TOXICOLOGICAL SCIENCES 89(1), 42–50 (2006)

doi:10.1093/toxsci/kfi339
Advance Access publication September 21, 2005

FORUM SERIES
Research Strategies for Safety Evaluation of Nanomaterials,
Part IV: Risk Assessment of Nanoparticles
Joyce S. Tsuji,*,1 Andrew D. Maynard,† Paul C. Howard,‡ John T. James,§ Chiu-wing Lam,§
David B. Warheit,¶ and Annette B. Santamariak
*Exponent, Bellevue, Washington 98007; †Woodrow Wilson International Center for Scholars, Washington, DC 20004–3027;

Downloaded from http://toxsci.oxfordjournals.org/ at Sussex Language Institute on December 26, 2012

‡National Center for Toxicological Research and National Toxicology Program Center for Phototoxicology, U.S. Food & Drug Administration,
Jefferson, Arkansas 72079; §National Aeronautics and Space Administration, Houston, Texas 77058; ¶DuPont Haskell Laboratory,
Newark, Delaware 19714; kEnviron International, Houston, Texas 77002

Received July 28, 2005; accepted September 20, 2005

Nanomaterials are a diverse class of small-scale (<100 nm)

Nanoparticles are small-scale substances (<100 nm) with unique
properties and, thus, complex exposure and health risk implica-
tions. This symposium review summarizes recent findings in
exposure and toxicity of nanoparticles and their application for
assessing human health risks. Characterization of airborne par-
ticles indicates that exposures will depend on particle behavior
(e.g., disperse or aggregate) and that accurate, portable, and cost-
effective measurement techniques are essential for understanding
exposure. Under many conditions, dermal penetration of nano-
particles may be limited for consumer products such as sunscreens,
although additional studies are needed on potential photooxidation
products, experimental methods, and the effect of skin condition
on penetration. Carbon nanotubes apparently have greater pulmo-
nary toxicity (inflammation, granuloma) in mice than fine-scale
carbon graphite, and their metal content may affect toxicity.
Studies on TiO2 and quartz illustrate the complex relationship be-
tween toxicity and particle characteristics, including surface coat-
ings, which make generalizations (e.g., smaller particles are always
more toxic) incorrect for some substances. These recent toxicity
and exposure data, combined with therapeutic and other related
literature, are beginning to shape risk assessments that will be used
to regulate the use of nanomaterials in consumer products.
Key Words: nanotechnology; nanoparticles; risk assessment;
exposure assessment.
The opinions and comments in this manuscript do not reflect the views of any
U.S. Government Agency nor do they reflect policy, regulatory positions, nor
any possible changes in regulatory positions. The views expressed in this
manuscript are those of the authors. The mention of any product should not be
considered an endorsement.
1
To whom correspondence should be addressed. 15375 SE 30th Place, Suite
250, Bellevue, WA 98007. Fax: (425) 519–8799. E-mail: tsujij@exponent.com.

The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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substances formed by molecular-level engineering to achieve
unique mechanical, optical, electrical, and magnetic properties.
Nanomaterials are expected to improve virtually all types of
products (Royal Society, 2004), and commercialization of
products that exploit these unique properties is increasing.
However, these same properties present new challenges to
understanding, predicting, and managing potential adverse
health effects following exposure (Hood, 2004).
Widespread application of nanomaterials confers enormous
potential for human exposure and environmental release.
Technological development and applications are out-pacing
research of health and environmental risks (Fig. 1). Like
genetically modified organisms, the future of nanotechnology
will depend on public acceptance of the risks versus benefits.
Even beyond public acceptance, experience with past ‘‘mira-
cle’’ materials (e.g., asbestos) advises caution in using novel
substances without fully evaluating potential health risks.
As with larger-scale substances, risk assessment will be the
basis of assessing and regulating nanomaterials to protect health
and the environment. Many applications will likely have limited
or manageable exposures and thus potential for effects; however,
nanoscale compounds are not the same as their larger counter-
parts, and risks will be product specific (Hood, 2004; Royal
Society, 2004). Although a precautionary moratorium on nano-
technology seems unwarranted, applications should have specific
assessments of potential hazards, exposure, and likelihood of
toxic effects. A symposium at the 2005 annual meeting of the
Society of Toxicology presented recent findings in exposure and
toxicity of nanoparticles that, with related research on fine-scale
and ultrafine particles, will help support critical assessments of
risk. These presentations summarized airborne exposures and
aerosol quantification, skin penetration and potential for toxicity,
inhalation toxicity of carbon nanotubes and other types of
particles, and implications for risk assessment.
