2011-Maynard New Tox of Soph Mats

8/2/2019 2011-Maynard New Tox of Soph Mats

1/21

TOXICOLOGICAL SCIENCES 120(S1), S109S129 (2011)

doi:10.1093/toxsci/kfq372

Advance Access publication December 22, 2010

The New Toxicology of Sophisticated Materials:Nanotoxicology and Beyond

Andrew D. Maynard,* David B. Warheit, and Martin A. Philbert,1

*Risk Science Center, University of Michigan School of Public Health, Ann Arbor Michigan 48019; DuPont Haskell La boratory for Heal th a nd Enviro nmental

Sciences, Newark, Delaware 19714-0050; and Toxicology Program, University of Michigan School of Public Health, Ann Arbor, Michigan 48019

1To whom correspondence should be addressed at Toxicology Program, University of Michigan School of Public Health, 1415 Washington Heights, Ann Arbor,

MI 48019. Fax: (734) 763-8095. E-mail: [email protected].

Received October 4, 2010; accepted December 1, 2010

It has long been recognized that the physical form of materials

can mediate their toxicitythe health impacts of asbestiform

materials, industrial aerosols, and ambient particulate matter are

prime examples. Yet over the past 20 years, toxicology research

has suggested complex and previously unrecognized associations

between material physicochemistry at the nanoscale and biological

interactions. With the rapid rise of the field of nanotechnology and

the design and production of increasingly complex nanoscale

materials, it has become ever more important to understand how

the physical form and chemical composition of these materials

interact synergistically to determine toxicity. As a result, a new field

of research has emergednanotoxicology. Research within this

field is highlighting the importance of material physicochemicalproperties in how dose is understood, how materials are

characterized in a manner that enables quantitative data inter-

pretation andcomparison, and how materials move within, interact

with, and are transformed by biological systems. Yet many of the

substances that are the focus of current nanotoxicology studies are

relatively simple materials that are at the vanguard of a new era of

complex materials. Over the next 50 years, there will be a need to

understand the toxicology of increasingly sophisticated materials

that exhibit novel, dynamic and multifaceted functionality. If the

toxicology community is to meet the challenge of ensuring the safe

use ofthisnew generation of substances,it will need to move beyond

nano toxicology and toward a new toxicology of sophisticated

materials. Here, we present a brief overview of the current state of

the science on the toxicology of nanoscale materials and focus onthree emerging toxicology-based challenges presented by sophisti-

cated materials that will become increasingly important over the

next 50 years: identifying relevant materials for study, physico-

chemical characterization, and biointeractions.

Key Words: nanotechnology; nanotoxicology; engineered

nanomaterials; biokinetics; biointeractions; dose; physicochemical

characterization.

In 1990, two consecutive articles appeared in the Journal of

Aerosol Science asking whether inhaled particles smaller than

100 nm in diameter elicit a greater than expected pulmonary

response (Ferin et al., 1990; Oberdorster et al., 1990). On

a mass for mass basis, nanometer-scale particles of TiO2 and

Al2O3 elicited a significantly greater inflammatory response in

the lungs of rats compared with larger particles with the same

chemical composition. The two studies were at the vanguard of

research challenging long-held assumptions that response to

particulate exposure can be understood in terms of chemical

composition and suggested unusual biological activity associ-

ated with nanometer-scale materials. Fourteen years later, this

growing field of research would be formalized as the field of

nanotoxicology (Donaldson et al., 2004).

The size-specific effects observed by Oberdorster, Ferin and

colleagues were attributed to an increased rate of interstitializa-

tion of nanometer-scale particles in the lungs. Oberdorsteret al.

concluded, Phagocytosis of particles in the alveoli counteracts

the translocation of particles into the interstitial space. Alveolar

macrophage death or dysfunction promotes translocation from

alveoli inter interstitium. Particles of about 0.020.03 lm in

diameter penetrate more easily than particles of ~0.20.5 lm.

Small particles usually form aggregates. Their aerodynamic size

determines the deposition in the airways. After deposition, they

may deagglomerate. If the primary particle size is~0.020.03 lm,deagglomeration may affect the translocation of the particles

more than for aggregates consisting of larger particles

(Oberdorster et al., 1990).

This simple statement outlined two emerging aspects of

materials that potentially mediated their toxicology: particle size

and dynamic behavior. In follow-up studies, further associa-

tions between material physicochemistry and effects were

uncoveredmost notably the role of particle surface area in

mediating pulmonary toxicity. Using TiO2 samples comprising

The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For permissions, please email: [email protected]


2/21

of two distinct sizes of primary particles, Oberdorster et al.

showed that, while inflammatory responses following inhala-

tion in rats depended on particle size, normalizing by surface

area led to a size-invariant dose-response function (Oberdorster,

2000). With surface area as the dosemetric instead of the more

conventional mass concentration, Maynard and Kuempel

(2005) and others showed that a range of insoluble materialstypically classified as nuisance dusts followed a similar dose-

response curve for pulmonary inflammation in rats. However,

more chemically active materials such as crystalline quartz

demonstrated a markedly different dose-response (Maynard and

Kuempel, 2005).

This early research was driven by occupational aerosol

exposures and concerns that the hazards associated with fine

dusts ranging from welding fume to metal and metal aerosol

powders were not predictable from the chemical composition of

these materials alone. What began to emerge was an un-

derstanding that the physicochemical nature of inhaled particles

was more relevant than previously thought in eliciting a re-

sponse and that materials with a nanometer-scale biologicallyaccessible structure (whether they were discrete nanometer-

scale particles or had a nanometer-scale surface structure, as in

the case of aggregates of nanoparticles) had the potential to

show previously unrecognized biological behavior. That this

new research on what were termed ultrafine aerosols came

out of occupational toxicology is perhaps not surprising, given

the fields long history of addressing hazards associated with

exposure to aerosol particles with varying sizes, shapes, and

compositions (Maynard, 2007a).

Although research into occupational exposure to ultrafine

aerosols was developing in the 1990s, environmental epidemi-

ology studies were beginning to uncover associations betweenambient aerosol particle size and morbidity and mortality.

Starting with the six-cities study (Dockery et al., 1993),

evidence emerged for ambient particles approximately smaller

than 2.5 lm (PM 2.5) having an elevated impact on human

health (Pope, 1996; Schwartz and Morris, 1995; Schwartz et al.,

1996). As small particles were implicated in eliciting more

pronounced pulmonary and cardiovascular effects following

inhalation exposure (Seaton et al., 1995), researchers began to

correlate impacts with exposure to ultrafine particles (Brown

et al., 2002; Chalupa et al., 2004; Pekkanen et al., 2002;

Wichmann and Peters, 2000). Although clear associations

between ultrafine particle exposure and health impacts remained

uncertain, this research was suggestive of a link between

aerosol inhalation and health impacts that was mediated by

particle size as well as chemistry, with smaller particles

exhibiting a higher degree of potency. In this respect,

epidemiological studies began to complement contemporary

toxicology studies on inhalation exposure to fine particles.

These two streams of research began to coincide in the late

1990s. But it was the formal advent of the field of

nanotechnology toward the end of the 1990s that galvanized

action toward developing a more complete understanding of

how material physicochemical characteristics impact on

material hazard and how nanoscale materials might lead to

previously unanticipated health impacts.

In the 1990s, federal research agencies in the United States

began looking to identify and nurture a new focus for science,

engineering, and technology that would stimulate research

funding and lead to economic growth. At the time, advancesacross the physical sciences were leading to breakthroughs in

understanding of how material structure at the near-atomic

scale influenced functionality and how this nanoscale structure

might be intentionally manipulated. Recognizing the potential

cross-disciplinary and cross-agency significance of these

breakthroughs, an Interagency Working Group on Nanotech-

nology was established to promote the science and technology

of understanding and manipulating matter at the nanometer

scale (IWGN, 1999).

