Authors:

1 2
Dr Barry Park and Dr Patricia Martin
1
Oxonica, 7 Begbroke Science Park, Sandy Lane, Yarnton, Oxon, OX5 1PF, UK
2
Product Assessment and Regulatory Compliance, 37 Sydney Road, West
Midlands, B64 5BA, UK

Correspondence to:
Dr Barry Park
gbarrypark@googlemail.com
Dr Patricia Martin
parc.pamartin@talktalk.net

Nanotechnology: applying the 3Rs to risk assessment

Available at www.nc3rs.org.uk

Abstract

Nanotechnology is the science used to manipulate atoms Keywords nanotechnology, cerium oxide, 3Rs, risk

and molecules on a nanometre scale to create new assessment, nanoparticle, in vitro screening assays, inhalation
toxicity, airborne particulate matter (PM10), EnviroxTM.
materials, structures and devices which often have novel
properties. The range of nanoparticulate material is Introduction
extensive. However there are currently few publicly
All living organisms and man-made materials are
available data relating to the potential short- and long-
comprised of atoms and molecules. The way in which
term effects of some nanomaterials on human health
these atoms and molecules are brought together
and the environment.
determines their form and complexity and also endows
Scientists, legislators, industry and public interest groups the organisms and materials with particular properties.
agree that there is a need to assess the safety of the
One nanometre is one billionth [10-9] of a metre, about
nanoparticulate forms of chemicals to identify any
one hundred thousandth of the width of a single human
potential risks at all stages from manufacture, through
hair. Nanotechnology is the science used to manipulate
downstream use to ultimate disposal. The risk
atoms and molecules on a nanometre scale to create
assessment for novel nanoproducts may therefore offer
new materials, structures and devices which have
new scientific and regulatory challenges and this in turn
varying and often novel properties and can be thought of
may have consequences for animal use and the 3Rs.
as “engineering on a very small scale” (1).
This article describes the scientifically robust risk
Nanoparticulate material is defined as solid particles or
assessment of EnviroxTM, a nanoparticulate cerium oxide
fibres in which at least one dimension is less than 100
diesel fuel additive. The risk characterisation took a 3Rs
nanometres (nm). The range of nanoparticulate material
approach using in vitro hazard data combined with
is extensive and includes various metals and their oxides,
exposure monitoring data and computer modelling
carbon black (similar to soot), and novel carbon entities
studies.
such as nanotubes and fullerenes – structures similar to
The authors believe that this is the first time a 3Rs graphite. The resulting applications of these
approach has been taken in the risk assessment of a nanomaterials are wide-ranging with the potential to
nanoparticulate material and as such may be viewed as bring benefits to many areas as diverse as information
a model for evaluating new materials or new forms of and communication technologies, medicine and personal
existing materials.

