Catherine Champmartin
email: catherine.champmartin@inrs.fr

Fabrice Marquet
email: fabrice.marquet@inrs.fr

Lisa Chedik
email: lisa.chedik@inrs.fr

Marie-Josèphe Décret
email: marie-josephe.decret@inrs.fr

Matthieu Aubertin
email: matthieu.aubertin@inrs.fr

Elisabeth Ferrari
email: elisabeth.ferrari@inrs.fr

Marie-Christine Grandclaude
email: marie-christine.grandclaude@inrs.fr

Frédéric Cosnier
email: frederic.cosnier@inrs.fr

Human in vitro percutaneous absorption of bisphenol S and bisphenol A: a comparative study

Keywords: BPA, bisphenol A, BPS, bisphenol S, BSA, bovine serum albumin, HPLC, high-performanceliquid-chromatography, MW, molecular weight, OECD, Organisation stratum corneum, TEWL, transepidermal water loss Bisphenol A (BPA), Bisphenol S (BPS), In vitro dermal absorption, In vitro dermal metabolism, fresh human skin, vehicle

Bisphenol A (BPA) is widely used in industrial products. Due to the toxicity of this compound, and to comply with restrictions and regulations, manufacturers have progressively replaced it by substitutes.

One of the main substitutes used is bisphenol S (BPS). Despite increasing use in many products, the effects of BPS on human health have been little investigated, and studies on percutaneous BPS absorption and particularly toxicokinetic data are lacking. However, the endocrine-disrupting activity of BPA and BPS appears comparable. Dermal contact is a significant source of occupational exposure and is the main route during handling of bisphenol-containing receipts by cashiers. Here, percutaneous BPS absorption was investigated and compared to that of BPA. Experiments were performed according to OECD guidelines. Test compounds dissolved in a vehicle -acetone, artificial sebum or water -were applied in vitro to fresh human skin samples in static Franz diffusion cells. Flux, cumulative absorbed dose and distribution of dose recovered were measured. BPA absorption was vehicle-dependentranging from 3% with sebum to 41% with water. BPS absorption was much lower than BPA absorption whatever the vehicle tested (less than 1% of applied dose). However, depending on the vehicle 20% to 47% of the applied BPS dose remained in the skin, and was consequently potentially absorbable.

Both BPA and BPS were mainly absorbed without biotransformation. Taken together, these results indicate that workers may be exposed to BPS through skin when handling products containing it. This exposure is of concern as its toxicity is currently incompletely understood.

Bisphenol A (BPA) is widely used as a monomer in polycarbonate plastic manufacturing and in epoxy resins lining food and beverage containers. It also acts as a color developer in thermal paper, such as till receipts. BPA has been extensively studied and the increasing concerns have been raised about its toxic effects. Among the adverse health effects noted, it can have endocrine-disrupting behavior. As a result, BPA is currently classified as a category 1B reproductive toxicant and also a skin sensitizer under the EU CLP Regulations (CE, 2016a). It has been demonstrated to be absorbed through the skin in in vitro assays [START_REF] Biedermann | Transfer of bisphenol A from thermal printer paper to the skin[END_REF][START_REF] Marquet | In vivo and ex vivo percutaneous absorption of [14C]-bisphenol A in rats: a possible extrapolation to human absorption?[END_REF][START_REF] Zalko | Viable skin efficiently absorbs and metabolizes bisphenol A[END_REF][START_REF] Toner | In vitro percutaneous absorption and metabolism of Bisphenol A (BPA) through fresh human skin[END_REF] and also in vivo during campaigns measuring occupational exposure in till operators [START_REF] Ndaw | Occupational exposure of cashiers to Bisphenol A via thermal paper: urinary biomonitoring study[END_REF][START_REF] Thayer | Bisphenol A, Bisphenol S, and 4-Hydroxyphenyl 4-Isoprooxyphenylsulfone (BPSIP) in Urine and Blood of Cashiers[END_REF][START_REF] Lee | Bisphenol A exposure through receipt handling and its association with insulin resistance among female cashiers[END_REF]. Consequently, the European Commission has banned its use in thermal paper at concentrations equal to or exceeding 0.02% by weight from 3 January 2020 (CE, 2016b). BPA will therefore need to be replaced by alternatives. As a substitute for BPA, a structural analogue, bisphenol S (BPS), is already commonly used in thermal papers used for till receipts [START_REF] Liao | Bisphenol S, a New Bisphenol Analogue, in Paper Products and Currency Bills and Its Association with Bisphenol A Residues[END_REF][START_REF] Goldinger | Endocrine activity of alternatives to BPA found in thermal paper in Switzerland[END_REF][START_REF] Rocha | High Levels of Bisphenol a and Bisphenol S in Brazilian Thermal Paper Receipts and Estimation of Daily Exposure[END_REF]Björnsdotter et al., 2017a;Björnsdotter et al., 2017b;[START_REF] Eckardt | Bisphenol A and alternatives in thermal paper receipts -a German market analysis from 2015 to 2017[END_REF][START_REF] Molina-Molina | Determination of bisphenol A and bisphenol S concentrations and assessment of estrogen-and anti-androgen-like activities in thermal paper receipts from Brazil, France, and Spain[END_REF][START_REF] Vervliet | Current-use of developers in thermal paper from 14 countries using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry[END_REF]. Because of its physicochemical properties and its low cost compared to other current potential alternatives, BPS should quickly replace BPA. However, BPS not only presents structural similarities with BPA, it also appears to share some of its toxic effects, including acting as an endocrine disruptor. Indeed, in vitro tests have shown the estrogenic and anti-androgenic activities BPS (binding to estrogen receptors, inducing cell proliferation in the MCF7 mammary line, inhibiting androgenic activity of dihydrotestosterone) to be only slightly lower or equivalent to those reported for BPA [START_REF] Kuruto-Niwa | Estrogenic activity of alkylphenols, bisphenol S, and their chlorinated derivatives using a GFP expression system[END_REF][START_REF] Molina-Molina | In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors[END_REF][START_REF] Eladak | A new chapter in the bisphenol A story: bisphenol S and bisphenol F are not safe alternatives to this compound[END_REF][START_REF] Goldinger | Endocrine activity of alternatives to BPA found in thermal paper in Switzerland[END_REF][START_REF] Rochester | Bisphenol S and F: A Systematic Review and Comparison of the Hormonal Activity of Bisphenol A Substitutes[END_REF][START_REF] Zalmanova | Bisphenol S instead of bisphenol A: a story of reproductive disruption by regretable substitution -a review[END_REF]Björnsdotter et al., 2017b;[START_REF] Molina-Molina | Determination of bisphenol A and bisphenol S concentrations and assessment of estrogen-and anti-androgen-like activities in thermal paper receipts from Brazil, France, and Spain[END_REF]. In vivo data corroborates these endocrine disruption effects [START_REF] Rochester | Bisphenol S and F: A Systematic Review and Comparison of the Hormonal Activity of Bisphenol A Substitutes[END_REF]. It should also be noted that although conjugated metabolites of BPS do not exhibit the endocrine-disrupting effects of the parent molecule, certain hydroxylated BPS metabolites have shown estrogenic and anti-thyroid activity in vitro [START_REF] Skledar | Influence of metabolism on endocrine activities of bisphenol S[END_REF]. The process of metabolizing BPS could, therefore, alterstrengthen or abolish -its endocrine activity.

