Sensitizing substances including isobornyl acrylate (IBOA) in diabetes devices are predicted as skin sensitizers by the non‐animal assay KeratinoSens™—Are better preventive approaches possible?

Substances released from diabetes devices (e.g., insulin pumps and glucose monitors) and other medical devices, including plasters, and ostomy devices, have caused allergic contact dermatitis (ACD).1-3 Such substances include colophonium and the acrylates isobornyl acrylate (IBOA) and Irganox 3052 (chemical structures, see Figure S1). Whereas colophonium (in oxidized form) and IBOA are classified as skin sensitizers based on animal and human data, Irganox 3052 is considered a new culprit allergen4, 5 (Table 1). It is known that numerous sensitizing substances are formed from colophonium by autooxidation on air exposure.7 The aim of this study was to test whether the selected substances would be indicated as skin-sensitizing by a validated non-animal assay, the KeratinoSens™.8 The luciferase gene induction was assessed in KeratinoSens™ cells (Givaudan SA, Switzerland), cultured and exposed for 48 h to the test chemicals and two positive controls (Table 1) according to the OECD test guideline.8 A slightly modified concentration range was used (fewer doses overall, but within a narrower concentration range) due to a steep dose–response in cytotoxicity. In parallel, cytotoxicity was measured with Alamar blue assay (Invitrogen). Luciferase activity was measured with One-Glo™ Luciferase assay kit (Promega), a plate reader (Tecan, Infinite F 200 Pro, Software: Magellan 7.2); the luciferase induction was calculated based on the luminescence values of blank and control. Three (n = 3) independent repetitions were performed, except for colophonium (n = 4), with each concentration tested in three wells per repetition, respectively. Following the OECD test guideline,8 a two-tailed Student's t test (p = 0.05) was used to assess if the induction of luciferase activity in sample wells was significantly different from negative control (dimethyl sulfoxide 1%). To estimate the concentration at which test substances positively activated KeratinoSens™, the lowest test concentration (LC1.5) was identified at which the mean value of all repetitions was 1.5-fold or more for the luciferase activity at cell viability above 70%. The interpolated concentration resulting in a 1.5-fold luciferase induction (EC1.5) was calculated according to OECD test guideline8 using values from positive repetitions (equation in Supporting Information). All substances proved positive in the KeratinoSens™ assay by statistically significantly inducing the luciferase activity by more than 1.5-fold above the control with a cell viability of >70%. The LC1.5 value for colophonium, IBOA, Irganox 3052 and the positive controls cinnamal and methylisothiazolinone was 10, 5, 2, 5 and 2.5 μg/mL, respectively (Figure 1, see shaded area, and Table 1). These values closely resemble the calculated EC1.5 values (Table 1). We are not aware of previous studies using KeratinoSens™ for testing colophonium and Irganox 3052, but our results on IBOA are in line with a recent study.9 Our findings indicate that a quick non-animal assay can be used to identify the skin sensitization potential of substances used in various medical devices with adhesives. This suggests a possibility for efficient screening of single substances or extracts from devices, to improve the products on the market and prevent lifelong suffering in children and adults with diabetes and other health issues.10 Madeleine Sjöberg: Investigation; formal analysis; writing – original draft. Ulrika Carlander: Conceptualization; formal analysis; writing – review and editing. Andrea Montano Montes: Methodology; supervision. Carola Lidén: Writing – review and editing. Hanna L. Karlsson: Conceptualization; supervision; writing – review and editing. The authors declare no conflicts of interest. Data S1. Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.