Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay

Introduction:
This article describes a study evaluating the interlaboratory transferability and performance of a multiplexed high information content in vitro genotoxicity assay, known as the MultiFlow VRDNa Damage Kit—p53, gh2aX, Phospho-histone h3. The assay combines several biomarkers relevant to DNa damage response pathways and is designed to predict a chemical's predominant genotoxic mode of action (Moa) by categorizing it as clastogenic, aneugenic, or non-genotoxicant.

Key Points:

* The study involved seven laboratories treating cells with a set of 84 chemicals, including aneugens, clastogens, and nongenotoxicants.
* The assay involves a one-step, add-and-read process in microtiter plate-format for high throughput analysis via flow cytometry.
* Biomarker responses include phosphorylation of histone h3 and histone h2aX, nuclear p53 content, frequency of cells, and absolute nuclei counts.
* Two data analysis strategies were used to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or nongenotoxic mode of action.
* Based on the aggregate 231 experiments, assay sensitivity, specificity, and concordance in relation to a priori Moa grouping were 92.1%, 96.2%, and 93.9%, respectively.
* The results suggest that the assay and data analysis strategies can reliably predict new chemicals' predominant genotoxic Moa.
* The study also explores multiple approaches for efficiently analyzing and interpreting the collection of biomarker responses generated by the assay.

Main Message:
The MultiFlow VRDNa Damage Kit—p53, gh2aX, Phospho-histone h3 assay has shown promising results in predicting the genotoxic mode of action of chemicals, with high sensitivity, specificity, and concordance. The study demonstrates the assay's transferability across laboratories and the robustness of two data analysis strategies. The high throughput and dimensionality of the assay make it a valuable tool for early safety assessments, reducing resources and test chemical requirements compared to conventional tests. Overall, the assay has the potential to significantly improve the efficiency and accuracy of genotoxicity testing.

Bryce, Steven M., Derek T. Bernacki, Jeffrey C. Bemis, Richard a. Spellman, Maria E. Engel, Maik Schuler, Elisabeth Lorge, et al. “Interlaboratory Evaluation of a Multiplexed high Information Content in Vitro Genotoxicity assay.” Environmental and Molecular Mutagenesis 58, no. 3 (april 2017): 146–61. https://doi.org/10.1002/em.22083.