355

Journal of Food Protection, Vol. 85, No. 2, 2022, Pages 355–372

https://doi.org/10.4315/JFP-21-184
Copyright Ó, International Association for Food Protection

Review

Nanoparticle Food Applications and Their Toxicity: Current Trends

and Needs in Risk Assessment Strategies
JEFFERSON DE OLIVEIRA MALLIA,1,2 RUSSELL GALEA,2 RAJAT NAG,3 ENDA CUMMINS,3 RUBEN GATT,2,4 AND
VASILIS VALDRAMIDIS https://orcid.org/0000-0001-6486-38901,4*

1Department of Food Sciences and Nutrition, Faculty of Health Sciences, 2Metamaterials Unit, Faculty of Science, and 4Centre for Molecular Medicine

and Biobanking, University of Malta, Msida MSD2080, Malta; and 3UCD School of Biosystems and Food Engineering, University College Dublin, Belfield,
Dublin 4, Ireland

MS 21-184: Received 3 May 2021/Accepted 5 October 2021/Published Online 6 October 2021

ABSTRACT
Nanotechnology has developed into one of the most groundbreaking scientific fields in the last few decades because it
exploits the enhanced reactivity of materials at the atomic scale. The current classification of nanoparticles (NPs) used in foods is
outlined in relation to the production and physicochemical characteristics. This review aims to concisely present the most
popular and widely used inorganic and organic NPs in food industries. Considering that the toxicity of NPs is often associated
with chemical reactivity, a series of in vitro toxicity studies are also summarized, integrating information on the type of NP
studies and reported specifications, type of cells used, exposure conditions, and assessed end points. The important role of the
digestive system in the absorption and distribution of nanoformulated foods within the body and how this affects the resultant
cytotoxicity. Examples of how NPs and their accumulation within different organs are presented in relation to the consumption of
specific foods. Finally, the role of developing human health risk assessments to characterize both the potential impact of the
hazard and the likelihood or level of human exposure is outlined. Uncertainties exist around risk and exposure assessments of
NPs due to limited information on several aspects, including toxicity, behavior, and bioaccumulation. Overall, this review
presents current trends and needs for future assessments in toxicity evaluation to ensure the safe application of NPs in the food
industry.

HIGHLIGHTS

The use and inclusion of NPs in food production is growing.

TiO2 NPs are widely used by the food industry.

Thorough NP hazard characterization by using more advanced in vitro models is required.

Individual and multimixture NP interactions require further hazard investigations.

Unified risk assessment approaches are needed to determine NP health risks.

Key words: Food industry; Hazard characterization; Nanoparticles; Risk assessment

The advent of nanotechnology, which involves the necessity for safe applications (6, 77). A “nanomaterial,” as
manufacture and use of materials of enhanced reactivity at defined by the European Parliament and the Council of the
the atomic scale, has brought great opportunities for the European Union (Off. J. Eur. Union L 275:40, 2011),
development of new materials. One of the characteristics of “means a natural, incidental or manufactured material
these materials is the antimicrobial properties, supporting containing particles, in an unbound state or as an aggregate
applications in several fields, such as medicine, agriculture, or as an agglomerate and where, for 50% or more of the
and food production. Several applications of metal nano- particles in the number size distribution, one or more
particles (NPs) are currently available, but their further external dimensions is in the size range 1 nm–100 nm.”
exploitation in the food sector requires thorough food safety Materials can exhibit new or altered physicochemical
and toxicity assessments. properties at nanoscale dimensions, enabling the develop-
The use of NPs in food-related applications has put ment of novel products (113). These materials are, therefore,
more pressure on regulatory bodies to assess and certify categorized separately from other materials, thus requiring
them for safe use. Current public awareness and concern distinct characterization and appropriate safety evaluation.
regarding NP use and consumption also increased the The application of nanomaterials by the food industry
has gathered much interest due to the potential in improving
* Author for correspondence. Tel: þ356-99648817; E-mail: food production efficiency, product shelf life, and sensory
[email protected]. properties (63). The most prevalent and consumed nano-
356 DE OLIVEIRA MALLIA ET AL. J. Food Prot., Vol. 85, No. 2

materials used within food products are titanium dioxide they provide protection from degradation and oxidation for
(TiO2) and iron (III) oxide (Fe2O3), acting as food colorants. sensitive compounds (10, 68) and, in some cases, allow
These colorants contain varying percentages of nano-sized release at target organs (55, 67, 102).
fractions that form part of these additives (54, 119). The NPs can be further classified into biological, physical,
inclusion of other nanomaterials within food products is and chemical NPs, depending on the synthesis mechanism
hindered, as more in-depth risk assessments are required to (100). Furthermore, according to their degradation nature,
support safe application and regulatory or policy reforms. NPs can be grouped into biodegradable and nonbiode-
The risk assessment process requires that nanomaterials be gradable NPs (8). The most popular and widely used
evaluated and monitored throughout their life cycle, inorganic NPs in food manufacturing are documented in
primarily in development, application, and disposal (108), Table 1.
requiring robust frameworks and platforms to thoroughly Clay (SiO44), cellulose-based, carbon nanotubes,
assess any associated risks. They also have to consider, SiO2, starch nanocrystals, and chitin or chitosan NPs act
evaluate, and expand current regulatory guidelines and as reinforcements to the biodegradable NPs (106). The most
policies. Studies conducted in the initial nanomaterial popular and widely used organic NPs are documented in
development stage can highlight possible issues for safe Table 2. Polymers most widely used in nanocomposites
applications. Thus, the requirement for studies to have a include gelatin, polylactic acid (PLA), isotactic polypro-
high degree of assessment and resemblance to the real-life pylene, and low-density polyethylene. Polylactic acid needs
environments these nanomaterials would experience is an associative compound such as polyethylene glycol to be
essential (35). The lack of resemblance would impede a successful in delivering active components.
precise evaluation; therefore, potential adverse effects might
not be determined. Studies must be developed, evaluated, EUROPEAN REGULATIONS FOR NP FOOD
and adapted accordingly to accurately assess potential risks APPLICATIONS
with the highest degree of real-life resemblance. Assess- Several reports and regulations provide detailed
ment strategies would also require the reevaluation of information on nanotechnology and its use in food
currently used approaches and the development of novel production (Table 3). The term “nanotechnology” first
and/or inclusion of other methodologies. appeared in legislation Regulation (EC) No 1333/2008 of
This review presents an overview of the kinds of NPs the European Commission (EC) on p. 17 and 23 (40).
being used in food products, how these are classified, and According to this legislation (p. 17), “a food additive
how current regulations affect categorization and evalua- already approved under this regulation but which is
tion. The potential effects that NPs might have within the prepared by production methods, or using starting materials
digestive system and studies that have assessed the significantly different (including nanotechnology) from
accumulations of NPs within humans are also discussed. those included in the risk assessment of the authority, or
The need for thorough human health risk assessment different from those covered by the specifications laid
strategies for NPs within foods is highlighted. Hazard down, should be submitted for evaluation by the authority.”
identification and characterization, as well as exposure Therefore, the uniqueness of the nanoscale state was
assessments, and how these are used in risk characteriza- acknowledged, and according to EC Regulation No 450/
tions are considered. The studies and presented strategies 2009 (41, p. 4), “new technologies that engineer substances
will assist in future assessments for the safe application of in particle size that exhibit chemical and physical properties
NPs in food products. that significantly differ from those of a larger scale, such as
NPs, should be assessed on a case-by-case basis for risk
NPS USED IN FOODS AND THEIR
until more information is known about this new technolo-
CLASSIFICATION
gy.”
Foods containing NPs can be classified as either natural The term “engineered nanomaterial” was further
or engineered (66). Engineered NPs can be further defined in Regulation EU No 1169/2011 of the EU (43, p.
categorized into three distinctive classes: organic, inorganic, 26), “as any intentionally produced material that has one or
and composite or hybrid NPs (97). The organic (lipid, more dimensions of the order of 100 nm or less, or that is
protein, and carbohydrate) NPs were observed to be more composed of discrete functional parts, either internally or at
quickly metabolized in the human body compared with the the surface, many of which have one or more dimensions of
inorganic ones (silver [Ag], Fe2O3, TiO2, silicon dioxide the order of 100 nm or less, including structures,
[SiO2], and zinc oxide [ZnO]) (78). This allows for organic agglomerates or aggregates, which may have a size above
NPs to be used extensively in nanoformulations to deliver the order of 100 nm but retain properties that are
drugs and nutraceuticals in humans (55, 67, 102). characteristic of the nanoscale. Properties that are charac-
Nanoformulations consist of suspensions of organic teristic of the nanoscale include (i) those related to the large
NPs that are partially encased by an encapsulant (56) and specific surface area of the materials considered; and/or (ii)
are divided into three broad categories on the basis of the specific physicochemical properties that are different from
encapsulant material: lipid and surfactant-based nanocar- those of the nonnano form of the same material” (43). In
riers, polysaccharide-based nanocarriers, and protein-based addition, according to the U.S. Food and Drug Administra-
nanocarriers (84, 94), with each type of encapsulant tion (FDA) (112, p. 5), the food industry was asked
imparting different properties, such as aqueous solubility, “whether a material or end product is engineered to have at
bioavailability, and absorption (102). In addition to this, least one external dimension, or an internal or surface
J. Food Prot., Vol. 85, No. 2 NANOPARTICLES AND FOOD TOXICITY 357

TABLE 1. Most popular and widely used inorganic NPs in food industries, use, and classificationa
NP(s) Purpose Solubility State Synthesis Degradability

TiO2 Color, lightness, and brightness Insoluble Stable Chemically Nonbiodegradable

additives (light-scattering
properties), binders for
composites
SiO2 Color additives, flavors, Insoluble Stable Chemically Nonbiodegradable
packaging, anticaking agents
in powdered foods
SiO2–gallic acid Antioxidants: scavenging Insoluble Stable Chemically Nonbiodegradable
capacity of 2,2-diphenyl-1-
picrylhydrazyl radicals
Silicate (SiO44) Nanosensors Insoluble Stable Chemically Nonbiodegradable
Clay (SiO44) Bottle industry: lighter and Insoluble Stable Natural, yet Nonbiodegradable
stronger than glass and also usually not
less likely to shatter, biological
increases shelf life, prevents
spoilage, prevents O2
absorption
ZnO UV light absorbers, active Insoluble Stable, can release Chemically Nonbiodegradable
packaging, an additive in ionic Zn
supplements, antimicrobial,
antifungal
Magnesium oxide (MgO) Active packaging, antifungal Insoluble Stable Chemically Nonbiodegradable
Copper (II) oxide (CuO) Active packaging Insoluble Stable Chemically Nonbiodegradable
Cu Active packaging Insoluble Both stable and ionic Chemically Nonbiodegradable
Fe2O3 Insoluble Stable Chemically Nonbiodegradable
Zn Antioxidants increase shelf life, Insoluble Stable and ionic Chemically Nonbiodegradable
packaging, food supplement,
colorant
Ag Disinfectant, antibacterial, Insoluble Both stable and ionic Chemically Nonbiodegradable
antifungal, packaging,
chopping boards, storage
containers, refrigerators, and
health supplements
Gold (Au), platinum (Pt), Packaging, metal-based Insoluble Stable Chemically Nonbiodegradable
palladium (Pd), nanosensors, food supplement
iridium (Ir)
Graphene or graphite Packaging Insoluble Stable Chemically Biodegradable/
oxide (compound nonbiodegradable
of carbon, oxygen,
and hydrogen in
variable ratios)
C nanotubes Nanosensors, active packaging, Insoluble Stable but can be Physically Nonbiodegradable
antibacterial or antifungal, (fullerene) used to form (vacuum (stable)
absorb undesirable flavors, multiwalled ionic or with
used in low-resistance nanocomposites process
conductors and catalytic gases)
reaction vessels, gelation,
and viscosifying agent
Ag-TiO2–SiO2, Packaging Insoluble Stable Chemically Nonbiodegradable
Ag-N–TiO2,
or Au-TiO2
Nanofilters (many)b Filters microorganisms (even Insoluble Stable Chemically Biodegradable/
viruses) nonbiodegradable
Nanoceramic Used for clustering of dirt Insoluble Stable Chemically Nonbiodegradable
particles molecules from a liquid
media
a
Sources: references 6, 49, 52, 59, 95.
b
Can be organic.
358 DE OLIVEIRA MALLIA ET AL. J. Food Prot., Vol. 85, No. 2

