Food and Chemical Toxicology 59 (2013) 754–765

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Food and Chemical Toxicology

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Dermal absorption and hydrolysis of methylparaben in different vehiclesthrough intact and damaged skin: Using a pig-ear model in vitro

0278-6915/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2013.07.025

Abbreviations: AD, applied dose; ANOVA, one-way analysis of variance; BP,butylparaben; BLOQ, below the limit of quantification; CIR, US Cosmetic IngredientReview; E, enhancer; EP, ethylparaben; FDA, Food and Drug Administration; FTS,full-thickness skin membrane; Jss, steady state flux; LOD, limit of detection; LOQ,limit of quantification; OECD, Organization for Economic Co-operation and Devel-opment; MP, methylparaben; PG, propylene glycol; PHBA, p-hydroxybenzoic acid;RF, receptor fluid; SC, stratum corneum; P, permeability coefficient; PP, propylpar-aben; SCCS, EU Scientific Committee on Consumer Safety; SD, standard deviation;SED, systemic exposure dosage; TC, Transcutol� CG; TEC, transcutaneous electricalconductivity; unmMP, unmetabolised MP; UR, urea.⇑ Corresponding author.

E-mail address: jarmila.hojerova@stuba.sk (J. Hojerová).

Silvia Pazoureková a, Jarmila Hojerová a,⇑, Zuzana Klimová a, Marianna Lucová b

a Slovak University of Technology, Faculty of Chemical and Food Technology, Laboratories of Cosmetology, Bratislava, Slovak Republicb Slovak Academy of Sciences, Institute of Experimental Pharmacology & Toxicology, Bratislava, Slovak Republic

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 April 2013Accepted 10 July 2013Available online 17 July 2013

Keywords:MetylparabenIn vitro dermal absorptionHydrolysisMetabolismIntact and damaged pig-ear skinPermeation enhancer

Currently, there is a trend to reduce of parabens use due to concern about the safety of their unmetab-olised forms. This paper focused on dermal absorption rate and effectiveness of first-pass biotransforma-tion of methylparaben (MP) under in-use conditions of skincare products. 24-h exposure of previouslyfrozen intact and tapestripped (20 strips) pig-ear skin to nine vehicles containing 0.1% MP (AD, applieddose of 10 lg/cm2), resulted in 2.0–5.8%AD and 2.9–7.6%AD of unmetabolised MP, and 37.0–73.0%ADand 56.0–95.0%AD of p-hydroxybenzoic acid, respectively, in the receptor fluid. The absorption rate ofMP was higher from emulsions than from hydrogels, from enhancer-containing vehicles than fromenhancer-free vehicles, and when skin was damaged. Experiments confirmed that the freezing of pig-ear skin slightly reduces hydrolysis of MP.After 4-h exposure of intact freshly excised and intact frozenstored skin, amount of <LOQ-2.3%AD and 2.3–3.3%AD unmetabolised MP, respectively, were found inthe receptor fluid. Taking into account the number of useful properties of MP, but also the potential ofsystemic availability of unmetabolised MP, we consider that MP is more suitable for preserving rinse-off topical products than for leave-on products. Risk of systemic absorption of parabens should also beexplored via the skin with damaged barrier.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Roughly 70 years, the parabens, alkyl esters of p-hydroxybenzo-ic acid (PHBA), were considered to be substances having low toxic-ity and some other properties of ideal preservatives. Parabenspossess a broad spectrum of antimicrobial activity and water/oilsolubility, excellent stability over a wide pH range, and low price(Soni et al., 2002, 2005; CIR, 2008). Furthermore, products contain-ing parabens can be autoclaved. Therefore, either alone or in com-bination with other preservatives, parabens are used in a widerange of cosmetic, pharmaceutical, and partially also foodproducts.

In our market survey conducted in 2011–2012 in the SlovakRepublic (SR), according to the ingredients listed on the label,among 430 evaluated leave-on cosmetics at least one parabenwas found in 39% products. Parabens were most frequently presentin emulsion such as body-care and sunscreen creams and milks(including products intended for baby), hydrogels, lotions, andmakeups. Among 159 topical medications registered in the SR, atleast one paraben was listed in 10% of prescription and 5% ofover-the-counter drugs. Methylparaben (MP) was present in 98%and 88%, propylparaben (PP) in 67% and 50%, respectively of para-ben-positive cosmetics and medicines, while ethylparaben (EP)and butylparaben (BP) were listed sporadically only (Hojerováet al., 2013).

At recent years, the safety of parabens has become question-able. However, studies investigating the health effects of parabensare conflicting. Using a wide variety of assay systems in vitro andin vivo, a large number studies have demonstrated, that parabensmay affect human health due to their endocrine disrupting activity(Routledge et al., 1998; Byford et al., 2002; Lemini et al., 2003;Pugazhendhi et al., 2005; Akomeah et al., 2007; Prusakiewiczet al., 2007; Darbre and Harvey, 2008; Boberg et al., 2010; Voet al., 2010, 2011; Hu et al., 2013). Other researchers (vanMeeuwen et al., 2008; Shaw and deCatanzaro, 2009; Witorschand Thomas, 2010; Sciali, 2011; Aubert et al., 2012; Kirchhof and

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de Gannes, 2013) have published a radical opposition toconcernsabout health risks from doses of parabens commonly used. In2004 Darbre’s team (Darbre et al., 2004; Harvey and Darbre,2004) firstly measured trace residues (nanograms/g of tissue) of in-tact parabens, particularly MP, in human breast cancer tissues, andsuggested that their presence in the human body might originatefrom topical application of body-care cosmetics such as under-arm deodorants and antiperspirants. Although some public healthauthorities (SCCP, 2005a,b, 2011a,b; FDA, 2007) and some cancerexperts (Gikas et al., 2004; Rageth, 2005; CIR, 2008; Namer et al.,2008) rejected these considerations, the study has sparked contro-versy and also stimulated new international research on parabenseffects on human health.

The Food and Drug Administration (FDA) in the USA, as well asthe European Scientific Committee on Consumer Safety (SCCS) re-opened the safety assessment for parabens, to request exposureestimates and risk assessment for cosmetic use. According to theopinion of the SCCS (2011a), parabens may really exert a weakestrogen-like activity but its potency is from 1000 (for MP) to1,000,000 (for BP) times below the potency of the positive control17b-estradiol. So the SCCS (2011a, 2013) considers that for generalcosmetic products containing parabens, excluding specific prod-ucts for the nappy area, there is no safety concern in children(any age group) and adult consumers. Regarding personal careproducts, FDA (2007) states parabens safe at concentrations up to0.8% (mixtures of parabens) or up to 0.4%(single paraben). TheSCCS recognises a mixture of parabens safe also at concentrationsup to 0.8%, MP and EP at concentrations up to 0.4% (single para-ben), but PP and BP up only to 0.19% individually or in combination(SCCS, 2011a,b, 2013).

