Bacterial reverse mutation test

The bacterial reverse mutation test (also called Ames test) was carried out according to the OECD - Guideline 471 for the testing of chemicals “Bacterial reverse mutation test” (26).

In the test, five strains such as TA98, TA100, TA1535, TA1537 of Salmonella typhimurium, and WP2uvrA of Escherichia coli were used. These strains are highly sensitive to mutagens, commonly used in mutagenicity studies and recommended in the regulatory guidelines (27). Salmonella typhimurium TA100, TA1535 and E. coli WP uvrA were used to detect the base-pair substitution type mutation, whereas TA98 and TA1537 were used to detect the frame-shift type mutation (28–30). The Salmonella strains were purchased from Molecular Toxicology Inc. and E. coli WP2 uvrA strain was obtained from Preclinical Research Center (ChemOn, Inc., Gyeonggi, Korea).

With metabolic activation system, Benzo[α]pyrene (Sigma-Aldrich, MO, USA) for TA98, 2-Aminoanthracene (Sigma-Aldrich) for TA100, TA1535, TA1537 and E. coli. Without metabolic activation system, Sodium azide (Sigma-Aldrich) for TA100 and TA1535, 4-Nitroquinoline N-oxide (Sigma-Aldrich) for TA98 and E. coli, 9-Aminoacridine (Sigma-Aldrich) for TA1537 were used.

As Salvia plebeian leaf extracts in KGC-03-PS was extracted with 30% ethanol, test article was diluted in 30% ethanol. 30% ethanol was used as negative control.

According to the results of a dose finding test, 5,000 μg/plate was selected as the highest concentration for all test strains both the absence and presence of S9 mixture.

For the test, two-fold serial dilutions were performed to yield five concentration (312.5, 625, 1,250, 2,500, and 5,000 μg/plate).

The test was performed based on the plate incorporation method with or out metabolic activation system S9 mixture (31,32). Each sample was assayed in triplicate. Each culture plate was placed in incubator for 48 hr at 37°C. After the incubation, the number of revertant colonies was counted by visual counting.

The test substance was considered as positive in the bacterial reverse mutation assay when there was an increase (≥ two-fold) of spontaneous revertant colonies compared with those in the negative control or a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation.