Review of health safety aspects of nanotechnologies in food production

Hans Bouwmeester
a,
*
, Susan Dekkers
b
, Maryvon Y. Noordam
a
, Werner I. Hagens
b
, Astrid S. Bulder
a
,
Cees de Heer
b
, Sandra E.C.G. ten Voorde
a
, Susan W.P. Wijnhoven
b
, Hans J.P. Marvin
a
,
Adrinne J.A.M. Sips
b
a
RIKILTInstitute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands
b
Centre for Substances and integrated Risk Assessment (SIR), National Institute of Public Health & The Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands
a r t i c l e i n f o
Article history:
Received 9 June 2008
Available online 6 November 2008
Keywords:
Nanoparticles
Nanotechnology
Food
Risk assessment
Detection
Kinetics
Toxicology
Exposure
Oral
a b s t r a c t
Due to new, previously unknown, properties attributed to engineered nanoparticles many new products
are introduced in the agro-food area. Nanotechnologies cover many aspects, such as disease treatment,
food security, new materials for pathogen detection, packaging materials and delivery systems. As with
most new and evolving technologies, potential benets are emphasized, while little is known on safety of
the application of nanotechnologies in the agro-food sector. This review gives an overview of scientic
issues that need to be addressed with priority in order to improve the risk assessment for nanoparticles
in food. The following research topics are considered to contribute pivotally to risk assessment of nano-
technologies and nanoparticles in food products.
Set a denition for NPs to facilitate regulatory discussions, prioritization of research and exchange of
study results.
Develop analytical tools for the characterization of nanoparticles in complex biological matrices like food.
Establish relevant dose metrics for nanoparticles used for both interpretation of scientic studies as well
as regulatory frameworks.
Search for deviant behavior (kinetics) and novel effects (toxicity) of nanoparticles and assess the validity
of currently used test systems following oral exposure.
Estimate the consumer exposure to nanoparticles.
2008 Elsevier Inc. All rights reserved.
1. Introduction
Due to new, previously unknown, properties attributed to engi-
neered nanoparticles (NP) many new consumer products contain-
ing these NPs have been launched to the market recently.
Application of NPs in electronics, medicine, textiles, defence, food,
agriculture, cosmetics, and other areas are already a reality and
applications are beginning to impact the food associated industries
(Chen et al., 2006a). The potential benets for consumers and pro-
ducers of these new products are widely emphasized. In food and
agricultural systems nanotechnologies cover many aspects, such as
food security, packaging materials, disease treatment, delivery sys-
tems, bioavailability, new tools for molecular and cellular biology
and new materials for pathogen detection (Maynard et al., 2006;
Chen et al., 2006a; Weiss et al., 2006).
Nanotechnology is generally seen as new and fast emerging
eld that involves the manufacture, processing and application
of structures, devices and systems by controlling shape and
size at the nanometer scale. Nanoparticles are dened as; a
discrete entity that has three dimensions of the order of
100 nm or less (SCENIHR, 2007b). It is this small size in com-
bination with the chemical composition and surface structure
that gives NPs its unique features and its huge potential for
applications.
Although potential benecial effects of nanotechnologies (NT)
are generally well described, the potential (eco)toxicological ef-
fects and impacts of NPs have so far received little attention. The
high speed of introduction of NP-based consumer products ob-
served nowadays urges the need to generate a better understand-
ing about the potential negative impacts that NPs may have to
biological systems. Some recent studies have shown that indeed
there are reasons to suspect that NPs may display toxicological ef-
fects on biological systems (Nel et al., 2006; Oberdorster et al.,
2007b; Donaldson and Seaton, 2007). As nanofood related products
are already on the market and uncertainty about potential risks is
0273-2300/$ - see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.yrtph.2008.10.008
* Corresponding author. Fax: +31 317 417717.
E-mail address: hans.bouwmeester@wur.nl (H. Bouwmeester).
Regulatory Toxicology and Pharmacology 53 (2009) 5262
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology
j our nal homepage: www. el sevi er . com/ l ocat e/ yr t ph
large (Morgan, 2005), the need for science-based adaptation of the
regulatory frameworks is high.
This review is focussed on application of engineered NPs and
nanotechnology in the agro-food production chain. The current
knowledge on possible consequences of oral exposure to NPs is dis-
cussed. Emphasis is put on those issues in toxicokinetic and toxico-
dynamic issues of NPs with importance for risk assessment. We
will suggest the most important knowledge gaps in human risk
assessment, that if resolved will bring the risk assessment of NPs
following oral exposure a step forward. It is acknowledged that
over the entire life-cycle of the products NPs will be released into
the environment, possibly resulting in human exposure via that
route, however, environmental contamination is not discussed in
this paper as we have focussed on food related consumer exposure
to NPs.
2. Nanotechnologies currently applied in the food chain
To improve the risk analyses of the application of nanotechnol-
ogy (NT) within food more insight is needed in the availability of
different types of food products containing engineered nanomate-
rials. Therefore, a survey on application of NT in the agro-food pro-
duction chain was performed for which various sources including
published literature and internet web pages were extensively
searched. Most products have been obtained from Google
TM
,
1
the
database of consumer products of the Nanotechnology project
(www.nanotechproject.org) of the Woodrow Wilson International
Center for Scholars, the Global New Products Database of Mintel
(www.gnpd.com), the Nanotechnology Product Directory (www.
nanoshop.com) and the report of nanoforum (Nanotechnology in
agriculture and food; www.nanoforum.org).
The survey clearly demonstrates that NT in the agro-food pro-
duction chain are claimed to be applied throughout all phases of
food production (Table 1).
Inclusion of products into Table 1 is based on labeling informa-
tion on the product as provided by the manufacturer. The claim
that these products contain nanotechnology cannot be veried
from the information presented. This also applies to the informa-
tion on the presence and/or type of NPs in these products. It can
be expected that the claim nanotechnology is wrongfully present
or absent on the label of some products. The latter situation might
even be more critical. Nevertheless, the table as shown gives direc-
tion on the type of products that are likely, on the market, includ-
ing areas of use. Both scientists and regulators should be aware of
this and use this information in the prioritization of research. In
general, NTs can be divided into several classes of applications,
including nanosensors, pesticides, packaging materials, supple-
ments, etc. For each application an indication of the likelihood of
consumer exposure to free nanoparticles (NPs) is given. Free, insol-
uble NPs including agglomerates are thought to be of the highest
concern to the consumer health (SCENIHR, 2007a).
