1.2.1 Materials
Four commercially available sunscreen products: Coppertone® ULTRA GUARD® SPF 70 (cream emulsion), Coppertone® Stick Kids SPF 50 (solid stick), Neutrogena® Age Shield® Face Oil-Free SPF 110 (lotion) and Banana Boat® Kids Clear UltraMist® SPF 100 (continuous spray) were purchased from Amazon.com, Inc. (Seattle, WA) and all contained 6% oxybenzone as one of the UV filters (Table 1). Oxybenzone-d5 internal standard (98+%), acetonitrile (99.9+%, High-Performance Liquid Chromatography (HPLC) grade) and methanol (99.9+%, HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO). Oxybenzone standard (98+%) was a gift from Sigma-Aldrich (St. Louis, MO). Glacial acetic acid (99.7+%) was purchased from AmericanBio, Inc. (Natick, MA). Acetonitrile (LC-MS grade), methanol (Liquid Chromatography–Mass-Spectrometry (LC-MS) grade), water (LC-MS grade), methyl tert-butyl ether, formic acid (OPTIMA LC-MS grade), Brij® 98, potassium phosphate monobasic and dibasic salts were purchased from Fisher Scientific, Inc (Fair Lawn, NJ). All reagents were analytical grade. Nanopure water from an in-house Milli-Q system (EMD Millipore; Billerica, MA) was sourced for buffers.
1.2.2 Skin Preparation
Ex vivo surgical-waste abdominal skin sections from four consenting human donors were provided by the NCI funded Cooperative Human Tissue Network (CHTN) skin repository (Charlottesville, VA). All skin samples were deidentified prior to next day delivery on ice to the University of Maryland, Baltimore where the stratum corneum (SC), viable epidermis and a small portion of the dermis were removed by dermatome to a resulting skin thickness of 295 ± 35 µm. Table 2 contains a summary of the demographic features for donors used for IVPT. All skin samples were stored at -20⁰C in aluminum foil and a sealed Ziploc bag until the morning of each experiment when it was thawed to room temperature, cut into 4.84 cm2 squares and placed into an In-Line cell (PermeGear, Inc.; Hellertown, PA) between the donor and receiver chamber with the epidermal side facing up. Prior to dosing, each skin piece was allowed to equilibrate for 30 min as receiver solution flowed under the dermis side. A heated circulating water bath maintained skin temperature at a baseline of 32 ± 1⁰C. Skin integrity was confirmed by measuring transepidermal water loss (TEWL) with a cyberDERM RG-1 open chamber evaporator (cyberDERM, Inc.; Broomall, PA). Any TEWL reading above 15 g/m2/h is outside the range reported for healthy skin and indicates a disruption of the skin barrier [40]. Any skin piece deemed unacceptable was removed, replaced and allowed to equilibrate again prior to reevaluating TEWL and dosing.
1.2.3 IVPT Studies
IVPT Method and Experimental Design
A PermeGear® flow-through In-Line diffusion system (Hellertown, PA) with cell membrane supports that offered a 0.95 cm2 permeation area was used for all IVPT experiments. The receiver solution was phosphate buffered saline at pH 7.4 with 0.1% Brij 98® as a surfactant. Surfactants are added at a low concentration to maintain the solubility of highly lipophilic compounds, like oxybenzone, in the receiver solution and preserve sink conditions throughout the 12 h study duration. Flow rate was set to 1 mL/h. After successful skin integrity verification, the semi-solid sunscreens were dosed by gently rubbing onto the skin surface using the flat bottom of a HPLC vial in a circular motion while the continuous spray sunscreen was dosed using a positive displacement pipette. Dosing occurred three times to mimic the harmonized in vivo protocol and simulate maximal dosing according to product labeling; once at 0 min and again at 80 min and 160 min. At each dosing time point, approximately 10 mg/cm2 of sunscreen was applied. Samples were collected at 1:00, 2:00, 3:00, 3:30, 4:00, 4:30, 5:00, 5:30, 6:00, 6:30, 7:00, 7:30, 8:00, 8:30, 9:00, 9:30, 10:00 and 12:00 h post sunscreen application. The resulting samples were diluted 1:1 with HPLC mobile phase and analyzed using a validated HPLC method. Skin was maintained at a temperature of 32 ± 1⁰C for the 12 h experiment with temperature monitoring conducted prior to dosing and at 2:00, 4:00 and 6:00 h post sunscreen application using a Traceable™ infrared thermometer (Fisherbrand™; Fair Lawn, NJ).
