`
`Available online at www.sciencedirect.com
`
`science )omnecr-
`
`Regulatory Toxicology and Pharmacology 39 (2004) 271-281
`
`Regulatory
`
`Toxicology and
`Pharmacology
`
`www.elsevier.com/locate/yrtph
`
`In vitro predictions of skin absorption of caffeine, testosterone,
`and benzoic acid: a multi-centre comparison study
`
`J.J.M. van de Sandt,** J.A. van Burgsteden,’ S. Cage,’ P.L. Carmichael,®! I. Dick,‘
`S. Kenyon,° G. Korinth," F. Larese,° J.C. Limasset,? W.J.M. Maas,* L. Montomoli,>
`J.B. Nielsen,® J.-P. Payan, E. Robinson,’ P. Sartorelli,” K.H. Schaller,"
`S.C. Wilkinson,’ and F.M. Williams!
`
`“TNO Nutrition and Food Research, Zeist, The Netherlands
`© Istituto di Medicina del Lavoro, Siena, Italy
`° Universita di Trieste, Italy
`“ Institut National de Recherche et de Sécurité, Vandoeuvre Cedex, France
`* Biological Chemistry, Faculty of Medicine, Imperial College London, London, UK
`" Health and Safety Laboratory, Sheffield, UK
`® University of Southern Denmark, Odense, Denmark
`h University of Erlangen-Nuremberg, Erlangen, Germany
`' Huntingdon Life Science Ltd, Eye, UK
`i The Medical School, University of Newcastle, Newcastle upon Tyne, 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 humanskin (9 laboratories) and rat skin (1 laboratory) was determined. The test materials were benzoic acid,
`caffeine, and testosterone, representing a range of different physico-chemical 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 humanskin, the
`highest ranking of the three compounds(overall mean flux of 16.54 + 11.87 g/cm7/h). The absorption ofcafleine and testosterone
`through humanskin wassimilar, having overall mean maximum absorptionrates of 2.24 + 1.43 ug/em?/h and 1.63 + 1.94 wg/em7/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 onerat study for benzoic acid and testosterone.
`Forcaffeine 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 wasclearly 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 effort was made to standardize the study performance, but, unlike in a formal validation study, not all variables were
`controlled. The variation observed may belargely attributed to human variability in dermal absorption and the skin source. For the
`most lipophilic compound, testosterone, skin thickness proved to be a critical variable.
`© 2004 Elsevier Inc. All rights reserved.
`
`
`* Corresponding author, Fax: +31-30-6960264.
`E-mail address: vandesandt@voeding.tno.nl (J.J.M. van de Sandt).
`' Present address: Unilever Colworth, Sharnbrook, UK
`;
`;
`0273-2300/$ - see front matter © 2004 Elsevier Inc. All rights reserved.
`doi:10.1016/j.yrtph.2004.02.004
`0001
`
`Noven Pharmaceuticals, Inc.
`EX2023
`Mylan Tech., Inc. v. Noven Pharma., Inc.
`IPR2018-01119
`
`

`

`272
`
`J.J-M. van de Sandt et al. | Regulatory Toxicology and Pharmacology 39 (2004) 271-281
`
`1. Introduction
`
`Reproducible data on percutaneous absorption in
`humansare required to predict the systemic risk from
`dermal exposure to chemicals, such as hazardous sub-
`stances at the workplace, agrochemicals, 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 wheneverscientifically feasible. Fur-
`thermore, owing to the difference in skin structure, an-
`imal studies do not always reflect the humansituation.
`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 absenceofreliable 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
`biotransformation exist between the oral and dermal
`route, excessive first pass effects occur and/or large dif-
`ferences in rate of absorption exist between the various
`routes of exposure. When no informationis available on
`percutaneous 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 percutaneous absorption
`measurements. It was concluded that evaluation of in
`vitro test methods from published literature wasdifficult
`(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 (Becket 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 methodologyare 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 10 independent lab-
`oratories from within the European Union, each testing
`the percutaneous absorption of three chemicals that are
`recommended by the OECDassuitable 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 ofthe guidelines proposed
`by the OECD.
`
`2. Materials and methods
`
`2.1. Test substances and preparation ofdose solutions
`
`The test substances were chosen on the basis of their
`range in physico-chemical properties (Table 1) 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, caffeine, 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-'*C]testosterone
`(53.6mCi/mmol) and [l-methyl-'*C]caffeine (51.2 mCi/
`mmol) were purchased from Perkin-Elmer Life Sci-
`ences, while [ring-UL-'*C]benzoic acid (6.2 mCi/mmol)
`was obtained from Sigma Chemical Company. The dose
`solutions were prepared freshly by each laboratory in
`ethanol/water (1:1, v/v), yielding a concentration of
`4.0mg/mL for each compound. Participants with a li-
`cense to handle radiochemicals prepared the dose solu-
`tions by mixing appropriate amounts of radiolabelled
`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
`
`Test substance
`
`Benzoic acid (benzenecarboxylic acid)
`Testosterone (4-androsten-17f-ol-3-one)
`Caffeine (3,7-dihydro-1,3,7-trimethyl-1 H-purine-2,6-dione)
`
`MW
`
`122.1
`288.4
`194.2
`
`log Po/w
`
`1.83
`3.32
`0.01
`
`CAS No.
`
`65-85-0
`58-22-0
`58-08-2
`
`0002
`
`

