Influence of artificial sebum on the dermal absorption of chemicals in excised human skin: A proof-of-concept study

Toxicology in Vitro 33 (2016) 23–28

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Influence of artificial sebum on the dermal absorption of chemicals in excised human skin: A proof-of-concept study Désirée Schneider, Kathrin Dennerlein, Thomas Göen, Karl Heinz Schaller, Hans Drexler ⁎, Gintautas Korinth Institute and Out-Patient Clinic of Occupational, Social and Environmental Medicine, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Schillerstrasse 25/29, 91054 Erlangen, Germany

a r t i c l e

i n f o

Article history: Received 20 October 2015 Received in revised form 2 February 2016 Accepted 17 February 2016 Available online 18 February 2016 Keywords: Percutaneous absorption Diffusion cell Artificial sebum Skin cream Ethanol Toluene

a b s t r a c t In an initial diffusion cell study, the influence of artificial sebum on dermal penetration and intradermal reservoir of ethanol and toluene was investigated in comparison with the effects of a skin cream (o/w- and w/o-emulsion) and untreated (control) skin. Human skin was exposed to neat ethanol and toluene for 4 h, respectively. During the experiments, the penetration of the compounds was assessed in the receptor fluid. The amounts of the test compounds in the skin were determined at the end of exposure. In the control experiments, 42% of the total resorbed ethanol amounts were found in the intradermal reservoir after 4 h, whereas 82% of the toluene amounts were found in the skin compartments. The treatment with artificial sebum showed no significant differences in dermal absorption of both test compounds compared to control skin. In contrast, the treatment with skin cream increased the percutaneous penetration (p b 0.001) and the intradermal reservoir of ethanol ~2-fold but not of toluene. In all exposure scenarios, a relevant intradermal reservoir was formed. The results indicate that sebum does not influence the percutaneous penetration and the intradermal reservoir of epidermally applied chemicals, whereas the application of skin creams may increase the dermal penetration of the compounds. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The epidermal barrier function is of interest in occupational medicine and hygiene, since in certain scenarios, the skin may be the predominant absorption pathway for several chemicals at workplaces (WHO, 2006). The lipid matrix of stratum corneum represents the main skin barrier against the systemic uptake of chemicals into the body (de Jager et al., 2006). It is reported that the skin appendage route, such as hair follicles (Liu et al., 2011) and glands (Schaefer and Redelmeier, 1996), also contributes to cumulative absorption of chemicals. Several functions of the sebaceous glands interact with skin protection (Zouboulis et al., 2008). The human skin surface lipid film mainly consists of sebum, a mixture of predominantly neutral lipids. The main function of sebum in fur-bearing animals is to form a hydrophobic barrier against water. However, little is known about the influence of sebum on the skin barrier function against a dermal absorption of chemicals in humans. The sebum amount on the skin surface ranges

Abbreviations: CAS, chemical abstract service; CV, relative coefficient of variation; GC, gas chromatography; KOH, potassium hydroxide; LogP, decadic logarithm of the octanol/ water partition coefficient; NaCl, sodium chloride; o/w, oil in water emulsion; SD, standard deviation; SEM, standard error of the mean; w/o, water in oil emulsion. ⁎ Corresponding author. E-mail address: [email protected] (H. Drexler).

http://dx.doi.org/10.1016/j.tiv.2016.02.010 0887-2333/© 2016 Elsevier B.V. All rights reserved.

