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Toxicology Letters 188 (2009) 119–129

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

ermal penetration and metabolism of p-aminophenol and p-phenylenediamine:pplication of the EpiDermTM human reconstructed epidermis model

ing Hua, Ruth E. Baileya, Stephen W. Morrall a, Marilyn J. Aardemaa, Lesley A. Stanleyb, Julie A. Skarec,∗

The Procter & Gamble Company, Miami Valley Innovation Center, Cincinnati, OH, 45253, United StatesConsultant in Investigative Toxicology, P.O. Box 29225, St. Andrews, Fife, KY16 9WS, UKThe Procter & Gamble Company, Sharon Woods Technical Center, Cincinnati, OH, 45241, United States

r t i c l e i n f o

rticle history:eceived 15 August 2008eceived in revised form 16 March 2009ccepted 23 March 2009vailable online 31 March 2009

eywords:-Aminophenol-Phenylenediamineeconstructed epidermis model

a b s t r a c t

To address the provision of the 7th Amendment to the EU Cosmetics Directive banning the use of in vivogenotoxicity assays for testing cosmetic ingredients in 2009, the 3D EpiDermTM reconstructed humanskin micronucleus assay has been developed. To further characterise the EpiDermTM tissue for poten-tial use in genotoxicity testing, we have evaluated the dermal penetration and metabolism of two hairdye ingredients, p-aminophenol (PAP) and p-phenylenediamine (PPD) in this reconstructed epidermismodel. When EpiDermTM tissue was topically exposed to PAP or PPD for 30 min (typical for a hair dyeexposure), the majority (80–>90%) of PAP or PPD was excluded from skin tissue and removed by rins-ing. After a 23.5 h recovery period, the PAP fraction that did penetrate was completely N-acetylated toacetaminophen (APAP). Similarly, 30 min topical application of PPD resulted in the formation of the N-

′

-acetyltransferase 1iotransformationytotoxicity

mono- and N,N -diacetylated metabolites of PPD. These results are consistent with published data onthe dermal metabolism of these compounds from other in vitro systems as well as from in vivo studies.When tissue was exposed topically (PAP) or via the culture media (PPD) for 24 h, there was good batch-to-batch and donor-to-donor reproducibility in the penetration and metabolism of PAP and PPD. Overall,the results demonstrate that these two aromatic amines are biotransformed in 3D EpiDermTM tissue viaN-acetylation. Characterising the metabolic capability of EpiDermTM tissue is important for the evaluation

enoto

of this model for use in g

. Introduction

The metabolism and potential toxicity of aromatic aminesre of considerable interest in ensuring safety for consumersecause of the widespread use of these compounds in the phar-aceutical, chemical, rubber, dye and photographic industries. In

articular, aromatic amines such as p-aminophenol (PAP) and p-

henylenediamine (PPD) are used as primary precursors in hairyes, leading to exposure of the scalp to these compounds (Nohynekt al., 2004a).

Abbreviations: APAP, acetaminophen; CYP, cytochrome P450; DAPPD, N,N′-iacetyl-p-phenylenediamine; d4-PAP, p-aminophenol-ring deuterium labelled;4-PPD, p-phenylenediamine sulphate-ring deuterium labelled; DMSO, dimethylulphoxide; DPBS, Dulbecco’s phosphate buffered saline; HPLC, High Perfor-ance Liquid Chromatography; LOD, limit of detection; MAPPD, N-monoacetyl-

-phenylenediamine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumromide; NAT1, N-acetyltransferase 1; NMM, new maintenance medium; PAP, p-minophenol; PPD, p-phenylenediamine; SD, standard deviation.∗ Corresponding author. Tel.: +1 513 626 1976; fax: +1 513 386 1572.

E-mail address: skare.ja@pg.com (J.A. Skare).

378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2009.03.019

xicity testing.© 2009 Elsevier Ireland Ltd. All rights reserved.

Studies of xenobiotic metabolism, including the biotransforma-tion of aromatic amines, have traditionally focussed upon the liverbecause this organ has the largest capacity for phases I and IImetabolism and plays a central role in the clearance of xenobioticsfollowing ingestion. However, the skin may play a key role in con-trolling the penetration and metabolism of hair dye componentswhen application of hair dyes results in skin exposure via the scalp.Therefore, the aim of this study was to establish an in vitro humanskin model in which to evaluate the metabolism of aromatic amineconstituents of hair dyes.

One model for studying aromatic amine metabolism in humanskin is primary ex vivo human skin explants. However, the use of thismodel has some disadvantages including the difficulty in obtainingfresh human skin for immediate experimental use and variabilityin tissue handling and tissue quality depending upon the source,which can affect the level of xenobiotic metabolising enzymes.

The limited availability and poor reproducibility of ex vivo

human skin samples have led various investigators to developthree-dimensional models to allow the metabolic behaviour ofhuman skin to be modelled (Roguet, 2002). Two such models are thereconstructed epidermis models EpiDermTM and EpiSkin®. Both ofthese models are derived from human keratinocyte cultures which

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were cultured for another 23.5 h. The culture media in the wells was collected andretained for analysis as the media sample at the end of desired exposure period; the

20 T. Hu et al. / Toxicology

ndergo differentiation to form three-dimensional structures thatlosely resemble human skin. The EpiDermTM model is a three-imensional multilayered skin model derived from human infantoreskin keratinocyte cultures which are induced to differentiate byir-lifting the growing cultures so that the apical surface is exposedo the atmosphere while the basal layers remain in contact withhe culture media. Following differentiation to form basal, spinous,ranular and cornified layers, the EpiDermTM model resemblesuman epidermis (Cannon et al., 1994). The Episkin® model iserived from normal adult human keratinocytes removed duringlastic surgery. These are grown on a specially developed dermalubstrate comprising oxidised Types I and II collagen covered withfilm made up of native and oxidised Type IV collagen (Tinois et

l., 1991). When exposed to the atmosphere, cells grown in thisay differentiate to form a stratified epithelium with an apparently

ontinuous basement membrane and a dense horny apical surface.oth EpiDermTM and Episkin® have previously been used for the initro evaluation of topically applied chemicals with respect to var-ous characteristics (Roguet, 2004), including cutaneous irritancyWelss et al., 2004), percutaneous absorption (Schafer-Korting etl., 2006), dermal xenobiotic metabolism (Nohynek et al., 2005)nd genotoxicity (Curren et al., 2006; Flamand et al., 2006).

The development of new genotoxicity assays in 3D human skinodels is directed at addressing the need for the replacement of in

ivo studies with in vitro assays as a result of the 7th Amendment tohe EU Cosmetics Directive and the Registration, Evaluation, Autho-isation and Restriction of Chemicals (REACh) regulations (Current al., 2006; Mun et al., 2009; Hu et al., 2009). Use of 3D human skinodels for genotoxicity testing may overcome many of the prob-

ems with standard in vitro genotoxicity assays that are thought toontribute to the high rate of false-positive results for the predictionf rodent carcinogenicity (Kirkland et al., 2005, 2006). Importantly,hese tissue models are comprised of normal human cells, and theyllow more physiologically relevant exposure conditions and moreelevant metabolism versus exogenous liver S9 added to current initro assays.

