Chromosome aberration test in Chinese hamster lung (CHL) cells

Chromosome aberration tests were conducted as described previously [17] with minor modifications as described by Ishidate [20] and by Dean and Danford [21]. Chinese hamster lung (CHL) cells were selected as the test system because they are sensitive to mutagens, their low chromosome number facilitates scoring and the same cell line can be used repeatedly. Furthermore, because CHL cells are a widely used test system for in vitro mutagenicity studies, a broad historical database exists. The modal chromosome number of this cell line is 25 with a doubling time of ~15–17 h. The cells are thawed in culture medium and then grown for more than 7 days as a monolayer. Sterility was checked by inverted microscopy to determine any gross mycoplasma contamination and confirmed by polymerase chain reaction amplification. Cells were cultured in reconstituted modified Eagle’s medium (Gibco-Invitrogen, Carlsbad, CA, USA) supplemented with Na₂HCO₃ (2.2 g), l-glutamine (292 mg), streptomycin sulfate (100 μg), penicillin G·Na (10⁵ units) and 10% (v/v) fetal bovine serum (Gibco-Invitrogen) per liter. The cultures were incubated at 37 °C under humidified 1.5% CO₂ in air. Based on the results, the dose range for the present study was designed considering the solubility and cytotoxicity of SST. Ethyl methanesulfonate (EMS) was used as a positive control chemical without metabolic activation using the S9 mix, and cyclophosphamide (CPA) was used as a positive control with metabolic activation. The present study was conducted in accordance with OECD guidelines for the Testing of Chemicals Section 4 Health Effects Test No. 473 about In vitro Mammalian Chromosome Aberration Test (21 July 1997).