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ProSafe Deliverable 4.4 Page 1 of 66

ProSafe

Grant Agreement Number 646325

Deliverable D4.4 Final Version

Inventory of the harmonized national regulation oriented tasks

Due date of deliverable, final version: 2017/01/31

Actual submission date: 2017/01/31

Author(s) and company:

Speranta Tanasescu, IPC (Institute of Physical Chemistry "Ilie Murgulescu" of the Romanian Academy)

Jürgen Höck, TEMAS AG

Work package/task: WP4 / Task 4.2

Document status: draft / final

Confidentiality: confidential / restricted / public

Key words:

DOCUMENT HISTORY

Version Date Reason of change

1 2015/08/22

2 2016/03/31 Update of Version 1 by S. Tanasescu, IPC and K. Hoehener, J.

Hoeck, TEMAS

3 2016/10/31 Update of Version 2 by S. Tanasescu, IPC and K. Hoehener, J.

Hoeck, TEMAS

4

2017/01/31 Update of Version 3 by S. Tanasescu, IPC and K. Hoehener, J. Hoeck, TEMAS

5 2017/07/19 Project Office harmonized lay-out

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Lead beneficiary for this deliverable: Institutul de Chimie Fizica Ilie Murgulescu, IPC, partner number 12

Owner(s) of this document

Owner of the content IPC, 12

Co-Owner 1 TEMAS, 7

…..


Table of Content

1 DESCRIPTION OF TASK ......................................................................................................................... 5

2 DESCRIPTION OF WORK & MAIN ACHIEVEMENTS ............................................................................ 5

2.1 SUMMARY ............................................................................................................................................ 5

2.2 BACKGROUND OF THE TASK .................................................................................................................. 5

2.3 DESCRIPTION OF THE WORK CARRIED OUT ............................................................................................. 6

3 RESULTS .................................................................................................................................................. 6

3.1 REQUIREMENTS AND TERMS OF THE CONTENT TO BE HARMONIZED ......................................................... 6

3.1.1 Requirements for the reengineering of substances .............................................................. 6

3.1.1.1 Provide key analysis and characterization tools ................................................................... 7

3.1.1.2 Developing Protocols for Measuring and Reporting a Minimum Set of Material Properties for MNMs Used in Nanotechnology-Related EHS Research: ............................. 8

3.1.1.3 Detection and Characterization of Nanomaterials in Complex Biologic and Environmental Samples ........................................................................................................ 9

3.1.1.4 Development of New Protocols or Modification of Existing Protocols for Toxicity Testing and Determination of Population and Ecosystem Effects ...................................... 10

3.1.1.5 Develop safe MNMs “by design” using principles similar to those of “green chemistry” ............................................................................................................................ 10

3.1.2 Exposure, Transformation and the Life Cycle..................................................................... 12

3.1.2.1 Research priorities related to MNM release, transformation, transport and exposure ....... 13

3.1.2.2 Research Needs for development of Protocols for Exposure Assessment ........................ 15

3.1.3 Hazard identification and characterisation .......................................................................... 16

3.1.3.1 The required research priorities on hazard assessment: ................................................... 16

3.1.3.2 Safe design of new MNM in a bottom-up approach............................................................ 17

3.1.4 Risk prediction and management tools ............................................................................... 18

3.1.4.1 Fundamental Processes important for the evaluation of risks posed by MNMs ................. 19

3.1.4.2 Challenges of Defining Potential EHS Risks of MNMs arising from the diverse properties of NMs (A Research Strategy for EHS Aspects, 2012; Nanosafety in Europe 2015-2025; 2013) ................................................................................................... 19

3.1.4.3 The required research priorities on risk assessment include: ............................................ 20

3.1.4.4 Applying the Principles to the Value Chain and Life Cycle of Nanomaterials and Products .............................................................................................................................. 22

3.1.4.5 Identifying health effects from MNM and potential biomarkers ........................................... 23

3.1.4.6 Databases ........................................................................................................................... 23

3.1.4.7 Steps to Ensure Progress toward Validated Models for Nanomaterial Risk ...................... 24

3.1.4.8 Risk management ............................................................................................................... 24

3.1.5 Search Call Document ........................................................................................................ 25

3.2 CALL ACROSS NANOREG AND THE PROJECTS OF THE NANO SAFETY CLUSTER TO ASK FOR PROCESSES, TOOLS INSTRUMENTS, ETC .............................................................................................. 28

3.2.1 Goal and Target groups ...................................................................................................... 28

3.2.2 Mailing ................................................................................................................................. 28

3.3 CONSOLIDATION OF THE RECEIVED FEEDBACKS ................................................................................... 29

3.3.1 Appraisal criteria ................................................................................................................. 29

3.3.2 Appraisal of existing DB/information ................................................................................... 29

3.3.3 Appraisal of DB/information coming from the “Search Call” ............................................... 29

3.4 HARMONISATION OF THE CONSOLIDATED FEEDBACKS AND PROVIDE A SET OF SUPPORTING ITEMS FOR THE SAFE-BY-DESIGN CONCEPT. .................................................................................................. 30


3.5 IDENTIFICATION OF GAPS .................................................................................................................... 32

3.6 PROVIDE INPUT TO NANOREG2 AND OTHER PROJECTS TO COVER THE GAPS ......................................... 60

4 EVALUATION AND CONCLUSIONS ..................................................................................................... 60

5 DEVIATIONS FROM THE WORK PLAN ................................................................................................ 61

6 PERFORMANCE OF THE PARTNERS ................................................................................................. 61

7 REFERENCES / SELECTED SOURCES OF INFORMATION .............................................................. 61

7.1 LITERATURE FOR CHAPTER 3.1 ........................................................................................................... 61

7.2 PROJECTS MENTIONED IN CHAPTER 3.1 ............................................................................................... 66


1 Description of task

The Task 4.2 is addressing two main fields of activity:

1. Definition of common call topics

2. Harmonization of national regulatory orientated tasks

This Deliverable D4.4 only addresses the second of the two fields, giving an overview of activities carried out in this respect within ProSafe.

In order to harmonize national and other regulation oriented protocols, procedures and data, ProSafe focuses on linking existing initiatives, promoting the acceptance, and uptake of safe-by-design approaches, regulatory conditions and precautionary principles within the MS and AS, as well as regional efforts. Measures address regulatory conditions (standards or recommendations for nanosafety R&D, production of nanomaterials and related products, and respective EHS risk management and assessment), precautionary measures and risk engineering.

2 Description of work & main achievements

2.1 Summary

The harmonisation of national and other regulation oriented protocols, procedures and data has been completed. To this end, the following steps of the harmonization process have been carried out:

The requirements and terms of the content to be harmonized have been identified, the attention being devoted to particular items derived from the previous analysis and overview on the advances in the safe-by-design approach, including fundamental and regulatory aspects, cross-cutting issues, as well as newly identified challenges and issues, and future related activities

Requirements for protocols, procedures, tools guidelines, instruments and the like have been collected and cross-checked with the state of the art in an internal process, carried out by S. Tanasescu (c.f. chapter 3.1)

Experts from NANoREG and the projects of the Nano Safety Cluster have been contacted and asked for critically reviewing, or, complementing processes, tools, instruments, etc which are available or under development, covering the requirements formulated in the first step (c.f. chapter 3.2)The experts' feedbacks were collected and appraised, and subsequently consolidated in the search call document (c.f. chapter 3.3)

The thus harmonised document was distributed to the experts involved, and has been made available on the ProSafe website for the stakeholders (c.f. chapter 3.4). The list of requirements for the implementation of SbD settles a basis of supporting items for further activities in the field of SbD

Gaps in current knowledge as well as missing tools for SbD have been identified and collected in an internal document, which has also been handed over to the NanoReg 2 project for further steps (c.f. chapters 3.5 and 3.6)

A SbD implementation concept has been developed. Building on the results of the search call and nitegrating them, an implementation concept has been elaborated. In the frame of this concept, a working tool is being created which is called the "Safety Dossier".

