Critical Examination of Selected Aspects of the ToxTracker In Vitro Genotoxicity Assay: Evaluation of S9 Metabolic Activation Protocols and Quantitative Interpretation of Dose-response Data
Creators: name: Boisvert, Lorrie
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2020
Subjects: genotoxicity
ToxTracker
Dose-response data
Quantitative

description: Genotoxic effects such as mutations and chromosome abnormalities can augment the risk of adverse health effects such as cancer and heritable genetic diseases; chemicals in commerce must be screened for genotoxic activity. To this end, Toxys B.V. developed the in vitro ToxTracker® assay, which detects (geno)toxicity by monitoring the activity of six reporter genes in cultured mES cells (murine embryonic stem cells), i.e., Rtkn, Bscl2, Btg2, Srxn1, Blvrb and Ddit3. The reporters respond to genotoxic stress, oxidative stress, and endoplasmic reticulum stress characterized by protein unfolding; reporter induction is monitored using flow cytometry. The ToxTracker® assay generates large amounts of multivariate concentration-response data; this study employed innovative quantitative methods to scrutinize ToxTracker® assay results. The work (i) defined a fold-change threshold for identification of a significant positive response, (ii) used two analytical approaches to define endpoint-specific Benchmark Response (BMR) values, (iii) used the BMD (Benchmark Dose) combined-covariate approach for potency ranking of assay validation compounds, and (iv) used PCA (Principal Component Analysis) to investigate functional and statistical relationships between the reporters. The results revealed fold-change cut-offs of 1.5 and 1.7 for identification of weak and strong positive responses, respectively. 1.5-fold is consistent with the value advocated by Toxys B.V.; 1.7-fold is more conservative than the Toxys-advocated 2-fold value. Potency ranking of the validation compounds permitted comparative identification of the most potent inducers of each reporter. The most potent compounds consistently included clastogens used for cancer chemotherapy. BMR values determined using the Zeller et al. (2017) approach ranged from 2.2% for Blvrb and Rtkn, to 7.0% for Ddit3, with an average of 3.9% across all the reporters. The Slob (2016) approach yielded values that ranged from 30% for Ddit3, to 52% for Rtkn, with an average of 43%. The PCA results indicated the Rtkn, Bscl2 and Btg2 reporters are functionally redundant; collectively indicative of genotoxic stress. The Blvrb and Ddit3 reporters are orthogonal indicators of oxidative stress and protein unfolding, respectively; they are essential for toxicological profiling using the ToxTracker® assay. PCA axis scores reflect the toxicological MOA (Mode of Action) of the tested compounds; hitherto unknown MOAs can be inferred using PCA axis-plot proximity to well-studied compounds. Like most in vitro (geno)toxicity assessment assays, ToxTracker® employs a material known as S9 to simulate mammalian hepatic metabolism. S9 is prepared from the livers of rats exposed to an inducer of microsomal CYP (Cytochrome P450) isozymes; the most common CYP inducer is the PCB (polychlorinated biphenyl) mixture known as Aroclor-1254. Due to restrictions in the availability of Aroclor-1254, this study also evaluated the utility of Phenobarbital (PB)/β-Naphthoflavone (BNF)-induced S9, a proposed substitute for Aroclor-induced S9. The results indicate that, despite differences in enzymatic profiles, a 24-hr protocol using 0.40% v/v PB/BNF-induced S9 yields results that are comparable to those obtained using 0.25% v/v Aroclor-induced S9. This study constitutes a significant step towards augmenting the utility of the ToxTracker® assay; it provides a foundation for eventual adoption of high-throughput reporter assays for routine regulatory screening of new and existing chemicals.