Towards Predicting Nano-Biointeractions:
An International Assessment of Nanotechnology Environment,
Health and Safety Research Needs
International Council on Nanotechnology
Number 4
May 1, 2008
Rice University, MS-63, P.O. Box 1892, Houston, Texas 77251-1892 USA
Main Office-1.713.348.8210 Fax-1.713.348.8218
Table of Contents
Foreword ...........................................................................................................................................5
Executive Summary ..........................................................................................................................6
Workshop 1: Towards Nanomaterial Classes .................................................................... 7
Key findings ..................................................................................................................................... 7
Workshop 2: Towards Predicting Nano-Biointeractions................................................... 8
Key findings ..................................................................................................................................... 8
Cross-Cutting Issues ............................................................................................................ 8
Next Steps.............................................................................................................................. 9
Workshop Recommendations .........................................................................................................10
Research to Predict Nano-Biointeractions ....................................................................... 10
Research to Meet Risk Management Needs ..................................................................... 11
Overview: Nanotechnology and its Implications ...........................................................................11
Related Reports ...............................................................................................................................13
1. Workshop 1: Towards Nanomaterial Classes ................................................. 1-14
1.1. Workshop Overview ............................................................................................................ 1-14
1.2. Summary of Workshop Findings ........................................................................................ 1-15
1.3. Next Steps ............................................................................................................................ 1-16
1.4. Workshop Steering Team and Sponsors ............................................................................ 1-16
1.5. Workgroup Summaries ........................................................................................................ 1-16
1.5.1 Oxide Workgroup Summary .................................................................................. 1-16
1.5.1.1 Introduction ...................................................................................................................1-16
1.5.1.2 Common Oxide Particles ..............................................................................................1-17
1.5.1.3 Nanomaterial Properties and Biointeraction ................................................................1-17
1.5.1.4 Synthesis, Formulation and Manufacture .....................................................................1-18
1.5.1.5 Potential Hot Spots in the Nanomaterial Life ...............................................................1-18
1.5.1.6 Research Priorities ........................................................................................................1-18
1.5.1.7 General Questions and Concerns .................................................................................1-19
1.5.1.8 Backup Information .......................................................................................................1-19
1.5.2 Metals Workgroup Summary ................................................................................ 1-20
1.5.2.1 Introduction ...................................................................................................................1-20
1.5.2.2 Common Metallic Nanomaterials and Applications ....................................................1-20
1.5.2.3 Nanomaterial Properties and Biointeraction ................................................................1-20
1.5.2.4 Synthesis, Formulation and Manufacture .....................................................................1-21
1.5.2.5 Potential Hot Spots in the Nanomaterial Life ...............................................................1-22
1.5.2.6 Research Priorities ........................................................................................................1-22
1.5.3 Semiconductor Workgroup Summary ................................................................ 1-22
1.5.3.1 Introduction ...................................................................................................................1-22
1.5.3.2 Synthesis .......................................................................................................................1-22
1.5.3.3 Applications ...................................................................................................................1-23
1.5.3.4 Hot Spots .......................................................................................................................1-23
1.5.4 Carbon Workgroup Summary ............................................................................... 1-24
1.5.4.1 Introduction ...................................................................................................................1-24
1.5.4.2 Carbon Nanomaterials ..................................................................................................1-24
1.5.4.3 Exposure Scenarios During Manufacture/Synthesis ....................................................1-24
1.5.4.4 By-Products and Contaminants ....................................................................................1-25
1.5.4.5 Applications and Uses for Carbon Nanomaterials .......................................................1-25
1.5.4.6 Exposure Scenarios During Use of Carbon Nanomaterials ........................................1-25
1.5.4.7 Improving Exposure Assessment ...................................................................................1-26
1.5.4.8 Prioritization of Research Needs ..................................................................................1-26
International Council on Nanotechnology
2007 Workshop Report
1.5.4.9 Carbon Nanoparticle Hazard ........................................................................................1-26
1.5.5 Macromolecule Workgroup Summary ................................................................. 1-27
1.5.5.1 Introduction ...................................................................................................................1-27
1.5.5.2 Nanomaterial Properties and Biointeraction ................................................................1-27
1.5.5.3 Synthesis, Formulation and Manufacture, and Application and Use ...........................1-28
1.5.5.4 Potential Hot Spots in the Nanomaterial Life ...............................................................1-29
1.5.5.5 Prioritized Research Needs Recommendations .............................................................1-29
1.5.6 Self-Assembly Workgroup Summary ................................................................... 1-30
1.5.6.1 Introduction ...................................................................................................................1-30
1.5.6.2 Lipid Assemblies ............................................................................................................1-30
1.5.6.3 Nanocomposite Assemblies ...........................................................................................1-32
1.5.6.4 Prioritized Research Needs Recommendations .............................................................1-34
1.6. References Cited ................................................................................................................... 1-34
Appendix A: Workshop 1 Agenda .............................................................................................. 1-35
Appendix B: Workshop 1 Attendees ........................................................................................... 1-36
2. Workshop 2: Towards Predicting Nano-Biointeractions ................................ 2-38
2.1. Workshop Overview ............................................................................................................. 2-38
2.2. Summary of Workshop Findings ........................................................................................ 2-40
2.2.1 Next Steps ............................................................................................................... 2-42
2.3. Summary Research Needs and Timetables ......................................................................... 2-42
2.3.1 Research to Predict Nano-Biointeractions .......................................................... 2-42
2.3.1.1 Characterization of Nanomaterials ................................................................................2-42
2.3.1.2 Standard Terminology ....................................................................................................2-43
2.3.1.3 Standard Reference Nanomaterials ...............................................................................2-43
2.3.1.4 Techniques for Detecting Nanomaterials in Biological Media ......................................2-43
2.3.1.5 In Vivo Tests and Correlation to In Vitro Tests .............................................................2-44
2.3.1.6 In Vitro Testing ...............................................................................................................2-44
2.3.1.7 Model Development ........................................................................................................2-45
2.3.2 Metrology for Risk Management ........................................................................... 2-45
2.3.2.1 Assessment of Bioavailability throughout the Lifecycle .................................................2-46
2.3.2.2 Characterization of Potential Mobility of Embedded Nanomaterials ...........................2-46
2.4. Common Themes ................................................................................................................. 2-46
2.4.1 Need to Correlate Nanoparticle Physicochemical Properties with Interactions
in Organisms and the Natural Environment ........................................................ 2-46
2.4.2 Importance of Dose and Dose Rate in Understanding Biointeractions ............ 2-47
2.4.3 Need for Well-Characterized Reference Materials and Standardized Assays,
and New Assay Development ............................................................................... 2-47
2.4.4 Reproducibility of Research and Better Documentation of Methods to
Improve the Quality and Comparability of the Science ...................................... 2-47
2.4.5 Methods for Predicting Potential Long-Term Effects ......................................... 2-48
2.4.6 Metrology for Locating and Characterizing Nanomaterials in Biological
Organisms and Samples ....................................................................................... 2-48
2.4.7 Metrology for Exposure Characterization ........................................................... 2-48
2.5. Workshop Steering Team and Sponsors ............................................................................. 2-48
2.6. Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of
Nanoparticles with Biological Organisms ......................................................................... 2-49
2.6.1 Breakout Group 1A: Oxidative Stress, Inflammation, and Immune Response 2-49
2.6.1.1 Background ...................................................................................................................2-49
Table of Contents
2
International Council on Nanotechnology
2007 Workshop Report
2.6.1.2 Important Considerations for Engineered Nanomaterial Biology Research ................2-50
2.6.1.3 Mechanisms of Cellular Response: The Signaling Pathways of Oxidative Stress,
Inflammation, and Immunity ..........................................................................................2-51
2.6.1.4 Research Needs .............................................................................................................2-52
2.6.2 Breakout Group 1B: Protein Misfolding/Biomolecules ...................................... 2-53
2.6.2.1 Background ...................................................................................................................2-53
2.6.2.2 Summary of Group Discussion ......................................................................................2-54
2.6.2.3 Research Needs .............................................................................................................2-56
2.6.3 Breakout Group 1C: Apoptosis and Necrosis ..................................................... 2-56
2.6.3.1 Background ...................................................................................................................2-56
2.6.3.2 Impact of Nanoparticles on Cell Integrity .....................................................................2-58
2.6.3.3 Research Needs ............................................................................................................2-59
2.6.4 Breakout Group 1D: Genotoxicity and Mutagenicity .......................................... 2-59
2.6.4.1 Background ...................................................................................................................2-59
2.6.4.2 Testing Strategies ..........................................................................................................2-60
2.6.4.3 How to Go Forward ......................................................................................................2-60
2.6.4.4 Research Needs .............................................................................................................2-61
2.6.5 Breakout Group 1E: Developmental Effects ........................................................ 2-62
2.6.5.1 Background ...................................................................................................................2-62
2.6.5.2 Summary of Discussions ................................................................................................2-63
2.7. Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living
Organisms ........................................................................................................................... 2-64
2.7.1 Breakout Group 2A: Nanoparticle-Biofluid Interactions/Target Cell
Interactions ............................................................................................................. 2-65
2.7.1.1 Background ...................................................................................................................2-65
2.7.1.2 Definitions .....................................................................................................................2-65
2.7.1.3 Research Needs .............................................................................................................2-66
2.7.2 Breakout Group 2B: Cell Signaling and Communication ................................... 2-67
2.7.2.1 Background ...................................................................................................................2-67
2.7.2.2 Summary of Discussions ................................................................................................2-67
2.7.3 Breakout Group 2C: Whole Animal Interactions—Biokinetics .......................... 2-68
2.7.3.1 Background ...................................................................................................................2-68
2.7.3.2 Research Needs .............................................................................................................2-69
2.7.3.3 Research Program .........................................................................................................2-69
2.7.3.4 Prioritized Objectives ....................................................................................................2-69
2.7.4 Breakout Group 2D: Ecotoxicology ..................................................................... 2-70
2.7.4.1 Background ...................................................................................................................2-70
2.7.4.2 Research Needs .............................................................................................................2-70
2.7.4.3 Sources and Routes of Release into the Environment ....................................................2-71
2.7.4.4 Detection and Quantification in the Environment .........................................................2-71
2.7.4.5 Characterization of Nanomaterial Physicochemical Properties ...................................2-72
2.7.4.6 Transportation, Transformation, Fate Leading to Exposure .........................................2-72
2.7.4.7 Effects on Organisms: Microorganisms, Invertebrates, Vertebrates, Plants ................2-72
2.7.4.8 Impact of Nanomaterials on the Development of Resistant Bacterial Strains ...............2-72
2.7.4.9 Nanomaterial’s Environmental Protection Capability ..................................................2-73
2.8. References Cited ................................................................................................................... 2-73
Appendix C: Workshop 2 Agenda .............................................................................................. 2-76
Appendix D: Workshop 2 Attendees ........................................................................................... 2-78
Table of Contents
3
Copyright © 2008, International Council on Nanotechnology
About the International Council on Nanotechnology (ICON)
ICON is an international, multistakeholder organization whose mission is to develop and communicate information regarding potential environmental and health risks of nanotechnology, thereby fostering
risk reduction while maximizing societal benefit. ICON was founded in 2004 as an extension of the U.S.
National Science Foundation Center for Biological and Environmental Nanotechnology (CBEN) at Rice
University in Houston, Texas. ICON is a knowledge-driven organization. It does not engage in advocacy or
commercial activities. More information about ICON can be found at http://icon.rice.edu.
Sponsorship
These workshops were jointly sponsored by the International Council on Nanotechnology, the U.S.
National Science Foundation [BES-0646107] with support from the U.S. National Institutes of Health and
the Swiss Reinsurance Company.
Distribution Information
This document is the work and property of the International Council on Nanotechnology. Text and
images may be copied and distributed freely as long as they are not modified and acknowledgement to the
ICON is made as follows: “Credit: the International Council on Nanotechnology, Rice University, Houston, Texas, http://icon.rice.edu.” Permission to reproduce all other images must be sought from the
originating source.
Suggested Citation
Towards Predicting Nano-Biointeractions: An International Assessment of Nanotechnology Environment, Health and Safety Research Needs. International Council on Nanotechnology, Rice University,
Houston, Texas. May 2008, No. 4.
Disclaimer
Unless otherwise stated, any views or opinions expressed in this statement do not necessarily represent those of a specific company, university, organization, or governmental agency. Individuals who
participated in these workshops did so as content experts and not as official representatives of their respective agencies, companies, or organizations.
International Council on Nanotechnology
2007 Workshop Report
Foreword
“Imagine you had the intellectual capital of all the nations on earth, strong support, and a large—
not infinite—budget to fund research that led to a deeper understanding of the impacts of nanotechnology.
What would you do?”
This was the fundamental question posed to the participants in two international workshops aimed at
defining a set of research needs for the nascent area of nanotechnology. The creation of new knowledge has
long been associated with technological innovation—without new ideas, how can society expect to benefit
from creative solutions to its most pressing problems? Perhaps less apparent is the critical role that the
research enterprise plays in defining and managing the possible environmental and health risks of its
inventions. No new technology has zero risk, and decision-makers, whether they are in a corporation,
supermarket, or government agency, understand this reality. But for them to take full advantage of the
potential of new technologies, they must have access to solid research results that permit them to
understand, quantify, and manage any risks.
Coordinated research into the risks of emerging technology, particularly nanotechnology, is
uncharted territory. Historically, the risks are assessed after technologies are deployed, when specific risks
are documented in defined settings and use patterns. Proactive research into risk is not about assessing a
single documented risk, but rather about filling out a more complex risk landscape that addresses a range of
possible scenarios of technology use and settings. Understanding how to construct and develop a research
program to accomplish this aim was the motivation for the work described in this report.
Nanotechnology was the specific focus area for the participants who helped define this report. Good
strategy depends critically on defining a desirable end goal, and as noted by groups before ours, there are
several overarching outcomes for nanotechnology and research into its impacts. To focus these workshops,
we oriented around one particular outcome: a framework for predictive models for nanotechnology’s
impacts. With the “what” being firmly established prior to the events, the groups were free to focus on the
“how”—that is, 2-, 5-, and 10-year goals that when taken together would result in tools to help all stakeholders characterize the impacts of emerging nanotechnologies possibly even before they are created. I
hope that the specific recommendations for strategy contained in this report can be used to structure various research programs around the world.
I also hope that readers of this document appreciate the “how” by which this document was produced—with open, equal, and truly shared ownership by stakeholders from many groups at all steps—
because the diversity of the participants and collaborative process in which they engaged could have significant implications for the future. Whether and how nanotechnology is used will not be the result of a single
decision made by a monolithic group. Rather it will be shaped by a thousand smaller decisions made by
individuals in academia, industry, government, and nongovernmental organizations (NGOs). Creating
common understanding, shared vision, and coordination among these diverse groups is thus essential to
defining a path for nanotechnology commercialization that earns the confidence of and acceptance by
many, if not all, international stakeholders.
The leadership of the International Council on Nanotechnology (ICON) in these workshops facilitated the engagement of multiple stakeholders—73 in all—that will be essential to the success of a
coordinated international research effort. All of ICON’s programs use diverse teams that follow transparent
processes to generate, plan, and ultimately implement projects of shared interest. The workshops whose
results are reported here were conceived in 2005, arising naturally out of other ICON activities toward
international coordination and research priorities for nanotechnology’s risk research. This report synthesizes the discussions, presentations, and identified research needs from these efforts.
Professor Vicki Colvin, Executive Director of ICON
Foreword
5
International Council on Nanotechnology
2007 Workshop Report
Executive Summary
This report presents the results and recommendations from two international workshops centered on a global research strategy for understanding nanotechnology’s environmental and health
impacts. More specifically, the overarching goal of the workshops was to develop a framework for predicting the interactions between engineered nanoparticles and biological systems at the molecular level so that
biocompatible nanomaterials can be developed and applied safely. Convened by ICON, the workshops
were held in Bethesda, Maryland, USA in January 2007, and in Rüschlikon, Switzerland in June 2007.
Each workshop brought together more than 50 experts, mostly scientists, representing diverse stakeholder
groups including academia, industry, governments, NGOs, and 13 countries. All participants are involved
in fields relevant to the workshop topics, including biology, computational modeling, toxicology, materials
science, biophysics, and environmental science. Cosponsors of the workshop included ICON and the
National Science Foundation (NSF) of the USA, with significant in-kind support provided by the U.S.
National Institutes of Health (NIH) and Swiss Reinsurance Company.
The grand challenge of producing computational models that predict interactions of engineered nanoparticles with organisms is a long-term challenge of at least 10 years. The ICON Research
Needs Assessment workshops were structured to approach the challenge systematically, breaking it down
into component areas that will ultimately produce predictive models that will enable all users of nanotechnology to better characterize its impacts, improve risk mitigation processes, and rationally design
biocompatible nanomaterials. Fundamental to the challenge, as defined by workshop organizers, is the
identification of the physicochemical properties of nanomaterials and establishment of links between such
properties and their biological impacts. Accordingly, the goals of the first ICON workshop (Towards Nanomaterial Classes) were to identify preliminary classes of nanomaterials with common properties and to
identify for these classes potential “hot spots” in their life cycle. The results from Workshop 1 served as
input to Workshop 2 (Towards Predicting Nano-Biointeractions), which had as its goal to define research
strategies for developing predictive models of engineered nanomaterials’ interactions with biological
systems.
No other research effort on potential nanotechnology risks has taken up this challenge, nor has
any other research strategy been a product of collaboration among such a diverse group of international participants. The workshop participants were charged with defining the research needs, or
milestones, required to produce predictive models of an engineered nanoparticle’s biological effects. The
research needs were constrained not to be so large as to require a decade or more of funding from multiple
sources. They were also written not to be so detailed as to define single investigator or even larger collaborative programs. In many ways, these research needs were developed to be at the level of (but not to serve
as actual) requests for proposals (RFPs) that could be developed by agencies worldwide—topically
focused with terms of several years, and engaging multiple investigators in distantly related activities.
The identified research needs and activities comprise recommendations for progressive
research within specific timeframes largely toward predictive models for nanomaterial risks. Altogether, 26 research needs for predicting nano-biointeractions were identified (Figure 1, page 10). In
addition, a second set of six research needs was identified for risk management (Figure 2, page 11). Such
research is necessary to inform policy makers about the adequacy of current detection and protection measures in workplaces where engineered nanomaterials are present and to inform researchers as to the
potential magnitude of exposure related to these materials. Identified research needs from the various
workgroups were combined and refined to avoid redundancy. Each research need in Figures 1 and 2 provides a link between the grand challenge and actionable programs for the near-term (2-year), mid-term (5year), and long-term (7- to 10-year). Details on research needs and milestones are contained in the workshop reports that start on page 14.
Executive Summary
6
International Council on Nanotechnology
2007 Workshop Report
The workshops were intended to explore and express the general views of a broad group of individuals rather than to achieve a consensus position. Drafts of the workshop report were generated by the
workshop steering teams, facilitators, and scribes, and then circulated among the participants for review.
The final product is a reflection of the wide-ranging discussions that occurred over the course of the two
events and should not be construed as expressing official positions of the organizations employing the
workshop participants.
The outputs of the ICON Research Needs Assessment workshops build on previous work toward
research agendas for biointeractions of nanomaterials. Several reports on such work are included in this
document’s references, among them Maynard et al.10 and Balbus et al.12
Workshop 1: Towards Nanomaterial Classes
Participants in this workshop began by discussing what is known about key classes of nanomaterials
and exploring whether physical and chemical properties are adequate for determining biointeractions. The
participating experts explored options for classifying the physical and chemical properties of engineered
nanomaterials that could affect biointeractions; assessed these properties for nanoparticles composed of
oxides, semiconductors, metals, carbon, macromolecules, and self-assembled materials; and identified
potential areas of concern (“hot spots”) for current and future applications, volumes, exposure, and hazard
throughout the nanoparticle life cycle.
Key findings
•
•
•
While participants of this workshop were asked to classify nanomaterials based on the
physicochemical properties that could affect bioactivity, it became clear that this was not possible
with the available body of knowledge. As the experts noted, subtle changes in structure, surface
structure, and composition could dramatically affect electronic and chemical properties over the
material’s life cycle. Many properties can be dramatically altered during nanomaterial synthesis,
functionalization, or at other points during the product-manufacturing process. Furthermore,
because nanoparticles change as they interact with living systems, it is unlikely that their
physicochemical properties at any one stage in the life cycle alone will predict biological
behavior. Therefore, the first recommendation from Workshop 1 was that tools and models must
be developed that can describe the dynamic nature of nanomaterials throughout their life cycle.
The best potential mechanisms to characterize nanomaterial properties at various stages of the life
cycle would be physical/chemical screens and select in vitro tests to determine chemical reactivity,
surface charge, surface composition, and solubility. These screens would need to be correlated to
transport properties and biointeractions in full biological tests in order to identify nanomaterials
that need detailed testing. In short, a set of screening tools is needed to correlate the functional
properties of nanomaterials with their potential for biological interaction.
Current information suggests some general conclusions regarding exposure potential to nanomaterials. For nanomaterials in a dry powder form, potential for exposure to high concentrations is
greatest during the cleaning of synthesis reactors, bagging operations, surface functionalization,
and formulation areas of manufacturing. Nanomaterials in liquid form present possible topical
application or inhalation exposures during manufacturing or product applications. Nanomaterials
bound in a liquid or solid matrix would have a lower potential for exposure than an unbound
nanomaterial. Little is known, however, about whether the physical form of a nanomaterial or its
chemical composition is most important in evaluating net dose for its various biointeractions.
Therefore, exposure assessment studies are needed to lead to predictions about physicochemical
properties and their implications for net dose.
Executive Summary
7
International Council on Nanotechnology
2007 Workshop Report
Workshop 2: Towards Predicting Nano-Biointeractions
The second workshop focused on identifying the research needs and milestones to inform predictions of an engineered nanoparticle’s biological effects, and on defining strategies to develop predictive
models of engineered nanomaterials’ interactions with biological and environmental systems. The first
breakout session focused on the mechanism of an organism’s response to stress induced by a nanomaterial,
and identified additional interactions of the nanomaterial with the recovery pathway. The second breakout
session identified the research needed to develop predictive models of interactions with biofluids, cells and
tissue, whole animals, and the environment. Detailed discussions focused on nano-biological interaction
mechanisms such as oxidative stress, inflammation and immune response, protein misfolding, apoptosis
and necrosis, genotoxicity and mutagenicity, and developmental effects at cell-free, cellular, tissue and
whole-animal levels. Research needs were identified for all of the areas of understanding and for determining interactions between in vivo and in vitro research to develop predictive models. Many of the needs are
not unique to nanomaterials but are required for progress in predictive chemical toxicology.
Key findings
•
When a nanoparticle is put into a biological fluid or the environment, it becomes coated with biomolecules in a complex and dynamic manner that is not well understood. For predictive modeling, nano-environmental, health, and safety (nano-EHS) researchers must be able to identify
what biomolecular interactions will dominate in a given environment and what the particle “identity” likely will be. To gain this information, quantitative models are needed to describe how the
physicochemical properties of nanoparticles control the nature and extent of biomolecular interactions at their surface.
•
With nanoparticles’ large ratio of surface area to mass, the traditional mass-based measure of
dose may result in higher than expected concentrations of nanoparticles at a cell membrane or
other biological structure. These high concentrations could result in new types of interactions
that would not occur at lower concentration. Therefore, it will be important to establish thresholds of interaction and to validate concentration measurements against them. Dose and dose rate
may need to be validated independently for nanomaterials.
•
Many of the research challenges for this area overlap with the larger needs of the toxicology
community. The workshop participants noted that for screening, especially given the sheer diversity of nanoparticles, in vitro assays would be essential. However, there was a strong recognition
that such datasets may not predict outcomes in animals. Specific research designed to develop
better biomarkers, or sets of biomarkers, is thus essential to address the vast diversity of nanoparticle types and to develop strong correlative models for predicting in vivo data based on in
vitro results.
Cross-Cutting Issues
Researchers in both workshops agreed that the most immediate barrier to realizing the grand challenge of creating predictive models for interactions between engineered nanoparticles and biological
systems is the lack of defined and shared research practice. Toward this, researchers must agree on a common language and general good practices for engineered nanoparticle characterization—especially with
respect to purity, biological endpoint assessment, and data-reporting structures. Reference materials were
widely discussed by participants as one way to address these issues; others recognized the importance of
workshops and face-to-face meetings of scientists to develop standard practices. Without agreement on
Executive Summary
8
International Council on Nanotechnology
2007 Workshop Report
definitions and common experimental practices, research across the world will be difficult to integrate and
interpret.