To evaluate the dermal metabolism of aromatic amine hairyes, we studied the biotransformation of PAP and PPD in thepiDermTM skin model. PAP and PPD are used as primary precursorsn oxidative hair dyes, and their toxicological and pharmacokineticroperties have been reviewed by Nohynek et al. (2004a). Since theetabolism of PAP and PPD has been characterised in several in

itro models (Kawakubo et al., 2000; Nohynek et al., 2005; Stanleyt al., 2005) as well as in vivo in rat and minipig after topical appli-ation (Dressler and Appelqvist, 2006), these compounds are usefuls model compounds for the evaluation of the metabolic capabilityf the EpiDermTM model.

In the present study, EpiDermTM tissue was exposed to PAP orPD either topically or from the bottom of the tissue via the cultureedia. The former exposure is more relevant to human exposurehile the latter allows comparison of the results with EpiDermTM

issue to results previously published for PPD with the EpiSkin®

odel (Nohynek et al., 2005). Following various incubation periods,he levels of parent compound and metabolites were determinedn the surface rinse (for topical exposures), the culture media andhe tissue itself. This experimental approach allowed an evaluationf the penetration and metabolism of the test compounds and aomparison of the EpiDermTM system with the EpiSkin® system asotentially useful models for skin metabolism of aromatic aminesollowing topical exposure.

. Materials and methods

.1. Chemicals and reagents

p-Aminophenol (free base, CAS# 123-30-8), p-phenylenediamine (freease, CAS# 106-50-3), N-monoacetyl-p-phenylenediamine, N,N′-diacetyl-p-

s 188 (2009) 119–129

phenylenediamine, acetonitrile (HPLC grade), and dimethyl sulphoxide (DMSO)were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetaminophen waspurchased from Avocado Research Chemicals Ltd. (Heysham, Lancashire, UK).p-Aminophenol-ring deuterium labelled (d4-PAP, CAS# 70237-44-4) and p-phenylenediamine sulphate-ring deuterium labelled (d4-PPD, CAS# 119516-83-5)were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA).Methanol (HPLC grade), ammonium acetate, and ammonium hydroxide for thepreparation of the HPLC mobile phase were purchased from Sigma–Aldrich (St.Louis, MO, USA), EMD (Gibbstown, NJ, USA) and J.T. Baker (Phillipsburg, NJ, USA).

2.2. EpiDermTM human skin model

The EpiDermTM EPI-200-MNA kit was obtained from MatTek Corporation(Ashland, MA). EpiDermTM EPI-200 tissue constructs are 0.64 cm2 human skin equiv-alents resembling the normal human epidermis (www.mattek.com). The kit includesnew maintenance medium (NMM) which is a proprietary DMEM-based mediumsold by MatTek that allows acceptable differentiated morphology of the EpiDermTM

EPI-200 tissue for at least five days upon receipt by end users. Experiments describedhere were conducted with EpiDermTM cultures constructed from two differentdonors (Donor 254 and Donor 4F1188) of foreskin-derived human epidermal ker-atinocytes. Upon receipt, tissues were cultured overnight in 6-well plates containing1 ml of NMM media at 5 ± 1% CO2 and 37 ± 1 ◦C before use in the experiments.

2.3. Preparation of aromatic amine dosing solutions

Stock solutions of unlabelled and deuterium-labelled aromatic amines (PAP, d4-PAP, PPD, and d4-PPD) were freshly prepared in acetonitrile prior to use. Thesesolutions were prepared as a 1:1 mix of unlabelled and deuterium-labelled (d4)PAP or PPD. An aliquot of PAP or PPD and its deuterated counterpart was transferredto a sterile test tube and the acetonitrile was removed under nitrogen. Immediatelybefore dosing, the 1:1 mix of PAP or PPD was reconstituted with the appropriatevolume of DMSO or warm Dulbecco’s phosphate buffered saline (DPBS). For the 24 hexposure studies, to allow comparison of the results with EpiDermTM tissue to resultspreviously published with the EpiSkin® model (Nohynek et al., 2005), PAP or PPDwas reconstituted with DMSO, then diluted with the appropriate volume of NMMmedia (final concentration of DMSO in the dosing solutions was 1% (v/v)) to give thedesired concentrations. For the 30 min exposure studies, PAP or PPD was reconsti-tuted with warm DPBS then diluted with the appropriate volume of NMM media(final concentration of 10% DPBS) to give the desired concentrations. Use of 10%DPBS in media as the solvent for the 30 min exposures eliminated any effect DMSOmight have on the metabolic capacity of the tissue. The use of deuterated materialserved as a metabolic tracer compound that would aid in distinguishing metabo-lites from background matrix interference. Both the unlabelled and d4-labelled testcompounds are metabolised, generating a distinctive mass spectrum in which unla-belled and deuterium-labelled metabolites produce ions that differ by four massunits.

2.4. Treatment of EpiDermTM constructs

Nohynek et al. (2005) investigated the biotransformation of PPD and PAP in theEpiSkin® model using a long duration of exposure (24 h) and, in the case of PPD, basalexposure via the culture media in the EpiSkin® model. Our initial studies replicatedthese exposure conditions. During an actual hair dyeing process the duration ofexposure is much shorter (∼30 min) and occurs via topical application. In order toassess more relevant topical exposure conditions, we also investigated EpiDermTM

tissue topically exposed to PAP and PPD for 30 min followed by a 23.5 h recoveryperiod.

For exposure via the culture media, the exposure volume was 1 ml of NMM media(as recommended by MatTek, the EpiDermTM tissue manufacturer). For topical expo-sure experiments, PAP and PPD were applied to the apical surface of the EpiDermTM

tissue in 87 �l of exposure media. For comparison with previously reported resultswith EpiSkin® (Nohynek et al., 2005), application of 87 �l of PAP/PPD solution tothe surface of an EpiDermTM construct (0.64 cm2) provides the same 136 �l/cm2

exposure to that obtained when 150 �l is applied to the surface of an Episkin® con-struct (1.1 cm2). The cultures were incubated at 37 ◦C for the desired period of time:30 min PPD and PAP, or 24 h PAP for topical exposure and 24 h PPD for media expo-sure. After the desired topical exposure periods, the media from the top surfaceof the tissues was collected and saved, the tissue surface was rinsed twice with200 �l of cold DPBS (400 �l total), and all were combined and retained for anal-ysis as the surface rinse sample. For 30 min topical exposure studies, the tissues

tissue was peeled off its support, placed in a tube with approximately 0.5 ml DPBSalong with a small steel bead and subjected to homogenization with a Retsch mixermill. The supernatant was collected after centrifugation for 10 min at 10,000 rpm.The residual tissue was extracted with 0.5 ml DPBS and the two supernatantsfrom each tissue were combined for further analysis as the tissue homogenatesample.

T. Hu et al. / Toxicology Letters 188 (2009) 119–129 121

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ig. 1. LC–MS analysis of PAP and APAP: (a) extracted ion chromatogram of a 5 nmom/z = 152) and d4-APAP (m/z = 156); (c) ESI+ mass spectrum of PAP standard showin

.5. Cytotoxicity assays

Cytotoxicity assays were carried out in triplicate tissues for each treatment con-ition by the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]ethod (Mosmann, 1983). The assay was performed according to instructions pro-

ided by the manufacturer (MatTek Corporation). Briefly, after treatment (either 24 h

xposure followed by DPBS rinsing or 30 min exposure followed by DPBS rinsing thenontinued incubation for 23.5 h), the EpiDermTM tissue was transferred into 24-welllate containing 300 �l of 1 mg/ml MTT solution in each well. After 3 h incubation,ach EpiDermTM tissue was transferred to a 24-well plate containing 2.0 mL of iso-ropanol in each well. After overnight isopropanol extraction at room temperature,he absorbance at 570 nm from each well was measured in triplicate at 200 �l/well

ig. 2. LC–MS analysis of PPD, MAPPD and DAPPD: (a) extracted ion chromatogram of a 10oth unlabelled DAPPD (m/z = 193) and d4-DAPPD (m/z = 197); (c) ESI+ mass spectrum of ad) ESI+ mass spectrum of a media sample with both unlabelled PPD (m/z = 109) and d4-P

P and APAP; (b) ESI+ mass spectrum of a media sample with both unlabelled APAPh unlabelled PAP (m/z = 110) and d4-PAP (m/z = 114).

in a 96-well plate with a Molecular Devices Vmax plate reader. Killed control tissues(inactivated by four freeze–thaw cycles) were included in all MTT assays to offsetpotential interference of the oxidative hair dye ingredients toward the MTT assay.

2.6. Preparation of calibration standards

Similar to the preparation of stock solutions described previously, stock solu-tions of APAP, MAPPD, and DAPPD were also prepared in acetonitrile. Calibrationstandards were prepared by transferring known amounts of the hair dyes, their d4counterparts, and their known metabolites from the stock solutions to glass testtubes. The acetonitrile solvent was removed under a stream of nitrogen. The stan-dards were reconstituted in 0.5 ml Milli-Q water, and then processed by solid phase

nmol of PPD, MAPPD and DAPPDP; (b) ESI+ mass spectrum of a media sample withmedia sample with both unlabelled MAPPD (m/z = 151) and d4-MAPPD (m/z = 155);PD (m/z = 113).

122 T. Hu et al. / Toxicology Letters 188 (2009) 119–129

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ig. 3. Toxicity of PAP and PPD to EpiDermTM tissues following 30 min or 24 h exollowing exposure to PAP or PPD, as described. (a) PAP, 24 h topical exposure: (� ) DF1188; (c) PPD, 30 min topical exposure: (�) Donor 254. Data shown are mean ± SD

xtraction, as described below. The amounts in the standards ranged from 0.01 to0 nmol for PAP, d4-PAP, and APAP; and from 0.01 to 600 nmol for PPD, d4-PPD,APPD, and DAPPD.

.7. Analytical sample preparation

Each sample and calibration standard was processed by solid phase extractionsing strata-X-C cation mixed mode extraction cartridges containing 30 mg of sor-ent (Phenomenex, Torrance, CA, USA). The cartridges were conditioned with 1.0 mlf methanol followed by 1.0 ml of Milli-Q water. The entire sample was then loadednto the cartridge. The cartridges were washed with 1.0 ml of 0.1N HCl (EMD, Gibb-town, NJ, USA). Finally, the analytes were eluted into glass test tubes using two.5 ml portions of 0.5% ammonium hydroxide in methanol. The solvent was removedrom the tubes under a stream of nitrogen using a manifold with a heating block. Prioro chromatographic analysis, each sample and calibration standard was reconsti-uted in 0.3 ml Milli-Q water. A single solid phase extraction method was developedor the analysis of PAP, PPD and their respective metabolites, APAP, MAPPD andAPPD. This method gave >85% mean recoveries for all five of the analytes of interest.

.8. HPLC and mass spectrometry conditions

The chromatographic system comprised an Alliance 2695 and 996 PDA detec-or (Waters Corp., Milford, MA, USA). A 5 �l injection of the prepared samples ortandards was made onto an X-Bridge phenyl (2.1 mm × 150 mm, 3.5 �m) columnWaters). The column temperature was set at 35 ◦C. The mobile phase consisted of0 mM ammonium acetate, pH 8 in Milli-Q water (A) and methanol (B). An isocraticow of 95% (A) and 5% (B) for 17 min at a flow rate of 0.25 ml/min was used for theeparation. All the effluent from the column was directed into a ZQ single quadrupoleass spectrometer (Waters). Electrospray ionization was performed in the positive

on mode. The mass range scanned was 80–500 m/z (mass-to-charge ratio). Process-ng masses selected for PAP were 110 (PAP), 114 (d4-PAP), 152 (APAP), 156 (d4-APAP),nd processing masses selected for PPD were 109 (PPD), 113 (d4-PPD), 151 (MAPPD),55 (d4-MAPPD), 193 (DAPPD), and 197 (d4-DAPPD). Peak integration and quantita-ion was performed using MassLynx 4.0 software. Quantitation curves ranged from.5 to 50 nmol for PAP and d4-PAP; 0.01–10 nmol for APAP; 1–600 nmol for PPD and4-PPD; and 0.1–60 nmol for MAPPD and DAPPD. Examples of the resulting analyt-

cal traces are presented in Figs. 1 and 2. The limit of detection (LoD) for all analytesas 0.01 nmol per total sample or less.

.9. Expression of results

Cytotoxicity experiments were carried out in triplicate while the results ofetabolism experiments are based on quadruplicate determinations in EpiDermTM

issues tested at each dose level. The metabolism results were normalized to 100%f the measured parent plus metabolites of either unlabelled PAP or unlabelled PPD,nd expressed as mean ± standard deviation (SD). The d4-labelled components wereot used for quantitation purposes, but served as a tracer for identification and ver-

fication of metabolites. A total mass balance was determined (sum of parent and alleasured metabolites as a percent of nominal parent dose) and the range of values

or media exposure of PPD were 105–150% at 20 �M, 67–103% at 100 �M, 74–82%t 500 �M, and 62–76% at 1000 �M. For the topical PAP exposure the mass balanceanged from 100 to 126% in experiments conducted at 100 �M. The reduced massalance values for PPD at higher dose concentrations are likely due to auto oxidationf the parent compound resulting in dimer and trimer formation and reaction of autoxidation products with other components in the test system (Aeby et al., 2009). Thehemical reaction products resulting from auto oxidation were not measurable withhe methods developed for sample preparation and analysis of metabolites.

e. The MTT assay was used to determine the viability of EpiDermTM preparations254, (�) Donor 4F1188; (b) PPD, 24 h exposure via media: (�) Donor 254, (�) Donorree tissues for each treatment condition.

