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In Vitro and In Silico Measurements of Sunscreen Protection

By Joe Stanfield
April, 2013

Sunscreens protect against sunburn by absorbing ultraviolet radiation energy (UVR) from sunlight before it penetrates the skin. The degree of protection by a sunscreen is described by the sun protection factor (SPF).

Commercially available SPFs range from 2 to more than 100. Typically, the SPF is measured on human volunteer subjects by applying 2 mg/cm2 of a sunscreen formula to an area of the mid-back, allowing the sunscreen to dry for 15 minutes, and administering a series of five increasing doses of UVR, simulating sunlight, to skin sites treated with the sunscreen. Another series of five increasing UVR doses is applied within a skin area without the sunscreen. After 16 to 24 hours, the irradiated skin sites are examined to determine the SPF. The SPF is the lowest dose of UVR that caused mild sunburn in the sunscreen-treated area divided by the lowest dose of UVR that caused mild sunburn in the area without sunscreen. The label SPF of a sunscreen formula is based on the average SPF for 10 volunteers. Label SPF values currently range from 8 to more than 100. See U.S. Food and Drug Administration, 21 C.F.R. Parts 201 and 310, Federal Register, Vol. 76, No. 117, Friday, June 17, 2011, 35620-35665.

Because SPF measurement requires administration of UVR to humans, and UVR is a known carcinogen, it is desirable to replace the current in vivo method of measuring SPF with non-invasive methods. See Cole, Forbes, & Davies, Photochem. Photobiol. 43: 275-284 (1986).

Current non-invasive methods for measurement of sunscreen SPF include in vitro measurements on artificial substrates that simulate the skin surface and computerized mathematical models based on the UVR absorbance spectra of active ingredients. The latter approach is known as “in silico” measurements, owing to the fact that computers are composed of silicon circuit components.

Current methods for in vitro measurements of sunscreen protection rely on polymethylmethacrylate (PMMA) or fused silica substrates, with application of weighed amounts of the sunscreen formula that are “spotted” over the surface in small droplets and rubbed with a bare finger that has been conditioned by immersion in the sunscreen formula so that ostensibly, no material is added or removed. Instructions for application typically specify 30 seconds of light rubbing and spreading, followed by 30 seconds of rubbing with high pressure. It is difficult to apply test products uniformly, and it is commonly accepted that the correct application technique is learned by intensive training and practice. There are an ISO Standard and several published methods for in vitro measurements of absorbance spectra and derived values of UVA protection factors and spectral ratios after the absorbance values are corrected for consistency with the SPF value measured on human subjects. However, there is no ISO Standard, regulatory agency protocol, or currently accepted method for in vitro measurements of SPF. See Rohr et al., Skin Pharmacol. Physiol. 7(23): 201-212 (2010); Diffey, Int. J. Cosmet. Sci. 16: 4 7-52 (1994); Broad Spectrum Test Procedure, U.S. Food and Drug Administration, 21 C.F.R. Parts 201 and 310, Federal Register, Vol. 76, No. 117, Friday, June 17, 2011, 35620-35665; Colipa Project Team IV, In vitro Photoprotection Methods, Method for the in vitro Determination of UVA Protection Provided by Sunscreen Products, Guideline, 2011; International Organization for Standardization (ISO), International Draft Standard, ISO/DIS 24443, Determination of sunscreen UVA photoprotection in vitro.

The basic in vitro measure of UV protection is the transmission spectrum, which permits computation of the SPF. The logarithmic transformation of the transmission spectrum yields the absorbance spectrum that is used for determination of the UVA/UVB absorbance ratio, the critical wavelength, and the spectral uniformity index. See Stanfield, In vitro techniques in sunscreen development in: Shaath, N. Sunscreens: Regulations and Commercial Development 3rd ed., Boca Raton, FL, Taylor & Francis Group (2005); Measurement of UVA:UVB ratio according to the Boots Star rating system (2011 revision) Boots UK Ltd, Nottingham, UK; Diffey, Int. J. Cosmet. Sci. 16: 47-52 (1994); Diffey, Int. J. Cosmet. Sci., 31: 63-68 (2009).

