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An integrated interdisciplinary approach to animal-free chemical and nanomaterial safety assessment

Periodic Reporting for period 1 - in3 (An integrated interdisciplinary approach to animal-free chemical and nanomaterial safety assessment)

Reporting period: 2017-01-01 to 2018-12-31

Chemicals, including drugs and cosmetics, reaching the marketplace need to be tested for their potential to harm the environment and humans. In addition, manufactured nanomaterials (NMs) are also of concern. Safety evaluation is currently achieved for the most part utilising non-human whole animal tests. While animal models can predict a large amount of human biology, they differ in significant ways in the handling of many chemicals and pharmaceuticals, primarily due to species differences in phase I and II metabolizing enzymes and in chemical transporters. Additionally, there are ethical concerns pertaining to the use of animals for safety evaluation and thus the EU is actively supporting animal alternatives via the Reduction Refinement and Replacement (3Rs) of animal use in testing.
Induced Pluripotent Stem Cells: a revolution in biology
Over the past decade there has been a major focus on human cells as tools for chemical safety assessment. Many cell lines however, exhibit cancerous phenotypes and thus have, by definition, an abnormal cell physiology. While primary cells recapitulate normal cell physiology to a greater extent, it is difficult to obtain a continuous source of high-quality material. Furthermore, it is practically impossible to collect some types of living human tissues such as the brain. In 2006, Takahashi and Yamanaka discovered a method to generate stem cells from somatic cells. The reprogrammed cells behaved like pluripotent stem cells, could self-renew indefinitely and could be differentiated into all three germ layers. The Nobel Prize in Physiology or Medicine 2012 was jointly awarded to John B. Gurdon and Shinya Yamanaka for this. The methods to induce stemness have improved over the years, using non-integrating techniques such as self-replicating RNA or Sendai virus. Also, much progress has been made in developing protocols to differentiate iPSC into specific cell types including neural, lung, liver, kidney and vasculature lineages. Thus, normal non-cancerous cells can now be acquired easily from skin biopsies, blood or even urine and re-programmed into iPSC from any individual. This has a huge advantage over embryonic stem cells, as the donors are living, they can be acquired from diseased individuals, there are less ethical issues and avenues such as patient-specific regenerative medicine and patient-specific drug design are now possible. The Innovative Medicines Initiative (IMI) has launched two large projects StemBANCC and EBiSC to create standards and iPSC biobanks as a resource of standardised, quality assured iPSC.
Toxicology: a modern data rich discipline
Toxicity is a complex process critically dependent on a number of factors including chemical structure/state, tissue concentration, inherent biological activity-reactivity of the chemical or metabolite, cellular defence and repair mechanisms and the function of the target tissue. The modern-day approach to toxicology is to focus on mechanisms, rather than the more traditional histopathological approach. This mechanistic approach has benefited greatly from the use of transcriptomics and related methodologies that allow hypothesis-free, unbiased experimentation with extremely high-content data. However, high-content data streams pose a problem for their application to risk assessment as specialist knowledge is often required. A potential solution is to create contextual frameworks of the evolution of a toxic event, from its molecular initiating event (MIE), to secondary, tertiary and downstream cascading key events (KEs), leading eventually to a pathology or adverse outcome. This concept of Adverse Outcome Pathways (AOP) is gaining momentum as a pragmatic method to enable the application of deeply mechanistic data to chemical risk assessment steered by the OECD and the EU regulatory agencies. The development of quantitative AOPs will require in vitro data, knowledge of disease progression and computer modelling approaches. in3 will build on previous knowledge by focusing to mechanistic studies, linking exposure to hazard, creating biokinetic models and integrating in vitro data with in silico tools.
Core scientific activities:
• Differentiation of well-characterised human iPSC into brain, lung, liver, kidney and vascular cells
• Delineation of tissue specific and donor specific effects of compound exposures (uptake, metabolism, extrusion, and mechanistic toxicity)
• Development and optimisation of quantitative adverse outcome pathways (qAOPs) for each target organ which will be unified in an organism-level model
• Optimisation of QSAR and read-across tools for safety assessment
• Ultimately to create a unified expandable integrated testing strategy for chemical and NM safety assessment
Fifteen highly motivated early stage researchers (ESRs), from 11 different countries, were recruited and relocated to the 13 European host institutions in the first 17 months. The host institutions represent academia (7), governmental bodies (1), SMEs (4) and non-profit organisations (1). The second year focused to ESR training, in specific-tasks at the local institute and in project-wide activities via lectures and training courses aimed at providing knowledge pertaining to chemical and nanosafety testing and state of the art in vitro and in silico methodologies. All ESRs have commenced their own projects and are starting collaborations with the other ESRs. A highlight of the first 2 years was Flash Presentations from all ESRs at the European Society of Toxicology in Berlin, October 2018.
Several of the core scientific activities have started in the labs: Delineation of tissue-specific and donor-specific effects of compound exposures (uptake, metabolism, extrusion, and mechanistic toxicity), as well as qAOP (quantitative adverse outcome pathway) development and optimisation for each target organ at organism-level model, and finally, the optimisation of QSAR and read-across tools for safety assessment are in progress. With the well-established bottom-up communication in the in3 project, the main experiments need to be performed in the coming year. By means of transcriptomics experiments (TempO-Seq) and analyses, the aim is to elucidate the toxicity-related differences and similarities in toxicity pathways in the various differentiated iPSC.
Socio-economic impact includes the training of a group of fifteen young scientists, from eight European - and three non-European countries, communicating intensively throughout Europe, all working towards the same goal. The societal implication of the project is to improve human safety assessment of chemicals and nanoparticles by the utilisation of human derived iPSC tissues and computational methods and thereby reduce the number of test animals in toxicity testing, eventually to zero. In addition, such activities will positively impact the pharmaceutical industry (better preclinical tools), cosmetic industry (improved safety assessment for systemic and repeat dose toxicity), and the chemical and manufacturing industry (identification of potential hazards and sensitive individuals).