1.0 Introduction
The rapid proliferation of many different engineered nanomaterials presents a dilemma to regulators regarding hazard identification. The screening strategy developed by the International Life Sciences Institute Research Foundation/Risk Science Institute (ILSI RF/RSI) Nanomaterial Toxicity Screening Working Group is an effort to make a significant contribution to the initial hazard identification process for nanomaterial risk assessment.
Engineered nanomaterials are commonly defined as materials designed and produced to have structural features with at least one dimension of 100 nanometers or less. Such materials typically possess nanostructure-dependent properties (e.g., chemical, mechanical, electrical, optical, magnetic, biological), which make them desirable for commercial or medical applications. However, these same properties potentially may lead to nanostructure-dependent biological activity that differs from and is not directly predicted by the bulk properties of the constituent chemicals and compounds. This report outlines the elements of a toxicological screening strategy for nanomaterials as the first step – i.e., hazard identification – in the risk assessment process. Both
Engineered nanomaterials encompass many forms and are derived from numerous bulk substances. Nanoparticles form a basis for many engineered nanomaterials, and are currently being produced in a wide variety of types for a variety of applications; fullerenes (C60 or Bucky Balls), carbon nanotubes (CNT), metal and metal oxide particles, polymer nanoparticles and quantum dots are among the most common.
Engineered nanomaterials are presenting new opportunities to increase the performance of traditional products, and to develop unique new products. "The ability to create unusual nanostructures such as bundles, sheets, and tubes holds promise for new and powerful drug delivery systems, electronic circuits, catalysts, and light-harvesting materials."
Many current efforts are predominantly focused on using relatively simple nanostructured materials such as metal oxide nanoparticles and carbon nanotubes in applications such as high performance materials, energy storage and conversion, self-cleaning surface coatings and stain-resistant textiles. Research into more complex nanomaterials is anticipated to lead to applications such as cellular-level medical diagnostics and treatment and advanced electronics. However, as nanotechnology blurs traditionally rigid boundaries between scientific disciplines, a rapid growth in unanticipated applications is to be expected over the next years and decades.
As new nanotechnology-based materials begin to emerge, it will be essential to have a framework in place within which their potential toxicity can be evaluated, particularly as indicators suggest traditional screening approaches may not be responsive to the nanostructure-related biological activity of these materials.
Several national and international organizations are currently developing standard definitions for comment terms in nanomaterial science including the International Association of Nanotechnology's Nomenclature and Terminology Subcommittee and the American National Standards Institute Nanotechnology Standards Panel (ANSI-NSP). The following key definitions are used throughout this document.
1. Nanoparticle
A particle with at least one dimension smaller than 100 nm including engineered nanoparticles, ambient ultrafine particles (UFPs) and biological nanoparticles.
2. Engineered/Manufactured Nanoparticle
A particle engineered or manufactured by humans on the nanoscale with specific physicochemical composition and structure to exploit properties and functions associated with its dimensions. Engineered nanoparticles include particles with a homogeneous composition and structure, compositionally and structurally heterogeneous particles (for instance, particles with core-shell structures) and multi-functional nanoparticles (for instance, 'smart' nanoparticles being developed for medical diagnostics and treatment).
3. Nanomaterial
A material having a physicochemical structure on a scale greater than typically atomic/ molecular dimensions but less than 100 nm (nanostructure), which exhibits physical, chemical and/or biological characteristics associated with its nanostructure.
4. Nanostructured Particle
A particle with a physicochemical structure on a scale greater than atomic/molecular dimensions but less than 100 nm, which exhibits physical, chemical and/or biological characteristics associated with its nanostructure. A nanostructured particle may be much larger than 100 nm. For example, agglomerates of TiO2 nanoparticles that are significantly larger than 100 nm in diameter may have a biological activity determined by their nanoscale sub-structure. Other examples include zeolites, meso-porous materials and multifunctional particulate probes.
