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Journal of Dermatological Science 56 (2009) 8998

Contents lists available at ScienceDirect

Journal of Dermat

e ls1. Introduction

Ultraviolet (UV) radiation is well known for exerting a variety ofdeleterious effects on human skin. Chronic and acute exposure toUV radiation increases the risk of developing skin cancer includingcarcinoma and malignant melanoma; furthermore UV radiationcauses skin aging and erythema. In recent decades scientists havewarned that the thin layer of ozone that protects life on earth fromthe suns ultraviolet radiation is being depleted. This allows moreUV radiation to get through the atmosphere, increasing the risk ofoverexposure. Consequently, there has been an increasing demandto protect the human skin against UV exposurewhich has led to thedevelopment of technologically advanced sunscreens.

Epidemiologic studies have demonstrated that sunscreens donot always prevent skin photo-damages since several factors mayaffect their efcacy, such as sweating, rubbing, water immersion

and photodegradation [1,2]. Therefore tests that simulate the realconditions of sunscreen use aremandatory, notably photo-stabilityand water resistance measurements.

In Europe, the ofcial and internationally accepted method forsunscreen tests is the in vivo SPF method which is based on theCOLIPA guidelines [3]. The in vivo method is expensive and timeconsuming; therefore it would not be suitable for routinely qualitycontrol in the event that such tests are compulsory.

In the last years, two main aspects of sunscreen tests have beeninvestigated: rening a method to assess SPF by in vitro tests andstandardizing a test procedure for UVA protection labelling.Recently COLIPA has published the guidelines for in vitro UVAprotection determination [4].

A sunscreen in vitro test is based on absorbance (calculatedfrom transmittance) or reectance measurements. It requires thedenition of both the instrumentation to be used and themeasurement procedure including substrate selection and relativeapplication criteria of the sunscreens. With regard to theinstrumentation, the absorption spectrum is the most relevantparameter for dening the protective performance of a sunscreen

Article history:

Received 13 December 2008

Received in revised form 16 July 2009

Accepted 23 July 2009

Keywords:

Sunscreen

In vitro tests

UV radiation

Photo-stability

Background: In vitro measurements of sunscreen products are used to assess their reliability in terms of

photoprotection and photo-stability.

Objective: In this study, several substrates have been fully characterized for in vitro sunscreen testing.

Methods: The following different substrates have been utilised in the study: roughened PMMA plates,

Transpore, Vitro-Skin, roughened quartz plate and a laboratory prepared roughened PTFE plate. The

suitability of these substrates for SPF evaluation has been investigated by performing total absorbance

measurements of seven sunscreen products with different SPF values produced by the same

manufacturer.

Results and conclusions: Application of sunscreen products on Transpore, roughened quartz plate, PMMA,

PTFE and Vitro-Skin was performed efciently. With regard to photo-stability of the substrate materials,

only PMMA plate, PTFE and roughened quartz plate showed to be photo-stable after UV irradiation.With

regard to SPF tests, our results indicate that Transpore, roughened quartz plate and Vitro-Skin are

preferable to assess SPF values because of a better correlation between in vitro and in vivomeasurements.

Our study also conrms that an initial calibration must be performed for sunscreen products labelled

with different SPF values. Finally, the results of our measurements demonstrate that, although a

correlation between in vitro and in vivo SPF results can be established, it is never exactly 1:1.

2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rightsreserved.

* Corresponding author.

E-mail address: mauro.alaibac@unipd.it (M. Alaibac).

0923-1811/$36.00 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jdermsci.2009.07.015Effectiveness of different substrate mat

Denis Garoli a,b, Maria Guglielmina Pelizzo a,b, PierElena Tonin d, Mauro Alaibac c,*aCNR-INFM LUXOR via Gradenigo, 6B, 35131 Padua, Italyb Information Engineering Department, University of Padua, Via Gradenigo, 6B, 35131cUnit of Dermatology, University of Padova, Via Battisti 206, 35128 Padua, ItalydMelanoma Unit, National Institute of Oncology, IOV, Via Gattamelata 21, 35128 Padu

A R T I C L E I N F O A B S T R A C T

journal homepage: www. int l .ials for in vitro sunscreen tests

rgio Nicolosi a,b, Andrea Peserico c,

a, Italy

aly

ological Science

ev ierhea l th .com/ journa ls / jods

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 899890product. The resulting curve gives two important attributes: theamplitude of the transmittance curve that reects the degree ofprotection and the shape of the curve that underlines the capabilityof the sunscreen to protect the different spectral regions. Asalready demonstrated [57], the use of a decient instrumentationcan be responsible of discrepancies in absorbance measurementsand, hence, in SPF evaluation. Moreover, to perform test on highSPF products, a spectrophotometer with a sufcient dynamicalrange is essential. With regard to the substrate, it should haveoptical and physicalchemical properties close to those of the skin.At present, however, a standard material has not yet been dened.Most commonly adopted substrates are: roughened PMMA plates,Transpore, Vitro-Skin and roughened quartz plate. Studiesperformed using excised human stratum corneum and excisedhuman epidermis have been reported, but the biological nature ofthese materials introduces signicant practical problems. Asalready reported, sunscreen tests performed on different sub-strates are not always reproducible [8], especially when thesubstrate/sunscreen is irradiated before or during the tests. Otherphysical effects related to the coupling between substrate andsunscreen can account for the discrepancy among tests performedon different materials. For instance, the roughness of the substrateand the uniformity of the application of the product can beresponsible of signicant differences [9,10].

