How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach

*Matheus M. Roberto and Cintya A. Christofoletti*

## **Abstract**

Nanomaterials had been used because of the properties they exert in such scale (<100 nm), and they have been used in a wide variety of products like paints, electronics, fabrics, and also personal care products. Recent manuscripts available in the literature demonstrate the potential benefits of nanotechnology with these products. However, when released in the environment or when in contact with the human body, it is hard to track their final destination and their influence over the living beings. So, since nanomaterials were considered an important technology, a concern about its risks also started. Due to the variety of sizes, physicochemical properties, and uses, many researchers are aiming to assess the possible toxicity of this class of particles. Because of that, the chapter objective is to gather which assay, performed in vivo and in vitro, is the most frequently used and recommended to measure nanomaterial toxicity. Also, it is important to know which is the most suitable test to evaluate the toxicity over the environment, through direct effect and after biodegradation, and also related to human health. This chapter presents a concise review about the accepted methods to assess nanomaterial toxicity and also discuss about the need for regulamentation.

**Keywords:** toxicity assays, test organisms, in vitro models, in vivo assays, nanoscale assessment

## **1. Introduction**

Emerging and promising nanotechnology represents a field of multidisciplinary knowledge responsible for development and application of materials, which measure less than 100 nm [1, 2]. The Royal Society and Royal Engineering Academy proposed this concept in 2004, which was associated to nanoscience as the branch responsible for studying the phenomenon of materials with atomic, molecular, and macromolecular scales, whose properties differ significantly from those with major scales [3, 4].

Nanoparticles can be generally described as ultrafine small material with 1–100 nm; however, several types of systems not limited only by small particles of certain material are included in this definition, as nanotubes, nanospheres, and nanocapsules [4, 5]. The properties exhibited by nanomaterials are unique and are being applied in many fields, from industrial to medicine [6, 7]. According to Arora et al. [8], the use of nanomaterials is increasing for commercial purposes as fillers,

opacifiers, water filtration agents, cosmetic ingredients, semiconductors, electronic parts, and others. However, these same authors report that nanomaterials are being used in the medical area, mainly as agents for drug delivery, biosensors, and imaging contrast, i.e., human contact can happen both indirectly and directly, also being administered by ingestion or injection [8]. Once nanomaterials are used, environmental releasing turns dependent on the incorporation form of this product in each matrix, intrinsic material properties and also environmental conditions [9]. When there is human exposure or direct intake of nanomaterials, nanoparticles' physicochemical properties and its possible modifications can influence absorption, distribution, and organism metabolism. Besides the potential to accumulate in some organs, relevant rates of nanomaterials are excreted, being released to the environment [10]. About the nanomaterials presence in the environment, a detailed description regarding its sources and fates can be found in the review of Part [11].

Due to the new scale of some materials, new physicochemical interactions may occur bringing unexpected and also adverse effects because these elements generally become highly reactive [12]. Physicochemical properties observed in engineered nanomaterials are attributed to small size, chemical composition (purity, crystallinity, electronics characteristics, etc.), structural surface (reactivity, organic or inorganic coating, etc.), solubility, form, and agglomeration potential [8].

In view of the properties that the nanomaterials present, studies that evaluate the toxicity, their behavior in different environments, and the interactions with the biological system are of extreme importance. According to Dusinska [6], the safety assessment of nanomaterials is based on principles of risk assessment of "bulk" chemical substances. However, it is known that the behavior of these materials, both in the environment and in the cells, is different from such crude samples, and therefore the monitoring needs to be more specific. Catalán et al. [13] emphasize that the damaging potential of biodurable nanomaterials is not well demonstrated, and thus the classical toxicity evaluation trials must undergo adaptations.

### **2. Brief history of nanotoxicology**

According to Maynard et al. [14], until the 1990s, many studies that focused on environmental epidemiology indicated a relationship between exposure to aerosols and increased mortality and morbidity of organisms. The relationships between particle size, chemical nature, and toxic effects were demonstrated, with the most pronounced effects observed in the lungs and heart due to exposure to smaller particles. These same authors argue that only in this decade has there been evidence that environmental particles with a diameter of less than 2.5 μm could cause deleterious health effects due to their reduced size [14]. Now it is known that engineered nanoparticles can perform these same activities [12].

Since the inception of this science, the studies and applications of nanoparticles have grown exponentially and, to the same extent, heightened concerns about environmental and health implications. In this context, the term nanotoxicology was formalized by a proposal of Donaldson et al. in 2004 [15] in an editorial in the journal *Occupational and Environmental Medicine* [5] and, since then, has been used to describe specifically the harmful effects of nanomaterials on environmental, animal, and human health. In 2005, nanotoxicology was consolidated as an area of expertise, with the launch of the journal *Nanotoxicology*, with the first article published by Oberdörster et al. in 2007 [16]. This article discusses the history of nanotoxicology as a science and presents some challenges to be faced by researchers.

Considering that nanoparticles have a greater potential to travel through the body than conventional-sized materials, researchers warn of the possibility of

**17**

**Table 1.**

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach*

and Development (OECD) and can be used for regulatory purposes.

numerous interactions with biological fluids, cells, and tissues. Therefore, in vitro tests are recommended for an initial evaluation of the cytotoxicity and genotoxicity of nanomaterials, as well as for the identification and understanding of cellular mechanisms of toxicity [3]. In vivo methods are also used and, for both, some methods have already been developed by the Organization for Economic Co-operation

According to Paschoalino et al. [3], the growing investment in nanoscience boosted the world market, as well as increased the use and consumption of products and processes aimed at this area. Despite this, it is true that research aimed at evaluating the toxicity of nanomaterials is still necessary, since the same properties that make nanomaterials so attractive may also be responsible for harmful effects on

In this context, there is a recommendation for the analysis of physicochemical properties of nanomaterials in relation to human health and environmental safety (**Table 1**). In 2006, the OECD established a working party on manufactured nanomaterials to determine the appropriate methods for evaluating nanomaterials. According to the guidance manual developed, 26 physicochemical properties should be considered [6]. However, according to these same authors, only a few

**Property Relevance** Particle size distribution Essential Degree/state of agglomeration Important Particle shape Important Chemical composition/purity Essential Solubility Essential (if

Density Matrix dependent Crystallinity Matrix dependent Microstructure Matrix dependent Optical and electronic properties Matrix dependent Bulk powder properties (important for dosimetry/exposure) Matrix dependent

Concentration (can be measured as mass, surface area, or number concentrations) Essential

Specific surface area/porosity Essential Surface chemistry/reactivity Essential Surface adsorbed species Important

Surface hydrophobicity Essential

Surface charge/Zeta potential (especially in aqueous biological environment— may

*Properties used for nanomaterial characterization regarding toxicity evaluation.*

applicable)

Important

*DOI: http://dx.doi.org/10.5772/intechopen.88970*

**3. Toxicological aspects of nanomaterials**

living organisms.

Physical properties

Surface properties

change according the environment)

*Adapted from Powers et al. [17]*

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach DOI: http://dx.doi.org/10.5772/intechopen.88970*

numerous interactions with biological fluids, cells, and tissues. Therefore, in vitro tests are recommended for an initial evaluation of the cytotoxicity and genotoxicity of nanomaterials, as well as for the identification and understanding of cellular mechanisms of toxicity [3]. In vivo methods are also used and, for both, some methods have already been developed by the Organization for Economic Co-operation and Development (OECD) and can be used for regulatory purposes.

## **3. Toxicological aspects of nanomaterials**

*Nanomaterials - Toxicity, Human Health and Environment*

opacifiers, water filtration agents, cosmetic ingredients, semiconductors, electronic parts, and others. However, these same authors report that nanomaterials are being used in the medical area, mainly as agents for drug delivery, biosensors, and imaging contrast, i.e., human contact can happen both indirectly and directly, also being administered by ingestion or injection [8]. Once nanomaterials are used, environmental releasing turns dependent on the incorporation form of this product in each matrix, intrinsic material properties and also environmental conditions [9]. When there is human exposure or direct intake of nanomaterials, nanoparticles' physicochemical properties and its possible modifications can influence absorption, distribution, and organism metabolism. Besides the potential to accumulate in some organs, relevant rates of nanomaterials are excreted, being released to the environment [10]. About the nanomaterials presence in the environment, a detailed description regarding its sources and fates can be found in the review of Part [11]. Due to the new scale of some materials, new physicochemical interactions may occur bringing unexpected and also adverse effects because these elements generally become highly reactive [12]. Physicochemical properties observed in engineered nanomaterials are attributed to small size, chemical composition (purity, crystallinity, electronics characteristics, etc.), structural surface (reactivity, organic

or inorganic coating, etc.), solubility, form, and agglomeration potential [8].

and thus the classical toxicity evaluation trials must undergo adaptations.

**2. Brief history of nanotoxicology**

nanoparticles can perform these same activities [12].

