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The inclusion of a read-out to detect functional consequences of craniofacial alterations in the zebrafish embryotoxicity test will allow to evaluate these alterations which are difficult to assess morphologically, and to detect alterations in cranial nerves functions leading to impairment of jaw movements. In this study we have established an ingestion test in zebrafish larvae younger than 120 hpf. To overcome the challenge of evaluating larvae which still do not present independent feeding behaviour, we have tested the ability of 72, 96 or 102 hpf larvae to ingest food mixed with fluorescent microspheres under several conditions (dark/light, with/without shaking) to find the best experimental set-up for the test. We have included the investigation of two substances as potential positive controls: ketoconazole and tricaine. Ketoconazole 10 μM exposure during development produced significant embryotoxic effects including a characteristic craniofacial alteration pattern consisting in impaired development of brain, nasal cavity, mouth opening and jaw, as well as a significant decrease in food intake. Tricaine exposure at 380 μM during the food availability period significantly decreased the food intake. The method proposed will be a useful alternative tool to animal testing to detect compounds inducing adverse effects on craniofacial development.

Epigallocatechin gallate (EGCG), the main catechin of green tea, is described to have potential health benefits in several fields like oncology, neurology or cardiology. Currently, it is also under pre-clinical investigation as a potential therapeutic or preventive treatment during pregnancy against developmental adverse effects induced by toxic substances. However, the safety of EGCG during pregnancy is unclear due to its proven adverse effects on neural progenitor cells' (NPCs) migration. As lately several strategies have arisen to generate new therapeutic agents derived from EGCG, we have used the rat ‘Neurosphere Assay’ to characterize and compare the effects of EGCG structurally related compounds and EGCG PEGylated PLGA nanoparticles on a neurodevelopmental key event: NPCs migration. Compounds structurally-related to EGCG induce the same pattern of NPCs migration alterations (decreased migration distance, decreased formation of migration corona, chaotic orientation of cellular processes and decreased migration of neurons at higher concentrations). The potency of the compounds does not depend on the number of galloyl groups, and small structure variations can imply large potency differences. Due to their lower toxicity observed in vitro in NPCs, 4,4′-bis[(3,4,5-trihydroxybenzoyl)oxy]-1,1′-biphenyl and EGCG PEGylated PLGA nanoparticles are suggested as potential future therapeutic or preventive alternatives to EGCG during prenatal period.