Development of alternative methods for toxicological risk assessment based on human Mesenchymal Stem Cells in the framework of REACH

Poisoning due to chemicals is generally recognized as a severe health problem. According to the Institute of Medicine (IOM), indeed, more than four million poisoning episodes occur annually in the United States. In order to ensure an adequate protection of human health and the environment, it is essential to evaluate if such products might cause adverse effects under normal and reasonably foreseeable use, and also to guarantee that reliable quality and adequate information on the adverse effects of exposure to such chemicals is provided. Toxicological testing is therefore an important part of the regulatory safety assessment for chemicals worldwide. Test methods rely largely on animal models, however, the validity of the data obtained in these in vivo tests has been often questioned. Recently many in vitro methods for toxicity testing have been developed as alternatives to whole animal tests, according to the 3Rs approach proposed by Russell and Burch. This research falls within the framework of the risk assessment of chemicals, and specifically in the framework of the REACH Regulation, giving specific relevance to the 3Rs concept. The activity performed during the PhD, indeed, concerns the development of novel in vitro models in order to test the potential toxicity of drugs and chemicals, following two different lines of research, both of them based for the first time on the use of human Mesenchymal Stem Cells (MSCs) isolated from bone marrow. From our best knowledge indeed, both animal and human MSCs have never been adopted for developing in vitro model systems for acute toxicity tests, but their unique proprieties, such as unlimited proliferation ability, plasticity to generate others cell types and even being a more readily available sources of human cells, clearly identify their potential benefits in toxicology. The first line of research focused on the application of the Neutral Red Uptake (NRU) assay to detect chemical toxicity on human MSCs. Specifically, the aim of this study was to evaluate the applicability of human MSCs as cell line for in vitro cytotoxicity tests to correctly predict LD50 and the hazard category of chemicals according to the Globally Harmonized System (GHS). By comparing the behavior of human MSCs with the already validated 3T3 and NHK NRU test methods, the results clearly show that the tested cells can be confidently used to perform in vitro acute toxicity tests. The second line of research, on the contrary, was mainly focused on the research of new in vitro methods in order to test the vascular toxicity of chemicals. It is based on the application of emerging tissue engineering concepts on the field of toxicology. For the moment, only preliminary studies were conducted, which regarded the application of decellularization techniques to animal blood vessels in order to obtain biological scaffolds. These scaffolds could be subsequently used for recellularization processes using human MSCs, and setting up of perfused in vitro human toxicity tests.