Nanotechnology and Nonanimal Testing Methods

What is nano?

The concept of fabricating materials at an atomic scale was introduced in 1959 by physicist Richard Feynman in his talk entitled “There’s Plenty of Room at the Bottom.” The term “nano” originates from the Greek word for “dwarf,” which represents the very essence of nanomaterials. In the International System of Units, the prefix “nano” means one-billionth, or 10-9; therefore, one nanometer is one-billionth of a meter, which is smaller than the thickness of a sheet of paper or a strand of hair. It is the small size of nanomaterials that can cause them to have properties different from their bulk counterparts. These differences arise because, as the size of a material decreases, the surface area increases and, therefore, the surface area to volume ratio increases (Figure 1). This significant difference of the surface area to volume ratio of bulk versus nanomaterials imparts unique properties to the latter. Owing to their relatively high surface area, nanomaterials have unique properties, such as high reactivity and strength, are lightweight, and have unique optical characteristics. Such properties have enabled the generation of novel tools in every area of advanced sciences, leading to the emergence of a number of interdisciplinary fields such as nanophysics, nanochemistry, and nanomedicine, which all fall under the umbrella of “nanotechnology.”

Figure 1 - nano webpageFigure 1. An illustration to depict the increase in surface area of a cube that accompanies a decrease in size. Adapted from BBC.

Nanomaterial types and uses

The ever increasing sophistication of nanomaterial synthesis has led to an exponential increase in the types of nanomaterials available, and Figure 2 lists just a few.

Figure 2a - nano webpage

Figure 2. Different classes of nanomaterials and their examples.

The unique properties of nanomaterials make them excellent candidates for a number of applications in the food industry (as additives and packaging), the pharmaceutical industry (as supplements and additives), and the construction industry (as additives for building materials and paint) (Figure 3). The specific use of nanomaterials depends on their physico-chemical properties. For example, the antimicrobial property of nanosilver makes it ideal for use in medicine and also in fabrics and food packaging, while the unique size- and shape-dependent optical properties of nanogold make it useful in the development of sensors for military operations and in the biomedical field.

Figure 3 - nano webpageFigure 3. Applications of nanomaterials

Toxicity testing of nanomaterials

The growing use of nanomaterials in consumer products has raised concerns about their health and environmental effects. Consequently, regulatory agencies require manufacturers of nanomaterials and nano-enabled products to test their products for possible risks before commercialization. Due to scientific and ethical considerations as well as time and monetary constraints, efforts are being channelled towards the use and development of nonanimal approaches to obtain the toxicity profile of nanomaterials. Such approaches require thorough evaluation of nanomaterials to obtain their physical and chemical profile (known as characterization), assessment of existing information to understand their potential toxicity and/or identify information gaps, and then in vitro toxicity testing to fill in any data gaps. The following points summarize the components required to assess the safety of nanomaterials.

Characterization of nanomaterials: It is critical to assess a nanomaterial’s physical and chemical properties, including shape, surface charge, surface chemistry, aggregation/agglomeration, and elemental composition. Table 1 lists key nano-parameters and the respective techniques used to analyze those.

Elemental CompositionAtomic absorption spectroscopy (AAS), energy-dispersive X-ray spectroscopy (EDX), inductively coupled plasma mass spectrometry (ICP-MS), fourier transform infrared spectroscopy (FTIR), mass spectrometry (MS), nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS)
Surface Chemistry/FunctionalisationAtomic absorption spectroscopy (AAS), FTIR, gas chromatography/liquid chromatography mass spectrometry (GC/LC-MS), high performance liquid chromatography (HPLC), NMR, ultraviolet visible spectroscopy (UV-Vis), XPS, X-ray diffraction (XRD), zeta potential
CrystallinityX-ray diffraction (XRD), Raman spectroscopy, thermogravimetric analysis (TGA)
MorphologyAtomic force microscopy (AFM), disc centrifugation, dynamic light scattering (DLS), field flow fractionation (FFF), HPLC, scanning electron microscopy (SEM), transmission electron microscopy (TEM)
Size/Aggregation StateAFM, TEM, SEM, small angle X-ray scattering (SAXS), NMR, XRD
Surface AreaBrunauer-Emmett-Teller (BET)
ConcentrationAAS, GC/LC-MS, HPLC, ICP-MS, TGA, UV-Vis
SolubilityDialysis, inductively coupled plasma (ICP), tangential flow filtration (TFF)

Table 1. Nano-properties and their respective characterization techniques.