5. Agglomerate/Aggregate
The terms "agglomerate" and "aggregate" are used differently and even interchangeably in different fields. In the context of this report, the term "agglomerate" is used exclusively to describe a collection of particles that are held together by both weak and strong forces, including van der Waals and electrostatic forces, and sintered bonds. In this document, the term is used interchangeably with 'aggregate'. However, the importance of understanding how the binding forces of an agglomerate affect the dispersibility of the component particles under different conditions – in essence how easily the agglomerate de-agglomerates – is noted.
6. Nanoporous Material
A material with particles that are larger than 100 nm may have significant structuring on the nanometer size scale, thereby providing properties based upon this smaller structuring that may be toxicologically relevant (e.g., dramatically increased surface area as compared to the bulk). Nanoporous materials, such as zeolites, are a significant class of materials which have porosity in the sub-100 nm size range but whose primary particles may be large.
2.0 Objectives and Scope
The objective of the ILSI RSI Nanomaterial Toxicity Screening Working Group, which was convened in February 2005, was to identify the key elements of a toxicity screening strategy for engineered nanomaterials. The group considered potential effects of exposure to nanomaterials by inhalation, dermal, oral, and injection routes; discussed how mechanisms of nanoparticle toxicity may differ from those exhibited by larger particles of the same chemical; and identified significant data needs for designing a robust screening strategy.
The elements of a screening strategy for nanomaterials presented by the Nanomaterial Toxicity Screening Working Group include an evaluation of the physicochemical characteristics and dose metrics; acellular assays;
This project was funded by the U.S. Environmental Protection Agency Office of Pollution Prevention and Toxics through a cooperative agreement with the ILSI Research Foundation/Risk Science Institute. It was an outgrowth of another project under the same cooperative agreement that proposed strategies for short-term toxicity testing of fibers
3.0 Literature Survey
The potential for human and ecological toxicity associated with nanomaterials and ultrafine particles is a growing area of investigation as more nanomaterials and products are developed and brought into commercial use. To date, few nanotoxicology studies have addressed the effects of nanomaterials in a variety of organisms and environments. However, the existing research raises some concerns about the safety of nanomaterials and has led to increased interest in studying the toxicity of nanomaterials for use in risk assessment and protection of human health and the environment. A new field of nanotoxicology has been developed to investigate the possibility of harmful effects due to exposure to nanomaterials
As materials reach the nanoscale, they often no longer display the same reactivity as the bulk compound. For example, even a traditionally inert bulk compound, such as gold, may elicit a biological response when it is introduced as a nanomaterial
The earliest studies investigating the toxicity of nanoparticles focused on atmospheric exposure of humans and environmentally relevant species to heterogeneous mixtures of environmentally produced ultrafine particulate matter (having a diameter <100 nm). These studies examined pulmonary toxicity associated with particulate matter deposition in the respiratory tract of target organisms
Laboratory-based studies have investigated the effects of a large range of ultrafine materials through
In contrast to the heterogeneous ultrafine materials produced incidentally by combustion or friction, manufactured nanomaterials can be synthesized in highly homogenous forms of desired sizes and shapes (e.g., spheres, fibers, tubes, rings, planes). Limited research on manufactured nanomaterials has investigated the interrelationship between the size, shape, and dose of a material and its biological effects, and whether a unique toxicological profile may be observed for these different properties within biological models.