However, general view on the safety of parabens is based on theassumption that the ester bonds in the parent compounds arequickly and nearly completely hydrolysed by carboxylesterases(EC 3.1.1.1) in the common metabolite, a non-specific PHBA (Soniet al., 2005; Boberg et al., 2010). Since PHBA is considered to bea compound without an endocrine effects (SCCS, 2011), carboxy-lesterases activity appears to be crucial for detoxification parabens.Several studies (Harville et al., 2007; Janjua et al., 2008; Boberget al., 2010; Shirai et al., 2013) confirmed that orally administeredparabens are indeed readily metabolised by carboxylesterases inthe intestines and liver and then excreted without significant accu-mulation in the body. However, metabolism of parabensadminis-tered dermally may be incomplete for some reasons. The mainreason is a lower capacity of skin carboxylesterases compared tomammalian liver carboxylesterases (Harville et al., 2007; Prus-akiewicz et al., 2007). Another reason may be the negative effectof skin esterase inhibitor (Bando et al., 1997; Seko et al., 1999;Prusakiewicz et al., 2007; Harville et al., 2007; Jewell et al.,2007a,b), long-term use of a wide range of paraben-positive topicalpreparations, as well as inter-individual variations of human skin(Darbre et al., 2004; Harvey and Darbre, 2004; Ishiwatari et al.,2007; Darbre and Harvey, 2008). So it is generally accepted thatunmetabolised (intact) forms of parabens in the body tissues aremore likely the result of dermal applications than oraladministra-tion (Oh et al., 2002; Prusakiewicz et al., 2007; El Hussein et al.,2007; Harville et al., 2007; Janjua et al., 2007; Darbre and Harvey,2008; Williams, 2008; Barr et al., 2012; Shirai et al., 2013).

Considerable number of studies through intact human and var-ious animal skin in vivo and in vitro have documented manyparameters influencing the overall dermal absorption rate of para-bens, i.e. without detection of individual quantities of unmetabo-lised parabens and their metabolite, PHBA (Pozzo and Pastori,1996; Kitagawa et al., 1997; Oh et al., 2002; El Hussein et al.,2007; Janjua et al., 2007, 2008; Jewell et al., 2007a; Mbah, 2007;Pedersen et al., 2007; Wilkinson et al., 2007; Caon et al., 2010;Romonchuk and Bunge, 2010). Unfortunately, the available studies

on the degree of hydrolysis of parabens during percutaneous per-meation are limited. Bando et al. (1997) reported that after appli-cation of BP and PP to the rat skin in vitro, about 4% of intact BPand about 30% of intact PP fromthe total permeants in the receptorfluid was detected. Ishiwatari et al. (2007) evaluated the influenceof daily exposure to MP containing formulations to human skin. At1 h after a single application of 0.15% MP in emulsion to the fore-arm of human volunteers, unmetabolised MP (unmMP) concentra-tions about 18% of the application quantity of the parent MP werefound in the stratum corneum (SC), but after 12 h the concentra-tion of unmMP was decreased at 10 pmol/cm2 (approximately0.028%). However, the authors confirmed that repeated applicationof MP containing topical products significantly increases theamount of unmMP in the SC. The same researchers studied alsothe metabolism of MP through Yucatan micropig skin in vitro. 2-h exposure to an aqueous solution (10 lg/cm2) containing 0.1%of MP resulted in 2.06 mgof unmMP and 0.36 mg of PHBA ex-pressed to 1 g tissue of full-thickness skin (Ishiwatari et al.,2007). Aubert et al. (2012) measured the content of unmMP in ex-cretes from rat following dermal application for 6 h at a dose of100 mg/kg of MP. No parent ester, only metabolite PHBA in the ur-ine and feces (14–27% and <2% of the applied dose, respectively)was determined.

According to Aubert et al. (2012) in line with our view, the piv-otal question of the safety assessment of parabens-containing top-ical products is the fate after human skin exposure, (a) their dermalabsorption rate and (b) whether they absorbed intact or after first-pass hydrolysis in the skin. So the first aim of this study was to as-sess systemic exposure of unmMP and its main metabolite PHBA asa result of single topical application of different MP-containingproducts to ex-vivo intact skin (frozen prior to the experiments).Because of anatomical, physiological and biochemical similarityto human skin (Sekkat et al., 2002; Singh et al., 2002; Godin andTouitou, 2007; Jacobi et al., 2007; Klang et al., 2012; Lau et al.,2012), excised pig-ear skin as a skin model was chosen.

When investigating dermal absorption values of substances,risk assessment is generally focused on intact skin. The OECD(2004), as well as the SCCS (2010) guidelines for studies onin vitro dermal absorption prescribe also optimal barrier integrityof the excised skin. Such membrane is suitable to predict dermalabsorption values through intact skin but may not be relevant forsituation, where the topical product is applied to barrier-impairedskin due to mechanical, physical, chemical or biological reasons.Several studies (Jacobi et al., 2007; Lademann et al., 2009; Weig-mann et al., 2009; Klang et al., 2012, 2013) have shown thatstripped ex-vivo pig-ear skin is very representative to the in vivohuman skin with barrier impaired due to mechanical trauma. Toour knowledge, skinbarrier damage due to stripping in relation tothe permeation of parabens has not been studied yet. Therefore,the second aim of this study was to assess the same objectives asin the first aim, but through stripped skin (frozen prior to theexperiments).

Conflicting reports concerning the effect of storage conditions ofthe skin on percutaneous absorption of chemicals and degree ofhydrolysis of ester bonds are published. While several reports sug-gest that the freezing of the skin alters the permeability of certaincompounds (Hadzija et al., 1992; Shaikh et al., 1996; Wester et al.,1998; Ahlstrom et al., 2007; Payne et al., 2013), Harrison et al.(1984) has shown that the permeability of human skin ex-vivo isno significantly affected after prolonged freezing at �20 �C up to466 days. Two studies have demonstrated reduced activity ofnon-specific esterases in the suspension of frozen stored (at�20 �C) pig ear skin towards retinyl ascorbate (Abdulmajed et al.,2006) and acetylsalicylate (Lau et al., 2012) compared to freshlyexcised pig-ear skin. Conversely, a number of studies have shownfor snake, human, rat, rabbit, guinea-pig, and mouse skin, that

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esterase activity can be almost or absolutely completely preservedfor frozen skin stored at �20 �C (Nghiem and Higuchi, 1988; He-witt et al., 2000; Beydon et al., 2010), and even at �70 �C (Jewellet al., 2007a) and at �80 �C (Stinchcomb et al., 2002). So the thirdaim of this study was to clarify whether storage by freezing of thepig-ear skin affects dermal absorption rate and hydrolysis degreeof MP.

2. Materials and methods

2.1. Chemicals

MP and PHBA (Table 1), both analytical-grade of P99%, were supplied by Sig-ma–Aldrich Chemie (Deisenhofen, Germany). Ingredients for cosmetic vehicleswere received from local cosmetic producers. Water, acetonitrile and methanol(all HPLC grade) were supplied from Fisher Scientific (Loughborough, UK). Transcu-tol� CG (CAS 111-90-0) from Gattefossè (Saint Priest, France), urea (CAS 57-13-6),propylene glycol (CAS 57-55-6), and all other chemicals from Mikrochem (Pezinok,Slovak Republic) were of reagent grade. The receptor fluid consisted from phos-phate-buffered saline (pH 7.4, own preparation) and 0.01% of Gentamicin-sulphate(Lek-Sandoz, Ljubljana, Slovenia) added for stabilization of the pig membranes.

