UV Transmission Assessment: Influence of Temperature on Substrate Surface

Editor’s note: This article is the first in a series of three considering the effects of certain test variables on SPF results. Here, the authors assess how variations in substrate surface temperature affect SPF. The next, to appear in September 2013, evaluates SPF results based on different substrates. The third will focus on the influence of pressure on SPF and is scheduled to appear in November 2013.

During outdoor recreation, protection against photo damage can be afforded by sunscreen products, which act by absorbing or scattering ultraviolet (UV) radiation. Historically, evaluating the level of protection afforded by sunscreen products against UVB, i.e., the 290–320 nm solar spectrum range, is based on an in vivo method1, 2 and universally expressed by its SPF. This method involves comparing the UV radiation dose required for the appearance of a biological endpoint, erythema, with and without protection. And although UVB radiation is mainly responsible for sunburn, recent studies have shown that UVA radiation (320-400 nm) can cause a number of detrimental effects in human skin. Until now, the in vivo method used for evaluating protection against UVA has been based on Persistent Pigment Darkening (PPD). However, in both cases, for ethical, economical and practical reasons, companies and health authorities want to substitute these in vivo methods with in vitro methods.

The determination of in vitro SPF by means of a spectrophotometer was initially described by Diffey and Robson,3 then modified and improved in view to evaluate the skin protection against UVB brought by a product. This method is based on the assessment of UV transmittance through a thin film of sunscreen sample spread onto a roughened substrate. Concerning in vitro UVA Protection Factor (UVA-PF) and critical wavelength (CW) evaluation, much work has been conducted4 in recent years to establish a reliable method. Similar to Diffey and Robson, the Cosmetics Europe, formerly COLIPA, and International Organization for Standardization (ISO) in vitro methods5, 6 are based on an assessment of the UV transmittance of a thin film of sunscreen sample spread on a roughened substrate after exposure to a controlled dose of UV radiation from a defined UV source.

Prior to in vivo/in vitro correlation, reproducibility appears to be crucial to the reliability of any of these alternative in vitro methods. Variations in parameters such as the amount of product applied, spreading protocol or properties of the substrate have been identified, studied and corrected in different methods in order to ensure reliable results. Conditions such as application and spreading, also essential to reliability, are not so easy to control. Here, the authors focus their work on a parameter that has not been yet considered: the substrate surface temperature during application, spreading and drying steps. Clearly it has been more or less define the condition of temperature of the room during testing also temperature of pre stocking either the products to be tested and the plates but considering thermodynamics, it is logical that the temperature of both the substrate surface and product while applying and resting may greatly influence the quality of film-forming. This paper thus presents an investigation of this specific temperature control on final in vitro values.

For this, sunscreen products were spread and the temperature of the substrate surfaces were controlled by means of a specific device developed for this purpose. Obviously, considering the previous comments on conditions for testing, all other conditions were kept strictly identical: using the same operator and quantity of product; the temperature of the product before application; substrate properties—i.e., roughness and surface energy; the spreading method, i.e., movement, pressure and drying time, realized under recordings and video controls; and the same transmittance analyzer. This work is part of a larger reproducibility optimization program that aims to identify, demonstrate and control all variables that can influence in vitro SPF. Clearly, whatever parameter is considered, its importance will depend on the products themselves. Note that for the present studies, products had not been exposed to UV radiation according to standardized method,5 although CW was still determined.

Materials and Methods

Temperature control: To assure the temperature of the substrate surface was controlled throughout application, spreading and drying, a temperature control device was developeda to maintain accuracy within approximately 0.3°C. In brief, this device consists of a heating element that heats a tray at a previously selected temperature, and a PT1000 measuring tube, which monitors the temperature of the substrate surface. The plate can be taken off the tray by means of a removable metallic support. The temperature of the substrate surface is maintained during the application and spreading steps based on thermal inertia. This support is designed for use with a specific polymethyl methacrylate (PMMA) substrateb. Images of this device are shown in Figure 1.

Substrate selection: A previous study7 showed the importance of substrate roughness on the reproducibility of in vitro SPF testing. Thus, molded polymethyl methacrylate (PMMA) platesb with one face roughened and measuring 47 mm x 47 mm x 1.5 mm, were used. The roughness parameter Ra, i.e., the mean arithmetic deviation of the assessed profile from the average profile, equaled 4.85 ± 0.29 µm. Other complementary surface topography parameters were also controlled by means of a profilometerc, which ensures the plates meet the defined specifications described in the ISO standard for UVA determination.6, 8

Sunscreen products: Thirty-seven sunscreen products of different types were chosen for this study. They included: o/w emulsions, i.e., cream, lotion or spray; w/o emulsions, i.e., lotion or spray; oils; alcohol-based sprays; sticks; and a foundation compact (see Table 1).

Sunscreen application: Before applying the test products, each researcher’s application finger (without finger cot) was pre-saturated with the product. Nine drops equal to 28.7 ± 0.5-mg of the test sunscreen product were applied by a 1-mL syringe across the whole PMMA plate surface maintained at a specific temperature by means of the device. To ensure the correct application rate of 1.3 mg/cm², the syringe was weighed before and after product application.

