2.2.1. Elemental Composition of Pristine Ingredients

Elements present in PS9 and G30 as well as ALI water has been addressed by Inductively Coupled Plasma mass spectrometry (ICP-MS) measurements. Solid samples were prepared by acid digestion in strong acids (HNO3 and HF at a 3:5 ratio) inside a Teflon reactor, thus the samples were subjected to high pressure and temperature by heating in a microwave oven (Milestone ETHOS ONE, Sorisole, Italy). The quantification of the elements was done by a NexION-300 ICP-MS spectrometer (Perkin Elmer, Waltham, MA, USA) equipped with a triple cone interface and a quadrupole ion deflector using argon for plasma formation. Standard solutions of 100 and 1000 ppb were prepared for each element (Multi-Element standards, Perkin Elmer, Waltham, MA, USA), and Rh was employed as an internal standard. All standards were prepared from ICP single-element standard solutions (Merck, Darmstadt, Germany) after dilution with 10% HNO3. Ultrapurified water (Milli-Q® grade, 18 MΩ·cm) was used during the whole experiment. The accuracy of the ICP-MS equipment used ranges between ±2 and ±5% for analyte concentrations between 50 and 5 ppm, respectively. The detection limits were <0.1 ppt for Ir and Ta; <1 ppt for Ba, Li, Cu, Mo, Sb, Sn, Ag, Au, Co, Ni, V, As, Cd, Pb, Zr, Be and Nd; <10 ppt for Cr, Hg and Te; < 1 ppb for P.

#### 2.2.2. In Vitro Release of Elemental Impurities from Hydrogels

The element mobility from nanoclay/spring water hydrogels (ALIPS9, ALIG30) was assessed by in vitro cation release studies performed in a Franz diffusion cell system FDC40020FF (BioScientific Inc., Phoenix, AZ, USA) [38]. This system is designed to recreate conditions of formulations placed over the skin and mucosa membranes. Particularly, the selected Franz diffusion cells possessed a contact area of 0.64 cm2 and an approximate total volume of 6.4 mL in the receptor chamber. In this study, the aim is to explore the potential number of elements that would be released by the formulation and that are potentially able to establish contact with the skin. To do so, dialysis membranes (cut-off 12–14 kDa (31.7 mm), Medicell International, London, UK) were placed and used to separate the donor and receptor chambers, just acting as physical support for the hydrogel and not as a permeation barrier. The membranes were boiled in ultra-purified water (Milli-Q® water) for 10 min in order to hydrate them. Over the membrane, in the donator chamber, 0.025 g of each hydrogel was placed. The receptor chamber of the Franz diffusion cells was filled with degassed, ultra-purified water, which was maintained at a constant temperature of 32 ± 0.5 ◦C (to reproduce human skin temperature) through a thermostatic bath circulation. The experiment lasted for 30 min, this being the typical time of topical nanoclay/spring water hydrogels application. At the end of the experiments, the receptor aqueous phase was withdrawn and filtered through 0.45 μm single-use, sterile filters. Then, the elemental composition on each sample was assessed by ICP-MS, following the same protocol previously described for the pristine materials. All samples (six replicates) were subjected to in vitro release experiments 48 h after the preparation and after one month. During the experiment, the manipulation of different materials and instruments could contaminate the ultra-purified water of the Franz cell receptor chamber. To eliminate this error, blanks were also analyzed in order to monitor the elements coming from the materials and the ultra-purified water itself. Briefly, it consisted of analyzing the ultra-purified water that was placed in the receptor chamber of the Franz cell device. The concentration of the elements detected in the Milli-Q water (which was considered the blank, data not shown) were subtracted from the concentration detected in the receptor chamber. This way, it will be possible to discern the real

number of elements exchanged/released from the semisolid formulation and, thus, able to establish contact with the patient skin.
