2.6. HET–CAM Test

The hen’s egg test chorioallantoic membrane (HET–CAM) assay was used for preliminary screening of ocular irritancy of the hydrogels according to the ICCVAM protocol [51], as previously described [34]. Briefly, fertilized hen’s eggs (Coren, San Cibrao das Viñas, Spain) were incubated at 37 °C with 60% relative humidity for 7 days and were manually rotated 180° three times a day for the duration of the incubation. On the eighth day, the eggs were cut on the wider extreme to extract the eggshell, remove the inner membrane, and expose the chorioallantoic membrane (CAM). Atorvastatin-loaded hydrogel discs and 300 µL of atorvastatin calcium (0.04 mg/mL in ethanol:water 20:80 v/v) were carefully placed on the CAM and the possible hemorrhage, vascular lysis, or coagulation of the vessels were monitored for 5 min. Two independent experiments were carried out for each hydrogel and the drug solution. The discs were previously loaded with atorvastatin calcium for 48 h, as described above. NaCl (0.9%) and NaOH (0.1 N) solutions were used as negative and positive controls, respectively. Then, the irritation score (IS) was estimated, as previously reported [35].