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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). [ABSTRACT FROM AUTHOR]

ToxTracker is an in vitro mammalian stem cell‐based reporter assay that detects activation of specific cellular signaling pathways (DNA damage, oxidative stress, and/or protein damage) upon chemical exposure using flow cytometry. Here we used quantitative methods to empirically analyze historical control data, and dose–response data across a wide range of reference chemicals. First, we analyzed historical control data to define a fold‐change threshold for identification of a significant positive response. Next, we used the benchmark dose (BMD) combined‐covariate approach for potency ranking of a set of more than 120 compounds; the BMD values were used for comparative identification of the most potent inducers of each reporter. Lastly, we used principal component analysis (PCA) to investigate functional and statistical relationships between the ToxTracker reporters. The PCA results, based on the BMD results for all substances, indicated that the DNA damage (Rtkn, Bscl2) and p53 (Btg2) reporters are functionally complementary and indicative of genotoxic stress. The oxidative stress (Srxn1 and Blvrb) and protein stress (Ddit3) reporters are independent indicators of cellular stress, and essential for toxicological profiling using the ToxTracker assay. Overall, dose–response modeling of multivariate ToxTracker data can be used for potency ranking and mode‐of‐action determination. In the future, IVIVE (in vitro to in vivo extrapolation) methods can be employed to determine in vivo AED (administered equivalent dose) values that can in turn be used for human health risk assessment. [ABSTRACT FROM AUTHOR]

Nucleoside analogues have long been designed and tested in cancer treatment and against viral infections. However, several early compounds were shown to have mutagenic properties as a consequence of their mode-of-action. This limited their use, and several have been discontinued for lengthy treatments or altogether. Nonetheless, nucleoside analogues remain an attractive modality for virally driven diseases, of which many still are without proper treatment options. To quantitatively assess the genotoxic mode-of-action of a panel of nucleoside analogues, we applied the ToxTracker® reporter assay. Many of the early nucleoside analogues showed a genotoxic response. The more recently developed nucleoside analogues, Remdesivir and Molnupiravir that are currently being repurposed for Covid-19 treatment, had a different profile in ToxTracker and did not induce the genotoxicity reporters. Our analyses support the metabolite GS-441524 over the parent analogue Remdesivir. In contrast, Molnupiravir was devoid of clear cellular toxicity while its active metabolite (EIDD-1931) was cytotoxic and induced several biomarkers. Nucleoside analogues continue to be attractive treatment options upon viral infections. ToxTracker readily distinguished between the genotoxic analogues and those with different profiles and provides a basis for clustering and potency ranking, offering a comprehensive tool to assess the toxicity of nucleoside analogues. • Nucleoside analogues can be mutagenic and carcinogenic because of their MoA. • We tested well-known nucleoside analogues using our genotoxicity assay ToxTracker. • > 50% of the nucleoside analogues were found to induce genotoxicity in ToxTracker. • The data are largely in line with in vivo micronucleus and/or carcinogenicity data. • Recently developed nucleoside analogues generally have better toxicity profiles. [ABSTRACT FROM AUTHOR]

Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment. [ABSTRACT FROM AUTHOR]

Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remain controversial. As GBHs are more cytotoxic than glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (United Kingdom), and MON 76207 (United States) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50, and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased miR-30, whereas miR-10 levels were increased. DNA methylation profiling of liver revealed 5727 and 4496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals, respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate. [ABSTRACT FROM AUTHOR]

Understanding the mode-of-action (MOA) of genotoxic compounds and differentiating between direct DNA interaction and indirect genotoxicity is crucial for their reliable safety assessment. ToxTracker is a stem cell-based reporter assay that detects activation of various cellular responses that are associated with genotoxicity and cancer. ToxTracker consists of 6 different GFP reporter cell lines that can detect the induction of DNA damage, oxidative stress, and protein damage in a single test. The assay can thereby provide insight into the MOA of compounds. Genotoxicity is detected in ToxTracker by activation of 2 independent GFP reporters. Activation of the Bscl2-GFP reporter is associated with induction of DNA adducts and subsequent inhibition of DNA replication and the Rtkn-GFP reporter is activated following the formation of DNA double-strand breaks. Here, we show that the differential activation of these 2 genotoxicity reporters could be used to further differentiate between a DNA reactive and clastogenic or a non-DNA-reactive aneugenic MOA of genotoxic compounds. For further classification of aneugenic and clastogenic compounds, the ToxTracker assay was extended with cell cycle analysis and aneuploidy assessment. The extension was validated using a selection of 16 (genotoxic) compounds with a well-established MOA. Furthermore, indirect genotoxicity related to the production of reactive oxygen species was investigated using the DNA damage and oxidative stress ToxTracker reporters in combination with different reactive oxygen species scavengers. With these new extensions, ToxTracker was able to accurately classify compounds as genotoxic or nongenotoxic and could discriminate between DNA-reactive compounds, aneugens, and indirect genotoxicity caused by oxidative stress. [ABSTRACT FROM AUTHOR]

The current generation of carcinogenicity tests is often insufficient to predict cancer outcomes from pesticide exposures. In order to facilitate health risk assessment, The International Agency for Research on Cancer identified 10 key characteristics which are commonly exhibited by human carcinogens. The ToxTracker panel of six validated GFP-based mouse embryonic stem reporter cell lines is designed to measure a number of these carcinogenic properties namely DNA damage, oxidative stress and the unfolded protein response. Here we present an evaluation of the carcinogenic potential of the herbicides glyphosate, 2,4-D and dicamba either alone or in combination, using the ToxTracker assay system. The pesticide 2,4-D was found to be a strong inducer of oxidative stress and an unfolded protein response. Dicamba induced a mild oxidative stress response, whilst glyphosate did not elicit a positive outcome in any of the assays. The results from a mixture of the three herbicides was primarily an oxidative stress response, which was most likely due to 2,4-D with dicamba or glyphosate only playing a minor role. These findings provide initial information regarding the risk assessment of carcinogenic effects arising from exposure to a mixture of these herbicides. [ABSTRACT FROM AUTHOR]

Cobalt metal and cobalt sulfate are carcinogenic in rodents following inhalation exposure. The pre-carcinogenic effects associated with exposure to these cobalt substances include oxidative stress and genotoxicity. Some, but not all, cobalt substances induce in vitro clastogenicity or an increase in micronuclei. As a result, these substances are classified genotoxic carcinogens, having major impacts on their risk assessment, e.g. assumption of a non-thresholded dose response. Here, we investigated the potential of nine cobalt substances to cause genotoxicity and oxidative stress using the ToxTracker assay, with an extension to measure biomarkers of hypoxia. None of the nine tested substances activated the DNA damage markers in ToxTracker, and five substances activated the oxidative stress response reporters. The same five substances also activated the expression of several hypoxia target genes. Consistent with the lower tier of testing found in the preceding paper of this series, these compounds can be grouped based on their ability to release bioavailable cobalt ion and to trigger subsequent key events. • Cobalt (Co) substances are potentially toxic and carcinogenic following inhalation. • Prediction of outcome by tiered testing of initiating and key events is described. • Two groups of Co substances with distinct effects and toxicities were identified. • The ToxTracker assay was used to investigate genotoxicity of cobalt substances. • Reactive Co substances caused oxidative stress and activated hypoxia target genes. [ABSTRACT FROM AUTHOR]