With regard to the technique and the amount of sunscreenapplication, measurements performed on a single type of substratechanging the quantity of the product have been reported in manystudies [7,11]. While in vivo test guidelines impose the uniformapplication of 2 mg/cm2 of product quantity on the substrate, thereis not a clear indication for in vitro SPF analyses. Goodcorrespondences between in vitro and in vivo SPF data have beenreported by using different rates of application (for instance 1, 1.2or 0.5 mg/cm2) [1113]. For in vitro UVA protection tests COLIPAguidelines recommend an amount of 0.75 mg/cm2. Moreover, ithas been demonstrated that the correlation between in vitro and invivo data depends not only from the quantity of the appliedproduct, but also from the formulation of the sunscreens [14].

The objective of this investigation has been to test differentmaterials as substrates for in vitro sunscreen tests and comparetheir performances in order to obtain a complete overview of theircharacteristics. Full systematic characterization of the substrateshas been performed in term of optical transmittance andreectance properties. Extensive photo-stability tests have beenperformed by exposing the substrate to different spectralwavebands. Suitability for SPF evaluation has been investigatedby performing total absorbance measurements on seven sunsc-reens. Results have then been correlated with the values declaredby themanufacturers. Evaluation of both photo-stability andwaterresistance of the sunscreens has not been performed as it was outof the scope of the study.

2. Materials and methods

2.1. Substrates

The following ve substrates have been utilised in the study:Transpore, Vitro-Skin, roughened quartz plate and roughenedPMMA plates, which are commonly used in several laboratories,and PTFE plate which has the properties of being very cheap,transparent to UV radiation and easy to be prepared [15].

The main characteristics of the substrates utilised in the studyare the following:

- Transpore1 (3M Company Health Care, Maine, USA) is a surgicaltape. It has been initially proposed as a readily available andinexpensive substrate [16], but now it is used less frequently. It isusually attached on a smooth quartz plate in order to obtain ahard support for an easier appliance of the sunscreen.

- Vitro-SkinTM (IMS Inc.) is a synthetic skin substrate that must beused following a specic hydration procedure. Published dataindicate that Vitro-Skin gives very good performances forsunscreen tests [17]. Nonetheless, the use of this materialpresents some disadvantages, notably a relatively high cost persample, the need to hydrate the substrate starting the day beforethe test and short lifetime of the hydrated sample. Both hydratedand not hydrated samples have been characterized in order toassess possible differences.

- Roughened quartz is largely used because of its high UVtransmittance. Its main disadvantage is the high cost, althoughit can be re-used following an appropriate cleaning procedure.

- PMMA plates are considered as a rst choice for UVA in vitrotesting [4]. It is very easy to handle and can be supplied with areproducible roughness. Suntest PMMA plates of 5 cm 5 cmhave been purchased from SCHONBERG GmbH (Munich,Germany). The plates have been cut from a large sheet of UV-stabilised Plexiglas1 and roughened on one side by an industrialsandblasting procedure (glasspearls, 90150mm, 30 cm, 6 bar).The roughness of our plates, as declared by the manufacturer,was about 2 mm, but PMMA plates with different roughness arealso available. Two additional reference plates have beenpurchased from the same manufacturer; the absorbance valuesmeasured by our instrumentation of the plates covered with aglycerol lm resulted in perfect agreement with the valuesreported in literature. Since it is known that transparency ofPMMA plate increases when a lm of product is deposited on theroughened surface, both PMMA plates alone or covered by aglycerol lm have been considered as substrates for transmissionmeasurements.

- Teon (PTFE) has been extensively used [18] for UV applicationdue to its good transmittance and its Lambertian properties.Samples of 7 cm2 area have been prepared in our laboratory froma standard sheet (0.8 mm thickness) previously treated with ablast sanding process in order to get a roughened surface. The useof PTFE as a substrates for sunscreen evaluation test has beenrecently proposed by our group [15].

Microscopic images of the substrates compared with an invivo skin image are reported in Fig. 1. These images have beenrealized by illuminating the samples with white light and byacquiring the reected images. As it can be observed, themorphology of the substrates is very different among thematerials. This affects the chemical and physical couplingbetween sunscreens and substrates. In particular, roughness ispartially responsible for the different optical properties. The RMS(roughness measurement system) surface roughness of thedifferent substrates has been measured using a Tencor 40Tprolometer. The values are derived from the mean roughnessmeasured on ve different samples. The results of thesemeasurements, with the relative error, are reported in Table 1.It is evident that there is a good reproducibility on the measuredroughness. The values are compared with the RMS roughnessobtained on a silicone mould of in vivo skin.