2.1. Food production phase: agricultural production
During primary production nano-formulated agro-chemicals
are employed to increase the efcacy of the agro-chemicals com-
pared to conventional formulations. Only some pesticides contain-
ing nano-sized or nano-formulated agro-chemicals were identied
as available on the market. Residues of these products might be
present in products as consumed. Furthermore, this survey indi-
cated applications of NPs for water and soil cleaning purposes. Car-
ry-over of the used NPs to crops cannot be excluded, resulting in
potential consumer exposure. Examples of used NPs are alumin-
ium oxide, lanthanum particles and nanoscale iron powder in the
process of water purication and/or soil cleaning (see Table 2).
2.2. Food production phase: processing
Nanotechnologies are applied in food production machinery.
Examples of this type of nanotechnology are coatings of machines
or the use of nano-sieves (e.g., to lter out bacteria). While direct
food contact is evident this application of nanotechnology is ex-
pected to have negligible additional safety concerns in comparison
with conventional techniques, as carry-over to food is expected to
be negligible. The type of material (and wear-off as result of the
use) of lters or coatings might requires some attention, but this
is not exclusively related to safety of nanotechnologies.
2.3. Food production phase: preservation and packaging
Indirect contamination of food can be expected when NPs or
nanotechnological devices are incorporated in packaging materials
or storage containers in order to lengthen the storage time while
keeping the products fresh. This type of application is seen as the
most important of nanotechnologies in the food area for the near
future (Chaudhry et al., 2008). Recently the Woodrow Wilson
International Center for Scholars published a review dealing with
both the applications and regulatory (and toxicological) issues re-
lated to NPs incorporated in food packaging materials (WWIS,
2008). NPs are for instance incorporated to increase the barrier
properties of packaging materials (e.g., silicate NPs, nanocompos-
ites, and nano-silver, magnesium- and zinc-oxide). When the NPs
are applied into the food packaging materials, direct contact with
food is only possible following migration of the NPs. The migration
of metals from biodegradable starch/clay nanocomposite lms
used in packaging materials for its gas barrier properties to vegeta-
ble samples, was shown to be minimal (Avella et al., 2005), but
more studies are needed to reach a conclusive statement.
NPs can also be applied as reactive particles in packaging mate-
rials. These so-called nanosensors are designed to respond to envi-
ronmental changes (e.g., temperature or moisture in storage
rooms), degradation products of the food commodities, or contam-
ination by micro-organisms. No data is available on possible
migration of NPs into food using these applications. However, it
is conceivable to assume that the use of active packaging releasing
NPs with antimicrobial functions into the food (e.g., nano-silver
and in rarer cases zinc-oxide NP), will lead to direct consumer
exposure to (free) NPs. Hence, this urges the need for information
on the effects of these NP to human health following chronic expo-
sure. Moreover, attention should be paid to life-cycle analysis
(LCA) and effects on the environment.
2.4. Functionalized foods: engineered NPs as a food additive or
supplement
The current survey revealed nano-formulated food additives or
supplements to be available on the market, e.g., regulatory
peptides from plants, nano-droplets/-clusters and nano-water
(see Table 2). The validity of the claimed effects and formulations
are low. The applicants claim increased bioavailability but valid
studies on relative bioavailability comparing these products with
similar non-nano-formulated products are lacking.
An important class of NPs for application within food are nano-
delivery systems (Letchford and Burt, 2007; Taylor et al., 2005).
Examples (see Table 2) range from novel pesticide formulations
(e.g., increased crop adherence) but also oral delivery systems for
bioactive compounds. The latter is created for targeted delivery
of bioactive compounds and to increase the bioavailability of these
1
using the search terms nano, nanotechnology, nanotubes, nanoparticles,
food, product in varying combinations.
H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262 53
compounds. When used as a food additive or supplement the
delivery systems are commonly build from peptide, carbohydrate
or lipid monomers (Chen et al., 2006b; Graveland-Bikker and de
Kruif, 2006; Mozafari et al., 2006). It is obvious that this application
of NPs might result in unusual high consumer exposure to the en-
closed compounds.
Summary of type of nanoparticles applied in the food produc-
tion chain.
2.5. Conclusions
It is difcult to get a clear overview of products either produced
by means of nanotechnology or products actually containing NPs.
Information on the type NPs applied and their claimed added value
for the product is essential for gaining insight in their potential
hazard and associated risks for humans might vary signicantly.
It is therefore important both for manufacturers as well as for reg-
ulatory bodies and consumers that valid information on relevant
characteristics of NPs as present in the product is at least known
to risk assessors. Consequently, products falsely claiming nano-
technology will be skipped from inventories of agro-food products.
On the other hand, special efforts are still required to expand these
inventories with products containing NPs but not claiming it.
From the information that is available we expect that consumer
exposure to NPs via food is likely to occur. Clearly, instruments
need to be developed for the verication of labeling information
and validation of databases of nano-products.
3. Applied types of nanoparticles in the agro-food production
chain
Current nanotechnology applications in the agro-food produc-
tion chain are focused on the development of nano-sized food
ingredients and additives, delivery systems for bioactive com-
pounds and innovative food packaging (Chaudhry et al., 2008). As
a consequence of their small size NPs show different physico-
chemical properties compared to their respective conventional-
sized materials, likely resulting in different biological interactions.
The physico-chemical properties are thus relevant for the assess-
ment of the possible associated hazards. The main characteristics
of the various types of NPs are introduced in this section.
3.1. Nanoparticles and nano-emulsions
NPs in food may appear in suspension (mostly solid in liquids)
or an emulsion (two liquid phases). Within the agro-food chain,
metal or metal-oxide NPs are applied (e.g., nano-Ag, nano-ZnO,
nano-Cu, nano-TiO
2
). These particles are known to have different
structures and shapes, which can be (homogeneous or heteroge-
neous) spherical, tubular, irregularly (non-spherical) shaped, or
can exist in fused aggregated or agglomerated forms (Fig. 1).
These characteristics are important with respect to potential
risks for health or environment and determine their fate and
behavior in the environment, humans and other organisms.
3.2. Nano-delivery systems
Nano-encapsulation involves the incorporation, absorption or
dispersion of bioactive compounds in/at or on small vesicles with
nano (or submicron) diameters. This type of application is espe-
cially important in food and is discussed here more in-depth. The
incorporated bioactive compounds may be protected against deg-
radation, have improved stability and solubility (e.g., solubilizing
a hydrophilic compound in hydrophobic matrices and vice versa)
and therefore might increase bioavailability and delivery to cells
and tissues (Letchford and Burt, 2007; Taylor et al., 2005). Reduc-
ing the size of the encapsulates into the nanoscale offers opportu-
nities related to prolonged gastrointestinal retention time caused
by bio-adhesive improvements in the mucus covering the intesti-
nal epithelium (Chen et al., 2006b; Medina et al., 2007). Modula-
tions of surface properties (e.g., coatings or bio-molecular ags)
can enable targeted delivery of compounds. The latter eld of
application is however, more developed in relation to biomedical
applications.