Endocrine disruptors that act on sex hormones, such as BPS, are likely to disrupt reproductive functions (fertility disorders including infertility, menstrual cycle abnormalities, pubertal disorders, menopause, etc.). The dangers of endocrine-disrupting toxins affect the entire population, but are particularly relevant to the health of pregnant or breastfeeding women, a population that deserves special attention. Since 85% of French cashiers are women [START_REF] Bodier | Travailler dans le commerce de détail ou l'artisanat commercial[END_REF], their occupational exposure to BPS is of particular concern [START_REF] Thayer | Bisphenol A, Bisphenol S, and 4-Hydroxyphenyl 4-Isoprooxyphenylsulfone (BPSIP) in Urine and Blood of Cashiers[END_REF][START_REF] Ndaw | Occupational exposure of cashiers to bisphenol S via thermal paper[END_REF][START_REF] Sogorb | Case study: Is bisphenol S safer than bisphenol A in thermal papers?[END_REF]. Dermal contact is a significant source of exposure in the work environment [START_REF] Semple | Dermal exposure to chemicals in the workplace: just how important is skin absorption?[END_REF], and is the main route during handling of bisphenol-containing receipts. However, we lack studies on percutaneous BPS absorption. For complete risk assessment, toxicity data must be completed by toxicokinetic and biotransformation data [START_REF] Sogorb | Case study: Is bisphenol S safer than bisphenol A in thermal papers?[END_REF]. This article is the first report on percutaneous absorption of BPS using freshly excised human skin samples. The aim of the study presented here was to compare in the same experimental conditions, the percutaneous flux, the cumulative absorbed dose and the distributions for BPA and BPS and to obtain toxicokinetic parameters. The skin metabolic activity was also assessed by determining the percentage of metabolized and unmetabolized-BPA or BPS in the receptor fluid.

-Materials and methods

Regulatory considerations

This study was conducted according to OECD TG 428 (Skin Absorption: In Vitro Method) (OECD, 2004a) and the guidance document for the conduct of skin absorption studies, OECD series on testing and assessment number 28 (OECD, 2004b).

Test, control substances and chemicals

Carbon-14 labeled bisphenol A ([ 14 C]-BPA) and Carbon-14 labeled bisphenol S ([ 14 C]-BPS) were supplied by Moravek Biochemicals, Inc. (California, USA) with a specific activity of 1.85 GBq/mmol (50 mCi/mmol) and of 2.53 GBq/mmol (68.3 mCi/mmol), respectively. Their radiochemical purity was 98.7% and 99.7% for BPA and BPS, according to the supplier. After dilution of the stock solutions, the radiochemical purities were nevertheless verified by HPLC combined with scintillation counting (Flow Scintillation Analyzer, Packard, USA). The radiolabeled compounds were stored at -20 °C. Unlabeled BPA [CAS: 80-05-7] and BPS [CAS: 80-09-1] (Sigma-Aldrich France) were stored at room temperature.