from 0.2 to 130 μg/cm2 (Wilhelm et al., 1991); the thickness of the sebum film varies from b0.5 μm in most body areas to N4 μm in the sebum-rich face skin (Sheu et al., 1999). Some components of the human sebum, like fatty acids and cholesterol (Stefaniak and Harvey, 2006), are main constituents of the stratum corneum lipid matrix as well (Jungersted et al., 2008) and are therefore involved in building the skin barrier. Percutaneous penetration data of chemicals is mostly obtained by diffusion cell studies using human skin from plastic surgeries (van de Sandt et al., 2004; OECD, 2011). However, experimental treatment of skin surface with 70% aqueous isopropyl alcohol demonstrated an initial removal of sebum of N 90% in vivo (Rode et al., 2000). Thus, it may be supposed that the integrity of the natural sebum film is affected by pre-surgical disinfection, too. Therefore, the question arises whether the lack of a topical sebum film can affect the results of dermal penetration experiments based on human skin ex vivo. The aim of this proof-of-concept study was to evaluate the influence of artificial sebum on the percutaneous penetration behaviour of two test compounds with different physicochemical properties, compared to the results for untreated skin. Moreover, the comparison with the effects of a regular skin cream application was included in the study, as it was reported that topical skin cream application can enhance the dermal absorption of the chemical compounds (van der Bijl et al., 2002; Korinth et al., 2003, 2008).

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D. Schneider et al. / Toxicology in Vitro 33 (2016) 23–28

2. Materials and methods 2.1. Test compounds Ethanol (CAS no. 64-17-5) and toluene (CAS no. 108-88-3) were used as test compounds for percutaneous penetration experiments. Ethanol (purity: absolute GR for analysis) and toluene (purity: SupraSolv® grade) were purchased from Merck (Darmstadt, Germany). 2.2. Preparation of the artificial sebum and ingredients of the skin cream Artificial sebum was prepared, considering the data on human sebum composition from literature (Stefaniak and Harvey, 2006). Fig. 1 shows the composition of artificial sebum. Squalene (CAS no. 111-024), lanolin (CAS no. 8006-54-0), glyceryl tripalmitate (tripalmitin; CAS no. 555-44-2), glyceryl tristearate (tristearin; CAS no. 555-43-1), stearic acid (CAS no. 57-11-4), palmitic acid (CAS no. 57-10-3), cholesteryl oleate (CAS no. 303-43-5) and free cholesterol (CAS no. 57-88-5) were purchased from Sigma-Aldrich (Munich, Germany); oleic acid (CAS no. 112-80-1) was supplied by Fisher Scientific (Schwerte, Germany). The purity of palmitic acid, cholesteryl oleate and free cholesterol was ≥ 99%; the purity of squalene was ≥ 98%, of stearic acid ≥98.5% and of tripalmitin ~90%. Tristearin was of technical grade purity (usually ≥90%), lanolin for laboratory research purposes (usually ≥ 95%) and oleic acid of general purpose grade (usually N95%). Considering the saturation state, equal amounts (v/v) of saturated (stearic acid:palmitic acid, 25:25%) and unsaturated (oleic acid, 50%) free fatty acids were chosen. The ingredients for artificial sebum were weighted using an analytical precision balance (XA105DU; Mettler Toledo®, Giessen, Germany), mixed and heated in a water bath at 60 °C for ~ 10 min under continuous stirring at 250 rpm (IKA® RET basis; IKA®-Werke, Staufen, Germany) until the mixture became a fluid consistence. Subsequently, the artificial sebum was cooled down over night to room temperature and stored at 4 °C for 27 days according to the literature (Wertz, 2009). The skin cream (Basiscreme DAC; NRF 11.104) was obtained from the pharmacy of the University Hospital Erlangen, which prepared it according to the German Drug Codex (DAC, Deutscher Arzneimittel

Fig. 1. Comparison of the composition of human (A) and our artificial sebum (B). *The composition of human sebum was obtained from Stefaniak and Harvey (2006). The amounts of the ingredients of both sebum types are expressed as a percentage. To represent the triglyceride fraction of human sebum, equal amounts of tristearin and tripalmitin were chosen. The fraction of free fatty acids is represented by equal aliquots of saturated (stearic:palmitic acids, 25:25%) and unsaturated (oleic acid, 50%) compounds.

