An open‐label clinical trial assessing the efficacy and safety of Bend Skincare Anti‐Aging Formula on minimal erythema dose in skin

Sunburn and other health risks associated with excess sun exposure place huge economic burdens on societies, and create discomfort and disease within susceptible individuals. Oral supplements that reduce sunburn may be advantageous. This study evaluated the safety and efficacy of Bend Skincare Anti‐Aging Formula to ameliorate sunburn induced with a solar simulator.


| INTRODUCTION
Sunburn erythema and other health risks associated with excess sun exposure including premature skin aging, damage to the immune system, cataracts, and nonmelanoma and melanoma skin cancers 1 place huge economic burdens on societies. In Canada, in 2004, the cost for treatment of melanoma alone was $444 million, and for nonmelanoma was $88 million, and is expected to rise by 2031 to $696 million and $226 million, respectively. 2 A study including beach goers in the United States (US) found that sunburn accounts for up to 92 720 lost workdays annually, making the economic burden for lost work plus treatment in excess of $10 million annually. 3 Erythema (skin redness) is an acute inflammatory skin response resulting from UV radiation overexposure. Both UVA (320-400 nm) and UVB (280-320 nm) contribute to this response known as sunburn, although UVB (280-320 nm) is more potent in this regard. 4 Sunburn response varies widely among individuals depending on skin type (ie people with lighter skin tone have a greater sunburn risk) and within the same person depending on factors such as age 5 and diet. 6,7 In experimental models, sunburn sensitivity is measured using the minimal erythema dose (MED), defined as the smallest UV dose producing perceptible skin redness within distinct borders in a given period of time after exposure. [8][9][10] The higher the MED, the more resistant the skin is to sunburn. Therefore, increased MED following an experimental treatment signifies photoprotection.
Experimental sunburn is achieved using solar stimulators providing a continuous spectral output in the UVB (290-320 nm), UVAII (320-340 nm) and UVAI (340-400 nm) ranges that are similar to sunlight. Instrument performance, calibration, and operation requirements are defined within the US Food and Drug Administration (FDA) Federal Register 8 and are necessary to achieve reliable test results.
In recent years, interest in using natural products including nutrients as photoprotectors has grown. [11][12][13] Orally administered nutrients showing benefit include omega-3 fatty acids derived from fish [14][15][16][17][18] and a variety of antioxidants, 13 while vitamin D exhibits anti-inflammatory effects 19 in vitro [20][21][22] and in vivo, 23 and reduces polymorphic light eruption when topically applied to human skin. 24 In addition, oral omega-6 fatty acids, frequently sourced from borage oil, are now recognized contributors to inflammation resolution, 25 and so may be valuable additions to products aimed at preventing/relieving inflammatory skin conditions like sunburn. To date, no clinical studies have reported the combined effects of these nutrients on lowering sunburn risk.
The primary objective of this pilot trial was to evaluate and compare changes in the MED following use of an oral supplement combining benefits of omega-6 and omega-3 fatty acids with the antioxidants zeaxanthin and lutein, and vitamin D. The secondary trial purpose was to assess the safety of the supplement under the conditions of use.

| METHODS
This open-label clinical trial, completed within a contract testing laboratory, included recruitment of at least 30 subjects from the general population in New Jersey, United States, aged 19-65 years, who met all of the following inclusion criteria and none of the exclusion criteria.

| Inclusion criteria
Subjects with a Fitzpatrick skin phototype I, II, or III (Table 1), aged 19-65 years, inclusive, who were considered suitable by a nurse or physician prior to their trial initiation; those who had protected the test site from sun for 4 weeks prior to trial initiation, had completed a medical history form, had understood and executed an informed consent form, and who were considered dependable and capable of understanding and following directions.

