Photodermatology, Photoimmunology & Photomedicine
REVIEW ARTICLE
Sunscreens – what is the ideal testing model? Curtis Cole
Johnson & Johnson Consumer and Personal Products Co. Inc., Skillman, NJ, USA.
Key words: diffuse reflectance spectroscopy; efficacy; SPF; sunscreen; UVA protection
Correspondence: Dr Curtis Cole, Ph.D., Johnson & Johnson Consumer Products Inc., 199 Grandview Rd., Skillman, NJ 08558, USA. Tel: 908 392 6165 e-mail: [email protected]
SUMMARY Sunscreen protection assessment methodologies have been evolving in tandem with the innovation and evolution of sunscreen products themselves; from initial human testing in the Swiss Alps, to laboratory testing with high intensity solar simulators, to spectrophotometers with modern CCD array photocells and diffuse reflectance spectroscopy techniques. The progress in the science leads regulatory development of standard methods, and provides new and improved ways to assess sunscreen protection properties. This review scans much of the history of the development of these methods and highlights the latest development in non-invasive sunscreen testing as an opportunity to improve accuracy while eliminating human UV exposures.
Photodermatol Photoimmunol Photomed 2014; 30: 81–87 HISTORICAL PERSPECTIVE
Accepted for publication: 3 December 2013
Conflicts of interest: None declared.
Sunscreen products are regulated differently in various countries as either drugs (US, Canada, Australian beach products for example) or as cosmetics (Europe, Latin America and Australia-daily wear products for example). They are however, all required to undergo testing to establish the Sun Protection Factor (SPF) that is used on the product packaging. Unlike other monographed drug or ‘positive list’ products that are permitted to make medicinal or health benefit claims by simply including an appropriate amount of the ‘active’ ingredient in the product, sunscreen products are require to undergo clinical performance testing to establish the level of efficacy of the product (for both SPF and Ultraviolet-A radiation (UVA) protection claims). While three individuals are often referenced as the ‘inventors’ of sunscreen products, one is clearly distinguished as the inventor of the SPF factor, Dr. Franz Greiter, who introduced the first products with an SPF indication in 1962. While the concept of SPF is thought to be a precise absolute value that indicates the number of multiples of the minimal amount of UV radiation that you can absorb while using the sunscreen before becoming sunburned, in fact, it should be regarded more as a measure for comparison of one product vs. another. It is quite impossible for someone to calculate the ‘safe time’ in sunlight using a specific SPF value sunscreen. The fact is that no one individual knows for themselves exactly what their ‘burn time’ is when unprotected as it varies with the season and their exposure history, nor can they accurately estimate the rate of sunburning intensity of sunlight that changes hour to hour, and month by month throughout the year – not to mention the variability introduced by fluctuating clouds and surface reflectivity. The concept of a ‘Protection Factor’ has been adopted by regulatory authorities around the globe and codified as an essential claim for any product promising to protect against
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/phpp.12095
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Fig. 1. SPF testing being conducted in the Swiss Alps in 1962 with Dr Franz Greiter, inventor of the SPF rating system for sunscreen protection.
sunburn and sun damage. As a result, consumers have had to learn the meaning and value of the SPF numbers for making product choices. This brings us to the heart of the topic. What is the ideal test method of evaluating sunscreen products? Is there a better way compared to the regulations we have in place now? If we could start from scratch, how would we approach it, knowing what we know now and with the tools now available to us? Perhaps the best way to approach these questions is to look at some of the learning from our collective experiences measuring sunscreen efficacy.
WHAT HAVE WE LEARNED ABOUT IN VIVO CLINICAL TESTING? Importance of the light source for clinical testing Fig. 1 documents the use or natural sunlight on human subjects as the original test methodology that is referenced 82
in the FDA 1978 Proposed Rule for Sunscreen Products (1, 2). While using real sunlight for SPF testing is admirable and ‘realistic’, it is quite unreliable for routine product testing. This approach to determining the ‘protective factor’ of a sunscreen was facilitated by the use of more reliable ‘solar simulators’ that could bring the ‘sun’ indoors to the laboratory for more controlled testing conditions. Laboratory tests for SPF have been were done with metal doped lamps (3), fluorescent lamps (4), and xenon lamps (5) with varying values as a result of the spectral differences and the interaction with the spectral qualities of the sunscreens tested (6–11). Today, the xenon arc solar simulator remains as the sole clinical light source for both SPF and UVA-PF testing of sunscreens. Daniel Berger developed the first clinical solar simulator (5) based on a compact arc xenon burner combined with a UV reflecting dichroic mirror, a visible light absorbing filter, and a shortwave UVC/UVB cutoff filter to shape the spectrum to provide only UVB and UVA radiation. This eliminates the energy from visible light and infrared radiation that is not contributing to the ultraviolet induced erythema and only contributes to heat induced erythema. Unfortunately, the spectral match to the UVB cut-off (12) and the longwave UVA cut-offs (10) do not match sunlight precisely, and can lead to overestimation of sunscreen performance in actual sunlight conditions. The European cosmetics trade association COLIPA developed its own sunscreen testing protocol for assessment of products distributed in Europe which further defined and the spectral distribution limits (13) of the xenon arc solar simulators that has been adopted and codified into the International Standard for Sunscreen SPF (14) testing, the US Federal Register monograph for sunscreen testing (15), as well as the newly developed ISO 24444 Standard (16) for sunscreen SPF testing that is being adopted globally. This refining of the definition of the spectrum breaks the spectrum into 10 nm bands in the short wave UV and limits the cumulative maximum and minimum proportions of the erythema contributing wavelengths within those ranges, helping to minimize the variability of solar simulator spectra used in clinical testing. A limitation was also introduced requiring a minimum amount of UVA1 radiation contained in the output of the solar simulators to eliminate sources without adequate output in this range. Unfortunately, the cutoff filters used to eliminate the visible portion of the spectrum also cuts away significant amounts of the very highest UVA wavelengths in the xenon arc solar spectrum leading to some overestimation of both SPF and UVA protection factors (10). Technical solutions to eliminate this characteristic in a practicable manner are still lacking. Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Sunscreen efficacy testing methods review
