## 4 Discussion  
Examining toxicity is a crucial step in the safety evaluation of chemical substances and their mixtures, aiming to identify potential adverse effects and determine the appropriate dosage for medicinal applications. Despite the discovered diverse beneficial biological effects of _C. vulgare_, its potential toxicity has not been thoroughly investigated. The main difficulty in the safety evaluation of wild-growing medicinal plants compared to cultivated plants is that their chemical composition and the concentration of the individual components vary widely depending on many different external factors, such as geographical and climate conditions, the presence of stress factors, time of collection, conditions of storage and others. In the case of cultivated plants, the controlled conditions of cultivation allow a higher consistency of the plant's phytochemical composition. In a previous study, the acute and subacute toxicity of an aqueous extract of wild-growing _C. vulgare_ plants was assessed through in vivo experiments in mice and rats [9]. The study demonstrated that the oral application of the extract did not induce any toxic side effects in the organs and systems of the experimental animals [9]. In our study, the cytotoxicity of the tested _C. vulgare_ extracts was evaluated on BALB/3T3 clone A31 murine embryo fibroblast cells. This cell line was chosen as it is one of the in vitro systems approved by the European Centre of Validation of  
Figure 6: Effects of a total aqueous extract of in vitro cultured _C. vulgare_ plants on the cell cycle of HeLa cervical carcinoma cells. (**A**) Histograms of control untreated cells and cells treated with the extract at concentrations of 600 \(\upmu\)g/mL and 800 \(\upmu\)g/mL; (**B**) graphic representation of the distribution of cells in the different phases of the cell cycle. Data are presented as mean \(\pm\) SD from three independent experiments. Stars indicate a difference with the respective phase in control cells at \(p<0.001\). Alternative Methods (ECVAM) as a suitable model for safety assessment studies of chemical compounds [33; 34]. The results of our study at the 24 th h demonstrate a complete absence of cytotoxicity of both tested extracts on the BALB/3T3 cells in the whole concentration range tested, except for the highest concentration (1000 \(\upmu\)g/mL). The extension of the time of exposure to the extract of wild-growing plants to 48 h, except at 1000 \(\upmu\)g/mL, did not lead to a reduction in cell viability compared to the untreated control cells, and cell viability was even slightly increased. The viability of the cell cultures treated for 48 h with the extract of in vitro cultivated plants, except at 1000 \(\upmu\)g/mL, was not decreased at the lowest concentrations of the extract (125 \(\upmu\)g/mL and 250 \(\upmu\)g/mL (it even slightly increased at 125 \(\upmu\)g/mL)) and slightly decreased at the higher concentration (500 \(\upmu\)g/mL). The results of our study are in agreement with the in vivo-obtained data indicating low toxicity of the aqueous _C. vulgaris_ extracts [9]. In addition to the study on BALB/3T3 cells, the cytotoxicity of both extracts was also evaluated using a panel of human tumor and non-tumor cell lines in order to assess their potential anticancer activity. Cervical, hepatocellular, colorectal and mammary carcinoma cell lines were used in the study as in vitro models of some of the most common types of cancer diseases in humans. The results of the analysis show that the tested _C. vulgaris_ extracts induced a statistically significant and concentration-dependent decrease in the cell viability of all tumor cell lines tested; however, the extent of the observed effects varied depending on the type of cancer cells used and the extract applied.