Establishment of a Human Cell-Based in Vitro Battery to Assess Developmental Neurotoxicity Hazard of Chemicals

Blum, John D.; Masjosthusmann, Stefan; Bartmann, Kristina; Bendt, Farina; Dolde, Xenia; Dönmez, Arif; Förster, Nils; Holzer, Anna‐Katharina; Hübenthal, Ulrike; Keßel, Hagen; Kilic, Sadiye; Klose, Jördis; Pahl, Melanie; Stürzl, Lynn‐Christin; Mangas, Iris; Terron, Andrea; Crofton, Kevin M.; Scholze, Martin; Mosig, Axel; Leist, Marcel; Fritsche, Ellen

doi:10.2139/ssrn.4161670

Cited by 2 publications

(4 citation statements)

References 96 publications

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“…Furthermore, Crofton and colleagues state, that the concentration range and the resulting concentration-response holds a very significant role in terms of comparison of sensitivity between different endpoints. For this study, we selected a concentration range that has already been chosen in other in vitro DNT assays and has elicited little to no cytotoxicity (Frank et al, 2017; Masjosthusmann et al, 2020; Blum et al, 2022). To discriminate specific from unspecific effects, we assessed the cytotoxicity of each compound on a weekly level during the 35-day culture period (P7 “Endpoint Selectivity”).…”

Section: Discussionmentioning

confidence: 99%

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A human iPSC-basedin vitroneural network formation assay to investigate neurodevelopmental toxicity of pesticides

Bartmann

Bendt

Dönmez

et al. 2023

Preprint

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Proper brain development is based on the orchestration of key neurodevelopmental processes, including the formation and function of neural networks. If at least one key neurodevelopmental process is affected by a chemical, an adverse outcome is expected. To allow a higher testing throughput than the guideline animal experiments, a developmental neurotoxicity (DNT) in vitro testing battery (DNT IVB) has been set up that includes a variety of assays, which model several key neurodevelopmental processes. Gap analyses of the DNT IVB revealed the need of a human-based assay to assess neural network formation and function (NNF). Therefore, here we established the human NNF (hNNF) assay. A co-culture comprised of human-induced pluripotent stem cell (hiPSC)-derived excitatory and inhibitory neurons, as well as primary human astroglia, was differentiated for 35 days on micro-electrode arrays (MEA) and spontaneous electrical activity, together with cytotoxicity, was assessed on a weekly basis after washout of the compounds 24 h prior to measurements. In addition to the characterization of the test system, the assay was challenged with 28 compounds, mainly pesticides, identifying their DNT potential by evaluation of specific spike-, burst- and network parameters. This approach confirmed the suitability of the assay for screening environmental chemicals. Comparison of benchmark concentrations (BMC) with an NNF in vitro assay (rNNF) based on primary rat cortical cells, revealed differences in sensitivity. Together with the successful implementation of hNNF data into a postulated stressor-specific adverse outcome pathway (AOP) network associated with a plausible molecular initiating event for deltamethrin, this study suggests the hNNF assay as a useful complement to the current DNT IVB.

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Section: Discussionmentioning

confidence: 99%

“…In the present study, 28 substances were tested with various concerns regarding their DNT potential. Compounds are part of a training set used for the current DNT in vitro battery (Blum et al, 2022; Carstens et al, 2022). More information on the compound selection can be found in Masjosthusmann et al (2020).…”

Section: Methodsmentioning

confidence: 99%

A human iPSC-basedin vitroneural network formation assay to investigate neurodevelopmental toxicity of pesticides

Bartmann

Bendt

Dönmez

et al. 2023

Preprint

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“…The particular value of immature LUHMES for neurotoxicity testing has been explored in depth (Delp 2019;Krug 2013;Stiegler 2011) and was used many times for neurotoxicity screening (Delp 2018b;Blum 2022;Krebs 2020b). However, some uncertainty remains that mature neurons would react in an entirely different way.…”

Section: 3mentioning

confidence: 99%

“…The clearly highest readiness level was found for the KE4 assay. This test has been evaluated by an international consortium (Krebs 2020b; Bal-Price 2018), used for OECD IATA case studies and been used and evaluated in the context of the EFSA in vitro battery study (Blum 2022;van der Stel 2021).…”

Section: Rationale For Pod Selectionmentioning

confidence: 99%

EFSA Pilot Project on New Approach Methodologies (NAMs) for Tebufenpyrad Risk Assessment. Part 2. Hazard characterisation and identification of the Reference Point

Alimohammadi

Meyburg

Ückert

et al. 2023

EFS3

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This Project was performed to provide an in vitro Point of Departure (PoD) for an Adverse Outcome Pathway (AOP)‐informed Integrated Approach to Testing and Assessment (IATA), concerning a potential risk of parkinsonian motor deficits after long‐term exposure to Tebufenpyrad. The AOP considered was AOP:3. Assays were performed for Key Event (KE) 1, KE2 (mitochondrial dysfunction) and KE4 of the AOP, based on the use of human dopaminergic neurons (LUHMES cells). KE1 (inhibition of complex I of the mitochondrial respiratory chain) was considered equivalent to the molecular initiating event (MIE). KE4 (dopaminergic cell degeneration) was considered as alternative AO (target cell degeneration). All necessary and linear steps of the AOP (i.e. KE2,3,4) were investigated by experimental test methods. The three major objectives were: (i) generation of potential PoD data from each assay; (ii) evaluation and optimization of the consistency of these data; (iii) selection of a PoD to be used for further risk assessment. During the optimization phase, assay conditions that reflect brain metabolism (MitoMet conditions) were implemented, and degeneration of neurites was considered the most relevant neuropathological endpoint. With this setting, the potential PoD ranged from 6 nM to 45 nM. The selection of the PoD put emphasis on more chronic effects and on the assay closest to the AO. Thus the KE4 assay, measuring neurite degeneration under MitoMet conditions was chosen, and the PoD was 8 nM. This value has a confidence interval of about one log10‐fold change from the lowest to the highest estimate (range: 3 nM – 30 nM), based on the experimental variations we observed. Some biokinetics measurements were undertaken to help estimating cell concentrations. These data had a high uncertainty concerning the measurements and the model assumptions. Based on the assumptions we consider most realistic and robust, we suggest a cell/tissue (brain) concentration of 8‐40 nM to be associated with a potential onset of toxicity of Tebufenpyrad.

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Establishment of a Human Cell-Based in Vitro Battery to Assess Developmental Neurotoxicity Hazard of Chemicals

Cited by 2 publications

References 96 publications

A human iPSC-basedin vitroneural network formation assay to investigate neurodevelopmental toxicity of pesticides

A human iPSC-basedin vitroneural network formation assay to investigate neurodevelopmental toxicity of pesticides

EFSA Pilot Project on New Approach Methodologies (NAMs) for Tebufenpyrad Risk Assessment. Part 2. Hazard characterisation and identification of the Reference Point

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