TABLE 2. Most popular and widely used organic NPs in food industries, use, and classificationa
NP(s) Purpose Solubility State Synthesis Degradability

Carbohydrate (digestible or Nanolaminates (extremely Lipid soluble Stable Biologically Biodegradable

indigestible polysaccharides, thin food-grade film):
such as starch, cellulose, flavors, colors,
xanthan, carrageenan, antimicrobials,
alginate, and pectin), antibrowning agents,
proteins, and lipids antioxidants, enzymes
(combination)
Lipid and liposomes Oral delivery systems, Lipid soluble Stable Biologically Biodegradable
liposomes are capable of
carrying both water-
soluble and oil- or fat-
soluble compounds within
a single particle
Protein Catalysis, materials Soluble-insoluble Stable Biologically Biodegradable
synthesis, drug and gene
delivery, and bioimaging
Nanofibers (globular proteins) A platform for bacterial Usually water Stable Physically Biodegradable or
cultures; structural matrix soluble or chemically: nonbiodegradable
for artificial foods and electrospinning
packaging; thermal
stability, increased shelf
life; formation of
transparent gel network
for use as a thickening
agent
Nanoemulsions (oil in water Stabilization of biologically Lipid soluble Stable Chemically Biodegradable
calcium) or dispersions, active ingredients, (organic CaCO3) or
emulsions (calcium carbonate delivery of active nonbiodegradable
[CaCO3]) compounds, extended (inorganic CaCO3)
shelf life, flavor release,
low-fat products;
increased solubility of
calcium carbonate, can be
used at higher addition
levels
a
Sources: references 6, 49, 52, 59, 95.

structure, in the nanoscale range (approximately 1 nm to The FDA also continues postmarket monitoring; however,
100 nm) and whether a material or end product is the industry remains responsible for ensuring that its
engineered to exhibit properties or phenomena, including products meet all applicable legal requirements, including
physical or chemical properties or biological effects, that are safety standards (113).
attributable to its dimension(s), even if these dimensions fall The provisions for engineered NPs stated in Regulation
outside the nanoscale range, up to one micrometre (1,000 EU No 10/2011 (42, p. 4) stated that these materials can
nm).” The agency applies these considerations broadly to all have “chemical and physical properties that are significantly
FDA-regulated products, including food substances (112). different from those at the macroscopic scale. These

TABLE 3. Current European regulations around NPs in food products

Regulation About Reference

(EC) No 258/97 Concerning novel foods and novel food ingredients 36

(EC) No 178/2002 Laying down the general principles and requirements of food law 37
(EC) No 1935/2004 On materials and articles intended to come into contact with food 38
(EC) No 1333/2008 Harmonizes the use of food additives in food products 40
(EC) No 282/2008 On recycled plastic materials and articles intended to come into contact with foods 39
(EC) No 450/2009 On active and intelligent materials and articles intended to come into contact with food 41
(EU) No 10/2011 Plastic materials and articles intended to come into contact with food 42
(EU) No 1169/2011 On the provision of food information to consumers 43
(EU) 2015/2283 On novel foods 44
J. Food Prot., Vol. 85, No. 2 NANOPARTICLES AND FOOD TOXICITY 359

FIGURE 1. A diagram illustrating organs

relevant to the digestive system and the
determined quantities of Si and Ti partic-
ulate matter found in the human liver,
spleen, jejunum, and ileum organs are
indicated, as reported by Peters et al. (89).

different properties may lead to different toxicological safety evaluations for these materials. Guidance documents
properties. These substances, therefore, should be assessed have also been issued to streamline and unify the various
on a case-by-case basis by the authority regarding their risk aspects to be considered in evaluating these NPs (62),
until more information is known about this new technolo- further emphasizing the need for more in-depth studies
gy.” It is made clear that authorizations on the basis of the investigating the toxicity effects of ingested NPs.
risk assessment of the conventional particle size of a
substance does not cover that of engineered NPs. Also, NPs NPS AND THE DIGESTIVE SYSTEM
should not be covered by the functional barrier concept The digestive system is responsible for the physiolog-
applied for food contact materials to prevent the migration ical processes of digestion, absorption, motility, and
of substances into foods (42). secretion, including that of NPs ingested through foods.
The most recent legislation Regulation EU 2015/2283 Daily, about 8 L of fluid passes through the gastrointestinal
(44, p. 2) states that “substances that give rise to significant tract in addition to 2 L of solids and liquids ingested by the
changes in the composition or structure of a food, affecting average adult (103). Almost 90% of this fluid is processed
its nutritional value, metabolism, or presence of undesirable and absorbed before entering the large intestine and is
substances” should also be considered as novel foods. It mostly absorbed before the colon. The remaining undigest-
further states that “limited information is available regard- ed solids and substances excreted are between 5 and 10% of
ing nanotoxicokinetics, the toxicology of engineered nano- the daily ingested quantities. Optimal metabolic function-
materials, and existing toxicity testing methods may need ality of the digestive system also depends on it being
methodological modifications” (44, p. 5). In this regard, the supplied with sufficient oxygenated and nutrient-rich blood
Organisation for Economic Co-operation and Development (103), which is supplied through the splanchnic circulation
concluded that approaches for the testing and assessment of through the celiac artery, the superior mesenteric artery, and
traditional chemicals are, in general, appropriate for the inferior mesenteric artery. The various digestive system
assessing the safety of nanomaterials but may have to be organs transfer this to the portal vein, which passes through
adapted to the specificities of nanomaterials (44). the liver. The portal vein supplies about 80% of blood to the
Other regulations that are relevant for the use of NPs in liver, while the remainder is from the celiac artery via the
foods are Regulations EC No 258/97 (36), EC No 178/2002 hepatic artery. The hepatic veins send back this blood to the
(37), EC No 1935/2004 (38), and EC No 282/2008 (39), heart via the vena cava, recirculated throughout the body.
which look at novel foods and ingredients and the role of Therefore, the digestive system plays an important role in
the European Food Safety Authority in implementing food the absorption and distribution of nanoformulated foods
safety laws and materials that come in contact with food, within the body. Some organs of note that are relevant to the
including recycled materials. The European Food Safety digestive system are shown in Figure 1.
Authority has issued scientific opinions and reevaluations Several studies have focused on the toxicity of organic
regarding food additives that might contain NPs of NPs due to their extensive use in food products and health
equivalent chemical composition. Since 2015, the scientific applications. The studies have confirmed that organic NPs,
opinions and reevaluations regarding TiO2 (E171) (4, 124, in general, are nontoxic, and do not accumulate in the body,
126), iron oxides and hydroxides (E172) (1), silver (E174) as they are biodegradable. In a study by Frenzel et al. (51),
(3), gold (E175) (2), and silicon dioxide (E551) (125) food quercetin-loaded, whey protein isolate–coated liposomes
additives suggested the need of further toxicological studies were observed to successfully deliver water insoluble core
due to the absence of toxicity data and characterization of materials, showing good stability and nontoxicity as a food
NPs present in these additives and to provide definitive additive. Proteins were also investigated in a study by Yi et
360 DE OLIVEIRA MALLIA ET AL. J. Food Prot., Vol. 85, No. 2

al. (122) in which a protein-based β-carotene nanocarrier of particular importance when considering the accumulation
was developed, showing very low cytotoxicity while of nonabsorbed foodborne NPs, as they can directly interact
yielding an increased cellular uptake of β-carotene. with the microorganisms present apart from the animal
However, in certain cases, the methods used to prepare cells.
NPs affect the resultant cytotoxicity. This was shown by In a study by Taylor et al. (109), cerium (IV) oxide
Shin et al. (101) when comparing the “ethanol injection” (CeO2), TiO2, and ZnO NPs were compared, and the effect
method and “dry thin film” method. The study showed that on the gut microbial community was investigated. This
toxic organic solvents used for the dry thin film method revealed significant differences in several phenotypic traits
restricted the use in food-grade liposomes. Similarly, in compared with an untreated community, including hydro-
nanoemulsions, the use of large quantities of surfactants phobicity and electrophoretic mobility. It was observed that
may result in increased cytotoxicity (46, 104). However, the community’s stability when treated with TiO2 NPs
this has been shown to depend on the surfactant used and caused extended changes in hydrophobicity. Similar studies
the amount, as shown by Sessa et al. (99). They produced a (16, 47) showed that the changes were deemed nonlethal yet
Tween 20 and glycerol monooleate–based nanoemulsion significant enough to affect the properties of the organisms
and evaluated them on Caco-2 cells with no cytotoxicity microbial community.
observed. Some studies have been undertaken to investigate The inorganic Ag NPs stand out as known antimicro-
the presence and impact of NPs that could have been bial agents and have been the focus of several studies. In a
derived through oral administration in humans. A study by study by Fröhlich and Fröhlich (53), the cytotoxicity of Ag
Jones et al. (70) showed no significant absorption of Ti after NPs and ZnO NPs on enterocytes and bacteria was
the oral administration of TiO2 NPs by nine human investigated and revealed Ag NPs to be antimicrobial
volunteers. The study reported that agglomeration of these agents that selectively damage Escherichia coli, while ZnO
NPs of various particles sizes in simulated gastric fluids NPs specifically damaged enterocytes more at lower
might have inhibited absorption during in vivo ingestion. concentrations than E. coli. Cueva et al. (22) managed to
Postdose Ti levels in urine and blood were not significantly create a model system simulating the gastrointestinal
different from predose levels. Another study by Rompelberg digestion of food in which Ag NPs were observed to
et al. (96) investigated the potential intake of TiO2 NPs undergo several modifications as they passed through
through several dietary and oral sources across the age simulated gastric fluids. It was observed that the composi-
groups of 2 to 6, 7 to 69, and 70þ years in the Dutch tion and activity of the intestinal microbiota were not
population. Chewing gum, coffee creamer, milk, and sauces noticeably affected. A similar study (13) focused on Ag NPs
were the main dietary contributors for the intake of TiO2 and their interaction with intestinal microbiota by using in
NPs, while for the age group of 2 to 6 years, old toothpaste vitro batch fermentation models inoculated with human
was the main contributor. The source of these particles was fecal matter. The core bacterial community was shown to be
through indirect environmental sources or feed to milk unaffected; however, a nonlethal concentration of Ag NPs
transfer. was shown to negatively affect the bacteria: Faecalibacte-
A recent study by Peters et al. (89) reported the rium prausnitzii and Clostridium coccoides–Eubacterium
amounts of SiO2 and TiO2 NPs in several postmortem rectales taxa in the fermentation cultures.
human organs. These particles were confirmed by using
scanning electron microscopy with energy dispersive x-ray NPS AND HUMAN HEALTH RISK ASSESSMENT
detection and inductively coupled plasma mass spectrom- Risk assessment typically consists of hazard identifi-
etry. Organs assessed were the liver, spleen, kidney, cation and characterization, exposure assessment, and risk
jejunum, and ileum. Particulate quantities determined in characterization (49). A pollutant or substance may be
the liver, spleen, jejunum, and ileum are shown in Figure 1. extraordinarily hazardous but have a small human exposure
It was reported that SiO2 particulates accounted for 10% of potential; therefore, the resultant risk may be small.
total Si content. These ranged between 0.2 to 25 mg of Si However, a hazard may have limited toxicity, but the
(in particulate form) per kg of tissue, with particle sizes human exposure is high, and over long periods, the pollutant
between 250 to 400 nm. Particles of TiO2 accounted for may pose a much greater risk. Hence, it is essential to
about 80% of the total Ti content within these tissues. These characterize both the potential impact of the hazard and the
ranged between 0.01 to 1.8 mg of Ti (in particulate form) likelihood or amount of human exposure for a risk
per kg of tissue, with particle sizes between 50 and 500 nm. assessment study (49). Uncertainties exist around the risk
These results reported for the liver and spleen were assessment and exposure assessment of NPs due to limited
comparable with those reported by Heringa et al. (65). information on several aspects, including toxicity, behavior,
The authors of both publications indicate that the main and bioaccumulation (23). It is, therefore, important when
source of these particles and the accumulation within the assessing and considering the potential toxicity of these
liver and spleen was most likely through food, toothpaste, or NPs; dosage, bioaccumulation, and metabolic rates have to
medicines. be synergistically regarded.
The toxicity of NPs is often associated with chemical
GUT CYTOTOXICITY OF NPS
reactivity; for example, some inorganic NPs dissolve and
The cytotoxicity of NPs within the gut system release ions that promote undesirable chemical or biochem-
encompasses both toxicities of the cells of the target animal ical reactions (e.g., Ag NPs), whereas others are relatively
and microbiota response populating these surfaces. This is inert (e.g., TiO2 NPs) (78). It has been reported that
J. Food Prot., Vol. 85, No. 2 NANOPARTICLES AND FOOD TOXICITY 361