Kodex, Eschborn, Germany). The cream consisted of glycerol monostearate 60 (4%), cetyl alcohol (6%), medium-chain triglycerides (7.5%), petrolatum (25.5%), polyethylene glycol (PEG)-1000–glycerol monostearate (7%), propylene glycol (7%) and purified water (40%). 2.3. Dermal application of the artificial sebum and the skin cream Both artificial sebum and the skin cream were applied on the skin surface by gentle wipes with one cotton swab per exposure area. They were coated with artificial sebum by wiping the sebum surface. The skin cream was applied on cotton swabs in a volume of 20 μl (weight (mean ± SD): 19.39 ± 0.63 mg) using a 1 ml syringe (Omnifix® — F 1 ml; B. Braun, Melsungen, Germany). The amounts of the artificial sebum and the skin cream applied on the skin surface were determined from the weight difference of the cotton swabs (before and after application). The weight difference was determined using an analytical precision balance (XA105DU; Mettler Toledo®, Giessen, Germany). 2.4. Percutaneous penetration experiments In this study, excised human skin of 4 female donors (age range: 30–59 years, mean age: 41 years) was used, which was anonymously obtained from a local clinic after reduction of abdominoplasty, according to the ethical guidelines of our university. After removing subcutaneous tissue with a scalpel, the skin was wrapped in aluminium foil, put in plastic bags and stored at −20 °C for up to 3 months until the beginning of diffusion cell experiments, as proposed in the study protocol of van de Sandt et al. (2004). Similar skin preparation and storage are proposed by Dennerlein et al. (2013) and OECD (2011). For the experiments, the skin was thawed at room temperature. It was prepared with a scalpel to a thickness of 0.9–1.2 mm (n = 48), using a precision vernier calliper before mounting on diffusion cells. The preparation of skin is in close agreement with recommendations of OECD (2011) for studies using full thickness skin. The physical integrity of the skin membranes was visually assessed. The skin surface temperature was considered. Percutaneous penetration experiments were carried out using static PermeGear® diffusion cells (vertical system, flat flange joint; 9 mm orifice; exposure area 0.64 cm2, receptor chamber volume 5 ml) (SES Analysesysteme, Bechenheim, Germany), which are similar to the model described by Franz (1975). A diffusion cell consists of an exposure chamber (upper compartment) and a receptor chamber (lower compartment), where excised skin can be fixed in between. Receptor chambers of diffusion cells were charged with 0.9% aqueous NaCl solution and heated during the experiments by a thermostatic circulating water bath (MV-4; Julabo®, Seelbach, Germany) at 35 °C. Receptor fluid was continuously stirred (500 rpm) with a teflon-coated magnetic bar. Before the treatment with artificial sebum or skin cream, the skin surface temperature was measured using a digital precision thermometer (GMH 1160 with GOF 500 universal probe, type K; Greisinger electronic, Regenstauf, Germany). To exclude background contamination, blank receptor fluid samples were taken from each diffusion cell before application of the test compounds. Percutaneous penetration of ethanol and toluene was compared between untreated and treated skin (with artificial sebum or skin cream). Here, the 2 skin membranes of the 4 donors (n = 8 per each exposure scenario) were used in parallel. 10 min after the treatment with artificial sebum or skin cream, 160 μl/0.64 cm2 (= 250 μl/cm2) of neat ethanol (dose: 126.3 mg) or toluene (dose: 139.2 mg) was applied on treated and control skin surface under occlusion. Receptor fluid samples of 500 μl (~ 10% of receptor chamber volume) were taken at 0.5, 1, 2, 3 and 4 h of exposure and stored deep frozen until chemical analyses several days later. The sampled volume was immediately replaced with fresh receptor fluid.

