Evaluation and conclusions Clause Samples

Evaluation and conclusions. The objectives presented in the deliverable description aiming to develop and validate SOPs for quantitative TEM analysis of MNM were reached. SOPs for quantitative TEM analysis of MNM were developed. These SOPs were validated by application on a series of nanomaterials of various types and sizes, by intra-laboratory and inter- laboratory validation based on the estimation of the measurement uncertainties. These SOPS allow to estimate the minimal external dimension of NM such that the EC definition for NM can be implemented for a wide selection of colloidal and aggregated fractal-like nanomaterials with known measurement uncertainties. Further a large panel of size and shape measurands with measurement uncertainties were determined for these materials, resulting in a detailed characterization required for e.g. risk analyses.  The SOP to prepare a TEM specimen suitable for qualitative and quantitative analysis from a dispersed NM ensures that the NM samples are suitable for TEM imaging and analysis. The examined materials were evenly distributed over the grids and the fraction of the attached NM represents the dispersed NM optimally.  The SOP to record a set of calibrated transmission electron micrographs showing NM that are representative for the NM on the EM grid ensures that the number of particles and the magnification of the micrographs are suitable for subsequent descriptive and quantitative image analyses.  The method for characterizing the primary particles and aggregates of a NM by describing their physical properties based on TEM micrographs provides a step-by-▇▇▇▇ ▇▇▇▇▇ for the descriptive characterization of nanomaterials.  The SOPs to analyze the 2D properties of the primary and aggregated/agglomerated particles on EM micrographs ensure that the primary particles are detected and that size and shape measurands are determined quantitatively. A modified version allows to measure the size and shape properties of the aggregates/agglomerates.  Data were analyzed and represented according to relevant ISO-norms. The EM-based results were related to the results obtained with alternative methods. These include ensemble techniques based on light scattering, such as dynamic light scattering (DLS) and particle tracking analysis (PTA), and single particle inductively coupled plasma-mass spectrometry (SP-ICP- MS) [1]. Our work illustrates that the size measurands measured with the different techniques are method- defined and cannot be directly compared ...

Evaluation and conclusions. The goal of this deliverable is to deliver a database of physicochemical properties that correlate with (eco)-toxicological endpoints by analysing research studies from literature that report information on both structural characteristics and toxicological profiles of nanomaterials. For this purpose a database structure has been developed and data has been prioritized and collected. A large amount of data has been collected that could serve this purpose. Additional activities such on database compatibility are ongoing. In a collaboration with the FP 7 eNanoMapper project the mapping of our database ontology to existing ontologies has been done. Due to small overlap with existing ontologies the eNanoMapper ontology will be expanded and an output report on the mapping will be formed by the eNanoMapper project. The full database has been shared with NANoREG partners and with the eNanoMapper project and will be made publically available as all other NANoREG data. The use and direct transfer of the WP6 literature database in the NanoReg2 and Calibrate project is currently under discussion. Focus of the work in these project is the transfer of the experimental data from NANoREG to the eNanoMapper instances. Finalization of this transfer would facilitate the transfer of the WP6 literature data to a large extend due to the strong overlap in database structure. In order to properly transfer all data and allow use of the data in these project the mapping of fields should be completed. Whether this will be done depends on the availability of resources and the expected impact of the literature data on these projects. Decisive conclusions on the use of the literature data in these project will be drawn in the first quarter of 2017. There are currently no ongoing data analysis activities.

