**2.2. Mutagenicity**

The mutagenicity is analyzed by using the method proposed by Maron and Ames [40]. The test charcoal and vinegars for this mutagenicity test, with or without S9 mix, are the same as for the cytotoxicity test, and the experimental procedure is referred by [37–39]. If the colony count of the TA98 and TA100 test group is larger than the control group by more than two times; that is, the mutagenicity ratio is larger than 2, the specimen for bamboo charcoal/vinegars is considered to have mutagenicity. The mutagenicity ratio is calculated as: mutagenicity ratio (MR) = induced revertants per plate/spontaneous revertants per plate (blank).

#### **2.3. Antimutagenic activity**

The test vinegars of the antimutagenic activity are assayed according to the Ames method [40]. The mutagens are 4-nitroquinoline-*N*-oxide (NQNO) and aflatoxin B<sup>1</sup> (AFB1 ) that are diluted with dimethyl sulfoxide (DMSO). NQNO (1 μg/plate for TA98 and TA100, respectively), a direct-acting mutagen and AFB1 (5 μg/plate for TA98 and TA100, respectively), an indirect mutagen which requires metabolic activation, require S9 mix for metabolic activation, respectively. A mutagen (0.1 mL; contained 1 μg NQNO or 5 μg AFB<sup>1</sup> ) is added to the mixture of a strain (TA98 or TA100), and 0.1 mL of each test vinegar is added to S9 mix for AFB<sup>1</sup> or to the phosphate buffer (0.1 mol/L, pH 7.4) for NQNO. The mutagenicity of each mutagen in the absence of an extract is defined as 100%. The inhibition (%) of mutagenicity of test vinegar is calculated as follows:

Inhibition (%) = [1–(number of His<sup>+</sup> revertants in the presence of the test vinegar—number of spontaneous revertants)/(number of His<sup>+</sup> revertants in the absence of the test vinegar—number of spontaneous revertants)] × 100.
