`
`_.-- .
`ELSEVLE
`
`Available online at wwwsciencedirectcom
`
`sclsnc2@nmec-rs
`
`Regulatory Toxicology and Pharmacology 39 [2004) 27] 281
`
`chylawry
`
`Toxncology and
`Pharmacology
`
`www.e|sevier.cotn!loca1cl'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.a S. Cage,i P.L. Carmichael,“l I. Dick,f
`S. Kenyon,C G. Korinth.l1 F. Larese,C .I.C. Limasset,d W.J.M. Maas,"l L. Montomoli,b
`J .B. Nielsen,g J .-P. Payan,d E. Robinson,f P. Sartorelli,b K.H. Schaller,h
`S.C. Wilkinson,J and EM. WilliamsJ
`
`‘1 TNO Nutrition our! Fraud Research. Zt’ixt‘. The Netherlmtrh'
`h Isn'mto th' Medicine rte! talcum. Sierra. lmlr
`“ Um'rersitn rh‘ Trieste. Italy
`'1 htstimt National dc Recherche ct (Fe Sécnrr'té. Vrmrlncm'rc Cedar. Front-e
`c Biological Chemist”: Faculty of Medicine. Imperial College burden. London. UK
`r Health and Stile-{r hilmrntory. Sheffield. UK
`3 University! tg'Somhern Demimrk, Oricnse, Denmark
`h Unit‘erxi'ry ofErInagar-Nuremberg. Eri'rmgcn. Germany
`i Hittitingrhm Life Science Ltd, Eye. UK
`j The Medical School. Unincrsitr of Newcastle. Newcastle norm Tyne UK
`Received I8 November 2003
`Available online 22 April 2004
`
`Abstract
`
`To obtain better insight into the robustness of ill 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 (1 laboratory) was determined. The test materials were benzoic acid.
`cafl'eine. and testosterone, representing a range of difl'erent 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 human skin. the
`highest ranking of the three compounds (overall mean flux of 16.543: 11.8?' uglcmzi’h}. The absorption of calleine and testosterone
`
`through human skill was similar. having overall mean maximum absorption rates of 2.24 :: [.43 ttgt'cmzt'h and L63 j: 1.94pgi’c1112l'h,
`respectively. In 7 out of 9 laboratories, the maximum absorption rates of eafieine 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 cafieine 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 dilTusion cell type and the ab—
`sorption of the test compounds. Skin thickness only slightly influenced the absorption of benzoie 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 skill 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 be largely attributed to human variability in dermal absorption and the skin source. For the
`most lipophilie compound, testosterone, skin thickness proved to be a critical variable.
`CC) 2004 Elsevier Inc. All rights reserved.
`
`
`' Corresponding author. Fax: +31-30-6960264.
`E—mm‘i’ address: va ndesandtgevoedingtnonl (.l .J. M. van dc Sandi).
`' Present address: Unilever Colworth. Sharnbrook. UK
`_
`.
`0273-23001'3: - see front matter © 2004 Elsevter Inc. All rights reserved.
`doi:lO.lO]6fj,y|-tph,2004_02_004
`0001
`
`Never] Pharmaceuticals, lnC_
`EX2023
`
`Mylan Tech., Inc. v. Noven Pharma, Inc.
`|PR2018—00173
`
`

`

`272
`
`1.}. M. can (It: South at of. f Regut'rttory Toxicofogy and Phartttrtr'ologr 39 {2W4} 2?! 481'
`
`1. Introduction
`
`Reproducible data on percutaneous absorption in
`humans are 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; BBC [99]; 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 difference in skin structure, ann
`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
`biotransformation exist between the oral and dermal
`
`route, excessive first pass effects occur andt'or large dif-
`ferences in rate of absorption exist between the various
`routes of exposure. When no information is 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 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 percutaneous 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. I. Test substances and preparation of dos-e solutions
`
`The test substances were chosen on the basis of their
`
`range in physico-chemical properties (Table I} 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.
`[4-14C]testosterone
`(53.6 mCit‘mmol) and [l-methyl-14C]cafl‘eine (51.2 mCit’
`mmol) were purchased from Perkin—Elmer Life Sci-
`ences, while [ring-UL-“Qbenzoic acid (6.2 mCiimmol)
`was obtained from Sigma Chemical Company. The dose
`solutions were prepared freshly by each laboratory in
`ethanoliwater {1:1, viv), yielding a concentration of
`4.0 mgimL for each compound. Participants with a li-
`cense to handle radiochemicals prepared the dose solu-
`tions by mixing appropriate amounts of radiolabclled
`and non-radiolabelied 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 I
`Test substances
`
`Test substa nee
`
`Benzoic acid (benzenecarboxylic acid)
`Tcsloslcronc (4-:1ndrosten-l TB-ol-3-one]
`Caffeine (3 .T-dihydro- | .3.7-trimcthyl-l H-purinc—lo-dionc)
`
`MW
`l22.1
`288.4
`I942
`
`log Po_.-\v
`1.83
`3.32
`0.01
`
`CAS No.
`65-85-0
`58-22-0
`58-08-2
`
`0002
`
`