Evaluation and conclusions. The NANoREG data platform is a complex ecosystem of components serving various purposes, as described in section 2 above. It was conceptually ready within a few months after the project start. The immediate choice of a free and ready to use CMS – CIRCABC – proved crucially beneficial to the implementation of the project. It also served as (temporary) repository of project experimental data. The development of the NANoREG database, the main component of the platform, and the related handling of experimental data required much more work from T1.5 and NANoREG partners than expected. The NANoREG management understood within the first year that a well-thought data management strategy inside the project was essential. T1.5 advocated the development of the 'ISA-TAB-Nano templates'. This proved to be a wise choice. That idea based in ISA-TAB-Nano was later used also by other projects and initiatives in the EU and beyond. The roadmap to implement the strategy was ready by November 2014. It was followed by T1.5 until the end of the project, leading to the population of the NANoREG database with well-structured data. That NANoREG experimental dataset, thanks to its good structure, is being exported to the NANoREG- eNanoMapper database, with the potential of full exploitation of its value by other EU-funded projects and possibly beyond, such as in the frame of the US-EU Communities of Research. This is possible only because ▇▇▇▇▇▇▇ was a front runner in the public release of its work and has been an active promoter of open access to EU-funded project results. As experienced by this task, it can be very difficult to get the buy-in of partner organisations when it comes to setting up a harmonised data management system in a project. Data recording was and will likely remain a tedious task for those conducting assays in their laboratories. Data fitness for publication in journals seems different from, or lighter compared to what was required within the project to be able to exploit cross-WP data links, and not merely publish results in papers. Things are likely to change independently of this data management streamlining effort in NANoREG, since publishers are requiring more insight in data supporting the manuscripts, following models like ISA-TAB-Nano when requesting data from authors. The ISA-TAB-Nano specifications were developed by T1.5, with the partners themselves. T1.5's leading principle in developing the templates was 'keep it simple' for the users. Hopefully th...

Evaluation and conclusions. The toxic effects observed differed among the four NFCs studied, but effects were also seen with the bulk- sized cellulose studied. As concerns inflammatory effects, the mice exposed to the NFC materials and the bulk-sized cellulose showed signs of recovery of inflammation during the 28-d study period, while the MWCNT-treated mice exhibited characteristics of long-term adverse health effects. Based on the results of the present study, it seems that exposure through the respiratory tract to NFC can cause acute inflammatory responses, which, however, are not anymore present 28 d later. Although NFCs appeared to be biopersistent during this follow-up time, they induced no pathological changes observable by histological examination of the lungs. In this sense, they markedly differed from MWCNTs which still showed clear inflammatory responses 28 d after the exposure and also produced various alterations in the histology of the lungs. All NFC materials, except one, caused DNA damage in lung or BAL cells, as determined by the comet assay. For one NCF, the effect was dose-dependent in lung cells both 24 h and 28 days after the exposure. The comparative materials, bulk-sized pulp and MWCNTs, were also able to induce DNA damage after 24 h and 28 days. None of the NFCs or comparative materials was shown to possess systemic genotoxic properties, as measured by the micronucleus assay in bone marrow. The outcome of the in vivo toxicity tests was not consistently predicted by the in vitro toxicity studies reported in WP5. None of the NFCs or the bulk-sized pulp were genotoxic in vitro and only one of the NFCs was able to induce inflammatory cytokines in vitro. This comparison suggested that the mechanisms responsible for the effects observed in vivo are not fully present in the in vitro cell systems used. In the present study, exposure via the respiratory route was chosen to mimic a tentative worst-case-scenario, where liquid nanocellulose is aerosolized in the atmosphere during the manufacturing process and inhaled by workers. As NFC production is presently in an experimental phase, it is presently not known how realistic this kind of exposure scenario could be in the future production of NFC. Possible exposure routes may include, in addition to inhalation of aerosols or dry fibres, also dermal and oral exposure. Toxicological data on nanocelluloses are still scarce, which limits comparison with existing literature. Although NFC is a ▇▇▇▇, nanocellulose fibres are flexi...