Although not fully realized until late in the 20th century (the

first documented coining of the term nanotechnology is often

credited to N. Taniguchi [Taniguchi, 1974]), the field of

nanotechnology had its roots in 20th century advances inmaterials science and high-resolution imaging and analytical

techniques. As techniques such as X-ray diffraction and

transmission electron microscopy began to illuminate the

structure of materials at the atomic scaleand how this

structure influenced functionalityinterest grew in improving

materials through manipulating this structure. The fields of

materials science and synthetic chemistry began to explore how

small changes in structure at the atomic and molecular level

could alter behavior at the macroscale. But it was perhaps the

physicist Richard Feynman who first articulated a grander vision

of nanoscale engineering. In a 1959 lecture at Caltech titled

Theres plenty of room at the bottom, Feynman speculated onthe revolutionary advances that could be made if scientists and

engineers developed increasingly sophisticated control over

how substances were built up at the nanoscale (Feynman,

1960)a level of control which at the time remained largely out

of reach. Despite Feynmans lecture often being considered the

foundation of modern nanotechnology, there is little evidence

that it had much impact at the time (Toumey, 2008, 2010).

However, the advent of Scanning Probe Microscopy in 1982

(Binnig et al., 1982), together with advances throughout the

physical and biological sciences in imaging and understanding

the nature of matter at the nanometer scale, began to open up the

possibility of altering the functionality of a wide range of

materials through nanoscale engineering.

Some of the more extreme and speculative possibilities of

building materials and even devices molecule by molecule

were captured in the popular book Engines of Creation by

Eric Drexler, inspired by shrinking human-scale materials

engineering down to the nanoscale (Drexler, 1986). Although

many of the ideas put forward by Drexler were treated with

caution and occasionally skepticism by the scientific commu-

nity, there was a ground swell of excitement through the 1980s

and 1990s over the possibilities that emerging techniques were

S110 MAYNARD, WARHEIT, AND PHILBERT
http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


3/21

opening up to systematically manipulating matter at the

nanoscale, allowing nanoscale structure-mediated functionality

to be exploited at the macroscale. This excitement was buoyed

up by the discovery of carbon nanotubes (Iijima, 1991)a new

and functionally unique allotrope of carbonand the demon-

stration of single-atom manipulation using scanning probe

microscopy (Eigler and Schweizer, 1990). Working at this scale,new opportunities were arising for enhancing the structure of

materials, for engineering materials tailored to exhibit specific

physical, chemical, and biological behavior, for exploiting

novel electron behavior in materials that begins to dominate at

nanometer length scales, and for building increasingly sophis-

ticated materials that could demonstrate multiple and context-

specific functionality. The door was being opened to a new era

of enhancing existing materials and products and creating

innovative new ones by intentionally manipulating the compo-

sition and physical form of substances at the nanoscale.

Riding the wave of this cross-disciplinary revolution in

science, engineering, and technology, President Clinton

announced a new U.S. initiative to explore and exploit thescience and technology of the nanoscale on January 21, 2000

(Clinton, 2000). In an address at Caltech on science and

technology, he asked his audience to imagine materials with

10 times the strength of steel and only a fraction of the weight;

shrinking all the information at the Library of Congress into

a device the size of a sugar cube; detecting cancerous tumors

that are only a few cells in size, and laid the foundation for the

U.S. National Nanotechnology Initiative (NNI). Since then, the

NNI has set the pace for national and international research and

development in nanoscale science and engineering and has led

the world in generating and using new knowledge in the field

of nanotechnology.As nanotechnology began to gain ground, it did not take

long for concerns to be raised over the potential health and

environmental implications of nanotechnology. In 2000, the

cofounder of Sun Microsystems Bill Joy wrote an influential

essay for Wired Magazine titled Why the Future Doesnt

Need Us in which he raised concerns about the impacts of

nanotechnology (Joy, 2000). This was followed by calls for

a moratorium on research until more was known about the

possible adverse impacts by one Civil Society group (ETC

Group, 2003). More scientifically, sound concerns were raised

by the reinsurance company Swiss Re in 2004 (Hett, 2004),

and later that year, the UK Royal Society and Royal Academy

of Engineering launched a highly influential report on the

opportunities and uncertainties of nanotechnology (RS/RAE,

2004). At the center of the Royal Society and Royal Academy

of Engineering report were concerns that engineered nanoscale

materials with unique functionality may lead to unexpected

exposure routes, may have access to unanticipated biological

compartments, and may exhibit unconventional biological

behavior associated with their size. In particular, concern was

expressed over materials intentionally engineered to have

nanoscale structurenanomaterialsand particles and fibers

with nanometer-scale dimensionsnanoparticles and nano-

fibers.

The Royal Society and Royal Academy of Engineering

report marked a move toward a more integrated approach to the

potential risks associated with nanotechnology. As global

investment in nanotechnology research and development has

grown (it has been estimated that global research anddevelopment investment in nanotechnologies exceeded $18

billion in 2008 and that the value of products utilizing these

technologies in some way has been projected to exceed $3

trillion by 2015 [Lux Research, 2009]), so has interest in

identifying, understanding, and addressing potential risks to

human health and the environment (Chemical Industry Vision

2020 technology Partnership and SRC, 2005; ICON, 2008a;

Luther, 2004; Maynard, 2006; Maynard et al., 2006; NNI,

2008; Oberdorster et al., 2005; PCAST, 2010; RCEP, 2008;

SCENIHR, 2005, 2009). This interest has been stimulated by

concerns that novel materials have the potential to lead to novel

hazards and risks. But fueling it has been the research noted

earlier on the role of particle size, physical form, and chemistryin mediating biological interactions and responses. With the

advent of nanotechnology and the production of increasingly

sophisticated engineered nanomaterials, research strands de-

veloping an understanding of the potential human health

impacts of fine particles were thrust into the mainstream and

became the basis of new thinking about how potential risks

associated with new materials can be addressed.

As research began to focus on the potential hazards presented

by engineered nanomaterials, the term nanotoxicology began

to be used informally to describe this growing area of study.

This was formalized in an editorial in Occupational and

Environmental Medicine by Donaldson et al. (2004). Writingabout the human health challenges presented by the emerging

field of nanotechnology, Donaldson et al. noted that:

NP [nanoparticles] have greater potential to travel through

the organism than other materials or larger particles. The various

interactions of NP with fluids, cells, and tissues need to be

considered, starting at the portal of entry and then via a range of

possible pathways towards target organs. The potential for

significant biological response at each of these sites requires

investigation. In addition, at the site of final retention in the

target organ(s), NP may trigger mediators which then may

activate inflammatory or immunological responses. Importantly

NP may also enter the blood or the central nervous system,

where they have the potential to directly affect cardiac and

cerebral functions. We therefore propose that a new subcategory

of toxicologynamely nanotoxicologybe defined to address

gaps in knowledge and to specifically address the special

problems likely to be caused by nanoparticles.

The new field was consolidated in 2005 with a highly cited

paper by Oberdorster et al. titled Nanotoxicology: an

emerging discipline evolving from studies of ultrafine

particles (Oberdorster et al., 2005), and the launch of the

journal Nanotoxicology in 2007.