2.2 Background of the task

The ProSafe Coordination and Support Action acknowledges the importance of the industrial implementation of the NANoREG Safe-by-Design concept (http://www.nanoreg.eu/media-and-downloads/publications). Through this implementation the currently used industrial management processes for innovations, risks, EHS and regulatory affairs are sought to be coherently integrated to improve the management of uncertainties and risks associated with nanomaterials and products containing nanomaterials. As a preparation step for this implementation, ProSafe has to carry out a harmonization of existing procedures and data. The work done with this focus is described in the present Deliverable 4.4.

http://www.nanoreg.eu/media-and-downloads/publications


2.3 Description of the work carried out

The concept of the Harmonization process is based on an integrated approach providing a bridging of data acquisition, evaluation and harmonization, aiming to promote the acceptance of Safe by Design at all levels of the MNMs value chain as well as the respective production processes.

The harmonisation process carried out within Task 4.2 is based on the following steps, all of which have been accomplished:

- Requirements and terms (Protocols, Procedures, Tools, Guidelines, Instruments, …) of the content to be harmonised, see chapter 3.1 of this Deliverable

- Call across NANoREG and the projects of the Nano Safety Cluster to ask for processes, tools instruments, etc which are available or under development covering the requirements, see chapter 3.2 of this Deliverable

- Consolidation of the received feedbacks. This task is described in chapter 3.3 of this Deliverable

- Harmonisation of the consolidated feedbacks and provision of a set of supporting items for the Safe-by-Design concept. This task is described in chapter 3.4 of this Deliverable

- Identification of gaps. This task is described in chapter 3.5 of this Deliverable

- Provide input to Nanoreg II and other projects to cover the gaps, see chapter 3.6 of this Deliverable.

3 Results

3.1 Requirements and terms of the content to be harmonized

In a first step, S. Tanasescu (IPC) has compiled a listing of requirements and terms with regard to the harmonization process. In the following sub-chapters the requirements for relevant fields of activities are explained.

3.1.1 Requirements for the reengineering of substances

Some substances can be reengineered to create safer, greener, and yet efficient products. It is important to discern the specific critical functionalities and physicochemical properties that make MNM harmful, then reengineer these properties to achieve safer products.

To do this, relationships between designed NP properties and their translocation at cellular and body level, as well as the toxicological response have to be established. Such information is available for a small number of MNM, but for most materials this information is lacking. Today, only a few EU funded nanosafety projects have, as their goal, the provision of a conceptual foundation, based on an in-depth understanding of the relationship between material characteristics and the mode of action (toxicity) of MNMs. However, this is a fundamental issue for the safe design of NPs. For instance, the (potential) ability to control surface chemistry or particle size by innovation processes is only helpful if toxicology studies have been performed on synthetic nanoparticles demonstrating which surface chemistry and which sizes are problematic and which are not.

The study of the toxicological response as a function of the uptake dose of NPs is also important. The linkage of MNM physicochemical characteristics to mechanism-based biological outcomes at the biomolecular and cellular levels allows the establishment of structure–activity relationships (SARs), which provides a robust platform on which to base:

a. safety assessment,

b. develop computerized prediction-making models, and

c. establish read-across categories and

d. safer-by-design’ approaches for the synthesis of new materials.

To characterize correlations between nanomaterial properties and the key interactions or end points in humans and the environment, several tools are needed, including:

1) adequately characterized materials that have different properties,


2) appropriate assays for examining interactions or end points,

3) experimental data of sufficient breadth and depth for assessing correlations between nanomaterial properties and the behaviour of the materials.

To develop these tools, the research needs have to be prioritized:

The first and most pressing need is for more and better analysis and characterization tools. These are a key input which is required to support the rest of the actions. They are needed for scientists who wish to understand the mechanisms of the reactions that produce MNMs in order to develop better synthesis methods. And they will allow for improved and more complete toxicological studies of green nanomaterials, which are required for better and smarter regulation. Secondly, improved mechanistic understanding is a key part of the foundation for developing green nanomaterial design guidelines. Finally, new regulations, as well as outreach to regulators must be based on the analysis, understanding, and design concepts that are the result of the first three items.

3.1.1.1 Provide key analysis and characterization tools

Materials which are basic for developing the correlations between MNM properties and the key effects and that help for developing more general design rules are in the following general categories:

Reference material defined by ISO (2006) is a “material, sufficiently homogenous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process”. The focus is on its physicochemical properties and its use in metrology when certified by national or international agencies (for example, NIST gold nanoparticles and TiO2 nanoparticles) (Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013, National Academy of Sciences). Reference materials are developed for hypothesis-driven research (A Research Strategy for Environmental, Health, and Safety Aspects of Engineered Nanomaterials, 2012). They are often highly purified to reduce or eliminate the effects of impurities on responses (Oostingh et al. 2011). They may not attain the same level of scrutiny as standards, but they require a smaller investment of time and resources to develop. Sources of these materials include academic and government research laboratories, commercial suppliers, and international harmonization efforts (such as the Organisation for Economic Co-operation and Development and the International Alliance for NanoEHS Harmonization). However, because these materials typically represent specific, narrow structural types that are not easily manipulated to access a broad range of structural features, it is difficult to develop more general design rules from studies of these materials.

Benchmark materials are well-characterized physico-chemically and toxicologically, and can serve as positive or negative controls for comparing exposure-dose-response relationships of nanomaterials in toxicologic tests and in risk assessment (Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013, National Academy of Sciences). Benchmarks are developed to compare results among various tests or assays or among laboratories (A Research Strategy for Environmental, Health, and Safety Aspects of Engineered Nanomaterials, 2012). They are designed and characterized so that material characteristics can be linked to nano-biologic or nano-ecologic interactions or end points. Generally accepted positive or negative benchmark materials for toxicologic purposes have not yet been identified, but suggestions have been made in some individual studies (Aitken et al. 2009; Stone et al. 2010). Well-characterized benchmark MNMs should serve as references against which new and untested MNMs can be ranked as an initial step in hazard identification. Such information, with exposure data, can serve as a basis of risk assessment.

Libraries are collections of reference materials in which structural or compositional variables are systematically varied throughout a series of members of the library. For example, the nanoparticle core material and size might be kept constant while a surface coating varies in its external charge—positively, negatively, or not at all. Libraries allow the influence of nanomaterial structure and composition on biologic or ecologic effects to be explored so that quantitative structure-activity relationships can be determined. Libraries also facilitate exploration of hypotheses related to material-effect correlations. Ideally, the materials in libraries have sufficient range and granularity across the structural or compositional measures of interest. Given the importance of detailed characterization for establishing cause-effect correlations, characterization data on each sample lot need to be provided with each sample.

To move high-priority research toward green, additional effort and coordination are required to develop appropriate nanomaterial libraries (Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013, National Academy of Sciences).

Standards are samples that have been thoroughly tested to support laboratory comparisons or to calibrate and harmonize measurements conducted in different laboratories (A Research Strategy for Environmental, Health, and Safety Aspects of Engineered Nanomaterials, 2012). They typically are prepared and provided


for by standard-setting organizations or agencies (for example, the National Institute of Standards and Technology). The benefits of developing standard materials that meet the criteria for definition and characterization are clear; however, the time (years) and expense of developing such standards sometimes restrict their use in EHS studies.

Commercial Samples. It is important to study the biologic and ecologic effects of the commercial MNMs, as such materials (and their impurities) have the greatest potential compared to other types of materials to be released into the environment (Alvarez et al. 2009; Gottschalk and Nowack 2011; A Research Strategy for Environmental, Health, and Safety Aspects of Engineered Nanomaterials, 2012). However, there are limitations to the use of commercial materials in the development of predictive models. The materials are generally insufficiently characterized; when they are studied in isolation, the polydispersity and lot-to-lot variation in their properties make them unsuitable for developing data that can be used for prediction. For greater utility in prediction, material characterization that is specific to EHS research should be conducted in addition to that carried out by material researchers or producers (Bouwmeester et al. 2011).

For Providing Reengineered Well-Characterized Nanomaterials for Nanotechnology-Related EHS Research, the following needs are identifying:

• Development of libraries of uniform, well-characterized reference materials of varied size, shape, aspect ratio, surface charge, and surface functionality.

• Development of standard materials for calibrating various assays and measurement tools.