2.2. Spectro-photometric measurements

All substrates have been characterized by transmittance andreectance measurements (total and diffuse). Measurements havebeen performed with a Varian Cary 5000 UV/VIS/NIR (ultravioletvisiblenear infrared) spectrophotometer using a UV/VIS/NIRintegrating sphere. The instrument spans a spectral wavelengthrange from 175 up to 3300 nm. Its optical system ensures amaximum level of light throughput that produces accurate

e of

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D. Garoli et al. / Journal of Dermatological Science 56 (2009) 8998 91Table 1RMS surface roughness of the different materials tested.

RMS surface roughness (mm)In Vivo Skin 16.3 0.2Transpore1 7.30.5Vitro-SkinTM 16.1 0.3Roughened quartz 1.80.1PMMA plates 2.60.1PTFE 4.7 0.1

Fig. 1. (20 optical zoom) (a) In vivomicroscopic image of skin; (b) microscopic imagof not-hydrated Vitro-Skin; (e) microscopic image of roughened quartz; (f) microsmeasurements at low transmission levels (up to 9 Abs). Moreover,with its variable and xed slits the spectrophotometer allowsoptimum control over data resolution (the spectral bandwidth canbe set down to 0.01 nm). In this experiment, spectra were recordedfrom 290 up to 400 nm using a spectral resolution and a datainterval of 1 nm. The samples were placed into the experimentalchamber at the entrance of the integrating sphere for thetransmittance analyses and at the exit port of the sphere for thereectance measurements. Two different measurement cong-urations have been adopted: one for the diffuse reectancemeasurements with the detector of the integrating spherecollecting only the diffuse part of the reected light; the otherone for the total reectance measurements, in which the detectorcollects both the specular and the diffuse reected light, in case of anormal incidence angle of 88. Additional measurements withoutthe integrating sphere have been performed in order to get theregular transmittance. In all cases, the sample has been illuminatedbymonochromatic radiation. For every sample tested three sampleareas (each of approximately 10 mm 10 mm) have beenmeasured.

Table 2Sunscreens main ingredients.

SPF 4 SPF 6

Bis-ethylhexyloxyphenol methoxyphenyl triazine

Butyl methoxydibenzoylmethane X X

Disodium phenyl dibenzimidazole tetrasulfonate X X

Ethylhexyl methoxycinnamate

Ethylhexyl triazone X X

Octocrylene

Titanium dioxide2.3. Photo-stability measurements of the substrate

Each substrate has been tested in order to assess its photo-stability under UV/vis/IR radiation. To the best of our knowledgethis type of characterization has never been reported before.Different samples for each material considered have been exposedby using an ORIEL 300 W full spectrum solar simulator equippedwith air mass 0 lter. It emits a uniform, collimated,50 mm 50 mm output beam with a close spectral match tosunlight. This simulator produces amaximumpower equivalent to2 suns, but with optional air mass lters it is possible to simulatevarious solar conditions and altitudes. When the sample has beenexposed to full UV + vis + IR spectrum, a WG 295 Edmund band-pass lter has been used to block contaminating wavelengthsbelow 290 nm. In order to expose samples only to vis + IRradiation, a GG 395 Melles Griot band-pass lter has been usedto cut all the wavelengths below 395 nm. In both cases, thesubstrates have been exposed for 1, 3, 5 and 10 min.

Transpore 3M; (c) microscopic image of hydrated Vitro-Skin; (d) microscopic image

c image of roughened PMMA plate; (g) microscopic image of one PTFE sample.The full spectrum exposures have been repeated by using othertwo standard band-pass lters: WG 305 and WG 320. They cut allthe wavelengths below 300 and 315 nm respectively.

The UV output irradiance (in presence of theWG295 lter) hasbeen measured by an HD9021 calibrated radiometer (DeltaOhmS.r.l.), equipped with two detector heads, one for UVB 280315 nm spectral region and another for UVA 315400 nm one.Both the UVB and UVA irradiances have been measured, beingthe results values 1.8 and 9.5 mW/cm2 respectively. Anerythemal irradiance of 0.5 mW/cm2 has been measured by anerythema like radiometer [19], to which correspond to a dose of3, 9, 15 and 30 SED for the four different exposure times

SPF 8 SPF 12 SPF 15 SPF 20 SPF 30

X X X X X

X X X X X

X X

X

X X X

X X X X

respectively. These doses are in accordance with values alreadystandardized by our group [20].