NANOTOXICOLOGY AND BEYOND S111
http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


4/21

Since the early 2000s, research into the potential impacts

of nanomaterials and nanoparticles in particular has increased

substantially. In the United States, the combined investment

across federal agencies in research and development addressing

environmental health and safety implications of nanotechnol-

ogy was $34.8 million in 2005 (NSET, 2006). In 2011, this

figure is estimated to rise to $116.9 million (NSET, 2010).Global publications addressing human health and environmen-

tal impacts of engineered nanomaterials have similarly in-

creased. In 2005, there were an estimated 179 articles

published on the potential environmental health and safety

implications of engineered nanomaterials. By 2009, that

number had risen to 791 publications (PCAST, 2010). Of

these, the majority address the potential hazards of engineered

nanomaterials. A search for publications with the key terms

nano* and toxic* between 2000 and 2010 shows a rapidly

increasing peer review literature in this area (Fig. 1)

Yet for all this activity, the field of nanotoxicology is

suffering from something of an identity crisis. There is a strong

sense that emerging, novel and complex materials that have been

engineered at the nanoscale may exhibit unusual or unantici-

pated toxicity from a conventional perspective and that research

is needed to understand and address how these designed

materials might cause harm in ways that are not readily

understood at present. This concern is supported by a growing

body of research which indicates that some nanometer-scale

materials do demonstrate biological behavior that is mediated by

physical form as well as chemical composition (Donaldson

et al., 2010; Nel et al., 2006; Oberdorster, 2010). Yet a clear

identification and formulation of the problems being faced

remain elusive. For example, what is meant by the nanoscale

is far from clear, meaning that there is considerable ambiguity

over which materials are embraced by nanotoxicology.

Widely accepted definitions of nanotechnology refer to a size

range of approximately 1100 nm where unique phenomena

enable novel applications (NSET, 2010). Yet these are largely

definitions of convenience, not of science. And while thedefinitions defining the field of nanotechnology have been

important in driving new science and technology innovation, it

is not clear how they apply to a new materials propensity to

cause harm in unexpected ways.

Within generally accepted definitions of nanotechnology,

there is considerable ambiguity over the terms uniqueness

and noveltyand how these attributes might lead to new

materials that raise new health concerns. To a degree, nano-

toxicology has been underpinned by an assumption that

materials engineered to utilize unique properties associated

with the nanoscale must, by definition, exhibit nanoscale-

specific toxicity. Yet this assumption is far from secure. Indeed,

a body of research has suggested that the toxicity of manynanomaterials is scalableand thus predictablefrom non-

nanoscale materials (Oberdorster et al., 2007), questioning the

uniqueness of the nanoscale. This does not of course negate the

importance of studying nanomaterial toxicologyit simply

brings into questions some of the blanket assumptions that direct

this research. One of these assumptions is that the toxicity of

nanomaterials is dominated by quantum effectsan assump-

tion that is currently not supported in simple terms by the

literature.

There is also uncertainty over the relationship between

emerging nanoscale materials and established nanomaterials,

including natural nanomaterials that have been presentthroughout human evolutionary history and anthropogenic

nanomaterials (whether engineered or produced as a by-

product) that have been part of human exposure for decades

and even centuries. Although the argument is often made that

engineered nanomaterials are unique by nature of their

intentionally designed functionality and their precise physico-

chemical form, the boundaries between engineered nanoscale

materials and other nanoscale materials in the real world can

become blurred very rapidly. For example, humans have

developed as a species in the presence of airborne carbona-

ceous nanoparticles from combustion, and our bodies have

evolved to handle exposure to such materials. Since before the

industrial revolution, people have been exposed to airborne

metal and metal oxide nanoscale particles from hot processes

(Maynard and Kuempel, 2005), and while these materials are

rarely innocuous, we have an understanding of how they

impact on human health. Even some forms of intentionally

engineered nanomaterials have been used for many deca-

desthe product Aerosil from Evonik (formally Degussa), for

instance, is a fumed silica intentionally engineered to have

a primary structure of the order of a few nanometers in scale.

Aerosil has been used commercially for over 60 years.

FIG. 1. .Publications related to nanotoxicology, 20002010. Source: ISI

Science Citation Index (Expanded). These data include research related to

environmental and human health impacts, as well as toxicology-related research

on nanoscale therapeutics, and thus provide an indicative rather than

quantitative perspective on publications addressing nanomaterial toxicity in

humans. Denotes data for 2010 were collected on September 19 and were

pro ratad for the full year to allow comparison with previous years. Actual

2010 data: nano* AND toxic*: 1364 publications; nanotoxicology: 64

publications.

http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


5/21

This context does not detract from the emerging challenges

presented by increasingly sophisticated new materials. But it

does demand that careful thought is given to the toxicity of these

materials and whether they are genuinely an unknown quantity

or whether we have a body of evidence and understanding from

which to address them. And it does require a distinction to be

made between the language and terminology that drives a newfield of technology innovation such as nanotechnology and that

which drives research into understanding potential health

impacts. History suggests that not every new technology leads

to new hazards and not every new hazard is associated with

a new technology.

Nevertheless, there is an array of increasingly sophisticated

materials that are emerging from advances in science, technol-

ogy, and engineering that do demand careful consideration of the

new risks they might pose. In this respect, a differential approach

to toxicology studies is requiredone which helps identify

where emerging materials and products deviate from established

ones in their potential to cause harm and focuses research on

narrowing the resulting knowledge gap. Undoubtedly, materialsintentionally designed and engineered to behave in specific ways

because of their fine structure are at the forefront of the new

challenges being faced in toxicology. These materials in-

creasingly demonstrate biological behavior that results from

a synergistic interaction between chemical composition and

physical form. But whether these new challenges can be

confined to a narrow size scale implied by nanotoxicology

is debatable. Rather, we would argue that a broader perspective

is needed on the challenges presented by novel and functional

materials that capture the idea of sophisticated materials.

These are substances that arise at the intersection of scientific

disciplines and technology platforms and demonstrate novel andeven time and context-dependent functionality based on their

engineered and increasingly complex physicochemical struc-

ture. Although many of these materials will depend on nanoscale

engineering, decoupling the materials from the underlying

technologyor technologiesis helpful in formulating sci-

ence-based questions regarding their toxicity. In this respect, the

toxicology challenge presented by sophisticated materials is to

understand and address the hazards presented by materials that

have the ability to enter the body, interact with it, and elicit an

adverse response in ways that are not adequately understood

through a conventional and chemical compositiondominated

perspective on toxicology.

In this review, we present a brief overview of the current

state of the science on the toxicology of nanoscale materials

and focus on three areas of emerging toxicology-based

challenges presented by sophisticated materials: identifying

relevant materials for study, physicochemical characterization,

and biointeractions. Given the rapidly increasing breadth of

research on the potential hazards and risks presented by

engineered nanomaterials, a comprehensive evaluation of the

field is beyond the scope of this review. It is also somewhat

redundant, given the large number of excellent previously

published reviews and analyses (Aitken et al., 2009; Balbus

et al., 2007; ICON, 2008b; Maynard and Kuempel, 2005;

Maynard et al., 2006; Oberdorster, 2010; Oberdorster et al.,

2005, 2007; SCENIHR, 2005, 2009; Warheit et al., 2007).

Rather, here we consider aspects of nanoscale materials that set

them apart from more conventional materials and build on

these to explore the emerging challenges of understanding thetoxicology of sophisticated materials.

THE TOXICOLOGY OF NANOSCALE MATERIALS

In 2005, the European Commission Scientific Committee on

Emerging and Newly Identified Health Risks (SCENIHR)

published a comprehensive assessment of the state of the

science regarding potential risks associated with engineered

and adventitious products of nanotechnologies (SCENIHR,

2005). It was one of the first in a long series of assessments and

reviews of the toxicology of nanoscale materials that have

helped identify emerging issues surrounding the potentialhealth impacts of these materials, and although the state of the

science has moved on since its publication, the overarching

issues identified by the committee remain contemporary.