Importance of common protocol and definitions After the first publication of the FDA Proposed Rule for SPF testing in 1978 (1), multiple protocols, primarily the German DIN method (3) and the COLIPA method (13) were developed for SPF testing using different light sources, definitions for the endpoint, and techniques for ‘color matching’ of the endpoint, as well as differing UV dosing intervals for the UV exposures. Companies wishing to market products globally were faced with the need to test the SPF of the same product several times and market with different SPF values depending on the local results. Collaborative industry efforts to evaluate the various test methods and unify on common test equipment, definitions, and procedures resulted in eliminating extraneous variables and processes and harmonizing on common standards and success criteria. Clinical testing showed that having 7 UV exposure sites narrower UV dose increments did not bring added precision to the result (17). Other testing determined that requiring more than 10 subjects to determine an SPF value did not bring a higher level of precision. Resolving these two variables in the SPF testing procedures allowed for alignment of protocols resulting in fewer unnecessary tests and test subjects to be conducted. There have been two successful sunscreen SPF trials used to demonstrate the capabilities of laboratories to measure SPF far above the SPF 15 values of the control sunscreen used to validate test procedures (17, 18). Both of these studies showed remarkable inter-laboratory reproducibility of SPF values between SPF 30 up to 85. Despite these clear test results, there can be inter-laboratory SPF variability that must be related to uncontrolled testing variables such as the spreading technique of product on the skin (pressure and duration variables), test subject sensitivity (19), and variability in the observation and determination of the biologic endpoint, the Minimal Erythema Dose response. Genetic variability in test subjects across the globe is unavoidable; however training for proper and consistent product application procedures has been shown to help reduce inter-laboratory variability. While there has been significant improvement in reproducibility and alignment of testing protocols for laboratory clinical testing, some publications have challenged the accuracy of laboratory generated SPF results when compared with ‘real sunlight’ spectral challenge or outdoor SPF testing results (20, 21). Given the high variability of the sun’s output and relatively low intensity compared to clinical UV sources, it is impossible to generate extensive outdoor SPF test results. Some of the impact of differences between solar simulator and solar spectra can and have Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
been estimated (11), but the interaction of UV with visible light and infrared radiation are missing from the laboratory testing experience and may have a significant effect on the estimate of erythema protection experienced in full outdoor sunlight over extended periods. Today the laboratory in vivo SPF test results remains the only validated way to determine and label products for SPF protection and is mandated across the globe. Nevertheless, the test requires irradiation of human subjects with damaging amounts of ultraviolet radiation, which ideally should be avoided if alternate methodology were established and validated. Which brings us to the question, why isn’t there a better method to establish a product’s SPF? Why haven’t in vitro test methods been validated?
IN VITRO TEST METHODS FOR SPF DETERMINATION A validated in vitro testing of sunscreens has been long sought ever since sunscreen products were first developed. The advantages over human clinical testing are obvious and compelling: no need to irradiate human subjects, objective laboratory measures can be used instead of subjective visual observations, imperfect solar simulators can be replaced with realistic solar spectra, and long and expensive clinical tests could be avoided. Regulatory agencies also desire to have a validated in vitro method in order to easily and inexpensively test product compliance. Despite the desire and decades of efforts the validated in vitro SPF test remains elusive and unfulfilled. Multiple articles over decades of time and efforts have shown the inability to determine reliable and repeatable SPF testing results based on in vitro test methods (22–25). Trade associations and regulatory/standardization authorities around the globe have conducted tests trying to validate a reliable in vitro SPF test without success. The reference by Rohr et al. (25) describes many of the issues that continue to require further refinement and standardization for the in vitro SPF testing. In vitro testing of sunscreens was initially done with dilute solutions of the UV filters in cuvettes and it was quickly apparent that the protection provided in vivo in products were far from the predicted in vitro values. Sunscreens are applied topically in layers that are approximately 10 to 20 microns thick when applied at an amount of 2 mg/cm2, the application rate prescribed by the first codified SPF test methods. This was considered to be the lowest application density that could be applied consistently for clinical SPF testing purposes. It was determined early on in sunscreen development that the vehicle and how it formed a film on the skin’s surface was critical to 83
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efficiency of the UV filters to provide protection. Artificial surfaces were evaluated in order to better mimic thin film sunscreen behavior on the skin including quartz plates (26, 27) mouse epidermis (20, 24, 28), Transpore™ tape (29), and Vitro-Skin® (30, 31). Each has its own advantages and disadvantages with none achieving a long-lived endorsement for in vitro SPF testing and validation. The most recent substrates that have been endorsed for official in vitro UVA test methods (32–34) are polymethylmethacrylate (PMMA) plates that have had surface modifications to provide various levels and topographical ‘roughness’ parameters. Some have surface roughness of 2 to 6 microns in average depth via a sandblasting procedure, and others have been molded with specific surface topographical features mimicking skin texture with skin roughness in similar ranges. The in vitro spectrophotometric measurements can accurately measure the shape of the absorbance curve, but the absolute magnitude of the absorption curve needs to be established by some other means given the high variability of thin film spectroscopy in vitro testing results and the non-uniform interaction between formulations and the plate surfaces. Without the ability to accurately measure the magnitude of the absorbance curves, it is impossible to qualify an in vitro SPF test method. The last element to be considered for the in vitro SPF tests with the artificial test substrate is the difficulty of finding a ‘universal’ substrate that provides the same surface interaction with the test sunscreen product. The level of UV protection relies critically on the uniformity and continuity of the sunscreen film formed on the test substrate surface. The thickness of product on the substrates is on the order of 10 microns (for full 2 mg/cm2 application density) and between 4 to 7 microns for lower application densities typically used in thin film spectroscopic test methods. This is approximately 1/10 to 1/20th the thickness of a human hair, and is expected to provide UV absorbance properties equivalent to 93–99% blockage of all the UV rays from 290 to roughly 400 nm. Any tiny hole or valley in protection acts like a huge window for UV photons to penetrate and dramatically reduce the protection beneath the sunscreen layer. Thus the uniformity of the film produced on the substrate surface can dramatically affect the overall protection and SPF value measured. Any mismatch of surface interactions between the sunscreen and the substrate can dramatically affect this film layer and provide erroneous measurements of the actual sunscreen protection that would be provided on human skin. Given the virtually infinite combination of sunscreen emollients, emulsifiers, humectants, and film forming polymers and water proofing agents, it has been difficult to
establish a ‘universally equivalent’ human test surface substrate for in vitro SPF testing. Solving the problems of finding a ‘universal’ test substrate, and the reproducibility of application, spreading, and rubbing techniques across laboratories still remain. More will be discussed on SPF testing shortly.
