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Periodic Report Summary 2 - NANOMILE (Engineered nanomaterial mechanisms of interactions with living systems and the environment: a universal framework for safe nanotechnology)

Project Context and Objectives:
The FP7 project NanoMILE ( is a unique partnership of 28 of the highest calibre European and US institutes in nanosafety, offering the full complement of expertise required to develop detailed mechanistic understanding of the interactions of manufactured nanomaterials (MNMs) with living systems. Test systems range from biofluids, simple unicellular through to multi-cellular organisms, whole animals and humans. Identification of conserved pathways across species and development of in vitro alternative test methods and high throughput approaches is a core aim, in line with the European Commission’s drive to reduce animal testing (the 3Rs) and to ensure safe and responsible implementation of nanotechnologies.
NanoMILE intends to revolutionise nanosafety research through its robust and novel approaches to the selection and development of test MNMs including environmentally aged variants to represent real-world materials, its technically and computationally advanced integration of systems biology, its thoughtfully balanced toxicological/ecotoxicological approaches, its development of novel high throughput platforms for screening and its feedback loops for development of MNMs that are safer by design. Together, these approaches will result in a robust framework for classification of MNMs according to their biological impacts. The workpackage (WP) structure is shown below in Figure 1.
The advanced scientific expertise offered by the academic partners has been matched by a complement of fully committed and well integrated industrial partners, capable of contributing to or advancing the innovations of NanoMILE to industrial applications. The partner map is shown in Figure 2. Of the 28 organisations, 10 are universities, 3 are research facilities, 5 are government bodies, 3 are multinational companies and 7 SMEs (3 technical consultants, 4 materials/instrumentation manufacturers). The two US partners were selected to add strength to the consortium by providing expertise at the highest technical level, thus matching and augmenting the capabilities of the European part of the consortium.

Project Results:
The middle period of any large project is where the bulk of the activitiy happens, and NanoMILE is no exception: all 8 technical WPs have been fully operational throughout the period, and all 28 partners have been actively engaged. All 6-monthly face to face meetings, in Copenhagen (M18), Brussels (M24), Athens (M30) and Edinburgh (M36) have been exceptionally well attended with over 60 NanoMILE researchers, faciliting internal WP discussions and planning, as well as cross-WP integration and alignment, and planning for the integrated NanoMILE grouping and classification framework that forms the final project outcome.

The 10 industry partners have been fully integrated into the NanoMILE research activities, contributing actively to WP2 (PROM, N4I; particle synthesis / manufacturing), WP5 (Malvern, Attana; method development for characterization of particle-biomolecule and particle-cell interactions), WP6 (Eurofins, ecotoxicity of pristine and aged NMs), WP7 (BASF, Vitrocell; inhalation exposure, including a new Air-liquid Exposure device); WP8/WP9 (Biomax, Novamechanics; data management and QSAR development) and WP10 (EU-vRI; dissemination and exploitation). A dedicated section of the NanoMILE website is industry-facing and devoted to showcasing NanoMILE’s products and services with and for industry.

For the 1st period, the public summary highlighted some of the key early publications from the project rather than giving an update on each WP. This approach was adopted because the different WPs they were at different stages of development. However, with 27 publications in this period, it was not possible to summarise them in 4 pages, so we provide a brief update on the highlights from each WP. The full list of the NanoMILE publications to date (39 and counting) is available via the NanoMILE website.

All workpackages have progressed well in the 18 months of the second period (Months 19-36) of the project, with a range of exciting outputs and scientific discoveries. Some highlights are provided below.

WP2 - MNM selection, acquisition, engineering, characterization has played a central role in the succees of the project to date. With regard to the work in Task 2.3 and Task 2.5 during the current reporting period all WP2 partners have continued to be active in contributing to the preparation of new or re-supply of nanomaterials for use in WP 3-8. In line with the planning in the DOW the contribution of all partners to the materials supply tasks has progressively decreased in the period up till M36. The majority of the materials supply work has now been completed. No new materials are planned for production in the next period although when necessary and agreed with the relevant partners re-supply of materials may be undertaken. With respect to the Task 2.4 "Characterisation of MNMs" all particles produced by UoB, as well as those received from industry partners have been subjected to basic physico-chemical characterisation by UoB to determine size, shape, zeta-potential and crystal structure. Where specifically requested by WP4-8 partners, tests of the stability of the different particles in the various media utilised in WPs 4-8 have been addressed. In the cases of materials prepared by JRC and CEA, basic characterisation was undertaken using their own in-house facilities. At this time the experimental studies for Task 2.4 have been largely completed and the results are being integrated into a single reference document for use by the consortium. This document which constitutes the Deliverable 2.5 "Characterization files for all 3 libraries of MNMs" currently contains over 650 pages of data characterizing the NanoMILE MNMs libraries.