Nano-encapsulates may consist of a core composed of one to
several types of compounds surrounded by a wall or barrier (Dins-
Table 1
Summary of applications of nanotechnology in the food production chain.
Chain phase Application Nanotechnology Function Likelihood
of free NPs
available to
consumers
Agricultural production Nanosensors Nanospray on food commodities Binds and colors micro-organisms +
Hand-held devices Detection of contaminants,
mycotoxins and microorganism

Pesticides Nano-emulsions, encapsulates Increased efcacy and water solubility ++

Triggered release nano-encapsulates Triggered (local) release ++
Water purication/
soil cleaning
Filters with nano-pores Pathogen/contaminant removal
NPs Removal/catalysation/oxidation of contaminants /+
Production and
processing of food
Food production Nano-ceramic devices Large reactive surface area /+
Refrigerators, storage
containers, food
preparation equipment
Incorporated nano-sized particles,
mostly silver, occasionally zinc-oxide
Anti-bacterial coating /+
Conservation Food products Nano-sized silver sprays Anti-bacterial action ++
Packaging materials Incorporated sensors Detection of food deterioration.
Monitoring storage conditions /+
Incorporated NPs Increasing barrier properties,
strength of materials
/+
Incorporated active NPs Oxygen scavenging, prevention of growth of pathogens /+
Functional food,
consumption
Supplements/additive Colloidal metal NPs Claimed enhanced desirable uptake of metal ++
Delivery systems Nano-clusters Protecting and (targeted) delivery of content ++
Nano-sized/-clustered food/drinks (nutrients) Claimed enhanced uptake ++
Legend: , no contact of nanotechnology with food; /+, contact with food product during production, but no direct consumer exposure to NPs is expected; +/++, NPs
directly added to consumer products.
54 H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262
more, 2002; Letchford and Burt, 2007) (see Fig. 1 for types of deliv-
ery systems relevant for food applications). These delivery systems
have its roots in the pharmaceutical industry, where often syn-
thetic polymeric nano-encapsulates are employed (Langer, 2003).
For application of nano-encapsulates into food, lipid- or natural-
polymer-based capsules are most often studied or applied (Chen
et al., 2006b). These are introduced in more detail in the sections
below.
3.2.1. Lipid-based nano-delivery systems
The main lipid-based nano-encapsulation systems that can
potentially be used in food and food supplements are nanolipo-
somes, archaeosomes and nanocochleates (see Fig. 2) (Mozafari
et al., 2006). Nanoliposomes are dened as bilayer lipid vesicles
(<30 or 30100 nm vesicles) possessing and maintaining nanomet-
ric size ranges during storage and application. Because of their un-
ique properties, e.g., hydrophilic and hydrophobic regions, they can
entrap, deliver and release both water-soluble and lipid-soluble
material (Mozafari et al., 2006). These may release their contents
into cells upon e.g., encountering specic cellular enzymes, due
to pH or thermo-sensitivity or after antigen-binding when anti-
body-tagged (Taylor et al., 2005).
Archaeosomes, which are liposomes made from Archaeobacte-
ria, are even more thermostable and stress resistant as compared
to normal liposomes. These are therefore considered ideal candi-
dates to protect i.e., antioxidants during food processing (Patel,
2000).
Nanocochleates have a multilayered structure consisting of a
continuous, solid, lipid layer sheet rolled up in a spiral fashion with
little or no internal aqueous space (Mozafari et al., 2006). Nanoc-
ochleates have been used to deliver proteins, peptides and DNA
for vaccine and gene therapy applications. They are resistant to
degradation in the gastrointestinal tract, which makes them ideal
candidates for oral delivery (Zarif, 2003). At this moment, it has
become possible to use liposomes to deliver functional compo-
nents such as nutraceuticals, antimicrobials, and avors to foods
(Hentschel et al., 2008; Hsieh et al., 2002; Laridi et al., 2003; Were
et al., 2003).
The actual use of these delivery systems in food and food sup-
plements is determined by their preparation procedure, which
may involve non-food grade solvents and detergents that might
leave residues in the delivery systems (Mozafari et al., 2006). Li-
pid-based nano-delivery systems were found most frequently in
the current survey.
3.2.2. Polymer-based nano-delivery systems
Nano-encapsulates based on polymers are obtained by the poly-
merization of more than one type of monomer, typically one
hydrophobic and one hydrophilic, so that the resulting molecule
is composed of regions that have opposite afnities for an aqueous
solvent. Natural polymers that are used are albumin (protein), gel-
atin (protein) (Zwiorek et al., 2004), alginate (saccharide), collagen
(protein), chitosan (saccharide) (des Rieux et al., 2006) and the
milk protein a-lactalbumin (Graveland-Bikker and de Kruif,
2006). Protein-based nano-encapsulates are particularly interest-
ing because they are relatively easy to prepare and can form com-
plexes with polysaccharides, lipids or other biopolymers. A wide
variety of nutrients can be incorporated (Chen et al., 2006b). In
addition, to date numerous copolymers have been synthesized
leading to the formation of micelles, nanospheres, polymersomes
and nanocapsules (see Fig. 2; Kabanov, 2006; Letchford and Burt,
2007).
Micelles are characterized by a core-shell architecture in which
the inner core is composed of the hydrophobic regions of the
amphiphilic molecules creating a cargo space for the lipophilic bio-
active compound (Chen et al., 2006b). Nanospheres can be dened
as a solid colloidal particles in which bioactive compounds are dis-
solved, entrapped, encapsulated, chemically bound or adsorbed to
the polymer matrix. However, the central core can become more or
less solid-like depending on the copolymer composition, makes it
difcult to have a clear distinction between micelles and nano-
spheres (Chen et al., 2006b). Nanocapsules and polymersomes
are colloidal-sized, vesicular systems in which the bioactive com-
pound is conned within a cavity surrounded by a polymer mem-
brane or coating. If the core is an oily liquid and the surrounding
polymer a single layer the vesicle is referred to as a nanocapsule;
these system have found utility in delivery of hydrophobic com-
pound. If the core of the vesicle is an aqueous phase and the sur-
rounding coating is a polymer bilayer, the particle is referred to
as a polymersome. These vesicles are analogous to liposomes and
nd utility in delivery of encapsulation of water-soluble com-
pound, but they differ from liposomes in that the external bilayer
is composed of amphiphilic copolymers. Variation in composition,
molecular geometry and relative monomer lengths results in vari-
ous physico-chemical properties and morphologies of the resulting
nano-encapsulates (Letchford and Burt, 2007).
