Research Progress on Environmental, Health, and Safety Aspects of Engineered Nanomaterials (2013)

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3 Assessment of Progress INTRODUCTION The committee’s first report identified indicators of research progress and implementation that could be used as benchmarks for gauging the extent of re- search and implementation in response to the report. In developing the indica- tors, the committee acknowledged that given the short timeframe between that report and this second one, there would not be measurable, long-term progress that could be assessed with the indicators. It considered, however, that there would be ample time for initiation of research and for initial development of the infrastructure needed for implementing the research strategy. In examining the extent of progress that has occurred, the committee is aware that many concomitant environmental, health, and safety (EHS) nano- technology reviews and planning efforts have occurred within the same period as its own work, including publication of the National Nanotechnology Initiative (NNI) EHS research strategy, other government assessments, international ini- tiatives, and continuing research efforts in general (see Chapter 2). It is neither possible nor useful to try to attribute progress to any particular effort, including this committee’s first report. Rather, we examine the trajectories of research and implementation to gauge whether steps have been made toward addressing the indicators identified by the committee and, if not, what efforts are needed to achieve progress. The committee used a color scheme for categorizing progress: green for substantial progress, yellow for moderate or mixed progress, and red for little progress. It adopted that qualitative approach as suitable for gauging progress given the scope and types of information available. It classified progress on the basis of a consensus of the committee. The assessment considered new activities since preparation of the committee’s first report and the trajectory of research progress. Thus, green implies that there are new activities and that sustained progress can be expected, red refers to a situation of limited activity and little expectation of change, and yellow refers to mixed scenarios. The committee recognizes that its assessment is not an exhaustive compilation and evaluation of 41 

42 Research Progress on EHS Aspects of Engineered Nanomaterials progress, rather it is intended to provide illustrative examples of progress. Chap- ter 4, “Getting to Green”, describes additional efforts and the pathways that are needed to achieve progress in the research and implementation indicators identi- fied by the committee in the context of the vision for the EHS nanotechnology research enterprise (Figure 1-2). The discussion below addresses advances made with regard to research and implementation progress indicators identified in the first report. The com- mittee considers that the indicators remain appropriate for evaluating progress. However, in certain cases (as noted), it has clarified the wording or modified the order of the indicators. Boxes 3-1 and 3-2 summarize the indicators, including the committee’s assessment of progress—green, yellow, or red. The following text identifies the indicators, discusses progress, and presents the rationale for selection of a particular assessment. BOX 3-1 Status of Indicators of Research Progress1 Adaptive Research and Knowledge for Accelerating Research Progress and Providing Rapid Feedback to Advance the Research  Extent of development of libraries of well-characterized nanomaterials, including those prevalent in commerce and reference and standard materials  Development of methods for detecting, characterizing, tracking, and monitoring nanomaterials and their transformations in simple, well-characterized media  Development of methods to quantify effects of nanomaterials in experimental systems  Extent of joining of existing databases, including development of common informatics ontologies  Advancement of systems for sharing the results of research and fostering development of predictive models of nanomaterial behaviors Quantifying and Characterizing the Origins of Nanomaterial Releases  Developing inventories of current and near-term production of nanomaterials  Developing inventories of intended uses of nanomaterials and value-chain transfers  Identifying critical release points along the value chain  Identifying critical populations or systems exposed  Characterizing released materials in complex environments  Modeling nanomaterial releases along the value chain (Continued) 1 The wording and ordering of some indicators have been modified from NRC (2012, pp. 181-182). Details of the modifications are noted in the descriptions of the indicators in this chapter. 

Assessment of Progress 43 BOX 3-1 Continued Processes That Affect Both Exposure and Hazard  Steps taken toward development of a knowledge infrastructure able to describe the diversity and dynamics of nanomaterials and their transformations in complex biologic and environmental media  Progress in developing instrumentation to measure key nanomaterial properties and changes in them in complex biologic and environmental media  Initiation of interdisciplinary research that can relate native nanomaterial structures to transformations that occur in organisms and as a result of biologic processes  Extent of use of experimental research results in initial models for predicting nanomaterial behavior in complex biologic and environmental settings Nanomaterial Interactions in Complex Systems Ranging from Subcellular Systems to Ecosystems  Extent of initiation of studies that address the impacts of nanomaterials on a variety of end points in complex systems, such as studies that link in vitro to in vivo observations, that examine effects on important biologic pathways, and that investigate ecosystem effects  Extent of adaptation of existing system-level tools (such as individual species tests, microcosms, and organ-system models) to support studies of nanomaterials in such systems  Development of a set of screening tools that reflect important characteristics or toxicity pathways of the complex systems described above  Steps toward development of models for exposure and potential ecologic effects  Identification of benchmark (positive and negative) and reference materials for use in studies and measurement tools and methods to estimate exposure and dose in complex systems INDICATORS OF RESEARCH PROGRESS Adaptive Research and Knowledge for Accelerating Research Progress and Providing Rapid Feedback to Advance the Research In the committee’s 2012 report (NRC 2012), the first set of research pri- orities involved establishing an adaptive infrastructure for research and knowledge generation to accelerate and advance EHS nanotechnology research. The components of this infrastructure include study and reference materials; nanomaterial libraries; instruments and methods for measuring nanomaterials and their transformations; methods or assays to quantify the effects of nano- materials; databases, ontologies, and tools for sharing research results; and mo- 

44 Research Progress on EHS Aspects of Engineered Nanomaterials dels to uncover relationships among the data. Progress toward those short-term and medium-term research priorities ranged from green for detecting and char- acterizing engineered nanomaterials (ENMs) in relatively well-characterized media to yellow for development of libraries of well-characterized ENMs, de- velopment of methods for quantifying effects of ENMs in experimental systems, and the extent of joining of existing databases, including the elements of an in- formatics infrastructure. It is expected that the integrated components of the infrastructure will need to be continuously improved to adapt to the growing needs of the research enterprise.  Extent of development of libraries of well-characterized nanomaterials, including those prevalent in commerce and reference and standard materials BOX 3-2 Status of Indicators of Progress in Implementation (NRC 2012, p. 183) Enhancing Interagency Coordination  Progress toward establishing a mechanism to ensure sufficient management and budgetary authority to develop and implement an EHS research strategy among NNI agencies  Extent to which the NNCO is annually identifying funding needs for interagency collaboration on critical high-priority research Providing for Stakeholder Engagement in the Research Strategy  Progress toward actively engaging diverse stakeholders in a continuing manner in all aspects of strategy development, implementation, and revision Conducting and Communicating the Results of Research Funded Through Public–Private Partnerships  Progress toward establishment of effective public-private partnerships, as measured by such steps as completion of partnership agreements, issuance of requests for proposal, and establishment of a sound governance structure Managing Potential Conflicts of Interest  Progress toward achieving a clear separation in management and budgetary authority and accountability between the functions of developing and promoting applications of nanotechnology and understanding and assessing its potential health and environmental implications  Continued separate tracking and reporting of EHS research activities and funding distinct from those for other, more basic or application-oriented research 

Assessment of Progress 45 The committee’s first report emphasized that libraries of well-characterized nanomaterials were needed to accelerate EHS nanotechnology research and that the libraries should include nanomaterials that meet the evolving needs of the re- search community. There has been progress in developing specific nanomaterials that have been appropriately characterized for nanotechnology EHS studies, in- cluding gold, silver, and carbon standards developed by the National Institute of Standards and Technology (NIST 2013a), Organisation for Economic Co- operation and Development reference materials characterized by the National In- stitutes of Health (NIH) Nanotechnology Characterization Laboratory (NCL) for the National Institute of Environmental Health Sciences Nanotechnology Consor- tium (AZoNano.com 2010), and materials developed in individual research groups and centers. Some of those materials are now available through commercial chan- nels (NanoComposix 2012). However, the composition, structure, properties, im- purities, and contaminants of a nanomaterial sample depend on the production, refinement, separation, and purification processes used to make them and can ex- hibit substantial lot-to-lot variation. In addition, the sample-preparation techniques used for different characterization methods are generally not well documented or reported. For example, the NCL reports (McNeil 2012) that up to 40% of samples submitted to it for characterization were contaminated with endotoxin even though they had been vetted for possible use in therapeutics. It will continue to be difficult to correlate published research results with nanomaterial types unless more detail is provided in publications or documentation of datasets regarding the manufactur- ing process, lot number, and sample-preparation and characterization methods used. For the last few years, it has been recognized that nanomaterials for EHS re- search need to be well characterized in the media in which they are used (Richman and Hutchison 2009; von der Kammer et al. 2012; Pettit and Lead 2013). Alt- hough there has been progress in that respect (for example, use of the same well- characterized materials in various studies to allow comparison of results), there still are no recommended standard materials for characterization. Nanomaterials produced for fundamental or applied research are rarely characterized adequately for EHS research. Therefore, new nanomaterials that are produced and developed for applied research typically cannot be used more broadly for EHS research, be- cause of the different types of characterization needed, which depend on the in- tended uses. With respect to developing materials libraries to support nanotechnology EHS research, the committee concludes that much work is needed. There has been an emphasis on nanomaterials that have been documented to be most prev- alent in commerce—including nanosilver, carbon nanotubes (CNTs), and zinc oxide (ZnO) (OECD 2008; PEN 2013)—although a recent survey of the patent literature suggests that there is probably a more diverse set of materials that are being and will be incorporated into products (Leitch et al. 2012). To accelerate research, a larger set of nanomaterials is needed to identify the structural fea- tures responsible for potential biologic and environmental effects. Specifically, ENMs should be selected to address hypotheses regarding the influences of in- 