3. Results

3.1. Cytotoxicity of PAP and PPD following long- andshort-exposure periods

The cytotoxicity of PPD and PAP at various concentrations wastested in three EpiDermTM tissues per concentration from twodifferent donors (Donor 254 and Donor 4F1188). The results indi-cated that PAP was non-toxic after a 24 h topical exposure at allthe concentrations tested (up to 1000 �M, Fig. 3a) in either tis-sue donor. PAP was also non-toxic after a 30 min topical exposureat all the concentrations tested (up to 1250 �M, data not shown)in Donor 254 tissue. In the case of PPD, mild cytotoxicity wasobserved when the tissue was exposed for 24 h via the culturemedia. There appeared to be a small difference in the responseof tissues from the two donors used (tissue viability was 88.2%at 1000 �M PPD when an EpiDermTM tissue from Donor 254 wasused and 72.8% when a tissue from Donor 4F1188 was used), butit is difficult to evaluate the biological significance of this findingbased upon results from only two donors (Fig. 3b). In contrast, nocytotoxicity was observed when EpiDermTM tissue was exposed toup to 2500 �M PPD for 30 min by topical application (Fig. 3c) inDonor 254 tissue. The exposure levels of PAP and PPD expressed asmicrograms per square centimetre of epidermis are summarized inTable 1.

3.2. Reproducibility of aromatic amine metabolism in theEpiDermTM model

In order to evaluate the batch-to-batch and donor-to-donorreproducibility of metabolism in the EpiDermTM skin model, twodifferent shipments (batches) of tissue prepared from one donor(Donor 254) were studied, and one experiment was conductedwith tissue prepared from a different donor (Donor 4F1188). Tis-sues were exposed to PAP (87 �l of 100 �M PAP, applied topically)and PPD (1 ml of 100 �M PPD, added to the culture media) for24 h. Each treatment was evaluated in four tissues from eachdonor/batch, and examples of the results obtained are illustrated inFigs. 4 and 5. In Fig. 4, d4-APAP (m/z = 156) is shown as an extractedion chromatogram. The d-label directly links this metabolite tothe parent material. The levels of PAP and APAP in surface rinse,tissue homogenate and culture media are shown in Table 2. Thelevel of PPD, MAPPD and DAPPD in tissue and media (there is no

surface rinse in this experiment because exposure was via the cul-ture media) are shown in Table 3. These results confirm that bothday-to-day and batch-to-batch reproducibility were excellent whenEpiDermTM constructs from the same donor (Donor 254) were used(Tables 2 and 3). In addition, a consistent profile of penetration

T. Hu et al. / Toxicology Letters 188 (2009) 119–129 123

Table 1Exposure of EpiDermTM constructs to PAP and PPD following topical exposure andexposure via the culture media.

PAP (�M) Volume applied Amount applied Dose/unit area

Topical exposure to PAP100 87 �l 0.96 �g 1.50 �g/cm2

500 87 �l 4.8 �g 7.5 �g/cm2

1000 87 �l 9.6 �g 15.0 �g/cm2

1250 87 �l 12 �g 18.8 �g/cm2

PAP (�M) Volume applied Amount applied Dose/ml

Exposure to PPD via culture media20 1 ml 2.2 �g 2.2 �g/ml100 1 ml 11 �g 11 �g/ml500 1 ml 55 �g 55 �g/ml1000 1 ml 110 �g 110 �g/ml

PAP (�M) Volume applied Amount applied Dose/unit area

Topical exposure to PPD100 87 �l 0.94 �g 1.47 �g/cm2

500 87 �l 4.7 �g 7.34 �g/cm2

1000 87 �l 9.4 �g 14.7 �g/cm2

2500 87 �l 23.5 �g 36.7 �g/cm2

These calculations are based upon the surface area of an EpiDermTM construct(0.64 cm2) assuming that the tissue is exposed to the entire amount of topicallyapplied compound.

Fig. 4. Detection of APAP following topical exposure of EpiDermTM tissue EpiDermTM

skin tissue was exposed to PAP (100 �M, topical) for 24 h. Overlay of extractedion mass chromatograms for d4-APAP (m/z = 156) in media, surface rinse, tissuehomogenate.

Fig. 5. Detection of PPD, MAPPD and DAPPD following basal exposure of EpiDermTM

tissue EpiDermTM skin tissue was exposed to PPD (100 �M in media) for 24 h. Overlayof extracted ion mass chromatograms for PPD (m/z = 109), MAPPD (m/z = 151) and

Table 2Batch-to-batch and donor-to-donor reproducibility of distribution of PAP and APAP follow

Donor 254

PAP (%) APAP (%) Total

Surface rinse 0.00 ± 0.00 18.35 ± 2.20 18.0.00 ± 0.00 19.38 ± 1.93 19.

Tissue homogenate 0.00 ± 0.00 2.71 ± 0.32 2.0.00 ± 0.00 1.70 ± 0.07 1.

Media 0.00 ± 0.00 78.95 ± 2.82 78.0.00 ± 0.00 78.91 ± 2.24 78.

Total 0.00 ± 0.00 100.00 ± 2.73 100.00.00 ± 0.00 100.00 ± 2.34 100.0

The distribution of PAP and its metabolites, expressed as percentage of the total amount asame donor (Donor 254) and in a third batch derived from a different donor (Donor 4F118The separate values reported for Donor 254 represent results from two batches of tissueeach experiment.

DAPPD (m/z = 193) in media and tissue homogenate (there is no surface rinse in thisexperiment because exposure was via the culture media).

and metabolism was observed with PAP in EpiDermTM constructsfrom a different donor (Donor 4F1188; Table 2). This experimentalso indicated that no parent compound was detected in any ofthe samples and that PAP was completely metabolised to APAPwithin 24 h following topical application of 87 �l of 100 �M PAP,equivalent to 1.5 �g/cm2 of epidermis. Approximately 20% of APAPwas found in the surface rinse, the remaining 80% being foundin the culture media; <3% of the APAP generated was retained intissue.

In the case of PPD, the results suggest that there can besmall quantitative differences between donors in the extent ofmetabolism when this compound is applied via the culturemedium, but the results were qualitatively similar (Table 3).Approximately 50–70% was metabolised to acetylated productswithin 24 h following exposure via the culture media with 1 mlof 100 �M PPD, equivalent to 11 �g/ml PPD. The major metabo-lite detected was DAPPD, the ratio of MAPPD:DAPPD being ∼1:3to ∼1:6. The vast majority of acetylated metabolites of PPD (∼97%)were found in the culture media.

The pattern of distribution and metabolism of PPD was con-sistent between the two batches and two donors over theconcentration range 20–1000 �M.

ing 24 h topical application of 87 �l of 100 �M PAP in the EpiDermTM model.

Donor 4F1188

(%) PAP (%) APAP (%) Total (%)

35 ± 2.20 0.00± 0.00

16.88± 5.41

16.88± 5.4138 ± 1.93

71 ± 0.32 0.00± 0.00

2.33± 0.38

2.33± 0.3870 ± 0.07

95 ± 2.82 0.00± 0.00

80.79± 13.60

80.79± 13.6091 ± 2.24

0 ± 2.73 0.00± 0.00

100.00± 8.34

100.00± 8.340 ± 2.34

dded, was determined using two different batches of EpiDermTM derived from the8). EpiDermTM preparations were exposed to aromatic amines as indicated for 24 h.run as separate experiments. The results shown are mean ± SD from four tissues in

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Table 3Batch-to-batch and donor-to-donor reproducibility of distribution of PPD, MAPPD and DAPPD following exposure of EpiDermTM tissues to various concentrations of PPD for 24 h via the media.