• Development of systematic sets of MNMs with properties varied in a stepwise manner that will allow assessment of the significance of each property for the toxicity of that MNM.

• Development of new synthetic methods and postsynthesis separation and purification methods for accessing the different types of materials, reducing polydispersity, and decreasing lot-to-lot variability and for efficiently removing undesirable impurities from nanomaterials without causing their decomposition or agglomeration.

Each type of materials must be characterized sufficiently for test results to be reproducible and for correlations between observed effects and material structure and composition to be established and ultimately used to predict effects of new materials on the basis of knowledge of their structure and composition.

3.1.1.2 Developing Protocols for Measuring and Reporting a Minimum Set of Material Properties for MNMs Used in Nanotechnology-Related EHS Research:

• To identify agreed-on minimum characterization principles to develop standardized descriptors for MNMs related to the key physical characteristics of the materials that can be used to describe materials for data-reporting and informatics and for cross-referencing nomenclatures.

Despite concerted efforts to establish a minimum set of standard properties to define MNMs, there is still lack of agreement in the research community as to what constitutes this minimum set of properties. However it is a consensus in demonstrating that there is overlap in their nanomaterial properties (MINChar Initiative 2009; Boverhof and David 2010).

• To identify criteria for grouping of nanomaterials as a basis for safer-by-design MNMs

The available concepts for the grouping of nanomaterials for human health risk assessment go beyond the determination of mere structure-activity relationships and are founded on different aspects of the nanomaterial life cycle (Arts, 2014). These include the MNM’s material properties and biophysical interactions, specific types of use and exposure, uptake and kinetics, and possible early and apical biological effects. None of the evaluated grouping concepts fully take into accounts all of these aspects. Some selected grouping concepts together the corresponding references are included in the “ProSafe Search Call” document described in the chapter 3.1.5 of the present Deliverable.

By 2020 there will be established norms for naming of MNMs and the properties that can be correlated with uptake and impact will have been identified and validated as predictors of toxicity for new materials. Based on these properties, strategies for nanomaterials grouping will have been established, on the basis of application of quantitative property-activity relationships (Savolainen et al 2013, Nanosafety in Europe 2015-2025: Towards Safe and Sustainable Nanomaterials and Nanotechnology Innovations). Looking for pragmatic approaches to deal with grouping (OECD, 2016a), the assessments should start from the study of the available data and the constraints they present, and then to consider tests that can help to fulfil the lacks and then the constraints.


• Determine best practices for characterizing groups of particle types (for example, by chemical composition or chemical-surface reactivity, for specific size ranges, for specific coating types or structures, and in relevant suspension media), including those to characterize reactive surface area, nanometer and sub nanometer surface features of MNMs, and adsorbed molecules and macromolecules on MNMs.

Most of the tools needed to accomplish that goal are available for pristine starting materials (Hassellöv et al. 2008). In addition to assessment of pristine material samples and dry powders, analytic methods should include characterization of MNMs in various reference suspension media that reflect real-world fluid suspension media and concentrations (for example, water, phosphate-buffered solution, lung fluid, and plasma) because MNM properties are determined in part by the dispersing fluid and MNM concentration (Oberdörster et al. 2005). Reactivity measurements are also needed and could include redox activity and reactive-oxygen species generation (EPA/600/R-14/244, 2014: Detection and Characterization of Engineered Nanomaterials in the Environment: Current State-of-the-Art and Future Directions Report, Annotated Bibliography, and Image Library, www.epa.gov/research). There is a lack of tools for characterizing the details of the surface chemistry of nanoparticles, including defects in surface layers, mixtures of bound molecules, and conformation of the adsorbed layer of organic macromolecules of high molecular weight. Tools and methods are needed to characterize the surface properties of MNMs better in situ or in vivo. Because the surface properties of MNMs will determine their interactions with environmental and biologic media, these properties will depend on the media in which they are dispersed so methods should be tailored to the exposure conditions. Many tools are available to characterize size, elemental composition, and structure, but fewer are capable of characterizing only the surfaces of MNMs. Surface curvature, roughness, crystal faces, and defects may all affect the physical, chemical, and toxicological properties of an MNM; it is not possible to characterize those features adequately with existing microscopic and spectroscopic techniques (for example, electron spectroscopy for chemical analysis, TEM, and FTIR). Surface functional groups—such as adsorbed or grafted surfactants, polymers, polyelectrolytes, proteins, and natural organic matter—can prevent or enhance agglomeration and deposition (Phenrat et al. 2008; Saleh et al. 2008; Jarvie et al. 2009), toxicity (Gao et al. 2005; Nel et al. 2009; Phenrat et al. 2009), and bioavailability (Kreuter 1991). Despite the influence of bound coatings on MNM behaviour, methods for readily measuring the distribution and, more important, the conformation of the bound species on the surface of MNMs are not widely available. Cryoelectron microscopy combined with computational methods can provide information on conformation of antibodies or other molecules, but these methods are time-consuming, and results can be influenced by sample-preparation methods. Methods for measuring those features in vivo, in vitro, or in situ do not exist and their development is necessary to begin to correlate the in situ properties of MNMs with their behaviour and effects.

• Develop standard reactivity measures and protocols for MNMs, including a standardized approach for measuring the sensitivity of methods to important variables (for example, pH, ionic strength, organic matter, and biomacromolecules).

Protocols and methods will need to be specific to a nanomaterial’s characteristics, including particle type, size, shape, coating type, and media type, because not all methods will be applicable to all types of MNMs. There is a need for widely accepted protocols for sample preparation and measurements, including methods for dispersing nanoparticles in media, protocols for reproducibly preparing samples for analysis and investigation, and approaches to using multiple instruments to cross-check and confirm results from techniques that may provide only partial answers (examples include the published assay cascade protocols NIST/NCL 2010). The sensitivity of the protocols to the array of variables that may affect their outcome (for example, solution pH and energy input for creating dispersion) should be determined and reported as part of the protocols.

3.1.1.3 Detection and Characterization of Nanomaterials in Complex Biologic and Environmental Samples

To promote SbD in this field, chemical and physical information on MNMs in environmental and biologic matrices is needed.

Many existing analytic techniques from material science and other disciplines are applicable to MNMs, but their use in measuring and characterizing low concentrations and heterogeneous matrices will require additional development or in some cases, development of completely new approaches (A Research Strategy for Environmental, Health, and Safety Aspects of Engineered Nanomaterials, 2012; EPA/600/R-14/244, 2014, Detection and Characterization of Engineered Nanomaterials in the Environment: Current State-of-the-Art and Future Directions Report, Annotated Bibliography, and Image Library, www.epa.gov/research). There are few analytic tools that can be used to quantify and characterize MNMs in situ (for example, in air, soil, or sediment samples), in vitro (for example, in cells or tissues), or in vivo at the low concentrations expected for most nanomaterials (in the low parts-per-billion to low parts-per-trillion range) (Hassellöv et al. 2008;


Gottschalk et al. 2009; Tiede et al. 2009). Some examples include radiolabeled materials (Hong et al. 2009; Gibson et al. 2011; Peterson et al. 2008); fluorescence (Schierz et al. 2010); mass spectrometry (MS) and single particle MS techniques (von der Kammer et al. 2012); spatially resolved X-ray analyses (von der Kammer 2012); and differential mobility analysis (Morawska et al. 2009), a well-developed technique used to quantify the number and size distribution of nanoparticles in air. Detection in vivo or in situ can be difficult because of the low concentrations of materials released into an organism or the environment. Even if the material has not been transformed, detection is difficult; if it has been transformed, detection is even more difficult. Strategies and tools for detecting and tracking materials are needed. These strategies should include combinations of techniques to detect and characterize MNMs in complex matrices and to differentiate between MNMs and naturally occurring nanomaterials (EPA/600/R-14/244, 2014, Detection and Characterization of Engineered Nanomaterials in the Environment: Current State-of-the-Art and Future Directions Report, Annotated Bibliography, and Image Library, www.epa.gov/research).