 RISK ASSESSMENT OF NANOPARTICLES
FIG. 1. National Nanotechnology Initiative total annual budget for various
sectors. Federal funding for environment, health, and safety is about 4% of the
total budget. Additional funding is also occurring secondarily through other
research (e.g., NIH); however, total funding for implications is still likely less
than 10% based on the NIH budget. Source: www.nano.gov.
CHARACTERIZING AIRBORNE EXPOSURES
TO NANOMATERIALS
While ingestion and skin penetration are potential exposure
routes for engineered nanomaterials (Oberdörster et al., 2005),
the inhalation route for airborne nanomaterials has perhaps
received the most attention (Maynard and Kuempel, in press).
For some materials, studies have shown that the toxicity of
inhaled particles increases as particle size becomes smaller and
as the overall surface area of inhaled material becomes larger
(Maynard and Kuempel, in press; Oberdörster et al., 2005). The
relevance of these findings to human health depends on the
amount of material released into air during research or
production, how its physical and chemical properties change
while in the air, the likelihood of the material being inhaled,
and the effectiveness of control measures to prevent inhalation.
For example, handling of carbon nanotubes resulted in very low
airborne concentrations of nanoparticles, consistent with the
tendency of this material to aggregate into unrespirable larger
masses (Maynard et al., 2004). Evaluation of the factors
affecting exposure requires measurement of aerosol properties
such as particle size, shape, surface area, and surface chemistry.
Although engineered nanomaterials rely on physicochemical
features smaller than approximately 100 nm, inhalation expo-
sures will potentially occur to airborne particles composed of
nanomaterials (including aggregates of nanoparticles) covering
a size range from a few nanometers to several micrometers in
43
diameter. These ‘‘nanostructured particles’’ are potentially of
concern if they can deposit in the respiratory system and have
nanostructure-influenced toxicity (e.g., high surface area, high
surface activity, unusual morphology, small diameters, or
disaggregation into smaller particles once deposited). Classes
of particles of interest, therefore, cover discrete nanometer-
diameter particles, agglomerates of nanoparticles, and droplets
of nanomaterial solutions, suspensions, or slurries. Particles
formed from the degradation or comminution of nanomaterials
may also present a potential risk if they exhibit nanostructure-
dependent biological activity.
Conventional exposure monitoring methods typically rely on
characterizing the mass and bulk chemistry of airborne
particles, two measures that do not seem particularly appro-
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priate to nanostructured particles. There may be ways to adapt
mass-based measurement approaches to evaluating exposures
to nanostructured particles, but solutions will most likely
represent a degree of compromise. Measurement of aerosol
number concentration (i.e., number of particles/volume) is a
relatively straightforward alternative. This method may be use-
ful for identifying sources of nanoparticle emissions (Brouwer
et al., 2004); however, the relevance and specificity of number
concentration measurements in isolation are questionable.
Alternatively, measuring the size distribution of airborne
particles provides a wealth of information on particle size,
surface area, mass, and number concentration, and can be used
to differentiate between background and process-specific
sources (Kuhlbusch et al., 2004; Maynard and Zimmer,
2002). However, current instruments are expensive, and mea-
surement of size distributions from the nanometer range to the
micrometer range is frequently a complex process (Zimmer
and Maynard, 2002).
Among methods for measuring surface area, isothermal
adsorption has been used for many decades to measure the
surface-area of powders; however, samples must be collected
and analyzed later, and applicability for low aerosol concen-
trations is unclear. Although time intensive, particle analysis in
the Transmission Electron Microscope (TEM) is capable of
providing detailed information on projected (two-dimensional)
surface area. Differential Mobility Analysis can be used to
estimate surface area with the added advantage of high
sensitivity at small particle diameters (Rogak et al., 1993).
Measuring the rate at which positive ions attach to airborne
particles provides a near real-time measure of surface area. The
rate at which ions diffuse to the surface of neutral nanometer-
diameter particles is proportional to particle surface area
(Keller et al., 2001). Diffusion charging-based aerosol mon-
itors rely on this relationship to estimate airborne particle
surface area. Ku and Maynard (2005) reported that a commer-
cial diffusion charger provides good estimates of aerosol
surface area for particles smaller than 100 nm in diameter over
a range of morphologies (Fig. 2), with increasing underestima-
tion for larger particles, consistent with diffusion charging
theory. Whether the degree of underestimation is significant for
44
FIG. 2. Measured diffusion charger response to monodisperse aerosols
with a range of particle morphologies (denoted by different symbols) (Ku and
Maynard, 2005). Mobility diameter refers to particle diameter as characterized
using differential mobility analysis.
health-related measurements depends on a number of factors
and is still under investigation. An alternative diffusion
charging-based device has recently been tested that showed
a different response to particles than the instruments tested by
Ku and Maynard. Intriguingly, the response of this device
correlates closely with aerosol surface-area dose that reaches
the lungs, as opposed to total surface area of the particles
(Wilson, 2004).