Currently, greater fundamental understanding of polymer
polymer and polymer-nutraceutical interactions at the molecular
level and their impact on functional properties of the delivery sys-
tems is required to ensure design of ideal nutraceutical carriers for
use in the food industry.
4. Knowledge gaps for risk assessment of nanotechnologies in
food
The remaining part of this paper will address the scientic
knowledge gaps that severely hinder the current risk assessment
of application of NPs in general but specically in food and related
products. Discussions on safety issues of nanoparticles (NPs) and
nanotechnological products can almost entirely be brought back
Table 2
Summary of type of nanoparticles applied in the food production chain.
Type of NP Application Function
Metal nanoparticles
(Silver, ZnO)
Food additive/supplement Claimed enhanced
gastrointestinal uptake of
metal
Packaging materials/storage Increase barrier properties
Food preparation devices Clean surface
Refrigerators, storage
containers
Anti-bacterial coating
Water purication/soil
cleaning
Removal/catalysation/
oxidation of contaminants
Sprays Anti-bacterial
Complex nanoscale
structures
Nanosensors in packaging Detection of food
deterioration
Hand-held devices Monitoring storage
conditions detection of
contaminants etc.
Incorporated active
nanoparticles
Mmigration out of
packaging materials
Oxygen scavenging,
prevention of growth of
pathogens
Filters with nano-pores Water purication Removal pathogens,
contaminants
Equal sized emulsions Product design (e.g., taste,
texture)
Nano-sized
nutrients /foods
Food additive/supplement Claimed enhanced uptake
Delivery systems
(nano-encapsulates)
Food additive/supplement Protecting and (targeted)
delivery of content
Pesticide Increased efcacy, water
solubility and crop
adherence, triggered (local)
release
H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262 55
to product related questions (e.g., which specic data are required
in order to come to proper insight into safety of the product) and
fundamental scientic questions (resulting in or based on the
development of new conceptual approaches).
Discussions on data requirements and expected performance of
current assays have demonstrated that it is important to focus the
question on what information is additionally required compared to
dossier requirements for conventional chemicals. Some research
agendas or roadmaps try to circumvent uncertainties which are ac-
cepted in risk assessment of conventional chemicals. For example,
questions like are in vitro tests applicable for NPs should rather
be formulated as are in vitro tests equally applicable for NPs as
for conventional chemicals, as the role of in vitro test results for
chemical in risk assessment is also still subject to many uncertain-
Particle type and shape Description
solid nanoparticles
Spherical or compact particles
compositionally homogeneous
Tubular particles
compositionally homogeneous
Complex non-spherical particles
compositionally homogeneous
Compositionally heterogeneous particles
compositional variation core - surface
Compositionally heterogeneous particles.
distributed compositional variation
Homogeneous aggregates/agglomerates
consisting of a single particle class
Heterogeneous aggregates/agglomerates
consisting of diverse particle types
Nano delivery systems: lipid based
Nanoliposomes /archaeosomes
bilayer lipid vesicles
Micelle
single layer lipid vesicles
Nanocochleates
lipid layer sheet rolled up in spiral fashion
Nano delivery systems: polymer based
Micelle
aggregated copolymers
Nanosphere
aggregated copolymers generating a solid
central core
Nanocapsule / Polymersome
polymer membrane surrounding a central
cavity:
Nanocapsule: oily liquid cavity, single layer
membrane
Polymersome: aqueous cavity, bilayer
membrane (similar to nanoliposome)
Adapted from Maynard and Aitken (2007) and Letchford and Burt (2007)
Fig. 1. Different forms and shapes of nano-structured particles.
56 H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262
ties. Also from a regulatory point of view the question has been
raised what information is additionally required and if the current
regulatory system within the EU is suited to deal with nanotech-
nology. The EUs approach to nanotechnology is safe, integrated
and responsible (Communication from the CommissionTowards
a European strategy for nanotechnology, 2004). To that end the EU
has commissioned its scientic committees and commission ser-
vices (amongst them the European Food Safety Authority in
2008) to perform a scientic and legislative review on the suitabil-
ity of the existing regulation for nanotechnologies. From a number
of regulatory reports it becomes clear that there is currently no
nano-specic regulation in the EU (Chaudhry et al., 2007), or other
countries (Hodge, 2007). The US Food and Drug Administration
was among the rst government agencies around the world having
dened nanotechnologies, but the FDA has no nanotechnology spe-
cic regulations, as it regulates products, not technologies. The
same holds true for other important nanotechnology innovative
worldwide regions like Japan and China (Chau et al., 2007). How-
ever, despite the lack of a nano-specic regulation, any new devel-
opments in nanotechnology will at the moment not be taking place
in a regulatory vacuum (Gergely, 2007). In principle the current EU
regulatory framework also covers nanotechnolgies as concluded by
the Scientic Committee on Emerging and Newly-Identied Health
Risks (SCENIHR, 2007a). In line with this the Health Council of the
Netherlands considered that: the best course of action would be
to modify existing laws and rules as and when developments with-
in the elds of nanoscience and nanotechnologies render such
measures necessary (Health Council Netherlands, 2006). SCENIHR
and others deemed adjustments of legislation, guidelines and guid-
ance documents concerning the testing of nanoparticles (NPs) of
the substance to be necessary (SCENIHR, 2007a). However, such
adaptations can currently not been performed due to the lack of
knowledge on this topic.
For regulatory purposes there is a need for a strict denition of
NPs. As mentioned recently SCENIHR (2007b) postulated a size def-
inition of the order of 100 nm or less. The exact size limit of
100 nm in the present denition of NPs is arbitrary due to lack of
knowledge on the relationship between particle size and kinetics
or toxicological effects. It will be relevant to explore the legal fea-
sibility of avoiding arbitrary size limits and establish a risk assess-
ment driven denition for NPs.
An important way of focusing the discussion is to keep in mind
what will really bring risk assessment to a higher level. In other
words, in an area where such an enormous amount of research
questions can be/are raised, it is essential to dene those questions
that represent the needs to know.
