The evaluation of mutagenesis is currently restricted to bacterial cells (Ames test), assays in yeasts and fruit flies or specific gene loci in mammalian cells (e.g. TK in the mouse lymphoma assay or Pig-a gene mutation assay) and/or transgenic gene mutation (TGR) assays such as Muta™mouse. As such, there is a need to develop assays that measure induced mutations holistically across the genome and that can be used to better understand the relationship between environmental chemical exposures and mutation induction across species. This would provide mechanistic insight and in a regulatory context, improve understanding for the potential for carcinogenic/heritable disease risk in humans.

As a novel genomic technology, next generation sequencing (NGS) allows the evaluation of mutation using genome-wide or genome-representative panels of loci to determine mutation frequency directly, in DNA from any given sample (i.e., bacteria, cultured cells, animal tissues and even humans). In addition, NGS protocols such as Duplex sequencing (Twinstrand) or NanoSeq (Sanger) can detect very low frequency variants, on the order of 1 in 10 million, allowing a precise estimate of mutation rates around the true spontaneous rate using single-molecule mutation calling. This level of precision also provides the opportunity to evaluate very early clonal expansion, as early markers of carcinogenesis processes, and many months before tumour formation occurs. Another great advantage of NGS is that the results across different samples can be standardized to a simple format for comparison and thereby provide mechanistic insight into the mutagenic mode of action by evaluating 1) mutation frequency 2) simple spectra and 3) trinucleotide spectra.

In recent years, NGS methods have been developed that can accurately detect and characterise mutations in any species/sample from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have applied NGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. NGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis in 3D organoid cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggests NGS can measure mutagenesis associated with clonal expansion as a mechanism-agnostic early marker of carcinogenic potential and there are potential applications for direct human biomonitoring.

UKEMS has established a special interest group (SIG) with a remit to maintain awareness, via its membership, and evaluate promising applications, challenges and key data initiatives needed to enable regulatory testing and adoption of ecNGS – including for advancing safety assessment, augment weight-of-evidence for mutagenicity and carcinogenicity mechanisms and identify early biomarkers of cancer risk and managing human and environmental health risks from chemical exposures. Potential Topics of Interest for the ecNGS SIG include the following:

• Application to mutational fingerprints in cancer – linking these to potential causal agents and correlating in vitro data with human tumour data.
• NGS in regulatory genotoxicity testing – what are the opportunities, where are we now and what are the gaps to build confidence and support discipline and regulatory acceptance? e.g. assay sensitivity; testing negative compounds; data modelling and interpretations for PDE/AI calculations, etc.
• NGS in regulatory carcinogenicity testing – what are the opportunities (e.g. revision of ICH S1), where are we now and what are the gaps to build confidence and support regulatory acceptance? Applications in other sectors e.g. consumer health, chemical industry, environmental genetox, etc.
• NGS in regulatory testing to support gene editing/therapy approaches and concerns for potential insertional effects, off-target effects, etc.
• Note: HESI CT-TRACS committee Tumorigenicity WG – developing methods to assess off-target gene editing (including using Duplex Sequencing), and tumorigenicity / carcinogenicity of edited cell products, among others.
• The US NIST genome editing consortium is interested in this topic also.
• Environmental exposures including the assessment of sentinel organisms for mutagenesis as biomarkers of environmental pollution and stress and population health.
• Comparison of emerging technology approaches and what are the barriers to advancing these in our science (costs, informatics, access to samples, UK funding etc., …)