Effects of skin washing frequency on the epidermal barrier function and inflammatory processes of the epidermis: An experimental study

Increased hand hygiene measures during the COVID‐19 pandemic have led to an increased quantum of hand eczema (HE).


| INTRODUCTION
For preventing transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and development of coronavirus disease 2019 (COVID- 19), hand hygiene measures must be followed strictly. 1 This applies to the general public and particularly specific professional groups, including healthcare workers (HCWs). 2 Hand hygiene recommendations during the COVID-19 pandemic entail longer durations and frequencies of hand washing, 3 which have been shown to go along with skin strain and adverse skin reactions. [4][5][6] The accumulating skin strain through water contactwhich alone may be skin irritating 7 -and the contact with soaps or detergents lead to impairments of the skin barrier function and subsequently irritant contact dermatitis of the hands. 8 Due to a high amount of wet work, the prevalence of hand eczema (HE) in HCWs was already about twice as high as in the general population before the COVID-19 pandemic. 9 Workers subjected to a high occupational skin strain 9,10 are now doubly burdened by the current hygiene measures which also take place in their private surroundings. Accordingly, the elevated COVID-19 associated hygiene measures have reportedly caused an increase of HE in the general population as well as high-risk occupations. [11][12][13] In view of individual suffering of HE patients and the health economical relevance of this disease, 14,15 preventative measures are needed in order to impede genesis of HE as well as to contain existing HE and to avoid chronification. Since hand hygiene measures will likely remain elevated momentarily, 16 demonstrably gentle hand washing regimes should be established. Against this background, this proof-of-concept study aims in an experimental set-up mimicking real-life conditions at assessing the effects of varying washing frequencies on the epidermal barrier function and inflammatory processes of the epidermis using a lipid-containing syndet compared to a standard skin cleanser. In addition, the effect of skin cream application after washing is to be determined.

| Trial design
This study was a non-invasive, experimental, skin bioengineering, biochemical, immunological trial. Ethic approval was obtained by the sub-

| Participants
Only skin-healthy volunteers without atopic diathesis according to the Erlangen Atopy Score 17 (score value <8) were included in the study. Detailed inclusion and exclusion criteria are listed in Table S1.
Informed consent in accordance with the Helsinki Declaration of 1975, and later revisions, was obtained from all participants prior to their inclusion in the study.

| Washing procedures
The practical study phase took place from February to April 2021.
Six test areas on the volar aspects of the lower arms and one control area on the volar aspects of the upper arm with a surface of 8 cm 2 each were marked. The test areas on the volar lower arms were randomized per participant. The test and control areas with the corresponding interventions are listed in Table 1. For each washing, a standardized amount of 3 mL cleanser +7 mL distilled water (aqua dest.) was used. If cream was used, a standardized amount of 24 mg cream was applied per test area ( Table 1). The cream was weighed by using an electronic precision scale and a plastic top as container. Weighing took place immediately prior to application. After weighing, the cream was completely taken off of the plastic top with the aid of finger gloves and evenly distributed in the test area. Standardized washings of the test areas were performed using the automated cleansing device (ACiD) with a contact pressure of the washing units of 150 g and a rotation speed of 100 rotations per minute ( Figure 1). The ACiD has been previously described in depth elsewhere. [18][19][20] The ingredients according to the International Nomenclature of Cosmetic Ingredients of the commercially available study products provided by Beiersdorf AG, Hamburg, Germany are listed in Table S2.

| Bioengineering measurements and sample collection
Before measurements were taken, participants were acclimatized to the standardized room air-conditioning (room temperature of 21 ± 1 C, relative air humidity [RH] of 50 ± 5% RH) for at least At T3, SC tape stripping (D-Squame sampling) was done to collect SC samples. 21 Sampling was conducted using round adhesive disks (22-mm Standard D-Squame Skin Sampling Discs; Monaderm) which were pressed onto the test areas for 10 s with the D500 D-Squame Pressure Instrument (CuDerm) in order to ensure a defined pressure. This procedure was conducted with eight discs. The fifth consecutive disk was used for natural moisturizing factor (NMF) analysis and the seventh consecutive disk was used for cytokine (interleukin-1α [IL-1α] as well as interleukin-1α receptor antagonist [IL-1αRA]) analysis. NMF analysis was conducted after extraction of NMF from the sampling discs using high-performance liquid chromatography with ultraviolet detection 22 and the analysis of the cytokines was conducted using electrochemiluminescence immunoassays (Mesoscale). 23 As the amount of SC on the tape varies, the amount of cytokine in the SC on each tape was normalized by the protein content, which was determined using the Pierce Micro BCA Protein Assay Kit (Thermo Fischer Scientific) with the bovine serum albumin supplied as standard. The results of NMF were corrected for SC protein content on the tape strips determined with the 850 nm absorption infrared densitometer SquameScan 850A (Heiland Electronic).

