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The Organisation for Economic Co-Operation and Development (OECD) coordinates international efforts to enhance developmental neurotoxicity (DNT) testing. In most regulatory sectors, including the ones dealing with pesticides and industrial chemicals registration, historical use of the in vivo DNT test guideline has been limited. Current challenges include a lack of DNT data and mechanistic information for thousands of chemicals, and difficulty in interpreting results. A series of workshops in the last decade has paved the way for a consensus among stakeholders that there is need for a DNT testing battery that relies on in vitro endpoints (proliferation, differentiation, synaptogenesis, etc.) and is complemented by alternative species (eg, zebrafish) assays. Preferably, a battery of in vitro and alternative assays should be anchored toward mechanistic relevance for applying an integrated approach for testing and assessment (IATA) framework. Specific activities have been initiated to facilitate this OECD project: the collation of available DNT in vitro methods and their scoring for readiness; the selection of these methods to form a DNT testing battery; the generation of a reference set of chemicals that will be tested using the battery; the case studies exemplifying how DNT in vitro data can be interpreted; and the development of an OECD guidance document. This manuscript highlights these international efforts and activities.

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).

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.

The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways., Competing Interests: Declaration of interest The employment affiliation of the authors is shown on the cover page. The authors have sole responsibility for the writing and content of this paper. The contributing authors were participants of the workshop organized by the EURL ECVAM. The external workshop participants were invited on the basis of a survey performed by EURL ECVAM neurotoxicity experts of the latest literature to identify those with specific expertise in the relevant research fields. The workshop organization was financially supported by the European Commission, including the cost of travelling and per diem of all invited external experts. The strategy for preparing the report, the literature selected for review, the conclusions drawn and the recommendations made are exclusively the collective scientific output of the workshop participants and do not necessarily represent the views of the participants’ employers.

Due to extensive use, human exposure to multiple pyrethroid insecticides occurs frequently. Studies of pyrethroid neurotoxicity suggest a common mode of toxicity and that pyrethroids should be considered cumulatively to model risk. The objective of this work was to use a pyrethroid mixture that reflects human exposure to common pyrethroids to develop comparative toxicokinetic profiles in rats, and then model the relationship between brain concentration and motor activity. Data from a national survey of child care centers were used to make a mixture reflecting proportions of the most prevalent pyrethroids: permethrin, cypermethrin, β-cyfluthrin, deltamethrin, and esfenvalerate. The mixture was administered orally at one of two concentrations (11.2 and 27.4 mg/kg) to adult male rats. At intervals from 1 to 24h, motor activity was assessed and the animals were sacrificed. Pyrethroid concentrations were measured in the blood, liver, fat, and brain. After controlling for dose, there were no differences in any tissue concentrations, except blood at the initial time point. Elimination half-lives for all pyrethroids in all tissues were < 7h. Brain concentrations of all pyrethroids (when cis- and trans-permethrin were pooled) at the initial time point were proportional to their relative doses. Decreases in motor activity indicated dose additivity, and the relationship between pyrethroid brain concentration and motor activity was described by a four-parameter sigmoidal E(max) model. This study links environmental data with toxicokinetic and neurobehavioral assays to support cumulative risk assessments of pyrethroid pesticides. The results support the additive model of pyrethroid effect on motor activity and suggest that variation in the neurotoxicity of individual pyrethroids is related to toxicodynamic rather than toxicokinetic differences.

Great progress has been made over the past 40 years in understanding the hazards of exposure to a small number of developmental neurotoxicants. Lead, polychlorinated biphenyls, and methylmercury are all good examples of science-based approaches to characterizing the hazard to the developing nervous systems from environmental contaminants. However, very little effort has been spent to address the challenge of assessing the potential developmental neurotoxic hazard of the thousands of other chemicals in common commercial use. The extensive time, financial and animal resource requirements for current regulatory testing guideline methods make this an untenable solution to this challenge. A new testing paradigm is needed that uses time and cost-efficient methods to screen large numbers of chemicals for developmental neurotoxicity (DNT). In silico models are needed to provide rapid chemical structure-based screening. In vitro techniques are being developed to provide rapid and efficient testing in cell-free and cell-based systems. In addition, the use of alternative species, such as zebrafish, will provide efficient models for testing the effects of chemicals in organisms with intact developing nervous systems. Finally, these methods and models need to be used in an integrated fashion to provide the data needs for hazard assessment in a manner that is problem-driven and cost-efficient. This paper summarizes discussions on these issues from the symposium 'Developmental neurotoxicity testing: Scientific approaches towards the next generation to protecting the developing nervous system of children' held at the 2011 annual meeting of the Japanese Teratology Society., (Published 2012. This article is a U.S. Government work and is in the public domain in the USA.)

