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Managing Risks of Nanoparticles

Final Report Summary - MARINA (Managing Risks of Nanoparticles)

Executive Summary:
Nanotechnology promises new materials for industrial applications by having new or enhanced physico-chemical properties that are different in comparison to their micron-sized counterparts. However, as in all industrial applications, the potential exposure of humans and the environment to these materials is inevitable. As these new materials go through their life-cycle – from development, to manufacture, to consumer usage, to final disposal – different human groups (workers, consumers), environmental compartments (e.g. air, soil, etc…) and species (e.g. worm, fish, etc…) will be exposed to these materials. Emerging data have shown a range of toxic effects from engineered nanoparticles, suggesting that exposure to these nanoparticles may result in a risk to human health or the environment. While standard methods exist for hazard and risk analysis of conventional chemicals, these tools need to be modified and verified before applied to nanomaterials. Similarly, current standard approaches to risk management, control and reduction need to be rendered relevant for nanomaterials. Thus, the development of nanotechnology-based products must be complemented with appropriate methods to assess, monitor, manage and reduce the potential risks of engineered nanomaterials (ENM) to human health and the environment. Not only good management tools are important, but public awareness is also important for industrial development and acceptance. Public mistrust of any new technology is often high, and demonstrating ‘safe’ products of nanotechnology will enhance public confidence. Hence, efficient communication strategies to the public and stakeholders, of significant progress are of high importance. The methods must be integrated in an overarching, coherent strategy for regulators and industry. Thus, a safe and environmentally responsible nanotechnology will safeguard current and future global investments and will be the key to the sustainability of this industry. While there are standard procedures for the material identification, exposure, hazard, and risk assessment for traditional chemicals, it is not yet clear how these procedures need to be modified to address all the novel properties of ENM. Thus, there is a need to evaluate and develop specific reference methods for all the fundamental steps in managing the potential risk of ENM. The aim of MARINA (MAnaging the RIsks of NAnomaterials) is to develop the Risk Management Methods for Nanomaterials by addressing the four central themes for the Risk Assessment and Management of Nanomaterials: Materials, Exposure, Hazard, and Risk. In MARINA we developed referential tools from each of these themes and integrate them into a Risk Management Toolbox and Strategy for both human and environmental health. These tools were also demonstrated by means of case studies. The fundamental achievements of MARINA are:
i. A well-tested set of reference nanomaterials with thoroughly validated referential characterisation methods.
ii. The methods to further understand the properties, interaction, exposure, and fate of ENM in relation to human health and the quality of the environment.
iii. The harmonised, and standardised reference methods for hazard assessment for both human and environmental health and an integrated/intelligent testing strategy.
iv. The risk assessment tools by combining elements of (i), (ii) and (iii); as well as strategies for monitoring ENM exposure for human health and environment (including accidental massive release, e.g. explosion or environmental spillage).
v. The MARINA database of experimental results to be shared with the Nanosafety Cluster and ongoing or future projects.
vi. Over 80 scientific papers published in peer-reviewed-journals.

Project Context and Objectives: Context
The European Commission had, at the time of the beginning of MARINA, funded some 15 projects relevant to health and safety issues regarding ENM. This commitment was set to continue in the future. At the national level, there were other similar efforts3,4. However, the valuable results generated from these projects had, in the main, been focused to generate concepts, methodology and data to be used for risk assessment and management of ENM. Thus, there was clearly a need to use the most up-to-date date available information and methodology for guidance on health and safety risk management to industry and regulators. To address this need, in MARINA, we created a consortium consisting of first class scientists and organisations with a track records for research in Health and Safety Issues of ENM. Most importantly,
• we had representatives from more than ten Framework Programme (FP) relevant projects . Our aim was to take the beyond the state-of-the-art results from these projects and use them for creating reference tools for Risk Assessment and Management
• we recognised the relevance of our results to industry therefore we involved the direct participation of the Nanotech Industries Association (NIA) and industrial key partners, big and small, such as BASF and Nanocyl.
• we also recognised the geopolitical and economical importance of countries such as China, Russia and Japan. The inclusion of the prestigious Academies of Sciences from China and Russia (for Toxicology) and the Japanese National Institute of Materials Science (for ENM synthesis, characterisation and Toxicology) as well as our existing US partners through current FP7 projects enabled MARINA to reflect a true global effort in addressing this important issue and promoting our strategy for risk management of ENM globally.
• we interconnected all MARINA activites with other relevant activities such as the EC European Technology Platform(s), Nanofutures, Infrastructure and Nanosafety cluster activities, the ERAnet, the OECD WPMN sponsorship programme as well as National Research programmes, such as NanoCare2 and NanoNature.