Changes in absorbance spectra associated with applied UV doses also permit quantitative assessment of the photostability of a sunscreen formula. See Stanfield, Osterwalder, & Herzog Photochem. Photobiol. Sci. 9: 489-494 (2010).

In vitro measurement of sunscreen protection presents a significant challenge: the set of film thicknesses used for determination of transmission and absorbance spectra must adequately match the final configuration of the sunscreen formula after application on the skin surface, rather than matching the properties of the skin itself. Measurement systems must provide an appropriate optical configuration and sufficient dynamic range and wavelength accuracy. Because many sunscreen formulas are not photostable, the measurement procedures and algorithms must account for changes in SPF and absorbance spectra during exposure to UVR. See Stanfield, Osterwalder, & Herzog Photochem. Photobiol. Sci. 9: 489-494 (2010).

For the simulation of realistic sunscreen film transmittance, the final film irregularity profile must be considered, by applying a relevant mathematical film profile model. See Ferrero et al., J. Cosmet. Sci. 54: 463-481 (2003).

To-date, the in vitro approach for measuring sunscreen protection has focused on matching skin surface parameters to construct substrates that partially simulate the topography of the skin surface, and provide a range of film thickness values. Ideally, a substrate for in vitro measurements would not necessarily resemble skin and its topography, but would simulate the final film thickness profile on skin, after an application of 2 mg/cm2 of sunscreen, as used for the in vivo SPF test. Because sunscreen films change during application to the skin and absorbance of UVR doses, successively measured absorbance spectra should simulate the dynamic behavior of sunscreens on skin. See Stanfield, SÖFW J. 132: 19-22 (2006).

Commercially available substrates for measuring sunscreen absorbance spectra are constructed of PMMA, with known roughness values (Sa) and include the Schönberg sandblasted plate, with a 2 μm roughness value (Schönberg GmbH & Co KG; Hamburg, Germany), the Helioscreen HD-6 molded plate, with a 6 μm roughness value (Helioscreen; Creil, France).and the “Skin-Mimicking” substrate, with a 17 μm roughness value (Shiseido, Yokohama, Japan). See Miura et al., Photochem. Photobiol. 88: 475-482 (2012).

Of the above substrates, only the “Skin-Mimicking Substrate” replicates the roughness value of skin topography, which is about 17 μm, and permits application of 2 mg/cm2 of sunscreen. Ferrero and coworkers have evaluated the performance of substrates with various roughness values. See Ferrero et al., IFSCC Magazine 9(2): 97-108 (2006). Miura has reported results of a ring test comparing SPF results for substrates with 6 μm and 16 μm roughness values. See Miura, Comparison of high and low roughness substrates. Presentation to ISO TC217 WG7, Baltimore, MD, June 22, 2009. All three substrates yield reasonable SPF estimates under a limited range of conditions. However, no known substrate has achieved consistently accurate measurements of in vivo SPF values.

Current substrates do not achieve consistently accurate measurements of in vivo SPF values for at least two reasons: First, current substrates do not adequately address the complex factors that determine the final configuration of the sunscreen film on skin. When a sunscreen film is applied to human skin, the multiple thickness values of the resulting film are determined by skin topography, the sheer forces and thixotropic behavior exhibited during product application and the viscoelastic properties of the skin. The resulting SPF depends on the final distribution of thickness values that determine the effective UVR absorbance of the film. Because the SPF is exponentially related to the thickness of the sunscreen film, thin areas protect much less than thicker areas, and thus have a greater influence on the SPF. When sunscreens are applied to artificial substrates by hand, there is a “waviness” of the top surface that strongly affects the absorbance value and the repeatability of measurements. See O’Neill, J. Pharmaceut. Sci. 73: 888-891 (1984). In order to replicate the actual thickness distribution of a sunscreen film when 2 mg/cm2 is applied to the skin, the substrate must not only have a roughness value (Sa) that is similar to that of the skin, but must simulate the final geometry of the sunscreen film on skin. Researchers have attempted to compensate for the multiple factors of application by employing special protocols for the product application procedure, such as rubbing for various lengths of time at various pressures, and intensive training of laboratory personnel. These measures have improved accuracy for particular formulas, but optimum application techniques differ for different types of formulas. No substrate or application technique is wholly effective for even a small subset of the wide range of sunscreen formula types and characteristics on the market.

