Cytotoxicity

The test was performed in accordance with ISO 10993-5:2009. For the test, extracts of PPG were prepared in Eagle minimum essential medium (EMEM) at 37 °C for 24 h. L929 cells were seeded in a 96-well plate at a density of 10,000 cells per well, treated with various concentrations of extracts (100, 50, 25, 12.5 and 6.25%) and stored at 37 °C in a 5% CO₂ incubator for a period of 24 h. Complete media was used as the negative control and 1% phenol in complete media was used as the positive control. After the incubation period, MTT assay was conducted to assess the cytotoxicity. For MTT, extract containing media was removed, 30 µL of MTT (1 mg/mL in EMEM) reagent was added to each well, and the plates were incubated for 3 h at 37 °C. The MTT containing media was removed and 100 μL DMSO was added to each well. After 10 min of mild mechanical shaking, the optical density (OD) was measured for each well at 570 nm keeping 600 nm as a reference wavelength in a plate reader (synergy 4 multimode microplate reader-BioTEK, VT, USA). The percentage cell viability was calculated using the following equation:

A material is considered as cytotoxic if the cell viability is <70% and non-cytotoxic if the cell viability is >70%.