Establishment of an a priori protocol for the implementation and interpretation of an in‐vitro testing battery for the assessment of developmental neurotoxicity

EFSA: European Food Safety Authority

Masjosthusmann S, Blum J, Bartmann K, Dolde X, Holzer A-K, Stürzl L-C, Keßel EH, Förster N, Dönmez A, Klose J, Pahl M, Waldmann T, Bendt F, Kisitu J, Suciu I, Hübenthal U, Mosig A, Leist M, Fritsche E.


Researchers at IUF and university of Konstanz were sponsored by the EFSA to propose a human cell based in vitro test battery of developmental neurotoxicology (DNT) assays covering neurodevelopmental processes essential for brain development in a temporal context.

A test battery comprising 5 test systems and 119 chemicals of known toxicity has been proposed to assess DNT-specific endpoints and evaluate cytotoxicity and viability (related parameters and is discuss in the report).

For properly representing the overall state-of-the-art concerning DNT in vitro testing for regulatory purposes, results from test methods established to assess neuronal network formation and activity have also been reported.

Axion's Maestro multielectrode array (MEA) system was used by Prof Fritsche and her group to develop a new assay, the hNNF (human Neural Network Formation) assay for the evaluation of the neurotoxic effects of compounds on the development of mature neuronal functional network.

A commercially available kit of human pre-differentiated hiPSC-based excitatory and inhibitory neurons and primary astroglia was plated on 48-well plates and baseline recordings were performed. After one week, the cultures were dosed with compounds at 7 different concentrations. Weekly recordings of electrical activity were taken and analyzed in AxIS software for network activity analysis, including number of spikes, number of active electrodes, burst duration, network burst frequency and different synchronicity parameters. These data were compared with results obtain from a rodent Neural Network Formation assay by Dr T Shafer at EPA-USA, also obtained with the Axion Maestro MEA platform on same 48-well plates type.

The comparison of  rodent with human cells based assays highlights how apparent species specific effects may lead to the development of hypothetical adverse outcome pathways (AOPs) that will require further integrated analysis for testing and assessment (IATA) assessment by the agency.