Typically, the biological activity of particles increases as the particle size decreases. Smaller particles occupy less volume, resulting in a larger number of particles with a greater surface area per unit mass and increased potential for biological interaction
Toxicological studies of fibrous and tubular nanostructures have shown that at extremely high doses these materials are associated with fibrotic lung responses and result in inflammation and an increased risk of carcinogenesis. Single-walled carbon nanotubes (SWCNT) have been shown to inhibit the proliferation of kidney cells in cell culture by inducing cell apoptosis and decreasing cellular adhesive ability. In addition, they cause inflammation in the lung upon instillation
Dermal exposure to MWCNT has been modeled through cell culture and points to the nanoparticles' ability to localize within and initiate an irritation response in target epithelial cells
Charge properties and the ability of carbon nanoparticles to affect the integrity of the blood-brain barrier as well as exhibit chemical effects within the brain have also been studied. Nanoparticles can overcome this physical and electrostatic barrier to the brain. In addition, high concentrations of anionic nanoparticles and cationic nanoparticles are capable of disrupting the integrity of the blood-brain barrier. The brain uptake rates of anionic nanoparticles at lower concentrations were greater than those of neutral or cationic formulations at the same concentrations. This work suggests that neutral nanoparticles and low concentration anionic nanoparticles can serve as carrier molecules providing chemicals direct access to the brain and that cationic nanoparticles have an immediate toxic effect at the blood-brain barrier
Tests with uncoated, water soluble, colloidal C60 fullerenes have shown that redox-active, lipophilic carbon nanoparticles are capable of producing oxidative damage in the brains of aquatic species
Oxidative stress as a common mechanism for cell damage induced by nano- and ultrafine particles is well documented; fullerenes are model compounds for producing superoxide. A wide range of nanomaterial species have been shown to create reactive oxygen species both
Quantum dots (QDs) such as CdSe QDs have been introduced as new fluorophores for use in bioimaging. When conjugated with antibodies, they are used for immunostaining due to their bright, photostable fluorescence.
To date, there is not sufficient analysis of the toxicity of quantum dots in the literature, but some current studies point to issues of concern when these nanomaterials are introduced into biological systems. Recently published research indicates that there is a range of concentrations where quantum dots used in bioimaging have the potential to decrease cell viability, or even cause cell death, thus suggesting that further toxicological evaluation is urgently needed
The range of approaches and methods used to reach conclusions regarding the effects of manufactured nanomaterials and ultrafine particles has led to different results. This inconsistency indicates a need for standardized tests in order to get comparable results in screening nanomaterials for potential adverse effects. As the field of nanotoxicology continues to grow, standard toxicology tests will aid those entering the field and allow for better comparisons and conclusions in determining the toxic effects of nanomaterials.
4.0 Elements of a screening strategy for nanomaterials
While the nanostructure-dependent properties of many engineered nanomaterials may place them in the category of potential hazards, the direct risk they present to human health will depend on the probability of exposures occurring, and the extent to which materials entering the body exhibit behavior associated with their nanostructure. Figure
Biokinetics of Nano-sized Particles
Biokinetics of Nano-sized Particles. While many uptake and translocation routes have been demonstrated, others still are hypothetical and need to be investigated. Largely unknown are translocation rates as well as accumulation and retention in critical target sites and their underlying mechanisms. These as well as potential adverse effects will be largely dependent on physicochemical characteristics of the surface and core of nano-sized particles. Both qualitative and quantitative changes in nano-sized particles' biokinetics in a diseased or compromised organism need also to be considered. Reproduced with permission from Environmental Health Perspectives.
In many cases, nanostructured materials will be components of large-scale products such as nano-composites, surface coatings and electronic circuits, and the potential for direct exposure will be negligible. However, if nanostructured materials may enter the body, toxicity screening strategies are required to ascertain the potential risk they present.
Nanoparticles are an obvious form of engineered nanomaterial presenting a significant exposure potential, because they can be readily deposited in the lungs or on the skin, and potentially translocate within the body. However, agglomerates of nanoparticles from a few hundred nanometers to a few micrometers in diameter may also be inhaled, ingested or deposited on the skin, and may have the potential to express toxicity associated with their nanostructure. Similarly, it is conceivable that nanostructured particles of a few micrometers in diameter and below (such as fragments of a nano-composite or a nanostructured surface coating) may exhibit nanostructure-dependent biological properties. In each of these cases, exposure potential exists for materials in air and in liquid suspensions or slurries.