46 Research Progress on EHS Aspects of Engineered Nanomaterials dividual structural parameters (for example, surface coating, surface functionali- ty, ion release rates from core material, core sizes, and material purity). Thus far, there has been little progress in producing structurally analogous sets (or librar- ies) of well-characterized nanomaterials (Harper et al. 2011). As a result, it is not possible to conduct systematic studies of families of structurally related na- nomaterials to determine how structure influences effects. Not surprisingly, the structural diversity of the materials that have been produced does not yet support the needed breadth of nanotechnology EHS studies. Thus, although there has been some progress in producing and characteriz- ing new nanomaterials to support EHS research, there are large gaps, and pro- gress toward this goal is categorized as yellow.  Development of methods for detecting, characterizing, tracking, and monitoring nanomaterials and their transformations in simple, well-characterized media In its first report, the committee gave high priority to research that pro- motes development of critical supporting tools, including methods of character- izing how the properties of ENMs affect their interactions with humans and the environment (NRC 2012). Those capabilities need to be developed in the short term and ramped up to become sustainable in the longer term. In simple and relatively well-characterized media (such as deionized water and physiologic buffer with known composition), substantial progress has been made in develop- ing analytic tools and methods for detecting and characterizing nanomaterials. (Detection and characterization of ENMs in more complex environmental media are discussed later in this chapter.) Several agencies—including NIST, the US Army Corps of Engineers Engineer Research and Development Center, and the NCL—have active research programs in place that are aimed at developing and validating the tools (NSET 2012a). Some components of activities in two re- search centers funded by the Environmental Protection Agency (EPA) and the National Science Foundation (NSF) are aimed at developing and validating ENM detection and characterization methods; in most cases, these are applica- tions of, or adaptations of, existing tools, including x-ray spectroscopy (Ma et al. 2012; Lawrence et al. 2012), spectrometry (Mitrano et al. 2012), and optical methods (Fatisson et al. 2012). Some new methods are being developed to measure important ENM properties, such as surface hydrophobicity of nanopar- ticles (Xiao and Wiesner 2012) and chirality of single-walled CNTs (Khan et al. 2013). In addition, the nanotechnology EHS research community now recogniz- es the dynamic nature of nanomaterials and the need to characterize nanomateri- al transformations and the transformed materials (Levard et al. 2012; Liu et al. 2012; Lowry et al. 2012a; Nowack et al. 2012). The committee classifies this indicator as green because of the number of programs initiated or under way in various agencies and the progress evident in the peer-reviewed literature (as described above). However, characterization 

Assessment of Progress 47 efforts are generally (not exclusively) limited to studies in well-controlled model media, and more work is needed to extend understanding to more complex sys- tems (discussed later in this chapter). Some ENM properties are still difficult to measure, such as the properties of adsorbed macromolecules and the structure of the outer surface layers of nanomaterials. Techniques for routine monitoring of nanomaterials in environmental media (for example, wastewater treatment-plant effluent) are not available (as discussed later). Finally, although there are many data on ENM characteristics and likely transformations, cross-validation and synthesis of the data to provide knowledge about ENM properties and the envi- ronmental properties that lead to the transformations have not occurred.  Development of methods to quantify effects of nanomaterials in experi- mental systems The committee’s first report identified the need for standardized methods for assessing environmental effects of nanomaterials in the environment and the need for markers for assessing toxicity. It also identified a lack of information on effects, especially ecosystem effects, of longer-term nanomaterial exposures of organisms and human populations. Studies have been published on the poten- tial effects of acute nanomaterial exposures of various organisms in aquatic and terrestrial environments. However, it is difficult to integrate the data to develop the information needed to predict the effects of ENMs, because of the lack of standardized assays, the variety of ENMs, the variety of organisms and experi- mental conditions used, and the fact that many studies have examined primarily acute mortality outcomes. More toxicity information on a greater variety of na- nomaterials is needed so that different ENM properties and different end points can be examined. Standardization of assays and development of reference mate- rials for positive and negative controls are also needed to ensure that the data gathered for toxicity assays are comparable and useful. The EPA, the Food and Drug Administration (FDA), and the National In- stitute for Occupational Safety and Health (NIOSH) have not identified assays targeted at specific outcomes to assess nanotoxicity. There is a need to standard- ize toxicity assays, both in vitro and in vivo, to reduce variability within and between laboratories and to improve consistency of results among different la- boratories. For example, a round-robin in vitro study involving 10 laboratories in the United States and Europe to characterize nanoparticles before toxicity testing revealed that although there was improved reproducibility between la- boratories because of adherence to strict protocols for shipping, measurement, and reporting, measurements of polydisperse suspensions of nanoparticle aggre- gates or agglomerates were not reproducible (Roebben et al. 2011). The use of ultrasonication increased variability among polydisperse suspensions. With re- spect to quantifying effects of nanomaterials in vivo, a 2013 round-robin study (Bonner et al. 2013) by four laboratories in the United States investigating pul- monary responses in mice and rats to three forms of nano-titanium dioxide 

48 Research Progress on EHS Aspects of Engineered Nanomaterials (nano-TiO2) and three forms of multiwalled CNTs (MWCNTs) showed some interlaboratory variability of the inflammatory response to TiO2, but the relative potency of the MWCNTs was similar among all laboratories. Although some agencies, such as NIST, are evaluating different protocols (NIST 2013b), the need for standard operating procedures has not been fully met. Establishing such procedures for all phases of ENM preparation and toxicity testing is required to increase consistency of results among laboratories. Several studies have identified acute ecotoxic effects of ENM exposures and issues associated with traditional nanotoxicity assays (see Klaine et al. 2008 and above references for review). However, there is little information on effects on ecologically relevant species or on ecosystem-level effects of the chronic low-dose exposures to ENMs that are expected in the environment (Bernhardt et al. 2010; Gottschalk and Nowack 2011). Investigations of perturbations in whole-organism systems are also lacking. Efforts have concentrated on oxida- tive stress, which may be a fleeting reaction of an organism to ENM exposures and may not be the sole mechanism of effects. The committee’s 2012 report called for targeted assays for assessing nanotoxicity. Efforts to assess toxicity by using high-throughput assays at the EPA–NSF funded centers (Lin et al. 2013; Nel et al. 2013) may provide some standard acute-toxicity information on se- lected nanomaterials. The relevance of those assays to more realistic chronic low-dose exposures and population-level effects has not been established. The committee specifically suggested development of a standard battery of assays and novel assays that may be required to describe the various effects of many types of nanomaterials, including ones that have new biologic activities. Stand- ardized assays for ecosystem effects of even standard chemicals are lacking. The EPA–NSF funded centers may be an indication of support for those types of assays, but this is the only direct support identified for this topic. The committee considered that there was some research progress in this category, but the progress was marked yellow because of the lack of identifica- tion of a set of methods to determine effects. More information on the variety of potential mechanisms and research that elucidates these mechanisms will move this indicator toward green.  Extent of joining of existing databases, including development of com- mon informatics ontologies Some progress has been made toward the development of informatics on- tologies and sharing of databases. For example, the Big Data Initiative was an- nounced in March 2012 “to greatly improve the tools and techniques needed to access, organize, and glean discoveries from huge volumes of digital data” with support from NSF, NIH, the Department of Energy (DOE), the Department of Defense (DOD), the Defense Advanced Research Projects Agency, and the US Geological Survey (OSTP 2012). The new program defines data as including data, publications, samples, physical collections, software, and models (NSF 

Assessment of Progress 49 2010). The same comprehensive definition underpins the new NSF Nanotech- nology Signature Initiative for a Nanotechnology Knowledge Infrastructure (NKI) with participation by the Consumer Product Safety Commission (CPSC), DOD, DOE, EPA, FDA, the National Aeronautics and Space Administration, NIH, NIOSH, NIST, NSF, and the Occupational Safety and Health Administra- tion (OSHA). In addition, the NKI will support the new Materials Genome Initi- ative (MGI) (NSET 2012b) so that informatics approaches, data curation work- flows, protocols, and standards developed through MGI activities may initially be explored for nanoscale activities by the NKI effort. Coordination of activities in the United States and the EU has been estab- lished through the Communities of Research (CoRs) by the National Nanotech- nology Coordination Office (NNCO) and the EU. The CoRs include “predictive modeling for human health, ecotoxicity testing and predictive models, exposure through the life cycle, databases and ontology, risk assessment, and risk man- agement and control” (Finnish Institute of Occupational Health 2012). The on- tology CoR is responsible for coordinating informatics needs for all the CoRs, and its databases provide a mechanism for developing prototype systems and applications to support information-sharing, annotation, validation, and curation for experimental, computational, and theoretical efforts in nanotechnology. The EU–US CoRs represent an important opportunity for international collaboration to develop an infrastructure that can serve both communities. Although those new programs are promising, progress in developing ele- ments of an informatics infrastructure has been less encouraging. The foregoing examples show the need for libraries of nanomaterials; for improved reporting on nanomaterial production processes and sample-preparation techniques; for new methods for characterizing, tracking, and monitoring nanomaterials and their transformations; for methods for quantifying the effects of nanomaterials; and for systems for sharing research results and the development of predictive models for nanomaterial behaviors. Core systems, services, and applications are not yet available or have been insufficiently adopted, and this gap impedes re- search and the translation of research findings into products. For example, a harmonized nomenclature system that facilitates and informs nanomaterial clas- sification and development does not exist; data and metadata standards are not established; reproducibility of methods (ruggedness testing) has not been estab- lished; and the sensitivity data are not shared and therefore cannot be used to improve the reproducibility of methods or to inform error propagation in risk analyses. The same general limitations are present for model development: fur- nishing accurate nanomaterial and nanoproduct structural models on the appro- priate scales; developing and validating the models and their sensitivity to input parameters, computer programs, the choice of run-time parameters, computer architectures, and compilers at the relevant dimensions and time scales; and ac- cessing and validating models for the physical, chemical, and biologic systems of interest, also at the appropriate dimensions and time scales. In that regard, NanoHUB constitutes a substantial and important start, providing a stable code for different users and assuming the burden of hosting the code; providing com- 