For Detection and Characterization of Nanomaterials in Complex Biologic and Environmental Samples the following research needs have been identified:

• Develop model MNMs that can be tracked without introduction of experimental artifacts in exposure and toxicity studies. Develop analytic tools and processes that can detect MNMs at low (relevant) concentrations in situ or in vivo, followed by tools to track and characterize MNM properties (for example, reactivity, reactive surface area, nanometer and subnanometer surface features, aggregation-agglomeration, and adsorption of organic macromolecules) in situ or in vivo. Develop tools and processes to assess the rate and degree of transformation of MNMs in vivo or in situ, especially alteration of surface properties of MNMs due to adsorption of proteins and lipids (corona formation) and natural organic matter.

3.1.1.4 Development of New Protocols or Modification of Existing Protocols for Toxicity Testing and Determination of Population and Ecosystem Effects

A focused, coordinated research effort is needed to identify and validate existing or newly developed toxicity-testing protocols and best practices, such as dosimetry, for an agreed-on set of toxicity end points for MNMs (A Research Strategy for Environmental, Health, and Safety Aspects of Engineered Nanomaterials, 2012). The protocols would include rigorous physicochemical characterization of particle types, use of relevant cell types or cell systems (for example, air-liquid interface) to simulate relevant in vivo exposures, relevant dose-response protocols, relevant time-course protocols, and assessments of biomarkers, such as inflammatory end points, that have relevance to in vivo pathway models (for example, sustained inflammation). For ecologic-health research, a set of sensitive species will need to be identified in the risk-characterization phase. The protocols for assessing ecotoxicity include those for assessing human toxicity but should also include protocols for predicting sensitive species and effects on communities and ecosystems if they are to be useful for risk assessment (Ankley et al. 2010). Those include effects on interactions among species, species community assemblages, biodiversity, and ecosystem function. There is no suite of standard tests for assessing community and ecosystem effects of chronic exposure to MNMs. That limitation is not peculiar to MNMs and presents a serious challenge to the modelling of ecologic effects.

Research Needs for Development of New Protocols or Modification of Existing Protocols for Toxicity Testing:

• Develop new standard toxicity-testing protocols or modify existing protocols for MNMs to include relevant cell types and organisms, appropriate dosimetrics, and appropriate toxicity end points (for example, chronic-toxicity end points) and validate those protocols.

• Identify and validate toxicity-pathway models and mechanisms to correlate in vitro end points with in vivo responses.

• Improve the interpretability of genomic tools by determining how gene expression and protein expression are related to MNM toxicity and mechanisms.

3.1.1.5 Develop safe MNMs “by design” using principles similar to those of “green chemistry”

An emerging topic related to the developing of safe MNMs “by design” is to create and apply design rules for “greener nanomaterials” with defined composition, structure, and purity (Hutchinson 2008) as well as for developing efficient and reproducible synthetic strategies using principles similar to those of “green chemistry”. Incorporating the 12 well-known principles of green chemistry (See Table 1) in the design,

http://www.epa.gov/research


production, and use of MNMs, “Green Nanoscience” can help to ensure that nanomaterials are designed to minimize risk whatever their application (Nel, 2010).

MNMs seem ideally suited to such approaches, given the ability to exert precise control over composition and structure. Such atomic-scale manipulation is the defining essence of nanotechnology and is what makes it possible to impart such materials with specific properties related to function and performance.

Green nanomaterials/processes can substitute for dangerous materials and processes shown to pose more risk. Nanotechnology-inspired production is likely to also lead to more efficient use of materials and lower energy needs and solve environmental problems. In principle, the same ability should extend to identifying and exerting control over the factors determining a nanomaterial’s potential for exposure, such as persistence, mobility, or bioavailability. Nanomaterial development, informed by an evolving risk assessment, presents the opportunity to identify and reduce, at the design stage, the inherent potential for exposure to and the hazards of nanomaterials (A Research Strategy for Environmental, Health, and Safety Aspects of Engineered Nanomaterials, 2012).

Table 1: Applying green chemistry principles to the practice of green nanoscience (adapted from ACS, 2011; Nel, 2010; Anastas, 1998)

Green Chemistry Principles Designing Greener MNMs

and MNM Production

Methods

Practicing Green Nanoscience

P1. Prevention

It is better to prevent waste than to treat or clean up waste after it has been created.

P2. Atom Economy

Synthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product

P3. Less Hazardous Chemical Syntheses

Wherever practicable, synthetic methods should be designed to use and generate substances that possess little or no toxicity to human health and the environment.

P4. Designing Safer Chemicals

Chemical products should be designed to effect their desired function while minimizing their toxicity.

P5. Safer Solvents and Auxiliaries

The use of auxiliary substances (e.g., solvents, separation agents, etc.) should be made unnecessary wherever possible and innocuous when used.

P6. Design for Energy Efficiency

Energy requirements of chemical processes should be recognized for their environmental and economic impacts and should be minimized. If possible, synthetic methods should be conducted at ambient temperature and pressure

P.7 Use of Renewable Feedstocks

A raw material or feedstock should be renewable rather than depleting whenever technically and economically practicable.

P.8 Reduce Derivatives

Unnecessary derivatization (use of blocking groups, protection/ deprotection, temporary modification of physical/chemical processes) should be minimized or avoided if possible, because such steps require additional reagents and can generate waste.

P.9 Catalysis

Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.

P.10 Design for Degradation

Design of safer MNMs

(P4, P12)

Determining the biological impacts of nanoparticle size, surface area, surface functionality; utilize this knowledge to design effective safer materials that possess desired physical properties; avoid incorporation of toxic elements in nanoparticle compositions.

Design for reduced

environmental impact

(P7, P10)

Study MNM degradation and fate in the environment; design material to degrade to harmless subunits of products. An important approach involves avoiding the use of hazardous elements in nanoparticle formulation; the use of hazard-less, bio-based nanoparticle feedstocks may be a key.

Design for waste reduction

(P1, P5, P8)

Eliminate solvent-intensive purification by utilizing selective nanosyntheses—resulting in great purity and nanodisparity; develop new purification methods (e.g., nanofiltration) that minimize solvent use, utilize bottom-up approaches to enhance material efficiency and eliminate steps.

Design for process safety

(P3, P5, P7, P12)

Design and develop advanced syntheses that utilize more benign reagents and solvents than used in the “discovery” preparations; utilize more benign feedstocks, derived from renewable sources, if possible; identify replacements for highly toxic and pyrophoric reagents.

Design for material efficiency

(P2, P5, P9, P11)

Develop new, compact synthetic strategies; optimize incorporation raw material in products through bottom-up approaches; use alternative reaction media and catalysis to enhance reaction selectivity; develop real-time monitoring to guide process


Chemical products should be designed so that at the end of their function they break down into innocuous degradation products and do not persist in the environment.

P.11 Real-time analysis for Pollution Prevention

Analytical methodologies need to be further developed to allow for real-time, in-process monitoring and control prior to the formation of hazardous substances.

P.12 Inherently Safer Chemistry for Accident Prevention

Substances and the form of a substance used in a chemical process should be chosen to minimize the potential for chemical accidents, including releases, explosions, and fires.

control in complex nanoparticle syntheses.

Design for energy efficiency

(P6, P9, P11)

Pursue efficient synthetic pathways that can be carried out at ambient temperature rather than elevated temperatures; utilize noncovalent and bottom-up assembly method near ambient temperature; utilize real-time monitoring to optimize reaction chemistry and minimize energy costs.

3.1.2 Exposure, Transformation and the Life Cycle

Exposure of humans and the environment is a result of many sequential or concurrent processes. These facts have emerged from research related to MNM production, MNM characterization, aging of products containing MNM, human and environmental induced release of MNM into the environment, transport, transformation, degradation and possibly accumulation of MNM in the environment or along the food chain.

The general processes and areas of possible release and exposure are defined in the following (Savolainen et al 2013, Nanosafety in Europe 2015-2025; OECD, 2016b):

1. Production

Possible release during production may occur through leaks into water and air in closed systems or open production processes. These have been studied in several European and national studies such as NANOSH, CarboSafe, and nanoGEM.

2. Handling and use

Handling and use covers several process-related stages e.g. handling of powders, diffuse emission from production plants, mechanical treatment of nanomaterials.

3. Aging

Aging encompasses all processes taking place in the environment such as selective degradation, wash-out, increased brittleness of the material.