The suppliers indicated purities of ≥ 99% and 98%, respectively. These compounds were used for isotopic dilution. Squalene [CAS: 11-02-4], oleic acid [CAS: 112-80-1], glycerol trioleate [CAS: 122-32-7] and jojoba oil [CAS: 61789-91-1], used to make up artificial sebum, were purchased from Sigma-Aldrich France. Purity of squalene and oleic acid was greater than 98%, and that of glycerol trioleate was given as >55% by the supplier. The artificial sebum formulation used for this study was inspired by the composition of human sebum and by various artificial sebum mixtures described in the literature [START_REF] Stefaniak | Dissolution of materials in artificial skin surface film liquids[END_REF][START_REF] Valiveti | Diffusion properties of model compounds in artificial sebum[END_REF][START_REF] Lu | Comparison of artificial sebum with human and hamster sebum samples[END_REF][START_REF] Wertz | Human synthetic sebum formulation and stability under conditions of use and storage[END_REF][START_REF] Stefaniak | Formulation and stability of a novel artificial sebum under conditions of storage and use[END_REF].

The final mixture contained 15% squalene, 25% jojoba oil, 30% glycerol trioleate, and 30% oleic acid.

Ultrapure water (18.2 MΩ.cm resistivity) was obtained from a water purification device Elga Purelab Classic (Veolia, France). Acetone [CAS: 67-64-1] (purity 99.8%) was purchased from Carlo Erba France.

Sodium acetate trihydrate (purity ≥ 99%), used for HPLC analysis, was provided by Sigma-Aldrich France, whereas sodium chloride (NaCl) 0.9% solution was supplied by Gilbert healthcare. All other reagents and solvents were from Sigma-Aldrich France, and were of the highest purity available.

After preparation, non-radiolabeled BPA and BPS solutions in acetone were stored at 4 °C; preparations in water were stored at room temperature. Working solutions were generally prepared a few hours before application to the skin (or, where appropriate, verified on the day of the experiment). Carbon-14 labeled bisphenol, the prepared unlabeled bisphenol solution and the vehicle (only for sebum deposits) were weighed on a precision balance verified as part of a quality assurance process. Liquid scintillation (Hionic-Fluor TM or Ultima-Flo TM from PerkinElmer USA) was used to determine the radiochemical concentrations in the different samples.

Human skin procurement

Human abdominal skin samples without visible defects were excised from patients undergoing abdominal plastic surgery. Patients provided written informed consent for use of their samples in this study. Samples obtained from six individual female donors were investigated. The volunteers were 28,38,38,41,54 and 64 years old. Immediately following surgery, the excised skin samples were transported refrigerated to the laboratory where they were rinsed with a saline solution (0.9% NaCl).

Adipose tissue was dissected off using a scalpel blade. Split-thickness membranes were prepared (targeted thickness: 500 µm) using an electric dermatome (Zimmer, Etupes, France). Their thickness was measured using a plate thickness gage. The split-thickness skin was used immediately and cut into disks (approximately 7 cm² area).

In vitro experiments

In vitro absorption through human skin was assessed using static Franz diffusion cells (Bercauverre, Lyon, France), with a diffusion area of 1.76 cm² and a receiver volume of 5.6 mL.

Skin samples were placed in diffusion cells with the stratum corneum facing up. The dermis side was in contact with the previously degassed receptor fluid, an RPMI 1640 solution (Gibco, France), supplemented with 0.2% gentamycin, 2.5 % penicillin-streptomycin and 2% bovine serum albumin (BSA). This medium has an osmolality between 270 to 310 mOs/kg H 2 O and a pH between 7 and 7.4.

The receptor fluid was continuously stirred with a Teflon-coated magnetic stirrer. The solubility of BPA and BPS in the RPMI mixture was not rate limiting in this study. Diffusion cells were maintained at 33 °C using a circulating water bath, this resulted in a temperature of 32 °C at the surface of the skin. The integrity of each skin sample was assessed by measuring transepidermal water loss (TEWL) with a Tewameter®TM300 (Courage+Khazaka, Monaderm, Monaco) equipped with a homemade adapter that could be mounted on diffusion cells. TEWL was measured for each sample before initiating exposure, after an equilibrium period of at least 1 h, and was measured again after exposure (except for samples with sebum deposits). Dermatomed skin was considered intact if the TEWL value was less than 10 g.m -2 .h -1 [START_REF] Guth | Suitability of skin integrity tests for dermal absorption studies in vitro[END_REF]. BPA or BPS solubilized in acetone, artificial sebum or ultrapure water (50 μL/cm² equivalent to 20 μg/cm²), corresponding to 88 nmol/cm² for BPA and 80 nmol/cm² for BPS, were applied to human skin samples in the same experimental conditions. The solubility of BPA and BPS in the different donor vehicles (experimental laboratory data) are indicated in table 1.

