Toxys scientists recently published an article in Toxicology entitled “ToxProfiler: A novel human-based reporter assay for in vitro chemical safety assessment”. In this article, ToxProfiler is presented as a robust platform that not only provides accurate toxicity profiles of chemicals but also reveals their toxicological mode-of-action (MoA). By testing 100 chemicals in ToxProfiler, we identified specific cellular pathways associated with each chemical’s toxicological profile.

In ToxProfiler, concentration response modeling and point-of-departure (PoD) estimation allowed to potency rank and identify primary toxicological MoA of chemicals. Furthermore, the assay was applied to group the toxicity liabilities of chemicals based on their ToxProfiler toxicity signatures and link them to a specific pathology.

ToxProfiler is an ideal in vitro safety testing assay

In vitro safety assessment of chemicals offers several advantages, including high throughput, the ability to capture relevant biological processes, and improved time and cost efficiency. However, a single test often cannot deliver all these benefits together. Moreover, in vitro chemical safety assessment often relies on simple and general cytotoxicity endpoint measurement, where simple yes/no results fall short of revealing mechanistic details and therefore, fail to adequately predict human toxicity.

ToxProfiler (Figure 1), on the other hand, is an ideal assay that captures these benefits:

• High throughput: Uses 384-well plates, can be applied in early screens
• Human relevant: Human cell lines with each reporting on different stress pathways
• Quantitative: Dose response modeling informs on primary MoA and potency
• Accurate: Identifies the cellular targets of chemicals and their links to a specific mechanism or pathology.

ToxProfiler accurately predicted toxicological MoA of chemicals

We selected 100 chemicals from previous studies including ToxCast, Tox21, and EUToxRisk representing pharmaceuticals, cosmetic ingredients, food additives, natural/plant-based compounds and agrochemicals. The selected chemicals were associated with different toxicological effects such as mitochondrial toxicity, genotoxicity, cardiotoxicity and liver toxicity.

Using a quantitative analysis, primary toxicological responses to chemical exposure were separated from secondary/tertiary responses. For this, we measured reporter activation for each biomarker in response to increasing chemical concentrations. The lowest ToxProfiler PoD determined the primary MoA. We observed a high correlation between the identified and predicted toxic MoAs (93 out of 100).

For instance, andrographolide is a natural product extracted from a plant with anti-neoplastic properties and was shown to induce ER stress, reactive oxygen species (ROS), and apoptosis. In ToxProfiler PoD analysis (Figure 2), we identified the primary MoA as oxidative stress, followed by protein stress, ER stress and finally cell cycle stress, which fits this chemical’s expected profile.

Finally, to link ToxProfiler signatures to a functional toxicological profile, we applied unsupervised hierarchical clustering. We identified 9 clusters representing a group of chemicals with known annotated toxicological targets or MoA. For example, andrographolide clustered together with bardoxolone methyl and sulforaphane. These chemicals are soft electrophiles causing strong induction of oxidative stress in combination with a weak ER stress, cell cycle stress and protein stress response (Figure 3).

The full article can be accessed here.

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