2.2. Vehicles with or without penetration enhancers

Because the most common paraben in our survey, for dermal absorption studymethylparaben was used. Nine formulations, representing the most frequentlytypes of MP-containing topical leave-on products, were prepared according to thecomposition showed in Table 2. Each formulation contained MP at the target con-centration of 0.1% (w/w), i.e. within the range of its using (a) in the skin-care cos-metics (0.1%, El Hussein et al., 2007; 0.007–0.409%, Eriksson et al., 2008; 0.03–0.3%, our unpublished results), (b) in the topical medications (0.02–0.3%, Soniet al., 2002). Three emulsions oil-in-water and three non-alcoholic hydrogels con-tained also one of the three chemicals known as penetration enhancers; Transcu-tol� CG (TC),urea (UR), and propylene glycol (PG), listed on the label of someproducts in our survey. Fourth emulsion and fourth hydrogel were without an en-hancer. For comparison a simple aqueous solution was prepared.

2.3. Preparation and storage of skin sheets

Fresh ears from 6 months old domestic pigs (Slovak large white) were obtainedfrom a local abattoir immediately post-mortem and prior to steam cleaning. Follow-ing brief cleaning with tap water, the sheet of the full-thickness skin (FTS, consist-ing of the SC, viable epidermis, and dermis) was separated from the underlyingcartilage on the upper half part of ear using a scalpel. Hairs were cropped to a lengthof 3 mm with an electric hair clipper. The FTS sheets with some visible imperfec-tions were excluded. For the experiments referred in the Section 2.8.2, four freshlyexcised FTS sheets were used in the same day and four those (stored at 4 �C for 18 h)in the next day. For all other experiments FTS sheets were wrapped individually inan aluminium foil and stored at �20 �C for up to 6 weeks before use. One hour priorto the experiment,frozen FTS sheet was allowed to thaw at room temperature.

2.4. Tape stripping

The aim of this process was to mimic in vivo conditions of topical medicationsand cosmetic products containing parabens applied to human skin with the SC im-paired due to various mechanical reasons. The previously frozen FTS sheets wereblotted dry with a soft tissue and the thickness was measured at ten different loca-tions of each of them using a micrometre (Digital micrometre SKW 1; Helios Mess-

Table 1Physicochemical characteristics of methylparaben and its metabolite, p-hydroxybenzoic a

INCI namea CASb CIDc MFd MWe (g/mol) Solubil

Methylparaben* 99-76-3 7456 C8H8O3 152.15 2.54-Hydroxybenzoic acid** 99-96-7 135 C7H6O3 138.12 6.0

* IUPAC name: Methyl 4-hydroxybenzoate (Pubchem, 2012).** IUPAC name: 4-Hydroxybenzoic acid (Pubchem, 2012).

a International Nomenclature of Cosmetic Ingredients (EC, 2006).b Chemical Abstracts Service (Pubchem, 2012).c Compound Identification Number – Chemical structure (Pubchem, 2012).d Molecular Formula (Pubchem, 2012).e Molecular Weight (Pubchem, 2012).f Jewell et al. (2007a).g Jouyban (2010).h Partition coefficient n-octanol/water (Jewell et al., 2007a).

technik, Niedernhall, Germany; readability of 1 lm). Only sheets with thicknessfrom 1.020 to 1.120 mm were used for further experiments. Subsequently, everysecond FTS sheet was stripped 20-times with the adhesive tape (3 M Scotch�) bythe same person according to the protocol by Klang et al. (2012). Then the thicknessof FTS sheet was measured again (Table 5). Distribution of corneocytes on 1st, 10th,and 20thtape-stripes (Fig. 1a–c) was examined under the Leica™ DM-1000 micro-scope and representative micrographs taken with a Leica™ digital camera (LeicaMicrosystems, Wetzlar, Germany).

2.5. Diffusion apparatus

The experiments were performed according to the Guideline for the in vitroassessment of dermal absorption of cosmetic ingredients SCCS (2010). Pre-cali-brated static unjacketed Franz-type diffusion cells (JM-Glass, Bratislava, SR) witha receptor chamber volume of 5.5 ± 1 mL and an area of 2.00 cm2 available for dif-fusion were used. The FTS sheet was cut into three circular discs with a diameter ofabout 3.3 cm. The FTS disc was mounted in the diffusion cell with the stratum cor-neum towards the donor chamber and fixed with a metal clip. The receptor cham-bers were filled with degassed receptor fluid. Then the cells were immersed in athermostatic water-bath to maintain the surface temperature of the FTS disc at32 ± 1 �C throughout the experiment. Thesolution in the receptor chamber was con-tinuously agitated at 600 rpm using a small Teflon-coated magnetic bar driven by asubmersible magnetic stirrer (Variomag; Thermo Scientific, Karlsruhe, Germany).After equilibrating for 1 h, the barrier integrity of the FTS disc was checked.

2.6. Skin barrier integrity

The integrity of the skin, crucial for experiment was determined by transdermalelectrical conductivity (TEC) across the FTS under the conditions of the method, asdescribed in our previous papers (Klimová et al., 2012; Lucova et al., 2013). Only FTSdiscs that passed the TEC test (the TEC value of 61.0 mS/cm for intact FTS and 1.5–3.0 mS/cm for FTS after 20 strips) were used for further experiments (Table 3).

2.7. Formulation dosing

Aqueous solution (20 lL) was introduced into the donor chamber using anadjustable micropipette. For dosing emulsions and hydrogels, the cells were dis-mounted. The FTS disc was blotted dry with a soft tissue and placed on an electronicbalance. A few small drops of formulation were deposited on the exposure area,homogeneously spread and accurately weighed to a quantity of 20 ± 0.1 mg. Thedisc was then mounted in the same place of the diffusion cell and fixed with a metalclip. Attention was paid to remove air bubbles. The exact time of application wasnoted (time zero). Then diffusion cell was immersed in a water-bath under theterms described in the Section 2.6. All experiments were conducted under unoc-cluded donor chambers since a vast majority of topical leave-on formulations is ap-plied open to the atmosphere.

2.8. Experimental design

Two sets of experiments were carried out as follows. First set, concerning thefate of MP after exposure of previously frozen intact FTS and stripped FTS, consistedfrom two steps: (a) 24-h kinetic studies of MP and PHBA for nine formulations, and(b) 4-h studies of dermal absorption rate and degree of MP hydrolysis for nine for-mulations and the mass balance of MP for four emulsions. Second set of experi-ments concerning the effect of skin storage by freezing on percutaneousabsorption of MP and degree of its hydrolysis, consisted from one step: 4-h studiesof dermal absorption rate, degree of MP hydrolysis and mass balance of MP for fouremulsions through freshly excised intact FTS and previously frozen intact FTS.

cid.

ityf in water 25 �C (g/l) Solubilityg in methanol 25 �C (g/l) Log Po/wh 25 �C

362.2 1.96340.3 1.56

Table 2Composition of formulations containing methylparaben mimicking leave-on topical preparations without or with chemical enhancers.

Ingredienta Aqueous solution (%, w/w) Hydrogel (%, w/w) Emulsion oil-in-water (%, w/w)

1 without E 2 with UR 3 with TC 4 with PG 1 without E 2 with UR 3 with TC 4 with PG

Methylparaben 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1Aqua q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100Urea – – 5 – – – 5 – –Ethoxydiglycolb – – – 5 – – – 5 –Propylene glycol – – – – 5 – – – 5Olea Europaea oil – – – – – 18 18 18 18Glyceryl stearate – – – – – 5 5 5 5C12-14 Pareth-3 – – – – – 3 3 3 3Cetyl alcohol – – – – – 2 2 2 2Carbomerc – 1 1 1 1 – – – –Sodium hydroxide to pH 5.5 to pH 5.5 to pH 5.5 to pH 5.5 – – – –Lactic acid to pH 5.5 – – – – to pH 5.5 to pH 5.5 to pH 5.5 to pH 5.5

E: enhancer; UR: urea; TC: Transcutol; PG: propylene glycol.a Name by the International Nomenclature of Cosmetic Ingredients (EC, 2006).b Commercial name Transcutol� CG.c Commercial name Carbopol� 940.

