OBJ1-Provide standardised protocols for quantification and characterisation of intrinsic and extrinsic Ad-NMs properties in complex matrices by the development of imaging protocols.
Protocols for the characterization, visualization, and quantification of intrinsic and extrinsic properties of Ad-MMs and NMPs were developed. Some standardized protocols were established for the characterization and quantification of material properties in biological and environmental fluids, including protocols based on advanced microscopy techniques, such as Raman Microspectrometry combined with confocal microscopy and Super-Resolution Imaging Microscopy.
OBJ2-Protocols and methods for isolating and characterizing the biomolecular corona formed on Ad-NMs and NMPs in biological and environmental fluids were established. The objective of developing reliable and exploitable procedures to correlate the biomolecular transformations of Ad-NMs and NMPs with their toxicity has been achieved.
OBJ3 -Develop cost-effective protocols for the quantitative determination of the environmental and health hazard of Ad-NMs reinforcing the 3Rs criteria.
Studies were conducted to characterize and develop an integrated platform for assessing the acute and subacute toxicity of Ad-NMs and MNPs. In particular, we employed 2D and 3D human cellular models to recapitulate the interaction of the materials with intestinal and lung epithelia in vitro, plant cellular models for ecotoxicity studies in vitro, and two animal models that can provide information on organ penetration and function.
OBJ4-Generate grouping and read-across approaches to support risk assessment and SSbD decision making
We have compiled an inventory of established and enhanced Ad-NMs known to affect health and pose ecological risks. There has been a particular focus on how materials' intrinsic characteristics affect their nanobio/eco interactions in complex biological matrices and environmental media. The potential impact of the properties on release, human biodistribution, environmental fate, and human and environmental toxicity has been assessed, and the confidence in this assessment has been evaluated. Moreover, we reviewed existing similarity assessment methods for chemicals and nanomaterials and prioritized methods such as the weighted ordered weighted average and machine-learning approaches, including Histogram-based Gradient Boosting Classification, for further advancement in POTENTIAL. In addition, the relevance of state-of-the-art approaches for grouping nanoforms (e.g. grouping hypotheses, IATA) to addressing the novel properties and interactions of Ad-NMs has been assessed, with a focus on the POTENTIAL case studies.
OBJ5 -Create a crosstalk with project results to international regulatory bodies for standardization and formal validation
We continued to exchange information with the European projects MACRAME, CHIASMA, the EFSA grant NAMS4NANO, and other EU projects. These projects are developing various aspects of animal-free testing for chemicals and nanomaterials. We have exchanged information, protocols, and approaches. As soon as we have significant results, we will engage the SGTA to discuss next steps and how to incorporate them into the regulatory framework.
