In vitro transdermal absorption of Al2O3 nanoparticles

In vitro transdermal absorption of Al2O3 nanoparticles

Accepted Manuscript In vitro transdermal absorption of Al2O3 nanoparticles Marcella Mauro, Matteo Crosera, Massimo Bovenzi, Gianpiero Adami, Giovanni...

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Accepted Manuscript In vitro transdermal absorption of Al2O3 nanoparticles

Marcella Mauro, Matteo Crosera, Massimo Bovenzi, Gianpiero Adami, Giovanni Maina, Elena Baracchini, Francesca Larese Filon PII: DOI: Reference:

S0887-2333(19)30301-7 https://doi.org/10.1016/j.tiv.2019.04.015 TIV 4524

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

24 January 2018 26 March 2019 15 April 2019

Please cite this article as: M. Mauro, M. Crosera, M. Bovenzi, et al., In vitro transdermal absorption of Al2O3 nanoparticles, Toxicology in Vitro, https://doi.org/10.1016/ j.tiv.2019.04.015

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ACCEPTED MANUSCRIPT

In vitro transdermal absorption of Al2O3 Nanoparticles Marcella Mauro, Post Doca,†, Matteo Crosera, Post Docb, †, Massimo Bovenzi, Prof.a, Gianpiero Adami, Prof.b, Giovanni Maina, Prof.c, Elena Baracchini, Drb, Francesca Larese Filon, Prof.a

Clinical Unit of Occupational Medicine, Department of Medical Sciences, University of

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a

Department of Chemical and Pharmaceutical Sciences, University of Trieste, via Giorgieri 1, 34127

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Trieste, Via della Pietà 19, 34100 Trieste, Italy

Trieste, Italy

Department of Clinical and Biological Sciences, University of Torino, Via Zuretti 29, 10126 Torino,

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c



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Italy

These authors contributed equally to this work.

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Corresponding author: Tel +39 040 3992409; fax: +39 040 368199, e-mail address:

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[email protected] (Mauro M.)

Declaration of interest: The authors report no current nor previous conflicts of interest.

ACCEPTED MANUSCRIPT Abstract: Dermal exposure to Aluminium nanoparticles (AlNPs) can occur in occupationally- and non occupationally exposed- population, due to the use of Al salts-based antiperspirants. No AlNPs transdermal permeation data exists. Our study investigated in vitro the permeation of 30-60 nm Al2O3NPs dispersed in synthetic sweat (20g/L) using excised human skin on Franz cells. Experiments were performed using intact

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(experiment 1) and needle-abraded skin (experiment 2). After 24 hours traces of Al were detectable in

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receiving solution of exposed cells (35.0 ± 6.0 ng/cm 2 for intact and 88.5 ± 34.2 ng/cm2 for damaged skin,

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mean and SD) and in blank cells (36.3 ± 7.0 ng/cm2), without statistical significance (p=0.08, Mann-Whitney test). The average amount of Al into intact and damaged skin samples was 3.96 ± 0.20 μg/cm 2 for intact and

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4.36 ± 0.47 μg/cm2 for damaged skin (p=0.08). Al content was similar in epidermal and dermal layers of intact and damaged skin (1.95 ± 0.13 μg/cm2 and 2.31 ± 0.12 μg/cm2 epidermal, 2.01 ± 0.25 μg/cm2 and

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2.05 ± 0.35 μg/cm2 dermal). Al is a trace element in human body and the amount found in receiving solutions could be due as background impurity. This data suggest a reassuring transdermal permeation

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profile for Al2O3NPs.

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Key terms: Franz cells, in vitro, Aluminium nanoparticles, transdermal absorption.

ACCEPTED MANUSCRIPT 1. Background: Aluminium oxide (Al2O3) is commonly used in manufacturing production of abrasives, refractories materials, ceramics, electrical insulators, catalysts, paper, spark plugs, light bulbs, alloys, glass and heat resistant fibres [1]. It has been estimated that working population can be exposed to aluminum (Al) up to three times higher compared to the general population [2-4], but data are lacking as regards exposure to nano Al,

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of which use keeps growing [5-13]. Concern is supported by the fact that nanoparticles (NPs) may behave in

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a different way compared to their homologous bulk material, due to their high surface/volume ratio, which

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confers different surface chemical properties [14], and therefore different toxicological potential after interaction with biological media.