PPD (�M) Donor 254 Donor 4F1188

PPD (%) MAPPD (%) DAPPD (%) Total (%) PPD (%) MAPPD (%) DAPPD (%) Total (%)

20 Tissue homogenate 3.84 ± 1.48 3.80 ± 1.30 3.86 ± 0.97 11.5 ± 3.73 0.00 ± 0.00 2.04 ± 0.13 0.00 ± 0.00 2.04 ± 0.130.00 ± 0.00 1.44 ± 0.15 0.00 ± 0.00 1.44 ± 0.15

Media 17.36 ± 2.80 12.29 ± 4.09 58.85 ± 7.28 88.50 ± 10.13 5.63 ± 4.33 3.63 ± 0.47 88.70 ± 4.35 97.96 ± 8.3818.70 ± 2.39 6.15 ± 2.04 73.72 ± 3.08 98.56 ± 1.76

Total 21.20 ± 3.60 16.09 ± 4.80 62.71 ± 7.61 100.00 ± 11.94 5.63 ± 4.33 5.63 ± 0.60 88.70 ± 4.35 100.00 ± 8.3618.70 ± 2.39 7.59 ± 1.98 73.72 ± 3.08 100.00 ± 1.90

100 Tissue homogenate 0.65 ± 0.49 0.37 ± 0.09 1.72 ± 0.32 2.73 ± 0.75 0.44 ± 0.53 0.25 ± 0.08 2.47 ± 0.50 3.16 ± 0.940.42 ± 0.85 0.37 ± 0.27 1.02 ± 0.04 1.81 ± 1.12

Media 52.96 ± 9.20 10.85 ± 2.13 33.45 ± 1.63 97.27 ± 10.42 27.00 ± 1.11 10.26 ± 1.37 59.59 ± 2.93 96.84 ± 2.3144.19 ± 2.28 11.87 ± 2.29 42.13 ± 0.84 98.19 ± 1.10

Total 53.61 ± 8.97 11.22 ± 2.04 35.17 ± 1.62 100.00 ± 9.95 27.44 ± 1.02 10.51 ± 1.39 62.06 ± 3.32 100.00 ± 2.2644.61 ± 2.36 12.24 ± 2.19 43.15 ± 0.87 100.00 ± 0.72

500 Tissue homogenate 2.40 ± 1.02 0.30 ± 0.10 0.26 ± 0.04 2.96 ± 1.13 2.40 ± 0.54 0.48 ± 0.08 0.92 ± 0.07 3.80 ± 0.68Media 75.17 ± 5.94 11.3 ± 0.56 10.34 ± 0.53 97.04 ± 6.17 63.31 ± 1.33 13.74 ± 0.73 19.15 ± 0.29 96.20 ± 0.80Total 77.7 ± 6.45 11.83 ± 0.57 10.60 ± 0.56 100.00 ± 6.74 65.71 ± 1.87 14.22 ± 0.82 20.07 ± 0.36 100.00 ± 0.46

1000 Tissue homogenate 3.63 ± 0.32 0.29 ± 0.02 0.10 ± 0.02 4.02. ± 0.35 5.20 ± 1.22 0.57 ± 0.14 0.44 ± 0.17 6.21 ± 1.522.44 ± 0.28 0.23 ± 0.02 0.12 ± 0.01 2.80 ± 0.30

Media 86.06 ± 2.22 6.76 ± 0.55 3.16 ± 0.20 95.98 ± 2.40 74.65 ± 2.44 9.99 ± 0.49 9.15 ± 0.36 93.79 ± 2.7683.96 ± 3.82 8.32 ± 0.53 4.93 ± 0.15 97.20 ± 4.08

Total 89.69 ± 2.51 7.05 ± 0.55 3.27 ± 0.22 100.00 ± 2.71 79.85 ± 3.66 10.56 ± 0.63 9.59 ± 0.53 100.00 ± 2.8486.41 ± 3.89 8.55 ± 0.55 5.05 ± 0.15 100.00 ± 4.17

The distribution of PPD and its metabolites, expressed as percentage of the total amount added, was determined using two different batches of EpiDermTM derived from the same donor (Donor 254) and in a third batch derivedfrom a different donor (Donor 4F1188). EpiDermTM preparations were exposed to PPD as indicated for 24 h. The separate values reported for Donor 254 represent results from two batches of tissue run as separate experiments.The results shown are mean ± SD from four tissues in each experiment.

T. Hu et al. / Toxicology Letters 188 (2009) 119–129 125

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ig. 6. Recovery of PPD and its acetylated metabolites in media and tissue followioncentrations of PPD for 24 h via the culture media. Culture media and EpiDermTM

ercentage recovery of PPD ( ), MAPPD ( ) and DAPPD (�): (a) recovery in medieparate batches). Data shown are mean ± SD in four tissues for each treatment con

.3. Dose response analysis of PPD and metabolites in media andissue following 24 h exposure via culture media

The dose dependency of PPD metabolism and the profile of dis-ribution in the media and tissue were evaluated from the resultsresented in Table 3 for EpiDermTM tissue preparations from Donor54 (two separate batches) exposed to PPD (20 100, 500 and000 �M) for 24 h via the culture media. The results are presentedraphically in Fig. 6. A similar profile of PPD metabolite distribution

as observed in tissues from Donor 4F188 (Table 3). The major-

ty of PPD was recovered in the culture media; the levels detectedn tissue were <6% of total PPD at all concentrations tested. Theesults of this experiment suggest that the metabolism of PPD is

ig. 7. Recovery of PAP, PPD and their acetylated metabolites in rinse and media followingo various concentrations of PAP or PPD. Chemicals were applied to the tissue surface, leftissue was then allowed to recover for a further 23.5 h, after which the media and EpiDerepresent percentage recovery of PAP or PPD: (a) recovery of PAP ( ) in surface rinse follof APAP (�) in media following 30 min exposure and 23.5 h recovery (recovery of PAP wasxposure (data was obtained from surface rinse for MAPPD and DAPPD, but is not graphed0.01%)); (d) recovery of PPD ( ), MAPPD ( ) and DAPPD (�) in media following 30 minreatment condition. Data was obtained from tissue homogenates but is not graphed bebelow LOD for all except DAPPD).

h exposure via cell culture media EpiDermTM constructs were exposed to variouse were recovered, processed and analysed as described. The data shown representor 254 (two separate batches); (b) recovery in tissue homogenate, Donor 254 (two.

saturable: as the concentration of PPD increased, the proportionrecovered from the culture media as parent compound increasedand the proportion recovered as DAPPD decreased. The proportionrecovered as MAPPD remained fairly constant, consistent with thisbeing an intermediate metabolite. The pattern of recovery of PPDand its metabolites from tissue homogenates was qualitatively sim-ilar to that in the culture media but the levels recovered were verylow. The data suggest that the amount of parent compound retainedin tissue increases with concentration and the amount present as

DAPPD decreases, although the levels detected remain low at all theconcentrations tested.