positively charged NPs were more toxic than negative or pastries, biscuits, sauces, dressings, spreads, cheese, or
neutral NPs (6). Insoluble NPs are more readily taken up even fish products, to give a white background color (7).
across the human body’s intestinal barrier and can be more Possible health effects are related to the use of E171 as a
immediately bioavailable. The uptake of NPs is determined food additive, which highlighted the importance of
mainly by particle solubility, charge, and size. Smaller examining immune toxicological effects in addition to
diameter NPs are more likely to be absorbed (49). Note that potential reprotoxicological effects (33). The FDA has
on the one hand, inorganic NPs have different tendencies to limited the total amount of E171 to 1% TiO2 per weight of
dissolve under specific solution conditions (pH and ionic food (114). In addition, the French Agency for Food,
strength) and chemical reactivities, significantly impacting Environmental and Occupational Health and Safety sus-
gastrointestinal fate and toxicity (6). pended the use of E171 (food-grade TiO2) in France (50).
More recently the European Food Safety Authority (33) has
Hazard identification. The physical and chemical updated its safety assessment of the food additive TiO2
behavior of NPs within the human body is not fully (E171), following a request by the EC in March 2020.
understood and requires further hazard investigation (75).
However, the primary pathways of human exposure were Exposure assessment. The sustainable development of
identified as inhalation (including intratracheal), ingestion nanotechnology requires a thorough knowledge of the life
(gavage or food), and dermal contact (75). The potential cycle of synthesized NPs, including environmental release,
health consequences of ingestion of NPs may cause Crohn’s deposition, exposure, and potential health risks (123). The
disease and colon cancer (6). Hemorheology suggests that life cycle of NPs can be assessed as fabrication stage .
once the NPs enter the bloodstream, blood circulation cells stabilization stage . effect of microenvironment during
and other components, such as serum proteins and application . degradation . metabolism . excretion (32).
coagulation factors, are exposed to NPs and may result in Human exposure to NPs through the digestive system is the
cardiovascular disease (30). Once within the bloodstream, it main focus of this section. Representative studies are
was determined that for medium- and high-dose groups presented in Table 4. Very few studies looked at the entire
(300 and 1,000 mg kg1), Ag NPs accumulated in the probabilistic approach of the quantitative human exposure
following descending order, stomach . liver . kidneys . model. Also, the experiment-based probabilistic models are
lungs . testis . brain . blood. When administrated with a mostly conducted on Ag, Cu, and TiO2 NPs only. Ag and
low dose (30 mg kg1), the following descending order was Cu NPs assessments are based on migration studies,
determined, stomach . kidneys . testis . liver . brain . whereas TiO2 assessments are based on the inherent
lungs . blood (75). concentration of engineered NPs in food and cosmetics
Systematic tools such as NanoRiskCat, a nanomaterial products. Thus, a more unified risk assessment model is
database developed by the Danish Ecological Council and required in this field.
Danish Consumer Council (27), and Hansen et al. (60, 61) In addition, a sensitivity analysis (24) can help a
can assist in the determination of NP hazard identification in probabilistic model investigate the influential parameters of
consumer products. A data filter strategy (NanoRiskCat . migration studies such as percent fill, storage duration, and
search database . filter; categories: food and beverage; temperature. Similar research can be conducted for other
potential exposure pathways . oral; CPDAT . food NPs. Peters et al. (90) raised the need to extend the exposure
contact) was used to narrow down the list of products in assessment nodes, as they indicated that the fate of TiO2
which NPs are used. (The Chemical and Product Database particles in the human digestive tract is unknown.
[CPDAT] is a filter search option available on the Nano- Therefore, the net and cumulative retention data after the
RiskCat Web site (27).) On the basis of the observation of 34 metabolism and excretion process can improve the
products, TiO2 and Ag NPs both appeared on 10 occasions. understanding of the fate of NPs inside the human body.
According to Regulation EC 1333/2008 (40), TiO2 is
authorized as a food additive (E171) in the EU in quantum Hazard characterization. NPs may cause significant
satis in 51 food categories. TiO2 is mostly used in chocolate reactive oxygen stress to the living cells, resulting in
products, and Ag NPs are used in packaging and surface cytotoxicity (127). Cytotoxicity is a vast domain, and it can
protection due to antimicrobial properties, as reported in the be categorized into specific hazard end points, such as
literature. Overall observation suggests that humans can be hepatotoxicity, nephrotoxicity, pulmonary toxicity, gastro-
exposed to NPs through food directly, and indirect exposure intestinal toxicity, cardiotoxicity neurotoxicity, reprotoxic-
includes cooking utensils, mostly the coating on the frying ity, and embryotoxicity (75). Therefore, there is a need for a
pan to make it nonstick and packaging products. With a few standard parameter to compare or rank different toxicities.
exceptions, TiO2 was categorized as high risk in both Human equivalent dose can be such a parameter; it can be
components of risk assessment, such as exposure (profes- derived from animal studies, and it is a function of animal
sional, consumers, environment) and effects (human, dose, animal correction factor, and human correction factor
environment). In contrast, the exposure to Ag NPs was (75). On the basis of the duration of exposure, toxicity can
classified as medium, whereas the effect was labeled as high. be categorized as acute (single dose), subacute (14 to 28
TiO2 is a white powder, mainly used in products days), subchronic (90 days), and chronic (180 days, rodent;
including chewing gum, ice cream, and confectionery 270 days, nonrodent) (29). Li and Cummins (75) collated
products, such as candies, chocolate products, cakes, acute, subacute, and subchronic toxicity data on Ag NPs on
TABLE 4. Representative exposure assessment studies of NPs through the food pathway
362

No. NP(s) Methodology Scope Migration Level on food Remarks Reference

3 Ag Multifactorial design, Migration of silver from Yes; percentage fill, time, Migration was found to occur Ag migration from the 24
probabilistic plasticized polyvinyl temperature dependent within a range of 0.03–8.4 mg nanocomposite to the food
DE

mathematical human chloride nanocomposites kg1 surface was influenced most by

exposure assessment to chicken meat, the percentage fill, followed by
model following varying storage time and storage
storage time and temperature (negative correlation)
temperature conditions
4 Ag, Cu Experimental design and Migration from Yes Migration of Ag 0.0030.005 mg Still considerable uncertainty 25
parameterization, polyethylene dm2, Cu 0.0240.049 m dm2; regarding the toxicity of materials
nanocomposite nanocomposites to food human exposure Ag 5.89 3 105 at the nanoscale; hence, exposure
preparation, migration and an associated 8.9 3 105 mg kg bw1 day1, estimates should be reevaluated
OLIVEIRA MALLIA ET AL.

test, probabilistic human exposure assessment Cu 2.26 3 1051.17 3 104 mg when more toxicity data become
exposure model kg bw1 day1 (95th percentile available
range)
5 Ag Ag quantification in Evaluate silver migration Yes Mean Ag migration from Agion Through careful polymer 26
food simulants after from nanosilver and composites (n ¼ 12) ,0.0011.50 engineering, composites that
migration tests with Agion (commercially 3 102, mg L1; mean Ag conform to European
laboratory-manufactured available filler) migration from Ag NP composites nonauthorized substance
composites polyethylene composites (laboratory) 4.65 3 1020.38 mg migration limits can be
to food simulants by L1, and 8.92 3 102 and 5.15 3 developed, while being cognizant
using analytical and 102 mg L1 for Hach-Lange of food pH and percentage fill
imaging techniques spectrophotometry and inductively rates
coupled plasma atomic emission
spectroscopy, respectively
7 Cu (also Probabilistic human Migration of NPs from Time-dependent Ag: 1.41, SD 0.29; Cu: 1.06, SD 0.22 Migration levels were found to 59
ionic form), exposure model looking at packaging to human migration: Ag (0.6%): (mg kg food1); partition exceed the EC regulatory limit
Ag (also NP migration from food exposure through food 0.46, SD 0.19; coefficient Ag: 2.18; Cu: 0.30 for unauthorized substances of
ionic form) packaging (EU 10/2011) consumption Cu (0.6%): 0.82, SD 0.01 mg kg1
0.08 (mg kg food1)
9 TiO2 Electron microscopy, mass 7 TiO2 materials, 24 food No 0.02–9.0 mg TiO2 g1 product, 5 The fate of TiO2 particles in the 90
spectrometry products, and 3 personal 10% of the particles in these human digestive tract is unknown
care products products had sizes below 100 nm
13 Ag Experiment-based NP Migration of NPs from Time-dependent Worst case: acute exposure to 4.2 μg of Negligible NP exposure in 117
migration study (food packaging to food migration: 11.9 (SD Ag can result from storage of 100 comparison with the background
and water) 2.4), 9.7 (SD 1.6), 23 mL of food in a new Ag-doped box exposure to Ag for the general
(SD 5.1), ,0.1, ,0.1, of normal size (calculated for 30 ng population
37.1 (SD 1.2) μg g1 cm2 migration from a 140-cm2
of polypropylene surface)
15 TiO2 Mechanistic human health Human exposure to NPs 6–12 mg g1 (dry weight) in some Consumption of selected food 123
exposure assessment of from selected seafood white-colored seafood products products may be an essential
NPs through selected products (squid and cuttlefish); relatively route for TiO2 NP uptake,
seafood products (jellyfish, low concn were observed in especially for younger people
J. Food Prot., Vol. 85, No. 2