In view of the properties that the nanomaterials present, studies that evaluate the toxicity, their behavior in different environments, and the interactions with the biological system are of extreme importance. According to Dusinska [6], the safety assessment of nanomaterials is based on principles of risk assessment of "bulk" chemical substances. However, it is known that the behavior of these materials, both in the environment and in the cells, is different from such crude samples, and therefore the monitoring needs to be more specific. Catalán et al. [13] emphasize that the damaging potential of biodurable nanomaterials is not well demonstrated,

According to Maynard et al. [14], until the 1990s, many studies that focused on environmental epidemiology indicated a relationship between exposure to aerosols and increased mortality and morbidity of organisms. The relationships between particle size, chemical nature, and toxic effects were demonstrated, with the most pronounced effects observed in the lungs and heart due to exposure to smaller particles. These same authors argue that only in this decade has there been evidence that environmental particles with a diameter of less than 2.5 μm could cause deleterious health effects due to their reduced size [14]. Now it is known that engineered

Since the inception of this science, the studies and applications of nanoparticles

have grown exponentially and, to the same extent, heightened concerns about environmental and health implications. In this context, the term nanotoxicology was formalized by a proposal of Donaldson et al. in 2004 [15] in an editorial in the journal *Occupational and Environmental Medicine* [5] and, since then, has been used to describe specifically the harmful effects of nanomaterials on environmental, animal, and human health. In 2005, nanotoxicology was consolidated as an area of expertise, with the launch of the journal *Nanotoxicology*, with the first article published by Oberdörster et al. in 2007 [16]. This article discusses the history of nanotoxicology as a science and presents some challenges to be faced by researchers. Considering that nanoparticles have a greater potential to travel through the body than conventional-sized materials, researchers warn of the possibility of

**16**

According to Paschoalino et al. [3], the growing investment in nanoscience boosted the world market, as well as increased the use and consumption of products and processes aimed at this area. Despite this, it is true that research aimed at evaluating the toxicity of nanomaterials is still necessary, since the same properties that make nanomaterials so attractive may also be responsible for harmful effects on living organisms.

In this context, there is a recommendation for the analysis of physicochemical properties of nanomaterials in relation to human health and environmental safety (**Table 1**). In 2006, the OECD established a working party on manufactured nanomaterials to determine the appropriate methods for evaluating nanomaterials. According to the guidance manual developed, 26 physicochemical properties should be considered [6]. However, according to these same authors, only a few


#### **Table 1.**

*Properties used for nanomaterial characterization regarding toxicity evaluation.*

methods are available for the characterization of the toxicological properties of the nanomaterials, and the association of the effects with the physicochemical characterization is still a challenge.

Nanomaterials encompass a broad spectrum of materials with different physical, chemical, and biological properties. Thus, they do not constitute a homogeneous group and are usually defined by the type of core, which may be organic, such as fullerenes (carbon derivatives) and carbon nanotubes (single and/or multilayer), or inorganic, such as those of metal oxides (iron, zinc, titanium, etc.), metals (mainly gold and silver), and quantum dots [4].

According to Ju-Nam and Lead [4], some nanomaterials can have their surfaces manipulated in order to introduce specific functionalities for new applications. Thus, a vast field of possibilities opens up for materials with different properties and, therefore, also for infinite interactions with organisms and environment. However, the major challenge of nanotoxicology is to understand and prevent the risk of the use and/or exposure to nanomaterials that can cause toxicity by mechanisms not yet known or not yet explained by traditional toxicology [5].

Concern about the toxicity of nanomaterials lies primarily in production and commercialization on such a large scale as at present. Thus, the risk of these compounds reaching the different environmental compartments (atmosphere, water, and soil), becoming bioavailable, is very large [3, 17].

Since 2005, the European Commission Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) has published reports on the impacts of nanoparticles on human health. The aforementioned committee focused its efforts on the evaluation of nanoparticles physically capable of entering the human body via inhalation, ingestion, and dermal absorption and reported that the size, shape, surface area, and chemical composition of the nanoparticle are closely associated with its toxicity. In addition, it has been explored how these characteristics affect bioavailability and interactions, as well as influence on exposure and dose. Therefore, the dose, the physicochemical properties, and the biokinetics are also important parameters to be evaluated when considering the toxicology of nanomaterials [14].

## **4. Deposition and interactions of nanomaterials with cells and the environment**

In view of the numerous properties and characteristics of nanomaterials, products that are increasingly light, resistant, and often of lower cost are daily produced and marketed by the most different segments, such as electronic, medical, pharmaceutical, cosmetic, food, and agricultural [3]. In this context, when considering the ecotoxicology of particles whose components are nontoxic in the micro- or macrometric scales, studies that elucidate the routes of exposure and effects of nanomaterials on environmental compartments and different organisms are fundamental.

According to Laux et al. [7], the entry of nanomaterials into the environment occurs by the release of their components during use and by final disposal, so it is important to track and understand the kinetics and transformation of these materials in organisms and the environment. Knowledge of the influence of biopersistence on biokinetics and environmental fate is of utmost importance when determining the toxic potential.

When a nanomaterial comes in contact with the human body or the environment, it is difficult to track it again. In the environment, some nanomaterials such as metallic (e.g., Ag and Cu) and metal oxides (e.g., ZnO and Fe2O3) can dissolve rapidly, while others are more persistent (e.g., TiO2, SiO2, carbon nanotubes, and

**19**

**Figure 1.**

*organisms (adapted from Paschoalino et al. [3]).*

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach*

graphene) [18]. However, according to these authors, soluble nanomaterials present the best scenario of toxicity evaluation, since their behavior is generally similar to that presented by their ions. However, when cells internalize them, they can solubilize and release toxic metals through a mechanism known as "Trojan Horse" [18]. The aquatic ecosystem is the main route of exposure to a nanomaterial, since this type of environment is usually the final destination of nanocomposites introduced in natural systems [19]. After the aquatic environment, the atmosphere (troposphere), soil, and sediment follow an order of priority as routes of exposure. When present in aquatic environments, nanomaterials can be absorbed by cells, especially during filtration by aquatic organisms, directly interfering with their physiology and/or their ability to feed and breathe [3]. According to these same authors, other pathways of entry of nanomaterials into receptor organisms occur through cellular

According to Bhaskar et al. [20] and Dusinska et al. [6], nanomaterials can enter

The first contact of the cell with any extracellular material occurs through the lipid (e.g., phospholipid) and protein components (e.g., membrane receptors) present in the cell membrane. For Paschoalino et al. [3], the nanomaterials present greater permeability to the skin, mucous membranes, and cell membranes due to

*Main sources, routes of exposure, and possible interactions between nanoparticles with the environment and* 

the cells actively or passively, overcoming any protective barrier of the organism, including the blood-brain barrier. The capture mechanisms are related to the intrinsic physicochemical characteristics of the nanomaterial, as well as its route of exposure. Considering human health, generally the main route of exposure is inhalation, in which smaller particles reaching the alveoli and depending on their physicochemical properties cross the blood-air barrier of the lungs and reach the liver, heart, spleen, and kidneys [7]. There is a challenge when it comes to nanoparticles developed to cross human body barriers, such as those applied in medicine. There are materials that are developed to pass through barriers, not to enter cells,

while there are others that are designed to act within them [10].

*DOI: http://dx.doi.org/10.5772/intechopen.88970*

uptake, inhalation, or ingestion (**Figure 1**).

their diminutive size.

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach DOI: http://dx.doi.org/10.5772/intechopen.88970*

graphene) [18]. However, according to these authors, soluble nanomaterials present the best scenario of toxicity evaluation, since their behavior is generally similar to that presented by their ions. However, when cells internalize them, they can solubilize and release toxic metals through a mechanism known as "Trojan Horse" [18].

The aquatic ecosystem is the main route of exposure to a nanomaterial, since this type of environment is usually the final destination of nanocomposites introduced in natural systems [19]. After the aquatic environment, the atmosphere (troposphere), soil, and sediment follow an order of priority as routes of exposure. When present in aquatic environments, nanomaterials can be absorbed by cells, especially during filtration by aquatic organisms, directly interfering with their physiology and/or their ability to feed and breathe [3]. According to these same authors, other pathways of entry of nanomaterials into receptor organisms occur through cellular uptake, inhalation, or ingestion (**Figure 1**).

According to Bhaskar et al. [20] and Dusinska et al. [6], nanomaterials can enter the cells actively or passively, overcoming any protective barrier of the organism, including the blood-brain barrier. The capture mechanisms are related to the intrinsic physicochemical characteristics of the nanomaterial, as well as its route of exposure. Considering human health, generally the main route of exposure is inhalation, in which smaller particles reaching the alveoli and depending on their physicochemical properties cross the blood-air barrier of the lungs and reach the liver, heart, spleen, and kidneys [7]. There is a challenge when it comes to nanoparticles developed to cross human body barriers, such as those applied in medicine. There are materials that are developed to pass through barriers, not to enter cells, while there are others that are designed to act within them [10].

