Design of an In Vitro System to Assess the Inhalation Toxicity of Nanomaterials

PHASE I (completed)

The PETA International Science Consortium convened an expert workshop that was held at the US EPA headquarters in Washington, DC on February 24-25, 2015. The workshop included experts from government, industry, academia and NGOs to discuss the development of an in vitro system to predict the development of pulmonary fibrosis in cells co-cultured at the air-liquid interface following exposure to aerosolized multi-walled carbon nanotubes (MWCNTs). During the workshop, experts made recommendations on cell types, exposure systems, endpoints and dosimetry considerations required to develop the in vitro model for hazard identification of MWCNTs.

Phase I Outputs


Sharma M., Nikota J., Halappanavar S., Castranova V., Rothen-Rutishauser B., Clippinger, A.J. (2016). Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs)Archives of Toxicology. 90(7):1605-22.

Clippinger, A.J., Ahluwalia A., Allen D., Bonner J.C., Casey W., Castranova V., David R.M., Halappanavar S., Hotchkiss J.A., Jarabek A.M., Maier M., Polk W., Rothen-Rutishauser B., Sayes C.M., Sayre P., Sharma M., Stone V. (2016). Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterialsArchives of Toxicology. 90(7):1769-83.

Polk W., Sharma M., Sayes C.M., Hotchkiss J.A., Clippinger A.J. (2016). Aerosol generation and characterization of multi-walled carbon nanotubes exposed to cells cultured at the air-liquid interfaceParticle and Fibre Toxicology. 13(1):20.

Workshop presentations
James Bonner: In vitro approaches for predicting pulmonary fibrosis in rodents and humans after exposure to carbon nanotubes.
Iris Camacho: Challenges of evaluating the human health risks of nanomaterials.
Maria Doa: Improving information used in decision-making.
Sabina Halappanavar: Adverse outcome pathways: a conceptual framework to support the evaluation and extrapolation of toxicological hazards of nanomaterials.
Annie Jarabek: “Mind the gap”: Dosimetry modelling to aid experimental design, evidence integration and inferences.
William Polk: Nanomaterial- and air-liquid interface (NanoALI)-enabled in vitro exposure systems.
Barbara Rothen-Rutishauser: Advanced in vitro lung models in nanotoxicology research – advantages and limitations.
Christie Sayes: State-of-the-science aerosol generation and characterization.
Vicki Stone: Longer term ideas for developing in vitro models for pulmonary toxicology.

PHASE II (in progress)

The Science Consortium is funding Professor Dr. Barbara Rothen-Rutishauser of the Adolphe Merkle Institute at the University of Fribourg, Switzerland and Professor Dr. Vicki Stone of the School of Life Sciences at Heriot-Watt University, Edinburgh, U.K. to jointly develop the system. The Consortium is also funding MatTek Corporation for the development of a three-dimensional reconstructed primary human lung tissue model to be used in Professors Rothen-Rutishauser and Stone’s work.

Phase II Outputs

2016 Society of Toxicology poster: Development of an In Vitro Test to Assess the Inhalation Toxicity of Nanomaterials.
2016 Nanotox Congress poster: Development of an In Vitro System to Assess the Inhalation Toxicity of Nanomaterials.


The system developed in Phase II will be tested in additional laboratories and using different nanomaterials (NMs). The method is intended to be included in a non-animal test battery to reduce and eventually replace the use of animals in studies to assess the inhalation toxicity of engineered NMs. The long-term vision is to develop a battery of in silico and in vitro assays that can be used in an integrated testing strategy, providing comprehensive information on biological endpoints relevant to inhalation exposure to NMs which could be used in the hazard ranking of substances in the risk assessment process.

Please direct any questions, to:
Monita Sharma, Ph.D.
Nanotoxicology Specialist
PETA International Science Consortium Ltd.
Email: [email protected]
Nano workshop 2015 collage pic2












What is “nano”?

The concept of fabricating materials at an atomic scale was introduced in 1959 by physicist Richard Feynman in his talk “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 in the surface-area-to-volume ratio of bulk materials versus that of nanomaterials imparts unique properties to the latter. Owing to their relatively high surface area, nanomaterials are lightweight and have unique optical characteristics and other properties, such as high reactivity and strength. Such properties have made it possible to generate 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 them for possible risks before commercialization. Because of scientific and ethical considerations as well as time and monetary constraints, a great deal of effort is being channelled towards the use and development of non-animal approaches to obtain the toxicity profile of nanomaterials. Such approaches require thorough evaluation of nanomaterials to obtain their physical and chemical profile (known as characterisation), 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 summarise the components required to assess the safety of nanomaterials.

Characterisation 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 analyse 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 characterisation techniques

Following characterisation, 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 in which information from a known material can be used to predict properties of a substance for which there are not enough experimental data, minimising 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, in order 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 then 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 characterisation, could provide useful information regarding nano-bio interaction and potential toxicity. A few examples of some of the non-animal methods that can be used to assess nanotoxicity can be found here. Additionally, 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 International Science Consortium

The PETA International Science Consortium identifies and promotes human-relevant non-animal methods for testing nanomaterials. Based on the current literature and trends, Consortium experts recognise the issues confronting the field of nanotechnology and promote the development and use of non-animal 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 non-animal tests; and (e) participating in standards-making organizations.

PISC Schematic_For Web_RGB72-01

One example of such a multi-faceted approach is demonstrated by the Consortium-funded project to develop 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 information about regulations and novel technology to researchers from government, academia, and industry in order to promote reliable and relevant strategies for replacing the use of animals in nanotoxicity assessment. The Consortium’s efforts are accompanied by participation in and presentations at scientific conferences (here).

Additionally, Consortium scientists participate in groups 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 in the US Technical Advisory Group to the International Organization for Standardization (ISO) Technical Committee (TC) 229 on Nanotechnologies. And as a member of the International Council on Animal Protection in OECD Programmes, the Consortium participates in the OECD’s Working Party on Manufactured Nanomaterials (WPMN). In this capacity, Consortium scientists help to develop and standardise in vitro nanotechnology testing strategies and work towards the implementation of these non-animal 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 non-animal methodologies.


For select publications and resources on nanotechnology and nanotoxicology, please click here.