Although the database that supports risk assessment and management continues to expand, the fundamental approaches have not changed significantly. Risk assessment and management must be based on the best available science, which is continually progressing. These changes, appearing in the nature and the interpretation of data, have prompted the MARINA approach.


1. In response to the likelihood of increasing restrictions and public acceptance of the use of animals for testing purposes in the EU, MARINA focussed on integrated test systems (ITS) targeting modules of hazard endpoints, fate and exposure, and monitoring.

2. The availability of data from new/rapidly advancing methodologies was fully acknowledged in MARINA - systems biology and early marker detection was used for integrated assessment schemes (IAS) for occupational and environmental exposure assessment and monitoring.

3. Advances in mode of action research and in the understanding of effects/disease mechanistic processes in MARINA resultedin hazard being addressed more specifically and the development of interconnecting module systems (IMS) for risk assessment and risk management as methodology for supporting decision making.

4. In order to have a long-lasting impact on the NANOSAFETY world MARINA placed significant emphasis on the training of the next generation of researchers in this area and on the dissemination of MARINA results to key stakeholders.

MARINA was a FLAGSHIP programme for health and safety issues related to ENM on the global scene due to it’s VISION, scientific EXCELLENCE and high IMPACT. Understanding and mitigating the risks of nanomaterials to human health and the environment is a GLOBAL challenge and we believe that our European platform has played a KEY ROLE in this important arena.
Our VISION was that the generation of ‘safe’ ENM will be the key to a sustainable global economic growth and societal acceptance of this industry sector. NANOSAFETY must be central and enabling instead of being a barrier for the development of Nanotechnology. MARINA aimed to articulate the concerns on the health and environment issues regarding ENM, addressing them constructively to all stakeholders in a continuing dialogue to promote the idea of ‘safe’ ENM as a challenge and a goal for future development of Nanotechnology.
To demonstrate the FLAGSHIP potential of MARINA, we successfully recruited the relevant competent authorities in relevant ministries and research centres of the EU (UK, DE, IT, ES, DK, FI, PL), China, Russia, Australia, Singapore, Korea and also the regulatory agencies of North America (Health Canada, US EPA, NIOSH) as well as their centres of excellence for NANOSAFETY research (Rochester (Prof Gunter Oberdorster), Duke (Dr Mark Weisner), California (Dr Andre Nel)) to support the MARINA project. Of equal importance, is the support by Industry, ensured by involving industrial partners in the project as well as linking with key stakeholders such as CEFIC. In response, the international community has given us positive support, reiterating the relevance and importance our approach.

In summary, MARINA stood for integrated testing, integrated assessment and modular interconnection of knowledge and information for science-based risk management methods. The approach was to translate scientific advancements and methodology in contribution to shifting from toxicological studies of specific individual nanomaterials towards a more systematic health and environmental safety assessment and management that handle the overall risks for types or classes of ENM based on their intrinsic, e.g. physico-chemical properties. Objectives

The specific objectives of MARINA were:

1. to develop a Risk Management toolbox for ENM (based on state of the art characterised particles), which includes:
a. referential tools for assessing the human and environmental risk (exposure and hazard) posed by ENM;
b. ENM monitoring systems for identifying, managing and reducing exposure following any workplace release, environmental spillage and especially massive accidential explosion;
2. to develop a strategy for Risk Management of ENM and to illustrate this strategy for a chosen ENM in a MARINA Case Study (MCS)

Assessing the Risk requires extensive information on the physical-chemical characteristics of the materials, exposure and hazard. Therefore, in MARINA, the essential elements of the Risk Assessment tools were based on the following :

(i) Development/implementation of ENM reference materials (WP3-4)
Some work was already ongoing in this area, eg via OECD WPMN and other EU-FP project. MARINA aimed to fill the gaps and to develop a full and coherent reference material and characterisation protocol. We have developed a panel of reference ENM to;
a. further develop validated methods for characterising the physico-chemical properties of ENM as pristine materials, in biological matrices, in environmental samples and for field detection;
b. collate data on the physico-chemical properties and (eco)-toxicity for each ENM.
c. use isotope-label ENM for their use in bio-distribution studies.