In this section, three key aspects of toxicity screening strategies are addressed: characterization of nanomaterials,
4.1 Physicochemical Characterization
4.1.1 Introduction
Unlike gases, liquids and many solid materials, the desirable properties of engineered nanomaterials closely depend on size, shape and structure (both physically and chemically) at the nanoscale. Similarly, there is a strong likelihood that biological activity will depend on physicochemical parameters not usually considered in toxicity screening studies. Although quantitative toxicity studies on engineered nanomaterials are still relatively sparse, published data on fullerenes, single walled carbon nanotubes, nanoscale metal oxides such as TiO2 and nanometer-diameter low solubility particles, support the need to carefully consider how nanomaterials are characterized when evaluating potential biological activity
Until the mechanistic associations between nanomaterial characteristics and toxicity are more fully understood, it will be necessary to ensure that all nanomaterial characteristics that are potentially significant are measured or can be derived in toxicity screening tests. In particular, in as far as it is possible; it is desirable to collect sufficient information to allow retrospective interpretation of toxicity data in the light of new findings. In this context, identifying a set of characterization criteria for nanomaterial toxicity screening presents a significant challenge. Clearly, the ideal of characterizing every possible aspect of a test material, while laudable, is impractical. In this document, we have therefore focused on the context under which characterization takes place and the minimum set of characterization parameters we consider essential within that context. Essential parameters have been supplemented with those considered desirable and those considered of interest but optional within a screening study. The two overarching characterization contexts discussed are human exposure studies and
4.1.2 Framework for Material Characterization
Material characterization for toxicity screening studies is most appropriately considered in the context of the studies being undertaken. Requirements for
• Human exposure characterization
• Characterization of material following administration
• Characterization of administered material
• Characterization of as-produced or supplied material
Human Exposure Characterization
Where exposure to a specific material is known to occur or is anticipated, exposure studies are desirable in developing and selecting appropriate toxicity screening tests. At present engineered nanomaterials are predominantly at the research or pre-production stage, and there are relatively few environments where exposures are known to occur. However, if commercialization of products using nanomaterials develops as anticipated, the potential for exposure is likely to increase dramatically over the coming decade. Therefore, estimates of future use and potential human exposures should be considered in the development of toxicity screening.
Nanomaterial Characterization after Administration
Characterizing delivered nanomaterial after administration in a test system or model provides the highest quality of data on dose and material properties that are related to observed responses, but this is limited by current methodological capabilities. Characterization after administration is particularly advantageous where the possibility of physicochemical changes in the material before and after administration exists. Examples of potential changes include aggregation state, physisorption or chemisorption of biomolecules and biochemically-induced changes in surface chemistry. In addition, possible physicochemical changes as a result of nanomaterial interactions with the surrounding biological systems such as rapid dissolution of water- or lipid soluble fractions of the nanomaterial need to be carefully considered. While characterization after administration is considered an ideal to work towards, it is recognized that in many cases, characterization at the point of administration will be a more realistic and feasible option. It is also recognized that in many cases, characterization at the point of administration will be essential for the intercomparison of studies, irrespective of whether characterization after administration is carried out.
Characterization of Administered Material
Characterization of administered material in toxicity screening studies is fundamental. This approach addresses potential physicochemical changes between the bulk material and the administered material (such as agglomeration state) and allows more robust causal associations between the material and observed responses to be developed. However, given the strong sensitivity of many nanomaterial properties to their local environment, it should be noted that biologically relevant changes in the physicochemical nature of a nanomaterial between administration and deposition may have a significant impact on observed responses in some instances.
Characterization of As-Produced or Supplied Material
Characterization of nanomaterials as-produced or as-supplied represents the most direct approach to obtaining physicochemical information and may provide useful baseline data on the material under test. Most engineered nanomaterials have a functionality based on their physicochemistry. It is therefore likely that information of relevance to toxicity screening studies will be available from suppliers or producers in many cases. However, due to the current lack of accepted nanomaterial characterization standards, it is strongly recommended that wherever possible, independent characterization of test nanomaterials be conducted.
Characterization of supplied nanomaterial may not appropriately represent physicochemical properties of the material during or following administration. For this reason exclusive reliance on this approach is discouraged, and is only recommended where characterization of material during or after administration is clearly not feasible.
