Exhaustive Extractions

At iuvo, we work hard to stay on the cutting edge of new developments in testing and regulatory changes affecting the medical device and pharmaceutical industries. We will share these updates regularly here in our blog - Exhaustive Extractions.

Genetox: Mutational Hazard Identification: Present to Future

As I begin to isolate single bacterial colonies for mutagenicity testing, I couldn’t help but wonder why the bacterial reverse mutation assay has evaded alternate and new technologies for detecting xenobiotic-induced mutation. Of course there is substantial literature and historical data on the salmonella and e. coli strains in wide use today, and there are internationally recognized testing strategies that have been harmonized throughout the past several decades. However, do the highly mutated and compromised bacterial test systems provide any insight into key events along the pathway to mutation? In this day and age, is this the only option to understand the potential for direct DNA reactivity?

 

We are now well into the 21st century and high throughput genomics technologies are becoming cost efficient and more robust. De novo mutations including indels and variants (copy number, single nucleotide and multi-site) are readily detected, which begs the question: why are we not actively developing new in vitro assays? If we consider the current climate on the use of animals in research testing and the fact that genetic toxicology has evolved to conduct exposure and/or mechanistic based risk assessment, a high-throughput mammalian cell assay capable of evaluating mutational events above and beyond point/frameshift mutations would be useful. Or are CNV and non-coding mutational events less important in the etiology of xeniobitic induced carcinogenesis?

Designing a new in vitro tool would not be difficult, as sets of test chemicals are published for use in assay validation, the timing and conduct of mammalian cell based cytogenetic assays could be leveraged as a starting point (chromosomal aberration and micronucleus), and cell lines are readily available. Creating a simple platform will permit various investigations to be conducted, as different endpoints may be needed to elucidate the mechanism of genotoxicity.

Hypothetical all-in-one assay

Allin1assay

Hypothetical all-in-one assay

Overall, the biggest hurdle is defining acceptability and positive response criteria for variant induction. It’s routine to “subtract out” existing germ line mutations, but what happens if a spontaneous mutation occurs in a high concentration? Could it be deemed biologically insignificant only if the event has been observed previously in vehicle-treated or negative-control cultures? How much historical data would this require? Or, perhaps if the variant is detected, but chromatin are stable (ie, no biomarker perturbation or micronucleus induction), we consider the assay to be negative? These are the conversations applied genetic toxicologists should be having, as the technology is there…now someone just has to jump in. In the meantime, I’m going to go back and label some vogell-bonner plates.

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