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Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
IN VIVO UVA TESTING Since the late 1980’s there has been immense debate and controversy on the ‘best way’ to measure and describe the UVA protective properties of sunscreen products. For a host of reasons, some philosophical, some academic, and some commercially biased, the debate has promoted a significant amount of research and development of testing methodology, much of which was captured in a review article in this same journal (35) in 2001. The main breakdown was into two approaches, an in vivo approach using either persistent pigment darkening as a biologic endpoint to asses a UVA Protection Factor (UVA-PF, or ‘PFA’ value), or an in vitro approach to evaluate more of the spectral absorbance ‘balance’ of sunscreens across the UVB and UVA range, described either with ‘ ’ ratings or with a ‘Critical Wavelength’ nm value. The first in vivo result provided more absolute UVA ‘protection’ information, while the latter in vitro approach provided more of a relative indication of the spectral ‘balance’ of the protection, and was intended to be used in conjunction with the SPF value to provide fuller knowledge of the sunscreen protection. The main difficulty lies (still today) in how to best communicate the UVA ‘quality’ of the product to the consumer in a manner that can be readily and accurately understood. For lack of consensus across the industry, academics, and regulatory authorities, the labeling scheme for sunscreen UVA protection has been limited to very simple ‘UVA’ in a circle logo (EU, Canada), and ‘Broad Spectrum’ labeling verbiage in US and Australia. In vivo UVA test protocols that result in a UVA-PF or ‘PFA’ value that have been accepted are the AFFSAPS (French) authorities (36), Japan (37), and a recently published ISO standard (38). These test methods all result in essentially the same numerical values and provide estimates of the relative UVA protection of one product relative to another and can be helpful for medical professionals to guide patients with UVA photosensitive conditions to appropriate sunscreen products. Performing these tests however is logistically and operationally quite challenging as long UVA exposure times are required to elicit the pigment darkening exposures, particularly when testing products with high UVA protection values. Product testing
Sunscreen efficacy testing methods review
Fig. 2. Optical schematic of DRS instrumentation for sunscreen evaluation on human skin.
procedures can require many hours to perform, and uncomfortable for the test subjects. In vitro test methods such as the Critical Wavelength test or the Boot’s Star Rating evaluations are used for ‘Broad Spectrum’ evaluations as they look only at the relative distribution of absorbance between the UVB and UVA regions and not at the absolute magnitude of the UVA protection. The recent acceptance of in vitro UVA test methods (32–34) by various regulatory agencies in several parts of the world has in large part obsoleted much of the need for in vivo UVA testing of sunscreens, and provided both UVA-PF estimates, as well as Critical Wavelength and spectral balance information These last referenced methods still however require reliance on the in vivo SPF test method to ‘calibrate’ the spectrophotometric measurements and set the absolute height of the in vitro absorbance curves. Currently in vivo SPF evaluations are globally required, at least until we have a validated alternative that can reliably evaluate SPF protection.
IDEAL TESTING MODEL: A WORK IN PROGRESS While the vast majority of sunscreen absorbance/ transmittance testing methodology was being developed using conventional spectrophotometers and spectroradiometer instruments, Dr. Nik Kollias and various other collaborators were developing a technique to evaluate sunscreen absorbance in vivo using diffuse reflectance spectroscopy instrumentation. Several publications have shown the applicability of this approach to evaluate UVA absorption of sunscreens on human skin, and to evaluate the UVA-PFs of various formulations (39–42). The technique utilizes two scanning monochromators (see Fig. 2), the first which provides monochromatic UV radiation to Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
the skin via one side of a bifurcated bundle of quartz optic fibers. The emitted energy passes through the sunscreen layer on the surface of the skin and is scattered and remitted within the skin, some of which reemerges from the skin and passes again through the sunscreen layer and is collected by quartz fibers on the second side of the bifurcated optical bundle. This energy is then passed through the second monochromator to a photomultiplier to measure the intensity of the remitted energy. Measurements taken before and after the sunscreen application are compared and the ratio of remitted energy is related to the square root of the effective transmittance of the sunscreen application (since the light has to travel twice through the sunscreen). The technique has been found to be highly predictive of the UVA-PF values of sunscreen products as determined by the conventional UVA irradiation techniques (36–38) used for UVA protectiveness of sunscreens. While useful to determine UVA protection and spectral absorbance characteristics, it is not usable to evaluate the sunscreen absorbance in the UVB region as the skin proteins effectively absorb most to all of the incident UV photons, and there is insufficient remitted UVB energy to measure to be able to calculate absorption properties of the sunscreen. Ruvolo, Kollias, and Cole (43) have taken the approach one step further to construct a hybrid model of the sunscreen absorption spectrum covering the entire UVA and UVB ranges in order to estimate the SPF of a sunscreen without having to irradiate the subjects and elicit the sunburn responses. First, the skin remittance properties of a subject’s skin is measured without the test sunscreen in place, and then again after sunscreen application. The absorbance spectrum of the sunscreen in the UVA portion of the spectrum is calculated. Second, the in vitro absorption of the sunscreen is measured using the conventional thin film spectroscopic techniques (32–34) to obtain a spectral shape of the sunscreen absorbance across both the UVB and UVA range. While the absolute magnitude of this spectroscopic scan may not be accurate, the spectral shape however is invariant, and the UVB portion of the curve can be ‘grafted’ onto the in vivo UVA absorbance curve using the common wavelength areas of the two overlapping spectra (330–340 nm) using the in vivo UVA absorbance values as the ‘absolute’ values and scaling the UVB curve values in the region to match them. The resulting full spectrum curve can then be used to calculate SPF protection factors with the use of UV simulator spectral curves and the erythema action spectra. Evaluations have been completed on a number of sunscreen test products and the correlation with in vivo SPF values has been remarkably predictive over a wide range of SPF values, up to and 85
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in vivo/vitro DRS SPF
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Y = B * xscale(X) B=1.007 R= 0.99 R2=0.98
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Fig. 3. Correlation between hybrid in vivo/in vitro sunscreen SPF test method and human clinical SPF test results.