Immediately after weighing, the sunscreen product was spread over the whole surface using a defined sequence of circular, linear and light linear strokes. Besides temperature control of the substrate surface, the pressure was controlled by means of a balance and movements by video. The video assured the same spreading movement and spreading time. After product spreading, the sample was allowed to dry and settle for 15 min at the previous fixed temperature on the device. After drying, UV transmittance measurements were performed on the samples.

Transmittance measurements: Before taking measurements, the transmittance analyzerd was validated and controlled by both the ISO and Cosmetics Europe standardse, and the linearity/additivity by calibrated reference standard HD0 PMMA calibration platesf in which UV filters were added. Measurements were taken from 290-400 nm at every 1 nm.

As a control, a PMMA plate covered with a film of white petrolatum, 15 mg applied, was used to obtain blank transmittance. Two different plates were used for each tested product and nine UV transmission spectras were recorded for each plate at the different application locations.

In vitro SPF calculation: By means of the sunscreen UV transmittance measurement T(λ), in vitro SPF could be calculated by combining the UV light attenuated by the sunscreen film, the erythema action spectrum, and a relevant solar emission spectrum according to Equation 1:

Eq. 1

where E(λ) is the relative effectiveness of UVR in producing erythema in human skin according to the erythema action spectrum (CIE-1987) at wavelength λ; I(λ) is the spectral irradiance received from the UV source at wavelength λ, in midday, midsummer sunlight; A(λ) is the monochromatic absorbance of the sunscreen layer at wavelength λ, with A(λ) = - log [T(λ)]; and d(λ) is the wavelength step, equal to 1 nm in this study. Finally, the in vitro SPF reported in this study is the arithmetical mean of the 18 in vitro calculated SPFn values for each n location. The range of variation of in vitro SPF values is characterized through standard deviation (SD).

Critical wavelength: The well-known critical wavelength (CW) index9 describes the range of protection over the entire spectrum of 290–400 nm. The λc is obtained when the integral of the absorbance spectrum from 290–400 nm reaches 90% by means of the following Equation 2. Although CW is also calculated from UV transmission after UV exposure at UV dose D, the authors used this method without UV radiation for practical reasons. Clearly, CW is, in a majority of cases, equal or lower after UV exposure than before, but one could consider the same influence of temperature regardless of the behavior of the product under UV exposure.

Eq. 2

Here, A(λ) is the monochromatic absorbance of sunscreen layer at wavelength λ; λc is the critical wavelength calculated to comply with the Equation 2; and d(λ) is the wavelength step, equal to 1 nm in this study.

Comparing in vitro values: To account for some variation in in vitro values, differences between two means, i.e., in vitro SPF or in vitro UVA-PF, were evaluated for significance according to a calculation described in a previous study.10 This method is based on a statistical explanation to compare the means of two values under the normality hypothesis according to the student’s t-test and with variance homogeneity according to Fisher’s F-test. Nevertheless, the authors considered in vitro SPF values above 100 as overestimated, and these should be taken with caution.

Correlation coefficient calculation: The relationship between temperature and in vitro indexes was calculated through Spearman’s rank correlation coefficient ρ, also signified by rs. This calculation method was chosen because even if a non-linear relationship exists, the relationship can be monotonic. In this case, analysis by Spearman’s correlation is more suitable than Pearson’s correlation. The authors used the procedure without tied ranks data to calculate rs according to Equation 3:

Eq. 3

where di is the difference between the two numbers in each pair of ranks, and n is the sample size.

Results and Discussion

Temperature control device evaluation: The first step of the study was to evaluate the performance of the temperature control device with and without its removable metallic support. For that, variations in temperature of the substrate surface were monitored over several minutes (see Figure 2) during the whole process of application, spreading and drying steps; the protocol is detailed in Figure 3.

These results confirmed very low temperature variations—approximately 4°C, of the substrate surface during the whole process at 35°C, mainly during the application and spreading steps, when the metallic support was used. Without the metallic support, the temperature varied up to 10°C. At 30°C or 25°C, use of the metallic support also maintained the temperature throughout the process better than without the support. Thus, by means of metallic support, temperature variation was reduced by half. For this first prototype, the metallic support was made of aluminum; thermal inertia performance was found to improve with the use of an isolator, although not used in this study. Figure 2 shows the accuracy of the temperature control device during the drying step. Based on these results, all further assays employed the temperature control device with its removable metallic support.

In vitro indices evaluation: Next, the in vitro SPFs and CWs for all 37 products were evaluated at graduated temperature levels, from 20°C to 35°C by steps of 5°C; detailed results are shown in Table 2. According to the differences calculation method described in a previous study,10 Table 3 was created with results cross-referenced between different temperatures according to codes: ▲ meaning significant differences were found in the in vitro SPF with increased temperatures; = meaning no significant differences in in vitro SPF; and ▼ meaning significant differences were found in the in vitro SPF with decreases in temperature.

