**3.2. Cytotoxicity of bamboo charcoal**

temperatures (80–150, over 80, 90–92, 99–102, 120–123, and 145–150°C), from the exit of chimney of earthen kiln are diluted to a percent content of 50, 33.33, 25, 20, 13.33, and 10%, respectively. Both of them are examined with *S. typhimurium* TA98 and TA100 for either S9 (+S9) or zero S9 (-S9) in accordance with the Ames test and the experimental procedure referred to [32, 37–39]. The colony count is calculated; if the bacterial count of the test group (+S9 or -S9) is larger than the bacterial count of the control group (no bamboo charcoal/vinegars) by 80% (the bacterial count rate, Survival), there is no toxicity [35]. The Survival (%) is the residual bacteria rate that is the percentage relative to the control (100%). The formula of the Survival of cytotoxicity is: Survival (%) = (the bacterial count of test group/the bacterial count of control group) × 100.

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

The test vinegars of the antimutagenic activity are assayed according to the Ames method

diluted with dimethyl sulfoxide (DMSO). NQNO (1 μg/plate for TA98 and TA100, respec-

indirect mutagen which requires metabolic activation, require S9 mix for metabolic activation,

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

The true density, BET specific surface area and average pore diameter of Moso bamboo char-

element of bamboo charcoal for Br, Pb, Hg, Cr and Cd is analyzed using X-ray Fluorescent Analyzer of XGT-1000WR [39]. Br and Cr are not detected because their amounts are probably

of a strain (TA98 or TA100), and 0.1 mL of each test vinegar is added to S9 mix for AFB<sup>1</sup>

(AFB1

) is added to the mixture

(5 μg/plate for TA98 and TA100, respectively), an

revertants in the presence of the test vinegar—number of

/g), and 2.41 (nm), respectively. Moreover, the heavy metal

revertants in the absence of the test vinegar—num-

) that are

or to

(MR) = induced revertants per plate/spontaneous revertants per plate (blank).

[40]. The mutagens are 4-nitroquinoline-*N*-oxide (NQNO) and aflatoxin B<sup>1</sup>

respectively. A mutagen (0.1 mL; contained 1 μg NQNO or 5 μg AFB<sup>1</sup>

**2.2. Mutagenicity**

52 Bamboo - Current and Future Prospects

**2.3. Antimutagenic activity**

calculated as follows:

coals are 1.68 (g/cm<sup>3</sup>

Inhibition (%) = [1–(number of His<sup>+</sup>

spontaneous revertants)/(number of His<sup>+</sup>

**3.1. Basic properties of bamboo charcoal**

**3. Safety evaluation of bamboo charcoal**

), 138.70 (m2

ber of spontaneous revertants)] × 100.

tively), a direct-acting mutagen and AFB1

The cytotoxicity test results with 1.0, 2.5, 5.0, 7.5 and 10.0 mg of Moso bamboo charcoal for *S. typhimurium* TA98 and TA100 are shown in **Figure 1**. The Survival (%) of the Moso bamboo charcoal with either zero S9 (-S9) or S9 (+S9) is higher than 80%. Waleh *et al*. [36] indicate that the amount of residual bacteria of S. *typhimurium* must be over 80% of the control group (Control) to determine that the test group has no cytotoxicity for S. *typhimurium* [36]. The Survival of the charcoals is higher than that of control by more than 80%, indicating that the Moso bamboo charcoal has no cytotoxicity for the test strains in the additional range of 1–10 mg/plate, and the dose for the mutagenicity test can be selected according to this range.