NC3Rs #17 Nanotechnology: applying the 3Rs June 2009 1

care products, materials engineering, electronics, and
energy sources.
Currently, there are few publicly available data relating
to the potential short- and long-term effects of some
nanomaterials on human health and the environment.
Companies wishing to place their nanoproducts on the
market are required to conduct an appropriate risk
assessment based upon robust safety testing. The risk
assessment for novel nanoproducts may therefore offer
both new scientific and regulatory challenges – this in
turn may have consequences for both animal use and
the 3Rs.
One nanoproduct which has been developed and
undergone toxicological testing applying the 3Rs is a
new environmentally-friendly, nanoparticulate
TM
additive called Envirox , manufactured by Oxonica, a
company which specialises in the design and application
of nanomaterials (see www.oxonica.com).
EnviroxTM, consisting of nanoparticulate cerium oxide
(CeO2; 2% w/v in a hydrocarbon carrier solution; see
Figures 1 and 2), is added to diesel fuel (5mg/L) where
it acts as a catalyst in the vehicle combustion chamber.
Studies have shown (2) that adding EnviroxTM to diesel
fuel decreased fuel consumption by 5–8%, and reduced
the harmful exhaust emissions of combustion-derived
nanoparticles (CDNP) and unburnt hydrocarbons by up to
15%. Decreasing fuel consumption by using EnviroxTM as
a diesel additive means that carbon dioxide (CO2)
emissions would potentially be reduced by 3 tonnes of
TM
CO2 for every tonne of Envirox -containing (additised)
fuel used.
Figure 1. Nanoparticulate cerium oxide.
NC3Rs #17 Nanotechnology: applying the 3Rs June 2009
Figure 2. Non-nanoparticulate cerium oxide.
Numerous epidemiological studies have demonstrated
an association between exposure to CDNP and adverse
health effects (3-7); it therefore follows that there could
fuel be significant benefits to health from new products
which can reduce harmful environmental emissions.
However, because EnviroxTM contains nanoparticulate
cerium oxide, it was also necessary to demonstrate that
there was no increase in the intrinsic toxicity of any
CDNP in the emissions from the exhaust of vehicles
using diesel fuel containing EnviroxTM compared with the
existing and known adverse effects associated with
emissions from unadditised diesel fuel.
Oxonica, which has a company policy of not testing any
of its products on live animals unless this is mandated by
law, conducted an extensive safety testing programme
using an in vitro approach that addressed those
endpoints of concern described for the hazard
identification of new chemicals in the current European
Union (EU) legislation (8).
Current regulatory controls in Europe and North America
(Notification of New Substances for the EU; Toxic
Substances Control Act [TSCA] for USA) do not require
specific safety testing and risk assessment of chemicals
based on particle size. However, a Royal Society
publication on nanoscience and nanotechnologies (9)
recommended that additional hazard characterisation of
nanoparticulate products is necessary since they may
have different properties and a robust safety assessment
could not be made based upon the properties of the
non-nanoparticulate form of a particular chemical alone.
2
Safety testing overview – the 3Rs approach
Oxonica’s safety testing strategy employed the classic
risk calculation paradigm:
Risk = ƒ Hazard x Exposure
The hazard assessment was initially informed by a
literature search for all available data in the public
domain, which indicated that non-nanoparticulate cerium
oxide was of low toxicity (10). This information was used
to develop a comprehensive testing programme to
assess the toxicity of nanoparticulate cerium oxide, as
well as CDNP emitted from engines. The test materials –
both nanoparticulate and non-nanoparticulate cerium
oxide had undergone a series of chemical analyses so
that any potential hazardous effects might be attributed
to the test material itself and not to any ‘impurity’.
The safety testing programme involved a range of
approaches and was carried out in a number of
laboratories with the appropriate expertise (see Figure
3). In vitro tissue culture and biochemical assays were
used to investigate the capacity, if any, of inhaled
nanoparticulate cerium oxide to induce biological signs of
oxidative stress and inflammation, both of which are
linked mechanistically to adverse health effects (11-14).
Environmental exposure assessment assays used data
from computer modelling studies as well as in situ
monitoring of ambient air samples from roadside sites in
the United Kingdom (UK) where vehicles using EnviroxTM-
additised diesel fuel operate.
The final risk characterisation involved comparing the
information gained from all the in vitro safety assays
(the hazard characterisation) with that of the exposure
assessment, i.e. the actual measured environmental data
from the monitoring studies and the computer
modelling.
As far as the authors are aware, this is the first time that
this multi-disciplinary approach employing the 3Rs has
been used for the risk assessment of a commercial
nanoparticulate product (15). This test case could
therefore provide a model for safety testing other similar
metal oxide nanomaterials.

Literature
Review

Toxicity Lung Inflammatory Exposure Exposure

Studies Slice Response Monitoring & Modelling
Assays Assays and RTLF
RTLF Assays Assays

Hazard characterisation Exposure assessment

X

Risk characterisation

Figure 3. Schematic representation of the in vitro safety testing approach used for EnviroxTM. (RTLF = Respiratory Tract Lining Fluid).

