2.6. In Vitro Tests for Irritation Potential

This test was carried out by QACS Ltd. Laboratory (Athens, Greece) according to the Organisation for Economic Co-operation and Development (OECD) Guideline No. 439 [10] and using the protocol In Vitro EpiDerm™ Skin Irritation Test [11]. Skin irritation refers to the generation of reversible damage to the skin following the exposure of the chemical to be evaluated, for up to 4 h [10]. The test consisted of a topical exposure of AKVANO formulation (intended for use as a skin disinfectant) to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT present in cell mitochondria into a blue formazan salt that was quantitatively measured photometrically after extraction from tissue. The reduction of the average viability of three tissues exposed to chemicals in comparison to average viability of three negative controls (treated with water) was used to predict the skin irritation potential. The negative control used was DPBS without Ca²⁺ and Mg²⁺ and 5% sodium dodecyl sulphate (SDS) solution was used as a positive control.

This study was carried out by Research Institutes of Sweden AB (RISE, Gothenburg, Sweden, according to the OECD guidelines No. 492 [12] and using the protocol In Vitro EpiOcular Eye Irritation Test [13]. The eye irritation test is based on the use of a reconstructed cornea epithelial model and the relevant materials were obtained from MatTek In Vitro Life Science Laboratories (Bratislava, Slovak Republic). The epithelia models are topically exposed to the product to be evaluated and after recovery, the viability of cells is measured via metabolic activity. Yellow water-soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is metabolically reduced in viable cells to a blue-violet insoluble formazan, and thus the number of viable cells correlates to the colour intensity determined by photometric measurements after dissolving the formazan in alcohol. For each treatment, the viability percentage relative to a negative control (cell culture water) is calculated. Positive control used was neat methyl acetate. Eye irritation is identified as the ability of the product to be evaluated to reduce the viability of the cells in the epithelial model system. Eye irritation potential of the product or formulation evaluated is predicted if the remaining relative cell viability is below 50% after exposure.

The AKVANO formulation, positive control and negative control were added to EpiOcular™ human cell construct models (MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic) pre-treated with DPBS, Dulbecco’s Phosphate Buffered Saline (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min, whereafter the tissues were thoroughly washed followed by a post-treatment immersion and then allowed to recover for 2 h. After the recovery period, MTT solution was added to the tissues which were incubated for an additional 3 h at 37 ± 1 °C in 5 ± 1% CO₂. Following incubation, the MTT solution was removed, 2-propanol was added, and the plate with the models was shaken rapidly for at least 2 h. The solutions for tissues were homogenized and transferred to a 96-well plate for absorbance measurement at 570 nm followed by calculation of the viability of the tissues.