squid, and cuttlefish) jellyfish 1–3 mg g1 aged 20–30 yr

J. Food Prot., Vol. 85, No. 2 NANOPARTICLES AND FOOD TOXICITY 363

the basis of animal studies available in the literature. It was regarding the use of animal in vivo assessments of novel
also the first study to provide a full picture of the dose- compounds (11, 93). The minimization for the use of animal
response relationships of potential hazards to humans models is becoming more relevant and in favor of the
resulting from Ag NP exposure. Oral exposure was found implementation of alternative approaches in line with the
to be the most critical exposure pathway for most of the replacement, reduction, and refinement principle (69, 71).
toxic end points. Other than acute toxicity on major organs, Several other nonhuman in vivo studies reported the
including the liver, kidney, and lungs, the potential for acute fates of NPs consumed through oral administration. For the
neurotoxicity is also an issue of concern, arising from small last 5 years, research efforts predominantly focused on
long-term doses (75). assessing TiO2 NPs characterization and toxicity (5, 14, 15,
For in vitro assessments that were carried out on NPs 17–20, 45, 57, 70, 79, 80, 107). There is interest in focusing
that can be consumed through the ingestion of foods, these on TiO2 because it is a common food additive E171 that has
studies are illustrated in Table 5 and mostly focus on TiO2 a fraction of about 36% consisting of NPs (120, 121).
NPs. However, interest in Ag and SiO2 NPs was noted.
Recent studies have reported that this could be higher than
Most studies report essential physicochemical characteris-
60% (54, 115). Several studies that evaluated TiO2 in vivo
tics, such as size, shape, crystal structure, elemental
are presented in Table 6. Although most studies provide the
composition, purity, hydrodynamic diameter, and zeta
required basic characterization specifications, such as size,
potential of the assessed NPs. Nevertheless, critical data,
shape, crystal structure, hydrodynamic diameter, and zeta
such as specific surface area, surface functionalization, and
metal ion leaching capacity, are not always reported. potential, specifications, such as specific surface area and
Differences between reactive surfaces of NPs with similar polydispersity index, are not always reported. Assessment
chemical composition might induce different responses and of the metal ion leaching capacity of the NPs should be
have been indicated as an essential parameter in comparing further investigated and reported due to the acidic
and understanding them (9, 72). Exposure conditions for the environments of the digestive system. The release of metal
studies illustrated in Table 5 mostly investigated concen- ions within these acidic environments might lead to a
tration ranges between 1 to 300 μg mL1, with exposures different mode of interaction. The further use of aggressive
lasting between 2 to 72 h. Note that a considerable number dispersion methods, such as ultrasound sonication, might
of studies predigested the NPs in liquids to mimic gastric also cause the release of metal ions. Note that within this
fluids and even assessed them in the presence of other food selection of studies, only one report was found that
compounds (12, 28, 76, 92, 105). Studies mostly reported investigates the interaction of TiO2 administered through
NP uptake, distribution, and cell viability as general end solid feed (80).
points for the toxicity effects of NPs. Other end points, such Throughout the studies in Table 6, it was observed that
as reactive oxygen species, DNA damage, cell cycle, a considerable elevated presence of Ti was found within the
oxidative stress, and gene expression, were reported to liver, stomach, intestines, and colon. Other organs that have
understand the observed toxicity interactions further. accumulated titanium were the spleen, pancreas, and
Cell lines reported in the various studies illustrated in kidneys. The most frequently assessed and reported toxicity
Table 5 mostly used the human colorectal adenocarcinoma end points were biochemical assessments of blood or tissue
Caco-2 cell line. An increasing interest was noted toward lysates to assess specific organ functionality within these
using Caco-2 cells in conjunction with human lymphocyte various studies. It is not always the case that changes in the
Raji B cells (73) and human mucus-secreting epithelial Ti content of blood or organs postexposure are reported,
HT29-MTX cells (12, 28, 31, 118) to obtain more complex limiting the comparisons between studies. The inclusion of
coculture model systems that resemble more closely in vivo DNA damage quantification assessments, such as the in
digestive cellular environments. Recently, studies (64, 91)
vivo mammalian erythrocyte micronucleus (85) and alka-
have been conducted by using the EpiIntestinal model to
line comet assay (86), would further add value to the
assess NP responses with a three-dimensional in vitro model
toxicity assessments of these NPs. These should be
that closely mimics the human in vivo intestine. This three-
complemented by oxidative stress and inflammatory
dimensional cellular model possesses a columnar epitheli-
cytokine assessments.
um with villi structures, brush borders, and tight junctions,
which closely imitate in vivo intestinal function. The
cellular morphology present within this model allows for Risk characterization. Risk characterization is the
spatial interactions and phenotypic crosstalk that is not final step of the risk assessment paradigm (Fig. 2A). It
present in two-dimensional cellular monolayers. Both combines exposure assessment and hazard characterization
studies reported that this closer in vivo resemblance makes stages and evaluates the final risk, conducting a series of
the reported end points more relevant to the risk assessment model analyses such as scenario and sensitivity analysis to
of the NPs assessed. The study by Henson et al. (64) also capture variability and uncertainty at each node of the
assessed NP responses by using rat small intestine epithelial model (48). A proposed risk assessment framework is given
IEC-6 cells and concluded that the different responses (Fig. 2B).
observed from using the EpiIntestinal model could have Assessment of the daily intake of NPs through food
been due to animal to human species differences. These products can also be assessed through the daily dietary
variations have been the subject of debate and concern index (DDI) on the basis of equation 1 (58, 111).
364 DE OLIVEIRA MALLIA ET AL. J. Food Prot., Vol. 85, No. 2

TABLE 5. In vitro studies focused on the potential effects of NPs might induce during ingestiona
NP studied Cells used Exposure conditions Assessed end points Reference

Type: TiO2 Caco-2 Concn used: 50 and 100 μg NP distribution 70

Reported specifications: of TiO2 per mL
Size and shape Medium: cell culture medium
Elemental composition and purity dosed with sonicated
Polydispersity index particulate suspension
Duration: 3 h
Type: Ag Caco-2 Concn used: 20–100 μg of Metal content in culture media and cells 76
Reported specifications: Ag per mL NP uptake and distribution in cells
Size and shape Medium: cell culture medium Cell viability
Metal ion leaching potential dosed with undigested and
digested particulate
suspensions and digested
with food components and
particulate suspensions
Duration: 24 h
Type: TiO2 Caco-2 Concn used: 50 and 200 μg Cell viability 105
Reported specifications: of TiO2 per mL Cell proliferation
Elemental composition and purity Medium: cell culture medium Reactive oxygen species generation
Size and shape dosed with digested food NP uptake and distribution in cells
Hydrodynamic diameter components and particulate
Zeta potential suspensions
Metal ion leaching potential Duration: 2, 4, and 24 h
Type: SiO2 Caco-2 Concn used: 250 μg of SiO2 NP distribution 73
Reported specifications: Raji B per mL Cell viability
Crystal structure Medium: cell culture medium
Size and shape dosed with particulate
Hydrodynamic diameter suspensions
Zeta potential Duration: 6 h
Surface functionalization
Type: Fe2O3 Caco-2 Concn used: 0.05 and 0.1% Metal content in culture media and cells 28
Reported specifications: Raji B (w/w) Fe2O3 NP uptake and distribution in cells
Crystal structure HT29-MTX Medium: cell culture medium Cell viability
Size and shape dosed with digested food Reactive oxygen species
Specific surface area components and sonicated
Hydrodynamic diameter particulate suspensions
Zeta potential Duration: 2 and 4 h
Type: Ag, Au, CuO, TiO2, and ZnO HT29 Concn used: 2–10 μg of Metal content in culture media and cells 98
Reported specifications: NP per mL NP uptake and distribution in cells
Elemental composition and purity Medium: cell culture medium Cell viability
Hydrodynamic diameter dosed with sonicated DNA damage
Zeta potential particulate suspension Cell cycle
Metal ion leaching potential Duration: 24 h
Type: Iron phosphate (FePO4), SiO2 HCECb Concn used: 37.5 and 75 μg Cell viability 118
Reported specifications: HT29 of NP per mL or 0.01–2.5 Cellular oxidative stress
Specific surface area HT29-MTX mM Fe NP uptake and distribution in cells
Crystal structure Medium: cell culture medium
Size and shape dosed with sonicated
Hydrodynamic diameter particulate suspension
Zeta potential Duration: 48 h
Type: TiO2 MKN-45 Concn used: 10–50 μg of Cell viability 83
Reported specifications: TiO2 per mL Cell cycle
Crystal structure Medium: cell culture medium Cell migration
dosed with sonicated
particulate suspension
Duration: 24, 48, and 72 h
Type: Ag Caco-2 Concn used: 1–100 μg of Ag Cell viability 116
Reported specifications: NP per mL NP uptake and distribution in cells
Size and shape Medium: cell culture medium DNA damage
Hydrodynamic diameter dosed with sonicated Gene expression
Zeta potential particulate suspension
Crystal structure Duration: 24 h
J. Food Prot., Vol. 85, No. 2 NANOPARTICLES AND FOOD TOXICITY 365

TABLE 5. Continued
NP studied Cells used Exposure conditions Assessed end points Reference

Type: TiO2 Caco-2-GFP Concn used: 10–250 μg Cell viability 31

Reported specifications: HT29-MTX of TiO2 per mL Reactive oxygen species
Size and shape Medium: cell culture medium DNA damage
Specific surface area dosed with particulate Gene expression
Hydrodynamic diameter suspension
Zeta potential Duration: 6, 48, and 72 h
Polydispersity index
Type: TiO2 Concn used: 42 and 84 μg of
Caco-2 NP uptake and distribution in cells 88
Reported specifications: TiO2 per mL Metal content in culture media and cells
Size and shape Medium: cell culture medium Cell viability
Specific surface area dosed with particulate Gene expression
suspension
Duration: 4, 24, and 48 h
Type: TiO2 Caco-2 Concn used: 150 and 300 μg Cell viability 12
Specifications reported in another Raji B of TiO2 per mL Reactive oxygen species
article (74): HT29-MTX Medium: cell culture medium Proteomic analysis
Size and shape dosed with digested food
Crystal structure components and sonicated
Specific surface area particulate suspensions
Elemental composition and purity Duration: 6 or 24 h
Surface functionalization
Type: TiO2 Caco-2 Concn used: 100 μg of TiO2 NP distribution 92
Reported specifications: HepG2 per mL Cell viability
Elemental composition NL20 Medium: cell culture medium DNA damage
Size and shape A-431 dosed with digested food Gene expression
Hydrodynamic diameter components and sonicated
Zeta potential particulate suspensions
Duration: 24 and 72 h
a
The table outlines the type of NP studies and reported specifications, type of cells used, exposure conditions, and assessed end points to
elucidate the potential in vitro toxicity effects.
b
HCEC, human corneal epithelial cells.

C PROPOSED RISK ASSESSMENT STRATEGIES

DDI ¼ A 3 B 3 ð1Þ
BW AND CONCEPTUAL MODELS
where A is the NP content in food products (mg kgdry
1 1. Future toxicity studies should integrate the effects of
weight ), B is the daily intake of food products (kgwet weight
day1), C is the conversion factor (0.085 is to convert fresh different properties of NPs in determining cytotoxicity.
Such properties include aggregation or agglomeration
vegetable weight to dry weight), and BW is the average
state, elemental composition, mass concentration, particle
human body mass (kg). The DDI can be compared with the
number concentration, shape (aspect ratio), size and size
reference dose (RfD), which is the oral reference dose,
distribution, (water) solubility and dispersibility, specia-
specific to the study (mg kg1 day1). Further, the health
tion, structure, surface area (and porosity), crystallite
risk index (HRI) can be calculated by equation 2 (58). If the phase crystallite size, surface charge, surface chemistry
value of HRI is ,1, the exposed population is said to be (32, 110).
safe. 2. For all in vivo applications, biodegradable and biocom-
DDI patible nanomaterials should be used with defined
HRI ¼ ð2Þ metabolism and excretion.
RfD
3. Nondegradable and nonbiocompatible NPs can be used
If the model’s inputs are variable instead of fixed value, for in vitro applications, such as diagnostics and
a probabilistic model may be used (82). A predictive industrial use. However, the potential for environmental
model’s most sensitive parameter can be identified in the contamination and hazards should be clearly defined.
risk characterization stage by analyzing Spearman’s rank The use of such NPs in food manufacturing has
order correlation coefficient in the sensitivity analysis limitations.
applications (21). This approach can be useful to understand 4. Nanomaterials for agricultural use must be assessed
the effects of natural variability and uncertainty of the carefully due to the possibility of entering the food chain
model input parameters on the model outputs. and affecting the environmental ecosystem.
TABLE 6. In vivo studies conducted for TiO2 NPs outlining specifications, in vivo animal model used, mode of oral administration, reported organs of concern, and assessed end points to elucidate
366

the potential NP in vivo toxicity effects

Reported organs
Reported TiO2 NP parameters In vivo animal model Mode of oral administration of concern Assessed end points Reference
DE

Size and shape 3-wk-old Sprague Mode: Oral gavage Liver, kidney, heart, Whole blood cell count 18
Crystal structure Dawley rats Medium: sonicated ultrapure spleen, ovaries, and Biochemical organ function assessments
Elemental composition and purity with occasional glucose testis Histopathological analysis
Size and shape supplementation
Hydrodynamic diameter
Specific surface area
Surface functionalization
Zeta potential
Size and shape 6-wk-old CD-1 mice Mode: oral injection via Liver, kidney and The titanium content in blood and organ tissues 57
OLIVEIRA MALLIA ET AL.

Hydrodynamic diameter syringe spleen, heart, Plasma glucose and insulin quantification
Crystal structure Medium: stirred unspecified stomach, and Inflammatory cytokine quantification
Zeta potential liquid suspension pancreas Oxidative stress quantification
Metal ion leaching potential Insulin resistance assessments
Histopathological analysis
Size and shape 10- to 12-wk-old Mode: Oral gavage Stomach The titanium content in organ tissues 79
Crystal structure Swiss Webster mice Medium: sonicated deionized DNA damage quantification
Hydrodynamic diameter distilled water Oxidative stress quantification
Zeta potential Histopathological analysis
Size and shape 7-wk-old CD-1 mice Mode: oral gavage Liver, spleen, lung, The titanium content in blood and organ tissues 15
Crystal structure Medium: unspecified liquid kidney, stomach, Whole blood cell count
Hydrodynamic diameter suspension duodenum, ileum, Disease activity index and colon length
Zeta potential jejunum, and colon Intestinal permeability
Gut microbiota
Histopathological analysis
Size Swiss albino mice Mode: oral administration Liver Biochemical organ function assessments 5
Shape, chemical purity, and forma Medium: dissolved in Oxidative stress quantification
physiological saline DNA damage quantification
Histopathological analysis
Size and shape 7- to 8-wk-old Swiss Mode: oral administration Lungs, heart, liver, Biochemical organ function assessments 14
albino mice Medium: water suspension and kidneys Whole blood cell count
DNA damage quantification
Histopathological analysis
Size and shape 3-wk-old Sprague Mode: oral gavage Liver Biochemical organ function assessments 19
Elemental composition and purity Dawley rats Medium: sonicated ultrapure Inflammatory cytokine quantification
Crystal structure water suspension Oxidative stress quantification
Specific surface area Gut microbiota
Hydrodynamic diameter Metabonomic analysis
Zeta potential Histopathological analysis
J. Food Prot., Vol. 85, No. 2
TABLE 6. Continued
Reported organs
Reported TiO2 NP parameters In vivo animal model Mode of oral administration of concern Assessed end points Reference

Size and shape 3-wk-old Sprague Mode: oral gavage Liver The titanium content in organ tissues 20
Elemental composition and purity Dawley rats Medium: sonicated ultrapure Biochemical organ function assessments
Crystal structure water suspension Whole blood cell count
Specific surface area Inflammatory cytokine quantification
J. Food Prot., Vol. 85, No. 2

Hydrodynamic diameter Oxidative stress quantification

Zeta potential Histopathological analysis
Size and shape 6- to 8-wk-old Swiss Mode: intraperitoneally Liver and kidney DNA damage quantification 45
Crystal structure mice Medium: sonicated distilled Oxidative stress quantification
Specific surface area water suspension
Hydrodynamic diameter
Zeta potential
Polydispersity index
Size and shape C57BL/6J mice Mode: ad libitum Mesenteric lymph Immune cell counts 80
Elemental composition and purity Medium: added in the solid nodes and colon Colon length
Crystal structure feed mix Gut microbiota
Polydispersity index
Size and shape 8-wk-old NFR mice Mode: oral administration Lung, liver, stomach, The titanium content in blood and organ tissues 107
Elemental composition and purity Medium: water suspension spleen, kidney, Oxidative stress quantification
Crystal structure brain, testes, and Inflammatory cytokine quantification
Hydrodynamic diameter whole intestine Histopathological analysis
Zeta potential
Polydispersity index
Size and shape 3-wk-old Sprague Mode: oral gavage No organs harvested Biochemical organ function assessments 17
Elemental composition and purity Dawley rats Medium: sonicated distilled Metabolomics analysis
Crystal structure water suspension Oxidative stress quantification
Specific surface area
Hydrodynamic diameter
Zeta potential
Size and shape C57BL/6JRj mice Mode: oral gavage and Lung, liver, spleen, The titanium content in blood and organ tissues 81
Hydrodynamic diameter oropharyngeal aspiration and kidney Whole blood cell count
Zeta potential Medium: liquid dispersion Biochemical organ function assessments
Polydispersity index Oxidative stress quantification
DNA damage quantification
a
Mentioned but not specified.
NANOPARTICLES AND FOOD TOXICITY
367
368 DE OLIVEIRA MALLIA ET AL. J. Food Prot., Vol. 85, No. 2

or vice versa, the resultant pH, NP concentration, ionic

strength, toxicity nature, dissolution or zeta potential of the
suspension changes (87). Not enough data are available to
bridge the relationship between exposure assessment and
hazard characterization. Therefore, limited studies are
available on exposure assessments only. In contrast, hazard
characterization, or the toxicity studies, especially for Ag
NPs have been extensively performed. Risk characterization
is the final step toward completing a risk assessment in
which scenario and sensitivity analysis of several NPs will
need to be performed before being included in food
applications. Developing a more unified risk assessment
methodology with further enhanced food-based NP model-
ing techniques and data sets would help advance knowledge
in this area.

REFERENCES
1. Aguilar, F., R. Crebelli, A. Di Domenico, B. Dusemund, M. J.
Frutos, P. Galtier, D. Gott, U. Gundert-Remy, C. Lambré, J.
Leblanc, O. Lindtner, P. Moldeus, A. Mortensen, P. Mosesso, A.
Oskarsson, D. Parent-Massin, I. Stankovic, I. Waalkens-Berendsen,
R. A. Woutersen, M. Wright, and Y. Maged. 2015. Scientific
Opinion on the re-evaluation of iron oxides and hydroxides (E 172)
as food additives. EFSA J. 13:4317.
2. Aguilar, F., R. Crebelli, A. Di Domenico, B. Dusemund, M. J.
Frutos, P. Galtier, D. Gott, U. Gundert-Remy, C. Lambré, J.
Leblanc, O. Lindtner, P. Moldeus, A. Mortensen, P. Mosesso, A.
Oskarsson, D. Parent-Massin, I. Stankovic, I. Waalkens-Berendsen,
R. A. Woutersen, M. Wright, and Y. Maged. 2016. Scientific
Opinion on the re-evaluation of gold (E 175) as a food additive.
EFSA J. 14:4362.
3. Aguilar, F., R. Crebelli, A. Di Domenico, B. Dusemund, M. J.
FIGURE 2. A framework for risk assessment of NPs through the Frutos, P. Galtier, D. Gott, U. Gundert-Remy, C. Lambré, J.
food pathway, showing the four-tier risk assessment approach (A) Leblanc, O. Lindtner, P. Moldeus, A. Mortensen, P. Mosesso, A.
and the proposed risk assessment framework for NPs (B). Oskarsson, D. Parent-Massin, I. Stankovic, I. Waalkens-Berendsen,
R. A. Woutersen, M. Wright, and Y. Maged. 2016. Scientific
opinion on the re-evaluation of silver (E 174) as food additive.
5. Coating NPs (e.g., with hydrophilic polymer polyethyl-
EFSA J. 14:4364.
ene glycol) can diminish NP toxic effects (6), and this 4. Aguilar, F., R. Crebelli, A. Di Domenico, B. Dusemund, M. J.
concept can be used as a remedial strategy for reducing Frutos, P. Galtier, D. Gott, U. Gundert-Remy, C. Lambré, J. C.
risk. Leblanc, O. Lindtner, P. Moldeus, A. Mortensen, P. Mosesso, D.
6. Inclusion of more advanced in vitro and in silico models Parent-Massin, A. Oskarsson, I. Stankovic, I. Waalkens-Berendsen,
are required to improve current risk assessment ap- R. A. Woutersen, M. Wright, and M. Younes. 2016. Re-evaluation
of titanium dioxide (E 171) as a food additive. EFSA J. 14:e04545.
proaches at the hazard identification and characterization
5. Ali, S. A., M. Z. Rizk, M. A. Hamed, E. I. Aboul-Ela, N. S. El-
stages (34). Rigal, H. F. Aly, and A. Z. Abdel-Hamid. 2019. Assessment of
titanium dioxide nanoparticles toxicity via oral exposure in mice:
CONCLUSIONS AND FUTURE CHALLENGES effect of dose and particle size. Biomarkers 24:492–498.
6. Ameta, S. K., A. K. Rai, D. Hiran, R. Ameta, and S. C. Ameta.
In the current situation, overregulation may drive away
2020. Use of nanomaterials in food science, p. 1–49. Springer,
further developments in nanoscience as vast areas of Singapore.
knowledge have not yet been investigated, while under- 7. Anastasi, E., G. Riviere, and B. Teste. 2019. Nanomaterials in
regulation may potentially expose humans to NPs with food—prioritisation & assessment. EFSA J. 7:10.
unintended adverse health effects. The ionic properties 8. Aragao-Santiago, L., H. Hillaireau, N. Grabowski, S. Mura, T. L.
(cations, anions) of NPs positively influence the nature of Nascimento, S. Dufort, J. L. Coll, N. Tsapis, and E. Fattal. 2016.
Compared in vivo toxicity in mice of lung delivered biodegradable
the toxicity pattern of NPs toward bacteria; however, the
and non-biodegradable nanoparticles. Nanotoxicology 10:292–302.
confirmation in an actual natural setting, such as in rivers 9. Bahl, A., B. Hellack, M. Wiemann, A. Giusti, K. Werle, A. Haase,
and lakes, needs to be established. Ag and ZnO are the most and W. Wohlleben. 2020. Nanomaterial categorization by surface
studied NPs in the laboratory, although TiO2, single or reactivity: a case study comparing 35 materials with four different
multiwalled C nanotubes, and fullerenes have the broadest test methods. NanoImpact 19:100234.
range of applications. There is also uncertainty around the 10. Bamrungsap, S., Z. Zhao, T. Chen, L. Wang, C. Li, T. Fu, and W.
Tan. 2012. Nanotechnology in therapeutics: a focus on nanoparti-
transport, partitioning, degradation, transformation, and
cles as a drug delivery system. Nanomedicine 7:1253–1271.
mutual interaction (environmental fate) of a multimixture 11. Brancato, V., J. M. Oliveira, V. M. Correlo, R. L. Reis, and S. C.
of NPs and the interaction with living organisms. Following Kundu. 2020. Could 3D models of cancer enhance drug screening?
the settling of NPs on natural organic matter or sediments, Biomaterials 232:119744.
J. Food Prot., Vol. 85, No. 2 NANOPARTICLES AND FOOD TOXICITY 369