(ii) Exposure assessment in human and environment settings (WP5-8) We developed tools for a tiered (human) exposure assessment approach (workers and consumers) and for assessing the fate and behaviour of ENM in soil/sediment/water. This included charactersation of the actually released ENM (eg aged ENM) compared to the pristine ENM.
Based on this work MARINA identified key ENM parameters (e.g. size, charge or coating) important for describing exposure and dose metrics by means of Quantitative-Structure-Activity Relationship modelling.
(iii) Hazard assessment for human and environment (WP9-11) We implemented in vivo dose-response models through repeated dosing via inhalation, ingestion, iv injection and dermal routes. Environmental hazard was assessed by evaluation, modification or new development of the present test guidelines. This included validation of existing (eco)-toxicology tests and the development of new, relevant ones. This work was done collaboration with other FP projects such as QNANO and NanoValid

(iv) A combination of (ii) and (iii) into a Risk Assessment protocol/toolbox and an Integrated/Intelligent Testing Strategy and Risk management strategy (WP12-16). MARINA developed a novel and flexible integrated risk assessment strategy for human and environmental exposure.

Training and dissemination of results, data and knowledge to stakeholders. Training initiatives were arranged in WP17 and WP18 developed and implemented an efficient communication strategy .

The relationship between the different WP’s and their integration within MARINA are illustrated in Figure 1.

All data generated from MARINA have been stored in the MARINA project database hosted by the NanoHUB at the JRC. This effort was part of the Nanosafety Cluster activities to centralise and harmonise all FP data. During the project, MARINA data were made available by following normal confidential requirements. After the project completion, the data will be continue to be available following registration with the NanoHUB.

Project Results:
MARINA was divided into four central themes. The major S & T achievements for each of these are described below. In all four themes and across the themes MARINA has lifted the knowledge in Nanosafety beyond the state of art. MARINA has been a global leading force in developing a well characterised standardised panel of ENM, new advanced methods for their detection and characterisation in complex media, protocols for exposure and hazard assessment and comprehensive strategies for risk assessment and monitoring. MARINA results were communicated through many peer reviewed publications, specially organised workshops, and meetings with relevant stakeholders. The data generated by MARINA have been incorporated in the MARINA database (see Figure 1 and 2) and transfered to the NanoHUB database at the JRC where it is stored with data of other completed projects (e.g. ENPRA) and will be part of an expanding body of information available to ongoing and future projects. OECD compatible material dossiers have also been compiled and made available to industry and other stakeholders.

Figure 1: Opening menu of the MARINA operational database

Figure 3. Schematic overview of content, operation of the database

Materials Theme

In MARINA well-characterised referential ENM were collected. The materials were distributed to all experimental MARINA partners and made available to other projects beyond MARINA. The ENM were characterised, labelled and verified with regard to homogeneity and stability and can be used as referential type materials and this has ensured that results are comparable across projects, so that progress in nano EHS studies can be as fast as possible. MARINA partners have also tested and developed new techniques and harmonised protocols for ENM characterisation in complex matrices (see e.g. Figure 4) including both protocols for measuring nanomaterial characteristics in pure and in complex matrices (for example, the measurement protocols included surface area of nanoparticles when they were suspended in liquid or number concentration in both simple and complex media). To enforce the global aspect of MARINA, a Best Practice Guide was produced in collaboration with the Japanese partners at NIMS, presented at the 3rd Workshop on the Risk Management of Engineered Nanomaterials at NIMS in Tsukuba (Japan), August 2014. Additionally NIMS launched an inter-laboratory test for the establishment of quantitative size and shape characterisation using AFM and other nanoscale microscopies such as SEM. To promote and share this knowledge, these results have been disseminated in peer-reviewed publications. Furthermore, to serve the need for teaching, a book “Nanomaterial characterisation: an Introduction’’ was produced by several MARINA partners and published by Wiley in 2015.

Figure 4. A schematic representation of STM nanoscale characterisation under extreme environments for novel nanofunctionality research

Exposure Theme

For human exposure, methods for the assessment of the release of ENM during production, processing and handling (by consumer / workers) were developed and new data were generated. The state of the art knowledge was established through a prototype of a searchable database tool of occupational exposure scenarios (included in the MARINA database), by reviewing existing exposure models and evaluating and proposing a standard protocol for workplace exposure measurement surveys. The reviewed exposure models were then included with a tiered exposure assessment approach for human risk assessment (see Figure 5). The performance of some tools such as the Advanced REACH Tool (ART) and Stoffenmanager-Nano model was evaluated. Standard methods for release of nanomaterials for different composites (e.g. natural rubber with carbon black or carbon nanotube as filler) were developed. This has also provided insight into the difference in the release mechanisms (Figure 6). MARINA experimental studies on deagglomeration behaviour have also provided complementary data with regard to the propensity of a given nanomaterial to become airborne and thus deliver valuable input for exposure assessment.