including SPF 85. Figure 3 shows the comparison of testing results for this hybrid test method compared with conventional SPF testing results for 15 sunscreens and two PVC film ‘surrogate’ sunscreen standards. This technique
appears to work well for these sunscreens, all of which are known to be photostable. It is expected that the accuracy of this technique will not be as reliable when nonphotostable products are utilized, as the sunscreens are not being subjected to the intense UV radiation challenge as would be experienced in in vivo SPF/UVA-PF testing. Further work is needed to assess the appropriate level of UV exposure is needed to challenge the in vitro test sample and to adjust downward the overall absorbance spectrum to reflect the impact of non-photostability on the sunscreen protectiveness and SPF value. With that said, this technique does represent in my opinion, our best path forward to determination of sunscreen SPF and UVA protectiveness indices and spectral characteristics that is both accurate and predictive of human test results – without having to irradiate and harm human test subjects. All the data to date indicate that the only reliable substrate for sunscreen testing appears to be the human himself, and with this new approach, we can attain meaningful evaluations of sunscreen protectiveness without the long and damaging UV exposure to the test subject. This in my opinion represents the best ‘ideal’ method to date and warrants further evaluation in more testing facilities and hopefully consideration for regulatory compliance use.
REFERENCES 1. Federal Register. Proposed rules: sunscreen drug products for over-the-counter human use. Fed Regist 1978; 43: 38207– 38269. 2. Greiter F. Sonnenschutzmittel-Typen und Andwendung. Parfum Kosmetik 1974; 55: 199–202. 3. Deutsches Institut fur Normung Normenausschuss Litchttechnik. Berlin, 1984. 4. Noda T, Kawada A, Hiruma M, Ishibashi A, Arai S. The comparison of sun protection factor values with different light sources. J Dermatol 1992; 19: 465– 469. 5. Berger DS. Specification and design of solar ultraviolet simulators. J Invest Dermatol 1969; 53: 192–199. 6. Sayre RM, Agin PP. Comparison of human sun protection factors to predict protection factors using different lamp spectra. J Soc Cosmet Chem 1984; 35: 439– 445. 7. LeVee GH, Sayre RM, Marlowe E. Sunscreen effectiveness can vary with different simulated solar ultraviolet spectra. J Soc Cosmet Chem 1980; 31: 173–177. 8. Kaidbey KH. Comparison of sun protection factors obtained with a xenon solar 86
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simulator and an Ultravitalux lamp. Photodermatol Photoimmunol Photomed 1987; 4: 154–159. Gabriel KL, Jackson EM, Davies RD, Forbes PD. Sun protection factor testing: comparison of FDA and DIN methods. J Toxicol Cutaneous Ocul Toxicol 1987; 6: 357–370. Sayre RM, Stanfield J, Bush AJ, Lott D. Sunscreen standards tested with differently filtered solar simulators. Photodermatol Photoimmunol Photomed 2001; 17: 278–283. Sayre RM, Dowdy JC. The FDA proposed solar simulator versus sunlight. Photochem Photobiol Sci 2010; 9: 535– 539. Sayre RM, Cole C, Billhimer W, Stanfield J, Ley RD. Spectral comparison of solar simulators and sunlight. Photodermatol Photoimmunol Photomed 1990; 7: 159– 165. COLIPA. Sun protection factor test method. Brussels: The European Cosmetic Toiletry and Perfumery Association, 21, 1994. European Cosmetic, Toiletry, and Perfumery Association (COLIPA): JCIA; CTFA-SA; CTFA. International Sun Protection Factor (SPF) test method, 2006.
15. Labeling and Effectiveness Testing. Sunscreen drug products for over-the-counter human use. Fed Regist 2011; 76: 35620– 356665. 16. ISO 24444:2010 cosmetics – sun protection test methods – in vivo determination of the sun protection factor (SPF). 17. Agin PP, Edmonds SH. Testing high SPF sunscreens: a demonstration of the accuracy and reproducibility of the results of high SPF formulations by two methods and at different testing sites. Photodermatol Photoimmunol Photomed 2002; 18: 169–174. 18. Stanfield J, Ou-Yang H, Chen T, Cole C, Appa Y. Multi-laboratory validation of very high sun protection factor values. Photodermatol Photoimmunol Photomed 2011; 27: 30–34. 19. Damian DL, Halliday GM, Barnetson R. Sun protection factor measurement of sunscreens is dependent on minimal erythema dose. Br J Dermatol 1999; 141: 502– 507. 20. Young AR, Boles J, Herzog B, Osterwalder U, Baschong W. A sunscreen’s labeled sun protection factor may overestimate at temperate latitudes: a human in vivo study. J Invest Dermatol 2010; 130: 2457–2462.
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21. Lott D. Testing SPF 15-100, indoor vs. outdoor. Cosmet Toil 2013; 128: 638–647. 22. Sayre RM, Agin PP, Desrochers DL, Marlowe E. Sunscreen testing methods: in vitro predictions of effectiveness. J Soc Cosmet Chem 1980; 31: 133–143. 23. Sayre RM, Agin PP, LeVee GJ, Marlowe E. A comparison of in vivo and in vitro testing of sunscreen formulations. Photochem Photobiol 1979; 29: 559–566. 24. Groves GA, Agin PP, Sayre RM. In vitro and in vivo methods to define sunscreen protection. Aust J Dermatol 1979; 20: 112– 119. 25. Rohr M, Klette E, Ruppert S et al. In vitro sun protection factor: still a challenge with no final answer. Skin Pharmacol Physiol 2012; 24: 201–212. 26. Groves G. The selection and evaluation of ultraviolet absorbers. Aust J Dermatol 1973; 14: 21–34. 27. Standards Association of Australia. Sunscreen products – evaluation and classification: Australian standard 2604-1986. 1986. 28. Cole C, Van Fossen R. Rapid in vitro evaluation of sunscreens: SPF and PFA. Photochem Photobiol 1988; 47: 73S. 29. Diffey BL, Robson J. A new substrate to measure protection factors throughout the ultraviolet spectrum. J Soc Cosmet Chem 1989; 40: 127–133. 30. Tokgoz NS, Marginean-Lazar G, Ponte A, Fructus AE. Use of synthetic skin for in vitro evaluation of photoprotective effi-
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cacy of sunscreens: applications to different types of emulsions. Proc. 20th IFSCC Cong. Paris, PF. Garoli D, Pelizzo MG, Nicolosi P, Peserico A, Tonin E, Alaibac M. Effectiveness of different substrate materials in vitro sunscreens. J Dermatol Sci 2009; 56: 89–98. ISO 24443:2012 Determination of sunscreen UVA protection in vitro. Matts PJ, Alard V, Brown MW et al. The COLIPA in vitro UVA method: a standard and reproducible measure of sunscreen UVA protection. Int J Cosmet Sci 2010; 32: 35–46. COLIPA (European Cosmetics Association). Method for the in vitro determination of UVA protection provided by sunscreen products, COLIPA Guideline, 2009. Cole C. Measurement of sunscreen UVA protectiveness. Review article. Photomed Photoallergy Photodermatol 2001; 17: 2–10. Agence Française de Sécurité Sanitaire des Produits de Santé (AFSSAPS). Determination of the UVA protection factor based on the principles recommended by the JCIA. 13 January 2006. Japan Cosmetic Industry Association Technical Bulletin. Measurement standards for UVA protection efficacy. Issued 21 November 1995 and effective as from 1 January 1996.
Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
38. ISO 24442:2011 Cosmetics: sun protection test – in vivo determination of sunscreen UVA protection. 39. Kollias N, Gillies R, Anderson R. The noninvasive determination of UVA sunscreen effectiveness in vivo. In: Urbach F, ed. Biological responses to ultraviolet a radiation. Overland Parks, KS: Valdenmar, 1992, 371–376. 40. Moyal D, Refrégier JL, Chardon A. In vivo measurement of the photostability of sunscreen products using diffuse reflectance spectroscopy. Photodermatol Photoimmunol Photomed 2002; 18: 14–22. 41. Gillies R, Moyal D, Forestier S, Kollias N. Non-invasive in vivo determination of UVA efficacy of sunscreens using diffuse reflectance spectroscopy. Photodermatol Photoimmunol Photomed 2003; 19: 190– 194. 42. Ruvolo E, Chu M, Grossman F, Cole C, Kollias N. Diffuse reflectance spectroscopy for ultraviolet A protection factor measurement: correlation studies between in vitro and in vivo measurements. Photodermatol Photoimmunol Photomed 2009; 25: 298–304. 43. Ruvolo E, Kollias N, Cole C. New noninvasive approach assessing in vivo Sun Protection Factor (SPF) using Diffuse Reflectance Spectroscopy and in vitro transmission. Photodermatol Photoimmunol Photomed, in press.
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REVIEW ARTICLE
Sunscreens – what is the ideal testing model? Curtis Cole
Johnson & Johnson Consumer and Personal Products Co. Inc., Skillman, NJ, USA.
Key words: diffuse reflectance spectroscopy; efficacy; SPF; sunscreen; UVA protection
Correspondence: Dr Curtis Cole, Ph.D., Johnson & Johnson Consumer Products Inc., 199 Grandview Rd., Skillman, NJ 08558, USA. Tel: 908 392 6165 e-mail: [email protected]
SUMMARY Sunscreen protection assessment methodologies have been evolving in tandem with the innovation and evolution of sunscreen products themselves; from initial human testing in the Swiss Alps, to laboratory testing with high intensity solar simulators, to spectrophotometers with modern CCD array photocells and diffuse reflectance spectroscopy techniques. The progress in the science leads regulatory development of standard methods, and provides new and improved ways to assess sunscreen protection properties. This review scans much of the history of the development of these methods and highlights the latest development in non-invasive sunscreen testing as an opportunity to improve accuracy while eliminating human UV exposures.
Photodermatol Photoimmunol Photomed 2014; 30: 81–87 HISTORICAL PERSPECTIVE
Accepted for publication: 3 December 2013
Conflicts of interest: None declared.
Sunscreen products are regulated differently in various countries as either drugs (US, Canada, Australian beach products for example) or as cosmetics (Europe, Latin America and Australia-daily wear products for example). They are however, all required to undergo testing to establish the Sun Protection Factor (SPF) that is used on the product packaging. Unlike other monographed drug or ‘positive list’ products that are permitted to make medicinal or health benefit claims by simply including an appropriate amount of the ‘active’ ingredient in the product, sunscreen products are require to undergo clinical performance testing to establish the level of efficacy of the product (for both SPF and Ultraviolet-A radiation (UVA) protection claims). While three individuals are often referenced as the ‘inventors’ of sunscreen products, one is clearly distinguished as the inventor of the SPF factor, Dr. Franz Greiter, who introduced the first products with an SPF indication in 1962. While the concept of SPF is thought to be a precise absolute value that indicates the number of multiples of the minimal amount of UV radiation that you can absorb while using the sunscreen before becoming sunburned, in fact, it should be regarded more as a measure for comparison of one product vs. another. It is quite impossible for someone to calculate the ‘safe time’ in sunlight using a specific SPF value sunscreen. The fact is that no one individual knows for themselves exactly what their ‘burn time’ is when unprotected as it varies with the season and their exposure history, nor can they accurately estimate the rate of sunburning intensity of sunlight that changes hour to hour, and month by month throughout the year – not to mention the variability introduced by fluctuating clouds and surface reflectivity. The concept of a ‘Protection Factor’ has been adopted by regulatory authorities around the globe and codified as an essential claim for any product promising to protect against
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/phpp.12095
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Fig. 1. SPF testing being conducted in the Swiss Alps in 1962 with Dr Franz Greiter, inventor of the SPF rating system for sunscreen protection.
sunburn and sun damage. As a result, consumers have had to learn the meaning and value of the SPF numbers for making product choices. This brings us to the heart of the topic. What is the ideal test method of evaluating sunscreen products? Is there a better way compared to the regulations we have in place now? If we could start from scratch, how would we approach it, knowing what we know now and with the tools now available to us? Perhaps the best way to approach these questions is to look at some of the learning from our collective experiences measuring sunscreen efficacy.
