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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
RADIOLABELLED TEST ITEM

SOURCE OF TEST MATERIAL
- Lot No. of test material: 372-058-0601-A-20150313-DRE

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.7 %
- Specific activity: 238.3 µCi/mg 60.1 mCi/mmol
- Locations of the label: [14C(U)]-

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: - 20 °C
- Stability under test conditions: The results of the radiochemical purity assessment confirmed that the test item was stable over the dosing period.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Ethanol (10 mL) was added to the radiolabelled test substance vieal. The contents were mixed by vortex. Three aliquots (10 µL) of the stock stolution were transferred into scintillation vials, mixed with methanol:scintillation fluid and analysed by liquid scintillation counting. The concentration of test item in the stock solution was determined to be 0.527 +/- 0.0141 mg/mL with a CV of 2.66 %.
- Final preparation of a solid:
Preparation at lower specific activity: The test item was added to a 2 mL volumetric flask. Stock solution was added to the flask. Ethanol was added up to the 2 mL calibration line and the sample was mixed by vortex for ca. 1 min. Three aliquots (10 µL) were taken, mixed with methanol:scintillation fluid and analysed by liquid scintillation counting. By radioactivity, the concentration of the radiolabelled test item in the solution was determined to be 2.31 mg/mL. The test item was homogeneously distributed in the solution with a CV of 0.91 %. The new specific activity determined was 13.7 µCi/mg.
Test Preparations: The test substance was added to a glass vial in aliquots. The solvent was removed under a gentle stream of nitrogen gas after the addition of each aliquot. Physiological saline was added to the vial in small aliquots and the contents were mixed by vortex. Six aliquots were taken, mixed with methanol:scintillation fluid and analysed by liquid scintillation counting.


NON-RADIOLABELLED TEST ITEM

SOURCE OF TEST MATERIAL
- Batch No.of test material: 161106
- Expiration date of the lot/batch: 16 November 2019
- Purity: 99.51 %
- Physical appearance: white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was predicted to have a water solubility of 1.1 g/L (Sponsor's supplied information). For an application of 500 µL/cm² over a 0.64 cm² application area, for the highest concentration formulation (200 mg/L, Test Preparation 3), 320 µL of the formulation (0.064 mg of BPS) would be applied to each skin sample. Theoretically, if 100 % of the test item was absorbed, this would result in a test item concentration in the receptor fluid of 12.8 mg/L (0.064 mg of BPS in 5 mL of receptor fluid). The solubility of BPS in the receptor fluid was assessed assuming 10 x 100 % absorption; therefore, the target concentration of BPS in the receptor fluid for the solubility assessment was 0.128 g/L.
Radiolabelling:
yes

Test animals

Species:
other: ex vivo study
Strain:
other: human skin stratum corneum samples
Sex:
female

Administration / exposure

Type of coverage:
open
Vehicle:
physiological saline
Duration of exposure:
8 h and 24 h
Doses:
1 and 102 µg/cm2
No. of animals per group:
8 split-thickness samples of human skin
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: The test substance was added to a glass vial in aliquots. The solvent was removed under a gentle stream of nitrogen gas after the addition of each aliquot. Physiological saline was added to the vial in small aliquots and the contents were mixed by vortex. Six aliquots were taken, mixed with methanol:scintillation fluid and analysed by liquid scintillation counting.

APPLICATION OF DOSE: The test substance preparations were applied to the surface of the exposed stratum corneum of 8 split-thickness samples of human skin, respectively, using a positive displacement pipette set. The donor chambers of the cells were not occluded and so were left open to the atmosphere. Seven representative aliquots in the test preparations were dispensed into vials at the time of dosing, mixed with methanol:scinitillation fluid and analysed by liquid scintillation counting.