50 Research Progress on EHS Aspects of Engineered Nanomaterials puters, storage, and user services; archiving and sharing data, metadata, and in- formation about results; and comparison with related model results. Finally, there is an overarching need for informatics to augment collabora- tion and accelerate research and translation by facilitating access to data. Exam- ples of the need for informatics include the accelerated adoption of models through NanoHUB and the increased amount of interlaboratory testing of meth- ods by various organizations (NIEHS 2012; ILSI 2013a). There are abundant examples of data that are not available through the publication process and that in many cases are not accessible on any database—such as sensitivity data on methods and validation data on models—but there are several areas of particular interest and activity. For example, high-throughput methods are increasingly used in nanotechnology-EHS research, and applications from EPA–NSF funded centers (Thomas et al. 2011; Mandrell et al. 2012) promise to generate large, correlated datasets obtained with standardized screening methods. ISA-TAB- Nano2, a new standard for data exchange, is emerging; its harmonized data for- mats incorporate high-throughput screening assays and methods for nanomateri- al characterization. Metadata capture will be possible through the NanoParticle Ontology (NPO) that builds on NIH’s Enterprise Vocabulary System. However, most important are the increasing informatics efforts (mentioned above) that promise new support and substantially increased collaboration—the NKI, col- laboration with the MGI, and the other NNI signature initiatives, particularly the EU-US CoRs. Those developments collectively signal heightened interest in increasing data quality throughout nanotechnology and nanoscience and height- ened activity in establishing a coherent infrastructure for increased collaborative research among all the disciplines. Additional data inputs are possible if databases are compiled from other studies. One potential mechanism, as mentioned in the committee’s first report, is NSF’s requirement that all grant proposals include a two-page plan for how data will be managed and shared publicly. However, modifications of that re- quirement through creation of a data commons could allow the collection of all nanotoxicity data from NSF-funded studies rather than siloed storage and re- trieval sites established by each researcher. On the basis of the still unmet need for more data-integration mechanisms, the committee has characterized this indicator as yellow.  Advancement of systems for sharing the results of research and foster- ing development of predictive models of nanomaterial behaviors 2 This format is an extension of the Investigation-Study-Assay (ISA) Tabular formats used for genomics and high-throughput screening (for example, MAGE-TAB) and adds a material file to permit transmission, linkage, and provenance of data on the nanomaterial samples being studied. This publication represents an initial step to providing one aspect of the needed infrastructure for sharing research data, and it is not yet clear how it will be received by the research community. 

Assessment of Progress 51 In its first report, the committee identified the need to develop predictive models for ENM behaviors and risk. However, the development of models can- not occur in isolation from data generation. Coordination is needed in the short term to ensure that experimental, modeling, and informatics efforts contribute to a coordinated, functional infrastructure. There is a need to collect, store, archive, and share data related to assessing the potential effects of ENMs (as described in the previous section) so that these data can be used to develop predictive models of ENM behavior. The goals of advancing systems for sharing and developing models of behavior are intimately related in that the models and data structures are both influenced by the specific questions related to exposure to ENMs and the resulting effects that need to be addressed. Therefore, the needs for models and infrastructure to support the models are assessed together. There has been some progress in development of models to predict nano- material exposures and toxicity (Gottschalk et al. 2011; Nel et al. 2013). Several government agencies have instituted specific programs to develop and test dif- ferent models to assess ENM behavior (for example, fate in the environment, releases from consumer products, plant uptake, and occupational exposure), including EPA, NIST, FDA, DOD, the US Department of Agriculture, and NIOSH (NSET 2012a). Efforts are also in place to develop computational mod- els for toxicity (for example, EPA’s ToxCast program). Finally, there has been progress towards the development of empirical predictive models as opposed to fully mechanistic models for behavior (Hou et al. 2013; Westerhoff and Nowack 2013). These models rely on empirical correlations (for example, partition coef- ficients) rather than complete mechanisms. The models can be developed in less time than fully mechanistic models, and can predict approximate behaviors (for example, in a wastewater treatment plant) and may be used to support regulatory decisions. The committee classifies progress in this category as yellow because, de- spite the development and use of the models in the nanotechnology-EHS com- munity, there is not yet a central repository for sharing the models (although NanoHub may be appropriate), and many needed models have not yet been de- veloped, such as models to predict the structure of ENM surfaces in various en- vironments. Most important, there is a paucity of data for calibrating and vali- dating models that have been developed; for example, there are very few data on ENM concentrations and speciation in environmental and biologic media that can be used to calibrate fate and transport or biodistribution models. The ab- sence of metadata and validation data for most models hampers their broad ac- ceptance and use because they are not deemed reliable and accurate. Some progress is being made in the collection, storage, and archiving of ENM physical and chemical properties. For example, the Nanomaterials Regis- try (NR) has been developed by the Research Triangle Institute with funding from NIH (Nanomaterialregistry 2013). The NR will provide a curated reposito- ry of ENM information (for example, ENM properties) from a wide array of studies that used the materials. The repository would allow researchers to com- pare model results for behaviors and effects of ENMs by using data on the na- 

52 Research Progress on EHS Aspects of Engineered Nanomaterials nomaterials stored in the NR. Incorporation of information on biologic and envi- ronmental interactions in the NR is also being considered. Other databases are being created (for example, the Nano-Bio Interactions Knowledgebase) with similar aims: to capture and store information about nanomaterial properties and behaviors that allow development of structure–activity relationships and other scientific synthesis using large datasets. Finally, the new standard data format, ISA-TAB-Nano, for sharing results obtained with analytic methods for charac- terization of nanomaterial properties and effects has recently been published (Thomas et al. 2013). The committee classifies progress in this category as yellow because de- spite initial efforts and models developed, the models and data are not yet wide- ly available and there is no agreement about the appropriate architecture for the databases, no agreement about ontology (although it is being developed through the NPO), and little discussion of interoperability and sharing among databases. Furthermore, the datasets are sparse, there is not a consistent level of variation in the collected data to allow rigorous scientific synthesis, and the breadth of data and metadata needed to make the datasets useful has not been determined or verified with a realistic “test bed” scenario. Quantifying and Characterizing the Origins of Nanomaterial Releases The quantities and characteristics of ENMs produced and the products that they enable influence human and ecosystem exposures. Even a thorough under- standing of ENM transport, transformation, and effects is not sufficient to de- scribe the effects of ENMs on human health and ecosystems if little is known about how the materials are produced and emitted and the forms in which they are introduced into the environment. Therefore, inventories3 are needed that describe what ENMs are being produced, how they are being used, and what their forms are along the value chain. However, the creation of inventories of nanomaterials is based on the notion that there is agreement as to what consti- tutes a nanomaterial. The committee returns to the issue of defining ENMs in Chapter 5. Progress in this research priority ranged from yellow to red; no priorities were classified as green. Yellow was the designation given for the extent of pro- gress in developing inventories of ENMs, in identifying critical release points 3 An inventory is a quantitative estimate of the location and amounts of nanomaterials produced or current production capacity, including properties of the nanomaterials pro- duced. Information on the nature of the systems into which nanomaterials might be re- leased during their production and the procedures for manufacturing the ENMs are im- portant for assessing the possible transformations that nanomaterials might undergo and the lifecycle impacts associated with energy and material use and waste generated. A broader definition also includes an enumeration of the amounts and uses of nanomaterials downstream in the value chain (that is, the types of products using nanomaterials, the fraction of ENMs by weight in the products, and the quantities of these products). 

Assessment of Progress 53 along the value chain, in identifying critical populations, and in characterizing released materials in complex environments. Because those priorities serve as a prerequisite to model development, the ability to model releases along the value chain was denoted as red.  Developing inventories of current and near-term production of nano- materials The committee identified efforts in academic and government laboratories to quantify and characterize the origins of nanomaterial releases and private- sector efforts focused on market reports. Production quantities, estimates of trends in production quantities, and the associated descriptions of what is being produced are components of what is referred to as inventories of nanomaterial production. The examples cited are not meant to be exhaustive but rather to pro- vide evidence that progress is being made. Work on estimating near-term inven- tories of nanomaterial production of many of the more commonly cited nano- materials (TiO2, CNTs, fullerenes, nanosilver, and nano-ZnO) at the base of the value chain has already been published by EPA–NSF funded researchers (Ro- bichaud et al. 2009; Hendren et al. 2011). Some of the materials were described by Michael Holman, of Lux Research, in the committee workshop (see Appen- dix C) as being the most likely to dominate in commercial products in the fore- seeable future. Whether that is the case and whether more advanced (for exam- ple, hybrid4) nanomaterials will grow in importance remain unclear inasmuch as estimates of nanomaterial production are subject to constant change and the un- certainties around production quantities are large. Such inventories are generally snapshots of nanomaterial production at a given time. The number of such in- ventories is quite small, but a related consideration is the lack of a systematic process that includes mechanisms and incentives for collecting such infor- mation; information-management plans for storage, dissemination, and interpre- tation of the data; and appropriate regulatory infrastructure. Progress in charac- terizing production amounts of nanomaterials is therefore likely to remain incomplete for some time and has been given an indicator status of yellow.  Developing inventories of intended uses of nanomaterials and value- chain transfers Research in NIST and EPA–NSF funded centers is quantifying releases of nanomaterials from composite matrices, a likely disposition of many nanomateri- als. Those centers are characterizing the release of CNTs, nanoclays, and nanosil- ver from porous foams and solid polymers through simulated abrasion and in vari- ous biologic fluids (Wohlleben et al. 2011; Liu et al. 2012; Nguyen et al. 2012). 4 A hybrid nanomaterial is one that results from combining different nanomaterials to form a new material that has characteristics different from those of the original materials. 

