Chemicals and materials
Dimethyl phthalate (CAS 131-11-3; molecular weight 194.18 g/mol; density 1.194 g/mL at 20°C, boiling point 283.7°C at 760 mm Hg; vapor pressure < 1 × 10− 2 mm Hg at 20°C, partition coefficient n-octanol/water logKow 1.47–1.6; water solubility 4.0-4.3 g/L at 20°C (NCBI 2022)) (≥ 99.0% pure) was obtained from Alfa Aesar (Kandel, Germany). Deuterated DMP (d4-DMP; 98.0% pure) was obtained from Sigma Aldrich Pty Ltd (Darmstadt, Germany). All GC-MS grade solvents used (dichloromethane, ethanol, methanol, acetone) and phosphate-buffered saline (PBS, pH = 7.4) were purchased from Sigma Aldrich Pty Ltd (Darmstadt, Germany). The following surfactants were used due to their frequent usage in cosmetics formulations: sodium dodecyl sulfate (ASAA; ≥99.0%) and hexadecyltrimethylammonium chloride (CSAA; ≥98.0%) from Sigma Aldrich Pty Ltd (Darmstadt, Germany), caprylyl/capryl oligoglucoside (O110) (NSAA; technical grade) from Logis-Tech (Mirków, Poland).
Experimental work has been conducted under conditions which limit contamination of glassware and other laboratory equipment with phthalates (treatment with ethanol, acetone and/or high temperature). Plastic containers were eliminated from protocols unless they had phthalate-free certificates.
In transdermal diffusion testing, three types of human skin models were used: Strat-M® membrane (SMM), in vitro epidermal model (RHE) and ex vivo human skin (HS). The synthetic Strat-M® membrane (25 mm, non-animal based model) was obtained from Merck KGaA, (Darmstadt, Germany). This membrane has already been used as a permeation barrier for predicting phthalate penetration through skin (e.g. diethyl phthalate, dibutyl phthalate, diisononyl phthalate) (Pan et al., 2014). RHE membranes (Fraunhofer ISC-TLC in vitro epidermal 25 mm models in 6-well format) with the supporting cell culture medium were ordered from the Translational Center Regenerative Therapies TLC-RT, Fraunhofer Institute for Silicate Research ISC (Würzburg, Germany). Frozen abdominal dermatomed human skin XenoSkin H (art. no. H-D20D-24, 24 mm) was obtained from Xenometrix AG (Allschwil, Switzerland) under strict ethical restrictions and with informed consent. No sensitive personal information regarding the patients was retained.
Franz Diffusion Cell Experiments
DMP permeation experiments, with and without the addition of surface active agents, were performed according to OECD guidelines (OECD 2004). A 6-cell manual diffusion cell system with 2mag-Magnetic-Drive (2mag-AG, München, Germany) and circulating waterbath HE4 (JULABO GmbH, Seelbach, Germany) was obtained from Hanson Research (Chatsworth, CA, USA). All studies were performed using 7 mL vertical diffusion cells (donor medium) with open cell top (1.8 cm2 diffusion area, 1 mL of donor medium) and cap. The temperature and stirring parameters were set to 32ºC and 350 rpm, respectively. Except for the Strat-M® membrane, skin models were hydrated before diffusion cell experiments (Sugino et al. 2017). PBS solution with addition of 10% of ethanol was used as donor and acceptor media for all tested mixtures, which had a positive effect on DMP solubility and the solvent is compatible with aqueous buffer (Katakam and Katari 2021). The applied DMP dose per cm2 of skin model was 1078 µg/cm2. The amounts of surface active agents added were based on average values used in cosmetics formulations. The composition of the tested mixtures can be seen in Table 1. Fresh mixtures were prepared for every round of diffusion experiments and measurements were carried out in three replicates with each mixture, for every type of membrane. After preparation, the diffusion cells (filled with acceptor medium, with mounted skin model, covered with the cell top) were checked to ensure that there were no air bubbles between the skin model and the receptor medium. Aliquots (500 µL) were collected from the receptor sections at specified time intervals (0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h) by injecting warmed PBS solution using a 1.0 mL chromatographic syringe. Next, samples were extracted using liquid-liquid extraction (LLE) and analyzed by gas chromatography coupled with mass spectrometry (GC-MS).
