Study Title: In vitro Toxicity Study of ION-ZCM1 on Human Cells Using an MTT Cytotoxicity Test Method
Author: INDEVION Biotechnology Research and Development Ltd. Principal Investigator Dr. Zsolt Keresztessy, Ph.D. (2018).
Methods: The data generated from the in vitro cytotoxicity assays were used to predict toxicity under cell culture conditions, based on the HACAT cell line. A standard Positive Control (PC), namely 10% (w/v) Sodium Lauryl Sulfate in PBS was used in the test and a dilution series of this PC was applied in the test together with the dilution series of the Test Compound (ION-ZCM1).
Results: ION-ZCM1 shows toxicity with IC50 at 0.0224%, which is at 119 uM of copper content (and at 359 uM of zinc content). This toxicity to keratinocyte cells is independent of the pH effect of the test compound (the pH effect in the assay medium is not relevant at or under 0.39%, since pH is >6), and independent of the osmolarity effect (the osmolarity effect is not relevant under 3.125%, (that is 165.625 mOsm/L from NaCl and ~200 mOsm/L from the cell culture medium, which equals 365.6 mOsm/L in total in the assay medium), as shown by NaCl dilution series used in the test as osmolarity control).
Conclusions: Evidence shows that the application of the test substance in a topical formulation at a concentration of 25% ION-ZCM1 is not toxic to animals/humans. This can be explained by the presence of the protective barrier, the keratin layer of the skin. In addition, the high-dose test compound, which diffuses through the skin, is diluted in the body/tissues and body/fluids. A safety profile was established for topical use at a concentration of 25% on human skin cells.