V79 in-vitro micronucleus test

OECD 487, Regulation (EG) Nr. 440/2008 B.49, ISO 21427-2, DIN EN ISO 21427

Principle

Genotoxicity test with micronuclei induction with V79 (lung tissue of Chinese hamsters)

Abstract

The micronucleus test allows detecting chromosome damage caused by clastogenic (chromosome breaking) and aneugenic (disrupting the spindle apparatus) substances. During cell division chromosome fragments are no longer integrated into the nuclei of the daughter cells. They remain in the cytoplasm, generating micronuclei, which can be detected by light-optical microscopy. Incidence of increased micronuclei frequency means irreparable damage of the DNA and thus manifested genetic damage which suggests a risk for succeeding cell generations.

Cells are sowed in well defined density and are allowed to adhere over a period of 6 hours to the surface of a microscope slide. The test item is applied in different dilutions and incubated for 4 hours (with S9-mix) or 24 hours (without S9-mix) together with the cells. The addition of the so-called S9-mix, a rat liver fraction, enables the simulation of metabolic activation of pro-mutagenic substances, as may happen in the mammalian liver. Giemsa staining of the cells is performed after washing and fixation. Using light-optical microscopy at least 1,000 cells per test concentration and controls are analysed for the occurrence of micronuclei.

Criteria

A genotoxic effect of the sample can be assumed if a significant increase in micronuclei frequency is detected after treatment of the cell culture with the test item.

Test substance properties

The Micronucleus Test is applicable for solid, soluble substances as well as liquids and water samples.