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

Periodic Reporting for period 2 - in3 (An integrated interdisciplinary approach to animal-free chemical and nanomaterial safety assessment)

Reporting period: 2019-01-01 to 2021-06-30

Chemicals and manufactured nanoparticles that we are exposed to need to be tested for their potential to cause harm. Safety evaluation of such products has been traditionally reliant on rodent based assays. While animal models are similar to human physiology in several ways, they can differ significantly in the handling of certain chemicals. 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 toxicological testing strategies.
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 human cells recapitulate normal cell physiology to a greater extent, it is difficult to obtain a continuous source of high-quality material or practically impossible for tissues such as the brain. In 2006, Takahashi and Yamanaka discovered a method to reprogrammed somatic cells into stem cells, termed induced Pluripotent Stem Cells (iPSC). iPSC permanently self-renew and cand be potentially differentiated into any cell type. Thus, it is now possible to generate normal non-cancerous pluripotent cells from any individual and differentiate them into target cells of interest.
Modern approaches to toxicology are mechanism focused and have benefited greatly from the use of transcriptomics and related methodologies that allow unbiased experimentation high-content data covering a multitude of potential pathways. However, high-content data streams pose a problem for their application to risk assessment as specialist knowledge is often required. A potential solution is the creation of contextual frameworks of the progression of a toxic event, from its molecular initiating event (MIE), to secondary, tertiary and further 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 in3 project aimed to build on previous knowledge and activities by focusing to mechanistic studies using iPSC, linking exposure to hazard, creating biokinetic models and integrating in vitro data with in silico tools.
15 early stage researchers (ESRs), from 11 different countries, were recruited and relocated to the 13 European host institutes. ESR training was focused to chemical safety testing and in vitro and in silico methodologies. Within their own specific project ESRs maintained a high level of collaboration to the other projects. This was particularly evident in the project-wide transcriptomic experiment, which was designed and executed by all ESRs generating 2880 samples with 3565 expressions levels per sample. The work was an in3 community effort at all stages including design, sample preparation, quality control, storage, shipment, tracking, meta-tagging and data analysis. A transcriptomic data analysis workshop (the final face to face meeting before COVID restrictions) took place in Ljubljana in February 2020 to facilitate the bioinformatic approaches.
The COVID-19 pandemic prevented travel within the in3 network from March 2020 onwards, which impacted on planned in3 face to face meetings, scheduled scientific conferences (international conferences were cancelled in 2020), and secondments. Despite these disruptions and facilitated with a 6 month project extension, the scientific and training goals were reached and even surpassed expectations. The rapid adoption to online dissemination tools likely also increased the projects penetration.
The core scientific achievements of the in3 project were:
• Extensive training on a theoretical and practical level in interdisciplinary aspects of computational and experimental toxicology to all in3 ESRs
• Development of 10 optimised protocols for the differentiation of human iPSC into brain (brain spheres and neuronal cells), lung (epithelium), liver (hepatocyte-like cells), kidney (podocyte-like and proximal tubular like), immune (alveolar macrophage like cells, monocyte like cells) and vascular endothelial cells (visceral and blood-brain barrier like)
• Characterisation and cross comparison of these models on a transcriptome level with and without exposure (2880 samples transcriptomic profiles were generated with the 10 model systems, 10 core compounds and 188 extension compounds).
• Examination and delineation of tissue specific effects of compound exposures (e.g. paraquat)
• Application of pharmaco/toxicokinetic modelling
• Advances in human p-glycoprotein (ABCB1) modelling approaches
• Further development of adverse outcome pathways and quantitative adverse outcome pathways (qAOPs)
• Further development and optimisation of QSAR and read-across tools

The project activities and results were well disseminated through social media (twitter, linkedin, youtube), scientific conferences, out-reach activities (e.g. to schools) and scientific peer-reviewed publications(18 to-date). In3 was represented at a total of 82 events with 84 oral communications and 41 poster presentations with 10 ESR awards. Dissemination highlights included dedicated in3 sessions at the European Society of Toxicology (ESTIV) conference Berlin, October 2018, where all 15 ESRs gave a flash presentation, the online in3 project meeting December 2020, where the presentations are available online, participation in the Science is Wonderful 2019 MSCA activity, the PYMCON 2020 meeting and presentation at House of Commons of the UK parliament for STEM 2020. An in3 inspired company called in3 solutions has also been set up by one ESR.
The in3 project had a significant impact on the evolution of biological and computational methods for animal-free safety assessments with solid developments and innovations in human pluripotent target cell development, toxicokinetics, transcriptional-based toxicodynamics, quantitative AOP development, quantitative structure analysis relationships and read across.
A major impact of the project has been the successful interdisciplinary (biological and computational) training. Fourteen ESRs completed the entire program, and three of these have already successfully defended their work and have been awarded a doctorate from their respective Universities. The remaining candidates are in the final parts of their PhD submission.
The hard and soft skills acquired together with the well-fostered science communication skills will be a major advantage to our trainees in their future careers. As a testament to this passion for communication several of the in3 ESRs have been actively involved in several outreach activities especially communicating with younger members of society. A short layman’s video was also developed by the ERS and is available in several languages. Finally, it is expected that the in3 graduates will continue to have a major impact in the field of ethical chemical safety assessment and related sciences, scientific public engagement and beyond.
Nicoleta Spînu (ESR at JM Liverpool University), presenting at the JRC during her secondment.
Caricature of the in3 consortium
Toxicity Assay hands on training. November 2018, Molecular and Computational Toxicity VU Amsterdam.
In3 ESRs give a flash presentation at ESTIV 2018, in Berlin.