Advances in nanotechnology have led to the emergence of a myriad of nanomaterials with unique properties and countless applications. The Danish Nanodatabase includes more than 4,000 consumer products containing nanomaterials.
As the number of different types of nanomaterials introduced to the global market grows, the likelihood of human exposure to them increases, leading to testing requests from regulatory agencies that often include tests on animals. Since animal tests are associated with ethical issues, are costly and time-consuming, and have limited relevance to humans, many agencies are moving towards using non-animal methods to fulfil regulatory requirements.
Sustainable, ethical, and scientifically sound alternatives to animal testing
Under European law, non-animal methods must be used wherever possible and tests on animals must be undertaken only as a last resort, with a final goal of fully replacing tests on animals. In the United States, the Environmental Protection Agency (EPA) plans to eliminate requests for and funding of all mammalian studies by 2035.
To fulfil the information requirements for nanomaterials and other chemicals, non-animal approaches can provide a sustainable, ethical, and scientifically sound solution. Since nanomaterials are covered under the regulatory framework that is in place for traditional chemicals, existing approaches and methods are generally applicable to them as long as nanospecific parameters are considered.
Grouping and read-across: maximising the use of existing data
One way to reduce reliance on animal testing and to test the growing number of nanomaterials efficiently is to group these materials based on different physicochemical attributes. Grouping and read-across can maximise the use of existing data to predict the effects of unknown substances, which could then facilitate the ranking and prioritisation of nanomaterials for further evaluation, if needed, and the identification of opportunities for waiving tests on animals.
Several grouping frameworks have been proposed for nanomaterials for which consideration of multiple criteria such as exposure, toxicokinetics, and lifecycle, in addition to the material's physicochemical properties, may be required.
Rats in inhalation tubes, Image: Environ Health Perspective. 2000; 108: A168-9
Approximately 1 million animals used worldwide every year for inhalation toxicity testing
Inhalation is one of the main routes by which exposure to nanomaterials can occur, and the respiratory tract may act as both the target tissue and the portal of entry.
The Organisation for Economic Co-operation and Development (OECD) test guidelines (TGs) for subacute (TG 412) and subchronic (TG 413) studies are frequently used to test nanomaterials. Each study conducted using these guidelines can include hundreds of animals – mostly rodents – which contributes to the use of an estimated 1 million animals worldwide for inhalation toxicity testing every year.
But owing to the physiological and anatomical differences between humans and rodents, these tests do not accurately predict human responses to the inhalation of these materials. Therefore, there is an urgent need for further development and use of human-based non-animal approaches that consider relevant exposure scenarios and the mechanism of action.
Adverse outcome pathways can predict complex human responses
Adverse outcome pathways can be used to organise existing information in order to gain a better understanding of the mechanism of action of chemicals, including nanomaterials. The measurable key events (KEs) leading to an adverse outcome can help design testing strategies based on non-animal methods.
An example is a proposed adverse outcome pathway for pulmonary fibrosis that is under review by the OECD. Its key events inform the choice of which assays to use in a study to predict the fibrotic potential of nanomaterials using the three-dimensional human co-culture model of the air-blood barrier EpiAlveolar™ (MatTek Life Sciences), whose development was funded in part by the PETA International Science Consortium Ltd..
The model comprises primary human alveolar epithelial cells, pulmonary endothelial cells, fibroblasts and optionally, alveolar macrophages. It successfully captures the aspects of pulmonary fibrosis as outlined in the adverse outcome pathway and is now being further assessed under the EU Horizon 2020 project Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment (PATROLS) for testing nanomaterials.
EpiAlveolar™ and other methods have recently been reviewed for their use in adverse outcome pathway-based approaches to inhalation toxicity testing. These examples of applying these approaches to the design of in vitro testing strategies demonstrate the way non-animal methods can be used to predict complex human outcomes.
EpiAlveolar™ model. Image: MatTek Life Sciences
Implementing non-animal approaches for regulatory use
The successful implementation of non-animal approaches for regulatory purposes relies on robust methods in addition to increased dialogue among stakeholders.
Committees such as the International Organization for Standardization's (ISO) Technical Committee 229: Nanotechnologies and the OECD Working Party on Manufactured Nanomaterials (WPMN) provide a platform for experts to work together to develop standards and guidance on harmonising testing to fulfil information requirements. Various government and privately funded consortia also play an important role in developing approaches to addressing specific data requirements.
Outputs from such collaborations not only lead to an increased uptake of human-relevant methods but also contribute to the design of approaches that support sustainable advancements in technology. With the growing popularity of "advanced materials", including nanomaterials and many other novel materials, the use of non-animal approaches is the only way for testing to keep pace with technological advancement.
Dr Monita Sharma
I received my PhD in biomedical sciences from Wright State University, with a focus on nanotoxicology. As the nanotoxicology specialist for the PETA International Science Consortium, I research and promote human-relevant, non-animal methods for assessing nanotoxicity. I also participate on the ISO Technical Committee 229: Nanotechnologies and the OECD WPMN to help ensure that the best non-animal methods are included in international standards and guidelines for nanomaterial testing.
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Dr Andreas Stucki
I have a PhD in biomedical sciences from the University of Bern, Switzerland. I have since worked in industry on in vitro respiratory toxicology testing before returning to the University of Bern for my postdoctoral research. My doctoral and postdoctoral research focused on the development and biological evaluation of a lung-on-a-chip – an advanced in vitro model of the human air-blood barrier. I advise the PETA International Science Consortium on inhalation toxicity and nanomaterial testing.
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