The revised COLIPA in vitro UVA method D. Moyal*, V. Alard † , C. Bertin ‡ , F. Boyer § , M.W. Brown ¶ , L. Kolbe**, P. Matts †† and M. Pissavini ‡‡ *L’Ore´al Research & Innovation, 25-29 Quai Aulagnier, 92665, Asnie`res Sur Seine, France, † LVMH Recherche, Branche Parfums et Cosme´tique, 185, Avenue de Verdun, 45804, St Jean de Baye, France, ‡ Johnson & Johnson Sante´ Beaute´ France, 1 Rue Camille Desmoulins, 92787, Issy-les- Mouline
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  The revised COLIPA  in vitro   UVA method D. Moyal*, V. Alard † , C. Bertin ‡ , F. Boyer § , M.W. Brown ¶  , L. Kolbe**, P. Matts †† and M. Pissavini ‡‡ *L’Ore´al Research & Innovation, 25-29 Quai Aulagnier, 92665, Asnie`res Sur Seine, France,  † LVMH Recherche, Branche Parfums et Cosme´tique,185, Avenue de Verdun, 45804, St Jean de Baye, France,  ‡  Johnson & Johnson Sante´ Beaute´ France, 1 Rue Camille Desmoulins, 92787, Issy-les-Moulineaux, France,  § Pierre Fabre Dermo Cosme´tique, 2 Rue Viguerie, 31025, Toulouse, France,  ¶  Alliance Boots Limited, Nottingham NG2 3AA,U.K.,  ** Beiersdorf AG, Forschung und Entwicklung, Troplowitzstraße 17, D  –  22529, Hamburg, Germany,  †† Procter & Gamble, London InnovationCentre, Egham, Surrey TW20 9NW, UK and  ‡‡ Coty-Lancaster, International Research & Development Center, 2 rue de la Lujerneta, 98000, MonacoReceived 25 June 2012, Accepted 30 July 2012 Keywords:  COLIPA,  in vitro , protection, UVA Synopsis A multicentred study derived from the COLIPA  in vitro  UVAmethod was performed to assess the influence of test conditions onUVA protection factor (UVAPF) values in terms of amplitude, repro-ducibility between laboratories and correlation with  in vivo  UVAresults. Eight products with a range of   in vivo  UVAPF from three to29 were used. Two different types of plates, namely high-roughness(5  l m) and low-roughness (2  l m) plates, were used with a differ-ent application rate for each (1.3 mg cm  2 and 0.75 mg cm  2 respectively). The UVR dose applied to both plate types followed thesame principle as the original test (1.2 J. cm  2 9  UVAPF0).Strong, significant correlations between  in vitro  and  in vivo  UVAPFvalues were observed for both plate types (Pearson correla-tion  >  0.9,  P    0.01). The correlation and slope obtained withthe low-roughness plates confirmed the previous results obtainedby COLIPA. Across all laboratories, higher UVAPF values wereobtained on the high-roughness plates ( P  <  0.01). Reproducibilityof UVAPF values between laboratories was comparable betweenthe two plate roughness values (low roughness, COV  =  8%; highroughness, COV  =  12%). Considering the  in vitro / in vivo  compari-sons, a regression slope of 0.83 was observed for the low-roughnessplates, in comparison with a value of 1.05 for the high-roughnessplates. The accuracy of the method was improved, therefore, withthe use of the high-roughness plates. With a constraint to recom-mend the use of only one plate type in the COLIPA UVA  in vitro Test, the high-roughness plate was selected on an on-going basis tolimit variability of results and to provide better accuracy with  invivo  data. Re´ sume´  Une e´tude multicentrique, base´e sur la me´thode UVA  in vitro  duCOLIPA a e´te´ re´alise´e pour e´valuer l’influence des conditions d’essaisur les valeurs du facteur de protection UVA (UVAPF) en termesd’amplitude, de reproductibilite´ entre les laboratoires et de corre´la-tion avec les re´sultats UVA  in vivo . 8 produits couvrant une gammede valeurs  in vivo  UVAPF de 3 a` 29 ont e´te´ utilise´s. Deux diffe´rentstypes de plaques, de rugosite´ e´leve´e (5  μ  m) et de faible rugosite´(2  μ  m) ont e´te´ utilise´s, avec des taux d’application diffe´rents pourchacune.