April 2 5 , 2011

1 

### CHARGE QUESTIONS : FIFRA Scientific Advisory Panel Consultation on “Integrated Approaches to Testing and Assessment Strategies: Use of New

Computational and Molecular Tools” May 24

- 26, 2011

A. Charge Questions About OPP’s Strategic Vision “ Integrated Approaches to Testing and Assessment Strategy: Use of New Computational and Molecular Tools”

EPA’s Office of Pesticide Programs (OPP) is committed to improving and transforming our risk assessment approach to stay abreast of the state of the science and take advantage of emerging technologies

. The goals are to increase the efficiency, effectiveness, and accuracy of the testing and assessment process by enhancing our abilities to target the effects of concern
and to base decisions on the information most r elevant to the assessment. The strategy to attain this goal is to enhance and advance our ability to use an integrated approach to testing and assessment (IATA)

. OPP views IATA as a logical way to transition from the current paradigm
of broad in vivo testing and assessment to a system of targeted testing based on a mechanistic understanding of the toxicity of a chemical

. T o use mechanistic data across various levels of biological organization to support chemical assessments

, there must be a meaningful translation and linkage of this information to adverse outcomes that are relevant to human and
environmental health . To accomplish this

, we are adopting the concept of an adverse outcome pathway (AOP) as the conceptual framework to organize knowledge in o rder to identify linkages at different levels of biological organization so that more efficient, hypothesis

- driven approach to chemical testing and assessment can be developed and supported (Ankley et al., 2010; Schultz 2011; SAP 2010). E stablishing the li nkages and dose

- response relationships between the key events 1 and the adverse outcome inform s an understanding of the magnitude of change needed to trigger the adverse consequences of a biologic perturbation as it relates to dose, timing and duration of t he perturbation, as well as the susceptibility of the host. We believe that by establishing the causal pathway we will be able to more reliably screen for potential adverse effects by quantifying such linkages to estimate effects to whole organisms or

pop ulations using data collected from lower levels of biological organization.

Development of the toxicological understanding across a range of AOPs, from their causal molecular initiating event and subsequent key events to the adverse outcome will establish the

1 “ K ey events” are biological events along a pathway that are obligatory and quantifiable steps leading to an adverse outcome. Pertinent observations include, for example, specific biochemical events, cell proliferation, perturbations in hormones or other signaling systems, receptor

– ligand interactions. To establish a series of key events along the causal path, EPA, the International Programme on Chemical Safety and the International Life Sciences Institut e (ILSI) have established a harmonized weight

- of

evidence approach using criteria based on those described by Bradford Hill, taking into account factors such as dose

- response and temporal concordance, biological plausibility, coherence and consistency. ( USEPA, 2005; Meek et al., 2003 ; Seed et al ., 2005 ; Boobis et al ., 2006; 2008 )April 2 5 , 2011

2 necessary knowledge to streamline the pesticide testing and assessment to more thoroughly integrate, in a tiered fashion, existing data, computer modeling, and in vitro models diagnostic of AOPs to target in vivo testing. Furthermore, on ce an AOP is established the information can be used to develop new quantitative predictive tools and qualitative read

- across methods. Thus, AOPs enable a shift from a chemical by chemical approach to a category based approach.

The current AOP framework e mploys a simplified depiction of a linear progression of events across levels of biological organization that translate a molecular perturbation to an adverse outcome (assuming the perturbation is sufficiently severe). However, it is well recognized that

A OPs actually operate as networks within a systems biology context and that various AOPs can share key events and interact with one another in a variety of ways. Reductionist experimentation has generally been characterized by a more linear mode of thinking . In contrast, because omics and HTS (viewed as a large suite or battery of assays implemented in a high

- throughput manner) can efficiently cover greater breadth of molecular initiating events and/or key events and associate them with a given chemical, the y are, perhaps, better equipped for promoting a network

- view. Ultimately, understanding of responses to stressors and effectively predicting toxicity, in a systems biology context that operates across multiple levels of biological organization, will requir e the kind of network

- thinking that HTS and omics promote. Consequently, effective integration of data from hypothesis

- driven, focused, testing, omics, and HTS represents an important foundation for an enhanced IATA.

The purpose of this consultation is to seek guidance from the FIFRA Scientific Advisory Panel (SAP) about OPP’s vision, strategy, plans, and initial efforts to incorporate advances in molecular science and emerging in silico and in vitro technologies (e.g., structure

- activity relationships (SA Rs), quantitative SARs [(Q)SARs], mechanistically

- based in vitro assays, high throughput screening (HTS) assays) as components of an enhanced IATA. OPP is requesting the
Panel’s input on whether plans to utilize emerging tools and methods represent a logic al progression that will ultimately achieve our vision of increased efficiencies

, effectiveness , and biological relevance in the assessment and management of risk associated with the use of pesticides. The Agency is also seeking input on a number issues s pecifically related to the development and evaluation of predictive tools and libraries of AOPs.

