If you understand the mechanisms of toxicity, you can form a rational basis for interpreting descriptive toxicity data. When you begin to understand that the effects of cellular mechanisms contribute to the toxicities and relate the series of events that began initially with exposure, and involving many further interactions between the organisms and invading toxicants to give the toxic effect shown.
Companies such as Gentronix offer a means to understand and work with such toxicology challenges and use a number of assay tools to establish a mode of action that can mitigate a positive assay test result and avoid the abandonment of otherwise promising molecules. By determining mechanism, you can be supported in managing hazards and risks.
If you are able to identify and understand the biological relevance of a positive result and demonstrate the toxicological mechanism and related mode of action, this could enable you to refine an exposure-based risk assessment. Rather than the need to abandon promising projects, you will be able to design appropriate follow-on studies, to investigate and possibly eradicate such risk or identify ways to manage it. There are now a number of assays that can assist in determining mechanism, help identify false positives and show when there may be demonstrable safe exposure thresholds, which can mean a project can continue within identified and permitted limits, whereas previously they may have been abandoned. We offer a brief overview of some of the assay tests available below.
With MultiFlow® Assay, it is possible to use multiple biomarkers within a single flow cytometry-based experiment that can distinguish between clastogenic and aneugenic modes of action. It will also identify potentially misleading positive results that might be secondary to cytotoxicity. Offering considerable practical use as a follow-up after a positive in vitro result. The MultiFlow® Assay is also a primary screening tool requiring low amounts of compound to provide fast predictions of the mode of action.
Fluorescence in situ hybridization (FISH) offers the ability for centromere detection to be incorporated into the in vitro micronucleus assay. This allows discrimination between an aneugenic mode of action and a clastogenic one. This is an important factor in determining whether a genotoxic substance may have a demonstrable safe exposure threshold and non-mutagenic mode of action by giving an improved understanding of the mechanism in relation to micronucleus induction.
An essay that uses fluorescently labeled specific antibodies for H2AX, and therefore allows for rapid flow cytometric detection with low compound requirements. Using phosphorylation of histone protein H2AX offers a biomarker for DNA double-strand breaks.
This assay offers a highly sensitive indicator for DNA damage. The in vitro nature of this assay can detect DNA damage resulting from a variety of events, including alkali labile sites, single and double-strand DNA breaks, and oxidative base damage. When a positive in vitro genotoxicity test result is received, this follow-up study can inform where a substance has the potential to be DNA reactive within in vitro mammalian cells.
Using flow cytometry to identify markers of cytotoxicity and apoptosis pathways including measuring Caspase 3/7 and Annexin-V the apoptosis assay may be used to identify the potential for misleading genotoxicity results that result from cytotoxic or non-biologically relevant conditions
With such additional assay tests available to identify modes of action, it can identify when a project may be successful in certain conditions. Positive assay results can be questioned, investigated further to define safe limits and the use of such assay testing designed to aid understanding of mechanism can then allow for a project to move forward.