NC3Rs #17 Nanotechnology: applying the 3Rs June 2009 3

A. Hazard assessment
1. Toxicity studies
Tests to investigate the potential intrinsic toxicity of
cerium oxide, in both nanoparticulate and non-
nanoparticulate forms were conducted and included:
(i) an initial in vitro screening assay to assess the
potential for any cytotoxic effects using mouse L929
fibroblast cells in culture (16)
(ii) evaluating localised acute toxicity indicated by skin
irritation using a reconstructed human epidermal tissue
model (Epiderm; 17-19)
(iii) assessing any potential to cause adverse effects on
the genetic material of cells using a bacterial cell assay
(the Ames test) involving genetically altered strains of
Salmonella typhimurium. The assay acts as a crude
assessment of the carcinogenic potential of chemical
compounds (20-22).
2. Lung slice assays
Slices of rat lung (23-25) were used to examine the
effects of exposure to a nanoparticulate cerium oxide
aerosol and subsequently, to emissions from vehicles
using diesel either with or without EnviroxTM. A range of
biological markers was examined to assess potential
lung damage, including changes in cell viability
(indicated by adenosine triphosphate [ATP]
intracellular glutathione [GSH] levels; see Glossary), pro-
inflammatory reactions (indicated by tumour necrosis
factor-alpha [TNF-α] induction) and antioxidant enzyme
activity (indicated by total glutathione peroxidase [GPx],
manganese-dependent superoxide dismutase [SOD] and
catalase levels; see Glossary).
The lung slice assay uses a precision-cut slice of lung
tissue which is maintained in a culture solution, which
can be challenged with test compounds
nanoparticulate cerium oxide or exhaust emissions from
diesel containing EnviroxTM) applied to the solution. The
tissue slice maintains its original cell-cell interactions to
allow both biochemical and pathological investigations of
the test compounds to be carried out, and give an
NC3Rs #17 Nanotechnology: applying the 3Rs June 2009
indication of the potential effects of exposure to EnviroxTM
emissions. Using slices of lung tissue possibly represents
a ‘worst case’ scenario in terms of the potential effects
following exposure since the lung tissue is not whole, i.e.
the organ is ‘damaged’ and may have increased pro-
inflammatory reactions and antioxidant enzyme activity
due to this process.
3. The respiratory tract lining fluid (RTLF) model
The respiratory tract epithelial lining fluid (RTLF) is the
first interface encountered by any atmospheric pollutants
inhaled into the lungs. As such, inhaled particles are a
potential source of oxidative stress to respiratory tract
epithelial cells. Human RTLF contains a wide range of
antioxidants including reduced glutathione [GSH], uric
acid and ascorbic acid as well as antioxidant enzymes
(e.g. SOD and catalase), which essentially form a first
defence against environmental toxins. The antioxidants
in the lining fluid react with the inhaled particles,
reducing their toxicity, thereby protecting underlying
lung epithelial cells from oxidative injury (26).
The synthetic RTLF in vitro model contains the major
water-soluble antioxidants present at the air-water
interface, such as ascorbate, urate and glutathione, at
concentrations known to occur physiologically. It has
been shown that these antioxidants are oxidised in the
presence of redox active metals, such as iron and
copper, as well as by quinone compounds associated
and
with ambient particulate matter (PM10; see Glossary and
Figure 4); and is designed to reproduce the events
occurring in vivo at the air-lung interface following
inhalation of particulate matter (27,28). The model was
used to examine any potentially harmful oxidative
changes or cytotoxicity induced by exposure to
nanoparticulate cerium oxide associated with other
airborne particulate matter. Effects on the lung tissue
were quantified based on the rate of depletion of
(e.g. ascorbate or glutathione which indicate antioxidant
activity.
The test materials included nanoparticulate cerium oxide,
a range of ‘standard’ particle controls including residual
oil fly ash (ROFA), carbon black and ambient diesel
particles, and oxides of two redox active metals, copper
4
and iron. Samples of ambient PM were collected from
sites alongside routes used by vehicles employing
EnviroxTM-additised diesel fuel. The ambient PM samples
