Evaluation of the Safety and Toxicological Profile of RheoSprayTM on Reconstructed Human Epidermis

The human epidermis is a common site for local and systemic delivery of medication. This study aims to compare the safety and toxicological profile of RheoSpray, a proprietary sprayable topical gel compounding base, to the profile of the positive control Triton X-100, using a 3D model of the human epidermis tissue. Results have demonstrated that RheoSpray presented an ET50 superior to 24 hr, as opposed to that of Triton X-100. Therefore, it may be concluded that RheoSpray does not cause toxicity to the epidermis tissue. Compounded medicines including this sprayable topical gel base may then be considered safe to be applied to the human epidermis for over 24 hr. Introduction: The epidermis is the outermost skin layer and it is increasingly used as a route of drug administration. Following topical application, medications may act locally on the skin surface or may penetrate the skin by percutaneous absorption into the microcirculation [1]. Topical compounded medications must be non-toxic and non-irritant to the skin and, therefore, it is important to guarantee the safety of the bases used in compounding. The aim of this study was to evaluate the safety and toxicological profile of RheoSpray, a proprietary sprayable topical gel compounding base, in comparison to the positive control Triton X-100, a nonionic surfactant that can be used as a solubilizer, stabilizer, and emulsifier [2], using a 3dimensional (3D) in vitro model of the human epidermis. Reconstructed Human Epidermis: EpiDermTM The reconstructed human epidermis tissue model – EpiDermTM – by MatTek Corporation (Ashland, MA) is a highly differentiated 3D model which consists of humanderived epidermal keratinocytes, cultured and differentiated to resemble the human epidermis. Its tissue structure and cellular physiology closely parallels in vivo human epidermis tissue. It is therefore an ideal in vitro research tool for safety and toxicological testing of topical products. (Figure 1) [3]. Figure 1. Illustration of the EpiDermTM tissue model. (Adapted from MatTek Corporation, 2017) Methodology: Upon receipt of the standard EpiDermTM kit, the EPI-200 cells (Lot 24956) were maintained in the supplied culture media and stored in accordance to the manufacturer’s protocol until the initiation of the study [4]. Following preparation of the cells, the EpiDermTM tissues were treated in triplicate with 100 μL of the test product (RheoSpray, Lot #7542733) and another set of tissues were treated with Triton X-100 (1%) for 1, 4 and 24 hr. A triplicate set of EpiDermTM tissues was also left untreated to serve as negative control. Following the exposure period, the dosing solutions were removed and the cells were analyzed for cell viability by the MTT Effective Time 50 (ET50) assay. The MTT ET50 assay consists of measuring the reduction of MTT (3-[4,5-dimethylthiazol-2yl]-2,5diphenyltetrazolium bromide) by the cells. Succinate dehydrogenase enzymes within the mitochondria of viable cells have the ability to reduce soluble yellow tetrazonium salt of MTT to an insoluble purple formazan derivative. MTT is therefore an indicator of cell viability as the tissues are evaluated for their ability to reduce soluble-MTT (yellow) to formazan-MTT (purple) [5]. The MTT solution was prepared in the provided medium and added to the basal side of each tissue, followed by an incubation period of the tissues for 3 hr at 37°C. The purple formazan product was then extracted using the provided extractant, which was previously applied to both the apical and basal side of the tissues. Sample absorbance was read at 570 nm and reference absorbance at 650 nm with CLARIOstar – BMG Labtech Plate reader. Results and Discussion: Viability of the epidermis tissue cells following exposure to the test products is represented by the absorbance of the respective extracts and expressed in percentage relative to the negative control (tissues left untreated), as follows: % Cell Viability=100 x [OD(test product) / OD(negative control)] ©2017 PCCA Science | 99380 | Page 1 of 2 T E C H N I C AL R E P O R T