MACRAMÉ – Advanced characterisation methodologies to assess and predict the health and environmental risks of advanced materials

Abstract

Ensuring the safety and sustainability of advanced materials (AdMas) is critical for fostering innovation while protecting human health and the environment. As industries integrate AdMas into commercial products to innovate in the next stage of the value chains, there is an urgent need for robust methodologies to detect, characterize, and assess their potential risks throughout their life cycle. The MACRAMÉ Project addresses this challenge by advancing standardized testing and regulatory frameworks, supporting the EU’s vision for a toxic-free environment. Through cutting-edge research and international collaboration, MACRAMÉ lays the groundwork for reliable hazard assessment, regulatory compliance, and the responsible development of next-generation materials. The MACRAMÉ Project aims to enhance the detection, characterization, and quantification of Advanced Materials (AdMas) throughout their life cycle, assessing potential human and environmental health impacts during exposure. By developing, demonstrating, and standardizing advanced methodologies, MACRAMÉ ensures their broad applicability across market-relevant AdMas-containing products. Fully aligned with EU strategies such as the Chemical Strategy for Sustainability and the European Green Deal, the project extends nanosafety approaches to the broader AdMas category, focusing on inhalable carbon-based materials - graphene-related materials, carbon nanofibers, and poly lactic-co-glycolic acid nanoparticles. Building on over 15 years of research, MACRAMÉ integrates knowledge from major European and international initiatives to establish harmonized test guidelines, guidance documents, and standards. Through five industrial Use-Cases, the project applies innovative sample preparation, detection, and toxicity assessment techniques to develop a tiered approach for AdMa safety testing. Centralized in the MACRAMÉ Information Hub, all data will support regulatory frameworks and future research. The project’s outcomes - harmonization and pre-standardization proposals - will contribute to a unified European assessment framework, reinforcing the continent’s leadership in safe and sustainable materials innovation.

Graphical Abstract

1. Introduction

Europe has committed to transitioning toward a sustainable, resilient, and toxic-free economy and to achieve climate neutrality by 2050. This transition is guided by key political strategies, including the European Green Deal [1] and the EU Chemical Strategy for Sustainability [2]. These initiatives aim to position Europe as a global leader in green technologies while ensuring the safety of human health and the environment. At the heart of these strategies are ambitious targets designed to address the dual challenge of advancing technological innovation and mitigating risks associated with new materials. Recognising the transformative potential of advanced materials (AdMas) — such as graphene-based materials, carbon nanofibers, and polymer-based nanocomposites selected as the focal materials for investigation within MACRAMÉ — the EU has set clear ambitions for their sustainable integration into industrial and consumer products. These materials hold the promise of enabling innovations across sectors [3], [4]. However, their complex nature presents unique challenges, particularly in terms of impact assessment across material and product lifecycles and regulatory safety testing.

Specific indicators and political targets of the EU’s industrial competitiveness and environmental policies include:

• •Climate Neutrality by 2050: As outlined in the European Green Deal, Europe aims to achieve net-zero greenhouse gas emissions by mid-century. AdMas, such as graphene and carbon nanofibers, are seen as key enablers in creating energy-efficient technologies and supporting decarbonization across industrial sectors.
• •Zero Pollution Ambition: The EU Chemical Strategy for Sustainability emphasizes the reduction of pollution from chemicals and materials, aiming for a "toxic-free environment." This includes eliminating hazardous substances from products, reducing the environmental release of harmful materials, and transitioning to safer alternatives through safe and sustainable (by) design (SSbD) principles.
• •One Substance – One Assessment: To streamline regulatory processes and enhance safety, the EU promotes an increasingly unified approach to chemical risk assessments. This initiative is a cornerstone of the Green Deal, simplifying compliance, improving transparency for stakeholders in regulatory, industrial, and scientific communities using a Common Data Platform on Chemicals to support regulatory risk assessment.
• •Support for the Circular Economy: AdMas are critical to achieving a circular economy, where resources are reused, and waste is minimized. Specific goals include increasing the recycling rate of materials, reducing landfill dependency, and designing products for durability and recyclability.

The MACRAMÉ project, funded under the EU’s Horizon Europe program [5], is a collaborative and strategic research and innovation (R&I) initiative addressing the urgent need for the safe and sustainable design, application, and management of AdMas. These AdMas, including nanomaterials and composites, are crucial in various industrial, medical, and consumer sectors. MACRAMÉ aims to shape regulatory frameworks rather than merely contributing to a voluntary SSbD approach. By focusing on carbon-based AdMas — such as graphene-related materials (GRMs), carbon nanofibers, and poly(lactic-co-glycolic acid) (PLGA) nanoparticles — the project advances methodologies to detect, characterise, and assess these materials in real-world applications and in stages at the end of their lifetime, thus addressing existing regulatory and technical gaps.

Aligning with the European Green Deal and the Chemical Strategy for Sustainability, MACRAMÉ builds on over 15 years of research in nanosafety, leveraging collaborations like the Graphene Flagship [6] and the Malta Initiative [7]. The Graphene Flagship is a major European Union research initiative launched in 2013 with a budget of €1 billion, aimed at taking graphene and related two-dimensional materials from academic laboratories into European society. It brings together over 170 academic and industrial partners from 22 countries, exploring various aspects of graphene and related materials to accelerate industry acceptance and develop applications in areas such as flexible electronics, telecommunications, aerospace, and medical technologies [8]. The Malta Initiative is a European-led effort aimed at improving the regulatory testing of chemicals under the framework of EU chemical safety laws, particularly REACH. Launched in 2018, it focuses on promoting the use of New Approach Methodologies (NAMs) - computational models amongst others - to enhance chemical and materials safety assessments.

The MACRAMÉ project addresses the critical need for robust, harmonised methodologies to ensure the safe and efficient integration of AdMas into global markets. Its central objectives include:

• •Developing reliable tools for the detection, characterisation, and quantification of AdMas during their lifecycle, focusing on handling, processing, and end-of-life stages;
• •Assessing health and environmental impacts of AdMas in real product formulations across diverse exposure scenarios;
• •Enhancing the applicability and validation of advanced testing methodologies in regulatory and industrial contexts; and
• •Supporting harmonisation and standardisation through collaboration with key regulatory bodies like the Organisation for Economic Co-operation and Development (OECD), Versailles Project on Advanced Materials and Standards (VAMAS), European Committee for Standardization (CEN) and the International Organization for Standardization (ISO).

Through its interdisciplinary approach, MACRAMÉ bridges gaps between scientific, industrial, and regulatory stakeholders, fostering innovation and ensuring that AdMas contribute positively to a sustainable and health-conscious future.

2. Project description

2.1. The MACRAMÉ R&I approach

The MACRAMÉ project focuses on the safe and sustainable integration of AdMas into industrial, medical, and consumer products. It addresses critical gaps in the detection, characterisation, and assessment of AdMas — specifically the above mentioned compounds. The project’s scope extends beyond traditional nanomaterial analysis, targeting real-world applications across diverse lifecycle stages including integration into multi-component products and interactions with biological systems, together labelled as complex matrices. Key industries covered include energy (storage), healthcare, transportation, and consumer products. One of the major targets is to contribute to policies, standards, and guidance towards safety and sustainability, aligning with the EU’s Green Deal strategic pillar of a zero-pollution ambition for a toxic-free environment (see Fig. 1).

Fig. 1.

Illustration of the MACRAMÉ R&I Approach (AdMa@CMs: Advanced Materials in complex matrices; CF: Characterisation Factor; GRM: graphene-related material; IATA: integrated approaches to testing and assessment; LCA: Life-Cycle Assessment; LCC: Life-Cycle-Costing; MFA: Material-Flow Analysis; RA: Risk-Assessment; SSbD: Safe and Sustainable -by-Design).