The erythemal irradiance has been measured also with the WG305 and WG 320 lters, given 0.51 and 0.08 mW/cm2. In order toexpose the substrates to the same doses, the duration of exposure

forWG305 has been xed to 1, 3, 5 and 10 min, while forWG320 ithas been xed to 6, 18, 30, 60 min.

From data provided by Oriel, an output irradiance of about200 mW/cm2 in the spectral range 4002500 nm has beenrecovered.

n T

-H

;

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 899892Fig. 2. (a) Transmittance measurements on Transpore; (b) reectance measurements oHyd. Sample Diffuse T; 3. Hyd. Sample Not-Diffuse T; 4. Not-Hyd. Sample Total T; 5. Not

hydrated Vitro-Skin: (1.Hyd. Sample Total R; 2.Hyd. Sample Diffuse R; 3.Hyd. Sample

88 R); (e) T% of roughened quartz; (f) R% of roughened quartz; (g) T% of PMMA plateranspore; (c) T% of hydrated and not hydrated Vitro-Skin: (1. Hyd. Sample Total T; 2.

yd. Sample Diffuse T; 6. Not-Hyd. Sample Not-Diffuse T); (d) R% of hydrated and not

88 R; 4.Not-Hyd. Sample Total R; 5.Not-Hyd. Sample Diffuse R; 6.Not-Hyd. Sample (h) R% of PMMA plate; (i) T% of PTFE (Teon); (j) R% of PTFE.

2.4. Total absorbance/transmittance measurements on sunscreens

Seven sunscreen products produced by the same manufacturerlabelled with different SPF protection level (from 4 up to 30). Themethod could be in principle extended to whatever SPF; it ishowever important to have instruments with high signal-to-noiseratio, because transmission of sunscreens with SPF50+ is really low.This measurement should be performed with a double gratingspectrophotometer (similar to the spectrophotometer used for thiswork), and not with a single grating spectrophotometer. The mainingredients of the sunscreens, as derived fromthe label, are reportedin Table 2. Only one sunscreen formulation (i.e. lait) was used. Eachproduct has been applied on each substrate material. Only onesunscreen formulation from one producer has been applied to each

case of a smooth transparent substrate with negligible surface/volume scattering, such as a polished plate of quartz. However, thisis an unrealistic situation because a smooth surface would notallow sunscreen adherence.

re 3M attached on polished Quartz and (b) reectance.

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 8998 93substrate at a time, in order to maintain the same chemicalcomposition and remove then the effects of possible differentchemical reactions. Sunscreen formulations have been appliedwithout UV pre-irradiation. The application of the product has beenrealized by spotting the sunscreen on several points of the substratewith a pipette, and by uniformly distributing it with a nger glove.After the application, the volatile compounds the product has beenallowed to evaporate. The lm of product applied on the substratehas been weighed with an analytical balance (max error< 0.2 mg)after the drying process; the weight has been recorded in order toevaluate possible dependences of the quantity evaporated from thesubstrate used. In all cases during the drying process the sunscreenlost about 4060% of its weight, and meaningful differences havebeen registered among different substrates. The amount of productinitially applied on the substrates corresponded to 1 mg/cm2whichhas been demonstrated to allow a good correlation between SPFvalues evaluated in vivoand in vitro. Total absorbance/transmittancemeasurements have been carried out using the apparatus alreadydescribed. The curves A(l) = Log T(l) =Log[1/PF(l)] [16] havebeen obtained by the ratio between the transmittance curvesmeasured with and without the sunscreen applied respectively. Inorder to increase statistic, for each sunscreen formulation severalsamples (about 10 for each formula) have been prepared and eachsample has been tested in three different areas to overcomepossibleerror due to non-uniformity of the product application.

2.5. In vitro Sun Protection Factor computation and correlation with

value reported on labels

The absorbance curves have been used to compute the SPFaccording to the following denition:

SPF P400

290 ElBlP400

290 ElBl 10Al

where E(l) is the spectral irradiance of the standard sun [21],B(l) is the erythema action spectrum. The nal in vitro SPF valueshave been calculated as the mean SPF values computed with the

Fig. 4. (a) Transmittance measurements on Transpodifferent A(l) curves measured, while the error bars associatedrepresent the standard deviation from the mean value.

3. Results and discussion

3.1. Transmittance and reectance measurements on substrates

Regular and diffuse total transmittance and reectance havebeen measured for all substrates (Fig. 2). Vitro-Skin not hydratedand PMMA covered by glycerine have also been investigated.

All tested materials had a certain amount of total reectance; itis important to considered that the back reected beam interactsagain with the sunscreen when applied. In particular, in case ofPTFE the reectance is higher than the transmittance. Thesubstrates presented different diffuse properties. While quartzand PMMA diffuse components were due only to the supercialroughness, in case of the othermaterials themain contributionwasdue to a volume scattering. PTFE was the only material with anegligible regular component. Transpore diffuse transmissionrepresented about 73% of the total transmission in the spectralrange considered, Vitro-Skin about 70%, roughened quartz plateabout 82% and PMMA plate about 80%. These optical propertieswere responsible of a very complex optical path of light in thesunscreensubstrate coupled system, which does not allow torecover the absolute transmittance curve of the sunscreen alone.As a matter of fact, if the absorbance signal of the referencesubstrate is subtracted from the absorbance signal of thesunscreensubstrate, the results do not correspond to thetransmittance of the sunscreen layer; this would be true only in

Fig. 3. Total transmittance measurements on PMMA plate, with or withoutglycerine applied.