4.1.3 Key Characteristics
Previous studies of asbestos and other fibers have shown that the dimension, durability and dose (the three D's) of fibrous particles are key parameters with respect to their pathogenicity. In general, fibers with a smaller diameter will penetrate deeper in the lungs. Long fibers (longer than the diameter of alveolar macrophages) stimulate macrophages to release inflammatory mediators and will only be cleared slowly. In addition to fiber length, chemical factors play an important role in fiber durability and biopersistence; fibers with high alkali or alkali earth oxide contents and low contents of Al2O3, Fe2O3, TiO2 tend to have low durability and hence low biopersistence
The unusual properties of nanomaterials are predominantly associated with their nanometer-scale structure, size and structure-dependent electronic configurations and an extremely large surface-to-volume ratio relative to bulk materials. Particles in the nanosize range can deposit in all regions of the respiratory tract including the distal lungs. Due to their small size, nanoparticles may pass into cells directly through the cell membrane or penetrate between or through cells and translocate to other parts of the body. Limited data have suggested possible translocation of inhaled nanoparticles to the nervous system and other organs/tissues
The size of nanoparticles alone may not be the critical factor determining their toxicity; the overall number and thus the total surface area may also be important. As a particle decreases in size, the surface area increases (per unit mass only; if you normalize to number of particles, the surface area decreases) and a greater proportion of atoms/molecules are found at the surface compared to those inside. Thus, nanoparticles have a much larger surface area per unit mass compared with larger particles. The increase in the surface-to-volume ratio results in the increase of the particle surface energy which may render them more biologically reactive.
Nano-scale materials are known to have various shapes and structures such as spheres, needles, tubes, plates, etc. Nanoporous materials are materials with defined pore-sizes in the nanometer range. The effects of the shape on the toxicity of nanomaterials are unknown. The shape of nanomaterials may have effects on the kinetics of deposition and absorption in the body. The results of a recent
Chemical composition is another important parameter for the characterization of nanomaterials, which comprise nearly all substance classes, e.g., metal/metal oxides, compounds, polymers as well as biomolecules. Some nanomaterials can also be a combination of the above components in core-shell or other complex structures. Dependent on the particle surface chemistry, reactive groups on a particle surface will certainly modify the biological effects. Under ambient conditions, some nanoparticles can form aggregates or agglomerates. These agglomerates have various forms, from dendritic structure to chain or spherical structures. To maintain the characteristics of nanoparticles, they are often stabilized with coatings or derivative surface to prevent agglomeration. The properties of nanoparticles can be significantly altered by surface modification and the distribution of nanoparticles in the body strongly depends upon the surface characteristics. Changes of surface properties by coating of nanoparticles to prevent aggregation or agglomeration with different types and concentrations of surfactants have been shown to change their body distribution and the effects on the biological systems significantly
Therefore, it is recommended that the following physicochemical properties of the test materials should be characterized:
• Size distribution
• Agglomeration state
• Shape
• Crystal structure
• Chemical composition – including spatially averaged (bulk) and spatially resolved heterogeneous composition
• Surface area
• Surface chemistry
• Surface charge
• Porosity
4.1.4 Dose Metrics
In any toxicity screening study, careful consideration should be given to the metric used to quantify dose. Although response may be associated with a wide range of physicochemical characteristics, measuring dose against a physical metric of mass, surface area or particle number for a well-characterized material will enable quantitative interpretation of data. Appropriate selection of the dose metric will depend on the hypothesized parameter most closely associated with anticipated response or the metric which may be most accurately measured. It is strongly recommended that in all cases, sufficient information is collected to enable dose against
Off-line mass concentration measurements using filter-based methods offer continuity with standard inhalation studies and are recommended as an essential component of inhalation nanomaterial screening tests. Likewise, on-line mass concentration measurements are recommended as an essential component of inhalation studies. Gravimetric and/or chemical analysis of filter samples will provide the most accurate characterization of exposure in many cases when compared to off-line surface area and number concentration analyses. With appropriate additional information, such measurements may be used to calculate aerosol surface area or number concentration. However, the diameter cubed relationship between particle size and mass can lead to large errors when transforming from mass to number concentration if the size distribution is broad or there are small numbers of excessively large particles present. On-line mass-concentration measurements using instruments such as the Tapered Element Oscillating Microbalance (TEOM®) potentially offer high precision and good accuracy
Aerosol size distribution measurements enable reasonably good calculation of exposure against all three physical metrics, if parameters such as particle shape and density are known. Off-line size distribution measurement methods such as Transmission Electron Microscopy (TEM) analysis offer detailed information on this distribution but are extremely time consuming, and frequently limited by the collection techniques and, in the case of TEM analysis, inference of 3-dimensional structure from 2-dimensional images. On-line size measurement techniques such as Differential Mobility Analysis
Off-line surface area characterization is possible using isothermal gas-adsorption, although techniques suited to filter samples need to be employed. There is also some possibility that the surface area of the collected material will differ from that of the airborne material due to compaction and surface occlusion. However, the extent to which this may occur is not well understood. Published studies have shown a good correlation between off-line surface area measurement and biological response
Number concentration may be measured on-line with relative ease using instruments such as Condensation Particle Counters
Table
Recommendations for measuring exposure during inhalation studies
Metric
Measurement Recommendation
Mass – off-line
E (coupled with on-line)
Mass – on-line
E
Size distribution – off line
E
Size distribution – on line
E/D
Surface area – off line
O
Surface area – on line
O
Number – off line
N
Number – on line
E
E: These measurements are considered to be essential.