The MACRAMÉ project aims to achieve several high-level objectives, including:

1.Community Integration and Methodological Streamlining: MACRAMÉ is establishing interfaces among regulatory, industrial, and scientific communities to align methodologies for assessing risks associated with AdMas in complex matrices (AdMa@CMs);

2.Lifecycle Exposure and Sampling Assessment: MACRAMÉ is evaluating AdMas across five industrial use cases to identify exposure points and lifecycle transformations, ensuring their safety in real-world applications;

3.Advanced Characterisation Techniques: MACRAMÉ is developing and harmonising state-of-the-art detection and imaging methods for AdMas, including physical, chemical, and biological characterisation;

4.Hazard and Toxicity Testing: MACRAMÉ is advancing in vitro and ex vivo models to assess inhalation risks of AdMas, focusing on tiered testing frameworks for regulatory applications; MACRAMÉ is also innovating in ecotoxicological assessment, extending the range of fish cell lines used for in vitro testing and assessing the adaptations to exposure scenarios needed for OECD tests in algae and daphnids;

• 5.Standardisation and Regulatory Readiness: MACRAMÉ is supporting global harmonisation by contributing pre-validated methods to organisations like the OECD, ISO, and CEN;
• 6.Data Stewardship and FAIR Principles: MACRAMÉ is implementing a centralized data hub to ensure adherence to FAIR (Findable, Accessible, Interoperable, and Reusable) principles, and implementing instance maps as a tool to track AdMas material and data provenance (for more details see Section 3 below) [17] thereby fostering transparency and knowledge sharing.

The resulting efficiency and effectiveness of the MACRAMÉ Methods are demonstrated through their application in Use-Case evaluations, using Life Cycle Assessment (LCA-), Life Cycle Costing (LCC-) and SSbD-based approaches [9], highlighting benefits like reduced costs of regulatory compliance, by following the process developed in the MACRAMÉ Safety & Sustainability Matrix. This matrix is a modular building block for MACRAMÉ ’s information-transfer interfaces for different scientific and regulatory communities, and will thus provide a stepping-stone for Europe’s route towards a ‘one substance – one assessment’ approach [10] and promote an open strategic autonomy [11] through key enabling and emerging technologies, including digital ones.

2.2. The MACRAMÉ ambition

The current state-of-the-art is to characterise and assess AdMas in their pristine form; MACRAMÉ surpasses this by looking at AdMas and AdMa-containing products in their market-relevant form in complex matrices. Based on this, one of MACRAMÉ ’s central challenges is posed by its focus on three carbon-based MACRAMÉ Material Families, which are hard to detect against a background of complex matrices. The following scientific & technical ambitions are pursued in MACRAMÉ to tackle this formidable challenge:

• •To support development, harmonisation, and benchmarking of testing methods applied within the Project, a MACRAMÉ Control Material Library (CML) was established. The CML contains established control/reference materials (of known properties) and new samples from the MACRAMÉ Material Families, prepared using standardised methods. The CML will be made available as a new service to collaborating and future projects, extending existing repositories (e.g., the EU Joint Research Centre (JRC) repository of certified reference materials [12] or representative nanomaterials [13], by adding well characterised AdMas and AdMa@CMs.
• •To measure the physical stability and aggregation propensity in biological and environmental liquid media, MACRAMÉ will go beyond current methods that are limited in their applicability to dark/opaque samples, such as GRMs. To assess the biological impact of AdMas upon inhalation, MACRAMÉ will further develop and optimise in vitro biological systems with increasing complexity representative of upper and lower airways, benchmarked against reference materials and AdMas from the MACRAMÉ CML, to demonstrate that these in vitro models are fit for purpose, start (pre-)evaluation for regulatory applications, and foster the development of a guidance document or test guideline for hazard assessment of inhalable AdMas.
• •To validate novel, robust methods to characterise AdMas in suspensions, including static multiple light scattering (SMLS), these are compared to complementary approaches;
• •To detect, spatially co-localise and characterise GRMs and CNTs in composites and biological matrices, innovative object localisation and correlative microscopic techniques are further developed to overlay scanning electron microscopy (SEM), SEM with energy dispersive spectroscopy (SEM/EDS), optical microscopy (OM) and confocal Raman chemical images with high accuracy by using image object recognition and pattern constellation matching algorithms (based on ISO-G-SCoPe results [14]);
• •To determine the surface oxygen content of GRMs and CNTs, high-sensitivity EDS (<100 nm lateral resolution) is used and complemented by X-ray photoelectron spectroscopy (XPS) and Raman analyses.
• •To quantitatively measure the uptake of PLGA doped with metal and/or metal oxide particles in cells and tissues from 3D cultures, state-of the-art LA-ICP-MS is used at subcellular resolution (< 5 µm). Combining histological images and spatially resolved results from LA-ICP-MS will allow for the evaluation of cellular uptake/tissue distribution of AdMas.
• •To provide harmonised approaches for the generation of comparable experimental results and, in this way, provide benefit to the entire inhalation toxicology field, MACRAMÉ is developing harmonised protocols for the generation and characterisation of aerosols for the in vitro exposure of biological systems for inhalation toxicology.
• •To assess the impact in the environment of the AdMas in relevant stages of the life cycles of the use cases, MACRAMÉ will further develop existing OECD ecotoxicity assays with increased complexity to allow assessment of acute, subchronic and chronic effects, and to assess internalised doses via in vitro gill barriers.
• •To support the evaluation of the environmental footprint impact of products and processes, MACRAMÉ will use in vitro / in vivo data extrapolation approaches and LCA inventories to produce data in a cost-efficient way.
• •To harmonise the simulation of the incineration processes and associated releases at different oxygen concentrations and advance the sampling and online/offline characterisation of gas and soot particles emitted during the thermal degradation process, a laboratory test bench is being developed by using a controlled-Atmosphere Cone Calorimeter and a tube furnace.

2.3. Workflow and collaborations within the MACRAMÉ consortium

The MACRAMÉ project consortium – comprising of scientists from 11 industry partners, 2 academic partners, 4 RTOs, and 2 institutions - combines a wide range of capabilities essential for advancing research and innovation in the safe and sustainable use of AdMas. The project brings together strong expertise in scientific coordination, research and innovation management, regulatory affairs, policy development, and standardisation. It includes advanced competencies in toxicology and risk assessment, including the development of Safe-by-Design frameworks and life-cycle-based evaluations such as LCA, LCC, and MFA.

Key technical capabilities lie in the development and validation of advanced physical-chemical characterisation methods, including aerosol generation, airborne particle analysis, end-of-life simulations, and surface analysis using state-of-the-art imaging techniques such as LA-ICP-MS, ToF-SIMS, and hyperspectral imaging. The project further integrates correlative microscopy and high-resolution imaging to track AdMas in complex matrices and biological systems.

Biological testing capabilities are equally robust, covering the development of in-vitro and ex-vivo models for inhalation toxicology, including tiered testing strategies for human health risk assessment. The project partners also provide comprehensive ecotoxicological testing using established and novel assays for regulatory purposes.

Another cornerstone of MACRAMÉ is its strong data management and informatics capacity. The project focuses on designing interoperable (meta)data formats, building a central information hub, and ensuring FAIR data principles through tailored data stewardship and curation services. Computational modelling, machine learning, and the development of ontologies for complex material matrices complement these efforts.

The consortium is also equipped with extensive expertise in regulatory alignment and stakeholder engagement, providing guidance for interfacing scientific, industrial, and regulatory communities at both national and international levels. These capabilities ensure that project outputs are not only scientifically sound but also aligned with current and future policy frameworks and ready for standardisation and exploitation. The collaboration between the MACRAMÉ project partners is structured around a tightly integrated, multi-layered approach. All partners jointly contribute to the development and implementation of a shared R&I strategy that serves as a foundation for the entire project. This is initially fine-tuned through joint planning sessions, where each partner helps define the workflows, timelines, and data exchange protocols that align with the project’s objectives across the different work packages. Key interactions are formalized early on through a shared strategy and later refined through Needs Assessments and engagement with external regulatory bodies.

The collaborative structure is cross-functional, as partners work together to map stakeholders, design sampling strategies, develop and validate characterisation methods, and set up advanced toxicity models. Cross-WP activities such as the generation of a Control Material Library (led by LNE with contributions from multiple partners) or the establishment of controlled aerosol generation (led by BAuA) illustrate how partners share resources and expertise to ensure harmonisation of methods. Additionally, platforms like the MACRAMÉ Information Hub, led by 7P9-SI and UoB, are central to facilitating the exchange of harmonised data across partners and work streams.

Coordination is enhanced through regular project-wide and WP-specific meetings, as well as through joint events such as the Risk Assessors Summits and stakeholder workshops, ensuring scientific alignment and regulatory relevance. This interconnected, iterative approach ensures that all technical, regulatory, and methodological advances are co-developed and co-evaluated across the consortium.

