**1. Introduction**

Nanoparticles (NPs) range from size 1–100 nm [1]. They are made from various materials like polymers, liposomes, dendrimers, and metals like Zinc, Titanium, Gold, and Aluminum [2]. NPs have been found to induce toxicity through the production of reactive oxidative species (ROS). Their small size, greater surface area to volume ratio, and ability to easily penetrate tissue cells, leading to higher chemical reactivity, cause increased ROS production when introduced into the body [3–5]. Also, due to their very small size, they can be ingested through inhalation and are able to pass through biological barriers into sensitive parts of the body like the lungs, brain, heart, liver, and spleens [6, 7]. A factor that Increases their toxicity is the solubility of the nanomaterial. NPs like zinc oxide and titanium oxide have been found to elicit more toxicity than ceria and titania, which are less soluble [8]. Also, due to the diversity in the use of these NPs in various industries like food, cosmetics, agriculture, biomedical, optics, and technology, it is easy for them to be absorbed and ingested into the human system, affecting the human gut microbiota. They have been observed to accumulate in the stomach, ileum, colon, and duodenum, which poses

**Figure 1.**

*Conventional methods of evaluating the cytotoxicity of the nanoparticles [12].*

serious risks and concerns [9, 10]. Hence, the need to study their toxicity in human systems has become necessary [11].

The two major ways of assessing nanoparticle toxicity are *in vitro* and *in vivo* methods; *ex vivo*, although not commonly used, is another form. As shown in **Figure 1**, here are the common assays used to measure nanoparticle toxicity. With the use of these methods, there has been observed variability in results and even obstruction from the NPs themselves in the assay. In this chapter, we will be considering the various limitations and advantages of the toxicity assays commonly used in nanoparticle toxicity studies to ensure scientists have adequate knowledge and make the best decision on the assays to use for their study.
