`
`"
`‘
`ELSEVIER
`
`Available online at www.5ciencedirect.com
`
`solch:@DlnncT®
`
`Regulatory Toxicology and Pharmacology 39 (2004) 271—281
`
`Regulatory
`
`Tomcology and
`Pharmacology
`
`wwwelseviercomtlocaletyrtph
`
`In vitro predictions of skin absorption of cafl‘eine, testosterone,
`and benzoic acid: a multi-centre comparison study
`
`J.J.M. van de Sandt,“"" J.A. van Burgsteden,“ S. Cage,‘ P.L. Carmichaelf“ I. Dick}
`S. Kenyon,E G. Korinth,h F. Larese,C .I.C. Limasset,d W..I.M. Maas.a L. Montomoli,b
`J.B. Nielsen,g J.-P. Payan,d E. Robinson,f P. Sartorelli,b K.H. Schallcr,h
`S.C. Wilkinson,J and EM. WilliamsJ
`
`" TNO Nntrt’tt’on and Food Research. Zrt‘st. The Netherlands
`b h'titnto (ti Mt'di't'inn (let Lorin-o, Sirntt. Hair
`‘ bittrcrsitit (ti Trieste.
`t'tttift'
`" lnstitnt Notional rte Recherche ct rte Sét'nrt'té. Vtttttlneni're Center. France
`° Biological Clienn'sn‘y. Faculty ochdi'r'itte. Ititperinl College London. London. UK
`I“ Heatth and Safety Ltthortttor't‘, Sheffield, UK
`g Li’rn'cerst'ty ofSrntthern Denmark, Onion-r. Denmark
`h Unircrst'tyofErlmtgcn—Nuremberg, Eri‘rtngen. Germany
`i Hitntingdon Lite Sr'ienrc Ltd, Eyc. UK
`1 The Medical School. University of Ncli'r'nstlt’. Nt'ii‘tw‘tle upon Tune, UK
`Received 18 November 2003
`
`Available online 22 April 2004
`
`Abstract
`
`To obtain better insight into the robustness of in vitro percutaneous absorption methodology. the intra- and inter-laboratory
`variation in this type of study was investigated in 10 European laboratories. To this purpose, the in vitro absorption of three
`compounds through human skin {9 laboratories} and rat skin [I laboratory) was determined. The test materials were benzoic acid.
`caffeine, and testosterone. representing a range of different physicoehemical properties. All laboratories performed their studies
`according to a detailed protocol in which all experimental details were described and each laboratory performed at least three
`independent experiments for each test chemical. All laboratories assigned the absorption of benzoic acid through human skin. the
`highest ranking of the three compounds {overall mean flux of 16.54i [1.87 tigfcmzih). The absorption of call'eine and testosterone
`through human skin was similar. having overall mean maximum absorption rates ol‘2.24 :I: 1.43 uglemth and 1.63 :I: 1.94 tigt'cmzt'h.
`respectively. In 7 out of 9 laboratories, the maximum absorption rates of caffeine were ranked higher than testosterone. No dif-
`ferences were observed between the mean absorption through human skin and the one rat study for benzoic acid and testosterone.
`For calleine the maximum absorption rate and the total penetration through rat skin were clearly higher than the mean value for
`human skin. When evaluating all data. it appeared that no consistent relation existed between the diffusion cell type and the ab-
`sorption of the test compounds. Skin thickness only slightly influenced the absorption of benzoic acid and caffeine. In contrast, the
`maximum absorption rate of testosterone was clearly higher in the laboratories using thin, dermatomed skin membranes. Testos—
`terone is the most lipophilic compound and showed also a higher presence in the skin membrane after 24h than the two other
`compounds. The results of this study indicate that the in vitro methodology for assessing skin absorption is relatively robust. A
`major elTort was made to standardize the study performance. but, unlike in a formal validation study. not all variables were
`controlled. The variation observed may be largely attributed to human variability in dermal absorption and the skin source. For the
`most Iipophilic compound. testosterone. skin thickness proved to be a critical variable.