Figure 5. The MARINA Exposure Assessment approach

The MARINA partners also implemented a MARINA information database on ENM fate-determining parameters in environment and biota. This work was complimented by the development of novel single-particle ICPMS methods and other methods to measure ENM in complex environmental matrices. Though this work the detection limit for nanomaterials was dramatically reduced, setting global standards and for the first time making it possible to differentiate man-made nanomaterials from natural occurring in the environment. To compliment this work mathematical and statistical model were developed describing the fate and behaviour of representative ENM in the environmental matrices surface water, sediment, and soil, as well as in sewage sludge. During the project it was realised that the fate models and current material measurement techniques do not quantify” the same “material concentrations”, hence it is presently impossible to validate fate models. Beside this, the ENM properties that cause them to be bio-accumulative were identified (cf. PBT-criteria for conventional chemicals). The work has been published in several peer-reviewed papers on model of environmental fate of ENM and methods for ENM analysis in environmental matrices.

Figure 6. Automatically controlled drilling setup developed at BASF

Hazard Theme

The hazard theme aimed to develop harmonized and standardized methods for hazard assessment of engineered nanomaterials for both human health and the environment culminating in an integrated testing strategy. Within the hazard theme, the panel of ENM selected in the Materials Theme, were in situ characterised and tested using a wide range of in vitro models representing different body systems (Figure 7). Further in vivo toxicity (inhalation and ingestion) were also performed for a subset of ENM. This work was carried out by wide range of laboratories and has resulted in a comprehensive database on ENM toxicity. Based on the results of the toxicology tests an Intelligent Testing Strategy was developed and fitted into the global risk assessment framework developed in the Risk theme.

MARINA researchers identified and evaluated existing in vitro test methods for the assessment of acute toxicity of a panel of representative ENM and assessed the reliability of existing and newly developed in vitro test methods. The in vitro testing approach included the use of twelve different cellular models representing six different target organs/systems.
Existing in vivo and ex vivo test methods were identified and evaluated. The responses to selected ENM were studied in terms of organ specific toxicity, biodistribution and barrier-crossing after inhalation and oral exposure.

Finally, the development of an intelligent/integrated testing strategy was done for human risk assessment utilizing in vitro and in vivo test systems according to a tiered approach.

Figure 7. Cellular models selected for in vitro screening of ENM – Farcal et al. PLoS-ONE 2015

For the environmental compartment, a wide range of OECD protocols where evaluated with regard to their suitability for nanomaterials and modifications were recommended. Moreover, a systematic investigation, of the most important impact-parameters on ecotoxicity, showed that environmental conditions do play a large role with regard to toxicity level reached. This work was widely disseminated therefore standardization organisations such as OECD and ISO had received substantial input from MARINA to implement guidelines for the testing of ENM. Besides the guideline orientated work, novel techniques were developed and data generated, identifying possible nano-specific mode of actions in a number of species, showing that indeed nanomaterials can have a different mode of action compared to the corresponding bulk material.

Figure 8. Overview of a tiered hazard assessment strategy for engineered nanomaterials adapted for the environment (Oomen et al, 2014).

The mode of action knowledge was further enhanced by detailed studies developed pipeline for automated incorporation of omics data across human and environmental species, involving genomics, proteomics and metabolomics (Figure 9). Based on detailed experimental system biology studies, comprehensive and fully automated pipelines for integraton of the different omics were also developed and implemented.

Figure 9. Developed pipeline for automated incorporation of omics data

Considerable attention has been devoted to the preparation of protocols in order to ensure high quality transcriptomics, metabolomics, and proteomics measurements of the samples from the MARINA (eco)-toxicology studies as well as the preparation of a data analysis platform that allows a parallel analysis of all the data sources. The experimental procedures were successfully applied to different samples generated in the MARINA (eco)-toxicology studies. This has yielded a first set of results for further computational analysis using an integrative approach. Future implementation of the MARINA methods for (eco)-toxicological profiling, using transcriptomics, proteomics and metabolomics for ENM, will enable the identification of (novel) ENM specific Modes of Action.
Risk Theme

MARINA has developed an integrated global risk-assessment and intelligent testing strategy, incorporating risk management and risk reduction paradigms with focus on nano-specific issues e.g. how to handle ENM size in a risk assessment and to monitor ENM in situ.
The global tiered risk assessment strategy integrated the human health and the environmental risk assessment into one framework including the characterisation of and assessment of the physical-chemical properties of materials, exposure, hazard, and risk (Figure 10). Based on this strategy, specific models were also developed (e.g. for the environment where the strategy took highly environmental specific issues into account) and included novel nano-specific probabilistic models.