(1.3 mg/cm 󰂲  et 0.75 mg/cm 󰂲 , respectivement). La doseUVR applique´e aux deux types de plaques a suivi le meˆme principeque l’e´preuve initiale (1.2 J/cm  󰂲  x UVAPF0). Des corre´lations forteset significatives entre les valeurs UVAPF  in vitro  et  in vivo  dans lesvaleurs UVAPF ont e´te´ observe´es pour les deux types de plaques(Corre´lation Pearson  >  0.9,  P    0.01). La corre´lation et la penteobtenue avec les plaques a` faible rugosite´ ont confirme´ les re´sultatsante´rieurs obtenus par le COLIPA. Sur l’ensemble des laboratoires,des valeurs UVAPF plus e´leve´es ont e´te´ obtenues sur les plaques derugosite´ e´leve´e ( P  <  0.01). La reproductibilite´ des valeurs UVAPFentre les laboratoires a e´te´ comparable quant aux valeurs de rugo-site´ des plaques (faible rugosite´, COV  =  8%; forte rugosite´, COV  = 12%). Conside´rant la comparaison  in vitro/in vivo , une pente dere´gression de 0.83 a e´te´ observe´e pour les plaques de rugosite´ faible, compare´e a` une valeur de 1.05 pour les plaques de rugosite´e´leve´e. La pre´cision de la me´thode a donc e´te´ ame´liore´e avec l’util-isation des plaques de rugosite´ e´leve´e. Avec la contrainte de recom-mander l’utilisation d’un seul type de plaque dans la me´thode detest UVA in vitro du COLIPA la plaque de forte rugosite´ a e´te´ se´lec-tionne´e afin de limiter la variabilite´ des re´sultats et de fournir unemeilleure concordance avec des donne´es  in vivo. Introduction In recent years, there have been concerted efforts around the globeto incorporate UVA protection into commercial sunscreens and todevelop methods to measure the photoprotection afforded by theseproducts. The COLIPA (European Cosmetics Trade Association) ‘ invitro  Sun Protection Methods’ Task Force was given the remit todevelop an  in vitro  measure of protection from UVA wavelengths,correlated with an  in vivo  measure of the same. The result was theCOLIPA technical guideline published in 2007, ‘Method for the  invitro  determination of UVA protection provided by sunscreenproducts’ [1].The test is based on the measurement of UV radiation (UVR)transmittance through a thin film of sunscreen sample spread ona UVR-transparent roughened substrate, before and after expo-sure to a controlled dose of UVR from a defined source of solar-simulated radiation. By convoluting the ensuing transmissiondata with the action spectrum for  in vivo  Persistent PigmentDarkening (PPD) and the spectral irradiance received from the Correspondence: P. Matts, Procter & Gamble, London InnovationCentre, Egham, Surrey, TW20 9NW, U.K. Tel.: +44 1784 474454; fax:+44 1784 474508; e-mail:   2012 Society of Cosmetic Scientists and the Socie´te´ Franc¸aise de Cosme´tologie 35 International Journal of Cosmetic Science , 2013,  35 , 35–40 doi: 10.1111/j.1468-2494.2012.00748.x  UVA source, an  in vitro  UVA protection factor (UVAPF) is pro-vided, which is correlated with its corresponding  in vivo  PPDvalue [2, 3].In a previous publication [4], we described and summarized themethods and results from the two ring studies critical in the devel-opment of the published COLIPA  in vitro  UVA Test Method [1].Eight laboratories tested a total of 13 sunscreens using this methodand established a very good correlation ( r  2 =  0.83; slope  =  0.84, P  <  0.0001) between resulting  in vitro  UVAPF values andcorresponding values derived from the  in vivo  PPD method. Wedemonstrate through these data that this new method can be usedto provide a reliable  in vitro  metric to describe and label UVA effi-cacy in sunscreen products, in line with the EU Commission recom-mendation 2006/247/EC [5].Substrate roughness and the corresponding dose of sunscreenapplied are two critical parameters in determining reproducibility of results and correlation with  in vivo  data. In the 2007 COLIPA  invitro  UVA guidelines, a dose rate of 0.75 mg cm  2 and a polym-ethylmethacrylate (PMMA) plate with a roughness of   S  a  =  2  l mwere defined. In continuing optimization of the method, the COLI-PA  in vitro  Methods Task Force