`

`J.JM. van de Sandt et al. | Regulatory Toxicology and Pharmacology 39 (2004) 271-281
`
`273
`
`1 MBq/mL for testosterone and caffeine and approxi-
`mately 4 MBq/mL for benzoic acid.
`
`2.4. Experimental design
`
`2.2. Preparation ofskin membranes
`
`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,
`Both human and rat skin membranes were prepared
`from frozen skin. Whole skin was cleaned of subcuta-
`preparation of the skin membranes, receptor fluid type,
`occlusion, temperature, sampling times, and number of
`neous fat and the skin was stored at approximately
`replicates were defined. Skin membranes were thawed,
`—20°C (participants 1 and 2 at approximately —70 °C)
`mounted in the diffusion cell and the skin integrity was
`for a maximum period of one year. The supply and use
`of human and animal tissue wasin full accordance with
`assessed by either visual assessment, permeation oftri-
`tiated water (cut-off K, > 3.5 x 10-7cm/h) or capaci-
`national ethical guidelines. Detailed information on the
`tance (cut-off: 55nF), depending on the participant.
`human skin source was recorded (Table 2). Most par-
`Subsequently,
`the test substances were applied at a
`ticipants used humanskin with a thickness between 0.7—
`concentration of 4.0mg/mL ethanol/water (1:1, v/v).
`1.1mm, while one participant used skin that was 0.8-
`The application volume was 25uL/cm? which is con-
`1.8mm. Three laboratories used dermatomed skin with
`sidered the minimum volume necessary to produce a
`a thickness of 0.5-0.7 mm (participants | and 7) or 0.3—
`homogeneous distribution on the skin surface. This
`0.4mm (participant 10). The range of skin thickness
`representedafinite dose (100 pg/cm?), in order to mimic
`used by the various participants allowed for the assess-
`mentof the influence of skin thickness on the absorption
`occupationally relevant situations. The exposure time
`characteristics of the test compounds. Skin from more
`was 24h, during which the donor compartment
`re-
`than one donor was used in each experiment and each
`mained occluded. Aliquots of the receptor fluid were
`experimental group consisted of 5-7 skin membranes
`collected at various time points (minimally at 1, 2, 4, 8,
`form different individuals. Rat full-thickness skin was
`and 24h post-dosing). Forstatic cells, the original vol-
`used by participant 5 and was collected from the back
`ume of the receptor fluid was restored by adding fresh
`(clipped carefully) of four weeks old male Sprague
`receptorfluid to the receptor compartmentdirectly after
`Dawley rats.
`each sampling. In case of non-radiolabelled test com-
`pounds, the receptor 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 ethanol/water (1:1, v/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 KOHin water/ethanol (1:4)] were
`analysed for presence of the test compound by B-
`counting. Each laboratory performed 3-5 independent
`experiments for each test chemical.
`
`2.3. Diffusion cells and receptor fluid
`
`Each participant used the diffusion cell that wases-
`tablished in their laboratory (details are shownin Table
`3). For experiments with caffeine and benzoic acid, the
`receptorfluid 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 systemsusing
`flow-through diffusion cells, the flow of receptor fluid
`was approximately 1.5 mL/h.
`
`Table 2
`Details of source of humanskin
`
`Participant Number of|Post-mortem/ Sex and age donor Bodysite Skin thickness
`
`
`
`
`
`
`
`donors
`surgical waste
`(mm)
`
`17
`6
`7
`6
`
`Surgical waste
`Post-mortem
`Post-mortem
`Surgical waste
`
`Female (20-59 y)
`Male (67-90 y)
`Male, female (67-89 y)
`Female (28-69 y)
`
`Breast
`Leg
`Abdomen
`Abdomen
`
`0.5
`0.7-0.9
`0.8-1.8
`0.7
`
`1. University of Newcastle, UK
`2. Instituto di Medicina del Lavoro, Italy
`3. Universita di Trieste, Italy
`4. TNO Nutrition and Food Research,
`The Netherlands
`0.9
`Abdomen
`Female (29-50 y)
`Surgical waste
`3
`6. Imperial College London, UK
`0.5-0.7
`Abdomen
`Female (26-60 y)
`Surgical waste
`3
`7. Health and Safety Laboratory, UK
`
`
`
`8. University of Southern Denmark, Surgical waste|Female (16-68 y)22 Breast, abdomen 0.7-1.1
`
`Denmark
`
`
`Germany
`
`10. Huntingdon Life Sciences, UK 0.3-0.4 5 Post-mortem Male, female (40-72 y) Abdomen, leg
`Participant No. 5 used rat skin.
`
`9. University of Erlangen-Nuremberg, Surgical waste Male, female (40-79 y)__Breast, leg2 0.9
`
`
`
`
`
`
`
`
`
`
`
`0003
`
`