54 Research Progress on EHS Aspects of Engineered Nanomaterials Such nano-composite materials impart antimicrobial, strength, or flame-retardant properties to fabrics, foams, and plastics that may be used in consumer products. Analysis of value-chain transfers of nanomaterials that enter commerce from primary production to integration into a multitude of consumer products is also being conducted in an EPA–NSF funded center and in an NSF-funded cen- ter. Such analyses remain inadequate, perhaps because few nanomaterials are widely used in commerce. Given the mixed picture of progress, the committee designated this item as yellow.  Identifying critical release points along the value chain As discussed in the first report, “each nanomaterial or product containing nanomaterials along the steps of the value chain has an associated life cycle of production, distribution, use, and end-of-life releases that may affect human health and the environment” (p. 56). There has been progress in developing in- ventories of a small number of key nanomaterials and in mapping key elements of the value associated with a subset of these materials, but actual modeling of releases of nanomaterials to the environment along the value chains does not appear to have been initiated to any important degree. Limited by progress in the prerequisite steps of compiling information on inventories in the value chains highlighted above, identification of likely release points that may result in direct exposure of humans in the workplace or during transportation, use and end-of- use of nanomaterial-containing products, and the associated points of release to ecosystems has not been quantitatively modeled. Since the preparation of the committee’s first report, additional commit- ments by federal agencies and their collaborators have been identified. Many of those efforts are summarized in the NNI budget supplement for 2013 (NSET 2012a). In 2013, NIST expanded its nanotechnology EHS program to focus on the safe manufacture, use, and disposal of ENM-containing products. Those activities include development of measurement methods and standards to detect ENMs in nanomaterial-enabled products and to assess their releases. NIST has indicated that this work will focus on the ENMs of greatest regulatory interest according to production volume (NSET 2012a, p. 17). Candidates have been reported to include silver TiO2, cerium oxide, CNTs, and clay-based composites. Release to all environmental media is of interest, but the focus appears to be on airborne releases, with NIST and CPSC implementing multiyear interagency agreements to cooperate in these efforts. EPA also has indicated an expansion of efforts to characterize properties of ENMs in products that affect their release, fate, and transport in the environment. EPA appears to be focusing its efforts on carbon-based, metal-based, and metal oxide–based products. This focus is likely to improve our understanding of the potential for release of ENMs from prod- ucts throughout the value chain. In support of those efforts, the EPA–NSF fund- ed centers are focused on increased understanding of human exposures to ENMs, including those released from products in commerce. CPSC staff are 

Assessment of Progress 55 reportedly also supporting such efforts in the centers. NIOSH and EPA are con- ducting testing to evaluate release of nanosilver from uses of nanomaterial- containing consumer products. Like NIST, CPSC, and EPA, NIOSH continues its focus on airborne releases of nanomaterials from products. An increasing number of studies are measuring releases of ENMs at manufacturing sites (Tsai et al. 2008, 2009, 2012; Methner et al. 2010; Kuhlbusch et al. 2011); however, there is a lack of data on consumer exposure along the value chain. Chen et al. (2010) simulated human exposure to a TiO2-containing aerosol in a spray that can be used as a cleaning agent. Federal agencies—including NIST, CPSC, EPA, NIOSH, and OSHA—are collaborating with nonprofit, industry, and in- ternational groups under the auspices of the International Life Sciences Institute (ILSI) Research Foundation’s Nanorelease Project (ILSI 2013a). In 2013, work on refinement of testing methods is expected to lead to a round-robin approach to testing products for release of ENMs. Although efforts to address release of ENMs from products are under way, additional efforts will be needed. In particular, the focus on large-production- volume ENMs may limit our ability to assess emerging materials before they enter the marketplace in consumer products. In addition, the focus on aerosol releases provides an incomplete perspective on all pathways of release to the environment. Expansion of these efforts will be critical for the assessment of potential EHS risks posed by ENMs throughout the value chain of nanomaterial- enabled products. Because a more comprehensive and comparative view of where nanomaterials may be released along the value chain is needed to identify where to mitigate risks, the indicator is yellow.  Identifying critical populations or systems exposed In its first report, the committee noted the importance of characterizing not only the quantity and nature of ENMs to which humans and ecosystems are ex- posed but possible changes in exposed populations and systems that occur dur- ing ENM releases throughout the life cycle and value chain. Understanding the complexity of ecosystems (that is, the interaction of the abiotic and biotic and the variety of environments and organisms) and of human populations (includ- ing such factors as age, socioeconomic status, health status, behavior and activi- ties, and exposure pathways) requires an integrative research structure involving collaboration among disciplines and among stakeholders. As a high-priority short-term research goal, the committee has suggested identifying exposed hu- man populations and the magnitude of exposure in different ecosystems after determination of critical release points along the ENM value chain. There has been some research activity but little progress in identifying critical human pop- ulations exposed to ENMs. NIOSH is developing public–private partnerships with companies manufacturing ENMs and has conducted exposure assessments at their manufacturing sites (for example, producers of titanium dioxide nano- particles, CNTs, and carbon nanofibers) (NIOSH 2012a). That activity is a use- 

56 Research Progress on EHS Aspects of Engineered Nanomaterials ful start in characterizing released ENMs in workplace environments. However, considerable work is needed to measure exposures throughout the ENM life cycle and value chain. For example, the influence of accumulation along the food chain on exposure and exposure to different types of CNTs during produc- tion, distribution, use, and disposal need to be evaluated (Helland et al. 2007). Some consumer products may be of greater concern than others (for ex- ample, cosmetics applied as sprays), given the form of the ENMs within prod- ucts. Initial efforts by ILSI (2013b) and the EU (Kuhlbusch et al. 2011) are lead- ing to strategies to assess potential ENM exposures from consumer products. Exposures from discarded products after disposal or end-of-life use (for exam- ple, from landfills) need to be measured. With respect to ecosystems, some individual research efforts to examine exposures to ENMs are supported by federal funding. Examples include exami- nation of titanium dioxide in wastewater outputs (Westerhoff et al. 2011), CNTs in aquatic systems (von der Kammer et al. 2012), and movement of ENMs through groundwater (Phenrat et al. 2010; Kim et al. 2012), and research into distribution of ENMs into model ecosystems (Lowry et al. 2012b; Schierz et al. 2012). EPA has also funded several projects to examine movement of ENMs in systems and to develop technologies for detecting nanomaterials (EPA 2012). However, for the majority of ENMs, important questions remain: What is their exposure potential in different environments, such as soil, water (aquifers), the food chain, and wastewater? How do alterations in the chemistry of ENMs in- fluence the potential for ecosystem exposure? Given the challenge of effectively measuring ENM exposures along the value chain and some, but limited progress in identifying both human popula- tions and ecosystems exposed, the committee has labeled this indicator yellow.  Characterizing released materials in complex environments5 Characterization of materials that are released into the environment re- mains a challenge because released materials are present at low concentrations, are often transformed during release, and must be analyzed within structurally and compositionally heterogeneous matrices (von der Kammer et al. 2012). For example, a nanoparticle released into a waterway may undergo a wide array of transformations that would render it difficult to detect and characterize:  The nanoparticle becomes highly diluted, and this makes detection and characterization difficult even if it is not transformed.  The nanoparticle surface coating or core may be fully or partially de- graded, and this can result in a complex mixture of unknown chemicals that are more difficult to detect and characterize. In addition, it may not be possible to 5 This indicator originally was phrased as “Characterizing released materials and asso- ciated receptor environments” (NRC 2012, p. 181). 

Assessment of Progress 57 relate whatever is detected to the primary nanoparticles released and to distin- guish between degradation products and naturally occurring nanoscale species.  The nanoparticle surface may be coated rapidly by natural organic mat- ter or proteins, and this can complicate detection and isolation and make it diffi- cult to characterize the surface chemistry. Releases have been tracked by monitoring the elemental compositions of macro- scopic products or the bulk environments into which nanomaterials are released. Such techniques as inductively coupled plasma mass spectroscopy (ICP-MS) are now widely used to gain information about elemental composition of ENMs in aqueous samples (for example, surface waters) (Heithmar 2011; Mitrano et al. 2012), but the measurements produced provide no information on the speciation of the released material. The detection of particle releases has also advanced, but gathering information on the compositions of those particles within the matrix into which they have been released remains challenging. Both the strong ele- mental signals from the matrix and the presence of naturally occurring nanopar- ticles complicate the analyses. Some progress has been made in assessing both particle release and com- position. For example, single-walled CNTs can be separated from soil and sedi- ment and quantified with near-infrared fluorescence spectroscopy (Schierz et al. 2012). C60 and C70 fullerenes have been extracted from soils using ultrasound and quantified by HPLC-MS (Perez et al. 2013), and from urine (Benn et al. 2011). Single-particle ICP-MS methods have been developed that have proved useful for metal nanoparticles, such as gold and silver (Heithmar 2011; Mitrano et al. 2012) in pore water extracted from soil. Those are examples of methods that rely on separation of nanomaterials from or within a matrix followed by analysis, but concern that the separation process itself can transform the materi- als remains (von der Kammer et al. 2012). Separation methods that lack station- ary phases, such as field-flow fractionation, show the most promise for separat- ing nanomaterials without altering them (Mitrano et al. 2012). Other approaches to monitoring environmental transformations that obviate separation include monitoring of the transformation of tethered nanoparticles (Glover et al. 2011) and use of x-ray–based spectroscopic methods that provide speciation infor- mation in media without the need to desiccate samples (Lawrence et al. 2012; Lombi et al. 2012; Lowry et al. 2012b). The generality of the new approaches and their relevance to real-world samples remain to be determined. There has been some progress toward this objective, but proper characteri- zation of nanomaterial releases will require additional progress in developing methods that can simultaneously determine the particulate characteristics and the nanomaterial composition (including surface chemistry) of the released ma- terial. Thus, the committee’s assessment is that progress toward this goal should be graded as yellow. 

