Developmental Neurotoxicity Testing Research Articles

New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project.

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

Recent advances and current challenges of new approach methodologies in developmental and adult neurotoxicity testing.

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.

Revolutionizing developmental neurotoxicity testing - a journey from animal models to advanced in vitro systems.

Developmental neurotoxicity (DNT) testing has seen enormous progress over the last two decades. Preceding even the publication of the animal-based OECD test guideline for DNT testing in 2007, a series of non-animal technology workshops and conferences (starting in 2005) shaped a community that has delivered a comprehensive battery of in vitro test methods (IVB). Its data interpretation is covered by a very recent OECD test guidance (No. 377). Here, we aim to overview the progress in the field, focusing on the evolution of testing strategies, the role of emerging technologies, and the impact of OECD test guidelines on DNT testing. In particular, this is an example of a targeted development of an animal-free testing approach for one of the most complex hazards of chemicals to human health. These developments started literally from a blank slate, with no proposed alternative methods available. Over two decades, cutting-edge science enabled the design of a testing approach that spares animals and enables throughput for this challenging hazard. While it is evident that the field needs guidance and regulation, the massive economic impact of decreased human cognitive capacity caused by chemical exposure should be prioritized more highly. Beyond this, the claim to fame of DNT in vitro testing is the enormous scientific progress it has brought for understanding the human brain, its development, and how it can be perturbed.

A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

Initial phase establishment of an in vitro method for developmental neurotoxicity test using Ki-67 in human neural progenitor cells.

Building a precise alternative neurotoxicological test is of great importance to respond to societal and ethical requirements. In this study, a new developmental neurotoxicity test (DNT) was established with the human neural progenitor cell line. ReNcell CX cells were exposed to neurotoxic chemicals (aphidicolin, hydroxyurea, cytosine arabinoside, 5-fluorouracil, and ochratoxin A) or non-neurotoxic chemicals (sodium gluconate, sodium bicarbonate, penicillin G, and saccharin). Propidium iodide (PI) was used to evaluate cell viability. BrdU and Ki-76 were employed to determine cell proliferation. Based on the cell viability and proliferation, mathematical models were built by linear discriminant analysis. Furthermore, the neurotoxic-considered chemicals inhibited cell cycle progression at the protein level, supporting the biomolecular rationale for the predictive model. Overall, these results show that the new test method can be used to determine the potential developmental neurotoxicants or new drug candidates.

Assessment of neurotransmitter release in human iPSC-derived neuronal/glial cells: a missing in vitro assay for regulatory developmental neurotoxicity testing.

Human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) and their differentiated neuronal/glial derivatives have been recently considered suitable to assess in vitro developmental neurotoxicity (DNT) triggered by exposure to environmental chemicals. The use of human-relevant test systems combined with in vitro assays specific for different neurodevelopmental events, enables a mechanistic understanding of the possible impact of environmental chemicals on the developing brain, avoiding extrapolation uncertainties associated with in vivo studies. Currently proposed in vitro battery for regulatory DNT testing accounts for several assays suitable to study key neurodevelopmental processes, including NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation. However, assays suitable to measure interference of compounds with neurotransmitter release or clearance are at present not included, which represents a clear gap of the biological applicability domain of such a testing battery. Here we applied a HPLC-based methodology to measure the release of neurotransmitters in a previously characterized hiPSC-derived NSC model undergoing differentiation towards neurons and glia. Glutamate release was assessed in control cultures and upon depolarization, as well as in cultures repeatedly exposed to some known neurotoxicants (BDE47 and lead) and chemical mixtures. Obtained data indicate that these cells have the ability to release glutamate in a vesicular manner, and that both glutamate clearance and vesicular release concur in the maintenance of extracellular glutamate levels. In conclusion, analysis of neurotransmitter release is a sensitive readout that should be included in the envisioned battery of in vitro assays for DNT testing.

Evaluation of Per- and Polyfluoroalkyl Substances (PFAS) In Vitro Toxicity Testing for Developmental Neurotoxicity.