`© 2004 Elsevier Inc. All rights reserved.
`
`‘ Corresponding author. Fax: +31-3n-69ans4.
`E—iimi‘l cttldrt‘ss: vandesandthgvoedingtnonl [J.J,M, van de Sandi).
`' Present address: Unilever Colworth. Sharnbrook. UK
`
`,
`_
`0273-23006 - see front matter © 2004 Elsevier Inc. All rights reserved.
`dot;tummyynphgomnzmtt
`0001
`
`Noven Pharmaceuticals, Inc.
`
`EX2023
`
`Mylan Tech., Inc. v. Noven Pharma, Inc.
`|PR2018—00174
`
`

`

`272
`
`J. J. M. can rile Sandi or al.
`
`4’ Regulatory Toxicologl' and Pharmacologv 39 (2004; 2?! 281
`
`1. Introduction
`
`Reproducible data on pcrcutaneous absorption in
`humans are required to predict the systemic risk from
`dermal exposure to chemicals, such as hazardous sub-
`stances at the workplace, agrochemieals, and cosmetic
`ingredients (EC 2002; EEC 1991; SCCNFP 2003).
`In
`this context, there is a need for reliable in vitro models
`
`since the European Union advocates this approach and
`national legislation stipulates that animal experiments
`should be avoided whenever scientifically feasible. Fur-
`thermore, owing to the dilference in skin structure, an-
`imal studies do not always reflect the human situation.
`Absorption through the skin is the primary route
`of exposure for most pesticides both occupationally
`(Benford et al. 1999) and in residential settings (Ross
`et al. 1992). Despite the often relatively high dermal
`(and inhalation) exposure in occupational settings, reg-
`ulations for pesticides and other chemical exposure have
`evolved from concern about the oral route of exposure.
`In the absence of reliable dermal absorption data, route—
`to-route extrapolation has been used to assess dermal
`risk. it should be noted that this extrapolation is not
`always straightforward in cases when differences in
`biotransforrnation exist between the oral and dermal
`
`route, excessive first pass effects occur andl‘or large dif-
`ferences in rate of absorption exist between the various
`routes of exposure. When no information is available on
`pcrcutaneous absorption, risk assessments may assume
`an absorption percentage of 100%, a worst case scenario
`(EC 2002). This is a very conservative approach and a
`more accurate measure of absorption would have a
`major impact on risk assessments for many chemicals in
`regulatory toxicology. The specific need for a valid
`method of assessing human dermal absorption has led
`the OECD (2000a,b,c) and EPA {1996, 1999) to produce
`guidelines for in vitro and in vivo assessment of percu-
`taneous absorption.
`A review of available data from published literature
`on in vitro dermal absorption was performed under the
`auspices of the OECD in order to evaluate the perfor-
`mance of in vitro and in vivo pcrcutaneous absorption
`measurements. It was concluded that evaluation of in
`
`vitro test methods from published literature was difficult
`(OECD 2000d) because studies containing direct com-
`parisons of in vitro and in vivo measurements were
`very limited. There were too many variables, such as
`different species, thickness and types of the skin, expo-
`
`sure duration, and vehicles. Also, very few multi-centre
`studies have been performed (Beck et al. 1994) and these
`studies were limited in their approach (e.g.._ with respect
`to the number of laboratories involved). Therefore, no
`proper data on the intra~ and inter-laboratory repro—
`ducibility of the in vitro methodology are available.
`The purpose of the present research was therefore to
`assess intra- and inter-laboratory variability in deter-
`mination of percutaneous penetration by in vitro
`methods on a larger scale than done previously. This
`report contains data generated by [0 independent lab-
`oratories from within the European Union. each testing
`the pcrcutaneous absorption of three chemicals that are
`recommended by the OECD as suitable reference com-
`pounds for regulatory studies (OECD 2000c). The ex-
`perimental conditions (amount applied, exposure time,
`vehicle, receptor fluid, preparation of membranes, and
`analysis) were standardized according to a detailed
`protocol that adopted many of the guidelines proposed
`by the OECD.