were collected routinely at three sites, two in London
and one in Newcastle, both prior to and after
introduction of EnviroxTM into the diesel fuel used by a
national bus company.
Cytotoxicity was investigated by incubating a human
lung epithelial cell line (A549 cells) with a range of
concentrations of nanoparticulate cerium oxide for 12
hours. A spectrophotometric assay using a coloured
formazan product (see Glossary) was used as a rapid test
of cell viability. Changes in colour (and therefore light
absorbance) were measured and found to correlate with
the number of viable cells when compared to a standard
concentration-response curve.
Figure 4. Proposed pathogenic sequence – from exposure to effects – indicating the institutions where the studies were conducted.
(Figure reproduced with kind permission of Professor Ken Donaldson, Professor of Respiratory Toxicology, MRC/University of Edinburgh
Centre for Inflammation Research, ELEGI Colt Laboratory, Queen's Medical Research Institute).
NC3Rs #17 Nanotechnology: applying the 3Rs June 2009
4. Inflammatory response
The final step in the hazard characterisation programme
involved examining the effects, if any, of cerium oxide
(both nanoparticulate and non-nanoparticulate) and soot
samples (following combustion of diesel alone and
diesel additised with EnviroxTM) on the inflammatory
process - the key process underlying adverse health
effects following exposure to inhaled particles (see
Figure 4; 29).
Inflammation is mediated by a variety of soluble factors,
including a group of secreted molecules known as
cytokines (see Glossary). Interleukin-8 (IL-8) is a cytokine
which draws inflammatory cells to the site of 'damage'
in a cell and is routinely used as a general measure of
the inflammatory process (30). Cultured A549 human
lung epithelial cells were again used to measure IL-8 and
lactate dehydrogenase (LDH) release from the cell
cytoplasm as biochemical indicators of cell membrane
damage and cytotoxicity.
5
B. Environmental exposure assessment
Environmental monitoring and computer modelling
studies were both used to estimate the potential for
airborne exposure to EnviroxTM as a result of adding it to
diesel fuel and any subsequent release as a composite in
vehicle exhaust emissions as measured with PM10
particles.
1. Monitoring
Information from the London Air Quality Network (LAQN)
was used to identify two relevant monitoring locations
which had archived samples of air quality and
monitoring data from before the introduction of
EnviroxTM-additised diesel fuel for public service vehicles
(i.e. buses). A third site in Newcastle was added to the
study, since this had a much larger number of buses
fuelled with EnviroxTM which passed within 100 metres of
the monitoring location daily. Ambient PM10 samples
were obtained from all three sites. The RTLF model was
used to assess the effect of exposure due to inhalation of
the ambient PM10 samples compared with 0 and 4 hour
particle-free antioxidant controls, as well as positive
(ROFA; 10-80nm diameter) and negative control particles
(inert carbon black: 50nm diameter).
Note: For further information on the monitoring
methods, filter archive and particle extraction, analysis
and measurement see Annex 1, and references 27 and
28.
2. Computer modelling
Two computer models – COPERT (COmputer Programme
to calculate Emissions from Road Traffic) and TRENDS
(Transport and Environmental Database System; 31,32) –
were used to calculate cerium oxide emissions from both
passenger cars and also light- and heavy-duty vehicles
using EnviroxTM–containing diesel following average
mileage over a full year. A ‘best’ and ‘worst’ case
approach was employed, where ‘best’ equated to
vehicles fitted with diesel particulate filters which
retained 100% of cerium oxide emissions and ‘worst’
assumed that all cerium oxide in additised diesel is
present in the atmospheric emissions. The two computer
models were also used to estimate cerium oxide
emissions and potential for soil contamination in two
NC3Rs #17 Nanotechnology: applying the 3Rs June 2009
high traffic scenarios – a highway (see Glossary) or a
street canyon (see Glossary) – over a range of varying
driving conditions.
3. Risk characterisation
Risk characterisation compares the hazard
characterisation test data, specifically the dose-response
relationship in terms of the No Observed Effect Level
(NOEL; see Glossary), with the potential environmental