WP3 - Life cycle evolution of MNMs was also in part a service WP, providing “aged” MNMs to other WPs. WP3 focused on ageing of MNMs in air, water and products, incuding sulfidation, phosphidation, role of fulvic acid (FA) and impact of UV. All WP3 tasks are completed. Highlights include the success of the ageing protocol developed for Ag-MNMs (EMPA) to achieve complete sulfidation of Ag-MNM, but the chemical composition was not the only parameter to be altered during this transformation process, as the MNMs were observed to agglomerate significantly, to form a spider-web structure after aging. Comparing / contrasting the effects of pristine to altered MNM produced under these conditions, it may not be the chemical transformations alone which are responsible for different biological or ecological effects but also the physical structure may play a role. Transformation of MNM with natural organic matter (UoGen), which confirms that a FA coating acquired around CeO2 MNMs is stable and irreversible, and is one of the key factors that will control, even in changing diluting conditions, transformation and toxicity of MNMs in aquatic systems. Moreover, when coated CeO2 MNMs are present even in systems with increasing ionic strength (e.g. passing from fresh to coastal or marine waters) they resist aggregation in particular when monovalent salt is considered.

WP4 - Development of a screening platform for MNMs has carried out systematic toxicity screening of up to 100 NanoMILE Phase 1 and 2 MNMs (with phase 3 MNMs ongoing currently) in a range of cell lines and zebrafish embryos against a range of endpoints to identify lead candidates for WP 6-8, and for interesting candidate MNMs is further developing novel, robust toxicity assays for HT/CS of MNMs as well as identifying common biomarker profiles (from WP 6-8) across multiple species. The first list of MNMs was selected on the basis of having similar size but different chemistry and coatings, while the second screening panel included the aged MNMs (from WP3), the series of Zr-doped CeO2 MNMs developed for WP7, and a series of 41 ultrasmall particles (diameter ≤ 20nm). The MNMs in the first list were tested in different mammalian cell lines and zebrafish embryos to link physico-chemical properties to multiple adverse effects in different biological systems. The cell lines represent different organs (liver, lung, colon, immune system). Dispersion and dilution of the MNMs was done according to an agreed standard operation procedure, and the doses tested ranged from 1-125 μg/ml which corresponds to cell surface area doses of 0.3-9.1 μg/cm². Pure medium was used as negative control and amine-modified polystyrene (PS-NH2) NPs as positive controls. The assays used are based on high-throughput/-content (HT/C) techniques. End points such as cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied in cells. The zebrafish embryos were tested for hatching rate, malformations and mortality. Integrated multi-partner publications are in preparation at present.
WP5 - MNM interactions with biomolecules and environmental factors is tasked with furthering our understanding of what is presented on the surface of the NanoMILE MNMs when dispersed in relevant biological media (e.g. the 10 % foetal calf serum used for the cell culture experiments and in vivo model fluids) in order to bridge from physico-chemical properties to biological effects. A significant focus has been on developing automated and higher throughout approaches to assess MNM-biomolecule interactions. One example is the development of automated handling of sample preparation and Fluorescence Correlation Spectroscopy (FCS) measurement and data analysis, whose workflow combines a Labcyte Echo Liquid handler with an automated FCS Setup and semi-automated data analysis. The setup allows preparation and readout of a 384 well plate in about one day with a 10 fold sample volume reduction (20µl per well). Measurement of a series of binding isotherms in a multi-component system is therefore possible. Preliminary experiments with TiO2 uncoated MNM and Bovine Serum Albumin (BSA, Alexa Fluor 488) were performed and optimized, suggesting that BSA interactions with TiO2 are not strong resulting in BSA having short residence times on the MNM surface. Other efforts include development of a surfactant titration method (see Figure 2) to study corona-MNM complex stability and allow selective separation of certain proteins from the corona.

WP6 - MNM bioavailability & biological effects in vitro/in vivo (ecotoxicology) has tested a range of sentinel organisms to investigate the relative toxicity and organisms sensitivity to selected nanoparticles (NPs) that are currently suspected to have biological effects (e.g. nano silver). The test organisms included a freshwater algae (Clamydomonas reinhardtii), a freshwater fish (Danio rerio), and a range of terrestrial invertebrates including Caenorhabditis elegans, earthworms (Eisenia fetida), springtail (Folsomia candida), soil mite (Hypoaspis aculeifer), and the isopod (Porcellio scaber). Some tests followed standardised OECD test guidelines but new protocols better suited for studies on MNMs for those specific test organisms were also developed. Effects analyses were focused on apical endpoints from mortality, to development, growth, reproduction, neurological function (e.g. behaviour assays) and photosynthetic yield (algae). Of the MNMs studied only AgNPs were found to show toxicity at concentrations modelled for surface waters and surface soils in Europe, across the wide range of study organisms tested. Our results, together with the existing literature indicate that the release of Ag+ and its uptake into the cell is the main cause of AgNP toxicity. For ZnO MNM dissoluton in the aquatic exposure medium was rapid and the predominant mode of toxicity of ZnO MNM is likely to be due to Zn2+ ions. However, no adverse effects were found for environmentally relevant concentrations of ZnO MNM in the terrestrial and aquatic organisms tested. No effects of CeO2 or for TiO2 were found on apical endpoints for any dosing level tested in the animals studied.