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: The applied dose was removed from the exposed skin surface from samples dosed with 200 mg/L test preparation only. Commercial hand wash soap (ca. 50 µL) was applied to the skin and the soap gently rubbed onto the skin with a tissue swab. The skin was then rinsed with ca. 5 mL of a ca. 2 % (v/v) commercial soap solution. The soap solution was applied in aliquots (0.5 mL) and each aliquot was aspirated three times with a pipette. The skin was dried with a tissue swab. The process was repeated and the skin was dried with an additional tissue swab.
- Time after start of exposure: 8 h

SAMPLE PREPARATION
- Storage procedure: in a freezer set to maintain a temperature of - 20 °C
- Preparation details: Four samples of full-thickness human skin (three samples from the abdominal site and one single sample obtained from abdomen and back region) were obtained from female donors aged 39 to 72 years old. Two of the skin samples were obtained NHS Lothian on ice. On arrival, the subcutaneous fat and connective tissue were removed from the skin samples using a scalpel. The samples were washed under cold running tap water and dried with paper towels. The samples were cut into pieces, vacuum packed and stored at - 20 °C until they were used for the study. Two of the samples were obtained from Tissue Solutions, Glasgow. The samples arrived deep frozen on dry ice and were stored in a freezer set to maintain a temperature of - 20 °C until they were used for the study. The age and sex of the donor and sites from which the skin samples were taken were recorded in the study records. Human skin samples were removed from - 20 °C storage and allowed to thaw at ambient temperature. The thickness of the full-thickness skin membranes was measured using a micrometer. Split-thickness membranes were prepared by pinning the full-thickness skin, stratum corneum uppermost, onto a raised cork board and cutting with an electric dermatome at a setting equivalent to 200 - 400 µm depth. The thickness of the split-thickness membranes was measured using a micrometer. Membranes were then wrapped in aluminium foil and stored in a freezer, set to maintain a temperature of - 20 °C, for a maximum period of two months.

ANALYSIS
- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC): Liquid scintillation, HPLC-UV
- Validation of analytical procedure: All samples were counted together with the representative blanks using a liquid scintillation analyser with automatic quench correction by external standard. Scintillation fluid (10 mL) or methanol:scintillation fluid (12 mL) was added to the samples. Representative blank sample values were substracted from sample count rates to give net dpm per sample. Prior to analysis, samples were allowed to stabilise with regard to light and temperature.
- Limits of detection and quantification: A limit of reliable measurement of 30 dpm above background has been instituted in these laboratories. Counts that are below 30 dpm above background represent a true value. This means that data are recorded with values that are less than the limit of reliable measurement.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: full-thickness human skin
- Ethical approval if human skin: female donors aged 39 to 72 years old
- Type of skin: three samples from the abdominal site and one single sample obtained from abdomen and back region
- Preparative technique: The thickness of the full-thickness skin membranes was measured using a micrometer. Split-thickness membranes were prepared by pinning the full-thickness skin, stratum corneum uppermost, onto a raised cork board and cutting with an electric dermatome at a setting equivalent to 200 - 400 µm depth. The thickness of the split-thickness membranes was measured using a micrometer.
- Thickness of skin (in mm): Full Thickness Skin: 1340 µm, 1420 - 2000 µm, 1200 µm, 1690 µm; Split-Thickness Skin: 400 µm, 390 - 400 µm, 400 µm, 400 µm
- Membrane integrity check: Skin samples were allowed to equilibrate at 32 °C +/- 1 °C for ca. 5 min. Phosphate buffered saline (1 mL) was then added to the donor chamber and the skin samples were allowed to equilibrate for a further ca. 30 min. The electrical resistance was then measured using a Tinsley Databridge (Model 6401) set at low voltage alternating current, 1000 Hz with a maximum voltage of 300 mV root-mean-squared (rms) in the parallel equivalent circuit mode. Any skin sample exhibiting a resistance less than 10.9 kΩ (threshold value) was excluded from subsequent absorption measurements. The phosphate buffered saline was removed from the skin surface, which was then rinsed with water and dried with tissue paper.
- Storage conditions: - 20 °C