4. End of Life (EoL)

End of Life activities refer to activities related to

i. Re-use or recycling, such as disassembling, and mechanical or thermal processes like crushing, melting, torch cutting;

ii. Waste treatment, e.g. incineration and

iii. Disposal. In particular, during high energy processes, the release of nano-objects may not be excluded.

For traditional chemicals and products has been created a framework by some major harmonized or regulated activities, e.g. OECD test guidelines for environmental fate studies, CEN standards such as dustiness tests for powdery materials and the regulation of chemicals Registration, Evaluation, Authorisation and Restriction of Chemical substances (REACH).

The current view is that the general existing regulatory frameworks are applicable but have to be adapted and extended for some MNM specific issues. It has been emphasized that MNMs are the subject of some special properties, especially those related to the transformation of materials during their life-cycle (LCA) or after their release into the environmental compartments which are known to alter their relevant substance characteristics e.g. size, shape, charge, state of agglomeration etc.

Research and development activities aimed at understanding processes relating to release of MNM. This research and development activities are likely to increase in the near future since this will allow a) detailed studies of processes, b) standardised testing for certain possibly relevant release mechanisms, c) international harmonisation, d) derivation of quantitative information of possible release rates, and e) good characterisation of the physico-chemical characteristics of the released material.


Based on recent developments in understanding of the behavior of MNM in different matrices and environmental compartments, as well as in mammalian and environmental organisms, a special emphasis have to be done to the foreseeable development of future types of MNM. Novel, emerging 2

nd generation

active nanomaterials, self-assembling 3rd

generation nanomaterials and nanosystems, and the 4th generation

systems of nanosystems and nanorobotics have to be considered (Savolainen et al 2013, Nanosafety in Europe 2015-2025). One can be noted that most of the current safety research deals with the 1

st generation

MNM and the data on the emerging materials are quickly required. As a matter of fact, these considerations could be extended to all thematic areas identified in this document.

3.1.2.1 Research priorities related to MNM release, transformation, transport and exposure

Release

The major obstacle in studying MNM release, transformation and exposure is the identification of the particles themselves. Some strategies and techniques have been developed and tested in workplaces (reviewed in Kuhlbusch et al., 2011). However, severe limitations exist even for those used for research purposes and the existing techniques cannot be employed in routine workplace measurements. In the other extreme case, the environment, it becomes very difficult to develop an appropriate and feasible analytical method as nanomaterials may undergo modifications e.g. aging processes. Another limitation is that currently there are very few measurement techniques that simulate aspiration efficiency and the deposition in the trachea-bronchial and alveolar regions resulting in mismatches between the concentrations measured, the concentrations inhaled, and the estimate of the deposited dose. In order to obtain health related exposure information, modelling techniques have to be applied to the data. This lack of health related exposure data, as well as some other factors, complicates the establishment of occupational exposure limits. Measurement techniques and strategies are crucial in studying nanomaterial properties, behaviour, transport, exposure, uptake and fate. A few established techniques are currently available for these studies and have been summarised in the literature (Kuhlbusch et al. (2008), Tiede et al. (2008), Stone et al. (2010). In summary, the main techniques currently employed are either microscopic methods for information on particle morphology, state of aggregation and chemical composition, or methods discriminating particles according to size in relevant media. The latter methods sometimes allow subsequent separate analysis for chemical composition.

Exposure in workplaces

The next steps to pursue with regard to workplace exposure will be the development and testing of personal monitoring devices delivering reliable results that can be used in health studies and/or for risk management. Focus should be placed on personal real time instruments that simulate uptake, e.g. deposition in the different areas of the respiratory tract. The development of realistic exposure scenarios is needed to allow a comparative assessment of different tasks and processes. They should be based on an extensive data set on workplace exposure, generated in a harmonized way as much as possible (Brouwer et al., 2012; OECD 2012). The data included should be accompanied by auxiliary contextual information that is required to interpret the measurement results for risk assessment and mediation purposes. The exposure scenarios are also needed since they can be useful in to derive information about uptake for combined assessments of hazard and exposure potential.

Exposure via consumer products

During consumer usage nanomaterials are subjected to mechanical, thermal and environmental stress situations. Studies based on the characterisation of airborne particles release due to individual processes can roughly be classified by the investigated nanomaterial used for coating and according to the nanomaterials used in composites. Coatings could be considered to be a thin layer of composite material, as the engineered nanoparticles are intentionally embedded in a matrix material. However, in exposure studies, composites and coating cannot be compared and have to be analysed in different ways. The relatively long duration of the current aerosol measurement has restricted the intensity of abrasion. This means that with higher abrasion intensity the coating could become worn off before the measurement finishes. Therefore only a limited simulation of exposure is possible. However, if one wishes to assess the real potential impact of nanomaterial on the environment and the human health, it will be necessary to characterize, with feasible techniques, the properties of the particles once they have been released into the environment.

Transport and Transformation

There are very rapid developments occurring in the fields of nanomaterial production and current technologies are not sufficiently well-developed to provide rapid assessments in a coherent manner. At present, some research groups are undertaking comprehensive research activities to develop some predictive models on how the material will interact with its surroundings, and how that may influence its subsequent transport, accumulation and reactivity; one must anticipate that there will be a huge increase in


the knowledge base relating to MNM transport and transformation - including predictive modelling - occurring within the next 10 years.

By the integration of safe-by-design to ensure the safe production, handling and use of MNM, the main goal to be achieved will be the development and implementation of integrated release to exposure models for nanomaterials in workplaces, consumer applications and the environment. The analysis made in the document Nanosafety in Europe 2015-2025: Towards Safe and Sustainable Nanomaterials and Nanotechnology Innovations (Savolainen et al 2013, Nanosafety in Europe 2015-2025) anticipates that these models (Figure 1) will be based on the following ‘building blocks’:

- Mechanistic understanding of processes determining the release of MNM.

• studies on the behaviour of MNM when processed, when worked with, when being used taking into consideration possible nanomaterial release, aerolised or the presence of MNM in liquids

• comprehensive release and emission inventories covering production and all subsequent processing, usage steps and recycling

- Understanding the transformation and transport of MNM.

• studies on environmental mobility and transformations during transport and storage, including environmental persistence of the corresponding nanomaterial

• effects of ageing on nanoparticles, including changes in their shape, surface morphology and chemistry induced by environmental factors such as weathering, electromagnetic fields, mechanical stress and chemical reactions

- Understanding workplace, consumer and environmental exposure.

• efficient exposure measurement approaches also which can be applied in epidemiological studies

• harmonized inventories, which can be utilized in the construction of exposure models

• development of personal devices to estimate deposition in the respiratory tract

• evaluation of information relevant to describe exposure and inclusion of these factors into risk assessment and mediation strategies as well as into exposure modelling efforts

Figure 1: Exposure models for release-transport-fate of nanomaterials (from Savolainen et al 2013, Nanosafety in Europe 2015-2025: Towards Safe and Sustainable Nanomaterials and Nanotechnology

Innovations)

A key outcome of the integration of all these data and information is development of a suite of models (Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013,


National Academy of Sciences). The models allow the application of new methods and instruments that reflect thinking regarding hypothesis testing and assessment. Such models may be used to predict physical characteristics of MNMs, outcomes of toxicity testing, and exposure potential in complex systems. In their initial forms, the models represent working assumptions that are refined with additional data. As confidence in a model increases, validation studies that involve comparisons of model outputs with results from experimental systems that use benchmark or unknown MNMs can be conducted. The process of data integration and model formulation and validation informs risk assessment. Given adequate knowledge, refined and validated models allow prediction of potential hazards associated with exposure to MNMs throughout their life cycle and value chain.

Mechanistic models should provide the greatest long-term benefit to the MHS nanotechnology research community with regard to anticipating risks. However identifying the critical elements of nanomaterial-environment and nanomaterial-biota interactions is a significant undertaking and will take time to develop. There is a near term need to predict behaviors of nanomaterials in relevant environmental and biologic matrices. Empirical predictive models that are parameterized appropriately (for example, partition coefficients between nanomaterials and bacteria in wastewater treatment plants or approximate dissolution rates and half times in specific media) may be sufficient to approximate behaviors of MNMs in selected matrices (RNC/RIP-oN2, 2011; Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013, National Academy of Sciences). The forms of these predictive models, their parameters, and appropriate assays to measure the values for these parameters in selected environmental and biologic media are still needed (Hou et al. 2013; Westerhoff and Nowack 2013).