Oxybenzone Skin Extraction
At the end of the study, each skin piece was wiped clean with an alcohol swab, removed from the IVPT cells and sliced into small pieces using a scalpel. The alcohol swab was placed in a 15 mL conical tube while the skin pieces and scalpel blade were placed in a separate 15 mL conical tube. Four mL of methanol was added to each conical tube to extract residual oxybenzone remaining on and within the skin at the end of the study. Conical tubes were capped, wrapped with Parafilm® and centrifuged for 10 min. After centrifugation, all samples were sonicated for 10 min and then placed on a shaker at 200 rpm for 24 h before an aliquot was removed for HPLC analysis.
HPLC Analysis of In Vitro Samples
The HPLC system was comprised of a Waters® Alliance e2695 separations module connected to a Waters® 2489 dual-wavelength absorbance detector and Waters® Empower software (Milford, MA). A Waters® Symmetry C18 column (5 µm, 4.6 x 150 mm) with Phenomenex SecurityGuard™ C18 cartridge guard column (5 µm, 4 x 3.0 mm) (Torrance, CA) was used for oxybenzone separation from other interference in each sample. The mobile phase was a mixture of acetonitrile, methanol and acidified water adjusted to pH 3.0 with glacial acetic acid at a ratio of 65:20:15 (v/v/v) under constant flow of 1 mL/min. All IVPT samples were diluted 1:1 with mobile phase prior to analysis and standards were created similarly in a 1:1 mixture of mobile phase and receiver solution prior to analysis. Oxybenzone elution occurred at 3.1 min with maximum UV detection at 287 nm. There were no interferences from any excipients or other UV filters from the commercial products or skin samples. The skin extraction and residual swab samples were diluted 4x and 50x, respectively, with a 1:1 mixture of methanol and mobile phase prior to HPLC analysis. Oxybenzone elution occurred at 4.5 min with maximum UV detection at 326 nm. The concentration of oxybenzone standards ranged from 0.05 to 10 µg/mL. All IVPT, extraction and quality control (QC) standards (20 µL) were injected in duplicate. The developed method had a limit of detection (LOD) at 0.025 \(\mu\)g/mL with a linearity range of 0.05 to 25 µg/mL. All QCs, including the lower limit of quantitation (LLOQ), showed inter-day precision within 5% of the calculated nominal value and accuracy between 99 to 102%.
1.2.4 In Vivo PK Study in Healthy Human Subjects
The in vivo PK study was approved by the University of Maryland, Baltimore Institutional Review Board and carried out in compliance with the ethical and scientific principles of the International Conference on Harmonization Good Clinical Practice E6 (ICH-GCP) at the General Clinical Research Center within the University of Maryland Medical Center.
Clinical Study Subjects
An open-label, randomized, four-way crossover study was completed by ten healthy adult subjects. Table 2 provides a summary of the demographic information. Study enrollment was open to both men and non-pregnant women from September 2020 to April 2021. Subject ethnicity was self-reported and collected by the clinical staff. Key eligibility criteria were ages 18 to 45 years with a body mass index (BMI) < 30 kg/m2, non-smoker/tobacco user, negative test results for drugs of abuse (cannabinoids, amphetamines, barbiturates, benzodiazepine, cocaine, methadone, opiates, PCP), normal screening laboratory values (white blood cells, complete blood count, hemoglobin, platelets, sodium, potassium, chloride, bicarbonate, blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, urine protein and urine glucose), mid thighs of ≥ 42 cm in circumference, legs that measure ≥ 46 cm in length from the iliac crest to the top of the patella or large enough to accommodate the products to be tested at an area of 400 cm2 per thigh, normal electrocardiogram readings as determined by the medically accountable investigator, and normal vital signs (temperature 35–37.9⁰C, systolic blood pressure (BP) 90–140 mmHg, diastolic BP 60–90 mmHg, heart rate (HR) 55–100 beats per minute, respiration rate 12–20 breaths per minute). Major exclusion criteria were subjects with a positive pregnancy test, positive hepatitis B, C, and/or HIV serologies, donation or loss of greater than one pint of blood within the past 60 days prior to study entry, any prior allergies to oxybenzone or other ingredients commonly found in applied sunscreens, history of infectious disease, skin infection or chronic skin condition (psoriasis, atopic dermatitis), history of diabetes, history of significant dermatologic cancers except basal cell carcinomas that were superficial and did not involve the thighs, and obvious differences in skin color, skin condition, excessive hair, sunburn, raised moles, scars, open sores, scar tissue, tattoos or discoloration that would interfere with product placement or skin assessment of both upper thighs. Additionally, subjects were instructed to refrain from using any products containing oxybenzone (makeup, lip balm, face creams, sunscreen, etc.) 0–7 days prior to the start of each procedure day as well as refrain from using any chronic prescription medications 0–30 days and over-the-counter (OTC) medications/short term prescription medications 0-3 days prior to the start of each procedure day with the exception of vitamins, herbal supplements and birth control.
Randomization
There were 24 combinations of procedure day orders that could be assigned to subjects. After screening and confirmation of eligibility, each subject was randomly assigned a different procedure day order to prevent any treatment bias. Once a procedure day order was used, it was not reassigned to any other subject.
Clinical PK Study Design
The IVPT and clinical PK studies were harmonized to the maximum possible extent, replicating sunscreen products, temperature, humidity, dosing times, sampling time points and study length. The duration of each clinical PK procedure day was 12 h with sunscreen applied at 0, 80 and 160 min. A minimum of one-week washout period between procedure days was required to remove residual oxybenzone from systemic circulation.
Clinical PK Study Procedures
At the beginning of each procedure day, female subjects were asked to provide a negative urine pregnancy test confirmed by a clinical staff member prior to dosing. Vital signs were recorded, and the subject was asked questions pertaining to changes in medical history and adherence to instructions regarding oxybenzone as well as medication restrictions. Once continuance in eligibility was confirmed, both upper thighs were examined for any signs of skin irritation, abrasions or sunburn and a template measuring 400 cm2 was marked as the dosing area on each thigh. An intravenous (IV) catheter was placed under sterile technique and approximately 4 mL of blood was drawn as a pre-dose blood sample. The application area on each thigh was cleaned with water, carefully dried and a temperature sensor (Novatemp® skin temperature sensors 400 series; NOVAMED USA; Elmsford, NY) was placed near the knee adjacent to the application area on one leg to measure skin temperature throughout the entire 12 h procedure day.
For each semi-solid sunscreen product (cream emulsion, lotion and solid stick (Table 1)), a teardrop silicone applicator (Qosmedix; Ronkonkoma, NY) covered by a XL nitrile finger cot was used to apply 800 mg of sunscreen onto the marked 400 cm2 location on each thigh simultaneously by two clinical staff until evenly distributed (~ 1 min). For the continuous spray product (Table 1), approximately 3 mL was sprayed into a glass scintillation vial and left to sit for 15 min so the propellant could evaporate. After 15 min, two 1 mL topical syringes were used to draw up 0.95 mL of liquid sunscreen. Similar to the semi-solid products, dosing occurred by releasing the liquid from the syringe onto the marked 400 cm2 application area on each thigh and rubbed in using a teardrop silicone applicator covered by a XL nitrile finger cot. Approximately 800 mg (2 mg/cm2) of sunscreen product was applied simultaneously to each thigh repeatedly at two additional times (80 and 160 min) during each procedure day, representing maximal usage and reapplication recommendation as stated on the respective sunscreen labels.
Before the scheduled dosing time of the first application, the environmental control chamber was set up and allowed to equilibrate for approximately 30 min so that the RH within the chamber measured between 35–55%. The individual components as well as the setup procedure of the environmental chamber are depicted in Fig. 1. The chamber consisted of a 3D printed dome (ARTLAB Virtual Studio; Lexington, KY) made of polyethylene terephthalate glycol filament with the dimensions 749.3 mm x 330.2 mm x 539.8 mm, designed to provide ample space underneath for the subjects’ upper thighs without touching the application area. Velcro was used to attach a far infrared tourmaline heating pad (UTK® Technology Store; China) to the underside of the dome. The hose and humidity monitor of an ultrasonic humidifier (Nebula®, River Systems; Campodarsego, Italy) were attached at the base of the dome and angled inward towards the subjects’ thighs. The humidifier was set to 45% RH and self-regulated to reach the set target based on the connected mini-USB humidity sensor. RH within the environmental chamber was also externally measured with a wireless humidity monitor (TP-60S Digital Hygrometer, ThermoPro; Duluth, GA) attached under the dome near the subjects’ feet. The entire 3D dome was covered by a polyester disposable aluminized rescue blanket (Dynarex Corporation; Grainger; Lake Forest, IL) and a hospital provided bed sheet.
After the initial sunscreen dose at time 0 min, the subjects’ upper thighs were covered with the environmental control chamber. The environmental chamber was only removed for subsequent dosing at 80 and 160 min or if the subject needed to use the restroom. During either event, skin temperature and RH were recorded prior to chamber removal, after chamber replacement, 5 min post replacement and 10 min post replacement to verify that skin temperature and RH returned to 32 ± 2⁰C and 45 ± 10%, respectively. The infrared heating pad was only turned on if subjects’ skin temperature fell below 30⁰C after the 10 min replacement window, otherwise it remained off.
At 12 h post initial application, the sunscreen was removed by clinical personnel. Each application site was wiped with a dry cotton gauze, followed by four alcohol swabs. Left and right application sites were processed separately for residual oxybenzone concentration. Alcohol swabs and gauze for each site were placed into 500 mL glass bottles containing 200 mL of methanol for extraction.