More detailed information on the milestones shown in Fig. 2 (Mx.x) can be found below: Confirmed & Specified R&I Strategy Agreed (M1.1): The consortium has reconfirmed and specified the project’s Research & Innovation Strategy. Benchmarks have been agreed upon to monitor and indicate progress throughout the project’s lifecycle.

Fig. 3.

In-vitro and ex-vivo Models that will be assessed within the Project, indicating the estimated cost that can be saved and MoA- Information that can be obtained at the different tiers.

Fig. 2.

Schematic overview of the MACRAMÉ project, its partners and their main roles, illustrating the workflow and interactions between the main project elements; milestones are shown in italics.

Presentation of Results at the 2nd Risk Assessors Summit (M1.2): Project results were presented at the 2nd Risk Assessors Summit. The presentation focused on addressing the needs and gaps identified, gathering feedback, and providing support to facilitate the integration of results into standards and policymaking.

Criteria for the MACRAMÉ CML Established (M2.1): A protocol had been finalized for the selection, sampling, and processing of samples for the Common Material Library (CML). The initial exchange of samples between partners marks an important step in the project's research activities.

In-vitro & Ex-vivo Models Established and Qualified at Respective Premises (M2.2): A suite of eight in-vitro methods had been established and verified across partner facilities. These methods served and will serve both for initial screening and ranking, as well as for more detailed hazard characterization, strengthening the scientific reliability of the project’s outputs.

Draft SOPs for Exposure and Characterisation Measurements (M2.3): Pre-final Standard Operating Procedures (SOPs) had been prepared for exposure and characterization of CML materials. These include two SOPs for controlled aerosol generation and two SOPs for cell exposure, helping to harmonize methodologies across the consortium.

MACRAMÉ Information Hub Fully Operational (M3.1): The Information Hub is fully operational, providing a platform for (meta)data exchange. It includes tools for efficient information management and sharing and establishes direct connections to external data sources, enhancing the project’s data interoperability.

Data Stewardship & Quality Control Established and Embedded (M3.2): Following a Data Stewardship Workshop, a data shepherd has been appointed and their role clearly defined. Data stewardship and quality control processes have been embedded within the consortium to ensure compliance with data reporting standards and to safeguard data quality.

Human- and Machine-readable Data-exchange Formats Defined & Integrated (M3.3): Standardized human- and machine-readable data-exchange formats have been defined and integrated into project activities. Training materials have been delivered, and partners have begun incorporating their datasets into the project’s information systems.

Sampling & Sample-provision Procedures Set (M4.1): Samples have been collected from exposure points and provided to project partners according to the established sampling protocols, ensuring consistency and traceability throughout the project.

LCI Dataset & Development of CFs Completed (M4.2): The Life Cycle Inventory (LCI) dataset and Characterisation Factors (CFs) had been completed and documented, covering all five project Use-Cases and their respective life-cycle stages. This dataset will support comprehensive environmental impact assessments.

Establishment of In-vitro Ecotox Models (M4.3): In-vitro ecotoxicology models will have been established for the five project Use-Cases. These models enable the development of decision trees, facilitating more structured and transparent environmental risk assessments.

MACRAMÉ Methods for TGs, GDs, Standards Mapped (M5.1): Five MACRAMÉ methods or methodologies have been mapped for potential use in the development of TGs, GDs, and relevant standards. These mappings had been shared with key stakeholders.

Draft Recommendations on Needs for TG & Standards Developments (M5.2): Draft recommendations regarding the needs for future Test Guidelines and standards development had been prepared. These recommendations had been distributed to stakeholders to collect feedback and refine the project’s contributions to regulatory and standardization efforts.

MACRAMÉ Corporate Identity Established & Dissemination Channels Set (M6.1): The MACRAMÉ corporate identity has been established, including the design of the project logo and the creation of a project website. These elements will ensure a unified visual identity and enhance the dissemination of project activities and results.

2.4. The MACRAMÉ drawing board - collaborative strategy setting & adoption

The MACRAMÉ workflow is highly complex, because it aims to analyse the physical-chemical characteristics and impact on humans and the environment of four different AdMas during the lifecycle of five products. The project started with a collaborative planning exercise, to establish collaborations and to re-confirm and further detail the underlying MACRAMÉ R&I Strategy, to set time-critical benchmarks to oversee the performance and achievements and to guarantee the maximisation of MACRAMÉ ’s highly interdisciplinary approach and its impacts. This approach guaranteed the resilience of the project delivery and enables the detailed consideration of the re-use of, and value-add to, previous project results, arrangements of collaborative approaches with sister projects, as well as the engagement of the wider relevant stakeholder community (elaborated in the MACRAMÉ Stakeholder Roadmap. During the first collaborative project meetings, and following the IndustryCommons initiative [15], MACRAMÉ has bridged between communities by emphasising the interdisciplinary knowledge exchange, acquisition of skills, innovation methodologies and new knowledge creation, demonstrated through the efficient and effective application of the MACRAMÉ results and outputs in various industry sectors (i.e., chemicals, healthcare, electronics, transportation, and consumer goods).

2.5. The MACRAMÉ foundations - use-case definitions & sample provision

The MACRAMÉ project identifies critical product- and life-cycle-relevant exposure points where AdMas may encounter humans or the environment. These exposure points span from production to the end-of-life of the materials and their host products. At each stage, specific sampling, characterisation, imaging, detection, and testing activities are performed to assess the potential for exposure and hazard under realistic scenarios.

At the production and manufacturing stage, exposure may occur during the handling of AdMa powders and their incorporation into polymer matrices or other products. Potential emissions include inhalable aerosols and workplace residues generated during processes such as compounding, machining, or mixing. At this stage, samples are collected from workplace air (e.g., using personal samplers or cascade impactors), residual dust, and waste streams. These samples are used to assess occupational exposure and emissions into indoor environments.

During the use and service life stage, materials embedded in consumer or industrial products may be released through mechanical wear, spraying, heating, or degradation. For example, aerosols and surface residues can result from normal product use or accidental scenarios such as overheating or mechanical failure. Sampling efforts at this stage focus on collecting airborne particles, droplets, surface swabs, and wastewater generated from product use. The collected samples help evaluate potential consumer exposure and environmental contamination under typical and extreme use conditions.

The end-of-life stage introduces further exposure scenarios as products are incinerated, landfilled, recycled, or undergo weathering and degradation. Processes such as shredding, pyrolysis, or combustion can lead to the generation of secondary emissions, including soot, char, and aerosols, as well as leachates from landfills. Sampling at this stage captures solid residues, gaseous emissions, and liquid leachates to understand how AdMas are transformed and released during waste treatment or disposal.

For each of these exposure points, a harmonized approach to sample collection is implemented across the consortium, based on the "Sampling & Sample-Provision Protocols for AdMas in Complex Matrices." These protocols ensure consistent sampling of aerosols, product matrices, biological matrices, and environmental media, enabling reliable downstream analysis. Collected samples undergo a broad suite of characterisation, imaging, and detection activities. The project also carries out human toxicity testing using advanced in-vitro models. Tiered lung models simulate realistic inhalation exposures to sampled aerosols and particulate matter. A range of toxicological endpoints are evaluated, including cytotoxicity, inflammation, oxidative stress, and genotoxicity. The project also incorporates chronic and repeated-dose testing to capture potential long-term health effects. Particle uptake and localization within cells are analysed using quantitative imaging methods.

Complementary ecotoxicity testing is conducted to assess environmental risks. Standardized aquatic bioassays using algae, Daphnia, and fish cell lines are applied to evaluate both acute and chronic toxic effects. Testing also considers environmental fate processes, such as agglomeration, sedimentation, and degradation of AdMas, along with ecotoxicological assessments of secondary materials like soot or char generated during end-of-life treatment.

All data generated across these exposure points are integrated into the MACRAMÉ Information Hub, which supports the broader objective of informing SSbD approaches. In doing so, MACRAMÉ delivers a comprehensive understanding of how advanced materials behave and interact with biological and environmental systems across their entire life cycle.

