Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

Read-across is one of the most frequently used alternative tools for hazard assessment, in particular for complex endpoints such as repeated dose or developmental and reproductive toxicity. Read-across extrapolates the outcome of a specific toxicological in vivo endpoint from tested (source) compounds to "similar" (target) compound(s). If appropriately applied, a read-across approach can be used instead of de novo animal testing. The read-across approach starts with structural/ physicochemical similarity between target and source compounds, assuming that similar structural characteristics lead to similar human hazards. In addition, similarity also has to be shown for the toxicokinetic and toxicodynamic properties of the grouped compounds. To date, many read-across cases fail to demonstrate toxicokinetic and toxicodynamic similarities. New concepts, in vitro and in silico tools are needed to better characterise these properties, collectively called new approach methodologies (NAMs). This white paper outlines a general read-across assessment concept using NAMs to support hazard characterization of the grouped compounds by generating data on their dynamic and kinetic properties. Based on the overarching read-across hypothesis, the read-across workflow suggests targeted or untargeted NAM testing also outlining how mechanistic knowledge such as adverse outcome pathways (AOPs) can be utilized. Toxicokinetic models (biokinetic and PBPK), enriched by in vitro parameters such as plasma protein binding and hepatocellular clearance, are proposed to show (dis)similarity of target and source compound toxicokinetics. Furthermore, in vitro to in vivo extrapolation is proposed to predict a human equivalent dose, as potential point of departure for risk assessment. Finally, the generated NAM data are anchored to the existing in vivo data of source compounds to predict the hazard of the target compound in a qualitative and/ or quantitative manner. To build this EU-ToxRisk read-across concept, case studies have been conducted and discussed with the regulatory community. These case studies are briefly outlined.

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BackgroundAndroid fat distribution (abdominal obesity) is associated with insulin resistance, hepatic steatosis, and greater secretion of large very low‐density lipoprotein (VLDL) particles in men. Since abdominal obesity is becoming increasingly prevalent in women, we aimed to investigate the relationship between android fat and hepatic lipid metabolism in pre‐ and postmenopausal women.Methods and ResultsWe used a combination of stable isotope tracer techniques to investigate intrahepatic fatty acid synthesis and partitioning in 29 lean and 29 abdominally obese women (android fat/total fat 0.065 [0.02 to 0.08] and 0.095 [0.08 to 0.11], respectively). Thirty women were premenopausal aged 35 to 45 and they were matched for abdominal obesity with 28 postmenopausal women aged 55 to 65. As anticipated, abdominal obese women were more insulin resistant with enhanced hepatic secretion of large (404±30 versus 268±26 mg/kg lean mass, P<0.001) but not small VLDL (160±11 versus 142±13). However, postmenopausal status had a pronounced effect on the characteristics of small VLDL particles, which were considerably triglyceride‐enriched (production ratio of VLDL 2‐ triglyceride:apolipoprotein B 30±5.3 versus 19±1.6, P<0.05). In contrast to postmenopausal women, there was a tight control of hepatic fatty acid metabolism and triglyceride production in premenopausal women, whereby oxidation (r s=−0.49, P=0.006), de novo lipogenesis (r s=0.55, P=0.003), and desaturation (r s=0.48, P=0.012) were closely correlated with abdominal obesity‐driven large VLDL‐triglyceride secretion rate.ConclusionsIn women, abdominal obesity is a major driver of hepatic large VLDL particle secretion, whereas postmenopausal status was characterized by increased small VLDL particle size. These data provide a mechanistic basis for the hyperlipidemia observed in postmenopausal obesity.