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Conflicting opinions are present in literature over whether nanoscale alumina presents health risks. The most investigated effects are cell viability and DNA damage, but results on citototoxicity and genotoxicity

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are not conclusive [15-16][17-21]. Zhang Q and coworkers [24] showed a higher genotoxicity of these NPs with respect to bulk alumina and Mirshafa and colleagues [25] showed a size-dependent neurotoxicity of

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Al2O3 , which seems to increase as particles size decrease, reaching highest effects in nano-size range. Al is a

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trace element in the human organism [26], but Al overload can be dangerous for human health. Al exposure may lead to the onset of occupational contact dermatitis [27-28] and irritant dermatitis [29],

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which have been reported among workers exposed to Al alloys and Al dust [1]. The possible onset of neurodegenerative diseases after chronic Al exposure, such as Alzheimer dementia and a possible linking of

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breast cancer to the use of underarm antiperspirant have been investigated but there is not consistent and convincing evidence to confirm these points (30). Almost all available in vitro studies on Al were performed using animal [31] or human skin [32] exposed to Al chlorohydrate (ACH) and found that the metal slightly penetrates the skin. Al forms complexes with skin proteins [33] and the greatest amount of the metal can ultimately be found in the sweat ducts and in the stratum corneum. On the other hand in vivo studies showed that after Al dermal application a statistically significant increase of the metal was detectable in urine and serum of exposed animals [34], while in humans an excretion of 0.012% was detectable in the urine after underarm single antiperspirants-

ACCEPTED MANUSCRIPT containing-Al application [35], meaning that a small absorption through skin happened. Pineau and colleagues [36] confirmed this data by means of an in vitro study using Al salts - derived from different commercial antiperspirants formulations - applied on human excised skin biopsies mounted on Franz diffusion cells. The results showed that Al concentrations in the receptor fluid corresponded to only 0.012% of the applied Al, while Al amount in stripped skin was significantly higher. But also a case report of

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systemic hyperaluminemia (plasma Al = 104.7 μg L-1) presenting with complaints of bone pain after 4 years

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of daily ACH deodorant application has been described [37].

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Data on Al and Al oxide nanoparticles skin penetration (Al into the skin) and permeation (Al that pass through the skin) are currently lacking [38] and the aim of the present study was to assess this point,

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investigating the transdermal absorption of Al2O3NPs through intact and damaged human skin using an in vitro method.

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2. Materials and Methods

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2.1. Chemicals

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All chemicals were analytical grade. Urea, sodium chloride, sodium hydrogenphosphate, potassium dihydrogenphosphate, were purchased from Carlo Erba (Milan, Italy); lactic acid (90% w/w) was bought

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from Acros Organics (Geel, Belgium); nitric acid (69% w/w), hydrogen peroxide (30% w/w), ammonium hydroxide (25% w/w), boric acid, hydrochloric acid (37% w/w) and hydrofluoric acid (48% w/w) from Sigma

system.

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Aldrich (Milan, Italy). Water reagent grade (milliQ water) was produced with a Millipore purification pack

The commercially available dispersion of aluminium oxide (Al2O3) nanoparticles (30-60 nm) was provided by Sigma Aldrich (Milan, Italy). The nanoparticle concentration was 20 wt % in water. 2.2. Nanoparticles Characterization Al2O3NPs have been visualized, once they were dispersed in synthetic sweat and at the end of the experiments (after the 24 h exposure time) to confirm the dimensions of the NPs and the aggregation state of the donor phase, by Transmission Electron Microscope (EM208; Philips, Heidhoven, The Netherlands)

ACCEPTED MANUSCRIPT with an high definition acquisition system SIS Quemesa (Olympus Soft Imaging Solutions) and a digital image acquisition system iTEM (FEI Italia, Milan, Italy). The synthetic sweat solution used as donor fluid consisted in 0.5% sodium chloride, 0.1% urea and 0.1% lactic acid in milliQ water [39]; pH 4.5 was adjusted with ammonia.

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2.3. Nanoparticles Dissolution In order to evaluate the ions release from NPs once they were put in synthetic sweat, 4 mL of the donor

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phase (the Al2O3NPs dispersion diluted ten times with synthetic sweat), have been ultrafiltered using

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Amicon Ultra-4 centrifugal filters (10K MWCO) supplied by Millipore Corporation (USA). The ultrafiltration has been performed in centrifuge at 5000 rpm for 30 min in order to remove the Al2O3NPs, but not

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aluminium ions, from the solution [40].

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The solution has been analyzed by ICP – OES (Inductively Coupled Plasma-Optical Emission Spectroscopy) to quantify the aluminium concentration. This operation has been repeated on three different aliquots of

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2.4. Preparation of Skin Membranes

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donor solution.