exposure, in order to assess the safety of the test
material – in this case nanoparticulate cerium oxide. The
Multiple Path Particle Dosimetry model (MPPD2; see
Glossary; 33) was used to assess the risk of pulmonary
inflammation due to the long-term (chronic) inhalation
of nanoparticulate cerium oxide and to simulate the build
up of cerium oxide in the lungs over a 20 year period at
levels that might be found in the environment if
EnviroxTM was widely used as a diesel fuel additive.
Results
A. Hazard assessment
1. Toxicity studies
The general toxicity studies found no difference in
intrinsic toxicity between nanoparticulate cerium oxide
and non-nanoparticulate cerium oxide (34). No
cytotoxicity was observed in mouse L929 fibroblast cells.
The human skin model (Epiderm) did not indicate any
potential for the tested materials to be in vivo skin
irritants. The bacterial gene cell mutation assay (Ames
test) was negative at dose levels up to the maximum of
5000µg per plate, thereby suggesting that there is no
potential for genotoxic carcinogenic response following
exposure to the tested materials.
2. Lung slice assays
There was no effect on lung slice viability following a 3
hour exposure to a range of different concentrations
(2%, 10% and 20% by mass) of nano-cerium oxide
aerosol (as measured by ATP and intracellular GSH),
antioxidant enzyme activity (indicated by SOD, catalase1
and total GPx levels), or pro-inflammatory factors (TNF-
α) – all indicators of oxidative stress (24). Catalase
activity was slightly increased but this was not
associated with a concomitant loss in cell viability. In
6
addition, vehicle exhaust emissions from EnviroxTM
additised diesel did not cause any increase in adverse
biological effects when compared with those from
exposure to emissions from unadditised diesel fuel.
3. The respiratory tract lining fluid (RTLF) model
Results of the RTLF assay found that there was no
increase in antioxidant activity (based on ascorbate
depletion) of PM10 samples from the Newcastle
monitoring site, even though there was a statistically
significant increase in the cerium oxide content of
ambient PM10 compared with air samples prior to the use
TM
of Envirox -additised fuel. Test samples from the two
London monitoring sites (15,35) showed no increase in
the cerium oxide content of ambient PM10 compared
with air samples taken before EnviroxTM use and no
increase in oxidative activity (based on ascorbate
depletion). The formazan colorimetric assay with A549
cells did not show any cytotoxicity following exposure to
nanoparticulate cerium oxide at concentrations up to
1000µM.
4. Inflammatory response
IL-8 levels (indicating inflammatory response) did not
increase following exposure of A549 cells to either
nanoparticulate or non-nanoparticulate cerium oxide at a
range of concentrations (6.25-100µg/ml). Soot from
cerium oxide-additised diesel produced an IL-8 response
that was no different from that produced by soot from
non-additised diesel fuel. Neither soot test material
caused stimulation of IL-8 release above that of the
control. There was also no detectable cytotoxicity
observed, based on the LDH leakage assay (15) in
human A549 lung epithelial cells when tested at
concentrations between 6.25-100µg/ml.
B. Environmental exposure assessment
1. Monitoring
Nanoparticulate-cerium oxide content in ambient PM10
samples from three UK locations over time (measured in
3
pg/m ambient air), was not associated with oxidative
activity using the RTLF model. However, the PM10
antioxidant depletion activity that was observed during
these monitoring studies was associated with copper
(Cu) and iron (Fe), both redox active metals, present in
NC3Rs #17 Nanotechnology: applying the 3Rs June 2009
the samples (at µg/m3 levels; 15,35). This was based on
correlating the oxidative activity of the PM10 samples
against the concentrations of these metal oxides found
in the PM10 samples
2. Computer modelling
Modelling data for a typical highway and along a street
canyon showed that no major soil contamination would
be expected and that soil levels of cerium oxide would
be similar to those found naturally. According to
information in the public domain (10) the amount of
naturally occurring cerium in the earth’s crust is 20–60
ppm (see Glossary). The worst case exposure figure for
nanoparticulate cerium oxide in air from this study was
