Cell-based test methods with a phenotypic readout are frequently used for toxicity screening. However, guidance on how to validate the hits and how to integrate this information with other data for purposes of risk assessment is missing. We present here such a procedure and exemplify it with a case study on neural crest cell (NCC)-based developmental toxicity of picoxystrobin. A library of potential environmental toxicants was screened in the UKN2 assay, which simultaneously measures migration and cytotoxicity in NCC. Several strobilurin fungicides, known as inhibitors of the mitochondrial respiratory chain complex III, emerged as specific hits. From these, picoxystrobin was chosen to exemplify a roadmap leading from cell-based testing towards toxicological predictions. Following a stringent confirmatory testing, an adverse outcome pathway was developed to provide a testable toxicity hypothesis. Mechanistic studies showed that the oxygen consumption rate was inhibited at sub-µM picoxystrobin concentrations after a 24 h pre-exposure. Migration was inhibited in the 100 nM range, under assay conditions forcing cells to rely on mitochondria. Biokinetic modeling was used to predict intracellular concentrations. Assuming an oral intake of picoxystrobin, consistent with the acceptable daily intake level, physiologically based kinetic modeling suggested that brain concentrations of 0.1–1 µM may be reached. Using this broad array of hazard and toxicokinetics data, we calculated a margin of exposure ≥ 80 between the lowest in vitro point of departure and the highest predicted tissue concentration. Thus, our study exemplifies a hit follow-up strategy and contributes to paving the way to next-generation risk assessment.

Human-relevant tests to predict developmental toxicity are urgently needed. A currently intensively studied approach makes use of differentiating human stem cells to measure chemically-induced deviations of the normal developmental program, as in a recent study based on cardiac differentiation (UKK2). Here, we (i) tested the performance of an assay modeling neuroepithelial differentiation (UKN1), and (ii) explored the benefit of combining assays (UKN1 and UKK2) that model different germ layers. Substance-induced cytotoxicity and genome-wide expression profiles of 23 teratogens and 16 non-teratogens at human-relevant concentrations were generated and used for statistical classification, resulting in accuracies of the UKN1 assay of 87–90%. A comparison to the UKK2 assay (accuracies of 90–92%) showed, in general, a high congruence in compound classification that may be explained by the fact that there was a high overlap of signaling pathways. Finally, the combination of both assays improved the prediction compared to each test alone, and reached accuracies of 92–95%. Although some compounds were misclassified by the individual tests, we conclude that UKN1 and UKK2 can be used for a reliable detection of teratogens in vitro, and that a combined analysis of tests that differentiate hiPSCs into different germ layers and cell types can even further improve the prediction of developmental toxicants.

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However, access to such cells is limited, and studies systematically mapping the spectrum of their inflammatory states are scarce. Here, we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust, accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling, revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli, including pro-inflammatory cytokines (TNFα, interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1), TLR3 (Poly(I:C)), TLR4 (lipopolysaccharide, LPS), and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g., oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally, the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1, LPS, or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism, highlighting the role of soluble mediators in the signal propagation. Altogether, this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation.

Compounds, which rely on metabolism to exhibit toxicity, pose a challenge for next-generation risk assessment (NGRA). Since many of the currently available non-animal new approach methods (NAMs) lack metabolic activity, their use may lead to an underestimation of the true hazard to humans (false negative predictions). We explored here strategies to deal with metabolite-mediated toxicity in assays for developmental neurotoxicity. First, we present an overview of substances that may serve as potential positive controls for metabolite-related neurotoxicity. Then, we demonstrate, using the MitoMet (UKN4b) assay, which assesses the adverse effects of chemicals on neurites of human neurons, that some metabolites have a higher toxic potency than their parent compound. Next, we designed a strategy to integrate elements of xenobiotic metabolism into assays used for (developmental) neurotoxicity testing. In the first step of this approach, hepatic post-mitochondrial fractions (S9) were used to generate metabolite mixtures (“metabolisation module”). In the second step, these were applied to a NAM (exemplified by the UKN4b assay) to identify metabolite-mediated toxicity. We demonstrate the applicability and transferability of these approaches to other assays, by an exemplary study on the basis of the cMINC (UKN2) assay, another NAM of the developmental neurotoxicity in vitro battery. Based on the experience gained from these experiments, we discuss key issues to be addressed if this approach is to be used more broadly for NAM in the NGRA context.

Little is known about how nicotinic signalling in human astrocytes may contribute to the functional neurotoxicity of compounds related to tobacco alkaloids and neonicotinoid pesticides. We generated a single-cell Ca²⁺-imaging assay in induced pluripotent stem cell (iPSC)-derived astrocytes, and profiled functional expressions of some neurotoxicologically relevant receptors. Responses to pharmacological tool compounds indicated the expression of nicotinic, muscarinic, purinergic, glutamatergic receptors and voltage-gated Na⁺/Ca²⁺ channels. Closer investigation of the nicotinic system, e.g., using the alpha7 nicotinic acetylcholine receptor (nAChR)-selective positive allosteric modulator PNU-120596 and alpha7-preferring agonist (AR-R17779) demonstrated that Ca²⁺ signals elicited by nicotine and neonicotinoids are dominated by alpha7 nAChRs and depend on the downstream activation of L-type Ca²⁺ channels and tetrodotoxin-sensitive Na⁺ channels. Crosstalk of nAChR activation/desensitization was not observed for the inflammatory response elicited by TNF or for activation of glutamatergic or purinergic signalling. However, pre-stimulation of nAChR by neonicotinoids significantly blunted the response to the neurotransmitter acetylcholine. Comparative experiments in the human neuronal cultures (LUHMES cells) revealed similar potency ranges and pharmacological fingerprints for several neonicotinoids and their human-relevant metabolites descyanothiacloprid and desnitroimidacloprid. The pesticide metabolites showed a high potency, compared with their respective parent compounds. After this basic system characterization, the hitherto data-poor pesticides cycloxaprid and flupyradifurone were comparatively profiled in astrocytic and neuronal test systems. They showed the typical features of alpha7 nAChR agonists. The disruption of cholinergic signalling in astrocytes suggests that neonicotinoids affect not only neurons in human brains. Therefore, future neurotoxicity screening approaches may need to consider astrocyte toxicity.