`

`J'J. M. mu n'e Sand! (31‘ of.
`
`! Regulatory Toxicology and Pharmacology 39 (2004) EFT—28!
`
`273
`
`] MBqlmL for testosterone and cafleine and approxi-
`mately 4MBqlmL for benzoic acid.
`
`2.4. Experimental design
`
`2.2. Preparation of’slrt‘n 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 —?0 °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—
`1.1 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 I and 7) 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—? skin membranes
`form different individuals. Rat full-thickness skin was
`
`used by participant 5 and was collected from the back
`(clipped carefully) of four weeks old male Sprague
`Dawley rats.
`
`2.3. Diffusion cells and reeept‘orflm‘d
`
`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)+S% Bovine Serum
`Albumin (BSA). adjusted to pH 7.4. For systems using
`flow-through diffusion cells, the flow of receptor fluid
`was approximately l.5 mIJh.
`
`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 diflusion cell and the skin integrity was
`assessed by either visual assessment, permeation of tri-
`tiated water {cut-off KP > 3.5x10‘3cmih) or capaci-
`tance (cut-off: SSnF), depending on the participant.
`Subsequently,
`the test substances were applied at a
`concentration of 4.0mglmL ethanollwater (|:l, viv).
`The application volume was 2511Ucm2 which is con-
`sidered the minimum volume necessary to produce a
`homogeneous distribution on the skin surface. This
`represented a finite dose {100 uglcmz), in order to mimic
`occupationally relevant situations. The exposure time
`was 24 h, 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 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 ethanoll'water (1:1, w’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 p-
`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-moment;
`surgical waste
`
`Sex and age donor
`
`Body site
`
`Skin thickness
`(min)
`
`I. University of Newcastle. UK
`2. Institute 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 Southem Denmark.
`Denmark
`9. University of Erlangen—Nuremberg.
`Germany
`
`It]. Huntingdon Life Sciences. UK 0.3 0.4 5 Post—moment Male. female [40- 72 y] Abdomen, leg
`Participant No. 5 used rat skin.
`
`Surgical waste
`
`Male. female [40- i") y)
`
`Breast. leg
`
`[1.9
`
`
`
`
`
`
`
`0003
`
`Surgical waste
`Post—morlem
`Post—morlcm
`Surgical waste
`
`Female (20 59 y)
`Male [6? 9!! y)
`Male. female (67- 89 y]
`Female (38— 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" | .8
`0.7
`
`0.9
`05—0.?
`[LT—l .|
`
`1?
`(a
`T
`6
`
`3
`3
`22
`
`2
`
`
`
`