Evaluation and conclusions. The data indicate that short/curved/highly agglomerated CNT are very differently distributed in the lung than straight longer thicker ones. Over time the latter seem to disperse and diffuse out into the lung tissue whereas the fate of the shorter agglomerated ones is determined by engulfment in macrophages and granulomas. The surface properties affect the infammogenicity of CNT. Nanocelluloses were unexpectedly rather inflammogenic compared to the CNT From the nose-only inhalation experiments, because of the differences in the shape and the size distribution of the aerosol, the pulmonary deposition of these two nanotubes was different (128- 192 μg/lung vs. 362-377 μg/lung for NM-403 and NM-401 respectively). However NM-403 was more inflammogenic than NM-401. Since NM-403 had a specific surface area 10 times greater than NM-401, the use of different metrics may be useful for the interpretation of the data. The genotoxicity of these CNTs has been assessed using the comet assay, even though a thorough analysis of the data is required, it is unclear whether NM-401 and NM-403 are able to induce significant DNA strand breaks in the lung or other organs such as spleen or liver. Nonetheless, in the lung, NM-403 may be more genotoxic than NM-401 and perhaps this may occur through the production of reactive oxygen species. Similar effects were seen after inhalation and instillation in rats as well as after instillation in mice. Despite the persistent presence of carbon nanotubes in lung tissues, no significant histopathological changes were observed. The use of more sensitive and high throughput methods like transcriptomics or proteomics would help understanding the molecular mechanisms triggered after the exposure to such nanomaterials. The interactions of ▇▇▇▇ materials the biological effects and the distribution in the lung is dependent on size and agglomerate size. Surface properties were less important but had an effect on the biological effects.

Evaluation and conclusions. Based on an extensive literature review and dialogue workshops with nanosafety researchers, this deliverable has created an inventory of the technical and social challenges that are currently inhibiting reliable safety assessment of manufactured nanomaterials, presented a characterisation of different types of uncertainty important in science for policy, and surveyed the potential of both new approaches to scientific research and to management and governance as ways to overcome the identified challenges and move forward in the face of uncertainties. In the review of two new approaches to safety testing of MNMs – high throughput screening and organ-on-a-chip – the deliverable concludes that although these approaches offer significant benefits such as a potential reduction of costs, time and animal use in safety testing, technical challenges and limitations remain concerning issues such as the regulatory acceptability of such approaches, the added value of the knowledge generated, and the relationship to real world scenarios. Having characterised various forms of uncertainty that are particularly relevant in safety assessment and that new technical approaches to safety testing cannot eliminate, the deliverable then provided a critical review of two new governance approaches seeking to manage complex risks in the face of uncertainty – Safe-by-Design and Responsible Research and Innovation. After highlighting the potential benefits and challenges associated with both approaches, this deliverable presented a way in which RRI and SbD could be combined within a stage-gate model of innovation for maximum benefit and a comprehensive assessment. This combination represents a unique contribution to the SbD stage gate model being developed within NANoREG. By including a broader range of actors and issues for consideration, it aims to enhance the potential for safe, sustainable and responsible innovation using MNMs, as called for by the European Commission. Even though challenges remain for the operationalisation and practical implementation of these new approaches to governance, the work in this deliverable offers a useful overview and critical review of the approaches and thereby a step forward towards the safe and responsible development of MNMs in the face of various forms of uncertainty. Finally this deliverable contributes in a distinctive way to the NANoREG project by offering an overview of how the challenges facing the safety assessment of MNMs that have been ...

Evaluation and conclusions. Notwithstanding the difficulties the T1.3 partners faced in collecting the scientific information from within NANoREG to compile elements of answers to the questions of regulatory relevance (D1.1), this deliverable, which was preceded by the interim overview of D1.3, shows that: i) Several important conclusions and elements of answer have been directly or indirectly produced by this large FP7 project, as can be seen by going through the "Summary Of The Findings And Elements Of Answers " of section A.3. ii) In several cases, procedures (SOPs) and a way to tackle an issue have been identified, developed and published, but the verification/validation process ('testing the tests') requires more time and resources than what the project had to offer. iii) The findings generally came to light toward the very end of the project. Nevertheless, all partners involved in WP1 were able to ensure that the output of T1.3 fed directly into the development of the NANoREG Framework for the safety assessment of nanomaterials (D.1.11 of T1.4) and the related NANoREG Toolbox (D1.12 of T1.7). iv) The information now included indirectly in the Framework and the Toolbox is an asset for other on-going nanoEHS initiatives at EU level, such as the ProSafe White Paper drafting, the development of a knowledge/database by former FP7 eNanoMapper project, and feeds into work at OECD level and into the US-EU scientific collaborations.