BPA or BPS application and collected samples

Three vehicles were used to prepare the test solutions. A volume of [ 14 C]-BPA or [ 14 C]-BPS solution in ethanol was weighed. Ethanol was then evaporated to dryness under a stream of air at 37 °C before adding the unlabeled BPA or BPS solutions in acetone or water to obtain the working solutions in acetone and water. The final concentration was 0.4 mg/mL, corresponding to 1.75 µmol/mL for BPA and 1.6 µmol/mL for BPS. Because of the low solubility of the bisphenols in sebum, it was more convenient to solubilize test compounds in acetone and then to evaporate the solvent after addition of the sebum. Thus, for these solutions, after evaporating the ethanol, an unlabeled BPA or BPS Maximum solubility (mg/mL) of Bisphenol A Bisphenol S

In acetone 600 400

In artificial sebum 40 0.7

In ultrapure water 0.38 0.74 solution in acetone was added to dissolve the [ 14 C]-BPA or [ 14 C]-BPS, then the artificial sebum was added. The acetone was finally evaporated under a stream of air at 70 °C; evaporation was controlled until only 2% was left, resulting in a working solution in sebum at 0.4 mg/mL. The BPA or BPS concentration and homogeneity of all unlabeled solutions were confirmed experimentally by HPLC before preparing the working solutions, and each working solution (except that in acetone) was verified after the experiments. The dose applied to skin samples was 20 µg/cm².

A total of 56 human split-thickness skin samples were obtained. After application of the working solution, percutaneous absorption was assessed by collecting 400-µL aliquots of receptor fluid at regular intervals during the exposure period: 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, 24 h, 32 h and 40 h, using an automatic fraction collector (Gilson GX-281, Middleton USA). The volume of receptor fluid was maintained constant by adding the same quantity of fresh receptor fluid to the cells automatically after receptor fluid sampling. At the end of the experiment, the receptor fluid was collected both to assess the absorbed dose of the test compound and to determine the percentage of metabolites. The surface of the skin was rinsed using 2 x 1 mL ethanol, and then dried with a cotton swab. The cell was dismantled and the donor chamber placed in pots containing ethanol/water (50/50). These three samples were analyzed to determine the dislodgeable dose. The skin was removed from the Franz cell, the unexposed skin was cut away from the exposed skin, and both samples were treated separately. The skin was digested in an 80% ethanol solution of KOH (1.5 M) for at least 24 h to obtain a solution of the target analyte.

Liquid scintillation was added to these different samples, and to the receptor fluid aliquots. Then samples were analyzed in a liquid scintillation counter (Tri-Carb 2900TR, Perkin Elmer). Liquid scintillation counting was also used to determine the radiochemical concentrations of triplicate samples of each working solution after application to the skin. The receptor fluid was stored at -20 °C until sample preparation and analysis by HPLC.

HPLC analysis of receptor fluid

Since the percutaneous flux of bisphenol is low, particularly for BPS, the radioactivity of the receptor fluid after 40 h of exposure was very weak. It was therefore necessary to concentrate this medium to detect the potential presence of metabolites. The fluid was first diluted 1:3 in sodium acetate trihydrate buffer 0.1 M pH = 1. This mixture was deposited on activated Sep-Pak® Plus C18 cartridges (Waters), at a maximum of 2 mL medium per cartridge. Cartridges were rinsed with 3 mL water and dried under a vacuum pump. The material retained on the cartridge was eluted with 3 mL methanol, and the eluate was concentrated under air flow at 70 °C. The dry extract was solubilized in acetonitrile/sodium acetate trihydrate buffer 0.01 M pH=2.7 (30/70) prior to centrifugation for 5 min at 14 000 rpm. Supernatant was collected and analyzed by HPLC (Waters alliance modular LC system with in-line UV absorbance detector). For both bisphenols, chromatographic separation involved application of a 2-eluent gradient on a Kinetex C18 column at a flow rate of 1 mL/min and a column temperature of 30 °C. Details of the chromatographic conditions are listed in table 1. The retention time for the peaks observed was compared to the reference time for standard BPA or BPS solutions.

Glossary of terms

Throughout this article, the following terms are used in accordance with the glossary published in OECD Guidance No.28 (OECD, 2004b).

Absorbable dose: "represents the dose which is present on or within the skin after washing"

Absorbed dose (in vitro): "mass of test substance reaching the receptor fluid or systemic circulation within a specified period of time". In this study, if not specified, it corresponds to the sum of the receptor fluid and receptor aliquots.

Dislodgeable dose: "mass of test substance which is removable from the application site". This takes into account the skin wash, the cotton swab and the donor chamber wash.

UV Various statistical procedures were implemented when analyzing results. These procedures were essentially based on analysis of variance (ANOVA). After checking the homogeneity of variance, the effects of skin donor, exposure time, vehicle, and substance were tested. When necessary, a general linear model, including both fixed effects and random effects (skin donor and replicates), was used to take the non-independent nature of data into account, and to test the statistical significance of each factor. Post-hoc analysis was performed by applying the Least Significant Difference (LSD) or Bonferroni methods. The 95% confidence interval was used as an indicator of statistical significance.

Significant variation of TEWL during the exposure using acetone or water as vehicle was assessed using paired-sample t-tests.