The optical properties of hydrated and not-hydrated samples ofVitro-Skin have been investigated in order to verify presence ofdifferences. The not-hydrated sample was suitable as a substrate,nonetheless it must be underlined that hydrating the substrate isnecessary in order to break down sunscreen emulsion in a similarway as in human skin [14,22]. The shape of transmittance andreectance curves was not affected by hydrating the substrate.However, an increase in transmittance, with a consequentdecreasing in reectance values, was observed in the hydratedsample. With regard to PMMA covered with glycerine, a smalldifference in the shape of the transmission curve was found whencompared with substrates without glycerine (Fig. 3). Nevertheless,it must be underlined that PMMA treated with glycerine is onlyused to recover the transmission of the plate without appliedsunscreen; therefore the impact of this difference is only in the SPF

data acquired; the reproducibility of the values has been foundwithin 10%. The absorbance values give the respective transmit-

nd (b) full spectrum photo-stability of not hydrated Vitro-Skin.

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 899894evaluation.Many authors do not use self-standing Transpore during their

measurement, since it breaks down easily, but they prefer to attachit on a smooth quartz plate in order to support it. We have alsocharacterized this composed system as reported in Fig. 4.

3.2. Photo-stability tests on substrates

In our study, we investigated for the rst time the photo-stability of substrate materials alone. The photo-stability of thesubstrates is fundamental to correctly understand the outcome ofexperiments, and some of the controversial outcomes can beexplained by the instability of the substrates [7].

All substrates investigated have been exposed under vis + NIR(visiblenear infrared) and full UV + vis + NIR (ultravioletvisiblenear infrared) radiation. In the case of full spectrum exposure thetests have been repeated by using three different low wavelengthband-pass lters. In three cases the same dose of exposure hasbeen used. Here we report the data relative only to the exposurewith the WG 295 lter. The results obtained with the other twolters were perfectly compatible.

The outcome is that PMMAwithout glycerine, Teon and quartzplates resulted photo-stable both to full spectrum exposure and tovis + IR spectral range irradiance. This fact suggests that thesesubstrates are suitable for sunscreen photo-stability testing. Incontrast, Transpore and hydrated Vitro-Skin are not suitable forsuch test.

Transpore resulted stable only to vis + IR exposure but not tofull spectrum exposure (Fig. 5). Therefore, we can assume that UVradiation alters Transpore properties. The transmittance variationwas mainly concentrated in the UVB part of the spectral rangewhich is an UV region that strongly affects the SPF computation;themaximumdifference of transmittance absolute valuewas 8% at310 nm for 10 min of exposure. This result can partially explainsome results reported in the literature using this substrate for UV

Fig. 6. (a) Full spectrum photo-stability of hydrated Vitro-Skin ain vitro testing. To this regard, Gers-Barlag et al. [6] demonstratedsignicant differences in their protection factor results usingquartz plates or Transpore, especially when the applied productwas irradiated before or during the tests. They also demonstratedthat Transpore is inappropriate for such tests because of anunspecied interaction between sunscreen lters and the sub-strate.

Vitro-Skin has been seldom used for photo-stability sunscreentests [23]. When exposed to either vis/IR or full spectrumirradiation, the hydrated samples of Vitro-Skin underwent atremendous damaging. In particular, its hydrating componentevaporated rapidly wrinkling the sample. Moreover, the transmit-tance curve of the sample changed rapidly during the exposure asillustrated in Fig. 6a. The not-hydrated sample did not undergo avisible damaging when exposed to vis/IR irradiation, but whenexposed to full spectrum radiation it resulted not photo-stable(Fig. 6b), with a maximum difference of transmittance absolutevalue of 10% at 300 nm for 10 min of exposure. Therefore, alsoVitro-Skin should not be used for photo-stability measurements.

In Fig. 7 the curves of absorption for each substrate considered,calculated as A = 1 R T where R and T are the total reectanceand transmittance as in Fig. 3, are reported. It is interesting tonotice that both Vitro-Skin and Transpore absorb UV radiationmainly in the spectral region in which they are photo-unstable.