Along with continued research on the toxicity of nano-
particles, ensuring the appropriate physicochemical character-
ization of materials is essential for drawing valid conclusions.
Currently available instruments and methods provide some
insight into relevant material properties. A range of tools are
available in diverse research fields that are relevant to health-
related nanoparticle characterization, and multidisciplinary
collaborations are encouraged to fully utilize these resources.
Long-term effective characterization of nanostructured particle
exposure will require further research and development of
portable and cost-effective instruments.
EVALUATION OF THE DERMAL PENETRATION
OF NANOMATERIALS
Skin can be exposed to solid nanoscale particles through
either intentional or nonintentional means. Intentional dermal
exposure to nanoscale materials may include the application of
lotions or creams containing nanoscale TiO2 or ZnO as
a sunscreen component or fibrous materials coated with
nanoscale substances for water or stain repellent properties.
Nonintentional exposure could involve dermal contact with
anthropomorphic substances generated during nanomaterial
manufacture or combustion (reviewed in Oberdörster et al.,
2005).
It is unclear whether nanoparticles will penetrate the skin
and have any toxicological impact. Concerns regarding dermal
penetration include (1) skin or other organ cytotoxicity, (2)
TSUJI ET AL.
accumulation in skin or other organs resulting in toxicity after
long-term exposure, (3) metabolism to even smaller particles
with potentially increased toxicity, or (4) toxicity of photo-
activated nanoparticles.
As an example of dermal contact with nanoparticles, nano-
scale TiO2 and ZnO (<100 nm) are being included in sunscreens
because of their ability to block ultraviolet (UV) light. TiO2
particles below approximately 200 microns do not scatter
visible light but will still scatter some UVA radiation. Thus
the inclusion of nanoscale TiO2 or ZnO in sunscreens has the
consumer-desired goal of a clear sunscreen with UV-absorbing
properties.
TiO2 can exist in several crystalline forms (e.g., anatase,
rutile, amorphorous). Anatase TiO2 is a semiconductor, and
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absorption of a photon of light below approximately 385 nm
(i.e., UVA or UVB) will result in the creation of an electron
hole pair. The electron hole pair will interact with water or
oxygen at the crystal surface, resulting in the generation of
reactive oxygen species (ROS) including hydroxyl radicals,
singlet oxygen, or superoxide (Konaka et al., 1999). ROS
generated following illumination of nanoscale anatase TiO2 are
bacteriocidal (Maness et al., 1999), oxidize water contaminants
(Moraes et al., 2004), are genotoxic in Chinese hamster and
mouse lymphoma cell lines (Nakagawa et al., 1997), and are
cytotoxic to cultured HeLa cells (Cai et al., 1992), human
fibroblasts (Wamer et al., 1997), Chinese hamster ovary cells
(Uchino et al., 2002), and human colon carcinoma Ls-174-t
cells (Zhang and Sun, 2004).
Coating the anatase TiO2 particle with inert oxides of silica,
alumina, or zirconium reduces or eliminates the generation of
ROS following UV irradiation (Mills and Le Hunte, 1997). As a
result, the use of coated nanoscale TiO2 or ZnO in commercial
products should eliminate the concern over the generation of
ROS; however, Brezová et al. (2005) recently demonstrated
that irradiation of commercial sunscreen products with light
>300 nm resulted in the formation of ROS. What remains to be
established regarding the use of TiO2 nanoparticles in commer-
cial products is whether there are any biological consequences
from the generation of ROS following UV irradiation.