PRINCIPLES OF ASSAY
- Diffusion cell: The glass static diffusion cells were placed in a manifold on a magnetic stirrer plate heated via a circulating water bath to maintain the skin surface temperature at 32 °C +/- 1 °C. The surface area of exposed skin within the cells was 0.64 cm². The receptor chamber volume was nominally 5 mL, with each receptor chamber individually marked with the actual volume by the manufacturer.
- Receptor fluid: Phosphate buffered saline containing polyoxyethylene 20 oleyl ether (PEG, ca. 6 %, w/v), sodium azide (ca. 0.01 %, w/v), streptomycin (ca. 0.1 mg/mL) and penicillin (ca. 100 units/mL). The pH of the receptor fluid was confirmed to be 7.36.
- Solubility of test substance in receptor fluid: The test substance was predicted to have a water solubility of 1.1 g/L (Sponsor's supplied information). For an application of 500 µL/cm² over a 0.64 cm² application area, for the highest concentration formulation (200 mg/L), 320 µL of the formulation (0.064 mg of BPS) would be applied to each skin sample. Theoretically, if 100 % of the test substance was absorbed, this would result in a test item concentration in the receptor fluid of 12.8 mg/L (0.064 mg of BPS in 5 mL of receptor fluid). The solubility of the test substance BPS in the receptor fluid was assessed assuming 10 x 100 % absorption; therefore, the target concentration of BPS in the receptor fluid for solubility assessment was 0.128 g/L.
- Static system: The receptor chambers were in a manifold connected to a circulating waterbath. Magnetic stirrer bars were placed in the receptor fluid chambers which were filled with receptor fluid. Split-thickness skin samples were removed from the freezer and allowed to thaw at ambient temperature. Sections of split-thickness skin (ca. 1.5 x 1.5 cm) were cut and mounted in the diffusion cells between the donor and receptor chamber. The donor chamber was tightened into place with a clamp. Cells were visually checked to ensure no cells were leaking and no air bubbles were present in the receptor fluid chamber.
- Test temperature: ambient
- Occlusion: open

Results and discussion

Absorption in different matrices:
- Absorption:
102 µg/cm2: The mean absorption rate through human split-thickness skin was 2.64 ng equiv./cm²/h during the 24 h experimental period
1 µg/cm2: The mean absorption rate through human split-thickness skin was 0.06 ng equiv./cm²/h during the 24 h experimental period
- Penetration:
102 µg/cm2: The amount penetrated at 24 h as measured in the receptor fluid was 63.4 ng equiv./cm², which is equivalent to 0.06 % of the applied dose
1 µg/cm2: The amount penetrated at 24 h as measured in the receptor fluid was 1.43 ng equiv./cm², which is equivalent to 0.14 % of the applied dose
- Stratum corneum (in vitro test system): tape strips
Percutaneous absorptionopen allclose all
Time point:
24 h
Dose:
102 µg/cm2
Parameter:
rate
Absorption:
2.64 other: ng equiv./cm2/h
Time point:
24 h
Dose:
102 µg/cm2
Parameter:
percentage
Absorption:
1.8 %
Time point:
24 h
Dose:
1 µg/cm2
Parameter:
rate
Absorption:
0.06 other: ng equiv./cm²/h
Time point:
24 h
Dose:
1 µg/cm2
Parameter:
percentage
Absorption:
8.79 %

Any other information on results incl. tables

Table 1: Cumulative absorption of all donors, 102 µg/cm2 test item

Time [h]

Mean cumulative penetration [µg equiv./cm²]

0

1

2

4

8

12

24

0.00000

0.00007

0.00186

0.00476

0.01067

0.02242

0.06336

Table 2: Cumulative absorption of all donors, 1 µg/cm2 test item

Time [h]

Mean cumulative penetration [µg equiv./cm²]

0

1

2

4

8

12

24

0.00

0.00

0.04

0.22

0.35

0.88

1.43

Following topical (infinite dose) application of 200 mg/L test preparation to human skin in vitro, the absorbed dose was 0.08 +/- 0.08 % (0.08 +/- 0.08 µg equiv./cm²) of the applied dose. The dermal delivery was 0.56 +/- 0.19 % (0.57 +/- 0.20 µg equiv./cm²) of the applied dose. The potentially absorbed dose was 1.80 +/- 0.38 % (1.84 +/- 0.39 µg eqiv./cm²) of the applied dose. The mass balance was 100.26 +/- 0.26 % (102 +/- 0.26 µg equiv./cm²) of the applied dose.

