Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: I. Unscheduled DNA synthesis assay in rat hepatocyte cultures

Genotoxicity of a Low-Dose Nitrosamine Mixture as Drinking Water Disinfection Byproducts in NIH3T3 Cells

“…After washing twice with PBS, the cells were continually cultured for seven days at 37°C, and the medium was refreshed every three days. Then, the cells were fixed with methyl alcohol and stained with 10% Giemsa, and the colonies with more than 50 cells were counted 21 , 28 . This was used to quantify colony-forming efficiency (CFE) and relative colony-forming efficiency (RCFE).…”

“…The NIH3T3 cells were seeded at a density of 2,000 cells/dish (10 cm), and the cells were cultured for 24 h. Cells were treated the same as the cell colony formation assay for 72 h. After rinsing with PBS, the cells were continually cultured for 14 days at 37°C, and the medium was replaced every three days. The cells were stained with 10% Giemsa, and the transformation frequency (TF) was calculated as follows 28 :…”

Assays for DNA Damage

“…Accordingly, this event has been termed unscheduled DNA synthesis (UDS) and is a component of excision repair of damaged DNA. Hence, UDS assays detect DNA repair following damage by a physical or chemical agent; these assays have been used to assess the genotoxicity of a number of chemicals (Mitchell et al, 1983;Williams et al, 1989), and there are several published guidelines for genotoxicity screening (Mitchell et al, 1983;Swierenga et al, 1991;Madle et al, 1994).…”

“…Experiments should be rejected if the positive controls are lower than the vehicle controls, if negative or solvent controls yield positive results, if cytoplasmic background grain counts are unacceptably high, or if there is low cell viability and/or poor cell attachment (Swierenga et al, 1991).…”

Evaluation of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) following treatment of rats and mice with p-dichlorobenzene

“…UDS is DNA synthesis that occurs in the process of repairing damaged DNA and is an indicator of DNA damage that could lead to mutation or cancer [13]. In vitro UDS assays have been used extensively to identify potential genotoxic carcinogens that act by inducing DNA damage [14][15][16][17]. The advantage of the in vivo/in vitro UDS protocol developed by Mirsalis and Butterworth [11] is that in vivo treatment provides a more realistic assessment of potential genotoxicity in the whole animal.…”