12. Cao, X., T. Zhang, G. M. DeLoid, M. J. Gaffrey, K. K. Weitz, B. D. and gastrointestinal effects on the biokinetics and cellular toxicity of
Thrall, W. J. Qian, and P. Demokritou. 2020. Evaluation of the ingested engineered nanomaterials. Part. Fibre Toxicol. 14:40.
cytotoxic and cellular proteome impacts of food-grade TiO2 (E171) 29. Denny, K. H., and C. W. Stewart. 2017. Acute, subacute,
using simulated gastrointestinal digestions and a tri-culture small subchronic, and chronic general toxicity testing for preclinical drug
intestinal epithelial model. NanoImpact 17:100202. development, p. 109–127. In S. A. Faqi (ed.), A comprehensive
13. Cattò, C., E. Garuglieri, L. Borruso, D. Erba, M. C. Casiraghi, F. guide to toxicology in nonclinical drug development. Academic
Cappitelli, F. Villa, S. Zecchin, and R. Zanchi. 2019. Impacts of Press, Boston.
dietary silver nanoparticles and probiotic administration on the 30. Dinda, A. 2020. Hemorheology of nanoparticles. nano-SAICM
microbiota of an in-vitro gut model. Environ. Pollut. 245:754–763. Workshop on “Toxicity.” New Delhi, India.
14. Chakrabarti, S., D. Goyary, S. Karmakar, and P. Chattopadhyay. 31. Dorier, M., C. Tisseyre, F. Dussert, D. Beal, M. E. Arnal, T. Douki,
2019. Exploration of cytotoxic and genotoxic endpoints following V. Valdiglesias, B. Laffon, S. Fraga, F. Brandao, N. Herlin-Boime,
sub-chronic oral exposure to titanium dioxide nanoparticles. F. Barreau, T. Rabilloud, and M. Carriere. 2019. Toxicological
Toxicol. Ind. Health 35:577–592. impact of acute exposure to E171 food additive and TiO2
15. Chen, H., R. Zhao, B. Wang, C. Cai, L. Zheng, H. Wang, M. Wang, nanoparticles on a co-culture of Caco-2 and HT29-MTX intestinal
H. Ouyang, X. Zhou, Z. Chai, Y. Zhao, and W. Feng. 2017. The cells. Mutat. Res. 845:402980.
effects of orally administered Ag, TiO2 and SiO2 nanoparticles on 32. European Commission, Directorate-General for Environment and
gut microbiota composition and colitis induction in mice. Nano- University of the West of England Science Communication Unit.
Impact 8:80–88. 2017. Assessing the environmental safety of manufactured nano-
16. Chen, J., S. Zhang, C. Chen, X. Jiang, J. Qiu, Y. Qiu, Y. Zhang, T. materials. European Commission, Directorate-General for Environ-
Wang, X. Qin, Z. Zou, and C. Chen. 2020. Crosstalk of gut ment by the Science Communication Unit, Bristol, UK.
microbiota and serum/hippocampus metabolites in neurobehavioral 33. European Food Safety Authority. 2021. Titanium dioxide: E171 no
impairments induced by zinc oxide nanoparticles. Nanoscale longer considered safe when used as a food additive. Available at:
12:21429–21439. https://www.efsa.europa.eu/en/news/titanium-dioxide-e171-no-
17. Chen, Z., S. Han, P. Zheng, D. Zhou, S. Zhou, and G. Jia. 2020. longer-considered-safe-when-used-food-additive. Accessed 15 July
Effect of oral exposure to titanium dioxide nanoparticles on lipid 2021.
metabolism in Sprague-Dawley rats. Nanoscale 12:5973–5986. 34. European Food Safety Authority, S. Bronzwaer, G. Kass, T.
18. Chen, Z., Y. Wang, L. Zhuo, S. Chen, L. Zhao, T. Chen, Y. Li, W. Robinson, J. Tarazona, H. Verhagen, D. Verloo, D. Vrbos, and M.
Zhang, X. Gao, P. Li, H. Wang, and G. Jia. 2015. Interaction of Hugas. 2019. Food safety regulatory research needs 2030. EFSA J.
titanium dioxide nanoparticles with glucose on young rats after oral 17:e170622.
administration. Nanomedicine 11:1633–1642. 35. European Food Safety Authority Scientific Committee, A. Hardy,
19. Chen, Z., D. Zhou, S. Han, S. Zhou, and G. Jia. 2019. D. Benford, T. Halldorsson, M. J. Jeger, H. K. Knutsen, S. More, H.
Hepatotoxicity and the role of the gut-liver axis in rats after oral Naegeli, H. Noteborn, C. Ockleford, A. Ricci, G. Rychen, J. R.
administration of titanium dioxide nanoparticles. Part. Fibre Schlatter, V. Silano, R. Solecki, D. Turck, M. Younes, Q. Chaudhry,
Toxicol. 16:48. F. Cubadda, D. Gott, A. Oomen, S. Weigel, M. Karamitrou, R.
20. Chen, Z., D. Zhou, S. Zhou, and G. Jia. 2019. Gender difference in Schoonjans, and A. Mortensen. 2018. Guidance on risk assessment
hepatic toxicity of titanium dioxide nanoparticles after subchronic of the application of nanoscience and nanotechnologies in the food
oral exposure in Sprague-Dawley rats. J. Appl. Toxicol. 39:807–819. and feed chain: part 1, human and animal health. EFSA J.
21. Clarke, R., M. G. Healy, O. Fenton, and E. Cummins. 2016. A 16:e05327.
quantitative risk ranking model to evaluate emerging organic 36. European Parliament and the Council of the European Union. 1997.
contaminants in biosolid amended land and potential transport to Regulation (EC) No 258/97 of the European Parliament and of the
drinking water. Hum. Ecol. Risk Assess. 22:958–990. Council of 27 January 1997 concerning novel foods and novel food
22. Cueva, C., I. Gil-Sánchez, A. Tamargo, B. Miralles, J. Crespo, B. ingredients. Off. J. Eur. Union L 43:1–6.
Bartolomé, and M. V. Moreno-Arribas. 2019. Gastrointestinal 37. European Parliament and the Council of the European Union. 2002.
digestion of food-use silver nanoparticles in the dynamic SIMulator Regulation (EC) No 178/2002 of the European Parliament and of
of the GastroIntestinal tract (simgit). Impact on human gut the Council of 28 January 2002 laying down the general principles
microbiota. Food Chem. Toxicol. 132:110657. and requirements of food law, establishing the European Food
23. Cushen, M., J. Kerry, M. Morris, M. Cruz-Romero, and E. Safety Authority and laying down procedures in matters of food
Cummins. 2012. Nanotechnologies in the food industry—recent safety. Off. J. Eur. Union L 31:1–24.
developments, risks and regulation. Trends Food Sci. Technol. 38. European Parliament and the Council of the European Union. 2004.
24:30–46. Regulation (EC) No 1935/2004 of the European Parliament and of
24. Cushen, M., J. Kerry, M. Morris, M. Cruz-Romero, and E. the Council of 27 October 2004 on materials and articles intended to
Cummins. 2013. Migration and exposure assessment of silver from come into contact with food and repealing Directives 80/590/EEC
a PVC nanocomposite. Food Chem. 139:389–397. and 89/109/EEC. Off. J. Eur. Union L 338:4–17.
25. Cushen, M., J. Kerry, M. Morris, M. Cruz-Romero, and E. 39. European Parliament and the Council of the European Union. 2008.
Cummins. 2014. Evaluation and simulation of silver and copper Commission Regulation (EC) No 282/2008 of 27 March 2008. Off.
nanoparticle migration from polyethylene nanocomposites to food J. Eur. Union L 86:9–18.
and an associated exposure assessment. J. Agric. Food Chem. 40. European Parliament and the Council of the European Union. 2008.
62:1403–1411. Regulation (EC) No 1333/2008 of the European Parliament and of
26. Cushen, M., J. Kerry, M. Morris, M. Cruz-Romero, and E. the Council of 16 December 2008 on food additives. Off. J. Eur.
Cummins. 2014. Silver migration from nanosilver and a commer- Union L 354:16–33.
cially available zeolite filler polyethylene composites to food 41. European Parliament and the Council of the European Union. 2009.
simulants. Food Addit. Contam. Part A Chem. Anal. Control Expo. Commission Regulation (EC) No. 450/2009 of 29 May 2009 on
Risk Assess. 31:1132–1140. active and intelligent materials and articles intended to come into
27. Danish Ecological Council and Danish Consumer Council. 2020. contact with food. Off. J. Eur. Union L 135:3–11.
NanoRiskCat. Available at: https://nanodb.dk/en/search-database/. 42. European Parliament and the Council of the European Union. 2011.
Accessed 16 December 2021. Commission Regulation (EU) No 10/2011 of 14 January 2011 on
28. DeLoid, G. M., Y. Wang, K. Kapronezai, L. R. Lorente, R. Zhang, plastic materials and articles intended to come into contact with
G. Pyrgiotakis, N. V. Konduru, M. Ericsson, J. C. White, R. De La food. Off. J. Eur. Union L 12:1–89.
Torre-Roche, H. Xiao, D. J. McClements, and P. Demokritou. 2017. 43. European Parliament and the Council of the European Union. 2011.
An integrated methodology for assessing the impact of food matrix Regulation (EU) No 1169/2011 of the European Parliament and of
370 DE OLIVEIRA MALLIA ET AL. J. Food Prot., Vol. 85, No. 2

