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Reference
Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July - October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
adopted April 13, 2004 (“Skin Absorption: in vitro Method”)
Deviations:
no
Remarks:
only minor deviations (determination of solubility of test-substance in the receptor fluid due to missing data; total recovery of radioactivity is 85 - 115% of applied dose according to SCCS criteria).
Qualifier:
according to guideline
Guideline:
other: SCCS/1358/10; Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance Document No. 28 for the conduct of skin absorption studies
Version / remarks:
March 2004
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Species:
human
Sex:
female
Details on test animals or test system and environmental conditions:
The study was carried out in vitro on skin preparations from human skin produced with a dermatome.
Donors: Open concerning gender and origin, age of donor <65 years
Localization: Surgically removed skin from abdomen.
Selection criteria: Only visually intact skin with a TEER (impedance value) above 2 kΩ and a thickness between 200 and 400 μm (± 10 %) was used.
Storage of skin preparations: Maximum storage time of 15 months at -20°C.
Type of coverage:
open
Vehicle:
other: blank body lotion (without Benzyl Salicylate)
Duration of exposure:
Single topical application.
The times of sampling of the receptor fluid lasted up to 24 hours (pre-dose (collection about 15 minutes), 0-0.5; 0.5-2; 2-4; 4-8;8-16; 16-24 hours after application).
Doses:
Target concentration of the test substance preparation: 0.5% or 5 mg/g (nominal concentration: 4.95 mg/g)
Target dose: 10 µg/cm² (actual nominal dose (mean) 9.5 μg/cm²)
No. of animals per group:
No animals were used in this study.
12 diffusion cells per dose group were used and 10 were considered valid in the experiment.
Control animals:
no
Remarks:
no animals were used in this study.
Details on study design:
Test-substance preparation:
The density of the blank body lotion determined by weighing 5 x 100 μL and was calculated to be 0.98 g/mL.
To obtain the desired concentration, a respective aliquot of the radiolabeled test substance was taken and the organic solvent (acetonitrile) was evaporated to dryness. An adequate amount of the blank formulation (body lotion) was added to the dried residue. The preparation was stirred to guarantee homogeneity. Based on the current data, the nominal concentration of the test substance in the test-substance preparation was 4.95 mg/g (corresponding to 4.87 mg/mL). The nominal specific activity in the test-substance preparation was 47.54 MBq/g (corresponding to 46.74 MBq/mL).
The density of the test substance preparation was determined by weighing 5 x 100 μL and was calculated to be 0.99 g/mL. A retain sample was taken and stored in a freezer but was not analyzed.

Analyses:
Samples for analysis were diluted in acetonitrile, ultrasonicated and filtrated (filter: Spartan 13/0.45 RC) before HPLC analyses.
The analyses of the radiolabeled test substance in the test-substance preparation were performed according to HPLC method (Agilent 1200 system and Dionex software (Chromeleon)).
The radiochemical purity of the radiolabeled test-substance was determined before test substance application. Additionally, the purity of the radiolabeled test substance was measured in the test-substance preparation at least before and after application.
The stability of the radiolabeled test substance in the test-substance preparations was determined by radio-HPLC before start and after application. The stability of the test-substance preparation was additionally verified by concentration control analysis at appropriate time points by means of HPLC-UV.
LSC and HPLC were applied to confirm the homogeneity and accuracy of the test-substance preparations in samples taken before and after the application procedure.
The solubility of Benzyl Salicylate in 5 % (w/v) Bovine Serum Albumin (BSA) in physiological saline with the addition of 0.01 % (w/v) NaN3 was determined: For this purpose, an aliquot of the radiolabeled test-substance solution was taken, and the organic solvent was evaporated to dryness. An adequate amount of the non-labeled test substance was added and thereafter, receptor medium (=5% BSA in 0.9% NaCl with 0.01 % (w/v) NaN3) was added. This mixture was stirred at about 32°C (for at least 2h) and was centrifuged at around 2000 g for about 5 min. The concentration of the test substance was determined in the supernatant via measurement of activity in two replicates. The solved amount of test substance was calculated based on the specific activity of the test substance and was expressed as mg/mL or mg/L. The absence of Benzyl Salicylate in the blank formulation was confirmed by HPLC-analysis.