`
`2. Materials and methods
`
`2. 1. Test substances and preparation (y'dose solutions
`
`The test substances were chosen on the basis of their
`
`range in physico-chemical properties (Table l} and their
`recommendation as reference compounds by the OECD
`(OECD 2000c). All participating laboratories used the
`same batches of test substances. Non-radiolabelled tes-
`
`tosterone, calfeine, and benzoic acid were purchased
`from Steraloids
`(Newport, RI, USA} and Sigma
`Chemical Company by the study coordinator and were
`then supplied to the participants.
`[4-‘4C]testosterone
`(53.6 mCilmmol) and [l—methyl—“Ckalfeine (51.2 mCil
`mmol) were purchased from Perkin—Elmer Life Sci-
`ences, while [ring-UL-14C]benzoic acid (6.2 mCilmmol)
`was obtained from Sigma Chemical Company. The dose
`solutions were prepared freshly by each laboratory in
`ethanoll‘water (1:1. vl'v), yielding a concentration of
`4.0 mgme for each compound. Participants with a li-
`eense to handle radiochemicals prepared the dose solu-
`tions by mixing appropriate amounts of radiolabellcd
`and non—radiolabelled test substances. The dose solu-
`
`tions were measured for exact total radioactivity prior to
`and directly after the application to the skin membranes.
`The
`radioactive
`concentration was
`approximately
`
`Table ]
`Test substances
`
`Tcsl substance
`
`Benmic acid (benmnecarboxylic acid]
`Tesloslerone (4—;mdroslen— | 70-01—3—one)
`Cafl‘eine (3.T—dihydro—l.3.T—t|'il11ethyl—1H—purine—2,6—dione)
`
`MW
`llll
`383.4
`194.3
`
`log Posw
`1.83
`3.32
`".01
`
`CAS No.
`65—854}
`58—2241
`58403—1
`
`0002
`
`

`

`J.J.M. tam dc Sand! er (if. ! Regulator]: Toxicology and Plitrrmnmlogi' 39 {2004} 2?} 281
`
`Is.) ‘-.1DJ
`
`lMqu‘mL for testosterone and cafleine and approxi-
`mately 4MBqlmL for benzoic acid.
`
`2.4. Experimental design
`
`2.2. Preparation qfskin membranes
`
`Both human and rat skin membranes were prepared
`from frozen skin. Whole skin was cleaned of subcuta-
`
`neous fat and the skin was stored at approximately
`—20 °C (participants 1 and 2 at approximately —70 °C)
`for a maximum period of one year. The supply and use
`of human and animal tissue was in full accordance with
`
`national ethical guidelines. Detailed information on the
`human skin source was recorded {Table 2). Most par-
`ticipants used human skin with a thickness between 0.7—
`].1 mm, while one participant used skin that was 0.8—
`1.8 mm. Three laboratories used dermatomed skin with
`
`a thickness of 0.5—0.7 mm (participants 1 and T) or 0.3—
`0.4mm {participant 10). The range of skin thickness
`used by the various participants allowed for the assess-
`ment of the influence of skin thickness on the absorption
`characteristics of the test compounds. Skin from more
`than one donor was used in each experiment and each
`experimental group consisted of 5—7 skin membranes
`form different individuals. Rat full-thickness skin was
`
`used by participant S and was collected from the back
`{clipped carefully) of four weeks old male Sprague
`Dawley rats.
`
`2.3. Dt'flitst'on cells and receptor fluid
`
`Each participant used the diffusion cell that was es-
`tablished in their laboratory (details are shown in Table
`3}. For experiments with cafleine and benzoic acid, the
`receptor fluid consisted of saline {0.9% NaCl), while for
`experiments with
`testosterone,
`the
`receptor
`fluid
`consisted of saline (0.9% NaCl}+ 5% Bovine Serum
`Albumin (BSA), adjusted to pH 7.4. For systems using
`flow-through diffusion cells, the flow of receptor fluid
`was approximately 1.5 leh.