investigated the use of plates witha higher roughness and this present article summarizes the resultsfrom a new multicentre ring study comparing the 2007 conditions[1] and new proposed conditions. Materials and methods Materials UV spectrophotometer  Six test laboratories used a variety of different UV spectrophotome-ters to conduct the transmission measurements, but it was ensuredthat they all conformed to minimum requirements, as outlined inthe COLIPA  in vitro  UVA Method [1,6,7].All UV spectrophotometers used, spanned the primary wave-band of interest, 290  –  400 nm. The wavelength accuracy of thedevices was within 1 nm, as checked using a mercury spectralstandard lamp or a xenon lamp with a specially doped filter.Device detectors were capable of collecting both the direct anddiffuse portions of UVR transmitted through the roughenedPMMA substrate, either with or without applied sunscreen. Thedynamic range of the device detectors was at least 2.2 absor-bance units. The maximum measured absorbance was  < 90% of the dynamic range of the device used.Lamp sources used by the spectrophotometers emitted continu-ous radiation with no peaks within the 290  –  400 nm wavebandand irradiance was low ( < 0.2 J cm  2 per measurement cycle), sothat photostability of the sunscreen did not become a factor duringspectrophotometric measurement. Monitoring of the UV spectrophotometer  UV spectrophotometers were tested at regular intervals (on amonthly basis) by the measurement of reference samples. A twofoldtest was recommended:(a) Monitoring the instrument’s response and dynamic range withstandard PMMA plates (see Appendix IIA in the published CO-LIPA  in vitro  UVA Method [1,6,7]).(b) Checking wavelength accuracy with an approved standardmaterial (e.g. holmium perchlorate, as recommended in Appen-dix IIB of the published COLIPA  in vitro  UVA Method [1,6,7]). Source for SSR irradiation The spectral irradiance of the artificial UVR source (at the sampleplane) that was used for irradiation was as similar as possible tothe irradiance at ground level under a standard zenith sun asdefined by COLIPA (2006) [8] or in DIN67501 (1999) [9]. TargetUV irradiance therefore was set within the following acceptancelimits (measured at the sample plane): Total UV irradiance (290  –  400 nm) 50  –  140 W m  2 Irradiance ratio of UVA (320  –  400 nm) to UVB (290  –  320 nm) 8  –  22 All laboratories used an Atlas Suntest TM insolator (Atlas MaterialTesting Technology GmbH, Linsengericht, Germany), type CPS/CPS +  or XLS/XLS + , fitted with the UV short cut-off filter and IRdichroic mirror with a temperature during irradiation of samplesmaintained below 40 ° C. Monitoring of the SSR source The emission of the SSR source was checked by a suitably qualifiedexpert using a calibrated spectroradiometer for compliance with thegiven acceptance limits. On an on-going basis, the SSR sourceemission was also monitored using a radiometer. All spectroradi-ometers and radiometers were calibrated according to COLIPArecommendations in the Guideline ‘Monitoring of UV light sources’(2007) [10]. The calibration of the radiometers provides acoefficient of correction between radiometry and spectroradiometrythat helps ensure that all laboratories are applying the same UVRdose. Substrates Each test sunscreen was applied to two types of polymethylmethac-rylate (PMMA) plate:(a) The first type of plate was that described in the COLIPA UVA  invitro  Method published in 2007 [1] and revised in 2009 [6].The average roughness value ( S  a ) of these sand-blasted PMMAplates (supplied by Schonberg GmbH, Hamburg) was approxi-mately 2  l m ( S  a  defined by EUR 15178 EN [11]) with dimen-sions of 5  9  5 cm (surface 25 cm 2 ).