the Council of 25 October 2011 on the provision of food particles on plasma glucose in mice. Food Chem. Toxicol. 86:124–
information to consumers, amending Regulations (EC) No 1924/ 131.
2006 and (EC) No 1925/2006 of the European Parliament and of the 58. Gupta, N., K. K. Yadav, V. Kumar, S. Kumar, R. P. Chadd, and A.
Council, and repealing Commission Directive 87/250/EEC, Council Kumar. 2019. Trace elements in soil-vegetables interface: translo-
Directive 90/496/EEC, Commission Directive 1999/10/EC, Direc- cation, bioaccumulation, toxicity and amelioration—a review. Sci.
tive 2000/13/EC of the European Parliament and of the Council, Total Environ. 651:2927–2942.
Commission Directives 2002/67/EC and 2008/5/EC and Commis- 59. Hannon, J. C., J. P. Kerry, M. Cruz-Romero, S. Azlin-Hasim, M.
sion Regulation (EC) No 608/2004. Off. J. Eur. Union L 304:18–63. Morris, and E. Cummins. 2016. Human exposure assessment of
44. European Parliament and the Council of the European Union. 2015. silver and copper migrating from an antimicrobial nanocoated
Regulation (EU) 2015/2283 of the European Parliament and of the packaging material into an acidic food simulant. Food Chem.
Council of 25 November 2015 on novel foods, amending Toxicol. 95:128–136.
Regulation (EU) No 1169/2011 of the European Parliament and 60. Hansen, S. F., A. Baun, and K. Alstrup-Jensen. 2011. Nano-
of the Council and repealing Regulation (EC) No 258/97 of the RiskCat—a conceptual decision support tool for nanomaterials.
European Parliament and of the Council and Commission Danish Ministry of the Environment, Copenhagen.
Regulation (EC) No 1852/2001. Off. J. Eur. Union L 327:1–22. 61. Hansen, S. F., K. A. Jensen, and A. Baun. 2013. NanoRiskCat: a
45. Fadoju, O., O. Ogunsuyi, O. Akanni, O. Alabi, C. Alimba, O. conceptual tool for categorization and communication of exposure
Adaramoye, S. Cambier, S. Eswara, A. C. Gutleb, and A. Bakare. potentials and hazards of nanomaterials in consumer products. J.
2019. Evaluation of cytogenotoxicity and oxidative stress param- Nanopart. Res. 16:2195.
eters in male Swiss mice co-exposed to titanium dioxide and zinc 62. Hardy, A., D. Benford, T. Halldorsson, M. J. Jeger, H. K. Knutsen,
oxide nanoparticles. Environ. Toxicol. Pharmacol. 70:103204. S. More, H. Naegeli, H. Noteborn, C. Ockleford, A. Ricci, G.
46. Fathi, M., M. R. Mozafari, and M. Mohebbi. 2012. Nano- Rychen, J. R. Schlatter, V. Silano, R. Solecki, D. Turck, M. Younes,
encapsulation of food ingredients using lipid based delivery Q. Chaudhry, F. Cubadda, D. Gott, A. Oomen, S. Weigel, M.
systems. Trends Food Sci. Technol. 23:13–27. Karamitrou, R. Schoonjans, and A. Mortensen. 2018. Guidance on
47. Feng, Y., L. Min, W. Zhang, J. Liu, Z. Hou, M. Chu, L. Li, W. Shen, risk assessment of the application of nanoscience and nanotech-
Y. Zhao, and H. Zhang. 2017. Zinc oxide nanoparticles influence nologies in the food and feed chain: part 1, human and animal
microflora in ileal digesta and correlate well with blood metabolites. health. EFSA J. 16:e05327.
Front. Microbiol. 8:992. 63. He, X., H. Deng, and H. M. Hwang. 2019. The current application
48. Food and Agriculture Organization of the United Nations. 2011. of nanotechnology in food and agriculture. J. Food Drug Anal.
27:1–21.
Guidelines for risk analysis of foodborne antimicrobial resis-
64. Henson, T. E., J. Navratilova, A. H. Tennant, K. D. Bradham, K. R.
t a n c e . Av a i l a b l e a t : h t t p s : / / w w w. f a o . o r g / f a o - w h o -
Rogers, and M. F. Hughes. 2019. In vitro intestinal toxicity of
codexalimentarius/sh-proxy/ar/?lnk=1&url=https%253A%252F%
copper oxide nanoparticles in rat and human cell models. Nano-
252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%
toxicology 13:795–811.
252FCXG%2B77-2011%252FCXG_077e.pdf. Accessed 15 De-
65. Heringa, M. B., R. J. B. Peters, R. Bleys, M. K. van der Lee, P. C.
cember 2021.
Tromp, P. C. E. van Kesteren, J. C. H. van Eijkeren, A. K. Undas,
49. Food Safety Authority of Ireland. 2008. The relevance for food
A. G. Oomen, and H. Bouwmeester. 2018. Detection of titanium
safety of applications of nanotechnology in the food and feed
particles in human liver and spleen and possible health implications.
industries. The relevance for food safety of applications of
Part. Fibre Toxicol. 15:15.
nanotechnology in the food and feed industries. Food Safety
66. Jafari, A., S. Hassanajili, M. B. Karimi, A. Emami, F. Ghaffari, and
Authority of Ireland, Dublin.
N. Azarpira. 2018. Effect of organic/inorganic nanoparticles on
50. French Agency for Food, Environmental and Occupational Health
performance of polyurethane nanocomposites for potential wound
and Safety. 2020. Nanomaterials in food: ANSES’s recommenda-
dressing applications. J. Mech. Behav. Biomed. Mater. 88:395–405.
tions for improving their identification and better assessing
67. Jampilek, J., J. Kos, and K. Kralova. 2019. Potential of nano-
consumer health risks. Available at: https://www.anses.fr/en/
material applications in dietary supplements and foods for special
content/nanomaterials-food-ansess-recommendations-improving- medical purposes. Nanomaterials 9:296.
their-identification-and-better. Accessed 15 July 2021. 68. Jampílek, J., and K. Králová. 2017. Nanomaterials for delivery of
51. Frenzel, M., E. Krolak, A. E. Wagner, and A. Steffen-Heins. 2015. nutrients and growth-promoting compounds to plants, p. 177–226.
Physicochemical properties of WPI coated liposomes serving as In R. Prassad, M. Kumar, and V. Kumar (ed.), Nanotechnology: an
stable transporters in a real food matrix. LWT - Food Sci. Technol. agricultural paradigm. Springer, Singapore.
63:527–534. 69. Jin, I. S., M. S. Yoon, C.-W. Park, J. T. Hong, Y. B. Chung, J.-S.
52. Froggett, S. J., S. F. Clancy, D. R. Boverhof, and R. A. Canady. Kim, and D. H. Shin. 2020. Replacement techniques to reduce
2014. A review and perspective of existing research on the release animal experiments in drug and nanoparticle development. J.
of nanomaterials from solid nanocomposites. Part. Fibre Toxicol. Pharm. Investig. 50:327–335.
11:1–28. 70. Jones, K., J. Morton, I. Smith, K. Jurkschat, A. H. Harding, and G.
53. Fröhlich, E., and E. Fröhlich. 2016. Cytotoxicity of nanoparticles Evans. 2015. Human in vivo and in vitro studies on gastrointestinal
contained in food on intestinal cells and the gut microbiota. Int. J. absorption of titanium dioxide nanoparticles. Toxicol. Lett. 233:95–
Mol. Sci. 17:509. 101.
54. Geiss, O., J. Ponti, C. Senaldi, I. Bianchi, D. Mehn, J. Barrero, D. 71. Kirk, R. G. W. 2018. Recovering The Principles of Humane
Gilliland, R. Matissek, and E. Anklam. 2020. Characterisation of Experimental Technique: the 3Rs and the human essence of animal
food grade titania with respect to nanoparticle content in pristine research. Sci. Technol. Hum. Values 43:622–648.
additives and in their related food products. Food Addit. Contam 72. Lamon, L., K. Aschberger, D. Asturiol, A. Richarz, and A. Worth.
Part A Anal. Control Expo. Risk Assess. 37:239–253. 2019. Grouping of nanomaterials to read-across hazard endpoints: a
55. George, A., P. A. Shah, and P. S. Shrivastav. 2019. Natural review. Nanotoxicology 13:100–118.
biodegradable polymers based nano-formulations for drug delivery: 73. Lee, J. A., M. K. Kim, J. H. Song, M. R. Jo, J. Yu, K. M. Kim, Y. R.
a review. Int. J. Pharm. 561:244–264. Kim, J. M. Oh, and S. J. Choi. 2017. Biokinetics of food additive
56. Graham, B., B. A. Piatt, J. J. Lind, J. E. Dvorsky, M. Bell, W. M. silica nanoparticles and their interactions with food components.
Prabodha, and S. Alavattam. April 2009. U.S. patent US 2009/ Colloids Surf. B Biointerfaces 150:384–392.
0,104,269 A1. 74. Lee, J. Y., H. Wang, G. Pyrgiotakis, G. M. DeLoid, Z. Zhang, J.
57. Gu, N., H. Hu, Q. Guo, S. Jin, C. Wang, Y. Oh, Y. Feng, and Q. Wu. Beltran-Huarac, P. Demokritou, and W. Zhong. 2018. Analysis of
2015. Effects of oral administration of titanium dioxide fine-sized lipid adsorption on nanoparticles by nanoflow liquid chromatogra-
J. Food Prot., Vol. 85, No. 2 NANOPARTICLES AND FOOD TOXICITY 371