2.6. The MACRAMÉ laboratory - characterisation & testing of AdMas in complex matrices

The ambitious MACRAMÉ objectives are being achieved through the development of (a) a MACRAMÉ CML and (b) MACRAMÉ (in-project) Validated Protocols. Based on exposure points defined by the five MACRAMÉ Use-Cases, the CML will provide reference materials and AdMas, matrices and their composites to partners to enable the R&I measurement- and test-transferability studies of the MACRAMÉ Project. The CML will contain samples of all combinations of AdMa@CMs described above. Once material quality, stability and traceability are established, the CML will be offered to other projects and standardisation and harmonisation communities. Library AdMas will be catalogued with respect to their life-cycle state and matrix; the AdMas will include platelet-shaped (2D) (i.e., GRMs, namely GO, FLG (4–5 layers), MLG (multi-layer graphene) (10–20 layers)), fibre-shaped (1D) (i.e., CNTs) and spherical (0D) (i.e., good manufacturing practice (GMP)-compliant polymer) (nano)particles. The selection of applied methods will be optimised and pre-validated for the different material classes. MACRAMÉ Validated Protocols will be defined and pre-validated with respect to robustness, repeatability and reproducibility.

The developed methods are focused on:

• •Measurement of critical attributes of GRMs, including (i) their stability, biotransformation and release in liquid media by SMLS for simultaneous screening of size, agglomeration state, stability and dosimetry of non-spherical particles like FLG in turbid solution [16], in relevant liquid mixtures (water-solvent), environmental matrices and biological fluids; (ii) measurement of the oxidation state of GRMs by high-sensitivity EDS detector complemented by XPS and Raman analyses;
• •Detection, spatial co-localisation, characterisation, and quantification of GRMs and CNTs in polymer composites and cells by correlative microscopic techniques. This will be achieved by spatially accurate overlaying SEM, SEM/EDS, AFM, optical microscopy (OM) and/or confocal Raman chemical images using image object recognition and constellation matching algorithms;
• •Approaches for controlled aerosol generation including wet techniques already established in the framework of inhalation studies, and new dry techniques that allow re-designing aerosols generation from dust and powder materials representative of real-life exposure scenarios as well as innovative techniques to deliver aerosols to cell models;
• •A modular approach for in-vitro cell exposure to aerosols of controlled morphology and dosimetry to ensure that the cell exposure dose is well defined and well characterised;
• •Approaches for detection and semi-quantitative measurement of AdMas in biological fluids, cells and tissue using a combination of advanced imaging techniques relying on light microscopy (i.e., enhanced darkfield microscopy, hyperspectral microscopy, confocal Raman microscopy), and mass spectrometry (i.e., LA-ICP-MS or Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS));
• •Controlled protocols for the simulation of end-of-life scenarios and further toxicological testing according to the reconfirmed product-relevant MACRAMÉ Exposure-Points and detailed during the LCA (e.g., incineration / pyrolysis / weathering, for the detection of released AdMas from finished products and for the characterisation of their degradation (i.e., applying different oxygen concentration in a controlled-Atmosphere Cone Calorimeter or in a tube furnace flushed to simulate incineration and/or pyrolysis));
• •Biological systems of increasing complexity (i.e., from single cell culture to advanced multi-cellular cultures and ex-vivo human tissue), to generate a MACRAMÉ Tiered Lung-Model Approach for hazard assessment of inhalable AdMas (see Figure 4).

• •Innovation in ecotoxicological models of increasing complexity, allowing assessment of acute, sub-chronic and chronic impacts leading to an improved and extended version of the Threshold Approach for Acute Fish Toxicity Testing [34] that accounts for the specific features of AdMas. The MACRAMÉ ecotoxicology approach, shown in Fig. 5, includes an innovative extension to the existing OECD TG 249 (Rainbow Trout Fish gill cell tests) over acute and sub-chronic timescales. (24 h - 2 weeks) supplemented with the existing acute OECD TG 201 and TG 202 tests (microalgae and daphnids, respectively), and the subchronic and chronic TG 211 (Daphnia reproduction test) utilising a conditioned medium approach to effectively disperse the materials.

Fígure 4.

Schematic illustration of the MACRAMÉ ecotoxicity testing approach and outcomes.

Fig. 5.

Schematic illustration of the flow- of and value-add to data within the MACRAMÉ Project. (TG=Technical Guideline, GD=Guidance Document, SOP=Standard Operating Procedures, LCA=Life Cycle Analysis, LCC=Life Cycle Costing, MFA=Material Flow Analysis, RA=Risk Assessor, AdMa@CM=Advanced Material in Complex Matrix, IATA=Integrated Approach to Testing and Assessment.

2.7. The MACRAMÉ processor – centralised knowledge-generation

To fully document the MACRAMÉ achievements in identification, characterisation and assessing AdMa@CMs including the need to clearly define the life-cycle stage (starting material, pristine, product, use and end-of-life) and the sample preparation, all information is managed and exchanged via the MACRAMÉ Processor, guaranteeing safeguarding of all MACRAMÉ (Meta)Data with full material, sample and data provenance trails. This data and knowledge exchange between the partners occurs in the form of materials and sampling plans, study designs translating needs into actions, method specifications describing the measurement principles, protocols and later SOPs, and finally the data itself (raw, transformed and robust summary), all which is called MACRAMÉ (Meta)Data in the following. Each MACRAMÉ method defines its applicability domains and constraints based on, and constantly updated with, the data produced in the Project including calibration measurements and negative/positive result samples. Full provenance trails to underlying data are generated simultaneously; to provide a seal of quality for each method, and to guide its selection and assay parameters, giving the most reliable result for the material, and ultimately guiding the development of standard operating procedures (SOPs) for standardisation / validation of the method.

For each UC, information is initially generated by the MACRAMÉ Foundations and Demonstrator (including materials flow analyses, exposure points, sampling points and protocols, and sample preparation SOPs). This MACRAMÉ data is being further enriched with information from a multitude of other sources and aggregated for knowledge discovery. This is done on different levels to:

• •provide information on materials study design and data analysis stage for specific MACRAMÉ methods;
• •test for the transferability of analysis approaches from one method to the other; and
• •evaluate data aggregation approaches applied in the UCs for automatisation and generalisation.

All of this information will be integrated, harmonised and handed back to the UCs for analysis against LCC criteria, and in a condensed (summary) form to the MACRAMÉ Interpreter and the MACRAMÉ Facilitator (see Fig. 2) as the basis for the development of updated test guidelines and standards, and for dissemination of the Project results to target stakeholders.

Collected data will ultimately also be applied in approaches for grouping and read-across. This will assess the relevance of the data produced in MACRAMÉ for setting the boundaries between nanoforms and alternative definitions for AdMA groupings. Grouping and read-across approaches have been recognised as very useful by ECHA and presented at the OECD respectively for assisting industry with preparing their registration dossiers as required under REACH for chemicals and nanomaterials. The project is not going beyond what is needed for regulatory validation and acceptance. However, by providing open data, the process is made more transparent and build data bases to be used as benchmarks for other labs implementing the methods and for comparison with results on new materials and/or chemicals. Even a link back to grouping and read-across might be possible since there also data-rich materials are needed, which are available in the form of materials used in the method validation. MACRAMÉ will go beyond the state of the art here by making all this information with the underlying data publicly accessible.

The data generation process just described is depicted in Fig. 6: it is based on a very intense two-way exchange of information and knowledge between all partners (working in different WPs) and documents all developments of new characterisation methods and their pre-validation via the Control Library and UCs.

Fig. 6.

Illustration of the abrasion of pure epoxy and epoxy-FLG composite, developed by EMPA.

To support this creation and usage of the method- and life-cycle-stage-specific documentation covering all aspects (method specifications, SOPs, data), a MACRAMÉ data shepherd has been established. A data shepherd is an enhanced version of a data steward, who not only oversees the data management, handling and quality control processes, but can mediate between parties in need of data management solutions and parties providing them and resolve any misunderstandings [17]. In this way, implementation of the FAIR principles and harmonisation with major European Initiatives (EU NanoSafety Cluster, European Materials Characterisation Council and IndustryCommons [18]) by reusing (at least in parts) and extending community interoperability approaches can be achieved. Curation, storage and sharing tools are selected and optimised for the Project and implemented and customised to generate the MACRAMÉ Information Hub as a one-stop interface to access all information, with each data point clearly associated with the lifecycle state of the material, the environment in which it was studied, as well as the method and protocol used to create it.