There are two principal challenges in quantifying and characterizing the average properties of MNMs in complex biologic and environmental matrices: the low concentrations of the MNMs in the matrices and the unknown history of the MNMs before analysis.

A critical research need that cuts across exposure and effects is the characterization of the properties of adsorbed macromolecules on MNMs, including the structure of the macromolecule and the outer surface layers of the MNMs (Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013, National Academy of Sciences). That information is needed to describe properties and changes of MNMs in relevant biologic and environmental media. It is also a prerequisite to development of appropriate models for predicting MNM behavior in complex systems (such as biouptake models) and effects. It is an extremely challenging task, especially in complex media, and will probably require new instrumentation with spatial resolution adequate for focusing on single particles and initial development in well-characterized systems before application in more complex media.

Another important component of this research is the ability to determine critical release points along the value chain and to identify exposed populations (Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013, National Academy of Sciences; Savolainen et al 2013, Nanosafety in Europe 2015-2025). Therefore, characterization in relevant complex matrices requires methods for characterizing MNMs and transformations in the matrix in which the MNMs are used. The matrix may affect the MNM properties that are used to measure pristine MNMs (such as fluorescence or absorption at a specific wavelength); therefore, development of new methods or validation of existing methods is needed to detect and characterize MNMs released from their matrices (Research progress on environmental, health, and safety aspects of engineered nanomaterials, 2013).

It is important that the measured properties and characteristics of transformed MNMs be captured in the knowledge commons. That requires ontology for describing such properties as the adsorbed macromolecular layer. Placing such data in the knowledge commons will allow the community to share them and to develop and update models for describing the behavior of the MNMs in complex environments.

Focusing on MNM exposure control rather on suppressing MNM intrinsic reactivity that contributes to toxicity might be a useful compromise for the nanosafety strategy. MNM coatings, and surface and functionality change can produce diverse properties that enhance or diminish certain types of exposure, depending on the application, chemistry, design and MNM properties. Identifying desired and undesired specific MNM functions and possible risks, and applying SbD principles to realize these properties while mitigating risks, represent attractive objectives for this strategy.

3.1.2.2 Research Needs for development of Protocols for Exposure Assessment

• Develop and validate standard protocols for measuring and reporting attachment of MNMs to biologic and environmental surfaces.

• Establish protocols that can be applied to pristine MNMs to identify and classify their stability in the environment. Develop protocols to provide “weathered,” transformed materials for study (for example, in transport and toxicity studies).


• Develop and validate protocols to assess bioavailability of MNMs to specific indicator organisms identified in a site-specific risk-assessment model.

• Develop standard protocols and analytic methods that measure number, surface area, and mass concentration to assess human inhalation exposures to MNMs.

3.1.3 Hazard identification and characterisation

Hazard assessment of MNMs has made good progress during recent years, but knowledge is still lacking in many areas including modes of action and mechanisms leading to toxicity, identification of susceptible populations and vulnerable conditions, and aspects of biokinetics and its impact on toxicity.

The EU large FP7 projects, NANOSOLUTIONS and NANOMILE are both dedicated to systems biology approaches to understanding the interactions of engineered nanomaterials with living organisms and the environment. A considerable amount of data on the potential hazard of MNMs has accrued while information on exposure to MNMs and on safety of nano-enabled products throughout their life cycle is still lacking. The problem with the hazard data obtained to date is that the results do not allow for any general conclusions. This is, in part, due to the lack of standardized methods and reference materials for toxicity assessment (Krug, 2011). In an attempt to resolve some of these issues, the European Commission has recently funded two large FP7 projects (MARINA and NanoValid) devoted to the development and validation of reference methods and materials for life cycle analysis (LCA), exposure, hazard identification, and risk assessment of MNMs.

It will be impossible to assess the risk of all MNM with all modifications and in all use scenarios using current case-by-case approaches. According to the new approaches for hazard assessment (Savolainen et al 2013, Nanosafety in Europe 2015-2025), it is important to move away from purely descriptive toxicology of MNM to a predictive toxicology/nanosafety assessment, based on a thorough understanding of the dynamics of the biological behaviour of MNM derived from an understanding of their material characteristics. In order to identify the most relevant hazard-associated features, as well as the most critical molecular signatures that predict the safety of the MNMs, biology and bioinformatics approaches will need to be utilized. These novel approaches are being actively developed and some have been successfully applied in bioinformatics. Mechanistic knowledge should be included in technology development, to help in the safe design of new MNMs in a bottom-up approach, and will feed directly into the development of a rational testing approach.

3.1.3.1 The required research priorities on hazard assessment:

Hazard assessment enabling grouping of MNMs

1) Scientifically established grouping criteria

2) Understanding the association between material characteristics and the subsequent cellular events

3) Utilizing systems biology approaches in the prediction of MNM safety

Biokinetics including translocation and clearance

1) Mechanistic knowledge resulting in groups of MNMs with marked similarities

2) Bioaccumulative properties of MNMs and biokinetics

Susceptible populations and vulnerable conditions

1) Systematic research of MNM effects on susceptible populations

2) Systematic research of the effects of MNM on individuals with vulnerable conditions

Environment

1) Fate of MNMs in complex media and life cycle

2) Improved prediction of the (bio)degradation rate of organic nanomaterials

3) Development of standardized test methods for water environments and soil

By 2020, the ultimate goal will be to develop a computational tool, i.e. MNM SAFETY CLASSIFIER (Savolainen et al 2013, Nanosafety in Europe 2015-2025). This tool will predict “MNM Safety” based on the evaluation of minimal but sufficient amounts of information to provide a robust manufactured nanomaterials safety classification. This novel prediction tool when available it will promote the utilization of safety-by-design principle, and also be capable of improving the speed of hazard identification and risk assessment.


This tool will be tested and validated in close collaboration with industry; it possesses enormous potential to promote marketing innovations based on nanotechnologies.

Although, MNM have been shown to undergo interactions with cellular systems in vitro (Lynch, 2008, 2015), this approach has only been partly successful to date. This will be a high research priority for the next years and it is anticipated that the correlation between properties of the materials and cellular functions will have been clarified by 2020. It should be possible to assign certain biological effects to specific material properties and group MNM based on these material characteristics.

As a complement, groupings may be based on similar biopersistence and biokinetic properties (See also the Chapter 3.1.5). Kinetics may also give a measure for grouping/summing nanomaterials. The same tissue distribution pattern may be one criterion for grouping of different nanomaterials. If a nanomaterial exhibits a different tissue distribution, this may result in different effects. Grouping may also be based on similar or common biological effects, including early effects and MNM-cell structure-interaction. Grouping based on early biological effects is tightly linked to the Adverse Outcome Pathway (AOP) concept of the OECD and the tox21c initiative in the USA (http://epa.gov/ncct/Tox21/).

It will be necessary to combine exposure, biokinetics and hazard data for the purposes of both risk assessment and grouping. The AOP approach will provide guidance regarding the integration of material properties, exposure, biokinetics and hazard data. This is a priority for future research on the safety of MNMs.

The key factors in developing knowledge and understanding the toxicity of MNMs are:

1. identification of the main modes of actions of toxicity for MNMs

2. understanding the transformation of MNMs during their life cycle and how this may influence their hazard potential

3. identification of the key physicochemical determinants that modulate MNM interactions and toxicity in biological systems

A concern-driven guidance for toxicity testing of nanomaterials will be developed. This will enable focused research on nanomaterials that may be of particular concern based on expected exposure routes, material-properties as well as hazard and biokinetic data (Savolainen et al, 2013, Nanosafety in Europe 2015-2025). This approach is in line with the 3R principle (Replacement, Reduction, Refinement), which focuses on the replacement of animal testing methods with alternatives that do not use animals, advocating that living animals are only to be used for crucial and focussed studies.

