Nanotechnology in food production:
a potential risk or a risky potential?
On the potential of emulsification techniques to
generate nanostructures for the use in food and the
risk and safety considerations
Aidan Noorman
Utrecht University
December 2008
Supervisors:
Raymond Pieters, PhD
Institute for Risk Assessment Sciences
Utrecht University, Utrecht, the Netherlands
Nico van Belzen, PhD
International Life Science Institute, Brussels, Belgium
Nanotechnology in food production: a potential risk or a risky potential? │ i
Nanotechnology in food production:
A potential risk or a risky potential?
On the potential of emulsification techniques to generate nanostructures
for the use in food and the risk and safety considerations
Master thesis by:
Aidan Noorman
MSc program Toxicology and Environmental Health
Faculty of Biomedical Sciences, Utrecht University, Utrecht, the Netherlands
e-mail: G.A.Noorman@students.uu.nl
Nanotechnology in food production: a potential risk or a risky potential? │ ii
Abstract
The purpose of this report was to investigate the safety assessment of emulsification techniques and the variety
of emulsion-based nanostructures (EBNS) that can be obtained and subsequently used in the food industry.
Environmental issues will not be addressed.
The number of nanotechnology applications is increasing and it is expected that the food industry will be the
newest field in which nanotechnology will be applied. A conservative size definition of 500 nm was chosen for
risk assessment purposes. Cells are capable of taking up nanostructures of up to 500 nm in size and
nanostructures can be engineered with certain properties that could mimic effects of smaller sized nanostructures.
Devices to produce emulsions and EBNS are already used in the food industry (homogenisers) while other
methods are still being developed which are more efficient (low-energy methods). A large variety of
nanostructures can be obtained with emulsification techniques such as simple emulsions, lipid nanostructures,
solid nanostructures etc.
Very little is known about the effects of nanostructures on the gastrointestinal tract. Nanostructures in the body
mostly accumulate in the liver and kidneys where the effects are the most pronounced. Surface properties are
very important as they can determine the fate, function and possible risks of nanostructures. More research is
needed in which other non-metallic and non-carbon-based nanostructures are (orally) tested.
Recommendations for a food nanostructure-specific risk assessment include the goal of the nanostructure
(intended or unintended exposure), consideration of physicochemical properties relevant to food nanostructures
(e.g. solubility), the history of safe use and a minimum amount of testing to ensure safety. A risk assessment
paradigm is proposed which incorporates these recommendations.
Nanotechnology in food production: a potential risk or a risky potential? │ iii
Table of contents
1. INTRODUCTION ----------------------------------------------------------------------- 1
1.1 Size definitions ........................................................................................... 2
2. EMULSIONS AND APPLICATIONS----------------------------------------------- 4
2.1 Nano fabrication ........................................................................................ 4
2.2 Emulsions as template............................................................................... 4
2.2.1 High-energy methods ................................................................................ 5
2.2.2 Low-energy methods ................................................................................. 6
2.3 Applications of emulsion based nanostructures ..................................... 7
2.3.1 Traditional emulsions ............................................................................... 7
2.3.2 Nano-emulsions as carriers: nanocapsules .............................................. 8
2.3.3 Nanoliposomes.......................................................................................... 8
2.3.4 Solid nanoparticles ................................................................................... 9
2.3.5 Colloidosomes........................................................................................... 9
2.4 Developments and trends........................................................................ 10
3. SAFETY ASSESSMENT OF NANOSTRUCTURES IN FOOD ------------- 11
3.1 Potential risks .......................................................................................... 11
3.1.1 Importance of preparation methods........................................................ 11
3.1.2 Nanostructures in the gastrointestinal tract ........................................... 11
3.1.3 Biodistribution ........................................................................................ 12
3.1.4 Effects of surface properties ................................................................... 12
3.1.5 Effects on the immune system ................................................................. 14
3.1.3 Other routes of exposure ........................................................................ 15
3.2 A food-specific risk assessment .............................................................. 15
4. CONCLUSIONS ----------------------------------------------------------------------- 21
5. REFERENCES ------------------------------------------------------------------------- 22
Introduction │ 1
1. Introduction
Nanotechnology and nanoscience deal with the development, application and production of materials with sizes
less than 100 nm. Materials with such dimensions posses unique properties that are not exhibited by larger
structures of the same material [Oberdörster et al., 2005b]. Research and development into nanotechnology is an
increasing business. In the US, the budget of the National Nanotechnology Initiative (NNI) has increased from
$464 million in 2001 to $1.4 billion in 2007 and is estimated to increase to $3 billion in 2009 [NNI, 2008]. In
Europe, funding is mostly provided by national governments and totals around €1 billion. Only a very small
percentage of the funding is invested in safety assessment, life cycle analysis, monitoring or tracking systems
[Powell et al., 2008]. For now, many scientists and companies are more interested in the technological and
economical benefits that nanotechnology may hold for the future [Renn and Roco, 2006].
Development of nanotechnology occurs in many different fields and disciplines such as electronics, cosmetics,
pharmaceutics, agriculture etc. One of the newer fields where nanotechnology is making an entrance is food. It is
known that a large part of the general population is already exposed to nanotechnology, via cosmetics [Thomas
et al., 2006] and now an even larger part may be exposed through the food chain. In February 2008 an online
survey by the Woodrow Wilson International Centre for Scholars (WWICS) identified over 600 products that
contain some form of nanomaterial [WWICS, 2008]. Of the 600 products 69 where found to be related with food,
this includes mostly supplements, storage devices and kitchen equipment but this is expected to be more, since
this is only an online survey and no supermarket products have been tested.
There are four major areas in food production to which nanotechnology can be applied: food processing,
packaging and storage, food safety and functional foods (food with additional heath-promoting functions). Food
packaging and storage is so far the most promising area for nanotechnology and some of the innovations include
oxygen barriers [Erlat et al., 2001; Sorrentino et al., 2007], antimicrobial films [Rhim et al., 2006] and thermal
buffering techniques for perishable foods [Johnston et al., 2008]. Food safety mostly includes detection and
elimination of foodborne pathogens and toxins [Branen et al., 2007; Yang et al., 2008]. Nanotechnology in food
processing and functional foods is raising both interests and concerns because this involves direct application
into the food itself. The use of nanostructures in food can affect the appearance, smell and taste of a product
[Graveland-Bikker and de Kruif, 2006; Rodríguez Patino et al., 2008] or reduce costs and/or environmental
burden [Vincze and Vatai, 2004].
Some of the techniques and principles to generate nanostructures are actually not new. An example is the
generation of nanofibers. One way to make nanofibers is through a process called electrospinning. The
technology for this was already invented in the early 1900s for polymer filaments, and in the 1990s the technique
was refined to produce nanofibers [Li and Xia, 2004]. With these fibres it was possible to produce nanofilters
[Ramakrishna et al., 2006] that eventually spawned the technology for membrane emulsification [Charcosset et
al., 2004; Kampers, 2007].
This report will focus on nanostructures that can be obtained through emulsification. Emulsification is the
mixing of two immiscible fluids and is a process that has been used for a very long time in food processing.
Emulsification is mainly used in dairy products and beverages but nowadays it is also possible to create
nanostructures with it. The capabilities of older techniques and principles to generate nano-emulsions and
emulsion-based nanostructures (EBNS) will be investigated for their use in food. As EBNS can have various
compositions and characteristics [Daufin et al., 2001] different risks have been associated with nanostructures.
