June 6, 2016 by Becky Brown

Adverse Outcome Pathways (AOPs) and their application for regulatory purposes is currently receiving considerable attention. In April the European Chemical Agency (ECHA) held a workshop on new approach methodologies (NAM) in read-across which concluded that acceptance of new methods such as those based on AOPs by regulators would be slow due to the cost, expertise and time required to evaluate them (ChemWatch 2016a).

The following week I attended a meeting hosted by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) on “Pathways-based approaches across the biosciences: Towards application in practice” which aimed to promote the use of AOPs for regulatory purposes.

An AOP is a structured representation of biological events starting from a molecular initiating event (MIE) leading to an adverse outcome linked by one or a series of causally connected key events (KE). The applications for AOPs in chemical assessment are potentially wide ranging from read across justification, prioritisation and screening, hazard and ultimately risk assessment. I presented a poster on how AOPs could be applied to effluent testing (Brown et al. 2016). The NC3Rs has been supporting the development of pathway based approaches as there is an expectation that AOPs will improve interpretation of in vitro test methods and ultimately result in the reduction in the number of animal tests required in pharmaceutical development and chemical risk assessment.

Recent interest in AOPs has included AOP Wiki, a database which forms part of the Organisation for Economic Co-operation and Development (OECD) sponsored knowledgebase where scientists can share and develop their information on AOPs. Of the AOPs currently in the database few have gone through a full peer review process and many vary considerably in detail (i.e. number of KEs) and specificity (e.g. many relate to a particular species or organism life stage). The holy grail is a fully validated AOP which is quantifiable i.e. where measurement of a KE can be clearly linked to a MIE which can be used to predict the magnitude of the adverse effect or population outcome. This concept has been demonstrated using the AOP “Aromatase inhibition leading to reproductive dysfunction (in fish)” (Villeneuve et al. 2014), however, due to the time and effort required to characterise such an AOP this is expected to be the exception rather than the rule.

At the NC3Rs meeting it was agreed that in vitro methods and AOP approaches were not perfect for risk assessment but that neither were the current methods. It was also considered that for many applications (e.g. read across or hazard assessments) the availability of a fully quantified AOP would not necessarily limit their application. Success stories were highlighted with AOPs related to endocrine activity being the most promising for use in risk assessment. This is supported by a recent UBA report (Muth-Koehne et al. 2016) although as highlighted at the NC3Rs meeting these AOPs for endocrine activity still need to be interpreted with caution (e.g. Wheeler and Coady 2016). AOPs have also been credited in providing the relevant information to provide support for the recent adoption by a REACH Committee of member state officials of in vitro models for assessing skin sensitisation (ChemWatch 2016b). This is based on the AOP for skin sensitisation initiated by covalent binding to proteins (OECD 2014).

In routine chemical assessment, however, AOPs or even mechanistic data are rarely taken into consideration and based on the outcomes from the ECHA workshop this is unlikely to change in the short term. To counter this, scientists at the NC3Rs meeting were encouraged to submit available information obtained using NAM or AOP methods alongside their traditional data to regulators to help improve understanding and acceptance. It was recommended that when presenting data from new approaches that the uncertainties in the methods are clearly detailed so that risk assessors can make objective assessments on how the data can be used.

The European Commission continues its investment in the AOP approach with the EU ToxRisk project focusing on “Mechanism-based Toxicity Testing and Risk Assessment for the 21st Century” a Horizon 2020 project starting in January 2016. The project aims to build on existing testing strategies and knowledge to establish novel alternative testing strategies that are fit for regulatory purposes (http://www.eu-toxrisk.eu/). Scientific confidence in AOPs could also be increased by using computer models to predict a pathway's possible molecular initiators (Perkins et al. 2015).

The use of AOPs for regulatory purposes will also be the subject of a Society of Environmental Toxicology and Chemistry (SETAC) Pellston Workshop® in 2017 and SETAC are currently looking for questions to be discussed at the workshop that will address the key outstanding challenges or limitations associated with using the AOP framework in research and regulatory decision-making. Questions should be submitted by the 30th June 2016 by following this link.


ChemWatch 2016a. New approach methodologies face huge barriers in supporting read-across. Article 28th April 2016.

ChemWatch. 2016b. REACH Committee approves annex amendments for skin sensitisation. In vitro and in chemico tests, with in vivo only as last resort. Article 21st April 2016

Muth-Koehne E, Teigeler M, Fenske M. 2016. Novel assessment methods in ecotoxicology for the identification of hormonal active substances: Combining the fish sexual development test with gene expression endpoints. Report on behalf of the Federal Environment Agency (Germany).

OECD. 2014. OECD Series on Testing and Assessment. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. DOI 10.1787/9789264221444-en

Perkins EJ, Antczak P, Burgoon L, Falciani F, Garcia-Reyero N, Gutsell G, Hodges G, Kienzler A, Knapen D, McBride M, Willett C. 2015. Adverse outcome pathways for regulatory applications: examination of four case studies with different degrees of completeness and scientific confidence. Toxicol. Sci., 148 (2015), pp. 14–25.

Villeneuve DL, Crump D, Garcia-Reyero N, Hecker M, Hutchinson TH, LaLone CA, Landesmann B, Lettieri T, Munn S, Nepelska M, Ottinger MA, Vergauwen L. Whelan M. 2014. Adverse Outcome Pathway Development II: Best Practices. Toxicol. Sci. 142:321-330.

Wheeler JR, Coady K. 2016. Are all chemicals endocrine disruptors? Integrated Environmental Assessment and Management 12:2:402–403