WHAT HAVE WE LEARNED ABOUT IN VIVO CLINICAL TESTING? Importance of the light source for clinical testing Fig. 1 documents the use or natural sunlight on human subjects as the original test methodology that is referenced 82
in the FDA 1978 Proposed Rule for Sunscreen Products (1, 2). While using real sunlight for SPF testing is admirable and ‘realistic’, it is quite unreliable for routine product testing. This approach to determining the ‘protective factor’ of a sunscreen was facilitated by the use of more reliable ‘solar simulators’ that could bring the ‘sun’ indoors to the laboratory for more controlled testing conditions. Laboratory tests for SPF have been were done with metal doped lamps (3), fluorescent lamps (4), and xenon lamps (5) with varying values as a result of the spectral differences and the interaction with the spectral qualities of the sunscreens tested (6–11). Today, the xenon arc solar simulator remains as the sole clinical light source for both SPF and UVA-PF testing of sunscreens. Daniel Berger developed the first clinical solar simulator (5) based on a compact arc xenon burner combined with a UV reflecting dichroic mirror, a visible light absorbing filter, and a shortwave UVC/UVB cutoff filter to shape the spectrum to provide only UVB and UVA radiation. This eliminates the energy from visible light and infrared radiation that is not contributing to the ultraviolet induced erythema and only contributes to heat induced erythema. Unfortunately, the spectral match to the UVB cut-off (12) and the longwave UVA cut-offs (10) do not match sunlight precisely, and can lead to overestimation of sunscreen performance in actual sunlight conditions. The European cosmetics trade association COLIPA developed its own sunscreen testing protocol for assessment of products distributed in Europe which further defined and the spectral distribution limits (13) of the xenon arc solar simulators that has been adopted and codified into the International Standard for Sunscreen SPF (14) testing, the US Federal Register monograph for sunscreen testing (15), as well as the newly developed ISO 24444 Standard (16) for sunscreen SPF testing that is being adopted globally. This refining of the definition of the spectrum breaks the spectrum into 10 nm bands in the short wave UV and limits the cumulative maximum and minimum proportions of the erythema contributing wavelengths within those ranges, helping to minimize the variability of solar simulator spectra used in clinical testing. A limitation was also introduced requiring a minimum amount of UVA1 radiation contained in the output of the solar simulators to eliminate sources without adequate output in this range. Unfortunately, the cutoff filters used to eliminate the visible portion of the spectrum also cuts away significant amounts of the very highest UVA wavelengths in the xenon arc solar spectrum leading to some overestimation of both SPF and UVA protection factors (10). Technical solutions to eliminate this characteristic in a practicable manner are still lacking. Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Importance of common protocol and definitions After the first publication of the FDA Proposed Rule for SPF testing in 1978 (1), multiple protocols, primarily the German DIN method (3) and the COLIPA method (13) were developed for SPF testing using different light sources, definitions for the endpoint, and techniques for ‘color matching’ of the endpoint, as well as differing UV dosing intervals for the UV exposures. Companies wishing to market products globally were faced with the need to test the SPF of the same product several times and market with different SPF values depending on the local results. Collaborative industry efforts to evaluate the various test methods and unify on common test equipment, definitions, and procedures resulted in eliminating extraneous variables and processes and harmonizing on common standards and success criteria. Clinical testing showed that having 7 UV exposure sites narrower UV dose increments did not bring added precision to the result (17). Other testing determined that requiring more than 10 subjects to determine an SPF value did not bring a higher level of precision. Resolving these two variables in the SPF testing procedures allowed for alignment of protocols resulting in fewer unnecessary tests and test subjects to be conducted. There have been two successful sunscreen SPF trials used to demonstrate the capabilities of laboratories to measure SPF far above the SPF 15 values of the control sunscreen used to validate test procedures (17, 18). Both of these studies showed remarkable inter-laboratory reproducibility of SPF values between SPF 30 up to 85. Despite these clear test results, there can be inter-laboratory SPF variability that must be related to uncontrolled testing variables such as the spreading technique of product on the skin (pressure and duration variables), test subject sensitivity (19), and variability in the observation and determination of the biologic endpoint, the Minimal Erythema Dose response. Genetic variability in test subjects across the globe is unavoidable; however training for proper and consistent product application procedures has been shown to help reduce inter-laboratory variability. While there has been significant improvement in reproducibility and alignment of testing protocols for laboratory clinical testing, some publications have challenged the accuracy of laboratory generated SPF results when compared with ‘real sunlight’ spectral challenge or outdoor SPF testing results (20, 21). Given the high variability of the sun’s output and relatively low intensity compared to clinical UV sources, it is impossible to generate extensive outdoor SPF test results. Some of the impact of differences between solar simulator and solar spectra can and have Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
been estimated (11), but the interaction of UV with visible light and infrared radiation are missing from the laboratory testing experience and may have a significant effect on the estimate of erythema protection experienced in full outdoor sunlight over extended periods. Today the laboratory in vivo SPF test results remains the only validated way to determine and label products for SPF protection and is mandated across the globe. Nevertheless, the test requires irradiation of human subjects with damaging amounts of ultraviolet radiation, which ideally should be avoided if alternate methodology were established and validated. Which brings us to the question, why isn’t there a better method to establish a product’s SPF? Why haven’t in vitro test methods been validated?