Therefore, the emphasis of this report will be on the safety assessment of EBNS. The first half of this report will
Introduction │ 2
provide a short elaboration of the methods to generate nanostructures and their possible impact on food. The
second half of this report is on the risk assessment aspect for nanotechnology in food. A risk assessment was
recently developed by the Scientific Committee on Emerging and Newly-Identified Health Risks (SCENIHR)
that applies to nanotechnology in general. Critical views and recommendations will be provided based on
possibilities of EBNS in order to make the tiered risk assessment more specific to the food industry.
1.1 Size definitions
In the scientific community there is a general tendency to define structures with at least one dimension that
measures less than 100 nm as a nanostructure. For toxicological and safety assessment purposes this definition
may not be sufficient. Cells are capable of taking up particles of 300 nm, this include modes not involving
receptors. Lately, a process has been described that can even take up 500 nm particles [Garnett and Kallinteri,
2006]. Also, the ‘engineered’ aspect of >100 nm engineered nanostructures can give similar capabilities as <100
nm nanostructures. So, in order to set a strict definition of the term ‘nano’ that can be used throughout the whole
scientific community, a consensus will have to be reached between toxicologists and scientists dealing with
structural engineering of nanostructures.
Making agreements on a general definition of the term nano is not easy, even in a specific field. There are
several reasons for this. One is the fact that the size definition of the word nano differs between scientists.
Emulsions are categorised based on their droplet sizes. From the commonly used definition of nano, we would
expect that ‘nano-emulsions’ are <100 nm in their droplet sizes. Several scientists do indeed use this definition
[Meleson et al., 2004] but there are also other scientists that employ a different size (range) such as 20-200 nm
[Solans et al., 2005; Tadros et al., 2004] or even 100-1000 nm [Windhab et al., 2005].
In the field of emulsion research the label ‘nano-emulsion’ where we assume approaches the general definition
of ‘nano’ (<100 nm), there are multiple synonyms such as mini-emulsion, submicron emulsions, ultrafine
emulsion and microemulsions [Solans et al., 2005]. Lately, the term nano-emulsion has been gaining popularity
due to the ‘nano’-hype in society and the scientific community. Thus, not only is the very definition of the term
nano being debated, also the terminology has not been standardised.
In light of all these aspects it will be hard to define a ‘true’ nano-emulsion. A search in the literature for the term
‘nano-emulsion’ and several of its synonyms yielded nanostructures with average sizes that were above the 100
nm. Considering this result, the uptake capacity of cells and the fact that nanostructures can be engineered to
have certain properties, it would seem appropriate to also include the structures that are 100 nm and larger in size.
The next step would be to determine a proper upper limit. From a toxicological and risk assessment point of
view, uptake sizes of 300 nm or even 500 nm are an important aspect to consider. Furthermore, a distinction is
often made between smaller (<500 nm) and larger (>500 nm) micron particles [Oberdörster et al., 2005b; VegaVilla et al., 2008] while others do consider 500 nm as the upper limit [Anton et al., 2008]. For now, a
conservative upper limit of 500 nm will be used for food risk assessment purposes.
The idea of implementing nanotechnology in food is new, and so, we can expect that the number of literature
sources with respect to engineered nanostructures in food to be limited. In this report we will describe EBNS less
than 500 nm in size that may be suitable for food applications. For the assessments of potential dangers both in
vitro and in vivo studies of EBNS (<500 nm) are used. However, other non-EBNS studies are used in order to
describe effects of various properties. Also, pharmaceutical studies with nano-emulsion formulations will be
considered. Pharmaceutical research on nanostructures has been going on longer than in food research, including
Introduction │ 3
oral formulations. For now, pharmaceutical studies may be the biggest source of information on engineered
nanostructures in the human body.
Emulsions and applications │ 4
2. Emulsions and applications
Emulsion is the mixture of two immiscible liquids, where one liquid is dispersed (dispersed phase, e.g. in
droplets) in the other (continuous phase). A nano-emulsion is an emulsion with droplet sizes that range up to
several hundred nm. For reasons discussed in the introduction the upper limit of 500 nm will be used. Nanoemulsions can present characteristics and properties which depend on the composition and preparation method.
In this chapter a general principle of nanostructure generation will be explained. Next, several methods to
generate nanostructures will be described. An elaboration will be given on the kind of nano-emulsions and
EBNS that can be obtained by these methods. The chapter will be concluded with a critical view on what aspects
of nanostructure generation and developments can be considered new.
2.1 Nano fabrication
In general, the production of nanostructures can be accomplished in two ways: via a bottom-up approach or a
top-down approach. The top-down approach is the down-sizing of macrostructures. This is usually accomplished
by applying a force in the form of shear and impact. Devices capable of this are milling devices and
homogenisers. Advantage of this method is that the amount of force, and thus the size can be easily controlled.
Top-down approaches are currently the most widely used methods in large scale industries [Sanguansri and
Augustin, 2006].
The bottom-up approach deals with the manipulation and/or controlled (self-)assembly of individual molecules
to create nanostructures. This method of production is not new in nature: in the body proteins and enzymes are
formed by amino acids which themselves are controlled by ribosomes. Examples of man-made assembled
nanostructures are the various carbon nanostructures such as nanotubes and fullerenes. This process is more
tedious and more susceptible to failure than the top-down approach due to the heavy dependence on the
physicochemical properties and the environment in which the reactions take place. The advantage, however, is
that there is a far greater chemical variety [Seeman and Belcher, 2002]. It is expected that the use of this
approach will further increase as nanotechnology matures [Sanguansri and Augustin, 2006].
2.2 Emulsions as template
The creation of nano-sized structures can be accomplished in many
different ways. One of them is by basing it on an emulsion-template.
Emulsions are a versatile system that can be used for many different
products, including standard food emulsions, formation of
nanospheres or powders, nanobeads for processing or flavour
enhancement,
etc.
[Vladisavljević
and
Williams,
2005].
Characteristics of an emulsion depend for a great deal on the size of
the droplets. For example emulsions with large droplets (>500 nm)
have a milky appearance whereas emulsions with small droplets
(<200 nm) are clear (see fig. 1).
There are many different kinds of emulsions and many different
ways to produce them. Here, an explanation is given of some of the
methods that are capable of producing nano-emulsions. Several of
Figure 1 Example of the difference between a
35 nm nano-emulsion (A) and a normal 1000
nm emulsion (B). Modified from [Solans et al.,
2005].
these methods are already in use by the food industry but explanations are also given of some new methods that
Emulsions and applications │ 5
can serve as a potential alternative to the current ones. Emulsions used in the explanations here are the standard
emulsion, consisting of water and oil.
2.2.1 High-energy methods
The methods of producing nano-emulsions can be roughly divided into two categories: high-energy and lowenergy methods. High-energy methods are now being used in the industry. The name is derived from the fact
that the energy that is applied to break up the droplets is very high but this method may not always be efficient
[Tadros et al., 2004]. Two high-energy methods will be explained that are currently used in the food industry.
2.2.1.1 High pressure homogenization
High pressure homogenisation is a technique that employs shear stresses to break up droplets. The fluid with
(preformed) emulsions is forced through microchannels where extreme shear stresses are generated due to the
sudden restriction of the flow under high pressure combined with acceleration, compression, turbulence, impact
and eventually rapid pressure drop. Figure 2 shows several different types of high pressure homogenisation
systems which all work with the same principles [Schultz et al., 2004]. Microfluidizer systems are the most
effective in producing nano-emulsions [Meleson et al., 2004; Pinnamaneni et al., 2003].
Figure 2 Different types of high pressure homogenisation
systems. Modified from [Schultz et al., 2004].