Single-factor ANOVA (time) on flux data (followed by LSD post-hoc analysis) was used for each condition (substance/vehicle) to determine the time required to reach a steady state.

Jss (flux at steady state) and, Tlag (lag time) were determined from the slope and the order at the origin of the established regression line [cumul=f(time)] using a least-squares regression procedure over the steady state period determined. Assay linearity was tested by ANOVA with Lack of Fit.

-Results

Mass balance

At the end of the exposure, mass balances were calculated by summing the radioactivity counts in the different compartments. Mean recovery rates for BPA deposited in acetone, sebum and water were 99%, 98% and 98% of the applied dose, respectively. Mean recovery rates for BPS deposited in acetone, sebum and water were similar, at 98%, 98% and 94% of the applied dose, respectively.

Absorption fluxes over time

All the TEWL values measured 40 h after exposure were less than 10 g.m -2 .h -1 ; these values were not significantly different from those measured before exposure (data not shown). The mean thickness of dermatomed skin sections was 476 ± 56 µm (n=56). In vitro percutaneous absorption flux of diluted BPA in acetone, artificial sebum and ultrapure water were measured as a function of time for solutions applied at 20 µg/cm² (Fig. 1).

A: B:

C: Although highly variable, statistical analysis of the BPS flux in acetone and in water revealed the steady state (from 1 or 8 hours, respectively). Jss were strikingly low, close to the ng/cm²/h with water and even lower with acetone.

BPS in water

With BPS in sebum, the flux increased gradually over the period of exposure without reaching a plateau. From a statistical point of view, the steady state could be assumed to be achieved from 24 h. At 40 h, the flux for BPS in sebum was 10-to 25-fold the flux measured in acetone and water, respectively.

Whatever the vehicle, the flux was always higher for BPA than for BPS; this was particularly notable when the test compounds were dissolved in acetone and water.

Cumulative absorbed dose

The cumulative absorbed dose corresponding to various fluxes were plotted (Fig. 3) and specific values are reported in table 3 for three different times: 8 h, 24 h and 40 h. This analysis clearly reveals two kinds of cumulative absorbed dose profiles. The profile in Fig. 3C presents an S-shaped curve with an inflection point at 8 h. All the other profiles showed an increase in bisphenol cumulative absorption that fit quadratic functions (cumulative amount = (a + b × time)²) throughout the exposure. Cumulative absorbed doses of BPA measured with water were significantly higher than those measured in acetone or sebum (from 7 to almost 180 times higher, depending on the conditions). Cumulative absorptions of BPA with acetone were also statistically significantly higher than those in sebum at all times. Compared to BPA, the cumulative absorption for BPS in acetone was 16 to 103 times lower (the ratio increased with exposure time); the BPA/BPS ratio with water as vehicle varied between 200 and 500. Although the difference between the compounds in sebum was lower BPS in water than that measured in acetone or water, cumulative absorption of BPA was statistically significantly higher from 8 h. Table 3 Mean cumulative absorbed dose (±SD) for BPA or BPS at 8 h, 24 h or 40 h exposure

Absorption parameters

For each condition, the type of scenario considered (finite or infinite dose), when the steady state was reached, and the associated parameters were determined (table 4). Table 4 Percutaneous absorption flux at steady state (Jss), Tlag and permeability coefficient (Kp) for infinite dose scenarios and percutaneous peak flux (J peak ) and peak time (t peak ) for the finite dose scenario had consequently a similar ratio. The two Tlag were similar, at 10 and 12 h. The fluxes measured at steady state were particularly low for BPS in water and acetone, at 1 ng/cm²/h and 0.3 ng/cm²/h, respectively. As a result, the permeability coefficients were also very low in these conditions (2.6 E-06 and 6.7 E-07, respectively). However, the two Tlag were very short, at less than 1 hour. In sebum, the BPS flux at steady state was 10-to 35-fold higher than the fluxes measured with water and acetone, respectively, and the Tlag was longer, at around 16 h.

14 C-BPA and 14 C-BPS distributions

Recovery in the three compartments: absorbed dose, exposed skin and dislodgeable dose, was determined after 40 h exposure as a percentage of the dose applied (Fig. 4.). For BPA, the absorbed dose was much higher when the vehicle was water (41%) compared to acetone (6%) and sebum (3%). This result is consistent with the respective flux and cumulative values. The proportion of product detected in the skin was very low with sebum (3%) compared to those measured when the compound was in acetone (24%) or water (27%). Except with water, the dislodgeable dose corresponded to the majority of the BPA applied. For BPS, the absorbed doses were very low in all cases (1% or less). In contrast, the proportion within the skin was high, at 20% and 27% for acetone absorbed dose exposed skin dislodgeable dose and sebum, respectively, and even 47% for water. Like for BPA, the majority of the BPS applied was present in the dislodgeable dose with acetone and sebum. With water, the proportion of BPS was equivalent in the dislodgeable dose and in the skin.

Whatever the selected vehicle, the absorbed dose was much higher for BPA than for BPS. The proportion of BPA and BPS in skin was equivalent when the vehicle was acetone; by contrast there was a considerable difference when comparing results with sebum: only 3% of the applied BPA was in the skin, whereas 27% of the BPS dose applied was detected in the skin.