0.08µg/m3 in a street canyon (15).
3. Risk characterisation
Utilising the MPPD2 model, it was concluded that it is
highly unlikely that nanoparticulate cerium oxide inhaled
from the atmosphere would elicit pulmonary
inflammation in humans, at the exposure levels
estimated based on use of EnviroxTM as a diesel fuel
additive (15). It should be noted that this assessment
assumes that the in vitro exposure and toxicity data can
be accurately projected to the in vivo situation. This
conclusion is based on the following calculations:
The maximum inhaled dose estimate of 30µg was
calculated based on the exposure modelling assessments
in a worst-case, long-term cerium oxide exposure
situation and was converted into surface area units (cm2)
using the specific surface area value for cerium oxide.
This surface area dose was then ‘normalised’ by
calculating the surface area of the proximal alveolar
region of the human lung (the area potentially affected
by particulates) to obtain a surface area dose per unit of
affected lung area; this was calculated to be 27cm2/cm2.
This figure is the highest NOEL – i.e. the highest dose
tested that resulted in no changes compared with
controls (see Glossary), and was calculated using the in
vitro data.
Using the exposure modelling assessment and the above
approach, the maximum internal dose of nanoparticulate
cerium oxide that could potentially be inhaled from the
atmosphere was calculated to be 4x10-7cm2/cm2. The
7
estimated internal lung dose in humans is therefore Based on the finding that the environmental
7 TM
7x10 times lower than the highest dose resulting in no concentrations of Envirox do not elicit pulmonary
effects in vitro (NOEL). Similarly, the highest theoretical inflammation in rat lung tissue or cause adverse effects
internal dose calculated from the computer modelling in mouse fibroblasts or human lung epithelial cells, the
-4 2 2 4
study is 1x10 cm /cm , which is 21x10 times lower study concluded that – under the conditions pertaining to
than the in vitro NOEL and also negligible in comparison. the use of diesel fuel containing EnviroxTM –
environmental exposure to nanoparticulate cerium oxide,
Conclusions
does not pose a human health risk.
The risk characterisation for EnviroxTM described in this
Applying the 3Rs is consonant with sound science and it
article did not involve tests on live animals at any point.
is hoped that the steps involved in safety testing and risk
Instead the risk assessment applied the 3Rs and
assessment outlined in this article may also be useful for
encompassed novel approaches to safety evaluation.
future risk assessments of other nanoparticulate
The robust testing strategy was underpinned by both materials.
literature data on the toxicology of cerium oxide
(nanoparticulate and non-nanoparticulate), and
epidemiological studies on the association between Acknowledgements
particulate matter and adverse health effects. Testing We would like to acknowledge the following
also involved in vitro assays using relevant human and organisations for their expertise: Harlan Laboratories
animal tissues and cell lines, as well as models which (formerly SafePharm Laboratories); Derby (intrinsic
are widely used in academia, industry, government and general toxicity tests); INSERM, France (studies using
other research organisations, and explored the perfused rat lung slices); University of Edinburgh, (lung
underlying mechanisms by which potential biological cytotoxicity assessments and IL-8 release); Kings College
effects might be induced in humans following London, KCL (RTLF and environmental monitoring
nanoparticulate material inhalation. information); University of Thessaloniki, Greece (for the
Toxicity studies compared the intrinsic toxicity potential environmental modelling data developed under contract
of nanoparticulate cerium oxide with non- for the European Commission).
nanoparticulate cerium oxide and demonstrated no
difference between the chemicals with respect to Glossary
cytotoxicity, skin irritation and mutagenicity. Other ATP: Adenosine triphosphate.
hazard characterisation studies examined the toxicity of
Ames test: A bacterial gene cell mutation assay used as a
vehicle engine emissions following combustion of pre-screen to indicate potential for carcinogenic activity of
EnviroxTM-additised diesel and targeted the primary route certain chemical compounds.