Human abdominal full thickness skin was obtained as surgical waste from 2 female healthy patients aged

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45–65 years, who underwent plastic surgery for esthetic reasons. The ethical committee approval was obtained. The use of this type of skin allows a possible comparison with other in vitro studies on metal

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permeation. After the skin excision, subcutaneous fat has been removed with a scalpel blade and hair was shaved from the epidermis, then skin samples were stored at −25 °C for a period up to, but not exceeding, 4 months. It has been demonstrated that this procedure does not damage skin barrier properties. At the day of the experiment skin samples had been defrosted in physiological solution at room temperature for a 30 min period and then 4 × 4 cm2 pieces were cut from each skin specimen and mounted separately on the diffusion cells. Thickness of the membranes were < 1mm. Skin integrity was tested before and after each experiment using electrical conductivity by means of a conductometer (Metrohm, 660, Metrohm AG Oberdorfstr. 68 CH-9100 Herisau) operating at 300 Hz and

ACCEPTED MANUSCRIPT connected to two stainless steel electrodes. The conductibility data in μS were converted into KΩ cm −2. Cells with a resistance lower than 3.95 ± 0.27 KΩ cm−2 were considered to be damaged and rejected, as suggested by Davies et al. [41]. 2.5. In Vitro Diffusion System

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Percutaneous absorption studies were performed using static diffusion cells following the Franz method [42], OECD test guideline 428 [43] and protocols suggested by EDETOX [44]. As the equipment used is

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static, there is no relationship among the cells; hence each of them represents an independent evaluation.

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The receptor compartment had a mean volume of 14.0 mL and was maintained at 32 °C by means of circulation of thermostated water in the jacket surrounding the cell. This temperature value was chosen in

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order to reproduce the hand physiological temperature at normal conditions. The physiological solution used as the receptor phase was prepared by dissolving 2.38 g of Na2HP04, 0.19 g of KH2P04 and 9 g of NaCl

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into 1 L of milliQ water (final pH 7.35). The salt concentration in the receiving fluid was approximately the same that can be found in blood. The physiological solution used as receiving phase was continuously

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stirred using a Teflon coated magnetic stirrer. Each piece of skin was clamped between the donor and the

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receptor compartment and skin samples from donor 1 and donor 2 were equally divided between experiment 1 and 2; the mean exposed skin area was 3.29 cm2 and the average membranes thickness was 1

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described below:

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mm. Two different experiments were conducted using intact (exp. 1) and damaged skin (exp. 2) as

2.5.1. Experiment 1

The donor solution was prepared just before the experiment, diluting the Al2O3NPs dispersion ten times with synthetic sweat (final concentration of 20 g L-1). The aluminium concentration in the donor phase was confirmed by ICP-OES analysis: the Al2O3NPs were dissolved by addition of 1 mL of hydrofluoric acid (48% w/w), than 6 mL of boric acid and diluted to 50 mL with milliQ water.

ACCEPTED MANUSCRIPT At time 0, the exposure chambers of 6 Franz diffusion cells were mounted with intact skin samples and filled with 1 mL of the donor suspension (6.06 mg cm−2) to ensure an infinite dose. The experiment was run for 24 h, when the receiving and the donor phases of each diffusion cell were recovered for analysis. 2.5.2. Experiment 2

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The same experimental conditions used in Experiment 1 were applied in parallel in Experiment 2 , with the only exception of the skin used, which was abraded by drawing the point of a 19-gauge hypodermic needle

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across the surface (20 marks in one direction and 20 perpendiculars) as suggested by Bronaugh and

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Steward was used [45].

Each donor contribute equally to both experiments ( exp 1 with intact skin: 3 samples from donor 1 and 3

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samples from donor 2; exp 2 with damaged skin: 3 samples from donor 1 and 3 samples from donor 2;

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blanks: 1 sample from donor 1 and 1 sample from donor 2 in each experiment). 2.5.3. Blanks

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For each experiment, two cells were added as blank. The blank cells were treated as the other cells with the

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exception that only synthetic sweat was used in the donor compartment.