Metabolism of BPA and BPS in the receptor fluid

Metabolism was investigated in most receptor fluids except those containing the lowest radioactivity levels, since even with the concentration steps the metabolites' concentrations were too low to be detected. Metabolism of BPA and BPS are reported in table 5, expressed as the percentage of radioactivity corresponding to non-metabolized bisphenol in the receptor fluid.

Table 5 Mean percentage of non-metabolized BPA or BPS (±SD) in receptor fluid after 40 h exposure Whatever the vehicle, the absorbed dose was mostly composed of non-metabolized bisphenol for both BPA and BPS. The proportion of metabolites of BPA and BPS was similar when sebum was used as the vehicle; it was significantly higher for BPS in acetone or water.

-Discussion

In this study, we performed a comparative investigation of the percutaneous absorption of BPA and its substitute BPS using an in vitro assay with human skin samples. The vehicles and the dose used for the in vitro experiments were chosen to get as close as possible to occupational exposure conditions. acetone

94.2 ± 2.0 90.5 ± 2.7 96.1 ± 0.9 84.7 ± 10.4 92.8 ± 3.5 88.2 ± 6.7

Non-metabolized BPA fraction (%) Non-metabolized BPS fraction (%)

Acetone, artificial sebum and ultrapure water were selected as vehicles to investigate three realistic exposure scenarios. Acetone evaporates rapidly and was thus used to mimic exposure to neat solid BPS or BPA. Artificial sebum was used to imitate human sebum, whereas water mimics exposure through moistened fingers. In terms of dose, the transfer of BPA to fingertips was has been quantified

after touching the BPA-containing side of a thermal paper for 30 seconds with the forefinger and middle finger [START_REF] Biedermann | Transfer of bisphenol A from thermal printer paper to the skin[END_REF]. That report indicated an average of 1.13 μg BPA transferred to a finger pad. The amount transferred increased up to 10-fold when the fingers were humid or greasy (maximum 23 μg/finger). As a worker may touch a receipt with all five finger pads of one hand, a total of 5.5 to 115 μg BPA could be transferred to the skin. Based on a skin contact area of 6 cm², this amount of BPA represents a dose of approximately 1 to 19 µg/cm², which is close to the surface density of 20 µg/cm² applied in the present study.

It has been verified that the absorption of bisphenols was not limited by the solubility in the receptor fluids. Indeed the maximum solubility is 1.3 and 3.7 mg/mL respectively for BPA and BPS (experimental determination, data not shown). In the worst case, if the total applied amount of BPA or BPS was found in the receptor fluid as non metabolized bisphenol, its concentration would be only around 6 µg/mL.

In the present study, the concentration of the permeant is necessarily well below 10% of its saturation solubility in the receptor medium.

Depending on the vehicle, a 20 µg/cm² dose of BPA can be either a finite or an infinite dose, determined by whether it became depleted or not in the donor compartment. As shown in figure 1 C (shape and values) and confirmed by the very low dislodgeable dose (28% of the applied dose) at the end of exposure, when using water as vehicle, BPA was depleted in the donor compartment. Thus, the corresponding cumulative absorption profile (Fig. 3.C) exhibited a characteristic shape for a finite dose, whereas the other absorption profiles were characteristic of an infinite dose [START_REF] Selzer | Finite and infinite dosing: difficulties in measurements, evaluations and predictions[END_REF]. In the present study, for a given substance (BPA) and a given amount of substance applied to the skin (20 µg/cm² ), the two types of scenario, finite or infinite dose, occurred depending on the vehicle tested. For BPS, the three cumulative absorption profiles (Fig. 3. D, E and F) were characteristic of an infinite dose. Indeed the applied dose was so high or more probably the percutaneous flux was so low.

Taking these data together, percutaneous absorption of BPS was low and, except in the case of sebum, much lower than absorption of BPA (more than 100-fold less with water and acetone at 40 h). The epidermal barrier function is mainly provided by the stratum corneum, which consists of corneocytes embedded in a lipid matrix. The low predicted lipophilicity of BPS (log Kow: 1.2) compared to BPA (log Kow: 3.3) could explain its lower percutaneous absorption.

For both BPA and BPS, the majority of the applied dose remained in the dislodgeable dose at the end of the experiment, except when the vehicle was water. Indeed for BPS, (in water), the percentage of applied dose was distributed evenly between the dislodgeable dose and the skin. When BPA was applied in water, a large proportion (41% of the applied dose) was absorbed and the finite dose condition was observed. The results obtained for BPA in water stand out from the other results.

Although this is probably not the only explanation, the difference of BPA absorption obtained with water and sebum is consistent with the difference of solubility in these two vehicles (Table 1), since in aqueous solutions the tendency of lipophilic substances to move to the more lipophilic SC will increase which in turn increases their absorption. These findings stress once again that the choice of vehicle when assessing a substances' skin permeability is a non-negligible criterion, and that using water as default vehicle could lead to results which may be very different to those obtained with other vehicles.

Depending on the vehicle, the proportions of BPA and BPS in the skin were either similar, as with acetone; or very different, as with sebum, with which 3% and 27% of the applied doses of BPA and BPS were found in the skin, respectively.