2.2.1.3 Ultrasound
Emulsification through ultrasound is also an effective way of producing nano-emulsions. Ultrasonication
generates high and low pressure waves which lead to the formation of vacuum bubbles in both fluids (i.e. water
and oil), which is also known as cavitation. These bubbles implode, causing extreme conditions with
temperatures of up to 5000 K and pressures of over 1000 atmosphere (≈101 MPa) [Patist and Bates, 2008]. The
implosions produce shockwaves, which in turn result in levels of highly localised turbulence. The heat, pressure
and turbulence are effective means of breaking up and dispersing large droplets in a liquid medium.
Emulsions and applications │ 6
Ultrasonication is a technology that has been used on a laboratory scale for many years. But recently (<5 years),
ultrasound devices have been developed for industrial uses.
2.2.2 Low-energy methods
Beside the more traditional high-energy methods for nano-emulsions, there are also methods that do not require
large amounts of applied energy, the so-called low energy methods. Three of such methods have been described
in the literature and will be briefly explained here as potential alternative to the high energy methods. Detailed
descriptions of the processes governing the reactions are beyond the scope of this report and only basic
explanations will be given.
2.2.2.1 The solvent displacement method
Solvent displacement utilises the physicochemical properties of components to form nano-emulsions. Solvent
displacement consists of three components. First, a solute (e.g. oil) is dissolved in a solvent (e.g. ethanol) and
afterwards a third component (e.g. water) is added. The third component is miscible with the solvent but not with
the solute. During the addition of the third component small droplets are formed containing the solute/solvent
mix. Spontaneous emulsification can occur when diffusion of the third component into the solute/solvent droplet
causes the solvent to be displaced from the droplet, leaving behind small nuclei of the solute. Since the third
component is immiscible with the solute nano-sized emulsions are rapidly formed.
This method of producing emulsions was termed the ‘ouzo effect’ because the original authors compared the
processes with the addition of water to ouzo, a common alcoholic beverage in Greece [Vitale and Katz, 2003].
The advantage of solvent displacement is that it does not require surfactants/emulsifiers. Also, the whole process
occurs rapidly and over the entire volume which makes it interesting for industrial applications.
2.2.2.2 Phase inversion temperature
Phase inversion temperature (PIT) utilises the physicochemical properties of the surfactant to obtain nanoemulsions or EBNS. It is based on the interruption of the transition of an oil-in-water (O/W) phase into a waterin-oil (W/O) phase. For instance, when the temperature rises, the surfactant is modified in such a way that it
exhibits equal affinity for both fluids (e.g. water and oil). This is when the O/W phase starts to go over into a
W/O phase. During the transition between phases nano-emulsions are formed due to low interfacial tension.
However, coalescence is extremely fast which will eventually result in a coarse emulsion [Solans et al., 2005].
To prevent coalescence, the temperature is suddenly and rapidly lowered in order to preserve the current state
(i.e. the transition state where nano-emulsions have formed). Due to the temperature change, the surfactant
exhibits normal affinity for one of the fluids [Anton et al., 2008].
2.2.2.3 Membrane emulsion
Membrane emulsification does not involve physicochemical properties of the components, but instead uses a
membrane with nanopores. A liquid-to-be-dispersed is slowly forced through the membrane. Mechanical
agitation, usually in the form of a flow, is used to detach the newly formed droplets at the membrane pores.
Membrane emulsification requires less energy than high pressure homogenisation and ultrasound, making it an
interesting option for large scale use. A disadvantage is the low flux of the liquid-to-be-dispersed through the
membrane, which means a longer production time [Charcosset et al., 2004; Vladisavljević and Williams, 2005].
Emulsions and applications │ 7
2.3 Applications of emulsion based nanostructures
The methods described above are capable of producing a wide variety of nanostructures. Aside from the usual
emulsions, various other nanostructures can be obtained. Figure 3 shows several nanostructures that can be
obtained based on an emulsion template. In this section an explanation is given of the general features of the
various EBNS and how they can be obtained.
Figure 3 Different types of emulsions and EBNS obtainable through various
methods of emulsification. Modified from [Vladisavljević and Williams, 2005].
2.3.1 Traditional emulsions
The traditional O/W and W/O emulsions are the most common product of emulsification techniques. Advantages
of nano-sized emulsions are improved stability, shelf life, digestion, taste, appearance (e.g. fig. 1) and flow
properties [Sanguansri and Augustin, 2006]. Consumer products that directly benefit from nano-emulsions are
baby foods, purees and beverages (through high-energy methods). W/O emulsions have been successfully used
in spreads and margarines [Joscelyne and Trägårdh, 1999].
More complex emulsions are the double emulsions, water-in-oil-in-water (W/O/W) and oil-in-water-in-oil
(O/W/O, fig. 3). These emulsions are used for fat reduction in food [Lobato-Calleros et al., 2006] and
Emulsions and applications │ 8
encapsulation purposes [Mellema et al., 2006; Su et al., 2008]. The preparation of such emulsions is first done
with high energy methods to obtain as small as possible inner-emulsions, and afterwards methods that are gentler
for the final emulsion are used. The use of gentle methods is done to prevent the ‘inner’ emulsion from
coalescing. Other fragilities in this system are risk of oil droplet coalescence, rupture of oil droplets and leakage
to and from the inner emulsion [Van Der Graaf et al., 2005]. Membrane emulsion seems to be the best suited
emulsion technique for the preparation of the final emulsion.
2.3.2 Nano-emulsions as carriers: nanocapsules
Nano-emulsions can be engineered in such a way that they can function as carriers. Aside from being used for
improved product features, nano-emulsions can now be used during preparation processes or as nutritional
additives. There are many different forms of nano-carriers, including the normal emulsions, double emulsions
and encapsulated emulsions.
Normal emulsions (O/W and W/O) are the simplest example of a carrier. The desired content can be easily
included within the emulsions, provided that the content is miscible with the to-be-dispersed phase. However,
most emulsions will not last long in the intestine due to the harsh conditions present there. Therefore, most
emulsions are encapsulated with proteins or polymers [Chen et al., 2006a].
There are several different techniques to encapsulate emulsions. One of them is the layer-by-layer technique
(LbL). LbL is based on application of oppositely charged molecules [Decher, 1997]. A primary emulsion is
made that exhibits a certain charge at the surface of the droplet. Oppositely charged molecules are then added
which will adsorb to the droplet surface due to electrostatic attraction. A second layer can be applied with an
opposite charge. The cycle can be repeated if necessary.
Other encapsulation techniques include the incorporation of the encapsulation-monomers or -proteins in the
droplet before emulsion formation. After an initiation signal, usually temperature, the monomers precipitate and
migrate to the water/oil interface where polymerisation occurs as a result of a reaction with the water or a
component in the water [Tiarks et al., 2001].
An advantage of these encapsulation techniques is that they can be applied after the emulsion has formed. Older
methods used solid particles on which the different layers were applied. To use the capsule the core had to be
dissolved which required harsh conditions. Removal of the dissolved core is also not always fully achieved. The
resulting shell should also be able to open up if it is to be used as a delivery system [Sukhorukov et al., 2004].
Encapsulation of emulsions avoids these problems by loading before the shell is formed.
2.3.3 Nanoliposomes
Nanocapsules can also be formed using lipid bilayers, i.e. nanoliposomes. In general, nanoliposomes can be
obtained through high- and low-energy techniques. For low energy techniques, liposome generation is based on
the PIT method where the addition of water has a temperature that is below the melting point of the surfactant.
