*3.2. In Vitro Release of Hazardous Elements from Hydrogels*

The results obtained after the Franz cell studies regarding the release of the elements from nanoclay hydrogels at 48 h and after one month are summarized in Table 3.


**Table 3.** Mobility of hazardous elements after Franz diffusion cell tests. The concentrations are expressed in μg/100 g of hydrogel. Elements have been placed in the same order as in Table 2. Mean values ± s.e. (*n* = 6). "ND" stands for "not detected".

Ba release was very variable between ALIPS9 and ALIG30, though the higher values detected in ALIG30 could be ascribed to higher Ba presence in the pristine material G30 in comparison with PS9 (Table 2). Cu mobility, which was higher in ALIG30 due to a higher amount in G30, significantly decreased after one month for both ALIG30 and ALIPS9.

The Li release was higher in ALIPS9 due to the higher Li levels in PS9 and maintained constant with time (no significant differences between 48h and 1 month). On the other hand, ALIG30 hydrogels showed a reduction in Li release as time passed. The highest Mo was found in ALIG30—48 h and significantly decreased after one month. On the other hand, ALIPS9 demonstrated a constant release of Mo through time. Sn, V, and Cd released from both hydrogels came from clay minerals since none of these elements were detected in ALI (Table 2). The V release increased with time, while Sn showed the opposite trend.

Heavy metals Hg, Pb, and Sb, though present in the pristine materials, were not released. Other not-allowed elements, such as Cr, Se, Tl, Ni, P, Be, Zr, Te, Nd, and Ta were neither release elements, which means that they do not pose any problem in terms of safety. On the other hand, Cd and As were slightly released, with higher results in the case of ALIG30 hydrogels. The absence of Cd in ALIPS9 is due to the extremely low amounts detected in PS9 and its absence in ALI. On the contrary, G30 possessed a higher amount of Cd (Table 2), which explains the release results (Table 3). In conclusion, Cd and As are the most crucial elements determining the safety of the hydrogels. It is worth mentioning that with respect to the As and Cd amounts and release, ALIPS9 hydrogel is considered the safest formulation.

#### **4. Discussion**

## *4.1. In Vitro Release of Elements: Safety Concerns and Doses*

The toxicity of elemental impurities obviously depends on the administration route of the dosage form. The studied hydrogels are topically administered, and the bioavailability of a certain element hardly reached 100%. Tateo et al. [9], in previous studies regarding elemental percutaneous mobility, stated that the major part of the elements could cross the skin. Nonetheless, they reported that "none of these elements reaches concentrations so high as to represent hazardous conditions". In the discussion, we systematically will consider a theoretical 100% bioavailability in order to guarantee safe doses in any case.

According to the results, the maximum amount of Ba released came from ALIG30, 48 h, and it counted for 8.3 μg/100 g of hydrogel. The oral PDE of barium was established as 730 μg/day [30]. If we consider the maximum mobility and a 100% bioavailability of Ba through the skin, the administration of ALIG30 and ALIPS9 hydrogels would be considered safe if doses are less than 8.79 kg hydrogel/day (Table 4). In view of the high amounts of hydrogels needed to pose a risk regarding Ba, it is possible to state that both ALIPS9 and ALIG30 are safe with respect to this element.

**Table 4.** Theoretical safe doses of ALIPS9 and ALIG30 hydrogels based on elements with defined parenteral Permitted Daily Exposure (PDE) levels. Calculations have been made by using the higher mobility value reported by Franz cells (either ALIPS9 or ALIG30). Additionally, safety doses are calculated assuming a theoretical dermal bioavailability of 100%.


Among the possible adverse effects associated with Cu, allergic dermatitis is the most commonly experienced [44]. Safe amounts of hydrogels regarding Cu release (Table 3) have been calculated according to parenteral PDE (Table 4). In view of the results, hydrogels aged for one month could be considered safe in terms of allergenic copper effects, since its mobility practically disappears. Moreover, ALIPS9 would be more advisable than ALIG30; the amount of Cu being lower in the former one. The amount of Cu released from extemporaneous formulated hydrogels could limit their use in general baths, as the calculated safe dose (Table 4) should be lesser than two kilograms of hydrogel.

Li is of relatively low toxicity by the oral route. Is a common metal present in animal tissues and is used in certain kinds of treatments, such as bipolar disorder or depression, among others. Recently, Yuan et al. [45] prepared a sponge scaffold with LiCl and evaluated wound healing activity in vitro. The presence of Li reduced inflammation and improved angiogenesis, re-epithelialization, and expression of β-catenin. Seborrheic dermatitis is another skin disorder that has been addressed by Li as an active lithium gluconate/succinate, with successful results [46–49]. The main problem of Li is the narrow therapeutic margin it possesses [50,51]. Parenteral Li PDE was established to be 280 μg/day [30]. Considering that all the released Li would be able to reach the bloodstream once the hydrogel is applied, the administration of ≤ 1.37 kg hydrogel/day would guarantee safe doses of Li (below the parenteral PDE, Table 4).

Mo could be considered as an essential element since its deficiencies have been related to night-blindness, nausea, disorientation, coma, tachycardia, tachypnea, and other biochemical abnormalities [30]. Nonetheless, excessive accumulation of Mo could also produce toxicity, so its limits need to be controlled. In particular, Mo could be accumulated in the skin, bound to dermal collagen. The amount of Mo released from ALIPS9 was constant with time, while it significantly reduced after one month in ALIG30 (Table 3). Parenteral PDE levels of Mo are 1700 μg/day. Considering the highest released amount of Mo (ALIG30—48 h), and supposing 100% of bioavailability, 94 kg/day of hydrogels would be necessary to reach PDE limits (Table 4). In view of these calculations, it is possible to guarantee that ALIPS9 and ALIG30 are safe with respect to Mo levels.