`
`All participating laboratories performed their studies
`according to a detailed study protocol in which the ex-
`perimental design and parameters such as the dose of
`the test chemical, vehicle, duration of the experiment.
`preparation of the skin membranes, receptor fluid type,
`occlusion, temperature. sampling times, and number of
`replicates were defined. Skin membranes were thawed.
`mounted in the diffusion cell and the skin integrity was
`assessed by either visual assessment, permeation of tri-
`tiated water (cut~ofl‘ KP > 3.5 x [0‘3cmi'hl or capaci-
`tanee (cut-off: 55 nF), depending on the participant.
`Subsequently,
`the test substances were applied at a
`concentration of 4.0mg/mL ethanolr'water (1:1, vlv).
`The application volume was 25 ttLl’cm2 which is con-
`sidered the minimum volume necessary to produce a
`homogeneous distribution on the skin surface. This
`represented a finite dose (100 ugfcmz), in order to mimic
`occupationally relevant situations. The exposure time
`was 24h, during which the donor compartment
`re—
`mained occluded. Aliquots of the receptor fluid were
`collected at various time points (minimally at l, 2, 4, 8,
`and 24h post-dosing). For static cells, the original vol-
`ume of the receptor fluid was restored by adding fresh
`receptor fluid to the receptor compartment directly after
`each sampling. In case of non-radiolabelled test com-
`pounds, the reeeptor fluid samples were stored at ap—
`proximately —20°C until analysis. At
`the end of the
`experiment, the test compound remaining at the appli»
`cation site was
`removed, using five cotton swabs
`dampened with ethanoll'water (l : l, vr‘v), followed by one
`dry cotton swab. When a radioactive test compound was
`used, the cotton swabs, donor compartment rinse,
`re—
`ceptor compartment rinse, and skin membranes [after
`digestion with 1.5 M KOH in waterlethanol (1:4)] were
`analysed for presence of the test compound by B-
`counting. Each laboratory performed 3—5 independent
`experiments for each test chemical.
`
`Table 2
`Details of source of human skin
`
`Participant
`
`Number of
`donors
`
`Post-mortetn,-"
`surgical waste
`
`Sex and age donor
`
`Body site
`
`Skin thickness
`(mm)
`
`I. University of Newcastle. UK
`2. lnstitulo di Medicina del Lavoro. Italy
`3. Universita di Trieste. Italy
`4. TNO Nutrition and Food Research.
`The Netherlands
`6. Imperial College London. UK
`7. Health and Safety Laboratory. UK
`8. University of Southern Denmark,
`Denmark
`9. University of Erlangen-Nut‘emberg.
`Germany
`10. Huntingdon Life Sciences. UK
`
`Participant No. 5 used rat skin.
`
`[7
`6
`”l
`6
`
`3
`3
`22
`
`2
`
`5
`
`Surgical waste
`Post—monern
`Post-inorlem
`Surgical waste
`
`Female (20—59 y)
`Male (6'?- 90 y)
`Male, female (Eli—89 y)
`Female (28 69 y)
`
`Breast
`Leg
`Abdomen
`Abdomen
`
`Surgical waste
`Surgical waste
`Surgical waste
`
`Female (29- 50 y)
`Female (26 60 y)
`Female ([6 68 y)
`
`Abdomen
`Abdomen
`Breast. abdomen
`
`0.5
`0.? 0.9
`0.8—1.8
`0.7
`
`0.9
`0.5- 0.?
`0.? LI
`
`Surgical waste
`
`Male. female (40—79 y)
`
`Breast. leg
`
`0.9
`
`Post-mortem
`
`Male. female (40—72 y)
`
`Abdomen. leg
`
`0.3—0.4
`
`0003
`
`

`

`274
`
`J. .l. M.
`
`t-‘ml' rle Semlr er (fl.