Fig. 1. Distribution of corneocytes on the removed tape strips. (A) 1st tape strip, (B) 10th tape strip, and (C) 20th tape strip. Micrographs taken through the Leica DM1000light microscope with a Leica digital camera driven by software Leica Application Suite V3 (both from Leica Microsystems, Wetzlar, Germany).

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2.8.1. Studies through previously frozen intact skin and stripped skin2.8.1.1. 24-h Kinetic studies. Three FTS discs were obtained from one previously fro-zen intact FTS sheet, as well as stripped FTS sheet. To minimize experimental errorsarising from possible variability of FTS quality, one disc for one of the three differentformulations was used in the same experiment. Further procedures were carriedout as described in the Sections 2.5–2.7. Formulation was left in contact with theskin for 24 h. At seven predetermined time intervals (1, 2, 3, 4, 5, 6, and 24 h),50 lL of the receptor chamber content (abbreviated as RF) was sampled and re-placed with the same volume of fresh RF. The sample was immediately assayedfor a concentration of MP and PHBA via HPLC. The process was repeated two more

times in other 2 days, so three cells with previously frozen intact FTS, as well asthree cells with stripped FTS for each formulation were used. The same scheduleof experiments was used for other sixformulations.

2.8.1.2. 4-h Permeation rate and degree of hydrolysis. Based on the results of kineticstudies (see Section 3.1 and Fig. 3), 4-h exposure of the FTS to nine formulationswere chosen for further experiments. Nine diffusion cells exposed to nine differentformulations were investigated in the same experiment. Formulation was left incontact with the skin for 4 h. For Hydrogels 1–4 and the aqueous solution, at the

Table 3The amount of unmetabolised methylparaben (MP) and its metabolite, p-hydroxybenzoic acid (PHBA) found in the receptor fluid (RF) after 24-h permeation through previouslyfrozen intact and stripped full-thickness pig-ear skin (FTS) in each of the nine formulations (10 ± 0.05 mg/cm2) containing 10 ± 0.05 lg/cm2 (0.1%) of the parent MP.

Membrane Permeant Aqueous solution (lg/cm2)

Hydrogel (lg/cm2) Emulsion oil-in-water (lg/cm2)

1 without E 2 with UR 3 with TC 4 with PG 1 without E 2 with UR 3 with TC 4 with PG

Intact FTS PHBA 3.85 ± 0.35 3.70 ± 0.47 4.05 ± 0.35 4.30 ± 0.42 4.15 ± 0.20 5.50 ± 0.45 6.20 ± 0.30 7.30 ± 0.41 6.85 ± 0.45MP 0.20 ± 0.03 0.22 ± 0.02 0.23 ± 0.03 0.26 ± 0.03 0.24 ± 0.02 0.34 ± 0.06 0.40 ± 0.05 0.58 ± 0.08 0.52 ± 0.04UnmMP 4.9% 5.6% 5.4% 5.2% 5.5% 5.8% 6.1% 7.4% 7.1%

StrippedFTS

PHBA 5.67* ± 0.56 5.60* ± 0.65 5.80* ± 0.42 6.60* ± 0.40 6.25* ± 0.45 8.00* ± 0.42 8.75* ± 0.55 9.50* ± 0.40 9.20* ± 0.35

MP 0.29* ± 0.04 0.29* ± 0.02 0.33* ± 0.03 0.39* ± 0.02 0.38* ± 0.04 0.50 ± 0.07 0.64* ± 0.04 0.76* ± 0.07 0.70* ± 0.03UnmMP 4.9% 4.9% 5.4% 5.6% 5.7% 6.3% 6.8% 7.4% 7.1%

E: enhancer; UR: urea (5%, w/w); TC: Transcutol� CG (5%, w/w); PG: propylene glycol (5%, w/w).UnmMP (%): a percentage of unmetabolised MP from the total amounts of permeants (MP + PHBA) in the RF (SD values are not included).Values are the mean ± SD (n = 3); no significant differences were found (p > 0.05).* Denotes the amount of MP or PHBA permeated through previously frozen stripped FTS significantly different (p < 0.05) from the amount of the same compound permeatedthrough previously frozen intact FTS in the RF.

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end of the exposure, only the RF was analysed. For emulsions 1–4, also the massbalance and the total recovery of MP were evaluated. This schedule was repeatedfive times in other days. So, 6 diffusion cells with previously frozen intact FTS, aswell as 6 cells with stripped FTS for each formulation were used.

After 4-h exposure to an emulsion, the skin surface was washed four times with0.5 mL of methanol. The rinsing solutions were collected into a vial, vigorously sha-ken with a vortex for 10 min and centrifuged at 6000 rpm for 10 min. The superna-tant was analysed via HPLC. To elute of MP absorbed in exposed skin area and PHBAincured as hydrolysis result, the FTS disc was cut into very small pieces using a scis-sors, introduced into a vial containing 5 mL of methanol, and subsequently pro-cessed as the rinsing solutions, with the exception of the vortexing time (6 h).The quantities of MP and PHBA in the supernatant were determined via HPLC.The extraction method was verified in blank experiments by spiking the FTS discwith a known amount of MP and PHBA; the total recovery percentages were in-cluded in the interval 96–101%. The RF was analysed via HPLC. Finally,the totalrecovery was calculated.

2.8.2. Studies through previously frozen intact skin and freshly excised intact skinThree FTS discs were obtained from one fresh FTS sheet, as well as from one pre-

viously frozen FTS sheet (stored at �20 �C for 41–42 days) and used for three of fouremulsions tested. For fresh FTS, the schedule was repeated three more times in thesame 4-h permeation experiments. Further procedures were carried out as de-scribed in the Section 2.8.1.2. The next day, the same schedule was repeated. In to-tal, 6 diffusion cells with fresh intact FTS and 2 cells with previously frozen intactFTS for each emulsion were used in the second set of experiments.

2.9. High performance liquid chromatography

HPLC analysis were performed using an Ecom instrument (Prague, CzechRepublic) and a Kromasil C 18 (5 lm) reversed-phase column 250 � 4 mm andVK1040 C 18 (5 lm) precolumn 10 � 4 mm (Macherey–Nagel, Düren, Germany).The UV detector Philips PU-4225 was set at 254 nm. The mobile phase used wasa mixture of acetate buffer and acetonitrile (30:70, v/v) pumped at 0.8 mL/min atroom temperature. Before each HPLC analysis, extraction solutions in methanoland samples of the receptor chamber were filtered through regenerated cellulosemembranes (0.45 lm; Millipore) and injected in the volume of 20 lL. The retentiontimes were 4.1 ± 0.1 min and 11.0 ± 0.1 min for PHBA and MP, respectively for themethanol extracts, and 4.4 ± 0.1 min and 12.1 ± 0.1 min for PHBA and MP, respec-tively for the RF samples (Fig. 2). The limit of detection (LOD) of 0.020 lg/mL and0.330 lg/mL and the limit of quantification (LOQ) of 0.066 lg/mL and 0.990 lg/mL for MP and PHBA, respectively were determined.