Following characterization, a nanomaterial can be tested for its effects on human and environmental health using a number of methods.

Non-testing methods: These are the methods which use existing data for assessment of nanomaterial toxicity and help expedite risk assessment and reduce overall costs of testing. Following are examples of non-testing methods:

  • Grouping and read-across are approaches where information from a known material can be used to predict properties of an substance for which there is not enough experimental data, and so can be used to minimize further testing.
  • Adverse outcome pathways (AOPs) are conceptual frameworks used to organise existing data into a logical sequence of events or processes within a biological system, to understand adverse effects. AOPs provide a clear mechanistic representation of critical toxicological events, starting with the molecular initiating event and ending with the adverse outcome. AOPs help to identify knowledge gaps, facilitate integration of newer ‘non-standard’ data, and reduce reliance on animal testing.

Toxicity testing: Tiered approaches using in vitro and in silico models should be used to determine the toxicity of nanomaterials. Assays should be carefully selected at each tier level, starting with assays to determine overt toxicity and moving to more complex mechanistic-based assays. There are a number of cell- and chip-based assays which, when used in conjunction with comprehensive material characterization, could provide useful information regarding nano-bio interaction and potential toxicity. A few examples of some of the nonanimal methods that can be used to assess nanotoxicity can be found here. In addition to the nonanimal methods, there are web-based tools that use existing data to model predictions of exposure and hazard from nano-enabled consumer products. Examples of such tools are:

Role of the PETA Science Consortium International

The PETA Science Consortium International identifies and promotes human-relevant nonanimal methods for testing nanomaterials. Based on the current literature and trends, Consortium experts recognize the issues confronting the field of nanotechnology and promote the development and use of nonanimal methods to fill potential data gaps. To do so, the Consortium has undertaken a multi-pronged approach that includes (a) hosting workshops and webinars, (b) publishing and presenting our work, (c) collaborating with industry, government, and academia, (d) funding the development and validation of nonanimal tests, and (e) participating in standards-making organizations.

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One example of such a multi-faceted approach is demonstrated by the consortium-funded project on the development of an in vitro system to assess the pulmonary toxicity of nanomaterials, especially multi-walled carbon nanotubes (MWCNTs). The project involves multiple phases including the organization of an expert workshop, method development, and publication of findings.

The Consortium also conveys regulations and novel technologies to researchers from government, academia, and industry to promote reliable and relevant strategies that replace the use of animals in nanotoxicity assessment. The consortium’s efforts are mirrored by participation and presentations in scientific conferences (here).

Additionally, Consortium scientists participate on groups such as the International Organization for Standardization (ISO) Technical Committee (TC) 229 on Nanotechnologies and the OECD’s Working Party on Manufactured Nanomaterials (WPMN) that develop standards and guidance documents based on the available scientific evidence, in order to shape the strategic and technical direction of nanotechnology development everywhere. Consortium member PETA US participates on the US Technical Advisory Group to ISO/TC 229 Nanotechnologies. As a member of the International Council on Animal Protection in OECD Programmes, the Consortium participates on the OECD WPMN. In this capacity, Consortium scientists help to develop and standardize in vitro nanotechnology testing strategies and work toward the implementation of these nonanimal approaches.

Consortium scientists also review and comment on government and private entity opinions and reports concerning the risks of nanomaterials, often providing information on current nonanimal methodologies.