Nanotechnology and Non-Animal 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. This concept is illustrated in Figure 1. This significant difference of the surface area to volume ratio of bulk versus nanomaterials imparts unique properties to the latter. Because of these properties, such as high reactivity, high strength, lightweight, and unique optical characteristics, nanomaterials are used in 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 fall under the umbrella of “nanotechnology.”

Nano pic1Figure 1: An illustration to depict the increase in surface area of a cube that accompanies a decrease in size. Adapted from BBC.

Are there different kinds of nano? nano pic table2

The possibility of controling biological processes using custom-synthesized materials at the nanoscale has intrigued researchers from different scientific fields. With the ever increasing sophistication of nanomaterial synthesis, there has been an exponential increase in the number and type of nanomaterials available or that can be custom synthesized. Table 1 lists some of the nanomaterials that are currently available.

How do we “see” nano?

Microscopy has been widely used for many decades to visualize objects that are beyond the limit of what our eyes can see. To see nanomaterials, scientists rely on high-powered microscopes, such as scanning electron microscopes, transmission electron microscopes, scanning tunneling microscopes, and atomic force microscopes. These microscopes provide precise measurements of size and dimensions of nanoscale objects.

What is “nano” used for? Nano pic2_2

The unique properties of nanomaterials, such as their strength, lightweight quality, greater chemical reactivity, and optical signatures, make them suitable for a number of applications. Areas where nanomaterials have already been incorporated in application-based tools are the food industry as additives and packaging, the pharmaceutical industry as supplements and additives, and the construction industry as additives for building materials and paints (Figure 2). The use of nanomaterials for technological advancement in different sectors depends on their physico-chemical properties. For instance, the antimicrobial property of nanosilver makes it ideal for use in medicine and also in fabrics and food packaging. Another example is nanogold, which, because of its unique size- and shape-dependent optical properties (referred to as surface plasmon resonance), is used in the development of sensors that have great use in military operations and in the biomedical field.

Toxicity testing of nanomaterials

The growing use of nanomaterials in consumer products necessitates an assessment of the ecological and biological safety of these engineered materials. It is critical to thoroughly characterize a nanomaterial’s physico-chemical properties before testing its toxicity in ecological and physiologically relevant systems. State-of-the-art techniques can precisely and accurately characterize the physico-chemical properties of nanomaterials, such as their shape, surface charge, surface chemistry, aggregation/agglomeration, and elemental composition. Table 2 lists key nano-parameters and the respective techniques used to analyze those.

Parameter

Method

Elemental composition Mass spectrometry (MS), atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR)
Surface chemistry/functionalization Ultraviolet visible spectroscopy (UV-Vis), high performance liquid chromatography (HPLC), gas chromatography/aiquid chromatography mass spectrometry (GC/LC-MS), AAS, FTIR, NMR, x-ray diffraction (XRD)
Morphology Field flow fractionation (FFF), HPLC, disc centrifugation, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), dynamic light scattering (DLS)
Size AFM, TEM, SEM, NMR, XRD
Surface area Brunauer Emmett Teller (BET)
Concentration UV-Vis, HPLC, GC/LC-MS, AAS, ICP-MS
Solubility Tangential flow filtration (TFF), dialysis

Table 2: Nano-parameters and their respective characterization techniques.

Following thorough characterization of a nanomaterial’s physico-chemical properties, a nanomaterial can be tested for its toxicity using in silico or in vitro systems. The diversity of nanomaterials, in addition to the vast number of materials awaiting testing, means that the traditional paradigm of testing one substance at a time in animals is neither feasible nor scientifically and ethically justified. Therefore, efforts are channeled towards the use and development of high throughput and high content testing methods to obtain the toxicity profile of nanomaterials. The following summarizes a tiered testing strategy that can be used to predict the toxic potential of nanomaterials in ecological and biological systems:

  • Characterization: Because nanomaterial properties play a crucial role in their intended applications and could contribute to toxicity, it is important to fully characterize the nanomaterial at all stages post-manufacturing, specifically, as it exists in its pristine form, as it is intended for use (as delivered), and as is present in its final biological and/or ecological system.
  • Toxicity testing: Tiered approaches using in vitro and in silico models should be employed to determine the toxicity of nanomaterials. Assays should be carefully selected at each tier level, starting with assays to determine overt toxicity 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.
  • Risk assessment and management: Based on the data generated using characterization and tiered testing approaches, regulators can make informed decisions regarding the risk/hazard potential of nanomaterials. These decisions are then made available for public dissemination in the form of guidance documents or standards.

Nano-resources

In addition to the use and development of novel methods to assess the toxicity potential of nanomaterials, efforts are being made to increase transparency and dialogue between researchers from government, industry, and academia to help streamline results from these studies. Many web-based repositories have been created that can be populated with a variety of nanomaterial testing data from materials characterization to toxicity results as well as the standards/guidances relevant to both.

The following are some of the web-based tools being used by nanotoxicologists and material scientists:

Role of the PETA International Science Consortium, Ltd.

Government, academic, and private entities have published voluntary standards and literature providing information on the current state of nanotechnology. Groups such as the International Organization for Standardization (ISO) Technical Committee (TC) 229 on Nanotechnologies and the Organization for Economic Co-operation and Development (OECD)’s Working Party on Manufactured Nanomaterials (WPMN), develop standards and guidance documents based on the available scientific evidence in order to shape the strategic and technical direction of nanotechnology development everywhere. PETA International Science Consortium, Ltd., 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 science 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. For example, the science consortium and its members have submitted comments on the following topics:

Based on the current literature and trends, science consortium experts recognize the issues confronting the field of nanotechnology and promote the development and use of nonanimal methods to fill potential data gaps. For instance, upon recognizing the duplicative toxicity testing of nanomaterials that occurs due to a lack of proper characterization, the science consortium sent a letter to the editors of nano-related scientific journals suggesting the addition of a basic characterization checklist as a requirement for all future publications.

In addition to providing technical support, the PETA International Science Consortium, Ltd., conveys regulations and novel technologies to researchers from different areas (government, academia, and industry) to promote reliable and relevant strategies that eliminate the use of animals in nanotoxicity assessment. The consortium’s efforts are mirrored by participation and presentations in scientific conferences (here).

The PETA International Science Consortium, Ltd., and its members also provide financial support directed toward the development and use of nonanimal methods and for the dissemination of information to researchers via workshops.
nano diagram ppt.

Figure 3: The PETA International Science Consortium, Ltd.’s role in nano-related knowledge transfer between researchers from different areas (click here for a larger image).

References

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  4. Bera, D.; Qian, L.; Tseng, T.-K.; Holloway, P. H. Materials 2010, 3, (4), 2260-2345.
  5. Mazumder, S.; Dey, R.; Mitra, M. K.; Mukherjee, S.; Das, G. C. Journal of Nanomaterials 2009, 2009.