§ 798.5375 In vitro mammalian cytogenetics.
(a) Purpose. The in vitro cytogenetics test is a mutagenicity test system for the detection of chromosomal aberrations in cultured mammalian cells. Chromosomal aberrations may be either structural or numerical. However, because cytogenetic assays are usually designed to analyse cells at their first post-treatment mitosis and numerical aberrations require at least one cell division to be visualized, this type of aberration is generally not observed in a routine cytogenetics assay. Structural aberrations may be of two types, chromosome or chromatid.
(b) Definitions. (1) Chromosome-type aberrations are changes which result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method—(1) Principle. In vitro cytogenetics assays may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance both with and without metabolic activation. Following exposure of cell cultures to test substances, they are treated with a spindle inhibitor (e.g., colchicine or Colcemid#) to arrest cells in a metaphase-like stage of mitosis (c-metaphase). Cells are then harvested and chromosome preparations made. Preparations are stained and metaphase cells are analyzed for chromosomal aberrations.
(2) Description. Cell cultures are exposed to test compounds and harvested at various intervals after treatment. Prior to harvesting, cells are treated with a spindle inhibitor (e.g., colchicine or Colcemid#) to accumulate cells in c-metaphase. Chromosome preparations from cells are made, stained and scored for chromosomal aberrations.
(3) Cells—(i) Type of cells used in the assay. There are a variety of cell lines or primary cell cultures, including human cells, which may be used in the assay. Established cell lines and strains should be checked for Mycoplasma contamination and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media, and incubation conditions (culture vessels CO2 concentrations, temperature and humidity) shall be used.