In this project we set up a human cell-based DNT in vitro testing strategy that is based on test methods with high readiness and data generated therefrom. The methods underwent a fit-for-purpose evaluation that considered four key elements: 1. The test system, 2. the exposure scheme, 3. the assay and analytical endpoint(s) and 4. the classification model. This testing battery was challenged with 119 chemicals for which rich toxicological information was available (for some of them also on their DNT hazard). Testing was performed in 5 test systems measuring 10 DNT-specific endpoints and additional 9 viability/ cytotoxicity-related parameters. For approximately half of the compounds, additional and complementary data from DNT in vitro tests was added by the US-EPA. This extended battery was also evaluated. Testing results revealed that the test methods of this current DNT in vitro battery are reliable and reproducible. The endpoints had to a large extent low redundancy. Battery performance, as assessed with compounds well-characterized for DNT hazard had a sensitivity of 82.7 % and a specificity of 88.2 %. Gap analyses suggested that radial, astro-and microglia as well as myelination endpoints may be added to the battery. Two case studies, one for screening and prioritization of 14 flame retardants, and one on hazard characterization of 2 pesticides, were presented. Hypothetical AOPs were developed based on the latter case study. In conclusion, the DNT testing strategy explored here is a very promising first approach for DNT hazard identification and characterization. The performance is encouraging and may be improved by inclusion of further tests. Some uncertainties in DNT in vitro battery testing outcomes could be reduced by incorporating test data and modelling approaches related to in vitro and in vivo toxicokinetics of test compounds.

Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2′,4,4′-tetrabromodiphenylether (BDE-47), 2,2′,4,4′,5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell–based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 μM (5 FRs), 1<10 μM (7 FRs) to the >10 μM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell–based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment. Graphical abstract

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Piempel u. a.: F.xperinientelle Befunde ii ber ciii neues, oral wirksames Antimykotikum Deutsche Medizinische Wochenschrift Experimentelle Befunde über ein neues, oral wirksames Antimykotikum mit breitem Wirkungsspektrum Nach den Blut-und Gewebespiegein, die mit verschiedenen Dosen von Bis-phenyl-(2-chlorphenyl)-1-imidazolyl-methan (BAY b 5097) bei Versuchstieren und Patienten erreicht werden, kann die Empfindlichkeitsgrenze pathogener Pilze in vivo mit 4-6 (-8) '/ml angenommen werden. In diesem Konzentrationsbereich in vivo sicher erreichbar sind 1. alle geprüften Candida-Arten, vor allem C. albicans und C. tropicalis, 2. die Dermatophyten der Gattungen Trichophyton und Microsporon, 3. die Sproßpilze der Gattungen Coccidioides immitis, Paracoccidioides brasiliensis, Histoplasma capsulatum und Cryptococcus neoformans, 4. die Aspergillen, 5. die Chromomyceten der Gattungen Phialophora und Hormodendrum, 6. Cladosporium carrioni. In vivo möglicherweise noch erreichbar sind die Allescherien, das biphasische Sporotrichon Schenckii sowie Schimmelpilze der Gattungen Mucor und Blakeslea, die nur ausnahmsweise pathogen sind. Nicht mehr im In-vivo-Wirkungsspektrum liegen Madurella-Arten und die aufgrund ihrer Zellwandstruktur zu den Bakterien zählenden, aerob wachsenden Strahlenpilze Nocardia asteroides und N. brasiliensis. Nach unseren tierexperimentellen Daten wirkt BAY b 5097 bei oraler Applikation bei System-und Dermatomykosen in der erforderlichen Dosierung zuverlässig. Die optimale Therapiedauer im Tierexperiment beträgt bei System-Mykosen 8-12 Tage, bei Dermatomykosen 12-16 Tage, eine kürzere Therapiedauer führt zu Rezidiven.

There is a call for a paradigm shift in developmental neurotoxicity (DNT) evaluation, which demands the implementation of faster, more cost-efficient, and human-relevant test systems than current in vivo guideline studies. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD), a guidance document is currently being prepared that instructs on the regulatory use of a DNT in vitro battery (DNT IVB) for fit-for-purpose applications. One crucial issue for OECD application of methods is validation, which for new approach methods (NAMs) requires novel approaches. Here, mechanistic information previously identified in vivo, as well as reported neurodevelopmental adversities in response to disturbances on the cellular and tissue level, are of central importance. In this study, we scientifically validate the Neurosphere Assay, which is based on human primary neural progenitor cells (hNPCs) and an integral part of the DNT IVB. It assesses neurodevelopmental key events (KEs) like NPC proliferation (NPC1ab), radial glia cell migration (NPC2a), neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5), and thyroid hormone-dependent oligodendrocyte maturation (NPC6). In addition, we extend our work from the hNPCs to human induced pluripotent stem cell-derived NPCs (hiNPCs) for the NPC proliferation (iNPC1ab) and radial glia assays (iNPC2a). The validation process we report for the endpoints studied with the Neurosphere Assays is based on 1) describing the relevance of the respective endpoints for brain development, 2) the confirmation of the cell type-specific morphologies observed in vitro, 3) expressions of cell type-specific markers consistent with those morphologies, 4) appropriate anticipated responses to physiological pertinent signaling stimuli and 5) alterations in specific in vitro endpoints upon challenges with confirmed DNT compounds. With these strong mechanistic underpinnings, we posit that the Neurosphere Assay as an integral part of the DNT in vitro screening battery is well poised for DNT evaluation for regulatory purposes.