The first contact of the cell with any extracellular material occurs through the lipid (e.g., phospholipid) and protein components (e.g., membrane receptors) present in the cell membrane. For Paschoalino et al. [3], the nanomaterials present greater permeability to the skin, mucous membranes, and cell membranes due to their diminutive size.

#### **Figure 1.**

*Main sources, routes of exposure, and possible interactions between nanoparticles with the environment and organisms (adapted from Paschoalino et al. [3]).*

*Nanomaterials - Toxicity, Human Health and Environment*

terization is still a challenge.

gold and silver), and quantum dots [4].

methods are available for the characterization of the toxicological properties of the nanomaterials, and the association of the effects with the physicochemical charac-

Nanomaterials encompass a broad spectrum of materials with different physical, chemical, and biological properties. Thus, they do not constitute a homogeneous group and are usually defined by the type of core, which may be organic, such as fullerenes (carbon derivatives) and carbon nanotubes (single and/or multilayer), or inorganic, such as those of metal oxides (iron, zinc, titanium, etc.), metals (mainly

According to Ju-Nam and Lead [4], some nanomaterials can have their surfaces manipulated in order to introduce specific functionalities for new applications. Thus, a vast field of possibilities opens up for materials with different properties and, therefore, also for infinite interactions with organisms and environment. However, the major challenge of nanotoxicology is to understand and prevent the risk of the use and/or exposure to nanomaterials that can cause toxicity by mecha-

Concern about the toxicity of nanomaterials lies primarily in production and commercialization on such a large scale as at present. Thus, the risk of these compounds reaching the different environmental compartments (atmosphere, water,

Since 2005, the European Commission Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) has published reports on the impacts of nanoparticles on human health. The aforementioned committee focused its efforts on the evaluation of nanoparticles physically capable of entering the human body via inhalation, ingestion, and dermal absorption and reported that the size, shape, surface area, and chemical composition of the nanoparticle are closely associated with its toxicity. In addition, it has been explored how these characteristics affect bioavailability and interactions, as well as influence on exposure and dose. Therefore, the dose, the physicochemical properties, and the biokinetics are also important parameters to be evaluated when considering the toxicology of nanoma-

**4. Deposition and interactions of nanomaterials with cells and the** 

In view of the numerous properties and characteristics of nanomaterials, products that are increasingly light, resistant, and often of lower cost are daily produced and marketed by the most different segments, such as electronic, medical, pharmaceutical, cosmetic, food, and agricultural [3]. In this context, when considering the ecotoxicology of particles whose components are nontoxic in the micro- or macrometric scales, studies that elucidate the routes of exposure and effects of nanomaterials on environmental compartments and different organisms are fundamental. According to Laux et al. [7], the entry of nanomaterials into the environment occurs by the release of their components during use and by final disposal, so it is important to track and understand the kinetics and transformation of these materials in organisms and the environment. Knowledge of the influence of biopersistence on biokinetics and environmental fate is of utmost importance when determining

When a nanomaterial comes in contact with the human body or the environment, it is difficult to track it again. In the environment, some nanomaterials such as metallic (e.g., Ag and Cu) and metal oxides (e.g., ZnO and Fe2O3) can dissolve rapidly, while others are more persistent (e.g., TiO2, SiO2, carbon nanotubes, and

nisms not yet known or not yet explained by traditional toxicology [5].

and soil), becoming bioavailable, is very large [3, 17].

**18**

terials [14].

**environment**

the toxic potential.

Conner and Schmid [21] stated that most nanomaterials are actively incorporated by cells through endocytosis. This is one of the most important mechanisms of cellular communication with the external environment, since it involves the transmembrane and bidirectional flow of vesicles, through the movement of extracellular content internalization [1]. According to Radaic et al. [1], the shape, size, characteristics (such as porosity) of the surface, surface charge, and composition of nanoparticles directly influence endocytosis. According to Drasler et al. [22], cell size, proliferation rate, and surface receptor growth and expression characteristics are the major factors involved in the entry of nanomaterials into cells. Generally, endocytic pathways are essential in this process, where large particles or agglomerates of nanomaterials are obtained by phagocytosis (diameter greater than 250 nm), whereas smaller particles (diameter smaller than 150 nm) are obtained by pinocytosis, specific or not. Valsami-Jones and Lynch [18] have beautifully illustrated the possible mechanisms of nanomaterial uptake by cells.

Collins et al. [23] have described that besides penetrating cells, many nanomaterials are able to cross nuclear membranes and gain access to chromatin at any stage of the cell cycle. Thus, in addition to direct damage to DNA, nanoparticles can induce the formation of reactive oxygen species (ROS), such as hydroxyl radicals (˙OH), causing oxidative stress (redox imbalance) and serious damage to the cell. Oxidative stress can be the result of the simple cellular response to the presence of the nanomaterial or a secondary effect of the inflammation generated by them [23]. It is also known that the dissolution of certain nanomaterials may be able to release toxic ions and/or other components, which may induce toxicity [22].

ROS are highly reactive molecules that disrupt intracellular medium homeostasis, since they interact with cellular macromolecules such as DNA, proteins, and lipids [24]. Singh et al. [24], Louro et al. [25], and Radaic et al. [1] stated that nanomaterials can induce genotoxic damage mediated by oxidative stress and through their interaction with cellular constituents, including mitochondria and NADPH oxidases bound to the cell membrane; by the depletion of antioxidants; or by the release of the metallic ions present in the constitution of many nanomaterials, which can promote the conversion of cellular oxygen metabolites into ROS. When considering the DNA molecule, the major damage induced by ROS is single-strand breaks, double-strand breaks, base modifications (such as the formation of 8-hydroxydeoxyguanosine adducts), and DNA cross-links. According to Singh et al. [24], all of the aforementioned damages have the potential to initiate and promote carcinogenesis. Once the DNA molecule has been damaged, several cellular processes can be triggered, such as cell cycle arrest, apoptosis, or DNA repair [24].

Apart from the oxidative stress-related lesions, other genotoxic effects may contribute significantly to the promotion of genetic instability, as nanomaterials can cross the pores of the nuclear envelope and interact directly with the genome of the cell and/or with nuclear proteins [25]. Under these conditions, Louro et al. [25] reported that some nanomaterials induce the formation of intranuclear protein aggregates, inhibiting the processes of cell replication, transcription, and proliferation. When the nanomaterials are not able to cross the nuclear envelope, there is still the possibility of interaction with the DNA molecule and nuclear proteins during the mitotic process, which can cause aneuploidies [24, 25].

Depending on the organism exposed to the nanomaterials, different interactions can be evidenced. Bielmeyer-Fraser et al. [26] demonstrated that nanoparticles of ZnO, AgO, and CuO were able to induce toxicity to algae bioindicator in a similar way to the respective solubilized metals. However, these researchers noted the metals accumulated in different regions of the cells, and the nanoparticles were retained mainly in the cell wall, while the metals were observed mainly in the organelles, as fragments of the endoplasmic reticulum. However, in both ways, the

**21**

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach*

cross physiological barriers, influencing its toxic potential [7].

type is actually affected by a certain type of nanomaterial).

physicochemical properties that nanomaterials can present.

ent efficient, cheap, and reliable results [22].

**5.1 In vivo methods**

organism [10].

to better characterize the toxicity of nanomaterials.

**5. Methods for evaluating the toxicity of nanomaterials**

authors point out that the accumulated metal could be transferred by the trophic

When a nanomaterial enters a living organism, several components can adhere to its surface, drastically modifying its interaction with cellular structures. Proteins are molecules that can adhere to the nanomaterial and form a type of coating, called biomolecular corona [27]. This corona may alter the ability of a nanomaterial to

According to Drasler et al. [22], in vitro assays performed with cell culture can indicate the biological fate of nanomaterials at the cellular and multicellular level, even in an excluding mode according to the cell type (i.e., to determine which cell

Within nanotoxicology there is an impasse on how best to assess the possible adverse effects of nanomaterials, both for human health and environmental monitoring. Toxicity tests may be performed employing live (in vivo) organisms, such as microcrustaceans, fishes, rodents, and other animals and/or cell cultures (in vitro). Several standardized toxicological tests are available to measure the biological response of an organism to a chemical. However, there is no standardization for the evaluation of the toxicity of nanomaterials, which hampers the comparison of results and the consensus about their toxicity. Most of the studies performed so far are adaptations of the standard procedures used for other substances [3]. Although some minimal combinations of assays have been proposed, Drasler et al. [22] have described that there is no standard evaluation protocol due to the wide range of

Animal tests are more predictive for human effects but have limitations, mainly

Understanding the demand for orientation and applicability, this chapter will address some of the main evaluation methods, developed both in vivo and in vitro,

In vitro evaluations have increased considerably, but in vivo validation still is necessary to understand and interpret its results. Furthermore, animal experimentation was also part of the NanoTEST project, whose purpose was to understand the effects on the physiology of organisms tested. Currently, the OECD presents some test guidelines on which biomarkers should be used for each test

In general, there are more researches on human toxicity, using rodent models, whereas few in vivo studies addressing the ecotoxicity of nanomaterials are available. Furthermore, most of those found in the literature consider the impact of nanomaterials on aquatic organisms, since the continental and marine waters end up being the main receiving compartment. Some scarce trials address the toxicity of nanomaterials in soil and in atmosphere, commonly as suspended particles. In general, bacteria (e.g., *Aliivibrio fischeri*), algae (e.g., *Raphidocelis subcapitata*),

because of physiological and biochemical differences between the species. In addition, there is a growing public and legal demand that ethically supports the substitution of animal testing for alternatives not based on in vivo testing. New concepts of experimentation have been based on strategies with primary culture of human cells and permanent cultures of well-established cell lines, since they pres-

*DOI: http://dx.doi.org/10.5772/intechopen.88970*

chain and carried to other organisms.