Per- and polyfluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors.

New Worm on the Block: Planarians in (Neuro)Toxicology.

Traditional mammalian testing is too time- and cost-intensive to keep up with the large number of environmental chemicals needing assessment. This has led to a dearth of information about the potential adverse effects of these chemicals, especially on the developing brain. Thus, there is an urgent need for rapid and cost-effective neurotoxicity and developmental neurotoxicity testing. Because of the complexity of the brain, metabolically competent organismal models are necessary to understand the effects of chemicals on nervous system development and function on a systems level. In this overview, we showcase asexual freshwater planarians as an alternative invertebrate ("non-animal") organismal model for neurotoxicology research. Planarians have long been used to study the effects of chemicals on regeneration and behavior. But they have only recently moved back into the spotlight because modern molecular and computational approaches now enable quantitative high-content and high-throughput toxicity studies. Here, we present a short history of the use of planarians in toxicology research, highlight current techniques to measure toxicity qualitatively and quantitatively in planarians, and discuss how to further promote this non-animal organismal system into mainstream toxicology research. The articles in this collection will help work towards this goal by providing detailed protocols that can be adopted by the community to standardize planarian toxicity testing. © 2022 Wiley Periodicals LLC.

Neuronal differentiation reporter mice as a new methodology for detecting in vivo developmental neurotoxicity

Current in vivo developmental neurotoxicity (DNT) tests are not performed routinely for chemical risk assessment because they are time and resource intensive and require many animals. Therefore, new methodologies are required that can detect and evaluate the DNT potential of chemicals in a more simple, quantitative, and objective manner. Toward this end, we generated transgenic mice expressing reporter genes (luciferase and lacZ) under the control of the rat synapsin 1 promoter (Syn-Rep mice) and evaluated their usefulness as a DNT detection tool. Brain luciferase expression levels in Syn-Rep mice increased dramatically from just before to after birth, peaked early in the postnatal period, subsequently decreased sharply, and then remained low after weaning. This pattern is analogous to the generally recognized temporal changes in synapse numbers in the developing mammal brain. To evaluate further the responsiveness of Syn-Rep mice during DNT induction, we administered valproic acid (VPA), a reference DNT-inducing chemical, to pregnant mice and evaluated its effect on reporter gene expression in the developing brains of Syn-Rep pups. In vivo luminescence in the brains of VPA-exposed pups was significantly lower than in controls from postnatal days 4 to 13. Moreover, luciferase activity in the prefrontal cortexes of 8-week-old VPA-exposed offspring was significantly lower than in controls, reflecting the reduced number of neurons in the prefrontal cortex. These results suggest that the Syn-Rep mice are potentially useful tools for streamlined detection of chemical-induced DNT in the developing mammalian brain.

Neuronal differentiation pathways and compound-induced developmental neurotoxicity in the human neural progenitor cell test (hNPT) revealed by RNA-seq.

There is an increased awareness that the use of animals for compound-induced developmental neurotoxicity (DNT) testing has limitations. Animal-free innovations, especially the ones based on human stem cell-based models are pivotal in studying DNT since they can mimic processes relevant to human brain development. Here we present the human neural progenitor test (hNPT), a 10-day protocol in which neural progenitor cells differentiate into a neuron-astrocyte co-culture. The study aimed to characterise differentiation over time and to find neurodevelopmental processes sensitive to compound exposure using transcriptomics. 3992 genes regulated in unexposed control cultures (p ≤ 0.001, log2FC ≥ 1) showed Gene Ontology (GO-) term enrichment for neuronal and glial differentiation, neurite extension, synaptogenesis, and synaptic transmission. Exposure to known or suspected DNT compounds (acrylamide, chlorpyrifos, fluoxetine, methyl mercury, or valproic acid) at concentrations resulting in 95% cell viability each regulated unique combinations of GO-terms relating to neural progenitor proliferation, neuronal and glial differentiation, axon development, synaptogenesis, synaptic transmission, and apoptosis. Investigation of the GO-terms ‘neuron apoptotic process’ and ‘axon development’ revealed common genes that were responsive across compounds, and might be used as biomarkers for DNT. The GO-term ‘synaptic signalling’, on the contrary, whilst also responsive to all compounds tested, showed little overlap in gene expression regulation patterns between the conditions. This GO-term may articulate compound-specific effects that may be relevant for revealing differences in mechanism of toxicity. Given its focus on neural progenitor cell to mature multilineage neuronal cell maturation and its detailed molecular readout based on gene expression analysis, hNPT might have added value as a tool for neurodevelopmental toxicity testing in vitro. Further assessment of DNT-specific biomarkers that represent these processes needs further studies.

