Carbon 44 (2006) 1028–1033

www.elsevier.com/locate/carbon

Toxicology of carbon nanomaterials: Status, trends,

and perspectives on the special issue
a,*
Robert H. Hurt , Marc Monthioux b, Agnes Kane c

a
Division of Engineering, Brown University, 182 Hope St., Providence, RI 02912, USA
b
Centre d’Elaboration des Matériaux et d’Etudes Structurales (CEMES), UPR-8011 CNRS, Toulouse, France
c
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA

Received 16 December 2005; accepted 17 December 2005

Abstract

This article introduces this special issue of Carbon devoted to the toxicology of new carbon nanomaterials. This lead article draws
from the ten contributed articles to discuss overall status, trends, and research needs in this new field. Common themes in the contributed
articles are highlighted and a special appeal is made for collaboration between toxicologists and materials scientists in the joint deve-
lopment of ‘‘green’’ nanomaterial formulations -those co-optimized for function and minimal health impact.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Carbon nanotubes; Carbon nanoparticles; Fullerenes; Biocompatibility; Bioactivity

1. Introduction
Two decades of nanotechnology research have led to a
dazzling array of new materials with one or more dimen-
sions in the 1–100 nm size range. It is increasingly recog-
nized that development of new nanomaterials should be
accompanied by parallel efforts to investigate and under-
stand their potential health and environmental effects.
Their small size and high surface area may produce greater
chemical reactivity, induce permeability or conductivity
changes in biological membranes, enhance lung penetra-
tion, or enhance cellular uptake leading to cytotoxicity
from the nanomaterial itself or from adsorbed species
transported into the cell during endocytosis of the nanoma-
terial (the ‘‘Trojan horse’’ effect). Early studies of nanoma-
terial toxicity have produced apparently conflicting results
and raised more questions than were answered.
*
Corresponding author. Tel.: +401 863 2685; fax: +401 863 1157.
E-mail address: Robert_Hurt@Brown.EDU (R.H. Hurt).
Our goal with this special issue was to compile a signif-
icant body of the most recent work by leading experts in
the emerging field of nanotoxicology with a special focus
on carbon. Carbon nanomaterials are arguably the most
celebrated products of nanotechnology to date, encom-
passing fullerenes, nanotubes, nanofibers, and a wide vari-
ety of related forms. These nanomaterials can enter the
human body through inhalation, dermal (skin) contact,
ingestion, or intentional injection, and may affect micro-
organisms, plants, or animals if released into the environ-
ment in significant quantities. This combination of new
materials, multiple exposure routes, and environmental
fate and transport issues creates a three-dimensional matrix
containing a rich set of research questions that this young
field has just begun to tackle.
The breadth and complexity of this new field is easily
seen in the articles selected for this special issue. Each arti-
cle was subjected to two reviews—one by a carbon scientist
from our normal reviewer pool and a second by a toxicol-
ogist, biologist, or biomedical engineer. The result we
believe is a unique collection of scientific papers, compre-
hensible to researchers in diverse fields that provide a