phy-tandem mass spectrometry. Anal. Bioanal. Chem. 410:6155– 92. Pogribna, M., N. A. Koonce, A. Mathew, B. Word, A. K. Patri, B.
6164. Lyn-Cook, and G. Hammons. 2020. Effect of titanium dioxide
75. Li, Y., and E. Cummins. 2020. Hazard characterization of silver nanoparticles on DNA methylation in multiple human cell lines.
nanoparticles for human exposure routes. J. Environ. Sci. Health A Nanotoxicology 14:534–553.
55:704–725. 93. Pound, P., and M. Ritskes-Hoitinga. 2018. Is it possible to overcome
76. Lichtenstein, D., J. Ebmeyer, P. Knappe, S. Juling, L. Bohmert, S. issues of external validity in preclinical animal research? Why most
Selve, B. Niemann, A. Braeuning, A. F. Thunemann, and A. animal models are bound to fail. J. Transl. Med. 16:304.
Lampen. 2015. Impact of food components during in vitro digestion 94. Pradhan, M., D. Singh, and M. R. Singh. 2013. Novel colloidal
of silver nanoparticles on cellular uptake and cytotoxicity in carriers for psoriasis: current issues, mechanistic insight and novel
intestinal cells. Biol. Chem. 396:1255–1264. delivery approaches. J. Control. Release 170:380–395.
77. McCarron, E. 2016. Nanotechnology and food: investigating 95. Ravichandran, K., P. K. Praseetha, T. Arun, and S. Gobalakrishnan.
consumers’ acceptance of foods produced using nanotechnology. 2018. Synthesis of nanocomposites, p. 141–168. In S. M.
M.S. thesis. Dublin Business School. Bhagyaraj, O. S. Oluwafemi, N. Kalarikkal, and S. Thomas (ed.),
78. McClements, D. J., and H. Xiao. 2017. Is nano safe in foods? Synthesis of inorganic nanomaterials. Woodhead Publishing,
Establishing the factors impacting the gastrointestinal fate and Duxford, UK.
toxicity of organic and inorganic food-grade nanoparticles. NPJ Sci. 96. Rompelberg, C., M. B. Heringa, G. van Donkersgoed, J. Drijvers,
Food 1:6. A. Roos, S. Westenbrink, R. Peters, G. van Bemmel, W. Brand, and
79. Mohamed, H. R. 2015. Estimation of TiO2 nanoparticle-induced A. G. Oomen. 2016. Oral intake of added titanium dioxide and its
genotoxicity persistence and possible chronic gastritis-induction in nanofraction from food products, food supplements and toothpaste
mice. Food Chem. Toxicol. 83:76–83. by the Dutch population. Nanotoxicology 10:1404–1414.
80. Mu, W., Y. Wang, C. Huang, Y. Fu, J. Li, H. Wang, X. Jia, and Q. 97. Saini, A., R. Kaur, N. Singh, A. Kuwar, and N. Kaur. 2019. High
Ba. 2019. Effect of long-term intake of dietary titanium dioxide performance fluorescent turn-on probe for amitriptyline based on
nanoparticles on intestine inflammation in mice. J. Agric. Food hybrid nanoassembly of organic–inorganic nanoparticles. ACS
Chem. 67:9382–9389. Appl. Bio Mater. 2:135–143.
81. Murugadoss, S., F. Brassinne, N. Sebaihi, J. Petry, S. M. Cokic, K. 98. Schneider, T., M. Westermann, and M. Glei. 2017. In vitro uptake
L. Van Landuyt, L. Godderis, J. Mast, D. Lison, P. H. Hoet, and S. and toxicity studies of metal nanoparticles and metal oxide
van den Brule. 2020. Agglomeration of titanium dioxide nanopar- nanoparticles in human HT29 cells. Arch. Toxicol. 91:3517–3527.
ticles increases toxicological responses in vitro and in vivo. Part. 99. Sessa, M., M. L. Balestrieri, G. Ferrari, L. Servillo, D. Castaldo, N.
Fibre Toxicol. 17:10. D’Onofrio, F. Donsì, and R. Tsao. 2014. Bioavailability of
82. Nag, R., A. Auer, B. K. Markey, P. Whyte, S. Nolan, V. O’Flaherty, encapsulated resveratrol into nanoemulsion-based delivery systems.
L. Russell, D. Bolton, O. Fenton, K. Richards, and E. Cummins. Food Chem. 147:42–50.
2019. Anaerobic digestion of agricultural manure and biomass— 100. Shah, M., D. Fawcett, S. Sharma, S. K. Tripathy, and G. E. J.
critical indicators of risk and knowledge gaps. Sci. Total Environ. Poinern. 2015. Green synthesis of metallic nanoparticles via
690:460–479. biological entities. Materials 8:7278–7308.
83. Nasr, R., H. Hasanzadeh, A. Khaleghian, A. Moshtaghian, A. 101. Shin, G. H., S. K. Chung, J. T. Kim, H. J. Joung, and H. J. Park.
Emadi, and S. Moshfegh. 2018. Induction of apoptosis and 2013. Preparation of chitosan-coated nanoliposomes for improving
inhibition of invasion in gastric cancer cells by titanium dioxide the mucoadhesive property of curcumin using the ethanol injection
nanoparticles. Oman Med. J. 33:111–117. method. J. Agric. Food Chem. 61:11119–11126.
84. Oehlke, K., M. Adamiuk, D. Behsnilian, V. Gräf, E. Mayer- 102. Shin, G. H., J. T. Kim, and H. J. Park. 2015. Recent developments
Miebach, E. Walz, and R. Greiner. 2014. Potential bioavailability in nanoformulations of lipophilic functional foods. Trends Food Sci.
enhancement of bioactive compounds using food-grade engineered Technol. 46:144–157.
nanomaterials: a review of the existing evidence. Food Funct. 103. Smith, M. E., and D. G. Morton. 2010. The digestive system: basic
5:1341–1359. science and clinical conditions. Churchill Livingstone, Edinburgh.
85. Organisation for Economic Co-operation and Development. 2016. 104. Solans, C., P. Izquierdo, J. Nolla, N. Azemar, and M. J. Garcia-
Test no. 474: mammalian erythrocyte micronucleus test. OECD Celma. 2005. Nano-emulsions. Curr. Opin. Colloid Interface Sci.
Publishing, Paris. 10:102–110.
86. Organisation for Economic Co-operation and Development. 2016. 105. Song, Z. M., N. Chen, J. H. Liu, H. Tang, X. Deng, W. S. Xi, K.
Test no. 489: in vivo mammalian alkaline comet assay. OECD Han, A. Cao, Y. Liu, and H. Wang. 2015. Biological effect of food
Publishing, Paris. additive titanium dioxide nanoparticles on intestine: an in vitro
87. Parsai, T., and A. Kumar. 2019. Understanding effect of solution study. J. Appl. Toxicol. 35:1169–1178.
chemistry on heteroaggregation of zinc oxide and copper oxide 106. Souza, V. G. L., and A. L. Fernando. 2016. Nanoparticles in food
nanoparticles. Chemosphere 235:457–469. packaging: biodegradability and potential migration to food—a
88. Pedata, P., G. Ricci, L. Malorni, A. Venezia, M. Cammarota, M. G. review. Food Packag. Shelf Life 8:63–70.
Volpe, N. Iannaccone, V. Guida, C. Schiraldi, M. Romano, and G. 107. Talamini, L., S. Gimondi, M. B. Violatto, F. Fiordaliso, F. Pedica,
Iacomino. 2019. In vitro intestinal epithelium responses to titanium N. L. Tran, G. Sitia, F. Aureli, A. Raggi, I. Nelissen, F. Cubadda, P.
dioxide nanoparticles. Food Res. Int. 119:634–642. Bigini, and L. Diomede. 2019. Repeated administration of the food
89. Peters, R. J. B., A. G. Oomen, G. van Bemmel, L. van Vliet, A. K. additive E171 to mice results in accumulation in intestine and liver
Undas, S. Munniks, R. Bleys, P. C. Tromp, W. Brand, and M. van and promotes an inflammatory status. Nanotoxicology 13:1087–
der Lee. 2020. Silicon dioxide and titanium dioxide particles found 1101.
in human tissues. Nanotoxicology 14:420–432. 108. Tarhan, Ö. 2020. Safety and regulatory issues of nanomaterials in
90. Peters, R. J. B., G. Van Bemmel, Z. Herrera-Rivera, H. P. F. G. foods, p. 655–703. In S. M. Jafari (ed.), Handbook of food
Helsper, H. J. P. Marvin, S. Weigel, P. C. Tromp, A. G. Oomen, A. nanotechnology. Academic Press, Boston.
G. Rietveld, and H. Bouwmeester. 2014. Characterization of 109. Taylor, A. A., I. M. Marcus, R. L. Guysi, and S. L. Walker. 2015.
titanium dioxide nanoparticles in food products: analytical methods Metal oxide nanoparticles induce minimal phenotypic changes in a
to define nanoparticles. J. Agric. Food Chem. 62:6285–6293. model colon gut microbiota. Environ. Eng. Sci. 32:602–612.
91. Pindakova, L., V. Kasparkova, K. Kejlova, M. Dvorakova, D. 110. Tiede, K., A. B. A. Boxall, S. P. Tear, J. Lewis, H. David, and M.
Krsek, D. Jirova, and L. Kasparova. 2017. Behaviour of silver Hassellöv. 2008. Detection and characterization of engineered
nanoparticles in simulated saliva and gastrointestinal fluids. Int. J. nanoparticles in food and the environment. Food Addit. Contam.
Pharm. 527:12–20. Part A Chem. Anal. Control Expo. Risk Assess. 25:795–821.
372 DE OLIVEIRA MALLIA ET AL. J. Food Prot., Vol. 85, No. 2

111. Tsafe, A., L. Hassan, D. Sahabi, Y. Alhassan, and B. Bala. 2012. A. Lampen, and H. Sieg. 2020. The presence of iron oxide
Evaluation of heavy metals uptake and risk assessment of nanoparticles in the food pigment E172. Food Chem. 327:127000.
vegetables grown in Yargalma of Northern Nigeria. J. Basic Appl. 120. Weir, A., P. Westerhoff, L. Fabricius, K. Hristovski, and N. von
Sci. Res. 2:6708–6714. Goetz. 2012. Titanium dioxide nanoparticles in food and personal
112. U.S. Food and Drug Administration. 2014. Guidance for industry: care products. Environ. Sci. Technol. 46:2242–2250.
Assessing the effects of significant manufacturing process changes, 121. Yang, Y., K. Doudrick, X. Bi, K. Hristovski, P. Herckes, P.
including emerging technologies, on the safety and regulatory status Westerhoff, and R. Kaegi. 2014. Characterization of food-grade
of food ingredients and food contact substances, including food titanium dioxide: the presence of nanosized particles. Environ. Sci.
ingredients that are color additives. Available at: https://www.fda. Technol. 48:6391–400.
gov/regulatory-information/search-fda-guidance-documents/ 122. Yi, J., T. I. Lam, W. Yokoyama, L. W. Cheng, and F. Zhong. 2015.
guidance-industry-assessing-effects-significant-manufacturing- Beta-carotene encapsulated in food protein nanoparticles reduces
process-changes-including-emerging. Accessed 15 July 2021. peroxyl radical oxidation in Caco-2 cells. Food Hydrocoll. 43:31–
113. U.S. Food and Drug Administration. 2018. FDA’s approach to 40.
regulation of nanotechnology products. Available at: https://www. 123. Yin, C., W. Zhao, R. Liu, R. Liu, Z. Wang, L. Zhu, W. Chen, and S.
fda.gov/science-research/nanotechnology-programs-fda/fdas- Liu. 2017. TiO2 particles in seafood and surimi products: attention
approach-regulation-nanotechnology-products. Accessed 15 July should be paid to their exposure and uptake through foods.
2021. Chemosphere 188:541–547.
124. Younes, M., P. Aggett, F. Aguilar, R. Crebelli, B. Dusemund, M.
114. U.S. Food and Drug Administration. 2020. 21 CFR 73.575. Title
Filipic, M. J. Frutos, P. Galtier, D. Gott, U. Gundert-Remy, G. G.
21—Food and drugs, part 73—Listing of color additives exempt
Kuhnle, C. Lambre, J. C. Leblanc, I. T. Lillegaard, P. Moldeus, A.
from certification, sec. 73.575—titanium dioxide. Available at:
Mortensen, A. Oskarsson, I. Stankovic, I. Waalkens-Berendsen, M.
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/
Wright, F. Lodi, A. M. Rincon, C. Smeraldi, and R. A. Woutersen.
CFRSearch.cfm?fr¼73.575. Accessed 15 July 2021.
2018. Evaluation of four new studies on the potential toxicity of
115. Verleysen, E., N. Waegeneers, F. Brassinne, S. De Vos, I. O.
titanium dioxide used as a food additive (E 171). EFSA J.
Jimenez, S. Mathioudaki, and J. Mast. 2020. Physicochemical
16:e05366.
characterization of the pristine E171 food additive by standardized
125. Younes, M., P. Aggett, F. Aguilar, R. Crebelli, B. Dusemund, M.
and validated methods. Nanomaterials 10:592.
Filipic, M. J. Frutos, P. Galtier, D. Gott, U. Gundert-Remy, G. G.
116. Vila, L., A. Garcia-Rodriguez, C. Cortes, R. Marcos, and A. Kuhnle, J. C. Leblanc, I. T. Lillegaard, P. Moldeus, A. Mortensen,
Hernandez. 2018. Assessing the effects of silver nanoparticles on A. Oskarsson, I. Stankovic, I. Waalkens-Berendsen, R. A.
monolayers of differentiated Caco-2 cells, as a model of intestinal Woutersen, M. Wright, P. Boon, D. Chrysafidis, R. Gurtler, P.
barrier. Food Chem. Toxicol. 116:1–10. Mosesso, D. Parent-Massin, P. Tobback, N. Kovalkovicova, A. M.
117. von Goetz, N., L. Fabricius, R. Glaus, V. Weitbrecht, D. Günther, Rincon, A. Tard, and C. Lambre. 2018. Re-evaluation of silicon
and K. Hungerbühler. 2013. Migration of silver from commercial dioxide (E 551) as a food additive. EFSA J. 16:e05088.
plastic food containers and implications for consumer exposure 126. Younes, M., G. Aquilina, L. Castle, K.-H. Engel, P. Fowler, M. J.
assessment. Food Addit. Contam. Part A Chem. Anal. Control Expo. Frutos Fernandez, R. Gürtler, U. Gundert-Remy, T. Husøy, W.
Risk Assess. 30:612–620. Mennes, P. M. Agneta Oskarsson, S. Rainieri, R. Shah, I. Waalkens-
118. von Moos, L. M., M. Schneider, F. M. Hilty, M. Hilbe, M. Arnold, Berendsen, D. Wölfle, E. Gaffet, J. Mast, R. Peters, A. M. Rincon,
N. Ziegler, D. S. Mato, H. Winkler, M. Tarik, C. Ludwig, H. and P. Fürst. 2019. Scientific opinion on the proposed amendment
Naegeli, W. Langhans, M. B. Zimmermann, S. J. Sturla, and I. A. of the EU specifications for titanium dioxide (E 171) with respect to
Trantakis. 2017. Iron phosphate nanoparticles for food fortification: the inclusion of additional parameters related to its particle size
biological effects in rats and human cell lines. Nanotoxicology distribution. EFSA J. 17:e05760.
11:496–506. 127. Zhou, F., F. Liao, L. Chen, Y. Liu, W. Wang, and S. Feng. 2019. The
119. Voss, L., I. L. Hsiao, M. Ebisch, J. Vidmar, N. Dreiack, L. Böhmert, size-dependent genotoxicity and oxidative stress of silica nanopar-
V. Stock, A. Braeuning, K. Loeschner, P. Laux, A. F. Thünemann, ticles on endothelial cells. Environ. Sci. Pollut. Res. 26:1911–1920.