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.

Astrocytes (ACs) do not only play a role in normal neurogenesis and brain homeostasis, but also in inflammatory and neurodevelopmental disorders. We studied here the different patterns of inflammatory activation triggered by cytokines in human induced pluripotent stem cell (iPSC)-derived ACs. An optimized differentiation protocol provided non-inflamed ACs. These cells reacted to TNFα with a rapid translocation of NFκB, while AC precursors showed little response. Transcriptome changes were quantified at seven time points (2–72 h) after stimulation with TNFα, IFNγ or TNFα plus IFNγ. TNFα triggered a strong response within 2 h. It peaked from 12–24 h and reverted towards the ground state after 72 h. Activation by IFNγ was also rapid, but the response pattern differed from that of TNFα. For instance, several chemokines up-regulated by TNFα were not affected by IFNγ. Instead, MHC-II-related antigen presentation was drastically enhanced. The combination of the two cytokines led to a stronger and more persistent response. For instance, TRIB3 up-regulation by the combination of TNFα plus IFNγ may have slowed NFκB inactivation. Additionally, highly synergistic regulation was observed for inflammation modifiers, such as CASP4, and for STAT1-controlled genes. The combination of the cytokines also increased oxidative stress markers (e.g., CHAC1), led to phenotypic changes in ACs and triggered markers related to cell death. In summary, these data demonstrate that there is a large bandwidth of pro-inflammatory AC states, and that single markers are not suitable to describe AC activation or their modulation in disease, development and therapy.

Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Here a new test format was established. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of the stopper barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of obtaining data on viability and migration by automated image processing was addressed by developing a freeware. Data on cell proliferation were obtained by labelling replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as an experimental confounder was tested experimentally by performing the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allows classification of toxicants as either inactive, leading to unspecific cytotoxicity, or specifically inhibiting NC migration at non-cytotoxic concentrations.