Preparation of diffusion cells:
Before the start of the experiment, the diffusion cells were monitored for potential radioactive contamination. It was ensured that the radioactive contamination was below 100 dpm. The diffusion cells were assembled using the receptor chamber, the donor chamber, the skin preparation and the stainless steel clamp as well as two three-port valves. For this purpose, the skin preparations were placed on the donor chamber. Subsequently, the donor chamber was placed on the receptor chamber and fixed with the stainless steel clamp. Thereafter the cell was filled with physiological saline with protease inhibitor avoiding air bubbles underneath.

Assessment of skin preparation integrity:
Before connecting the penetration cell, equipped with skin preparations, to the experimental setup, the integrity of the skin preparation was determined by measuring its electrical resistance (TEER). For this purpose, about 0.8 mL physiological saline solution with protease inhibitor was pipetted onto the skin preparation and an electrode of stainless-steel wire (donor electrode diameter of 2.05 mm) was suspended into the fluid column to a depth of ca. 0.5 cm. A further electrode (receptor electrode diameter of ca. 0.95 mm) was introduced as far as possible into the receptor chamber filled with physiological saline with protease inhibitor via the inlet of the Franz cell. After the two electrodes had been connected to the EVOM2 measuring bridge, the impedance was detected and documented. Measured values above 2 kΩ were expected for intact skin preparations. After the resistance measurement, the penetration cells were covered with Fixomull® Stretch (semiocclusive adhesive fleece) and were stored in the refrigerator overnight. The integrity of the skin preparations was additionally checked visually immediately before the application of the test-substance preparation and immediately before sampling procedures for balancing.
The skin preparations used within this study showed a TEER above 2 kΩ (evaluated before test substance application) and no physiological damage of the skin membranes before application and during exposure period (before 24h skin wash) or leakage of receptor medium to the surface. The cells were assessed to be valid, if total recoveries generally fulfilled SCCS criteria (85 ± 115 %), if aberrant or insufficient skin wash could be excluded and if absorption kinetics showed no aberrant behavior in relation to other skin samples. Epidermal membranes were reported but excluded from calculations if they fail for one of the
above-mentioned validity criteria.

Integration of the diffusion cells into the experimental setup:
On the day of application, the cells were placed into the cell warmer. The appropriate tube connections were attached to the supply vessel or the peristaltic pump and to the fraction collector. The receptor fluid was changed and the receptor system was brought to 36°C by means of the water bath thermostat (equilibration time at least 15 minutes). If a diffusion cell leaked after the connection or if the surface of the skin was still moist after swabbing (and low impedance was measured), the cell was exchanged. After the setup was completed, a test run was performed until no air bubbles were observed in the tubes. This run was performed to check the system before application and to change the physiological saline solution with protease inhibitor against the defined receptor medium. The flow rate was adjusted by the system to about 2.3 mL / h. The test run also helped to detect any defects of the tube connections and ensured the
presence of pure receptor fluid in the receptor chamber.

Preparation of the fraction collector:
The sampling time points and the flow rate were defined in the software of the fraction collector which is integrated in the system. The specific collection vessels were positioned in the vessel racks according to the number and position of the diffusion cells to be measured in parallel and to the number of sampling times. The arrangement of the vessels in the racks corresponds to the numbering of the diffusion cells and the sampling times.

Application:
The test-substance preparation was distributed uniformly on the exposed skin preparation using a displacement pipette. The exact amount applied per diffusion cell was determined by reweighing the pipette after application, the weight of which had been previously determined after filling. The application time was documented for each cell. The test-substance preparation was stirred during application. In addition so called exemplary applications (=“test applications”) were performed by parallel sampling of 5×2 μL of the test-substance preparation(s) and consequent LSC measurement. The results of these exemplary applications are not taken into account for calculations unless preliminary results indicate that (depending on e.g. the phys./chem. properties of the test-substance
preparation) the weights taken as described before and therefrom derived actual nominal doses may be unacceptable in respect of variability. In this case the dose can be also calculated from the so called “exemplary applications”. For the current test-substance preparation in body lotion, preliminary results indicated that the application weights taken as described before yielded results with higher variability than the performed “exemplary applications”. Therefore, a mean
applied dose (and standard deviation) was calculated from these so called “exemplary applications”. The mean value was used for data assessment.
The application area / diffusion cell was 1 cm² exposed skin / diffusion cell.
The target amount of test-substance preparation applied was about 2 μL of the test-substance preparation / cm² corresponding to about 2 μL / diffusion cell.
The frequency of application was a single topical application. After application of all diffusion chambers, the fraction collector was started for the automatic
sampling at the sampling times