From the results shown in Table 2, the in vitro spreading method with the same operator was found to be repeatable within two plates for each temperature and with a low average coefficient of variation for in vitro SPF of 11.1%, and 10.0% for in vitro UVA-PF. It is important to note that the influence of temperature will depend on the product and temperature range. The percentages of products influenced by temperature during spreading and drying steps are summarized in Figure 4.

These results indicate how an increase of about 5°C can change the in vitro SPF values for about 81.1% of products. This percentage increases as the temperature difference increases. The authors selected two particular products, P5 and P29, which were influenced the most by temperature variations and observed the impact of just a 2°C difference during spreading and drying steps. Results confirmed this influence of temperature (see Table 4 and Figure 5).

The relationship between temperature and in vitro values was calculated using Spearman’s correlation, rs; nevertheless, statistical results must be taken with caution, considering the small amount of data involved (temperature, n = 4) and cases of SPF overestimated (SPF > 100). Thus, the goal of this part of the study was only to show the qualitative relationship between in vitro indexes and temperature.

Three types of correlation were considered, including: weak = - 0.5 < rs < 0.0 or 0.0 < rs < 0.5; moderate = -0.5 < rs < - 0.8 or 0.5 < rs < 0.8; and strong = - 0.8 < r < - 1.0 or 0.8 < r < 1.0. From correlation results (see Figure 6), the existence of a dose-effect relationship between temperature and in vitro indexes was shown for a majority of products; i.e., moderate and strong correlations.

By means of results of difference calculation method (see Table 3) and correlation (see Figure 6), the influence of temperature only on in vitro SPF is presented in Figure 7, Figure 8 and Figure 9 according to three different behaviors: in vitro SPF decrease, identical SPFs, and SPF increase with temperature elevation.

Finally, it was observed that temperature of substrate surface had an influence on the CW value. Indeed, in more than half of the cases, CW changed when the temperature rose from 20°C to 25°C. The mean difference for CW results due to temperature is 1.2 nm, with a maximum of 4.0 nm. The mean difference between 20°C to 30°C and 20°C to 35°C were, respectively, 1.5 nm with a maximum of 5.0 nm and 1.6 nm with a maximum of 4.0 nm.


The measurements of 37 products applied on substrate surfaces were elevated to a range of temperatures and demonstrated a significant impact on in vitro SPF and CW values. Therefore, based on the data obtained, it is not demonstrated there is an ideal temperature and as it is product dependant we can suppose the usual range seems to be correct but it obviously highly recommended to fix and control the temperature of substrate surfaces during whole UV protection assessment process to avoid variability of results intra or inter laboratory and finally, to improve reproducibility.

Very low temperature variations (2°C) can change in vitro results. Further, choice and control of the temperature during the UV exposure with a solar simulator should be also considered and equal to the spreading and resting step for the case of in vitro UVA0-PF with an incidence on the final UVA-PF and the irradiation dose. Finally, the temperature level during all procedure should be considered as a key parameter, just as substrate topography parameters, product amount and spreading protocol to assure a future reliable harmonized method.

Acknowledgements: The authors would like to thank Anthony Jouny of XE-Solutions for his technical support and development of the temperature control device.


  1. US Food and Drug Administration, Sunscreen drug products for over-the-counter human use, Final Rule 21 CFR parts 201 and 310, Federal Register 76 (117) (Jun 17, 2011)
  2. ISO 24444:2010, Cosmetics—Sun protection test method—In vivo determination of the sun protection factor (SPF), available at www.iso.org/iso/catalogue_detail.htm?csnumber=46523 (2010)
  3. BL Diffey and J Robson, A new substrate to measure sunscreen protection factors throughout the ultraviolet spectrum, J Soc Cosmet Chem 40 127–133 (1989)
  4. D Moyal, UVA protection labeling and in vitro testing methods, Photochem Photobiol Sci 9 516–523 (2010)
  5. Colipa Method for in vitro determination of UVA protection, available at www.cosmeticseurope.eu/publications-cosmetics-europe-association/guidelines.html?view=item&id=33 (2011)
  6. ISO 24443, Cosmetics—Sun protection test method—Determination of sunscreen UVA photoprotection in vitro, ISO/FDIS 24443:2011(E) (2011)
  7. L Ferrero, M Pissavini, A Dehais, S Marguerie and L Zastrow, Importance of substrate roughness for in vitro sun protection assessment, IFSCC 9(2) 1–13 (Apr/Jun 2006)
  8. M Pissavini, S Marguerie, A Dehais, L Ferrero and L Zastrow, Characterizing roughness: A new substrate to measure SPF, Cosm & Toil 124(9) 56–64 (Sep 2009)
  9. BL Diffey, A method for broad-spectrum classification of sunscreens, Intl J Cos Sci, 16 47-52 (1994)
  10. M Pissavini, O Doucet and O Brack, Interpretation of SPF in vivo results: analysis and statistical explanation, Cosm & Toil 126(3) 172-184 (Mar 2011)
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