Safety
Adverse events (AE) were recorded during each procedure day with vital sign monitoring (temperature, BP, HR, respiratory rate) prior to initial sunscreen application and periodically throughout the 12 h visit. Sunscreen application and temperature sensor sites were assessed subjectively on a scale of 0–4 (0: no evidence of irritation, 1: faint but definite erythema, 2: moderate erythema, 3: severe erythema, 4: generalized vesicles or eschar formations) by clinical staff for signs of erythema, itching, hyperpigmentation and edema prior to initial sunscreen application and at the end of the 12 h study after sunscreen and temperature sensor removal. Subjects were asked about oxybenzone allergies prior to enrollment and to confirm that the heating pad within the environmental chamber was not in contact with the skin; therefore, sunscreen application was not anticipated to cause any health concerns. Any subject with ongoing AEs at the end of a procedure day was contacted by phone until resolution was reported.
Serum Sample Collection
Collection of approximately 4 mL of whole blood with a vacutainer (BD Vacutainer® Plus plastic serum tube; BD; Franklin, NJ) from an IV catheter occurred prior to initial application of sunscreen, and at 1:00, 2:00, 3:00, 3:30, 4:00, 4:30, 5:00, 5:30, 6:00, 6:30, 7:00, 7:30, 8:00, 8:30, 9:00, 9:30, 10:00 and 12:00 h post initial sunscreen application. The sample was allowed to clot for 30–60 min, centrifuged at 1300xg for 20 min at 4⁰C and supernatant serum was aliquoted into two identical cryovials. Cryovials were immediately placed in an ultralow (-80⁰C) freezer until analysis with a validated ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
1.2.5 LC-MS/MS Assay for Serum Oxybenzone Concentrations
For oxybenzone serum extraction, 200 µL of each sample was placed in a glass screw-top culture tube along with 50 µL of oxybenzone-d5 (50 ng/mL) as an internal standard (IS). Two mL of methyl tert-butyl ether were added to each culture tube with a glass pipette as an extraction solvent. Samples were vortexed for 5 sec, placed on a high-speed shaker (Fisher Scientific, Inc; Fair Lawn, NJ) at 2,500 rpm for 20 min and centrifuged for 5 min. The upper organic layer (1.5 mL) was transferred to a clean glass culture tube while a second extraction was performed repeating the above procedure. After centrifuging the second extraction, 1.5 mL of the upper organic layer were combined with the previous extraction and the sample was evaporated to dryness under nitrogen gas (N-EVAP; Organomation Associates, Inc; Berlin, MA) at ambient temperature. Residue was reconstituted in 100 µL of mobile phase consisting of methanol and water with 0.1% formic acid (73:27 v/v), centrifuged for 5 min and 5 µL was injected into the LC-MS/MS for analysis.
Analysis was conducted on a Waters® LC-MS/MS system consisting of a Waters® Acquity H class UPLC connected to a Waters® Xevo TQ-XS mass spectrometer (Milford, MA). All samples were injected by an autosampler onto a Waters® BEH C18 column 130 Å (1.7 µm, 2.1 x 100 mm) with a Waters® VanGuard™ BEH C18 guard column (1.7 µm, 2.1 x 5 mm). Mobile phase was a mixture of methanol and water with 0.1% formic acid (73:27, v/v) held constant for 3 min at a flow rate of 0.3 mL/min. Each sample was proceeded by a gradient column wash. From 3–8 min, mobile phase was primarily organic (99:1, v/v). From 8–12.5 min, initial conditions (73:27, v/v) were reestablished allowing the column to return to equilibrium. Multiple reaction monitoring was carried out with the parent to daughter ion transition of 229.06→150.62 at a cone voltage of 4 V and collision energy of 20 eV for oxybenzone. The IS had similar monitoring with a parent to daughter ion transition of 234.06→150.75 at a cone voltage of 2 V and a collision energy of 18 eV. Calibration standards had a linearity range from 0.1 to 100 ng/mL. Method validation was conducted according to the Bioanalytical Method Validation Guidance for Industry [41]. All QCs, including the LLOQ, showed inter-day precision within 11% of the calculated nominal value and accuracy between 96 to 101%. Freeze-thaw, bench top, processed sample, stock solution and long-term stability (6 months) of both the low QC (0.3 ng/mL) and high QC (50 ng/mL) had accuracy between 90 to 103%. Recovery at all QC levels was high (91–94%) with minimal matrix effect. During clinical sample analysis, the deviation from nominal values did not exceed 15% for any standards or QCs except for the LLOQ with a maximum allowable error of 20%.