A recent review highlights the significant impact of dispersion methods on the outcomes of toxicity tests involving nanoparticles. The authors found that variations in dispersion methods, such as sonication settings and the composition of the dispersion medium, can affect the agglomerate particle size and, consequently, the toxicity of nanoparticles. The authors emphasise the need for better guidance, which has  recently been published in the updated OECD guidance on sample preparation and dosimetry. 

Dispersion methods can affect toxicity test outcomes

A recent review conducted by researchers from the UK Heriot-Watt University found that different methods are used to disperse nanoparticles in an aqueous medium for use in toxicity studies. These differences affect how the particles behave in the medium, especially in terms of agglomeration, i.e., the extent to which the particles stick together and form a cluster. This, in turn, affects the outcome of toxicity studies.

Dispersion methods vary among researchers

To study the toxicity of nanoparticles in animal studies or cell-based models, it is often necessary to first disperse the particles in an aqueous medium. The dispersion method used can vary considerably among researchers. For example, researchers may use different types of medium and different sonication settings. Sonication is a technique using high frequency sound waves to agitate the particles. . However, the process of dispersion affects how particles stick together, the extent to which they dissolve, and changes their physicochemical properties. These factors, in turn, affect how nanoparticles adhere to surfaces, interact with other particles or biomolecules, and are taken up by cells. 

Dispersion factors affecting the toxicity of titanium dioxide

In the review,  the authors used studies with titanium dioxide (TiO₂) particles as an example to compare different dispersion methods and the impact on physicochemical identity and toxicity. They considered both in vitro and in vivo studies. Overall, the authors found that variations in dispersion methods, particularly those related to sonication and the composition of the dispersion medium, significantly influenced the agglomerate particle size, and consequently, the toxicity of TiO2 particles. The authors found that in some cases, an increase in agglomerate size increased toxicity, while in other cases, it led to decreased toxicity. 

Sonication settings and its reporting are not standardized

Sonication is used to overcome the interparticle adhesion forces that lead to agglomeration. There is significant variation in the type of sonication used by researchers in their studies, including differences in the duration and power settings. At present, there is no standardized way to report the sonication settings, resulting in a varying level of details being provided by the researchers. 

Different studies require different exposure media

The choice of dispersion medium depends on the type of study being conducted (whether animal or cell-based), as well as the specific characteristics of the model and the particles involved. For example, many cell lines require the presence of fetal bovine serum in the medium. This complex biological fluid is obtained from the blood of bovine fetuses and can form a “protein corona” on the particle surface, which may affect their dispersibility. Other substances may also be added to improve dispersion. It is essential to confirm that these additives do not exert toxic effects themselves. 

There is no one-size-fits-all dispersion protocol

The authors reason that a one-size-fits-all dispersion protocol does not exist and is not feasible. However, they plead for more guidance on factors that should be considered when preparing particle dispersions for hazard testing. They also recommend conducting pilot studies to identify the most appropriate dispersion protocol before starting a toxicity study. 

Reflections by RIVM

The review highlights the need for better guidance on the considerations for making a nanoparticle dispersion. The RIVM has been actively contributing to the OECD’s updated guidance on sample preparation and dosimetry (document will be  available here). This document provides general recommendations for preparing samples used in testing manufactured nanomaterials. Understanding the effect of dispersion protocol differences is important, not only for comparing different toxicity studies, but also for interpreting the results of these studies in the context of risk assessment of particles and for comparison with real-life exposure situations.