Reliable and simple spectrophotometric determination of sun protection factor: A case study using organic UV filter‐based sunscreen products

Current in vitro SPF screening method for plant oil body (oleosome)‐based SPF products possesses significant inconsistency and low reliability in the SPF rating.

the skin through accelerated aging processes. 2 UV radiation induces a malfunction of the body's protective systems, such as increased inflammation, decreased tissue repair functions, and serious damage to cutaneous DNA, which elevates the occurrence of skin cancer. 3 UV radiation damages on the skin are race, skin type, and climate dependent with, for example, the highest development rate of cutaneous malignant melanoma occurring in Australia compared to other geographic regions. 4 In the general public, skin cancer accounts for approximately 40% of all cancers, comprising basal skin cancer (80%), squamous cell carcinomas (16%), and melanomas (4%). 5,6 The mode of UV-induced destructive actions is governed by dosages that are higher than the tolerance limits of the body. 7 UVB irradiation induces the formation of reactive oxygen species (ROS) in the skin and increases the oxidative stress on the skin's important macromolecules, inducing destructive damages and loss of cellular function. In addition, UVB induces more fundamental and serious damages: genetic mutation such as pyrimidine dimer formation, inflammation, and skin cancer. 5 UVA is a deeper penetrating spectral range of UV radiation compared to UVB and is also believed to induce an increased level of oxidative stress in skin tissue. The dermis is the most sensitive area for UVA irradiation damage. Among UVA absorbing tissues and molecules, collagen is the most sensitive tissue to UVA radiation in the dermis and it causes wrinkle formations on the skin or aging effects. The mode of action has been postulated to be direct cellular damage, formation of oxygen radicals, and a reduction of antioxidant defense mechanisms, all of which cause damage to the skin. 8 It is recommended that people wear sunscreen products as well as physical protection such as using protective clothing regularly to prevent damage to the skin. Better protection through higher sun protection factor (SPF)-labeled products is commonly believed to provide enhanced protective benefits. 9 A thorough evaluation of the data collected from a cohort study over 147,900 people conducted in Norway between 1991 and 2007 demonstrated the beneficial effects of using higher SPF products (ie >SPF 15) over lower SPF products (ie SPF≤15) with respect to melanoma risk. 10 Nevertheless, a lack of public awareness on how to use sunscreen products is one of major problems facing their effective and regular use. As a result, regulatory bodies throughout the world have put more pressure on product labeling and SPF testing instructions, for example US FDA and Health Canada sunscreen product rules. 11,12 With the recent stricter regulations on SPF products in the market for both labeling and testing methods, the demand for an economical and reliable means to determine SPF from the research and development stage has increased significantly. Despite efforts to develop a reliable and compatible in vitro SPF test method, inevitable human errors and technical challenges have posed a hurdle to development of a reliable testing method. In addition to technical challenges, the cost and complexity of currently available spreading methods are obstacles in the in vitro SPF determination. This study will report a simple and readily available spectrophotometric method for SPF determination that can support fast screening and evaluation of approximate SPF values for sunscreen products.
A spectrophotometric method using organic UV filter-containing plant oil body (oleosome)-based products has been developed. The oleosomes are composed of individual oil droplets and the phospholipid with anchored structural surface proteins, oleosins. As the oleosome surface is covered with unique amphiphilic oleosin proteins with the hydrophobic conserved domain of the protein at the triglyceride core, the oleosome possesses unique emulsifying capability. 13 We used purified safflower seed oil bodies in approximately 35% water emulsion. This methodological development was aimed to provide a fast, cost-efficient, but reliable tool to screen and estimate the SPF values of sunscreen products. To build a more applicable SPF data pool, we used the most popular SPF label claim, SPF 30 products, as a model system. The spectrophotometric observation was compared to a 2-subject in vivo clinical measurement to correlate the two data sets and to draw better comparison to real application cases. The 2-person assays were further supported by using a 10-person in vivo SPF study to fully validate the results of the 2person and spectrophotometric study results.