Figure10. MARINA Risk Assessment Strategy: Flexible Strategy for Efficient Information Collection and Risk Assessment of Nanomaterials – Bos et al. (submitted)

Combined with this risk assessment a framework for read-across of hazard based on the physical-chemical properties of the materials was also developed (Figure 11). The RA strategy considered the impact of the varying properties of an ENM during the different life cycle stages. In parallel, an ITS for the environmental compartment was also developed (the ITS developed in MARINA was constructed in collaboration with the FP7 project ITS_NANO).

Figure 11. Grouping and Read-Across Approaches for Risk Assessment of Nanomaterials – Oomen et al. (submitted)

MARINA was the first FP project to explicitly focus on risk from release due to spillage and explosion of nanomaterials. An approach was developed to evaluate the potential impacts of massive release and explosion of nanomaterials on infrastructures, the environment and human health. Small to medium scale laboratory experiments were designed and tested to allow extrapolation to larger industrial sites. MARINA partners have extended the explosivity database on nanopowders. They also created a framework to pre-assess pyrophoricity of nanopowders together with an experimental framework for assessing explosivity of nanopowders. The physico-chemical parameters of interest for nanopowders have been identified through the development of a thermal flame propagation model. Experimentally, MARINA has highlighted the influence of agglomeration, the thermal propagation mode of the flame and concentration by modelling the specific pattern of the evolution of explosivity. MARINA has also proposed a classification diagram of explosion hazard according to the level of agglomeration. A modelling approach was linked with standardised explosivity results (20 L sphere), burner measurements available in the literature and direct flame velocity measurements (determined from explosion safety parameters).

Monitoring methods for ENM were also developed. These tools relied upon a tiered method that was unambiguous, logically proceeding step by step and standardised which guaranteed monitoring measurements that are comparable with each other. The results can be used as information source for future monitoring needs when stored, e.g. in a monitoring database. MARINA has found that the distribution behaviour of ENM determined which environmental compartment has to be considered in the monitoring process. For example, for the environmental hazard, due to their ecotoxicity ion-releasing ENM seem to be of higher relevance for monitoring compared to “stable” ENM, and monitoring should focus on sites with low sorption capacity environments. The monitoring methods and data generated by MARINA can be used as information source for future monitoring needs when stored in a monitoring database, such as MARINA Exposure Scenario library or NECID database (developed by the PEROSH group). The MARINA monitoring and measurement strategy describes the requirements for monitoring measurements at different scale industrial surroundings.
To develop safer nanomaterial and risk reductions strategies nano-(Q)SAR and other models were developed. The models included among others heat maps (R), parallel coordinates plots (CVE) and principal components (SIMCA). A Physiologically-Based-Pharmaco-Kinetics (PBPK) approach to model the translocation of ENM throughout the body was extended to include other exposure routes - oral, intravenous injection – other than inhalation. A control banding approach was also developed together with the guidance for the preparation of safety data sheets (SDS) for ENM and also an SDS template. The first document complemented the ECHA guidance for the preparation of SDS for bulk chemicals while the second was a more practical tool. To demonstrate how the MARINA tools were applied, case studies with two ENM, TiO2 and Silver were constructed. These case studies illustrated how the MARINA results of exposure, hazard assessments were combined for a risk assessment of nano TiO2 and silver.
In summary, ENM relevant methods in relation to risk assessment and risk management are required to reduce the risk uncertainty when using ENM. MARINA has established the first coherent set of novel ENM relevant methods in the area of materials, exposure and hazard prediction and assessment. These methods can be applied in the developed ENM targeted risk assessment and management strategy. This implementation was ensured by passing on the knowledge to the research community, to other European projects, and most importantly to major relevant stakeholders.

