Integrated assessment and Advanced Characterisation of Neuro-Nanotoxicity

Project Information

iCare

Grant agreement ID: 101092971

DOI

10.3030/101092971

EC signature date 24 November 2022

Start date 1 January 2023

End date 31 December 2026

Funded under

• Digital, Industry and Space

Total cost

€ 2 744 655,00

EU contribution

€ 2 744 654,75

Coordinated by

INTERNATIONAL IBERIAN NANOTECHNOLOGY LABORATORY
Portugal

Periodic Reporting for period 1 - iCare (Integrated assessment and Advanced Characterisation of Neuro-Nanotoxicity)

Reporting period: 2023-01-01 to 2024-06-30

Summary of the context and overall objectives of the project

The key issue addressed by the iCare project is the lack of guidelines and regulations to assess the neurotoxicity of ANMC, particularly their ability to cross the blood-brain barrier and potentially cause irreversible damage to the central nervous system. Currently, there are no OECD or ISO guidelines to evaluate in vitro neuro-nanotoxicity or the effects of ANMC on the central nervous system. iCare aims to develop the world's first integrated approach for the comprehensive assessment of neuro-nanotoxicity. This approach will involve three innovative technologies: (1) high-throughput/high-content analysis, (2) multiparametric analysis, and (3) harmonized in vivo-in vitro assessment. iCare will evaluate the transformations that ANMC undergoes during manufacturing, use, and release into the environment. This information will support the grouping and read-across schemes proposed by ECHA, which are important for cost-effective data collection for REACH dossiers. The key elements of iCare include developing advanced imaging and characterization methods for ANMC in complex matrices, creating new realistic in vitro models and high-throughput assays for neuro-nanotoxicity studies, establishing in vitro-in vivo bridging models, and conducting read-across and life cycle assessment studies. The project aims to reduce the need for animal testing, shorten time to market for innovators, and potentially reduce the incidence of neurodegenerative diseases, contributing to healthy aging. Overall, iCare addresses a critical gap in the regulation of ANMC, particularly regarding their potential neurotoxicity, and proposes an integrated approach to improve the assessment and management of these materials in the context of the European Union's sustainability goals.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

iCare is divided into four phases to address the challenges of nanomaterial characterization and toxicological assessment. This integrated framework combines advanced laboratory investigations, new in vitro models, and bridging between in vitro and in vivo studies to generate robust data for the eNanoMapper database. In brief, the different phases are:
- Phase 1 focuses on enhanced high-resolution imaging techniques. The project partners will develop and validate novel imaging methods capable of providing detailed characterization of engineered nanomaterials (ANMCs) at the lab scale during the first 36 months of the project. These advanced imaging technologies will enable researchers to gain a deeper understanding of the physicochemical properties and behavior of nanomaterials, crucial for accurately assessing their potential toxicological impacts.
- Phase 2 involves the creation of new in vitro models, including those that bridge the gap between in vitro and in vivo studies. These models will be designed to more closely mimic the complex biological environments and interactions that occur in living organisms, allowing for more reliable and relevant toxicological assessments. Data generated from these in vitro investigations will be fed into the eNanoMapper database, contributing to the growing knowledge base on nanomaterial safety.
-Phase 3 integrates the technologies and methods developed in the first two phases and applies them to real-world industrial use cases. During months 24 to 36, iCare will test the advanced imaging techniques and in vitro models on samples collected from points along the value chains where the risk of ANMC release and exposure is considered greatest. This demonstration of the methods under realistic conditions will validate their performance for nanomaterial risk assessment.
- Phase 4 focuses on preparing and lobbying for new dedicated test guidelines and standards. The consortium will work towards the development of updated OECD test guidelines and new standards. This critical step will help ensure that the innovative approaches and methods developed in iCare can be widely adopted and integrated into the regulatory framework for nanomaterial safety assessment.

iCare’s methodology aims to accelerate the development and implementation of robust, reliable, and reproducible tools and techniques for the characterization and toxicological evaluation of engineered nanomaterials. By combining cutting-edge laboratory investigations, advanced in vitro models, and a focus on regulatory standards, iCare aims to drive progress in the field of nanosafety and contribute to the safe and responsible development of nanomaterials.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

1) Super-resolution imaging of nanoparticle behavior: Current imaging techniques either have high spatial resolution but cannot monitor dynamic behavior, or can track particle movement but with poor resolution. iCare will adapt super-resolution fluorescent imaging to enable unprecedented insights into how nanoparticles aggregate, decompose, and interact in complex biological matrices.
2) High-throughput, multi-modal imaging of cell mechanics: Existing methods for evaluating the mechanical properties of cells are precise but time and resource-intensive. iCare will adapt nanoindentation technology to enable high-throughput, high-resolution imaging of cell mechanics as a marker of nanotoxicity and blood-brain barrier permeability. This will provide a faster, more efficient way to assess these important toxicity endpoints.
3) High-content fluorescent imaging assays for nanotoxicity: While high-content phenotypic imaging has been used to study small molecule toxicity, it has not been widely applied to nanomaterials. iCare will adapt these techniques to provide a more time- and cost-effective way to evaluate nanotoxicity while also gaining insights into potential toxicity mechanisms.
4) Integration of real-time multiplex sensing with advanced in vitro models: iCare will integrate a real-time, multi-sensing device with their advanced blood-brain barrier and brain models to continuously monitor the release of biomarkers in response to nanomaterial exposure, providing detailed information on the initiation and progression of cytotoxic processes.
5) Development of advanced in vitro models: iCare will create a novel, realistic in vitro blood-brain barrier model using a multi-chamber dynamic setup with various neurovascular unit components. They will also develop brain organoids for acute and long-term neurotoxicity studies, providing more realistic and cost-effective alternatives to animal models.
6) Bridging in vitro and in vivo models: iCare will establish the use of C. elegans and planarians as bridging models between in vitro and in vivo systems for evaluating neuro-nanotoxicity and ecotoxicity, respectively. These invertebrate models are more cost-effective and address ethical concerns associated with mammalian models.
7)Industry-relevant sampling and testing protocols: iCare will systematically study nanoparticle release mechanisms and develop sampling protocols that better mimic industrial scenarios, addressing the current reliance on simple, pristine samples that may not accurately represent real-world conditions.