Second, several widely used sunscreen ingredients are not photostable, and almost all sunscreen formulas degrade and/or “settle” on the skin to some extent, which means that their ability to absorb UVR changes as UVR is absorbed and the temperature is increased. The time course and extent of photodegradation and other changes, such as evaporation of volatile ingredients and skin penetration, depends on the thickness of the sunscreen film, but with different mechanisms than the thickness dependence of UVR absorbance. Therefore, the substrate must simulate the final film thickness distribution on skin, not only to duplicate the UVR absorbance on skin, but also to account for potential photodegradation and other changes, as well as the multiple factors of application.

Successful In Vitro and In Silico measurements of sunscreen protection present formidable challenges. At Suncare Research Laboratories, we are pursuing new approaches to meet these challenges.

US FDA announces final rule on sunscreen labeling

Brianne Rothrock
Suncare Research Laboratories, LLC
July 2011

Like in many years before, as the dawn of summer 2011 approached, consumers were left to their own devices to decide which sunscreen products are most effective. With manufacturers, dermatologists, politicians, scientists, advocacy groups and the media all contributing their own sometimes conflicting opinions, the consumer has been left in the dark in determining which products are truly effective.

That is until 10am on June 14, when the FDA held a press conference to announce the much anticipated regulations on sunscreens. The FDA’s announcements included:

  • A Final Rule for sunscreen labeling and testing;
  • A Proposed Rule;
  • An Advance Notice of Proposed Rulemaking (ANPR) for Dosage Forms; and
  • A Draft Enforcement Guidance for Industry.

Final Rule for Labeling

Sunscreen products that meet the current standards for effectiveness can now be labeled with information that assists consumers in selecting the best products that, when used with other sun protection measures, help prevent sunburn as well as reduce the risk of skin cancer and premature skin aging. The final regulation allows for sunscreen products that pass the FDA’s test for protection against both ultraviolet A (UVA) and ultraviolet B (UVB) rays, which both contribute to sunburn, skin cancer, and premature skin aging, to be labeled as “Broad Spectrum.” Under the new standards for labeling, sunscreens that meet the criteria for both “Broad Spectrum” labeling and have a SPF of 15 or greater, can claim that “sunscreen reduces the risk of skin cancer and early skin again when used as directed” (U.S. FDA). Manufacturers must comply with these label changes by June 2012.

Other important label changes address application and water resistance. Now, regardless of the formula, no label can contain the terms “waterproof,” “sweat proof,” “sunblock,” or “all day protection” (U.S. FDA).  Now, labels must also state the time (40 or 80 minutes) that the product remains water resistant and that reapplication is required every two hours. The improved, informative Drug Facts Box and new product labeling are a welcome improvement.  

Changes in Testing

Testing requirements for UVA protection claims have also been revised. In vivo UVA testing is no longer required; and the four star UVA labeling system and descriptors have been done away with. A simple critical wavelength spectrometer reading has replaced the previous extensive in-vitro UVA testing. Procedures for SPF testing have also been modified. The required number of test subjects has been dropped from 20-25 to 10.

The Proposed Rule, ANPR, and Draft Enforcement Guidance for Industry

In addition to the final rule on sunscreen labeling, three additional important regulatory documents were released. The proposed rule would limit the maximum SPF value to “50+” on sunscreen labels because there is no significant data to demonstrate that products with SPF values greater than 50 provide better protection for consumers than products with a SPF value of 50. According to Shelly Burgess of the FDA, the ANPR “will allow the public a period of time to submit requested data addressing the effectiveness and the safety of sunscreen sprays and to comment on possible directions and warnings for sprays that the FDA may pursue in the future, among other issues regarding dosage forms for sunscreens.” The Draft Enforcement Guidance for Industry provides information to assist sunscreen product manufacturers understand how to properly test and label their products in accordance with the new final rule and other regulations.