`

`274
`
`J.J.M. van de Sandt et al. | Regulatory Toxicology and Pharmacology 39 (2004) 271-281
`
`Table 3
`
`Details of diffusion cell systems
`
`Participant
`
`Diffusion
`cell type
`
`Exposed skin
`area (cm*)
`
`Receptor
`compartment
`
`Reference
`
`1. University of Newcastle, UK
`
`Flow-through
`
`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 de Recherche et de
`Sécurité, France
`6. Imperial College London, UK
`
`Flow-through
`
`Static
`
`Flow-through
`
`7. Health and Safety Laboratory, UK
`
`Flow-through
`
`8. University of Southern Denmark,
`Denmark
`9. University of Erlangen-Nuremberg,
`Germany
`10. Huntingdon Life Sciences Ltd., UK
`
`Static
`
`Static
`
`Flow-through
`
`0.64
`
`0.95
`
`3.14
`
`0.64
`
`1.76
`
`0.32
`
`2.12
`
`0.64
`
`0.64
`
`Volume: 0.25 mL;
`stirrer bar: yes
`Volume: 3.5 mL;
`stirrer bar: yes
`Volume: 15mL;
`stirrer bar: yes
`Volume: 0.2 mL;
`stirrer bar: no
`Volume: 5.15mL;
`stirrer bar: yes
`Volume: 0.4mL;
`stirrer bar: no
`Volume: 0.35 mL;
`stirrer bar: yes
`Volume: 17.7 mL;
`stirrer bar: yes
`Volume: 5.0mL;
`stirrer bar: yes
`Volume: 0.25 mL;
`stirrer bar: yes
`
`Cloweset al. (1994)
`
`Reifenrath et al. (1994)
`
`Larese Filon et al. (1999)
`
`Bronaugh and Stewart (1985)
`
`—
`
`Bronaugh and Stewart (1985)
`
`Nielsen and Nielsen (2000)
`
`Franz (1975)
`
`Cloweset al. (1994)
`
`2.5. Analysis of non-radiolabelled test 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:metha-
`nol:phosphate buffer
`(pH 6)
`(4:6),
`flow 1 mL/min,
`2 =229nm), caffeine (Hypersil ODS column, eluent:
`methanol:water (1:3), flow 1 mL/min, 4 = 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 ofradiolabelled test substances
`
`Radioactivity measurements were madeby individual
`participating laboratories. Radioactivity in the various
`samples (receptor
`fluid,
`skin,
`skin swabs, and cell
`washings) was determined byliquid scintillation count-
`ing. Receptor fluid samples were added directly to an
`appropriate scintillation fluid. For analysis of the skin
`membranes, an aliquotofthe tissue digest (1.5 M KOH
`in 20% aqueous ethanol) was used.
`
`time course was constructed from the amount oftest
`substance in the receptor fluid and the maximum ab-
`sorption rate was determined from thesteepest, linear
`portion of the curve. The time to maximum rate,
`the
`percentage of the dose recovered in the receptorfluid 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 +standard deviation, together with the
`coefficient of variation (CV). The presence of the test
`compoundin the skin membrane after washing the ap-
`plication area at 24h was expressed by the ratio between
`the percentage of the dose in skin and receptor fluid
`[total penetration (TP)] and the percentageof the dose in
`receptor fluid (RF).
`
`3. Results
`
`The absorption of caffeine, benzoic acid, and testos-
`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
`
`3.1. Benzoic 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 16.54+ 11.87 pg/
`cm?/h, while the amountin the receptorfluid after 24h
`
`0004
`
`

`

`JLJLM. van de Sandt et al. | Regulatory Toxicelagy and Pharmacology 39 (2004) 271-28]
`
`275
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`