While not formally on the agenda, risk management practices emerged repeatedly in both workshops, especially as they relate to potential exposures and exposure assessment. Identified by participants
were needs for metrology and tools to characterize and measure nanomaterials, and to monitor their presence in the environment and in biological media used in research. Test methodologies to characterize the
potential mobility of embedded nanomaterials also were called for, as was the basic need for characterization of nanomaterials, evaluation of the appropriateness of in vitro tests to characterize nanomaterial
interactions more broadly, standardization for biological materials used in testing, and the creation of a
data-sharing framework to accelerate development of models. Further discussion of these crosscutting
issues can be found in Common Themes section of Workshop 2 (page 46). Because time frames for
research needs also were addressed in Workshop 2, please refer to page 42 for further discussion and detail
related to risk management.
Next Steps
A coordinated international research effort will be needed to formulate tools for predicting engineered nanoparticle interactions with organisms. The scope of the materials under consideration, the
diversity of possible exposure scenarios, and the quantification of biological response are all daunting challenges to achieving this outcome. Furthermore, efforts should be established to coordinate the collection
and dissemination of biointeraction knowledge from research in such diverse areas as medical diagnostics
and treatment applications and interaction studies for consumer applications.
Workshop 1 participants noted that further deliberative efforts with similar programming would be
required to provide detail for many of the specific research needs identified as significant obstacles to producing predictive models (Figure 1). Among the areas that need further investigation are 1) reviewing the
individual workgroup research priorities to assess their broader applicability, 2) identifying screening strategies to measure physical (size, shape, nanostructure) and chemical (composition, chemical reactivity,
particle surface charge, solubility, surface composition) properties, 3) establishing agreement on a set of
functional screens and identifying tests to determine whether these properties correlate to biointeraction
potential, 4) assessing potential for nano-biointeractions with a wider range of biological systems, and 5)
identifying research needed to develop predictive models of nano-biointeraction.
At the end of the second workshop, participants also brainstormed on the subject of potential future
directions. Some of these recommendations were for activities that ICON might be in a position to organize, including future workshops that begin to address one or more specific needs identified by the group,
and others for activities that might be taken up by other groups or organizations. The following activities
were advanced as potential future directions:
•
•
•
•
•
an international effort to identify promising metrology and methodologies for monitoring nanomaterials in the workplace and the environment
an international effort to identify tools for detecting the presence and characteristics of nanomaterials in biological systems
an effort to develop a minimum set of experimental data to be submitted with a technical manuscript (such as the Minimum Information About a Microarray Experiment [MIAME] protocols)
to allow for greater reproducibility and comparison of nano-biointeractions research
an effort to identify model biological systems and model nanoparticles for nano-biointeractions
research
a workshop to identify a number of functional materials properties, including chemical screens,
reactivity, charge, and solubility.
Executive Summary
9
International Council on Nanotechnology
2007 Workshop Report
Workshop Recommendations
Research to Predict Nano-Biointeractions
NM = Nanomaterial
Figure 1.
Workshop Recommendations
10
International Council on Nanotechnology
2007 Workshop Report
The ICON workshops identified 26 types of research needed to predict nano-biointeractions. All of
these identified areas will contribute to the ultimate, 10-year goal of this effort—also referred to as the
grand challenge—to produce computational models for predicting nano-biointeractions.
Research to Meet Risk Management Needs
NM = Nanomaterial
Figure 2.
Figure 2 lists the research needed to enable improved risk management of nanomaterials and enable
predictive models of nanomaterial biointeractions based on nanomaterial physicochemical properties.
Resulting information will inform policy makers about the adequacy of current detection and protection
measures in workplaces where engineered nanomaterials are present and will inform researchers as to the
potential magnitude of exposure related to these materials, a key component of predictive models.
Overview: Nanotechnology and its Implications
Nanotechnology is an emerging area of technology development involving structures that measure
from 1–100 nm in one or more dimensions. While precise definitions are still somewhat variable, most recognize that nanotechnology involves science and engineering of matter at the nanoscale where properties
may change with size or new properties may emerge. Nanoparticles are small pieces of matter within the
nanoscale range and constitute an important area of nanotechnology research and development. A subset of
this research involves engineered nanoparticles, which are intentionally manufactured to have specific
properties or composition, in contrast to incidental nanoparticles, which are generated inadvertently or
through natural processes. Engineered nanoparticles include such materials as quantum dots (QDs—semiconductor nanoparticles), nanotubes (carbon nanoparticles), and nanotitania (oxide nanoparticles), among
many others. Smaller than microscale particles, yet larger than atoms and many molecules, nanoparticles
occupy a transitional regime between classical and quantum physics where physical and chemical properties are somewhat tunable with changes in size, structure, composition, surface structure, and surface
composition.
This flexibility presents the nanomaterials scientist with a toolbox for tailoring material properties to
a specific application. For example, through some rather rudimentary chemistry we can make a nanoparticle glow one color or another, make it electrically conductive or semiconductive, enable or hinder its
Overview: Nanotechnology and its Implications
11
International Council on Nanotechnology
2007 Workshop Report
penetration through a cell membrane, or enable multiple properties simultaneously. Thus, engineered
nanoparticles are now making their way into the marketplace in familiar consumer products such as sporting goods and personal care products, as well as more high-end applications such as targeted drug
therapies. Numerous predictions have been made about the total market for products containing engineered
nanoparticles, all of which forecast enormous growth.
The prospect of rapid and massive scale-up of the use of engineered nanoparticles has raised some
concerns about the potential for new problems to emerge, in particular with respect to EHS issues. A relatively small but growing body of scientific research has demonstrated that engineered nanoparticles can
interact in biological organisms and the natural environment in ways that cannot easily be predicted. Moreover, the very properties that application developers seek to exploit, such as the ability to enhance
photocatalysis or cross a cell membrane, may cause unanticipated effects that have yet to be fully documented. If engineered nanoparticles have the potential to impact living organisms or the environment, we
must understand better how to maximize the beneficial aspects and minimize undesirable outcomes of
these interactions through improved risk assessment and management processes. Doing so will enable
technology developers to exploit the full promise of nanotechnology and to solve existing problems in
medicine or environmental remediation, as well as other application areas, without introducing new modes
of toxicity or contamination.
The unique properties and potential mobility of engineered nanoparticles, along with the lack of
mobile monitors to detect their presence, pose significant challenges to the development of best practices
for nanomaterial handling throughout the life cycle. Extrapolating from health and safety data available for
a larger-scale material may fail to capture the nanoscale analog’s interactions. Nanoparticles’ diversity and
tunability make it difficult to predict their behavior. The interaction of an engineered nanoparticle with a
cell, for example, can change dramatically with small changes in size, shape, or surface properties, such as
may occur during the nanoparticle’s incorporation into a product or as a result of introduction into the
body, even if the chemical composition of the base nanoparticle is constant. Testing each different variant
of a nanoparticle, even if limited to those of commercial relevance, is impractical. A better understanding is
needed of the structure-activity relationships (SARs) of nanoparticles themselves, particularly those with
potential for high exposure or high-volume application in current and future products, so that we can proceed with greater confidence that the EHS issues have been identified and can be managed.
As nanoparticles with new properties are discovered and designed with more complex functionality,
a scientifically based hierarchy of risk assessment is needed to develop handling protocols that expand in
scope as the nanomaterial progresses from research to development to product. Until predictive models are
developed, risk assessors will need knowledge of the potential interaction of the nanomaterial with biological organisms and the environment at each stage of the life cycle. Thus, an understanding of the functional
properties that correlate with the biological response is needed. There is also a need for metrology and
monitoring equipment to readily detect the presence or absence of nanoparticles in the laboratory, workplace, and environment.
Overview: Nanotechnology and its Implications
12
International Council on Nanotechnology
2007 Workshop Report
Related Reports
1.
Dowling, A. et al. Nanoscience and nanotechnologies: Opportunities and uncertainties. The Royal
Society and the Royal Academy of Engineering, (2004). http://www.nanotec.org.uk/finalReport.htm
(accessed July 29, 2004).
2.
Aitken, R.J., K.S. Creely, and C.L. Tran. Nanoparticles: An occupational hygiene review (Research
Report 274). (2004). http://www.hse.gov.uk/research/rrpdf/rr274.pdf (accessed December 1, 2004).
3.
Hett, A. Nanotechnology: Small matter, many unknowns. Swiss Reinsurance Company, Zurich,
Switzerland (2004). http://www.swissre.com/resources/31598080455c7a3fb154bb80a45d76a0Publ04_Nano_en.pdf
4.
Small sizes that matter: Opportunities and risks of nanotechnologies. Allianz Center for Technology,
(2005). http://www.oecd.org/dataoecd/4/38/35081968.pdf (accessed June 18, 2005).
5.
Strategic plan for NIOSH nanotechnology research: Filling the knowledge gaps. National Institute
for Occupational Safety and Health (2005). http://www.cdc.gov/niosh/topics/nanotech/pdfs/
NIOSH_Nanotech_Strategic_Plan.pdf (accessed September 28, 2005).
6.
Oberdörster, G. et al. Principles for characterizing the potential human health effects from exposure
to nanomaterials: Elements of a screening strategy. Particle and Fibre Toxicology 2 (8) (2005).
7.
Opinion on the appropriateness of existing methodologies to assess the potential risks associated
with engineered and adventitious products of nanotechnologies. Scientific Committee on Emerging
and Newly Identified Health Risks (2005). http://ec.europa.eu/health/ph_risk/committees/
04_scenihr/docs/scenihr_o_003.pdf (accessed September 2005).
8.
National Nanotechnology Initiative. Environmental, health, and safety research needs for engineered
nanoscale materials. Nanotechnology Environmental and Health Implications Working Group,
National
Science
and
Technology
Council
(2006).
http://www.nano.gov/
NNI_EHS_research_needs.pdf
9.
Wooldridge, R. and J. Solomon. Joint NNI-ChI CBAN and SRC CWG5 nanotechnology research
needs recommendations. Vision 2020 Chemical Industry Technology Partnership and Semiconductor Research Corporation (2006). http://www.chemicalvision2020.org/pdfs/chem-semi%20ESH%20
recommendations.pdf (accessed January 1, 2006).
10.
Maynard, A.D. et al. Safe handling of nanotechnology. Nature 444 (7117) 267-269 (2006).
11.
Nanoscale Science, Engineering, and Technology Subcommittee. Prioritization of environmental,
health and safety research needs for engineered nanoscale materials: An interim document for public
comment. Nanotechnology Environmental and Health Implications Working Group, National Science and Technology Council (2007). http://www.nano.gov/Prioritization_EHS_Research_Needs_
Engineered_Nanoscale_Materials.pdf
12.
Balbus, J.M. et al. Hazard assessment for nanoparticles: Report from an interdisciplinary workshop.
Environmental Health Perspectives 115 (11) (2007). http://dx.doi.org/doi:10.1289/ehp.10327
(accessed August 14, 2007).
Related Reports
13
1.
Workshop 1: Towards Nanomaterial Classes
January 9–10, 2007
National Institutes for Health
Bethesda, Maryland, USA
1.1.
Workshop Overview
The goals of the first ICON Research Needs Assessment Workshop (Workshop 1), held from January 9–10, 2007, in Bethesda, Maryland, were to identify preliminary classes of nanomaterials with
common properties, to identify for these classes potential “hot spots”a in their life cycle, and to identify the
research needed to improve our ability to classify materials according to functional properties and exposure
potential. Workshop 1 included 65 participants from diverse international stakeholder groups in academia,
government, industry, and NGOs.
The meeting started with several presentations to set the stage for the workgroup discussions. Steve
Brown identified the importance of establishing principles of interaction for different classes of nanomaterials with biological organisms to enable development of improved practices for assessing the risk of new
nanomaterials. Andrew Maynard presented an overview of nanomaterial properties that may be important
in biointeraction based on early research results. Dr. Maynard’s presentation highlighted the potential
importance of size, shape, nanostructure, composition, surface charge, chemical reactivity, and other properties in biotransport and reactions with biological organisms. He also identified the need for establishing
definitions for nanomaterial classifications based on physical, structural, and chemical properties of nanomaterials. Vicki Colvin presented a review of unique nanomaterial properties that arise as a result of their
size and structure. Michael Holman identified the range of applications for a number of materials and the
volumes of the materials being used. His presentation highlighted that oxide-based nanomaterials are being
used in high volume in a broad range of applications, and that other nanomaterials were used in more specialized applications in generally lower volumes.
The participants then split into six workgroups to identify nanomaterials, their common applications,
potential hot spots in the life cycle of nanomaterials, and properties that would be important to their biointeraction. These groups were 1) oxides, 2) metals, 3) semiconductors (or QDs), 4) carbon, 5)
macromolecules, and 6) self-assembling materials. The groups were arbitrarily established and their organization was not meant to offer a materials classification scheme. The fluidity of such classification at this
point in time was demonstrated by the recommendation of the metals workgroup to be merged into the
oxides group, because most metals would oxidize or be used in nanotube synthesis or embedded catalyst
applications where exposure potential would not be high. Furthermore, the range of nanomaterial applications in the different workgroups varied significantly; application could affect exposure potential and life
cycle properties, and thus how nanomaterials ultimately are classified.
Breakout sessions were to address the following topics: what is known about the nanomaterials
being explored by the groups; whether physical and chemical properties are adequate for determining biointeractions; options for classifying the physical and chemical properties of engineered nanomaterials that
could affect biointeractions; assessment of properties for the six types of nanomaterials; and potential areas
of concern (hot spots) for current and future applications, volumes, exposure, and hazard throughout the
nanoparticle life cycle.
Workshop 1 participants were successful in synthesizing available research regarding the classes and
properties of nanomaterials, advancing discussion regarding various properties and potential for biointeractions, and in identifying potential hot spots in nanomaterial life. However, the consensus of the group was
that it was premature to establish classes of nanomaterials based on properties that will affect biointeraca. A “hot spot” is defined as a process or application in which there is a potential for direct exposure to high
concentrations, or prolonged exposure to moderate concentrations.
International Council on Nanotechnology
2007 Workshop Report
tions. The results from Workshop 1 were used as input to the second workshop, which was more focused
on research related to biological interactions and hazard prediction. Risk management needs related to
exposure were discussed in both workshops, and are summarized in the Workshop 2 report of this document (Section 2.3), where the needs were clearly defined and corresponding research timelines were
established.
1.2.
Summary of Workshop Findings
While an understanding of properties that affect biointeraction is emerging from research, these
properties are not unique to any of the material types. Furthermore, many physicochemical properties can
be designed into the material and changed by synthesis and formulation, so it will not be possible to establish classes of nanomaterials based on composition or a limited set of structural properties. Nanomaterial
biointeractions were identified to be dependent on some combination of size, shape, surface charge, chemical reactivity, chemical toxicity, surface composition, and concentration that may change depending on the
type of material involved. Therefore, it may be important to establish a set of physical, chemical and in
vitro screening methodologies that determine the biointeraction class of materials based on physicochemical properties, and on the functional performance of the nanomaterials that indicate the relative chemical
reactivity, surface charge, solubility, and surface composition. Tools and models must be developed that
can describe the dynamic nature of nanomaterials throughout their lifecyle.
Based on discussions of macromolecules and self-assembled materials, future nanomaterials will be
more complex and the potential for unplanned assembly should be explored as these and all nanomaterials
are placed in complex environments. It is important to develop an understanding of the principles that govern biointeractions with existing and future nanomaterials.
Drawing from preliminary research findings, expert participants identified nanomaterial properties
that may affect their transport, including size, shape, surface charge, solubility, surface chemistry and
chemical reactivity, and concentration. Because many of these properties can be dramatically altered during the synthesis of the material, and because the surface charge, surface chemical reactivity, and surface
chemistry can be modified during the functionalization and formulation of these materials, a set of screening tools is needed to correlate the functional properties of nanomaterials with their transport properties
and potential for biological interaction, which would identify nanomaterials that require detailed testing.
Also emerging from the workgroups was a set of common hot spots for nanomaterials in dry powder
form and a different set for those in liquid form. For nanomaterials in dry powder form, the primary potential hot spots were in the cleaning of synthesis reactors, bagging operations, surface functionalization and
formulation areas of manufacturing, and in applications where materials are topically applied or formulated in aerosol delivery systems. Similarly, nanomaterials in liquid form had potential hot spots when the
material was topically applied or aerosolized in manufacturing or product applications. To better understand exposure potential, research is clearly needed into the 1) bioactivity of nanomaterials in their native
form, 2) stability and mobility of engineered nanomaterials in different formulations, and 3) biointeractions of aerosolized formulations and liquids. Research also is needed into the effectiveness of filters and
engineering controls in reducing exposure to engineered nanomaterials, whether in dry or liquid form.
Finally, exposure assessment studies are needed to lead to predictions about physicochemical properties
and their implications for net dose.
Because the applications of the materials in each workgroup were so diverse, each team was often
studying different properties that potentially affect biointeraction. For this reason, research needs identified
by the workgroups are very different and are included in the individual workgroup summaries that follow.
Summary of Workshop Findings
1-15
International Council on Nanotechnology
1.3.
2007 Workshop Report
Next Steps
Workshop 1 participants noted that further deliberative efforts with similar programming would be
required to provide detail for research needs toward the significant challenges of producing predictive
modeling for nanomaterial biointeractions. Among the areas that need further investigation are 1) reviewing the individual workgroup research priorities to assess their broader applicability, 2) identifying
screening strategies to measure physical (size, shape, nanostructure) and chemical (composition, chemical
reactivity, particle surface charge, solubility, surface composition) properties, 3) establishing agreement on
a set of functional screens and tests to determine whether these properties correlate to biointeraction potential, 4) assessing potential for nanointeractions with a wider range of biological systems, and 5) identifying
research needed to develop predictive models of nano-biointeraction.
Workshop 1 outputs were shared with participants of Workshop 2: Towards Predicting Nano-Biointeractions, which had the goal of identifying the research needed to develop predictive models of nano-biointeractions.
1.4.
Workshop Steering Team and Sponsors
Workshop 1 was jointly sponsored by ICON, the U.S. NSF (BES-0646107) and the U.S. NIH.
The steering team included:
Cate Alexander, National Nanotechnology Coordination Office
David Berube, University of South Carolina
Vicki Colvin, Rice University
Scott Cumberland, Clorox Company
Michael Garner, Intel Corporation
Tracy Hester, Bracewell & Giuliani, LLP
David Johnson, Rice University
Kristen Kulinowski, Rice University
Andrew Maynard, Woodrow Wilson International Center for Scholars
Günter Oberdörster, University of Rochester
Jennifer Sass, Natural Resources Defense Council
Hideo Shindo, New Energy and Industrial Technology Development Organization, Washington
Vicki Stone, Napier University
Sally Tinkle, National Institute of Environmental Health Sciences
1.5.
Workgroup Summaries
1.5.1
Oxide Workgroup Summary
Breakout group leaders: Steve Brown (Facilitator), Mike Holman (Facilitator), Richard
Canady (Scribe)
1.5.1.1 Introduction
The meeting started with presentations by Barry Park and Fred Klaessig, who provided the background required for understanding the physical properties of the materials and applications. Then the team
reviewed the nano-oxide materials and their applications. It was agreed that these oxide materials have a
wide variety of properties that may make classification challenging. In many cases, materials are being
doped with other metal oxides to change properties. Also, the properties are often dependent on the syntheNext Steps
1-16
International Council on Nanotechnology
2007 Workshop Report
sis technique and postsynthesis thermal treatment. Thus, it may be very difficult to identify nanomaterials
classes based on composition. It was proposed that a number of chemical screens and in vitro tests be
developed to determine potential for biological interaction, and correlate these to physical and chemical
properties. Because the applications for different oxide particles are very diverse, it is difficult to assess for
potential hot spots in the applications. Exposure to high concentrations of engineered nanomaterials in dry
form was identified as a potential hot spot, which could occur while cleaning dry synthesis reactors and
while handling powders during bagging and formulation.
1.5.1.2 Common Oxide Particles
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Titanium oxide
Zinc oxide
Cerium oxide
Iron oxide
Silicon oxide (silica)
Copper oxide
Zirconia
Alumina
Nickel oxide
Antimony pentoxide
Yttria
Barium sulphate
Hydroxyapatite
Calcium carbonate (paper mills, filler, probably in the nanoscale)
Pigments, including particles in the nanoscale (manganese oxide), pigments used in inkjets
Nano-clays (including talc).
1.5.1.3 Nanomaterial Properties and Biointeraction
The properties affecting biointeraction include size, shape, composition, structure, surface composition, chemical reactivity, solubility, and surface charge of the nanomaterial. While many of these properties
can be characterized with existing metrology, the chemical reactivity can be dramatically changed by small
changes in composition or nanostructure occurring in synthesis or processing, so correlation to measurable
properties is very difficult. Similarly, the surface charge of a nanomaterial can be changed during processing
or by environmental conditions, so characterization of the surface charge needs to be conducted in an environment similar to that found in biological fluids. Thus, functional tests may need to be developed to
determine the relative magnitude of chemical reactivity and surface charge for nanomaterials.
Chemical Reactivity
While different chemical reactions can occur, the electrons in the highest occupied states in one
material must have sufficient energy to either transfer electrons to an unoccupied excited state in a molecule, or form a bond with a molecule. In nanomaterials, a material must have electrons at the surface with
adequate energy. Introducing impurities to the surface of an oxide may dramatically change the availability
of electrons with energies capable of either transferring an electron or forming a bond. Similarly, the nanostructure of a surface may change the electron energy levels and electron concentration on a surface.
Therefore, subtle changes in composition may have dramatic affect on chemical reactivity. Thus, developing a test of the functional ability of a nanomaterial to either transfer electrons or form a chemical bond
Workgroup Summaries
1-17
International Council on Nanotechnology
2007 Workshop Report
with biological molecules may better establish the potential for a nanomaterial to interact with biological
organisms.
Surface Charge
Surface charge can be established by the chemical functionalization of the surface, defects in the surface or near the surface, or impurities introduced on the surface. Furthermore, exposure to light can
generate electrons and holes and either of these could migrate to the surface of the material. In addition,
changing the pH of a solution may change the surface charge of the material. Therefore, it will be important to have a monitor that determines the surface charge over a range of pH that would be common in a
biological organism in light and dark conditions.
1.5.1.4 Synthesis, Formulation and Manufacture
Synthesis techniques:
• combustion synthesis
• plasma synthesis
• wet-phase processing
• chemical precipitation
• sol-gel processing
• mechanical processing
• mechanochemical processing
• high-energy ball milling
• chemical vapor deposition
• laser ablation
• plasma synthesis or arc methods.
Formulation
Almost all applications are placed in a liquid or polymer with surfactants or coupling agents. The
range of chemistries is quite broad. Common matrices include water, silicone, rubber, plastics, and oils.
1.5.1.5 Potential Hot Spots in the Nanomaterial Life
The highest risk areas in the life of these materials are in the handling of powders: bagging and
unbagging, maintaining pyrolysis reactors, cleaning bagging houses, and accidental spilling. Because these
materials are used in such diverse applications, it was difficult in a short time to assess the potential hot
spots in applications and disposal. Precipitated materials processes are likely to predominate for synthesis
(sol-gel, chemical precipitation, arc). Flame pyrolysis is used for SiO2 and TiO2, so data from “incidental”
nanoparticles in soot may provide information useful to fumed silica.
1.5.1.6 Research Priorities
Because these materials are used in such diverse applications, it was difficult in a short time to assess
the potential hot spots in applications and disposal. It was felt that precipitated materials processes are
likely to predominate for synthesis from sol-gel or chemical precipitation processes. Flame pyrolysis is
used for SiO2 and TiO2, so data from “incidental” nanoparticles in soot may provide information useful to
understanding potential issues with fumed silica:
• intrinsic chemical toxicity
• chemical reactivity (and factors that control) for generations of oxygen radicals
• charge state.
Workgroup Summaries
1-18
International Council on Nanotechnology
2007 Workshop Report
Applications research priorities included
•
•
•
applications where the engineered nanomaterials may be released from the matrix
dispersed applications (sunscreens)
application formulations with human exposure potential (paints).
The highest research priority issues were to
•
•
establish a suite of validated tests to determine potential bioactivity response of different nanomaterials and correlate to physical properties
establish standardized test methodologies to assess the potential for release of engineered nanomaterials from different matrices.
1.5.1.7 General Questions and Concerns
•
•
•
•
Can we identify a list of likely and less likely physical/chemical properties that correlate to bioactivity?