A few studies have investigated skin penetration by nanoscale
TiO2. A sunscreen containing 8% microfine TiO2 (10–50 nm)
was applied for 2–6 weeks to the skin of human volunteers, and
the penetration of TiO2 into the epidermis was evaluated using
tape stripping (Tan et al., 1996). The depth of penetration was
related to the number of tape strips required to remove the TiO2
from skin. TiO2 did not penetrate the skin of treated subjects
(16 samples) when compared to TiO2 levels in cadaver skin
(9 samples); however, removal of one high value in the cadaver
group resulted in a statistically significant increase in TiO2 in
the epidermis of treated subjects, providing the first evidence of
skin penetration by nanoscale TiO2. The authors noted that
a larger sample size would be necessary to establish this
difference. Lansdown and Taylor (1997) demonstrated appar-
ent penetration when microfine TiO2 was applied as a castor oil
 RISK ASSESSMENT OF NANOPARTICLES
suspension to the backs of rabbits. Using light and electron
microscopy, Schulz et al. (2002) and Pflücker et al. (2001)
concluded that TiO2 nanoparticles did not penetrate the skin of
human volunteers. Bennat and Müller-Goymann (2000) ap-
plied TiO2 to human skin either as an aqueous suspension or
oil-in-water emulsion and evaluated skin penetration using the
tape stripping method. They observed that TiO2 apparently
penetrated skin when applied as an oil-in-water emulsion, and
that penetration was greater when applied to hairy skin, sug-
gesting surface penetration through hair follicles or pores. The
observation that the hair follicles could be a ‘‘repository’’ of
TiO2 was further established by Lademann et al. (1999), where
TiO2 penetrated into the orifices of hair follicles. The pene-
tration of submicron sized (0.75–6.0 lm) fluorescent spheres
into hair follicles has also been reported (Toll et al., 2004).
Tinkle et al. (2003) provide compelling evidence that
nanoparticles could penetrate the skin in demonstrating
epidermal and dermal penetration by fluorescent microspheres
(0.5–1.0 lm) in human skin in an in vitro flexed skin model.
To assess the dermal penetration and toxicological impact of
nanoparticles, future studies will need to focus on (1) charac-
terization of the materials being tested (e.g., monodispersed or
aggregated, anatase versus rutile TiO2), (2) the analytical
method used to detect the nanoparticle (i.e., needs to be able
to discriminate between epidermis, dermis, and hair follicles),
and (3) the condition of the skin (e.g., stratum corneum) before
and after testing. Dermal application and intradermal injection
with CdSe quantum dots may assist in further investigating
dermal penetration and systemic absorption of nanoscale
materials.
PULMONARY TOXICITY OF CARBON NANOTUBES
AND IMPLICATIONS FOR RISK ASSESSMENT
Nanotubes hold great promise for many applications for
aerospace and other industries, including miniature electronics
such as efficient wires and semiconductors; space-efficient
computer memory; light-weight, exceptionally strong struc-
tures with excellent heat dissipation; miniature electromechan-
ical devices; and highly efficient air purification systems. In
addition to new technologies, space missions may encounter
nanoparticles in lunar or Martian dusts. Release of nano-
particles in an enclosed environment is a great concern for crew
health and for hardware engineering.
For many nanotube manufacturing and use settings, the
actual amount of material reaching the lungs will likely be
negligible; however, under some conditions nanotubes can be
extremely small and buoyant (Maynard et al., 2004) and, thus,
penetrate deep into the lung. Research sponsored by NASA
investigated the potential hazard associated with three types of
single-walled carbon nanotubes (SWNT) to assess the response
of the lower respiratory system (Lam et al., 2004). Findings of
this research relevant for conducting risk assessments are
summarized below.
45
Properties of SWNT that Affect Toxicity
Single-walled nanotubes are graphite sheets rolled into tubes
on the order of 1 nm in diameter and 1000 nm or more in
length. They may be capped at either end by half-fullerene
domes and achieve great strength by the sp2 bonds between
carbon atoms. Some nanotubes have a strong tendency to
agglomerate by van der Waals forces into tattered ropes much
larger than individual fibers; whereas, others remain as a fine
powder, much like carbon black. Both intentional and un-
avoidable impurities can affect the electrical, chemical, and
mechanical properties of the particles and, thereby, their
toxicity. The toxicity of the particles will also depend on
whether they are persistent or cleared from the lung and
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whether the host can mount an effective response to sequester
or dispose of the particles.
Intratracheal Instillation of SWNT into Mice
Lam et al. (2004) used intratracheal instillation to minimize
the amount of material required, given the expense or limited
availability for some of the SWNTs, and because of known
dosage to the lungs and ease of comparison to compounds of
known toxicity. Disadvantages included an unnatural delivery
that could exaggerate the effects on the lungs, missing any
effects on the upper airways, anesthetization of animals, and
vehicle effects on the delivery and properties of the particles.