`
`2‘ Regttl(l'i‘ur_l-‘ Toxicologt' and Pharmacologr 39 [2004; 235’ SM
`
`Table 3
`Details of diffusion cell systems
`
`Participant
`
`1. University of Newcastle, UK
`
`Ditl‘usion
`cell type
`
`Flow-through
`
`Exposed skin
`area (cmz)
`0.64
`
`2. Instituto di Medicina del Lavoro. Italy
`
`Flow-through
`
`3. Universita di Trieste. Italy
`
`Static
`
`4. TNO Nutrition and Food Research.
`The Netherlands
`5. Institut National dc Recherche et de
`Se'cttrité. France
`6. [mperial College London. UK
`
`Flow-through
`
`Static
`
`Flow—through
`
`”t. Health and Safety Laboratory. UK
`
`Flow—through
`
`8. University of Sottthern Denmark.
`Denmark
`9. University of Erlangcn-Nuremberg.
`Germany
`It). Huntingdon Life Sciences Ltd. UK
`
`Static
`
`Static
`
`Flow—through
`
`0.95
`
`3.14
`
`0.64
`
`1.76
`
`[1.32
`
`2.12
`
`0.64
`
`[1.64
`
`Receptor
`compartment
`Volume: 0.25mL:
`stirrer har: yes
`Volume: 3.5mL;
`stirrer bar: yes
`Volume: ISmL;
`stirrer bar: yes
`Volume: 0.2 tnL:
`stirrer bar: no
`Volume: 5.15mL:
`stirrer bar: yes
`Volume: 0.4mL:
`stirrer bar: no
`Volume: 0.3SmL;
`stirrer bar: yes
`Volume: 17.7mL:
`
`stirrer bar: yes
`Volume: 5.0tnL;
`stirrer har: yes
`Volume: 0.25mL:
`stirrer bar: yes
`
`Reference
`
`Clowes el al. “994)
`
`Reifenrath et al. ([994)
`
`Larese Filon et al. (1999)
`
`Bronaugh and Stewart (1985)
`
`—
`
`Bronaugh and Stewart (1985}
`
`Nielsen and Nielsen (2000)
`
`Franz (I915)
`
`Clowes el al. “994)
`
`2.5. Analysis of 11'tilt-i'flt’llolflbe'lle‘d res! substances
`
`The analysis of non-radiolabelled test substances in
`the dose solutions and receptor fluid samples was per»
`formed centrally: benzoic acid by the Health and Safety
`Laboratory (UK), caffeine by the University of Trieste
`(Italy), and testosterone by TNO Nutrition and Food
`Research (The Netherlands). Established protocols were
`used for the HPLC-UV analysis of benzoic acid {Phe-
`nomenex column, SphereClone ODS (2), eluent:methan
`nol:phosphate buffer
`(pH 6)
`(4:6),
`flow llemin,
`2222‘) nm), cafleine (Hypersil ODS column, eluent:
`methanol:water (1:3). flow I mUmin, x“. = 276 nm), and
`testosterone (according to Bogaards et al. 1995). The
`amount of non-radiolabelled test substance was not
`determined in the skin tissue and therefore total recov-
`
`ery values were not calculated.
`
`2.6. Analysis of t'adt'olabelled rest substances
`
`Radioactivity measurements were made by individual
`participating laboratories. Radioactivity in the various
`samples (receptor
`fluid, skin, skin swabs, and cell
`washings) was determined by liquid scintillation count-
`ing. Receptor fluid samples were added directly to an
`appropriate scintillation fluid. For analysis of the skin
`membranes, an aliquot of the tissue digest (1.5M KOH
`in 20% aqueous ethanol) was used.
`
`time course was constructed from the amount of test
`
`substance in the receptor fluid and the maximum ab-
`sorption rate was determined from the steepest, linear
`portion of the curve. The time to maximum rate, the
`percentage of the dose recovered in the receptor fluid in
`24h,
`the percentage in the skin membrane, and the
`percentage total recovery (for radiolabelled studies) was
`also calculated. The data of each laboratory were pre-
`sented as mean :t standard deviation, together with the
`coefficient of variation (CV). The presence of the test
`compound in the skin membrane after washing the ap»
`piication area at 24 h was expressed by the ratio between
`the percentage of the dose in skin and receptor fluid
`[total penetration (TP)] and the percentage of the dose in
`receptor fluid (RF).