In addition to the expected acetylated metabolites of PPD,a peak with m/z values of 137/141 (representing the unla-

30 min topical exposure EpiDermTM constructs (Donor 254) were exposed topicallyin contact for 30 min and then rinsed off (the rinse being retained for analysis). ThemTM tissue were recovered, processed and analysed as described. The data shownwing 30 min exposure (recovery of APAP was below the LOD (0.02%)); (b) recoverybelow the LOD (0.02%)); (c) recovery of PPD ( ) in surface rinse following 30 minbecause the level of MAPPD was <0.5% and the level of DAPPD was below the LODexposure and 23.5 h recovery. Data shown are mean ± SD in four tissues for each

cause the levels detected were <0.2% for all analytes at all applied concentrations

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elled and deuterium-labelled forms, respectively) was detectedn the culture media samples following 24 h media exposureo 100–1000 �M of PPD in both live EpiDermTM tissues andilled control EpiDermTM tissues (inactivated by four freeze–thawycles). Accurate mass MS/MS experiments identified the peaks having the formula C7H8N2O, which is structurally consistentith 4-aminophenylformamide. Another peak with m/z values

f 179/183 (representing the unlabelled and deuterium-labelledorms, respectively) was also detected in culture media samplesollected from live EpiDermTM tissues only, not from killed controlpiDermTM tissues, following 24 h media exposure to 100–1000 �Mf PPD. This peak was later identified by accurate mass MS/MSxperiments as having the formula C9H10N2O2, which is struc-urally consistent with N-[4-(formylamino)phenyl]acetamide (i.e.,he N-acetylated derivative of 4-aminophenylformamide) (dataot shown). These peaks could be identified as arising from PPDecause of the use of the deuterated PPD as a metabolic tracer, butere not quantitated due to the lack of a calibration standard. The

onditions under which these peaks were detected indicate non-nzymatic reaction of PPD to form 4-aminophenylformamide inhe culture medium followed by enzyme-mediated N-acetylationf this compound by the EpiDermTM tissue. No other metabolitesf PPD were detected in either the culture media or tissue samples.

.4. Recovery of PAP, PPD and their acetylated metabolites frominse and media following 30 min topical exposure

To study the penetration and metabolism of PAP and PPD underxposure conditions more similar to those relevant to hair dye use,AP (100, 500, 1000 and 1250 �M) and PPD (100, 500, 1000 and500 �M) were applied directly to the surface of EpiDermTM tis-ue, left on the tissue for 30 min, and then rinsed off. The rinseas retained for analysis. The tissues were then incubated for a

urther 23.5 h, after which the culture media and tissues were alsoampled. Under these conditions PPD was not cytotoxic (Fig. 3c).he results of this experiment are illustrated in Fig. 7. It shoulde noted that data from the tissue homogenates has not beenraphed because the values detected were <0.2% for all analytest all applied concentrations (levels were actually below the limitf detection (LOD) for all analytes except for DAPPD, which waslways <0.2%).

In the case of PAP, >90% of the material added was recovereds parent compound in the 30 min surface rinse (Fig. 7a). No APAPas detected from the 30 min surface rinse. In the culture media

sampled after a 23.5 h recovery period), the remaining ∼10% wasecovered as APAP; no PAP parent compound was recovered inhe culture media at this time point (Fig. 7b). These results sug-est that the majority of PAP does not enter skin tissue during a0 min topical application, but remains on the surface as parentompound. All the PAP which does enter the tissue is metabolisedo APAP over the following 23.5 h. This appears to be a highapacity process: there was no evidence for saturation over theoncentration range 100–1250 �M, equivalent to 1.5–18.8 �g/cm2

f epidermis.In the case of PPD, 80–90% of the added material was recov-

red as parent compound in the 30 min rinse. The level of MAPPDas <0.5% and the level of DAPPD was below the limit of detec-

ion (LOD = 0.01%) (Fig. 7c). The metabolism of the PPD that didenetrate through the skin tissue was concentration-dependent:ecovery of unmetabolised PPD in media after a 23.5 h recoveryeriod increased over the range 100–2500 �M (but was always <6%)

Fig. 7d). The proportion of the entire amount of PPD recovereds MAPPD remained fairly constant (0.5–1.2%) over the range of00–2500 �M, consistent with this being an intermediate metabo-ite. It would appear that at 100 �M the acetylation capacity ofhe tissue is sufficient to convert all the absorbed PPD to MAPPD

s 188 (2009) 119–129

or DAPPD so that no parent compound is detected in the cul-ture media. At 500–1000 �M it is sufficient to convert most of theabsorbed PPD to MAPPD or DAPPD so that only approximately 1% ofthe total recovered PPD-equivalents can be detected in the culturemedia as unmetabolised parent compound. However, at 2500 �Mapproximately 5% of the total recovered PPD-equivalents remainsunmetabolised and can be detected in the culture media.

4. Discussion

The aim of the studies presented here was to characterise themetabolism of two aromatic amine hair dye ingredients, PAP andPPD in the EpiDermTM model. The experimental conditions for theinitial studies replicated those used in previous studies with theEpiSkin® model, i.e., 24 h topical PAP exposure to the apical sur-face of the tissue or basal PPD exposure via the culture media(Nohynek et al., 2005). In addition, variability between batches ofEpiDermTM tissue obtained from the same donor and variabilitybetween donors was evaluated in these studies.

The results obtained in these studies demonstrate the repro-ducibility of the EpiDermTM tissue model for studies on the skinmetabolism of aromatic amines such as PAP and PPD. Both day-to-day and batch-to-batch reproducibility were found to be good whenEpiDermTM constructs from the same donor were used. In addi-tion, a consistent profile of metabolism was observed in EpiDermTM

constructs from two different donors.Our results showed complete metabolism of PAP to the N-

acetylated metabolite APAP in EpiDermTM tissue and penetrationof the metabolite into the culture media following a 24 h exposurevia topical application of 100 �M PAP, equivalent to 1.5 �g/cm2 ofepidermis. These results were comparable to those reported pre-viously by Nohynek et al. (2005), who used the EpiSkin® modelto characterise the in vitro biotransformation of PAP. They foundthat PAP was non-toxic to the EpiSkin® keratinocyte model over theconcentration range of 25–1000 �M and selected a concentrationof 100 �M for the study of metabolism in vitro. At this concentra-tion essentially 100% of PAP was metabolised to APAP and foundin the culture media of EpiSkin® preparations [Fig. 3 of Nohyneket al., 2005, “A” should read Culture Medium (instead of Epider-mis) and “B” should read Epidermis (instead of Culture Medium);G. Nohynek, personal communication].