(b) The second type of plate used in the study (PMMA plates HD6from Helioscreen, France; dimensions 4.7 cm  9  4.7 cm andsurface area of 22.1 cm 2 ) had a moulded surface topography,providing both lower intra- and inter-batch variation in surfaceroughness [12]. The average Ra value was determined as4.85  l m (referred to as 5- l m plates from hereon). Reference plate Reference plates were produced by spreading a few microlitres of glycerine on the roughened side of the plate, using just enough tothinly coat the entire plate surface (approximately 15  l L for bothtypes of plate). Amount of applied product An application rate of 0.75 mg cm  2 was used on the 2- l m plates(total amount 18.75 mg), and an application rate of 1.3 mg cm  2 was used for the 5- l m plates (total amount 28.7 mg). The1.3 mg cm  2 dose was chosen to ensure a sufficient film thicknesson the higher roughness 5- l m plates [13].   2012 Society of Cosmetic Scientists and the Socie´te´ Franc¸aise de Cosme´tologie International Journal of Cosmetic Science ,  35 , 35–4036The Revised COLIPA  in vitro  UVA method  D. Moyal  et al.  Sample application Product was applied to the roughened side of PMMA plates as alarge number of small droplets of approximate equal volume,distributed evenly over the whole surface of the plate. Positive-displacement automatic pipettes were found ideal for this purpose.To check for the correct application rate, pipettes and/or plateswere weighed before and after dispensing the product.After application and check weighing, the sunscreen productwas spread immediately over the whole surface using light strokeswith a naked fingertip (no finger-cot) ‘pre-saturated’ with theproduct. Spreading was completed in two phases: (a) the productwas first distributed over the entire plate using light pressure, in < 30 s and (b) the distributed sample was then rubbed into therough surface using stronger pressure over a period of 20  –  30 s.Treated samples were then allowed to equilibrate in the dark, atambient temperature, for at least 15 min to help facilitate forma-tion of a standard stabilized product film. Transmission measurements through product-treated plates The UV transmission (monochromatic absorbance data over 290  –  400 nm with 1 nm steps) of at least three PMMA plates, treatedwith product in the manner described above, was measured usinga calibrated UV spectrophotometer. Depending on the spectropho-tometer type, each plate was measured either on a large spot areaor at a number of different sites to ensure that an area of at least2.0 cm 2 was measured in total. Care was taken to ensure thatexactly the same sites on each plate were measured before andafter irradiation, to help reduce variability. Each laboratory appliedeach test product to at least three plates of each type. Exposure to SSR Care was taken to ensure that samples were not exposed to temper-atures of   > 40 ° C during irradiation. In the Atlas Suntest TM insola-tors, for example, air-conditioning units or water-cooled trays wereemployed for this purpose. PMMA plates were also fixed in placeusing suitable means (e.g. the use of a template with wells in thesurface to accommodate the plates) and placed against a non-UVreflective background to minimize reflection of UVR back throughthe sample.The irradiation dose Dx used for exposure of the samples was cal-culated using the same principle for the two different plate types:Dx  =  UVAPF 0  9  1.2 J cm  2 Summary of the different procedure steps After standardized sunscreen film application on roughened PMMAplates (Calculations are detailed in Appendix)(i) transmission measurement with a UV spectrophotometer(ii) iterative adjustment of the absorbance values by coefficient Csuch that  in vitro  SPF now equals  in vivo  SPF(iii) calculation of UVAPF 0  from the corrected spectra(iv) determination of the irradiation dose D;D = UVAPF 0  9  1.2 J cm  2 (v) irradiation of the sunscreen samples with dose D(vi) calculation of UVAPF from the absorption spectra after irradiation(vii) calculation of critical wavelength value after