Sampling for determination of the penetration rate:
Method: flow through
Sampling: The sampling of fractions of the receptor medium was carried out automatically using the fraction collector that is controlled by the software of the system. After application to all cells, the program was started as described above. Fractions were sampled in defined time intervals and were recorded
automatically in log files. At each time interval, fractions were sampled into separate vessels assigned to specific diffusion cells.
Times of sampling of receptor fluid: pre-dose (collection about 15 minutes), 0-0.5; 0.5-2; 2-4; 4-8; 8-16; 16-24 hours after application

Samples for the mass balance:
Terminal procedures: After the last sampling of receptor fluid, the refill function of the receptor medium was stopped and the contents of the individual receptor cells was sampled by emptying the receptor chambers with the fraction collector. The diffusion cells were dismantled step by step. All parts (with the exception of the stainless-steel clamp) were washed with tap water followed by 50 % ethanol and extracted in ethanol or Soluene®-350.
Terminal skin wash: The skin surface was thoroughly washed with washing fluid [Estesol® HAIR&BODY 3 % w/v in tap water] and was gently swabbed with a cotton swab soaked with washing solution. This step was repeated. Finally, the membrane was rinsed once with tap water, and the skin was wiped with one cotton swab soaked with water and dried with one dry cotton swab. The skin was removed from the diffusion cell and put onto a Styrofoam support covered with aluminum foil. The skin surface was washed using two cotton swabs soaked with the above mentioned washing fluid, other cotton swabs (one dry and one soaked with washing fluid) were used to wash the bottom side of the donor chamber. Thereafter the skin surface was washed with one cotton swab soaked with water. To be sure that the skin was dry before tape stripping, one additional cotton swab was used to dry the skin after the last washing procedure. The washing fluid, tap water, pipette tips and cotton swabs were stored for analysis.
Tape Stripping: When the skin surface had dried, the upper layers from stratum corneum were removed by tape stripping. In total 20 Scotch Crystal Tape 600 were used for following process: The skin was fixed with pins between a cork tile covered with aluminum foil and an aluminum plate with a piercing of exactly 1 cm2 so that the application area of the skin was located exactly under the piercing. The tape was adhered on the application site and the weight (1000 g) was placed on this area for about 5 seconds. Then the tape was removed and was coated with glass beads. This procedure was repeated with twenty tape strips in total that were stripped in different directions each time. The tapes were pooled into five samples for analysis. The remaining skin was heat separated to epidermis and dermis and analyzed separately. First tape strip samples are considered to represent the upper layers of stratum corneum which desquamate from the skin surface in due time. The test-substance content of tape strip samples was reported as dose associated to tape strips. For calculating the balance, the net weights of the extracting agent were determined from the gross weight and tare weight of the parts determined beforehand.
Details on in vitro test system (if applicable):
Technical equipment:
Skin preparations: Dermatomed frozen skin was used as delivered
Tape strips: Scotch Crystal Clear Tape 600
Glass beads: Diameter 3 mm, are used to prevent the tape strips from sticking together and to improve the extraction
Aluminum plate: With a piercing of 1 cm2 to standardize tape stripping procedure
Brass weight: 1000 g for tape stripping
Diffusion cell: Modified Franz cell (11.28 mm in-line diffusion cell) consisting of an upper donor chamber and a wide opening at the top and a lower receptor chamber. After introducing the skin preparation, the two chambers were assembled and fixed with a stainless-steel clamp and screws. The prepared modified Franz cells were placed in a cell warmer which brings the receptor medium to the right temperature. Sampling and replacement of the removed receptor fluid was carried out via an outlet and inlet tube connection. The exposed area of the skin is about 1 cm².
Cell warmer: 16 diffusion cells can be positioned in parallel in the rack in holders. Cell warmer was operated by a water thermostat pump. The temperature was checked daily and measured once within an experiment by a thermometer.
Water thermostat pump: Electric water bath with circulating thermostat. The temperature of the water was adjusted to 36 °C to achieve a skin temperature of
about 32 ± 1 °C at the skin surface.
Refillment of receptor medium: A regulating vessel with an electronically controlled fluid level of the receptor medium was used. The amount of receptor fluid in the container was kept constant by controlled feed from a supply vessel. The regulating vessel was connected to the inflow of each receptor chamber via tube connections and a stopcock. If the stopcock was open, any receptor fluid that was removed was continuously replaced hydrostatically and the fluid level was kept constant in the receptor chamber.
IPC-16 cassette peristaltic pump: Multi-channel peristaltic pump with 16 channels. For the study, the rotation speed was regulated by the system.
Tube and connection material: A cassette tube extended bilaterally (color: orange/green), inner Ø: 0.38 mm, material: PVC (solvent resistance flex tubing) and
Tygon tubes were used to connect to the system. The total length of the tube connection from the outlet (bottom) of the receptor chamber to the cannulas of the fraction collector was 200 cm. The sampling on the outlet tube connections take place by means of a multi-way tap and injection cannulas placed on the tubes.
Fraction collector: The collection is performed by a robot which is controlled by the system. The robot arm was equipped with injection cannulas which are connected to the tubes by means of stainless-steel tube connections.
Plastic collection vessels: 15 and 50 mL reaction vessels
TEER measurement: EVOM2
LSC: Perkin Elmer Tri-Carb 2910TR