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2.5.4. Skin Digestion after the Experiment

After the experiment, the skin pieces were washed three times with physiological solution to remove

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residual Al2O3NPs from the skin surface, then removed from the diffusion cells and separated into epidermis and dermis by heat shock, immerging in water at 60 °C for 1 min before freezing at −25 °C. At the time of the analysis, the skin membranes were dried for 2 h at room temperature, weighed and aciddigested in a closed microwave system (Multiwave PRO, Anton Paar) using a mixture of 4 mL of HNO 3 (69 %) and 0.5 mL of HF (48%). The mineralization was performed through two heating steps. In the second step, H3BO3 was added in order to buffer the excess of HF. The solutions obtained from the mineralization process were diluted up to a volume of 15 mL by adding Milli-Q water before ICP-OES analysis. 2.6. Analytical Measurements

ACCEPTED MANUSCRIPT ICP-MS 7500 CE Agilent Technologies Inc., Santa Clara, CA, USA instrument (with integrated autosampler) was used to determinate the total aluminum concentration in the receiver phases. A six-points standard curve, obtained by dilution of aluminium standard solution for ICP-MS analyses (by Merck, Germany) was used for ICP-MS measurements (0.1, 0.5, 1, 5, 10, and 100 μg L-1, ion mass 27 u.m.a.). The limit of detection of aluminum was 0.1 μg L-1 for ICP-MS and the precision of the measurements as

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repeatability (RSD %) for the analysis was always <5%. Total aluminium concentration in the donor phases

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and in the solutions resulting from the mineralization of the skin samples were performed by (ICP-OES) using an Optima 8000 Spectrometer (PerkinElmer, U.S.A.), equipped with an S10 Autosampler. Analysis

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were conducted using a calibration curve obtained by dilution (range: 0 - 10 mg L-1) of aluminium standard

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solution for ICP-OES analyses (by Sigma Aldrich, Milan, Italy). The limit of detection (LOD) at the operative wavelength of 396.153 nm was 0.01 mg/L.

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The precision of the measurements expressed as relative standard deviation (RSD %) for the analysis was

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always less than 5%.

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2.7. Statistical Analysis

Aluminium concentration data (μg cm−2) in the receptor solution were converted to the total amount that

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penetrated (μg cm−2), with a correction for dilution due to sample removal. Data analysis was performed with Excel for Windows, release 2007 and Stata Software, version 11.0

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(StataCorp LP, College Station, TX, USA). Skin absorption data were reported as mean ± standard deviation (SD). The difference among independent data and donors were assessed by means of the Mann-Whitney test. A p value <0.05 was considered significant. 3. Results 3.1 Nanoparticles characterization Representative TEM images of Al2O3NPs suspension, diluted in synthetic sweat, before and after the experiments, and their size distribution, are presented in Fig. 1 (a, b, c, respectively). The mean size of the

ACCEPTED MANUSCRIPT NPs was 53 ± 15 nm in accordance with the declared dimension (number of NP measured = 100), but tended to form bigger aggregates reaching the micrometer range. Aggregation was similar before and after experiments. NPs were stable in synthetic sweat and results derived from the ultrafiltration of the NPs suspension showed that the free aluminium concentration was always lower than 0.1% of the total NPs dispersion., meaning that dissolution of NP or ions release from NP is very low

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3.2 Franz diffusion cells experiments

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After 24 hours of exposure only traces of Al were detectable in the receiving solution of cells exposed to

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NPs (35.0 ± 6.0 ng cm-2 for intact and 88.5 ± 34.2 ng cm-2 for damaged skin, results expressed as mean and SD) and in blank cells too (36.3 ± 7.0 ng cm-2). The difference between damaged skin samples exposed to

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NPs and blanks did not reach statistical significance (p = 0.08, Mann-Whitney test), meaning that the average amount of the metal found could be present as impurity (Figure 2). The percentage of the applied

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dose that permeate the skin was 0.0011% for intact skin and 0.0028% for the damaged skin. The average amount of Al in intact and damaged whole thickness skin samples was 3.96 ± 0.20 μg cm -2

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(0.12% of the applied dose) and 4.36 ± 0.47 μg cm-2 (0.13% of the applied dose), respectively (p = 0.08). Al content was similar in epidermal and dermal layers of both intact and damaged skin samples (1.95 ± 0.13 μg cm-2 and 2.31 ± 0.12 μg cm-2 for epidermal layer, 2.01 ± 0.25 μg cm-2 and 2.05 ± 0.35 μg cm-2 for dermal

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layer). Al penetration in blank cells was 2.16 μg cm-2 (1.70 for epidermal layer and 0.46 μg cm-2 for dermal