| Preparation of sunscreen product formulation
The formulation protocols varied depending on the target SPF levels desired, with a typical standard method summarized below. All the formulation samples were prepared in duplicate. The SPF 15, 30, and 50 formulated products were prepared as shown in Table 1.
• Place the required amount of safflower oleosome emulsion into a 500-mL beaker.
• Add octyl methoxycinnamate and mix at 400 rpm for 20 minutes. This is Phase A.
• Prepare Phase B at 40-50°C with gentle mixing in a separate beaker and then add Phase B into Phase A when it reaches to room temperature (23 AE 3°C).
• Add Phase C and increase the mixing speed to 700-750 rpm.
• Add Phase D and continue mixing for a total of 20 minutes. • Add Phase E and decrease the mixing speed to 100-140 rpm for 30 minutes.
• Adjust the pH to pH 5-6 with 10 N NaOH and continue mixing for a total of 40 minutes.
After the formulation was complete in duplicate, it was stored at room temperature for at least 24 hours to allow the emulsion to stabilize, and, all the samples were evaluated using spectrophotometric methods within 30 days of sample preparation. UV absorbance from 320 to 290 nm was continuously recorded with a 5-nm step size.
The spectrophotometric SPF was calculated shown in calculation of spectrophotometric SPF section. One hundred gram of each sample was also sent to AMA laboratories Inc, AMA Research Laboratories Inc, New City, NY, USA, for the 2-person and 10-person in vivo SPF determinations.

| Calculation of spectrophotometric SPF
The Equation (1) to determine the SPF values spectrophotometrically was modified from the work done by Mansur et al. 14 For the determination of SPF 50, the numerical correction factor 6.65 needs to be removed to agree with the reported in vivo results. This has been determined to minimize the deviation between the calculated mean values and the in vivo results. (1) In the equation, EE (k), I (k), and Abs (k) are erythemal action spectrum, solar intensity spectrum, and UV absorbance of the sample, respectively. The EE (k) and I (k) are given as constants as reported and shown in Table 2. 15 2.4 | Sample preparation for spectrophotometric

SPF measurement
The spectrophotometric sample preparation was modified from previous work in the following ways. 16 One hundred micrograms of SPF product was transferred into 10 mL of 40% ethanol and followed by vigorous vortexing. A visual inspection of the homogeneous sample dispersion without any aggregation was confirmed before further processing was conducted. 500 lL from the prepared sample solution was transferred using a pipette into 4.5 mL of 40% ethanol. The second solution was gently mixed by pipetting, and a 1 mL aliquot was transferred into 4 mL of 40% ethanol, followed again by a gentle pipet mixing. The prepared samples were scanned within 20 minutes of the preparation.

| In vivo SPF determination
The in vivo tests were carried out at AMA Laboratories, New City, NY, USA, on 2-subject panels as instructed in the 2011 US Food and Drug Administration (FDA) final rule for the sunscreen drug products. 11 The average SPF values were quoted for comparison against the spectrophotometric SPF results. In addition, the full 10-person study was also conducted at AMA Laboratories. The phototypes of panelists were selected based on skin types I-III according to FDA monograph. 11

| Statistical analysis
The statistical processing of the data was carried out with one-way analysis of variance (ANOVA), Student's t test and P-value, using the package available in Microsoft Excel program. This further demonstrates the high level of reliability and reproducibility of the spectrophotometric SPF method presented here.
These observations are very encouraging due to low reproducibility of currently available in vitro SPF test methods. 17 The current in vitro test methods are popular but not accepted by any regulatory bodies in the world. 18 The rubbing on the plate type in vitro SPF methods requires intensive training and care to increase the accuracy of the results, mainly due to sample preparation and intrinsic drawbacks in the techniques. With respect to reported in vitro SPF testing methodologies, the SPF values are prone to potential experimental errors, depending on the skill levels of the operators. Even and homogeneous distribution of the sample on the substrate plate using a finger cot is another area for potential error, considering the small amount of sample applied (2 mgÁcm À2 or various amounts suggested for the tests) prior to UV irradiation of the sample. 11 Therefore, SPF ratings from reputable clinical laboratories are currently the sole SPF tests accepted by the regulatory bodies around the world. In this aspect, our SPF screening method for oleosome-based formulas is an efficient means to alleviate cost and time required for the routine screening.

| Preliminary evaluation of SPF 15 and 50 product
To expand the compatibility of the spectrophotometric SPF test method, SPF 15 and 50 formulated products were also evaluated using both in vivo and spectrophotometric testing methods (Table 7).  Table 4 Duplicate sample preparations were carried out (sample 1 and 2) with duplicate samplings (sample number-1 and-2). UV absorbance from 320 to 290 nm was continuously recorded with a 5-nm step size. The spectrophotometric SPF was calculated using Equation (1). The mean values of in vivo and spectrophotometric SPF test results with standard deviations (SD) are shown. "in vivo Ref #" and "Spec SPF" are the sample ID numbers from a clinical laboratory and spectrophotometric SPF, respectively. b SPF was calculated without considering 6.65 correction constant shown in Equation (1) (see "Calculation of spectrophotometric SPF" section).