Receptor Fluid:
Fluid for storage under the skin until application: physiological saline solution with protease inhibitor (1 tablet protease inhibitor in 100 mL physiological saline)
Receptor fluid: 5 % (w/v) bovine serum albumin (=BSA) in physiological saline with the addition of 0.01 % (w/v) NaN3
Reason for selection of receptor fluid: Within the current study, the solubility of Benzyl Salicylate in 5 % (w/v) BSA in physiological saline with the addition of 0.01 % (w/v) NaN3 was determined (see section 3.6.) and was taken into consideration to demonstrate that the concentration of Benzyl Salicylate after dermal absorption was below 10 % of its solubility. 0.01 % (w/v) NaN3 was added to suppress potential biological contamination. The addition of these low amounts of NaN3 should have no impact on dermal absorption.

Reagents:
Skin washing solution: mild soap solution: Estesol® HAIR&BODY 3 % w:v in tap water.
Extraction media: Soluene®-350, ethanol, ethanol/deionized water (50 %)
Scintillation cocktail: Hionic Fluor™

Test system:
-Preparation of human split thickness skin:
Skin preparations of 4 donors were used within the study with a thickness of 278 – 400 μm, from which each donor yielded valid results.
-Hydration of skin preparations:
Each skin preparation was hydrated in physiological saline for about 10 minutes before mounting to the diffusion cells which were filled up with physiological saline with protease inhibitor (one tablet (SigmaFASTTM Protease Inhibitor Tablet) protease inhibitor in 100 mL saline solution). The prepared diffusion cells were stored overnight in a refrigerator.
The surface of the skin samples was checked visually before application and was dried with a cotton swab, if necessary.
Absorption in different matrices:
The mean absorption rate of all cells corresponding to the steepest slope of the cumulative absorbed dose curve over time was 0.0085 μg/(cm²×h). Taking the applied concentration into account, this is equivalent to a permeability constant of 0.18×10-5 cm/h. Furthermore, the evaluation of the cumulative absorption curves indicated a mean lag time of 9.78 hours. Based on an exposure period of 24 hours (during the complete penetration experiment), the cumulative absorption increased steadily over time. Under these test conditions, 7.36 % of the applied dose of 14C-Benzyl Salicylate were recovered as absorbed dose. 9.01 ± 1.46 % of the applied dose of 14C-Benzyl Salicylate were recovered as the sum of absorbed dose and dose associated to remaining skin.
To take into account the standard deviation of the result (± 1.46 %), an absorption of ca. 10.5% is considered from this skin penetration study.
Total recovery:
The mean total recovery measured in diffusion cells equipped with human skin fulfilled the OECD and SCCS quality criteria.
The individual values ranged between 91.08 and 97.30 % of dose. The mean total recovery resulted in a mean total recovery of 95.23 ± 2.02 %.
Major amounts of supplied test substance (85.11 ± 3.10 % of dose) were recovered from the skin preparation washings (membrane washing after 24 hours) and donor chamber washing. Recovery from the tape strips was calculated to be 1.11 ± 0.87 % of dose. Recovery from the receptor fluid, receptor chamber and receptor chamber washing, that is assessed to represent the absorbed dose, was 7.36 ± 1.51 % of dose. The recovery from the sum of absorbed dose and dose associated to remaining skin was 9.01 ± 1.46 % of applied dose.
Key result
Time point:
24 h
Dose:
nominal mean dose of 9.5 μg/cm², test substance formulated in a representative body lotion
Parameter:
percentage
Absorption:
ca. 10.5 %
Remarks on result:
other: sum of absorbed dose and dose associated to remaining skin