The addition of water allows for the crystallisation of the shell, thus creating a capsule around the emulsion
[Heurtault et al., 2002].
Advantages of nanoliposomes are that they can be readily made from natural or food-grade lipids. They can also
be used to target specific areas in the food matrix [Mozafari et al., 2006]. Nanoliposomes are already used as a
food processing aid [Piard et al., 1986] and newer applications will mainly be as carriers in the food additives
business. More advanced forms of lipid nanocapsules are multi-vesicular lipid capsules. These are similar to the
double emulsions but now in the form of liposomes [Mozafari et al., 2006].
Emulsions and applications │ 9
2.3.4 Solid nanoparticles
High- and low-energy methods are also produce solid nano-sized particles. This class of structures consists of a
homogeneous structure throughout the whole particle whereas the nanocapsules have a (hollow) core-shell
structure. Nanoparticles can function as carriers by imbedding the desired substance into the particle itself
[Ishihara et al., 2008] or by attaching it to the surface of the particle [Atyabi et al., 2008; Chauvierre et al., 2003].
Production of solid nanoparticles is done by dissolving monomers which are used as the hydrophobic component
of the mixture, just like oil is in water. Application of a high-energy method will cause the monomers to
polymerise which will then form droplets. Polymerisation is accomplished by molecules that are added to the
mixture. These molecules start reacting with the monomers causing them to form polymers [Thickett and Gilbert,
2007]. Droplets are entirely composed of polymerised monomers and are stabilised by surfactants, thereby
becoming (solid) nanoparticles.
Another type of solid nanoparticles is the solid lipid nanoparticle (SLN). These particles already have a large
potential in the pharmaceutical industry as carrier. Production is simple and is usually done with high-energy
methods [Vladisavljević and Williams, 2005]. Basically, the lipid is mixed with water or another immiscible
fluid at a temperature that is above the melting point of the lipid. The mixture is put through a high-energy
device and subsequently cooled down to crystallize the lipids. Advantages of SLN are similar to the solid
nanoparticles with respect to carrier capabilities. SLN can be made from lipids which makes them potentially
less biotoxic than solid nanoparticles.
2.3.5 Colloidosomes
Colloidosomes are emulsions with nanoparticles locked together on the interface forming a shell (fig. 4)
[Dinsmore et al., 2002]. Both components (inner-emulsion and shell nanoparticles) can be made from emulsions.
Shell formation can be prepared through electrostatic interactions. This preparation can be considered surfactantfree to a certain extent [Sakai, 2008]. Colloidosomes as a whole structure cannot be considered a nanostructure
because their size is far greater than 500 nm and can even range into the 10-100 μm. But the nanoparticles that
form the shell can be well below 500 nm.
Colloidosomes can be, theoretically, multifunctional, with the nanoparticles on the shell as well as the inneremulsion itself acting as a delivery system. However, reports of its use have not yet appeared which is most
likely due to this being a recent development.
Inner-emulsion
Nanoparticle
Figure 4 Colloidosome, an advanced product of emulsions. On the left is a schematic view of a colloidosome and on the
right scanning electron microscope image of a 10 μm vacuum dried colloidosome. Modified from [Dinsmore et al., 2002].
Emulsions and applications │ 10
2.4 Developments and trends
Nanostructures in our food is not a new phenomenon. Food contains many natural nano-sized structures such as
globular proteins, one molecule thick layers (e.g. around emulsions) and crystalline lamellae. Such structures are
naturally present or formed during production. Size-reduction of emulsions occurred because of attempts to
improve food characteristics and not necessarily on the fact that nano-sizing has added advantages. Nanoemulsions may have already been present in our food, especially after the introduction of high pressure
homogenisation in the 1950’s [Niro-Soavi, 2008].
The difference between natural nanostructures in our food and the ones discussed in the previous section is that
the ones presented here can be engineered to perform certain functions. In other words, the ability to manipulate
nano-sized structures for certain tasks is considered to be the innovating part. Nano-emulsions, nanoparticles,
lipid nanocapsules and nanofibers are structures that have contributed to the innovation of food ingredients and
processing due to specific functions that they could perform.
What also can be considered new is the increasing number of methods to generate nanostructures. As
nanostructures are being associated with many benefits, several industries are developing new and efficient ways
to produce nanostructures. Interestingly, the current methods (high-energy) of producing nano-emulsions are
based on old principles and updated techniques. Ultrasound is a relatively new form of production. Ultrasound in
the food industry was already used for extraction, viscosity alteration, fermentation, enzyme and microbial
inactivation [Patist and Bates, 2008]. Nano-emulsion production is a potentially new candidate to this list.
The low-energy methods are also not new. Emulsification through solvent displacement has already been used in
the field of radical polymerisation thirty years ago but the mechanism has only recently been described [Vitale
and Katz, 2003]. A concern with solvent displacement is that the solvent may not always be fully removed. Nonfood grade ingredients can be used and, if not fully removed, may introduce new risks [Mozafari et al., 2006].
Nanostructures are becoming more complex in order to perform certain functions. Nanostructured coatings,
dispersion (e.g. emulsions) and nano-bulk materials such as nanometals, -ceramics and -polymers are fairly
simple compared to the structures that are currently being designed. So-called second generation nanostructures
are more complex forms of the older nanostructures [Renn and Roco, 2006; Weiss, 2007]. These include the
double emulsions, multi vesicular lipid nanocapsules, SLN and colloidosomes. Most of these systems can be
distinguished from the first generation by their multi-functional capability and because of this they are most
likely capable of more complex interactions during food production and/or in the body [Renn and Roco, 2006;
Sanvicens and Marco, 2008].
In summary, nanostructures are naturally present in our food. In the past, attempts to improve food
characteristics unintentionally lead to increased nanostructure exposure, whereas nowadays nanostructures are
actively introduced in order to improve food characteristics. New methods are being developed to generate
increasingly more complex nanostructures. Due to the possibility of more complex interactions of nanostructures
extra attention may be needed for their safety assessment, especially in the food industry where EBNS can be
used as food additives, processing aids or as a nutritional supplement.
Safety assessment of nanostructures in food │ 11
3. Safety assessment of nanostructures in food
3.1 Potential risks
Many people have doubts about nanotechnology concerning its safety because so little is known. If
nanotechnology is going to be used on a large scale in the food industry then the risks that can be associated with
nanostructures will need to be characterised. This section will focus on the identification of possible risks of
nanostructures that can be used in the food industry. Possible risks and known effects will be identified, from the
preparation methods up to the immune system. In recent years, numerous papers have described the risks of
engineered nanomaterials. Examples of these materials are carbon nanotubes, fullerenes and metallic
nanoparticles. Also in the pharmaceutical field are there many reviews on risk assessment of nanomaterials and
delivery systems. Assessments of nanoformulations used in the food industry, however, have hardly been
performed. Due to the lack of food risk assessments of nanostructures, hazard assessments of nanoformulations
in the (oral) pharmaceutical research will also be used.
3.1.1 Importance of preparation methods
The composition and components of the nanostructures are important determinants of toxicity. A comparison of
EBNS made of non-toxic components and potentially toxic components, such as cationic molecules, indicate
toxicity related to the materials and not to the fact that it is an EBNS [Weyenberg et al., 2007].
Surfactants are an important component of EBNS, which influence particle size and distribution. High pressure
homogenisation and ultrasound generally have a broad size distribution which is often overcome by adding more
surfactant. If the surfactant is known to have adverse effects at relevant doses then the preparation method may
be a cause for concern. Certain preparation and purification methods can help reduce toxicity mainly by
removing excessive amounts [Heydenreich et al., 2003]. One disadvantage is that these preparation methods
themselves can affect the size properties of the EBNS, so careful consideration is needed for a balance between
risks and benefits.