D. Schneider et al. / Toxicology in Vitro 33 (2016) 23–28

After 4 h of exposure, the test compounds, sebum, and skin cream were removed from the skin surface using 2 dry cotton swabs per exposure chamber. Residual amounts of test compounds and the upper part of stratum corneum were removed by 5 tape strips (tesa Film no. 57,329, kristall-klar; tesa AG®, Hamburg, Germany) with a width of 19 mm to prepare comparable skin membranes for the assessment of intradermal reservoir of ethanol and toluene. For chemical analysis, circular skin punches (ø1 cm) were taken from the exposure area and digested in 2 ml aqueous 1.5 M KOH solution. Skin digestion was achieved in closed flange glass vials protecting the evaporation of test compounds after ~8 days at room temperature. 2.5. Chemical analysis Chemical analyses of ethanol and toluene in receptor fluid samples and in digested skin were carried out by static headspace gas chromatography (Varian 3300; Varian, Darmstadt, Germany; DANI 3950 HSS Sampling Unit, Dani Instruments S.p.A., Cologno Monzese, Italy) according to slightly modified methods described by Angerer et al. (1994, 1997). The samples for the GC analysis were taken from the vapour phases at a vapour–liquid equilibrium in sampling vials. Separation was performed on a DB-624 capillary column of 60 m × 0.32 mm and 1.8 μm thickness film (J&W Scientific Products, Folsom, CA, USA). Nitrogen was used as a carrier gas, with 410 kPa and a constant flow of 10 ml/min. The temperature programme started with a column temperature of 60 °C, which was held for 10 min, increased by a rate of 10 °C/min to 145 °C and held for further 5 min. Then the temperature was raised by rates of 20 °C/min to 220 °C and maintained for 1 min. The compounds were detected with a flame ionisation detector at 300 °C using hydrogen (30 ml/min) and oxygen (300 ml/min). Calibration was carried out with standard solutions of 1, 2, 4, 7 and 10 μl toluene (55.65 mg/25 ml ethanol) and ethanol (51.33 mg/25 ml double distilled water) solved in 2 ml 0.9% aqueous NaCl solution. The limit of quantification of ethanol and toluene in the receptor fluid and in an aqueous 1.5 M KOH solution was 0.4 and 0.01 mg/l, respectively. The relative coefficients of variation (CV) were 4.7 and 15.4%. 2.6. Calculations and statistics The percutaneous penetration kinetics for ethanol and toluene were calculated from the concentrations in receptor fluid samples, considering the dilution factor by the sampling procedure. The intradermal reservoir was calculated from the concentrations of the test compounds in the samples of known volume. The percutaneous penetration rates (fluxes) were calculated from the slopes of the linear regression of the cumulative amounts of the compounds, penetrated into the receptor fluid per square cm of skin surface versus time (unit: μg cm−2 h−1) within the period of 2–4 h after exposure. The lag times are the intersections of the same regression lines with the time (X) axes. The percutaneous penetration data are presented as means and standard errors of the means (SEM). Statistical analysis was performed using Excel 2010 (Microsoft Co., Redmond, WA, USA). The kinetic penetration data between different exposure scenarios were compared using a one-way analysis of variance. The skin surface temperature and the other dermal absorption data between the different exposure scenarios were compared with the Mann–Whitney-U-test using SPSS® 21.0 for Windows (IBM, Armonk, NY, USA). Statistical significance was assessed for p b 0.05. 3. Results The close range of skin surface temperature (mean ± SD: 32.6 ± 0.5 °C; n = 48) indicates similar experimental conditions between the different exposure scenarios. The amounts (mean ± SD; n = 8) of artificial sebum and skin cream applied on the skin surface were 4.48 ± 2.08 mg/0.64 cm2 for ethanol vs. 2.78 ± 1.54 mg/0.64 cm2 for toluene,