1) D oes the Panel believe that OPP’s strategic vision clearly articulate s a sound scientific basis

to making the risk assessment process more efficient and informative? To what extent have

we described the critical components ( i.e., knowledge bases and databases of existing information, predictive tools (e.g., (Q)SAR, in

- vitro methods, HTS assays and AOPs ) and the progression of events ( i.e., research and the use of research) to support the evolution from the current testing and assessment paradigm to an improved IATA of targeted tiered testing based on an understanding of AOPs ( i.e., the molecular and cellular events leading to adverse effects at the whole organism level) ? Are the re any key science issues that the Agency has not captured that are necessary elements to consider in an IATA approach ?April 2 5 , 2011

3 2)

a) Given that there could be hundreds of AOPs and that varying levels of AOP information are needed depending on the decision being made ( i.e ., the tier of the risk assessment), please discuss principles for scientific acceptance ( i.e ., tools for gauging the reliability and robustness) of AOPs and the use of AOPs to m ove away from chemical by chemical approaches.

b) OPP plans to evaluate new tools and information using available criteria and methods such as the OECD principles for (Q)SAR expert systems and the EPA

- IPCS (International Progra m m e on Chemical Safety) mode of action framework. Please discuss these and
other criteri a the Agency can use to reliably integrate predictive tools earlier in the

process.

c) How can the foundation of knowledge generated through historical and on

- going
reductionist ( i.e ., making use of information from lower levels of biological organization to understand adverse outcomes)

, hypothesis

- driven, biological experimentation be effectively mined and integrated with the capabilities of omics (including toxicogenomics

, transcriptomics, proteomic s, and metabolomics) and high

- throughput screening to speed the discovery and development of AOPs and their subsequent acceptance as a credible foundation for predictive risk assessment?

3) OPP is a t the beginning stages of contemplating metrics to demon strate success in achieving our vision. What is the Panel’s initial thinking regarding the concepts, considerations, and
factors OPP should take into account as we evaluate our progress ? What methodologies are best suited to measure success in achieving o ur vision? For example, what methods would you recommend to quantify the development of AOPs and th e qualitative and quantitative application of AOPs in risk assessment?April 2 5 , 2011

4 B. Charge Questions for the Case Studies

Case Study 1: “ Use of Toxicogenomic Technology to Inform the Risk Assessment A Case Study:

Propiconazole ”

In this case study we began with the identification of an adverse outcome. We incorporated omic and molecular technologies to develop a mode of action (MOA) for propiconazole based on apical, omic and molecular endpoints. The challenge for the future will be to use diagnostic assays of Key Events MOA in the opposite direction, i.e ., as a tool for predicting specific toxicological responses. This will require an increased understandi ng of adverse outcome pathways (AOP)/MOA, and most importantly the quantitative relationships among the key events within the pathway. We envision that diagnostic in vitro assays for early key events will be sufficient to predict toxicological outcomes, o r limit the need for extensive in vivo studies. Over time, the building of libraries of AOPs will make greater use of existing data and enable a
shift from chemical

- by

- chemical approaches to the formation of toxicological categories based on key events alo ng the pathway rather than intrinsic chemical activity. Thus, “read across” to fill data gaps would be based on MOA/AOP information permitting the application of data from a chemical(s) that has been well

- characterized to similar untested chemicals.

1) Ple ase comment on the use of omic and related technologies employed to develop and link Key Events to create the MOA/AOP of propiconazole. Was the resultant MOA/AOP for propiconazole logical and scientifically sound?

2) Are there other approaches/technologies that could be used to develop and link Key Events to develop MOAs/AOPs for propiconazole and/or chemicals?

3) Please comment on the general utility of AOPs/MOAs or Key events to read across chemicals to provide a more defensible weight of evidence support f or the traditional risk assessment approaches that OPP will continue to use in the near

- and mid

- term?

4) Please comment on the potential use of omic and related technologies to inform AOPs

in non

- mammalian taxa for use in ecological risk assessment?April 2 5 , 2011

5 Case Study 2: Use of an Adverse Outcome Pathway (AOP) to Inform Risk Assessment

-

Triclosan as a Case Study

The case study with triclosan presents an example of development of an adverse outcome pathway (AOP) as the conceptual framework to organize kno wledge and identify linkages at different levels of biological organization to support a more efficient, hypothesis

driven approach to chemical testing and assessment (Ankley et al., 2010; Schultz 2011; SAP 2010). Establishing the linkages and dose

- respons e relationships between the key events and the adverse outcome informs an understanding of the magnitude of change needed to trigger the adverse consequences of a biologic perturbation as it relates to dose, timing and duration of the perturbation, as well as the susceptibility of the host. In this respect, the magnitude of the change in thyroid hormone level and susceptibility of the adult and developing mammalian organism to adverse consequences resulting from this perturbation are relevant areas of

cons ideration for this case study. We believe that by quantifying such linkages we will be able to reliably estimate effects to whole organisms or populations using data collected from lower levels of biological organization.

The purpose of this consultation is to seek guidance from the FIFRA Scientific Advisory Panel (SAP) about the Agency’s use and interpretation of the in silico, in vitro, and in vivo technologies as components of the adverse outcome pathway for thyroid hormone perturbation. The Agency is r equesting your input on the following questions with respect to the Triclosan Case Study :

1) Use of the adverse outcome pathway concept to characterize the impact of triclosan on the thyroid hormone system promotes a research and risk focus on toxicodynamic and kinetic differences in thyroid homeostasis between (1) the study species (e.g.., rats) and the population of interest (e.g., pregnant women/fetus and young children) and (2) potential species similarities and/or differences in the molecular initiating events. Please comment on scientific knowledge and types of data considered to be informative in selecting appropriate factors for interspecies differences.

2) Please comment on the factors most responsible for the variability of toxicokinetics and toxicodynamics of compounds affecting thyroid hormone homeostasis among humans (particularly susceptible subpopulations such as pregnant women and children).

3) Benchmark dose analysis involves selection of a benchmark response (BMR). The consequences of a perturbation of a key event depend on the magnitude, timing, and duration of the perturbation, which in turn depends on dose. Subtle perturbations of key events may be damped out and have little effect on downstream biology due to the operation of homeos tatic processes. Please comment on the current understanding of the magnitude of perturbation of thyroid hormone levels in humans that may be required to lead to adverse outcomes.