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach DOI: http://dx.doi.org/10.5772/intechopen.88970*

authors point out that the accumulated metal could be transferred by the trophic chain and carried to other organisms.

When a nanomaterial enters a living organism, several components can adhere to its surface, drastically modifying its interaction with cellular structures. Proteins are molecules that can adhere to the nanomaterial and form a type of coating, called biomolecular corona [27]. This corona may alter the ability of a nanomaterial to cross physiological barriers, influencing its toxic potential [7].

According to Drasler et al. [22], in vitro assays performed with cell culture can indicate the biological fate of nanomaterials at the cellular and multicellular level, even in an excluding mode according to the cell type (i.e., to determine which cell type is actually affected by a certain type of nanomaterial).

### **5. Methods for evaluating the toxicity of nanomaterials**

Within nanotoxicology there is an impasse on how best to assess the possible adverse effects of nanomaterials, both for human health and environmental monitoring. Toxicity tests may be performed employing live (in vivo) organisms, such as microcrustaceans, fishes, rodents, and other animals and/or cell cultures (in vitro). Several standardized toxicological tests are available to measure the biological response of an organism to a chemical. However, there is no standardization for the evaluation of the toxicity of nanomaterials, which hampers the comparison of results and the consensus about their toxicity. Most of the studies performed so far are adaptations of the standard procedures used for other substances [3]. Although some minimal combinations of assays have been proposed, Drasler et al. [22] have described that there is no standard evaluation protocol due to the wide range of physicochemical properties that nanomaterials can present.

Animal tests are more predictive for human effects but have limitations, mainly because of physiological and biochemical differences between the species. In addition, there is a growing public and legal demand that ethically supports the substitution of animal testing for alternatives not based on in vivo testing. New concepts of experimentation have been based on strategies with primary culture of human cells and permanent cultures of well-established cell lines, since they present efficient, cheap, and reliable results [22].

Understanding the demand for orientation and applicability, this chapter will address some of the main evaluation methods, developed both in vivo and in vitro, to better characterize the toxicity of nanomaterials.

#### **5.1 In vivo methods**

In vitro evaluations have increased considerably, but in vivo validation still is necessary to understand and interpret its results. Furthermore, animal experimentation was also part of the NanoTEST project, whose purpose was to understand the effects on the physiology of organisms tested. Currently, the OECD presents some test guidelines on which biomarkers should be used for each test organism [10].

In general, there are more researches on human toxicity, using rodent models, whereas few in vivo studies addressing the ecotoxicity of nanomaterials are available. Furthermore, most of those found in the literature consider the impact of nanomaterials on aquatic organisms, since the continental and marine waters end up being the main receiving compartment. Some scarce trials address the toxicity of nanomaterials in soil and in atmosphere, commonly as suspended particles. In general, bacteria (e.g., *Aliivibrio fischeri*), algae (e.g., *Raphidocelis subcapitata*),

*Nanomaterials - Toxicity, Human Health and Environment*

possible mechanisms of nanomaterial uptake by cells.

Conner and Schmid [21] stated that most nanomaterials are actively incorporated by cells through endocytosis. This is one of the most important mechanisms of cellular communication with the external environment, since it involves the transmembrane and bidirectional flow of vesicles, through the movement of extracellular content internalization [1]. According to Radaic et al. [1], the shape, size, characteristics (such as porosity) of the surface, surface charge, and composition of nanoparticles directly influence endocytosis. According to Drasler et al. [22], cell size, proliferation rate, and surface receptor growth and expression characteristics are the major factors involved in the entry of nanomaterials into cells. Generally, endocytic pathways are essential in this process, where large particles or agglomerates of nanomaterials are obtained by phagocytosis (diameter greater than 250 nm), whereas smaller particles (diameter smaller than 150 nm) are obtained by pinocytosis, specific or not. Valsami-Jones and Lynch [18] have beautifully illustrated the

Collins et al. [23] have described that besides penetrating cells, many nanomaterials are able to cross nuclear membranes and gain access to chromatin at any stage of the cell cycle. Thus, in addition to direct damage to DNA, nanoparticles can induce the formation of reactive oxygen species (ROS), such as hydroxyl radicals (˙OH), causing oxidative stress (redox imbalance) and serious damage to the cell. Oxidative stress can be the result of the simple cellular response to the presence of the nanomaterial or a secondary effect of the inflammation generated by them [23]. It is also known that the dissolution of certain nanomaterials may be able to release

ROS are highly reactive molecules that disrupt intracellular medium homeostasis, since they interact with cellular macromolecules such as DNA, proteins, and lipids [24]. Singh et al. [24], Louro et al. [25], and Radaic et al. [1] stated that nanomaterials can induce genotoxic damage mediated by oxidative stress and through their interaction with cellular constituents, including mitochondria and NADPH oxidases bound to the cell membrane; by the depletion of antioxidants; or by the release of the metallic ions present in the constitution of many nanomaterials, which can promote the conversion of cellular oxygen metabolites into ROS. When considering the DNA molecule, the major damage induced by ROS is single-strand breaks, double-strand breaks, base modifications (such as the formation of

8-hydroxydeoxyguanosine adducts), and DNA cross-links. According to Singh et al. [24], all of the aforementioned damages have the potential to initiate and promote carcinogenesis. Once the DNA molecule has been damaged, several cellular processes can be triggered, such as cell cycle arrest, apoptosis, or DNA repair [24]. Apart from the oxidative stress-related lesions, other genotoxic effects may contribute significantly to the promotion of genetic instability, as nanomaterials can cross the pores of the nuclear envelope and interact directly with the genome of the cell and/or with nuclear proteins [25]. Under these conditions, Louro et al. [25] reported that some nanomaterials induce the formation of intranuclear protein aggregates, inhibiting the processes of cell replication, transcription, and proliferation. When the nanomaterials are not able to cross the nuclear envelope, there is still the possibility of interaction with the DNA molecule and nuclear proteins during

Depending on the organism exposed to the nanomaterials, different interactions can be evidenced. Bielmeyer-Fraser et al. [26] demonstrated that nanoparticles of ZnO, AgO, and CuO were able to induce toxicity to algae bioindicator in a similar way to the respective solubilized metals. However, these researchers noted the metals accumulated in different regions of the cells, and the nanoparticles were retained mainly in the cell wall, while the metals were observed mainly in the organelles, as fragments of the endoplasmic reticulum. However, in both ways, the

toxic ions and/or other components, which may induce toxicity [22].

the mitotic process, which can cause aneuploidies [24, 25].

**20**

nematodes (e.g., *Caenorhabditis elegans*), microcrustaceans (e.g., *Daphnia magna*, *D. pulex*, *Ceriodaphnia dubia*), mollusks (e.g., *Lymnaea stagnalis*), fish (e.g., *Danio rerio*), and rodents (Wistar rat and mice) are the most used test organisms for the evaluation of acute toxicity (**Table 2**).

#### **5.2 In vitro methods**

According to Drasler et al. [22], assays can be performed with primary cultures or eternal cell lines. According to these authors, cell lines are preferably chosen because they present great homogeneity and stability, which favors reliability in the results, especially in initial tests. For more specific tests, these same researchers recommend the use of 3D co-cultures, to better understand the mechanisms of action of nanomaterials on tissues.

For the nanomaterial toxicity evaluation, the use of epithelial cell lines (skin, gastrointestinal tract, or lung) is usually indicated as these cells present characteristics of real barriers against harmful agents and are therefore the first to suffer the influence of these compounds [37]. However, it is important to note that some strains may not be responsive to the effects of nanomaterials and, in this case, primary cultures may be more indicated [22].

Aiming at the reproducibility of in vitro assays with culture of cell lines, it is necessary to record details that are generally missing from the publications. The origin of the cells, the number of the passage, the detailed method of cell culture, the brand of plastics, and reagents used during the cultivation/exposure, besides the description of the morphology, growth, and cell differentiation, before and after the test, are the information that should be included in the results' publication [22]. Among the in vitro assays, those performed with mammalian cells are considered to be more important than those performed with other cell types [13].

