TK6 MNT – Assay Principles
For the TK6 Micronucleus Test (MNT), individual cell cultures 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. Cells are only harvested once cultures have reached between 1.5 and 2 population doublings to ensure that the majority of cells have undergone at least one cell division. Harvested and stained cells are analysed for the presence of micronuclei.
Gentronix offers the TK6 MNT in both a GLP and rapid turn around screening format.
Gentronix obtained it’s authenticated, quality controlled TK6 cell line from the European Collection of Cell Cultures (ECACC).
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 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. 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.