Impact of Skin Temperature and Intradermal pH on Transdermal Fluoride Absorption Following Hydrofluoric Acid Exposure: An Ex Vivo Diffusion Cell Study in Human Skin.

4. Discussion

4.1. Temperature- and pH-dependency of fluoride penetration and influencing factors

In our study, we were able to demonstrate that a higher skin temperature and an intradermal pH shift into the acidic range resulted in a significantly increased cumulatively penetrated amount of fluoride. Several mechanisms may be at play here:

The diffusion coefficient of a substance is temperature-dependent, such that changes in temperature can significantly influence its transdermal movement. Blank et al. (1967) first identified a polar route for substances to traverse the hydrated membranes of the stratum corneum (SC), involving what they termed “bound water”. Researchers later identified extracellular lacunar domains within the SC, contributing to this porous pathway (Menon and Elias, 1997). As skin temperature rises, the penetration of substances through this porous route tends to increase (Peck et al., 1995). Additionally, elevated temperatures raise the water content within both the SC and the corneocytes themselves, further promoting penetration (Kodiweera et al., 2018, Spencer et al., 1975).

The SC of human skin shows a pH gradient with surface values typically in the acidic range of pH 4.1–5.8 (Lambers et al., 2006, Segger et al., 2008) and deeper intradermal layers near the living keratinocytes exhibiting a more neutral pH range (pH 7–7.4) (Hanson et al., 2002, Proksch, 2018, Schreml et al., 2011, Turner et al., 1998). This gradient plays a critical role in skin function, supporting pH-sensitive enzymes such as ?-glucocerebrosidase and acid sphingomyelinase (Mavon et al., 1998), both of which are essential for maintaining the structural integrity of the SC’s lipid layers and the differentiation of corneocytes. The architecture of these lipid layers, influenced by the position of fatty acids through pH-dependent head-group repulsion (Lieckfeldt et al., 1995), is key to a healthy skin barrier. Physiological pH values, both on the surface of the skin and in the deeper intradermal layers, are therefore necessary for optimal skin keratinization and barrier function (Elias, 1996, Fluhr et al., 2001, Vahlquist, 1999). Adjusting intradermal pH from neutral to slightly acidic (e.g. pH ~6.5) could disrupt pH balance, potentially impairing keratinocyte and lipid-layer functions essential for maintaining a robust skin barrier.

When applying the results of our study to real-life workplace scenarios, other factors associated with skin temperature and the skin barrier must be considered. First, the temperature of the skin can be altered as part of the body’s homeostatic thermoregulation, which adjusts temperature by modifying peripheral circulation (Romanovsky, 2014, Arens and Zhang, 2006). Additionally, infrared and ultraviolet radiation from the sun and its heat affect skin temperature (Blazejczyk, 1999). Second, skin surface pH is influenced by both endogenous and exogenous factors. Endogenous factors include age, anatomical site, genetic predisposition, ethnic differences, sebum production, skin-moisture content, and sweat production. Exposure to detergents, cosmetic products, soaps, occlusive dressings, skin irritants, and topical antibacterials are important exogenous factors for the determination of skin pH (Parra et al., 2003, Rippke et al., 2003, Yosipovitch and Maibach, 1996). As stated above, changes in the skin surface pH affect its physiological pH gradient, thus interfering with the structural integrity of the SC’s lipid layers and impairing the barrier function of the skin. Third, it must be considered that real-life skin is not necessarily intact and healthy. Common skin conditions, such as eczema or sunburn, involve inflammatory processes that lead to extracellular acidification in the dermis and can therefore enhance transdermal penetration (Nafisi and Maibach, 2018, Rizi et al., 2011).

