Report of the 1 st and 2 nd Mystery of Reactive Oxygen Species Conferences

events (KEs) to an adverse outcome (AO), via key event relation-ships (KERs). KERs describe the causal linkages between each KE using biological knowledge, empirical evidence, and quan-titative understanding. This modular framework increases effi-ciency in AOP development and facilitates the development of AOP networks, the ultimate tool for AOP application. A central tenet of AOP development is sharing KEs across pathways. This enables an enhanced understanding of cross-talk across pathways to represent the complexity of biology.


Introduction
To reduce or replace animal experimentation, approaches are needed where computational networking of tissue and cellular biological events addresses disease mechanisms. One important tool for this is the adverse outcome pathway (AOP) framework, which describes how molecular and cellular events cause adverse health effects. The AOP framework is compiled by the scientific community in the Adverse Outcome Pathway Knowledge Base (AOP-KB) as part of Organisation for Economic Co-operation and Development (OECD) projects 1 .
AOPs begin with a molecular initiating event (MIE), which is the starting point describing how stressors interact with specific molecules in cells. AOPs then progress through a series of key events (KEs) to an adverse outcome (AO), via key event relationships (KERs). KERs describe the causal linkages between each KE using biological knowledge, empirical evidence, and quantitative understanding. This modular framework increases efficiency in AOP development and facilitates the development of AOP networks, the ultimate tool for AOP application. A central tenet of AOP development is sharing KEs across pathways. This enables an enhanced understanding of cross-talk across pathways to represent the complexity of biology.
Reactive oxygen species (ROS) play crucial roles in a variety of diseases and physiological conditions. ROS are produced by various stressors such as chemicals, particles or radiation energy, and endogenous peptides or the enzymatic machinery. Tran- initial concept for a ROS-related KE group was introduced, which led to the creation of harmonized KE1940 3 "Up-regulation of ROS" in a collaborative effort of conference attendees (Fig. 1). The Mystery of ROS collaboration discussed the harmonization of the existing ROS-related KEs and associated AOPs in the AOP-Wiki ( Fig. 1; Tab. S1 4 ). A similar discussion on inflammation-related KEs has resulted in a consensus around a hub of three modular events, which was presented at the Mystery of ROS (II) as a model (Villeneuve et al., 2018).

Report of the 1 st and 2 nd Mystery of Reactive Oxygen Species Conferences
The Mystery of ROS consortium carefully considered many critical aspects of the creation of ROS-related KEs. It was identified that ROS are needed for critical cellular functions at optimal levels; however, excessively high levels of ROS production over prolonged durations lead to adverse effects. Thus, the cellular balance between ROS production and scavenging to maintain homeostasis needs to be represented within the KEs.
The second conference on the Mystery of ROS (II) included four presentations and panel discussions on DNA damage and ROS and on nanomaterial-related oxidative stress (Tab. 2). The  Shihori Tanabe, PhD stanabe@nihs.go.jp

Plans of the consortium
Members of the consortium agreed to work toward harmonization of the molecular-level KEs focused on up-regulation of ROS production under the umbrella of KE1940 3 "Up-regulation of ROS". KE1940 focuses on the production of ROS per se, while the reduction of protective enzyme activity that also results in ROS increase will be the focus of the KE1869 5 "Depletion of protective oxidative stress response" (Short name "Depleted Protective Response to ROS") at "molecular" and "cellular" level KEs for ROS.

Conclusion
The consortium had fruitful discussions on harmonizing ROS-related AOPs. Although no definitive consensus on the KEs was reached, there was agreement that further conversations are needed to elucidate how best to represent the complexity of oxidative stress. Since oxidative stress is caused by excessive ROS production and depletion of scavenging machinery, all aspects of ROS should be considered for harmonizing AOPs. These discussions of the Mystery of ROS consortium will strengthen AOPs and facilitate the development of AOP networks to support the design and justification of new approach methodologies.
Representing the process of inflammation as key events in adverse outcome pathways.