Despite various media claims that sunscreens cause vitamin D deficiency and assorted other dangers that leave us vulnerable to medical problems and cancers, there is no evidence that sunscreen causes any adverse effects. According to Warwick Morison, MD, MB and Steven Wang, MD,  “When used as directed, [sunscreen] can reduce the risk of actinic keratosis, the most common skin precancer, and squamous cell carcinoma, the second most common skin cancer” (Morison). The jury is still out on basal cell carcinoma and melanoma, the most deadly form of skin cancer. Because current sunscreen products have been on the market for many years without proven danger, the FDA does not have any reason to believe that these products are not safe for consumer use. However, the FDA is currently re-examining the safety information that is available for active ingredients in current sunscreen products.

The Sunscreen Labeling Protection Act of 2011

The FDA’s announcement came after many years of years of pressure from six U.S. senators. In a May 25 letter to Commissioner Hamburg and Director Lew of the FDA, Senators Jack Reed (RI), Tom Harkin (IA), Charles Schumer (NY), John Kerry (MA), Patrick Leahy (VT), and Kirsten Gillibrand (NY) urged the FDA to release the final monograph that they promised to release by Fall of 2010. The primary concern of the senators’ letter, urging for “clearly and accurately labeled sunscreen products, containing comprehensive information that includes UVA and UVB protection,” was, surprisingly enough, what the FDA delivered just shy of a month later (Reed).  Senator Jack Reed has served as the leader of the cause, pressuring the FDA to “finalize [the] monograph without further delay so that Americans will no longer be deceived into thinking they are truly protected from the sun when that isn’t the case” (Reed). Reed released a statement June 14, the day of the FDA’s press conference, saying, “This is a victory for consumers that is long overdue…These new sunscreen standards are going to give consumers better, more accurate information” (Reed).

The FDA’s final rule on labeling, however, did not satisfy the senators in their quest for a final rule on sunscreen products.  On the same day as the release of the letter to the FDA, Senators Reed, Schumer, Kerry, Leahy, and Al Franken (MN) submitted a bill to “make effective the proposed rule of the Food and Drug Administration relating to sunscreen drug products, and for other purposes” (US Congress). If passed, the SUN Act will make the FDA’s proposed rules become law 180 days from passage of the Act, unless the FDA issues the final rules.



References:


“Labeling and Effectiveness Testing; Sunscreen and Drug Products for Over-the-
Counter Human Use (Final Rule).” Federal Register 76.117 1-15. Web. 05 Jul
2011.

Morison, Warwick L. and Wang, Steven Q. “Sunscreens: Safe and Effective?” The Skin
Cancer Foundation Journal. 29 (2011): 55-58.

Reed, Jack. “After Years of Prodding from Reed, FDA Strengthens Sunscreen
Standards.” 14 Jun 2011. Senator Jack Reed. Web. 08 Jul 2011.

Reed, Jack; Harkin, Tom; Schumer, Charles; Kerry, John; Leahy, Patrick; Gillibrand,
Kirsten. Letter. 25 May 2011. United States Senate. Print. 08 Jul 2011.

Shaath, Nadim. "Finally...The Final Rule On Sunscreen Labeling." The Sunscreen Filter.
Household And Personal Products Industry, July 2011. Web. 05 July 2011.

United States. Cong. Senate. 112th Congress, 1st Session. S.1064, Sunscreen Labeling
            Protection (SUN) Act of 2011. 112th Cong, 1st sess. Congressional Bills, GPO
            Access. Web. 08 Jul 2011.

United States Government. Food and Drug Administration. FDA Announces Changes to
            Better Inform Consumers About Sunscreen. U S Food and Drug Administration
            Home Page. US Food and Drug Administration, 14 June 2011. Web. 06 July
2011.


