The degradation of two-dimensional (2D) materials throughout their life cycle can result in the formation of degradation products with different properties and effects on human health and the environment than the original materials. A recent review highlights the importance of understanding these degradation processes for the safety assessment of these materials. However, appropriate regulatory testing methods are currently not available.

The importance of understanding the degradation of 2D materials

A recent review highlights several challenges in assessing the degradation and transformation of 2D materials, such as graphene. These materials, which have a thickness of one or several atom layers, can degrade both in the environment and within the human body. This degradation can lead to the formation of products that alter how these materials behave in target organs, potentially affecting human and environmental toxicity. Therefore, understanding the degradation and transformation of these materials is essential for evaluating risks to both human health and the environment.

Types of degradation

Environmental degradation occurs under natural conditions, through exposure to air, sunlight, water, or through the action of primary decomposers like bacteria, fungi, and some insects. In addition to environmental degradation and transformation, 2D materials can also degrade within the human body. The degradation products may affect cells differently than the intact 2D material. The review's authors emphasise the importance of studying these degradation products and their kinetics to understand and predict potential health effects. Non-degraded or slowly degrading material may accumulate within cells, which could ultimately lead to accumulation in vital organs and impair their function.

Experimental approaches to assess degradation in the body

Most degradation studies are performed in test tubes, where materials are incubated with enzymes, buffers at varying pH levels, salts and ions, or in simulated body fluids. These methods are beneficial for understanding the mechanisms and kinetics of material degradation and investigating specific interactions between (transformed) materials and biological molecules, such as proteins. Degradation can also be studied using cell cultures or animal studies. The review showed that different types of cells exhibit distinct mechanisms of degradation. For example, graphene-related 2D materials are degraded intracellularly in macrophages, while neutrophils stimulate extracellular degradation. These mechanisms result in the formation of distinct degradation products, including hydroxyl radicals which are formed during degradation by macrophages.

Knowledge gap on the long-term fate of 2D materials

The review also shows that there is limited research on the long-term intracellular fate of 2D materials. Studies on degradation in mammals have primarily focused on mice. In addition, while there has been some research on graphene-related 2D materials, there remains a significant gap in understanding the degradation kinetics of other 2D materials in organisms, the chemical identity of their degradation products, and their effects on tissues and organs. The authors highlight that understanding the prolonged internalisation of these materials and the formation of degradation products in mammalian cells will significantly improve our understanding and prediction of long-term effects.

Novel experimental approaches

The authors state that physiologically relevant advanced human in vitro models that mimic human tissue architecture and cellular complexity of key human organs, e.g. spleen, liver may help study intracellular degradation. To improve the biological relevance of the models, it is important to incorporate functional immune system components, as they play an important role in capturing and degrading 2D materials.

Monitoring degradation processes

A final point in the review involves the importance of advanced analytical techniques for gaining a deeper understanding of the degradation processes of 2D materials. For instance, a highly specialised technique such as synchrotron radiation-based X-ray absorption can be beneficial for tracking the chemical valence of elements within these materials. While electrical impedance spectroscopy is traditionally used for assessing the oxidation or corrosion of metals, it also shows potential for application in 2D materials. The analysis of degradation processes of 2D materials is complicated by their dynamic nature in cells and tissues, which necessitates analytical tools capable of providing continuous, longitudinal read-outs.

Reflections by RIVM

The comprehensive review shows that degradation products is a critical step in the life cycle of any material, but assessing degradation processes remains challenging, especially for 2D materials. While environmental degradation in general is addressed to some extent in several OECD test guidelines, it is important to note that these test guidelines were developed and validated for assessing organic chemicals. As highlighted in the Malta Initiative priority list, these methods may face limitations when applied to nanomaterials and 2D materials.

Research into degradation within the body or cells is limited, especially for materials other than graphene-related 2D materials according to the review. Aside from very general toxicokinetic information (which includes but is not limited to metabolism of substances/particles), there are no regulatory requirements under REACH for studying the effects of biodegradation in cells or tissues on human health. Nevertheless, the degradation of materials within the body or cells may affect human health. If nanomaterials are part of medicines or medical devices, there are regulatory requirements for safety testing, including effects of degradation. Guidance about testing is available, see for example the guidance for medical devices. There is no specific guidance for testing degradation of 2D nanomaterials.

The authors have identified several areas needing improvement, but RIVM emphasizes two critical developmental needs: the creation of more complex cell system methods that include immune system components and the development of analytical methods to monitor degradation processes. RIVM considers these priorities crucial for advancing this topic. Implementing complex test systems and analytical methods in a regulatory context will be challenging and may take multiple years. Initiatives like the Malta initiative have the potential to help speed up the development and implementation process of next generation testing methods. This can be achieved by prioritizing actions to support the development and amendment of OECD Test Guidelines or Guidance Documents. A prioritisation that is accepted by scientists, regulators and industry will encourage scientists to develop the required OECD Test Guidelines and provide direction for funders to fund the required next generation of Test Guidelines.