4.1. Physico-chemical characterization of NPs in food
As indicated in previous sections engineered NPs encompass
many forms. They can be made bottom up, through assembling
molecules into NPs, or derived top down by down sizing conven-
tional substances. A complete and accurate characterization of
NPs (Oberdorster et al., 2005a; Powers et al., 2006) is an essential
part of both understanding the possible benets as well as the po-
tential toxicity of NPs in biological systems (The Royal Society and
the Royal Academy of Engineering, 2004). Whereas the character-
ization of chemicals is usually relatively straightforward (e.g., com-
position, purity), characterization of NPs in biological matrices is
more complex from an analytical point of view but also regarding
lack of knowledge on which characteristics that need to be identi-
ed (Powers et al., 2007).
It might however, not always be possible to fully characterize
the NPs. In an attempt to give some guidance on prioritization of
characterization of NPs Oberdorster and coworkers (Oberdorster
et al., 2005a) proposed three criteria:
the context within which a material is being evaluated;
the importance of measuring a specic parameter within that
context;
the feasibility of measuring the parameter within a specic
context.
Because of the complexity of food it is especially important that
the characterization is performed in the matrix containing the NPs
as administered to test systems (or as consumed by the consumer).
It is clear that the functionalities of the NPs (e.g., particle size, size
distribution, potential agglomeration and surface charge), can
change in different biological matrices (Powers et al., 2006),
depending on compounds that are present in the matrix and ther-
modynamic conditions (Borm and Kreyling, 2004). In addition, NP
interactions with the matrix can change as a result of dilution
(Oberdorster et al., 2005a). In practice this means that the appear-
ance of a NP can be expected to change following sample process-
ing (e.g., freezethaw cycles, heating, dilution).
At present there is a vast array of analytical techniques to char-
acterize NPs (Oberdorster et al., 2005a; Powers et al., 2006; Tho-
mas and Sayre, 2005; Tiede et al., 2008), but methods for in situ
characterization of NPs are currently lacking as well as the detec-
tion of nano-delivery systems (Luykx et al., 2008). Therefore prior-
ity research should focus on methods that are able of in situ
detection and characterization of NPs. Ideally with methods that
are relatively easily performed with equipment that is currently
present at laboratories suited for detection of chemicals in food.
The development of routine analytical techniques for the char-
acterization of NPs might be very difcult to achieve. In analogy
with screening approaches used for complex mixtures of chemicals
and products derived from genetically modied organisms in food,
a completely different approach could be to determine the pres-
ence of NPs by means of effect screening (e.g., in vitro). In this ap-
proach the presence of NPs can be detected using developed
assay systems that focus on biomarkers of exposure or effects.
The in vitro assays could be used in a rst tier of detection of NPs
in food. However, suspected samples would have to be further
characterized by means of analytical techniques.
4.2. Dose metrics
Up to now it has not been possible to establish a single dose-
describing parameter that best describes the possible toxicity. It
is likely that mass alone is not the good metric (SCENIHR,
2007a). It has become clear that the size will not be the only critical
factor to consider, the total surface area may also be relevant, as
Fig. 2. Classication of nano-delivery systems. Adapted from Letchford and Burt,
2007.
H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262 57
well as the number of particles per particle size and perhaps other
characteristics. As long as it is not known which metrics should be
used to describe the dose (e.g., particle size distribution, number of
particles, particle charge, total surface) (Hagens et al., 2007; Ober-
dorster et al., 2007a), toxicity tests will have to be analyzed case by
case using different dose-describing parameters. This has led in lit-
erature to a general recommendation that NPs used for (toxicolog-
ical) studies should be characterized as completely as possible
(Oberdorster et al., 2005b; Powers et al., 2006; Thomas and Sayre,
2005). For risk assessors it is important to have access to a clear
description of the analytical methods that were used to determine
the physico-chemical properties of the respective NP, to the (raw)
experimental data and a sound description of the statistical proce-
dure used to analyze the data.
Discussions on denitions and dose metrics are important for
several reasons. First it is important for adaptation of the regula-
tory framework that is in place to ensure the safety of food and
food ingredients. A proper denition and dose metrics will help
researchers to compare study results and will help regulators to
formulate health-based limit values. It will also enable risk asses-
sors to compare and combine exposure and hazard information
and conclude on the likelihood of health risks. Currently pragmatic
denitions are postulated by scientists and regulators. In future,
better understanding of the mechanisms of action and dosere-
sponse relations of NPs with biological systems will support the
formulation of new denitions. This requires priority research.
4.3. Assessment of consumer exposure to engineered NPs
Exposure assessment is dened as the qualitative and/or quan-
titative evaluation of the likely intake of biological, chemical or
physical agents via food as well as exposure from other sources if
relevant (FAO/WHO, 1997).
Basically, the principle of exposure assessment of NPs (via food)
will be comparable to the exposure assessment of conventional
chemicals. Issues like food sampling and variability within com-
posite samples, variation in concentrations between samples and
consumption data on specic food products are not different from
the exposure assessment of conventional chemicals. However,
some aspects do require specic attention.
4.3.1. Detection of engineered NPs in food
The actual determination of the amount and characterization of
the NPs present in the food in its consumable form will be difcult
because of the earlier mentioned lack of methods for the detection
of engineered NPs in food matrices. Special attention is needed for
the determination of nanoscale delivery systems loaded with bio-
active compounds or bioactive compounds themselves in nano-
scale formulations. For nanoscale delivery systems, both the
amount of bioactive compounds within the capsules as well as
the free form in the food matrix has to be determined, because
these factors determine the bioavailability.
The presence of NP in a food matrix might result in increased
bioavailability of other substances (both nutrients and contami-
nants) normally present in food. For example, food containing
NPs with actively charged surfaces can absorb biomolecules as
they pass through the GI tract (Govers et al., 1994). These so-called
Trojan horses (Lomer et al., 2002) may transport toxins into the
intestinal mucosa, resulting in changed exposure of the cellular lin-
ing of the intestine (Borm and Kreyling, 2004). It is advisable to
consider such nutritional implications when NP are present in
food.
It will not always be feasible to measure chemicals and NPs in
the food matrix as consumed. If chemicals are measured at an early
stage of the food chain or at the site of production, effects of pro-
cessing should be considered in exposure assessment (Kroes
et al., 2002). However, processing factors as used for determination
of exposure assessment of conventional chemicals (e.g., pesticides
[Joint FAO/WHO meeting on pesticide residues]), are not (yet)
available for NPs. These might be even more important for NPs be-
cause the functionalities of the NPs (e.g., particle size, size distribu-
tion, potential agglomeration and surface charge) can change in
different biological matrices (Powers et al., 2006), depending on
compounds that are present in the matrix and thermodynamic
conditions (Borm and Kreyling, 2004).An alternative approach next
to the search for NPs by actual measuring food products could be to
rely on information from producers. This information could be use-
ful for an initial exposure assessment, but also to prioritize those
NPs that are most frequently used at the moment in order to start
predicting health effects. However, it can be questioned if reliable
information on what NPs are being produced and in which prod-
ucts they are applied will become available for risk assessors. No-
vel governance approaches are needed to address this basic lack of
data in order to make this information available to risk assessors.