D: These measurements are considered to provide valuable information, but are not recommended as essential due to constraints associated with complexity, cost and availability.
O: These measurements are considered to provide valuable but non-essential exposure information.
N: These measurements are not considered to be of significant value to inhalation studies.
4.1.5 Characterization Prioritization
In developing recommendations on material characterizations for nanomaterial toxicity screening studies, three specific factors have been taken into consideration: the context within which a material is being evaluated, the importance of measuring a specific parameter within that context, and the feasibility of measuring the parameter within a specific context. Recommendations on off-line material characterizations for nanomaterial toxicity screening studies are presented in Table
Recommendations on material characterization
Characterization (Off-line)
Human exposure
Toxicity Screening Studies
Supplied material
Administered material
Material
Size distribution (primary particles)
E (Combine with agglomeration state)
E
D
D
Shape
E
E
O
O
Surface area
D
E
D
O
Composition
E
E
O
O
Surface chemistry
D
E
D
D/O
Surface contamination
D
N
D
N
Surface charge – suspension/solution
O
E
E
O
Surface charge – powder (use bio fluid surrogate)
O
E
N
O
Crystal structure
O
E
O
O
Particle physicochemical structure
E
E
D
D
Agglomeration state
E
N
E
D
Porosity
D
D
N
N
Method of production
E
E
--
--
Preparation process
--
--
E
--
Heterogeneity
D
E
E
D
Prior storage of material
E
E
E
--
Concentration
E
--
E
D
E: These characterizations are considered to be essential.
D: These characterizations are considered to provide valuable information, but are not recommended as essential due to constraints associated with complexity, cost and availability.
O: These characterizations are considered to provide valuable but non-essential information.
N: These characterizations are not considered to be of significant value to screening studies.
In addition, recommendations have been made on recording information on nanomaterial production, preparation, storage, heterogeneity, and agglomeration state. To enable retrospective interpretation of toxicity data and replication of tests, it is strongly recommended that all information on the production and processing of nanomaterials be recorded. Fully documenting storage time and conditions (including temperature, humidity, exposure to light and atmosphere composition) is essential, as physicochemical changes may take place over time. If possible, the physicochemical stability of samples over time should be demonstrated. Where a test material is a heterogeneous mixture of different components, information is required on the relative abundance of the different components, and whether associations in the bulk material are maintained in the administered material, or whether different components are preferentially administered with specific delivery mechanisms.
The agglomeration state of a nanomaterial during and following administration may have a significant impact on its biological activity. Agglomeration state at different structure scales should be characterized, including primary (primary particles), secondary (primary particle agglomerates and self-assembled structures) and tertiary (assemblies of secondary structures) scales. Ideally, agglomeration state in the biological environment following administration should be evaluated. If possible, some insight into the binding forces within agglomerates (e.g. relatively weak van der Waals forces or relatively strong sintered bonds) should be obtained. Material agglomeration or de-agglomeration in different liquid media should also be investigated where possible.