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and 7.06 ± 1.72 mg/0.64 cm2 for ethanol vs. 7.75 ± 1.89 mg/0.64 cm2 for toluene, respectively. The differences between the amounts of artificial sebum and skin cream for ethanol and toluene were significant (p b 0.05), but not for the artificial sebum or skin cream application (p N 0.05). 3.1. Percutaneous penetration kinetics The percutaneous penetration kinetics of the test compounds, after different skin treatments, is shown in Fig. 2. In all experiments, the penetration equilibration phase was achieved or nearly achieved 2 h after exposure. There was no significant divergence between ethanol penetration kinetics of skin treated with artificial sebum compared to control skin, whereas skin cream application led to a significantly different course of percutaneous penetration (Fig. 2a). Considering the course of toluene penetration (Fig. 2b), both artificial sebum and skin cream applications did not affect the penetration compared to the control experiments. 3.2. Percutaneous penetration parameters and intradermal reservoir The dermal absorption data for the test compounds are presented in Table 1. Ethanol penetrated through the skin in a 30 times higher amount than toluene. The pre-exposure treatment of the skin with the cream enhanced the dermal penetration of ethanol 2.3 times compared with the control skin (p b 0.001). For the treatment with artificial sebum, the percutaneous penetration of ethanol was not significantly higher. For toluene, no significant differences in penetrated amounts were found between both treatments and untreated skin. Comparing the amounts penetrated into receptor fluid to the amounts that remained in the skin at the end of exposure (4 h), a large intradermal reservoir for toluene was determined (factors: 4.3–4.5; p b 0.001). The formation of intradermal reservoir was independent from skin treatment. The intradermal reservoir for ethanol was less (58–73%; p = 0.16–0.05) than for toluene. Whereas the reservoir was not influenced by sebum, the treatment with skin cream increased it about 2-fold. The behaviour of dermal fluxes was similar to the penetrated amounts of the compounds in all application types. The lag times for both compounds were the shortest in experiments with skin cream application, although the differences were not statistically significant. 4. Discussion In the present study, the influence of sebum pre-treatment on the percutaneous penetration of occupational relevant chemicals was investigated for the first time according to our best knowledge. The natural human sebum consists of 31.7% of triglycerides, 27.6% of free fatty acids, 24.4% of wax esters, 10.4% of squalene, 3.9% of free cholesterol and 2.0% of cholesteryl oleate (Fig. 1A). In this study, we applied an artificial sebum in which the triglyceride fraction was reproduced by tripalmitin and tristearin (each 15.9%), the free fatty acid fraction by oleic acid (13.8%), palmitic acid (6.9%) and stearic acid (6.9%) and the wax ester fraction by lanolin (Fig. 1B). Prior investigations have revealed that a comparable artificial sebum offers similar properties like the natural human sebum with respect to the barrier function. Lu et al. (2009) demonstrated a similar partition into natural human and most types of artificial sebum for 3 homologous compounds of 4hydroxybenzoate. Although the selected test compounds are commonly used industrial solvents, information on the penetration into and through human skin is poor. In the present study, the majority of ethanol penetrated through the skin into the receptor phase, whereas a minor part of the compound was found in the skin compartments after 4 h of exposure. In contrast, the intradermal reservoir was 4.3–4.5 times higher than the amounts penetrated into the receptor fluid for toluene (Table 1).

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Fig. 2. Cumulative penetrated amounts of ethanol (A) and toluene (B) through untreated (control) and treated skin with artificial sebum or skin cream (mean ± SEM; n = 8). For ethanol, the differences between control and treated skin are significant (p b 0.001).

The different behaviours of the two test compounds can be explained by their different physicochemical properties. The lipophilic toluene (LogP: 2.73) may tend to remain in the lipophilic skin compartment, whereas the hydrophilic ethanol (LogP: − 0.31) is prone to penetrate into the aqueous receptor fluid (Korinth et al., 2012b). Moreover, the dermal flux (found for ethanol) was 16-fold higher than for toluene, which supports this explanation. The dermal flux of 535 μg cm− 2 h− 1 for ethanol is comparable with the flux of 1490 μg cm−2 h−1 found in a previous study with similar conditions, but over an exposure period which was twice as long (Korinth et al., 2012a). Additionally, the same study confirms an extremely low ability of toluene to penetrate the human skin (Korinth et al., 2012a). However, that study showed a 13 times lower dermal flux of toluene compared to the present study, which may be explained by the higher thickness of human skin (~ 2.5 vs. ~0.9 mm). The thicker skin implies a lower penetration but may serve as a higher intradermal reservoir. In the present study, we did not find any effect of sebum application on the dermal absorption and penetration of both test compounds. However, the treatment with skin cream significantly increased both the intradermal reservoir as well as the penetration of ethanol into the receptor phase. For toluene, such a penetration enhancement was not found (Table 1). The negative result for a probable enhancement effect of skin cream on toluene penetration is in agreement with the findings of an in vivo microdialysis study (Klede et al., 2005). The result of the study, in which rats were epicutaneously exposed to toluene with and without pre-treatment by a similar skin cream (“cremor basalis”) also did not show any effect of the pre-treatment on the dermal penetration of toluene. According to the manufacturer, the skin cream, applied in the present study, featured properties of o/w (oil in water) as well as w/o (water in oil) emulsion. Here, the o/w- and w/o-emulsion type of the skin cream implicates that it could function as a transport barrier against lipophilic as well as hydrophilic chemicals. However, there is some evidence suggesting a promoted penetration of chemicals into the skin instead. Previous diffusion cell studies demonstrated penetration enhancement of hydrophilic (ethylene glycol, isopropanol) as