of exposure for humans – inhalation. Studies using rat CDNP: Combustion derived nanoparticles – one form of
lung slices, and a range of biological endpoints for cell which is found in the exhaust emissions from vehicles.

viability, pro-inflammatory reaction and antioxidant Catalase: An enzyme which rapidly converts hydrogen
peroxide – a potentially harmful by-product of many
activity, clearly demonstrated that adding
normal metabolic processes in the cell – into harmless
nanoparticulate cerium oxide to fuel did not increase the oxygen and water molecules.
toxicity of the emissions compared with non-additised Epiderm: The commercial name of an in vitro product used
diesel. Nanoparticulate cerium oxide did not cause an to assess the skin irritation potential of applied materials.

increase in oxidative activity, cytotoxicity or pro-
inflammatory changes in human lung epithelial cells,
despite a significant increase in cerium content of
atmospheric PM10.
Formazan dyes: Are artificial coloured (chromogenic)
products formed when tetrazolium salts undergo a
chemical reaction and are reduced by dehydrogenase and
reductase enzymes. The dyes, which only stain living cells,
can have a variety of colours from dark blue to deep red or
orange depending on the original tetrazolium salt substrate
used for the reaction.

NC3Rs #17 Nanotechnology: applying the 3Rs June 2009 8

 Annex 1 – Methods
Glossary
Environmental PM sampling method: Ambient PM10
Glutathione peroxidase (GPx): An enzyme in the body with
the primary function of cell defence; acts to prevent oxidative samples (from all three sites) were obtained using a
damage. Tapered Element Oscillating Microbalance (TEOM)
GSH: Glutathione (reduced form) is an antioxidant which is a connected to a sampling pump which passed heated air
defence material found in cells; it is converted to oxidised
(500C) over the microbalance containing a pre-weighed
glutathione (GSSH) in the presence of free radicals.
filter. Filters were collected over a specified time period.
Highway: Typically a road with four lanes and an overall
width of about 32m and no buildings on either side.
Particulates were extracted from the filter matrix and
analysed for metal content and separate aliquots of each
ICP-AES: Inductively coupled plasma atomic emission
spectroscopy; an analytical tool. particle suspension were assessed for oxidative activity
of ambient PM10 using the RTLF model after a 4 hour
Interleukin-8 (IL-8): Is a protein (a chemokine) secreted by
cells in response to pro-inflammatory effects typically to incubation at 370C. Assays were done in triplicate and
promote immune cells to the site. compared against 0 and 4 hour particle-free antioxidant
L929 cells: Mouse fibroblast derived cell line used to test controls as well as positive (ROFA; 10-80nm diameter)
cytotoxicity of various solid test materials.
and negative control particles (inert carbon black: 50nm
Lactate dehydrogenase (LDH): Tests for lactate diameter). Metal analyses of ambient PM10 samples was
dehydrogenase are typically used to indicate adverse effects
i.e. LDH is released following cell membrane disruption.
undertaken via acid extraction of the filters followed by
using Inductively Coupled Plasma Atomic Emission
MPPD2 model: The Multiple Path Particle Dosimetry model
allows calculation of PM deposition fractions and exposure Spectroscopy (ICP-AES) for copper, zinc and iron and ICP-
doses for humans. Mass Spectrometry (ICP-MS) for cerium. Further
Monodisperse: All particles have approximately uniform size, information on the monitoring methods, filter archive
shape and mass. and particle extractions and measurement can be found
No Observed Effect Level (NOEL): The largest amount of a in references 24 and 36.
substance which causes no adverse changes in morphology,
functional capacity, growth, development, or life span of the References
test organisms compared with those observed in control
organisms under the same conditions. See
www.inchem.org/documents/ehc/ehc/ehc104.htm. 1. Institute of Nanotechnology (2007) What is
nanotechnology? See www.nano.org.uk/whatis.
PPM: Parts per million.

Particulate matter (PM10): Particulate matter with an 2. Oxonica Technical information. See
aerodynamic diameter less than 10µm; this particulate size
www.oxonica.com/energy/energy_home.
can be inhaled by humans.

Respiratory tract lung fluid (RTLF) model: Synthetic model
constituted to resemble the human respiratory tract lining fluid
which contains physiological concentrations of a range of
antioxidants.
3. Dockery DW, Luttmann-Gibson H, Rich DQ, Link MS,
Mittleman MA, Gold DR et al. (2005) Association of
air pollution with increased incidence of ventricular

Street Canyon: Typically an urban city street bordered on tachyarrhythmias recorded by implanted
both sides by multi-story buildings which causes the wind to cardioverter defibrillators 2. Environmental Health
blow in turbulent eddies which partially trap airborne
pollutants within the ‘canyon’ preventing their dispersion and Perspectives 113: 670-674.
often leading to high airborne concentrations of the pollutants.
4. Ibald-Mulli A, Wichmann HE, Kreyling W, Peters A
Superoxide dismutase (SOD): An enzyme which catalyses
(2002) Epidemiological evidence on health effects of
the breakdown of the superoxide radical (O2−) into oxygen
and hydrogen; an important antioxidant defence mechanism ultrafine particles. Journal of Aerosol Medicine 15:
found in cells.
189-201.
TNF-alpha (TNF-α): Tumour necrosis factor alpha is a
cytokine involved in immune cell regulation and systemic 5. Peters A, von Klot S, Heier M, Trentinaglia I,
inflammatory processes.
Hormann A, Wichmann HE et al. (2004) Exposure to

NC3Rs #17 Nanotechnology: applying the 3Rs June 2009 9

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