For the in vitro comet assay with mammalian cell culture, Collins et al. [23] make some recommendations: (1) use non-cytotoxic concentrations (less than 20% of cell viability loss; if the nanomaterial is not cytotoxic, concentrations below 100–150 μg/mL are recommended); (2) choose the cell type according to the exposure scenario (based on exposure route and target organ); (3) determine both short (2–3 h) and long (24 h) tests to obtain a better understanding of the mode of action of the nanomaterial; and (4) determine if the genotoxic damage evidenced is a result of the direct effect with the DNA or due to the oxidation of the DNA. According to Drasler et al. [22], the exposure period is one of the main factors related to contradictory toxicity results for identical nanomaterials, as this involves transformations and the aging of their components.

In vitro assays can cover specific endpoints, such as dermal absorption, skin and eye irritation, endocrine disruption, and genotoxicity, among others. Among the tests, most nanomaterial evaluation protocols align the main routes of exposure, being dermal, oral, and inhalation [22].

According to Catalán et al. [13], the relevance and limitations of genotoxicity/ mutagenicity assays should be taken into account when choosing the most appropriate monitoring method. According to these authors, the tests considered in the evaluation should be based on three categories, following the importance order: (1) gene mutation, (2) chromosomal damage, and (3) DNA damage. DNA damage is considered a mild effect because of the possibility of repair, while chromosomal damage and gene mutation are considered to be severe effects because they are irreparable changes.

Regarding the mutagenic potential of nanomaterials to humans, the effects observed in vivo should be considered more relevant than those observed in vitro,

**23**

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach*

(Mollusca)

*marinoi* (Diatom*—* Skeletomataceae), *Thalassiosira pseudonana* (Diatom*—* Thalassiosiraceae), *Dunaliella tertiolecta* (Algae*—* Dunaliellaceae), *Isochrysis galbana* (Algae— Isochrysidaceae)

15–30 nm *Skeletonema* 

— *Caenorhabditis elegans* (Nematoda)

> *Thalassiosira weissflogii* (Diatom— Thalassiosiraceae)

— *Danio rerio* (Chordata) Metal oxide

(Chordata)

*Xenopus laevis* (Chordata)

**Test organism Main results References**

Growth alteration and bioaccumulation

Decreased reproduction rates

Decreased diatom population growth in similar way to respective dissolved metals. Bioaccumulation of nanoparticles in cell wall and possible transfer through trophic chain

nanoparticles induced different toxic effects in zebrafish development according to each metal. ZnO delayed larvae and embryo development and also induced serious ulceration in larvae

Fish

immunotoxicity and gene expression alteration

Significantly affected tadpole growth. The highest concentration caused mortality, suppressed tadpole body length, and delayed animal development

Only nanoparticles of ZnO have decreased growth rate of diatom and algae population

Croteau et al. [28]

Miller et al. [29]

Chatterjee et al.

Bielmeyer-Fraser et al. [26]

Zhu et al. [31]

Jovanović et al.

Zhang et al. [33]

[32]

[30]

*DOI: http://dx.doi.org/10.5772/intechopen.88970*

**diameter of the particles (nm)**

Ag 13–17 nm *Lymnaea stagnalis*

ZnO: 20–30 nm CuO: 20–100 nm AgO: 20–70 nm

TiO2 ~43 nm *Pimephales promelas*

32 nm

TiO2 5, 10, and

**Nanomaterial Mean** 

ZnO TiO2

Graphene family nanoparticles

ZnO CuO AgO

ZnO Al2O3 TiO2


*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach DOI: http://dx.doi.org/10.5772/intechopen.88970*

*Nanomaterials - Toxicity, Human Health and Environment*

evaluation of acute toxicity (**Table 2**).

action of nanomaterials on tissues.

primary cultures may be more indicated [22].

transformations and the aging of their components.

being dermal, oral, and inhalation [22].

**5.2 In vitro methods**

nematodes (e.g., *Caenorhabditis elegans*), microcrustaceans (e.g., *Daphnia magna*, *D. pulex*, *Ceriodaphnia dubia*), mollusks (e.g., *Lymnaea stagnalis*), fish (e.g., *Danio rerio*), and rodents (Wistar rat and mice) are the most used test organisms for the

According to Drasler et al. [22], assays can be performed with primary cultures or eternal cell lines. According to these authors, cell lines are preferably chosen because they present great homogeneity and stability, which favors reliability in the results, especially in initial tests. For more specific tests, these same researchers recommend the use of 3D co-cultures, to better understand the mechanisms of

For the nanomaterial toxicity evaluation, the use of epithelial cell lines (skin, gastrointestinal tract, or lung) is usually indicated as these cells present characteristics of real barriers against harmful agents and are therefore the first to suffer the influence of these compounds [37]. However, it is important to note that some strains may not be responsive to the effects of nanomaterials and, in this case,

Aiming at the reproducibility of in vitro assays with culture of cell lines, it is necessary to record details that are generally missing from the publications. The origin of the cells, the number of the passage, the detailed method of cell culture, the brand of plastics, and reagents used during the cultivation/exposure, besides the description of the morphology, growth, and cell differentiation, before and after the test, are the information that should be included in the results' publication [22]. Among the in vitro assays, those performed with mammalian cells are considered to

For the in vitro comet assay with mammalian cell culture, Collins et al. [23] make some recommendations: (1) use non-cytotoxic concentrations (less than 20% of cell viability loss; if the nanomaterial is not cytotoxic, concentrations below 100–150 μg/mL are recommended); (2) choose the cell type according to the exposure scenario (based on exposure route and target organ); (3) determine both short (2–3 h) and long (24 h) tests to obtain a better understanding of the mode of action of the nanomaterial; and (4) determine if the genotoxic damage evidenced is a result of the direct effect with the DNA or due to the oxidation of the DNA. According to Drasler et al. [22], the exposure period is one of the main factors related to contradictory toxicity results for identical nanomaterials, as this involves

In vitro assays can cover specific endpoints, such as dermal absorption, skin and eye irritation, endocrine disruption, and genotoxicity, among others. Among the tests, most nanomaterial evaluation protocols align the main routes of exposure,

According to Catalán et al. [13], the relevance and limitations of genotoxicity/ mutagenicity assays should be taken into account when choosing the most appropriate monitoring method. According to these authors, the tests considered in the evaluation should be based on three categories, following the importance order: (1) gene mutation, (2) chromosomal damage, and (3) DNA damage. DNA damage is considered a mild effect because of the possibility of repair, while chromosomal damage and gene mutation are considered to be severe effects because they are

Regarding the mutagenic potential of nanomaterials to humans, the effects observed in vivo should be considered more relevant than those observed in vitro,

be more important than those performed with other cell types [13].

**22**

irreparable changes.


**25**

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach*

since the first allow the detection of inflammation and, therefore, secondary genotoxic effects [13]. Although more predictive for human effects, animal tests still have limitations, mainly because of the physiological and biochemical differences between species. Also, there is a trend to substitute animal testing for suitable

Among the tests recognized by the scientific community, those with certified guidelines for nanomaterial assessment have greater "weight" than others that have not been validated in the determination of genotoxicity/mutagenicity [13]. Although they cannot be used to determine mutagenicity, the remaining assays can

There are several recommended tests to assess nanomaterials, especially those described by the OECD. In accordance with the OECD guidelines [38], in order to select a test and evaluate the genotoxicity of a nanoform, exposure, absorption, solubility, metabolites, and other derivatives should be considered, as well as pos-

Comparing the genotoxicity tests for chemical substances, the comet assay and the micronucleus test are also the most indicated and used by the researchers [13]. The comet assay (single cell gel electrophoresis) is a common method of DNA damage evaluation, which can be performed with very diverse cell types. Briefly, a suspension of individualized cells is mixed with agarose and placed on a pregelatinized slide. Then, cell lysis on Triton X-100 removes membranes and soluble cellular components, while NaCl removes the histones from the DNA, promoting a superadhesion of this material to a matrix, forming a structure known as a nucleoid. When there are breaks in DNA strands (single or double), the fragments tend to move toward the anode during electrophoresis. When there is damage and it is observed by fluorescence microscopy, a comet-like image is noted. The percentage of DNA in the tail is proportional to the frequency of breaks, that is, the damage

As described by Collins et al. [23], several nanomaterials (e.g., TiO2, ZnO, Au, Ag, Co3O4, Fe3O4, SiO2, ZrO2, and others) have already been evaluated by variations of the comet assay with specific endonucleases for some lesions, which increase the power of this tool. Among these enzymes, formamidopyrimidine DNA glycosylase (FPG) recognizes lesions of the 8-oxo-7,8-dihydroguanine (8-oxoG) and formamidopyrimidine type (open-ring purines) and is therefore widely used to estimate oxidative damages to DNA caused by

However, other famous trials are not recommended, such as the Ames test [13]. Catalán et al. [13] discourage the use of this test to evaluate nanomaterials, since some compounds are unable to cross the bacterial wall, while others have bacteri-

Undoubtedly, nanoscience and nanotechnology offer the prospect of great advances to the most different sectors of industry and medicine. However, as any area of technology that makes intensive use of new materials/structures, it brings some risks to the health of organisms and the environment. Generally, toxicological studies involving nanomaterials are still scarce, with results often controversial when compared to each other, mainly due to incipient standardization. In this context, the combination of in vitro and in vivo methods in a battery of tests is still

the best way to assess the toxicity of nanomaterials [22, 23].