Is it appropriate to have an arbitrary threshold for nanotechnology of 100 nm?
Can we identify where in the life of nanomaterials primary particles will arise when they are
placed in complex matrices that will involve agglomerates?
Should we consider the benefits of nanotechnology in assessment?
1.5.1.8 Backup Information
To establish a validated suite of bioactivity tests and to correlate results of such tests with specific
physical properties, we will need to
• arrive at and test a standard set of assays
• independently confirm assays in different labs
• use standard reference materials to compare against those that have been validated in standard
assays
• compare and validate by in vivo tests
• correlate assay results with physical characteristics, especially dynamic characteristics (towards
SARs)
• modify assays where appropriate.
Materials characterization should comprise the following as appropriate:
•
•
•
•
•
•
•
•
•
•
chemical composition
aggregation/agglomeration state
number of particles per unit mass
physical form
median size and size distribution
surface area
surface charge
solubility/miscibility
state of dissolution
partition coefficient.
Workgroup Summaries
1-19
International Council on Nanotechnology
1.5.2
2007 Workshop Report
Metals Workgroup Summary
Breakout group leaders: Andre Nel (Facilitator), Mbhuti Hlophe (Facilitator), Jennifer Sass
(Scribe)
1.5.2.1 Introduction
The meeting started with presentations by Frank DiStefano and Michael Thompson, who provided
the background required for understanding the physical properties of the materials and applications. Then
the team reviewed the common nanometals, their applications, and potential hot spots in the life of these
nanomaterials and the properties that could contribute to biointeraction. After the first day the team was, at
their request, integrated into the oxide nanomaterials group, as most metals would oxidize or be used in
nanotube synthesis or embedded catalyst applications (catalytic converters, fuel cells).
1.5.2.2 Common Metallic Nanomaterials and Applications
Common metallic nanomaterials and their applications are shown in the following table.
Nanometal
Common Applications
Silver
Antimicrobial uses
Cobalt, nickel, iron
Catalysts for carbon nanomaterial synthesis
Platinum, palladium, rhodium
Catalytic converters
Gold
Medical diagnostics
Aluminum
Propellant
Copper
Electronics
Iron
Metal doping
1.5.2.3 Nanomaterial Properties and Biointeraction
The most common applications of nanometals are catalysis or antimicrobial applications. For silver,
the most important properties are electrochemical potential, surface area, surface texture, edge texture, size,
and size distribution. These properties may be applicable to other metals. Surface or edge texture has been
demonstrated to dramatically increase the catalytic properties of silver, which highlights the critical importance of surface structure. Research should be directed toward understanding the relationship between
catalytic activity and surface topography at the atomic level, and to developing metrology for characterizing
catalytic activity. Surface properties may be important to the activity of other metals in generating oxygen
radicals, but the relationship is not clear.
The metals used for growth of engineered nanomaterials have properties that could affect biointeraction, including chemical composition, surface chemistry, valence of the charge states, electrochemical
potential, and redox cycling potential. For nanocatalysts incorporated with nanotubes, the bonding of the
carbon to the surface may affect the combined bioactivity potential. Therefore, research is needed to distinguish between the bioactivity arising from the nanotubes themselves and that arising from the presence of
the embedded cobalt, nickel, and iron catalyst particles.
For catalytic converters, the catalyst is inert to adiabatic reactions, but the potential for bioreactions
is unknown. With the number of applications for catalysis rapidly increasing, there is a critical need to
develop catalysts from new materials that are not resource-limited, as are platinum and rhodium. As the
Workgroup Summaries
1-20
International Council on Nanotechnology
2007 Workshop Report
range of materials used in this field increases, so will the need to predict and monitor their potential
bioactivity.
1.5.2.4 Synthesis, Formulation and Manufacture
Synthesis
The most common synthesis technique for nanosilver and nanocatalysts used in catalytic converters
is wet chemical precipitation, and the group felt that this technique should have a low risk for human exposure.
Many forms of nanocatalyst are used to grow carbon nanomaterials, most often in a closed reactor.
This issue should be addressed by the carbon nanomaterial breakout group.
Formulation (applies to most engineered nanomaterials)
The steps after synthesis include functionalization, dispersion, and formulation.
Functionalization is done to
•
•
•
•
•
•
•
enable dispersion
improve matrix compatibility
passivate the nanomaterial
reduce particle solubility
introduce reactive sites
minimize photocatalysis
improve biocompatibility.
Common functional groups employed include
•
•
•
organic carboxylic acids, amines, phosphonates, or mercaptans
organosilanes and siloxanes
inorganic oxide coatings, such as before SiO2 and Al2O3.
Dispersion mechanisms include
•
•
•
•
direct-charging (MOH2+) electrostatic techniques
silane treatment (M-OSiR)
polymeric dispersal (steric)
use of surfactant bilayers.
The direct-charging and surfactant layers may introduce particle charging.
Formulation Applications
The functionalized engineered nanomaterials are mixed with multiple materials depending on the
application. Examples of these are
•
•
•
architectural coatings: anionic materials, wetting agents, surfactants and thickeners
personal care: oils, thickeners, emollients, emulsifiers, polymers, and amines
plastics and elastomers: polymers, antioxidants, curing agents, plasticizers, pigments and fillers,
thermal stabilizers, and flame retardants.
The chemistry of these formulations is quite complex, so risk assessment must consider the specific
formulation and application. In most applications, the goal is to keep the particles in the formulation
matrix.
Workgroup Summaries
1-21
International Council on Nanotechnology
2007 Workshop Report
1.5.2.5 Potential Hot Spots in the Nanomaterial Life
The workgroup felt that metals used in embedded catalytic applications presented low risk for
human or environmental exposure though their life cycle. Nanosilver and nanocopper may have risk as
ecotoxins in the disposal of waste. Most metal nanoparticles will form oxides and the potential hot spots
for these will be addressed in the oxide breakout group.
1.5.2.6 Research Priorities
Significant research is needed in materials to correlate properties including electrochemical potential,
surface area, surface structure, oxidation state, surface composition with chemical reactivity, and catalytic
properties. It is also important to determine which of these properties may correlate with the ability to generate oxygen radicals or other biointeractions. New parameters may need to capture the bioactivity of
surfaces as there may be no direct correlation between chemically active sites and surface area.
1.5.3
Semiconductor Workgroup Summary
Breakout group leaders: Vicki Colvin (Facilitator), Amy Cannon (Scribe)
1.5.3.1 Introduction
Fluorescent crystalline semiconductor nanoparticles, also known as QDs, are being developed for
use in biolabels and in vitro diagnostics; optoelectronic applications such as light-emitting diode (LED)
displays, and solar cells; and inks and paints for identification or brand protection. Lux Research estimated
the 2005 market for QDs to be approximately $4.3 million, growing to $38 million by 2010.13 Given the
small market volume and relatively high cost of production, QDs can be fairly characterized as boutique
nanomaterials.
1.5.3.2 Synthesis
The synthesis and processing of QDs are done primarily in solution phase and are to a great extent
performed manually rather than through automated processes, especially in the purification stage. Therefore, the primary route of exposure is expected to be dermal, although inhalation exposure should be ruled
out definitively through testing of ambient air around the breathing zone, particularly for those QDs that
are generated as powders after undergoing a coating process.
QDs have shown the ability to dissolve and release their constituent components into solution over
time.14 Therefore, any assessment of potential hazard from QD exposure should distinguish between QDs
whose constituents are known to be highly toxic, such as cadmium (Cd), and those with lower intrinsic toxicity, such as selenium (Se) or zinc (Zn). In addition, the bioavailability and distribution of the constituents
should be considered.
Synthesis and processing of QDs generally involves the use of organic solvents, highly volatile metal
precursors (e.g., dimethyl cadmium), and temperatures between 100–400°C. Under these conditions,
explosion presents a possible hazard, though new processing techniques are beginning to utilize salts of
metals rather than the highly volatile precursors. The formation of aerosols or sublimed metals should be
ruled out by sampling the breathing zone during a typical synthesis and processing procedure. Current
techniques generate significant volumes of waste solvent contaminated with heavy metals.
Purification of QD solutions generally involves centrifugation, phase transfer with an amphiphilic
polymer, and sedimentation. Large volumes of solvent are used, posing an explosion hazard and generating
Workgroup Summaries
1-22
International Council on Nanotechnology
2007 Workshop Report
hazardous waste that requires special handling. Because these processes are still done manually, effectiveness of personal protective equipment (PPE), particularly gloves, should be validated.
A key point was that the scale of material-biomarker synthesis was 100-mg quantities (minute
amounts) whereas, for solar cells, much larger quantities of materials would be fabricated, and their synthesis would generate 10-g to 1-kg quantities, which would increase exposure risk.
1.5.3.3 Applications
Two of the primary applications of QDs are solar cells and biomarkers. Each raises different questions with respect to possible hazards during their intended use. QDs used in solar cells will generally be
encapsulated and thus not offer a high exposure potential despite their much greater volume. QDs used in
biomarkers pose the possibility of bioaccumulation, which has not been explored in great depth in the technical literature.
At the end of life, QDs in solar cells will likely not be recovered from the product and will therefore
become part of the waste stream with unknown long-term consequences. QDs in biomarkers would be used
in extremely small quantities, and these small quantities used are unlikely to pose a great exposure potential
in the long-term.
Measuring exposure to workers could involve periodic (e.g., 6-month) serum tests for high heavymetal concentration or the use of fluorescent techniques to detect the QDs themselves. However, it remains
unknown whether the QD fluorescence will persist long enough in the body to render this an effective
technique.
1.5.3.4 Hot Spots
The primary hot spots for QDs occur during synthesis and processing through the potential for
explosion and dermal exposure, which is related to the use of high-temperature solvents, not the nanomaterials. Therefore, the emphasis should be on developing engineering controls that limit personal exposure
and the risk of explosion from the use of volatile materials.
The disposal of used solar panels at the end of their life would require controls to be implemented to
protect the workers and the environment.
Critical Material Properties
Chemical toxicity:
Many of the elements used in these materials (e.g., cadmium, mercury, selenium) have inherent
chemical toxicity.
Energy levels:
These materials have a ground state (valence band) and an excited state (conduction band).
Direct bandgap materials:
As the size of the semiconductor nanoparticles decreases, the energy levels in both the bands become
split and usually the separation between the conduction and valence band increases (band gap increases).
For luminescent nanomaterials, e.g., “direct bandgap,” the wavelength of light shifts to shorter wavelength
(higher energy) as the particle size decreases.
Workgroup Summaries
1-23
International Council on Nanotechnology
2007 Workshop Report
Indirect bandgap materials:
These materials are generally not luminescent, but absorb energy above the bandgap and create an
electron (negative charge) and hole (positive charge). If the energy of electrons at or close to the surface is
higher than the energy to excite oxygen, they could create some level of oxygen radicals.
Work function:
The work function of the material is the energy required to excite an electron from the Fermi level to
vacuum, which depends on the dopant levels in the semiconductor.
1.5.4
Carbon Workgroup Summary
Breakout group leaders: Vicki Stone (Facilitator), David Berube (Scribe)
1.5.4.1 Introduction
It is worth noting that Risk = Hazard x Exposure. This document refers to exposure (hot spots) and to
the intrinsic properties of the carbon nanomaterials that might influence hazard. Hazard per se will be considered in the next workshop.
1.5.4.2 Carbon Nanomaterials
Common carbon nanomaterials identified included carbon nanotubes ([CNTs]–single, double and
multiwalled), fullerenes, carbon nanofibers, graphene sheets, and carbon black. Even within these general
categories there is great diversity among size, structure, physical aggregation forms, and residual species
associated with the carbon. Most of the discussion focused on CNTs/nanofibers and fullerenes because
these are produced in relatively large quantities and have many applications. Carbon black was recognized
as a useful reference material for which toxicology and epidemiology data are available.
CNT production usually involves the reaction at high temperature of a source of carbon with a catalyst particle either 1) floating in a reaction fluid or gas, 2) in a porous microparticle, or 3) on a surface.
Fullerene production involves a carbon source reacted using either an arc or combustion method. Both carbon nanotubes and fullerenes are produced on large scales by industry, and in small scales in university and
research settings. In many applications nanoparticles are part of a composite material, and are strongly
bound to the host polymer. In some materials (such as single- and double-walled nanotubes) the carbon
materials can stick tenaciously to each other to form large aggregates, and do not provide a ready source of
nanoparticles on their own. Alternatively, in applications like drug delivery, the carbon materials are treated
to remain suspended in water and resist aggregation.
1.5.4.3 Exposure Scenarios During Manufacture/Synthesis
A number of activities have the potential to result in relatively high exposure. During manufacturing
and synthesis of nanomaterials, processes considered able to generate high exposure included anything that
disrupts the normal process, maintenance, cleaning, vacuuming, and failure of PPE. Source materials (such
as catalysts) may have low density and be in a relatively weak physical form, providing the potential for
generation of dust. Following manufacture, transport, and transfer from transport vessels into a subsequent
product may potentially generate high exposure. Carbon nanomaterials are produced in a multitude of different forms for subsequent use. Examples include powders of various sizes, pressed fibers, suspensions in
fluids, and dry coatings on surfaces. Waste disposal and unintended use were also considered to be potentially high-exposure scenarios.
Workgroup Summaries
1-24
International Council on Nanotechnology
2007 Workshop Report
1.5.4.4 By-Products and Contaminants
It is likely that CNT and fullerene samples will generate by-products as well as a product that contains contaminants unless specifically purified/cleaned. Because the CNT and fullerenes are made by
combustion processes, contamination with carcinogenic polyaromatic hydrocarbons (PAHs) is possible,
though by-products depend strongly on the synthetic process used. The metal catalysts are also contaminants of unpurified CNT. Amorphous carbon is found in many fullerene and CNT samples. Fullerene
production also produces polymer C60 and oxidized products. CNT purification requires acids, and so acid
waste containing trace amounts of CNT occurs.
1.5.4.5 Applications and Uses for Carbon Nanomaterials
Uses for CNTs and nanofibers are diverse, including high-performance, light-weight fibers, highthermal-conductivity fibers, wires of low-loss electricity transmission over 1000s kw, multifunction fibers
and materials (enhanced polymers), imaging of diseases, treatment of diseases (ablation), scaffolds for biological applications, lithium ion batteries, fuel cells, coatings on films for electronic applications such as
flat-panel displays, photovoltatic cells, touch panels, sensors (environmental monitoring), food packaging
(smart packaging), and even concepts like a space elevator. Some of these were identified as potentially
generating a high exposure to either the consumer (especially those used in medical applications) or the
environment.
Uses for fullerenes overlap with those of nanotubes and nanofibers with respect to lithium ion batteries, information technology, and medical applications, but also includes coatings and cosmetics.
1.5.4.6 Exposure Scenarios During Use of Carbon Nanomaterials
Exposure to humans and the environment during use can be broken down into the occupational scenario, where raw nanomaterials are used and incorporated into products, and a consumer scenario, where
the general public uses a product containing nanomaterials. Most exposure scenarios have already been
covered in an occupational setting in relation to manufacture and synthesis, with the exception of exposure
during unloading of nanomaterials from storage vessels. For consumers, exposure could occur via multiple
routes, such as personal care products, food, clothing, and medical treatment. Consideration of the wide
range of uses lead the group to believe that exposure via ingestion, inhalation, and dermal absorption (as
well as injection for medical applications) are all conceivable.
Accidents (fires, traffic accidents) also provide a risk for high exposure. Multiple risks during disposal include comminution (cutting, grinding), incineration/burning, changing filters, waste handling,
cleaning of reactors, and accidents at reactor/manufacturing sites (fires, spillages, uncontrolled removal of
materials). During disposal, aerosol, liquid, powder, and solid materials were all considered. Aerosolization of carbon nanoparticles could occur by accidental release, but during controlled processes could be
minimized by filtration. Liquids containing carbon nanoparticles could gain access to landfills, potable and
waste water (shower, drugs), marine environments, and estuarine environments. Release into these environments could occur via spills and transport accidents, as well as purposeful disposal. Powders are most
likely to be recycled during the production process, but cleaning (release into waste water), scraping, filtering, and vacuuming could all lead to human and environmental exposure. Vacuumed product has the
potential to be contaminanted. Powders are likely to be disposed of via landfill and incineration and via
waste disposal companies that are not well regulated.
Finally, there are additional potential exposures that are difficult to quantify, such as those that could
result from terrorism.
Workgroup Summaries
1-25
International Council on Nanotechnology
2007 Workshop Report
1.5.4.7 Improving Exposure Assessment
To understand exposure and risk more adequately, a variety of information is required. For example,
to evaluate workplace risks, we require exposure assessment linked to hazard assessment. An understanding of the dispersal of carbon nanomaterials during uncontrolled combustion and incineration for different
media (powder, liquid, composites) is required. An understanding of whether nanoparticles can be released
from composites by comminution is required. Whether the nanoparticles exist as free particles or are
strongly aggregated within a host material depends on the material set and application. The ability of maintenance and cleaning procedures to release nanoparticles into the air is currently not understood. Little is
known with respect to human and environmental exposure through release during degradation of products
or during the transportation of raw materials and products. The environmental fate of consumer products is
also poorly understood. There is very little information on the behavior of released nanoparticles in waste
water or groundwater.
1.5.4.8 Prioritization of Research Needs
The following research priorities were set:
• gather data relating to the quantity of carbon nanomaterials made over time; e.g., multiwall
CNTs–1750 metric tons (2005). C60–specific data 40 tons per year.15
• gather information relating to potential applications
• improve the assessment of workplace and consumer exposure
• identify and differentiate the environmental and bioavailability by material type
• prioritize high-hazard and high-volume materials
• determine the role of contaminants in influencing hazard
• define structure function relationships
• link occupational health screening (registries) to epidemiology studies.
1.5.4.9 Carbon Nanoparticle Hazard
The intrinsic toxicity of carbon was recognized as being low, but factors that may enhance toxicity
include particle size (small being more toxic) and contamination with other materials (metal contamination
of CNTs), and certain characteristics of size and shape. Metals, fullerenes, and CNTs have all been shown
to be redox active, with the suggestion that metals are more potent than fullerenes, which are more potent
than nanotubes, although this requires further investigation. Because of their large surface area, these particles were proposed to be potential carriers of other materials that may influence toxicity. The charge of
carbon nanomaterials varies according to the media in which they are dispersed; e.g., in water, nano-C60 is
negatively charged and changes its charge with functionalization.
The group conducted a survey of which particle properties need to be characterized as part of the
EHS process. Each member had three votes; the number of votes were then used to rank the characteristics
in terms of importance.
The results were
• particle surface area (7)
• redox activity (6)
• composition/contamination (metals, organics) (6)
• solubility (water and organics) (3)
• durability (biopersistence) (3)
• particle count (2)
• particle size distribution (2)
Workgroup Summaries
1-26
International Council on Nanotechnology
•
•
•
•
•
•
2007 Workshop Report
defect density (1)
general characteristics from material science (1)
length (aspect ratio) of CNT affecting inhalation, transport, filtering, and toxicity
charge
degree of agglomeration
environmentally relevant characterization.
1.5.5
Macromolecule Workgroup Summary
Breakout group leaders: Yuliang Zhao (Facilitator), Scott Cumberland (Scribe)
1.5.5.1 Introduction
The Macromolecules group focused on nanomaterials generally engineered from organic molecules
to have a precise size, shape, and surface functionality. Such materials include dendrimers, dendrons, and
dendrigrafts of various generations, hyperbranched polymers and nanoengineered classical polymers. Naturally occurring macromolecules such as deoxyribonucleic acid (DNA) constructs, peptides/peptoids/
proteins, carbohydrates, and biopolymer/synthetic polymer constructs were also discussed because of their
similarities to the engineered macromolecules.
The group included technical experts in the field, such as Donald Tomalia and Mark Banaszak Holl,
who provided the background required for understanding the physical properties of the materials.
1.5.5.2 Nanomaterial Properties and Biointeraction
The group identified a number of measurable critical properties that are engineered into the design of
macromolecules that may lead to increased EHS risks, including
• intrinsic chemical toxicity of monomers (acrylates as neurotoxins)
• shape
• size/molecular weight
• surface area
• surface chemistry
- charge
- intermolecular forces
- chemical reactivity (redox chemistry).
Cationic macromolecules have been observed to bind to the negatively charged heparin sulfate proteoglycan molecules on the surfaces of cells through electrostatic interactions, which lead to the rapid
uptake of the macromolecules by the cells through an actin filament-mediated endocytosis process.16 Similarly, amine-terminated dendrimers have been observed to generate holes in lipid bilayers, leading to
disruption of membrane functions and leaking of cytosolic enzymes out of the cells.17,18 This research also
suggested that larger G7 dendrimers had a greater effect on membrane disruption than smaller G5 dendrimers. Although amine-terminated dendrimers show greater association with tissue, anionic dendrimers
functionalized with carboxylic moieties have been observed to rapidly cross the intestinal membrane of
adult rats.19
Other physical properties discussed that may contribute to transport and toxic response in biological
systems that warranted capturing include
• primary sequence (base structure)
• secondary structure (internal structure built off the base)
• tertiary structure (external structure that interacts with environment)
Workgroup Summaries
1-27
International Council on Nanotechnology
•
•
•
•
•
2007 Workshop Report
- crystallinity
topology/architecture
branching
amphoterism
alteration of transport properties of other materials (i.e., serve as delivery systems)
dynamics—ability to rearrange structural characteristics.
1.5.5.3 Synthesis, Formulation and Manufacture, and Application and Use
Techniques for the synthesis of macromolecules include
•
•
•
•
•
step-growth/chain polymerization (liquid phase)
gas-phase polymerization
grafting
reactive coextrusion
electrospinning.
Common formulation processes include
•
•
•
•
•
•
batch mixing
ultrafiltration
ball milling/jet milling
extrusion/thermoforming
coating (spin, spray, dip)
microfluidics.
Common formulation chemistries include
•
•
surfactants
additives (stabilizers, inhibitors, antioxidants).
Common matrices in which the macromolecules can be found include
•
•
•
aqueous/solvent solutions
creams/gels
solids/powders.
Potential applications for macromolecules include
•
•
•
•
•
•
•
•
•
•
•
delivery systems (drugs, platforms, therapeutics, nutraceuticals)
bioassays
image contrast agents
transfection polymers
inkjet printers
ion exchange resins/metal chelation
coatings
cosmetics
engineering materials
formulation viscosity modifier
environmental remediation.
Workgroup Summaries
1-28
International Council on Nanotechnology
2007 Workshop Report
1.5.5.4 Potential Hot Spots in the Nanomaterial Life
For potential near-term commercial applications of macromolecules, the liquid phase step-growth/
chain polymerization technique is the most commonly employed synthetic technique. Within this process,
the greatest potential activities of high exposure include synthetic steps in the purification of the materials
when they are at their highest concentration. However, the greatest exposure potential to free nanomaterials
will occur with synthetic techniques and applications in which the materials are aerosolized. Examples of
these techniques include spray drying and ball or jet milling. Unexpected hot spots for exposure potential
may occur from degradation by-products of these materials resulting from temperature, oxidative, or photochemical degradation.
Applications with the greatest potential for intentional human exposure include their uses as delivery
agents for pharmaceuticals, nutraceuticals, contrast agents, and cosmetics.
There is less of a concern around potential for high exposure at the end of life of products containing
macromolecules because of their high propensity for degradation in the environment. The greatest concern
addressed was the difficulty in removing these materials at waste treatment plants.
Hot Spots Summary
Nanomaterial
Synthesis
Formulation
Application
Disposal
Cosmetics
Macromolecule
Purification
Milling, spraying, and
Delivery systems
aerosolizing
Remove at
treatment plants
Inkjet printing
To isolate and identify macromolecules at these hot spots for high exposure potentials, the characteristics of the materials that need to be understood include
• material size and shape (includes molecular weight)
• polyvalency/dynamics
• amphiphilic character
• charge state
• monomer chemistry
• surface functional groups
• formulation chemistry (accompanying compounds).
The size of the material includes the nanometrics, hydrodynamic diameter, and radius of gyration of
the particles. The functional groups on the material surface influence the charge, hydrophobicity/hydrophilicity, and receptor-specific characteristics of the material. The architecture of the material is its shape as
defined by topology; primary, secondary, and tertiary structure; aggregation; and dynamic characteristics.
Understanding the materials’ physical properties will help develop appropriate metrology protocols for
detecting them in the environment.
