In the project the following centers experienced in alternative toxicity testing take part – NIOM – Coordinator (Poland), VUB (Belgium), NILU (Norway) and UMIL (Italy).
NIOM is a large, one of the leading research institutes (+300 full time employees) in the field of occupational and environmental health in Poland having status of WHO Collaborating Centre. NIOM maintains links with many international organizations and institutions (ICOH, MEDICHEM, IARC, EUROTOX, ACGIH, EUPHA, ASPHER, EASOM, IUPAC). Presently, NIOM provides background research and expertise to the Polish Ministry of Health and serves as an advisory body to the Polish Ministry of Environmental Protection and numerous governmental agencies, including State Sanitary Inspectorate and State Labour Inspectorate, and also to local administration, trade unions and industry. For many years toxicologists from NIOM have been involved in safety assessment of genotoxic/mutagenic, carcinogenic, pulmonary, and pro-inflammatory effects of chemicals including manufactured nanomaterials.
The researchers cooperate with national (e.g. Bureau for Chemical Substances) and international organizations (e.g. Joint Research Centre in Ispra, Italy), which are responsible for regulatory and safety assessment issues of chemicals including manufactured nanomaterials. For many years the researchers from Toxicology and Carcinogenesis Department (TCD) have been striving for implementing and maintaining the highest standards of research. TCD has Good Laboratory Practice status since 2008 for conducting basic cytotoxicity, mutagenicity and in vivo toxicity testing (according to OECD and ISO guidelines) (scan of the certificate is attached in Annex 1 below). Since 2008 they performed many studies for clients from the cosmetics, pharmaceutical or chemical industry from Poland and abroad. Since 2000 researchers from TCD actively participated in dissemination of 3R’ principles on the Polish ground. In 1999 they established the National Centre for Alternative Methods to Toxicity Assessment, issued their periodical called “Vitryna” (scans of the early issues are attached in Annex 1), organized many meetings and lectures on the alternatives. Moreover, until 2009 they participated in “ecopa” (European consensus-platform for alternatives) activity. Molecular Toxicology Laboratory (MTL) in the Toxicology and Carcinogenesis Dept has been a member of the EU-NETVAL (European Union Network of Laboratories for the Validation of Alternative Methods) since 2015 (scan of the letter of invitation in Annex 1).
In 2012-2013 the scientists from MTL participated in a validation study on Colony Forming Efficiency assay sponsored by JRC (JRC Science and Policy Reports. Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials. DOI: 10.2788/406937. 2014). In 2017 NIOM has been assigned by EURL-ECVAM a task for validation of one of the methods for screening of chemicals towards thyroid disrupting activity. In October 2019 the Thyroid Disruption Validation Study started in NIOM (scan of both letters in Annex 1). In September 2018 NIOM established the Platform for cooperation on alternatives with National Ethics Committee on Animal Experimentation and Polish regulatory bodies encouraging using alternative methods for educational and regulatory testing (scan of the document in Annex 1). The scientists from MTL have been co-authors of patent: “Tools and methods useful in characterizing the immunotoxic activity of xenobiotic substances” Patent Number: US 08361711. Official Gazette of the United States Patent and Trademark Office Patents Published: JAN 29 2013. The research in MTL is mainly focused on molecular mechanisms of toxicity of chemicals in both bulk and nanosized forms. The researchers have considerable experience in cell culturing and assessment of molecular mechanisms of chemical toxicity that together with a very well equipped two cell culture labs gives a great opportunity for twinning in TWINALT project.
Among the methods routinely used in MTL are the following: – basal cytotoxicity test: Neutral Red Uptake (NRU) test/ ISO 10993-5:2009, MTT reduction, WST-1 test, LDH measurement, propidium iodide staining/flow cytometry – cellular mechanisms of cytotoxicity: apoptosis&necrosis/ROS/GSH by flow cytometry, ELISA tests for cytokines – genotoxicity tests: comet assay; Mouse Lymphoma Assay/OECD TG490; In vitro Micronucleus Test/OECD TG 487 – nanotoxicology: Dynamic Light Scattering (DLS) to measure size distribution of nanoparticles before and during in vitro exposure; internalization of nanoparticles by DualBeam FIB/SEM (FEI Scios) or fluorescently labelled nanoparticles by flow cytometry (BD FACS Canto II) and confocal microscopy (Leica SP8). The general vision of the development of MTL is to be able to characterize cell morphology at different levels, starting from ultrastructural organisation, through imaging cells cultured in monolayers, up to studying cells growing in 3D models. To this end, in the laboratory, modern equipment has been collected such as scanning transmission electron microscopy (STEM) with Energy-dispersive detector (EDS) and Focused Ion Beam (FIB), OperettaTM–High-Content Imaging System, or confocal microscopy Leica SP-8. Researchers from MTL realize great potential of the twinning project and capacity of the partners involved. In the project the equipment in MTL will be fully open for training and after ending of the project will be used for future joint research projects with the partners.