## 3 Materials and Methods
### Measurements
#### 3.6.3 Cytotoxicity and Genotoxicity Assay  
Cytotoxicity of the PCEC matrices was evaluated by the NRU test using BALB/c T3T clone A31 mice fibroblasts cell line (American Type Culture Collection), according to ISO 10993-5:2009 Annex A guideline [43]. The cells were seeded in 96-well plates (15,000/100 \(\mu\)L) and incubated for 24 h (5% CO2, 37 \({}^{\circ}\)C, \(>\) 90% humidity) in DMEM medium with 10% bovine serum and 100 IU/mL penicillin and 0.1 mg/mL streptomycin. After incubation, the cells monolayer was assessed under a microscope. The supernatant medium was replaced by the PCEC extracts prepared by incubating the material in a of 1 mg/mL DMEM medium with 5% bovine serum for 24 h at 37 \({}^{\circ}\)C, with stirring; the extracts were sterilized on a syringe filter. The cytotoxicity of each extract was determined in four two-fold dilutions, each in triplicate. The cells were incubated for 24 h. Subsequently, the medium was removed from the cells, the wells were washed with PBS and 100 \(\mu\)L of fresh medium with neutral red was added to each well. After 2 h the medium was removed, the wells were washed with PBS and ethanol and acetic acid aqueous solution was added to each well to release the dye from the cells. The plates were shaken for 15 min and afterwards the red color intensity was measured spectrophotometrically (\(\lambda\) = 540 nm). Polyethylene film and latex were used as reference materials (with no cytotoxicity and highly cytotoxic, respectively). Samples were considered cytotoxic if they reduced cell survival below 70% when compared to the untreated cells (a baseline cell viability).  
Genotoxicity of the PCEC matrices was evaluated by the umu-test using _Salmonella typhimurium_ TA3515/psk1002 (Deutsche Sammlung von Mikroorganismen Und Zellkulturen GmbH, Braunschweig, Germany) cell lines according to ISO 13829:2000 guideline [44]. The experiment was conducted with and without metabolic activation (hepatic fraction S9, Xenometrics). Further, 2-Aminoanthracene and 4-nitroquinoline N-oxide were used as positive controls. In response to various types of DNA damage, the umuC gene in bacterial cells, which is a part of the SOS system, was induced. The cell strains were genetically modified and the activity of the umuC gene was linked to \(\beta\)-galactosidase synthesis; the other DNA regions connected with this synthesis were deleted. In the presence of the enzyme, colorless substrate ortho-nitrophenyl-\(\beta\)-galactosidase was converted to yellow product which can be quantified colorimetrically (at 420 nm). Moreover, the growth factor (G) was evaluated by optical density measurements; the cytotoxicity of tested samples was assessed. The genotoxic potential was characterized by an induction ratio (IR), the ratio of \(\beta\)-galactosidase activity between tested sample and the negative control. The sample was considered genotoxic when the IR value was \(\geq\)1.5.  
All tested polymers were incubated in PBS buffer (GIBCO) for 24 h at 37 \({}^{\circ}\)C with shaking. All of the extracts were sterilised by filtration prior to the assay. Each sample was examined in two three-concentration dilution series (three data points for each concentration). The PBS solvent control was prepared in the same manner.