The SCENIHR committee was tasked with addressing three

questions: Are existing methodologies appropriate to assess

potential and plausible risks associated with different kinds of

nanotechnologies and processes associated with nanosized

materials as well as the engineered and adventitious products of

nanotechnologies? If existing methodologies are not appropri-

ate to assess the hypothetical and potential risks associated with

certain kinds of nanotechnologies and their engineered and

adventitious products, how should existing methodologies beadapted and/or completed? And in general terms, what are the

major gaps in knowledge necessary to underpin risk assessment

in the areas of concern?

In common with most other reviews addressing the toxicity of

nanomaterials, SCENIHR focused on materials that are

physically able to enter the body via inhalation, ingestion, and

potentially dermal penetration, leading to an emphasis on the

particulate form of nanomaterialsand nanometer-scale par-

ticles (nanoparticles) in particular. In reviewing the literature,

the committee identified three nanoscale mediators of toxicity:

particle size, shape, and chemical composition. Drawing on

evidence of material toxicity that was influenced by physical

form as well as chemical composition, SCENIHR explored how

these three mediators potentially affect bioavailability and

biointeractions and influence exposure and dose. Specific

mechanisms of toxicity highlighted included epithelial tissue

injury, inflammation, oxidative stress, and allergy. Concluding

that there is insufficient data available to identify any generic

rules governing the likely toxicology and ecotoxicology of

nanoparticles in general, the committee identified a number

of major knowledge gaps that prevented a complete risk

assessment of engineered nanomaterials. These included

http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


6/21

understanding the mechanisms and kinetics governing nano-

material release, quantifying the range of potential exposures,

developing an understanding of the extent to which data from

non-nanosized materials can be extrapolated to the nanoscale,

generating toxicokinetic data associated with various portals of

entry to the body, and addressing worker health.

Although the SCENIHR report was published nearly 6 yearsago, it outlined issues associated with synergistic interaction

between the chemical composition and physical form of

nanoscale materials and their biological interactions that

continue to be relevant. Although the state of the science has

moved on since the reports publication, the key themes that

the committee laid out remain central to understanding and

addressing the toxicology of nanoscale materials.

These themes are reflected and expanded on in one of the

more recent reviews of the field by Oberdorster (Oberdorster,

2010). Although there is a growing literature on the toxicology

of nanoscale materials and many reviews of the potential risks

presented by such materials (Aitken et al., 2009; Balbus et al.,2007; Donaldson and Poland, 2009; Maynard, 2006, 2007a,

2007b; Maynard and Kuempel, 2005; Maynard et al., 2006;

Nel et al., 2006; Oberdorster et al., 2005), much of this is

captured in Oberdorsters review.

Oberdorster considers the toxicology of nanoparticles (as

a special but biologically important case of nanomaterials) in

terms of their physicochemical characteristics, their biokinetics,

and their effects. Specifically, he focuses on nanoparticles that

are likely to be biopersistent and therefore show prolonged

behavior that is governed by their physicochemistry. Relatively

transient nanoparticle such as nanoscale micelles and lip-

osomes are not addressedwhereas the temporal physical

form of these and similar soft materials may influence their

toxicity, it remains unclear the extent to which their impact isdominated by chemistry or form.

Comparing particles smaller than 100 nm in diameter to

those > 500 nm in diameter, Oberdorster identifies 22 aspects

that are potentially important to influencing size-related

biological impact (Table 1). In doing so, he begins to develop

a framework for a differential toxicology approach to nano-

materials, where the toxicology of nanoscale materials is

understood in the context of chemically similar but physically

different materials. Importantly, this approach acknowledges

the fuzzy transition between large and small particles that is not

always governed by well-defined size boundaries and abrupt

changes in behavior.Within this framework, Oberdorster highlights three areas

which are significant in understanding nanomaterial toxicity

compared with that of macroscale materials and/or constituent

chemicals: dose, biokinetics, and the significance of physico-

chemical properties. Although these are not the only issues of

significance in addressing nanomaterials, they provide a useful

framework for summarizing the current state of the science.

TABLE 1

Comparing the Characteristics, Biokinetics, and Effects of Inhaled Nanoparticles versus Larger Particles (Oberdorster, 2010)

Nanoparticles (< 100 nm) Larger particles (> 500 nm)

General characteristics

Ratio: Particle number/mass or surface area/mass High Low

Agglomeration/aggregation in air and/or liquids Likely (dependant on medium) Less likely

Deposition in respiratory tract Diffusion dominates Sedimentation, impaction and interception dominate

Protein/lipid adsorption in vitro Yes; important for biokinetics Less important

Translocation to secondary target organs

Clearance Yes Generally not

Mucociliary Probably yes Efficient

Alveolar macrophages Poor Efficient

Epithelial cells Yes Mainly under overload

Lymphatic circulation Yes Under overload

Blood circulation Yes Under overload

Sensory neurons (uptake and transport) Yes No

Protein/lipid adsorption in vivo Yes SomeCell entry/uptake Yes (caveolae, clathrin, l ipid rafts, diffusion) Primarily phagocytic cells

Mitochondria Yes No

Nucleus Yes (< 40 nm) No

Direct effects (chemistry and dose dependent)

At secondary target organs Yes No

At portal of entry (respiratory tract) Yes Yes

Inflammation Yes Yes

Oxidative stress Yes Yes

Activation of signaling pathways Yes Yes

Primary genotoxicity Some No

Carcinogenicity Yes Yes

http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


7/21

Dose

Over the past 20 years, questions surrounding dose,

including how it is characterized and quantified, have been

central to addressing the toxicity of nanomaterials. As was

highlighted earlier, evidence has emerged that, for some

materials, the use of mass concentration alone as a dose metric

can obscure associations between the material and biological

behavior. If response is mediated by particle number

concentration, the disparity between what is measured and

what leads to an affect is potentially large if mass is the dose

metric used as the number of particles in a given mass of

material increases inversely with diameter cubed. For example,

1 m g of 10 lm diameter spherical carbonaceous particles

would consist of approximately 1012 particles; the same mass

of 10-nm diameter particles would consist of approximately

1021 particles. A smaller but still potentially significant disparity

exists if mass is used as a dose metric where surface area

mediates response. For a given mass of material, surface area

varies inversely with particle diameter (assuming sphericalparticles). So whereas 1 mg of 10-lm diameter spherical

carbonaceous particles has a surface area of approximately 270

m2, the same mass of 10-nm diameter particles has a surface area

of 270,000 m2. As a result, although Paracelsus observation that

the dose makes the poison still holds in contemporary

toxicology, there is considerable uncertainty over what is meant

by dose when it comes to nanomaterials.

A number of studies have suggested that particle surface area

is a relevant metric for small, insoluble inhaled particles

(Maynard and Kuempel, 2005; Oberdorster, 2000). Yet it is by

no means clear whether this is a general rule for a wide range of

materials and exposure routes. Even with well-studiedmaterials such as TiO2, there is research, suggesting that

surface area alone may not provide a good indicator of

response (Warheit et al., 2006). It is also possible that

conventional metrics of mass concentration and chemical

composition may be used as surrogate measures of dose, even

when effects are not driven by the measured quantity per se

(Maynard and Aitken, 2007). For instance, in a highly

monodisperse suspension of nanoparticles, dose characterized

by mass, surface area, or particle number are highly correlated

and probably interchangeable.

In addressing how dose is most appropriately characterized,

there remains limited understanding of the underlying

mechanisms of interaction and impact. For instance, where

surface area correlates well with response, there is uncertainty

whether (in specific cases) this is governed by dissolution,

surface reactivity, or other mechanisms. A greater understand-

ing is needed of these mechanisms before empirical findings on

dose-response for engineered nanomaterials can be placed on

a more systematic and mechanistic footing. This will become

increasingly important as more sophisticated materials are

engineered with complex and multifunctional components at

the material-biological interface.

