Human Lymphocyte MNT – Assay Principles
For the Human Lymphocyte Micronucleus Test (MNT), cultures of freshly isolated lymphocytes are exposed to the test item both with and without an exogenous source of metabolic activation (S9 fraction). Solvent/vehicle and positive controls are included in all tests. During exposure to the test item, the cells are grown for a period sufficient to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. Harvested and stained cells are analysed for the presence of micronuclei.
Micronuclei are only scored in cells that have completed mitosis during exposure to the test item. In this version of the assay cytokinesis is blocked using cytochalasin B.
Whole blood is drawn from young, healthy, non-smoking volunteers with no known recent exposures to genotoxic chemicals or radiation. Typically at least 2 donor samples are pooled for each assay. Lymphocyte cells are isolated using standard methods and are cultured in the presence of a mitogen for between 44 and 48 hours prior to exposure to the test item.
Cells are exposed to the test item along with solvent/vehicle and positive controls. A preliminary dose range finding assay can be set up to assess the toxicity of the test item. From the information provided by the initial test, at lease 3 -6 analysable concentrations are evaluated, in duplicate, with the highest concentration aiming to produce 55 ±5% cytotoxicity.
The assay is carried out both in the presence and absence of a rat-liver S9-mediated metabolic activation system. Typical treatment periods are outlined in the table below.
Following the treatment period, cells are washed, enumerated and adjusted to the same cell density. A thin monolayer of cells is produced on a glass slide which is then fixed and stained using a DNA specific stain to allow the straightforward detection of nuclear material.
All slides are independently coded prior to analysis to ensure there is no operator bias. The cytokinesis-block proliferation index (CBPI) is determined to demonstrate cell proliferation using at least 500 cells per test item concentration, vehicle and positive control. Micronucleus frequencies are analysed in at least 2000 binuclear cells per test item dose level.
Data and Reporting
The final test report generated includes all of the information required by OECD Guideline 487. Individual culture data are provided and all data are summarized in tabular form. There are several criteria for the determination of a positive result such as a concentration-related increase of a statistically significant increase in the number of cells containing micronuclei. In addition, the biological relevance of the results is evaluated by comparing the test data to the appropriate historical control ranges.