Evaluation of non-clinical toxicity studies of COVID-19 vaccines.

In this study we evaluated the outcomes of non-clinical toxicity studies of various SARS-CoV-2 vaccines produced with different manufacturing technologies, with focus on Repeated Dose Toxicity (RDT) and Developmental and Reproductive Toxicity (DART) studies. We found that RDT and DART studies at doses relevant for human treatment showed no adverse effects while remaining observations were expected findings including local reactogenicity, immune response and macroscopic findings at the injection site. We have also reviewed the European Medicines Agency (EMA) nonclinical assessment reports for market authorization. Regardless of utilized vaccine manufacturing technology EMA assessment of the non-clinical studies consisted most frequently of comments related to study design, species selection and missing data. Sponsors have often submitted platform studies (vaccine studies with the same technology/construct but using other antigens) as supplementary data. Animal model-based toxicity testing has shown rather small effects, which have been never serious adverse effects. The translational value to support clinical development is mainly to inflammatory effects, indicative of the primary action of the vaccines. From a 3R perspective supportive platform technology data consisting of previously executed RDT and DART studies from the same platform technology are encouraged to be implemented in the vaccine assessment process.

Toward a comparative retrospective analysis of rat and rabbit developmental toxicity studies for pharmaceutical compounds.

Based on a proposal made at the ICH Workshop in Tallinn, Estonia (2010), the value of the rabbit embryo-fetal development (EFD) versus the rodent EFD was examined by the HESI DART group. A cross-industry data survey provided anonymised EFD and toxicokinetic data from EFD studies on over 400 marketed and unmarketed drugs (over 800 studies) that were entered by experts at RIVM into US EPA's ToxRefDB style database. The nature and severity of findings at the lowest observed adverse effect level (LOAEL) are being reviewed to quantitate the frequency with which lesser signs of embryo-fetal effects (e.g., delays in ossification, minor changes in frequency of variants) are driving the LOAELs. Interpretation was based on exposure rather than administered dose. This paper provides an update of this ongoing project as discussed during a workshop of the European Teratology Society in Ispra, Italy (2013). This was the first presentation of the initial data set, allowing debate on future directions, to provide a better understanding of the implications of either delaying a rabbit EFD or waiving the need in particular circumstances.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn).

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

An abbreviated protocol for multilineage neural differentiation of murine embryonic stem cells and its perturbation by methyl mercury.

Alternative assays are highly desirable to reduce the extensive experimental animal use in developmental toxicity testing. In the present study, we developed an improved test system for assessing neurodevelopmental toxicity using differentiating embryonic stem cells. We advanced previously established methods by merging, modifying and abbreviating the original 20-day protocol into a more efficient 13-day neural differentiation protocol. Using morphological observation, immunocytochemistry, gene expression and flow cytometry, it was shown predominantly multiple lineages of neuroectodermal cells were formed in our protocol and to a lower extent, endodermal and mesodermal differentiated cell types. This abbreviated protocol should lead to an advanced screening method using morphology in combination with selected differentiation markers aimed at predicting neurodevelopmental toxicity. Finally, the assay was shown to express differential sensitivity to a model developmental neurotoxicant, methyl mercury.