irradiationCalculations are detailed in Appendix. Products tested  Eight products were tested in total, of which seven were marketedproducts of different types with labelled SPF values from 6 to 50 + .Mean  in vitro  UVAPF values for each of these products were deter-mined on 10 subjects. Measured values ranged from 2.9 to 29.4,as shown in Table I.Reference sunscreen S2 (proposed in the COLIPA UVA  in vitro Method issued in 2011 [7]) was also tested. The SPF  in vivo  value usedfor calculations was 16. The  in vivo  UVAPF acceptance range for thereference formulation S2 was from 10.7 to 14.7 (mean value, 12.7). Statistical analysis Normality and homogeneity of variance were checked using aShapiro  –  Wilk test. Intra-laboratory comparisons between the twoplate types were performed using a Student’s  t -test (withsignificance set at  P  <  0.05). For each product, inter-laboratorycomparisons between the two types of plates were performed usinga Student’s  t -test (with significance set at  P  <  0.05). The relation-ship of   in vitro  and  in vivo  UVAPF values obtained for the eightproducts was modelled using simple linear regression for both platetypes, to yield slope, significance and Pearson correlation coefficientmetrics [correlation is significant at the 0.01 level (2-tailed)]. Results Mean UVAPF 0 , UVAPF and critical wavelength results are summa-rized in Table II for the 2- l m plate and in Table III for the 5- l mplate. Table I  Labelled SPF,  in vivo  UVA protection factor values, mean and 95%confidence intervals on 10 subjects, types of products Products Labelled SPF  In vivo   UVAPF Mean (95% CI) Type of product UVA1 SPF50 +  29.4 (26.5  –  32.3) Gel creamUVA2 SPF50 +  25.4 (20.5  –  30.3) LotionUVA3 SPF50 +  21.4 (18.0  –  24.8) CreamUVA4 SPF30 11.9 (10.8  –  13.0) Fluid lotionUVA5 SPF6 2.9 (2.4  –  3.4) LotionUVA6 SPF10 4.9 (4.0  –  5.8) OilUVA7 SPF30 18.5 (15.5  –  21.5) LotionS2 SPF16 12.7 (10.7  –  14.7) * Lotion*Acceptance rangeof in vivo   UVAPF values forS2proposed reference formulation. Table II  UVAPF 0 , UVAPF and critical wavelength results on the 2- l mplates, mean values obtained across six laboratories, standard deviation (SD)and % variance between laboratories ProductUVAPF 0  UVAPF  k cMean SD % Var Mean SD % Var Mean SD % Var UVA1 31.8 3.3 10.5 24.8 3.2 12.8 378 1.3 0.3UVA2 23.2 1.8 7.9 19.9 2.1 10.6 376 1.3 0.3UVA3 22.3 2.1 9.4 18.1 1.1 6.1 377 1.3 0.3UVA4 15.8 1.2 7.7 13.6 1.3 9.2 378 1.3 0.3UVA5 3.8 0.2 5.1 3.5 0.2 4.6 376 1.2 0.3UVA6 4.4 0.3 6.6 4.3 0.3 7.7 373 1.5 0.4UVA7 18.4 1.6 8.9 13.5 1.4 10.7 379 1 0.3S2 14.2 1 7.3 11.5 0.9 7.9 378 2 0.5   2012 Society of Cosmetic Scientists and the Socie´te´ Franc¸aise de Cosme´tologie International Journal of Cosmetic Science ,  35 , 35–40 37The Revised COLIPA  in vitro  UVA method  D. Moyal  et al.  The mean UVAPF values obtained for the reference formulationS2 using the 2- l m or the 5- l m plates were both in the acceptancerange, based on  in vivo  UVAPF values.Both before and after irradiation, mean UVAPF 0  values acrossall laboratories for the 2- l m plates were lower than those for the5- l m plates, whatever the product ( P  <  0.01).The percentage of variability in data between laboratories, beforeand after irradiation, across the eight products was approximately8% using the 2- l m plates and approximately 12% using the 5- l mplates.A summary of   in vitro  UVAPF values obtained using the 2- l m and the 5- l m plates and corresponding  in vivo  UVAPF val-ues is presented in Fig. 1, and a plot  in vitro  values against cor-responding  in vivo  values, along with fitted linear regressioncurves, are presented in Fig. 2. The correlation between  in vitro UVAPF values and  in vivo  UVAPF values was highly significant( P  <  0.01) for both plate plates (Pearson  r  2 correlation values of 0.98 for the 2- l m plates and 0.98 for the 5- l m plates). Interest-ingly, the