3.3. Total absorbance/transmittance measurements of sunscreen

Each substrate has been tested with the application of sevendifferent sunscreen products. The total absorbances A(l) arereported in Fig. 8. The curves are calculated as the mean of several

Fig. 5. Full spectrum photo-stability of Transpore (3M).

tance values spanning from 0.001 up to 0.99 (0.199%). In order torecord correctly the low values, it is essential to use a spectro-photometer with an high dynamic range, as the case of a doublemonochromator system. The absorbance values A(l), and equiva-lently, the PF(l) values showed a high degree of variation amongsubstrates. This result is consistentwith several studies. Pearse and

Edwards [8], using human stratum corneum and Transpore assubstrates for SPF evaluation, observed that in 10 out of 11sunscreens studied the SPF values measured with Transpore weresystematically higher than those obtained using human stratumcorneum. In another study, Lott et al. [9] adopting three differentsubstrates obtained signicant differences in the SPF values; theseresults have been related to the non-uniformity of productapplication. Other physical effects related to the coupling betweensubstrate and sunscreen can partially account for such discre-pancy, as suggested by Ferrero et al. [10], who showed a correlationbetween substrates roughness and SPF outcomes in case of PMMAplates.

We have analyzed the variability in term of absorbance valuesby using different substrates as follow. Lets try to design theinteraction between two media, A (sunscreen) and B (thesubstrate), and light taking into account different physical effects.If the two media have both smooth faces and low scatteringproperties, the transmitted radiation path is straight forwardwhilepart of radiation is straight back reected (Fig. 9a). If medium A isdiffusing, both the in-coming radiation and the back reected atinterface are then scattered in all directions, and part of the last oneis again re-directed in the substrate (Fig. 9b); if the substrate has aroughened surface, as in case of PMMA plates and roughened

Fig. 7. Absorption, computed as A = 1 T R, of the different substrates.

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 8998 95Fig. 8. Sunscreen absorbance measurements on the seven sunscreens tested. (a) Sunschydrated Vitro-Skin; (c) sunscreen applied on roughened quartz; (d) sunscreen applied

Transpore.reen applied on Transpore attached on polished quartz; (b) sunscreen applied on

on PMMA; (e) sunscreen applied on PTFE; (f) sunscreen applied on self-standing

um

um

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 899896Fig. 9. Schematic visualization of light interaction into two differentmaterials. (a) Medidiffusing; (c) medium A volume diffusing and medium B surface diffusing; (d) mediquartz plate (Fig. 9c), the radiation at the interface is back-scattered in all directions. Amore complicated situation happens ifbothmedia are volume diffuser, as in case of sunscreens applied onPTFE, Vitro-Skin and Transpore (Fig. 9d); in this case, it is verydifcult to realize a model to predict the optical path of radiationand the measurement results.

As it can be seen in Fig. 8 and in detail for selected sunscreens(SPF 4 and 12) in Fig. 10, the absolute value of the absorbance varyconsiderably among substrates; this is also because, according towhat explained, the amount of back reected radiation withrespect to the transmitted radiation is very different. Besides thesephysical aspects related to surface/volume scatting of light,another fundamental event that can affect the result is thedifferent chemical interaction of sunscreen with substratematerials. A further possible physical effect that can justify the

Fig. 10. Sunscreen absorbance curves comparison. The curves represent theabsorbance values measured on different substrates for the same sunscreen.A andmediumB not-diffusing; (b)mediumA volume diffusing andmediumB not-

A and medium B volume diffusing.differences is the uorescence. The spectrophotometer used forour absorbancemeasurements has not lters on the input after theintegrating sphere to block out sunscreen product uorescence.This could alter the absorbance spectrum. In order to check foruorescence effect, additional measurements of sunscreen absor-bance have been performed by introducing different interferentiallters between the sunscreen/substrate and the integratingsphere. In all these cases the results were compatible with thoseobtained without the lters. This conrms that uorescence doesnot affect the measured absorbance values. Moreover, thechemical composition of the sunscreens tested has been analyzedand no uorescence ingredients have been found.

Due to all the effects described, the absorbance curves A(l)recovered by the measurement of the substrates with and withoutthe sunscreen were very different among them both in shape andabsolute value. For these reasons, absorbance and reectancemeasurement curves are not meaningful by itself for labelling,unless they are used to calculate parameters that correlates to invivo measurements or they are used only to make a relativecomparison between different products. Generally, the trendamong different SPF productswasmaintained, except in the case ofthe product labelled SPF 20 when applied on Transpore (Fig. 8a)and product labelled SPF 15 when applied on PMMA (Fig. 8d). Inthese cases, the transmittance curves intersected those of productswith higher or lower SPF values. These curves have beenmeasureddifferent times and results have been always conrmed. In the caseof Transpore, a chemical reaction between sunscreen and thesubstrate could be the cause of this result.

Some measurements have been performed also on Transporeself-standing. As already mentioned, the application of the creamon this substrate is hard to perform due to the fragility of the tape.Results show that, in comparison with Transpore on quartz, theshape of the curves was maintained, but the values of transmit-

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 8998 97tance were 10 times higher (Fig. 8f). Therefore, with lowperformance instrumentation, self-standing Transpore should bechosen, but if the dynamic range of the instrument is good enoughTranspore on quartz should be preferred.