An issue related to dose metrics raised by Oberdorster is that

of dosimetry. Oberdorster argues that an increasingly sophis-

ticated understanding of dosimetry is neededone that not

only recognizes different mediators of response but also one

that is related to real-world exposures and is responsive to

localized dose within the body. There have been a number of

instances where in vitro studies have been publisheddemonstrating a response to nanoparticles, but at doses that

far exceed those reproducible in vivo (Oberdorster,

2010)resulting in headline-catching data that is difficult to

interpret and near-impossible to apply to human exposures.

Similarly, there have been in vivo studies that elicit responses at

extremely high doses but are again difficult to relate to real-

world conditions precisely because of this. As Oberdorster

notes, these studies are valuable in exploring proofs of

principle but are limited in terms of their ability to develop

a clear and predictive understanding of nanomaterial toxicity.

This becomes all the more difficult if the dose metric of

relevance is not the one that is measured, leading to the

possibility of unintended high dosing in studies.Questions surrounding dosimetry also relate to localized and

temporal dose. If nonlinear associations exist between dose and

response, significant spatial and time variations in dose within

an animal model or cell culture have the potential to confound

studies. For example, the administration of an aerosol as a high

concentration bolus in inhalation studies has the potential to

influence response and lead to data misinterpretation. Ober-

dorster cites a study where a total dose of 7.5 mg of nanoscale

TiO2 was instilled intranasally in mice and resulted in

significant oxidative stress and inflammation in the brain

(Wang et al., 2008). The study was subsequently highlighted in

the media, where it was misrepresented as an inhalation studyshowing that nanoparticles can damage brain cells (Benningh-

off and Hessler, 2008). As Oberdorster points out, the dose in

this case was the equivalent of intranasally instilling ~17.5 g of

the material into a human subject.

In addition, localized dose hot spots often drive response

following aerosol inhalation. Particles preferentially deposit at

bifurcations in the airwayslarge particles through inertial

deposition and small particles through diffusion within the

stagnation zone that develops at bifurcations. These localized

doseswhich can be a hundredfold higher than the mean dose

for larger particlesare frequently used to justify high dose

in vitro assays. Yet the local dose enhancement for nano-

particles is somewhat differentranging from around a 5-fold

to a 60-fold increase in dose (Balashazy et al., 2003).

Misrepresenting these dose hot spots as they relate to

particle size has the potential to confound the extension of

in vitro studies to in vivo exposures.

The question of dose also becomes important when

comparing studies and when developing predictive models of

nanoparticle toxicity. This is particularly significant when

comparing in vitro and in vivo studies, where physicochemical

parameters make simple comparisons difficult. Rushton et al.

http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


8/21

(2010) have proposed a novel approach where studies are

compared using the steepest part of the dose-response curve.

Using this approach, Rushton et al. have reported good

predictive power between in vitro cell-free studies and in vivo

studies looking at inflammatory response. Building on this

work, the authors have looked at using the maximum rate of

response as a function of dose (the steepest part of the dose-response curve) as an approach to categorize nanomaterial

hazard based on reactivity per unit surface area (Rushton et al.,

2010).

As a final reflection dose, there is increasing evidence that

particulate dose may need to be rethought in in vitro studies as

well as in vivo studies. Teeguarden et al. (2007) have identified

discrepancies between the amount of a material introduced to

in vitro cell culturesnominally considered to be the

doseand the amount of material cells are able to interact

with. As particles form a dynamic concentration gradient

within the suspension medium, there are indications that over

short time periods actual doses of material experienced by cells

may be orders of magnitude lower than assumed, suggesting

that further work is needed in characterizing particle doses

in vitro.

Physicochemical Properties

The biological nature of nanoscale materials is intimately

associated with their physical form and chemical composition,

leading to toxicologic responses that are associated with a wide

range of physicochemical parameters and that are affected by

dynamic changes in materials. Understanding the association

between physicochemical properties, biological interactions,and hazard is a significant challenge as it requires new

approaches to think about how physical formwhich may

vary with time and between batches of materialcan modulate

biological response beyond what is anticipated from chemical

composition alone. In 2005, Oberdorster et al. proposed 17

physicochemical material characteristics that potentially affect

nanomaterial toxicity and which ideally need to be character-

ized in studies (Oberdorster et al., 2005). Recognizing the

dynamic nature of these materials, characterization in situ was

recommended where possible, as well as characterization of the

as-supplied material and the as-administered material. This list

of parameters formed the basis of a reduced list developed at

a workshop held in Washington, DC, in 2008, and made public

through the Minimum Information for Nanomaterial Charac-

terization (MINChar) Initiative (2008). Similar lists have been

proposed in the literature (Card and Magnuson, 2009; Warheit,

2008).

Particle aggregation and agglomeration present particular

challenges in toxicology studies. The process of particles

joining together to form weak bonds (agglomeration) or strong

bonds (aggregation) changes profoundly the size, dynamics,

and properties of the resultant clusters. In air, changes in

particle size through agglomeration influence transport, de-

position, whether the material can be inhaled, where it deposits

within the respiratory tract, whether it can translocate from the

lungs to other parts of the body, and how it is cleared from the

body. Likewise, agglomeration and aggregation in liquids

affects how a material is transported, where it goes, and how it

interacts with its environment. Agglomeration/aggregation (oreven de-agglomeration) between material release, exposure,

and transport within the body (or preparation, administration,

and transport in toxicology studies) may lead to significant

changes in hazard potential. For instance, where transport

between organs, across cell barriers, and along neuronal

pathways is mediated by particle size, an understanding of

agglomeration/aggregation state is essential to understanding

potential impact (Oberdorster, 2010). The rate at which

particles will aggregate or agglomerate is dependent on

concentration and sizethe smaller the particles and the

higher the concentration, the greater the aggregation/agglom-

eration rate (Hinds, 1999).

Internal particle structure has also been shown to influencetoxicity. Jiang et al. (2008) and Sayes et al. (2006) have shown

for instance that the crystal structure of TiO2 nanoparticles can

have a significant impact on particle toxicity. In both studies,

anatase TiO2 was found to be more potent than the rutile form

of the material. Mixtures of anatase and rutile TiO2 had an

intermediate potency. Using a cell-free assay designed to probe

a materials capacity to generate reactive oxygen species

(ROS), Jiang et al. also indicated a significant dependence

between particle size and capacity to generate ROS, with

a clear transition in behavior with anatase nanoparticles

between ~10 and 40 nm. What was particularly interesting in

this study was that the smallest particles demonstrated a reducedcapacity to generate ROS. However, as the surface structure of

materials can change markedly at very small sizes (Jefferson,

2000), it is unclear whether this transition was size mediated or

surface chemistry mediated. The authors speculated that the

findings might be associated with the density of defects on the

surface of the particles, suggesting another physicochemical

parameter of potential interest in understanding the toxicity of

nanomaterials.

As well as particle size, particle shape has also been

indicated as a key parameter in determining biological impact.