well as lipophilic (aniline, benz[a]pyrene, o-toluidine) compounds through human skin by so-called skin barrier creams (van der Bijl et al., 2002; Korinth et al., 2003, 2008). In these experiments, neither the o/w nor the w/o emulsion type of skin creams achieved a retardation of the dermal penetration of the test compounds. In contrast, the application of an o/w-emulsion cream most pronouncedly increased the percutaneous penetration of aniline and o-toluidine (Korinth et al., 2008). The skin cream, applied in the present study, contained glycerol monostearate and propylene glycol. Such compounds are capable of interacting with the lipid matrix of stratum corneum and disrupting its structure, facilitating the penetration of chemicals (Williams and Barry, 1992). Especially propylene glycol is very well known to enhance the dermal penetration of chemicals and, moreover, to amplify the effect of other enhancement agents (Williams and Barry, 1992). Furthermore, ethanol and toluene can act as skin irritants (Suhonen et al., 1999; Wigger-Alberti et al., 2000) like many other solvents (Fartasch et al., 2012), too. One mode of action may have the potential to dissolve stratum corneum lipids (Goldsmith et al., 1988) and may result in a subsequent increase of their own dermal absorption. This effect can appear not only in vivo but in ex vivo as well. Nevertheless, the most plausible explanation for the enhancement of dermal ethanol absorption in experiments with the skin cream could be the probable rise of thermodynamic activity by the considerable aliquot of water (40%) in the cream. Our previous study (Korinth et al., 2012b) demonstrated that such a water effect is very strong in amphiphilic compounds, leading to a higher ethanol flux from aqueous solutions even when the compound dose amounted only by a half. The explanation that ethanol penetration enhancement may primarily be generated by a water-mediated effect of the skin cream, fits very well with the result that such an effect was not observed after artificial sebum application. The applied artificial sebum also contained fatty acid esters but did not contain any water and resolving agents, e.g. propylene glycol. The ingredients of sebum show predominantly lipophilic properties, which could potentially form a transport barrier against the diffusion of hydrophilic compounds like ethanol. On the other hand, for the sebum constituents oleic acid and cholesterol, an enhanced penetration of

Table 1 Percutaneous penetration parameters for ethanol and toluene in control and in treated skin (mean ± SEM; n = 8). Application type

Amount (μg/0.64 cm2) [% of applied dose]

Flux (μg cm−2 h−1)

Lag time (h)

905.3 ± 119.1a [0.72] 1034.0 ± 161.4b [0.82] 2090.5 ± 223.2a/b [1.65]

535.0 ± 70.0a 576.0 ± 85.7b 1149.0 ± 141.8a/b

1.4 ± 0.1 1.3 ± 0.1 1.1 ± 0.1

29.6 ± 4.8 [0.02] 29.8 ± 4.8 [0.02] 28.2 ± 5.6 [0.02]

20.7 ± 2.3 19.7 ± 2.5 18.6 ± 2.9

1.8 ± 0.1 1.5 ± 0.1 1.5 ± 0.2

Intradermal reservoir

Receptor fluid

Ethanol Control skin Skin treated with sebum Skin treated with skin cream

658.8 ± 112.9 [0.52] 598.2 ± 116.8a [0.48] 1406.2 ± 269.0a [1.12]

Toluene Control skin Skin treated with sebum Skin treated with skin cream

134.3 ± 3.3a [0.09] 132.2 ± 4.7 [0.09] 122. 2 ± 3.7a [0.09]

Note: Values in the same column and application subgroup sharing the same subscript are significantly different at p b 0.05 (intradermal reservoir) and p b 0.01 (receptor fluid and flux).