| Exclusion criteria
Subjects who had a history of abnormal response to sunlight, such as lupus erythematosus or skin cancer; subjects who had a sunburn, suntan, uneven skin color or visible disease that would interfere with test result evaluations; subjects with known allergy or intolerance to the test material ingredients (ie fish oil, soy); subjects who had nevi, blemishes, or moles, which, in the opinion of the principal investigator (PI), would interfere with the trial results, but including subjects with excessive hair who clipped their hair; subjects who were in ill health or taking medication other than birth control, which could influence the purpose, integrity or outcome of the trial; subjects who used a tanning bed or overexposed themselves to sunlight on the skin test site; females who were pregnant, planning to become pregnant, or nursing during the course of the trial; subjects who had participated in testing procedures that precluded a sufficient area being clear of all previous skin tanning and subjects who had experimented on the skin test site within the previous 2 months.

| Intervention
Participants were instructed to consume 4 Bend Skincare Anti-Aging

| Outcomes
The primary outcome was a statistically significant increase (P < .05) in MED following 4-and 8-week treatment relative to baseline, and between 4-week treatment and 8-week treatment. There were no changes to the trial's primary outcome after the trial commenced. The secondary outcome was a progressive positive percentage change in MED from baseline to after 4-and 8-week treatment. Unsolicited patient-reported outcomes were recorded in the daily diaries.

| Determination of MED
The MED of each subject was determined within the first 7 days of the initial time point (when supplementation began), and again at 4 and 8 weeks following supplementation, using instrumentation specifications, qualifications, and calibrations as well as MED assessment scoring requirements defined by the US FDA. 8 Subjects were seated upright during the UV irradiation and MED assessments. During irradiation, unprotected skin on each subject's back was exposed to fullspectrum UV radiation with a continuous spectral output similar to sunlight (UVB range 290 nm-320 nm, UVAII range 320 nm-340 nm, and UVAI range 340-400 nm) using a solar simulator (Solar Light Company, Philadelphia, PA, USA).
At baseline, different sections of each subject's back were exposed to a progressive sequence of timed UV radiation exposures, each of which was graduated incrementally by 25% over the previous exposure. The initial dose of radiation varied according to their Fitzpatrick skin phototype (eg those with Type I received a lower dose than Type III) such that different subjects were exposed to different dosage sequences.
A trained grader evaluated the skin test sites for erythema between 16 and 24 hours after irradiation using the MED Scoring Scale described in Table 2. The MED was defined as the quantity of erythema-effective energy (mJ/cm 2 ) that produced mild but definite erythema within clearly defined borders (ie "1" on the MED Scoring Scale). If a baseline MED could not be determined by the grader during the first irradiation sequence, as second irradiation occurred. Subjects were removed from the study if the second irradiation sequence did not yield a baseline MED.
After 4 weeks of supplementation, the MED was determined again as previously described on a skin test site in close proximity to the baseline MED skin test site. However, this time the starting dose of UV radiation was adjusted such that the baseline MED became the midpoint irradiation dose within the sequence of 25% incremental doses applied. If the MED could not be determined after the first irradiation sequence, a second sequence was conducted. If the MED could not be determined with the second irradiation sequence, the subject was allowed to remain in the study, but they were not included in the statistical analysis. Similarly, subjects returned to the test facility after 8 weeks of supplementation. This time the MED was determined using an irradiation sequence in which the week 4 MED was the midpoint. Evaluation of the irradiated test site was conducted as described for the baseline and week 4 time points.
Throughout the study, subjects were required to maintain a daily diary of test material use and to record comments pertaining to their experiences with the test material. to discontinue due to a clinical event for which the PI did not consider removal from the trial to be necessary or any other nonspecific, subject-initiated reason.

| Statistical methods
Per-protocol analysis was performed using 3 paired t tests to determine if any statistically significant differences in MED occurred be-  Any actions taken (eg discontinuation of test material, administration of treatment, etc.) and the resulting outcome of the AE were recorded.
Subjects who withdrew from the trial due to an AE were followed until the outcome of the event was determined, and a written summary of the event and follow-up was maintained. The Allendale Institutional Review Board (an independent group of professionals utilized by the contract testing laboratory, as required by FDA federal regulations) was informed of any serious AEs as instructed in the study protocol.