In particular, the fiber-like morphology of some carbon

nanotubes has prompted concerns over possible asbestos-like

behavior following inhalation, including the potential de-

velopment of mesothelioma (Coles, 1992; Maynard et al.,

2006; RS/RAE, 2004). Takagi et al. induced mesothelioma and

reduced mortality in p53/ mice through ip injection of

multiwalled carbon nanotubes (Takagi et al., 2008), although

this study was subsequently criticized for the use of extremely

high doses and poor material characterization (Ichihara et al.,

2008). To confirm the possibility of mesothelioma resulting

from exposure to carbon nanotubes, Poland et al. exposed the

mesothelial lining of the peritoneal cavity of mice to long

http://toxsci.oxfordjournals.org/


9/21

multiwalled carbon nanotubes via ip injection and concluded

that the early pathological effects were characteristic of

asbestos-like events in producing inflammation (Poland et al.,

2008). Subsequently, Ryman-Rasmussen et al. (2009) sub-

jected mice to a single inhalation exposure of multiwalled

carbon nanotubes and reported that, at a subsequent post-

exposure period, the nanotubes translocated from airspace tosites outside the respiratory tract and embedded in the

subpleural wall and within subpleural macrophages. This

finding served to provide an indirect confirmation of the

possibility of inhaled multiwalled carbon nanotubes producing

effects both inside and outside the respiratory tractsimilar to

asbestos fibers. It is interesting to note, however, that two 90-

day inhalation studies with multiwall carbon nanotubes

conducted in rats, reported by Ma-Hock et al. (2009) (Nanocyl

multiwalled carbon nanotubes) and Pauluhn (2010) (Baytubes

multiwalled carbon nanotubes), failed to find pathological

effects outside the respiratory tract. Either there is a difference

among species, the pleural effect is not particularly pronouncedor a greater focus needs to be implemented to investigate the

potential and relevance of this pleural effect (Warheit, 2009).

To add further complexity to the biological actions of

nanotubes, Kagan et al. (2010a) recently reported that carbon

nanotubes may be biodegraded via a neutrophil myeloperox-

idase mechanism under conditions of inflammation, although it

remains unclear how relevant the results of this in vitro study

are to conditions in vivo.

The question over carbon nanotube toxicity is dominated by

the physicochemical nature of the material. Carbon nanotubes

are not a homogeneous material category but rather represent

an extremely wide array of material chemistries and morphol-

ogies, determined by the number of concentric graphene walls

constituting the nanotubes, their chirality, their diameter, their

length, the density of surface defects, surface functionalization,

the presence of trace elements and other contaminants,

nanotube straightness, the degree of nanotube entanglement,

and so on. Poland et al. demonstrated the potential of one

subset of this materiallong, straight multiwalled carbon

nanotubesto show fiber-like behavior in a biological envi-

ronment (Donaldson et al., 2010). However, many forms of the

material are too short, too long, or too tangled to demonstrate

similar behavior. Nevertheless, these non-fiberlike forms of

carbon nanotubes may present their own distinct hazards

(Shvedova et al., 2003, 2008). Given evidence that themorphology of carbon nanotube material released into the air

during handling can vary markedly from batch to batch, the

challenges of relating relevant characteristics to hazard are

complex (Maynard et al., 2007). This is a material that cannot

be adequately characterized by chemistry alone, or as a simple

fiber, in determining its potential toxicity. Rather, it epitomizes

the need for a detailed and sophisticated understanding of

nanomaterial physicochemical characteristic in understanding

potential hazard.

Biokinetics

Unlike free or loosely bound molecules, the transport,

accumulation, transformation, and clearance of nanomaterials

in the body is intimately associated with physical form as well

as chemical composition. Understanding the biokinetics of

nanomaterials provides information on internal doses to

secondary organs and is essential to designing and interpreting

in vitro studies. Oberdorster cites the well-documented

tendency of nanoparticles to translocate from primary de-

position sites to secondary organs (Oberdorster, 2010) but

cautions that uninformed interpretation of these data can lead to

misunderstanding of potential risk. Inhalation studies using 15

and 80 nm iridium nanoparticles have demonstrated the

translocation of inhaled particles to extrapulmonary organs.

However, translocation rates were the order of~12%, with the

rate decreasing rapidly at larger particle sizes (Kreyling et al.,

2002, 2009). Nevertheless, there is mounting evidence that

changes in physical and chemical nature at the nanoscale can

have a significant impact on biodistribution. For example,Semmler-Behnke et al. (2008) have demonstrated a marked

difference in biodistribution of 1.4-nm diameter Au55 clusters

and 18-nm diameter gold particles administered to rats via

injection and intratracheal instillation; 24 h following iv

injection, 18-nm diameter gold particles were cleared from

the blood and predominantly accumulated in the liver and

spleen; 0.5% of the injected dose was excreted via that

hepatobiliary system, but renal excretion was extremely low. In

comparison, the 1.4-nm diameter gold clusters were excreted

by the kidneys as well as by the hepatobiliary system.

Of particular concern in recent years has been the nature of

interactions between nanoparticles and the central nervoussystem (Yang et al., 2010). There is evidence that inhaled

nanoparticles can translocate to the central nervous system via

olfactory neurons following nasal deposition (Oberdorster

et al., 2004) and induce significant inflammation-related effects

(Elder et al., 2006). This appears to be a particle size and

chemistry transport route that is unique to nanometer-scale

particles and raises the possibility of previously unidentified

organ-specific doses and responses. Although data remain

inconclusive, Oberdorster hypothesizes that differential protein

adsorption on nanoparticles will affect their uptake and

transport within the central nervous system (Oberdorster,

2010). Preliminary data generated using Apolipoprotein E

coated gold nanoparticles are consistent with increased nano-

particle translocation to the central nervous system in rats

following iv administration. However, in this study, less than

0.01% of injected particles were translocated, leaving the

authors to conclude that further confirmatory studies are

needed (Oberdorster, 2010).

Nanoparticle translocation to the central nervous system is

indicative of research on a number of fronts looking at

nanoparticle movement across tight barriers. For a number for

years, there has been concern over the ability of blood-borne

http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


10/21

nanoparticles to cross the placental barrier (Saunders, 2009).

Recently, Bhabra et al. (2009) have indicated using an

in vitro model that blood-borne nanoparticles may be able to

exert an influence across the placental barrier without

physically crossing it. Using an in vitro system designed to

investigate cellular barriers, Bhaba et al. showed that high

concentrations of Cobalt-Chromium alloy nanoparticles on oneside of a tightly meshed layer of cells can cause measurable

DNA damage to cells on the other side. However, it remains

uncertain how relevant these data are to in vivo exposures.

The skin represents another tight barrier that has received

a high level of attention in recent years, as concerns over the

ingress of mineral nanoparticle such as TiO2 and ZnO from

sunscreens and cosmetics into the body have been raised. Early

research suggested that the potential exists for sub-micrometer

diameter particles to penetrate across the dermal barrier under

some circumstances, depending on their size and chemistry

(Ryman-Rasmussen et al., 2006; Tinkle et al., 2003). However,

the majority of studies to date suggest that under mostconditions healthy skin is an effective barrier to nanometer-

scale particles entering the body and causing adverse effects

(Choksi et al., 2010; Newman et al., 2009; Nohynek et al.,

2008, 2010; Osmond and McCall, 2010; Stern and McNeil,

2008). However, there remain some uncertainties surrounding

the tightness of the skin as a barrier against nanoparticles under

varying conditions, and the impacts and clearance of particles

that may cross the barrier. Recently, Gulson et al. (2010)

exposed human volunteers to sunscreens formulated with ZnO

particles tagged with the stable isotope 68Zn. Traces of 68Zn

were found in blood and urine samples of volunteers exposed

to nanometer-scale and non-nanoscale particles, providingevidence of Zn transport into the body. However 68Zn levels

were orders of magnitude below normal blood-borne Zn

concentrations. It also remains uncertain whether these findings

were associated with particle translocation, or particle disso-

lution and subsequent ion transport.