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chemicals has been reported in diffusion cell studies. In rat skin, oleic acid increased the penetration of the drugs tenoxicam 14–23 times (Larrucea et al., 2001) and of carvedilol ~ 3 times (Amin et al., 2008). In experiments with human skin, the addition of a small cholesterol fraction (2%) to a w/o-emulsion led to a penetration increase of the hydrophilic diphenhydramine hydrochloride (Schmalfuß et al., 1997). Positive effects of sebum on skin, such as moisturising and maintaining of suppleness, are well known (Man et al., 2009). Artificial sebum may, similarly to skin creams, be absorbed into the skin. In humans, ~ 60% of artificial sebum applied on the forearm was percutaneously absorbed within only 15 min of exposure (Blanc et al., 1989). In a recent study, sebum was used as a vehicle investigating the dermal absorption of 4-cyanophenol and cimetidine in volunteers (Tsai et al., 2012). However, in the latter study, the sebum was applied in an aqueous solution, where enhancing effects of the vehicle on the dermal drug penetration have not been evaluated. In our study, however, the sebum was applied not as a vehicle but as a coating prior to exposure. The small surface area of the appendage route (including sebaceous glands), covering only 0.01–0.1% of the skin surface (Scheuplein and Blank, 1971; Schaefer and Redelmeier, 1996), may also support the conclusion that sebum does not influence the dermal uptake of chemicals. For 30 chemicals, Valiveti and Lu (2007) showed fluxes through separated artificial sebum, ranging from 1.2 μg cm− 2 h−1 for betamethasone to 114.8 mg cm−2 h−1 for lidocaine. Here significant differences in the capability of chemicals to diffuse through a sebum film of similar composition (as in our present study) were indicated. However, such data do not provide any information on the interaction of sebum with stratum corneum to form a permeability barrier against chemicals. To investigate this effect, we prepared artificial sebum, since the collection of sufficient amounts of human sebum is highly sophisticated. Many types of artificial sebum have been developed in the past several years (Blanc et al., 1989; Lu et al., 2009; Stefaniak and Harvey, 2006; Wertz, 2009). Considering the composition, there are no fundamental differences between all these sebum types, also not for our artificial sebum and the natural human sebum (Fig. 1). The skin, used in our study, was disinfected during surgeries with cutasept® (BODE Chemie, Hamburg, Germany) containing 63% aqueous isopropyl alcohol, distilled water, and benzalkonium chloride (b 0.1%). Though benzalkonium chloride may act as surfactant, the effect on skin barrier should be marginal, due to the low concentration of b0.1%. The sebum amount in the abdominal area is ~ 3.1 μg/cm2 of skin surface in young adults (Wilhelm et al., 1991). Rode et al. (2000) reported that skin treatment with 70% aqueous isopropyl alcohol almost completely removes the natural sebum film in humans. Therefore, it is to assume that sebum was also removed during the surgical disinfection of our skin specimens. Although we applied as little as manually possible of artificial sebum to cover the skin surface (0.64 cm2), our amount was ~1800 times larger compared to the sebum amount in humans, but still in close agreement with ~3 mg/cm2 as performed in human studies testing skin creams (Fartasch et al., 2015). Nevertheless, in our study the skin treatment with artificial sebum did not increase the percutaneous penetration and the intradermal reservoir of ethanol and toluene. Moreover, it reduced significantly the intradermal reservoir of ethanol compared to control skin (Table 1). The penetration kinetics also showed no different course (Fig. 2a–b). Skin creams for workers are used to prevent contact dermatitis and to support skin regeneration. This may be the best approach to avoid a dermal penetration of chemicals (Fartasch et al., 2015). The creams are beneficial for workers when the use of gloves is banned, e.g. while operating rotating machines with cutting fluids, which are considered to be strong skin hazards. However, the actual application of skin creams by metalworkers is very low with 29% (Kütting et al., 2009). Due to the solid nature of sebum, it could remain longer on the workers' skin at such workplaces. Here, a more effective barrier without occlusive effects, like silicon-based skin creams, is formed and perhaps shows a higher acceptance because of its affinity to biological substances.