A good preparation can also avoid adverse effects due to impurities. Non-food grade materials are often used to
solubilise or dissolve ingredients which may become entrapped together with the active ingredient [Mozafari et
al., 2006]. It has been shown in vitro that metal impurities in carbon nanotubes are responsible for the generation
of reactive oxidative species and inflammatory reactions in lung cells and macrophages. Purified nanotubes did
not display such effects [Pulskamp et al., 2007]. Thus, beside the intrinsic toxicological properties of the
components, preparation methods of EBNS are equally important to consider.
3.1.2 Nanostructures in the gastrointestinal tract
The gastrointestinal tract (GIT) is the first major contact point with nanostructures. The first large obstacle is the
stomach where the high acidity, cleavage enzymes and the mucus layer can prevent nanostructures from entering
the body. But this does not provide a full protection. Uptake of various nanostructures was observed throughout
the GIT [Desai et al., 1996; Jani et al., 1994; Wang et al., 2006; Wang et al., 2007]. Uptake occurs mostly
through specialised membranous epithelial (M) cells in the Peyer’s Patches [Desai et al., 1996; Jani et al., 1994].
Other cells and mechanisms such as enterocytes and paracellular transport, respectively, also make it possible
for nanostructures to pass the intestinal epithelium [Lomer et al., 2002].
The GIT is capable of taking up particles of up to 150 μm in size [Hussain et al., 2001], which is well above the
size of nanostructures. However, uptake of nanostructures is dependent on the nanostructure and its surface
Safety assessment of nanostructures in food │ 12
properties (see subsection 3.1.4). Oral administration of carbon-based nanotubes, fullerenes and quantum dots
were found not to be taken up by the intestines and were eventually recovered in the faeces [Deng et al., 2007;
Nel et al., 2006], indicating that size is not the main factor driving uptake.
Effects of nanostructures on the GIT are not yet fully known. Only slight inflammation of the stomach and
intestines in mice was observed after oral administration of nano zinc and titanium dioxide (TiO 2) [Wang et al.,
2006; Wang et al., 2007]. In humans, it is thought that exposure to nano-sized structures can contribute to GIT
diseases such as inflammatory bowel disease and Crohn’s disease [Lomer et al., 2002]. Other than some
inflammation, no other adverse effects of nanostructures in the GIT are known. Detailed hazard characterisations
of the GIT and the knowledge of the effects on susceptible people will be essential before engineered
nanostructures are allowed to be used in food.
3.1.3 Biodistribution
After being taken up in the GIT, nanostructures will distribute throughout the body. Studies of nano TiO 2 and
zinc indicate that the liver is the primary site of accumulation [Jani et al., 1994; Sugibayashi et al., 2008; Wang
et al., 2006]. Prominent effects are hydropic degeneration and slight necrosis of hepatocytes. Furthermore,
increased levels of ALT, AST, ALP, LDH and cholesterol esters indicated liver injury and dysfunction in mice
[Wang et al., 2006; Wang et al., 2007]. TiO2, however, is easily eliminated from the body [Sugibayashi et al.,
2008]. In a safety evaluation of TiO2 nanoparticles in food most of the TiO2 accumulated in the liver and was
gradually eliminated from the body (mice, 30% decrease 1 month after administration). No symptoms or
noticeable behaviour changes were observed.
Kidneys are the second largest site of accumulation for TiO2 and zinc nanoparticle. Histopathological effects are
swelling of the renal glomerulus and proteinic liquid in the renal tubules [Wang et al., 2006; Wang et al., 2007].
Other minor accumulation sites include the spleen, heart and lung tissues. The heart tissue showed no
morphological alterations after oral exposure of TiO2 and zinc but blood serum levels of LDH and α-HBDH
were higher compared to controls [Wang et al., 2006; Wang et al., 2007]. LDH and α-HBDH are often used as
markers of cardiovascular damage. TiO2 and zinc also accumulated in the spleen and lung but no
histopathological effects were observed [Jani et al., 1994; Wang et al., 2007].
In summary, the main sites of accumulation are the liver and kidneys where the effects are also the most
prominent. Other organs may also be affected to a lesser degree. TiO 2 was shown to be gradually eliminated
from the body and thus effects may be reversible. These studies were done with nano TiO2 and zinc but no
chronic oral exposure studies of other food nanostructures and EBNS are known.
3.1.4 Effects of surface properties
Surface properties of nanostructures are a special issue. First of all, the smaller the particle, the higher its
surface-to-volume ratio becomes i.e. smaller nanostructures have relatively more surface area than larger
structures. This makes them potentially more reactive. Secondly, the surface can be engineered to exhibit certain
properties or functions. The combination of these two aspects is a reason why nanostructures in food raise so
many concerns. A few examples of adverse effects due to surface properties are discussed below.
An important factor for uptake or internalisation of the particles is surface charge. To enhance the uptake
potential of nanostructures their surfaces are often modified with molecules with a certain charge. Particles
Safety assessment of nanostructures in food │ 13
[Florence, 1997; Mao et al., 2005] and emulsions [Yang and Benita, 2000] with a positive charge are more easily
absorbed by the GIT. The positive charge, however, can have a toxic effect on cells. Positive charged or cationic
molecules can enter cells through various cell mechanisms [Xia et al., 2008] or by disrupting the lipid membrane
[Leroueil et al., 2008]. A wide variety of commonly used nanostructures was investigated for the potential to
create (nano)holes in the lipid bilayer of cells. It seems that regardless of the shape, size, composition, charge
density and deformability, all tested cationic nanostructures were capable of disrupting the bilayer by either
membrane thinning/erosion, hole formation or expansion [Leroueil et al., 2008]. Hole formation/expansion can
lead to leakage of the cell content and even cell death. Whether cell death is attributed to the leakage or to the
nanostructure itself is still unclear.
Uptake of cationic nanostructures by cells disrupts certain cell processes and leads to cell death. Recently, 60 nm
cationic polystyrene nanospheres have been shown in vitro to induce mitochondrial damage in macrophages and
bronchial epithelial cells, followed by apoptosis and necrosis, respectively [Xia et al., 2008]. Uptake and cell
death mechanisms differed between the two cell lines. Xia et al. also tested polystyrene nanospheres on cells of
the liver, adrenal gland and microvascular endothelium. Nanospheres were taken up by these cells but in
contrary to macrophages and bronchial epithelial cells, did not induce mitochondrial damage, increased Ca 2+ flux
or lysosomal disruption, indicating cell-specific toxicities and mechanisms.
The severity of adverse effects can have a charge (as in dose)-response relation. Direct cell-polymer contact
assays demonstrated increasing cytotoxicity with increasing charge density of polyesters in mouse fibroblasts
[Unger et al., 2007]. Thus, a certain dose-response relationship of nanostructures may be set up based on the
charge.
The use of cationic molecules as building blocks for nanostructures can still be toxic despite it going through
several processes. Processing cationic lipids into 100-500 nm SLN does not reduce their capability to decrease
cell viability [Heydenreich et al., 2003]. Though, caution is needed when inferring in vitro results. Comparative
studies between in vitro and in vivo pulmonary toxicity results of nanoparticles demonstrated little correlation
[Sayes et al., 2007]. Whether this holds true for other non-pulmonary toxicity studies is not known.