*DOI: http://dx.doi.org/10.5772/intechopen.88970*

sible side effects (e.g., generation of ROS).

inferred to the genetic material [23].

nanomaterials [23].

**6. Final considerations**

cidal effect.

alternatives that do not promote pain and suffering [22].

be used to demonstrate the genotoxic potential of nanomaterials.

#### **Table 2.**

*Experimental conditions and obtained results through in vivo tests.*

#### *How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach DOI: http://dx.doi.org/10.5772/intechopen.88970*

since the first allow the detection of inflammation and, therefore, secondary genotoxic effects [13]. Although more predictive for human effects, animal tests still have limitations, mainly because of the physiological and biochemical differences between species. Also, there is a trend to substitute animal testing for suitable alternatives that do not promote pain and suffering [22].

Among the tests recognized by the scientific community, those with certified guidelines for nanomaterial assessment have greater "weight" than others that have not been validated in the determination of genotoxicity/mutagenicity [13]. Although they cannot be used to determine mutagenicity, the remaining assays can be used to demonstrate the genotoxic potential of nanomaterials.

There are several recommended tests to assess nanomaterials, especially those described by the OECD. In accordance with the OECD guidelines [38], in order to select a test and evaluate the genotoxicity of a nanoform, exposure, absorption, solubility, metabolites, and other derivatives should be considered, as well as possible side effects (e.g., generation of ROS).

Comparing the genotoxicity tests for chemical substances, the comet assay and the micronucleus test are also the most indicated and used by the researchers [13]. The comet assay (single cell gel electrophoresis) is a common method of DNA damage evaluation, which can be performed with very diverse cell types. Briefly, a suspension of individualized cells is mixed with agarose and placed on a pregelatinized slide. Then, cell lysis on Triton X-100 removes membranes and soluble cellular components, while NaCl removes the histones from the DNA, promoting a superadhesion of this material to a matrix, forming a structure known as a nucleoid. When there are breaks in DNA strands (single or double), the fragments tend to move toward the anode during electrophoresis. When there is damage and it is observed by fluorescence microscopy, a comet-like image is noted. The percentage of DNA in the tail is proportional to the frequency of breaks, that is, the damage inferred to the genetic material [23].

As described by Collins et al. [23], several nanomaterials (e.g., TiO2, ZnO, Au, Ag, Co3O4, Fe3O4, SiO2, ZrO2, and others) have already been evaluated by variations of the comet assay with specific endonucleases for some lesions, which increase the power of this tool. Among these enzymes, formamidopyrimidine DNA glycosylase (FPG) recognizes lesions of the 8-oxo-7,8-dihydroguanine (8-oxoG) and formamidopyrimidine type (open-ring purines) and is therefore widely used to estimate oxidative damages to DNA caused by nanomaterials [23].

However, other famous trials are not recommended, such as the Ames test [13]. Catalán et al. [13] discourage the use of this test to evaluate nanomaterials, since some compounds are unable to cross the bacterial wall, while others have bactericidal effect.

## **6. Final considerations**

Undoubtedly, nanoscience and nanotechnology offer the prospect of great advances to the most different sectors of industry and medicine. However, as any area of technology that makes intensive use of new materials/structures, it brings some risks to the health of organisms and the environment. Generally, toxicological studies involving nanomaterials are still scarce, with results often controversial when compared to each other, mainly due to incipient standardization. In this context, the combination of in vitro and in vivo methods in a battery of tests is still the best way to assess the toxicity of nanomaterials [22, 23].

*Nanomaterials - Toxicity, Human Health and Environment*

**Test organism Main results References**

The highest concentration (100 mg L−1) did not induce toxic effects under experimental conditions. A mixture of TiO2 forms induced toxic effects by ROS generation when exposed to UVA

Marcone et al. [34]

Heinlaan et al. [35]

Griffitt et al. [19]

Hund-Rinke et al. [36]

light

Suspensions of nano- and bulk TiO2 were not toxic. A nano-ZnO formulation was very toxic to *V. fischeri*, *D. magna*, and *T. platyurus*. Cu compound also showed toxicity; however, for Daphnia magna were less bioavailable than for bacteria

Nanometals caused acute toxicity in multiple aquatic organisms, but the effect was different according to the metal particle and the species used. Since *R. subcapitata*, *C. dubia*, and *D. pulex* were susceptible to nanometals, trophic chain could be compromised

Ag and Cu nanoparticles affected all organisms; ZnO was toxic to algae and daphnids; TiO2 and CeO2 were toxic only to algae

**diameter of the particles (nm)**

TiO2 — *Daphnia similis*

TiO2: 25–70 nm ZnO: 50–70 nm CuO: 30 nm

Ag (20– 30 nm), Cu (15– 45 nm), Al (51 nm), Co (10– 20 nm), Ni (5–20 nm), and TiO2 (30 nm)

Ag (15 nm) ZnO (34–42 nm) TiO2 (10–23 nm) CeO2 (10–33 nm) Cu (76 nm)

*Experimental conditions and obtained results through in vivo tests.*

(Crustacea)

*Vibrio fischeri*

*Raphidocelis subcapitata* (Algae— Selenastraceae), *Ceriodaphnia dubia* (Crustacea), *Daphnia pulex* (Crustacea), *Danio rerio* (Chordata)

*Raphidocelis subcapitata* (Algae— Selenastraceae), *Daphnia magna* (Crustacea), *Danio rerio*

(Chordata)

(*Gammaproteobacteria*), *Daphnia magna* (Crustacea), *Thamnocephalus platyurus* (Crustacea)

**Nanomaterial Mean** 

TiO2 ZnO CuO

Metallic nanoparticles of Ag, Cu, Al, Co, Ni and TiO2

Ag ZnO TiO2 CeO2 Cu

**24**

**Table 2.**

One of the major concerns is the choice of dose/concentration range of nanomaterials to be tested. The inclusion of excessively high doses/concentrations may generate false positives, while excessively low doses may prevent detection or may underestimate the genotoxic potential [23]. Drasler et al. [22] provide all guidelines to be considered in evaluating the toxicity of nanomaterials by cell culture, but in vivo evaluation must not be overlooked. Paschoalino et al. [3] state that the environmental risk analysis of nanomaterials depends mainly on the regulatory structure, which involves the generation of protocols, which must be based on a multidisciplinary interaction, in order to obtain a more risk assessment possible.

As demonstrated by Valsami-Jones and Lynch [18], harmonization of methods and approaches could benefit this young science, as there is still no consensus on basic assessment protocols. Current protocols involve specific techniques and methods to collect and analyze data sufficient to quantitatively describe the release, destination, transport, transformation, exposure, and toxicity of chemicals. Furthermore, in order to be more precise about the toxicity and mechanism of action of nanomaterials on living organisms, the physicochemical characteristics must be sufficiently detailed. So far, a great effort has been made by the OECD to try to standardize test methods that can correctly evidence the risk of nanomaterials. There are a number of internationally accepted test guidelines that are used for toxicity assessment involving trials with organisms for aquatic, soil, and sediment monitoring. Since 2013, experts from all over the world hold strategic meetings to determine what directions the OECD should take regarding the assessment of nanomaterials, as explored in the Petersen et al. [39].

Also, there is a lot of potential in computational models to help elucidate the possible effects of nanomaterials on humans and the environment. Currently, the quantitative structure-activity relationship (QSAR) model seems to be quite adequate because it can relate the structural, physical, and chemical characteristics to the behavior that some nanomaterial can present. To date, the combination of field, laboratory, and computational work still is the most promising technique to ensure reliable responses to the issues involved with nanomaterial toxicity.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Matheus M. Roberto\* and Cintya A. Christofoletti Hermínio Ometto Foundation's University Center, Araras, Brazil

\*Address all correspondence to: mmr@fho.edu.br

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**27**

1):797-811

2017;**6**:69-80

[8] Arora S, Rajwade JM,

2012;**258**(2):151-165

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach*

[9] Suzuki S, Part F, Matsufuji Y, Huber-Humer M. Modeling the fate and end-of-life phase of engineered nanomaterials in the Japanese construction sector. Waste Management. 2018;**72**:389-398

[10] Juillerat-Jeanneret L, Dusinska M, Fjellsbø LM, Collins AR, Handy RD, Riediker M, et al. Biological impact assessment of nanomaterial used in nanomedicine. Introduction to the NanoTEST project. Nanotoxicology.

