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Ethanol extracted propolis (EEP) solution was prepared by extracting Korean propolis. Raw propolis 1kg was extracted with 80% ethanol 3.5 L (Woo et al., 2012). EEP solution was filtered with Whatman No. 2 filter paper and then concentrated to 18% concentration.

Acacia honey was purchased from Korean Beekeeping agricultural cooperative. The moisture content was 18.5%.

Honeypolis was made with honey and EEP solution. We prepared 1kg acacia honey in beaker, poured 100ml EEP solution, and stirred with stirrer (Hei-torque200, Germany) over 1 hour. It was used as the test material.

Controls were set up in parallel to honeypolis in order to confirm the validity of the test. Negative Control was performed with Dulbecco’s phosphate buffered saline (DPBS; Gibco, USA), for the positive Control, 5% sodium dodecyl sulfate (TC-SDS-5%; MatTek, USA) was used.

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm^TM (MatTek, USA). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell^Ⓡ, USA). The EpiDerm^TM epidermis model exhibits in vivo like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

To check the non-specific MTT-reducing capability of 30μL of honeypolis were mixed per 1mL MTT medium and incubated for 60 min at 37±1°C in the incubator.

To check the colouring potential of 30μL of honeypolis were mixed per 300μL aqua dest, and per 300μL isopropanol each in a transparent recipient and incubated at 37±1°C for 60 min.

Upon receipt of the EpiDerm^TM, the tissues were inspected visually and transferred into 6-well plates containing 0.9mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37±1°C, 5.0% CO₂ for 60±5 min. Subsequently the tissues were transferred into new wells containing 0.9mL pre-warmed assay medium per well and were incubated for 18±3 h in a humidified incubator at 37±1°C, 5.0% CO₂.

After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. After dosing of all tissues, all plates were transferred to the incubator for 35±1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60±1 min incubation time of the first dosed tissue was over. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150mL DPBS and shaken to remove rests of honeypolis. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37±1°C, 5.0% CO₂, humidified to 95%, for 24±2 h. Following this incubation the tissues were transferred to new wells containing 0.9mL fresh assay medium and incubated for additional 18±2 h.

After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300μL pre-warmed MTT medium. This plate was incubated for 3 h±5 min at 37±1°C, 5.0% CO₂, humidified to 95%.

After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 h with gentle shaking on a plate shaker.

Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2×200μL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570nm with a filter band pass of maximum ±30nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

Irritant potential of honeypolis was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. honeypolis is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”) (EURL-ECVAM, 2009; EC, 2008), if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance (OECD, 2014). The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.