Susanne Hougaard Bennekou

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes

Abstract A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Guidance on dermal absorption

Abstract This guidance on the assessment of dermal absorption has been developed to assist notifiers, users of test facilities and Member State authorities on critical aspects related to the setting of dermal absorption values to be used in risk assessments of active substances in Plant Protection Products (PPPs). It is based on the ‘scientific opinion on the science behind the revision of the guidance document on dermal absorption’ issued in 2011 by the EFSA Panel on Plant Protection Products and their Residues (PPR). The guidance refers to the EFSA PPR opinion in many instances. In addition, the first version of this guidance, issued in 2012 by the EFSA PPR Panel, has been revised in 2017 on the basis of new available data on human in vitro dermal absorption for PPPs and wherever clarifications were needed. Basic details of experimental design, available in the respective test guidelines and accompanying guidance for the conduct of studies, have not been addressed but recommendations specific to performing and interpreting dermal absorption studies with PPPs are given. Issues discussed include a brief description of the skin and its properties affecting dermal absorption. To facilitate use of the guidance, flow charts are included. Guidance is also provided, for example, when there are no data on dermal absorption for the product under evaluation. Elements for a tiered approach are presented including use of default values, data on closely related products, in vitro studies with human skin (regarded to provide the best estimate), data from experimental animals (rats) in vitro and in vivo, and the so called ‘triple pack’ approach. Various elements of study design and reporting that reduce experimental variation and aid consistent interpretation are presented. A proposal for reporting data for assessment reports is also provided. The issue of nanoparticles in PPPs is not addressed. Data from volunteer studies have not been discussed since their use is not allowed in EU for risk assessment of PPPs.

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

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.

Towards a regulatory use of alternative developmental neurotoxicity testing (DNT)

&NA; There is a need for a more effective Developmental Neurotoxicity (DNT) screening which is scientifically driven by the fact that the developing nervous system might be more sensitive to exposures to some hazardous chemical. Additional concern comes from the recent societal concerns that toxic chemicals can contribute to the prevalence of neurodevelopment disabilities. Consequently, hazard identification and actions to reduce exposure to these chemicals is a priority in chemical risk assessment. To reach this goal a cost‐efficient testing strategy based on a reliable in‐vitro testing battery should be developed. Although this goal is representing a huge challenge in risk assessment, available data and methodologies are supporting the ultimate aim of developing a predictive model able to respond to different regulatory based problem formulations. HighlightsEpidemiological observations indicate exposure to chemicals contributing to neurodevelopmental disabilities.Insufficient information on DNT.In vivo toxicological studies are not adequate to evaluate a large number of chemicals.A cost‐effective in vitro testing battery should be developed.The ultimate aim is to build a predictive, valid and flexible model.

Scientific Opinion about the Guidance of the Chemical Regulation Directorate (UK) on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessments

Abstract The EFSA Panel on Plant Protection Products and their Residues reviewed the guidance on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessment. The inclusion of aged sorption is a higher tier in the groundwater leaching assessment. The Panel based its review on a test with three substances taken from a data set provided by the European Crop Protection Association. Particular points of attention were the quality of the data provided, the proposed fitting procedure of aged sorption experiments and the proposed method for combining results obtained from aged sorption studies and lower‐tier studies on degradation and adsorption. Aged sorption was a relevant process in all cases studied. The test revealed that the guidance could generally be well applied and resulted in robust and plausible results. The Panel considers the guidance suitable for use in the groundwater leaching assessment after the recommendations in this Scientific Opinion have been implemented, with the exception of the use of field data to derive aged sorption parameters. The Panel noted that the draft guidance could only be used by experienced users because there is no software tool that fully supports the work flow in the guidance document. It is therefore recommended that a user‐friendly software tool be developed. Aged sorption lowered the predicted concentration in groundwater. However, because aged sorption experiments may be conducted in different soils than lower‐tier degradation and adsorption experiments, it cannot be guaranteed that the higher tier predicts lower concentrations than the lower tier, while lower tiers should be more conservative than higher tiers. To mitigate this problem, the Panel recommends using all available higher‐ and lower‐tier data in the leaching assessment. The Panel further recommends that aged sorption parameters for metabolites be derived only from metabolite‐dosed studies. The formation fraction can be derived from parent‐dosed degradation studies, provided that the parent and metabolite are fitted with the best‐fit model, which is the double first‐order in parallel model in the case of aged sorption.

Scientific opinion on pesticides in foods for infants and young children

Abstract Following a request from the European Commission, the EFSA Panel on Plant Protection Products and their Residues (PPR Panel) prepared a scientific opinion to provide a comprehensive evaluation of pesticide residues in foods for infants and young children. In its approach to develop this scientific opinion, the EFSA PPR Panel took into account, among the others, (i) the relevant opinions of the Scientific Committee for Food setting a default maximum residue level (MRL) of 0.01 mg/kg for pesticide residues in foods for infants and young children; (ii) the recommendations provided by EFSA Scientific Committee in a guidance on risk assessment of substances present in food intended for infants below 16 weeks of age; (iii) the knowledge on organ/system development in infants and young children. For infants below 16 weeks of age, the EFSA PPR Panel concluded that pesticide residues at the default MRL of 0.01 mg/kg for food for infants and young children are not likely to result in an unacceptable exposure for active substances for which a health‐based guidance value (HBGV) of 0.0026 mg/kg body weight (bw) per day or higher applies. Lower MRLs are recommended for active substances with HBGVs below this value. For infants above 16 weeks of age and young children, the established approach for setting HBGVs is considered appropriate. For infants below 16 weeks of age the approach may not be appropriate and the application of the EFSA guidance on risk assessment of substances present in food intended for infants below 16 weeks of age is recommended. The contribution of conventional food to the total exposure to pesticide residues is much higher than that from foods intended for infants and young children. Because of the increased intake of conventional food by young children, these have the highest exposure to pesticide residues, whereas infants 3–6 months of age generally have lower exposure. The impact of cumulative exposure to pesticide residues on infants and young children is not different from the general population and the EFSA cumulative risk assessment methodology is also applicable to these age groups. Residue definitions established under Regulation (EC) No 396/2005 are in general considered appropriate also for foods for infants and young children. However, based on a tier 1 analysis of the hydrolysis potential of pesticides simulating processing, the particular appropriateness of existing residue definitions for monitoring to cover processed food, both intended for infants and young children as well as conventional food, is questionable.