Potential Impact:
MARINA has made a significant impact on the European objectives for the safe, integrated and responsible development of Nanotechnology. Specifically, for the development of comprehensive understanding of the properties, interaction and fate of ENM in relation to human health and environment, results generated within MARINA are providing an overarching understanding of the interaction of ENM with humans and the environment and are therefore helping to assess and manage the potential risk of ENM. MARINA developed validated reference methods for managing the risks of ENM. MARINA has generated a very substantial dataset covering the four Themes of Materials, Exposure, Hazard and Risk. The information has been organised into referential toolboxes for each of the themes.
Almost all of the work has been published in peer-reviewed journals, been disseminated at meetings with other projects, at NanoSafety Cluster and Community of Research meetings, and at relevant regulatory bodies such as ECHA, who handles REACH

For the Material Theme
MARINA had used reference nanomaterials selected from JRC repository, which overlaps with the ones used in the OECD Sponsorship Programme. Hence this has ensured a high degree of compatibility of MARINA results with the initiatives in OECD. In fact, MARINA has obtained a set of reference type material that have been extensively characterised in regard to physico-chemical characters, to fate and exposure properties, and to biological effects. In connection to this the reference dossiers for the nanomaterials has been finalised. The format of the dossiers is similar to the OECD dossiers. The materials used in MARINA have further been offered to other projects such as NanoValid, NANoREG, NanoMile, QNANO, to ensure coherency across the European efforts. Discussions on MARINA dispersion protocols has been presented to OECD, ECHA and integrated in NANoREG work. This activity contributed to closing one of the major gaps in the process for ENM risk assessment. Critical appraisals of some protocols have taken place. Papers have been produced and published in peer-reviews journals. Through discussions with our Japanese partner at NIMS (The Third Workshop on the Risk Management of Engineered Nanomaterials, NIMS, Tsukuba, Japan September 2014), NIMS has launched the inter-laboratory test for the establishment of quantitative size and shape characterisation using AFM and other nanoscale microscopies such as SEM; the activity has been proposed to VAMAS TWA2 for surface chemical analysis. Another invaluable exploitation route for this Theme was the publication of a book (“Nanomaterial characterisation: an Introduction”) that was edited and co-authored by MARINA partners and published by Wiley in 2015. The MARINA WP 4 leader was the main editor for this multi-author book with contributions not only from MARINA partners but also from NanoVALID, NANoREG as well as US partners. The material presented in the book is written in such a way that both students and experts in other fields of science will find the information useful and will thus appeal to a wider range of audience, whether they are in academia, industry or part of nanoregulatory framework chain. Thus, it can be used as a nanoscience textbook for undergraduate students (useful in experimental research setting) or as a quick guide for regulators e.g. to understand physicochemical properties of nanomaterials. The book will be promoted through Wiley, one of the biggest publishinghouse for science.

For Exposure Theme
Experimental investigations regarding release from specific composite nanomaterials. For the composite materials, the two round robin tests on mechanically induced released together with the weathering and incineration studies, have provided a comprehensive investigation of a possible nanomaterial release. A second matrix material, natural rubber with CB and CNT as nano-fillers, was furthermore investigated and has provided insight on the possible difference regarding the release mechanisms of these two material classes. The experimental studies on de-agglomeration behaviour have provided complementary data with regard to the propensity of a given nanomaterial to become airborne and thus deliver invaluable input for exposure assessment following an accidental release. We developed a prototype of the online Exposure Scenario (~ 70 scenarios) Library; eEvaluated the performance of existing Exposure Assessment models ART and Stoffenmanager-Nano; nd developed the framework for the tiered exposure assessment approach in accordance with the risk assessment evaluation cycle. The results from this Theme have been disseminated to other projects, namely, GUIDENano, SUN and specially NANoREG. A substantial number of publications were achieved and more papers are under preparation.

For the environmental part the work had, in several ways, developed novel aproaches and set reference points for future research. High impact work were devoted on the creation of an information database of ENM fate-determining parameters in the environment and biota which future research projects and regulators can use as a solid baseline of information. The dramatically lowering the detection limit of the Single-Particle ICP-MS analyses and the ability to measure ENM in nature is a fundamental step for future validation of fate and exposure in complex environments. The probabilistic fate modelling developed through MARINA will likely be the way forward in fate and exposure assessment, and has already been used in other projects. It is also especially useful for regulatory setting. Moreover, the identification of the ENM properties that drive their bio-accumulation will influence the direction of future research. The environmental fate and exposure results from MARINA have already had a pronounced impact on the research in environmental nanosafety and the tools created will be useful for future regulations of ENM.

