Utility of ToxTracker in animal alternative testing strategy for fragrance materials

Abstract

To determine the utility of the ToxTracker assay in animal alternative testing strategies, the genotoxic potential of four fragrance materials (2‐octen‐4‐one, lauric aldehyde, veratraldehyde, and p‐methoxy cinnamaldehyde) were tested in the ToxTracker assay. These materials have been previously evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per OECD guidelines. In addition to these studies, reconstructed human skin micronucleus studies were conducted on all four materials. All four materials were positive in an in vitro micronucleus assay but were negative in both in vivo and 3D skin micronucleus assays. The ToxTracker assay, in combination with in silico methods to predict metabolism was used to identify mechanisms for the misleading positive outcomes observed in the in vitro micronucleus assays. The results show that the ToxTracker assay, in conjunction with in silico predictions, can provide the information needed to aid in the identification of an appropriate animal alternative follow‐up assay, for substances with positive results in the standard in vitro test battery. Thus, the ToxTracker assay is a valuable tool to identify the genotoxic potential of fragrance materials and can aid with replacing animal‐based follow‐up testing with appropriate animal alternative assay(s).

Interlaboratory validation of the ToxTracker assay: An in vitro reporter assay for mechanistic genotoxicity assessment

“…To date, ToxTracker has proven to be a valuable addition to the standard battery of in vitro genotoxicity assays for both internal decision making and regulatory applications since it has the unique ability to reveal both genotoxic and non‐genotoxic modes of action. Data from ToxTracker have already been included successfully in regulatory submissions as part of a WoE approach across different industrial sectors (Baltazar et al, 2020; Czekala et al, 2021; Osimitz et al, 2022; Thakkar et al, 2023).…”

“…The general protocol for conducting the ToxTracker assay has been published previously (Hendriks et al, 2016; Thakkar et al, 2023). Briefly, all assays were performed in 96‐well plates, cells were seeded 24 h prior to exposure, and typically five concentrations were evaluated per compound with concurrent negative and positive controls.…”

“…The general protocol for conducting the ToxTracker assay has been published previously (Hendriks et al, 2016;Thakkar et al, 2023).…”

Currently Used Methods to Evaluate the Efficacy of Therapeutic Drugs and Kidney Safety

“…In addition, alternative methods have become essential for animal experimental replacement to provide more ethical and humane research approaches [24,25]. Advanced alternative methods, such as in vitro cell culture, human organoids, and in silico methods, provide unique opportunities for the safety assessment of drug candidates and for studying human disease to overcome these limitations [26][27][28]. For example, integrated testing strategies using computer models and some instruments (e.g., mass spectrometry, functional and pharmacological magnetic resonance imaging, and flow cytometry) have facilitated measurable progress to produce valuable data [29].…”

Next generation risk assessment of androgens and estrogens

“…A broad range of other CALUX reporter gene assays is available that cover receptor binding MIEs, like the progesterone receptor (PR)-CALUX assay to quantify progestin activity (Sonneveld et al, 2011), which could be used in a DCR approach to evaluate progestins. Other examples which can be implemented in the DCR approach are in vitro bioactivity data from ToxTracker (Thakkar et al, 2023) to assess in vivo genotoxicity or from the embryonic stem cell test (EST) (Genschow et al, 2002(Genschow et al, , 2004 to assess in vivo developmental toxicity in humans, demonstrating the broad applicability domain of the DCR approach.…”