| Ethics Approval
The study was conducted in accordance with the current Declaration

| RESULTS
This Thirty-three subjects were enrolled and 28 completed the study.
The reasons for subject drop out/withdrawal were as follows: Two did not meet the inclusion criteria, one withdrew consent, one was disqualified due to excessive sun exposure in the test area and one took exclusionary medication. The demographics for the subjects included in the study at baseline are presented in

| Outcomes
Results for the paired t tests are presented in Table 3. The amount of energy needed to produce the MED significantly increased (P < .001) compared with baseline after 4 and 8 weeks of supplementation. The mean MED at baseline was 23.5 mJ/cm 2 , and increased to 33.9 and 43.8 mJ/cm 2 after 4 and 8 weeks of supplementation, respectively.
There was also a significant increase in energy required to achieve a MED between 4 and 8 weeks (P < .001). These results corresponded to a mean increase of 39% in MED at 4 weeks and an 84% mean increase in MED at 8 weeks, compared with baseline (Table 3, Figure 1).
In addition, these increases were observed throughout the test population with 85.7% and 100% of the subjects requiring an increase in energy to produce a MED at week 4 and week 8, respectively ( Figure 2).
Results of the linear mixed models analyzes confirmed our conclusion based on the paired t test and showed that the MED significantly increased after treatment (Table 3). Adding the Week covariate had a significant effect on the MED (P < .001), adding both the Week and the Skin Type covariates had a significant effect on the MED (P < .001), and adding the Skin Type covariate had a weaker but still significant effect on the MED (P < .001). Combined, these results confirm that the MED does increase significantly due to treatment effects.
A few patients reported outcomes within their daily diaries (Table 4) including 5 mild side effects and 5 perceived enhanced skin, hair, or overall health effects.
No serious AEs related to the supplement were reported by the subjects or noted by the trial monitors. There were 2 AEs unrelated to supplement use as follow: One subject developed a cough and took a cough medicine on 1 day and another subject rolled her ankle. The latter subsequently consumed anti-inflammatory medication that prevented continuation within the trial.