UVA Protection: An Update

Joe Stanfield
Suncare Research Laboratories
December, 2010


UVA Radiation
The longer ultraviolet wavelengths in sunlight are known as UVA radiation (320-400 nm). Until a few years ago, the shorter wavelengths, known as UVB (290-320 nm), were considered the most important contributor to skin damage resulting from excessive sun exposure. New studies have provided evidence of greater UVA involvement in skin tumor development, suppression of immune function and premature aging, than previously realized. [1]

UVA comprises 90-95% of terrestrial radiation, and penetrates deeply into the dermis, whereas the shorter UVB wavelengths, are up to a thousand times more effective in producing sunburn, but penetrate the more superficial epidermis only. UVB is associated primarily with damaging to cellular DNA, while UVA damages DNA to a lesser extent, and is primarily associated with indirect damage to skin cells through production of free radicals. Sunlight is always a mixture of UVA and UVB, and laboratory tests that separate the effects of UVA from those of UVB can distort the relative importance of UVA protection. Perhaps the most important reason for "balanced" UVA and UVB protection is to assure that sunscreen products protect over the broad range of solar spectra that occur with varying sun angles due to latitude, season and time of day. [2]

The new marketed sunscreen products, with SPFs up to 100 offer vastly improved protection against solar UVA, as well as UVB, yet the FDA has not published a Final Rule for SPF labeling or UVA protection labeling.

Recommendation of the European Communities (EC)
In 2006 the Commission of the European Communities (EC) recommended that sunscreen products have a UVA protection factor (UVAPF) that is at least 1/3 the SPF and a critical wavelength [3] of at least 370 nm.[4] The Commission also endorsed the use of in vitro methods for measuring the UVA protection factor. The European Cosmetics Association (Colipa) has since published a Guideline for in vitro measurements of the UVAPF/SPF and critical wavelength. [5]

UVA Protection Test Methods
Earlier proposed methods, in addition to the Critical Wavelength, include the persistent pigment darkening (PPD) test [6] and the Boots Star Rating, which is still used in the UK. [7] The Critical Wavelength method proposed by Diffey requires mathematical integration of the in vitro product absorbance spectrum from 290 to 400 nm to determine the wavelength below which 90 percent of the cumulative area of the absorbance curve resides. If the Critical wavelength is 370 nm or greater, the product is considered "broad spectrum," which denotes balanced protection throughout the UVB and UVA ranges.

Persistent Pigment Darkening (PPD) Test
The PPD test is a laboratory evaluation on human subjects that yields the ratio of the UVA dose required to produce a defined skin darkening response (Minimal Persistent Pigment Darkening Dose, MPPDD), with and without a sunscreen product on the skin. The response is evaluated 2 to 4 hours after administration of UV doses. The ratio of the MPPDD with the sunscreen product on the skin to that without the sunscreen is called the UVAPF, or the PFA.

The PPD test produces rapid results with moderately low doses of UVA. Product UVA protection may be categorized based on the UVAPF. The PPD response is stable and reproducible; however its clinical significance is questionable, because the action spectrum for PPD is not defined for wavelengths shorter than 320 nm, and the response is masked during outdoor sun exposure by other skin responses to UV. Thus it is impossible to relate the PPD protection factor directly to the degree of protection is sunlight.

Boots Star Rating
The Boots Star Rating [7] involves in vitro measurement of the product absorbance spectrum and calculation of a ratio of average UV absorbance to average UVB absorbance. The ratio is used to assign products among 3 categories of "broad spectrum," protection, denoted by stars. (Now 3 to 5 stars, depending on the UVA/UVB ratio before and after irradiation with a UV dose of 17.5 J/cm2.