2.10. Statistical analysis

The statistical significance of the data (p < 0.05) was computed with a one-wayanalysis of variance (ANOVA) using the software program Microsoft� Office Excel2007 for Windows.

3. Results

The quantities of PHBA and unmMP detected based on theHPLC-calibration curves were recalculated. The precise receptorchamber volume of 5.5 ± 0.1 mL for the RF sample, the volume of5 mL for methanol extract from FTS discs, and the volume of2 mL for methanol extract from unabsorbed emulsion in the calcu-

lation for each analyte were included. To express the amount ofPHBA as a percentage of an applied dose (%AD) of the parent MP,the conversion was done with regard to the molecular weight ofboth compounds according to

PHBA amount as the parent MP

¼ 152:15138:12

� PHBA amount determined ðlg=cm2Þ ð1Þ

3.1. Results of the first set of experiments

3.1.1. 24-h Kinetic studies (Fig. 3, Tables 3 and 4)The total amounts of PHBA and unmMP after 24-h permeation

through previously frozen intact and stripped FTS in the RF fromnine vehicles are summarised in Table 3. The amount of MP andPHBA in the RF, which permeated the skin per unit surface areaduring 24 h, was plotted against time. Four extreme permeationprofiles of them are shown in Fig. 3. Among hydrogels, the lowestpermeation rate was observed for Hydrogel 1 (without E) and thehighest for Hydrogel 3 (with TC), among emulsions the lowest per-meation rate was found for Emulsion 1 (without E) and the abso-lutely highest rate for Emulsion 3 (with TC).

To illustrate, the permeability parameters for Emulsion 3 weredetermined (Table 4). The lag time and the steady state flux (Jss)were calculated by linear regression from the plot using the Table-Curve 2D software program for Windows. The permeability coeffi-cient (P) was calculated according to

P ¼ Jss

Cð2Þ

where P is permeation coefficient (cm/h �103), Jss is Flux steadystate (lg/cm2 x h), and C is Concentration of the parent MP addedto the donor compartment (mg/cm3).

Since the concentration of PHBA P LOQ in the RF was detect-able first time after 4-h skin exposure to Hydrogel 1 (Fig. 3), for fur-ther experiments just this 4-h exposure was chosen.

3.1.2. 4-h Permeation rate and degree of hydrolysis (Fig. 4)The concentrations of PHBA and unmMP (expressed as %AD of

the parent MP) measured in the RF after 4-h exposure of previouslyfrozen intact and stripped FTS to nine vehicles is summarised inFig. 4. Only negligible amounts (above LOD, below LOQ; markedas point B in Fig. 4) of unmMP after permeation from the aqueoussolution, Hydrogel 1, and Hydrogel 2 through both intact FTS andstripped FTS, as well as from Hydrogel 4 through intact skin werefound. However, quantifiable amounts of MP from other vehicleswere determined; 0.20 lg/cm2 (i.e. 2.0%AD) of MP from Hydrogel

Table 4In vitro skin permeation parameters of unmetabolised methylparaben (MP) and its metabolite, p-hydroxybenzoic acid (PHBA) through previously frozen intact and stripped full-thickness pig-ear skin (FTS) from Emulsion 3 (10 ± 0.05 mg/cm2, containing 10 ± 0.05 lg/cm2 of MP).

Membrane Permeant Lag time (h) Jss (lg/cm2 h) C (mg/cm3) P (cm/h �103) P (cm/s)

Intact FTS PHBA N.d. 2.16 1.00 2.16 0.600MP 1.00 0.13 1.00 0.13 0.036

Stripped FTS PHBA N.d. 2.52 1.00 2.52 0.700MP N.d. 0.18 1.00 0.18 0.050

N.d.: Not detectable; Jss: Steady state flux; C: Concentration of the parent MP (0.1% w/w, i.e. approx. 1 mg/cm3); P: Permeability coefficient.Data are calculated from three replicates (Fig. 3).

Table 5Distribution of unmetabolised methylparaben (MP) and its metabolite, p-hydroxybenzoic acid (PHBA, expressed as MP) in a compartment of the diffusion system after 4-hexposure of intact freshly excised, intact frozen and stripped frozen stored (at �20 �C, maximum 6 weeks) full-thickness pig-ear skin (FTS) to each of the four emulsionscontaining 0.1% of MP (dose of MP 10 ± 0.05 lg/cm2).

Membrane Compartment of thediffusion system

Emulsion 1 without E Emulsion 2 with UR Emulsion 3 with TC Emulsion 4 with PG

MP PHBAc MP PHBAc MP PHBAc MP PHBAc

Intact freshlyexcised FTSa

Surface (lg/cm2) 2.10 ± 0.25 <LOD 2.01 ± 0.31 <LOD 1.59 ± 0.19 <LOD 1.69 ± 0.28 <LOD

Skin (lg/cm2) 1.19 ± 0.10 2.84 ± 0.10 1.13 ± 0.42 <LOQ 0.98 ± 0.31 <LOQ 1.00 ± 0.29 <LOQTEC 0.21–0.40

(mS/cm)Receptor fluid (lg/cm2) <LOQ 4.06 ± 0.56 <LOQ 4.55 ± 0.46 0.23 ± 0.02 5.70 ± 0.49 0.21 ± 0.02 5.45 ± 0.67

Thickness 1.025–1.200 (mm)

MP from (MP + PHBAc) inreceptor fluid (%)

Incalculable Incalculable 3.9 3.7

Total recovery(MP + PHBAc) (%)

101.9 76.9 85.0 83.5

Intact frozenstored FTSb

Surface (lg/cm2) 1.28 ± 0.33* <LOD 1.26 ± 0.30* <LOD 1.07 ± 0.18* <LOD 1.24 ± 0.22 <LOD

Skin (lg/cm2) 2.41 ± 0.65 <LOQ 2.33 ± 0.72* <LOQ 1.66 ± 0.39* <LOQ 1.49 ± 0.51 <LOQTEC 0.24–0.42

(mS/cm)Receptor fluid (lg/cm2) 0.23 ± 0.05* 4.58 ± 0.39* 0.23 ± 0.04* 4.66 ± 0.53 0.33 ± 0.09 6.09 ± 0.55 0.28 ± 0.07 5.59 ± 0.72*

Thickness 1.020–1.195 (mm)

MP from (MP + PHBAc) inreceptor fluid (%)

4.8 4.7 5.1 4.8

Total recovery(MP + PHBAc) (%)

85.0 84.8 91.5 86.0

Stripped frozenstored FTSa

Surface (lg/cm2) 1.08 ± 0.22 <LOD 0.98 ± 0.18 <LOD 1.02 ± 0.05 <LOD 0.98 ± 0,05 <LOD

Skin (lg/cm2) 1.32 ± 0.48 <LOQ 1.02 ± 0.14 <LOQ 1.05 ± 0.16** <LOQ 1.13 ± 0.23 <LOQTEC 1.85–2.81

(mS/cm)Receptor fluid (lg/cm2) 0.32 ± 0.08** 6.10 ± 0.93** 0.37 ± 0.05** 6.52 ± 0.42** 0.55 ± 0.15** 7.26 ± 0.60** 0.42 ± 0.05 6.60 ± 0.90**

Thickness 1.000–1.160 (mm)

MP from (MP + PHBAc) inreceptor fluid (%)

5.0 5.4 7.0 6.0

Total recovery(MP + PHBAc) (%)