However, the most crucial factor influencing intradermal pH during HF exposure is most likely hydrofluoric acid itself. Due to its acidic effects, HF inflicts damage on the skin architecture, denaturing proteins and destroying cell-cell contacts (Proksch, 2018). It can easily advance to deeper skin layers, such as the dermis, where its protons additionally cause a pH shift towards more acidic values. In light of the results reported in our study, it might therefore be reasonably assumed that hydrofluoric acid is able to enhance its own penetration through the skin. This hypothesis aligns with previous studies conducted in our laboratory (Kilo et al., 2020), in which the influence of skin temperature on the absorption of model lipophilic compounds like anisole and the hydrophilic compounds 1,4-dioxane and ethanol through human skin was investigated. Different substances may react differently to skin temperature and pH variations. Therefore, in our present study, we aimed to validate our hypothesis on the influence of skin pH and temperature stemming from these previous studies for hydrofluoric acid. Further research using more precise methodologies may add evidence to further confirm the hypothesis (Nielsen et al., 2016, Dennerlein et al., 2015).4.2. Selection of experimental setup and resulting strengths and limitations of the present study

The Franz diffusion cell is a long-established and widely recognized experimental setup designed to simulate dermal absorption under controlled conditions, with standard protocols especially for detecting hazardous chemicals in vitro (Howes et al., 1996, Nesvadbova et al., 2015, OECD, 2011, COLIPA, 1997).

HF is a highly corrosive substance known to cause severe dermal injury. It can penetrate intact skin, allowing its ions to damage deeper tissues while the surface may appear unaffected (Dennerlein, 2016). Thus, prompt decontamination is critical to mitigate HF’s corrosive effects and limit systemic absorption. A 30% (v/v) HF solution was used in this study, representative of concentrations commonly encountered in industrial and pharmaceutical settings (NICNAS., 2001). A 3-minute exposure duration was selected to simulate workplace accidents. Given the widespread use of HF in industries like semiconductor manufacturing, understanding its dermal penetration is crucial for improving occupational safety (Dennerlein et al., 2015).

Previous studies (Dennerlein et al., 2013, Hodge and Smith, 1981, Barbero and Frasch, 2016) indicate that fluoride absorption plateaus after 6 hours under similar conditions (WHO,1996; Lund et al., 1997). In the present study an observation period of 12 hours was selected for the assessment of short-term effects within the first 8 to 12 hours which simulates work shifts/settings. According to the OECD, which recommends monitoring up to 24 hours post-wash but does not require a 24-hour exposure, 8 hours was deemed sufficient to characterize fluoride absorption without causing unnecessary tissue damage (OECD., 2004).

We included donor skin from both male and female donors as well as from donors of different ages, which may have affected penetration. Significant intra- and inter-individual differences in dermal absorption were observed, consistent with previous studies on skin penetration variability (Polak et al., 2012, Larsen et al., 2003). These differences are important for data interpretation and highlight the need for caution when extrapolating results to broader populations. The study design included a minimum of three replicates per donor to address intra-donor variability. To account for possible inter-donor variability, five donor skins were used in the present study. Increasing the number of donors (e.g., four in duplicate) can reduce inter-donor variability and improve statistical significance (OECD., 2011). However, the variability in cumulative penetration values across donors remains a limitation (Schaefer et al., 2013). According to Hopf et al. (2020), the number of replicates and donors should be based on the study’s specific goals. In risk assessment studies like this, fewer replicates and donors may be acceptable unlike in bioequivalence studies for topical pharmaceuticals, which require greater statistical range.

A skin surface pH in the slightly acidic range contributes to the skin’s protective barrier (Schmid-Wendtner & Korting, 2006), whereas intradermally, the physiological pH lies in the neutral range (Hachem et al., 2003). The skin’s robust buffering system helps maintain its protective function under varying conditions. While barrier function was not explicitly assessed through specialized tests in the present study, visual inspection before and after the experiment showed no signs of irritation or disruption, consistent with similar studies reporting no significant effects on skin barrier function under mildly acidic conditions (Rawlings & Harding, 2004). However, more comprehensive testing to assess the effect of pH changes on skin integrity should be performed in the future for confirmation.