Based on the above information, integrated testing strategies will be developed for different types of MNM, starting by non-testing and existing toxicological data, proceeding with tests using a cellular systems, and further proceeding through cellular systems to in vivo, long-term testing approaches when necessary.

These strategies will be based on a thorough understanding of the matrix- dependent MNM biokinetics enabled by continuous sampling, analyses and characterization paradigm. The strategies will be based on validated methods with proven predictive power and they will be designed both to evaluate human health and environmental safety/risk assessment.

In 2020, guidance will be developed to determine how best nanomaterials can be grouped and how these groupings should be constructed. In addition to avoiding extensive hazard testing of nanomaterials, this will also provide insights when information on exposure and hazard for nanomaterials can be used for risk assessment purposes.

3.1.3.2 Safe design of new MNM in a bottom-up approach

The development and implementation of Safety by Design (SbD) strategies with its “primary” prevention value of risk management, represents one of the biggest challenge of nanotechnology that should guarantee its sustainable development. Surface engineering has opened the doors to the development of a second, third, and fourth generation of MNM. Self-assembling bottom-up techniques have been widely developed at industrial scale, to create, manipulate and integrate nanophases into more complex nanomaterials with new or improved technological features (Savolainen et al, 2013, Nanosafety in Europe 2015-2025).

Materials scientists have the chance to address such knowledge to the control of hazard specific properties by preserving nanoscale reactivity, towards the integration of safe-by-design approaches into the development stages of new nanomaterials and their applications.

The conceptual framework to identify key features that drive the design of safe nanomaterials (Roco et al., 2012) is reported in Figure 2. It includes a first level of data generation/gathering. The understanding of the mechanism that governs both the adverse effects of NMs on biological system and the emission/exposure

http://epa.gov/ncct/Tox21/


potential in terms of fate from nano-aereosolization to bio-uptake is, in fact, fundamental to implement a rational approach for the safe design of nanomaterials. At a second level the observed evidences on nano-bio interaction mechanism should be supported by predicting models. Finally, at a third level the safer by design NMs should be implemented within real industrial processing lines (Leszczynski, 2012), allowing cost-

benefit analysis and the promotion of primary prevention based risk control measurements.

Figure 2: Components of “Nano design” framework (From Savolainen et al, 2013, Nanosafety in Europe 2015-2025)

A full understanding of the key descriptors for characterising MNM along with validated methods to identify and quantify MNM in complex matrices is vital in order to identify crucial parameters relevant for risk assessment (Lynch, 2014) and is also important for the measurement of the relevant MNM properties that correlate exposure with biological impacts. This will require agreed reference states for NMs characterization, libraries of reference materials, and a framework for understanding later generation nanomaterials.

The required research priorities to achieve these objectives are to:

1. Develop systematic sets of MNMs with properties varied in a stepwise manner that will allow assessment of the significance of each property for toxicity.

2. Describe “reference” states and agreed media compositions to enable identification of significant biomarkers and enable a move towards a predictive toxicity assessment.

3. Understand the longer term fate of particles following their interaction with living systems.

Nanomaterial identification and classification approaches to determine the key descriptors that can be used to reveal correlations associated with impacts. The inter-relationship between the nanomaterials’ identification and classification is a cross-cutting topic and it feeds into the other cross-cutting nanosafety research themes.

3.1.4 Risk prediction and management tools

Traditional risk assessment frameworks (NRC 2009) follow the four-step paradigm: 1) hazard identification; 2) hazard assessment; 3) exposure assessment; and 4) risk assessment.

In the context of the development of an EHS risk-research strategy for MNMs, a high emphasis has been given to issues related to risk assessment and management of MNM and the general considerations of NRC (NRC, 2009) are translated into specific considerations related to MNMs. The conventional RA framework may fail to estimate the risks from MNMs due to overwhelming methodological limitations and epistemic uncertainties (Savolainen et al 2013, Nanosafety in Europe 2015-2025). The present paucity of quantitative nano-EHS data will lead to ambiguous, qualitative risk estimations, based on expert judgments that may fail to be reflected in appropriate and timely regulatory decisions. Currently, quantitative risk assessment has been translated into operational requirements by using risk ratios, i.e. observed dose of exposure divided by a reference dose or exposure limit. To date, for nanomaterials, only a few reference doses or health-based limit values have been proposed (Schulte et al., 2010; NIOSH, 2010; 2013).

Alternative risk analysis tools and frameworks as well as modifications to existing risk assessment approaches have been proposed for NMs. The high priority of novel risk assessment paradigms and the need to obtain human data have been considered in this context. Informatics (databases) and epidemiological or health studies can be considered as enabling ‘tools’, supporting the processes of Risk


assessment (RA) and risk management (RM) (Savolainen et al 2013, Nanosafety in Europe 2015-2025; 2013). For example database studies which enable predictive hazard modeling, e.g (Q)SAR, PBPK or exposure modeling (RNC/RIP-oN3, 2011) (See also the point 3.1.4.6). Epidemiological studies can provide data supporting both hazard and risk assessment. Clusters of a given hazard in an epidemiological study can serve hazard identification or hypothesis formulation, and odds ratios or risk ratios found in epidemiological studies may serve the evaluation of the relevance of toxicological findings to human health. Furthermore, epidemiological studies may serve hazard and risk assessment by identifying unexpected potential biological adverse effects, or finding vulnerable (sub) populations.

If one wishes to conduct a realistic risk assessment of nanoparticles, it is extremely important to identify the physico-chemical properties that predict different toxicological outcomes. MNM are complex groups of materials with diverse physicochemical properties, which not only can affect their biological activities but also their underlying mechanisms of action. For the same reason, it is as important to identify the behavior of MNM interacting with biological systems. They behave totally different than larger (micron) particles.

The conventional Risk Assessment framework should be supplemented with non-conventional tools (Savolainen et al 2013, Nanosafety in Europe 2015-2025) like Weight of Evidence (WoE) and Multi Criteria Decision Analysis (MCDA). In addition, a holistic (and if possible a probabilistic) approach should be explored for human health risk assessment (HHRA) and freshwater/terrestrial ecotoxicological risk assessment (FTERA) by bridging the gaps between the current state of the art and the conventional quantitative risk assessment approaches. This approach should involve a material life cycle perspective to enable comparison and aggregation of the health impact over the material’s life cycle stages. The applicability of a general approach to estimate the human effect factors for both linear and non-linear dose response relations for different health endpoints, or alternative developed indictors for hazard values, resulting in compatible output values of HHRA (different human health metrics) will need to be explored.

3.1.4.1 Fundamental Processes important for the evaluation of risks posed by MNMs

Research in this category occurs both in the laboratory world and the real world and involves experimental approaches to understand the physical, chemical, and biologic processes that affect exposure and hazard. Hypothesized MNM properties are scrutinized in well-defined laboratory experiments and in observations of MNM behavior in complex systems, from in vivo experiments to models of ecosystem interactions. The research is informed by development of methods and instrumentation that are needed for understanding MNM transformations, distribution, and effects.

Continued efforts to elucidate mechanisms of MNM interactions with organisms and ecosystems are critical for achieving the long-term goal of predicting MNM effects. The ability to make such predictions will allow evaluation of risks posed by MNMs at the design stage, in model predictions, and in validated screening assays. Continued progress in understanding mechanisms of MNM behavior will require advances in instrument development and an improved data-integration infrastructure.

3.1.4.2 Challenges of Defining Potential EHS Risks of MNMs arising from the diverse properties of NMs (A Research Strategy for EHS Aspects, 2012; Nanosafety in Europe 2015-2025; 2013)

• It is difficult to specify the composition of MNMs, because of the variety of material types and variation within types. Countless assemblages of atoms and structures and a plethora of inorganic and organic macromolecular coatings affect their surface chemistry and therefore their behavior in the environment and their potential for biologic impact.

