Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Where will genetic toxicology testing be in 30 years' time? Summary report of the 25th Industrial Genotoxicity Group Meeting, Royal Society of Medicine, London, November 9, 2011.

A number of influences including legislation, industry and academia have encouraged advances in computational toxicology and high-throughput testing to probe more broadly putative toxicity pathways. The aim of the 25th United Kingdom Mutagen Society (UKEMS) Industrial Genotoxicity Group Annual Meeting 2011 was to explore current and upcoming research tools that may provide new cancer risk estimation approaches and discuss the genotoxicity testing paradigm of the future. The meeting considered whether computer modelling, molecular biology systems and/or adverse outcome pathway approaches can provide more accurate toxicity predictions and whether high-content study data, pluripotent stem cells or new scientific disciplines, such as epigenetics and adductomics, could be integrated into the risk assessment process. With close collaboration between industry, academia and regulators next generation predictive models and high-content tools have the potential to transform genetic toxicology testing in the 21st century.

The mouse lymphoma assay.

The mouse lymphoma TK assay (MLA) is part of an in vitro battery of tests designed to predict risk assessment prior to in vivo testing. The test has the potential to detect mutagenic and clastogenic events at the thymidine kinase (tk) locus of L5178Y mouse lymphoma tk ( +/- ) cells by measuring resistance to the lethal nucleoside analogue triflurothymidine (TFT). Cells may be plated for viability and mutation in semi-solid agar (agar assay) or in 96-well microtitre plates (microwell assay). When added to selective medium containing TFT, wild-type tk ( +/- ) cells die, but TFT cannot be incorporated into the DNA of mutant tk ( -/- ) cells, which survive to form colonies that may be large (indicative of gene mutation) or small (indicative of chromosomal mutation) in nature. Mutant frequency is expressed as the number of mutants per 10(6) viable cells.

Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test.

With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(®) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these investigations demonstrate the suitability of the multi-endpoint design.

Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe and the United States, met on September 9, 2005, in San Francisco, CA, USA. This meeting of the MLA Workgroup was devoted to reaching a consensus on issues involved with 24-h treatment. Recommendations were made concerning the acceptable values for the negative/solvent control (mutant frequency, cloning efficiency and suspension growth) and the criteria to define an acceptable positive control response. Consensus was also reached concerning the use of the global evaluation factor (GEF) and appropriate statistical trend analysis to define positive and negative responses for the 24-h treatment. The Workgroup agreed to continue their support of the International Committee on Harmonization (ICH) recommendation that the MLA assay should include a 24-h treatment (without S-9) in those situations where the short treatment (3-4 h) gives negative results.