1.5.5.5 Prioritized Research Needs Recommendations
The criteria for prioritizing research needs to understand the EHS impact of these materials includes
understanding the association of the materials’ physical properties to their biointeraction and facilitating
the development of metrology, standards, and data to correlate the SAR of the materials. The recommended research priorities are focused on the pharmacological and environmental impact of the materials’
physical properties and the manufacturing processes. These research priorities include
Workgroup Summaries
1-29
International Council on Nanotechnology
•
•
•
2007 Workshop Report
SAR of size, topology/architecture, and functional groups to pharmacokinetics, pharmacodynamics, and pharmacology
- size*–nanometrics, hydrodynamic diameter, radius of gyration
- functional groups*–charge, receptor specific, hydrophobicity/hydrophilicity
- architecture*–shape as defined by topology; primary, secondary, tertiary structure;
aggregation; and dynamics
- pharmacology/pharmacodynamics§–toxicology and therapeutics
- pharmacokinetics§–absorption, distribution, metabolism, excretion
SAR of size, topology/architecture, and functional groups to environmental fate and transport
- size*–nanometrics, hydrodynamic diameter, radius of gyration
- functional groups*–charge, receptor specific, hydrophobicity/hydrophilicity
- architecture*–shape as defined by topology; primary, secondary, tertiary structure;
aggregation; and dynamics
- environmental fate and transport–diffusion, degradation, persistence, bioaccumulation
toxicological and environmental impacts of manufacturing processes (green chemistry)
where * represents the potential for interactions among combinations of these properties, and § represents
the potential for synergies of macromolecules changing the pharmacological properties of other small or
biomolecules.
In addition, it is recognized that these studies need to be conducted across a wide range of organisms,
as their response to these materials may differ.
1.5.6
Self-Assembly Workgroup Summary
Breakout group leaders: Kenneth Dawson (Facilitator), Sally Tinkle (Scribe)
1.5.6.1 Introduction
The Self-Assembly group was charged with identifying potential physical, chemical, and biological
properties of engineered, self-assembling nanostructures and conceptualizing the points in their production, use, and disposal that might merit special attention to hazard.
Self-assembled nanomaterials are composed of even smaller nanoscale building blocks such as lipids
and metal oxide nanoparticles, and may include modifying components such as surfactants, inorganic
materials, and organic molecules. Self-organizing nanostructures are designed to assemble into ordered
functional or structural units by maximizing colloidal, electrostatic, and noncovalent properties and minimizing human intervention. Self-assembling nanoscale structures display interesting and potentially useful
properties, such as optical transparency, enhanced diffusive transport, structural flexibility, and improved
stability of nanoemulsions. Examples of self-assembling nanomaterials include nanoemulsions, lattices,
hollow spheres, tubes, and capsules.
This meeting started with presentations by Tom Mason, UCLA, and Mike Wong, Rice University.
Two major categories of self-assembled nanomaterials were discussed—lipid assemblies and nanocomposite assemblies—and these will be reviewed separately.
1.5.6.2 Lipid Assemblies
Synthesis and Formulation
Common formulation process:
•
emulsification with sufficient shear to produce nanoscale assemblies.
Workgroup Summaries
1-30
International Council on Nanotechnology
2007 Workshop Report
Common formulation chemistries may include
•
•
surfactants
additives (stabilizers, inhibitors, antioxidants).
Common matrices in which lipid assemblies can be found include
•
•
•
aerosol
solution
cream/gel.
Potential Applications
Primarily as delivery systems:
•
•
therapeutics, contrast agents, and nutraceuticals
cosmetics and personal care products.
Other advanced research and development applications.
Properties and Biointeraction
Critical characteristics:
•
•
•
•
size and morphology dependent on conditions of synthesis
- primary particle size: 10–1000 nm
- agglomerate size: nanoscale to macroscale
shape: compact, fibrous, tubular, and multilamellar
surface chemistry
- charge state: positive, negative, or neutral
- surface functionalized for specific applications; e.g., targeting to a cell type or organ system
- nonspecific surface-abstraction of molecules from the environment
permeability.
Biological interactions:
•
•
•
•
•
biocompatibility of lipids, the primary building blocks
toxicity derived from nonspecific adsorption of molecules from the microenvironment or inappropriate functionalization
primary routes of exposure: lung and skin, although ingestion and ocular uptake also possible
uptake dependent on the biological context
simple or complex response to stimuli.
Dose metrics should consider three parameters:
•
•
•
particle number per cell
volume fraction of the contents (i.e., the concentration of molecular species per vesicle)
concentration of liposomes in the delivery system.
Potential Hot Spots in Lipid Assembly Life Cycle
Exposure to materials over their life cycle—manufacture, use and disposal—all present different
issues for safety assessment and are largely unknown.
Lipid assemblies have high potential for environmental transformation. They may bind nonspecifically to entities in the environment that would change their shape, chemistry, and propensity for
environmental transport. Inaccurate assembly could permit inappropriate systemic transport; e.g., across
the blood-brain barrier, or through the environment.
Workgroup Summaries
1-31
International Council on Nanotechnology
2007 Workshop Report
The potential for and consequences of disassembly and inappropriate reassembly, especially in postuse, is unknown. This could occur in the body or in the environment and lead to inappropriate structures,
unanticipated uptake, and transport.
Increased potential for stealth entry into undesirable locations in the body and inappropriate reassembly represents a significant concern.
1.5.6.3 Nanocomposite Assemblies
Two forms were discussed: nanoparticle polymer assemblies and organic-inorganic hybrid
assemblies.
Synthesis and Formulation
Common formulation processes:
•
•
self-assembly driven by thermodynamics and/or kinetics
- evaporative self-assembly
- electrostatic assembly
• nanoparticle assembly
• layer-by-layer assembly
aggregation prevented by charge repulsion and steric repulsion (polymer and surfactant coating).
Common formulation chemistries may include
•
•
surfactants, polymers
organic molecules.
Common matrices in which nanocomposite assemblies can be found include
•
•
•
•
•
aerosol
solution
cream/gel
solid organic composites
ceramics.
Potential Applications
Delivery systems:
•
•
therapeutics, contrast agents, and nutraceuticals
cosmetics and personal care products.
Other advanced research and development applications.
Environmental applications:
•
•
energy harvesting
catalysis.
Other applications:
•
•
structural materials and nanoceramics
rheological modifiers for nanocomposites.
Properties and Biointeraction
• Critical characteristics, size, and morphology are dependent on conditions of synthesis:
- primary particle size: 1–100 nm
- assembly size: 10–>1000 nm
Workgroup Summaries
1-32
International Council on Nanotechnology
2007 Workshop Report
-
•
•
•
•
agglomerate size: morphology is condition-dependent and size is microenvironmentdependent
shape: compact, fibrous (e.g., self-assembling polypeptides), capsular structures, two-dimensional sheets, hollow spheres, crystals, wires
crystal structure: higher-order crystallinity
functionalized surface chemistry
- charge state: positive, negative, or neutral
- coating composition: organic (surfactant), polymers (polyethylene glycol, polylysine),
inorganic (native nanoparticle)
- surface coating functionalized for specific application
- nonspecific surface-abstraction of molecules from the environment
porosity and permeability.
Biological interactions:
•
•
•
•
toxicity derived from toxicity of components in the assembly and from nonspecific adsorption of
molecules from the microenvironment onto the assembly or inappropriate functionalization
primary routes of exposure: lung and skin, although ingestion and ocular uptake also possible
uptake dependent on the biological context
simple (disassembly: pH, temperature, pressure, ionic strength) or complex response to stimuli.
Dose metrics should consider three parameters:
•
•
•
number of assembly units per cell
volume fraction of the contents (i.e., the concentration of molecular species in each assembly)
concentration of assemblies in the delivery system.
Potential Hot Spots in Nanocomposite Assembly Life Cycle
Potential hot spots identified were
•
•
•
•
•
•
•
•
•
toxicity of the nanocomposite assembly components during manufacture
toxicity and potential transformation of components during degradation and disassembly
assemblies as inappropriate carriers of manufacturing byproducts; e.g., toxic solvents
inaccurate assembly that may inappropriately enhance systemic transport; e.g., across the bloodbrain barrier, or through the environment
exposure to materials over their life cycle—manufacture, use and disposal—that may all present
different, largely unknown issues for safety assessment
nanocomposite assemblies that bind nonspecifically to entities in the environment that would
change their shape, chemistry, and propensity for environmental transport
unknown potential for and consequences of disassembly and inappropriate reassembly, especially in postuse, which could occur in the body or in the environment and lead to inappropriate
structures, unanticipated uptake, and transport
increased potential for stealth entry into undesirable locations in the body and inappropriate reassembly
use of nanocomposite assemblies in industry that may lead to an industry sector mismatch
between the required material science expertise to create the nanocomposite assemblies and the
applications expertise; e.g., material scientists making drug delivery systems.
Workgroup Summaries
1-33
International Council on Nanotechnology
2007 Workshop Report
1.5.6.4 Prioritized Research Needs Recommendations
The criteria for prioritizing research needs to understand the EHS impact of these materials are similar
to other categories of nanomaterials; they include understanding the association of the materials’ physical
properties to their biointeraction and facilitating the development of metrology, standards, and data to correlate the SAR of the materials. In addition, self-assembly imposes additional critical research needs to
address the potential for inappropriate assembly and inappropriate disassembly and reassembly.
1.6.
13.
14.
15.
16.
17.
18.
19.
References Cited
http://www.luxresearchinc.com/tnr.php
Derfus, A.M., W.C.W. Chan, and S.N. Bhatia. Probing the cytotoxicity of semiconductor quantum
dots. Nano Letters 4 (1) 11-18 (2004).
http://www.smalltimes.com/Articles/Article_Display.cfm?ARTICLE_ID=267721&p=109
Kopatz, I., J.S. Remy, and J.P Behr. A model for non-viral gene delivery: through syndecan adhesion
molecules and powered by actin. J. Gene Med. 6 769-776 (2004).
Mecke, A. et al. Lipid bilayer disruption by polyamidoamine dendrimers: The role of generation and
capping group. Langmuir 21, 10348-10354 (2005).
Hong, S. et al. The interaction of polyamidoamine (PAMAM) dendrimers with supported lipid bilayers and cells: Hole formation and the relation to transport. Bioconjugate Chemistry 15, 774-782
(2004).
Wiwattanapatapee R., B. Carreño-Gómez, N. Malik, and R. Duncan. Anionic PAMAM dendrimers
rapidly cross adult rat intestine in vitro: A potential oral delivery system? Pharm. Res. 17 (8) 99-998
(2000).
References Cited
1-34
International Council on Nanotechnology
2007 Workshop Report
Appendix A: Workshop 1 Agenda
Tuesday, January 9, 2007
7:30
8:00
8:20
8:45
9:15
9:45
10:00
10:30
10:50
11:00
11:30
12:00
3:00
4:30
6:00
6:30
Registration
Welcomes and Opening Remarks–Jeremy Berg, NIGMS Director; Samuel Wilson,
NIEHS Deputy Director; Mihail Roco, Senior Advisor on Nanotechnology, NSF-ENG/
OAD
ICON Director’s Welcome and Overview–Kristen Kulinowski
Research Needs for Future Development of EHS Nanomaterial Standards and Practices–
Steve Brown
Physical Properties of Nanomaterials: Towards Predictive Assessments of Risk–
Vicki Colvin
Break
Assessing the Risks of Engineered Nanomaterials: Setting the Scene–Andrew Maynard
Nanomaterial Forecast: Volumes and Applications–Michael Holman
ICON International NanoEHS Research Needs Assessment Goals–Michael Garner
Charge to Workgroups–Michael Garner
Lunch
Workgroups Convene (Six workgroups: Oxide, Metal, Semiconductor, Carbon, Macromolecules, Emerging Nanomaterials)
Break
Reconvene for Report Backs
Adjourn
Reception and Dinner
Wednesday, January 10, 2007
8:00
8:15
9:45
11:00
12:00
1:00
2:00
References Cited
Reconvene: Charge to Breakout Groups
Workgroup Breakout Sessions
Break
Reconvene for Report Backs
Lunch
Report Outs, Summary, and Next Steps
Adjourn
1-35
International Council on Nanotechnology
2007 Workshop Report
Appendix B: Workshop 1 Attendees
ICON Staff:
Vicki Colvin, Executive Director (Rice University–USA)
Kristen Kulinowski, Director (Rice University–USA)
David Johnson, Operations Manager (Rice University–USA)
David Berube, Communications Director (U South Carolina–USA)
Eric Amis (National Institute of Standards and Technology–USA)
John Balbus (Environmental Defense–USA)
Bob Bronaugh (Food and Drug Administration–USA)
Steven Brown (Intel Corporation–USA)
Richard Canady (Food and Drug Administration–USA)
Amy Cannon (Center for Green Chemistry–University of Massachusetts–USA)
Vince Castronova (National Institute for Occupational Safety and Health–USA)
Wei Chen (Chinese Academy of Sciences–China)
Scott Cumberland (Clorox Company–USA)
Ken Dawson (University College Dublin–Ireland)
Frank Distefano (Air Products and Chemicals, Inc.–USA)
Thomas Epprecht (Swiss Reinsurance Company–Switzerland)
Mike Garner (Intel Corporation–USA)
Robert Glenn (Crowell & Moring LLP–USA)
Mbhuti Hlophe (North West University–South Africa)
Mark Banaszak Holl (University of Michigan - USA)
Mike Holman (Lux Research Inc.–USA)
Matthew Jaffe (Crowell & Moring LLP–USA)
Guibin Jiang (Chinese Academy of Sciences–China)
Fred Klaessig (Degussa–USA)
Bill Kojola (AFL-CIO–USA)
Stephen Lehrman (ASME Fellow–Senator Mark Pryor–USA)
Thomas Mason (University of California–Los Angeles–USA)
Andrew Maynard (Wilson Center–USA)
Scott McNeil (NCI Nanotechnology Characterization Lab–USA)
Cyrus Mody (Chemical Heritage Foundation–USA)
Hideki Murayama (Frontier Carbon Corp.–Japan)
Chris Murray (IBM–USA)
Imad Naasani (Invitrogen–USA)
André Nel (University of California, Los Angeles–USA)
Günter Oberdörster (University of Rochester–USA)
Barry Park (Oxonica–UK)
Matteo Pasquali (Rice University–USA)
Juergen Pauluhn (Bayer Health Care–Germany)
Francis Quinn (L’Oréal–France)
John Randall (Zyvex Corporation–USA)
Mike Roco (National Science Foundation–USA)
Marc Saner (Council of Canadian Academies–Canada)
Jennifer Sass (Natural Resources Defense Council–USA)
Ted Schettler (Greater Boston Physicians for Social Responsibility–USA)
Jo Anne Shatkin (Cadmus Group–USA)
References Cited
1-36
International Council on Nanotechnology
2007 Workshop Report
John Small (National Institute of Standards and Technology–USA)
Vicki Stone (Napier University–UK)
Robert Tanguay (Oregon State University–USA)
Clayton Teague (National Nanotechnology Coordination Office–USA)
Adam Teepe (ICF International–USA)
Treye Thomas (Consumer Product Safety Commission–USA)
Mike Thompson (FEI Company–USA)
Sally Tinkle (National Institute of Environmental Health Sciences–USA)
Donald Tomalia (Dendritic Nanotechnologies–USA)
Nigel Walker (National Institute of Environmental Health Sciences–USA)
David Warheit (DuPont–USA)
John Warner (Center for Green Chemistry, University of Massachusetts–USA)
Michael Wong (Rice University–USA)
Yuliang Zhao (Chinese Academy of Sciences–China)
References Cited
1-37
2.
Workshop 2: Towards Predicting Nano-Biointeractions
June 5–7, 2007
Swiss Re Centre for Global Dialogue
Rüschlikon, Switzerland
2.1. Workshop Overview
The second of the workshop series, Towards Predicting Nano-Biointeractions, was held in
Rüschlikon, Switzerland, June 5-7, 2007, at the Swiss Reinsurance Company’s Centre for Global Dialogue. Drawing upon the knowledge of 53 participants with expertise in areas such as biology, computational modeling, toxicology, materials science, biophysics, and environmental science, the goal of this
workshop was to identify research needs for determining engineered nanomaterials’ interactions with biological and environmental systems and to define research strategies for developing predictive models of
engineered nanomaterials’ interactions with biological systems.
The workshop started with an introduction by ICON Director Kristen Kulinowski of Rice University
that summarized the results of the first Research Needs Assessment workshop (Workshop 1), including
considerations for creating classes of nanomaterials related to their bioactivity and potential “hot spots” for
exposure in the workplace and throughout the material’s life cycle. Dr. Kulinowski explained the goal of
Workshop 2 to be identifying the research needs and strategies to establish predictive models of nano-biointeraction. Andrew Worth, from the European Commission, presented the current state of computational
toxicology with particular emphasis on quantitative structure-activity relationships (QSARs) for chemicals
and identified potential approaches for extending this to engineered nanomaterials. His presentation identified that mechanistic understanding of protein nanoparticles can be derived from using molecular modeling
techniques, but prior knowledge of interactions is needed, so he suggested that a weight-of-evidence
approach be applied. Sally Tinkle of NIEHS identified the biological response mechanisms that must be
elucidated to understand SARs. Upon exposure, molecular and cellular responses are activated proportionally to the exposure and work to return the biological system to baseline.
The Mechanisms breakout sessions were charged by ICON Executive Director Vicki Colvin of Rice
University to discuss the current state of knowledge on the mechanisms by which a toxicant interacts with
a biological organism, whether or not these mechanisms are well established in toxicology generally, and
what the most common tests are to probe the mechanism. Participants were encouraged to identify whether
existing tests could be confounded by the presence of nanoparticles, where in a mechanistic pathway nanoparticles might have the potential to be disruptive or deleterious, and then to identify the short-,
intermediate-, and long-term research needed to answer the most pressing outstanding questions (Figure
3). The Mechanisms breakout sessions were organized around the topics of oxidative stress, protein misfolding, apoptosis and necrosis, developmental effects, and genotoxicity and mutagenicity. The next day
the conclusions from the sessions were reported and discussed with the full group. Summaries of these
conclusions are found in the breakout reports.
International Council on Nanotechnology
2007 Workshop Report
Oxidative Stress / Inflammation and Immune Response
Protein Misfolding
Mechanisms
Apoptosis and Necrosis
Genotoxicity and Mutagenicity
Developmental Effects
Figure 3. The first breakout session focused on five mechanisms of potential nano-biointeraction.
On Day 2, Kenneth Dawson of University College Dublin presented his latest findings regarding the
identity taken on by a nanoparticle when it comes into contact with biofluids. His research demonstrates
that biomolecules adsorb to and desorb from a nanoparticle surface in a complex and dynamic fashion that
is still poorly understood. The nanoparticle-biomolecule composite object is what interacts with cells,
organs, and fluids rather than the native nanoparticle; therefore, understanding the nature and time-dependence of this composite material will be vital to predicting nano-biointeractions. Nanomaterial properties
that can affect the adsorption and desorption of biomolecules include size, shape, agglomeration state, and
other physicochemical properties. Dawson also presented preliminary evidence that nanoparticles, under
some circumstances, can change the conformation of the biomolecule and induce protein fibrillation. This
has potential implications for the use of nanoparticles in medical applications.
Sally Tinkle presented the charge to the second breakout sessions on Interactions. She instructed the
teams to highlight research needed to identify the potential sources of stress and potential interactions or
disruptions of the nanomaterial with the biological response pathway, and to develop an RFP that would
identify research required to enable development of models. As a nanomaterial interacts with an organism,
the nanomaterial can induce stress directly or indirectly in the organism. The organism then initiates a number of responses, which are proportional to the stress, to return the organism to an “equilibrium” condition
through different biological response pathways. Breakout sessions were organized around biofluids, cell and
tissue constructs, whole animal interactions, and environmental interactions. The breakout sessions reported
their findings and discussed these with the full session. The summaries of these discussions are found in the
breakout reports. The meeting participants then discussed common themes, missing themes, and next steps,
topics that are covered in the following pages.
Workshop Overview
2-39
International Council on Nanotechnology
2007 Workshop Report
Nanoparticle-Biofluid Interactions / Target Cell Interactions
Tissue Constructs
Interactions
Whole-Animal Interactions / Biokinetics
Ecotoxicology
Figure 4. The second breakout session focused on these potential modes of nanoparticle-biointeraction.
2.2. Summary of Workshop Findings
Throughout the workshop, in both the Interactions and Mechanisms discussions, there was agreement on the need for a more fundamental understanding of the interaction of nanomaterials with living
organisms and the natural environment. Key findings were that it is critical to correlate nanoparticle physicochemical properties with interactions in organisms and the natural environment. Second, determining the
dose and dose-rate dependence of biointeractions is critical to understanding biological interactions. Third,
specific research is needed to develop better biomarkers, or sets of biomarkers, to address the vast diversity
of nanoparticle types and to develop strong correlative models for predicting in vivo endpoints based on in
vitro results.
A number of common research needs were identified by multiple groups and are captured in the
Common Themes section (Section 2.4). Identified in discussions in Workshop 1 and Workshop 2 was the
lack of well-characterized reference materials, which is limiting progress in understanding nano-biointeractions. Another commonly identified need was for standardized assays, as well as new assay development
as appropriate. Better documentation of biointeraction test methods in publications is needed to improve
the quality and comparability of the science. Metrology is needed for locating and characterizing nanomaterials in biological organisms and samples, and for monitoring exposure concentration, size distribution,
and dose.
Specific research was identified for characterizing different interactions between engineered nanomaterials and biomolecules, cells, cellular communication processes, animals, and the environment.
Because some interactions occur in a sequential fashion, the summary of this research with timelines (Section 2.3) highlights the interactions and mechanisms that could occur as a nanomaterial comes in contact
with an organism. Furthermore, because some of the interactions are cumulative, the summary highlights
the coupling between different levels of interactions.
To develop predictive models of interactions between engineered nanomaterials and biological
organisms, the nanomaterial state and properties must be carefully characterized, and a detailed understanding of the mechanisms and interactions with biomolecules, cells, organs, and systems must be
developed. Because engineered nanomaterials in biological fluids become coated to some degree with biomolecules, it is important to understand the nanomaterial physicochemical properties and biological factors
that determine which of the more than 3500 biomolecules will coat the nanoparticle. As is highlighted in
Summary of Workshop Findings
2-40
International Council on Nanotechnology
2007 Workshop Report
several breakout report sections, it is important to identify fluids and cell lines for use in in vitro studies
that are appropriate to the relevant exposure routes and for those in vitro studies needed to characterize the
state of the nanomaterials after they have reached an equilibrium coating.
It is furthermore critical to develop a fundamental correlation between the physicochemical properties of the nanomaterial, the agglomeration state, and the resulting biomolecular coating, because the
composition of this “corona” (full definition on page 66) nanomaterial composite may be significant in
determining the nature of the interaction. While the composition of the corona may vary with time and biological conditions, the composition may be critical in determining cell and organ interactions. As the
organism responds to the changes induced by the nanomaterial, the nanomaterial may also interact with the
response pathways. Several breakout sessions noted that monitoring of intra- and intercell signaling might
give insights to these interactions. More details of these discussions are found in the breakout session
reports.
While the individual workgroup reports present more detailed recommendations, the following highlevel research is needed to establish the fundamental knowledge required to develop predictive models:
1. Determine the fate of nanomaterials for different exposure paths. Experiments are required to
monitor and determine the distribution of nanomaterials in different organs at both low and high
doses and dose rates. It is also important to recover nanomaterials from fluids and organs and
characterize the agglomeration state of the nanomaterials and the composition of the biomolecular coatings. This is important to determine relevant biological media for use in tissue constructs
and fluids for in vitro experiments.
2. Correlate physicochemical properties of nanomaterials with the biomolecular corona and the
properties of this composite structure in relevant fluids. Because the composite nanomaterial/biomolecular corona will be interacting with fluids, membranes, and cells, research is needed to
understand how the nanomaterial properties affect the composition and coverage of the composite structure in a range of fluid conditions. This also requires characterization of the physicochemical properties of the composite structure, including surface composition and charge state,
as these may affect interactions with cells and membranes.
3. Understand the interactions of nanomaterial-biomolecule composite structures with relevant
membranes, cells, and cellular components, including DNA. Research is needed to understand
the direct and indirect interactions between the composite nanomaterial corona with membranes
and cells. This is required to understand the mechanisms for transport through the membranes
and cell walls, nuclear walls, and the interactions within these. It is also important to study the
interactions of these components with DNA, and to determine how the properties of the composite structure affect these interactions.