Stringfellow A, Sun W, Bartelt-Hunt S, et al. A review of the fate of engineered nanomaterials in municipal solid waste streams. Waste Management.

[12] Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environmental Health Perspectives.

[13] Catalán J, Stockmann-Juvala H, Norppa H. A theoretical approach for a weighted assessment of the mutagenic potential of nanomaterials. Nanotoxicology. 2017;**11**(8):964-977

[14] Maynard AD, Warheit DB, Philbert MA. The new toxicology of sophisticated materials: Nanotoxicology and beyond. Toxicological Sciences.

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*DOI: http://dx.doi.org/10.5772/intechopen.88970*

[1] Radaic A, Pugliese GO, Campese GC, Pessine FBT, Bispo de Jesus M. Como estudar interações entre nanopartículas e sistemas biológicos. Química Nova.

**References**

2016;**39**(10):1236-1244

2017. pp. 51-82

2010;**33**(2):421-430

2008;**400**(1-3):396-414

[2] Dusinska M, Rundén-Pran E, Schnekenburger J, Kanno J. Toxicity tests: In vitro and in vivo. In: Adverse Effects of Engineered Nanomaterials. 2nd ed. Cambridge: Academic Press;

[3] Paschoalino MP, Marcone GPS, Jardim WF. Os nanomateriais e a questão ambiental. Quimica Nova.

[4] Ju-Nam Y, Lead JR. Manufactured nanoparticles: An overview of their chemistry, interactions and potential environmental implications. Science of the Total Environment.

[5] Consiglieri VO. Biossegurança em nanotecnologia. In: Hirata MH, Hirata RDC, Mancini Filho J, editors. Manual de Biossegurança. 2nd ed. Barueri: Manole; 2012. pp. 331-339

[6] Dusinska M, Tulinska J, Yamani El N, Kuricova M, Liskova A, Rollerova E, et al. Immunotoxicity, genotoxicity and epigenetic toxicity of nanomaterials: New strategies for toxicity testing? Food and Chemical Toxicology. 2017;**109**(Part

[7] Laux P, Riebeling C, Booth AM, Brain JD, Brunner J, Cerrillo C, et al. Biokinetics of nanomaterials: The role of biopersistence. NanoImpact.

Paknikar KM. Nanotoxicology and in vitro studies: The need of the hour. Toxicology and Applied Pharmacology. *How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach DOI: http://dx.doi.org/10.5772/intechopen.88970*

## **References**

*Nanomaterials - Toxicity, Human Health and Environment*

nanomaterials, as explored in the Petersen et al. [39].

The authors declare no conflict of interest.

Matheus M. Roberto\* and Cintya A. Christofoletti

\*Address all correspondence to: mmr@fho.edu.br

provided the original work is properly cited.

Hermínio Ometto Foundation's University Center, Araras, Brazil

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**Conflict of interest**

**Author details**

One of the major concerns is the choice of dose/concentration range of nanomaterials to be tested. The inclusion of excessively high doses/concentrations may generate false positives, while excessively low doses may prevent detection or may underestimate the genotoxic potential [23]. Drasler et al. [22] provide all guidelines to be considered in evaluating the toxicity of nanomaterials by cell culture, but in vivo evaluation must not be overlooked. Paschoalino et al. [3] state that the environmental risk analysis of nanomaterials depends mainly on the regulatory structure, which involves the generation of protocols, which must be based on a multidisciplinary interaction, in order to obtain a more risk assessment possible. As demonstrated by Valsami-Jones and Lynch [18], harmonization of methods

and approaches could benefit this young science, as there is still no consensus on basic assessment protocols. Current protocols involve specific techniques and methods to collect and analyze data sufficient to quantitatively describe the release, destination, transport, transformation, exposure, and toxicity of chemicals. Furthermore, in order to be more precise about the toxicity and mechanism of action of nanomaterials on living organisms, the physicochemical characteristics must be sufficiently detailed. So far, a great effort has been made by the OECD to try to standardize test methods that can correctly evidence the risk of nanomaterials. There are a number of internationally accepted test guidelines that are used for toxicity assessment involving trials with organisms for aquatic, soil, and sediment monitoring. Since 2013, experts from all over the world hold strategic meetings to determine what directions the OECD should take regarding the assessment of

Also, there is a lot of potential in computational models to help elucidate the possible effects of nanomaterials on humans and the environment. Currently, the quantitative structure-activity relationship (QSAR) model seems to be quite adequate because it can relate the structural, physical, and chemical characteristics to the behavior that some nanomaterial can present. To date, the combination of field, laboratory, and computational work still is the most promising technique to

ensure reliable responses to the issues involved with nanomaterial toxicity.

**26**

[1] Radaic A, Pugliese GO, Campese GC, Pessine FBT, Bispo de Jesus M. Como estudar interações entre nanopartículas e sistemas biológicos. Química Nova. 2016;**39**(10):1236-1244

[2] Dusinska M, Rundén-Pran E, Schnekenburger J, Kanno J. Toxicity tests: In vitro and in vivo. In: Adverse Effects of Engineered Nanomaterials. 2nd ed. Cambridge: Academic Press; 2017. pp. 51-82

[3] Paschoalino MP, Marcone GPS, Jardim WF. Os nanomateriais e a questão ambiental. Quimica Nova. 2010;**33**(2):421-430

[4] Ju-Nam Y, Lead JR. Manufactured nanoparticles: An overview of their chemistry, interactions and potential environmental implications. Science of the Total Environment. 2008;**400**(1-3):396-414

[5] Consiglieri VO. Biossegurança em nanotecnologia. In: Hirata MH, Hirata RDC, Mancini Filho J, editors. Manual de Biossegurança. 2nd ed. Barueri: Manole; 2012. pp. 331-339

[6] Dusinska M, Tulinska J, Yamani El N, Kuricova M, Liskova A, Rollerova E, et al. Immunotoxicity, genotoxicity and epigenetic toxicity of nanomaterials: New strategies for toxicity testing? Food and Chemical Toxicology. 2017;**109**(Part 1):797-811

[7] Laux P, Riebeling C, Booth AM, Brain JD, Brunner J, Cerrillo C, et al. Biokinetics of nanomaterials: The role of biopersistence. NanoImpact. 2017;**6**:69-80

[8] Arora S, Rajwade JM, Paknikar KM. Nanotoxicology and in vitro studies: The need of the hour. Toxicology and Applied Pharmacology. 2012;**258**(2):151-165

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[21] Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;**422**(6927):37-44

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[23] Collins A, Yamani El N, Dusinska M. Sensitive detection of DNA oxidation damage induced by nanomaterials. Free Radical Biology and Medicine. 2017;**107**:69-76

[24] Singh N, Manshian B, Jenkins GJS, Griffiths SM, Williams PM, Maffeis TGG, et al. NanoGenotoxicology: The DNA damaging potential of engineered nanomaterials. Biomaterials. 2009;**30**(23-24):3891-3914

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[27] Emer M, Cardoso MB. Biomolecular corona formation: Nature and bactericidal impact on surface-modified silica nanoparticles. Journal of Materials Chemistry B. 2017;**5**(40):8052-8059

[28] Croteau M-N, Misra SK, Luoma SN, Valsami-Jones E. Silver bioaccumulation dynamics in a freshwater invertebrate after aqueous and dietary exposures to nanosized and ionic Ag. Environmental Science & Technology. 2011;**45**(15):6600-6607

[29] Miller RJ, Lenihan HS, Muller EB, Tseng N, Hanna SK, Keller AA. Impacts of metal oxide nanoparticles on marine phytoplankton. Environmental Science & Technology. 2010;**44**(19):7329-7334

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[32] Jovanović B, Anastasova L, Rowe EW, Zhang Y, Clapp AR, Palić D. Effects of nanosized titanium dioxide on innate immune system of fathead minnow

**29**

*How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach*

et al. Adapting OECD aquatic toxicity tests for use with manufactured nanomaterials: Key issues and consensus recommendations.

Environmental Science & Technology.

2015;**49**(16):9532-9547

*DOI: http://dx.doi.org/10.5772/intechopen.88970*

(*Pimephales promelas* Rafinesque, 1820). Ecotoxicology and Environmental Safety.

[33] Zhang J, Wages M, Cox SB, Maul JD, Li Y, Barnes M, et al. Effect of titanium dioxide nanomaterials and ultraviolet light coexposure on African clawed frogs (*Xenopus laevis*). Environmental

2011;**74**(4):675-683

2012;**31**(1):176-183

Toxicology and Chemistry.