FDA Star Ratings
The FDA does not permit a numeric UVA protection at present. On August 27, 2007, the FDA published a proposed Sunscreen Monograph Amendment (8) that outlines a UVA rating system based on "stars" similar to the Boots Star Ratings (See above). To label a product with one or more stars, the sunscreen must be tested on 20 human subjects for protection against PPD (See above). The proposed ratings are as follows:

  • For one star (Low) the UVAPF must be 2 to under 4.
  • For 2 stars (Medium)the UVAPF must be 4 to under 8.
  • For 3 stars (High) the UVAPF must be 8 to under 12.
  • For 4 stars(Highest) the UVAPF must be 12 or more.
In addition to the in vivo test, the FDA proposal also requires an in vitro test on roughened quartz plates that measures the ratio of the area under the absorbance curve from 340 to 400 nm (UVAI) to that under the full UV absorbance curve (290-400 nm). The measurement is made after irradiation with 2/3 the labeled SPF in MEDs (1 MED = 20 effective mJ/cm2). The proposed ratings are as follows:
  • For 1 star (Low) the ratio must be 0.20 to 0.39.
  • For 2 stars (Medium) the ratio is 0.40 to 0.69.
  • For 3 stars (High) the ratio is 0.70 to 0.95
  • For 4 stars (Highest) the ratio must be greater than 0.95.
Both the in vivo and the in vitro tests must be performed and the lower rating of the two methods is used for labeling. The FDA ratings are very stringent, and achieving a 4 star rating will be a significant challenge for manufacturers in the US, with our limited choices of active ingredients, for years to come—that is IF and WHEN the FDA finalizes the regulations.

FDA Final Monograph
The original proposed FDA Sunscreen Monograph was issued in 1978. There are still no final label ratings for SPF or UVA protection. (Although SPF numbers and UVA claims such as “Broad Spectrum” are widely used.) As of this posting, the Final Monograph is expected to be released before the end of 2010, and will contain detailed labeling requirements for SPF and UVA protection.

Photostability
At present, Avobenzone is the only significant, broad spectrum UVA protective active sunscreen ingredient available for general use by sunscreen manufacturers in the U.S. Lack of photostability is a major problem for sunscreens containing Avobenzone, particularly in combination with Octinoxate. (9,10) There are successful strategies for stabilizing Avobenzone, including the use of commercially available stabilizers and avoiding Octinoxate [11], but achieving photostable, broad spectrum sun protection is a significant challenge in the U.S. at present.

References
  1. Fourtanier A, Bernerd F, Bouillon C, Marrot L, Moyal D, Seité S, Protection of skin biological targets by different types of sunscreens. Photodermatol Photoimmunol Photomed 2006; 22: 22-32
  2. Lim H, Rigel D. UVA: Grasping a better understanding of this formidable opponent. Skin & Aging. 2007;15:62-67.
  3. BL Diffey. A method for broad spectrum classification of sunscreens. Int J Cosmet Sci 16:47-52, 1994.
  4. The Commission of the European Communities. Commission Recommendation of 22 September 2006 on the efficacy of sunscreen products and the claims made relating thereto. Official Journal of the European Union, L265/39, 26.9.2006.
  5. In vitro UV Protection Method Task Force, In vitro method for the determination of the UVA protection factor and critical wavelength values of sunscreen products, Colipa, 2009.
  6. Chardon A, Moyal D, Hourseau C. Persistent pigment darkening as a method for the UVA protection assessment of sunscreens. In: Protection of the Skin Against Ultraviolet Radiations, Rougier A, Schaefer H, eds. John Libbey Eurotext, Paris 1998, pp. 131-136.
  7. Measurement of UVA:UVB ratio according to the Boots Star rating system (2008 revision). Boots UK Limited, Nottingham, NG2 3AA, UK. January 2008.
  8. U. S. Food and Drug Administration. Sunscreen Drug Products for Over-the-Counter Human Use; Proposed Amendment of Final Monograph; Proposed Rule; 21CRF Parts 347 and 352. Federal Register 72 (165) August 27, 2007. 49070-49122.
  9. Sayre R, Dowdy J. Photostability Testing of Avobenzone. Cosmetics & Toiletries. 1999;114 (1):85-91.
  10. Sayre R, Dowdy J,Gerwig A, Shields W, Lloyd R. Unexpected photolysis of the sunscreen octinoxate in the presence of the sunscreen Avobenzone. Photochem Photobiol, 2005;81:452-456.
  11. Bonda C, Pavlovic A, Hanson K, Bardeen C. Singlet quenching proves faster is better for photostability. Cosmetics & Toiletries 2010;125(2): 40-48.

 

 
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