58 Research Progress on EHS Aspects of Engineered Nanomaterials  Modeling nanomaterial releases along the value chain Models are needed for accurate prediction of nanomaterial releases, envi- ronmental concentrations, and human and ecosystem exposures. Models of re- leases also are needed to identify the form and speciation of released nano- materials. To date, modeling efforts appear to be confined to specific release points and routes of exposure (such as inhalation exposure in the workplace and releases in wastewater-treatment plant effluent discharges). Further progress has not been initiated, because of the lack of information on inventories in the value chains highlighted above. Because of the lack of quantitative information throughout the life cycle of ENMs on which to build such models, progress in this indicator is denoted as red. Processes That Affect Both Exposure and Hazard In its first report, the committee highlighted the need to identify the criti- cal nanomaterial interactions that affect ENM behaviors. It recommended identi- fying cross-cutting processes (for example, agglomeration, aggregation, dissolu- tion, and deposition) that are common to assessing exposure and assessing hazard. Identifying nanomaterial interactions requires cataloging the types of ENM transformations in complex matrices and the time scales associated with the transformations, developing instrumentation to monitor transformations in vivo or in complex environmental media, and developing models to predict ENM behaviors. Integral to these efforts are the need to develop the ontology to describe “transformed” nanomaterials and the need to develop the infrastructure to archive data that enables model development and identification of these pro- cesses. Progress ranged from yellow for initiation of basic studies that are be- ginning to characterize likely types of ENM transformations and to require addi- tional study and for studies that begin to relate ENM properties to observed effects in more complex systems to red for development of new instrumentation to measure transformations in situ, in vivo, or on single particles. The committee also notes that the data generated have not been effectively used to develop and validate the models, because of the absence of a central structured database for consistent documentation of research results.  Steps taken toward development of a knowledge infrastructure able to describe the diversity and dynamics of nanomaterials and their transformations in complex biologic and environmental media In its first report, the committee indicated the need to develop a knowledge infrastructure to measure and describe nanomaterial behaviors, including trans- formations that affect exposure and hazard. The types and nature of the transfor- mations that affect both exposure and toxicity studies (for example, aggregation, 

Assessment of Progress 59 agglomeration, oxidation, reduction, dissolution, adsorption of macromolecules, and interactions of ENMs with cell membranes) have been documented in many studies and review articles (Verma and Stellacci 2009; Wiesner et al. 2009; Levard et al. 2012; Lowry et al. 2012a; Moghadam et al. 2012; Mu et al. 2012; Nowack et al. 2012; Cheng et al. 2013; Zhu et al. 2013). The importance of the components of the media in which ENMs are dispersed is also well established (Maiorano et al. 2010). Despite a large volume of laboratory-based research, the committee classi- fies progress as yellow because no knowledge base exists to describe and under- stand these transformations in general. Most studies have examined specific condi- tions, so current understanding of ENM behaviors is system-specific. Knowledge of the mechanisms behind the transformations is also often incomplete. Several nascent efforts are under way to characterize systematically and precisely how various solution conditions (for example, dissolved solutes, pH, and redox state) and ENM properties affect transformations that will allow the development of predictive models (Ottofuelling et al. 2011; Nel et al. 2013). However, the data infrastructure needed to share the results is not yet widely available, as highlighted above. Development of the infrastructure and data-sharing are complicated by the highly variable nature of the transformations and by the lack of an ontology to describe the “state” of a fully or partially transformed ENM. Finally, there is no way to characterize many of the transformations in relevant media at realistic con- centrations. In some cases, that will require the development of new instrumenta- tion, as described below. The lack of an ontology and a mechanism for data syn- thesis led to categorizing this indicator as yellow.  Progress in developing instrumentation to measure key nanomaterial properties and changes in them in complex biologic and environmental media Measurement of nanomaterial transformations in relevant biologic and en- vironmental media has high priority. In those complex media, a wide array of substantial or subtle changes involving material composition and structure may occur. Adsorption of natural organic matter, proteins, and lipids may change the surface coating. Etching, degradation, or agglomeration of nanoparticle cores may transform the material. Oxidation and dissolution or sulfidation may occur (Liu et al. 2012). Measurements of the materials are further complicated by their presence at low concentrations and in a wide variety of compartments. Little progress has been made in this indicator despite its importance and the recognition that such measurements are crucial for accelerating nanotech- nology EHS research. Several sections in this chapter describe how analytic techniques are being adapted and used in combination to gain information about the composition and structure of ENMs in simple well-characterized media. This indicator is focused on the development of new instrumentation that can meas- ure core and surface compositions and physical dimensions in complex biologic or environmental matrices and in some cases at a single particle resolution. The optimal methods would permit measurement of size and composition in the ma- 

60 Research Progress on EHS Aspects of Engineered Nanomaterials trices. Some publications have called for improved detectors to enable single- particle ICP-MS and to improve the spatial resolution of x-ray microprobes (von der Kammer et al. 2012). Instruments for measuring airborne ENMs are being developed. J. Wang et al. (2011) have developed a universal nanoparticle analyzer to measure and characterize airborne nanoparticle agglomerates. Rhoads et al. (2003) designed an instrument (rapid single-particle mass spectrometry, RSMS-11) to analyze the chemical composition of airborne nanoparticles (less than 20-nm), and efforts are directed at developing an instrument to measure nanoparticle-bound reactive oxygen species in real time (Y. Wang et al. 2011). Despite those developments, overall progress is insufficient. Considerable progress is required to meet current and future needs in the nanotechnology-EHS field, and little headway has been made toward the neces- sary instrumentation. Therefore, this objective is labeled red by the committee.  Initiation of interdisciplinary research that can relate native nano- material structures to transformations that occur in organisms and as a result of biologic processes In its first report, the committee emphasized the importance of processes that lead to transformations of ENMs in organisms or ecosystems. Adsorption of proteins, lipids, and organic materials may alter surface properties of ENMs, form a corona, affect mechanisms of cell interactions, and alter ENM biokinetics. (The corona, a coating that binds to the surface of ENMs, influences the biodistribution and effects of ENMs [Walczyk et al. 2010].) Although the concept of “differential adsorption” of lipids and proteins has been described (Müller and Heinemann 1989) and has been developed in vitro (Cedervall et al. 2007; Walcyzyk et al. 2010), the committee identified a major gap in understanding of the effects, par- ticularly in vivo effects, of the types and amounts of adsorbed lipids and proteins. Some progress has been made by several laboratories in the United States and Europe that are investigating the adsorption of lipids and proteins on ENMs introduced into organisms or when interacting with biologic media in vitro. Alt- hough in vitro studies have advanced this field of research considerably (for example, showing that modifying ENM surfaces by coating them with proteins or surfactants can result in altered cellular responses), confirmatory in vivo stud- ies are lacking. Despite the progress, much research is needed. The importance of other ENM transformations (altered surfaces, agglomeration, deagglomera- tion, aggregation, and solubilization) for biokinetics and effects needs to be con- sidered. In addition, studies have focused on acute short-term effects, and little is known about the persistence of such effects in vivo. Moreover, effects of chronic low-dose exposure are not well established. Phenomena observed at high doses may not be entirely relevant in vivo inasmuch as dose may influence mechanisms (Slikker et al. 2004). 

Assessment of Progress 61 Formulation of time-course studies will be essential for in vivo and in vitro evaluations. They are necessary for documenting the possible transfor- mations of ENM characteristics within the life cycle and for assessing the persis- tence of measured responses in organisms. The latter issue will be essential for identifying and characterizing hazards. The mixed progress in these subjects led the committee to assess this indicator as yellow.  Extent of use of experimental research results in initial models for pre- dicting nanomaterial behavior in complex biologic and environmental settings The fate and effects of ENMs in complex environments will be determined by a set of interactions between the materials and the properties of the environ- ments. Identifying mechanisms by which those interactions occur requires integra- tion of the mechanistic understanding gained from studies on a laboratory scale in well-controlled environments and the understanding obtained from research con- ducted in environments of varied complexity. Predictive models can then be de- veloped on the basis of the mechanisms identified in relevant exposure scenarios. In contrast with the development of models for specific behaviors (such as aggre- gation and agglomeration) in relatively well-characterized environments, devel- opment of models for predicting nanomaterial behavior in complex biologic and environmental systems has seen little progress. One key limitation is the lack of resources for conducting long-term experiments in large-scale environmental sys- tems, such as mesocosms6, or for performing in vivo studies. Another is the ab- sence of a central structured database for consistent documentation of research results that permits datasets to be compared and used in models. Some efforts are under way in EPA–NSF funded centers to develop models for predicting nano- material behavior in complex biologic and environmental systems, but they are disjointed. Data collected in the various systems (environments and organisms) are not characterized in the same manner and are therefore not readily usable for mod- eling. In addition, the focus has been on only a few ENMs, so comparisons among ENMs that have different chemical composition are not possible. The committee therefore identified this indicator as red. Nanomaterial Interactions in Complex Systems Ranging from Subcellular Systems to Ecosystems In its first report, the committee recognized the need to investigate and in- crease the understanding of interactions of ENMs in a variety of complex sys- tems. Complex systems can range from subcellular organelles to cells to organ- isms to ecosystems. These elements may act independently, synergistically, or antagonistically in response to ENM exposures. Research efforts that focus on system-level approaches to investigate potential ENM effects on human health 6 A means of studying the natural environment under controlled conditions. 