Analyses:

The stability of the test substance in the test-substance preparation over the application period was verified by analyses. The concentration and the homogeneous distribution of the test substance in the test-substance preparation was confirmed by

analysis. After 2 hours shaken at 32°C, the solubility of test substance in the receptor fluid (physiologic saline with 5% BSA)

was measured with 0.89 mg/mL.

Taking into account the amounts of active ingredient (a.i) (target dose: 10.0 μg/cm²), the amounts of test substance penetrated through the skin during the experiments as well as the volume of receptor fluid used (flow: 2.3 mL/h; corresponds to 55.2 mL over 24 h), no rate limiting effects on the diffusion process by saturation of the receptor fluid were present. Therefore, physiological saline solution with about 5 % BSA (with 0.01 % (w/v) NaN3) was an appropriate receptor medium to guarantee sufficient solubility of the test substance. The nominal concentration (4.95 mg/g) of the test-substance preparations is in accordance with the target value (5 mg/g). The control analyses performed by HPLC-UV of aliquots taken before and after administration confirmed the nominal concentration. Standard deviations below 5 % confirmed the homogenous distribution of the test substance in the test-substance preparation. Since only radiolabeled 14C-Benzyl Salicylate was used for the preparation, the activity analysis performed by LSC and the analysis of the radiochemical purity performed by Radio-HPLC were additionally able to confirm the nominal values.

The measured specific activities of the test-substance preparations compared to their target values are assessed to be acceptable within the precision of the method. Radio-HPLC analyses confirmed the stability of the radiolabeled test substance over the application period by radiochemical purities of 14C-Benzyl Salicylate > 98 %.

Discussion:

In the study 12 diffusion cells were used and yielded results that were assessed to be valid. Two cells were assessed to be invalid due to failed application (resulting in valid recoveries that were calculated to be >120 %). Therefore, these cells were excluded from statistics.

No penetration-relevant observations were detected for the selected skin preparations. Skin preparations of cells 3 and 10 showed visually irregular dermatomed areas. However, it should be mentioned that for these skin preparations the integrity parameters (visual observation and TEER measurement) passed and no abnormalities were detected during the experiment. Therefore, the results of this skin preparations are assessed to be valid. Minor detachments of epidermis were observed during the tape strip procedure for skin preparations of cells 2, 3 und 5. However, for these cells, obtained results are assessed to be comparable to results of parallel diffusion experiments. Therewith, the observed detachments of epidermis / parts of epidermis did not “negatively impact” the overall results and these cells were assessed to yield valid results.

Before the 24h-skin wash was performed, few small air bubbles were observed underneath the skin preparation of cell 1. However, since the overall results and all determined values for this cell were within the range observed for the other valid cells, it can be concluded that the observed air bubbles did not “negatively impact” the results of this replicate. Therefore, the

results of skin preparation 1 are assessed to be valid. The target dose of about 2 mg or μL/cm² was achieved well. The target dose 10 μg/cm² 14CBenzyl Salicylate was well achieved as indicated by a mean actual nominal dose 9.5 μg/cm². The applied dose was calculated from so called “exemplary applications” that were performed in parallel to the applications on the skin membrane. A mean value of the applied dose was calculated based on the measured activities in these “test applications”. This procedure yielded lower variability than the gravimetrically measured application weights (based on the weight of the pipette with test-substance preparation in the tip subtracted by the tare weight of the pipette). The receptor samples started to show quantifiable amounts of radioactivity (test substance) 2 hours after application (related to cumulative absorption of test substance [μg/cm2]). The individual total recoveries ranged between 91.08 % and 97.30 % of the applied dose while the

mean total recovery was 95.23 % of the applied dose. The mean lag time for the onset of dermal penetration was 9.78 hours. The presence of a lag time of penetration underlines the functional diffusion barrier of the skin against the tested substance preparations.