4. Characterize interactions of nanomaterial-biomolecule composite structures with response pathways. Research and metrology are needed to characterize interactions of the nanomaterial corona
structures with the biological response pathways as a function of concentration and biological
conditions.
5. Predict chronic effects through long-term testing. Because standard toxicology test methods
commonly employ high concentration and high dose rates, many of the effects that manifest will
be acute responses. Other effects that result from chronic exposure may not be manifest in shortterm experiments. Methodologies are needed to correlate interactions between nanomaterials and
organisms that could result in long-term health effects or diseases such as cancer. Understanding
these interactions will require a combination of in vivo and in vitro experiments, with metrology
capable of analyzing the composition of these structures.
Summary of Workshop Findings
2-41
International Council on Nanotechnology
2007 Workshop Report
Each of the workgroups has more detailed discussions in its report on the factors that are important
for research, and this summary identifies only high-level issues. For a more thorough understanding of
these issues, please review the workgroup reports.
[NOTE: Outside the scope of these workshops were the subjects of testing protocols and the development of standard libraries of well-characterized nanoparticles. While these were recognized by most
participants as important, even critical, drivers of a successful nano-EHS research agenda, the organizers
felt that these topics are worthy of greater exploration than could be accommodated in these workshops
and merit focused and individual attention in the future. Instead, Workshop 2 participants in particular were
asked to assume that well-characterized reference nanomaterials are readily available and that government
funding agencies have the necessary resources to implement the full research strategy.]
2.2.1
Next Steps
At the end of the meeting, workshop participants brainstormed on the subject of potential future
directions. Some of these recommendations were for activities that ICON might be in a position to organize, including future workshops that begin to address one or more specific needs identified by the group,
and others for activities that might be taken up by other groups or organizations. The following activities
were advanced as potential future directions:
•
•
•
•
•
an international effort to identify promising metrology and methodologies for monitoring nanomaterials in the workplace and the environment
an international effort to identify tools for detecting the presence and characteristics of nanomaterials in biological systems
an effort to develop a minimum set of experimental data to be submitted with a technical manuscript (such as the MIAME protocols) to allow for greater reproducibility and comparison of
nano-biointeractions research
an effort to identify model biological systems and model nanoparticles for nano-biointeractions
research
a workshop to identify a number of functional materials properties, including chemical screens,
reactivity, charge, and solubility.
2.3. Summary Research Needs and Timetables
10-Year Goal: Computational models that predict nano-biointeractions and estimate safety and hazard with validation screens.
The following is a more detailed description of the needs identified in Figures 1 (page 10) and 2
(page 11).
2.3.1
Research to Predict Nano-Biointeractions
2.3.1.1 Characterization of Nanomaterials
There is limited understanding at present of the correlation between nanomaterial physicochemical
properties and biointeractions. While several of these properties, including size, shape, and composition,
can be readily characterized with currently available tools, multiple physicochemical properties may contribute to the chemical reactivity, solubility, and charge state of the nanomaterial. Thus, tests need to be
established that characterize the effective chemical potential of the nanomaterial for interactions and that
determine the charge state and solubility.
Summary Research Needs and Timetables
2-42
International Council on Nanotechnology
2007 Workshop Report
2-Year Goals
•
Establish minimum nanomaterial physicochemical properties for characterization to enable prediction for biointeractions
5-Year Goals
•
Establish validated correlation between physiochemical properties and biointeractions
2.3.1.2 Standard Terminology
While standards development is underway, there is yet no widely adopted standard terminology for
describing nanomaterials, biological media, and testing protocols. Thus, it is difficult to determine whether
research performed by different groups used the same materials or protocols. The research community
should start using available standard terminology and establish consensus on common vocabulary for items
not covered by existing standards.
2-Year Goals
•
Establish common vocabulary/terminology for materials and assays
2.3.1.3 Standard Reference Nanomaterials
Although the identification of standard reference materials was outside the scope of the workshops,
participants felt strongly that the availability of reference materials would greatly accelerate progress in
understanding nano-biointeractions. It is at present difficult to determine whether disparities in research
outcomes are because of differences in the materials, testing protocols, or the biological media. Standard
reference nanomaterials whose biointeractions have been validated via testing in multiple laboratories will
enable comparative analyses of experimental results. This will require developing a library of materials
that exhibits clearly specified material size, shape, and properties.
2-Year Goals
•
Establish validated reference nanomaterials that have been tested in vitro and in vivo
5-Year Goals
•
Establish well characterized and controlled nanoparticle reference materials with a statistical distribution of physicochemical properties
2.3.1.4 Techniques for Detecting Nanomaterials in Biological Media
The ability to monitor the movement and location of nanomaterials in a biological system is critical
to understanding biointeractions and transport for in vivo and in vitro testing. Currently, the composition,
radiation reaction, and fluorescence of certain nanoparticles can be modified to enable easy detection. In
the case of fluorescence, for example, the emissive molecules that are attached to the surface of nanoparticles may change their interaction with biomolecules, cells, and organs. Techniques are needed that enable
the interactions and movement of a wide range of nanomaterials to be monitored in organisms and cell cultures without interference. Furthermore, there is a need to extract nanomaterials from biological media and
analyze the surface composition resulting from the biointeractions.
2-Year Goals
•
Develop new techniques for imaging nanomaterials in biological media and organisms to supplement transmission electron microscopy (TEM)
Summary Research Needs and Timetables
2-43
International Council on Nanotechnology
2007 Workshop Report
2.3.1.5 In Vivo Tests and Correlation to In Vitro Tests
A critical challenge is to determine whether the biological media and protocols used for in vitro testing are appropriate for characterizing nanomaterial interactions more broadly. As engineered
nanomaterials are introduced into organisms, it is imperative to determine which of the thousands of biomolecules in the organism will coat the nanoparticle, as this may determine their fate. Nanoparticles may
affect intra- and intercell signaling and it is important to correlate this to mechanisms such as inflammation. A better understanding is needed of the ability of nanomaterials to accumulate in organs and cells. It
is also critical to determine the dose and dose-rate dependence to understand whether new phenomena
occur at higher concentrations and rates.
2-Year Goals
• Quantitatively determine the fate and interactions of engineered nanomaterials within reference
organisms, including dose and dose-rate effects:
- determine the state of engineered nanomaterials (unbound, agglomerated)
- elucidate role of surface modification of nanomaterials on biocompatibility
- characterize interactions in fluids (biomolecule coatings, fibrillation)
- characterize interactions with cells (cell membrane, nucleus, location)
- identify potential correlations between cell signaling and cell responses such as inflammation
- identify fate and interactions of nanoparticles with organs
5-Year Goals
• Determine properties of nanomaterials that contribute to biointeractions, and develop preliminary
models for potential interactions
- develop a fundamental understanding of nanoparticle interaction with cell-signalling
pathways
7–10-Year Goals
• Identify nano-biointeractions for chronic exposure
• Validate SARs based on in vitro and in vivo data
2.3.1.6 In Vitro Testing
Once the relevant biomolecules, fluids, organs, and tissues have been identified, standard biological
media will need to be established and validated with reference to the relevant in vivo tests. This may be
best accomplished via interlaboratory tests. Sources of new biological media will need to be established
and validated as needed. New tests may be needed to assess the interaction of nanomaterials with the
nucleus when cells are replicating and differentiating.
2-Year Goals
• Identify useful in vitro systems that are predictive of in vivo responses
- identify fluids, cell membranes, cells, tissue constructs
- identify cell-signaling interruption media and tests
- develop tests that replicate nuclear membrane disruption (as during cell replication)
• Evaluate interactions of a range of engineered nanomaterials with standard in vitro tests and
compare with in vivo tests
- evaluate fluids, cell membranes, cells, tissue constructs
5-Year Goals
• Establish validated correlation between physicochemical properties and biointeractions
Summary Research Needs and Timetables
2-44
International Council on Nanotechnology
•
•
•
•
2007 Workshop Report
Validate standard in vitro biological media and assays, compare with in vivo tests
Correlate engineered nanomaterials for model in vitro and in vivo systems
Explore interactions of a broad range of engineered nanomaterials with complex coatings in standardized in vitro tests
Complete mechanistically based QSAR studies
7-Year Goals
• Develop engineered nanomaterial-specific, high-throughput screening methods with supplemental modeling
• Validate SARs based on in vitro and in vivo data
2.3.1.7 Model Development
To accelerate development of models, it is important to establish a data-sharing framework that
includes a minimum set of nanomaterial physicochemical properties, and the biological tests and biointeractions that are observed. The database should be searchable and available to a wide range of researchers to
enable comparison to their experiments.
2-Year Goals
• Design framework(s) for data sharing and ontologies
• Explore applicability of established modeling algorithms
5-Year Goals
• Establish data-sharing structures
• Establish mechanistically based QSAR studies
- develop algorithms for computational models
7-Year Goals
• Validate SARs based on in vitro and in vivo data
• Validate algorithms and training sets for computational models
• Develop engineered nanomaterial-specific, high-throughput screening methods with supplemental modeling
• Research to meet risk management needsa
2.3.2
Metrology for Risk Management
Tools for measuring the presence of engineered nanomaterials in occupational settings and the natural environment and for validating the effectiveness of PPE remain outstanding needs. Manufacture,
bagging, and clean-up of airborne engineered nanomaterials and aerosolization of liquid-form nanomaterials were identified as potential hot spots for exposure; therefore, better tools are needed to detect the
presence of engineered nanomaterials during normal workplace operations. For workplaces where nanoparticle exposure is possible, PPE must be validated as effective in limiting worker exposure. Research by
the Industry Nanoparticle Occupational Safety and Health Consortium and others is underway to evaluate
the effectiveness of filters, but efforts to establish commercial, portable nanoparticle monitors capable of
operating in the workplace and environment should be accelerated.
a. These needs emerged in workgroup discussions in both Workshops 1 and 2 and are reported here with
timelines established in the second workshop.
Summary Research Needs and Timetables
2-45
International Council on Nanotechnology
2007 Workshop Report
2-Year Goals
• Identify metrology techniques capable of detecting the presence of engineered nanomaterials in
the workplace and environment
• Validate the effectiveness of PPE in limiting exposure
5-Year Goals
• Develop portable tools to monitor a wide range of nanomaterials in the workplace and environment
2.3.2.1 Assessment of Bioavailability throughout the Lifecycle
The form that a nanoparticle takes can vary dramatically throughout its life cycle, with important
implications for its bioavailability. Certain types of carbon nanotubes, for example, aggregate into strongly
bound bundles that may be less likely to serve as a source of free nanoscale particles, whereas other types
may be more bioavailable. The bioavailability of the nanoparticle likely depends on a number of physicochemical properties, many of which will vary throughout the product life cycle. Better correlation is
needed between the nanomaterial’s physicochemical properties and its bioavailability.
5-Year Goal
Determine the bioavailability of nanomaterials throughout the life cycle
2.3.2.2 Characterization of Potential Mobility of Embedded Nanomaterials
In many applications, nanomaterials are suspended in liquids or embedded in solid matrices. The stability and mobility of nanomaterials in different matrices and their potential to break down or become
mobile under different environmental exposure conditions are largely unknown for many nanomaterials.
2-Year Goals
• Establish test methodologies to evaluate the stability and mobility of nanomaterials in liquid and
solid matrices
5-Year Goals
• Complete evaluation of stability and mobility of nanomaterials in common liquid and solid
matrices
2.4. Common Themes
2.4.1
Need to Correlate Nanoparticle Physicochemical Properties with Interactions
in Organisms and the Natural Environment
As a nanoparticle interacts with the environment or a biological organism, there is evidence that it
may become coated to varying degrees with natural organic matter or biomolecules. The extent to which
the characteristics of the coating depend on the physicochemical properties of the nanoparticle has yet to
be determined for most engineered nanomaterials, though it is expected that the precise nature of the nanoparticle surface will be an important factor. The composite structures that result from these interactions
may then interact with the organism or environment in ways that differ from the interactions of their constituent components. Moreover, it is possible that even without the formation of a composite structure, the
presence of a nanoparticle could induce changes in the local environment that result in an alteration of biological function. Thus, research is needed to correlate the physicochemical properties of engineered
nanomaterials to their interactions with biomolecules or material found in the environment, to understand
Common Themes
2-46
International Council on Nanotechnology
2007 Workshop Report
the interactions of the resultant composite structures with biological organisms (e.g., cells, tissues, and
organs), and to understand the full range of potential of nanoparticle interactions with natural systems,
regardless of whether binding occurs.
2.4.2
Importance of Dose and Dose Rate in Understanding Biointeractions
Common methods of toxicology testing employ a high dose rate and increase the dose until a biological reaction results. Because engineered nanomaterials have such a small mass, high doses would
potentially also result in high concentrations of nanoparticles in fluids and cells, so it is important to determine whether high concentrations resulting from high doses and dose rates are producing new interactions
that would not occur at lower concentrations. Similarly, engineered nanomaterials become concentrated on
membranes, so it is important to understand 1) whether inflammation and other outcomes are enhanced by
the proximity of other particles, and 2) the concentration-dependence of these interactions. It is also important to develop correlations between concentration-dependent in vitro and in vivo effects.
Because the concentration-dependence of nanoparticle interaction may be difficult to establish, it is
important to determine whether thresholds of interactions occur, which may require documentation of null
results.
2.4.3
Need for Well-Characterized Reference Materials and Standardized Assays,
and New Assay Development
Because the bioreactivity of engineered nanomaterials may depend on their synthesis, structure, and
physicochemical properties, it is very important that a line of reference nanomaterials be established where
the biointeractions are well documented and reproducible. Similarly, it is important to establish sets of biological media where the biointeractions of reference nanomaterials are reproducible. Because the
engineered nanomaterials may interact with biomolecules, and some combinations of biomolecules may
coat the nanoparticles, the biological test media need to have a controlled composition and distribution of
biomolecules. Furthermore, chemical indicators to highlight reactions need to have controlled composition
and properties. As new interactions are identified, new assays will need to be developed, validated, and
demonstrated to be reproducible.
There was some discussion of, but no resolution on, which organization should take the lead in
developing, validating, and documenting the reproducibility of the nanomaterials, media, chemicals, and
the assay. Some workshop participants felt that it may be premature to establish standard assays, because
doing so may limit the identification of new biointeractions. This may require development of standard
assays for specific tests, but not for others. Further discussion is needed on this issue.
2.4.4
Reproducibility of Research and Better Documentation of Methods to
Improve the Quality and Comparability of the Science
As many factors, some of which are yet unknown, may affect the interactions of engineered nanomaterials with biofluids and organisms, it is important that experimental details be available to enable
validation by other research groups. In some cases, technical journals only allow high-level experimental
details to be published, and those trying to reproduce results are unable to validate the earlier reported
results. It is important to establish a minimum set of data on the engineered nanomaterials, biological
media, and assays, which should be characterized and documented to allow comparisons to other experiments. If technical journals are unable to publish adequate technical details on the nanomaterial properties,
biological media, assays, and test conditions, other organizations should establish publicly accessible
repositories of experimental data.
Common Themes
2-47
International Council on Nanotechnology
2007 Workshop Report
One possibility would be to expand the ICON Nano-EHS Database to include a template for
researchers to provide information on the nanomaterials, biological media, assay, and test conditions that
would be linked to the paper’s entry in the database.
2.4.5
Methods for Predicting Potential Long-Term Effects
Testing that is done at high dose and high dose rate may elicit acute responses but fail to interrogate
chronic effects that could result in long-term health effects or diseases such as cancer. Methodologies are
needed for correlating the mechanisms associated with these diseases with the physicochemical properties
of nanomaterials that elicit these interactions. Clearly, developing models of mechanisms for long-term
interactions requires the engagement of the broader research community, but new nanomaterial tracers may
enable study of interactions that produce long-term effects. This may also require development of methods
to correlate in vitro effects to chronic diseases and cancer.
2.4.6
Metrology for Locating and Characterizing Nanomaterials in Biological
Organisms and Samples
The ability to detect the presence and concentration of nanomaterials in biological organisms is critical to developing a fundamental understanding of their biointeractions. Currently, it is possible to trace the
movement in vivo of a limited set of engineered nanomaterials with unique composition or luminescence
profiles. Tracking nanoparticles that lack these characteristics will require new tools or techniques that
enable detection without altering the properties of the nanomaterials.
2.4.7
Metrology for Exposure Characterization
Metrology is needed to detect and quantify the concentrations of nanomaterials to which workers and
consumers can be exposed. Existing methods for detecting nanoparticles in situ require large laboratory
instruments that are not practical for use in a workplace or in less controlled settings such as during consumer use. Thus, there is a need for mobile metrology tools and methodologies to monitor nanoparticle
concentrations and size distributions in the workplace, and eventually in areas where consumers use nanomaterials. The information developed in these studies would be used to improve risk assessment
methodologies and identify where hazard research is needed because of high-concentration exposure. Some
engineered nanomaterials may stay airborne at small sizes but not at larger ones, so it is important to understand how this would occur in different work environments. In the workplace, this could initially take the
form of an exposure badge that is “developed” periodically to quantify particle numbers and size distributions.
2.5. Workshop Steering Team and Sponsors
Workshop 2 was jointly sponsored by ICON, the U.S. NSF [BES-0646107] and the Swiss Reinsurance Company. The steering team included
John Balbus, Environmental Defense
Vicki Colvin, Rice University
Kenneth Dawson, University College Dublin
Thomas Epprecht, Swiss Reinsurance Company
Mike Garner, Intel Corporation
David Johnson, Rice University
Kristen Kulinowski, Rice University
Andrew Maynard, Woodrow Wilson International Center for Scholars
Workshop Steering Team and Sponsors
2-48
International Council on Nanotechnology
2007 Workshop Report
Günter Oberdörster, University of Rochester
Vicki Stone, Napier University
Sally Tinkle, NIEHS
2.6. Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of
Nanoparticles with Biological Organisms
Each breakout group was charged to produce a one- to two-page document that lays out the research
needed to develop predictive models for the mechanisms noted in the breakout group title. These should
include acellular, cellular, and whole organism considerations. For example, the oxidative stress group
(1A) was to consider the tests and research needed to evaluate the potential and potency for engineered
nanomaterials to create or neutralize reactive oxygen species in acellular matrices, how these properties
predict their activity in cellular systems, and ultimately lead to the manifestation and/or prevention of disease. Emphasis was to be on a conceptual approach and methods and was to set forth intermediate research
milestones over a 2-, 5-, and 10-year timeline.
Breakout groups in Session 1:
•
•
•
•
•
oxidative stress, inflammation, and immune response
protein misfolding/biomolecules
apoptosis and necrosis
genotoxicity and mutagenicity
developmental effects (reproductive, young, aged, susceptibility issues).
2.6.1
Breakout Group 1A: Oxidative Stress, Inflammation, and Immune Response
Breakout group leaders: Gérard Riviere (Facilitator), Sally Tinkle (Scribe and Presenter)
Goal: The goal of this breakout group was to identify the research needed to establish a structureactivity correlation between the physicochemical properties of engineered nanomaterials and the
response pathways of oxidative stress, inflammation, and the immune response.
2.6.1.1 Background
Oxidative stress, inflammation,20 and immunity are sensitive pathways that respond to environmental exposures.21 Cell biologists have been highly successful in delineating the characteristics of pathway
activation and the cellular and molecular events underlying these mechanisms. While several studies demonstrate that addition of engineered nanomaterials to cells or administration to animal models activate all
three pathways, the characteristics of engineered nanomaterials that are critical to activation and the point
of engineered nanomaterials interaction with these response pathways are not fully understood.
Identification of engineered nanomaterial-pathway interaction is challenging because of the molecular and cellular overlap between these response mechanisms, and because the physical and chemical status
of the engineered nanomaterials are dependent upon their biological microenvironment.22,23 Therefore,
Group 1A identified a set of well-established molecular and cellular endpoints in these pathways for which
assays are readily available. While validation of the assays for use with nanomaterials is necessary, these
endpoints will be useful in addressing the critical research needs for the development of SARs and predictive models of engineered nanomaterial interactions with biological systems, or nano-biointeractions.
The endpoints identified for oxidative stress include lipid peroxidation, protein oxidation, DNA oxidation, and glutathione depletion.24,25 For endpoints of inflammation, the group chose in vitro and in vivo
markers and cellular and biochemical markers. They include cell differentiation, actin smooth-muscle difCharge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
ferentiation, and epithelial-mesenchymal transition markers, as well as cytokines and chemokines,
adhesion proteins, and cell-specific markers of activation; e.g., neutrophils and elastase production. For in
vivo research, neutrophil influx and inflammatory tissue pathology were identified. Multiple endpoints of
innate, adaptive, and humoral immunity are well established; however, B cell Ig production and T helper
cell IL4/IFNγ ratios were discussed. Markers of innate immunity include p65/RelA translocation and production of cytokines on immediate time course (as opposed to the later time course of adaptive immunity).
2.6.1.2 Important Considerations for Engineered Nanomaterial Biology Research
Group 1A accepted the assumptions given in the Charge to the Breakout Groups. The group assumed
that an unlimited supply of pure, homogeneous, dispersed and well-characterized engineered nanomaterials and sufficient funding would be available for the research. However, several overarching concerns were
identified and discussed.
First, given the high surface reactivity of engineered nanomaterials, the study of agglomerated engineered nanomaterials may be unavoidable and many of the points addressed in this section will apply
directly to particle agglomerates. Therefore, analysis of particle size distribution and agglomeration status, pre- and postexperiment, is essential to obtain relevant and reproducible results.
Second, while we are aware that this may cause a controversy among different research communities, it is critical to evaluate and compare data across multiple laboratories to improve the quality and
reliability of results. It is well established that several engineered nanomaterials are able to adsorb proteins
and/or chemicals that are used in assay detection systems, thus compromising the results and the validity of
the assay. In addition, several of the nanomaterials (e.g., QDs) have color. While this in itself can be a
desirable property for tracking their fate by microscopy, it may severely limit their utility in colorimetric
assays. As a consequence of these possibilities, it becomes imperative to pretest engineered nanomaterials in biological assays to assess potential interference prior to extensive use in biological experiments.
Furthermore, it is important to validate the cell-based in vitro assays against in vivo models. While this step
is not feasible for all nanomaterials that require testing, validation of in vitro assays against animal models
may reduce the use of animals and accelerate establishment of engineered nanomaterial training sets for
the computational modeling of nanoparticle and biosystems interaction. The development of animal and
tissue culture models should be conducted in parallel.
Third, because immortalized cell lines frequently have altered protein expression and signaling pathways, validation of commonly used tissue culture models is essential. The identification and/or
development of reference cell lines was considered essential for understanding the SARs of engineered
nanomaterials in biological systems. The central questions—what constitutes a reference cell line and how
does one characterize a reference cell line—will require consideration by the nanotechnology research
community. Furthermore, reference cells lines should be validated against multiple primary cell lines and,
if possible, against in vivo models. An engineered nanomaterial training set to query and validate reference
cell lines is highly desirable.
A further important issue concerns the route of exposure. Route of exposure not only dictates the cell
type or in vivo models to be used, but is also important for studies of biokinetics and biodistribution.
These parameters are tightly linked to exposure route, as well as dose and dose rate of engineered nanomaterials in tissue culture and animal models. The range of doses should include no effects and a moderate
effect level where possible (dose metrics).
Following contact with or uptake of the engineered nanomaterials in a biological system, the engineered nanomaterials are exposed to biological fluids (biofluids) that contain more than 3500 different
molecules. Many of these molecules have the potential to interact with engineered nanomaterials on the
basis of physicochemical properties of both entities. The structure and status of the engineered nanomateriCharge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
als as a consequence of interaction with molecular entities will fundamentally alter the properties of the
engineered nanomaterials under investigation, as well as the biochemical and cellular response. This phenomenon obviously needs to be considered when selecting, for example, buffer or media for tissue culture
experiments. It also raises the question of how tissue culture experiments can be correlated across different
cell lines, because some lines require fetal bovine serum in the medium while others do not. Correlation of
tissue culture experiments to animal experiments is also challenging because media composition will vary
greatly from the molecular composition of rodent biofluids, and again from rodents to humans.
Finally, because time points and endpoints differ for in vivo and in vitro systems, it is important
that they are relevant to the model system being used and the endpoint being interrogated. This is not only
significant for the differentiation between acute and chronic effects, but also for validation of assays across
different laboratories.
2.6.1.3 Mechanisms of Cellular Response: The Signaling Pathways of Oxidative Stress,
Inflammation, and Immunity
For practical reasons, this section will first present several pathways and endpoints that measure the initial impact of engineered
nanomaterials on biological systems followed by elucidation of a more
integrated response. Given the limitations of in vitro systems, only signaling pathways activated during the acute response phase will be
discussed.