value of the slope of the regression curve was 0.83for the 2- l m plates and 1.05 for the 5- l m plates (indicating acloser agreement with absolute  in vivo  UVAPF values for the 5- l m plates).After irradiation, critical wavelength values on the 2- l m plateswere lower compared with values obtained using the 5- l m platesacross all laboratories, whatever the product ( P  <  0.01). Variabilityof results between laboratories was very low for either plate type( < 1.0%). Discussion Different types of sunscreen formulation, including creams, lotions,fluids and oils with  in vivo  UVAPF values from 3 to 29, were testedusing two different types of plates and application rate. Using 5- l mroughness plates and a 1.3 mg cm  2 dose rate, UVAPF 0  valueswere higher than those obtained using the 2- l m plates and a0.75 mg cm  2 dose rate which, we hypothesize, demonstrates thesignificant influence of these different test conditions on the shapeof the absorbance curves. The UVR dose applied in the irradiationstep (using D  =  1.2 J cm  2 9  UVAPF 0 ) to the 5- l m plates was onaverage 15% higher than that applied to the 2- l m plates. Becauseof the relative photostability of the formulations tested, however,measured UVAPF values for the 5- l m plates were higher thanthose measured for the 2- l m plates.The variability of the results between laboratories was higher forthe 5- l m plates compared with the 2- l m plates (12% vs. 8%respectively), possibly due to less familiarity with the new platetype by technicians (a significant factor to consider in  in vitro  test-ing). Notwithstanding, the slightly higher variance seen with the5- l m plates is still lower than that achieved with the  in vivo method and, we believe, acceptable. These results agree with previ-ous observations made by Ferrero  et al.  [13].The slope and correlation (0.83;  P  <  0.01) obtained with thelow-roughness plates were in good agreement with previous resultsobtained for this plate type by COLIPA [4]. Conclusion Based on the results obtained, the COLIPA  in vitro  Task Force hasrecommended using only one type of plate to limit possible variabil-ity. The group has selected the higher roughness (5  l m) because of further improved accuracy against the  in vivo  method. Based onthese data, therefore, COLIPA published revised guidelines for themethod for  in vitro  determination of UVA protection in April 2011[7], specifying new specifications for plates’ roughness (5  l m) andapplication rate (1.3 mg cm  2 ). 16111621263136 UVA1 UVA2 UVA3 UVA4 UVA5 UVA6 UVA7 S2       U      V      A      P      F Figure 1  Mean  in vitro  UVA protection factor (UVAPF) values obtained onthe low ( )- and high ( )-roughness plates compared with correspondingmean  in vivo  UVAPF values ( ). y   = 0.8358x y   = 1.0519x 6 11 16 21 26 31    I   n   v   i   t   r   o   U   V   A   P   F In vivo UVAPF Figure 2  Correlation between mean  in vitro  UVA protection factor (UVAPF)values for the high ( )- and low ( )-roughness plates and correspondingmean  in vivo  UVAPF values. Table III  UVAPF 0 , UVAPF and critical wavelength results on the 5- l mplates, mean values obtained across six laboratories, standard deviation (SD)and % variance between laboratories ProductUVAPF 0  UVAPF  k cMean SD % Var Mean SD % Var Mean SD % Var UVA1 34 5.1 15.1 32.1 4.5 14.1 379 1.6 0.4UVA2 26.1 3.6 13.7 23.5 1.8 7.8 377 0.9 0.2UVA3 28.4 5.7 20.2 22.2 4 18 379 1.6 0.4UVA4 17.5 3.2 18.3 15.8 2.7 16.9 379 1.8 0.5UVA5 4.3 0.2 4.8 3.9 0.3 7.5 378 1 0.3UVA6 6.8 0.7 10.7 6.6 0.8 12.5 378 1 0.3UVA7 21.8 2.7 12.3 19.1 2.6 13.8 380 2 0.4S2 15.2 0.7 4.3 14.1 1.2 8.1 381 1 0.3   2012 Society of Cosmetic Scientists and the Socie´te´ Franc¸aise de Cosme´tologie International Journal of Cosmetic Science ,  35 , 35–4038The Revised COLIPA  in vitro  UVA method  D. Moyal  et al.
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