4.3.2. Consumption data
Various sources of consumption data are currently utilized
ranging from standardized food baskets used in pre-marketing
authorizations to household or individual dietary surveys used in
post-marketing studies (Kroes et al., 2002). There are no additional
requirements on consumption data to perform an exposure assess-
ment of NPs. However, the use of NPs as additive or in specic food
products (novel foods or food supplements) might require addi-
tional data on consumption of these specic food products, be-
cause this information is generally lacking in the regular
consumption databases. This is of course a general problem for
exposure assessment of food products, but more prominent in
evaluating NPs because these particles are likely to be incorporated
more frequently in food supplements.
4.3.3. Exposure assessment
The last step in performing exposure assessment is the integra-
tion of food consumption and amount of chemicals or NPs present
in food. Usually one of the following three approaches is applied
for integration of data: (1) point estimated; (2) simple distribu-
tions; (3) probabilistic analyses (Kroes et al., 2002). In the end con-
sumer exposure is usually compared to a toxicological reference
value (e.g., tolerable daily intake, acceptable daily intake or acute
reference dose) or to a nutritional reference value (e.g., recom-
mended daily intake or upper safe intake levels). These reference
values are currently lacking for NPs and need to be established.
4.4. Toxicokinetics
Available experimental data so far indicate that the characteris-
tics of NPs (e.g., size, surface charge, functionalizedgroups) are likely
to inuence the absorption, metabolism, distribution and excretion
(ADME) (Ballou et al., 2004; des Rieux et al., 2006; Florence, 2005;
Jani et al., 1990; Roszek et al., 2005; Singhet al., 2006) of NPs present
in food. However, not much is known on the relationship between
these physico-chemical characteristics and the behavior of NPs in
the body. The kinetics of NPs following various exposure routes have
recentlybe reviewedby Hagens andcolleagues (Hagens et al., 2007).
In this section, current knowledge and focus on ADME characteris-
tics following oral exposure only is discussed, with emphasis on
peculiarities of special agro-food related NPs, like nano-encapsu-
lates. Subsequently knowledge gaps are identied that currently
hinder the risk assessment of NPs in food.
4.4.1. Gastrointestinal absorption
Uptake of NPs (or particles in general) in the gastrointestinal
tract depends on diffusion and accessibility through mucus, initial
58 H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262
contact with the gut epithelium and various uptake and transloca-
tion processes. There seems to be a tendency that that smaller par-
ticles are able to diffuse faster through the mucus layer than larger
particles. The diffusion rate also depends on the charge of the par-
ticle; anionic particles have been shown to reach the epithelial sur-
face, whereas cationic particles were trapped in the mucus
(Szentkuti, 1997) The mucus layer can thus be considered the rst
barrier NPs have to pass before entering the body.
The gastrointestinal epithelium represents the second barrier. A
rst possible route of passage is between the cells (e.g., paracellu-
lar route). There is a body of evidence that indicates that the intes-
tinal epithelium is permeable to large proteins and polypeptides.
Cells of the gastrointestinal epithelium are tightly connected to
each other by means of tight junctions. The permeability of the
tight junctions can be modulated for instance by specic polymers.
These polymers can act as expanders for the tight junctions there-
by introducing a port of entry for many particles including toxins,
bacteria and immunogens (Salamat-Miller and Johnston, 2005).
Therefore, passage of nano-encapsulates (and NPs in general) via
tight junctions cannot be excluded before hand, they should be
considered by risk assessors.
Another uptake route is the transcellular route. This route de-
scribes the process by which particles are taken up at the apical
side of the intestinal epithelium (by endocytocis), are transported
through the M-cells in the Peyers Patches and/or the enterocytes
and subsequently released at the basolateral side of the intestinal
epithelium (Aprahamian et al., 1987; Jani et al., 1990; Hillery
et al., 1994; Carr et al., 1996; Hoet et al., 2004; Florence, 2005;
des Rieux et al., 2006). Specic NP characteristics, such as particle
size, the surface charge of particles, attachment of ligands or coat-
ing with surfactants, may inuence the transcellular uptake of
particles in the gastrointestinal tract (Hoet et al., 2004;
Russell-Jones et al., 1999). If the encapsulates or NPs are pro-
tected/prevented from local degradation or metabolism due to
the modication, they will enter both the blood and lymphoid cir-
culation intact (Gabor et al., 2004) and can be further distributed
in the body.
4.4.2. Distribution, metabolism and excretion
Once nano-encapsulates or NPs pass the gastrointestinal epithe-
lium and end up in the blood circulation, they can interact with
various blood-components (i.e., plasma-proteins, coagulation fac-
tors, platelets and red and white blood cells) depending on the sur-
face chemistry of the particle (Nemmar et al., 2002). This
interaction may have a substantial effect on the distribution and
excretion of the NP (Dobrovolskaia, 2007). For instance, the hydro-
phobic surfaces of nanospheres are highly susceptible to opsoniza-
tion and clearance by the reticulo-endothelial system, resulting in
sequestering of the particles within organs such as the liver and
spleen (Letchford and Burt, 2007).
A widespread distribution of NPs inside the (animal) body has
been identied. In addition, the smallest NPs revealed a more di-
verse distribution to e.g., brain, bone marrow, spleen and liver
compared to the larger counterparts. (Jani et al., 1990; Hillery
et al., 1994; Hillyer and Albrecht, 2001; Hoet et al., 2004; De Jong
et al., 2008). Crucial for the risk assessor is the potential passage of
natural barriers like the cellular barriers, bloodbrain barrier, pla-
cental barrier and the bloodmilk barrier.