Recently social concerns have been increasing about the effects of environmental factors on children's health, especially on their nervous systems. The U.S. Environmental Protection Agency (EPA) and the Organisation for Economic Co-operation and Development (OECD) have published testing guidelines for developmental neurotoxicity (DNT). Approximately 110 guideline studies have been conducted to date. Importantly, information from these studies has provided data critical for regulatory decisions for a number of chemicals. However, the DNT guidelines do not always satisfy all stakeholders because of some uncertainties in their methodology, evaluation, and regulation. Methodological issues include incomplete harmonization between EPA and OECD guidelines, criticisms of the methodology for learning and memory testing, and unspecified positive control substances. Potential artifacts in morphometric neuropathological measures, criteria for observation measures, uncertainty of postnatal offspring exposure, especially in feeding studies, and extrapolation of data from rats to humans are major evaluation issues. In addition, there is some uncertainty in the use of an additional safety factor for susceptibility of infants and children. Moreover, the DNT guidelines have extensive time and cost requirements, use large numbers of animals, and there is a limited set of laboratories that can conduct the study. This paper reviews some of these issues and summarizes discussions from the symposium 'Developmental neurotoxicity testing: Scientific approaches towards the next generation to protecting the developing nervous system of children' held at the 2011 annual meeting of the Japanese Teratology Society., (© 2012 The Authors. Congenital Anomalies © 2012 Japanese Teratology Society.)

Developmental neurotoxicity testing (DNT) is perceived by many stakeholders to be an area in critical need of alternative methods to current animal testing protocols and guidelines. An immediate goal is to develop test methods that are capable of screening large numbers of chemicals. This document provides recommendations for developing alternative DNT approaches that will generate the type of data required for evaluating and comparing predictive capacity and efficiency across test methods and laboratories. These recommendations were originally drafted to stimulate and focus discussions of alternative testing methods and models for DNT at the TestSmart DNT II meeting (http://caat.jhsph.edu/programs/workshops/dnt2.html) and this document reflects critical feedback from all stakeholders that participated in this meeting. The intent of this document is to serve as a catalyst for engaging the research community in the development of DNT alternatives and it is expected that these recommendations will continue to evolve with the science.

Developmental neurotoxicity testing involves functional and neurohistological assessments in offspring during and following maternal and/or neonatal exposure. Data from positive control studies are an integral component in developmental neurotoxicity risk assessments. Positive control data are crucial for evaluating a laboratory's capability to detect chemical-induced changes in measured endpoints. Positive control data are also valuable in a weight-of-evidence approach to help determine the biological significance of results and provide confidence in negative results from developmental neurotoxicity (DNT) studies. This review is a practical guide for the selection and use of positive control agents in developmental neurotoxicology. The advantages and disadvantages of various positive control agents are discussed for the endpoints in developmental neurotoxicity studies. Design issues specific to positive control studies in developmental neurotoxicity are considered and recommendations on how to interpret and report positive control data are made. Positive control studies should be conducted as an integral component of the incorporation and use of developmental neurotoxicity testing methods in laboratories that generate data used in risk decisions.

Pyrethroids are pesticides with high insecticidal activity and relatively low potency in mammals. The influence of dosing volume on the neurobehavioral syndrome following oral acute exposure to the Type-I pyrethroid insecticide bifenthrin in corn oil was evaluated in adult male Long Evans rats. We tested bifenthrin effects at 1 and 5 ml/kg, two commonly used dose volumes in toxicological studies. Two testing times (4 and 7 h) were used in motor activity and functional observational battery (FOB) assessments. Four to eight doses were examined at either dosing condition (up to 20 or 26 mg/kg, at 1 and 5 ml/kg, respectively). Acute oral bifenthrin exposure produced toxic signs typical of Type I pyrethroids, with dose-related increases in fine tremor, decreased motor activity and grip strength, and increased pawing, head shaking, click response, and body temperature. Bifenthrin effects on motor activity and pyrethroid-specific clinical signs were approximately 2-fold more potent at 1 ml/kg than 5 ml/kg. This difference was clearly evident at 4 h and slightly attenuated at 7 h post-dosing. Benchmark dose (BMD) modeling estimated similar 2-fold potency differences in motor activity and pyrethroid-specific FOB data. These findings demonstrate that dose volume, in studies using corn oil as the vehicle influences bifenthrin potency. Further, these data suggest that inconsistent estimates of pyrethroid potency between laboratories are at least partially due to differences in dosing volume.