Transient MPTP exposure at a sensitive developmental window altered gut microbiome and led to male-biased motor and social behavioral deficits in adult zebrafish

Zebrafish is an economical alternative model for developmental neurotoxicity (DNT) testing. DNT studies in zebrafish have been focused on acute effects; few studies explore enduring neurotoxicity in adults. More recently, gut microbiome has emerged as an important modulator between chemical exposure and neurotoxicity, rendering its necessity to be included in DNT testing. The present study used a well-known dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model chemical to explore long-lasting neurotoxicity in adults after transient exposure during early development. We demonstrated that transient MPTP exposure at 1 μM during a sensitive developmental window of 48–96 h post-fertilization (hpf) altered gut microbiome and led to male-biased locomotion and behavioral deficits in adult fish. The locomotion deficit was manifested as hypoactivity observed in adult males under light conditions or specifically the reduction of fast swim bouts. The social behavioral deficits were characterized by the reduced number of times fish crossed the mirror zone in the mirror response assay and the reduced percent time fish spent at the area proximal to conspecific fish shoal in the social preference test. Gut microbiome analysis revealed that transient MPTP exposure during early development might render fish more susceptible to the colonization of the pathogenic Vibrio.In conclusion, our study revealed that transient MPTP exposure during early development could lead to long-lasting neurotoxicity in adult fish and cause altered gut microbiome composition in both larval and adult fish.

Characterization of Developmental Neurobehavioral Toxicity in a Zebrafish MPTP-Induced Model: A Novel Mechanism Involving Anemia.

Zebrafish represent an economical alternative to rodents for developmental neurotoxicity (DNT) testing. Mechanistic understanding is the key to successfully translating zebrafish findings to humans. In the present study, we used a well-known dopaminergic (DA) neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model chemical to uncover the molecular pathways for observed DNT effects. To enhance the specificity of potential molecular targets, we restricted our exposure to a concentration that is nonteratogenic yet exhibits high DNT effects and an exposure window sensitive to MPTP. Our DNT assessment based on a battery of motor and social behavioral tests revealed an effective concentration of 1 μM and a sensitive window of 48-96 h postfertilization (hpf) for MPTP-induced hypoactivity. It is worth noting that this hypoactivity persisted into later larval development until 28 dpf. We observed increased cell apoptosis, oxidative stress, and decreased ATP levels in larvae immediately after exposure at 96 hpf. Significant reductions of DA neurons were found in the retina at 72, 96, and 120 hpf. No visible deformity was found in motoneurons at 72, 96, and 120 hpf. Transcriptome analysis uncovered a novel pathway manifested by significant upregulation of genes enriched with erythropoiesis. Sensitive window exposure of MPTP and other DA neurotoxins rotenone and paraquat exhibited a concentration-dependent effect on transcriptional changes of embryonic hemoglobins and anemia. Given that anemia is a significant risk factor for Parkinson's disease and MPTP is known to cause parkinsonism in humans, we concluded that anemia resulting from dysregulation of primitive erythropoiesis during embryonic development might serve as a common mechanism underlying DA neurotoxin-induced DNT effects between zebrafish and humans.