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layer) (Figure 3). The two donors specific values of Al whole skin content and Al in receiving solution in intact and damaged skin samples are showed in table 1. Statistical analysis did not show differences between them (Mann-Whitney test between donors: intact skin p= 0.82, damaged skin p= 0.83, receiving solutions intact skin p = 0.52, receiving solutions damaged skin p = 0.126). 4. Discussion Dermal exposure to Al can occur in some occupational scenarios and using personal care products (PCP). In the first case Al micro- and nano-powders (diameter in the range of 10-300 nm) can be intentionally added in the production of automobile paints [46], as fuel additives in propellants, explosives and pyrotechnics

ACCEPTED MANUSCRIPT [47-49], or can represent a waste product in some occupational activities such as friction stir, flame, and solid-state welding on Al [50-54], sanding painted surfaces [55] and in electric arc thermal sprays [56]. In the second case, Al can be found in PCP in the form of salts, mostly as ACH, because its capacity to block secretion of sweat is exploited. Experimental data on Al dermal absorption are limited and all of them investigated the behavior of Al salts,

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while actually no data at all exist regarding transdermal permeation of Al and Al oxides nanoparticles.

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Our study investigated in vitro on human excised skin samples - intact and damaged – the penetration and

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permeation of 30-60 nm Al2O3NPs and results showed that the average Al amount found in the receiver chambers of exposed cells was very low and similar to the one found in blank cells. This result can be

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justified by the fact that Al is a trace element in human body and a small background level of Al is expected to be found in receiving solution, without this meaning that in cells exposed to Al2O3NPs a metal

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permeation through skin did occur.

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This is in line with results of Putterman and colleagues [31], whom investigated in vitro on guinea pigs

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stratum corneum the absorption and desorption of Al chloride and Al chlorohydrate solutions. Their results showed moreover that the desorption of Al chlorohydrate is lower compared to Al chloride, which means that this form is retained longer inside the outer skin layer. Blank and colleagues [32] investigated in vitro

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on human excised skin a 23h exposure to 20% Al chlorohydrate solution by means of tape stripping and

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found that very little Al reached the dermis. They hypothesized a possible strong binding of Al complexes to the outer stratum corneum layers. In vivo studies on humans and animals showed different results between acute and chronic exposure. Lansdown and coauthors [57] investigated in vivo on animals a 5 consecutive days dermal exposure to six different Al salts (aluminum chloride 10%, nitrate 10%, sulphate 10%, hydroxide 10%, acetate 10% and chlorhydrate 10% and 25%) and found different kind of inflammatory skin reactions only for exposure to Al chloride and nitrate, but no data on transdermal absorption were collected. Anane and colleagues [34] investigated in vivo on mice chronic exposure (130 days) to Al chloride. The Al amount tested was similar to

ACCEPTED MANUSCRIPT the one expected in a one day antiperspirants use. Results showed a significant increase of Al content in urine and serum of treated mice, meaning that a small fraction of topical antiperspirants might produce measurable increase in urine and tissue Al levels. Flarend L. and colleagues [35], investigated in vivo on two volunteers (underarm), the absorption of a single radiolabeled Al chlorohydrate (26Al) by means of blood and urine samples collection for 7 weeks . Results indicated that only 0.012% of the applied Al was

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absorbed through the skin, which is about 2.5% of the Al typically absorbed by the gut from food over the same time period. Pineau and colleagues [36], studying the permeation of Al salts contained within

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commercial antiperspirants through excised human skin using Franz cells apparatus, found the same Al

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transdermal permeation percentage.

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Comparing our results with the latter cited study [36], we found a percentage of metal permeated through the skin after 24 hours approximately five orders of magnitude lower using intact skin and approximately

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four orders of magnitude lower using damaged skin. This could be due to different reasons, the most important of which is the different dissolution potential in biological media. Al salts are by definition water

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soluble, while our results showed an Al2O3NPs dissolution value in donor solution lower than 0.1%. This is in

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line with other metal oxides NPs [58-59]. Al salts are the main ingredient inside antiperspirants, since they can form hydroxide precipitates of denatured keratin in the cornified layer that surrounds and occludes the

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opening of sweat ducts, leading to a sweating reduction [60]. Exposure to Al oxide NPs can occur in workers employed in industrial processes where this NP can be used (production of abrasives, refractories

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materials, ceramics, electrical insulators, catalysts, etc.) or in waste treatments. However, our study demonstrated that skin is a negligible route of penetration and permeation of Al2O3NPs and that only a damaged skin barrier can cause a very low Al permeation. It can be interesting to compare Al2O3NPs penetration and permeation results with the ones obtained in previous studies conducted using other metal oxide NPs - Co3O4NPs and TiO2NPs - using similar experimental protocol [58-59]. Both Al2O3NPs and Co3O4NPs slightly permeate the skin, reaching fully comparable final values through damaged skin, while permeation through damaged skin was lightly higher for Al2O3NPs, but still in the same order of magnitude of ng/cm2. Instead, TiO2NPs do not permeate and only barely penetrate the skin.