Tin is an element widely used nowadays [52]. The PDE limits of Sn have been established since it has been reported to increase in vitro oxidative stress or DNA breakage [53]. In view of tin's released amounts in ALIPS9 and ALIG30, its toxicity and PDE limits should be borne in mind. In particular, this element showed lower release from both hydrogels after one month of preparation. The inhalation and oral consumption of Sn are the main routes for Sn intoxication [54–56], thus meaning that the topical application of these hydrogels would be a safe administration route. In fact, in vitro cytotoxicity studies of these hydrogels were not shown to hinder normal dermal human fibroblast growth nor cell motility during in vitro wound healing [57].

Ni has been widely detected in cosmetic products, together with Co and Cr, among others [8,58]. The attention paid to Ni, Co, and Cr is based mainly on skin conditions, such as contact allergic dermatitis, itching, and edema, among others [59–64]. What is more, these elements can be solubilized by sweat during prolonged contact [60,65]. Pristine materials possessed higher amounts of Ni than Co. Nonetheless, no Ni and Cr mobility was detected in Franz cell tests, thus reducing the risk of contact skin alterations produced by ALIPS9 and ALIG30 hydrogels. The Co mobility in ALIPS9 increased with time, while in ALIG30, it maintained constant (Table 3). The application of young hydrogels (ALIPS9) would entail the lowest risk of skin allergies related to cobalt. Co is an integral component of vitamin B12, which means that it is essential for the human body. It is estimated that the average person receives about 11 μg Co/day with normal diet, and the parenteral PDE is established to be 5 μg/day [3]. Additionally, it has been demonstrated that Co is able to pass the skin [65,66], though its percutaneous absorption was found to be very low (0.0123 <sup>μ</sup>g·cm−2·h<sup>−</sup>1). Time of hydrogels application in thermal stations takes at about 20–30 min, which is not enough time for all the mobile Co (Table 3) to cross the human skin, thus making the "hydrogel safe dose/day" (calculated assuming a 100% of bioavailability, Table 4) to be remarkably higher in real conditions.

V is a ubiquitous element in the human body, though no essential role has been found yet for this element. Although systemic toxicity of V has already been accepted, its deficiency has also proved to be problematic since it is associated to thyroid, glucose, and lipid metabolism malfunctions. It also participates in the regulation of several genes and has been demonstrated to influence cancer development, including skin cancers such as malignant melanoma [67,68]. V was a mobile element in both hydrogels prepared. No V was detected in the ALI, Franz cells results ascribable to pristine clay minerals composition (Table 2). In ALIG30, the mobility of V was higher due to the higher amount of this element in G30 in comparison with ALIPS9 (Table 3). Although antiproliferative properties of V ions have been found [68], neither ALIPS9 nor ALIG30 hydrogels impaired normal human fibroblasts in vitro proliferation, according to previous studies [57].

As is a forbidden element in cosmetics [3]. Arsenic mobility is reported to be very low in both hydrogels. It maintains constant through time in ALIG30, while it increases in ALIPS9 after one month. This element is very ubiquitous in the environment, so it is expected to be present in natural ingredients such as clay minerals. This element is classified in the category 1A in view of its carcinogenicity, as reported in the European Regulation EC 1272/2008 [69]. For greater clarity, substances belonging to the 1A category are known to have carcinogenic potential to humans. It possesses a pronounced affinity for skin and keratinizing structures, although it does not act as a sensitizer due to poor skin penetrating ability. According to article 1 of the EC 1223/2009, "prohibited substances should be acceptable at trace levels only if they are technologically inevitable with correct manufacturing processes and provided that the product is safe". Assuming 100% bioavailability and using parenteral PDE levels, the maximum dose of hydrogels that could be used without exceeding these levels is 3.75 kg·day−<sup>1</sup> (Table 4) [30]. Moreover, the maximum mobility of As is far less than the maximum permissible concentration of inorganic As in drinking water (10 μg/L [70,71] vs. 4.24 μg/L in ALIPS9 one month).

Cd is another element whose presence in cosmetics is forbidden. Cd did not show mobility from ALIPS9. Higher amounts of Cd in G30 justified the higher mobility of this element in ALIG30. The low release of Cd from fibrous clay minerals is in agreement with previous studies, which reported irreversible interaction between Cd and the solid phase, thus hindering the mobility of this element [72]. Regarding toxicity, Cd is classified in the category 2 (suspected human carcinogen). It can accumulate in the skin, having deleterious effects on this organ, as recently demonstrated by an in vivo study [73]. Nonetheless, the in vitro percutaneous bioavailability of cadmium chloride salt tested through human skin was determined to be among 0.07% (from water) and 0.01% (from soil) [74].

Other heavy metals such as Pb and Sb, though present in the pristine materials (Table 2), were not mobile (Table 3). Hg and Au were not detected in the pristine materials, and their absence after Franz cell diffusion guaranteed the absence of contamination during preparation, conservation, packaging, and manipulation of the hydrogels (art. 17 of the EC 1223/2009 [3]). Additionally, the cytotoxicity of these hydrogels has been previously tested in vitro [57], obtaining very positive results, which supports the hypothesis of product safety.