IN VITRO TEST METHODS FOR SPF DETERMINATION A validated in vitro testing of sunscreens has been long sought ever since sunscreen products were first developed. The advantages over human clinical testing are obvious and compelling: no need to irradiate human subjects, objective laboratory measures can be used instead of subjective visual observations, imperfect solar simulators can be replaced with realistic solar spectra, and long and expensive clinical tests could be avoided. Regulatory agencies also desire to have a validated in vitro method in order to easily and inexpensively test product compliance. Despite the desire and decades of efforts the validated in vitro SPF test remains elusive and unfulfilled. Multiple articles over decades of time and efforts have shown the inability to determine reliable and repeatable SPF testing results based on in vitro test methods (22–25). Trade associations and regulatory/standardization authorities around the globe have conducted tests trying to validate a reliable in vitro SPF test without success. The reference by Rohr et al. (25) describes many of the issues that continue to require further refinement and standardization for the in vitro SPF testing. In vitro testing of sunscreens was initially done with dilute solutions of the UV filters in cuvettes and it was quickly apparent that the protection provided in vivo in products were far from the predicted in vitro values. Sunscreens are applied topically in layers that are approximately 10 to 20 microns thick when applied at an amount of 2 mg/cm2, the application rate prescribed by the first codified SPF test methods. This was considered to be the lowest application density that could be applied consistently for clinical SPF testing purposes. It was determined early on in sunscreen development that the vehicle and how it formed a film on the skin’s surface was critical to 83
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efficiency of the UV filters to provide protection. Artificial surfaces were evaluated in order to better mimic thin film sunscreen behavior on the skin including quartz plates (26, 27) mouse epidermis (20, 24, 28), Transpore™ tape (29), and Vitro-Skin® (30, 31). Each has its own advantages and disadvantages with none achieving a long-lived endorsement for in vitro SPF testing and validation. The most recent substrates that have been endorsed for official in vitro UVA test methods (32–34) are polymethylmethacrylate (PMMA) plates that have had surface modifications to provide various levels and topographical ‘roughness’ parameters. Some have surface roughness of 2 to 6 microns in average depth via a sandblasting procedure, and others have been molded with specific surface topographical features mimicking skin texture with skin roughness in similar ranges. The in vitro spectrophotometric measurements can accurately measure the shape of the absorbance curve, but the absolute magnitude of the absorption curve needs to be established by some other means given the high variability of thin film spectroscopy in vitro testing results and the non-uniform interaction between formulations and the plate surfaces. Without the ability to accurately measure the magnitude of the absorbance curves, it is impossible to qualify an in vitro SPF test method. The last element to be considered for the in vitro SPF tests with the artificial test substrate is the difficulty of finding a ‘universal’ substrate that provides the same surface interaction with the test sunscreen product. The level of UV protection relies critically on the uniformity and continuity of the sunscreen film formed on the test substrate surface. The thickness of product on the substrates is on the order of 10 microns (for full 2 mg/cm2 application density) and between 4 to 7 microns for lower application densities typically used in thin film spectroscopic test methods. This is approximately 1/10 to 1/20th the thickness of a human hair, and is expected to provide UV absorbance properties equivalent to 93–99% blockage of all the UV rays from 290 to roughly 400 nm. Any tiny hole or valley in protection acts like a huge window for UV photons to penetrate and dramatically reduce the protection beneath the sunscreen layer. Thus the uniformity of the film produced on the substrate surface can dramatically affect the overall protection and SPF value measured. Any mismatch of surface interactions between the sunscreen and the substrate can dramatically affect this film layer and provide erroneous measurements of the actual sunscreen protection that would be provided on human skin. Given the virtually infinite combination of sunscreen emollients, emulsifiers, humectants, and film forming polymers and water proofing agents, it has been difficult to
establish a ‘universally equivalent’ human test surface substrate for in vitro SPF testing. Solving the problems of finding a ‘universal’ test substrate, and the reproducibility of application, spreading, and rubbing techniques across laboratories still remain. More will be discussed on SPF testing shortly.
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IN VIVO UVA TESTING Since the late 1980’s there has been immense debate and controversy on the ‘best way’ to measure and describe the UVA protective properties of sunscreen products. For a host of reasons, some philosophical, some academic, and some commercially biased, the debate has promoted a significant amount of research and development of testing methodology, much of which was captured in a review article in this same journal (35) in 2001. The main breakdown was into two approaches, an in vivo approach using either persistent pigment darkening as a biologic endpoint to asses a UVA Protection Factor (UVA-PF, or ‘PFA’ value), or an in vitro approach to evaluate more of the spectral absorbance ‘balance’ of sunscreens across the UVB and UVA range, described either with ‘ ’ ratings or with a ‘Critical Wavelength’ nm value. The first in vivo result provided more absolute UVA ‘protection’ information, while the latter in vitro approach provided more of a relative indication of the spectral ‘balance’ of the protection, and was intended to be used in conjunction with the SPF value to provide fuller knowledge of the sunscreen protection. The main difficulty lies (still today) in how to best communicate the UVA ‘quality’ of the product to the consumer in a manner that can be readily and accurately understood. For lack of consensus across the industry, academics, and regulatory authorities, the labeling scheme for sunscreen UVA protection has been limited to very simple ‘UVA’ in a circle logo (EU, Canada), and ‘Broad Spectrum’ labeling verbiage in US and Australia. In vivo UVA test protocols that result in a UVA-PF or ‘PFA’ value that have been accepted are the AFFSAPS (French) authorities (36), Japan (37), and a recently published ISO standard (38). These test methods all result in essentially the same numerical values and provide estimates of the relative UVA protection of one product relative to another and can be helpful for medical professionals to guide patients with UVA photosensitive conditions to appropriate sunscreen products. Performing these tests however is logistically and operationally quite challenging as long UVA exposure times are required to elicit the pigment darkening exposures, particularly when testing products with high UVA protection values. Product testing
Sunscreen efficacy testing methods review
Fig. 2. Optical schematic of DRS instrumentation for sunscreen evaluation on human skin.
procedures can require many hours to perform, and uncomfortable for the test subjects. In vitro test methods such as the Critical Wavelength test or the Boot’s Star Rating evaluations are used for ‘Broad Spectrum’ evaluations as they look only at the relative distribution of absorbance between the UVB and UVA regions and not at the absolute magnitude of the UVA protection. The recent acceptance of in vitro UVA test methods (32–34) by various regulatory agencies in several parts of the world has in large part obsoleted much of the need for in vivo UVA testing of sunscreens, and provided both UVA-PF estimates, as well as Critical Wavelength and spectral balance information These last referenced methods still however require reliance on the in vivo SPF test method to ‘calibrate’ the spectrophotometric measurements and set the absolute height of the in vitro absorbance curves. Currently in vivo SPF evaluations are globally required, at least until we have a validated alternative that can reliably evaluate SPF protection.