For Hazard Theme
The in vitro and in vivo toxicology studies have been completed and the results compiled and published or in the process of being published in peer-reviewed journals. The in vitro and in vivo samples were supplied to the ‘omics’ studies and the results were shared with other ongoing projects specially NanoSolutions. MARINA has developed and intelligent toxicity testing strategy serving human risk assessment by utilizing in vitro and in vivo test systems in a tiered approach. This strategy is delivered to the ongoing project NANoREG. The systematic investigation of the most important impact-parameters on ecotoxicity, show that environmental conditions do play a large role in regard to toxicity level reached. This means that the standardization organisations such as OECD and ISO receive substantial input to publish test guidelines on the testing of nanomaterials. As several partners of this Theme are engaged in the OECD WPMN work, this information is transferred to this forum, besides this the work is presented to ECHA and will be incorporated in the revision of REACh, early 2016.

For Risk Theme
The MARINA Risk Assessment framework is now fully developed. Several workshops have been held with other projects to disseminate the MARINA results. The latest workshop (Lisbon, November 2014) was organised with project NANoREG with the participation of other partners from GUIDENano and SUN. The following presentations were made at relevant regulatory meetings: ECHA 11-12th November 2015; ENM workshop: 19th May 2014, Washington, USA; OECD meeting: 17th-19th September 2014, Washington, USA; ECHA: 24th October 2014, Helsinki, Finland; GUIDEnano meeting: 9th October 2014, Bilthoven, the Netherlands; CoR teleconference presentation: 10th September 2013. MARINA was in charge of the WG 5 RISK of the Nanosafety Cluster and represents the EU in the US-EU community of Research (CoR).
Close collaboration with FP7 BUONAPARTE has yielded fruitful results in the Explosion and Accidental release risk theme. MARINA was able to advise projects like BUONAPARTE on minimizing the risks of explosion in nanomaterial production plan.
For monitoring of continual exposure to Nanomaterials in the environment, the distribution behaviour of ENMs determines which environmental compartment has to be considered. Due to their ecotoxicity, ion-releasing ENM seem to be of higher relevance for monitoring compared to “stable” ENM. There are indications that monitoring with respect to ecotoxicity should focus on sites with low sorption capacity (e.g. high sand content). For ion releasing ENMs, sites with a low pH have a higher potential for undesired effects. The monitoring method/strategy developed is a tiered method to guarantee measurement results that are comparable with each other, and that may be used as information source for future monitoring needs when stored in a monitoring database, such as MARINA Exposure Scenario Library or the NECID database. For risk reduction methods, quantitative methods, QSAR and PBPK models, have been developed to describe the relationship between the physico-chemical characteristics of nanoparticles and the biological responses as well as the distribution of these materials in the body following inhalation, injection and oral exposure. A control banding approach has also been developed together with experiments to modify the different physical aspects of nanomaterials with the views to reduce its toxicity – the safe by design approach. A guidance for the preparation of safety data sheets (SDS) for ENMs and also an SDS template have been developed. The first document complements the ECHA guidance for the preparation of SDS for bulk chemicals. The second is a more practical tool. Peer reviewed papers have been published in this area.

In summary, MARINA has developed a considerable number of tools within the 4 Themes. These tools have been actively shared with other projects. This will help in establishing them as Referential tools. MARINA has also actively disseminated these tools through collaborative workshops organised with other projects and through various EU and international activities (e.g the US-EU CoR). The results supporting these tools have been and are still being published extensively in the peer-reviewed domain.
MARINA is providing a unique resource of information and methodology in support to regulatory policy and decision-making activity. It supports the shifting from case-by-case evaluation of individual ENM risk to a more holistic health and environmental safety assessment and management that addresses overall risks. With the past and current dissemination activities aimed at the regulatory agencies (e.g. OECD, REACH), decision makers and Industry would find information directly in a format, which allows analysis across endpoints, across material types or preparation forms. Apart from the direct MARINA contribution through participation at OECD, another important contribution that MARINA made was the collaboration with the projects NANoREG (I and II). The referential tools developed in MARINA were reported and made available to NANoREG and MARINA partners who are currently NANoREG partners are performing the work needed beyond MARINA. Since NANoREG is directly relevant to the regulatory world, MARINA’s contribution is indeed substantial. Contribution to the future definition of appropriate measures, where needed MARINA has developed standard, reference methods covering a wide range of themes, from Materials to Risk as described above.

MARINA has contributed to an overarching strategy for risk management and reduction and enabling the EU regulatory bodies, agencies and authorities to make informed decisions and policies to safeguard consumers while taking full advantage of the advancements that nanotechnologies will bring to the economy and competitiveness of EU industry. There are clear national and international requests for strategies to improve the understanding of nanosafety across diverse industry sectors. Efficient use of the nanosafety information generated, and the incorporation in the business models of industries and regulators is absent and needs to be improved to fully exploit the product value chains.