62 Research Progress on EHS Aspects of Engineered Nanomaterials and the environment are needed. Indirect effects may also result from direct in- teractions with ENMs. For example, ENM transformations that occur in envi- ronmental systems—for example, through weathering in ecosystems or metabo- lism in organisms and ecosystems—may have unexpected effects on other organisms along the food chain or indirectly in organ systems. Specifically quantum dots have been found to be toxic to a variety of systems, but weather- ing of quantum dots can induce antibiotic resistance in some bacterial strains that could in turn affect organisms that are susceptible to these bacteria (Yang et al. 2012). In mammals, inhaled ENMs that are deposited in the distal lung or alveolar epithelial sites may interact with lung lining fluids to form nanomateri- al–corona complexes that may alter the disposition and biologic activity of the ENM. Therefore, a first step is to identify relevant exposure sources, concentra- tions, and cellular and ecologic targets so that potential effects on complex sys- tems can be addressed. Research progress indicators for this category ranged from yellow to red; no indicators were denoted as green. Indicators were yellow for extent of initiation of studies that address effects of ENMs in complex sys- tems, adaptation of system-level tools to support studies in these systems, and steps toward development of models for assessing ecologic exposures and ef- fects. Indicators were red for developing screening tools that reflect important toxicity pathways and identifying benchmark and reference ENMs for use in studies to estimate exposure or dose.  Extent of initiation of studies that address the impacts of nanomaterials on a variety of end points in complex systems, such as studies that link in vitro to in vivo observations, that examine effects on important biologic pathways, and that investigate ecosystem effects7 The majority of toxicology studies involving ENMs have examined only a few end points, including acute mortality and acute oxidative stress. Although valuable, these studies provide little information that is useful for examining the effects of ENM exposures on organisms. Historical studies of chemicals have demonstrated that evaluation of outcomes—such as reproductive, developmen- tal, and endocrine effects—is critical for understanding human health and eco- logic impacts. In vitro analyses, although potentially useful as an initial screen for gross effects, have not been shown to predict in vivo effects adequately. Ecosystem effects, which are difficult to measure, are often not considered in chemical assessments, but such information is essential for understanding changes in community and abiotic interactions that may lead to detrimental ef- fects. Initial model ecosystem studies on mescocosms can begin to address changes that may occur on a larger scale due to ENM releases. In addition, mo- 7 This indicator was formerly titled “Extent of initiation of studies that address hereto- fore underrepresented fields of research, such as those seeking to relate in vitro to in vivo observations, to predict ecosystem effects, or to examine effects on the endocrine or de- velopmental systems” in NRC (2012, p. 182). 

Assessment of Progress 63 lecular studies can provide the basis to predict potential larger scale impacts. Conduct of these more complex studies, rather than reliance on data from more simplified assays, is critical for comprehensive understanding of the potential effects of ENMs on humans and ecosystems. Nanotoxicology studies to determine the suitability of in vitro study results for predicting responses in vivo had been published before the release of the committee’s first report (Sayes et al. 2007; Gerde 2008; Lu et al. 2009; Rushton et al. 2010; Han et al. 2012; Zhang et al. 2012), but the report identified several subjects that required further in-depth study. Continuing concerns include do- simetry-related issues and the need for further in vivo validation of effects and underlying mechanisms. The committee also identified a dearth of exposure- assessment studies—that is, studies of workplace exposures and consumer expo- sures to ENMs. Some studies have addressed the latter subject, including workplace- exposure studies and ecosystem studies that were conducted by EPA–NSF fund- ed centers. NIOSH and academic institutions (Bello et al. 2009; Methner et al. 2010; NIOSH 2012a; Tsai et al. 2012) have increased efforts to engage with industry to perform workplace monitoring. The European NanoCare Program also includes exposure-assessment studies (Kuhlbusch et al. 2011). With respect to in vitro–in vivo correlations, several studies have com- pared results of in vitro and in vivo toxicity testing for their predictive power. The comparisons have provided findings that encompass good and poor con- cordance between in vitro and in vivo results (for example, Sayes et al. 2007; Rushton et al. 2010); this indicates a need for improved approaches to the design of comparative studies with the goal of predicting hazards. High-throughput screening (HTS) assays allow hazard ranking of many ENMs simultaneously on the basis of mechanistic information about cellular activation pathways of injury (Meng et al. 2009). However, Thomas et al. (2012a, b) concluded from an eval- uation of HTS assays of numerous chemicals that these assays have little ability to predict in vivo hazards. They can, however, be useful for setting priorities among materials for further testing (Dix et al. 2012). Validation of the predictive value of HTS assays for assessing in vivo haz- ards of ENMs is essential, including consideration of and differences between short-term, intermediate, and chronic exposures. Study designs should focus on developing tests with relevant ENM dosimetry and realistic doses (based on exposure data) and on time-course assessment to gauge the persistence of meas- ured end points. The cell types used should simulate in vivo point-of-entry ex- posure routes. Selection of relevant doses for cell types of secondary organs should be based on results of biokinetic studies. In the aggregate, those integrat- ed components are necessary for developing science-based in vivo predictability and extrapolation. With regard to acute-hazard ranking, HTS assays can be powerful, but present approaches for short-term and long-term hazard assess- ments and corresponding risk characterization have serious limitations. Fur- thermore, only a few long-term in vivo studies have examined more sensitive end points, such as reproduction and growth, and few funded studies other than 

64 Research Progress on EHS Aspects of Engineered Nanomaterials those supported through EPA–NSF funded centers have examined ecosystem effects. Some other subjects that remain underrepresented are toxicity mechanisms and pathways examined under realistic exposure conditions, exposure to mix- tures of contaminants, genotoxicity, and ecosystem effects of ENM exposures. Therefore, the committee designates progress in these fields as yellow.  Extent of adaptation of existing system-level tools (such as individual species tests, microcosms, and organ-system models) to support studies of na- nomaterials in such systems In its first report, the committee noted inadequate activity in this indicator. Specific studies would contribute to a better understanding of system-level ef- fects that can be induced by ENMs in an organism or in the environment. Adap- tation of existing system-level tools to support studies of isolated organ sys- tems—isolated perfused heart or lung, explant models (isolated vessels, including coronary vessels and aorta, and muscle), in vitro double- and triple- cell models, and complete constructs of airway epithelium—have been devel- oped and used in nanotoxicologic research, either through exposure of live or- ganisms to ENMs or through exposure of the isolated model systems directly (Nurkiewicz et al. 2008). Such models are useful for exploring mechanisms of specific effects, preferably if appropriate doses have been selected for exposure of the organism or the explant. Whole-organism environmental studies have been adapted to be used in nanotoxicology (Lovern and Klaper 2006; Bai et al. 2010; Galloway et al. 2010; H. Wang et al. 2009; S. Wang et al. 2011). Specific projects that are addressing systemwide effects of ENM exposures include studies of the use of microcosms and mesocosms to examine organism and ecosystem-level effects (Priester et al. 2012; Colman et al. 2013). Those studies demonstrate that steps toward meeting this objective have been initiated, but progress is confined to a few studies, and system-level effects remain largely unknown. The committee therefore deter- mined that this indicator is yellow.  Development of a set of screening tools that reflect important charac- teristics or toxicity pathways of the complex systems described above8 As noted in the committee’s first report, hazard-identification studies of a variety of ENMs have used both in vivo and in vitro methods. Development of a set of reliable and validated screening tools is critical in that adequate testing of individual ENMs used in commerce, each with different functionalities and ap- 8 This indicator was originally phrased as “Extent of refinement of a set of screening tools that reflect important characteristics or toxicity pathways of the complex systems described above” (NRC 2012, p. 182). 