Inflammatory/oxidative stress responses can be activated by several distinct cell-engineered nanomaterial interactions. Although these
studies provide interesting data about nano-biointeractions, the mechanisms of pathway activation and examination of critical endpoints may
Figure 5. Carbon nanotubes and
prove more important. Activation of classic signaling pathways through
QDs inside cells.46
toll-like receptors and the nuclear factor-kappa B-cascade provides one
such example. This signaling cascade activates several downstream components, such as the mitogen-activated kinases (MAPK).7 MAPKs are also activated by other signaling cascades that are under the control of
growth factors such as epidermal growth factor, or cytokines such as tumor necrosis factor-alpha. Other
molecules involved in these signaling cascades translocate, upon activation, to the nucleus and act as gene
transcription factors. Transcription factors initiate transcription and translation of relevant proteins, such as
cytokines and chemokines. The translocation of transcription factors, such as RelA/p45 or AP1, may provide additional endpoints that could be measured by standard biochemical assays (gel shift assays,
enzyme-linked immunosorbent assays [ELISAs]), microscopy (e.g., green fluorescent protein-tagged tag
RelA/p65) and/or luciferase-based reporter assays. Furthermore, several signaling cascades lead to changes
in calcium concentrations, an event with profound cellular and molecular consequences. Many of these
changes are routinely measured in in vitro models and are important for developing experimentally based
engineered nanomaterial trainings sets.
AProinflammatory molecules, such as cytokines or chemokines, provide important endpoints for
measuring biological response, and many commercial kits are available. These assays are well established
for biofluids and tissue homogenates obtained from in vivo experiments, as well as media and cell homogenates obtained from in vitro systems. Changes in regulation of proinflammatory mediators have been
reported for several classes of engineered nanomaterials and will be a valuable tool in elucidating engineered nanomaterials interaction with inflammatory and immune pathways. The use of tissue samples also
allows the assessment of histopathological changes, such as changes in cell populations within an organ.
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
For example, IL-8 is upregulated by environmental exposures and induces neutrophil accumulation in the
exposure organ. Other changes may pertain to cell differentiation, such as seen in muscle or epithelial cells.
Bench-top experiments are essential for elucidation of engineered nanomaterial SARs, and the data
from these experiments must be collected, curated, and organized for use in the development of predictive
models of nano-biointeractions.
2.6.1.4 Research Needs
Breakout Group 1A identified the specific research that will be necessary to develop SARs for engineered nanomaterials over the next decade. The group acknowledged the complexity and interdigitating
nature of scientific research, the multidisciplinary integration that would need to occur, and the need for
flexibility in the timeline. To achieve all of these aims, the group established a 10-year goal and then
worked backward to determine what research would be needed to reach the 10-year goal. They also
expanded their timeline to include 7-year goals, as well as the requested 2-, 5-, and 10-year goals. Finally,
in addition to the assumptions outlined in the Workshop Scope and Assumptions presentation, Group 1A
assumed that funding would permit research to advance in parallel on multiple research tracks.
10-Year goal: To have developed computational models that assess the structure-activity behavior of
engineered nanomaterials in the oxidative stress, inflammatory, and immune responses, and that
provide estimates of hazard to human health and the environment.
2-Year Goals
•
Establish common vocabulary/terminology for engineered nanomaterials so that distinctions in
composition and structure can be readily identified
•
Develop, validate, and make available engineered nanomaterial reference materials for performance of the experiments
•
Validate existing toxicity assays for use with engineered nanomaterials and, as necessary, begin
to modify existing, or develop new, assays
•
Develop abiotic and biotic model systems from which the structure-activity data can be obtained:
-
cell-free systems for biochemical analysis
-
simple model systems, such as bilayer models, layers of receptors in culture dish
-
complex in vitro cell models that have been shown to mimic in vivo conditions accurately
•
Focus research efforts on understanding the oxidative stress, inflammatory, and immune
responses to acute engineered nanomaterial exposures
•
Identify components of a network to link laboratories focused on similar questions and explore
mechanisms to establish the linkages
•
Begin database development:
•
-
design framework(s) for data sharing that are appropriate to engineered nanomaterials
-
develop ontologies for the selection and categorization of classes of molecules and molecule
types to be examined
-
explore applicability of established algorithms used for computational modeling of chemical
exposure
Develop multidisciplinary training opportunities for graduate students and established scientists
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
5-Year Goals
• Validate newly developed assays and assay materials
• Validate abiotic and biotic model systems from which the structure-activity data can be obtained:
- cell-free systems for biochemical analysis
- simple model systems, such as bilayer models, layers of receptors in culture dish
- complex in vitro cell models that have been shown to mimic in vivo conditions accurately
• Examine abiotic and biotic data for biological correlations that confirm the accuracy of the model
systems and extrapolate to toxicity assays
• Develop and organize research data from abiotic and biotic model systems into temporary
datasharing frameworks for future use in model development
• Correlate exposure with body burden and effect
• Begin to establish data-sharing structures designed in the first 2 years
• Develop algorithms specific to the computational modeling of engineered nanomaterials
7-Year Goals
• Begin meta analysis of the biological and biochemical data to identify simple SARs that can be
used in the engineered nanomaterial modeling algorithms
• Validate algorithms using a training set of engineered nanomaterials and anticipated biological
response parameters
• Focus research efforts on understanding the oxidative stress, inflammatory, and immune
responses to acute engineered nanomaterial exposures
• Develop engineered nanomaterial-specific, high-throughput screening methods
• Develop mechanisms for integrating research data into the computational models
The development of computational models that assess the structure-activity behavior of engineered
nanomaterials for oxidative stress, inflammation, and the immune response will be challenging. Group 1A
acknowledges the optimism inherent in the 10-year timeframe that has been outlined; however, we believe
that this aggressive approach is warranted to support the safe development of this outstanding technology.
2.6.2
Breakout Group 1B: Protein Misfolding/Biomolecules
Breakout group leaders: Kenneth Dawson (Facilitator), Michael Wong (Scribe), Vyvyan
Howard (Presenter)
Question: If the human body responds to a certain dosage and exposure to nanoparticles (nominally
1–100 nm in diameter) of certain size, shape, composition, and surface coating by undergoing protein misfolding (leading to some sort of protein misfolding disease), how does the human body do so?
Do nanoparticles induce or accelerate the onset of amyloid disease?
The objective of this breakout group was to lay out the research (i.e., conceptual approach and methods) needed to develop predictive models for protein misfolding promoted by nanoparticles. Given what is
known about protein misfolding diseases and about the implied role of nanoparticles in protein misfolding
diseases, what are the chemical tests needed to evaluate the potency of nanoparticles in causing protein
misfolding (through direct or nondirect intereactions with proteins)?
2.6.2.1 Background
There are approximately 26 protein misfolding diseases, which can be categorized into neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, ALS (Lou
Gehrig’s disease), and Creutzfeldt-Jakob disease, and nonneurodegenerative diseases, such as diabetes 2
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
and dialysis-related amyloidosis (DRA). Many neurodegenerative diseases are characterized by the accumulation of abnormal protein deposits (referred collectively as amyloid deposits) in the brain and other
organs in the central nervous system. “…approximately 20 distinct proteins are involved in amyloidoses
(extracellular deposits). A number of diseases are also associated with the accumulation of fibrillar intracellular deposits or are associated with the buildup of nonfibrillar deposits.”28
Protein molecules normally exist in the properly folded, soluble form. As a disorder of protein
metabolism, protein misfolding can result from the intracellular disruption of chaperoning and the disruption of the protein degradation pathway. It is not clear what causes proteins to misfold to form protein
aggregates and fibrils in their interaction with nanoparticles.
While there is strong evidence that protein fibrillation is the origin of several of these diseases, there
are cases in which it is unclear that fibrils are the cause or consequence of the disease. In several cases,
reactive oxygen species (ROS) have been associated with the disease, though the role as a cofactor or spectator species is not known. In yet other cases (for example, Alzheimer’s), oligomers (or protofibrillar
structures) have been identified as vectors for the disease.
2.6.2.2 Summary of Group Discussion
Breakout Group 1B began the discussion by building up the case that nanoparticles are connected to
protein misfolding diseases, based on several pieces of literature evidence. There is already in vitro evidence that nanoparticles increase protein misfolding and fibril formation.30 There is also evidence that
nanoparticles smaller than 40 nm can pass through the blood-brain barrier. And, there is epidemiological
work by Lilian Calderón-Garcidueñas and coworkers30 that correlated a higher incidence in Alzheimer’s
disease in Mexico City with air quality in dogs.
To address the question “Do nanoparticles induce/accelerate the onset of amyloid disease?,” the
breakout group considered two general pathways through which nanoparticles can lead to this: 1) the direct
route, in which nanoparticles somehow cause proteins to misfold and aggregate into fibrils, and 2) the indirect route, in which nanoparticles somehow interfere with protein metabolism mechanisms. The breakout
group focused on the direct route as the main protein misfolding pathway.
Direct Route
In vitro fibril formation in the presence of several nanoparticle types was recently described. Linse
and coworkers30 showed that several nanoparticle types (of different compositions and sizes) can cause
fibril formation in test-tube studies (see figure below). Once the fibrils nucleate, their growth continues
unabated. Their results indicate that nanoparticles act to catalyze the nucleation of fibrils, suggesting the
possible in vivo nanoparticle-induced fibril formation as a new pathway to protein-misfolding disease.
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
Figure 6. Schematic of (A) nanoparticles (blue) and amyloid protein molecules (green), and (B) nanoparticles
with the protein molecules in the form of surface-adsorbed larger protein fibrils. From Colvin and Kulinowski,
2007.31
Indirect Route
The breakout group acknowledged the potential interference of nanoparticles on protein metabolism.
It was suggested that nanoparticles could cause the failure of the various biochemical reactions that affect
protein structure (such as the well-described proteolysis, well-described chaperoning, and post-translational modification), thereby leading to proteins that are more susceptible to misfolding or to accumulation
of misfolded proteins in the body.
The most common tests for amyloid diseases are cognitive function testing and pathology (postmortem). These tests are not diagnostic, unfortunately. Also, there are no generally accepted diagnostics for
these diseases. Thus, only when the patient exhibits function impairment or loss is there any indication of
the disease, which is too late in disease prevention or management.
Concerning the level of published literature that links nanoparticles to amyloid disease, there is the
above-cited papers by Lilian Calderón-Garcidueñas on air quality and Alzheimer’s disease, suggesting the
active role of aerosol-originating nanoparticles. Linse and coworkers have published work describing fibril
formation induced by nanoparticles.29 It was noted that there are papers that indicate that nanoparticles can
retard fibril formation also, suggesting a possible amyloid disease treatment modality.32-34 Such work indicates that fibrils are not the cause of the disease. It also suggests that an understanding of how these
aggregates cause the disease can lead to new insights into the role of nanoparticles in amyloid disease,
because the aggregates can be considered to be a type of nanoparticle (naturally occurring, but abnormal).
Given the paucity of published research, the breakout group considered there to be significant challenges to identifying the protein-misfolding mechanisms induced by nanoparticles. It is reasonable to
assume that the direct and indirect pathways can occur simultaneously, thus making more difficult the formulation of a predictive model for nanoparticle-induced protein misfolding.
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
2.6.2.3 Research Needs
2-Year Goals
•
•
Qualitative determination of the fate of nanoparticles within the human body (biodistribution,
pharmacokinetics)
In vitro assessment of the likelihood of fibrillation (if it doesn’t happen in vitro, then it doesn’t
happen in vivo [most likely])
5-Year Goals
•
•
•
Quantitative determination of the fate of nanoparticles within the human body (biodistribution,
pharmacokinetics)
Computational modeling of protein-nanoparticle interactions, molecular dynamic (MD) simulation
Detailed biochemical assay to inform on indirect nanoparticle effects
10-Year Goals
•
•
•
Epidemiological studies (occupational health in nanoparticle manufacturing)
In vivo mechanistic studies
Advanced microscopy techniques (TEM, positron emission tomography [PET])
2.6.3
Breakout Group 1C: Apoptosis and Necrosis
Breakout group leaders: Agnes Kane (Facilitator and Presenter), Scott McNeil (Scribe)
Objective: The goal of this breakout group was to identify research needed to establish a correlation
between the physicochemical properties of engineered nanomaterials and the activation of apoptosis
or necrosis.
2.6.3.1 Background
Exposure to nanoparticles could potentially impact cells and tissues, leading to disruption of normal
homeostasis accompanied by adaptive physiological responses, reversible injury, or irreversible injury and
cell death. Depending on the severity and duration of external stress or altered homeostasis, cells can adapt
by hypertrophy, hyperplasia, or metaplasia. When the capacity for adaptation is exceeded, cells may
undergo reversible injury or progressive, irreversible injury leading to the morphologic manifestations of
cell death recognized as apoptosis or necrosis.
Apoptosis is a normal physiological occurrence in immune reactions, during normal embryonic
development and following hormonal withdrawal. Inhibition of apoptosis can lead to exaggerated immune
responses, developmental malformations, and development of tumors, while excess apoptosis can lead to
tissue injury following ischemia and reperfusion or neurodegenerative disease. Pathologic apoptosis can be
induced by exogenous stimuli, such as cytotoxic drugs or viral infections.
Two distinct pathways lead to apoptosis or necrosis. Activation of apoptotic pathways occurs rapidly
over a period of minutes to hours. Ultrastructurally, apoptosis is characterized by condensation of cellular
organelles, chromatin condensation, DNA fragmentation, and loss of membrane asymmetry, followed by
plasma membrane blebbing, cell fragmentation, and release of apoptotic bodies. The end result of apoptosis is phagocytosis of apoptotic bodies by adjacent cells with no accompanying inflammatory or immune
response. Apoptosis may occur through caspase-dependent mechanistic pathways that may be extrinsic;
for example, receptor mediated, or intrinsic and mediated by mitochondria, as illustrated in Figure 7.
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
Figure 7. Left: Pathways leading to apoptosis. Right: TEM illustrating apoptosis induced in a murine macrophage exposed to asbestos fibers in vitro. Photograph prepared by Vanesa Sanchez and Paula Weston at
Brown University. Both images from Majno and Joris, 2004.35
Necrosis is linked to accidental or pathologic cell death. The early stages leading to necrosis are
potentially reversible and involve damage to the cell membrane or adenosine triphosphate (ATP) depletion,
which leads to decreased activity of ion pumps and cell swelling. The late stages of necrosis are characterized by nuclear shrinkage and dissolution, followed by cell lysis, which evolve over a period of 24 hours to
several days. The biochemical alterations responsible for necrosis involve irreversible mitochondrial damage, lysosomal disruption, loss of the plasma membrane permeability barrier, elevated levels of
intracellular Ca2+, activation of phospholipases and proteases, and breakdown of membrane phospholipids,
as illustrated below.36 The consequences of cell death characterized by necrosis may be severe, resulting in
compromised tissue function, inflammation, and fibrous scarring.
Figure 8. Left: Pathways leading to necrosis. Right: TEM illustrating necrosis (upper right) induced in a
murine macrophage exposed to crystalline silica particles in vitro. Photograph prepared by Vanesa Sanchez
and Paula Weston at Brown University. Both images from Kumar et al., 2005.36
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
2.6.3.2 Impact of Nanoparticles on Cell Integrity
Exposure to nanoparticles could potentially impact cells or tissues at any point along the continuum
of normal homeostasis, reversible adaptive responses, reversible injury, and irreversible injury or cell
death. Under some conditions or at sublethal doses, exposure to nanoparticles may induce cell proliferation, reproductive cell death, or cellular senescence.
The potential interactions between nanoparticles and these apoptotic or necrotic pathways have yet
to be determined. Nanoparticles may penetrate cells and subcellular organelles more readily than larger
particles or agglomerated nanostructures, leading to potential disruption of the cell membrane, blockage of
ion channels, and alteration of redox potential. Binding of nanoparticles to surface receptors may alter
intracellular signalling or disrupt cellular adhesion. Alternatively, external nanoparticles or agglomerates
may cause injury indirectly by releasing toxic components (e.g., metals) or catalyzing generations of ROS,
resulting in oxidative stress.37,38 Recent publications have described several examples of interactions
between nanoparticles and subcellular organelles that may lead to cell death by activation of apoptotic or
necrotic pathways, as illustrated in the following table.
Cellular Targets of Nanoparticle Toxicity
Mechanism of
Injury
Example
Reference
Lipid peroxidation
Nano-C60, SWNTs
Sayes et al., 200539
Kagan et al., 200637
Ion channel blockers
Pore formation
SWNTs
Dendrimers
Park et al., 200340
Hong et al., 200641
Mitochondria
Physical disruption
Oxidative stress
Ultrafine ambient
particulates
Polystyrene nanoparticles
Li et al., 200342
Xia et al., 200638
Nucleus
Protein aggregation
DNA damage
SiO2 nanoparticles
QDs
Chen and von Mikecz, 200543
Green and Howman, 200544
Cellular Target
Plasma
membrane
Diagnostic assays for apoptosis and necrosis are technically difficult, particularly in vivo. A battery
of assays is usually indicated rather than relying on a single endpoint. Single-cell assays are desirable,
especially TEM, which is the gold standard for recognizing apoptosis.
Additional techniques to identify apoptotic cells include DNA extraction and visualization of a DNA
ladder pattern using gel electrophoresis, detection of double-strand DNA breaks using a terminal uridine
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, biochemical or immunocytochemical assays for caspase or transglutaminase activation, and fluorescence staining for annexin-V using flow
cytometry.
Quantitative morphometry at the ultrastructural level is required to determine the dose of nanoparticles delivered to target cells. For non-TEM-based assays, a dose-response curve is generated based on the
average response of a cell population; however, nanoparticles may specifically target one type of cell in a
mixed population. Extracellular dose may be expressed in terms of surface area, mass, particle number, and
agglomeration state of the nanoparticle, while the intracellular dose refers to the dose of nanoparticles
delivered to the target subcellular site.45 Nanoparticle toxicity assays must be calibrated with negative and
positive reference samples. Secondary modification of nanoparticles during systemic exposure may alter
cellular toxicity in vivo, so caution must be applied in extrapolation of short-term cellular toxicity assays to
chronic exposure in vivo.
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
2.6.3.3 Research Needs
2-Year Goals
• Develop methodology and generate data
• Measure in vitro toxicity quantitatively
• Correlate multiple assays to confirm cell function and loss of viability
• Develop new techniques for live cellular imaging at the nanoscale to supplement TEM
• Assess high content/multiparameter cellular responses to nanoparticles
• Elucidate role of surface modification of nanoparticles on biocompatibility
• Weight physicochemical properties that influence biocompatibility/toxicity
5-Year Goals
• Validate multiparameter cellular toxicity assays
• Engineer particles with tight control of specific properties
• Extrapolate in vitro to in vivo effects
• Study dose response and kinetics to elucidate mechanistic pathways leading to toxicity
• Study mechanistically based QSAR
• Incorporate other relevant cellular endpoints into models: cell proliferation, state of differentiation, senescence
10-Year Goals
• Implement engineering and predictive models
• Develop modeling tools for predictive assessment of nanoparticle-induced cell injury and death
• Engineer nanoparticles with beneficial properties: biocompatible medical implants and devices,
improved diagnostic and imaging tools, and novel drug delivery strategies
2.6.4
Breakout Group 1D: Genotoxicity and Mutagenicity
Breakout group leaders: Flemming Cassee (Facilitator, Presenter, Scribe), Bruno Orthen (Scribe)
Objective: The goal of this breakout group was to identify the research needed to establish a correlation between the physicochemical properties of engineered nanomaterials and the activation of genotoxicity and mutagenicity.
2.6.4.1 Background
Genotoxicity is defined as damage to a DNA molecule that can sometimes result in mutations. Substances that are genotoxic may bind directly to DNA or act indirectly, leading to DNA damage by affecting
enzymes involved in DNA replication, thereby causing mutations that may or may not lead to cancer or
birth defects (inheritable damage). Mutagenicity is the capacity of a chemical or physical agent to cause
permanent alteration of the genetic material within living cells. Fixation of damage to DNA in the form of
gene mutations, larger-scale chromosomal damage, recombination, and numerical chromosome changes is
generally considered to be essential for heritable effects and in the multistep process of malignancy, a complex process in which genetic changes may play only a part.
Mechanisms by which nanoparticles may lead to genotoxicity include
• induction of oxidative stress-producing ROS and reactive nitrogen species (NOS) that can signal
various processes or directly interact with the DNA
• direct interaction of nanoparticles with DNA to form, for example, bulky adducts
• interaction with receptors and receptor mediated uptake
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
•
2007 Workshop Report
deposition of particles on cells with subsequent release of active components or phagocytosis and
dissolution to become toxic.
In addition, genotoxicity and mutations can ultimately lead to carcinogenicity or reproductive and
teratological disorders.
Papageorgiou et al.47 compared genotoxic effects of nanoparticles and micron-sized particles of
cobalt chrome. Nanoparticles induced more DNA damage than micron-sized particles using the alkaline
comet assay. They induced more aneuploidy and more cytotoxicity at equivalent volumetric dose. Nanoparticles appeared to disintegrate within the cells faster than microparticles with the creation of electrondense deposits in the cell, which were enriched in cobalt. On the other hand, dimercaptosuccinic acid(DMSA-) coated maghemite nanoparticles did not appear to have genotoxic potential when tested on
human dermal fibroblasts.48
2.6.4.2 Testing Strategiesb
Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds that
induce genetic damage directly or indirectly by various mechanisms. These tests should enable hazard
identification with respect to damage to DNA (genotoxicity) and its fixation (mutagenicity). Compounds
that are positive in tests that detect such kinds of damage have the potential to be human carcinogens or
mutagens; i.e., may induce cancer or heritable defects. A number of standardized tests are available and
already in use for chemical risk assessment; e.g., induction of DNA strand breaks measured by the comet
assay, and mutagenicity using the standardized Ames test on the Salmonella typhimurium strains. The
chromosome aberration (CA) and micronucleus (MN) are two other examples of assays that can be applied
using in vitro and in vivo exposure. In case of in vitro exposures in cell systems, one should take care that
nanoparticles indeed reach the cell when suspended in culture media.
Examples of requirements for testing:
•
•
•
a test for gene mutation in bacteria
an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an
in vitro mouse lymphoma tk assay (detect gross chromosomal damage, chromosomal aberrations
or loss of chromosomes, breakage and balanced or unbalanced rejoining of DNA)
an in vivo test for chromosomal damage using rodent hematopoietic cells.
Modifications of these strategies are warranted if
•
•
•
•
use of bacterial test organisms is problematic, for example because of high cytotoxicity
despite negative findings, the compounds bear structural alerts for genotoxic activity
in vitro tests are of limited use in replicating standard in vivo tests activity due to toxicokinetics
effects
additional carcinogenicity has been demonstrated in vivo.
2.6.4.3 How to Go Forward
Two areas of outstanding research must be explored to more fully understand the potential for nanoparticles to induce genotoxicity or mutagenicity:
•
•
nanoparticles might damage cell membranes, resulting in access to the DNA (in the nucleus)
nanoparticles might penetrate the nucleus.
b.
Based on an FDA report: http://www.fda.gov/CDER/GUIDANCE/1856fnl.pdf
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
What’s the difference? In each case the nanoparticle would have to get to the nucleus to affect the
DNA.
Nanoparticle penetration of the nucleus has potential implications for reproductive toxicology and
teratology as well as child development. Yet little is known about whether nanoparticles can get across the
cell and nuclear membranes and what physicochemical factors (size, coating, charge) might influence penetrability. Developing or identifying tests wherein the nuclear membrane disappears (during cell
replication) would enable researchers to explore directly the impact of nanoparticles on the nucleus.
The other outstanding question is whether nanoparticles might damage a cell membrane, resulting in
access to the DNA. Because direct interaction is the less well-known mechanism, the group identified that
there is a need to find out under what conditions and with what kind of particle properties a nanoparticle
can translocate to the cell nucleus. We may learn from studies that have identified certain compounds as
genotoxic in the absence of oxidative stress or in which it has actually been demonstrated that a chemical
reaches the DNA. What were the intrinsic properties of these components?
Because most, if not all, of these tests are in vitro, there is a need to identify realistic in vivo exposure
pathways. In other words, if in vivo tests are performed with single nanoparticles but in vivo these nanoparticles agglomerate prior to interacting with a cell, the predictive value of the in vitro tests is limited and
more realistic tests should be developed.
