TH1, + for TH2 Keratinocyte stimulation of + for TH1, - for TH2 Keap1-Nrf2-ARE cellular Pathway Dendritic cell expression of IL4 - for TH1, + for TH2 Dendritic cell expression of IL8 + for TH1, - for TH2

 The sequence of events from chemical structure through the MIE event to the in vivo outcome.  Designed to avoid mixing data from multiple mechanisms, which can cause the same in vivo outcome.  An organizing principle for hazard assessment, especially for elaborate endpoints.

 Describes a technique for grouping chemicals based on both up-stream chemical and down- stream biological processes.  Shifts emphasis from just intrinsic chemical activity to chemical activity plus the cascade of events that occur across the different levels of biological organization.

 Allows a shift from animal testing to hypothesis testing.  Provides a basis for chemical extrapolation.  Provides for comparisons across level of biological organization.  Provides for consideration of life form & life stage at exposure.  Provides a basis for species extrapolation.

Chemical Reactivity Profiles Receptor, DNA, Protein Interactions Mechanistic Profiling Biological Responses Current OECD Toolbox T oxicant Macro -Molecular Interactions Molecular Initiating Event

Lethality Sensitization Birth Defect Reproductive Impairment Cancer Altered Function Altered Development Gene Activation Protein Production Signal Alteration Chemical Reactivity Profiles Receptor, DNA, Protein Interactions Structure Extinction Cellular Organ Mechanistic Profiling In Vivo Testing Biological Responses ITS and Adverse Outcome Pathway T oxicant Organism Macro -Molecular Interactions Molecular Initiating Event Population Cellular & In Vitro Testing