| Statistical analyses
Calculations were performed by using Prism 9 software (GraphPad).
Distribution of data was tested using the Shapiro-Wilk normality test.
Results for all investigated parameters at T3 were analysed by using two-way repeated measures analysis of variance (ANOVA) and uncorrected Fisher's least significant difference (LSD) followed by post hoc correction for multiple comparisons carried out by the false discovery rate (FDR) method of Benjamini Hochberg (BH). Difference in data on skin bioengineering parameters TEWL, SCH, erythema and SC pH measured at T0 to T3 were tested by two-way repeated measures ANOVA. Association between all measured parameters was performed by using Spearman correlation analysis. P < .05 was considered significant.

| Characteristics of the participants
Twenty-five healthy volunteers (23 women) with an age ranging from 22 to 55 years (mean ± SD: 33.8 ± 10.7 years) and an Erlangen Atopy Score 17 ranging from 0 to 7 (mean ± SD: 3.6 ± 1.9) were included.
Nineteen participants (76.0%) had a Fitzpatrick 24 skin phototype II and 6 (24.0%) had a Fitzpatrick skin phototype III. None of the participants had used any topical immunosuppressants in the test areas in the weeks before the study.

| Effects of washing frequency and cream application
Two-way repeated measures ANOVA and uncorrected Fisher's LSD followed by post hoc correction for multiple comparisons carried out by the FDR method of BH revealed that for TEWL, SCH, erythema, SC pH, NMF, IL-1α, and IL-1αRA there were no statistically significant differences at T3 comparing the various test areas (Table 1) with their respective control sites (Figures S1-S7). Full results of TEWL, SCH, erythema, SC pH, NMF, IL-1α and IL-1αRA are provided in Tables S3-S7, respectively. Two-way repeated measures ANOVA did not reveal significant differences from T0 to T3 in any of investigated parameters.

| DISCUSSION
To the best of our knowledge, this study is the first study to examine the effects of varying washing frequencies on the epidermal barrier function and inflammatory processes of the epidermis using a variation of cleansing agents with and without the application of a rehydrating cream under laboratory conditions using a skin cleansing device. The mimicking of almost life-like conditions and the standardization of procedures by using the ACiD can be considered as a strength of this study.
To analyse effects on the epidermal barrier function and inflammatory processes of the epidermis, measurements taken at T3 (24 h after the last washing procedure) were examined primarily. It was shown that the applied interventions (Table 1) had a mild effect on the examined skin bioengineering parameters TEWL, SCH, erythema, and SC pH which was not statistically significant nor of practical importance. This observation leads to the conclusion that all of the interventions did not lead to detectable impairment of the epidermal barrier function or inflammatory processes of the epidermis. Furthermore, this finding is sustained by the reported results on biochemical/immunological parameters since there were no statistically significant changes in NMF, IL-1α, and IL-1αRA. The positive effect of emollient application on the epidermal barrier function after skin washing has, however, already been described in previous studies. 25,26 Potentially damaging effect of cleansing agents on the skin barrier function might be reduced by the application of emollients, which seems advisable after hand washingespecially when higher hand washing frequencies are applied.

Correlation analysis of skin barrier and inflammatory parameters
has shown that TEWL is inversely correlated with NMF levels. Due to its hygroscopic properties, NMF contributes largely to the retention of water in the SC. Reduced NMF values would result in an increase in epidermal water loss concomitant with higher TEWL. 27,28 Positive association was observed between TEWL and IL-1α levels, which corroborates earlier studies which have shown that skin barrier damage-depicted by higher TEWL-is associated with lower IL-1α concentrations. 29,30 COVID-19-associated hygiene regimes comprise elevated hand washing frequencies and longer washing durations. 3 The chosen duration (60 s) for each washing was thus considered appropriate for mimicking current hand washing durations in pandemic times. However, the applied washing frequencies of 5 times and 11 times within 4 h might not reflect the actual skin strain HCWs are exposed to during working days, particularly considering additive effects over several days. Whilst designing this study, infection control regulations needed to be followed to protect study participants as well as the examiners.
To generate results even closer to the exposure HCWs are subjected to in their working environment, it seems recommendable to conduct skin washings within a longer time period of 8 h as well as for consecutive days when the pandemic situation allows such a study design.
It should be considered to include not only skin-healthy subjects in following studies but also individuals with (a history of) atopic dermatitis who have a reduced skin barrier function 31 and are more susceptible to developing contact dermatitis 32,33 providing a more representative design for a real-life situation in the workplace. It can be suspected that the study design of the present study might generate different results in individuals with an atopic skin diathesis or sensitive skin rather than in individuals with normal, healthy skin. Christoph Skudlik: Conceptualization (equal); funding acquisition (lead); resources (lead); writingreview and editing (equal). Swen Malte John: Conceptualization (lead); formal analysis (equal); funding acquisition (lead); methodology (lead); project administration (lead); resources (lead); writingreview and editing (lead). Richard Brans: Conceptualization (lead); investigation (equal); methodology (lead); project administration (equal); supervision (lead); writingreview and editing (lead). Flora Karla Sonsmann: Conceptualization (lead); formal analysis (equal); investigation (lead); methodology (lead); project administration (equal); supervision (lead); writingreview and editing (lead).

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the supplementary material of this article.