WP7 - MNM biokinetics and toxicity testing in vitro/in vivo (toxicology) is investigating selected MNMs to identify molecular mechanisms and pathways of toxicity. One hypothesis used in these studies is that the redox potential of the MNMs governs the toxicity. CeO2 was chosen due to the fact that it can cycle between two redox states, Ce3+ and Ce4+, which endows this MNM with catalytic properties, and suggests a mechanism of activity based on oxidative stress. The use of CeO2 MNMs in vehicle catalysts makes it relevant for exposure via inhalation. Doping with Zr was used to alter the redox activity by incorporating ZrO in the crystal structure of the CeO2 MNM (prepared by PROM, as per Table 1). The differences in crystal structure and redox potential did not result in large differences in to xicity (in in vitro and in vivo studies, papers in preparation) as the toxicity of the original CeO2 MNM was relatively low. For the second phase, Fe2O3 MNMs doped with different amounts of cobalt. In vitro and in vivo studies are underway.

WP8 - Systems biology approaches to reveal mechanisms of MNM activity comprises the collection and deep analysis of “omics” Big Data associated with the biological responses of four model systems (Daphnia, Chlamydomonas, zebra fish embryos and A549 human cell line) to selected MNMs. Following earlier delays in the project related to the selection of MNMs and provision of aged MNMs for the deeper mechanistic investigation in WP8, progress in the wet laboratory exposures and omics data generation has been good. An array of omics responses have been measured, including gene expression profiling (using RNA Seq), metabolic changes (using mass spectrometry metabolomics) and lipid changes (using mass spectrometry lipidomics), as well as traditional phenotypic (apical) endpoints, for the four model systems. Data analysis is underway and further details will be provided in the report on the final period of activity.
WP9 - Data integration/QPARs, risk assessment, safe MNM designs has focussed on the development of a robust and predictive model to quantitatively define the correlation of the cell association of a set of gold NPs with their physicochemical properties and available data on protein corona fingerprints. We have computationally explored a data set that consists of 105 chemically diverse gold NPs with different surface modifiers and three different core sizes, namely 15, 30 and 60 nm. In the formulations 67 organic surface modifiers were used, including small molecules, polymers, peptides, surfactants and lipids that can be characterized as “neutral”, “anionic” and “cationic” based on their chemical structure and net charge at physiological pH (pH 7.4). KNIME (Konstanz Information Miner) platform, a freely available and open source tool that is increasingly used for solving chemoinformatics problems (, was used to implement all steps required for model development and validation.
Potential Impact:
Dissemination activities (WP10)
NanoMILE partners have been active in terms of disseminating their activities within NanoMILE from the outset of the project, with period 2 being especially active: The table shows the period 2 and total dissemination activities, including 39 papers and 107 conference presentations. NanoMILE partners chaired several NanoSafety cluster (NSC) working groups (WGs) including Hazard (WG2), Standardisaiton Sub-group (WG7), the new Safety-by-design (WG9), and represent WG7 (Dissemination) on the Steering Committee.
Expected impact: NanoMILE will contribute significantly to the efforts to reduce the many uncertainties about the potential impact of MNMs on health and the environment, which is urgently needed for the development of a sound regulatory framework. It is crucial to learn what combinations of physico-chemical parameters govern the toxicity of nano-sized objects and what the underlying mechanisms are for the sustainable development of MNMs, and to finalise a set of approaches and tools to assess the impacts of MNMs across species and levels of complexity.
The NanoMILE consortium has already identified a number of key outputs that will have significant impact for the various stakeholders involved in the nanosafety and nano-commercialisation question, as follows:
- Descriptors for grouping / classification of MNMs (including aged MNMs)
- Algorithms and predictive models (& the associated Standard Operating Procedures, SOPs)
- New High-throughput (HT) assays for screening MNM impacts (cell-based, cell free) (& the associated SOPs), including 2 industry-led demonstration models
- Data management tools to link physico-bio-impact data from data generation to mining ability
- ‘Omics’ datasets for the 4 test species in response to systematic sets of MNMs
- Design rules for tailoring MNM impacts – novel MNMs as Reference Materials etc.
- Data on controlled human and organism exposure & comparison to models of increasing complexity
- MNM libraries (with synthesis, functionalization, purification SOPs) and safety dossiers for SME partners on their MNMs for use in business to business marketing.
Based on our selction criteria (existence of a need, e.g. for industry and regulators; technical readiness; organisational readiness) 3 work items have been identified for Standardisation: Stable-isotope labelling of MNMs, Isopods as a model for bioaccumulation of MNMs, and Air Liquid Exposure Systems as a replacement for animal testing.

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Record Number: 187889 / Last updated on: 2016-08-24