`
`3. Results
`
`The absorption of caffeine. benzoic acid, and testes—
`terone through the skin was defined on the basis of
`
`maximum absorption rate, time to maximum rate, per-
`centage dose recovered in the skin membrane (at 24h
`post-dosing), and percentage dose recovered in the
`receptor
`fluid (at 24h post-dosing). The results of
`individual
`laboratory measurements
`are
`shown in
`Tables 4—6 and overviews of the mean values are given
`in Figs. 1—4.
`
`2. 7. Calculation of results
`
`3. l. Benz-ore acid
`
`The calculations were performed using a standardized
`Excel spreadsheet prepared by the study coordinator. A
`cumulative amount absorbed per unit skin area versus
`
`The mean maximum absorption rate of benzoic acid
`through human skin membranes was l6.54i 11.87 ug/
`cmllh, while the amount in the receptor fluid after 24 h
`
`0004
`
`

`

`1.1M. van de Sand! 9! a}. I Regtdafwy Toxic-ofogy and Pharmm‘oiogy 3 9 {2004) 2?1—281
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`I Regulatory Toxicofogy and Pharmacology 39 (2004) 2?} £81
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`

`

`J.J.M. arm dc Swirl! er (:1. I Regulatory Tdvi't'ologL‘ and Pflflrlilflt‘0f(}g_l' 3 9 {26104.1 2?1' 28}
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`27“)
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`was 70.6 i 17.2% of the dose applied (8 laboratories}.
`The mean maximum absorption rate of benzoic acid
`through rat skin {1 laboratory) was 21.21 ngi‘cmzlh and
`the amount in the receptor fluid after 24h was 89.8%.
`For both human and rat skin,
`the ratio TP:RF was
`
`approximately 1.0, indicating that almost no benzoic
`acid remained in the skin membrane after washing the
`application area. The total recovery of the radioactivity
`ranged between 53.6 and 98.5% (7 laboratories).
`Each laboratory performed 3—5 independent experi-
`ments. The coefficient of variation (CV) of the maxi-
`mum absorption rate varied from 6.3% (lab 4) to 52.2%
`(lab 2). For the percentage in the receptor fluid (at 24 h),
`the CV values ranged between 1.6% (lab 4) and 52.1%
`(lab 2).
`
`3. 2. Caffeine
`
`The mean maximum absorption rate of caffeine
`through human skin membranes was 2.24 :1: 1.43 ng/cmzl'
`h. while the amount in the receptor fluid after 24h was
`245111.694: of the dose applied {9 laboratories). The
`mean maximum absorption rate of caffeine through rat
`skin (1 laboratory) was 6.82 pglcmli'h and the amount in
`the receptor fluid after 24h was 53.7%. For both human
`and rat skin, the ratio TP:RF was only slightly higher
`than 1.0, indicating that only a small amount caffeine
`remained in the skin membrane after washing the ap-
`plication area. The total recovery of the radioactivity
`ranged between 66.4 and 100.6% [7 laboratories).
`Each laboratory performed 3—5 independent experi-
`ments. The CV value of the maximum absorption rate
`varied from 12.0% (lab 5) to 91.4% (lab 1). For the
`percentage in the receptor fluid {at 24 h), the CV values
`ranged between 5.4% (lab 5) and 66.0% (lab 1).
`
`3. 3. Testosterone
`
`The mean maximum absorption rate of testosterone
`through human skin was 1.63il.94 uglcmzl'h, while
`the amount
`in the receptor
`fluid after 24h was
`11.8:t 10.9% of the dose applied (9 laboratories). The
`mean maximum absorption
`rate
`of
`testosterone
`through rat skin (1 laboratory) was 1.84 ug/cmzih and
`the amount in the receptor fluid after 24h was 21.4%.