When EpiDermTM tissue was exposed to PPD at various con-centrations (20–1000 �M) for 24 h via the culture media, themetabolism of PPD was found to be saturable: as the concentra-tion of PPD increased, the proportion recovered from the culturemedia as parent compound increased and the proportion recov-ered as DAPPD decreased. The proportion recovered as MAPPDremained fairly constant, consistent with this being an interme-diate metabolite. These results are comparable with those reportedwith the EpiSkin® model in which the extent of metabolism ofPPD also decreased with increasing concentration in the media(Nohynek et al., 2005). Interestingly, Nohynek et al. (2005) alsoreported a peak with m/z value of 137 present in the culturemedia after 24 h incubation of PPD in the presence and absenceof EpiSkin® preparations. Their results as well as ours support theconclusion that this compound, which we have identified is 4-aminophenylformamide, is formed by non-enzymatic reaction ofPPD in the culture media. Thus, the formation of this compoundappears to be an artefact of extended (24 h) incubation of PPD inculture media.

In our subsequent experiments we utilised exposure conditions

more similar to human exposure to hair dyes, i.e., 30 min topicalexposure to the apical surface followed by rinsing. The incuba-tions were then continued for 23.5 h. The total incubation timeof 24 h was chosen to allow for maximal penetration into/throughthe tissue, based on ex vivo dermal penetration studies with pig

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T. Hu et al. / Toxicology

r human skin which indicate that dermal penetration is essen-ially complete after 24 h when PPD or PAP are topically appliednder these conditions (Steiling et al., 2001; SCCP, 2005). To selectoses for these experiments, the results of published in vitro der-al penetration studies with PPD in human and pig skin were

onsidered (Steiling et al., 2001; Hueber-Becker et al., 2004). Fromhese studies the dose to the skin after application of a hair dyeest formulation containing 2% PPD (the maximum concentrationsed in black shade hair dye formulations; concentrations in lighterhades are much lower), can be calculated as the sum of the amountn the stratum corneum, the epidermis/dermis, and the receptoruid (16–37 �g/cm2). Therefore, the dermal doses of PPD applied inur EpiDermTM experiments (1.47–36.7 �g/cm2, corresponding todosing solution of 100–2500 �M) are in a range that is representa-

ive of doses achieved in the skin under conditions that are relevanto hair dye use. The maximum dermal dose we selected for PAP18.8 �g/cm2) was lower because the maximum use concentrationf PAP in hair dye formulations is 0.9%.

After topical application, both PAP and PPD were non-toxic atll doses tested. The results obtained suggested that the majorityf PAP does not enter skin tissue during a 30 min topical applica-ion, but remains on the surface as parent compound. All the PAPhich did penetrate the tissue was N-acetylated and recovered in

he media as APAP. This appears to be a high capacity process; thereas no evidence for saturation over the range of doses tested. In the

ase of PPD, the extent of metabolism was dose-dependent and athe highest dose tested (36.7 �g/cm2, corresponding to 2500 �MPD) approximately 5% of the total recovered PPD-equivalents wasound in the media as unmetabolised compound.

Our results identify N-acetylation as the route of metabolismf PAP and PPD in EpiDermTM tissue. N-acetylation is catalysedy N-acetyltransferase (NAT). Humans have two NAT isozymes,AT1 and NAT2, both of which have the capacity to N-acetylateromatic amines such as PAP and PPD and are known to be poly-orphic (Vatsis et al., 1995; Sim et al., 2007). Although human

kin expresses genes for both NAT1 and NAT2, only NAT1 showsatalytic activity in the skin (Kawakubo et al., 2000). Studies innimal models and human volunteers suggest that after topi-al application aromatic amine hair dye ingredients such as PPDnd PAP are N-acetylated in the skin by NAT1 prior to sys-emic absorption (Nohynek et al., 2004b; Dressler and Appelqvist,006). N-acetylation of PPD in humans after topical applicationf a PPD-containing hair dye was found to be independent ofAT2 genotype (Nohynek et al., 2004b). Therefore, these resultsuggest that NAT2 genetic polymorphisms are not an impor-ant consideration in evaluating the capacity of the EpiDermTM

odel for metabolism of topically applied aromatic amines. Withegard to NAT1 polymorphism, both of the EpiDermTM constructssed in this study were derived from heterozygous donors withhe NAT1*4/NAT1*10 genotype (data not shown). There is as yeto consensus on whether or not NAT1*10 is a rapid variantnd this question remains a topic of ongoing studies (Walravent al., 2008). However, skin cells from individuals with theAT1*4/NAT1*10 genotype should have N-acetylation capability

epresentative of the human population (D. Hein, personal com-unication).The metabolism results obtained for PAP and PPD in these stud-

es with EpiDermTM tissue are consistent with a growing bodyf evidence identifying N-acetylation in the skin as the majoroute of metabolism of PPD and PAP following topical administra-ion. Investigations of the metabolism of PPD by human skin have

emonstrated that PPD is acetylated to both MAPPD and DAPPD

n cytosolic fractions and keratinocytes prepared from human skinnd that this activity is attributable to NAT1 (Kawakubo et al., 2000).tudies of the toxicokinetics and metabolism of PPD and PAP inat and minipig following dermal application show that both of

s 188 (2009) 119–129 127

these compounds are metabolised by N-acetylation (Dressler andAppelqvist, 2006). These investigators reported that DAPPD wasthe only metabolite detected in plasma after dermal exposure toPPD in the rat. Similarly, the only plasma metabolites detectedafter dermal application of PAP were the N-acetylated metabo-lite (acetaminophen) and sulphate or glucuronic acid conjugatesof acetaminophen, which are likely produced by conjugation ofthe N-acetylated metabolite in the liver after formation in theskin and entry into the systemic circulation. Results from clinicalstudies in human volunteers after topical application of PPD alsohave identified N-acetylation as the major route of metabolismof PPD (Goetz et al., 1988; Wang and Tsai, 2003; Nohynek et al.,2004b).

The metabolism of PAP and PPD has also been studied in humanhepatocytes in vitro. As in the skin, hepatic metabolism of PPDinvolves N-acetylation to form both MAPPD and DAPPD (Stanleyet al., 2005; Nohynek et al., 2005). No evidence of CYP-mediatedmetabolism of PPD has been observed in human CYPs (1A1, 1A2,1B1, 2C9, 2C19, 2D6, and 3A4), human hepatic microsomes, orhuman hepatocytes (Stanley et al., 2005). Human hepatocytesmetabolise PAP to sulphate and glucuronic acid conjugates of bothPAP and APAP (Nohynek et al., 2005). We have found no evidenceof CYP-mediated metabolism of PAP in human hepatic microsomes(Skare et al., 2008).

The potential of reconstructed skin epidermis models to reducethe need for animal testing and their advantages in terms of avail-ability, batch-to-batch reproducibility and similarity to human skinhave been recognized for a number of years. An extensive pro-gram of research, supported by the European Commission wasundertaken with the aim of establishing scientific guidelines fortheir routine use in the evaluation of cutaneous irritancy, dermalmetabolism, and percutaneous absorption (Roguet, 2002).