88.2 88.9 98.8 91.3

E: enhancer; UR: urea (5%, w/w); TC: Transcutol� CG (5%, w/w); PG: propylene glycol (5%, w/w).<LOD: below the limit of detection; LOD for MP: 0.020 lg/mL; LOD for PHBA: 0.330 lg/mL (see Section 2.9).<LOQ: below the limit of quantification;LOQ for MP: 0.066 lg/mL, i.e. 0.066 lg/cm2 on surface; 0.165 lg/cm2 in skin; 0.182 lg/cm2 in receptor fluid (see Section 3).LOQ for PHBA: 0.990 lg/mL, i.e. LOQ for PHBAc: 1.089 lg/cm2 on surface; 2.475 lg/cm2 in skin; 2.995 lg/cm2 in receptor fluid (see Section 3).* Denotes the amount of MP and PHBAc permeated through intact frozen stored FTS significantly different (p < 0.05) from the amount of the same compound through intactfreshly excised FTS in a given compartment of the diffusion system.** Denotes the amount of MP and PHBAc permeated through stripped frozen stored FTS significantly different (p < 0.05) from the amount of the same compound throughintact frozen stored FTS in a given compartment of the diffusion system.

a Values are the mean ± SD (n = 6); no significant differences were found (p > 0.05) in a given compartment of the diffusion system (surface, epidermis plus dermis, andreceptor fluid).

b Values are the mean ± SD (n = 6 + 2); no significant differences were found (p > 0.05) in a given compartment of the diffusion system (surface, epidermis plus dermis, andreceptor fluid).

c PHBA is expressed as MP, i.e. PHBA � 1.1 (see Section 3).

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4 through stripped skin and 0.18 and 0.22 lg/cm2 of MP fromHydrogel 3 through both intact skin and stripped skin, respectively.In the Section 3.1.3, the results concerning the dermal absorptionsof MP from Emulsions 1–4through previously frozen intact skinand stripped skin are given. In contrast to MP, after 4-h exposureof previously frozen intact FTS and stripped FTS from all nineformulations into the RF, considerable amounts of PHBA (27.0–72.6%AD expressed as MP) were determined. The stripping proce-dure resulted in significantly greater permeation rates of PHBA

from all formulations tested relative to permeation rates throughintact skin from the same formulation (Fig. 4).

3.1.3. Mass balance analysis for previously frozen intact skin andstripped skin (Table 5)

The total recovery of the parent MP was evaluated in 4-h per-meation experiments for Emulsions 1–4 only. Concentration ofPHBA and unmMP (lg/cm2) distributed in the compartments ofthe diffusion system for previously frozen intact FTS (6 plus 2

Fig. 2. (A) HPLC chromatogram of reference standards, methylparaben (MP) and p-hydroxybenzoic acid (PHBA) dissolved in the receptor fluid and their correspondingretention times at k = 254 nm (two HPLC chromatograms put together). (B) HPLCchromatogram of MP and PHBA in the sample from the receptor fluid and in themethanol extract from the skin after 4-permeation through previously frozenstripped full-thickness pig-ear skin exposed to Emulsion 3 with Transcutol� CG(two HPLC chromatograms put together). BLOQ: below the limit of quantification(LOQ), over the limit of detection (LOD).

760 S. Pazoureková et al. / Food and Chemical Toxicology 59 (2013) 754–765

replicates) and stripped FTS (6 replicates) are reported in Table 5.As expected, the main compound in the RF, PHBA was found;45.8–60.9%AD for previously frozen intact FTS and 61.0–72.6%ADfor stripped FTS. In contrast to PHBA, only small quantities of MP(2.3–3.3%AD and 3.2–5.5%AD) in the RF after passage through pre-viously frozen intact FTS and stripped FTS, respectively were found.A considerable amount of the parent MP remained on the surfaceof intact FTS (10.7–12.8%AD) and stripped FTS (9.8–10.8%AD) discs.If certain amount of PHBA on the surface of both intact andstripped FTS was present, it was <LOD in all emulsions. Comparedwith the amount ofemulsion remaining on the surface, even great-er quantity of MP was accumulated in intact FTS (14.9–24.1%AD)and stripped FTS (10.2–13.2%AD). For all dosing emulsions, onlyamounts of PHBA > LOD and <LOQ in all extracts from the intactand stripped FTS were detected. We were initially surprised at thisfinding because the extraction method has been verified (see Sec-tion 2.8.1.2). However, as the LOQ value of 2.722 lg/cm2 for PHBA(expressed as the parent MP) in the skin was approximately 16-fold more than LOQ value of 0.165 lg/cm2 for MP in the skin (seeTable 5), if a certain amount of PHBA was absorbed in the skin, thiswas below of LOQ.

Following conversion of PHBA quantities to the parent MP (Ta-ble 5) according to Eq. (1), the mass balance was calculated. The totalrecovery of the parent MP for Emulsion 1–4 exposed of previouslyfrozen intact and stripped FTS ranged from 84.8 to 91.5%AD and from88.2 to 98.8%AD, respectively, which are percentages within therange of 85–115% recommended by SCCS (2012).

3.2. Results from the second set of experiments

3.2.1. Mass balance analysis for previously frozen intact skin andfreshly excised intact skin (Table 5)

Concentration of PHBA and unmMP (lg/cm2) distributed in thecompartments of the diffusion system for previously frozen intactFTS (6 plus 2 replicates) and freshly excised intact FTS (6 repli-cates) are reported in Table 5. The quantities of PHBA and unmMPfound in the RF were slightly lower after permeation throughfreshly excised intact FTS (40.6–57.0%AD and <LOQ–2.3%AD,respectively) than through previously frozen intact FTS (45.8–60.9%AD and 2.3–3.3%AD, respectively). A considerable amountof the parent MP remained on the surface of previously frozen in-tact FTS (10.7–12.8%AD) and freshly excised FTS (11.6–20.1%AD). Ifsmall amounts of PHBA were present in the unabsorbed dose onthe surface of both frozen stored and fresh intact FTS, these werebelow LOD in all emulsions. Compared with the amounts of emul-sions remaining on the surface, even greater quantities of MP wereaccumulated in frozenstored intact FTS (14.9–24.1%AD) and freshFTS (9.8–11.9%AD). For all dosing emulsions, only negligibleamounts of PHBA (above LOD and below LOQ) were detected inall extracts from intact previously frozen and fresh FTS, with oneexception. For Emulsion 1 without enhancer the amount of28.4%AD of PHBA were measured in fresh intact skin. The totalrecovery of the parent MP for Emulsion 1–4 exposed of intactfreshly excised FTS ranged from 76.9% to 101.9%.