2.8. The MACRAMÉ demonstrator – analyses, assessments & validations in industrial value-chains

Based on the identified exposure points and the characterisation of AdMas in complex media performed, the MACRAMÉ Laboratory, in collaboration with the MACRAMÉ Demonstrator, will conduct a detailed Material flow analysis, to obtain an overall assessment of the releases of materials at each stage of the AdMas lifecycle for all possible applications and the potential exposure to humans. The evaluation of AdMas degradation or transformation in the matrix of the final product, or during use and at the end-of-life or ageing under environmental conditions is, although extremely challenging, highly relevant, as currently a worst-case scenario is assumed, where AdMas stay in their pristine form.

The application of LCA to novel materials such as AdMas is hampered by the absence of characterisation factors which allow comparison of the environmental impacts of various substances. Information collected in MACRAMÉ will support the 3Rs strategy on reduction, refine and replacement of animal experimentation and will feed risk assessment and LCA, which traditionally rely on in vivo data. The MACRAMÉ Demonstrator thus defines, demonstrates and documents the validity of the advanced MACRAMÉ Methods (i.e., characterisation and (quantitative) imaging and detection techniques): methodologies developed in the MACRAMÉ Demonstrator will both embed existing SSbD approaches [19] and support the SSbD strategies developed in previous and ongoing EU projects (e.g., HARMLESS [20], SUNSHINE [21] and IRISS [22]), extending them to real-life industrial settings, and enabling the conceptualisation of a novel ‘MACRAMÉ Safety & Sustainability Matrix’ – a mathematical matrix combining several parameters from RA and LCA with sustainability criteria to derive a value, which can be visualised by consumers in a simple colour bar scale.

2.9. The MACRAMÉ interpreter – supporting science towards harmonisation & standardisation

The results of the MACRAMÉ Project are being documented in a fashion that is specifically useful and relevant to the standardisation community (i.e., via standardisation project-item proposals), and the policy-making and policy-informing communities (such as the OECD Working party on manufactured materials (WPMN) and the working party on national coordinators of the Test Guideline programme (WNT)). The characterisation and test methods developed by MACRAMÉ are at a relatively low Technology readiness level (TRL3–5), and some cannot yet be validated by an interlaboratory comparison or brought forward to the standardisation bodies or OECD. Therefore, MACRAMÉ is preparing strategic Roadmaps for the harmonisation and standardisation of MACRAMÉ Methods, building on the MACRAMÉ (Meta)Data, and advancing them within the Project as far as possible. MACRAMÉ partners are feeding into new and ongoing test guidelines, guidance documents and standard developments. As MACRAMÉ ’s method development can only cover some aspects of AdMas characterisation and testing, informed recommendations are being developed on future R&I needs with a focus on the MACRAMÉ Material Families and the needs for testing in the (nano)medicine market context for relevant AdMas in their complex matrices and value-chains. Risk-Assessors Summits and (online) workshops are used to make MACRAMÉ results attractive and accessible to regulatory risk assessors.

2.10. The MACRAMÉ facilitator – streamlined project coordination & impact maximisation

In order to efficiently support this short, but highly ambitious R&I Project through both optimum management and impact-enhancing measures, the MACRAMÉ Facilitator has been streamlined to provide combined scientific coordination, project management and dissemination, communication & exploitation measures for the Project, thus maximising the performance and project outputs.

2.11. External collaborations of the MACRAMÉ project

The MACRAMÉ project builds on extensive collaboration with international harmonisation and standardisation bodies and initiatives, including:

• •OECD: For developing harmonized test guidelines and methodologies;
• •Malta Initiative: Updating of safety and regulatory frameworks for nanomaterials;
• •EU projects NANOHARMONY [23] and REFINE [24]: Provide insights into harmonization and regulatory validation strategies;
• •EU sister projects ACCORD [25]), iCARE [26]) and NANOPASS [27]
• •MACRAMÉ is funded under the Horizon Europe program (grant agreement No. 101092686), specifically within the HORIZON-CL4–2022-DIGITAL-EMERGING-01–35 call, dedicated to advancing digital and emerging technologies. The project is financially supported through:
• •European Commission: Funding the core research and innovation activities.
• •National Contributions: Participating national agencies contribute additional resources to ensure the project's objectives align with local regulatory and industrial priorities.
• •Industrial Co-Funding: Industrial consortium members, such as BASF and Medica, provide in-kind support, including access to proprietary materials, expertise, and facilities.

3. Impact

The MACRAMÉ project already has achieved significant milestones in progressing the MACRAMÉ Drawing Board, which focuses on "Bridging Communities and Refining MACRAMÉ Strategies”. The primary activities focussed on aligning methodologies for AdMas across regulatory, industrial, and scientific communities.

The Needs Assessment Report (deliverable D1.2) [28], finalized in November 2024, provides a comprehensive evaluation of European and international regulatory frameworks applicable to and relevant for AdMas. This report identifies critical gaps and offers strategic recommendations to harmonise approaches for LCA and risk governance. The findings indicate that while some regulations (e.g., REACH for nanomaterials) are in place, many advanced materials fall outside these specific frameworks due to their diverse properties and applications. The lack of clear definitions, harmonised test methods, and comprehensive life cycle assessments (LCAs) complicates risk assessment and regulation. Sustainability evaluations are further hindered by limited data, particularly regarding material transformations during product life cycles and waste phases.

The report outlines several specific needs:

• •Develop clear definitions and categories for advanced materials to improve regulatory clarity.
• •Enhance and harmonise test methods and risk assessment tools tailored to the specificities of advanced materials.
• •Promote and operationalise SSbD principles with sector-specific tools and incentives.
• •Foster robust data management practices aligned with FAIR principles to facilitate data sharing and reuse across the value chain.
• •Expand methodologies for evaluating sustainability, especially life cycle thinking and prospective LCAs for emerging technologies

The “Summary Report on the 1st Regulatory Risk Assessors Summit” (deliverable D1.3) [29] details outcomes from the summit held in November 2023. This event facilitated a productive dialogue among 43 stakeholders, including representatives from regulatory bodies like the European Chemicals Agency (ECHA), industry leaders, and academic researchers. Discussions emphasized challenges in assessing AdMas' safety and highlighted opportunities to refine regulatory approaches. One of the central findings was the pressing need to adapt regulatory frameworks to the dynamic and rapidly evolving nature of AdMas, ensuring that policies remain responsive and fit for purpose. Furthermore, participants emphasized the necessity of standardizing terminologies across different disciplines and stakeholder groups, which would enhance communication, facilitate the development of coherent regulatory instruments, and support effective risk assessment. Harmonization of testing and assessment methodologies along the entire lifecycle of AdMas emerged as another priority to enable consistent and comparable evaluations of potential risks. Current exposure models are often insufficiently adapted to the specificities of AdMas, particularly due to their unique physico-chemical properties, such as their size, shape, surface reactivity, and potential to transform throughout the life cycle. There is a lack of validated models that can accurately capture exposure scenarios for AdMas across different stages, including production, processing, use, and end-of-life (waste and recycling). Existing models may not fully integrate key processes like agglomeration, sedimentation, or aerosolization behavior of AdMas, leading to uncertainty in exposure assessments. There is also limited consideration of the release mechanisms of AdMas, including secondary releases during product degradation or mechanical wear (e.g., nanoparticle release during recycling).

A strong consensus was reached on the importance of fostering collaborative innovation within the AdMas value chain. The creation of innovation hubs and multi-stakeholder platforms was highlighted as essential for bringing together diverse expertise, including industry, regulators, researchers, and consumers. Such collaboration is crucial not only to address the heterogeneous needs of various actors but also to drive the development of safer and more sustainable materials. Equally, enhancing traceability and transparency in the handling of AdMas was seen as a cornerstone for trust-building. In this context, digital solutions such as the Digital Product Passport (DPP) were identified as promising tools for improving data management and enabling transparent information sharing across the entire value chain [30].

The “MACRAMÉ Engagement Roadmap” (deliverable D1.4) outlines a strategic three-phase plan to ensure broad stakeholder engagement and the successful dissemination of project methodologies and outcomes:

• •Phase I: Connect & Inform – Focused on establishing communication channels, mapping of all AdMa value chains and life cycles, documentation of production processes and planned workflows for distribution among MACRAMÉ partners for characterisation, toxicity or ecotoxicity assessment and LCA, and launching the centralized MACRAMÉ Information Hub.
• •Phase II: Collect & Integrate – Centered on gathering feedback to refine methodologies and validate protocols.
• •Phase III: Rollout & Transfer – Aims to disseminate findings to all relevant stakeholders including follow-up projects, and to provide actionable recommendations for integrating regulatory frameworks.