The amount of BPS found in the receptor fluid (absorbed substance), considered to be systematically available, was consistently low, at 1% or less. However, the proportion of BPS present in the treated skin at the end of the experiment was far from negligible since it represented between 20% and 47%

of the dose applied. The amount of substance present in the skin after washing is considered absorbable or absorbed as it can be released from the skin at a later time, and is thus available for systemic absorption. Consequently, it must be included in the total amount absorbed (OECD, 2004a;OECD, 2004b;SCCS, 2010;EFSA, 2012). However, debate is ongoing as to whether or not skin levels of a substance remaining in the stratum corneum following an in vitro study should be totally or partially included in or excluded from the overall estimation of absorbed material.

In the absence of consensus, analysis of the individual skin layers -stratum corneum (with separate tape stripping analysis), viable epidermis, and dermis -would allow comparison of the results, and could be used to produce a concentration-depth profile of permeant at the different levels.

Nevertheless, it would be necessary to agree on the methods used to separate the layers and perform tape stripping, and to be sure that analysis is not impacted by surface contamination due to substantivity and binding of compounds to the skin [START_REF] Law | Twenty Clinically Pertinent Factors/Observations for Percutaneous Absorption in Humans[END_REF]. Here, the distribution in the various skin layers was not assessed.

From a prevention perspective, we will consider that the large proportion of BPS contained in the skin could diffuse through the skin and enter the blood stream. Consequently, occupational exposure following dermal contact with BPS-containing thermal receipts cannot be ruled out. Furthermore, dermal exposure of till operators to BPS was highlighted in biomonitoring studies [START_REF] Thayer | Bisphenol A, Bisphenol S, and 4-Hydroxyphenyl 4-Isoprooxyphenylsulfone (BPSIP) in Urine and Blood of Cashiers[END_REF][START_REF] Ndaw | Occupational exposure of cashiers to bisphenol S via thermal paper[END_REF]. This exposure is all the more worrying since the toxicological properties of BPS have yet to be fully evaluated, and it is not clear if adverse health effects linked to BPS are lower than those associated with BPA.

Some previous studies reported in vitro percutaneous absorption of BPA [START_REF] Morck | Placental transport and in vitro effects of Bisphenol A[END_REF][START_REF] Marquet | In vivo and ex vivo percutaneous absorption of [14C]-bisphenol A in rats: a possible extrapolation to human absorption?[END_REF][START_REF] Zalko | Viable skin efficiently absorbs and metabolizes bisphenol A[END_REF][START_REF] Demierre | Dermal penetration of bisphenol A in human skin contributes marginally to total exposure[END_REF][START_REF] Toner | In vitro percutaneous absorption and metabolism of Bisphenol A (BPA) through fresh human skin[END_REF][START_REF] Liu | Comparison of Bisphenol A and Bisphenol S Percutaneous Absorption and Biotransformation[END_REF]. However, it is not easy to compare the BPA data presented here with other published data because of numerous differences in study design: the origin or the characteristics of the skin used, the vehicles, the conditions in which the substance was applied, etc. Nevertheless, the Kp values obtained here for BPA (4.7 E-05 cm/h and 9.3 E-05 cm/h) were of the same order of magnitude as three values previously reported in the literature for this substance: 1.1 E-05 cm/h, 3 E-05 cm/h and 11 E-05 cm/h. A fourth Kp was much lower, at 2.8 E-03 cm/h (EFSA, 2015;[START_REF] Reale | Influence of experimental parameters on Bisphenol A skin permeation kinetics[END_REF]. The only other study investigating in vitro percutaneous absorption of BPS compared to BPA [START_REF] Liu | Comparison of Bisphenol A and Bisphenol S Percutaneous Absorption and Biotransformation[END_REF], was not performed following the OECD guidelines (OECD, 2004b): in particular 6-well plates were used instead of diffusion cells, a reconstructed human epidermis was tested instead of real human skin, and the two substances of interest, BPA and BPS, were applied simultaneously. Despite these limitations, and the numerous differences in study design, these authors also observed that in vitro percutaneous absorption of BPS was much lower than BPA absorption, and low proportions of conjugated metabolites were detected in the receptor fluid. In contrast, the percentage of BPA or BPS recovered in the reconstructed human epidermis after exposure to aqueous BPA and BPS solutions for 25 h was similar for both compounds (14-17%), whereas we found a higher percentage of both compounds in the skin, that was distinct for each substance (27% for BPA or 47% for BPS). This difference may be explained by the absence of dermis in the reconstructed skin samples, but it could also be due to other differences in materials and methods.