Assessment of Progress 65 plications, is not practical. In general, results obtained from in vivo studies may have limited value for assessing health risks due to use of higher doses of ENMs than might be expected from real-world exposures and a focus primarily on acute responses. However, implementation of a spectrum of in vitro investiga- tions may ultimately hold promise for revealing important mechanistic insights into toxicity pathways. Optimizing the relevance of in vitro studies to toxicity considerations would require experimental designs that involve dose–response behavior over a full range of doses (very low to high) in relevant cell types, in- cluding time-course assessments and validation of findings with corresponding in vivo systems. Chapter 3 of the first report, “Critical Questions for Understanding Human and Environmental Effects of Engineered Nanomaterials”, posed the question (p. 91), What biologic effects occur at realistic ENM doses and dose rates, and how do ENM properties influence the magnitude of these effects? The report noted that a long-term goal is to develop simple in vitro assays that predict in vivo effects at the organism level and may eventually be used for HTS assays. To address that long-term goal, it was concluded that a key requirement should be that any in vitro assay used as a predictive tool needs to have been validated with appropriate and pertinent in vivo data (with particular relevance to expo- sure routes). The results of simple assays have been proposed for identifying potential effects and possibly establishing a hazard scale (Rushton et al. 2010), although some comparative studies have reported a lack of convergence between in vivo–related (inhalation or intratracheal instillation) findings and in vitro data on the same nanoparticle test materials, perhaps partly because of mechanisms that are dose-dependent (Slikker et al. 2004; Sayes et al. 2007, 2009; Warheit et al. 2009) or because of differential ENM transformations that depend on the exposure vehicle (medium) used (Lowry et al. 2012a). Finally, as currently de- signed, in vitro studies are limited by their inherent measurement of acute re- sponses. Even if they are conducted under relevant dose conditions, in vitro re- sults generally reflect early (acute) effects of exposures and may not predict long-term (chronic) effects. Research activity to correlate in vivo mechanistic toxicity studies system- atically at relevant concentrations with in vitro screening assays that use relevant exposure concentrations, ENMs, cell types, and appropriate routes of exposures (such as inhalation, oral, dermal, and intravenous exposure) is central to pro- gress. Some initial efforts have been proposed to address that issue in the Tox- Cast and NIEHS U199 programs. Laboratories are pursuing such research, and important insights into mechanisms of toxicity are being generated, but these efforts are not sufficient to provide the information necessary for adequate un- 9 U19 is part the National Institute of Environmental Health Sciences Centers for Nan- otechnology Health Implications Research. It is an interdisciplinary program that com- prises five U19 and three cooperative centers and other grantees and is intended to in- crease understanding of how the properties of ENMs influence their interactions with biologic systems and potential health risks. 

66 Research Progress on EHS Aspects of Engineered Nanomaterials derstanding of toxicity pathways in cell and organ systems. The committee des- ignates this indicator as red given the limited progress in appropriately designed studies.  Steps toward development of models for exposure and potential ecolog- ic effects10 Work on modeling exposure to and effects of nanomaterials in ecosys- tems, including food webs, is in its infancy. Important first steps have been tak- en to understand the phenomena of uptake, bioaccumulation, and trophic trans- fer (Werlin et al. 2011; Unrine et al. 2012), and this mixed progress gives this indicator a yellow rating. However, more work is required to understand the mechanisms of bi- ouptake. Lack of progress in modeling the transfer of materials between organ- isms can be attributed in part to the relatively low priority that this topic has received, as measured by publications, relative to work on direct health effects (see Figure 3-3 in NRC 2012). In addition, the modeling required is predicated on fundamental discovery concerning the mechanisms of biouptake and assimi- lation of ENMs in organisms. Greater focus on modeling of biouptake, bioac- cumulation, and trophic transfer is essential not only for predicting the fate of nanomaterials in ecosystems but for interpreting the growing body of literature on nanomaterial effects associated with ambient concentrations introduced in laboratory studies. In addition to uptake, more information is needed on the ef- fects of chronic, low-level realistic exposure scenarios in complex ecologic sys- tems. Effects of ENMs in a simplified assay may not accurately reflect the gross effects on a system of interconnected species. Alterations in uptake in the pres- ence of multiple species, population and community effects, changes in interac- tions among organisms, transformation of ENMs, and such changes in abiotic factors as nutrients because of nanomaterials are all important variables that need more research attention.  Identification of benchmark (positive and negative) and reference ma- terials11 for use in studies and measurement tools and methods to estimate expo- sure and dose in complex systems. 10 The indicator originally titled “Steps toward development of models for exposure and potential effects along the ecologic food chain” (NRC 2012, p. 182) was rephrased to broaden its scope to include all ecologic effects (both biotic and abiotic). 11 The committee differentiates between benchmark materials and reference materials. 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). Benchmark materials are well-characterized physicochemically and toxicologically, and can serve as positive or 

Assessment of Progress 67 The committee’s first report identified a pressing need to establish refer- ence materials for all aspects of nanomaterial-related research. Availability of toxicity benchmark materials (positive and negative) and reference materials for metrology are of high value for hazard ranking and risk assessment. NIST has issued two well-characterized, certified reference nanomaterials, TiO2 (P-2512) and gold (10, 30, and 60 nm), that could be used in toxicologic studies. The International Organization for Standardization defines a reference ma- terial as a “material, sufficiently homogeneous 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” (ISO 2006). Therefore, existing reference mate- rials are standards solely for material characterization (for example, NIST gold nanoparticles, standard reference material RM 8011, 8012, and 8013) or for standardizing measurement instrumentation. Benchmark materials for use in toxicologic and ecotoxicologic research need to be carefully characterized with respect to both physicochemical and toxicologic properties. The latter probably depend on several physicochemical properties (for example ENM size, charge, or in vivo solubility) that may make it necessary to establish more than one ref- erence material for hazard ranking. 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. 2008; Stone et al. 2010). Well-characterized benchmark ENMs should serve as refer- ences against which new and untested ENMs can be ranked as an initial step in hazard identification. Such information, with exposure data, can serve as a basis of risk assessment. The committee therefore believes that overall progress in this objective is inadequate and therefore has designated it red. INDICATORS OF PROGRESS IN IMPLEMENTATION Enhancing Interagency Coordination In its first report, the committee acknowledged the value of the coordinat- ing role played by the NNI and pointed to some changes that have enhanced interagency coordination, including the naming of an NNCO EHS coordinator by the Office of Science and Technology Policy (OSTP). However, the commit- tee concluded in its first report (p. 169) and continues to believe that accounta- bility for implementation of the NNI EHS research strategy is limited and ham- pered by the absence of an entity that has sufficient management and budgetary authority to direct implementation throughout NNI agencies and to ensure its integration with EHS research being undertaken in the private sector, the aca- demic community, and international organizations. Ensuring implementation of negative controls for comparing exposure-dose-response relationships of nanomaterials in toxicologic tests and in risk assessment. 12 P-25 is the product number for titanium dioxide. 

68 Research Progress on EHS Aspects of Engineered Nanomaterials the strategy and gauging progress in high-priority research also requires an as- sessment of the effectiveness of available mechanisms for interagency collabo- ration and frequent periodic identification—not just of recent, current, or newly initiated interagency research collaborations but also of funding needs. The committee’s assessment of progress against its two indicators for interagency collaboration is as follows.  Progress toward establishing a mechanism to ensure sufficient man- agement and budgetary authority to develop and implement an EHS research strategy among NNI agencies The committee reviewed the NNI’s 2011 research strategy and its 2013 budget supplement, which show considerable progress in coordination among NNI agencies on EHS research. Favorable developments include the addition of FDA and CPSC research programs to the NNI’s EHS budget “crosscut”, an in- creased focus by the Nanotechnology Environmental Health Implications work- ing group on identifying opportunities for cross-agency collaborations, joint solicitations and funding of research by multiple NNI agencies, and clearer tracking of research against the NNI’s broad goals and designated program component areas. The NNI strategy and budget documents also identify numer- ous plans to foster additional interagency collaboration, although the extent to which the plans have been or are being implemented appears to be limited on the basis of input received by the committee at its November 2012 workshop and discussions with NNCO staff. However, as highlighted in the committee’s first report, the need extends well beyond better coordination among NNI agencies, a role that the NNCO is fulfilling. Therefore, the committee identified as an indicator of progress the establishment of a mechanism that would have sufficient management and budgetary authority to ensure implementation of the NNI’s EHS research strate- gy. The committee has not discerned substantial progress on this indicator, so it is marked red. The committee is not alone in raising the need for a more centralized and accountable authority. In its fourth assessment of the NNI, the President’s Council of Advisors on Science and Technology (PCAST 2012) noted the “lack of integration between nanotechnology-related EHS research . . . and the kind of information policymakers need to effectively manage potential risks from na- nomaterials” (p. vi); it called on the NNI to establish “high-level, cross-agency authoritative and accountable governance” (p. viii) even as it acknowledged changes made to enhance coordination of research efforts among NNI agencies. Similarly, a recent Government Accountability Office report (GAO 2012) that reviewed the NNI’s research strategy and associated activities identified sub- 

Assessment of Progress 69 stantial instances of interagency research collaborations13 but also the absence of “outcome-related performance measures, targets or time frames that allow for monitoring and reporting on progress toward meeting the research needs” (p. 46). The 2012 PCAST review of the NNI made several specific recommenda- tions for OSTP to strengthen the NNCO “to broaden its impact and efficacy and improve its ability to coordinate and develop NNI programs and policies related to those programs” (p. 19; italics added). With respect to program management, the review noted that “PCAST is concerned that the agency representatives ap- pointed to the NSET Subcommittee do not have a level of authority within their agencies to influence budget allocations needed to meet NNI objectives” (p. vi), reiterating the 2010 PCAST recommendation that OSTP “require each agency in the NNI to have senior representatives with decision-making authority partici- pate in coordination activities of the NNI” (p. 39). The NNI’s response to the 2010 recommendation is included in its 2013 budget supplement (NSET 2012a) under the heading “Recommendations considered but actions unlikely or not needed” (p. 61); this indicates that the NNI considers its current structure to be sufficient and intends to maintain it. The committee remains concerned about the absence of a clear, central convening authority in the NNI structure and considers it a serious gap in the NNI’s ability to implement an effective EHS research strategy.  Extent to which the NNCO is annually identifying funding needs for in- teragency collaboration on critical high-priority research The NNI and its member agencies have made considerable progress to- ward increased collaboration in EHS research, including issuance of its 2011 EHS research strategy. As noted above, the committee’s review of the NNI’s strategy and the NNI’s 2013 budget supplement identified numerous examples of current and planned cross-agency collaborations. The examples demonstrate that multiple NNI agencies are and plan to continue jointly conducting intramu- ral research and jointly sponsor and fund solicitations for extramural research. The NNI has also instituted a clearer means of tracking in its budget supplement how current research aligns with its broad goals and new strategy. However, concerns have been raised by committee members that jointly funded research is not being managed jointly and that joint research solicitations have been rela- tively open-ended and not sufficiently strategically aligned to key research needs. The NNI’s signature initiatives (NNI 2012) offer another potential means of fostering collaboration in EHS research; although focused now only on nano- technology development, they encompass such efforts as the multiagency Nano- 13 A helpful list of collaborative agreements between NNI agencies is provided as Ap- pendix II of the GAO report. It is notable, however, that many of the agreements date back several years, and none is listed as having been initiated in 2012; this suggests that momentum in the activity may have waned recently. 