Key progress managed via the MACRAMÉ Drawing Board includes the successful launch of the MACRAMÉ Information Hub, a platform that facilitates data exchange and stakeholder interaction.

Excerpts highlighting key messages and quotes from the round table of the first day of the 1st Harmonisation & Standardisation Workshop:

“Dedicated calls for harmonisation efforts as well as for validation studies seem to be a good trigger to support this. A further incentive could be changes in regulation (see animal free testing for cosmetics.”

“Exchange between regulators and developers of test methods is needed from early on and continuously. Developers need to learn how methods are used for regulatory purposes and regulators need training on NAMs.”

Excerpt key messages and quotes from the round table of the second day:

“We need to distinguish between standards for quality control and safety assessment/ regulation. The safety of [graphene-related 2D materials] GR2M needs to be considered. Therefore, we need exchange with manufactures and regulators.”

“The same hurdles we had a few years ahead for nanomaterials now apply for graphene. Look over the EU plate and work with other communities.”

The work undertaken as part of the MACRAMÉ Drawing Board has revealed several preliminary findings: A notable regulatory gap is that current frameworks, such as REACH, are insufficient to address the unique lifecycle complexities, transformations, and risks associated with AdMas. Furthermore, the lack of a unified definition for AdMas complicates regulatory integration and adaptations.

From a stakeholder perspective, the industry has expressed the need for robust LCA and risk evaluation protocols against which to ensure compliance and sustainability. Policymakers emphasize integrating SSbD principles into risk assessor frameworks for AdMas.

Opportunities for progress include fostering collaborations with standardization bodies such as the OECD, ISO, and CEN, and leveraging initiatives like the Malta Initiative to harmonize global methodologies. Data-sharing initiatives aligned with the FAIR principles can enhance the accessibility and reliability of safety data across the value chain, and while FAIR doesn’t directly assess data quality or completeness community agreement around rich metadata descriptions for datasets can address quality aspects of assays and the resulting datasets.

However, several challenges persist. The lack of standardized definitions and testing methods for AdMas creates regulatory ambiguity, while insufficient lifecycle inventory data hinders comprehensive RA. Moreover, diverse stakeholder priorities among regulators, industries, and non-governmental organizations (NGOs) complicate the establishment of unified methodologies and actionable outcomes. Engagement metrics from Phase I of the engagement roadmap reflect interaction with over 20 stakeholder organisations, leading to preliminary recommendations for integrating SSbD principles into LCA. Additionally, tools like Digital Product Passports (DPPs) are being proposed to enhance traceability and lifecycle data management, fostering consumer trust and ensuring regulatory transparency.

The MACRAMÉ Laboratory focuses on developing methodologies and protocols for the safe characterization and assessment of AdMas; it serves as the experimental backbone of the project, facilitating the harmonization of procedures, validation of toxicological frameworks, and comprehensive understanding of AdMas lifecycle impacts. At the core of these efforts is the establishment of the CML [31], a repository of representative AdMas integral to MACRAMÉ ’s experimental and validation workflows. The CML includes benchmark AdMas like GO and PLGA nanoparticles, as well as "end-of-life" particles derived from controlled mechanical abrasion and incineration processes.

Additionally, 12 Standard Operating Procedures (SOPs) have been developed and pre-validated covering critical workflows such as Raman spectroscopy for graphene characterization, aerosol generation, and toxicological assay preparation. The use of CML materials ensures reproducibility and validity in these workflows. An example is shown in Fig. 7.

Fig. 7.

Five phases of the data management life cycle. In contrast to other depictions where FAIRification is a sixth phase, FAIRification in MACRAMÉ is starting in the Plan & Design phase and then continues throughout all other phases continuously enhancing the FAIRness of the data (on-the-fly FAIR).

Materials from specific use cases — such as GO-based water filters and graphene-enhanced epoxy resins — have been integrated into the CML for lifecycle transformation and toxicological studies. Initial validation using advanced imaging techniques, including dark field microscopy and confocal Raman spectroscopy, have begun to quantify AdMas in biological matrices effectively.

Preliminary findings highlight several critical insights into the characterization, lifecycle transformation, and toxicological impacts of AdMas. Raman spectroscopy has proven to be an effective tool for distinguishing GRMs from biological matrices, allowing for the assessment of key properties such as defect densities and layer thicknesses. Additionally, biodegradable particles like PLGA and polycaprolactone exhibit enhanced stability and reliable dispersion in biological media, making them well-suited for use in toxicological studies. Lifecycle transformation studies have shown that abrasion and incineration processes generate respirable particles that are highly suitable for hazard evaluations. These particles exhibit minimal endotoxin contamination (< 0.5 EU mL−1), making them ideal candidates for macrophage uptake studies. Furthermore, initial toxicological evaluations using macrophage studies revealed dose-dependent cytotoxic effects of materials such as silicon carbide (SiC) nanowires and tungsten carbide nanoparticles, underscoring the potential hazards associated with these materials.

Despite these advances, the project has encountered several challenges. The heterogeneous properties of AdMas pose significant difficulties in developing standardized protocols capable of accounting for variations in morphology and composition.

The CML includes benchmark particles such as ZnO, TiO₂, graphene composites, and PLGA nanoparticles. Detailed characterization data within the CML encompass parameters like particle size, morphology, and elemental composition. Experimental results validate the effectiveness of SOPs, with Raman spectra for GRMs ensuring consistent implementation across partner institutions. Dispersion protocols for fibrous materials achieve optimal de-agglomeration, enhancing their suitability for toxicological studies. Toxicological data confirm that abraded particles, such as FLG-reinforced epoxy composite particles with a hydrodynamic size of 1164 ± 292 nm (respirable size range), are appropriate for macrophage uptake studies. Cytotoxicity assays further demonstrated dose-dependent increases in lactate dehydrogenase (LDH) release, validating the relevance of safety test methods for use with AdMas. These findings collectively contribute to advancing the understanding and management of AdMas in diverse applications.

The MACRAMÉ Processor focusses on the topic of Information and (Meta) Data Harmonization, Processing and Sharing; among its major achievements is the development of the MACRAMÉ Registry [32]), a central data hub designed to index research outputs, including sampling plans, study designs, protocols, and experimental data. The registry fosters that all project-related data is properly documented, accessible, and traceable. Complementing this effort, the Research Output Management Plan (ROMP) has been introduced to provide structured guidelines to partners for managing and sharing data. This plan lays out a roadmap for harmonising data formats and ensuring alignment with the FAIR principles throughout the project lifecycle.

The MACRAMÉ Processor has also introduced tools like Instance Maps, which visualise data flows and dependencies among project partners [17]. These maps facilitate the coordination of material handling, sampling, and experimental workflows, improving collaboration across teams. From the outset, the FAIR principles have been embedded into the project to ensure that all generated data is shared and easily accessible within the project as well as systematically prepared for long-term accessibility and reuse.

Several important preliminary findings have emerged from the MACRAMÉ Processor: the MACRAMÉ Registry has significantly enhanced transparency and accessibility among project partners, improving data flow and reducing inefficiencies in knowledge exchange. Instance Maps have proven especially valuable in planning complex workflows, enabling partners to track data dependencies and manage projects in real time. By integrating the FAIR principles early in the project lifecycle, MACRAMÉ ’s approach to data management has enabled smoother data exchange internally and alignment with European open data initiatives for external collaboration.

The MACRAMÉ Registry currently indexes a wide array of resources, including protocols and processed data, with metadata that includes unique identifiers, resource descriptions, and documentation of data provenance. Instance Maps have streamlined the organization of sampling and testing workflows, particularly for the CML. The ROMP has provided structured guidelines for managing research outputs, ensuring consistent data documentation and alignment with open science policies. Integration with tools like Zenodo and DataCite is planned for the future.

