2.4. In Vitro Mammalian Chromosomal Aberration Assay

A BLE0-induced chromosomal aberration test was performed based on OECD guideline TG473 [27] and guidelines of NIFDS of Korea (No. 2014-136) [26] using Chinese hamster lung fibroblast (CHL/IU) cells. CHL/IU cells (CRL-1935) were procured from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in minimum essential medium (Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum. Cells were cultured by adding test or control substances in the absence and presence of an S9 metabolic activation system (S9 mix: 200 μL of rat liver S9 fraction in 1 mL of S9 mix, 5 µM glucose-6-phosphate, 8 µM MgCl₂·6H₂O, 33 µM KCl, 5 µM NADP). A solution of colchicine (final concentration 1 μM) was added approximately 2 h before the end of the culture. After incubation, the culture medium containing metaphase cells was centrifuged at 200 g for 5 min followed by the addition of a fixing solution (glacial acetic acid: methanol = 1:3 v/v). The cell suspension was placed on a glass slide to prepare a slide sample. After drying, the samples were stained for 20 min with a Giemsa staining solution (5%) and analyzed for chromosomal abnormalities under a 1000-fold microscope.