70 Research Progress on EHS Aspects of Engineered Nanomaterials technology Knowledge Infrastructure and the Nanoinformatics 2020 Roadmap, both of which are aimed at developing the infrastructure needed to collect, ana- lyze, and share nanotechnology-related information (NSET 2012b)—which could readily be extended to include EHS-related information. The committee continues to believe that accountability for fostering inter- agency collaboration in implementing the strategy requires not only identifying what collaborative research is under way or contemplated, but having in place a rigorous means of estimating periodically (ideally at least annually) the levels and sources of funding needed to ensure that interagency research efforts have sufficient funding to meet specific goals and complete high-priority research. That need echoes the calls by PCAST (2010, 2012) and GAO (2012) for the NNI to develop and implement better performance metrics that can be used to track progress against core objectives. The committee’s progress indicator fo- cuses on identifying funding needs for collaborative efforts between agencies to accelerate and enhance progress in high-priority research. The committee has not been made aware of any effort by the NNCO to develop such a mechanism and renews its call for the NNCO to do so. Because of the limited progress made in addressing this indicator, the committee denotes it yellow. Providing for Stakeholder Engagement in the Research Strategy  Progress toward actively engaging diverse stakeholders in a continuing manner in all aspects of strategy development, implementation, and revision This indicator represents a very high but achievable bar for stakeholder engagement, seeking broad engagement both in a continuing manner and in all aspects of the strategy. It seems clear that this high bar has not been cleared alt- hough the committee notes examples of progress. The stakeholder community includes government and academic researchers, nongovernment organizations, regulators, industry, nanotechnology workers, and of course consumers. The committee’s workshop sought to hear from representatives of each of those communities and did so successfully. Representatives of the various groups were well informed of the process and need but also frustrated by the lack of pull from the NNI EHS community for their involvement. Certainly, none could point to a recurring and inclusive forum for their involvement and participation. The committee, however, notes several examples of progress toward the goals. The committee workshop was one such example. Another, more promi- nent case is the NIOSH-sponsored Safe Nano by Design Conference, which took place in Albany, NY, in August 2012 (NIOSH 2012b) that was specifically fo- cused on NIOSH priorities. The creation and recurrence of similar forums that engage the range of EHS stakeholders would be a positive step. This indicator is yellow given that some progress has been observed. However, further engage- ment through such forums on all other aspects of strategy development, imple- 

Assessment of Progress 71 mentation, and revision with a more complete set of stakeholders would be a marker of substantial progress. Conducting and Communicating the Results of Research Funded Through Public–Private Partnerships  Progress toward establishment of effective public–private partnerships, as measured by such steps as completion of partnership agreements, issuance of requests for proposal, and establishment of a sound governance structure In its first report, the committee identified the need for public–private partnerships (PPPs) to help to implement the four broad, high-priority research categories of its research strategy. The need for PPPs is driven by the need to supplement and leverage federal funding and by the importance of having pri- vate stakeholders (such as manufacturers) actively involved in the research. For example, data on reference materials, nanomaterial product inventories, and the release of nanomaterials through the value chain are critical inputs into the re- search; one good way to provide such information accurately is to establish for- mal partnerships between government agencies, manufacturers, and other key stakeholders—such as academe—that are involved in implementing the research strategy. Progress in creating well-defined, effective partnerships as measured by partnership agreements, issuance of requests for proposals, and the establish- ment of governance structure is poor, so this indicator is red. NIOSH provides the closest examples. A summary report by NIOSH (2012a) covering the period 2004–2011 describes accomplishments and research findings from surveys in a research and development laboratory, in commercial nanoscale metal oxide pro- duction facilities, in a facility engaged in development of optical products with quantum dot coatings, and in a facility that spins nylon nanofibers. Other sur- veys included MWCNT manufacturers, metal oxide manufacturers, nano- enhanced silica iron absorbent manufacturers and additional diverse nanoscale- material producing laboratories. Those surveys do not represent formal PPPs, but they were performed on the basis of a NIOSH–manufacturer collaborative effort by conducting over 40 field assessments in nanomaterial manufacturer and user facilities. In another example, the Nanoparticle Occupational Safety and Health consortium—comprising 16 members in industry (for example, Procter and Gamble and DuPont), federal agencies (for example, NIOSH), and nonprofit organizations (Environmental Defense Fund)—tackled issues of the measure- ment of nanoparticles and the efficiency of filtration materials for engineered nanoparticles, evaluation of bioactivity of silicon nanowires in the consortium’s partnership with IBM, and understanding of and improvement in exposure con- trols for fullerenes and other engineered nanoparticles in its partnership with Luna Nano (NOSH 2007). Overall, the main impediments to creating PPPs are the lack of agreement on needed elements of a governance structure, disparate core objectives between 

72 Research Progress on EHS Aspects of Engineered Nanomaterials public and private entities, insufficient funding commitments from both gov- ernment and industry, and confidentiality concerns. In Chapter 4, the committee provides recommendations and examples of best practices to alleviate those road blocks. Managing Potential Conflicts of Interest In its first report, the committee noted that the NNI’s dual functions— developing and promoting nanotechnology and its applications and mitigating risks arising from such applications—pose tensions or even actual conflicts be- tween its goals. Manifestations of the tension previously noted by the committee included the vastly disparate allocation of resources between the two functions, the inadequacy of EHS risk research funding, and the NNI’s classification of research projects with respect to their “EHS relevance”. The committee believes that the tension can also affect the extramural research community, especially EHS risk researchers in large centers, the bulk of whose research funding is fo- cused on applications. To address what it saw as an inherent conflict, the com- mittee concluded that a clear separation in the management and budgetary au- thority and accountability between the functions was needed, and it identified two indicators for tracking progress in managing conflicts of interest. That con- clusion echoed that of a previous National Research Council report (2009), which noted that “a clear separation of accountability for development of appli- cations and assessment of potential implication of nanotechnology would help ensure that the public health implications has appropriate priority” (NRC 2009, p. 11).  Progress toward achieving a clear separation in management and budgetary authority and accountability between the functions of developing and promoting applications of nanotechnology and understanding and assessing its potential health and environmental implications The committee sees little progress in establishing clear and discernibly separate management and budgetary structure between the two potentially con- flicting functions in the NNI itself or the agencies that pursue or fund research on both applications and EHS risk implications of engineered nanomaterials. Therefore, this indicator is red. Both functions continue to operate under the same management and budget structures in the NNI and in its member agencies. In its first report, the committee noted possible models and mechanisms that could be used to separate accountability for the NNI’s dual functions, for exam- ple, elevating oversight of the EHS research portfolio in OSTP (NRC 2012; pp. 166–169), assigning responsibility and comparable authority for the two func- tions to different offices or to senior staff members in individual agencies or in the NNI itself (NRC 2012; pp.167, 173–174), or separating the two functions into independent entities—a model used elsewhere to address potentially con- flicting issues related to nuclear power (p. 174). The committee acknowledges 

Assessment of Progress 73 that in the absence of a change in its statutory mandate, the NNI would be hard- pressed to establish wholly separate management and budgetary structures and authorities for its dual functions. In the absence of such a change, the committee encourages the NNI and participating agencies to consider other approaches for managing perceived or actual conflicts of interest and biases. If not adequately addressed, such perceptions could undermine public trust and confidence in the research, technology, and government processes that are meant to ensure the health, and safety of ENMs.  Continued separate tracking and reporting of EHS research activities and funding distinct from those for other, more basic or application-oriented research The NNI has made considerable progress on this issue, commencing be- fore the committee issued its first report. That progress constitutes an impressive step toward creating the transparency noted above. The Office of Management and Budget (OMB) call to NNI agencies for detailed information on FY 2009 EHS research project funding facilitated easier identification of research projects most directly relevant to EHS risk. That data call helped to inform the NNI’s Environmental, Health, and Safety Research Strategy (NSET 2011). The NNI supplement to the president’s 2013 budget (NSET 2012a) also provides narra- tive information on agency-specific EHS research activities and projects. Despite the impressive progress, the tracking of EHS research progress and performance between and within NNI agencies remains challenging. As noted by GAO in its May 2012 report, performance information— such as out- comes, outputs, quality, timeliness, customer satisfaction, and efficiency—can inform such critical management decisions as priority-setting and resource allo- cation. Without project-specific information, researchers and other stakeholders have only a vague understanding of the research questions, methods, materials, and study populations being addressed through the NNI. Although periodic OMB data calls for EHS research project funding are helpful and could be made even more helpful if they included clearer guidance on how agencies should differentiate research directly relevant to EHS risk from applications research with EHS implications, they cannot address the need for a continuing (ideally annual) system for identifying and tracking EHS research projects and their per- formance. REFERENCES Aitken, R.J., S.M. Hankin, C.L. Tran, K. Donaldson, V. Stone, P. Cumpson, J. Johnstone, Q. Chaudhry, S. Cash, and J. Garrod. 2008. A multidisciplinary approach to the identification of reference materials for engineered nanoparticle toxicology. Nan- toxicology 2(2):71-78. 

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