4. Discussion

First of all we would like to express our opinion on the potentialcriticism that the previously frozen skin was used in most experi-ments of our study. The skin permeation experiments can be car-ried out using freshly excised or frozen stored skin, but forassessment of the metabolism the fresh skin is preferred (OECD,2004; SCCS, 2010). It is undisputed that fresh skin best mimicsthe metabolic conditions in human skin in vivo. However, whenlong-term comparative permeation experiments are performed, itis no possible to obtain day-to-day freshly excised pig ear withthe same qualitative properties. In addition, as it has been alreadymentioned in the introduction, there is a considerable inconsis-tency in the results of studies regarding the ability of esterases tohydrolyse ester compounds in previously frozen skin comparedto fresh skin. Thus, to clarify the effects of freezing on carboxylest-eraseactivity in pig ear skin, we have assessed the permeation ofMP through intact frozen stored (41–42 days at �20 �C), as wellas freshly excised FTS for four different emulsions oil-in-water.According to the results summarised in Table 5, freezing of pig-ear skin only slightly reduces the hydrolytic activity of esterases.This result is in an agreement with the observation by Abla et al.(2006) that pig-ear skin stored frozen at �20 �C for up to 2 monthsis still able to verify hydrolysis of an ester prodrug in the skin.Compared to intact previously frozen FTS in our study, the amountof unmMP permeated in the RF through intact freshly excised FTSwas not statistically significant for Emulsion 3 with TC and Emul-sion 4 with PG, but was significantly lower (p < 0.05) for Emulsion1 without enhancer and Emulsion 2 with UR. Insufficient totalrecovery for Emulsion 2 with UR (76.9%) and Emulsion 4 with PG(83.5%) after exposure of intact freshly excised FTS, than recom-mended by SCCS (2012) was probably due to the relatively largenumber of PHBA absorbed in the skin, but still lower than LOQ.Our results about percentages from the applied dose of unmMPin the RF (2.1–3.3%) for fresh, as well as previously frozen intactskin are in accordance with the SCCS (2011), which the 3.7% der-mal absorption value of unmetabolised paraben uses in final calcu-lations of the Margin of Safety (MoS).

Fig. 3. Absorption-time profiles of methylparaben (10 ± 0.05 lg/cm2) during the 24-h exposure of previously frozen intact and stripped full-thickness pig-ear skin (FTS). Theamount (lg/cm2) of unmetabolised MP and its metabolite, p-hydroxybenzoic acid (PHBA) found in the receptor fluid (RF) from Hydrogel 1 and Emulsion 1 (both withoutenhancer) showed the lowest dermal absorption rate and Hydrogel 3 and Emulsion 3 (both with Transcutol� CG) showed the highest dermal absorption rate. Values are themean ± SD (n = 3). B: below the limit of quantification (LOQ), over the limit of detection (LOD); LOD < B < LOQ.

S. Pazoureková et al. / Food and Chemical Toxicology 59 (2013) 754–765 761

Significantly decreasing amounts of corneocytes with anincreasing tape number after tape-stripping of FTS sheets are doc-umented in Fig. 1a–c. Although the skin damage by stripping in ourexperiment was more drastic than a normal skin mechanical dam-age in everyday life. However, we assume that due to repeateddepilation, shaving, or skin injured for various reasons, the processis close to skin conditions in some consumers and patients.

Compared to intact FTS sheet, the stripping procedure causedonly a slight decrease in the thickness of FTS sheet (on average of11 ± 3 lm, i.e. approx. of 1%) but dramatically higher TEC value(on average 7-fold). Latest fact resulted in higher permeation rateof the parent MP and its metabolite from all nine formulationstested (Tables 3 and 5). After 24-h, there were found 2.9–7.6%ADof unmMP for previously frozen stripped FTS compared to 2.0–5.8%AD of MP for intact FTS in the RF (Table 3). For Emulsion 3the values of the steady state flux and permeability coefficient ofMP and PHBA show that the parent MP passed 1.4, and 1.2 times,respectively, faster through stripped FTS than through intact FTS.

The lag time of MP was not detectable through stripped FTS, butwas detectable through intact FTS(1.0 h; Table 4). Pedersen et al.(2007) in the in vitro study through intact rabbit ear skin foundno detectable lag time for MP, comparable with our results forstripped skin. Akomeah et al. (2007) in the in vitro study throughintact human epidermis determined a lag time of MP (0.3 h) lessthan those obtained by us, which could be the result of the useof different membranes. However, Barbero and Frasch (2009)based on the review study (concerning substances other than para-bens) reported that there is a relatively weak correlation betweenthe lag times of pig skin and human skin. Despite this fact, theauthors recommend pig skin as a good model for human skin per-meability, due to it shows less variability than human skinmodel.Unfortunately, we did not find any scientific studies regard-ing to the lag time of MP through damaged skin.

Quantities of unmMP and PHBA in the RF after 24-h skin expo-sure to the four emulsions were significantly greater than to fourhydrogels of similar composition; i.e. from 1.4 to 2.2-fold and 1.5

Fig. 4. Dermal absorption rate and hydrolysis degree. The amount of unhydrolysed methylparaben (MP) and its metabolite, p-hydroxybenzoic acid (PHBA; expressed as % ofapplied dose of the parent MP) found in the receptor fluid after 4-h exposure of previously frozen intact and stripped full-thickness pig-ear skin to 10 ± 0.05 lg/cm2 of theparent MP in each of a given vehicle (10 ± 0.05 mg/cm2). Values are the mean ± SD (n = 6 for stripped and n = 8 for intact skin). E: enhancer; UR: urea (5%, w/w); TC:Transcutol� CG (5%, w/w); PG: propylene glycol (5%, w/w). B: below the limit of quantification (LOQ), over the limit of detection(LOD); LOD < B < LOQ.

762 S. Pazoureková et al. / Food and Chemical Toxicology 59 (2013) 754–765

to 1.7-fold, respectively, through intact FTS and from 1.8 to 2.2-foldand 1.4 to 1.5-fold, respectively, through stripped FTS. This findingis consistent with the assumption that the more a substance is sol-uble in the vehicle, the more likely it will be transported (Baker,1986). As shown in the octanol/water partition coefficient (Log-Ko/w) of MP (1.96, Table 1), the nature of the hydrophilic–lipophilicMP is closer to emulsion o/w than to high hydrophilic hydrogel andaqueous solution. In accordance with the results of the study byWatrobska-Swietlikowska and Sznitowska(2006) we assume thata high content of the parent MP was accumulated in the interfaceregion of the emulsion. Our results are in an agreement with theobservation by Esposito et al. (2003) that the permeability coeffi-cient of parabens increases with paraben water solubility for emul-sions, whereas it decreases in the case of gels. The permeation ratesof MP from emulsions were greater also probably due to the phys-ical form of an emulsion is closer to the natural skin film as a formof a hydrogel or a solution. The Jss values of PHBA and MP obtainedin our study for Emulsion 3 were 2.16 and 0.13 lg/cm2 h throughintact skin and 2.52 and 0.18 lg/cm2 h through stripped skin.Esposito et al. (2003) investigated the diffusionof MP in topicalproducts containing 0.05% of MP through a sandwich of two mem-branes; (a) polydimethylsiloxane-based membrane and (b) a ny-lon-based membrane (150 lm) with 0.22 lm pore size. Theresearchers determined the Jss value of MP (total amount of MPand PHBA) at 4.87 lg/cm2 h from o/w emulsion and 3.95 lg/cm2 h from hydrogel. Caon et al. (2010) in the 6-h kinetic studythrough intact full-thickness pig-ear skin exposed to 0.1% parabens(in an ethanol/PBS mixture 50:50) determined the Jss value of MPof 20 lg/cm2 h (total quantities of MP and PHBA). Unfortunately,the two above-mentioned groups of researchers did not assessmetabolism of the parentMP.