0008-6223/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbon.2005.12.023
 R.H. Hurt et al. / Carbon 44 (2006) 1028–1033
critical body of information that can serve as a guide for
continuing work in this field. These brief introductory
comments summarize the main contributions of our con-
tributing authors, highlight general themes that are echoed
in multiple papers, and make a general plea for close col-
laboration between toxicologists and materials scientists
to move this field forward.
2. Overview of our contributed articles
The special issue begins with a comprehensive review of
the existing literature on carbon nanotube biocompatibility
by Smart and coworkers. This article opens with a discus-
sion of traditional carbon materials used in biomedical
implants and coatings as a prelude to the main discussion
of recent literature on the toxicity of nanotubes. The article
surveys nanotube skin exposure, inhalation, cellular toxic-
ity (cytotoxicity), and the application of nanotubes as
nanostructured surfaces for bone growth or neuronal scaf-
folds. The unified discussion of toxicity and biocompatibil-
ity highlights the close connection between the basic
scientific principles that underlie these two fields. In closing
the authors point out the potential role of catalytic metal
impurities in toxicity, and point to poor nanotube disper-
sion in aqueous media as a persistent challenge for acquir-
ing consistent and reproducible data in toxicological
assays.
Muller and coworkers also review the nanotube litera-
ture but with a special focus on inhalation toxicology.
The authors believe that the current literature is limited
and fragmentary, but nevertheless sufficient to raise con-
cern that carbon nanotubes may cause serious inflamma-
tory and fibrotic reactions if they reach the lung. They
point to the need for more realistic exposure methods,
namely inhalation rather than instillation (the latter being
a liquid spray method), before firm scientific conclusions
can be reached. Muller et al. also point to the need for bet-
ter materials characterization and for comprehensive toxi-
cological studies correlating dose, physical and chemical
properties to pave the way for possible design of less toxic
materials—themes that are echoed in many of the contribu-
tions that follow.
This special issue contains two studies of chronic lung
effects using animal models thereby revealing this problem
as one of the major concerns. Grubek-Jaworska and
coworkers bring new data to this special issue in a study
of three types of fibrous nanocarbons delivered to the lungs
of guinea pigs. Intratracheal instillation of carbon nano-
tubes/nanofibers induced significant pathology in the form
of bronchiolitis obliterans organizing pneumonia (organiz-
ing pneumonitis) with or without peribronchial fibrosis.
David Warheit provides an update of his seminal 2004
study on single-wall nanotube toxicology in the rat lung
along with his perspectives on hazard identification and
risk. Although his studies show the formation of multifocal
granulomas following intratracheal installation in the rat
lung, nanomaterial risk assessment requires information
1029
on realistic exposure scenarios and this is highly uncer-
tain due to the apparent difficulty with which nanotubes
can be disentangled and aerosolized in the respirable
size range. The results point to the need for complete
chronic toxicity studies based on inhalation rather than
instillation.
The special issue moves away from inhalation toxicol-
ogy to skin exposure (Monteiro-Riviere and Inman) and
subcutaneous implantation (Koyama et al.). Monteiro-
Riviere and Inman provide a useful introduction to the
issues that arise when nanomaterials contact human skin.
Toxicity is determined by the probability of penetration
and transport through the lipid matrix in the outer skin
layers (the stratum corneum) and by subsequent interac-
tions with a variety of cell types in the subsurface layers
or by further migration and systemic toxicity. In addition
to reviewing this literature, the authors present new data
examining the role of carbon black commonly used as a
control material in toxicity assays. The authors point out
potential interferences associated with adsorption of fluo-
rescent dyes and other reagents on the high-specific-area,
nanoparticulate carbon.
Koyama examined four types of high-aspect ratio car-
bon nanoforms implanted for up to 3 months in mice
and studied the biological responses in tissue and periphe-
ral blood relative to asbestos as a positive control with
known toxicity. This study includes an unusually thorough
materials characterization of the carbon forms and their
impurities. The authors observe the formation of granulo-
mas, time-dependent changes in peripheral T-cells, no ani-
mal mortality and reduced fibrosis than asbestos fibers,
which they feel is encouraging for large scale commercial
production and application of carbon nanomaterials.
Articles seven and eight are mechanistic studies of
cytotoxicity using human endothelial cells and murine
(mouse-derived) macrophages. Flahaut and coworkers
evaluated the toxicity of three samples of CCVD carbon
nanotubes using human umbilical vein endothelial cells in
culture to evaluate toxicity following injection of carbon
nanotubes used for drug delivery or diagnostic imaging.
Confluent cultures were exposed to different dilutions of
test samples in culture medium supplemented with 10%
fetal calf serum for 24 h. In comparison with phenol used
as a toxic control, none of these samples decreased cell
viability or metabolic activity greater than 75% of
untreated control cultures. These results suggest that
direct exposure of confluent endothelial cells to these car-
bon nanotube samples in the presence of serum does not