Test particles were suspended (sonicated) in heat-inactivated
mouse serum to deliver doses of 0.1 or 0.5 mg/mouse in a 50 ll
volume. Three types of SWNT, along with carbon black and
quartz (reference dusts of known toxicity) were tested. The
lungs were removed from euthanized animals and subjected to
histopathology study 7 or 90 days after instillation. The types
of nanotubes tested varied according to the method of prepa-
ration (electric arc discharge or vapor deposition) and the
content of metals. Raw SWNTs contained 27% Fe, 1% Mo,
0.8% Ni, and 0.4% Cu; purified SWNTs contained 2% Fe; and
Carbolex SWNTs contained 26% Ni, 5% Y, and 0.5% Fe.
Response of the Lung to Instillation of SWNTs
The highest dose of Carbolex SWNTs caused death in 5/9
mice, most likely due to Ni release from the SWNTs during
sonication, which would not likely occur by aerosol inhalation.
No other lethality occurred, although the high-dose Carbolex
group lost weight, and the raw (but not purified) SWNT group
had transient signs of acute toxicity. Mice showed a dose-
dependent incidence of focal lesions in their lungs 90 days after
instillation. Microscopically, these lesions contained bundles of
SWNTs within macrophages surrounded by fibrous tissue
forming granulomas with some necrotic cells. In some areas
the granulomas were discrete, while in other areas the fibrotic
tissue was widespread and contiguous. Alveolar wall thicken-
ing occurred in areas without obvious granulomas. Multifocal
granulomas were also observed in the lungs of rats that
46 TSUJI ET AL.
received instillations of SWNT containing 5% Ni and 5% Co
(Warheit et al., 2004).
Lungs instilled with carbon black showed the presence of
opaque particles even after 90 days, but no tissue reaction.
The Fe-containing nanotubes elicited inflammation that was
still present after 90 days, whereas the inflammatory response
had subsided in the lungs instilled with Ni-containing Carbolex
nanotubes. Lungs instilled with quartz showed inflammation,
but no granuloma formation or fibrosis.
Nanotubes were thus at least as toxic as quartz and much
more toxic than carbon black, with some indication of the
effect of metal content on toxicity. The redox properties of iron
in SWNT were implicated in oxidative stress and cytotoxicity
in cell cultures of human keratinocytes (Shvedova et al., 2003).
Nickel is also a redox-active metal with potential to influence
toxicity.
Implications for Human Risk Assessment
These findings indicate that, if inhaled into the lungs,
nanotubes (depending on their content) are capable of eliciting
an inflammatory, granulomatous, and fibrogenic response, and
that the mass-based permissible exposure level (PEL) for
respirable graphite dust may be inadequately protective for
exposure to SWNTs. If a 30-g mouse were exposed to airborne
nanotubes at a concentration of 5 mg/m3, the PEL for respirable
graphite dust, and 40% of the respired nanotubes deposited in
the pulmonary region, the lungs would accumulate a mass of
nanotubes equivalent to the low dose within 4 working days
and a mass equivalent to the high dose within 17 working days.
Moreover, because SWNTs were more toxic than quartz based
on histopathology, assuming similar relative toxicity in hu-
mans, a PEL below that for quartz dust (0.05 mg/m3) is
suggested until further characterization of nanotube toxicity,
assessment of the fraction of airborne nanotubes in the
respirable size range, and determination of whether a mass-
based standard is even applicable given evidence for a surface-
area based standard (Maynard and Zimmer, 2002).
Additional questions of relevance for human health assess-
ment include under what conditions are the nanotubes respira-
ble; how quickly are inhaled nanotubes cleared from the lungs
and what is their fate; what are the long-term tissue responses
to persisting nanotubes; and how do shape (e.g., varying tube
lengths) and metal impurities such as Ni and Fe influence
toxicity?
IMPACT OF EXPOSURES TO NANOPARTICLES ON
RESPIRATORY HEALTH: PARTICLE SIZE
MAY NOT BE MORE IMPORTANT THAN
SURFACE CHARACTERISTICS
Several toxicology studies in rats have reported that ultrafine
or nanoparticles cause increased lung toxicity compared to
larger particles of similar chemical composition at equivalent
mass concentrations (reviewed by Oberdörster et al., 2005).
Surface area and particle number metrics are thus postulated to
play important roles in ultrafine particle lung toxicity.