With such potential toxic effects in mind certain aspects of nanostructures will need extra caution. One technique
that deals with surface charge is the LbL technique, which makes it possible to coat emulsions and EBNS
[Decher, 1997; Gil et al., 2008]. The application of this technique may unintentionally increase the toxicity of
nanostructures and possibly nullifying the intended effect of carrier systems, e.g. reduced toxicity. It is
interesting to note that the application of positively charged molecules on nanostructures may decrease oral
bioavailability. Mucus in the stomach is negatively charged, and so, positively charged molecules may become
entrapped in it [Hoet et al., 2004]. Alternatively, some scientists believe that this can also increase intestinal
lifetime and facilitate uptake by presenting nanostructures to the GIT epithelial cells [Arbós et al., 2002; Chen et
al., 2006a; Hussain et al., 2001]. This issue is still under debate. Important characteristics of nanostructures,
especially surface properties, are to a certain extent accompanied with negative effects.
The intestinal environment with its high acidity and cleavage enzymes may change the nanostructure surfaces in
a certain way. Inflammatory effects and cell death were induced by pathogen-associated molecular patterns
(PAMP) conjugated to food additive-grade nano-TiO2. PAMP may inadvertently adsorb to the surface of TiO 2
[Ashwood et al., 2007]. The gut flora is a potentially large source of PAMPs. Thus, even though a nanostructure
can be harmless at first, in or ex vivo events may change its current status. Table 1 summarises a few of the
potential risks that can be induced as a result of in or ex vivo (metabolic) processes and interactions. Ultimately,
Safety assessment of nanostructures in food │ 14
the proteins that are present or adsorbed to the surface determine what is actually presented to cells [Cedervall et
al., 2007].
Nanostructures in the GIT do not necessarily have to be taken up to exert an effect on other organs. One example
is the effect on the pH buffering system [Chen et al., 2006b]. Chen et al. investigated the effects of nano- and
micro-copper. Severe effects (LD50 was 413 mg/kg body weight for nano-copper as compared to >5000 mg/kg
for micro-sized copper) and damage to liver, kidney and spleen where observed. Chen et al. hypothesized that
depletion of H+ in the gastric juice due to higher reactivity of nano-copper lead to the formation of bicarbonate
ions (HCO3-). HCO3- is hardly excreted by the kidney due to renal disorders and so accumulation occurs that
could lead to other metabolic and physiologic states/disorders, such as alkalosis in this case.
Table 1 Examples of materials used in food and in nanostructures that can induce undesired effects due to interactions.
References are in vivo studies unless indicated otherwise.
Material
PEG
Food use
Food additive
Possible effect
Immune evasion, may
enhance unwanted
biodistribution
Reference
[Oberdörster et al.,
2005b; Sanvicens and
Marco, 2008]
Thiamine
Natural vitamin in food (e.g.
whole cereal grains)
Immunogenic, adjuvant
properties
[Salman et al., 2007]
Chitosan
Edible films, additive
Immune suppression
[Roy et al., 1999]
Cell membrane damage
[Leroueil et al., 2008]
(in vitro)
Binds with
lipopolysaccharide and
induces proinflammatory
effects and cell death.
[Ashwood et al., 2007]
(in vitro)
Cationic molecules
TiO2
Food additive
Abbreviations: PEG, polyethylene glycol; TiO2, titanium dioxide.
3.1.5 Effects on the immune system
Nanostructures may also affect the immune system in several ways. This can be increased or decreased reactivity,
alteration of immune response (e.g. Th1 versus Th2) or adjuvant activity.
Nanostructures can be potent adjuvants. With a nanostructure as adjuvant, antigens were able to stimulate the
immune system better than when the antigen is presented alone [Salman et al., 2007]. When compared with
other adjuvants, including aluminium hydroxide, nanoparticles are capable of inducing higher antibody titres
[Khatri et al., 2008; Seferian and Martinez, 2000; Stieneker et al., 1991]. Moreover, smaller nanostructures are
associated with higher antibody levels than larger nanostructures [Xiang et al., 2006].
Surface modification of nanostructures can also play a major role. For example, Salman et al. [2007] tested
orally administered thiamine-coated and non-coated nanoparticles in mice and showed that the thiamine-coating
elicited stronger IgG1 and IgG2a titres. Non-coated nanoparticles induced a typical Th2 response, indicating that
coated nanoparticles are capable of shifting towards a more balanced response. This also further indicates the
importance of surface properties, i.e. surface can determine functionality of the nanostructure.
Nanostructures may also affect diseases based on deregulation of the immune system. Nano metals and ceramics
can bias the defence/inflammatory capabilities of macrophages [Lucarelli et al., 2004]. SiO2 nanoparticles biased
naive macrophages towards pro-inflammatory reactions by selectively inducing production of IL-1β and TNF-α.
Safety assessment of nanostructures in food │ 15
TiO2 was shown to increase expression of viral TLR receptors TLR3 and TLR7. Nanoparticles made from cobalt
induced a cytokine profile that resembles experimental and clinical autoimmunity in vitro [Petrarca et al., 2006].
No reports are known that focus on nanostructures in food and the effect on susceptible subjects, e.g., GIT
disease models.
On the other hand, there are reports describing immune suppression by nanostructures. In a mouse model of
peanut allergy, nanoparticles of plasmid DNA and chitosan reduced IgE levels, plasma histamine and vascular
leakage. Secretory IgA and serum IgG2a were found after treatment [Roy et al., 1999]. This may imply a
positive effect of (functionalised) nanostructures that can be used in food. However, this feature may also raise
concerns about undesired and excessive immune suppression. To date there are no extensive reports on this
matter, further research is needed.
3.1.3 Other routes of exposure
Other routes such as inhalation may become increasingly more relevant as more food applications are going to
be developed for nanostructures. Before food ends up on our plates it will undergo several processes. Exposure
through inhalation can occur with cooking processes or when the nanostructure is in a powder form. Effects of
nanostructures on the respiratory tract have been extensively described [Gill et al., 2007; Mühlfeld et al., 2008;
Oberdörster et al., 2005b]. Interestingly, it was already known sixty years ago that nanostructures (<500 nm) can
be taken up and transported up the olfactory bulb and nerve [Oberdörster et al., 2005b]. Oberdörster et al. [2005]
noted that these studies were performed on rodents and questioned the relevance to humans. Indeed, the human
olfactory mucosa comprises only 5% of the total nasal mucosal surface as opposed to 50% in rats. Thus, relative
exposure would seem to be far less for humans. Nevertheless, this still may be a concern for people with pets.
Other important effects on the lungs are, in general, inflammation and fibrosis, generation of reactive oxidative
species, various effects on macrophages and cardiovascular effects. Again, surface properties can have a large
influence [Oberdörster et al., 2005b]. Most of the assessment studies have used metallic or carbon-based
nanostructures and not much emphasis is given to EBNS that are used in the pharmaceutical or food industry.
More research is needed using EBNS that can also be used in the food industry.
A third major route of potential uptake is through dermal exposure. Skin contact with nanostructures (in food)
can occur during handling or preparation of food. Risk assessments of cosmetics, mainly sunscreens and lotions,
have shown however, that nanostructures do not penetrate the living skin [Nohynek et al., 2007]. Moreover, it
was concluded that nano-sized structures do not significantly penetrate deeper than micro-sized structures,
although the definition of micro-sized structures was not clearly explained by Nohynek et al.