• Nanoscale structures include materials (e.g. particles, fibers, or sheets) and macromolecules (e.g. proteins or DNA). Many NMs are particles or designed structures, not molecules. The heterogeneity of the materials profoundly affects efforts to detect or to measure the MNMs or to assess their potential to cause harm. Large biomolecules that are labeled as MNMs may be detected with high specificity. Spectroscopic approaches may provide certifiable identification for some large molecular MNMs. Such approaches will frequently fail with the more complex structures. These materials may have highly uniform properties, while many of the more complex structures will lead to a range of possible interactions. The magnitude of forces and the resulting bond strengths induced by interactions with MNMs may be different from those for molecules. In addition to forces that show size dependence (e.g. van der Waals interactions), the presence of a separate phase introduces surface energies and boundary effects (e.g. discontinuity of crystal lattices at a particle surface and resultant surface charge) that are not present with molecules in solution. Also, the relative impacts of kinetic and thermodynamic factors in controlling the environmental behavior of NPs may be expected to differ from conventional chemical species for which there has been success in predicting phenomena, such as bioaccumulation or transport (e.g. use of structure-function relationships to calculate fugacity).


• Like many “conventional” contaminants, chemical transformations of the MNMs and their coatings will occur in the environment and in organisms, and such transformations are not well characterized or readily predictable.

• The surface properties of NMs are defined in part by the media in which they are dispersed; surface water, lung fluid, salt water, and air may affect these properties differently. Because the NMs behavior may be controlled largely by surface properties, general predictions about environmental behavior and effects cannot be readily made. The lack of a clear and stable material identity makes it difficult to group materials or classes of materials that may behave similarly with respect to fate, transport, toxicity, and risk. Moreover, because most NMs can be thought of not only as chemical entities but as having separate phases, there is considerable doubt regarding the appropriateness of applying or interpreting some of the conventional parameters used in exposure assessment, such as octanol-water partition coefficients and volatility.

• Risk assessment (RA) for MNM with respect to the life cycle of these materials is challenging for several reasons. After their production, NMs may be transformed, e.g. by agglomeration or de-agglomeration, or by loss, change or development of coatings, which may have impact on the uptake and biological effect after uptake. The information about the level of exposure to MNM is fragmented, most of the studies are rather explorative and the results cannot realistically be used for an estimation of the exposed dose. Predictive exposure models are mass-based and this parameter might be less appropriate in cases where one wishes to evaluate the risks associated with a NM. To estimate the impact of a health risk, one needs to be aware of the number of people that could be exposed. This number relates very much to the penetration of NM-based products in the value chain and for the time being, accurate information is lacking (Pronk et al., 2011). Clearly, most of the parameters of the risk assessment process involve uncertainties and these results in high uncertainties when one tries to estimate the overall outcome of this process. The same issues also apply to environmental risks, where during and after release, transformation reactions are even more important in changing the properties of the pristine nanomaterials (Gottschalk and Nowack, 2011).

3.1.4.3 The required research priorities on risk assessment include:

1. Development of ‘grouping’ strategies and nano-QSARs to identify high concern MNMs and predict relevant endpoints of toxicity and ecotoxicity

2. Development of standard test methods and validation of relevant in vivo/in vitro models.

3. Characterization of the hazard in terms of quantitative dose-response relationships, relevant for the threshold limits values.

4. Characterization of the hazard in terms of quantitative time-response relationships, relevant for the development of a reaction.

5. Globally harmonized epidemiological studies to validate biomarkers and to prevent / assess health effects in a longer perspective, and the field study approaches to assess potential effects of MNM at the population level of different environmental organisms.

6. Extrapolation from in vitro to in vivo (animals and man) and vice versa

The new conceptual framework (Figure 3) focused on EHS risk assessment (A Research Strategy for EHS Aspects of ENMs, 2012) reflects a coordinated, strategic research effort characterized by three key features:

• A reliance on principles that help to identify emergent, plausible, and severe risks resulting from designing and engineering materials at the nanoscale, rather than an adherence to rigid definitions of MNMs.

• A value-chain and life-cycle perspective that considers the potential harm originating in the production and use of NMs, nanomaterial-containing products, and the wastes generated.

• A focus on determining how nanomaterial properties affect key biologic processes that are relevant to predicting both hazard and exposure; for example, nanomaterial-macromolecular interactions that govern processes ranging from protein folding (a basis for toxicity) to the adsorption of substances (that may influence mobility or bioavailability of the materials).


Figure 3: A conceptual framework linked to risk assessment (A Research Strategy for EHS Aspects of Engineered Nanomaterials, 2012)

In the context of the new conceptual framework, the EHS research priorities can be established on the basis of judgments regarding the relationships between NM properties and the processes that govern their interactions with organisms and ecosystems (Auffan, 2008; A Research Strategy for EHS Aspects of Engineered Nanomaterials, 2012). The nature of the interactions will ultimately define the risk posed by the materials.

Based on this idea, the new conceptual framework (A Research Strategy for EHS Aspects of Engineered Nanomaterials, 2012) focuses on a set of principles in lieu of definitions to help identify nanomaterials and associated processes on which research is needed to ensure the responsible development and use of the materials. The principles were adopted in part because of concern about the use of rigid definitions of MNMs that drive EHS research and risk-based decisions (Maynard 2011; Maynard et al. 2011a). The principles are technology-independent and can therefore be used as a long-term driver of nanomaterial risk research. They help in identifying materials that require closer scrutiny regarding risk irrespective of whether they are established, emerging, or experimental MNMs.

The principles are built on three concepts: emergent risk, plausibility, and severity; the principles are based on proposals articulated by Maynard et al. (Maynard, 2011b).

Emergent risk refers to the likelihood that a new material will cause harm in ways that are not apparent, assessable, or manageable with current risk-assessment and risk-management approaches. Examples of emergent risk include the ability of some nanoscale particles to penetrate to biologically relevant areas that are inaccessible to larger particles, the failure of some established toxicity assays to indicate accurately the hazard posed by some NMs, scalable behavior that is not captured by conventional hazard assessments (such as behavior that scales with surface area, not mass), and the possibility of abrupt changes in the nature of material-biologic interactions associated with specific length scales. Identifying emergent risk depends on new research that assesses a novel material’s behavior and potential to cause harm. Emergent risk is defined in terms of the potential of a material to cause harm in unanticipated or poorly understood ways rather than being based solely on its physical structure or physicochemical properties. Thus, it is not bound by rigid definitions of nanotechnology or NMs. Instead, the principle of emergence enables MNMs that present unanticipated risks to human health and the environment to be distinguished from materials that probably do not. It also removes considerable confusion over how nanoscale atoms, molecules, and internal material structures should be considered from a risk perspective, by focusing on behavior rather than size. Many of the MNMs of concern in recent years have shown a potential to lead to emergent risks and thus require further investigation. But the concept also allows more complex NMs to be considered—those in the early stages of development or yet to be developed. These include active and self-assembling nanomaterials.

Plausibility refers in qualitative terms to the science-based likelihood that a new material, product, or process will present a risk to humans or the environment. It combines the possible hazard associated with a material


and the potential for exposure or release to occur. Plausibility also refers to the likelihood that a particular technology will be developed and commercialized and thus leads to emergent risks. For example, the self-replicating nanobots envisaged by some writers in the field of nanotechnology might legitimately be considered an emergent risk; if it occurs, the risk would lie outside the bounds of conventional risk assessment. But this scenario is not plausible, clearly lying more appropriately in the realm of science fiction than in science. The principle of plausibility can act as a crude but important filter to distinguish between speculative risks and credible risks.

The principle of severity refers to the extent and magnitude of harm that might result from a poorly managed nanomaterial. It also helps to capture the reduction in harm that may result from research on the identification, assessment, and management of emergent risk. The principle offers a qualitative reality check that helps to guard against extensive research efforts that are unlikely to have a substantial effect on human health or environmental protection. It also helps to ensure that research that has the potential to make an important difference is identified and supported.

Together, those three broad principles provide a basis for developing an informed strategy for selecting materials that have the greatest potential to present risks. They can be used to separate new materials that raise safety concerns from materials that, although they may be novel from an application perspective, do not present undetected, unexpected, or enhanced risks. They contribute to providing a framework for guiding a prioritized risk-research agenda. In this respect, the principles were used by the committee as it considered the pressing risk challenges presented by MNMs.

When the principles are applied to existing and emerging MNMs, various groups of materials that may warrant further study are evident. These group

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