produce significant acute toxicity. Of note in comparing
this study with previously-published results are significant
differences in the length of carbon nanotubes and surface
functionalization.
Fiorito and coworkers compared the inflammatory
responses of murine and human macrophages exposed to
purified carbon single-wall nanotubes and fullerenes. Syn-
thetic graphite particles (1 lm in diameter) in combination
with lipopolysaccharide were used as a positive stimulus
1030 R.H. Hurt et al. / Carbon 44 (2006) 1028–1033
for production of an inflammatory mediator (nitric oxide)
after 24, 48, and 72 h of exposure in the presence of 5%
fetal calf serum. Phagocytosis, cell morphology, and viabil-
ity were also assessed. In combination with lipopolysaccha-
ride, graphite particles stimulated NO production by the
J774 murine macrophage cell line while purified SWNTs
or fullerenes had no effect. In contrast to rapid internaliza-
tion of 1 lm graphite particles, in cultures of human mac-
rophages, fewer SWNTs and fullerenes were internalized
and minimal cell activation and toxicity were observed.
These investigators used highly-purified carbon nanomate-
rials and conclude that the lower levels of residual metal
catalysts reduced their toxicity and production of this
inflammatory mediator.
The article by Chlopek and coworkers differs from the
other contributions by focusing on the potential positive
health effects of intentional exposure to carbon nanotubes.
The paper reviews the biocompatibility literature and then
examines the interactions of fibroblasts, osteoblasts and
osteocalcin with HCl-purified multiwalled nanotubes con-
taining small quantities of residual Mg and Co (0.02 and
2 wt%, respectively). The nanomaterials show a limited
reactivity, within the range of convential biomaterials.
The article discusses the induction of collagen production
by fibroblasts and osteoblasts and its role in tissue
regeneration.
Finally, Oberdorster and coworkers complete our series
with a glimpse into the world of environmental effects. The
authors study the effect of fullerenes on five environmen-
tally relevant organisms including two fish species and
fresh and salt water crustaceans. One of the motivations
cited for studying fullerenes at this early stage in the field
is their simplicity relative to nanotubes, whose test samples
are typically complex mixtures containing metals and mul-
tiple carbon forms. Fullerene environmental studies are not
without complications, however, most notably the very
poor solubility of free C60 in aqueous media. For this rea-
son Oberdoster et al. focus on so-called nC60—fullerene
nanoparticles that self assemble following long duration
stirring or by solvent exchange as the most likely C60 form
to be encountered by marine organisms. Lethal doses were
not achieved due to limited solubility. Species-dependent
sub-lethal effects were observed in the freshwater zooplank-
ton Daphnia magna.
3. Issues and research needs in carbon nanomaterial
toxicology
It is the universal consensus of our special issue authors,
who represent both academia and industry, that more work
is needed on all of the new carbon nanomaterials to ade-
quately assess their toxicity and health risks. Another uni-
versal theme is the need for caution when interpreting data
obtained using these highly complex bio/nanomaterial sys-
tems. Our contributing experts also cite a large number of
specific issues and research needs that may be helpful to
readers entering this new field. Here, we highlight a few.
3.1. Need for detailed materials characterization
There is broad agreement that future studies should put
more emphasis on detailed characterization of test nanom-
aterials. Most practical carbon nanomaterials are now
understood to be complex mixtures containing multiple
carbon forms and metal residues of variable chemistry,
particle size and mode of association with carbon. In many
cases the observed toxicity indices may reflect these
byproducts or residues rather than the primary material
structure. Meeting this need requires collaboration between
toxicologists and materials scientists—a point so important
that we revisit it in the last section on the Role for Materials
Science.
3.2. Need for realistic exposure scenarios
Essentially all studies to date have measured toxicity
indicators when nanomaterials are placed in contact with
living systems by an artificial mechanism: implantation,
instillation (lung spray), or incubation with cells in vitro,
and sometimes using dose levels that unlikely reflect most
of what would be expected on a routine basis of nanoma-
terials handling and use. Risk is the product of hazard and
exposure, and little is currently known about realistic
exposure levels, especially for lung exposure. Although
nanoparticles behave aerodynamically in a way that may
allow deep lung penetration, the propensity of nanotubes
to form persistent agglomerates can make their aerosoliza-
tion difficult and the size of the suspended particles much
greater than the individual tube size—often in the supe-
rmicron range. Generally speaking, exposure of whole ani-
mals to nanomaterials by direct contact can overlook the
role of natural defenses that may discriminate, segregate,
modify, or even eliminate subsequent distribution of
nanomaterials throughout the body. More research is
needed on the generation and characterization of nano-
tube aerosols, as well as chronic animal inhalation studies,
which are typically expensive but necessary for reliable
risk assessment.
3.3. Need for methods to track nanomaterials in biological
experiments
Light microscopies are favored techniques in the life
sciences but are difficult to adapt for nanomaterial studies
as the materials structures in question lie below optical res-