3.4. In vitro determination of SPF: correlationwith the values declared

by the manufacturer

In Fig. 11 the x-axis represents the SPF value as indicated on theproduct label, whereas the y-axis registers the value obtained fromour measurements. It can be easily observed that the linearcorrespondences among in vitro and label values have not alwaysbeen satised. In the case of roughened quartz, Vitro-Skin andTranspore correlations between the in vitro SPF and label valuescan be established, but they are not 1:1. The slope of the linearregression curves results higher than 1 (1.5; 1.55 and 5.9respectively); probably the slope could be modied by applyingdifferent quantities instead of 1 mg/cm2 as demonstrated by otherauthors [13]. The results obtained with PTFE and PMMA plates aremore critical. For PTFE, in vitro SPF/Label correlation seems to bedifferent for low and high SPF values; it has been alreadydemonstrated that better results are obtained when a smalleramount of sunscreen is applied [15]. In case of PMMA, SPF valueshave been calculated assuming that the substrate used for A(l)recovering was with or without glycerine; results were almost

Fig. 11. Comparison of in vitro derived SPF values calculated from sunscreen transmittanclabel correlation for Transpore attached on polished quartz; (b) in vitro SPF/label correlatio

in vitro SPF/label correlation for PMMA plate; (e) in vitro SPF/label correlation for PTFEequivalent.While PMMA appear to be a rst choice for UVA in vitrotesting [4], our experimental results suggest that for in vitro SPFother substrates could provide better performances. In our study,we have performed testswith a rate of application of 1 mg/cm2 andno other amounts of product have been evaluated; we choose anunique quantity of application in order to compare the absorbancecurves obtained with the different substrates. For PMMA plates, itis probably possible to improve the correspondence between invitro and label values by using a different amount of product ordifferent roughness. Heinrich and co-workers [13] obtained thebest correlation in vitro/in vivo by using an amount of 1 mg/cm2 foremulsion type screen; for other formulations (as sprays and oils),the best correlation was obtained using an amount of 0.5 mg/cm2.In the COLIPA guideline for the in vitro determination of UVAprotection factor [4], an application rate of 0.75 mg/cm2 is indicatedfor PMMA plates used for UVA labelling; this amount could also beevaluated for SPF determination. In our laboratory, we are alsotesting PTFE with different rate of applications. So far, we haveobtained a good correspondence between in vitro and label valueswith an amount of 0.65 mg/cm2. From the results reported above,it is possible to conclude that different substrates can be suitablefor in vitro SPF evaluation, but a calibration must be performed inorder to dene the regression curve. Calibration should beperformed by each laboratory, since the result depends on methodand instrumentation used. Calibration should be performed also

e spectra versus label SPF values reported by productmanufacturers. (a) In vitro SPF/

n for hydrated Vitro-Skin; (c) in vitro SPF/label correlation for roughened quartz; (d)

; (f) in vitro SPF/label correlation for self-standing Transpore.

for each type of product formulation. This variability is wellrecognized when testing various types of products with differentformulation bases which yield different correlation slopes. Here,we have demonstrated that differences in correlation slopes canalso derive from the use of different substrate materials.

4. Conclusions

In this work different substrate materials for sunscreens testinghave been investigated. Transpore and laboratory prepared PTFEare cheap and easily available on the market, while Vitro-Skin,roughened quartz plate and roughened PMMA plate are moreexpensive. Roughened quartz and PMMA plate can be re-used, butthe cleaning process is difcult and very time consuming. Vitro-

approach for the evaluation of sunscreen efcacy and validation oflabelling).

References

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[2] Epstein H. Sunscreen savvy: why do sunscreens fail to protect? Clin Dermatol2006;24:4634.

[3] COLIPA (The European Cosmetic Toiletry and Perfumery Association). SunProtection Factor test method; 2006, http://www.colipa.com.

[4] COLIPA. In-vitro photo-protection methods task force. Method for the in-vitrodetermination of UVA protection provided by sunscreen products. Guideline;2007, http://www.colipa.com.

[5] Klette E, Wendel V, Wittern KP, Gers-Barlag H, Quick A. A practical testprocedure to evaluate the performance of instruments used for in vitro UVprotection measurements. Int J Cosmet Sci 2002;24:3239.

[6] Gers-Barlag H, Klette E, Bimczok R. In vitro testing to assess the UVA protectionperformance of sun care products. Int J Cosmet Sci 2001;23:314.