The results of our study clearly showed that the addition of pen-etration enhancers to a formulation increases the amount of MPand PHBA in the RF and therefore also a potential their entry intothe bloodstream. Transcutol� CG was seen to have greater abilityto enhance epidermal-dermal diffusivity of MP than vehicles withUR and PG. After 24-h passage, the amount of MP and PHBA fromTC-containing Emulsion 3 through intact skin was 1.7 and 1.3-foldgreater and through stripped skin 1.5 and 1.2-fold greater, respec-tively than from Emulsion 1 without enhancer. We consider thatTC as solvent was acting by fluidizing lipids within the SC that al-lowed greater solute movement within the epidermal membrane.Due to this fact, when topical leave-on products preserved withMP are formulating, producer should avoid the use of penetration

enhancers, except when it is necessary. When examined the pro-portion of MP from total flux of permeants in the RF (sum of MPand PHBA; seeTable 4), the findings are notable. Regardless ofthe skin condition, the proportion of MP was approximately thesame for certain formulation and ranged from 4.9% (for the aque-ous solution) to 7.4% (for Emulsion 3).

Finally, despite the possible differences between the humanskin and previously frozen pig-ear skin in the dermal rate andeffectiveness of hydrolysis of MP, we calculated the Systemic Expo-sure Dosage (SED) of both MP and PHBA for humans treated withthe finished cosmetic product. The SED value is the amount of acosmetic ingredient expected to enter the blood stream (and there-fore be systemically available) per kg body weight and per day(SCCS, 2012) and calculated according to

SED ¼ DA ðlg=cm2Þ � SSA ðcm2Þ � F ðday�1Þ60 ðkgÞ ð3Þ

where SED (lg/kg bw/day) is the Systemic Exposure Dosage; DA isdermal absorption of the substance reported as amount (lg/cm2);SSA (cm2) is the skin surface area expected to be treated with thecosmetic product; F (day�1) is the usual frequency of applicationof the cosmetic product; 60 kg is default human body weight (SCCS,2012).

In a classical in vitro dermal absorption setting, the totalamount measured in the epidermis (without the SC), dermis andthe receptor fluid is considered to be systemically available and ta-ken into account for further calculations (SCCS, 2010, 2012; OECD,2011). As mentioned above, after our experiments we were unableto separate quantitatively the SC from the rest of the FTS disc, bothintact and stripped skin, mainly due the hair follicles. Also OECDGD 28 (OECD, 2011) states that fractionation of the skin afterexperiments can be difficult in some cases. However, if we hypoth-esise that a majority of the SC was removed as the consequence ofthe tape-stripping procedure (20-times), the amount of MP andPHBA found in stripped skin, was absorbed mainly in its living epi-dermal-dermal layers. Seko et al.(1999) studied the effect of cuta-neous metabolism of PP and BP using intact and stripped rat skinin vitro. Consistent with our hypothesis, they also considered theamount of PP and BP found in the skin after tape-stripping proce-dure (15 times) as absorbed in the living layers of the skin.

Therefore, in our calculations, the SED values were evaluated forpermeation only through stripped skin and under the two case sce-narios. In a best case scenario, only the amount of MP in the RFafter 4-h exposure was considered to be systemically available

Table 6Estimated daily Systemic Exposure Dosage (SED) of unhydrolysed methylparaben (MP) and its metabolite, p-hydroxybenzoic acid (PHBA), after 4-h exposure of damaged humanskin in vivo to a leave-on emulsion oil-in-water (10 mg/cm2) containing 0.1% of MP and 5% of propylene glycol (PG).

Cosmetic product Localitya SSAa

(cm2)Fa

(day�1)DAb (lg/cm2) DAb (lg/cm2) SEDc (lg/kg bw/day) SEDc (lg/kg bw/day)

MP MP PHBA PHBA MP MP PHBA PHBAIn theRF

In theSkin

In theRF

In theSkin

Best casescenario

Worst casescenario

Best casescenario

Worst casescenario

Body lotion withPG

Area body plus areahead female

15,670 2.28/day

0.42 1.13 6.60 BLOQ 250.10 922.97 3930.04 3930.04

Face cream withPG

1/2 Area head female 565 2.14/day

0.42 1.13 6.60 BLOQ 8.46 31.23 133.00 133.00

Hand cream withPG

Area hands 860 2/day 0.42 1.13 6.60 BLOQ 12.04 44.43 189.20 189.20

After-shave lotionwith PG

1/4 Area head male 305 1/day 0.42 1.13 6.60 BLOQ 2.14 7.88 33.55 33.55

Deo non-spraywith PG

Both axillae 200 2/day 0.42 1.13 6.60 BLOQ 2.80 10.33 44.00 44.00

SSA: skin surface area (expected to be treated with a cosmetic product); F: frequency (of the application of the cosmetic product); DA: dermal absorption (in the receptor fluidonly); Best case scenario: the amount of MP or PHBA only in the RF was used in the exposure calculations; Worst case scenario: the total amount (sum of the quantities in theRF and skin) of MP or PHBA was used in the exposure calculations.

a Parameter is consonant with the notes of guidance for testing of cosmetic ingredients and their safety evaluation by the European Scientific Committee on ConsumerSafety (SCCS, 2012).

b Experimental value from Tables 3 in this study: the amount measured only in the receptor fluid considered as systemically absorbed.c Calculated data from the experimental value according to the Eq. (2) (see Section 4).

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and used in the exposure calculations. In a worst case scenario, thetotal amount of MP (in the FTS disc and the RF) after 4-h exposurewas considered to be systemically available and used in the expo-sure calculations. The same scenarios were used for PHBA.

Since propylene glycol is widely used in many types of cosmeticproducts including emulsions, Emulsion 4 with PG was selected asan illustrative example for the calculation of SED values for MP andPHBA after 4-h exposure of mechanically damaged skin (Table 6).Here we discuss two extremes among them. First, assuming that,a body milk with 5% PG containing 0.1% MP is applied to the femaleface and whole body with damaged skin barrier, in the best casescenario the amount of 250 and 3930 lg/kg bw/day, in the worstcase scenario 923 and 3930 lg/kg bw/day of unmMP and PHBA,respectively, become systemically available. Second, assuming thata deo-emulsion with 5% PG containing 0.1% MP is used for treat-ment of both axillae shortly after hair removal, in the best case sce-nario the amounts of 2.8 and 44.0 lg/kg bw/day, in the worst casescenario 10.3 and 44.0 lg/kg bw/day of unmMP and PHBA, respec-tively, could be systemically available.

5. Conclusions

In this paper, the potential for systemic absorption of methyl-paraben through intact skin and mechanically damaged skin wasstudied. Four major findings can be drawn. Firstly, after a singleskin exposure to MP containing products similar to the formula-tions tested, MP is not hydrolysed completely and certain amountof unmMP may be systemically available, even if at a low concen-tration. Secondly, the mechanical damage of the skin increases arate of systemic availability of both MP and PHBA from hydrophilicvehicles. Thirdly, when formulating topical preparations contain-ing MP, the penetration enhancers should not be used if it is notnecessary. Fourthly, freezing of pig-ear skin prior to the experi-ments slightly reduces the hydrolysis of the ester bonds.

Overall, taking into account, on the one hand, the number of use-ful properties of MP and on the other hand, the results of thesein vitro studies, we consider that MP is more suitable for preservingrinse-off topical products than for leave-on products. MP should notbe added to products intended for skin with damaged barrier, espe-cially when the product is used repeatedly. And finally, we considerthat systemic absorption of cosmetic and pharmaceutical raw mate-

rials should also be assessed through the skin with impaired barrier,since many of topical medications and certain cosmetic products areapplied to the skin with disturbed condition.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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