`

`274
`
`JIM. [rm de Sand! (’1' a]. I Regulatory THATI'OIOg'I' and Phanrmcoiogt' 39 {2004) EFT—281'
`
`Table 3
`Details of diffusion cell systems
`Diffusion
`Reference
`Participant
`Exposed skin
`Receptor
`cell type
`com pa rtment
`area (cmz)
`0.64
`Volume: 0.25mL:
`stirrer bar: yes
`Volume: 3.3mL:
`stirrer bar: yes
`Volume: lSmL:
`stirrer bar: yes
`Volume: 0.2 mL:
`stirrer bar: no
`Volume: 5.15mL:
`stirrer bar: yes
`Volume: 0.4 mL:
`stirrer bar: no
`Volume: 0.3SmL:
`stirrer ba r‘. yes
`Volume: 17".?Ir mL:
`stirrer bar: yes
`Volume: 5.0 mL:
`stirrer bar: yes
`Volume: 0.25 mL:
`stirrer bar: yes
`
`0.95
`
`3.14
`
`0.64
`
`1.76
`
`0.32
`
`2. l2
`
`0.64
`
`0.64
`
`Clowes et al. ([994)
`
`Reifenrath et al. (1994)
`
`Larese Filon et al. {1999)
`
`Bronaugh and Stewart “985)
`
`-—
`
`Bronaugh and Stewart ([985)
`
`Nielsen and Nielsen (2000)
`
`Franz (197'5)
`
`Clowes et al. “994)
`
`1. University ol‘ Newcastle. UK
`
`F low-throu gh
`
`2. Institute di Medicina deI Lavorn, Italyr
`
`Flow—through
`
`3. Universita di Trieste. Italy
`
`Static
`
`4. TNO Nutrition and Food Research.
`The Netherlands
`5. Institut National de Recherche et de
`Se'curité. France
`6. Imperial College London. UK
`
`Flow—through
`
`Static
`
`Flow-through
`
`'r'. Health and Safety Laboratory. UK
`
`Flow—through
`
`S. University ol‘ Southern Denmark.
`Denmark
`9. University of Erlangen-Nuretnberg.
`Germany
`[0. Huntingdon Life Sciences Ltd.. UK
`
`Static
`
`Static
`
`Flow-through
`
`2.5. Ahab-art's ofnon-rrtdioktheiled rest substances
`
`The analysis of non-radiolabelled test substances in
`the dose solutions and receptor fluid samples was pern
`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
`(pl-I 6)
`(4:6),
`flow 1 er‘min,
`,1. = 229 nm), caffeine {Hypersil ODS column, elucnt:
`methanol:water (1:3), flow 1 mLimin, x". = 2?6 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 i'adior'abet’led rest substances
`
`Radioactivity measuretnents 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 (1.5 M 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-
`plication 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 ol‘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 oft-earths
`
`3.1. Benz-nit: 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 I6.54j: l [.87 ug.’
`cmzih. while the amount in the receptor fluid after 24h
`
`0004
`
`

`

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`11M. van de Sand! er al. I Regulatory Toxicology and Pharmmwlagv 39 {2004) 271—28}
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`

`278
`
`1.1M. mm de Sand! .9! al.
`
`2’ Regulatory Toxicology and Pharmacologv 39 {2004) 271—281
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`13.0
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`

`

`JJ. M. can do Sandi c1 of.
`
`I Regulatory Toxi'coiogv and Pharmacology 39 (2JNJ4) 221—28!
`
`23‘"?
`
`was 70.6i1?.2”/E. of the dose applied (8 laboratories).
`The mean maximum absorption rate of benzoic acid
`through rat skin {1 laboratory} was 21.21 uglemzlh and
`the amount in the receptor fluid after 24h was 39.8%.
`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 ratc varied from 6.3" 0 (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 57.1%
`{lab 2).
`
`3.2. Caffeine
`
`The mean maximum absorption rate of caffeine
`
`through human skin membranes was 2.24 :: 1.43 pgt‘cmh’
`11, while the amount in the receptor fluid after 24h was
`24.5 :t l 1.6% of the dose applied (9 laboratories). The
`mean maximum absorption rate of cafleine through rat
`skin {1 laboratory) was 6.82 tiglcmZI'h and the amount in
`the receptor fluid after 24 h was 53.2%. For both human
`and rat skin, the ratio TPzRF 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 recoveny 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 1.63i 1.94 ug/cmzt'h, while
`the amount
`in the receptor
`fluid after 24h was
`11.8 i10.9% of the dose applied (9 laboratories). The
`mean maximum absorption
`rate of
`testosterone
`through rat skin (I laboratory} was 1.84 pg/cmlfh 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%
`{2 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. On 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.. difierent exposure
`times, dose levels, and vehiclet‘formulations.
`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 difiusion 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 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, cafleine. and benzoic acid) have a
`wide spread in their physico-chemical properties and
`have been recommended as reference compounds by the
`OECD (20000). 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 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 (10011004. for OECD and 100:: 15% for
`SCCNFP). 01‘ the T laboratories that determined mass
`balance, 3 (benzoic acid), 4 (cafleine), and 5 (testoster-
`
`0009
`
`

`

`280
`
`.f.J.M. can dc Sandi at (if. 1 Regulatory Toxit'ofogy and Phammr'ol'ogr 39 {2W4} 2‘?! 178!
`
`one) obtained a mass balance larger than 85" n. 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 poCH'lz). 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 st