Characterization of material as administered is recommended as the highest priority, supplemented by characterization after
4.1.6 Analysis Methods
Many analytical techniques, both established and developmental, are available for characterizing the nanomaterial properties listed in Table
Applicability of a range of analytical techniques to providing specific physicochemical information on engineered nanomaterials, in the context of toxicity screening studies
Analytical technique
Transmission Electron Microscopy (TEM)
Scanning Electron Microscopy (SEM)
X-Ray Diffraction (XRD)
X-ray Photon Spectroscopy (XPS)
Auger Spectroscopy (AES)
Secondary Ion Mass Spectrometry (SIMS)
Scanning Probe Microscopy
Dynamic Light Scattering (DLS)
Zeta potential
Size Exclusion Chromatography
Analytical Ultracentrifugation
Differential Mobility Analysis (DMA)
Isothermal Adsorption (e.g. BET)
Spectroscopic techniques (UV vis, IR, Raman, NMR)
Elemental analysis (eg ICP-MS/AA etc)
Physicochemical Characteristic
Size distribution (primary particles)
▲
●
●
●
●
●
●
●
Shape
▲
●
●
Surface area
●
◇
◇
◇
◇
●
Composition
●
●
●
▲
●
▲
Surface chemistry
●
●
●
◇
Surface contamination
●
●
Surface charge – suspension/solution
▲
Surface charge – powder (use bio fluid surrogate)
▲
Crystal structure
●
◇
▲
Particle physicochemical structure
▲
●
Agglomeration state
▲
●
●
●
Porosity
◇
●
●
Heterogeneity
▲
●
◇
Other applicable techniques are available that have not been listed.
▲Highly applicable
● Capable of providing information in some cases
Capable of providing information in some cases, with validation from more accurate/applicable techniques
◇ Capable of providing qualitative or semi-quantitative information
Given the wide range of analytical techniques available in many disciplines associated with nanotechnology, multidisciplinary collaborations with research and analysis groups offering state of the art nanomaterial characterization capabilities are strongly recommended when carrying out nanomaterial toxicity screening studies.
4.1.7 Research Gaps
1. The development of viable
2. The development and production of inexpensive real-time monitoring instruments and methods for aerosol mass concentration (low concentrations, nanoscale particles), surface area concentration and size distribution.
3. The development of standardized, well characterized nanomaterial samples.
4. The development of radio-labeled nanomaterial samples, and samples that can be tracked and detected through neutron-activation.
5. The development of more advanced surface chemistry characterization techniques, in particular techniques capable of detecting and speciating biological molecules on the surface of nanoparticles and nanomaterials.
6. The development of electron microscopy techniques for biologically-relevant nanoscale analysis.
4.1.8 Recommendations
1. All nanomaterial physicochemical characteristics that are potentially significant should be measured or be derivable in toxicity screening tests.
2. Characterization of nanomaterial as administered is strongly recommended, supplemented by characterization following administration where it is technically feasible and practicable. Characterization of the bulk material as-produced or supplied to the exclusion of the above is not recommended, except where more appropriate measurements are not feasible.
3. It is recommended that independent characterizations of nanomaterials (beyond information provided by producers and suppliers) are carried out where possible.
4. It is recommended that the following physicochemical properties of nanomaterials should be characterized in the context of toxicity screening tests: Particle size distribution, agglomeration state, particle shape, crystal structure, chemical composition (bulk and spatial), surface area, surface chemistry, surface charge, and porosity.
5. It is recommended that in all cases, sufficient information be collected to enable derivation of the delivered dose against all three primary physical metrics (number, surface area and mass concentration).
6. Off-line mass concentration measurements using filter-based methods are recommended as an essential component of inhalation nanomaterial screening tests. In addition, off-line measurement of aerosol size distribution is recommended.
7. On-line mass concentration and number measurements are recommended as an essential component of inhalation studies.
8. Multidisciplinary collaborations between research and analysis groups offering state of the art nanomaterial characterization capabilities are strongly recommended.
9. It is recommended that information on nanomaterial production, preparation, storage, heterogeneity and agglomeration state be recorded for all nanomaterial toxicity screening studies.