•
•
•
•
Dose rate and high-dose situations need to be addressed in more detail (toxicokinetics)
Well defined, validated, and standardized tests are probably appropriate for nanoparticles, but
other tests may need to be developed as new effects are identified.
Accumulation may lead to inactivation of gene expression. Also, what happens with phagocytosed particles?
There is a need for reference material (such as standardized quartz for inhalation toxicity); also
reference coatings (having an influence on cellular incorporation and toxicity) that have been
demonstrated to have genotoxic and/or mutatgenic properties. Reference test methods are also
needed.
2.6.4.4 Research Needs
2-Year Goals
• In vitro assessment of nanoparticle-DNA and nanoparticle-ribonucleic acid (RNA) interactions
• In vitro assessment of the likelihood that nanoparticles cross cell and nucleus membranes
• Evaluation of mutagenicity and genotoxicity of a small set of frequently used nanoparticles
5-Year Goals
• In vivo assessment of kinetics of a wide range of nanoparticles with different physicochemical
properties
• In vivo assessment of genotoxic and mutagenic effects in target organs
• Validation of novel high-capacity screening tools such as photoelectrochemistry-based DNA
sensors49
• Well-defined reference materials
10-Year Goals
• Epidemiological studies
• In vivo mechanistic studies
Other relevant works appear in the citation list at the end of the document.50,52-55
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2.6.5
2007 Workshop Report
Breakout Group 1E: Developmental Effects
Breakout group leaders: Michael Riediker (Facilitator, Presenter), Robert Tanguay (Scribe)
2.6.5.1 Background
There is likely to be unique susceptibility of the organism from conception through senescence in
regard to interactions with nanomaterials. All cells of the developing organism have identical genetic information, but differential gene expression leads to differences in cell fate. A normal and complete organism is
established via a multistep process in which cells become committed to their fate and then work collectively.
This process is characterized by the sequential expression of unique repertoires of structural and functional
molecules (see Figure 9). At any of the stages, there is a potential for interactions with nanomaterial. Even
minor alterations, such as changes in ion gradients or hormones, can lead to developmental changes if the
interactions occur at a sensitive stage. Such sensitivity has been previously shown with toxicants such as tetrachlorodibenzo-p-dioxin (TCDD), thalidomide, ethanol, retinoids, endocrine disruptors, valproic acid,
lithium, and diethylstilbestrol (DES). Like the developing organism, the aged or senescent organism is also
likely to represent a sensitive population following exposure to nanomaterial because of altered oxidant/
antioxidant balance, slower injury repair mechanisms, and underlying degenerative diseases. The sensitivity
of the aged, compromised organisms has been shown in studies on the effects of ambient particulate matter.
A basic assumption made by the group in order to move forward with discussions of mechanisms of
nanomaterial interactions with developmental targets was that the nanomaterial would reach those targets
irrespective of the route of exposure (e.g., nanomaterial will cross the placenta). However, it was also
acknowledged that nanomaterial may not have to be physically present at the target to have effects. For
example, neurodegenerative processes in the aging organism could be exacerbated after translocation of
nanomaterial to the brain, as is suggested by current literature, or via neurohormonal or inflammatory
mediators. Another assumption was that different organ systems have different susceptibilities, both during
development and senescence. To help focus the discussion, it was also acknowledged that the key mechanistic pathways discussed by the other groups (oxidative stress/inflammation, immune response, protein
misfolding, apoptosis/necrosis, genotoxicity/mutagenicity) could be operative in producing developmental
effects if they occur at sensitive time points. The key, however, was that nanomaterial must reversibly or
irreversibly cause structural or functional alterations that interfere with homeostasis, normal growth, cellular differentiation, tissue repair, or behavior to be considered to have developmental effects.
(6-7 d)
CONCEPTION
1-3 d
Cleavage
9 WEEKS
3 MONTHS
IMPLANTATION
4-6 d
Blastula
20-28 d
Neurula
Gastrula
7-13 d
36-56 d
Embryo
26 WEEKS
6 MONTHS
BIRTH
38 WEEKS
9 MONTHS
Labor
Tailbud
29-35
Pre-implantation
Period
Perinatal
Period
Organogenesis
Nursing Period
EMBRYONIC PERIOD
WEANING
Figure 9. Diagram illustrating the timing of developmental events. Notice
that an animal is in a unique state at
each time point and expresses a
unique repertoire of molecular targets.
Fetal Period
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
2.6.5.2 Summary of Discussions
Research Needs and Examples
First, there are several hypothetical mechanisms that need to be tested by which developmental
effects might occur following nanomaterial interactions with key targets, but very little is known about
such interactions. Early studies with encapsulated semiconductor nanocrystals, for example, showed that
Xenopus embryos retained the fluorescent materials and developed normally following microinjection
exposures during the blastula stage of development.56 There is the potential for nanomaterial to alter
molecular signaling via inhibition, inappropriate activation, or modulation. Such alterations could occur
via the adsorption of key signaling molecules to nanomaterial (i.e., nanomaterial acting like a sponge)
thereby lowering the concentration of the signal or altering a gradient. Likewise, if signaling molecules
adsorb to nanomaterial, they could effectively be delivered to inappropriate targets or to their targets in an
inappropriate time frame (i.e., a “moving sponge”). Included in this concept is the idea that ion transport
can be altered; e.g., Ca2+ signaling. There are several stages of development where Ca2+ signaling is critical and several scenarios are conceivable by which nanomaterial could alter ion concentrations. This makes
altered molecular signaling a plausible mechanism by which nanomaterial can have developmental effects.
Nanomaterial could also change intercellular interactions, possibly via binding to matrix or cytoskeletal
proteins, which could ultimately lead to changes in cellular migration. In vitro studies demonstrated the
binding of actin and intermediate filament proteins to 200-m polystyrene spheres.57 Changes in cell migration could have disastrous consequences in the developing organism. It is also possible that nanomaterial
interactions with cells could result in altered cell surface charge or mechanical properties related to the
maintenance of membrane integrity and the distribution of membrane-bound macromolecules (e.g., receptors). All of these processes could affect the discrete and highly controlled communication between cells
that controls development as well as injury repair.
Second, the senescent organism may be uniquely susceptible to the effects of nanomaterial. One contributing factor could be the accumulation of biopersistent nanomaterial over a lifetime of exposures. In
addition, because there appears to be changes in metabolism as well as antioxidant status, adaptation in the
senescent organism could be affected such that responses to nanomaterial are enhanced. Along these lines,
the degradation and efficacy of therapeutic interventions employing nanosized materials should be investigated. Slower injury repair processes could also contribute to increased susceptibility of the senescent
organism. Susceptibility may also be caused by “multiple insults.” Underlying neurodegenerative, cardiovascular, or pulmonary diseases could be exacerbated by nanomaterial. Such increased susceptibility has
been shown in epidemiological studies of aged individuals with preexisting cardiopulmonary disease who
are exposed to fine-mode (containing nanosized particles) ambient air particulate matter.58,59
Third, although it is unclear exactly how such effects might occur, nanomaterial could cause reproductive toxicity. This cannot be excluded from a theoretical standpoint, however, given the paucity of data.
Gametogenesis is one stage at which reproductive effects could occur. The stages of sexual development
and maturation could also represent sensitive windows of opportunity for nanomaterial interactions. If
nanomaterials bind to hormones and, therefore, effectively remove their signal or create an inappropriate
one, as discussed above, this particular developmental stage may not be so implausible a target. Lastly, if
nanomaterials are retained in the tissues of the developing organism following in utero exposures, it is
remotely possible that effects could be observed in the subsequent generation.
The largest information gap in assessing the life stage effects of nanomaterial is the lack of both
descriptive and mechanistic data regarding the fate and effects of the materials in developing systems. For
this type of assessment, there are some serious limitations to the knowledge that can be attained using cell
culture model systems, and caution should be exercised to prevent inappropriate generalizations from such
studies. Examples of the issues that can best be addressed in vivo and are critically needed for effective risk
Charge to the Breakout Groups—Session 1: Mechanisms for Interaction of Nanoparticles with Biological Organisms2-
International Council on Nanotechnology
2007 Workshop Report
assessment include nanomaterial metabolism, translocation, intercellular signaling and interactions, and
effects in tissues that are distant from the site of deposition. A few well-defined in vivo studies will also
help place into context the results from in vitro studies, particularly in regard to dose and response relevance. Finally, it may be possible to leverage nanomaterial research regarding susceptible populations with
existing programs focusing on ambient particulate matter.
Time Frames
In the short term, there is a critical need for the rapid assessment of nanomaterial life stage interactions and effects using diverse in vivo indicator model systems. In the time frame of 2-5 years, the field
should be advanced to the point that some defining mechanisms of response are elucidated. Using prior
knowledge from the study of endocrine disruptors or metals, for example, it may be feasible to determine if
a given developmental process or molecular event is perturbed by nanomaterials using in vitro systems. If a
signaling pathway is disrupted, then hypotheses could be tested using animal models to determine dose and
response relevance. A reasonable goal for the long-term is to start prospective studies of occupationally
exposed populations and their offspring. Another long-term goal is the development of predictive models
that arise from solid mechanistic- and effects-based research.
•
Especially for the evaluation of developmental effects and for identifying sensitive subpopulations (e.g., senescent individuals, people with underlying inflammatory conditions or exposures
to pathogens), it is essential that animal studies be included because of the complex cellular interactions that are likely to play a role in altered sensitivities.
•
In vitro models need to be well validated (e.g., in regard to dose relevance, response relevance).
2.7. Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles
with Living Organisms
Each group was charged to consider crosscutting issues related to nanoparticles and biological systems generally. Each group focused on a particular type of interaction (e.g., biomolecular-nanoparticle
interactions outside of cellular systems) and developed a model RFP that outlines the research steps necessary to provide predictive models by 2018. Topics for discussion were to include what near-term
correlative studies are needed to test important hypotheses, best research strategies for proving general
mechanisms for nanoparticle-biological interactions, and how best to develop informative in vitro tests that
predict in vivo responses to nanoparticles. Ultimately, the output of this group was to be an RFP-type document that lays out research areas for prioritization over the 2-, 5-, and 10-year time frames and integrates
discussions from both breakout sessions.
Breakout groups in Session 2:
•
nanoparticle-biofluid interactions/target cell interactions
•
cell signaling/tissue constructs
•
whole-animal interactions/biokinetics
•
ecotoxicology.
[Note: The RFP assignment was designed to stimulate creative thinking about research strategies and
was not intended to provide an actual governmental call for proposals.]
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-64
International Council on Nanotechnology
2.7.1
2007 Workshop Report
Breakout Group 2A: Nanoparticle-Biofluid Interactions/Target Cell
Interactions
Breakout group leaders: Kenneth Dawson (Facilitator), Michael Garner (Scribe), Michael
Wong (Presenter)
2.7.1.1 Background
Nanoparticles placed in biological fluids can interact with biomolecules including proteins, nucleic
acids, and lipids, which can deposit on the surface of the particle in a dynamic interaction. Evidence shows
that these biomolecules have a residence time on the particle surface determined by the particle properties,
molecule, and fluid conditions. Some molecules have a higher affinity for adhesion and longer residence
times on the surface than others, but molecular coverage and conformation may change dynamically, based
on fluid conditions and other factors. In cases where the particle is coated to a large degree with biomolecules, this is referred to as a corona. Because the interaction between the biomolecule and particle also
depends on the properties of the particle surface, different biomolecular species may be deposited to differing degrees on the surface of particles with different compositions, electronic properties, or nanostructure.
For particles that are coated with a biomolecular “corona,” their interaction within the fluid and with cells
may be influenced by the properties of the species in the corona in addition to the properties of the nanoparticle surface. Thus, it is important to understand the role of nanoparticle surface chemistry and structure
on the biomolecular adhesion profile and how this is changed by the fluid chemistry.
It is also possible that some particle surfaces may not be coated or may be incompletely coated.
Because the particle may be imperfectly coated with a biomolecule corona, the fluid may interact directly
with the particle, and reactions or dissolution could occur on exposed particle surfaces. Soluble nanoparticles may release atoms or ions into the biological fluid depending on coverage and the fluid chemistry, and
the released atoms or ions may interact with the fluid or cells independently.
The properties of the particle-corona may change depending on the molecule conformation on the
surface of the particle, which may be changed by fluid conditions such as pH and temperature. Thus, interactions of the particle-corona with cells and other biomolecules will be affected by the conformational
state of the biomolecules in the corona, which can be changed by fluid conditions. Furthermore, molecules
not bound to the particle may be conformationally changed by the interaction with some corona structures
or with exposed regions of partially covered particles. The biomolecule coating on nanoparticles may vary
with different animal species and in their fluids. Also, correlations need to be established between biomolecular coatings determined in vitro and coatings occurring in living organisms, in vivo.
The scope of this assessment is for engineered nanoparticles to be used in beneficial applications
(biological apps) and unintended interactions. The corona of biomolecules on nanoparticles may influence
their behavior for potential beneficial applications as well as in unintended interactions. Thus, it will be
important to understand the interactions of the nanoparticle in the biofluid, potential coating with biomolecules, the formation of a corona, changes of the biomolecule coating or corona with time and fluid
conditions, and how these biomolecule-coated particles interact with other biomolecules and cells.
2.7.1.2 Definitions
Biomolecule = proteins, nucleic acids, lipids
Nanoparticle-biomolecule interaction = chemical and physical interactions, including ionic, covalent,
hydrogen bonding, van der Waals, and other effects resulting in conformational changes
Nanoparticle-cell interactions (with or without biopolymer corona) = physical/chemical contact between
the complex nanoparticle that may or may not produce a change in cell function, structure, or sensitivity
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-65
International Council on Nanotechnology
2007 Workshop Report
Corona = a biomolecular coating on a nanoparticle that covers most of the particle and may have multiple
biomolecule types on the surface.
2.7.1.3 Research Needs
Objective: Understand and model the important concepts that define nano-biointeraction (e.g., particle/agglomerate-corona-cell interactions) with in vitro effects validated in living organisms, in vivo.
2-Year Goals
• Experiments to understand the important concepts that define nano-biointeraction (e.g., particlecorona-cell interactions)
- characterization of size, shape, physicochemical properties of selected nanomaterials
- categorization of material properties that correlate to protein and other biomolecule corona
composition: identification of trends in corona composition with physicochemical particle
properties
• Metrology
- feasibility study of corona reading with aberration-corrected TEM and other single-particle
imaging techniques
- OMICs (genomics, transcriptomics, proteomics, and metabolomics) profiling of cell particle
interactions
- consensus on strategy to measure the corona composition, structure, and conformation
- consensus on measuring physicochemical properties
• Standards
- standard cell lines (e.g., macrophage, dendritic cells, Kupffer cells) from single source
- standardized biological media (e.g., protein representative, lavage)
- interlaboratory comparisons of standard cells, biological media, and biological endpoints
(potential crosscutting issue)
• Informatics
- simulations to evaluate simple biomolecule-surface interactions
- experimental conditions documented with publications
5-Year Goals
• Systematic variation of chosen physicochemical parameters to establish the relationship between
the physical, structural, and chemical characteristics of nanoparticles and key nanoparticle-biological interactions, and to assess the feasibility of deriving QSAR models for defined activities
- study nanoparticle agglomeration state in model biofluids (simplified and multicomponent)
• thermodynamic interactions and kinetic control
- screening of material properties that correlate to biomolecule corona composition
- determine the interactions of the model nanoparticle-corona structures with reference/
standard cell lines
• multiple biological endpoint correlations with protein corona
• Metrology to characterize critical structure-property biointeractions
- analytical tools and methods to quantitatively characterize the corona’s composition,
coverage, and structure (as a function of pH, temperature, ionic strength, and other
representative biofluid properties)
• small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS)
- successful corona reading with aberration-corrected TEM and other single-particle imaging
techniques
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-66
International Council on Nanotechnology
2007 Workshop Report
-
•
•
quantitative OMIC (transcriptomics, proteomics) profiling of corona and cell-particle
correlations
- well-characterized metrology to monitor agglomeration state and biointeractions
Standards
- reference cell lines (e.g., macrophage, dendritic cells, Kupffer cells)
- reference biological media
Bioinformatics capability that enables
- kinetic modeling to capture particle-biomolecule-cell interaction (activation energies, rate
constants)
- molecular modeling to simulate and evaluate particle-biomolecule interactions, and
biomolecule-biomolecule interactions
10-Year Goals
•
•
•
Model the physical, structural, and chemical evolution of the particle-corona (temporal location
of the particle and other life-cycle issues) (dynamics and kinetics)
Understand the important concepts that define nano-biointeraction (e.g., particle-corona-cell
interactions)
Understand the effect of nanoparticle aggregation on biointeractions
2.7.2
Breakout Group 2B: Cell Signaling and Communication
Breakout group leaders: Joel Pounds (Facilitator), Alison Elder (Scribe and Presenter)
2.7.2.1 Background
The cell membrane and its receptors (e.g., pattern-recognition sequences) may be the first structural
components with which nanomaterials interact. Signaling events that occur within and between cells coordinate diverse processes related to cell division, senescence, and adaptation or response to physiological
and toxicological stimuli. These events are critical during developmental, inflammatory, and immune
responses. By examining these signaling processes, we can gain insight regarding changes within the cellular environment that are caused by nanomaterials.
2.7.2.2 Summary of Discussions
Research Needs
The main goal of future research efforts should be to better understand how nanomaterials acutely
affect inter- and intracellular signaling pathways and cell-cell interactions in relevant organ systems. Proposals should implement well-validated experimental approaches for assessing the biocompatibility of
nanoparticles to support the creation of predictive models for assessing nanoparticle effects in vertebrates.
In vitro model systems are appropriate if well validated for gaining information about mechanistic
aspects of response and inter-/intracellular signaling. There are, however, significant limitations with existing systems using either single-cell types or constructs with multiple cell types that are related to dose and
response relevance. There is, therefore, a need to improve current in vitro methods to make them reliable
predictors of nanoparticle interactions with and effects in cells. Similarly, in vivo studies could be informative regarding cell signaling and communication. Tissues harvested from acutely exposed animals for
manipulations in ex vivo systems could be particularly useful. However, in vivo studies should focus on
appropriate target organs (e.g., skin, lung, gut, kidney, liver) to reduce the total number of animals used.
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-67
International Council on Nanotechnology
2007 Workshop Report
It can be envisioned that projects undertaken to address the issues regarding nanomaterial-cell interactions can take on many different forms, including both single-investigator and multiple investigatormultidisciplinary projects with budgets adjusted appropriately.
Examples of Needed Research. Several research areas were identified as being critical to both effective risk assessment and the successful construction of predictive models. First, studies regarding
nanoparticle-induced changes in normal inter- and intracellular communication leading to perturbations or
enhancements in cell cycle progression, cell motility and migration, intracellular signaling (proapoptotic,
prosurvival, proinflammatory, autophagic, response to intracellular pathogens), bactericidal activity, and
cytokine networking should be a priority. Results from studies about nanoparticle-induced changes in cell
signaling and communication would have the potential to be predictive of inflammatory/immune, apoptotic, mutagenic/genotoxic, and developmental responses following human exposures. These studies
should be done in the near term.
Another critical area is the need to understand how nanoparticles might interfere with or promote
wound healing, regeneration, or fibrosis following tissue injury. Ultimately, such studies would focus on
cell proliferation and differentiation and the inflammatory or immune responses following injury. Studies
should also investigate effects of nanoparticle exposure on signaling and trafficking of tissue and bone
marrow-derived stem cells in the normal state or following tissue injury or inflammation.
The use of adapted or stressed systems may be useful for elucidating different types of biological
responses to engineered nanomaterials. In general, two paradigms of sequential or concurrent exposures
could be relevant. First, research efforts should be directed toward characterizing how the adaptation of
cells to inflammatory/oxidative stimuli (e.g., endotoxin exposure, vitamin deficiency) or pathogenic organisms (e.g., Escherichia coli [E. coli], Salmonella ssp.; Pseudomonas aeruginosa) modulates the
intracellular and cell-cell signaling pathways in response to nanoparticle exposure. Conversely, studies
should characterize how the adaptation of cells to nanoparticle modulates the intracellular and cell-cell signaling pathways in response to proinflammatory/oxidative stressors and pathogens.
Research efforts should not be focused on in vitro model systems that cannot be validated conceptually or experimentally for the in vivo situation. In addition, it would be the most time- and cost-effective to
focus efforts on projects that use engineered nanoparticles instead of naturally occurring ones (e.g., ambient air pollution ultrafine particles). Finally, the field is not yet mature enough to support clinical trials.
2.7.3
Breakout Group 2C: Whole Animal Interactions—Biokinetics
Breakout group leaders: David Warheit (Facilitator and Presenter), Mark Banaszak Holl (Scribe)
2.7.3.1 Background
An understanding of the biokinetics following nanoparticle exposures may be particularly important
given the potential of engineered nanomaterials to transmigrate from the original port of entry to systemic
compartments in the body. In this regard, a number of investigators have reported that inhaled engineered
nanomaterials that deposit in the lung can translocate from airspace to vasculature and therefore circulate
systemically throughout the body. Other reports have indicated that inhaled engineered nanomaterials can
deposit in the olfactory tubercle in rats and migrate via axonal transport to the brain. Similar effects could
be operative following oral exposures, with engineered nanomaterials migrating out of the gastrointestinal
tract to become distributed in the bloodstream. Intravenous administration of engineered nanomaterials for
diagnostic or medicinal applications clearly would lead to a widespread distribution throughout the body.
Finally, it is possible that chronic exposures of engineered nanomaterials to the skin could potentially lead
to systemic distribution of the nanoparticles. This is conceivable because, although the epidermis and corCharge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-68
International Council on Nanotechnology
2007 Workshop Report
responding stratum corneum are not vascularized, the dermal compartment of the skin has a limited blood
supply.
Therefore, when considering all of the routes of potential exposures, ADME (absorption, distribution, metabolism, excretion) studies are critical to understanding the biological effects of engineered
nanomaterials in the organism. Unfortunately, the techniques for assessing the biokinetics of engineered
nanomaterials following route-of-entry exposures require further development. Some of the fundamental
issues are associated with labeling of engineered nanomaterials and ensuring that surface chemistry
changes do not alter the biological activity of the nanoparticulate.
2.7.3.2 Research Needs
Objective: Quantify biokinetics and biodistribution of engineered nanomaterials in animals. It is
envisioned that these data could be employed for the development of predictive models. Studies
should explicitly include nanoparticle physical and chemical characterization and effects of dose and
dose rate. Assessing correlations to health outcomes are desirable, but not required. Key exposure
routes envisioned include inhalation, dermal, intravenous, oral, and implants.
2.7.3.3 Research Program
Scope
It is anticipated that research programs will systematically vary nanoparticle properties, routes of
exposure, effects of dose and dose schedule, and identify susceptibility factors affecting biokinetics and
biodistribution. Medical, occupational, consumer, and environmental exposure sources are all anticipated.
1. Nanoparticles. Comparative studies through systematic changes of particle properties (e.g., size,
surface chemistry [including charge], and shape) are needed for a given route of exposure and
resulting retention kinetics in key organs.
2. Routes of exposure. Comparison of routes of exposure (inhalation, dermal, intravenous, oral,
implants) are needed with respect to organ retention, including quantification of translocation
rates and—very importantly—excretory pathways (mass balance studies, ideally).
3. Effect of dose (dose rate and dose schedule) upon biokinetics and biodistribution (e.g., retention,
accumulation, and elimination).
4. Role of susceptibility factors influencing biokinetics and biodistribution (e.g., gender, age, disease, genetic differences, environmental interactions).
Nanoparticles must be characterized prior to administration to a degree that allows correlation of
properties with biological responses. It is also desirable to characterize after exposure in key organs as well
as excreted engineered nanomaterials, if possible. Physical-chemical stability of the nanoparticle should be
known.