Evaluation and conclusions. There is an ongoing growth in Nanotechnology where MNMs are more often coming into contact with humans and the environment. The interface between any type of nanomaterial with the surrounding environment either proteins/cells in a culture medium or bound to a matrix/composite or in a solvent depends on colloidal forces as well as dynamic biophysicochemical interactions. The development of predictive relationships between structure (key parameter) of nanomaterials and activity (functionality) are determined by nanomaterial properties. The key parameters of size, shape, composition, surface charge, aggregation, test medium (in our case water) are the priority parameters affecting all the functionalities; while the rest of parameters are of importance. The understanding of these relationships is very important from the perspective of safe use of nano materials. It is not possible to describe all the biophysicochemical interactions at the interface, but we made an effort to assemble knowledge and information to provide a framework to guide this exploration. The literature study consisted of about 140 papers, mainly reporting on CNTs; ZnO, SiO2, Ag and then the rest of MNMs; a priority list was formulated focusing on the core nanomaterials of NANoREG project; the publication year in order to have as recent as possible literature data; and the key physicochemical parameters as showed in other deliverables of WP6 including toxicity. The main finding from D6.3 was a list of risk potentials, which in our case involve our functionalities such as solubility/dissolution, that needs to be screened during the development of toxicity testing. D6.6 is part of the NANoREG project aimed to provide some consensus on which key parameters of MNMs affect specific functionalities in an attempt to establish a relation between them and to propose a workable data model. This is one of the first attempts to identify the main key properties of the MNMs that in relation to their functionalities play a dominant role on the safe by design. This proposed data model is considered as one of the possible approaches towards a decision tree strategy for (re)designing safe MNMs for humans and the environment which will be established in NanoReg2 project. The optimal design of safe nanomaterials is a challenge with multiple compromises between functionality and safety characteristics.

Evaluation and conclusions. Inhalation is considered the gold standard for dosing the respiratory tract. The major advantage of inhalation exposure is that it mimics human pulmonary exposure by distributing the inhaled material throughout the whole respiratory tract in a physiological way over time. The work was done along two axes: - A literature review conducted in an objective to propose recommendations in terms of aerosol measurement strategy. The first objective was conducted mainly by LNE and INRS. - Experimental work with the aim to characterize different test aerosols using different generation methods to be used for inhalation studies. The second objective was conducted by the four partners INRS, LNE, LTH and NRCWE. The results clearly show several things: - For dry-based method, where mechanical forces are added to dry nanomaterial powder, and the resulting separated particles are entrained in air, the test aerosols obtained cover the particle size range from about few tens of nanometers up to several micrometers. Test aerosols are mainly composed or aggregates and/or agglomerates that composed the nanomaterial powder. The conditions required for inhalation studies (stability, repeatability, level of concentrations) are sometimes difficult to obtain, but possible. Few generation devices are commercially available. Nevertheless, it is necessary to carry out preliminary tests in order to ascertain the performances of the chosen devices and their suitability for the inhalation facility. The dry-based method, apparently easy to implement, actually requires a good level of know-how and expertise in terms of generation and characterization. - For direct-synthesis method, where airborne nanoparticles are synthesized in the gas phase and delivered to inhalation ▇▇▇▇▇▇▇▇ as they remain suspended, the test aerosols obtained cover the particle size range from about few nanometers up to several hundred of nanometers. Higher concentrations in number can be obtained with this method. Also stability and repeatability is generally very good. Very few generation devices are commercially available. The development of such home-made generation system is a long-term task requiring a high level of expertise in terms of generation and aerosol characterization. Altogether, the work carried out show that the generation and the characterization of the test aerosols for inhalation studies is an essential step but remains complex if one wishes to obtain robust results.