olution limits. Thin section TEM analysis can be very effec-
tive but is difficult and time consuming. Fluorescence
 R.H. Hurt et al. / Carbon 44 (2006) 1028–1033
detection has been shown to be feasible but research is
needed to identify the best ways of covalently or adsorp-
tively linking fluorophores to nanomaterials as well as com-
plementary studies to assess whether these fluorophores
alter nanomaterial surface chemistry and thus alter their
toxicity and fate/transport in organisms. Secondary modi-
fication of the chemical or surface properties of carbon
nanomaterials by host cells or tissues should be considered
in experimental design. Nanoparticles may be translocated
from the site of entry and sensitive methods of detection
are required to quantitate the extent of systemic transport
and persistence at distant organs following dermal
exposure, inhalation, ingestion, injection, or implantation.
This aspect of the field is wide open for the development
of new experimental tools at the interface of biology and
materials science.
3.4. Issue of adsorptive interferences with
fluorescent assays
Biology and mechanistic toxicology make widespread
use of fluorescent probes as indicators of cell death, oxi-
dant production, and synthesis of proteins or small mole-
cules involved in cell signaling and immune responses.
Monteiro-Riviere and coworkers in this issue raise the
question whether carbon blacks, used as a negative toxicity
control, may interfere with these fluorescent assays through
adsorption or other means. As our readers know, carbons
are nearly universal sorbents for organic compounds from
aqueous solutions, and although most nanomaterials are
not designed as sorbents (i.e. not optimized for surface
area) most do have high areas by virtue of their small size.
It is reasonable to imagine that carbon black particles, with
surface areas from 20 to 200 m2/g could adsorb not only
the fluorescent dyes (which often contain polyaromatic
groups) but also a variety of proteins, DNA, reductants
(e.g. ascorbic acid), and chelating agents, and even salts
that are used in such assays. We may further expect the
problem not to be limited to carbon black, but to be an
issue for other nanomaterial test samples. Carbon nano-
tubes, fibers, and particles all have significant surfaces
areas coupled often to hydrophobic surface chemistry that
makes them good sinks for the nonspecific adsorption of a
wide variety of organic molecules and macromolecules
from water. Care is needed when carrying out even estab-
lished toxicity assays in the presence of significant quanti-
ties of fine carbon.
3.5. Dose metrics
Toxicologic studies require complete dose-response
curves and sufficient information about the mass, number
of nanoparticles or nanotubes, and surface area to calcu-
late doses using multiple dose metrics. In in vitro cellular
toxicity assays, a full description of the target cells, culture
1031
medium, and plating density per surface area must be pro-
vided. Similar to the considerations described in Section
3.4, adsorption of serum or cellular proteins to the test
materials should be considered in interpretation of the
results. As emphasized by Monteiro-Riviere and cowork-
ers, intra-cellular localization of nanomaterials should be
established by TEM or other sensitive detection methods.
This information is essential in determining the dose deliv-
ered to the target cell; however, it is possible that lipophilic
nanomaterials (e.g. fullerenes) may interact with plasma
membrane lipids and exert toxicity directly in the absence
of cellular uptake. It is also likely that native or modified
carbon nanomaterials interact with cell membrane recep-
tors and may trigger or alter intracellular signal transduc-
tion pathways.
3.6. What are the most important indicators of toxicity?
Most of the published toxicologic studies use short-term
indicators of toxicity, altered cellular function, or inflam-
mation as endpoints. Following intratracheal instillation
or pharyngeal aspiration, these toxicity endpoints are com-
pared with accepted toxic (e.g. quartz or crystalline silica)
and nontoxic (e.g. fine titanium dioxide) particles. How-
ever, native and modified carbon nanomaterials may elicit
additional types of pathologic reactions including granulo-
mas and immune or hypersensitivity reactions. Nanomate-
rials may also interfere with endogenous metabolic or
signal transduction pathways and perturb cellular bio-
chemical functions in more subtle ways that would not be
readily apparent in short-term toxicity assays. Both
short-term assays and subchronic exposures must be con-
ducted in well-characterized in vitro and in vivo models
and calibrated using validated negative and positive con-
trols. There is ongoing debate among toxicologists, indus-
try and regulatory agencies about the utility of in vitro
cellular toxicity assay vs. sub-chronic animal testing in
screening for potential adverse health effects of
nanomaterials.
4. What is the role of materials science?
Toxicology is the core science most critical to this
emerging field, and indeed toxicologists can now readily
purchase nanomaterial samples and carry out their studies
independently. What role is there, then, for materials scien-
tists? It is particularly appropriate for us to deal with this
question on the pages of the journal Carbon.
One role for materials scientists is the full characteriza-
tion of the biologically relevant properties of nanomateri-
als as a standard accompaniment to toxicological studies.
As discussed above, most practical carbon nanomaterial
samples are highly complex, containing the desired struc-
ture (e.g. single-wall nanotubes) mixed with amorphous
1032 R.H. Hurt et al. / Carbon 44 (2006) 1028–1033