There are indications that cellular barriers like membranes may
not constitute an obstacle for some NPs. Several different NPs (gold
and titanium-oxide) have been identied inside human red blood
cells (Rothen-Rutishauser et al., 2006). Interestingly, this cellular
uptake of NPs did not involve endocytosis or phagocytosis (Geiser
et al., 2005) since erythrocytes do not have phagocytotic receptors
(Rothen-Rutishauser et al., 2006). This suggests that some NPs are
able to cross the cell membrane by processes other than phagocy-
tosis and endocytosis. The permeability of the bloodbrain barrier
is highly restricted to molecules which are either lipophillic, ac-
tively transported or are small soluble molecules (<500 Da). This
barrier may therefore represent a strict defense mechanism from
blood borne particle exposure that limits the distribution of NPs
to the brain. However, evidence exists that distribution to the brain
might occur for some NPs (Hillyer and Albrecht, 2001; De Jong
et al., 2008). Literature on the effectiveness of the placental barrier
in relation to NPs is scarce as is information on passage of the
bloodmilk barrier.
One of the primary aims of applying nano-encapsulates next to
increased bioavailability, is to protect the loaded compounds from
liver metabolism and excretion by bile. At this moment, little is
known on the prospect and capacity of the liver to metabolize
and excrete NPs. However, a size-dependent excretion of NPs has
been suggested via bile. In rats, intravenously administrated poly-
styrene NPs were taken up by the liver and subsequently excreted
in the bile (Ogawara et al., 1999). The smaller particles (50 nm)
were phagocytosed by Kupffer cells partly and partly taken up by
the hepatocytes, whereas polystyrene microparticles (500 nm)
were taken up predominantly by the non-parenchymal cells (Kupf-
fercells and endothelial cells) (Ogawara et al., 1999). Although it is
unlikely that the inert NP itself, such as gold and silver particles,
fullerenes and carbon nanotubes, can be metabolized effectively
by enzymes in the body, there are some indications that functional
groups added to the inert nanoparticle are sensitive for metabo-
lism. For instance, the protein cap of a functionalized quantum
dot could be cleaved by proteases (Hardman, 2006). Also the
metallic core of quantum dots (and other metal-oxides) could be
bound by metallothionein and excreted via liver and kidney (Coyle
et al., 2002).
4.4.3. Importance of kinetics for the risk assessment of NPs
Although there are some data on kinetics of NPs following
oral exposure, there is also a clear need for fundamental re-
search on the absorption, distribution, metabolism and excretion
(ADME) of NPs to elucidate the driving forces and mechanisms
behind these processes. This knowledge would greatly facilitate
the extrapolation and modeling approaches. Moreover, especially
important for the risk assessment, this kinetic knowledge will
guide the search for target tissues where the NPs could exert
a toxic effect. Due to the potential impact of toxicological effects
special attention needs to be paid to the possibility that certain
NP can cross the barriers (e.g., gastrointestinal barrier, cellular
barrier, bloodbrain barrier, placenta barrier, bloodmilk
barrier).
4.5. Toxicity of NPs
As the application of NPs in consumer products is a phenome-
non of recent years, knowledge on the potential toxicity of NPs is
limited, but rapidly growing. Several studies suggest that NPs
may have a deviating toxicity prole when compared to their con-
ventional chemical analogs (Donaldson et al., 2001; Nel et al.,
2006; Oberdorster et al., 2005a). The most important question for
risk assessment is the sensitivity and validity of currently existing
test systems. It is generally thought that the standard battery will
sufce, but special attention is needed for specic endpoints (Bal-
bus et al., 2007). These will be discussed in the following para-
graphs, with a focus on toxic effects following oral exposure and
if not available also from other exposure routes.
While the literature on toxicity of NPs is rapidly growing it
should be kept in mind that results are often obtained for only
one type and size of NPs. Furthermore test animals are generally
exposed to high concentrations under articial conditions. This
limits the usefulness of obtained data for risk assessment. Extrap-
H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262 59
olation from one type of NPs to another or from one size to another
is on the basis of present knowledge still impossible. At this mo-
ment there is too few data to determine which type of effects are
to be expected for which type of NP.
4.5.1. In vitro toxicity
While results of in vivo studies might provide relevant informa-
tion for the hazard identication of the studied NP, caution has to
be exercised when extrapolating results or mechanisms for the
hazard characterization and subsequent human risk assessment
(Oberdorster et al., 2007b). The search for mechanistic explana-
tions is only now starting. For this in vitro models can be very
important. Numerous in vitro studies using various NPs are being
published. Many NPs can trigger the release of reactive oxygen spe-
cies and cause oxidative stress and subsequent inammation by
means of interaction with the reticulo-endothelial system (Nel
et al., 2006; Donaldson et al., 2007; Donaldson and Seaton, 2007;
Oberdorster et al., 2007b).
Cells are generally exposed under articial conditions, and a se-
lected number of endpoints are studies. Special attention is re-
quired for cellular interactions in order to better understand and
predict cellular toxicity and the validity of currently used in vitro
models (e.g., for the gastrointestinal absorption). While in vitro
studies might be useful in a tiered screening approach it is recom-
mended to develop validated assays and assess sub-lethal changes
for example by means of proling studies (Balbus et al., 2007; Lew-
inski et al., 2008).
4.5.2. Acute toxicity
Acute, subacute and subchronic toxicity following oral exposure
have been investigated in rodents for several different NPs (e.g.,
copper, selenium, zinc and zinc-oxide and titanium dioxide NPs).
The results of the available oral toxicity studies indicate that,
depending on the particle size, coating and chemical composition
of the NPs, acute toxicity at high doses may occur (Jia et al.,
2005; Zhang et al., 2005; Chen et al., 2006c; Wang et al., 2006;
Wang et al., 2007; Wang et al., 2008).
4.5.3. Long term toxicity
No information on the toxicity after chronic or acute low dose
oral exposure is currently available. Information from toxicity
studies with other routes of exposure indicate that several sys-
temic effects on different organ systems may occur after long term
exposure to NPs, including the immune, inammatory and cardio-
vascular system. Effects on the immune and inammatory systems
may include oxidative stress and/or activation of pro-inammatory
cytokines in the lungs, liver, heart and brain. Effects on the cardio-
vascular system may include pro-thrombotic effects and adverse
effects on the cardiac function (acute myocardial infarction and ad-
verse effects on the heart rate). Furthermore, genotoxicity, and
possible carcinogenesis and teratogenicity may occur, but no data
on these latter endpoints are available yet.
For the following endpoints research is needed.
4.5.3.1. Neurotoxicity. There is evidence from ADME studies that NP
may pass the bloodbrain barrier following systemic availability of
NPs (Hillyer and Albrecht, 2001; Borm et al., 2006; Silva, 2007). It is
not clear if this is a generic effect of all NPs or only a subgroup. This
emphasizes the need of kinetic studies, and increased attention of
toxicologists to neurotoxicity in their search for potential effects in
target tissues. Toxic effects due to the presence (or even accumula-
tion) of NPs in the brain have not been studied so far, but risk
assessors should be aware of possible neurological effects when
assessing toxicology experiments. Possibly, current guideline tests
will need to be adapted to render these tests more sensitive for
neurotoxic effects of NPs.