IDEAL TESTING MODEL: A WORK IN PROGRESS While the vast majority of sunscreen absorbance/ transmittance testing methodology was being developed using conventional spectrophotometers and spectroradiometer instruments, Dr. Nik Kollias and various other collaborators were developing a technique to evaluate sunscreen absorbance in vivo using diffuse reflectance spectroscopy instrumentation. Several publications have shown the applicability of this approach to evaluate UVA absorption of sunscreens on human skin, and to evaluate the UVA-PFs of various formulations (39–42). The technique utilizes two scanning monochromators (see Fig. 2), the first which provides monochromatic UV radiation to Photodermatol Photoimmunol Photomed 2014; 30: 81–87 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
the skin via one side of a bifurcated bundle of quartz optic fibers. The emitted energy passes through the sunscreen layer on the surface of the skin and is scattered and remitted within the skin, some of which reemerges from the skin and passes again through the sunscreen layer and is collected by quartz fibers on the second side of the bifurcated optical bundle. This energy is then passed through the second monochromator to a photomultiplier to measure the intensity of the remitted energy. Measurements taken before and after the sunscreen application are compared and the ratio of remitted energy is related to the square root of the effective transmittance of the sunscreen application (since the light has to travel twice through the sunscreen). The technique has been found to be highly predictive of the UVA-PF values of sunscreen products as determined by the conventional UVA irradiation techniques (36–38) used for UVA protectiveness of sunscreens. While useful to determine UVA protection and spectral absorbance characteristics, it is not usable to evaluate the sunscreen absorbance in the UVB region as the skin proteins effectively absorb most to all of the incident UV photons, and there is insufficient remitted UVB energy to measure to be able to calculate absorption properties of the sunscreen. Ruvolo, Kollias, and Cole (43) have taken the approach one step further to construct a hybrid model of the sunscreen absorption spectrum covering the entire UVA and UVB ranges in order to estimate the SPF of a sunscreen without having to irradiate the subjects and elicit the sunburn responses. First, the skin remittance properties of a subject’s skin is measured without the test sunscreen in place, and then again after sunscreen application. The absorbance spectrum of the sunscreen in the UVA portion of the spectrum is calculated. Second, the in vitro absorption of the sunscreen is measured using the conventional thin film spectroscopic techniques (32–34) to obtain a spectral shape of the sunscreen absorbance across both the UVB and UVA range. While the absolute magnitude of this spectroscopic scan may not be accurate, the spectral shape however is invariant, and the UVB portion of the curve can be ‘grafted’ onto the in vivo UVA absorbance curve using the common wavelength areas of the two overlapping spectra (330–340 nm) using the in vivo UVA absorbance values as the ‘absolute’ values and scaling the UVB curve values in the region to match them. The resulting full spectrum curve can then be used to calculate SPF protection factors with the use of UV simulator spectral curves and the erythema action spectra. Evaluations have been completed on a number of sunscreen test products and the correlation with in vivo SPF values has been remarkably predictive over a wide range of SPF values, up to and 85
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Fig. 3. Correlation between hybrid in vivo/in vitro sunscreen SPF test method and human clinical SPF test results.
including SPF 85. Figure 3 shows the comparison of testing results for this hybrid test method compared with conventional SPF testing results for 15 sunscreens and two PVC film ‘surrogate’ sunscreen standards. This technique
appears to work well for these sunscreens, all of which are known to be photostable. It is expected that the accuracy of this technique will not be as reliable when nonphotostable products are utilized, as the sunscreens are not being subjected to the intense UV radiation challenge as would be experienced in in vivo SPF/UVA-PF testing. Further work is needed to assess the appropriate level of UV exposure is needed to challenge the in vitro test sample and to adjust downward the overall absorbance spectrum to reflect the impact of non-photostability on the sunscreen protectiveness and SPF value. With that said, this technique does represent in my opinion, our best path forward to determination of sunscreen SPF and UVA protectiveness indices and spectral characteristics that is both accurate and predictive of human test results – without having to irradiate and harm human test subjects. All the data to date indicate that the only reliable substrate for sunscreen testing appears to be the human himself, and with this new approach, we can attain meaningful evaluations of sunscreen protectiveness without the long and damaging UV exposure to the test subject. This in my opinion represents the best ‘ideal’ method to date and warrants further evaluation in more testing facilities and hopefully consideration for regulatory compliance use.
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21. Lott D. Testing SPF 15-100, indoor vs. outdoor. Cosmet Toil 2013; 128: 638–647. 22. Sayre RM, Agin PP, Desrochers DL, Marlowe E. Sunscreen testing methods: in vitro predictions of effectiveness. J Soc Cosmet Chem 1980; 31: 133–143. 23. Sayre RM, Agin PP, LeVee GJ, Marlowe E. A comparison of in vivo and in vitro testing of sunscreen formulations. Photochem Photobiol 1979; 29: 559–566. 24. Groves GA, Agin PP, Sayre RM. In vitro and in vivo methods to define sunscreen protection. Aust J Dermatol 1979; 20: 112– 119. 25. Rohr M, Klette E, Ruppert S et al. In vitro sun protection factor: still a challenge with no final answer. Skin Pharmacol Physiol 2012; 24: 201–212. 26. Groves G. The selection and evaluation of ultraviolet absorbers. Aust J Dermatol 1973; 14: 21–34. 27. Standards Association of Australia. Sunscreen products – evaluation and classification: Australian standard 2604-1986. 1986. 28. Cole C, Van Fossen R. Rapid in vitro evaluation of sunscreens: SPF and PFA. Photochem Photobiol 1988; 47: 73S. 29. Diffey BL, Robson J. A new substrate to measure protection factors throughout the ultraviolet spectrum. J Soc Cosmet Chem 1989; 40: 127–133. 30. Tokgoz NS, Marginean-Lazar G, Ponte A, Fructus AE. Use of synthetic skin for in vitro evaluation of photoprotective effi-
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38. ISO 24442:2011 Cosmetics: sun protection test – in vivo determination of sunscreen UVA protection. 39. Kollias N, Gillies R, Anderson R. The noninvasive determination of UVA sunscreen effectiveness in vivo. In: Urbach F, ed. Biological responses to ultraviolet a radiation. Overland Parks, KS: Valdenmar, 1992, 371–376. 40. Moyal D, Refrégier JL, Chardon A. In vivo measurement of the photostability of sunscreen products using diffuse reflectance spectroscopy. Photodermatol Photoimmunol Photomed 2002; 18: 14–22. 41. Gillies R, Moyal D, Forestier S, Kollias N. Non-invasive in vivo determination of UVA efficacy of sunscreens using diffuse reflectance spectroscopy. Photodermatol Photoimmunol Photomed 2003; 19: 190– 194. 42. Ruvolo E, Chu M, Grossman F, Cole C, Kollias N. Diffuse reflectance spectroscopy for ultraviolet A protection factor measurement: correlation studies between in vitro and in vivo measurements. Photodermatol Photoimmunol Photomed 2009; 25: 298–304. 43. Ruvolo E, Kollias N, Cole C. New noninvasive approach assessing in vivo Sun Protection Factor (SPF) using Diffuse Reflectance Spectroscopy and in vitro transmission. Photodermatol Photoimmunol Photomed, in press.
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