or graphitic nanoparticulate byproducts, other nanotube synthesis and purification steps. This manner of close col-
varieties, and metallic catalyst residues of unknown chem- laboration between materials scientists and toxicologists
ical form (metal, oxide, carbide), and in complex associa- has the potential to create a suite of ‘‘green’’ nanomaterials
tion with carbon (within nanotube cavities or as particles with intrinsic low toxicity as an engineered property.
partially or wholly encapsulated by carbon shells). The tox-
icity indicators associated with any given material may just
5. Conclusions
as well be related to these impurities as to the properties of
the nanomaterials in question. The catalyst residues in
We hope that this special issue will contribute in some
vapor-grown carbon nanotubes include Fe, Ni, Co, and
small way to defining and guiding the new field of carbon
less often Y, Mo, and other metals, many of which would
nanotoxicology. We also hope it serves to motivate closer
pose health risks of their own in the form of nanophase
collaboration between toxicologists and carbon materials
powders. Ideally toxicological assays would be accompa-
scientists, as both skill sets are needed to unravel complex
nied by characterization of all the biologically relevant
phenomena at the bio/nano-interface and to advance this
properties of these complex materials—size, shape, surface
field further.
chemistry (hydrophobicity/philicity), and the amount,
We also hope the Carbon journal continues to be a useful
form, and encapsulation state of metals. Some of these
forum for discussions of nanotoxicology issues, and a pre-
properties can be measured by routine techniques (overall
ferred journal for those papers with strong materials science
metal contents by atomic adsorption or emission tech-
components. We specifically propose that future submis-
niques; size and shape by scanning and transmission elec-
sions in this area include the following minimal materials
tron microscopies) but others are not standard (surface
characterization: complete bulk chemical composition (spe-
chemistry, metal form and encapsulation) and require
cifically including metals and heteroatom content >0.1%),
experts. Materials scientists have a key role to play to char-
specific surface area, and detailed descriptions of morphol-
acterizing these materials fully to allow the toxicity assays
ogy (aspect ratios, secondary carbon forms, metals loca-
to be properly interpreted and (hopefully) associated with
tion) by electron microscopy examination of multiple
the correct material features.
fields. Further desirable characterization would include sur-
A second role for materials scientists is in the synthesis
face chemical composition (by energy dispersive or X-ray
of custom or developmental nanomaterials that are not
photoelectron spectroscopies), texture (spatial arrangement
available commercially. These materials may be novel
of graphene layers) and the degree of crystallinity, or perfec-
forms (nanohorns, or double-wall nanotubes) or model
tion of the graphene layers.
nanomaterials whose homogeneity, purity, and monodis-
Finally, we hope that the work presented herein contrib-
persity make them excellent candidates for mechanistic tox-
utes to a realistic societal view of nanomaterial health risks.
icology (thus avoiding the mixture effects described above).
There is little support for either extreme viewpoint—that
Materials scientists must also provide important informa-
nanomaterials pose no health risks, or that nanotechnology
tion regarding post-synthesis stability, storage, and aging
presents extreme risks that warrant cessation of develop-
of materials, which are often in nonequilibrium states
ment activities. Instead we need to continue our commit-
under ambient conditions.
ment to systematic research on toxicity and safety with
The third and perhaps most important role for materials
the goal of understanding and managing health risks for
scientists is the engineering of nanomaterials for low toxic-
a wide range of new material formulations. A worthy goal
ity. Unlike many other toxicants, nanomaterials are fabri-
for toxicologists and materials scientists is the joint devel-
cated, not natural, so there are opportunities to modify
opment of ‘‘green’’ nanomaterial formulations—those
synthesis and purification procedures to avoid the specific
co-optimized for function and minimal health impact. Only
material features that cause toxicity. Can we remove or
this approach has the potential to protect human health
passivate all accessible metal? Can we reduce hydrophobic-
and environment while preserving the potential benefits
ity and thus lipid interactions by surface oxidation (e.g.
that nanotechnology offers to society in the form of new
fullerenes/fullerols) or by surfactant coating? Are there
products and therapies.
critical nano-size ranges that activate macrophages by frus-
trated phagocytosis similar to long asbestos fibers, and can
nanofibers in this size range be re-engineered by length con- Acknowledgments
trol? The necessary modifications may not face large com-
mercial barriers since most nanomaterials are under The editors wish to thank our contributing expert
development rather than in mass production. One can envi- authors for their efforts and timely cooperation, and Prof.
sion development as an iterative process, in which synthesis Peter Thrower for his support for this special issue. Marc
and purification are followed by performance testing and Monthioux specifically acknowledges the preliminary dis-
toxicological testing, and the toxicity information fed back cussion with the Scientific Board of the European Research
to the development team for intelligent modification of the Group ‘‘Science and Application of Carbon Nanotubes’’
 R.H. Hurt et al. / Carbon 44 (2006) 1028–1033 1033

(CNRS GDR-I #2756) from which the need for such a P42 ES013660 and R01 ES03721), EPA (STAR Grant
Special Issue first arose. The North American authors wish RD83171901), and NSF (DMI-0506661) for financial
to thank the NIEHS (Superfund Basic Research Program, support.