A pH-dependency of fluoride penetration has already been reported in previous studies. Our findings align with these previous results, which have shown that fluoride absorption is enhanced in low pH environments (Kabir et al., 2020). HF penetrates the SC and releases protons (H?) within the skin, reducing intradermal pH and promoting further penetration. The skin’s inherent buffer capacity helps maintain pH under most conditions, but during HF exposure, fluoride ions penetrate tissue while protons remain unbound, leading to localized pH drops (Johnston & Strobel., 2020). Although directly modifying intradermal pH is challenging, some interventions might reduce proton availability in the skin, like the topical application of buffering decontaminant gels with good skin penetration properties during the post-acute phase of first-aid measures after HF accidents. Buffering intradermal pH may help mitigate the self-reinforcing mechanism of HF penetration (Bajraktarova-Valjakova et al., 2018) and should be considered in future research on decontamination strategies.

Although OECD., 2004 recommends a skin thickness between 200–400 µm for penetration studies, for the present experiments a thickness of 0.9 mm was selected to ensure accurate pH measurements using microminiature needle electrodes and to better simulate systemic absorption. This approach aligns with OECD., 2011, which permits the use of full-thickness skin up to 1000 µm when scientifically justified. However, formal barrier integrity testing was not conducted. The skin was visually inspected before and after the experiment, and absorption time curve analysis was performed, but this method may not fully capture all aspects of skin barrier function (Monteiro-Riviere, 2016). More advanced techniques, such as transepidermal water loss (TEWL) measurements, electrical resistance testing, or the use of tritiated water, could offer more precise insights into barrier integrity (Wester & Maibach, 2005 Due to laboratory constraints, tritiated water was not feasible, and electrical resistance measurements were discontinued due to interference with the stratum corneum, particularly when using cotton swabs for decontamination (Menon & Elias, 1997). Future studies should include barrier integrity assessment to enhance data accuracy and robustness regarding skin permeability and damage (Zhai & Maibach, 2001). Although visual inspection is most likely not sufficient to confirm skin integrity – considering the corrosive nature of HF – previous studies in our laboratory with a similar experimental approach have shown only insignificant visible damage after 3 minutes of exposure to 30% HF at 8- and 12-hours follow-up (Dennerlein et al., 2016).

What is more, our study’s focus was to evaluate the influence of skin parameters on the systemic absorption of HF due to its penetration through the skin in a real-world occupational safety scenario. The possible damage inflicted on the skin’s barrier integrity under laboratory conditions as well as its effect on penetration will likewise occur in practice, for example in workplace accidents. In a real-life workplace accident, it is of no consequence if the systemic absorption that occurs is solely caused by penetration (strictly speaking) or also by the disruption of the skin barrier integrity. The only important factor for the patient’s systemic outcome is the cumulative systemic absorption of HF and therefore the combined effect of penetration itself and skin barrier disruption. Thus, in our approach to emulate a real-life situation, TEWL-measurement results would not have changed the conclusions from our experiments.

The focus on receptor fluid sampling to assess the total amount of fluoride absorbed through the skin aligns with the OECD, which emphasizes the relevance of receptor fluid in vitro dermal absorption studies (OECD., 2004). A full mass balance assessment was not conducted in the current study. However, previous studies (for example Dennerlein et al.; 2013) analyzed dermal storage in the skin and retention, reporting a fluoride recovery rate of about 79%. Although this slightly falls outside the 80–120% range recommended by the OECD., 2004; ECHA, 2021), the recovery rate is considered acceptable for volatile substances like fluoride, as seen in similar studies.

Decontamination by one dry cotton swab, as used in the present experiments, does not fully reflect best practices for decontamination after HF exposure in real world scenarios, but was chosen intentionally as our study focused on the influence of physiological skin parameters on fluoride absorption rather than the influence of the decontamination procedure. The results provide valuable insights into the relationship between skin temperature, pH, and dermal penetration in the context of HF exposure, which may have clinical significance in occupational and emergency settings (Shin et al., 2024).

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