A few studies have assessed lung toxicity in rats following
aerosol exposures to ultrafine particles at very high particle
concentrations. The results of 2-year inhalation studies with
ultrafine TiO2 (P-25) or fine-sized TiO2 particles (average pri-
mary particle sizes ~25 and ~300 nm, respectively) have indi-
cated that less than one-tenth (10 vs. 250 mg/m3) of the inhaled
exposure concentrations of the ultrafine particle-types, com-
pared to fine-sized particle-types, produced equivalent numbers
of lung tumors in rats (approximately 16–30%) (Heinrich et al.,
1995; Lee et al., 1985). In addition, shorter-term pulmonary
toxicity studies with ultrafine and fine carbon black, as well as
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TiO2 particles in rats (Bermudez et al., 2004; Oberdörster et al.,
1994), have demonstrated increased pulmonary inflammatory
potency of the ultrafine particles when compared to fine-sized
particulates of respective similar composition.
However, the common perception that all nanoparticle-types
are more toxic and inflammogenic than fine-sized particle-
types of similar content is based upon a limited number of
studies; in fact, a systematic comparison of only two particle-
types (TiO2 particles and carbon black particles) and essentially
only one type of TiO2: P25 (80% anatase, 20% rutile). A variety
of physical characteristics, other than particle size, may
play significant roles in modifying toxicity of nanoparticles
(Yamamoto et al., 2003). These include, but are not limited to
surface treatments of particles (Warheit et al., 2003b), the
tendency of aerosolized particles to aggregate/disaggregate, the
method of synthesis of particles (i.e., whether the particle was
generated in the gas or liquid phase), particle shape, and/or
surface charge.
In considering the influence of particle size and surface
treatments on lung toxicity, three sets of pulmonary bioassay
studies are currently being conducted and the preliminary
findings are described below.
First, because fine-sized Min-U-Sil quartz particles are
classified by the International Agency for Research on Cancer
(IARC) as Category 1 (i.e., human carcinogen), one would
expect that nanoscale quartz particles could be even more
potent pulmonary toxicants than highly cytotoxic fine-sized
quartz particles. Fine-sized quartz particles (Min-U-Sil, aver-
age diameter ¼ 1.6 lm) or nanoscale quartz particles (mean
particle size ~50 nm) were intratracheally instilled into the
lungs of rats at doses of 1 or 5 mg/kg. Following exposures,
assessments of bronchoalveolar lavage fluid biomarkers of lung
toxicity were made at 1 day, 1 week, and 1 and 3 months.
Pulmonary exposures to the Min-U-Sil quartz particles pro-
duced significantly enhanced and sustained lung inflammatory
responses compared to the effects produced by the nanoscale
quartz particles. These studies were repeated with smaller
nanoscale quartz (range ¼ 10–20 nm) samples or fine-sized
quartz sample (400 nm), with Min-U-Sil quartz particles as
a positive reference quartz particle-type. Although the study
 RISK ASSESSMENT OF NANOPARTICLES
FIG. 3. Pulmonary inflammation in Min-U-Sil quartz, nanoscale quartz,
fine-sized quartz, or iron particle-exposed rats and controls as evidenced by
percent neutrophils (PMN) in BAL fluids at 24 h, 1 week, 1 month, and
3 months postexposure. Values given are means ± SD. Exposures to Min-U-Sil
or nanoscale quartz particles produced sustained, significant pulmonary
inflammatory responses at all time points, as measured through 3 months
postexposure. Exposures to fine-sized (400 nm) quartz particles also produced
lung inflammatory responses, but were not as potent as with the Min-U-Sil and
nanoscale quartz particles.
has not been completed, the results thus far indicate that the
order of inflammatory potency was nanoscale quartz ¼ Min-U-
Sil > fine-sized quartz particles (Fig. 3).
In a second set of studies, rats were exposed to fine-sized TiO2
particles (300 nm), TiO2 nanoscale rods (250 nm 3 30 nm), or to
TiO2 nanoscale dot particles (10 nm) at intratracheal instillation
doses of 1 or 5 mg/kg, with lung assessments conducted at 1 day,
1 week, and 1 and 3 months postexposure. TiO2 dust is known
have low toxicity and solubility. Preliminary results have thus
far demonstrated no significant differences among any of the
particle-exposed groups compared to vehicle controls with
regard to inflammatory or cytotoxic lung responses at any
postexposure time periods. Moreover, following a transient (24-h
post exposure) neutrophil-associated inflammatory response,
subsequent immunological responses were macrophage medi-
ated for all three particle types. By contrast, Min-U-Sil quartz
particles produced persistent inflammatory response (through
3 months postexposure) characterized by the presence of neu-
trophils, lymphocytes, and macrophages. Interim conclusions
suggest that particle size was not a determinant of toxicity, in
contrast to previous studies of fine-sized TiO2 and ultrafine P25
TiO2 (Bermudez et al., 2004; Heinrich et al., 1995; Lee et al.,
1985). Thus, P25 may not be representative of all nanoscale
TiO2 particle-types, and pulmonary toxicity for each fine-sized
and nano-sized particle types should be evaluated on a case-by-
case basis.
