*4.5. Cytotoxicity*

To evaluate the possible cytotoxic e ffect of the experimental dental resin, human keratinocytes (HaCaT, CLS Cell Lines Service GmbH, Eppelheim, Germany) were used in this test [40]. All reagents used to evaluate the possible cytotoxicity of the dental resins were purchased from Aldrich Chemical Company (St. Louis, MO, USA). The keratinocytes were kept in contact (5 × 10<sup>3</sup> cells/per well) with 100 μL of Dulbecco's modified Eagle's medium (DMEM) in 96-well plates for 24 h at 37 ◦C. On the same day, five samples per group (1 mm thickness and 4 mm diameter) were placed separately in Eppendorf tubes containing 1 mL of DMEM and kept for 24 h at 37 ◦C. Thus, eluates from possible leaching from the samples were formed. These eluates (100 μL) were kept in contact with the keratinocytes in the

96-well plates for 72 h at 37 ◦C. There was one group that was maintained without eluates with 100 μL of pure DMEM that was used as a control for the test. In addition to using five samples per group, the eluates were applied in five replications, totalizing 100 wells containing eluates from the samples. After 72 h, 50 μL of trichloroacetic acid/distilled water solution (50:50) was added in each well and kept at 4 ◦C for 1 hour to fix the cells on the bottom. Running water (30 s) was used to wash the 96-well plates six times. The 96-well plates were kept at room temperature until drying.

Sulforhodamine B at 0.4% (50 μL) was added in each well, and the 96-well plates were kept at room temperature for 30 min. The 96-well plates were washed four times with acetic acid at 1% and kept at room temperature until drying. Trizma solution at 10 mM (100 μL) was added in each well, and the 96-well plates were incubated for 1 hour at room temperature. The absorbance of plates' wells was analyzed at 560 nm, and the cell viability of the wells that contained eluates was compared to the wells without eluates. The viability of the keratinocytes used was normalized against the viability of cells without contact with eluates (negative control). The results were expressed in percentage using the viability of negative control as 100%.

## *4.6. Statistical Analysis*

Data normality was evaluated by the Shapiro–Wilk test. One-way ANOVA and Tukey's post-hoc test was used to compare groups for all tests at a level of 0.05 of significance.