The use of ozonation in the purification of drinking water can lead to the formation of bromate. The current regulatory challenges for bromate contamination of drinking water include the need to assess potential human health risks. One health risk of concern is developmental neurotoxicity. Currently, the need for a developmental neurotoxicity study for bromate, based on the weight of evidence, is uncertain. Bromate induces neurotoxicity in adults at high acute exposures and produces hearing loss and structural damage in the cochlea in humans and rodents. However, there is a wide margin of exposure in these studies compared to environmental levels of bromate in water supplies. Data on the effects of bromate on thyroid hormone levels is not consistent and thyroid endocrine disruption is not likely a causative factor in thyroid tumor formation. There is no evidence that bromate caused central nervous system malformations, brain weight changes in developmental studies, nor are there any known structure-activity relationships to other known neurotoxicants. A prudent approach to reduce the uncertainty in the need for a developmental neurotoxicity study of exposure to bromate in drinking water should include determinations of whether bromate ototoxicity occurs with extended duration, low concentration exposures. These studies would provide invaluable data for the weight-of-evidence approach used to determine the necessity of a developmental neurotoxicity study of bromate.

A one and a half day workshop on behavioral testing was conducted in order to discuss experimental procedures and practices that may help enhance the utility of behavioral data as a reliable index of neurotoxicity and in the safety evaluation of chemical substances. The workshop was open to participation by all sectors of the neuroscience community including academia, government, testing laboratories, and industry. The level of confidence with which changes in behavior can reliably signal adverse effects on the nervous system depends, in part, on the scientific quality of the data generated. With an emphasis on education and problem solving, the workshop focused on the practical aspects and scientific rationale underlying valid and high quality testing. In behavioral testing, there are numerous experimental factors that may impact on the quality of data. These include such elements as experimental design, selection of test methods, the care and precision in the conduct of behavioral testing, procedures to minimize bias and potential confounds, appropriateness of statistical analyses, and data interpretation. In plenary session investigators experienced in behavioral testing discussed the significance of these various experimental factors to data quality, outlined problematic issues, and presented a synopsis of approaches for addressing each of the factors as outlined in a draft of a primer developed by the Interagency Committee on Neurotoxicology (ICON). During the remainder of the workshop, open discussions in small breakout groups were used to address the problematic issues identified by the plenary speakers and explore alternative approaches for dealing with them. Finally, all workshop participants were reconvened in plenary session for summation of breakout group discussions and final recommendations. Information from the workshop was used to form the basis of this manuscript and will be used to help finalize a behavioral test methods primer being drafted by the ICON. The overall conclusions from the workshop were that consensus can be reached on the fundamentals of behavioral assessment, and that aspects of behavioral assessment including experimental design, test method selection, training, validation, control of confounds, data variability, data analysis, and data interpretation need to be carefully considered in the planning and conduct of behavioral safety assessments.

Pyrethroid insecticides have been used for more than 40 years and account for 25% of the worldwide insecticide market. Although their acute neurotoxicity to adults has been well characterized, information regarding the potential developmental neurotoxicity of this class of compounds is limited. There is a large age dependence to the acute toxicity of pyrethroids in which neonatal rats are at least an order of magnitude more sensitive than adults to two pyrethroids. There is no information on age-dependent toxicity for most pyrethroids. In the present review we examine the scientific data related to potential for age-dependent and developmental neurotoxicity of pyrethroids. As a basis for understanding this neurotoxicity, we discuss the heterogeneity and ontogeny of voltage-sensitive sodium channels, a primary neuronal target of pyrethroids. We also summarize 22 studies of the developmental neurotoxicity of pyrethroids and review the strengths and limitations of these studies. These studies examined numerous end points, with changes in motor activity and muscarinic acetylcholine receptor density the most common. Many of the developmental neurotoxicity studies suffer from inadequate study design, problematic statistical analyses, use of formulated products, and/or inadequate controls. These factors confound interpretation of results. To better understand the potential for developmental exposure to pyrethroids to cause neurotoxicity, additional, well-designed and well-executed developmental neurotoxicity studies are needed. These studies should employ state-of-the-science methods to promote a greater understanding of the mode of action of pyrethroids in the developing nervous system.