Based on the observed variation of the lag times of the individual cells, the experiments are assessed to be consistent and acceptable. Skin integrity was proven by measured TEER values of > 2kΩ. The low absorption of 14CBenzyl Salicylate in the experiment further confirmed the functional barrier of the skin samples used.

Conclusions:
The diffusion of 14C-Benzyl Salicylate into and through human skin was assessed by single topical application of a target dose of 10.0 μg/cm² of test substance, formulated in a representative body lotion to split thickness human skin preparations mounted on Franz-type diffusion cells.
Under these test conditions, 7.36 % of the applied dose of 14C-Benzyl Salicylate were recovered as absorbed dose. 9.01 ± 1.46 % of the applied dose of 14C-Benzyl Salicylate were recovered as the sum of absorbed dose and dose associated to remaining skin.
Executive summary:

The diffusion of 14C-Benzylsalicylate (=14C-Benzyl Salicylate) into and through human skin was assessed by single topical application of a target dose of 10 μg/cm² of test substance formulated in body lotion to split thickness skin preparations mounted on Franz-type diffusion cells. The study was performed using 12 diffusion cells, from which 10 cells were assessed to be valid. 2 cells were assessed to be invalid due to failed application (resulting in invalid recoveries that were calculated to be >120 %). Therefore, these cells were excluded from statistics.
Diffusion cells were operated in the flow-through mode using 5 % (w/v) bovine serum albumin (BSA) in physiological saline with the addition of 0.01 % (w/v) NaN3 as receptor fluid. The system was set to a continuous flow of 2.3 mL/h. Receptor fluid was collected from each cell in fractions by continuous collection for defined intervals over 24 h. Kinetic parameters (lag phase, absorption rate and Kp) were determined. 24 h post application, the skin membranes were washed with a mild soap solution followed by tap water. The test substance was recovered from all compartments of each diffusion cell. The results of recovery are summarized as non-absorbed dose (donor chamber and skin washings), amount in tape strips ((tape strips 1-2 (=tape strip sample 1), tape strips 3-5 (=tape strip sample 2), tape strips 6-10 (=tape strip sample 3), tape strips 11-15 (=tape strip sample 4), and tape strips 16-20 (=tape strip sample 5)), amount in skin preparation (remaining skin separated in dermis and epidermis) and absorbed dose (receptor fluid, receptor chamber washing, receptor samples).

Mean recoveries and kinetic parameters:

dose group / human

 

1

 

target concentration

[mg/mL]

5.0

target dose level of test substance

[µg/cm²]

10.0

mean actual nominal dose level of test substance

 

[µg/cm²]

 

9.5

recovery

[% of applied dose]

 

dislodgeable dose

(donor chamber washing, skin washes)

 

 

85.11

 

± 3.10

dose associated to tape strips

 

 

 

tape strips 1-20

 

1.11

± 0.87

dose associated to remaining skin

 

 

 

(epidermis excluding tape strips, dermis)

 

1.64

± 0.79

absorbed dose

(receptor fluid, receptor chamber washing)

 

 

7.36

 

± 1.51

sum of absorbed dose and dose associated to remaining skin                                             9.01   ± 1.46

total recovery

 

95.23

± 2.02

absorption kinetics

 

 

 

Kp

[×10-5cm/h]

0.18

± 0.04

absorption rate

[µg/(cm²×h)]

0.01

± 0.002

lag time

[ h ]

9.78

± 0.58

1mean values calculated from cells 1-7 and 9-11. Cells 8 and 12 were excluded from statistics due to failed application (resulting in invalid recoveries that were calculated to be >120 %); application weight calculated based on detected radioactivity in so called “test applications”, performed in parallel to the applications on the skin preparations (mean value); 2absorption rate was calculated to be 0.0085 µg/(cm²×h)