3.2 A food-specific risk assessment
In 2007 the EU Scientific Committee on Emerging and Newly-Identified Health Risks (SCENIHR) developed a
tiered risk assessment methodology for nanomaterials based on an exposure assessment algorithm (fig. 5)
[SCENIHR, 2007]. The SCENIHR acknowledges that both assessments deal with nanostructures in general and
that there may be a need for more industry-specific risk assessments. Here, several aspects will be discussed that
can make the exposure assessment algorithm by the SCENIHR more specific to the food industry.
Safety assessment of nanostructures in food │ 16
Figure 5 Exposure assessment algorithm from SCENIHR [SCENIHR, 2007].
Several of the guiding questions proposed by the SCENIHR in figure 5 are directed to nanostructures not
intended for food. For example, in the SCENIHR report nanostructures do not need to be considered as such if
they are found to be soluble in water. However, water solubility is an important aspect for uptake in the GIT
[Bronner, 1993]. Many nutrients are required to be soluble in water or oil in order to be taken up efficiently.
Nutrients that are insoluble in water and/or oil such as β-carotene can be delivered with nanostructures that have
been made soluble [Yuan et al., 2008]. Thus, water soluble as well as water insoluble nanostructures need to be
considered in food risk assessments.
Food nanostructures larger than 100 nm should also be considered in risk assessments. Nanoparticles in the air
can penetrate deeper into the lungs depending on their size. Nanostructures in food do not have this limitation,
making it easier to come in contact with cells. Though, a proper upper limit still needs to be determined.
Another physicochemical property, agglomeration, may be a potential concern. Agglomeration (or coalescence)
can cause nano-emulsions to lose their beneficial effects. Food manufacturers can avoid this by applying
molecules capable of repulsing other particles [Studart et al., 2007]. However, such procedures can alter other
properties of the nanostructures [Kirchner et al., 2005] and in vivo behaviours (e.g. increased circulation
capabilities) [Garnett and Kallinteri, 2006], consequently introducing new risks. In food risk assessments it may
therefore be important to assess the effect of anti-agglomeration measures.
One important aspect of nanotechnology in the food industry is that there can be an intended exposure. Currently,
manufacturers try to minimise the amount of processing and enhance food nutrition. For instance, by reducing
the transport medium the uptake of nutrients in the gastrointestinal tract will be improved [Desai et al., 1997;
Jani et al., 1990].
Safety assessment of nanostructures in food │ 17
There are two types of nanostructures with an intended exposure: 1) nanostructures which are involved in
nutritional enhancement. This is mostly in the form of carrier systems. The goal of such nanostructures is to have
a high as possible bioavailability. These structures will concern both occupational and consumer safety.
2) Nanostructures which are present in the end-product but have a food-improvement or cosmetic related
function such as enhancement of taste or smell. This also includes food spoilage and antimicrobial protection
[An et al., 2008]. These nanostructures should not be taken up into the body. This may raise concerns for
manufacturers. Nano fragrance molecules, for example, can be taken up by cells in the lung and nasal cavity
[Oberdörster et al., 2005b]. Bioavailability of these nanostructures should be as low as possible.
Nanostructures that do not have any intended exposures will most likely be used as processing aids or as a
functionalised coating on equipment. This can be for example nanocapsules protecting an enzyme during the
production process and/or allowing gradual release of the enzyme afterwards [Piard et al., 1986]. When these
nanostructures remain in the end-product, measures may be necessary to insure that consumer exposure (and
bioavailability) is kept at a minimum, especially when adverse effects are known.
In vitro testing is well suited as an initial screening for adverse effects but a recent comparison of in vitro and in
vivo pulmonary tests indicated little correlation [Sayes et al., 2007]. Characteristics of nanostructures (e.g. size,
size distribution, charge etc.) can be altered by the food matrix, processing of the food matrix and the GIT. To
describe the effects as accurately as possible, in vivo tests need to be carried out with the matrices that (will)
house the nanostructures [Oberdörster et al., 2005a]. Although, with this approach many complications arise that
may obscure the effects of the nanostructure. Feeding high levels of whole foods to test animals can lead to
nutritional imbalances and difficulties with establishing causal relations [Constable et al., 2007]. To reduce
unnecessary animal testing in vitro studies should get priority over in vivo studies. If in vitro studies indicate
adverse effects at relevant doses, then in vivo experiments should be done to confirm results. In vitro and in vivo
studies need to be developed that correlate with each other.
Effects of chronic exposure are harder to determine than acute effects. To provide a certain level of safety on the
long term, measures have been devised that take into account a lifetime exposure. One measure discussed by the
SCENIHR is the threshold of toxicological concern (TTC). It provides guidance in the form of a level of
exposure or intake per person per day below which no significant risk to humans is expected to exist. The
determination of a TTC value is done by comparing the molecular structure with other structural analogues and
the knowledge gained from the general toxicity database from the past sixty years [Kroes et al., 2005; Kroes and
Kozianowski, 2002; Munro et al., 2008]. This method is beneficial with respect to limited resources, time, and
expertise and is very useful when there is little or no data available of the substance. For nanostructures, however,
the TTC may not be an appropriate guidance value. Nanomaterials are a relatively new phenomenon, and so,
there are no other substances to compare with. If nanostructures are made from existing components such as
chitosan then a TTC value can be derived from chitosan toxicity (bulk) data. But the effect of nano-sizing
chitosan is not known and therefore some minimal amount of (in vivo) testing may still be required. Also, due to
their complexity different forms of nanostructures can present different risks depending on their manufacturing
process [Lin, 2007]. The lack of knowledge of nanostructures will make it difficult to apply a TTC concept in
risk assessments. Knowledge on nanostructure characteristics and preferably also dose-response relations will
therefore need to be generated first.
Because comparison with structural analogues may not always be possible, an alternative to the TTC may be
needed. The acceptable daily intake (ADI) may fulfil this role for now. The ADI is also a guidance value for the
daily intake of a substance over the entire lifetime [Lu, 1988]. The difference between TTC and ADI is that the
Safety assessment of nanostructures in food │ 18
ADI relies on identification of the highest dose level where no effect occurs and subsequent application of a
safety margin, whereas the TTC assures safety based on sufficiently low exposure (in the absence of specific
toxicity data) [Munro et al., 2008]. Thus, the advantage of an ADI over the TTC is that it is more specific to a
substance. The ADI is likely to be a precursor to the TTC because it is derived after the determination of adverse
effects. The TTC is more of a predictive form of hazard assessment and can be more efficient than the ADI if the
nanostructure is composed of structures with already existing toxicity data. ADI values are currently being used
for food additives and judging of the potentials of nanostructures, the first applications will most likely be as
food additives.
One issue that may need addressing in the future but is not explicitly mentioned in the SCENIHR report is the
possibility of multi-functionality. The currently known nanostructures (nanotubes, metallic nanoparticles) are
relatively simple but future nanostructures are expected to be more complex (delivery systems; e.g.
colloidosomes, fig. 6) [Renn and Roco, 2006; Sanvicens and Marco, 2008; Weiss, 2007]. Renn and Roco [2006]
predict that the next generation of nanostructures will involve assembly and networking systems, adding up to
complexity and multi-functionality. In nano-food processing this can possibly include miniature ‘factories’
capable of several (enzymatic) processing steps at the same time. But should we therefore be afraid of ‘multitoxicity’? This will most likely depend on the nanostructure capabilities. It may be that the safety of multifunctional nanostructures will have to be judged on each separate function and its capability as a whole.