European stakeholders' workshop on new approach methodologies (NAMs) for developmental neurotoxicity (DNT) and their use in the regulatory risk assessment of chemicals

Characterization of potential chemical-induced developmental neurotoxicity (DNT) hazard is considered for risk assessment purposes by many regulatory sectors. To address challenging endpoints such as DNT, EFSA, in collaboration with the Organisation for Economic Co-Operation and Development (OECD), the Danish EPA and the US EPA, has been working toward the development of integrated approaches for testing and assessment (IATA) that rely on a combination of multiple layers of data (e.g. in vitro, in silico and non-mammalian in vivo models) that are supported by mechanistic knowledge organized according to the adverse outcome pathway (AOP) framework. This international effort culminated in the development of an OECD guidance on the use and interpretation of NAMs for DNT; this guidance is currently in an advanced draft status along the OECD process for approval and publication. This workshop was organized by EFSA to update European stakeholders on the progress and receive comments thereof. The workshop provided an overview of this international effort and represented an opportunity to discuss the current DNT testing paradigm and the regulatory hurdles for the implementation of DNT testing of chemicals by incorporation of a mechanistic understanding and inclusion in the regulatory process.

Pre-validation study of alternative developmental neurotoxicity test using Sox1-GFP cell.

Pre-validation study of alternative developmental neurotoxicity test using Sox1-GFP cell.

Pre-validation of an alternative test method for prediction of developmental neurotoxicity

Exposure to neurodevelopmental toxicants can cause permanent brain injury. Hance, determining the neurotoxicity of unknown substances is essential for the safety of substance. As an alternative method to animal studies, developmental neurotoxicity test (DNT) and the first discriminant function (DF) were established in previous study. This study aimed to increase the predictability of the DNT method and perform a mobility test. Two endpoints of 29 newly investigated substances were used to establish a second-generation DF (2nd GDF). As two endpoints, the half-inhibitory concentration of the cell viability (IC50) was determined using a cell counting kit-8 assay. The half-inhibitory concentration of differentiation (ID50) was determined by measuring the green fluorescent protein (GFP) intensity in 46C cells. The substances were treated dose-dependently to measure IC50 and ID50. The 2nd GDF classified 29 chemicals accurately as toxic and non-toxic. Four participants of three independent laboratories were enrolled to test the mobility. The results of the test set were highly accurate in reproducibility (100% of accuracy, sensitivity, and specificity) and mobility (accuracy 93.33%, sensitivity 90.91%, and specificity 100%). In conclusion, the protocol is transferable, reproducible, and accurate. Therefore, this could be a standardizing method for determining a neurotoxicant as an alternative for animal experiments.

Integrating Data From In Vitro New Approach Methodologies for Developmental Neurotoxicity.

In vivo developmental neurotoxicity (DNT) testing is resource intensive and lacks information on cellular processes affected by chemicals. To address this, DNT new approach methodologies (NAMs) are being evaluated, including: the microelectrode array neuronal network formation assay; and high-content imaging to evaluate proliferation, apoptosis, neurite outgrowth, and synaptogenesis. This work addresses 3 hypotheses: (1) a broad screening battery provides a sensitive marker of DNT bioactivity; (2) selective bioactivity (occurring at noncytotoxic concentrations) may indicate functional processes disrupted; and, (3) a subset of endpoints may optimally classify chemicals with in vivo evidence for DNT. The dataset was comprised of 92 chemicals screened in all 57 assay endpoints sourced from publicly available data, including a set of DNT NAM evaluation chemicals with putative positives (53) and negatives (13). The DNT NAM battery provides a sensitive marker of DNT bioactivity, particularly in cytotoxicity and network connectivity parameters. Hierarchical clustering suggested potency (including cytotoxicity) was important for classifying positive chemicals with high sensitivity (93%) but failed to distinguish patterns of disrupted functional processes. In contrast, clustering of selective values revealed informative patterns of differential activity but demonstrated lower sensitivity (74%). The false negatives were associated with several limitations, such as the maximal concentration tested or gaps in the biology captured by the current battery. This work demonstrates that this multi-dimensional assay suite provides a sensitive biomarker for DNT bioactivity, with selective activity providing possible insight into specific functional processes affected by chemical exposure and a basis for further research.