D. Garoli et al. / Journal of Dermatological Science 56 (2009) 899898Skin must be hydrated and this must be performed 24 h before theuse. Application of sunscreen products at a rate of 1 mg/cm2 onTranspore, Quartz, PMMA, PTFE and Vitro-Skin have beenperformed quite easily, although with hydrated Vitro-Skin theapplicationmust be performed quickly in order to weight correctlythe amount applied. Controlling the amount of product applied onroughened quartz plate is difcult to achieve because of the lowadherence of the sunscreen. Self-standing Transpore tends to breakdown and, for this reason, it is used coupled to a smooth quartzplate; this results in lower transmittance values. Not all substrateswere photo-stable and only PMMA plate and roughened quartzplate can be recommended for photo-stability tests. PTFE alsoshowed to be suitable for photo-stability tests and can be regardedas an alternative option.

From our results, self-standing Transpore, quartz plate andVitro-Skin are preferable for SPF tests due to a better correlationbetween in vitro and in vivo measurements. To recover the linearregression slope, an initial calibration must be performed fordifferent sunscreen products labelled with different SPF values.Calibration should be performed for each instrumentation used,each method adopted (substrates, amount of sunscreen appliedetc.), each type of sunscreen formula (lait, gel, etc.). Finally, theresults of our measurements demonstrate that, while a correlationbetween in vitro and in vivo SPF results can be established, thiscorrelation is never exactly 1:1; the linear regression slopemust bealways used to correct the data in order to use them for labelling.Therefore in vitro SPF testing, regardless of substrate, is not a viablesubstitute for in vivo SPF testing, and should not be used as such byany regulatory bodies for quality control of sunscreens. Furtherstudies are needed in order to achieve a 1:1 correlation.

Acknowledgment

This work has been supported by a grant to M.A. from theUniversity of Padua (Ateneo Project Development of a new in vitro[7] Stokes R, Diffey B. In vitro assessment of sunscreen photostability: the effect ofradiation source, sunscreen application thickness and substrate. Int J CosmetSci 1999;21:34151.

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[10] Ferrero L, Pissavini M, Dehais A, Marguerie S, Zastrow L. Importance ofsubstrate roughness for in vitro sun protection Assessment. IFSCC Mag2006;9(2).

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[12] Ferrero L, Pissavini M, Marguerie, Zastrow L. Sunscreen in vitro spectroscopy:an application to UVA protection assessment and correlation with in vivopersistent pigment darkening. Int J Cosmet Sci 2002;24:6370.

[13] Pissavini M, Ferrero L, Alard V, Heinrich U, Tronnier H, Kockott D, et al.Determination of the in vitro SPF. Cosmet Toiletries 2003;118:6372.

[14] Heinrich U, Tronnier H, Kockott D, Kuckuk R, Heise HM. Comparison of sunprotection factors determined by an in vivo and different in vitro methodol-ogies: a study with 58 different commercially available sunscreen products.Int J Cosmet Sci 2004;26:7989.

[15] Garoli D, Pelizzo MG, Benardini B, Nicolosi P, Alaibac M. Sunscreen tests:correspondence between in vitro data and values reported by the manufac-ture. J Dermatol Sci 2008;52:193204.

[16] Diffey BL, Robson J. A new substrate to measure sunscreen protection factorsthroughout the ultraviolet spectrum. J Soc Cosmet Chem 1989;40:12733.

[17] Tokgoz NS, Marginean-Lazar G, Ponte A, Fructus AE. Use of synthetic skin forin-vitro evaluation of photoprotective efcacy of sunscreens: application todifferent type of emulsions. In: Proceedings of the 20th IFSCC congress; 1998.

[18] Martin CJ, Currie GD, Pye SD. The importance of radiometer angular responsefor ultraviolet phototherapy dosimetry. Phys Med Biol 1999;44:84355.

[19] http://www.deltaohm.com/ver05_06/pdf_ante05/depliant/hd9021.pdf.[20] Pelizzo MG, Garoli D, Nicolosi P. Realization of a radiometric head for mea-

surements of ultraviolet total erythemal effective irradiance. Appl Opt2007;46:497784.

[21] Springsteen A, Yurek R, Franzier M, Carr KF. In vitro measurements of sunprotection factor of sunscreens by diffuse transmittance. Anal Chim Acta1999;380:15564.

[22] Ferrero L, Golz K, Bouttes S, Zastrow L. Sunscreen O1/W/O2, multiple emulsionwith organic and mineral UV absorbers. In: Proceedings of the 20th IFSCCcongress; 1998.

[23] Diffey BL, Tanner PR, Matts PJ, Nash JF. In vitro assessment of the broad-spectrum ultraviolet protection of sunscreen products. J Am Acad Dermatol2000;43:102435.

Effectiveness of different substrate materials for in vitro sunscreen testsIntroductionMaterials and methodsSubstratesSpectro-photometric measurementsPhoto-stability measurements of the substrateTotal absorbance/transmittance measurements on sunscreensIn vitro Sun Protection Factor computation and correlation with value reported on labels

Results and discussionTransmittance and reflectance measurements on substratesPhoto-stability tests on substratesTotal absorbance/transmittance measurements of sunscreenIn vitro determination of SPF: correlation with the values declared by the manufacturer

ConclusionsAcknowledgmentReferences