Figure 6 Example of a fictitious multifunctional nanostructure. Figure from
[Sanvicens and Marco, 2008]
Laws have been implemented to ensure food safety. But current food laws may have unintentionally created
opportunities for manufactures to circumvent testing of nanostructures. This mainly concerns nanostructures that
have been made through a top-down approach. Existing substances have usually already been through extensive
testing, but in all cases the effect of size has not been considered. Therefore, nanostructures can be unjustly
deemed safe due to the existence of a Safety Data Sheet of its bulk form. TiO 2 is used as a whiting agent in food
and was recently deemed safe for use according to EU legislation [HSE, 2006]. Such regulations may
unintentionally help nanostructures and manufacturers circumvent nano-specific risk assessments. This can be a
concern if adverse effects are size-dependent.
Figure 7 proposes a food nanotechnology risk assessment paradigm that incorporates the aspects discussed above.
A sequence of questions is asked to determine the need for further extensive testing. This paradigm builds on the
existing risk assessment by the SCENIHR (fig. 5) and incorporates several of its elements. Furthermore, this
Safety assessment of nanostructures in food │ 19
paradigm is only intended for nanostructures that are in contact with food and does not address environmental
issues.
The risk assessments set up by the SCENIHR focuses more on physicochemical properties as a rationale for
safety testing (fig. 5). The proposed risk assessment, on the other hand, starts by identifying the purpose of the
nanostructure (i.e. intended versus unintended). If a nanostructure is only used during food production and
removed afterwards from the end-product (i.e. unintended), then no detailed assessments are needed (Fig 7, left
column). This is to prevent unnecessary and costly testing. The only other concern of these types of
nanostructures is limited to occupational safety.
For nanostructures that remain in the end-product, further testing may depend on assessment of the known data
on the physicochemical properties (fig.7, second column). This is a similar procedure as in figure 5. Enhanced
solubility or reduced agglomeration potential can increase bioavailability. These and other properties can give a
good indication of the bioavailability potential without doing any extensive testing.
Alterations to the nanostructure throughout its life should also be taken into consideration. This includes physical
changes but also adsorption of uptake enhancing molecules. A changing environment like the GIT or (domestic)
heating can change such properties and can even create different routes of exposure. Therefore also other routes
such as inhalation should be considered. Besides adverse effects due to uptake, effects not related to uptake such
as irritation or inflammation may also need to be considered.
Nanostructures with high bioavailability should be subject to at least some (initial) tests to ensure a minimum
level of consumer safety (fig. 7, third column). The initial testing should also include further characterisation of
other unknown physicochemical properties. Effects can be compared to the corresponding bulk material. Bulk
material guidelines can be followed only when effects are of the same kind and less severe at the same mass
concentration. This is to avoid manufacturers bypassing further testing as a result of existing bulk form data.
Gaps in the legislation should not be a reason to avoid testing even though the bulk form of the nanostructure has
been in use for a long time (e.g. GRAS, history of safe use). A full risk assessment should be performed when
adverse effects of the nanostructure are more severe than the bulk form or when tests indicate different (new)
kinds of adverse effects.
Safety assessment of nanostructures in food │ 20
Is there an intended
exposure?
YES
NO
Will the nanostructure
be removed from the
end-product?
NO
YES
NO/
Unknown
YES
Is or can the
nanostructure be fully
contained during
production?
Intended exposure but
no/low bioavailability?
YES
Does the nanostructure or its components have a GRAS-status or a
history of safe use (not counting in as bulk form)?*
NO
NO/
Unknown
YES
Based on physicochemical
properties and/or
structural features, is
there potential for uptake?
NO
YES/
Unknown
No further tests may
be necessary.
NO
Initial tests: in vitro
studies. Are there any
adverse effects?
YES
Are the nanostructures
also in bulk form?
Is there uptake possible
due to alterations by the
food matrix, processing of
the food matrix and/or
GIT?
NO
NO
YES
YES
YES/
Unknown
NO
No further tests may
be necessary.
Intended exposure with
high bioavailability
YES
YES
Validate initial tests with
specialized in vitro, in vivo
and ADME studies. Are
effects similar/worse than
in initial tests?
Are there any other
exposure routes possible?
NO
YES
No further tests may be
necessary.
Do in vivo effects occur at
relevant doses?
Are effects more severe
than corresponding
bulk material?
NO
Guidelines of the bulk
material may suffice.
NO
NO
No further tests may
be necessary.
YES
Derive ADI or, if possible,
TTC
YES
Are there any other
exposure routes possible?
NO
Complete risk assessment
Figure 7 Proposed risk assessment paradigm for nanostructures that can be used in the food industry. A series of questions are
asked of which the answers determine the need for further studies. *The large box about GRAS-status or a history of safe use
should not be considered as an interconnecting box between the second and third column but instead as two separate boxes
with similar content. This box was depicted as one large box solely for graphical reasons. Abbreviations: GRAS, generally
recognised as safe; GIT, gastrointestinal tract; ADME, absorption, distribution, metabolism and excretion; ADI, acceptable daily
intake; TTC threshold of toxicological concern.
Conclusions │ 21
4. Conclusions
One of the purposes of this report was to investigate the possibilities to generate various nanostructures through
emulsification methods that can be used in the food industry. Methods to produce nanostructures are based on
old techniques and principles. Manufacturers are developing more efficient methods to produce EBNS, thereby
increasing consumer exposure.
A wide variety of EBNS can be obtained through emulsification. These nanostructures can be functionalised to
perform certain functions such as enzyme delivery. This aspect differentiates engineered nanostructures from
natural nanostructures which are already present in food in the form of proteins, bacteria, single molecules (e.g.
vitamins) etc. Nanostructures that are currently being developed are becoming more complex which enables
them to perform multiple functions. These multi-functional EBNS may require special attention in risk
assessments.
The other purpose and main focus of this report was to identify possible risks that may arise due to
nanostructures in food and to determine important aspects for a risk assessment that is specific to food
nanostructures. Surface properties are probably the most important aspect of nanostructures to consider. Surface
molecules can determine the fate, function and possible risks of nanostructures. There is still very little known on
the risks of nanostructures, especially when it comes to effects on the GIT. There have been a lot of in vitro
studies conducted and relatively few (oral) in vivo studies. Pulmonary toxicity studies of nanoparticles indicated
little correlation. Similar investigations need to be conducted for other non-pulmonary studies. Also, most
studies have used metallic or carbon-based nanoparticles and no extensive studies have been conducted using
other kinds of nanostructures. Effect of nanostructures on hypersensitivity and disease models (as susceptible
subjects) will also need more research.
Recommendations for a food nanostructures-specific risk assessment are: 1) to base assessments on the purpose
of the nanostructure i.e. intended versus unintended exposure. Some nanostructures are intended to be taken up
whereas others are only used during production, which does not need extensive testing.
2) The need for testing based on physicochemical properties differs for nanostructures in food compared to those
used in other industries. Solubility can increase uptake and, depending on the purpose may be undesired.
Assessments should also include nanostructures larger than 100 nm because cells are capable of taking up larger
sized nanostructures. Nanostructures can be ‘engineered’ with certain properties which may have similar adverse
effects as smaller sized nanostructures.
3) A minimum amount of testing for nanostructures with a potential of being taken up (based on
physicochemical properties), whether it is intended or not. This is to provide a minimum level of safety. Further
testing if needed, can provide guidance values such as ADI and may even set up a toxicity database that can be
used for other nanostructures. Such a database will be helpful for a TTC concept, which may help in further
reducing (animal) testing.
Implementation of these recommendations in a risk assessment will make it more specific to for nanostructures
in food. The risk assessment paradigm presented in figure 7 incorporates these recommendations in the form of a
series of questions of which the answers determine the need for further testing.
References │ 22
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