However, the variation in data management practices among project partners has to be respected when creating data harmonisation workflows to minimise the additional workload for the data producers. Additionally, the increased data documentation needs for not (yet) fully standardized and validated test methods have often to go beyond standardized (meta)data formats and reporting templates. Developing universally accepted metadata schemas for diverse research outputs, such as sampling plans and computational models, remains a complex task. While allowing partners the flexibility to use their preferred tools, ongoing coordination and technical adjustments are needed to ensure interoperability and compliance with the FAIR principles across partners first and then across projects and disciplines to be able to establish a common (European) data ecosystem. MACRAMÉ is working with many projects including but not limited to BIO-SUSHY, PINK, CHIASMA, INSIGHT and DigiPass to achieve this goal.

In the MACRAMÉ Foundations and the MACRAMÉ Demonstrator, which focus on the Identification, Demonstration and Validation of MACRAMÉ Methods in UCs, significant strides in defining, demonstrating, and validating advanced methodologies for assessing AdMas in complex industrial and consumer use cases have been made. The project has successfully developed and implemented comprehensive sampling and lifecycle evaluation protocols across five industrial use cases, which involve key material families such as GO, FLG, CNTs, and PLGA. These efforts are geared toward ensuring the safety and sustainability of AdMas across their lifecycle.

The development of sampling protocols has been a cornerstone of this work. Customised procedures have been established for collecting pristine and transformed AdMas from various use cases, with protocols integrated into downstream toxicity and LCA workflows. The project also employs Dynamic Probabilistic Material Flow Analysis to simulate the fate of AdMas across product life cycles, including use and end-of-life phases. This approach has been applied to specific use cases, including GO-based water filters, where release potential during incineration is being investigated, FLG in battery systems, where material release through abrasion has been analysed, and CNT-infused car seats, where shredding and incineration have been evaluated for particle release dynamics.

Preliminary findings indicate key insights into material release dynamics, lifecycle analysis, and environmental concentrations. GO-based water filters demonstrated minimal detectable release during incineration under current analysis limits, while the abrasion of FLG-epoxy composites generated respirable particles requiring further toxicological testing. CNT-containing polyurethane foils displayed stability during incineration but released particles during shredding. Results from lifecycle analysis simulations suggest that a significant fraction of AdMas remains embedded in matrices throughout the product lifecycle, with minimal release into environmental compartments. Additionally, predicted environmental concentrations for AdMas in soil and water indicate low risks under current recycling and waste management scenarios.

Insufficient data on AdMas release rates during specific recycling processes limits predictive accuracy, and the variability in product lifespans and recycling pathways complicates the modelling of consistent material flows. Harmonising sampling and lifecycle evaluation protocols across diverse UCs requires extensive coordination, presenting further challenges in standardisation.

The project has produced valuable data and results to support these efforts. Samples from GO-based water filters include pristine GO, polyurethane components, and end-of-life filters for incineration analysis. Abrasion processes in FLG-based batteries have generated particles suitable for inhalation and ecotoxicity testing, while post-shredding and incineration samples from CNT-laden polymer foils have been collected to evaluate release dynamics. Decision trees have been developed for waste stream analysis, highlighting critical stages where material releases occur, while materials flow modelling results confirm that most AdMas are retained in recycling streams or transformed during incineration. PEC values for GO in water filters and CNTs in car seats remain below regulatory thresholds, suggesting negligible risks under current disposal and recycling practices.

The MACRAMÉ Interpreter has focused on advancing the harmonisation and standardisation of methodologies for the RA and sustainability evaluation of AdMas. The primary deliverable, “Harmonization and Standardisation Roadmap” (D5.1) [33], outlines strategic approaches to align MACRAMÉ -developed protocols and methods with regulatory standards. Key achievements include the development of five roadmaps addressing critical areas: characterization and dispersion protocols for graphene-based materials, aerosol generation for inhalation studies, advanced biological models for in vitro and ex vivo inhalation toxicology, ecotoxicity assessment of AdMas, and sustainability analysis focusing on improved LCA frameworks.

Fig. 8.

Bar chart representation of the workshop participants’ opinions regarding the identified priorities for robust toxicological and ecotoxicological assessment. Participants have been requested to rank the proposed options between 1 and 5. The average of 42 answers is reported.

The project has also engaged extensively with regulatory bodies such as the OECD and ISO/TC 229 committees to promote the adoption of these methods. This engagement has included discussions on addressing industrial and regulatory standardization needs, supported by community feedback gathered during the MACRAMÉ Harmonization and Standardization Workshop in November 2023 [34]. The workshop provided valuable insights, guiding the prioritisation of methods for GRMs and other AdMas.

Preliminary findings highlight several critical aspects. Stakeholders have emphasized the importance of addressing GRM attributes such as surface functionalization, chemical composition, and stability in media to develop robust toxicity protocols. The development of effective aerosol generation methods for inhalation studies has been identified as essential to ensure dose reproducibility and regulatory relevance. Progress has been made in creating in vitro inhalation toxicology models, demonstrating their potential to replace animal studies and align with the European emphasis on non-animal testing methods. In the area of ecotoxicity, strategies such as the use of conditional media and eco-coronas have been developed to improve material dispersion in aquatic toxicity tests, ensuring environmental relevance.

Significant standardization gaps still exist, particularly in adapting current OECD Test Guidelines to accommodate the unique properties of AdMas, such as GRMs and complex product matrices. The complexity of multi-phase testing, involving diverse materials and product lifecycles, presents a further challenge. Additionally, ensuring that methods meet the stringent requirements of reproducibility, robustness, and scalability for regulatory approval is resource-intensive and requires continued effort.

Protocols for GRMs include optimised dispersion workflows using stabilisers like bovine serum albumin and dissolved organic matter for in vitro and ecotoxicological assays. Modular workflows for aerosol generation have been established to ensure precise dose control in both in vitro and in vivo studies, with flowcharts and decision trees guiding sample preparation, characterization, and dose application. Advanced biological models, such as alveolar macrophage assays and epithelial cell co-cultures, have demonstrated initial success in hazard assessments for inhalable AdMas. In ecotoxicity tests, adapted OECD TG 249 protocols have shown promise for fish cell line testing, with proposed extensions for rainbow trout liver cells to evaluate long term toxicity and additional endpoints like inflammation and ROS production. Besides, fit-for-purpose requirements and remaining challenges for the successful implementation of in vitro data into AdMas SSbD have been identified. In addition, decision trees for LCA are being further developed for investigating AdMas' lifecycle impacts and identifying critical stages for assessing environmental and human health risks.

4. Discussion

The MACRAMÉ project delivers groundbreaking advancements in the safe and sustainable use of AdMas, focusing on three material families: GRMs, CNFs, and PLGA nanoparticles. Its achievements lie in developing tools and methodologies to detect, characterize, and assess lifecycle risks associated with AdMa@CMs, such as industrial or consumer products. This work builds upon the foundational achievements of MACRAMÉ Laboratory, which established a CML for distributing and characterizing AdMas. Moving forward, the project aims to validate these materials in its research strategies and create a roadmap for making them accessible to the broader scientific community, ensuring a long-term impact.

The MACRAMÉ Processor has further reinforced the project’s outcomes by establishing a robust foundation for data management and knowledge sharing. Tools like the MACRAMÉ Registry and Instance Maps have prioritized the FAIR principles, ensuring long-term usability of research outputs. However, ongoing efforts to harmonize data practices and refine metadata standards remain vital to sustaining these achievements.

The results from MACRAMÉ Demonstrator highlight the project’s ability to integrate advanced sampling and lifecycle evaluation methodologies into real-world use cases. The project has successfully implemented sampling and lifecycle protocols, revealing key insights into material dynamics during product use and disposal. For example, mechanical abrasion processes produced respirable particles, and lifecycle transformation studies confirmed minimal endotoxin contamination during incineration and abrasion. While these findings establish a robust foundation for future toxicological testing and regulatory assessments, data gaps and standardization challenges must still be addressed.

The MACRAMÉ Interpreter plays a critical role in aligning MACRAMÉ ’s methodologies with international regulatory standards. Frameworks such as characterization protocols for GRMs, advanced inhalation toxicology models, and ecotoxicity assessments have been developed to ensure global applicability. Significant progress has been made in ensuring AdMas safety and sustainability, but further work is required to finalize protocols, validate models, and secure regulatory adoption. Continued collaboration with standardisation bodies and stakeholders will be essential to achieving these goals. Collectively, these results align the project with EU strategies and policy frameworks like the European Green Deal and SSbD, driving forward the EU’s vision of a toxic-free, climate-neutral economy.