#### **3.3. Mutagenicity of bamboo charcoal**

**Figure 2** shows the mutagenicity test results of the Moso bamboo charcoal for *S. typhimurium* TA98 and TA100. The bamboo charcoal, without or with S9, in the test range (1–10 mg/plate) does not exceed spontaneous revertants by more than two times for TA98 and TA100, that is, the mutagenicity ratio (MR) is smaller than 2. According to the standards proposed by Ames *et al*. [32], if the number of spontaneous revertants induced by the specimen is larger than the spontaneous revertants of the control group by more than two times, the specimen has mutagenicity. Therefore, the bamboo charcoals have no mutagenicity toward *S. typhimurium* TA98 and TA100. Furthermore, Weng reports that the maximum percent weight of Moso bamboo at RH 40% is 6.7% and at RH 90% is 9.9% [39]. The water activity of the bamboo charcoal (0.57), compared to Aw for the growth of a microorganism environment, is below 6.0. The heavy metal contents (as Pb ppm base) of charcoal are below 40 ppm and meet the Sanitation Standard for Edible Natural Colorants, Food Sanitation Standards as well. For the Ames test, the bamboo charcoal has no toxicity and mutagenicity toward *S. typhimurium* TA98 and TA100. In the scope of this report, the above results indicate that the bamboo charcoal can be expected to be the same as the materials of edible natural colorants.

90–92°C, the residual bacterial count without S9 for diverse diluting percent contents is 0–1830 for TA98 and 87–1641 for TA100; for those with S9, it is 1605–2000 for TA98 and 1736–2249 for TA100. Waleh *et al*. indicate that the residual bacteria rate of *S. typhimurium* must be over 80% of the control group to determine that the test group has no cytotoxicity for *S. typhimurium* [36]. The residual bacteria rate (Survival, %) toward TA98 and TA100 for bamboo vinegar without S9 mix at a diluting percent content 33.3% or less, and with S9 mix at 25.00% or less is greater than 80%. In other words, the bamboo vinegars collected at temperatures of 90–92°C has cytotoxicity for *S. typhimurium* at a diluting 33.3% less. For vinegars collected at all differ-

**Table 1.** Cytotoxicity of bamboo vinegar collected at the temperature of 90–92oC toward *Salmonella typhimurium* TA98

**Bamboo vinegar (90–92°C)**

−S9 Original vinegar 0 0.00 87 5.82

S9 Blank 2165 100.00 2585 100.00

Survival (%) = (the bacterial count of test group/the bacterial count of control group) × 100.

**Blank2 18383 100.00 1500 100.00**

50.00 336 18.26 285 19.00 33.33 1652 89.86 1284 85.58 25.00 1822 99.13 1201 80.04 20.00 1736 94.43 1264 84.27 13.33 1829 99.53 1353 90.20 10.00 1830 99.56 1641 109.42

33.33 1605 74.16 1736 67.15 25.00 1960 90.53 2209 85.46 20.00 1938 89.53 2243 86.75 13.33 2046 94.53 2885 111.60 10.00 2000 92.39 2249 87.00

**TA98 Survival1 (%) TA100 Survival (%)**

Preliminary Safety Evaluation of Bamboo Pyrolysis Products: Charcoal and Vinegar

http://dx.doi.org/10.5772/intechopen.68542

55

The Survival of all bamboo vinegars collected from 80 to 150, over 80, 90–92, 99–102, 120–123, and 145–150°C without S9 mix at a diluting percent content of 33.33% or less is all higher than those for Blank by more than 80%. However, the Survival of the bamboo vinegar collected at temperatures of 99–102°C at a diluting percent content of 25% shows that the cytotoxicity toward *S. typhimurium* TA98 and TA100 with S9 mix is lower than 80%, indicating "with toxicity." It is asserted that the Survival of bamboo vinegars collected at all different temperatures at a diluting percent content of 20.00% less for either with or without S9 mix is no cytotoxicity absolutely, and the dose for the mutagenicity test can be selected according to the

ent temperatures, the Survivals are shown in **Table 2**.

Blank (the control group) was added without bamboo vinegars.

and TA100 without or with S9 mix.

**S9 mixture Diluting percent** 

**content (%)**

results (**Table 2**).

1

2

3 Mean.

**Figure 2.** Mutagenicity of Moso bamboo charcoal toward *S. tyhpimurium* TA98 and TA100 without or with S9 mix.
