The colony forming assay is an in vitro cytotoxicity test used to assess the cytotoxic effects of leachable substances released from a test item. Cytotoxicity is evaluated by measuring the colony-forming ability of cells following treatment with various concentrations of the test item extract compared with untreated controls.

The dilution that reduces colony formation to 50% (IC50) can be determined by plotting the relative colony-forming rate (%) against the dilution of the test extracts (%).

The mean number of colonies formed in treated cultures represents the colony-forming ability of the cells and reflects cell survival following treatment. Cultures incubated with fresh culture medium serve as the 100% control.

Assessment of cytotoxicity by colony-forming ability

• The test item is extracted for a defined period in cell culture medium at 37 ± 1°C, and V79 cells are incubated with the extract for several days. 
• Following incubation, the cultures are examined microscopically and carefully washed with PBS buffer. 
• After fixation with methanol, colonies are stained using Giemsa staining solution. 
• The stained colonies are dried at room temperature and colonies consisting of more than 50 cells are counted manually.

Test item acceptance criteria

• IC50 acceptance limit according to MHLW: ≥ 90%
• A reduction of relative plating efficiency at 100% test solution concentration of more than 30% is considered a cytotoxic effect.

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