One key result is the establishment of pre-validated protocols and SSOPs for detecting and quantifying AdMas, such as Raman spectroscopy for GRMs, and SMLS for in-situ quantification. These tools provide critical insights into the transformations and potential exposure risks of AdMas. For example, lifecycle transformation studies revealed that abrasion processes produce respirable particles, while incineration results in minimal endotoxin contamination, and this knowledge can inform subsequent design of AdMas.

In addition to its technical contributions, MACRAMÉ bridges gaps between regulatory bodies, industry, and scientific communities by aligning with EU policy frameworks like the European Green Deal and the Chemical Strategy for Sustainability. This alignment ensures that the project's outcomes, such as Control Material Libraries and pre-validated test protocols, directly contribute to achieving Europe's goal of a toxic-free, climate-neutral economy.

The MACRAMÉ project builds upon a rich foundation of nanosafety research, including collaborative initiatives like the Graphene Flagship and the Malta Initiative. Existing literature in the field of AdMas has largely focused on individual lifecycle stages (e.g., synthesis or end-of-life), leaving significant gaps in understanding the transformation and risks of AdMas across their entire lifecycle. MACRAMÉ addresses these gaps by providing holistic assessments that integrate material characterization, lifecycle exposure studies, and toxicological evaluations.

AdMas present unique challenges in safety assessment, particularly in capturing their dynamic transformations across the lifecycle. While previous studies made significant strides in detecting and characterizing individual life stages or simpler particles, they often lacked harmonized methods suited for industrial and regulatory applications. MACRAMÉ leverages this foundational work, enhancing it with advanced imaging and analytical tools to quantify AdMas transformations in real-world conditions. By integrating techniques such as Raman spectroscopy for defect characterization in GRMs, MACRAMÉ extends beyond traditional approaches, enabling a more comprehensive understanding of AdMas behaviour. In the regulatory domain, prior work often highlighted the need for harmonized guidelines but lacked alignment with global standardization bodies like the OECD or ISO. MACRAMÉ directly engages with these organizations, contributing pre-standardisation methodologies for detecting, characterizing, and evaluating AdMa@CMs. This collaboration ensures that the project's outcomes are positioned for widespread adoption, addressing regulatory gaps identified in earlier works.

Moreover, unlike previous research focused solely on nanoscale materials, MACRAMÉ ’s extended scope includes hybrid and larger-scale AdMas. This broader scope positions the project at the forefront of addressing the challenges of emerging AdMas, which increasingly feature complex chemistries and multifunctional properties.

While the MACRAMÉ project has achieved significant progress, certain limitations remain that need to be addressed in future work. One key challenge lies in the heterogeneity of AdMas, which complicates the development of universally applicable protocols. For example, the physical and chemical properties of GRMs vary widely depending on their source, synthesis methods, and applications. This variability necessitates material-specific protocols, which can limit the scalability of MACRAMÉ 's methodologies across different AdMas.

Another limitation is the availability of comprehensive lifecycle inventory data. Although the project integrates LCA into its methodologies, a lack of detailed data on material release rates, transformation mechanisms, and environmental exposure pathways restricts the accuracy of its risk predictions. Similarly, gaps in data on the long-term environmental and health impacts of end-of-life AdMas transformations hinder the project's ability to propose definitive safety guidelines.

From a regulatory perspective, while MACRAMÉ engages with organizations like the OECD and ISO, the process of translating its findings into globally accepted standards remains time-intensive and complex. Differences in regulatory frameworks between regions (e.g., the EU and North America) present additional hurdles for harmonizing AdMas safety standards worldwide.

Future directions for MACRAMÉ include expanding its focus on data stewardship and FAIR implementation. Enhancing the MACRAMÉ Information Hub and integrating machine learning models could help to automate data analysis and streamline protocol validation. Additionally, efforts to extend the CML to include more diverse and emerging AdMas will be crucial for broadening the project's impact in future activities.

Another critical area for future work is the refinement of advanced inhalation toxicology models. While in vitro and ex vivo systems show promise, their sensitivity and reproducibility must be further validated to enable them to replace animal testing fully. Collaborative efforts with industrial partners could also help scale these models for broader applications in consumer product testing and environmental monitoring.

Finally, increased outreach to international regulatory bodies and industrial stakeholders will be essential to maximize the adoption of MACRAMÉ ’s methodologies. By fostering collaborations beyond the EU and expanding engagement with developing markets, the project can ensure its findings contribute to the global push for safer, more sustainable material innovations.

5. Conclusion

The MACRAMÉ project has and will have achieved a significant impact on the safe and sustainable development of AdMas by developing reliable, pre-validated, and scalable methods for characterizing AdMa within their different value-chains, assessing, and managing impacts across their lifecycles. A central accomplishment is the establishment of a CML, which has become the foundation for experimental and regulatory assessments. Furthermore, the project integrates SSbD principles into its methodologies, aligning closely with the EU’s Green Deal objectives.

MACRAMÉ 's use-case evaluations, spanning diverse industrial and consumer applications, provide practical frameworks for lifecycle assessments. For instance, protocols for detecting material release during incineration and abrasion have proven critical for understanding environmental and health risks. Through dynamic collaboration with standardization bodies like the OECD and ISO, the project has also laid the groundwork for global regulatory adoption of the methods developed and optimised, positioning Europe at the forefront of AdMas innovation.

The practical implications of MACRAMÉ 's work extend beyond the immediate project outcomes, providing a pathway for long-term innovation in material science and environmental safety. The integration of advanced imaging techniques and lifecycle models for AdMas into regulatory processes will accelerate the adoption of safer and more sustainable practices across industries, from automotive to electronics.

One of the most promising innovations is the emphasis on non-animal testing methods for toxicological assessments, which aligns with global shifts toward ethical and sustainable practices. The adoption of in vitro inhalation models and the transition from traditional nanomaterial-specific methods to adaptable AdMas protocols demonstrate the project's forward-thinking approach.

The project’s dissemination and communication strategies further enhance its impact, fostering collaborations among stakeholders and creating open, accessible platforms for knowledge sharing. This ensures that MACRAMÉ ’s methodologies are scalable, transferable, and adaptable to future innovations, reinforcing its potential to drive a global transformation in the design, use, and regulation of AdMas.

CRediT authorship contribution statement

Lynch Iseult: Writing – review & editing, Validation, Methodology, Investigation, Data curation, Conceptualization. Buerki-Thurnherr Tina: Writing – review & editing, Investigation, Data curation. Exner Thomas: Writing – review & editing, Validation, Software, Funding acquisition, Data curation, Conceptualization. Gupta Govind: Writing – review & editing, Validation, Investigation, Data curation. Hagenhoff Birgit: Writing – review & editing, Investigation, Data curation. Wick Peter: Writing – review & editing, Supervision, Investigation, Conceptualization. Breitenstein Daniel: Writing – review & editing, Validation, Investigation, Formal analysis, Data curation. Ortiz-Galvez Luis Mauricio: Writing – review & editing, Investigation, Data curation. Seitz Christian: Writing – review & editing, Writing – original draft. Heunisch Elisabeth: Writing – review & editing, Investigation, Data curation, Conceptualization. Duistermaat Evert: Writing – review & editing, Investigation, Conceptualization. Pohl Anna: Writing – review & editing, Investigation, Data curation, Conceptualization. Wiemann Martin: Writing – review & editing, Investigation. Reilly Katie: Writing – review & editing. Vennemann Antje: Writing – review & editing, Investigation, Data curation. Guo Zhiling: Writing – review & editing. Katsumiti Alberto: Writing – review & editing, Investigation, Data curation. Friedrichs Steffi: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Weltring Klaus-Michael: Writing – review & editing, Supervision, Investigation, Data curation, Conceptualization. Haase Daniel: Writing – review & editing, Investigation. Ouhajji Samia: Writing – review & editing, Investigation, Conceptualization. Wu Jimeng: Writing – review & editing, Investigation. Bleeker Eric: Writing – review & editing, Methodology, Funding acquisition, Data curation, Conceptualization. Vandebriel Rob: Writing – review & editing, Investigation.

Declaration of Competing Interest

The authors declare to have no conflicting interests with the content of the study, have read and revised the manuscript carefully, agreed to its submission, and accepted their responsibility for the content.