Table 1
Composition of tested dimethyl phthalate mixtures with surfactants
Sample name
|
Amount of applied chemicals
|
PBS with 10% ethanol
|
DMP1
|
ASAA2
|
CSAA3
|
NSAA4
|
DMP
|
10 mM
|
-
|
-
|
-
|
filled to 10 mL
|
DMP + ASAA
|
10%
|
-
|
-
|
DMP + CSAA
|
-
|
2%
|
-
|
DMP + NSAA
|
-
|
-
|
10%
|
1 DMP - dimethyl phthalate |
2 ASAA - anionic surface active agent |
3 CSAA - cationic surface active agent |
4 NSAA - non-ionic surface active agent |
Chromatographic Analysis
The determination of DMP migration through the skin models was performed based on analytical methodologies proposed by Guo et al. (2010). Stock standard solutions of DMP and d4-DMP (internal standard) at 1000 µg/mL were prepared separately. A calibration curve for both analytes, ranging from 0.025 µg/mL to 50 µg/mL, was prepared by dilution of the mentioned stock solution. Validation samples at concentrations of 1, 10 and 25 µg/mL were also prepared as above. Each sample was analyzed in triplicate with methanol injections between every three samples.
The amount of DMP in acceptor and donor samples, after LLE (double extraction with 1 mL of dichloromethane after addition of 10 µL d4-DMP at 5 µg/mL), was investigated with a GC-2010 PLUS gas chromatograph coupled with an AOC-20ia auto injector and MS-TQ8040 mass spectrometer from Shimadzu Corp. (Kyoto, Japan). Before analysis, the extracts were evaporated and the residue was dissolved in 500 µL of methanol. The injection temperature was set to 280°C in splitless mode. The GC oven temperature was programmed as follows: at 50°C initially and held for 1 min, then ramped to 310°C and held for 2.5 min. The separation was carried out on a fused silica capillary column GC Zebron ZB-5MS (30 m, 0.25 mm, 0.25 mm) with helium as carrier gas (99.999995% pure, flow rate of 1.0 mL/min). The ion source temperature was maintained at 220°C and the transfer line was heated to 310°C. The MS was operated in electron impact mode with electron energy of 70 eV. The target compounds were determined in full scan (SCAN) and selected ion monitoring (SIM) mode. LabSolution Analysis software (Shimadzu Corp., Kyoto, Japan) was used for GC-MS control and data acquisition. The identification was performed by using similarity search in the National Institute of Standards and Technology MS database (NIST 11).
Quality Assurance And Data Analysis
The analytical method has been evaluated using the following validation parameters: detection limit (DL), quantification limit (QL), linearity, recovery, and precision (ICH 2021). DL and QL were calculated based on the standard deviation of the linear response and the slope of the calibration curve as well as on a signal to-noise ratio of 3:1 and 10:1, respectively. The linearity was evaluated based on the coefficient of determination (R2) of a 6-point calibration curve. Recovery of the target compound was performed at three different levels to evaluate the accuracy of the proposed protocol. Precision (as percent relative standard deviation, %RSD) was investigated by carrying out six independent sample analyses for three consecutive days.
The skin permeability of DMP was calculated from the quantity of target analyte, which permeated through the skin membrane, divided by the membrane surface and the time duration. The permeability coefficient (kp) was determined from the steady-state flux (Jss) and DMP levels in the donor phase. Phthalate flux was calculated from the slope of the penetration amount vs. time profile. Additionally, the ratio of total amount of DMP (Eq. 1) in the receptor fluid was compared to the amount of DMP in the donor phase to determine the total absorption rate (Hopf et al. 2014; Neri et al. 2022):
total absorption (%) = amount of DMP acceptor/total amount DMP in donor × 100 (1)
Total absorption of the test compounds can be used as one of the exposure parameters in human HRA. The obtained results were used in non-cancer risk assessment of DMP connected with its potential occurrence in cosmetics or personal care products (MFDS 2017; SCCS 2016; Kim et al. 2020). The systemic exposure dose (SED) was calculated using Eq. (2).
SED (mg/kg/day) = B × 1000 mg/g × C/100 × A/100/BW (2)
where:
SED - systematic exposure dosage for cosmetic ingredients (estimated amount of exposure, per body weight, per day) [mg/kg body weight/day];
B - amount of cosmetic products used in one day [g/day];
C - concentration of target ingredient in evaluated cosmetic products [%];
A - skin absorption rate expressed in real use conditions [%];
BW – average body weight (60 kg) [kg].
The results of the non-cancer HRA were taken as the margin of safety (MoS) (Eq. 3), with values above 100 indicating a safe value (MFDS 2017; SCCS 2016; Kim et al. 2020). If MoS is < 100 for an investigated compound, the ingredient is considered to be a potential cause of adverse health effects and there are safety concerns in terms of its use.
MoS = NOAEL/SED (3)
where:
MoS – margin of safety [-];
NOAEL - no observed adverse effect level [mg/kg body weight/day];
SED - systematic exposure dosage for cosmetic ingredients [mg/kg body weight/day].
All calculations were done using Microsoft® Excel® 2016 MSO.