10. It is recommended that nanomaterial preparation methods are fully documented, including the selection of appropriate dispersion media, methods of dispersion in the medium and agglomeration state within the medium. Specific preparation techniques are not recommended, as these will depend on the material and test protocols being used. However, caution is advised when using ultrasonic agitation to disperse materials, as at high energies the method may be sufficiently aggressive to alter the material characteristics (see section 4.3.1.1).
4.2
4.2.1 Introduction
Before considering the application of specific
1)
- Advantages and disadvantages
2)
- Control particles
3)
- Expression of dose
4)
- Adsorption of proteins by nanoparticles
The
4.2.2 Portals of Entry
4.2.2.1 Lungs
The lungs represent a potential target for any airborne particles, and many
The Epithelium
The epithelium is the first barrier that confronts particles that deposit in either the conducting airways or the alveolar region. Therefore, both bronchial and alveolar epithelial cells should be considered as target cells for
If cancer is an endpoint that is under consideration, then direct measures of genotoxicity can be quantified by methods that include COMET assay and 8-hydroxy-deoxyguanosine measurement
Macrophages
Macrophages play a key role in the cellular response to particles that deposit in the lungs. Macrophages could be affected by nanoparticles in various ways that can be studied
Endothelial cells
Although these are found in the lungs, they are considered a part of the cardiovascular system and are dealt with below.
Fibroblasts
Fibroblasts are found in the interstitium and are liable to be affected by any particle that gains access to this site. At least two important modes of response could be activated by nanoparticle/fibroblast interactions and both modes constitute relevant endpoints for
The Immune System
Immunopathological effects could be envisaged if particles interact with lymphocytes, or as a consequence of their predilection for entering the interstitium, they modulate dendritic cell function. The effects of nanoparticles on immunological functions including antigen presentation by macrophages and dendritic cells and the subsequent effects on immune responses
Co-Cultures
In addition to monocultures of lung cells, co-cultures such as epithelial cells/macrophages or epithelial cells/endothelial cells may more closely represent the
Lung Slices
Methodology to culture whole lung tissue slices is available, such that multiple pulmonary cell types can be exposed
Cell Lines vs. Freshly-Derived Cells
If possible, freshly-derived primary cells should be used. Where cell lines are used, these should preferably not be cancer cells. Where cancer cells are used, the endpoint response under study should be carefully compared to non-cancer cells to ensure that, for that endpoint, the fact that the cell is a cancer cell does not greatly modify the response compared to a non-cancer cell.
Whole Heart-Lung Preparation
The Langendorff heart-lung preparation may provide the opportunity to study the behavior of nanoparticles under highly controlled conditions. In this model the exsanguinated heart and lungs are maintained by perfusion and so transport between the lungs and the vascular space can be studied in the absence of blood
4.2.2.2 Skin
Skin or the integument is the largest organ of the body and is unique because it is a potential route for exposure to nanoparticles during their manufacture and also provides an environment within the avascular epidermis where particles could potentially lodge and not be susceptible to removal by phagocytosis
Research should address the effects of dermal and systemic exposure to a number of types of nanoparticles in the skin. The skin is a primary route of potential exposure to toxicants, including novel nanoparticles. However, there is no information on whether particles are absorbed across the stratum corneum barrier or whether systemically administered particles can accumulate in dermal tissue. Nanoparticles may traverse through the stratum corneum layers at varying rates due to particle size or become sequestered within the epidermis to increase their exposure time to viable epidermal keratinocytes.
Nanomaterials are difficult to obtain in large quantities; therefore, it is best to conduct
Cell Culture
Human epidermal keratinocyte (HEK) monolayers can be affected by nanoparticle interactions. It has already been shown that changes in biomarkers of viability and toxicity can occur with exposure to multi-wall carbon nanotubes
Three dimensional skin cell cultures are also available commercially. They have shown to be able to predict irritation but may significantly overestimate absorption or penetration
Flow-through Diffusion Cell Studies
Diffusion cell system consists of flow-through diffusion blocks each containing multiple Teflon cells perfused by a constant temperature circulator through a Silastic oxygenator, an automatic fraction collector, and a desiccant. Circular fresh skin from pigs (pig skin mimics human skin and eliminates the extreme variability seen with