ACCEPTED MANUSCRIPT Therefore, albeit with the limitations due to the “in vitro” test procedure - which cannot take into account any active uptake mechanism - our study supports the hypothesis of a reassuring permeation profile as regards transdermal penetration and permeation of Al2O3NPs, which may be generated in different working

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scenarios.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-

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for-profit sectors.

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54. Pfefferkorn FE, Bello D, Haddad G, Park J-Y, Powell M, McCarthy J, et al. Characterization of exposures to nanoscale particles during friction stir welding of aluminum. Ann Occup Hyg. 2010; 54:486–503. [PubMed: 20453001] 55. Koponen IK, Jensen KA, Schneider T. Comparison of dust released from sanding conventional and nanoparticle-doped wall and wood coatings. J Expos Sci Environ Epidemiol. 2010; 21:450–63. 56. Bémer D, Régnier R, Subra I, Sutter B, Lecler MT, Morele Y. Ultrafine particles emitted by flame and electric arc guns for thermal spraying of metals. Ann Occup Hyg. 2010; 54:607–14. [PubMed: 20685717] 57. Lansdown AB. Production of epidermal damage in mammalian skins by some simple aluminum compounds. Br J Dermatol 1973;89:67–76. [PubMed: 4788320] 58. Crosera M, Prodi A, Mauro M, Pelin M, Florio C, Bellomo F, Adami G, Apostoli P, De Palma G, Bovenzi M, Campanini M, Filon FL. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells. Int J Environ Res Public Health. 2015; 12(8), 9282-9297. 59. Mauro M, Crosera M, Pelin M, Florio C, Bellomo F, Adami G, Apostoli P, De Palma G, Bovenzi M, Campanini M, Filon FL. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity. Int J Environ Res Public Health. 2015; 12(7), 8263-8280. 60. Quartrale RP. In: Laden K, Felger CB, editors. The mechanism of antiperspirant action in eccrine sweat glands. New York: Marcel Dekker; 1988. p. 89–118.

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FIGURES LEGENDS:

Figure 1. Representative TEM images of Al2O3NPs (a, b) and NPs size distribution (c).

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Figure 2. Aluminium content (ng cm-2) in receiving solutions of cells exposed to a concentration of 6.06 mg cm-2 Al2O3NPs dispersion (intact skin and damaged skin) and in cells exposed to physiological solution

used as blanks.

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(blanks). Mean and standard deviation of 6 cells with intact skin , 6 cells with damaged skin and 4 cells

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Figure 3. Aluminium content (µg cm-2) inside the skin (whole thickness, epidermis, dermis) of blank cells exposed to physiological solution (4 cells), intact skin and damaged skin exposed to a concentration of 6.06 mg cm-2 Al2O3NPs dispersion (6 cells each). Results expressed as mean and standard deviation. Table 4. Donor specific values of Al Skin content (ug/cm 2) and Al in receiving solution (ng/cm2)of intact and damaged skin samples as mean and SD.

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FIGURES

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Figure 3.

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Al Skin content (ug/cm2)

Donor

(mean ± SD)

Intact

(mean ± SD)

Damaged

(mean ± SD)

4.14

3.95 ± 0.17

4.75

4.35 ± 0.50

30.90

35.25 ± 6,90

98.30

64.60 ± 30.56

4.51

43.20

3.80

3.79

31.65

3.64

3.96 ± 0.29

4.82

4.38 ± 0.57

30.75

3.73

42.57

4.21

4.58

31.30

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4.36 ± 0.48

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3.96 ± 0.21

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Table 1.

34.87 ± 6.67

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4.03

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3.91

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Damaged

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Total

(mean ± SD)

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#2

Intact

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#1

Al in receiving solution (ng/cm2)

35.06 ± 6.07

38.70 56.80 116.30

112.39 ± 16.80

93.98 126.90 88.5 ± 34.23

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No data on skin permeation of Aluminium oxides NPs are available In vitro permeation of Al2O3NPs (53 ± 15 nm) has been assessed using Franz cells Intact and damaged human full thickness skin samples have been used Only traces of Al were detected in receiving solution in all types of exposed cells

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