As has been alluded to, a possible confounding factor in

understanding the biokinetics of nanoparticles is their differ-

ential interactions with proteins within biological environ-

ments. Nanoparticles within a biological environment rapidly

acquire a coating or corona of protein molecules and there is

increasing evidence that this dynamic coating mediates the

transport of, and first order interactions with, nanoparticleswithin the body (Cedervall et al., 2007a, 2007b; Ehrenberg

et al., 2009). Furthermore, there is evidence that the corona

and thus particle biokinetics is influenced by particle size and

chemistry (Lundqvist et al., 2008). This relatively new area of

research suggests that interactions between nanoparticles and

biological systems may be more complex and dynamic than

previously thought, requiring a more holistic understanding of

how biokinetics are influenced by particle physicochemistry

and their local environments over time.

EMERGING CHALLENGES

Although we have touched on just some of the more

prominent developments in the science of nanoscale materials

toxicology, it is clear that as understanding of how these

materials interact with biological systems increases, new

questions are being raised as to how to understand and quantify

the toxicity of increasingly sophisticated materials in the context

of identifying, assessing, and managing risks. It is also be-

coming clear that, although new questions are being prompted

by the development and commercial use of engineered nano-

materials, the challenges being faced by toxicology are not

solely confined to materials or particles with physical structure

in the range of 1100 nm. Rather, the emergence of new

nanomaterials is highlighting the importance of material

physicochemistry in mediating biological interactions that result

in toxicity. Research to date suggests that synergism between

particle chemistry and physical form becomes increasingly

important as the features and dimensions of materials entering

the body become increasingly small. But beyond this, there arefew indicators of generalized sharp size-specific transitions in

behavior. Aufann et al. (2009). have attempted to define

a particle size region where size-specific biological behavior

unique to nanoparticles might occur. Reviewing the literature,

they concluded that particles smaller than ~30 nm in diameter

are more likely to demonstrate dramatic changes in behavior

with size. However, particle sizes at which abrupt changes in

behavior occur are clearly material dependentas was shown

by Semmler-Behnke et al. in contrasting the biokinetics of 1.4

and 18 nm diameter gold nanoparticles (Semmler-Behnke et al.,

2008). And there is little reason not to suppose that some

materials may exhibit abrupt changes in behavior above 100 nm.Concerns over the possible novel toxicity of nanomaterials

are frequently driven by abrupt size-specific changes in

functionality that are governed by size-constrained electron

behavioroften referred to as quantum effects. Yet very

few studies have shown a clear association between nanoscale

phenomena such as quantum confinement or surface plasmon

resonances and toxicity. Instead, studies have tended to

highlight the importance of decreasing particle size and

increasing specific surface area for specific particle chemistries

in altering biological behavior. In many cases, these are

scalable effectssmall particles show greater or less tendency

to behave in a certain way compared with large particles, but

their behavior is predictable from larger particles. This is the

case for most studies correlating toxicity with surface area. In

other cases, nonscalable effects are seen, such as with size-

specific translocation. Yet even here, it is unclear whether the

unusual biological behavior observed is related to the

functionality these materials are designed to exhibit or simply

a function of small size.

Yet the field of toxicology is undoubtedly facing a new and

growing challenge: How to understand and address the hazard

of intentionally engineered materials where physical form and

http://toxsci.oxfordjournals.org/


11/21

chemical composition interact synergistically to determine

biological behavior. As collaborations across diverse fields of

research lead to increasingly sophisticated new materials

many of which will be engineered with nanoscale features

this challenge will only grow in magnitude. Up to now,

research has been driven by small particles of conventional

materials such as TiO2, ZnO, and Ag and the occasional newmaterial such as carbon nanotubes. But as the science and

technology of new materials becomes increasingly sophisti-

cated, toxicologists will be faced with complex multicompo-

nent materials, hybrid materials that blur the boundaries of

biological and nonbiological components and active materials

that are designed to change behavior according to their

environment or a received set of signals (Subramanian et al.,

2010). These new sophisticated materials will require a new

toxicology that recognizes the significance of physicochemistry

and dynamic (and possibly remote activated) behavior, that is

cognizant of but not constrained by the importance of the

nanoscale, and that is focused on the potential biological

impacts of the materials rather than their commercially relevant

functionality.

Within this context, we highlight three emerging challenges

to addressing the toxicology of sophisticated materials:

identifying materials that have the potential to exhibit novel

and significant toxicity, characterizing materials appropriately,

and biointeractions.

Identifying Relevant Materials

Effective problem formulation is a cornerstone of contem-

porary risk assessment and, by association, toxicology

(National Academy of Science, 2008). Nevertheless, formulat-ing the environmental health and safety impact problems

posed by sophisticated materials is not trivial. A key question is

how to delineate between the materials and products that are of

concern and those that are not. In regard to engineered

nanomaterials, the conventional approach has been to use

established definitions of nanotechnology and engineered

nanomaterials. These debates typically focus on material

functionality within a narrow size range and are designed

primarily to stimulate research and innovation leading to

economically and socially beneficial new products (NSET,

2010). However, these simple function-oriented definitions do

not always lend themselves to supporting well-defined problem

statements that frame relevant toxicology research on engi-

neered nanomaterials. For example, they do not allow easy

differentiation between functionally unique materials and

products that do not present new toxicology challenges and

functionally mundane materials and products that do present

new hazards. An example of the former might be cadmium

selenide-based quantum dots, where functionality is associated

with size-dependent quantum confinement, but hazard is more

likely associated with the composition of the quantum dots.

And an example of the latter might be the use of nanoscale

particles in a product simply on the grounds of convenience or

economy, but where particle size leads to new exposures, doses,

and hazards. This disconnect between definitions driving

research and innovation and hazard-based problem formulation

is likely to become increasingly important in the face of

increasingly sophisticated materials.

An alternative approach to addressing potential hazardspresented by sophisticated materials is to use principles that

guide scientifically grounded problem formulation. Three

principles that go some way to support science-based and

socially relevant problem formulation address emergent risk,

plausibility, and impact.

Emergent Risk

The idea of emergent risk reflects the likelihood of a new

material causing harm in a manner that is not apparent,

assessable, or manageable based on current approaches to risk

assessment and management. Examples of emergent risk

include the ability of small particles to penetrate to normallyinaccessible places, the inapplicability of established toxicol-

ogy assays to some materials, scalable behavior that is not

addressed by conventional approaches to assessing hazard, and

the possibility of abrupt scale-dependent changes in material

interactions within biological systems. This understanding of

emergence is dependent on the potential of a material to

cause harm in unanticipated or poorly understood ways, rather

than its physical structure or properties per se. As such, it is not

bound by rigid definitions such as those used to define

nanotechnology or nanomaterials. Rather, it enables sophisti-

cated materials that potentially present emergent and un-

anticipated risks to human health and the environment to bedistinguished from those that probably do not.

Many of the engineered nanomaterials that have raised

concerns in recent years have shown potential to lead to

emergent risks and thus would be classified as requiring further

investigation under this principle. But the principle also

embraces more complex nanomaterials that are either in the

early stages of development, or have yet to be developed,

including active nanomaterials and self-assembling materials.

Plausibility

Plausibility capturesin qualitative termsthe science-

informed likelihood of a new material or product presentinga risk to humans. It is based on the possible hazard of a material

and potential for exposure or release to occur. But it also

addresses the likelihood of a technology being developed and

commercialized, and it leading to emergent risks. For example,

the gray goo of self-replicating nanobots envisaged by some

(Joy, 2000) might legitimately be considered an emergent risk

but is clearly not a plausible risk. In this way, plausibility acts

as a crude but effective filter to distinguish between speculative

riskswhich are legionand credible riskswhich are not.

http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


12/21

Impact

Impact is an indicator of the extent to which a poorly

managed sophisticated material might cause harm or the

possible reduction in harm resulting from new research into

identifying, assessing, and managing emergent risks. It helps

provide a qualitative reality check to guard against extensive

research efforts that are unlikely to have a significant impact onprotecting human health, while ensuring that research having

the potential to make a significant difference is identified and

supported.

