Andrea Terron

Adverse outcome pathways: opportunities, limitations and open questions

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.

OECD/EFSA workshop on developmental neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes

311 cluding: neural proliferation, differentiation, migration, neurite outgrowth, synaptogenesis, myelin formation, and neural network formation and function. Many of these human cell-based assays have been used to study small numbers of chemicals (n < 15; e.g., Harrill et al., 2011; He et al., 2012; Rempel et al., 2015; Baumann et al., 2016; Brown et al., 2016) or to derive mechanistic information for limited numbers of chemicals (e.g., Gassmann et al., 2010; Balmer et al., 2012; Balmer and Leist, 2014; Barenys et al., 2016). Only a few have been utilized to screen larger numbers (n > 15) of compounds (e.g., Stiegler et al., 2011; Zimmer et al., 2012; Culbreth et al., 2012; McConnell et al., 2012; Krug et al., 2013; Valdivia et al., 2014; Mundy et al., 2015; Hoelting et al., 2016; Nyffeler et al., 2016). On the scientific premise that alternative methods are available and can be assembled into a larger DNT screening battery, a joint OECD/EFSA workshop was held in Brussels on October 18 and 19, 2016 that aimed to facilitate the use of such methods in regulatory decision making. Specific objectives of this workshop were: 1. Development of a consensus that the proposed testing battery of alternative DNT methods is ready to be applied right now, and could be used in a fit-for-purpose manner for either screening and prioritization, or as a first starting point to conduct targeted testing in a tiered testing approach in the process of hazard identification and characterization for specific chemical risk assessment. 2. Identification of the next steps necessary to encourage the regulatory use of the alternative methods depending on their level of readiness. 3. Outline what could become an integrated approach to testing and assessment (IATA) for the purposes of screening and prioritization or hazard assessment. The meeting was co-chaired by Ellen Fritsche (Leibniz Research Institute for Environmental Medicine, IUF) and Kevin Crofton (US Environmental Protection Agency, US EPA). Meeting participants and their affiliations are reported in the supplementary file at https://doi.org/10.14573/altex.1701171s. Scientists from 15 countries across the world, representing stakeholders from regulatory agencies, non-governmental organizations (NGOs), academia and industry, reached a consensus that current data requirements for in vivo developmental neurotoxicity (DNT) testing are not sufficient to screen and characterize potentially hazardous compounds. In addition, there was agreement on the need to develop a standardized in vitro testing battery to generate additional data on the effects of chemicals on the developing nervous system. The need for more effective DNT screening is driven by the scientific fact that the developing nervous system might be more sensitive to exposures to some chemical classes of hazardous substances. In addition, recent societal concerns have been raised linking the rise in children’s neurodevelopmental impairments (e.g., learning disabilities) to chemical exposures. Despite a clear deficit in knowledge concerning DNT effects, only approximately 140 in vivo guideline studies (according to OECD 426 & EPA OPPTS 870.630) have been conducted to date, leaving a huge data gap on the DNT potential of chemicals within the universe of thousands of compounds present in industrial, agricultural and consumer products. This deficit is mainly due to the fact that currently accepted guideline studies are at present not mandatory data requirements and are extremely timeand costintensive. Additionally, they can result in methodological and scientific uncertainties. This includes the challenges in extrapolation of findings from rats to humans that result from timing differences in brain development, toxicokinetics, and inherent difficulties in the use of non-homologous functional tests (Tsuji and Crofton, 2012; Dorman et al., 2001; Kaufmann, 2003). For these reasons, DNT has been regarded as an area in need of the development of alternative methods in order to establish a timeand cost-efficient predictive testing strategy. A series of workshops held over the past decade (Lein et al., 2007; Crofton et al., 2011; Bal-Price et al., 2012, 2015a) have fostered the development of in vitro assays or methods using alternative model organisms that assess the impact of chemicals on cellular processes critical to normal brain development, inMeeting report

Guidance on the use of the weight of evidence approach in scientific assessments

Abstract EFSA requested the Scientific Committee to develop a guidance document on the use of the weight of evidence approach in scientific assessments for use in all areas under EFSAs remit. The guidance document addresses the use of weight of evidence approaches in scientific assessments using both qualitative and quantitative approaches. Several case studies covering the various areas under EFSAs remit are annexed to the guidance document to illustrate the applicability of the proposed approach. Weight of evidence assessment is defined in this guidance as a process in which evidence is integrated to determine the relative support for possible answers to a question. This document considers the weight of evidence assessment as comprising three basic steps: (1) assembling the evidence into lines of evidence of similar type, (2) weighing the evidence, (3) integrating the evidence. The present document identifies reliability, relevance and consistency as three basic considerations for weighing evidence.

