An evaluation of 25 selected ToxCast chemicals in medium‐throughput assays to detect genotoxicity

Abstract

ToxCast is a multiyear effort to develop a cost-effective approach for the US EPA to prioritize chemicals for toxicity testing. Initial evaluation of more than 500 high-throughput (HT) microwell-based assays without metabolic activation showed that most lacked high specificity and sensitivity for detecting genotoxicants. Thus, EPA initiated a pilot project to investigate the use of standard genotoxicity endpoints using medium-throughput genotoxicity (MTG) assays in the context of a large testing program. Twenty-five chemicals were selected from the ToxCast program based in part on their known genotoxicity. The two MTG assays used were the Ames II(™) assay and 96-well In Vitro MicroFlow(®) Micronucleus (MN) assay. The Ames II assay showed a reasonable correlation with published Ames test data and industry submissions, though specificity was much better than sensitivity due to restraints on top concentrations as prescribed by ToxCast. Overall concordance was 73% both with and without metabolic activation. The flow MN assay had concordances of 71% and 58% with and without metabolic activation, respectively, when compared to published data and submissions. Importantly, a comparison of results without S9 from the MTG assays to an HT ToxCast p53 activation assay showed a fairly good degree of concordance (67%). The results reported here indicate that assays for genotoxicity endpoints can be conducted in a MT format and have the potential to add to the interpretation of results from large-scale testing programs such as EPA's ToxCast program. Inherent limitations such as the top concentrations used in large scale testing programs are discussed. Environ. Mol. Mutagen. 56:468-476, 2015. © 2014 Wiley Periodicals, Inc.

Application of the TGx‐28.65 transcriptomic biomarker to classify genotoxic and non‐genotoxic chemicals in human TK6 cells in the presence of rat liver S9

“…Genotoxicity testing is used to predict potential carcinogenicity or risk of inducing heritable genetic effects. There is increasing emphasis on the use of high‐throughput in vitro screening in human cells in culture as the first tier of chemical screening [Kavlock et al, ; Thomas et al, ; Kligerman et al, ], with growing interest in using toxicogenomics signatures to predict adverse effects and mode of action, and for prioritizing further chemical testing [Thomas et al, ; Zhu et al, ]. Shortcomings of existing in vitro high‐throughput tests include the lack of: (a) metabolic capacity of many of the cell models used; (b) validated toxicogenomics signatures to predict toxicity; and (c) sensitive biomarkers of genotoxicity for high‐throughput screening [Kligerman et al, ].…”

“…There is increasing emphasis on the use of high‐throughput in vitro screening in human cells in culture as the first tier of chemical screening [Kavlock et al, ; Thomas et al, ; Kligerman et al, ], with growing interest in using toxicogenomics signatures to predict adverse effects and mode of action, and for prioritizing further chemical testing [Thomas et al, ; Zhu et al, ]. Shortcomings of existing in vitro high‐throughput tests include the lack of: (a) metabolic capacity of many of the cell models used; (b) validated toxicogenomics signatures to predict toxicity; and (c) sensitive biomarkers of genotoxicity for high‐throughput screening [Kligerman et al, ]. Our previous work established a protocol for assessment of genotoxicity using the TGx‐28.65 biomarker [Li et al, ], and confirmed that the biomarker accurately classified two established mutagens (benzo[a]pyrene and aflatoxin B1) and two non‐mutagens (dexamethasone and phenobarbital) in TK6 cells in the presence of 1% BF/PB‐induced rat liver S9 [Buick et al, ].…”

Bioanalytical Approaches in Assessing Transformation Products

“…Several novel cell based assays have been developed to measure these activities in medium to HTP manners. [42][43] For direct measurement of oxidative stress, a popular assay is the AREc32, which is based on the Nrf2-Keap-ARE32 pathway. 22 This assay has worked well in wastewater applications and is relatively easy to perform.…”

Micronuclei Formation by Promutagens in Metabolism‐Incompetent V79 Cells Interacting With Activation‐Proficient Cells in Various Experimental Settings

“…Many organic carcinogens/mutagens are chemically stable with no bioreactivity by themselves, although they may become reactive only after metabolic activation, a process usually dependent on the activity of relevant biotransformation enzymes, such as cytochromes P450 (CYPs) and sulfotransferases (SULTs) (Rendic and Guengerich, 2012). As target cell lines commonly used in in vitro mutagenicity studies do often not adequately express relevant activating enzymes, an exogenous bioactivating system, typically rat liver S9 mix, is introduced to the in vitro assays, for effective detection of promutagens-a procedure required in the guidelines for mutagenicity assays in the European Union and United States of America (FDA, 2012;Kligerman et al, 2015;OECD TG 487, 2016;OECD TG 490, 2016). The commercially available rat liver S9 fraction prepared from rats pretreated with phenobarbital, β-naphthoflavone, or Aroclor-1254 has been observed to activate many promutagens, such as benzo[a]pyrene (Machanoff et al, 1981;Salassidis et al, 1991), cyclophosphamide, 7,12-dimethylbenz [a]anthracene, aflatoxin B1 (Salassidis et al, 1991), and several nitrosamines (Wagner et al, 2012), in which gene mutations or DNA damage is used as endpoints.…”