 

The mean total recovery fulfilled the quality criteria put forward in the test guidelines, resulting in a mean total recovery of 95.23 ± 2.02 %.
Major amounts of the applied test substance were recovered as dislodgeable, calculated as the sum of skin wash and donor chamber washing (85.11 ± 3.10 % of dose). Dose associated to tape strips resulted in 1.11 ± 0.87 % of dose. Minor amounts of the test substance were associated with the skin after the exposure period. This amount accounted to 1.64 ± 0.79 % of dose. The recovery from the sum of absorbed dose and dose associated to remaining skin was 9.01 ± 1.46 % of applied dose.
Mean absorption lag time was 9.78 ± 0.58 hours and demonstrates the presence of a functional barrier in the skin samples used.

Description of key information

An in vitro skin penetration study was conducted with radiolabeled Benzyl Salicylate using human dermatomed skin, according to OECD 428 guideline and GLP. 14C-Benzyl salicylate was applied to 12 human skin samples (from 4 donors) at a target dose of 10 µg/cm2 (actual 9.5 µg/cm2) in application of representative body lotion formulation at 2mg/cm2 in Franz-type diffusion cells in flow-through mode (surface skin area approximately 1cm2), using receptor fluid (physiological saline with 5% BSA and 0.01% (w/v) sodium azide (NaN3)). The recovery from the sum of absorbed dose and dose associated to remaining skin was 9.01 ± 1.46% of applied dose. The mean total recovery fulfilled the quality criteria put forward in the test guidelines, resulting in a mean total recovery of 95.23 ± 2.02 %. A value for skin absorption can be calculated from using the mean + 1SD i.e. 9 + 1.5 = 10.5%.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
10.5
Absorption rate - inhalation (%):
100

Additional information

Although REACH does not specifically require generation of toxicokinetic information, it does require that all relevant available information is used to assess the toxicokinetic behaviour of a substance, and that human health hazard assessment considers the toxicokinetic profile of the substance.

 

Benzyl Salicylate is a small organic molecule and the physico-chemical properties suggest it is likely to be absorbed via dermal, inhalation and gastric routes following exposure.

Acute and subchronic toxicity data indicate that Benzyl Salicylate is absorbed following administration by gavage and metabolised by the liver.

An in vitro skin penetration study was conducted with radiolabeled Benzyl Salicylate using human dermatomed skin, according to OECD 428 guideline and GLP. The study showed a dermal absorption of 9.01 ± 1.46% of applied dose, hence 10.5% as a maximum.

This dermal absorption value of 10.5% was not considered in the risk assessment of Benzyl Salicylate, in the Chemical Safety Report. The Chemical Safety Report, which was done before the generation of the in vitro skin absorption study, relied on a worst-case dermal absorption value of 100% and the risk assessment showed that all the RCRs were below 1, for both the workers and the consumers on the human health side, ensuring that Benzyl Salicylate could be used safely. Therefore, it was not deemed necessary to refine the exposure scenarios of the Chemical Safety Report with the new experimental dermal absorption value.

No other absorption, distribution, metabolism and elimination (ADME) studies are available. However, the very low toxicity of Benzyl Salicylate suggests that it may be considered inappropriate at this time to conduct further animal work to support ADME.

 

The main hydrolysis product of all the salicylate substances is salicylic acid (Belsito et al, 2007). The alcohols and acids that are formed as metabolites of salicylates are without significant toxicity. The hydrolysed side chains are metabolized by common and well characterized metabolic pathways. These primary alcohols (butanol, pentanol, hexanol, octanol, and propanol) and their corresponding aldehydes and acids have also been evaluated by JECFA (2001) who found them to have no safety concerns based on their current levels as food flavours.

 

Further, the reproductive and developmental toxicity of alcohol products that are formed upon hydrolysis of salicylates was evaluated by the Maximum workplace concentration (MAK) commission and concluded that 2- ethyl hexanol, methanol, ethanol, butyl alcohol, Octanol and isobutyl alcohol show no reproductive/developmental potential when used at levels ranging from 200–8000 ml/m3 for inhalation studies and 130–300 mg/kg for dietary studies.

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