Following topical (finite dose) application of 100 mg/L to human skin in vitro, the absorbed dose was 0.19 +/- 0.21 % (1.94 +/- 2.15 ng equiv./cm²) of the applied dose. The dermal delivery was 1.66 +/- 1.00 % (17.2 +/- 10.3 ng equiv./cm²) of the applied dose. The potentially absorbed dose was 8.79 +/- 3.24 % (90.8 +/- 33.4 ng equiv./cm²) of the applied dose. The mass balance was 100.40 +/- 0.96 % (1037 +/- 9.87 ng equiv./cm²) of the applied dose.

Applicant's summary and conclusion

Executive summary:

The test item is used in a variety of products commercially.

The aim of this study was to determine the rate and extent of absorption of the test substance following topical application to human skin.

Split-thickness human skin membranes were mounted into static diffusion cells in vitro. Receptor fluid was added to the receptor chamber (nominal volume 5 mL, nominal exposure are 0.64 cm²). The skin surface temperature was maintained at 32 °C +/- 1 °C throughout the experiment. An electrical resistance barrier integrity assessment was performed and any skin sample exhibiting resistance lower than 10.9 kΩ was excluded from subsequent absorption measurements.

The test preparations were applied (500 µL/cm² or 10 µL/cm²) to human split-thickness skin membranes obtained from four different donors and the cells were left open to the atmosphere. The concentration of the test substance in the test preparations was 200 mg/L for infinite dose and 100 mg/L for finite dose applications. The application rate of the test item was 102 µg/cm2 and 1 µg/cm2. The stability throughout the application of all test preparations was confirmed by high performance liquid chromatography (HPLC).

Percutaneous absorption was assessed by collecting receptor fluid samples prior to dosing and at 1, 2, 4, 8, 12 and 24 h post dose. At 8 h post dose, the exposure period was terminated by removing the applied test preparation from the skin surface (where applicable) and washing the skin surface with a concentrated commercial hand wash soap followed by rinsing with a dilute soap solution (2 %, v/v) and drying the surface with a tissue swab. At 24 h post dose, the skin was removed from the static cells, the stratum corneum tape stripped and the skin divided into epidermis, dermis and unexposed skin samples. The skin samples were dissolved with Solvable tissue solubiliser. All samples were analysed by liquid scintillation counting.

Following topical (infinite dose) application of 200 mg/L test preparation to human skin in vitro, the absorbed dose was 0.08 +/- 0.08 % (0.08 +/- 0.08 µg equiv./cm²) of the applied dose. The dermal delivery was 0.56 +/- 0.19 % (0.57 +/- 0.20 µg equiv./cm²) of the applied dose. The potentially absorbed dose was 1.80 +/- 0.38 % (1.84 +/- 0.39 µg eqiv./cm²) of the applied dose. The mass balance was 100.26 +/- 0.26 % (102 +/- 0.26 µg equiv./cm²) of the applied dose.

Following topical (finite dose) application of 100 mg/L to human skin in vitro, the absorbed dose was 0.19 +/- 0.21 % (1.94 +/- 2.15 ng equiv./cm²) of the applied dose. The dermal delivery was 1.66 +/- 1.00 % (17.2 +/- 10.3 ng equiv./cm²) of the applied dose. The potentially absorbed dose was 8.79 +/- 3.24 % (90.8 +/- 33.4 ng equiv./cm²) of the applied dose. The mass balance was 100.40 +/- 0.96 % (1037 +/- 9.87 ng equiv./cm²) of the applied dose.