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The findings of this proof-of-concept study are based on the results for two chemicals. For general recommendations, it is necessary to support the results by further investigations. We used frozen abdominal skin according to the conditions described by Dennerlein et al. (2013), where no impact on skin barrier functions was observed by freezing. To ensure the resistance of skin for mechanical pressure caused by the application and even distribution of the sebum and the skin cream, full-thickness skin was used in the present study. For studies using pre-treatment, the skin thickness of ~ 0.9 mm was established in our laboratory previously (Korinth et al., 2008). With the percutaneous penetration data being in all experimental series within three standard deviations of the mean (data not shown), the integrity of skin membranes used in this study can be assumed according to proposals in Howes et al. (1996). Infinite doses of the test compounds were used to simulate a worstcase exposure scenario, which is preferred in occupational risk assessment applying ex vivo models. In our previous studies applying pretreatment of skin with different creams, the exposure of infinite doses of solvents has been demonstrated successfully (Korinth et al., 2008). Applying finite doses of ethanol and toluene without occlusion evaporation of the compounds and vehicle might not have been effectively controlled. The experiments in the present study were conducted with respect to the evaluation of occupationally relevant skin exposure. Compared to 24 h as usually applied in studies testing cosmetic ingredients we limited the exposure duration to 4 h, representing most reliably a continuous occupational exposure during a half work shift without hand washing. 5. Conclusions Although the sebum layer in our study was significantly thicker than in vivo, it neither formed an impermeable skin barrier against the hydrophilic ethanol and the lipophilic toluene nor enhanced their absorption. In experimental studies, investigating the dermal absorption of chemicals using human skin from surgeries, a reconstitution of the sebum film seems therefore, not to be necessary. In contrast, the application of a commonly used skin cream enables an enhanced penetration of ethanol through human skin. To avoid the adverse effect of dermal penetration enhancement of chemicals in workers, skin creams could be manufactured with a similar composition to human sebum. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgement The study was financially supported by the Foundation UNIBUND (Friedrich-Alexander University Erlangen-Nürnberg (FAU) Germany). We would like to thank Johannes Müller for assistance in the chemical analyses performed in our laboratories. References Amin, S., Kohli, K., Khar, R.K., Mir, S.R., Pillai, K.K., 2008. Mechanism of in vitro percutaneous absorption enhancement of carvedilol by penetration enhancers. Pharm. Dev. Technol. 13, 533–539. http://dx.doi.org/10.1080/10837450802309646. Angerer, J., Gündel, J., Heinrich-Ramm, R., Blaszkewicz, M., Working Group Analyses in Biological Materials, 1997. Alcohols and ketones — determination in blood and urine. In: Angerer, J., Schaller, K.H., Greim, H. (Eds.), Analyses of Hazardous Substances in Biological Materials. Deutsche Forschungsgemeinschaft, Wiley-VCH, Weinheim vol. 5, pp. 1–33. http://dx.doi.org/10.1002/3527600418.bi6764e0005. Angerer, J., Gündel, J., Knecht, U., Korn, M., Working Group Analyses in Biological Materials, 1994. Benzene and alkylbenzenes (BTX aromatics) — determination in blood. In: Angerer, J., Schaller, K.H., Greim, H. (Eds.), Analyses of Hazardous Substances in Biological Materials. Deutsche Forschungsgemeinschaft, Wiley-VCH, Weinheim vol. 4, pp. 107–130. http://dx.doi.org/10.1002/3527600418.bi7143e0004. van der Bijl, P., Gareis, A., Lee, H., van Eyk, A.D., Stander, I.A., Cilliers, J., 2002. Effects of two barrier creams on the diffusion of benzo[a]pyrene across human skin. SADJ 57, 49–52.

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