47
A third set of studies (Warheit et al., 2005) investigated the
effects of various surface treatments (0–6% alumina [Al2O3]
and/or 0–11% amorphous silica [SiO2]) on the toxicity of
commercial TiO2 particle formulations. Pulmonary bioassay
data from instillation exposures in rats to TiO2 particle-type
formulations were compared to reference base TiO2 particle
types, along with vehicle controls. The TiO2 particle formula-
tions with the largest concentrations of both alumina and
amorphous silica surface treatments produced mildly enhanced
adverse pulmonary effects when compared to the reference
base control particles. These results along with other studies
(Derfus et al., 2004; Warheit et al., 2003a,b) indicate that
surface treatments on particles (likely of more relevance for
human exposure than reference particle-types) can modify
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(either up or down) particle toxicity.
Based on the available literature and these preliminary
findings, a number of additional factors are likely to influence
the pulmonary toxicity of nanoparticles, in addition to particle
size and surface area. These include but are not limited to the
following:
(1) Particle number and size distribution;
(2) Dose of particles to target tissue;
(3) Surface treatments on particles, particularly for engi-
neered nanoparticulates;
(4) The degree to which engineered nanoparticles aggre-
gate/agglomerate—which strongly influences particle deposi-
tion characteristics in the lung;
(5) Surface charges on particles;
(6) Particle shape and/or electrostatic attraction potential;
(7) Method of particle synthesis—i.e., whether formed by
gas phase (fumed) or liquid phase (colloidal/precipitated)
synthesis and post-synthetic modifications, which likely in-
fluence the aggregation behavior of particles.
FRAMEWORK FOR RISK ASSESSMENT
Recent studies on nanoscale substances, together with
considerable related research on ultrafine particles (UFP) from
air pollution (e.g., Englert, 2004; Oberdörster et al., 2005),
metal fume (e.g., Kuschner et al., 1997a,b), and mineral fibers
(e.g., Cugell and Kamp, 2004) provide an initial basis for
evaluating the primary issues in a risk assessment framework of
nanomaterials (Fig. 4).
Identification of hazards depends on the diverse character-
istics of these particles discussed above. Hazards of novel
structures will be less predictable than smaller-scale versions of
previously studied substances (e.g., metal oxides such as TiO2
or ZnO). Encapsulation of the material either by surface
coatings or within a matrix affects the reactivity and biological
mobility of nanoparticles (Warheit et al., 2003a,b), but the
durability of such encapsulation needs consideration.
Exposure assessment includes the entire life cycle of nano-
materials from synthesis to disposal. Exposure is likely highest
48
FIG. 4. Risk assessment framework for nanomaterials.
for workers, although product specific evaluations should
consider consumer uses, wear, disposal, and potential for
environmental release and fate for products containing nano-
materials (Fig. 5). As noted above, several nanomaterials
(e.g., carbon nanotubes [Maynard et al., 2004], metal oxides
[Bennat and Muller-Goymann, 2000]) tend to aggregate, which
may reduce their ability to penetrate membranes, reach deep
airways, or disperse. However, aggregation by fullerenes in
water has been associated with increased solubility and
antibacterial properties (Fortner et al., 2005). Determining
whether nanoparticle exposure would occur for specific
products is a critical initial step in assessing potential risks.
Inhalation and dermal routes have been the primary focus for
health effects research of nanoparticles, as described above.
Research on medical device (e.g., Yamamoto et al., 2003),
diagnostic, and therapeutic (e.g., Lockman et al., 2004)
applications is also contributing to understanding the toxico-
kinetics and toxicodynamics of nanomaterials. Such studies
indicate that skin (see above) and the blood–brain barrier do
not appear to be readily penetrated, and penetration depends on
specific conditions. As in the lung, phagocytic cells (macro-
phages, monocytes) of the reticuloendothelial system (RES)
rapidly uptake nanoparticles after intravenous injection. Vari-
ous nanoparticle coatings have thus been evaluated in drug
delivery and imaging research to evade the RES and keep
particles in circulation to reach targeted sites (e.g., tumors)
(Jain et al., 2005).