4.5.3.2. Reprotoxicity. Transfer of NPs across the placenta cannot be
excluded (including excretion via breast milk (the blood milk bar-
rier), which could lead to embryotoxicity as a result of exposure to
NPs (Fujimoto et al., 2005). Data addressing the distribution of NPs
to the reproductive cells is currently unavailable. In addition, no
clear data showing the distribution of NPs in the fetus are available
(Tran et al., 2005). This leads to the recommendation that repro-
toxicity needs to be considered carefully when there is evidence
for NP passage of the placenta.
4.5.3.3. Mutagenicity. Intracellular NPs do not appear to be mem-
brane bound and might have direct access to the intracellular pro-
teins, organelles and DNA of the cell, which might imply enhanced
toxic potential (Geiser et al., 2005; Kabanov, 2006). However, pos-
sible interactions of NPs with cell components are poorly under-
stood and validated assays with meaningful endpoints for
genotoxicity are needed (SCENIHR, 2007a).
4.5.3.4. Allergenicity (or sensitization). Even for conventional chem-
icals little is known on the induction of food allergy and the type of
exposure required to induce such responses. In the case of NPs this
becomes extra prominent for two reasons. First of all it is the pos-
sible adjuvant activity of NPs that introduces additional uncer-
tainty (Dobrovolskaia, 2007). And secondly, because of the
actively charged surfaces of NPs it can absorb biomolecules as they
pass through the GI tract (Govers et al., 1994). This might result in
changed exposure of the cellular lining of the intestine (Borm and
Kreyling, 2004). In addition the surface properties (e.g., coatings)
are important determinants for the active uptake of encapsulates,
but might also be a reason for concern. For example lectins used
for coatings are highly immunogenic, can be cytotoxic or induce
inammatory responses and gastrointestinal irritation (Gabor
et al., 2004; des Rieux et al., 2006;Hong et al., 2006; Kabanov,
2006).
4.6. Setting health-based guidance values
The last step in the hazard characterization may involve the set-
ting of health-based guidance values such as acceptable or tolera-
ble daily intakes. These are generally based on animal toxicity
studies. Reference points (e.g., the no-observed-adverse-effect-le-
vel or benchmark-dose-level) for the critical effect of a substance
form the starting point of the risk assessment. This is a general ap-
proach for all substances either being in a conventional form or at a
nano-sized scale. For NPs, using only mass as the dose metric for
health-based guidance values will not be sufcient. This is exem-
plied by the ongoing discussions on dose metrics as mentioned
before.
Furthermore, guidance values are based on toxicological studies
performed with a substance (or NP) with a given bioavailability. Of
special concern is the increased bioavailability of bioactive com-
pounds loaded in nano-delivery systems (or nano-sized bioactive
compounds).
Extrapolation of a health-based guidance value between formu-
lations with different bioavailability might therefore not be possi-
ble. Ultimately, this might require setting of separate health-based
guidance values depending on the formulation (e.g. nano-formu-
lated or not).
5. Consequences for risk assessment of NPs
As indicated, engineered nanoparticles (NPs) have novel or dis-
tinct properties that are attributed to a combination of their small
size, physiochemical properties, chemical composition and surface
structure (Nel et al., 2006). It is the added functionality of NPs that
makes the engineered NPs different from natural small sized parti-
60 H. Bouwmeester et al. / Regulatory Toxicology and Pharmacology 53 (2009) 5262
cles, but also from their conventional counterparts. Because of this,
unexpected toxicological effects might occur, that need to be ad-
dressed by risk assessors.
To improve the existing risk assessment methodology, good
governance and regulatory framework of the application of nano-
technology within food, the following issues should be addressed:
Establish a risk assessment driven denition for NPs to facilitate
regulatory discussions, prioritization of research and exchange
of study results.
Develop analytical tools for the detection and characterisation of
NPs in food matrices to estimate exposure, kinetics and toxico-
logical doseresponse relationships.
Establish a dose metrics to facilitate the interpretation of scien-
tic studies as well as regulatory frameworks.
Investigate kinetics (especially oral bioavailability) and (oral)
toxicity of the different types of NPs, with special attention to
those parts of the body that are normally protected by barriers
like the bloodbrain barrier and placenta.
Assess the validity if currently used toxicological assays for
detecting the effects caused by NPs.
Identify products containing nanoparticles that are on the mar-
ket (or being developed), including the type of NPs are (or will
be) used and the estimated consumption of these products.
Elaborate regulatory/policy approaches for the disclosure of
information on NPs being produced including the products they
are incorporated in.
This request for extra information is not to be considered solely
as a request for additional newly developed studies. It can also im-
ply that conventional study approaches need to be redesigned. The
use of novel technologies (e.g., proling approaches) and the more
frequent use of in vitro approaches for risk assessment need to be
studied and used in parallel with conventional techniques.
Logically, present safety and risk assessment requirements are
based on knowledge gathered for conventional chemicals. Also in
the risk assessment of conventional chemicals assumptions have
to be made because of knowledge gaps. However, uncertainties
in these assumptions for example extrapolations from one com-
pound to another are approached on a sound basis of general
knowledge. For NPs such a basis is lacking, moreover uncertainties
in the safety assessment are expected to be larger (Morgan, 2005).
To reduce the uncertainties it is important that currently devel-
oped quality-controlled databases are also suitable to obtain infor-
mation on the occurrence of NPs in food. Such a database can be
used to identify certain patterns of behavior and/or toxicity of
NPs, such as for example toxicological size-thresholds.
At this stage of (lack of) knowledge of nanotoxicology it is
unavoidable that risk assessors need as much information as pos-
sible about NPs and their appearance and behavior in biological
matrices and organisms. Over time it will be possible to evaluate
the data and look for the set of pivotal information. This clearly
needs close collaborations between NP (and products) developers,
risk assessors, regulators and researchers.
Conict of Interest statements
The authors declare that there are no conicts of interest.
Acknowledgments
Dr. W. de Jong (RIVM), Prof. Dr. I.M.C.M. Rietjens (Wageningen
University) and Dr. A.C.M. Peijnenburg (RIKILTWUR) are thanked
for their contribution to discussions that formed the basis of this
paper. The research described in this paper was commissioned
and funded by the Dutch and Consumer Products Safety Authority,
Ofce for Risk Assessment.
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