2.7.3.4 Prioritized Objectives
5-Year Goals
• Quantify biokinetics and biodistribution of engineered nanomaterials in animals, including the
influence of dose (comparative dosimetry: what is anticipated human exposure and associated
dose, including dose rate and dose schedule [acute vs. chronic exposure])
• Assess the role of particle properties in biokinetics and biodistribution
10-Year Goals
• Relate biokinetics/biodistribution to health outcomes
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-69
International Council on Nanotechnology
•
•
•
2007 Workshop Report
Identify the susceptibility factors influencing biokinetics and biodistribution (e.g., gender, age,
disease, genetic differences, environmental interactions)
Examples of research include, but are not limited to
- the role of surface coatings (of biological/physiological relevance) for engineered
nanomaterials with cores of identical composition and structure for one route of exposure
- biokinetics and biodistribution of identical engineered nanomaterials by two or more
exposure routes
- identification of translocation pathways and mechanisms from the portal of entry to distal
organs (e.g., brain, spleen) and subsequent excretion
- characterization of engineered nanomaterials during the biokinetics time course
Exclusions
- Ambient ultrafines or nonengineered nanosize particles (e.g., from combustion processes)
- Native biological species (proteins and nucleic acids) and genetically modified proteins
- In vitro models
- Invertebrate models
- Environmental fate and distribution
- Nonphysiological modes of exposure (e.g., aspiration, instillation) provided
- Funds and availability of engineered nanomaterials are unlimited. Otherwise, justify why
exposure mode is chosen to achieve specific goal and discuss relevancy to biological relevant
exposure modes.
2.7.4
Breakout Group 2D: Ecotoxicology
Breakout group leaders: Vicki Stone (Facilitator and Presenter), Elise McCarthy (Scribe)
2.7.4.1 Background
The following strategy is based on the assumptions that engineered nanomaterials are currently
entering the environment and that, as a consequence, nano-biointeractions are likely to occur. It is also recognized that the field of nano-ecotoxicology is a nascent discipline, as is the regulation of nanotechnology
in the environment.
There are approximately 30 existing reports on this topic, including the U.S. Environmental Protection Agency (EPA) 100/B-07/001 Nanotechnology White Paper,61 and reports by the Royal Society and
the Royal Academy of Engineering,62 and the Scientific Committee on Emerging and Newly Identified
Health Risks (SCENIHR).51
However, significant data gaps remain in relation to where, when, and how there may be nanomaterial releases into the environment; what the materials do in that context, how they travel, and where they
go; whether there is an environmental hazard associated with their presence; and ultimately what their life
cycle is. These data gaps are closely related to broader needs to identify and classify risk-relevant criteria
for nanotechnology.
2.7.4.2 Research Needs
Against this background, what research is needed to allow the safe, sustainable, and profitable development of the field of nanotechnology?
The research needed might be summarized as that providing information that will set the foundation
for understanding and managing the ecotoxicology of nanotechnology, specifically that which allows a
deepened understanding of
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-70
International Council on Nanotechnology
•
•
•
2007 Workshop Report
how nanomaterials behave in the environment (air, water, terrestrial)
the safety of priority substances already in the environment—such as nanosilver, titanium dioxides, and nanoformulations in pesticides
the safety of those nanomaterials that may be released in the future.
Ultimately these kinds of data should be a scientific platform with which to guide regulatory review.
The following research areas are prioritized in terms of pursuing research programs essential to
enabling subsequent research, facilitating immediate understanding of nano-biointeractions in the most
important/prevalent products to inform regulatory review and growing consumer interest, and providing
information for industry to develop standards or best practices—such as the European REACH (Registration, Evaluation, Authorisation and Restriction of Chemical substances) model.
Research would ideally be addressed by multidisciplinary teams and, where possible, the approximate time frame is included.
2.7.4.3 Sources and Routes of Release into the Environment
Sources and routes of nanomaterial release into the environment should be approached in terms of
hazard potential and quantity. The quantity should be expressed not just as weight, which is in line with
current regulatory requirements, but also taking into account the unique character of nanotechnology and
considering priorities such as volume, surface area, or other appropriate dose metrics. This work should be
established within the next 2 years and maintained indefinitely.
Central to this goal is the development of methodologies to estimate and monitor on an ongoing
basis the sources of release into the environment of nanomaterials. Key considerations here would include
information management systems and legal and regulatory frameworks.
2-Year Goals
• Begin to estimate current and future releases of different types of nanomaterials into the environment based on existing information throughout the entire life cycle
5-Year Goals
• Revise and update release scenarios
10-Year Goals
• Develop a comprehensive Web-accessible database of aggregated release totals or predictive
models based on production volumes or relevant available data
2.7.4.4 Detection and Quantification in the Environment
This research area prioritizes the development of methods to detect and quantify nanomaterials in the
environment.
2-Year Goals
• Develop chemical and physical methods of detection
5-Year Goals
• Develop biosensor technology for detection
• Develop and validate technical detection equipment and protocols
10-Year Goals
• Develop uses of nanotechnology to detect nanomaterials the environment
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-71
International Council on Nanotechnology
2007 Workshop Report
2.7.4.5 Characterization of Nanomaterial Physicochemical Properties
2-Year Goals
•
Characterize and compare nanomaterials in both laboratory settings and environmental settings,
such as fresh water, marine environments, and soil media
2.7.4.6 Transportation, Transformation, Fate Leading to Exposure
This research priority considers the movement of nanomaterials and its implications for both the
nanomaterials and the environment.
2-Year Goals
•
•
Evaluate the transport of nanomaterials in different environments (e.g., air, water, terrestrial)
Evaluate the transport of nanomaterials in terms of the complete life cycle (e.g., titanium oxide),
from cradle to grave, including its toxicity, secondary effects, and persistence. The comparable
standard model for this is ISO 14000.
5-Year Goals
•
Assess whether the nanomaterial is released into the environment in a pure form or in a matrix
10-Year Goals
•
Produce a validated tool for estimating the environmental background levels of both natural and
incidental nanoparticles
2.7.4.7 Effects on Organisms: Microorganisms, Invertebrates, Vertebrates, Plants
A key research focus should be epidemiological studies or descriptive studies that look for anomalies
among indicator species in the exposed environment (2–10 years).
2-Year Goals
•
•
Develop and validate existing test methods
Develop additional tests for effects that include mechanistic studies, bioaccumulation and bioconcentration studies, and effects on waste-water treatment (dependent on bacterial action but
which may be affected by nanosilver, for example)
5-Year Goals
•
Create new test methods that consider acute and chronic conditions and include lethal and sublethal studies with foci such as reproduction, development, and behavior, if necessary
2.7.4.8 Impact of Nanomaterials on the Development of Resistant Bacterial Strains
Investigations of the role of nanomaterials in relation to current resistant strains of bacteria should be
pursued. For example, evidence of silver-resistant bacterial strains has been found, making understanding
the role of nanosilver in this process a short-term priority. The development of new resistances should also
be investigated and monitored on an ongoing basis.
2-Year Goals
•
Investigate the potential for nanomaterials to facilitate the development of resistant bacterial
strains
Charge to the Breakout Groups—Session 2: Interactions of Nanoparticles with Living Organisms
2-72
International Council on Nanotechnology
2007 Workshop Report
2.7.4.9 Nanomaterial’s Environmental Protection Capability
In addition to these prioritized goals the workgroup considered other aspects of nanomaterial
impacts on the environment outside the context of a prioritized research agenda. These included understanding the impacts of nanomaterial on atmospheric chemistry and aquatic chemistry and further
exploring the use of nanotechnology as an environmental technology—for example, for the detection of
environmental problems, and for its potential in processes of remediation, waste-water treatment, filtration
techniques, and sensing. Furthermore, investigations should explore the engineering of biodegradable
nanotechnology.
2.8. References Cited
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
Kim, S.B., T. Ozawa, H. Tao, and Y. Umezawa. A proinflammatory cytokine sensor cell for assaying
inflammatory activities of nanoparticles. Anal Biochem 362 (1) 148-150 (2007).
Oberdörster, G., E. Oberdörster, and J. Oberdörster. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113 (7) 823-39 (2005).
Lynch, I., T. Cedervall, M. Lundqvist, C. Cabaleiro-Lago, S. Linse, and K.A. Dawson, K.A. The
nanoparticle-protein complex as a biological entity; A complex fluids and surface science challenge
for the 21st century. Adv Colloid Interface Sci, doi:10.1016/j.cis.2007.04.021 (2007).
Cedervall, T. et al. Understanding the nanoparticle-protein corona using methods to quantify
exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad Sci USA 104 (7) 20502055 (2005).
Henry, T.B., F.M. Menn, J.T. Fleming, J. Wilgus, R.N. Compton, and G.S. Sayler. Attributing effects
of aqueous C60 nano-aggregates to tetrahydrofuran decomposition products in larval zebrafish by
assessment of gene expression. Environ Health Perspect 115 (7) 1059-65 (2007).
Zhu, S., E. Oberdörster, and M.L. Haasch. Toxicity of an engineered nanoparticle (fullerene, C60) in
two aquatic species, Daphnia and fathead minnow. Mar Environ Res 62 Suppl, S5-9 (2006).
Wang, X.B., H.Y. Gao, B.L. Hou, J. Huang, R.G. Xi, and L.J. Wu. Nanoparticle realgar powders
induce apoptosis in U937 cells through caspase MAPK and mitochondrial pathways. Arch Pharm
Res 30 (5) 653-8 (2007).
Service, R.F. NANOTOXICOLOGY: Nanotechnology Grows Up. Science 18 304 (5678) 1732-1734
(2004).
Uversky, V.N., A.V. Kabanov, and Y.L. Lyubchenko. Nanotools for megaproblems: Probing protein
misfolding diseases using nanomedicine. Modus Operandi. Journal of Proteome Research 5, 25052522 (2006).
Linse S., C. Cabaleiro-Lago, W-F. Xue, I. Lynch, S. Lindman, E. Thulin, S.E. Radford, K.A. Dawson. Nucleation of protein fibrillation by nanoparticles. Proc Natl Acad Sci USA 104, 8691-8696
(2007).
Calderón-Garcidueñas, L., R.R. Maronpot, R. Torres-Jardon, C. Henríquez-Roldán, R.
Schoonhoven, H. Acuña-Ayala, A. Villarreal-Calderón, J. Nakamura, R. Fernando, W. Reed, B.
Azzarelli, and J.A. Swenberg. DNA damage in nasal and brain tissues of canines exposed to air pollutants is associated with evidence of chronic brain inflammation and neurodegeeration. Toxic Path
31 (5): 524-538 (2003).
Colvin, V.L. and K.M. Kulinowski. Nanoparticles as catalysts for protein fibrillation. Proc Natl Acad
Sci USA 104, 8679-8680 (2007).
References Cited
2-73
International Council on Nanotechnology
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
2007 Workshop Report
Cui, Z.R., P.R. Lockman, C.S. Atwood, C.H. Hsu, A. Gupte, D.D. Allen, and R.J. Mumper. Novel Dpenicillamine carrying nanoparticles for metal chelation therapy in Alzheimer’s and other CNS diseases. Eur J Pharm Biopharm 59, 263-272 (2005).
Ji, X., D. Naistat, C. Li, J. Orbulesco, and R.M. Leblanc. An alternative approach to amyloid fibrils
morphology: CdSe/ZnS quantum dots labelled -amyloid peptide fragments A (31–35), A (1–40) and
A (1–42). Colloids Surf B 50, 104-111(2006).
Pai, A.S., I. Rubinstein, H. Onyuksel. PEGylated phospholipid nanomicelles interact with β -amyloid(1–42) and mitigate its β -sheet formation, aggregation and neurotoxicity in vitro. Peptides 27,
2858-2866 (2006).
Majno, G. and I. Joris. Chapter 5: Cell Injury and Cell Death. Cells, Tissues, and Disease: Principles
of General Pathology, 2nd ed., Oxford University Press, New York, 186-245 (2004).
Kumar, V., A.K. Abbas, and N. Fausto. Chapter 1: Cellular Adaptations, Cell Injury, and Cell Death.
Robbins and Cotran Pathologic Basis of Disease, 7th ed., Elsevier, Philadelphia, 3-46 (2005).
Kagan, V.E., Y.Y. Tyurina, V.A. Yyurin, N.V. Konduru, A.I. Potpovich, A.N. Osipov, E.R. Kissin, D.
Schwegler-Berry, R. Mercer, V. Castranova, and A.A. Shvedova. Direct and indirect effects of single
walled carbon nanotubees on RAW 264.7 macrophages: Role of iron. Toxicol Letts 165, 88-100,
(2006).
Xia, T., M. Kovichich, J. Brant, M. Hotze, J. Sempf, T. Oberley, C. Sioutas, J.I. Yeh, M.R. Wiesner,
and A.E. Nel. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett. 6, 1794-1807 (2006).
Sayes, C.M., A.M. Gobin, K.D. Ausman, J. Medez, J.L. West, and V.L. Colvin. Nano-C60 cytotoxicity is due to lipid peroxidation. Biomaterials 26. 7587-7595 (2005).
Park, K.H., M. Chhowalla, Z. Iqbal, and F. Sesti. Single-walled carbon nanotubes are a new class of
ion channel blockers. J Biol Chem 278, 50212-50216 (2003).
Hong, S., P.R. Leroueil, E.K. Janus, J.L. Peters, M-M. Kober, M.T. Islam, B.G. Orr, J.R. Baker Jr.
and M.M.B. Holl. Interaction of polycationic polymers with supported lipid bilayers and cells:
Nanoscale hole formation and enhanced membrane permeability. Bioconjugate Chem 17, 728-734
(2006).
Li, N., C. Sioutus, A. Cho, D. Schmitz, C. Misra, J. Sempf, M. Wang, T. Oberley, J. Froines, and A.
Nel. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ.
Health Perspect 111, 455-4600 (2003).
Chen, M. and A. von Mikecz. Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles. Exp. Cell Res 305, 51-62 (2005).
Green, M. and E. Howman. Semiconductor quantum dots and free radical induced DNA nicking.
Chem. Commun 1, 121-123 (2005).
Teeguarden, J.G., P.M. Hinderliter, G. Orr, B.D. Thrall, and J.G. Pounds. Particokinetics in vitro:
Dosimetry considerations for in vitro nanoparticle toxicity assessments. Toxicol. Sci 95, 300-312
(2007).
Unfried, K., C. Albrecht, L-O. Klotz, A. von Mikecz, S. Grether-Beck, and R.P.F. Schins. Cellular
responses to nanoparticles: Target structures and mechanisms. Nanotoxicology 1, 52-71 (2007).
Papageorgiou I., C. Brow, R. Schins, S. Singh, R. Newson, S. Davis, J. Fisher, E. Ingham, and C.P.
Case. The effect of nano- and micron-sized particles of cobalt-chromium alloy on human fibroblasts
in vitro. Biomaterials 28 (19) 2946-58 (2007).
Auffan M., L. Decome, J. Rose, T. Orsiere, M. De Meo, V. Briois, C. Chaneac, L. Olivi, J.L. BergeLefranc, A. Botta, M.R. Wiesner, and J.Y. Bottero. In vitro interactions between DMSA-coated
References Cited
2-74
International Council on Nanotechnology
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
2007 Workshop Report
maghemite nanoparticles and human fibroblasts: A physicochemical and cyto-genotoxical study.
Environ Sci Technol. 40 (14) 4367-4373 (2006).
Liang M. and L.H. Guo. Photoelectrochemical DNA sensor for the rapid detection of DNA damage
induced by styrene oxide and the Fenton reaction. Environ Sci Technol. 41 (2) 658-64 (2007).
Orsiere T., M. De Meo, V. Briois, C. Chaneac, L. Olivi, J.L. Berge-Lefranc, A. Botta, M.R. Wiesner,
and J.Y. Bottero. In vitro interactions between DMSA-coated maghemite nanoparticles and human
fibroblasts: A physicochemical and cyto-genotoxical study. Environ Sci Technol. 40 (14) 4367-4373
(2006).
SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks). Modified Opinion on the appropriateness of existing methodologies to assess the potential risks associated with
engineered and adventitious products of nanotechnologies. http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_003b.pdf, (2006).
NRCG Task Force 3: Report on in vitro methods for assessing the toxicity of nanoparticles. Characterising the potential risks posed by engineered nanoparticles, UK Government research - a progress
report (2006) http://www.defra.gov.uk/environment/nanotech/research/pdf/nanoparticles-progressreport.pdf
Borm, P.J.A., Robbins, D., Haubold, S., Kuhlbusch, T., Fissan, H., Donaldson, K., Schins, R., Stone,
V., Kreyling, W., Lademann, J., Krutmann, J., Warheit, D., and Oberdorster, E. The potential risks of
nanomaterials: a review carried out for ECETOC. Particle and Fibre Toxicology 3 (11) (2006) http:/
/www.particleandfibretoxicology.com/content/3/1/11
Oberdörster, G., A. Maynard, K. Donaldson, V. Castranova, J. Fitzpatrick, K. Ausman, J. Carter, B.
Karn, W. Kreyling, D. Lai, S. Olin, N. Monteiro-Riviere, D. Warheit, and H. Yang. Principles for
characterizing the potential human health effects from exposure to nanomaterials: elements of a
screening strategy. ILSI Research Foundation/Risk Science Institute Nanomaterial Toxicity Screening Working Group. (2005) Particle and Fibre Toxicology 2 (8) http://www.particleandfibretoxicology.com/content/2/1/8
Nel, A., Xia, T., Madler, L., et al. Toxic potential of materials at the nanolevel. Science 311 (5761)
622-627 (2006).
Dubertret, B., P. Skourides, D.J. Norris, V. Noireaux, A.H. Brivanlou, and A. Libchaber. In Vivo
Imaging of Quantum Dots Encapsulated in Phospholipid Micelles. Science 29, 298 (5599) 17591762 (2002).
Ehrenberg, M. and J.L. McGrath. Binding between particles and proteins in extracts: implications for
microrheology and toxicity. Acta Biomaterialia 1, 305-315 (2005).
Burnett, R.T., R.E. Dales, D. Krewski, R. Vincent, T. Dann, and J.R. Brook. Associations between
ambient particulate sulfate and admissions to Ontario hospitals for cardiac and respiratory diseases.
Am J Epidemiol 142 (1) 15-22 (1995).
Schwartz, J. and D.W. Dockery. Particulate Air Pollution and Daily Mortality in Steubenville, Ohio.
Am J Epidemiol 135 (1) 12-19 (1992).
Schwartz J. and R. Morris. Air pollution and hospital admissions for cardiovascular disease in
Detroit, Michigan. Am J Epidemiol 142, 23–35 (1995).
U.S. Environmental Protection Agency 100/B-07/001 Nanotechnology White Paper, http://
www.epa.gov/osa/pdfs/nanotech/epa-nanotechnology-whitepaper-0207.pdf (2007).
The Royal Society and the Royal Academy of Engineering. Nanoscience and nanotechnologies:
Opportunities and uncertainties. http://www.nanotec.org.uk/finalReport.htm (2004; cited 2004 July
29).
References Cited
2-75
International Council on Nanotechnology
2007 Workshop Report
Appendix C: Workshop 2 Agenda
Tuesday, 5 June 2007
1:15
1:25
1:45
2:15
2:45
3:00
3:15
5:15
6:15
6:30
7:00
9:30
WORKSHOP COMMENCES (Forum A) Welcome and Opening Remarks—Thomas
Epprecht, Swiss Re
ICON Welcome and Workshop Objectives—Kristen Kulinowski, ICON
Plenary 1: Computational toxicology/Computational modeling—Andrew Worth, JRC
Plenary 2: Mechanisms of biological response—Sally Tinkle, NIEHS
Charge to working groups—Vicki Colvin, ICON
Break (Foyer)
Convene Mechanisms breakout groups (five breakout rooms as assigned)
Risk Talk Session convenes in parallel to breakout groups (Forum A)
ADJOURN breakout groups
Apéro for Risk Talk and ICON guests (Foyer)
Dinner at CGD Ruschlikon (Dining Room)
Shuttle bus from CDG to Hotel Ascot
Wednesday, 6 June 2007
7:30 am
7:45
8:15
8:30 am
Gather in lobby of Hotel Ascot
Shuttle bus Hotel Ascot to CGD
Welcome coffee (Foyer)
Reconvene Mechanisms breakout groups, finalize reports (five breakout rooms as
assigned)
9:30
Mechanisms breakout session reports (Forum A)
10:15
Break (Foyer)
10:30
Reconvene breakout session reports (Forum A)
11:15
Plenary 3: Nano-biointeractions—Kenneth Dawson, UCD
11:45
Charge to Interactions breakout groups—Sally Tinkle, NIEHS
12:00 pm Lunch (Foyer)
1:00
Convene Interactions breakout groups (four breakout rooms as assigned)
3:15
Break (Foyer)
3:45
Reconvene Interactions breakout groups, finalize reports (four breakout rooms as
assigned)
5:30
ADJOURN—Apéro for ICON guests (Foyer)
6:00
Shuttle bus transfer to Hotel Schwan in Horgen
6:30
Dinner at Hotel Schwan
8:30
Walk to Harbor Horgen
9:00
Cruise on Lake Zurich to Zurich Burkliplatz
9:45
Walk to Hotel Ascot from Zurich Burkliplatz (10 minutes)
Thursday, 7 June 2007
7:30 am
8:00
8:30
8:45
References Cited
Checkout of Hotel Ascot (if applicable)
Shuttle bus Hotel Ascot to CGD
Welcome coffee (Foyer)
Interactions breakout groups reports (Forum A)
2-76
International Council on Nanotechnology
2007 Workshop Report
10:15
Break
10:30
Wrap-up discussion
12:00 pm WORKSHOP CONCLUDES
Lunch at Ruschlikon (Foyer)
1:00
Shuttle bus from CGD to Hotel Ascot
1:00
Convene writing session with team leaders and assigned others
4:00
Writing session concludes
4:15
Shuttle bus from CGD to Hotel Ascot
References Cited
2-77
International Council on Nanotechnology
2007 Workshop Report
Appendix D: Workshop 2 Attendees
ICON Staff:
Vicki Colvin, Executive Director (Rice University–USA)
Kristen Kulinowski, Director (Rice University–USA)
David Johnson, Operations Manager (Rice University–USA)
Tilman Butz (University of Leipzig–Germany)
Flemming Cassee (National Institute for Public Health and the Environment–The Netherlands)
Fanqing Frank Chen (Lawrence Berkeley National Laboratory–USA)
Kenneth Dawson (University College Dublin–Ireland)
Seamas Donnelly (University College Dublin–Ireland)
Kevin Dreher (Environmental Protection Agency–USA)
Alison Elder (University of Rochester–USA)
Thomas Epprecht (Swiss Reinsurance Company–Switzerland)
Mike Garner (Intel Corporation–USA)
Marianne Geiser (University of Bern–Switzerland)
Mar Gonzales (Organization for Economic Co-operation and Development–Europe)
Hans-Joachim Guentherodt (University of Basel–Switzerland)
Peter Hoet (Katholieke Universiteit Leuven–Belgium)
Mark Banaszak Holl (University of Michigan–USA)
Vyvyan Howard (University of Ulster–Ireland)
Gaku Ichihara (Nagoya University–Japan)
Agnes Kane (Brown University–USA)
Harald Krug (Empa–Switzerland)
Nastassja Lewinski (Rice University–USA)
Philippe Martin (European Commission–DG SANCO–Europe)
Elise McCarthy (Rice University–USA)
Scott McNeil (NCI Nanotechnology Characterization Lab–USA)
Christoph Meili (University of St Gallen–Switzerland)
Brent Miller (AAAS/NSF Science Policy Fellow–USA)
Winfried Moeller (GSF–National Research Center for Environment and Health–Germany)
Nancy Monteiro-Riviere (North Carolina State University–USA)
Hideki Murayama (Frontier Carbon Corp.–Japan)
Imad Naasani (Invitrogen–USA)
Bruno Orthen (Federal Institute for Occupational Safety and Health–Germany)
Günter Oberdörster (University of Rochester–USA)
Adrian Parsegian–(National Institutes of Health–USA)
Jens Poschet (Sandia National Laboratories–USA)
Joel Pounds (Pacific Northwest National Laboratory–USA)
Joachim Raedler (Ludwig-Maximilians University–Germany)
Michael Riediker (Institute for Work and Health–Switzerland)
Gérard Riviere (European Committee for Standardisation and Research–Belgium)
Marc Saner (Council of Canadian Academies–Canada)
Alan Shakesheff (QinetiQ Nanomaterials Limited–UK)
Anna Shvedova (National Institute for Occupational Safety and Health–USA)
Del Stark (European Nanotechnology Trade Association–Europe)
Vicki Stone (Napier University–UK)
Robert Tanguay (Oregon State University–USA)
References Cited
2-78
International Council on Nanotechnology
2007 Workshop Report
Treye Thomas (Consumer Product Safety Commission–USA)
Mike Thompson (FEI Company–USA)
Sally Tinkle (National Institute of Environmental Health Sciences–USA)
David Warheit (DuPont–USA)
Peter Wick (Empa–Switzerland)
Michael Wong (Rice University–USA)
Andrew Worth (European Commission–Joint Research Centre–Italy)
Robert Yokel (University of Kentucky–USA)
References Cited
2-79