An adverse outcome pathway for parkinsonian motor deficits associated with mitochondrial complex I inhibition

Epidemiological studies have observed an association between pesticide exposure and the development of Parkinson’s disease, but have not established causality. The concept of an adverse outcome pathway (AOP) has been developed as a framework for the organization of available information linking the modulation of a molecular target [molecular initiating event (MIE)], via a sequence of essential biological key events (KEs), with an adverse outcome (AO). Here, we present an AOP covering the toxicological pathways that link the binding of an inhibitor to mitochondrial complex I (i.e., the MIE) with the onset of parkinsonian motor deficits (i.e., the AO). This AOP was developed according to the Organisation for Economic Co-operation and Development guidelines and uploaded to the AOP database. The KEs linking complex I inhibition to parkinsonian motor deficits are mitochondrial dysfunction, impaired proteostasis, neuroinflammation, and the degeneration of dopaminergic neurons of the substantia nigra. These KEs, by convention, were linearly organized. However, there was also evidence of additional feed-forward connections and shortcuts between the KEs, possibly depending on the intensity of the insult and the model system applied. The present AOP demonstrates mechanistic plausibility for epidemiological observations on a relationship between pesticide exposure and an elevated risk for Parkinson’s disease development.

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

Peer review of the pesticide risk assessment for the triazole derivative metabolites in light of confirmatory data submitted

Abstract The conclusions of EFSA following the peer review of the initial risk assessment carried out by the competent authority of the rapporteur Member State, the United Kingdom, for the pesticide risk assessment for the triazole derivative metabolites are reported. The context of the peer review was that requested by the European Commission following the submission and evaluation of confirmatory data in relation to mammalian toxicology, metabolism and residue data. The conclusions were reached on the basis of the evaluation of various uses for a number of triazole fungicides. Recommendations are proposed. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.

Peer review of the targeted hazard assessment of the pesticide active substance quinoxyfen

Abstract The conclusions of EFSA following the peer review of the initial assessments carried out by the competent authorities of the rapporteur Member State, the United Kingdom, and co‐rapporteur Member State, Austria, for the pesticide active substance quinoxyfen are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of information targeted at the assessment of the potential persistent, bioaccumulative and toxic (PBT), very persistent and very bioaccumulative (vPvB) and persistent organic pollutant (POP) properties of quinoxyfen according to Article 11(2) of Regulation (EC) No 1107/2009. The reliable end points, appropriate for use in these regulatory hazard cut off assessments are presented. Missing information identified as being required by the regulatory framework is listed. The concern is identified that quinoxyfen may be considered to exhibit the hazard properties of both a PBT and vPvB substance considering the triggers specified in Annex II of Regulation (EC) No 1107/2009.

Peer review of the pesticide risk assessment of the active substance pethoxamid

Abstract The conclusions of the EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Austria, and co‐rapporteur Member State, the Czech Republic, for the pesticide active substance pethoxamid are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of pethoxamid as a herbicide on maize and soya bean. The reliable endpoints, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.

Peer review of the pesticide risk assessment of the active substance mepanipyrim

Abstract The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Belgium, and co‐rapporteur Member State, Greece, for the pesticide active substance mepanipyrim are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of mepanipyrim as a fungicide on table and wine grapes, and in field and protected strawberries and tomatoes. The reliable end points, appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.

Peer review of the pesticide risk assessment for the active substance terbuthylazine in light of confirmatory data submitted

Abstract The conclusions of EFSA following the peer review of the initial risk assessment carried out by the competent authority of the rapporteur Member State, the United Kingdom, for the pesticide active substance metazachlor are reported. The context of the peer review was that requested by the European Commission following the submission and evaluation of confirmatory data regarding the groundwater exposure of metabolites and their toxicological relevance triggering an assessment. The conclusions were reached on the basis of the evaluation of the representative uses of metazachlor as a herbicide on winter and spring rapeseed and on ornamental trees and shrubs. The reliable endpoints concluded as being appropriate for use in regulatory risk assessment, derived from the available studies and literature in the dossier peer reviewed, are presented. Concerns are identified.