`

`278
`
`Jd. M. van de Sandt et al. | Regulatory Toxicology and Pharmacology 39 (2004) 271-281
`
`
`
`Flux(ug/em’h)
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`Participant no.
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`*
`%,
`%
`
`Fig. 1. Overview of the maximum absorption rates of benzoic acid (grey), caffeine (white) and testosterone (black). ND is not determined.
`
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`Fig. 2. In vitro skin absorption of benzoic acid, expressed as percentage of the dose present in the receptor fluid (grey) or present in the receptor
`fluid + skin membrane(total penetration—white). ND is not determined.
`
`Participant no,
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`Fig. 3. In vitro skin absorption of caffeine, expressed as percentage of the dose present in the receptor fluid (grey) or present in the receptor
`fluid +skin membrane(total penetration—white). ND is not determined.
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`Fig. 4. In vitro skin absorption of testosterone, expressed as percentage of the dose present in the receptorfluid (grey) or present in the receptor
`fluid + skin membrane(total penetration—white). ND is not determined.
`
`0008
`
`

`

`J.JM. van de Sandt et al. | Regulatory Toxicology and Pharmacology 39 (2004) 271-281
`
`279
`
`was 70.64 17.2%of the dose applied (8 laboratories).
`The mean maximum absorption rate of benzoic acid
`through rat skin (1 laboratory) was 21.21 j1g/em?/h and
`the amountin 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 receptorfluid (at 24h),
`the CV values ranged between 1.6%(lab 4) and 57.1%
`(lab 2).
`
`3.2. Caffeine
`
`The mean maximum absorption rate of caffeine
`through human skin membraneswas2.24 + 1.43 p.g/cm?/
`h, while the amountin the receptor fluid after 24h was
`24.5+11.6% of the dose applied (9 laboratories). The
`mean maximum absorption rate of caffeine through rat
`skin (1 laboratory) was 6.82 .g/cm?/h and the amountin
`the receptorfluid after 24h was 53.7%. For both human
`and rat skin, the ratio TP:RF wasonly slightly higher
`than 1.0, indicating that only a small amountcaffeine
`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 24h), 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 1.63+1.94ug/em*/h, while
`the amount
`in the receptor
`fluid after 24h was
`11.8+10.9% of the dose applied (9 laboratories). The
`mean maximum absorption
`rate of
`testosterone
`through rat skin (1 laboratory) was 1.84 g/em7/h 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 24h), 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. On the other hand, the
`guidelines allow for certain flexibility in order to study
`compounds with widely
`differing physicochemical
`properties and undercircumstances which are the most
`relevant for its use, resulting in e.g., different exposure
`times, dose levels, and vehicle/formulations.
`In the
`OECD guidance document (OECD 2000c), useful
`in-
`formation is provided on howto 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 1.0mm (epidermis
`and dermis) is not recommended and mustbejustified
`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, caffeine, and benzoic acid) have a
`wide spread in their physico-chemical properties and
`have been recommendedas reference compoundsby the
`OECD (2000c). The studies were performed according
`to a very detailed protocol. Two participants were GLP-
`compliant while the other laboratories adhered to this
`quality system as much aspossible. Analysis of samples
`from studies using non-radiolabelled test compounds
`was performed centrally in order to limit analytical
`variation and data analysis ofall 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 (L00+10% for OECD and 100415%for
`SCCNFP). Of the 7 laboratories that determined mass
`balance, 3 (benzoic acid), 4 (caffeine), and 5 (testoster-
`
`0009
`
`

`

`280
`
`JIM. van de Sandt et al. | Regulatory Toxicology and Pharmacology 39 (2004) 271-281
`
`The absorption of caffeine and testosterone through
`one) obtained a massbalance larger than 85%. The most
`humanskin wassimilar, having overall mean maximum
`probable cause of the low recovery observed in some
`absorption rates of 2.24 and 1.63 1g/cm?7/h, respectively.
`cases is the technical difficulty of evenly spreading the
`small volume of the dose solution on the skin surface
`In 7 out of 9 laboratories,
`the maximum absorption
`(25uL/cm?). It may be that part of the dose solution
`rates of caffeine were ranked higher than testosterone.
`The absorption differences between the compoundsfit
`may have adhered to the pipet tip and therefore was not
`well in with those reported in human volunteers, where
`applied to the skin. It is important to mention that for
`the absorption of benzoic acid was found higher than
`regulatory studies most often very small volumes should
`that of caffeine and testosterone (Bronaugh and Franz
`be applied which are relevant for the in-use situation: 2—
`5mg/cm* for solid and semi-solid preparations and
`1986). It should be noted, however, that the concentra-
`10L/em? for liquids (OECD 2000a; SCCNFP 2003).
`tions used in the in vivo study were not the same for
`each compound, which makes a direct comparison of
`Although information on the mass balance is often
`the ranking to ourresults difficult.
`lacking in the open literature, we present these data in
`One of the participating laboratories tested the ab-
`order to give a complete overview of the sources of
`sorption of compounds throug