Most in vitro percutaneous absorption studies performed on human skin are conducted on abdominal skins. However, in the case of occupational exposure to BPA or BPS in till operators, it is generally skin on the palm (and fingers) that is exposed. The much thicker stratum corneum layer on the palm (400 µm) compared to on the abdomen (15 µm) could represent a greater barrier, and could thus reduce the absorption of chemicals. In vivo experiments on volunteers were carried out to assess percutaneous absorption of two lipophilic substances: the pesticides parathion (log Kow: 3.8, MW:

291 g/mol) and malathion (log Kow: 2.4, MW: 330 g/mol) [START_REF] Maibach | Regional variation in percutaneous penetration in man[END_REF]. Results show that absorption was higher through the abdominal skin than through the skin on the palm, but unexpectedly, for both compounds, there was less than a two-fold difference between the absorbed doses recovered in the urine (expressed as a percentage of the applied dose). Thus, for these two lipophilic compounds, the thickness of the stratum corneum only slightly influenced their absorption, unless the difference in thickness was compensated by an unknown phenomenon. In any case, the use of abdominal skins to predict the absorption of a substance following contact with the palm does not seem to be nonsensical given the experimental uncertainties, and in addition, it is protective for the workers.

Our results indicate that the absorbed dose mostly corresponds to unmetabolized BPA and BPS.

However, the method used in this study has limitations, since it could only be used to analyze receptor fluid samples, and even with the concentration steps, the metabolite concentrations were too low to be detectable in some samples. Further experiments and analytical developments will be necessary to better characterize the skin's metabolic activity toward BPS.

-Conclusions

Overall, in the present study, percutaneous absorption of BPS measured in the receptor fluid was low, whatever the vehicle used. BPS absorption was much lower than BPA absorption, with at least a 100fold difference when the vehicle used was acetone or water. With sebum, BPA absorption was also higher than BPS absorption, although there was only a 2-fold difference. However, a large amount of BPS (20 to 47% of the applied dose) remained in the skin at the end of the experiment and must be (at least partly) taken into account when calculating the overall absorbed dose. From a prevention perspective, potential exposure during dermal contact with BPS cannot be excluded. This potential exposure is all the more worrying since today, we cannot state that BPS is less toxic than BPA. Indeed, very few studies have yet been performed to determine the human health effects of this molecule.

Moreover, to assess the overall toxicity of BPS absorbed through the skin, it would be interesting to identify the BPS metabolites, even if they account for less than 15% of the absorbed dose. Finally, this study highlights the influence of the vehicle on BPA and BPS absorption. For in vitro experiments, the choice of vehicle must be carefully considered in order to reflect the targeted occupational exposure situation as closely as possible.

-Acknowledgments

Gradient 0 -

 0 10 min: A/B= 70/30 10-10.5 min: A/B = 60/40 10.5-16.5 min: A/B= 0/100 16.5-20 min: A/B= 70/30 0-8 min: A/B= 85/15 8-8.5 min: A/B = 65/35 8.5-13.5 min: A/B= 0/100 13.5-20 min: A/B= 85/15 t R : retention timeAs the in-line radio-detection detector was insufficiently sensitive, the concentrated medium had to be analyzed in two steps. Fractions were collected every 30 s over the entire duration of the HPLC analysis. These fractions were then individually counted by liquid scintillation. The radioactivity detected in the fluid corresponds to the bisphenol which has been absorbed in the receptor fluid, as non-metabolized or metabolized bisphenol. Therefore radioactive peaks detected at retention times other than 7 min or 7.3 min (BPA and BPS, respectively) indicated the presence of metabolites. This method was used to determine the percentage of radioactivity corresponding to metabolized and nonmetabolized bisphenol in the receptor fluid.

Fig. 1 .

 1 Fig.1. Skin permeation flux profiles for bisphenol A following the topical application of 20 µg/cm²

Fig. 3 .

 3 Fig.3. Cumulative absorbed dose over time for BPA or BPS following the topical application of

Fig. 4 .

 4 Fig.4. Distribution of dose recovered after 40 h of topical exposure to 20 µg/cm² of [ 14 C]-bisphenol A or [ 14 C]-bisphenol S applied to human split-thickness skins.

Table 1 :

 1 Solubility of BPA and BPS in different donor vehicles (experimental laboratory data)

Table 2

 2 Chromatographic conditions for analysis of BPA or BPS and their metabolites in receptor fluid.

		BPA	BPS
	Eluents	A: sodium acetate trihydrate	A: sodium acetate trihydrate
		buffer 0.01 M pH=2.7	buffer 0.01 M pH=2.7
		B: acetonitrile	B: acetonitrile

  Finite dose: "amount of test preparation applied to the skin where a maximum absorption rate (noted J peak ) of the test substance may be achieved during a certain time interval (t peak ) but is not

	detection (nm)	220	260
	t R (min)	7	7.3
	maintained."
	Flux or absorption rate: "mass of a test substance passing through a unit area of skin per unit of
	time" (in µg/cm²/h). Under steady state conditions, it is noted Jss.
	Lag time or Tlag: "derived from a graph of cumulative absorbed dose and time. Intercept of the
	tangent of the linear part of the absorption profile with the x-axis."

Permeability coefficient (Kp): "a value, in units of cm/h, that represents the rate at which a chemical penetrates the skin. This is calculated from the flux divided by the applied concentration."

Steady state: "the part of an absorption profile where the absorption rate remains constant."

2.8. Statistics

All statistical analyses were performed using Statgraphics Centurion XVIII Software (Version 18.1.06) (StatPoint Technologies, Inc., Warrenton, VA, USA).