MARINA has developed (published and submitted) the fundamental tools for such risk governance, developing both the underlying fundamental tools (e.g. test guidelines and protocols) and the risk assessment/management framework (e.g. how to combine the underlying tools into a relevant and reliable risk evaluation). The approach taken in MARINA thus builds on the formalised structure of guidelines and guidance documents, combined with a more novel flexible frame for risk evaluation, allowing for grouping of materials and possible read-across between materials. Hence, the structure of the approaches follows the regulatory requirements; hence it is directly useful for regulatory bodies. This allow for a strong impact on regulation, since it is the first coherent tool development. Further, since the guidance and framework has been developed in collaboration with the industrial partners within MARINA and the MARINA case study was led by the Industry, it is likely that there will be a broad acceptance. Hence, MARINA clearly has contributed to an overarching strategy for risk management and reduction and enabling the EU regulatory bodies, agencies and authorities to make informed decisions and policies to safeguard nanotechnology, and to bring to the economy and competitiveness for EU industry.

Support to pre and co-normative activities, such as with reference to the implementation of REACH

MARINA has contributed to the REACH process through our activities and the ITS. MARINA has also worked with the Commission and global (e.g. OECD) services involved in development of adaptations of guidance documents concerning nanomaterials relevant for REACH. There has continuously been a clear focus within MARINA to support the pre and co-normative activities within EU, which is why the tools developed have at all levels referred to the current risk assessment approach as outlined in REACH, even when de novo approaches have been developed. At the same time this close relationship to the normative activities has been pursued through meetings with the relevant authorities, e.g. inviting ECHA to meeting and participating in ECHA meetings. Further, the structure of the MARINA framework has been evaluated in comparison to the co-normative approach, and the nanospecific changes been highlighted.
Support to the safe, integrated and responsible approach as lay down in "Nanosciences and Nanotechnologies: An action plan for Europe", the risk management recommendations has been developed in cooperation between MARINA scientists and industrial stakeholders.
As outlined an essential element of this responsible strategy for Nanotechnologies is to integrate health, safety and environmental aspects to the technological development and to establish an effective dialogue with all stakeholders, MARINA consisted of many industrial partners and regulatory bodies, which have been involved in the development of the framework in order to ensure that specific stakeholder concerns are considered. The work has continuously been communicated to all stakeholders, including the NGOs, via yearly workshops to ensure an effective dialogue and to avoid negative societal impact. This work has also included the education of young researchers, through yearly Nanosafety summer schools, in the risk assessment and management tools for nanomaterials. Finally, MARINA has emphasised the international dimension though extensive international collaboration, partly through the partners form Russia, China and US connected directly to MARINA, partly through the NanoSafety Cluster and Community of Research Work, led by MARINA within the Risk area. The extensive network of the individual partners has complemented this. The focus of this works has been to promote, based on the integrated strategy developed within MARINA, the dialogue at international level with a view to adopting a declaration or a ‘code of good conduct’ for the responsible development and use of Nanotechnology.
The above outlined impact has been achieved through communicating the extensive work performed with MARINA through targeted workshops involving all stakeholders, through education of young researchers and through publishing the result in peer reviewed journals.

Impact on participating Small and Medium Enterprises (SME)

The MARINA consortium included SMEs in Nanotechnology such as NanoCYL and Colorobbia and SME’s in research such as the IOM. The tools developed by MARINA were directly relevant and useful to industrial SMEs because these enterprises usually do not have enough funds and resources to develop the tools themselves. Furthermore, some of the materials tested in MARINA were from the SME (such as silver (Colorobbia) and multi-wall carbon nanotubes (NanoCYL)) so the tools developed by MARINA were directly relevant and useful to these enterprises. |For research, MARINA has offered the opportunity for IOM to coordinate a large research programme. This opportunity was unique for a research SME and has generated much visibility, which otherwise IOM may not have acquired. Therefore the outcomes of MARINA have made considerable impacts on all the participating SME.

Exploitation potential for the participating SME

The success of MARINA was exploited by the participating SME. For IOM, this meant the ability to participate in more projects in Nanosafety (e.g. NanoREG I &II, Prosafe, etc…). MARINA has given IOM the opportunity to develop scientific skills in Exposure Assessment, mathematical modelling. These tools are readily exploited by IOM for use in current and future projects. For industrial SMEs, the hazard assessment and associated tools were applied to their materials. These SMEs can claim that their materials were fully tested as part of a large European Research Programme. This is a great exploitation potential item for these SMEs which would not be been available to them had they not been participating in MARINA.

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