Differences among compounds and studies are apparent in
systemic absorption of particles from the lung. Nemmar et al.
(2002) but not Brown et al. (2002) reported that inhaled
nanocarbon particles in humans readily pass into systemic
TSUJI ET AL.
Downloaded from http://toxsci.oxfordjournals.org/ at Sussex Language Institute on December 26, 2012
FIG. 5. Potential for release and exposure to nanoscale substances.
circulation, where they are a concern for cardiovascular
toxicity. Iridium nanoparticles administered to rats by endotra-
cheal tube were cleared by the airways to the gastrointestinal
tract and eliminated, with less than 1% translocating to other
organs (Kreyling et al., 2002). For systemically absorbed
particles, the liver may be a primary translocation site with
spleen, bone marrow, heart, kidney, bladder, and brain as
secondary sites (Nemmar et al., 2001, 2002; Oberdorster
et al., 2005; Takenaka et al., 2004). Uptake and translocation
via olfactory and trigeminal nerve axons to the brain of rodents,
as reported for airborne UFP (Oberdörster et al., 2005) and
manganese, but not iron (Rao et al., 2003), could be a more
direct route to the brain than across the blood–brain barrier
(Oberdörster et al., 2005).
As noted above, the relevant inhalation dosimetry in risk as-
sessments of nanoparticles may be surface area or particle
number rather than mass per volume or per body weight, although
the complexity of other properties preclude generalizations to
all nanoparticles (described above; Yamamoto et al., 2003).
Despite this complexity, some patterns are emerging for the
more studied substances. The primary mechanism of action by
inhalation or dermal routes appears to be free radical gener-
ation and oxidative stress associated with surface reactivity
(Oberdorster et al., 2005). Oxidative stress associated with
TiO2 nanoparticles, for example, results in early inflammatory
responses such as an increase in polymorphonuclear cells,
impaired macrophage phagocytosis, and/or fibroproliferative
changes in rodents (Bermudez et al., 2004).
An inverse relationship has been observed between lung
clearance rate and nano P25 TiO2 toxicity in rodent species
(i.e., order of increasing clearance and decreasing lung toxicity
in a 90-day inhalation study: rats > mice > hamsters;
Bermudez et al., 2004). Lung clearance of particles in humans
is greater than in rats. Moreover, lung cancer in rats exposed
to high levels of inert particles is thought to occur by
a lung-overload mechanism that is not relevant for humans
(Borm et al., 2004). On the other hand, translocation to other
 RISK ASSESSMENT OF NANOPARTICLES
organs may be a greater concern for humans. Such species
differences are a concern in extrapolating results from animal
models to humans (Borm et al., 2004).
Intratracheal inhalation studies in rats indicate that ultrafine
or nano TiO2 is less inflammatory than quartz (Rehn et al.,
2003), nickel, or cobalt (Zhang et al., 1998), or carbon black
(Renwick et al., 2004). Studies in humans exposed to ZnO
metal fume (including nanoscale particles) have shown a rela-
tively low order of toxicity associated with signs of pulmonary
inflammation and metal fume fever that does not progress to
pulmonary disease (ACGIH, 2001; Kuschner et al., 1997a). By
contrast, greater toxicity is expected for more reactive nano-
particles such as fullerenes, carbon nanotubes, or perhaps UFP.
Although most toxicological studies with nanomaterials
have been in vitro, or short-term in vivo studies involving
unnatural delivery (e.g., intratracheal instillation) in limited
species and types of nanoparticles, the National Toxicology
Program is planning short and long-term studies, including
oral, dermal, and inhalation exposures for some nanoparticles
(http://ntp-server.niehs.nih.gov/files/nanoscale05.pdf). Nano-
material research and risk assessments will ultimately need to
address multiple potential health effects including cardiovas-
cular, carcinogenicity, reproductive/developmental, immuno-
logical, and neurological.
Screening assessments of exposures to the more studied
metal oxides could be conducted by developing toxicity
benchmarks using the weight of evidence from studies of (1)
nanoscale metal oxides in the toxicological and pharmacolog-
ical literature, (2) fine-scale forms corrected for the propor-
tionally greater surface area of nano-scale particles, (3) more
toxic particles such as UFP, and (4) the toxicology and
epidemiology of metal fume. Uncertainties in such assessments
will have to be considered given data limitations; however,
collectively, the available studies are beginning to reveal
important features necessary for initial risk assessments of
specific nanoparticles.
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