Xenobiotic metabolism in 3D human skin reconstructs hasbeen recently reviewed by Gibbs et al. (2007). Reconstructedepidermis models, including EpiDermTM and EpiSkin®, havebeen demonstrated to exhibit NADPH:quinone reductase activityand glutathione-S-transferase-mediated metabolism of chloro-dinitrobenzene (Harris et al., 2002a; Harris et al., 2002b).7-Ethoxyresorufin-O-deethylase (EROD) activity was below thelevel of detection in all the models unless induced by treat-ment with 3-methylcholanthrene (10 �M for 18 h); the EpiDermTM

model gave the most consistent results in response to 3-methylcholanthrene induction (Harris et al., 2002a). Basal levelsof 7-ethoxycoumarin-O-deethylase (ECOD) activity were exhibitedin reconstructed epidermis models and this was also inducible by3-methylcholanthrene (Harris et al., 2002a). In EpiSkin® the imi-dazoles econazole and clotrimazole induced ECOD activity at lowconcentrations and inhibited at high concentrations (Cotovio et al.,1996). Since studies with recombinant human CYPs and humanhepatocytes have demonstrated no evidence of CYP-mediatedmetabolism of either PPD or PAP (Stanley et al., 2005; Nohynek etal., 2005; Skare et al., 2008), the low levels of CYP enzyme activity innon-induced EpiDerm® tissue do not limit the utility of EpiDerm®

tissue as a model for reflecting relevant metabolism of these aro-matic amines.

Our results add to the characterisation of the metabolic compe-tency of the EpiDermTM model by demonstrating its capacity forN-acetylation of aromatic amines. Regarding other phase II con-jugation enzymes, hepatic metabolism of PAP results in sulphateand glucuronic acid conjugates of both PAP and its N-acetylatedmetabolite, APAP. We saw no evidence of phase II metabolism

to these conjugates in EpiDermTM tissue. Sulphotransferase andUDP-glucuronyltransferase activities have been reported in ex vivohuman skin preparations (reviewed by Gibbs et al., 2007). However,in EpiDermTM tissue (our results) and EpiSkin® tissue (Nohynek etal., 2005) only N-acetylation of PAP has been observed. These results

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28 T. Hu et al. / Toxicology

re consistent with the observations of Dressler and Appelqvist2006) whose in vivo metabolism results in rats and pigs suggesthat topically applied PAP is N-acetylated in the skin so that sys-emic exposure is to the N-acetylated metabolite APAP and not tohe parent compound. Sulphate and glucuronic acid conjugationf APAP then occurs in the liver, resulting in conjugates of APAPut not PAP being detected in plasma. The absence of conjuga-ion of PAP via sulphotransferase and UDP-glucuronyltransferasen EpiDermTM tissue is therefore consistent with the conclusions ofressler and Appelqvist (2006).

With respect to percutaneous absorption, a series of in vitroptake studies indicated that both EpiDermTM and EpiSkin® wereble to predict rank orders of solute permeability correctly, butt was noted that absolute values did not necessarily agree withhose obtained using ex vivo human skin (the reconstructed epider-

is models had greater permeability to xenobiotics) and that smallffects such as those due to changes of vehicle were less predictablesing these models (Dreher et al., 2002a,b; Lotte et al., 2002). Arevalidation study comparing the uptake of the model compoundsaffeine and testosterone by EpiDermTM, EpiSkin® and SkinEthicSchafer-Korting et al., 2006) indicated that lag times for uptakeere shorter and less variable in the reconstructed skin models

han in ex vivo human skin, consistent with the incomplete barrierunction of these models.

Our results with PPD also suggest an incomplete barrier func-ion for the EpiDermTM model since the extent of penetration withhe EpiDermTM tissue is higher than dermal penetration data forPD obtained from studies in human volunteers. The total recoveryf PPD-equivalents in culture media after a 30 min topical expo-ure in the EpiDermTM model was 12.5% (5.32% as PPD, 1.24% asAPPD and 5.93% as DAPPD) when 2500 �M PPD was applied

Fig. 7d). Reported systemic exposure in humans after exposureia hair dye use is much lower. Goetz et al. (1988) reported thathe fraction of PPD-equivalents excreted in urine was 0.04–0.25%f the amount applied during hair dyeing. More recently, Nohynekt al. (2004b) reported that the fraction of PPD-equivalents appliedn a hair dye that was excreted in the urine was 0.43%. The majorxcreted metabolites in the latter study were DAPPD (48.9%) andAPPD (44.6%) (Nohynek et al., 2004b). Unmetabolised PPD was

ot found in the urine, and because the urinary metabolite profileas independent of the NAT2 genotype status, it was concluded that

he urinary metabolites of PPD were most likely due to metabolismy epidermal NAT1 prior to systemic absorption (Nohynek et al.,004b). In contrast, the EpiDermTM results reported here suggesthat some unmetabolised PPD penetrates the skin. However, ithould be noted that, even if a small proportion of PPD does enterhe systemic circulation as the parent compound, this is likely tondergo rapid acetylation via hepatic NAT2 (Nohynek et al., 2005;tanley et al., 2005).

In summary, our studies have evaluated the dermal penetrationnd metabolism of two widely used aromatic amine hair dyes inhe EpiDermTM tissue model. The metabolism results are consis-ent with other in vitro and in vivo data for these compounds whichndicate that NAT1-mediated metabolism in the skin is a majoroute of aromatic amine detoxification following dermal exposure.espite the known fact that reconstructed skin models such aspiDermTM and EpiSkin® have higher permeability for xenobioticshan intact skin, the vast majority of a topically applied dose ofAP or PPD is excluded from skin tissue, consistent with the in vivoituation. Importantly, EpiDermTM exhibited good batch-to-batchnd donor-to-donor reproducibility, and biotransformation perfor-

ance of the tested chemicals was comparable to the EpiSkin®

odel. Additional studies to investigate the expression/activity ofenobiotic metabolising enzymes in the EpiDermTM model com-ared to human skin will be important to further characterise thetility of this model.

s 188 (2009) 119–129

Conflict of interest statement

Five authors (Ting Hu, Ruth Bailey, Stephen Morrall, Julie Skare,and Marilyn Aardema) are employees of the Procter & Gamble Com-pany. The author Lesley Stanley participated as a paid consultantto the Procter & Gamble Company. p-Phenylenediamine and p-aminophenol, the compounds studied in this paper, are currentlyused as ingredients in commercial hair dyes marketed by the Proc-ter & Gamble Company.

The Procter & Gamble Company funded the studies describedin the manuscript. The authors who are employees of P&G wereinvolved in study design; collection, analysis, and interpretation ofthe data; writing the manuscript; and the decision to submit it tothis journal. The author who is a consultant to Procter & Gamblewas involved in analysis and interpretation of the data and writingthe manuscript.

Acknowledgements

The authors thank Mr. Mark Doll (University of Louisville) forperforming NAT1 genotyping. Our thanks go to Drs. Daniel Duche(L’Oreal), Carsten Goebel (P&G/Wella), Stefan Pfuhler (P&G/Wella),Kim Rich (P&G), Heike Scheffler (P&G/Wella), and David Hein (Uni-versity of Louisville) for their technical input and constructivecomments on this manuscript.

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