| DISCUSSION
The need for effective UV skin protection is important from both an economic and personal perspective. Human skin exposed to UV radiation produces reactive oxygen species leading to DNA, cell, and tissue damage that can alter immune function and generate health issues ranging in severity from skin pigmentation and photoaging 4 T A B L E 3 Patient demographics and clinical characteristics at baseline of patients who completed the study and their minimal erythema dose (MED) results and statistical analysis following treatment To the author's knowledge, no human intervention studies have investigated the protective effects of GLA against UV damage in skin. However, GLA has proven anti-inflammatory effects in skin as evidenced in atopic eczema trials, 26-28 possibly due to its metabolic conversion to anti-inflammatory PGE1 29 and lipoxin, 30 a specialized proresolving mediator derived from arachidonic acid (AA) that activates inflammation resolution. 25 Hence, GLA may lessen sunburn associated inflammation.
The fish oil derived omega-3s, EPA, and DHA, inhibit IL-8 production that may partly account for their anti-inflammatory effects yielding UV-protection in skin. 31 In human epidemiological studies, omega-3 intake is associated with reduced UVB-erythemal sensitivity and photoaging. 32 Omega-3s also alter the skin EPA:AA ratio, which during the acute stage of inflammation, reduces pro-inflammatory prostaglandin and leukotriene production 14 thereby reducing blood vessel dilation, 16 under basal as well as inflammatory insult conditions. 14 Numerous randomized, double-blind, placebo-controlled, 14,17,18 and open trials 15,16 have reported significantly increased UV-induced sunburn threshold (a defined dose causing perceptible erythema similar to a MED) following 1800 mg-5 g/d EPA alone 14,17 or with 1200 mg/d DHA 15,16,18 for as little as 4 weeks. 18 In addition, sunburn threshold rose progressively throughout treatment, but fell 10 weeks after treatment cessation, indicating that constant supplementation is needed for continuous protection. 15 This enhanced efficacy with prolonged use is consistent with the results obtained in our study, even though our dosage of total omega-3s was slightly less (ie 1400 mg EPA + DHA daily).
The current United States daily dietary intake of EPA + DHA is only 40 mg in children and teens, and 90 mg in adults. 33  This intake data, combined with the significant efficacy of omega-3s against UV skin damage, lead one to speculate that nearly the entire North American population could benefit from photoprotection through omega-3 supplementation.
UV radiation promotes skin cancer development through mutations, immunosuppression, and inducing oxidative stress. 6 Carotenoids are important components of the antioxidant network and help protect light-exposed tissue. 43,44 Zeaxanthin and lutein are 2 such carotenoids that work by scavenging singlet oxygen molecules and quenching free radicals responsible for oxidative tissue damage. 45 They cannot be de novo synthesized and therefore must be diet derived. 46 Zeaxanthin is not only an effective antioxidant in vivo, it also reduces hydroperoxide formation in dietary oils, making it an attractive ingredient to reduce oxidative degradation within fatty acid supplements. 45 The antioxidant capability of zeaxanthin has been compared to that of the highly potent synthetic antioxidant, Trolox. In one study, testing 3 different zeaxanthin isomers, 2 had greater free radical scavenging capacity, and prevented oxidation better than Trolox, while one isomer was less effective. 47 In a second study, zeaxanthin was reportedly more effective than Trolox. 48  None of these side effects, or cautions, warning, contraindication or known adverse reactions, are required to be declared on product labels by the Canadian Natural and Non-Prescription Health Products Directorate (NNHPD) associated with either fish oil 52 or borage 53 oil use. Therefore, the side effects observed in this study are considered to be of no significant health consequence.
The strengths of this study included rigorous monitoring of UV radiation instrument performance, subject's product dosing compliance, and only minor deviations to the study protocol. Failure to address subject ethnicity within the inclusion/exclusion criteria may be perceived as a trial weakness. However, although there may be real differences in the skin biology of different demographic groups, sorting people effectively into categories has been problematic given the prevailing definitions of race, ethnicity, photo skin type, and pigmentation, 54 making it difficult to define those that should have been included and those that should have been excluded from the study based on their ethnic background. The main weakness of the study was the subjective nature of the MED scoring scale and that the trial design was open label. In addition, a control group of individuals receiving placebo or no intervention and graders being blinded to the intervention when reading MEDs would have made the results of this study more convincing. A randomized, double-blind, placebo-controlled trial, including a nonsubjective assessment of skin redness using a devise such as a spectrophotometer 9 rather than visual inspection, might be appropriate to confirm the beneficial results achieved in this trial. It should also include quantification of pro-vs anti-inflammatory cytokines to pinpoint the mechanisms underlying the apparent antiinflammatory effects indicated by changes in MED, as well as markers for UV-induced DHA damage such as cyclobutane pyrimidine dimers 55 or oxidative DNA damage such as 8oxo-guanine, 56 to determine the extent of UV protection against DNA damage, if any. Such future studies are necessary because pure suppression of inflammation without any substantial protection against damage raises the question whether mechanisms of skin adaptation against UV radiation 57 could be adversely affected.

| CONCLUSION
Bend Skincare Anti-Aging Formula statistically significantly increased the amount of energy needed to produce a MED in subjects treated for as little as 4 weeks. This increase indicated a resistance to UVinduced redness characteristic of sunburn. In addition, this photoprotection increased with continued product use. Oral supplementation with omega-6 and omega-3 fatty acids combined with lutein, zeaxanthin, and vitamin D may effectively reduce sunburn risk.