Looking at Developmental Neurotoxicity Testing from the Perspective of an Invertebrate Embryo.

Developmental neurotoxicity (DNT) of chemical compounds disrupts the formation of a normal brain. There is impressive progress in the development of alternative testing methods for DNT potential in chemicals, some of which also incorporate invertebrate animals. This review briefly touches upon studies on the genetically tractable model organisms of Caenorhabditis elegans and Drosophila melanogaster about the action of specific developmental neurotoxicants. The formation of a functional nervous system requires precisely timed axonal pathfinding to the correct cellular targets. To address this complex key event, our lab developed an alternative assay using a serum-free culture of intact locust embryos. The first neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons which use guidance cues from membrane-bound and diffusible semaphorin proteins. In a systematic approach according to recommendations for alternative testing, the embryo assay quantifies defects in pioneer navigation after exposure to a panel of recognized test compounds for DNT. The outcome indicates a high predictability for test-compound classification. Since the pyramidal neurons of the mammalian cortex also use a semaphorin gradient for neurite guidance, the assay is based on evolutionary conserved cellular mechanisms, supporting its relevance for cortical development.

The Future of 3D Brain Cultures in Developmental Neurotoxicity Testing.

Human brain is undoubtedly the most complex organ in the body. Thus, it is difficult to develop adequate and at the same time human relevant test systems and models to cover the aspects of brain homeostasis and even more challenging to address brain development. Animal tests for Developmental Neurotoxicity (DNT) have been devised, but because of complex underlying mechanisms of neural development, and interspecies differences, there are many limitations of animal-based approaches. The high costs, high number of animals used per test and technical difficulties of these tests are prohibitive for routine DNT chemical screening. Therefore, many potential DNT chemicals remain unidentified. New approach methodologies (NAMs) are needed to change this. Experts in the field have recommended the use of a battery of human in vitro tests to be used for the initial prioritization of high-risk environmental chemicals for DNT testing. Microphysiological systems (MPS) of the brain mimic the in vivo counterpart in terms of cellular composition, recapitulation of regional architecture and functionality. These systems amendable to use in a DNT test battery with promising features such as (i) complexity, (ii) closer recapitulation of in vivo response and (iii) possibility to multiplex many assays in one test system, which can increase throughput and predictivity for human health. The resent progress in 3D brain MPS research, advantages, limitations and future perspectives are discussed in this review.

Zebrafish embryo neonicotinoid developmental neurotoxicity in the FET test and behavioral assays.

The need for reliable, sensitive (developmental) neurotoxicity testing of chemicals has steadily increased. Given the limited capacities for routine testing according to accepted regulatory guidelines, there is potential risk to human health and the environment. Most toxicity studies are based on mammalian test systems, which have been questioned for low sensitivity, limited relevance for humans, and animal welfare considerations. This increased the need for alternative models, one of which is the zebrafish (Danio rerio) embryo. This study assessed selected neonicotinoids at sub-lethal concentrations for their effects on embryonic development and behavior. The fish embryo acute toxicity test (OECD TG 236) determined the lowest observable effective concentrations, which were used as the highest test concentrations in subsequent behavioral assays. In the FET test, no severe compound-induced sublethal effects were seen at < 100 µM. In the coiling assay, exposure to ≥ 1.25 µM nicotine (positive control) affected both the burst duration and burst count per minute, whereas ≥ 50 µM thiacloprid affected the mean burst duration. Exposure to ≥ 50 µM acetamiprid and imidacloprid induced significant alterations in both mean burst duration and burst count per minute. In the swimming assay, 100 µM acetamiprid induced alterations in the frequency and extent of movements, whilst nicotine exposure only induced non-significant changes. All behavioral changes could be correlated to findings in mammalian studies. Given the quest for alternative test methods of (developmental) neurotoxicity, zebrafish embryo behavior testing could be integrated into a future tiered testing scheme.

Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs.

ABSTRACTBranaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects, we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMNΔ7), and in juvenile Wistar Hannover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image-analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.