`For both human and rat skin, the ratio TP:RF ranged
`between 1.35 and 3.54, indicating that a considerable
`amount testosterone remained in the skin membrane
`
`after washing the application area. The total recovery
`of the radioactivity ranged between 52.3 and 103.5%
`(7 laboratories).
`Each laboratory performed 3—5 independent experi-
`ments. The CV value of the maximum absorption rate
`ranged from 6.3% (lab 7) to 111.0% (lab 8). For the
`percentage in the receptor fluid (at 24 h), the CV values
`ranged between 12.6% (lab 7) and 111.7% {lab 8).
`
`4. Discussion
`
`The presence of international guidelines has led to a
`partial standardization of in vitro skin absorption
`studies for regulatory purposes. 0n the other hand, the
`guidelines allow for certain flexibility in order to study
`compounds with widely
`difl‘ering physicochemical
`properties and under circumstances which are the most
`relevant for its use, resulting in e.g., diflerent exposure
`times, dose levels, and vehiclelformulations.
`In the
`OECD guidance document (OECD 2000c), useful
`in-
`formation is provided on how to properly design in vitro
`and in vivo skin absorption studies. Both static and
`flow-through diffusion cell types are considered suitable.
`In order to prevent underestimation of skin absorption,
`the test compound should be soluble in the receptor
`fluid, but the receptor fluid should not alter the barrier
`properties of the skin membrane. Skin membranes can
`be prepared in various ways. but the use of skin mem-
`branes with a thickness of more than 1.0 mm (epidermis
`and dermis) is not recommended and must be justified
`by the researcher, since the absorption of lipophilic
`compounds may be impeded by a thick dermis. This
`guidance has been proved useful for both investigators
`in the laboratory and for regulatory agencies which
`evaluate this type of data for risk assessment purposes.
`Only very limited data exist on the intra-laboratory
`and inter-laboratory variation of in vitro skin absorp—
`tion studies. In 1994, Beck et al. reported a good cor-
`relation of in vitro absorption of hair dyes through full-
`thickness pig skin in 2 laboratories. Recently, using an
`artificial (silicone rubber} membrane, the intra-labora-
`tory and inter-laboratory variation of methyl paraben
`absorption was assessed in 18 laboratories [Chilcott
`et al. submitted). In their study, the CV values between
`laboratories were approximately 35%, while the intra-
`laboratory variation averaged 10%.
`In the study presented here, the in vitro absorption of
`three compounds through human skin {9 laboratories}
`and rat skin (1 laboratory) was investigated. The com-
`pounds (testosterone, cal’feine, and benzoic acid) have a
`wide spread in their physico-chemical properties and
`have been recommended as reference compounds by the
`OECD (2000c). The studies were performed according
`to a very detailed protocol. Two participants were GLPn
`compliant while the other laboratories adhered to this
`quality system as much as possible. Analysis of samples
`from studies using non-radiolabelled test compounds
`was performed centrally in order to limit analytical
`variation and data analysis of all laboratories was car-
`ried out according to a study—specific Excel spreadsheet.
`The total recovery of the radioactivity at the end of the
`experiment was not always as high as required by the
`guidelines (1003: 10% for OECD and 100:1: 15% for
`SCCNFP}. Of the 7 laboratories that determined mass
`balance, 3 (benzoic acid}, 4 (caffeine), and 5 (testoster-
`
`0009
`
`

`

`380
`
`J.J.M. van de Sand! er of.
`
`4’ Regulatory Toxicologt' min“ Pfim'rimeoi’ogr 39 (2004; 2?! 281
`
`one) obtained a mass balance larger than 85%. The most
`probable cause of the low recovery observed in some
`cases is the technical difficulty of evenly spreading the
`small volume of the dose solution on the skin surface
`
`(25 uLlcmz). It may be that part of the dose solution
`may have adhered to the pipet tip and therefore was not
`applied to the skin. It is important to mention that for
`regulatory studies most often very small volumes should
`be applied which are relevant for the in~us