[34] Marcone GPS, Oliveira ÁC,

Materials. 2012;**211-212**:436-442

[36] Hund-Rinke K, Schlich K, Kühnel D, Hellack B, Kaminski H, Nickel C. Grouping concept for metal and metal oxide nanomaterials with regard to their ecotoxicological effects on algae, daphnids and fish embryos.

NanoImpact. 2018;**9**:52-60

cells in vitro – Are they an

2015;**4**(1):1-19

[37] Rothen-Rutishauser B, Clift MJD, Jud C, Fink A, Wick P. Human epithelial

advantageous tool to help understand the nanomaterial-biological barrier interaction? EURO-NanoTox-Letters.

[38] OECD. Draft guidance document on the use of OECD test guidelines on genotoxicity testing for manufactured

nanomaterials. Organization for Economic Co-operation and Development. 2014. pp. 1-3. http:// www.oecd.org/env/ehs/testing/Draft\_ GD\_nano\_and\_genotox\_rev2.pdf

[39] Petersen EJ, Diamond SA, Kennedy AJ, Goss GG, Ho K, Lead J,

[35] Heinlaan M, Ivask A, Blinova I, Dubourguier H-C, Kahru A. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria *Vibrio fischeri* and crustaceans Daphnia magna and *Thamnocephalus platyurus*. Chemosphere. 2008;**71**(7):1308-1316

Almeida G, Umbuzeiro GA, Jardim WF. Ecotoxicity of TiO2 to Daphnia similis under irradiation. Journal of Hazardous *How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach DOI: http://dx.doi.org/10.5772/intechopen.88970*

(*Pimephales promelas* Rafinesque, 1820). Ecotoxicology and Environmental Safety. 2011;**74**(4):675-683

*Nanomaterials - Toxicity, Human Health and Environment*

[25] Louro H, Borges T, Silva MJ. Nanomateriais manufaturados: Novos

Revista Portuguesa de Saúde Pública.

[26] Bielmyer-Fraser GK, Jarvis TA, Lenihan HS, Miller RJ. Cellular partitioning of nanoparticulate versus dissolved metals in marine phytoplankton. Environmental Science & Technology. 2014;**48**(22):13443-13450

[27] Emer M, Cardoso MB. Biomolecular

bactericidal impact on surface-modified silica nanoparticles. Journal of Materials Chemistry B. 2017;**5**(40):8052-8059

[28] Croteau M-N, Misra SK, Luoma SN, Valsami-Jones E. Silver bioaccumulation dynamics in a freshwater invertebrate after aqueous and dietary exposures

Environmental Science & Technology.

[29] Miller RJ, Lenihan HS, Muller EB, Tseng N, Hanna SK, Keller AA. Impacts of metal oxide nanoparticles on marine phytoplankton. Environmental Science & Technology. 2010;**44**(19):7329-7334

[30] Chatterjee DK, Fong LS, Zhang Y. Nanoparticles in photodynamic therapy: An emerging paradigm. Advanced Drug Delivery Reviews.

[31] Zhu X, Zhu L, Duan Z, Qi R, Li Y, Lang Y. Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Zebrafish (*Danio rerio*) early developmental stage. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances & Environmental Engineering.

[32] Jovanović B, Anastasova L, Rowe EW, Zhang Y, Clapp AR, Palić D. Effects of nanosized titanium dioxide on innate immune system of fathead minnow

corona formation: Nature and

to nanosized and ionic Ag.

2011;**45**(15):6600-6607

2008;**60**(15):1627-1637

2008;**43**(3):278-284

desafios para a saúde pública.

2013;**31**(2):188-200

[17] Powers KW, Palazuelos M, Brown SC, Roberts SM. Characterization of nanomaterials for toxicological evaluation. In: Sahu SC, Casciano DA, editors. Nanotoxicity: From In Vivo And In Vitro Models To Health Risks. New Jersey: John Wiley & Sons. 2009.

[18] Valsami-Jones E, Lynch I. NANOSAFETY. How safe are nanomaterials? Science. 2015;**350**(6259):388-389

[19] Griffitt RJ, Luo J, Gao J, Bonzongo J-C, Barber DS. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environmental Toxicology and Chemistry. 2008;**27**(9):1972-1978

[20] Bhaskar S, Tian F, Stoeger T, Kreyling W, la Fuente de JM, Grazú V, et al. Multifunctional nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: Perspectives on tracking and neuroimaging. Particle and Fibre

[21] Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature.

[22] Drasler B, Sayre P, Steinhäuser KG, Petri-Fink A, Rothen-Rutishauser B. In vitro approaches to assess the hazard of nanomaterials. NanoImpact.

Toxicology. 2010;**7**(1):3

2003;**422**(6927):37-44

[23] Collins A, Yamani El N, Dusinska M. Sensitive detection of DNA oxidation damage induced by nanomaterials. Free Radical Biology and Medicine. 2017;**107**:69-76

[24] Singh N, Manshian B, Jenkins GJS, Griffiths SM, Williams PM, Maffeis TGG, et al. NanoGenotoxicology: The DNA damaging potential of engineered nanomaterials. Biomaterials. 2009;**30**(23-24):3891-3914

2017;**8**:99-116

pp. 1-27

**28**

[33] Zhang J, Wages M, Cox SB, Maul JD, Li Y, Barnes M, et al. Effect of titanium dioxide nanomaterials and ultraviolet light coexposure on African clawed frogs (*Xenopus laevis*). Environmental Toxicology and Chemistry. 2012;**31**(1):176-183

[34] Marcone GPS, Oliveira ÁC, Almeida G, Umbuzeiro GA, Jardim WF. Ecotoxicity of TiO2 to Daphnia similis under irradiation. Journal of Hazardous Materials. 2012;**211-212**:436-442

[35] Heinlaan M, Ivask A, Blinova I, Dubourguier H-C, Kahru A. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria *Vibrio fischeri* and crustaceans Daphnia magna and *Thamnocephalus platyurus*. Chemosphere. 2008;**71**(7):1308-1316

[36] Hund-Rinke K, Schlich K, Kühnel D, Hellack B, Kaminski H, Nickel C. Grouping concept for metal and metal oxide nanomaterials with regard to their ecotoxicological effects on algae, daphnids and fish embryos. NanoImpact. 2018;**9**:52-60

[37] Rothen-Rutishauser B, Clift MJD, Jud C, Fink A, Wick P. Human epithelial cells in vitro – Are they an advantageous tool to help understand the nanomaterial-biological barrier interaction? EURO-NanoTox-Letters. 2015;**4**(1):1-19

[38] OECD. Draft guidance document on the use of OECD test guidelines on genotoxicity testing for manufactured nanomaterials. Organization for Economic Co-operation and Development. 2014. pp. 1-3. http:// www.oecd.org/env/ehs/testing/Draft\_ GD\_nano\_and\_genotox\_rev2.pdf

[39] Petersen EJ, Diamond SA, Kennedy AJ, Goss GG, Ho K, Lead J, et al. Adapting OECD aquatic toxicity tests for use with manufactured nanomaterials: Key issues and consensus recommendations. Environmental Science & Technology. 2015;**49**(16):9532-9547

**31**

**Chapter 3**

**Abstract**

Risk Assessment and Health,

*Guilherme Lenz e Silva, Camila Viana, Danieli Domingues*

The management of health, safety, and environmental (HSE) aspects during production, manipulation, storage, incorporation, and disposal of carbon nanomaterials is the key factor for the development of a safe-by-design work based on nanotechnology. The almost endless possibility of functionalization, chemical interaction, and addition of nanomaterials into new products implies a new management approach of HSE. Low amount of reliable toxicity and ecotoxicity data of nanomaterials and nanomaterial composites is available. As complete exposure/ release assessments are a challenging task, recommendation for control measurements is still based on the precautionary point of view. There is an incomplete understanding of environmental fate- and time-related exposure, and of consumer- and worker-related risks and hazards. Control banding and risk evaluation matrix tools can be used to mitigate labor and environment impacts of carbon nanomaterials. This chapter presents new tools and methodologies for exposure assessment and risk evaluation of hazards used on HSE management system of

**Keywords:** risk assessment, risk analysis, risk evaluation, nanocarbons,

Looking into the last 20 years—since the classical publications from the Royal Society, ETC Group, OECD/Allianz, European Commission, Swiss Reinsurance, and studies and think tanks related to risks and uncertainties of nanotechnology [1–7]—without any doubt, we are now in a better position concerning risks (assessment, analysis, evaluation, management, communication, policies, monitoring, and treatment) and benefits of nanotechnologies. However, new nanomaterials and new nanoproducts are achieving several markets, and the global changes on the business environment—including, fast, small and short-term life enterprises, start-ups and spin-offs based on new technologies—underline the need of special attention from the society regarding safety and environmental issues. Sometimes,

Safety, and Environmental

Management of Carbon

Nanomaterials

*and Fernanda Vieira*

carbon nanomaterials.

nanotechnology, HSE

**1. Introduction**

## **Chapter 3**