Together, these three principles provide a basis for de-

veloping informed and relevant approaches to problem

formulation when faced with evaluating the hazards associated

with emerging sophisticated materials. They are tools that allow

new materials which raise safety concerns to be differentiated

from those that, while they may be novel from an applications

perspective, do not present undetected, unanticipated, or

enhanced risks. The principles are technology independent

and therefore can be used to guide research independently of thesophistication of the materials being produced or shifts in

terminology and emphasis underlying technology innovation.

Applying the principles to increasingly sophisticated materi-

als that are being envisaged, a number of groups of materials

begin to emerge that may require further study:

Materials demonstrating abrupt scale-specific changes in

biological or environmental behavior. Materials that undergo

rapid size-dependent changes in physical and chemical

properties, which in turn affect biological behavior, may present

a hazard that is not predictable from larger scale materials of the

same composition. In this case, size and form at the nanoscale

may increase or decrease hazard in a way that is currently notwell understood.

Materials capable of penetrating to normally inaccessible

places. Materials that, by their size, shape, and/or surface

chemistry, are able to persist in or penetrate to places in the body

that are not anticipated based on current understanding may

present emergent risks. Where there is a credible possibility of

accumulation of, exposure to, or organ/system-specific dose

associated with a material that is not expected from how the

dissolved material or larger particles of the material behave,

a plausible and emergent risk is possible.

Active materials. Materials that undergo a change in their

biological behavior in response to their local environment or

a received signal (Subramanian et al., 2010), potentially

present dynamic risks that are currently not well understood.

Self-assembling materials. Materials designed to assemble

into new structures in the body once released raise issues that

may not be captured well within current approaches to hazard

assessment.

Materials exhibiting scalable hazard that is not captured by

conventional hazard assessments. Where hazard scales

according to parameters other than those normally associated

with an assessment, emergent risks may arise as dose-response

relationships are inappropriately quantified. For instance, if the

hazard presented by an inhaled material scales with the surface

area of the material and the dose-response relationship is

evaluated in terms of mass concentration, the hazard will remain

ill quantified.In each of these examples (they are not exclusive), new

research is needed if emergent and plausible risks associated

with new sophisticated materials are to be identified,

characterized, assessed, and managed.

Physicochemical Characterization

Relevant physicochemical characterization is essential to

interpreting data from toxicity studies on sophisticated materials

if generated data are to be useful. The early research by

Oberdorsteret al. on inhalation exposure to TiO2 particles using

rats showed that chemistry alone could not explain differencesin dose-response relationships for two distinct sizes of particles

with the same composition (Oberdorster, 2000); it was only

when the physical structure of the two materials was included in

the assessment that the data were reconcilableand a single

dose response relationship relative to material surface area

emerged. But relevant physicochemical characterization is also

necessary if different studies are to be reproduced and

compared. Without it, vital information is lacking that can

prevent a robust picture of material toxicity from emerging. In

the case of the Oberdorster study, material surface area was

measured, but not discrete particle size or aggregation state. As

a result, it was initially difficult to evaluate or validate whetherthe effects observed were simply because of an elevated material

surface area or were associated with the presence of discrete

nanometer-scale particles. A clearer case of data confounding

through a lack of physicochemical characterization can be

found in studies on carbon nanotubes. Despite the enormous

variation in physical and chemical properties among carbon

nanotubes from different sources (or even the same source at

different times), early toxicology studies were remarkably

vague on the precise nature of the materials being studied,

leading to irreproducible, conflicting, and ultimately uninter-

pretable data (Lam et al., 2006).

The relevance of physicochemical characterization in un-

derstanding and assessing material toxicity has received

considerable attention in recent years. A workshop in 2004,

organized by the National Institute of Environmental Health

Sciences and the University of Florida placed a strong emphasis

on the need for highly detailed materials characterization for

instance (Moudgil, 2004). These recommendationsdriven in

part by materials scientistswere considered at the time to be

beyond the scope of many toxicologists. In 2005, an influential

review led by Oberdorster proposed a reducedbut still

extensiveset of physicochemical parameters that should be

http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/http://toxsci.oxfordjournals.org/


13/21

included in nanomaterial toxicology studies (Oberdorsteret al.,

2005). The recommendations listed 17 parameters as either

essential or desirable in studies. Importantly, they also

considered characterization at three distinct points, recognizing

the dynamic nature of physicochemical characteristics: in the

bulk material, as-prepared for administration, and in situ. Of

these, the importance of characterizing materials in situ wasstressed as materials are capable of undergoing significant

alterations in properties once they are introduced to a biological

environment. However, it was also recognized that few

technologies currently exist that enable detailed materials

characterization within in vitro and in vivo test systems.

Although the Oberdorster recommendations were less chal-

lenging than those from Florida, they were still seen as presenting

near-insurmountable barriers to toxicologists. As a result,

discussions within the community continued to focus on

a minimum characterization set that would be both feasible and

readily adoptable. A 2008 workshop was held at the Woodrow

Wilson International Center for Scholars in Washington, DC, was

influential in mapping out such a minimum characterization set(Table 2) (MINChar Initiative, 2008). The list was one of the first

pragmatic sets of physicochemical characterization requirements

and reflects similar thinking published elsewhere (Boverhof and

David, 2010; Card and Magnuson, 2009; Warheit, 2008).

As a result of these and other efforts, there is movement

toward expecting and including detailed physicochemical

characterization data in nanomaterial toxicology studies. This

is becoming increasingly important as methodologies are

developed to develop predictive models for engineered nano-

materials andby extensionsophisticated materials in gen-

eral. In 2006, Maynard et al. challenged the scientific

community to work towards predictive models for nano-material impact (Maynard et al., 2006). Four years later,

a number of initiatives are beginning to work toward this goal

(Alvarez et al., 2009; Meng et al., 2009). These approaches

depend on associating key material properties with mecha-

nisms of biological interaction and ultimately effects. To be

successful, they will depend on detailed physicochemical

characterization of the materials under test.

However, knowing what needs to be measured is only part of

the challenge, it is complemented by the need for tools to make

appropriate measurements. Here, progress is still lacking, with

even the minimum characterization requirements proposed by

initiatives like MINChar challenging toxicologists. Three

challenges in particular face the toxicology community as

increasingly sophisticated materials are developed: presenting

samples to test systems that are well-characterized, evaluating

key physicochemical properties in situ, and developing

analytical techniques that can provide useful information into

tools that are accessible to the toxicology community. Each

comes with its own challenges and it is likely to be many years

before substantial progress is made. Yet evaluating and

quantifying the toxicology of sophisticated materials will

depend on new tools and methodologies in each of these areas.

One final significant challenge exists here: developing an

understanding of the tolerance within which physicochemical

characteristics show similar or markedly different biological

behavior (National Academies, 2009). Most sophisticated

materials will be manufactured within a certain range ofphysicochemical properties, ensuring functionality is achieved

without resulting in over-costly production processes. As a

result, materials will demonstrate variation in particle size,

shape, surface properties, and other characteristics, both

between batches and within samples. Understanding the

association between variations in physicochemical character-

istics and toxicity will be essential in developing the tools and

methodologies to quantify and address risks presented by

sophisticated materials. Central to this are three questions: (1)

How precisely do physicochemical characteristics need to be

measured? (2) What constitutes a significant change in

characteristics such as particle size, shape, or compositionwhen evaluating toxicity? and (3) How should the hazard

associated with a material representing a distribution of

physicochemical characteristic be evaluated? So far, ve

Documents

2011-Maynard New Tox of Soph Mats