Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. certificate)
Remarks:
Charles River Laboratories, Edinburgh, UK

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
Please refer to confidential details on test material.
Radiolabelling:
no

Test animals

Species:
other: Split-thickness (12 samples from 4 human donors)
Strain:
other: not applicable
Details on test animals and environmental conditions:
not applicable

Administration / exposure

Vehicle:
other: disolved in a cream formulation
Duration of exposure:
24 h
Doses:
3.0% (w/w) or approx. 2 mg/cm²
No. of animals per group:
not applicable
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: NHS Lothian, St. John’s Hospital, Livingston, UK and NHS Greater Glasgow & Clyde, Biobank, Glasgow, UK
- Ethical approval if human skin: All patients gave informed consent prior to undergoing surgery, for their excised skin to be used for scientific purposes.
- Type of skin: full-thickness human skin
- Preparative technique: Split-thickness membranes were prepared by pinning the full-thickness skin, stratum corneum uppermost, onto a raised cork board and cutting at a setting equivalent to 200-400 μm depth using a Zimmer® electric dermatome
- Thickness of skin (in µm): Full thickness skin: 900-1700; split-thickness skin: 350-400
- Membrane integrity check: by electrical resistance determination. An electrical resistance barrier integrity test was performed and any human skin sample exhibiting a resistance < 4 kΩ was excluded from absorption measurements. No samples were rejected.
- Storage conditions: The split-thickness membranes were stored in a freezer set to maintain a temperature of -20°C.


PRINCIPLES OF ASSAY
- Diffusion cell: A static diffusion cell system was used (PermeGear Inc). The static diffusion cells were placed in a steel manifold 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 3.14 cm². The receptor chamber volume was nominally 10 mL, with each receptor chamber individually marked with the actual volume by the manufacturer.
- Receptor fluid: Phosphate buffered saline supplemented with bovine serum albumin (5%, w/v) was used as the receptor fluid.
- Solubility of test substance in receptor fluid:
The solubility of C-1701 B_C_3 in Receptor Fluid and methanol was determined to be 0.207 mg/mL and 0.246 mg/mL, respectively. This was equivalent to 108.77% and 127.50% of the target concentration 0.190 and 0.193 mg/mL, respectively.
Therefore, based on these results, the receptor fluid was determined to be suitable for use in the test item absorption study.
For an application of 2 mg/cm² over a 3.14 cm² application area, 6.28 mg/cell was applied. If 100% of the test item, in the 3% (w/w) test preparation penetrated, it would result in 18.84 μg/mL of test item. The maximum concentration of test item in the receptor fluid and positive control (methanol) was estimated at a level assuming 10 x 100% absorption following application of the suncare formulation to skin. Therefore, the solubility of C-1701 B_C_3 was acceptable in receptor fluid, and was accepted for use.
- Static system:
Split-thickness skin was removed from storage in a freezer set to maintain a temperature of -20°C and allowed to reach ambient temperature. Sections of split-thickness skin, ca 3 x 3 cm, were cut out and positioned on the receptor chamber of the diffusion cell, which
contained receptor fluid and a magnetic stirring bar. The donor chamber was tightened into place with a clamp. The prepared cells were then placed in the heated manifold and the magnetic stirrer was switched on to mix the contents of the receptor chamber. No air bubbles were present in the receptor fluid chamber
- Application of test preparation to human skin:
A single dose of test preparation was applied over the stratum corneum surface of the exposed skin. A pipette was used, set to deliver a target dose of 6.28 mg (2 mg/cm²). The donor chambers were left open to the atmosphere. To accurately quantify the dose of the test item applied in the test preparation to the skin samples, 7 representative weighed aliquots (mock doses) of the test preparation were collected for analysis at the time of dosing. Methanol (10 mL) was added to each mock dose sample and mixed by vortex mixing prior to analysis by LC-MS/MS. By LC-MS/MS, the mean application of the formulation, mean application of the test item and homogeneity (CV, %) of the formulation were 6.41 mg, 204 μg and 4.74%, respectively.
- Recepor fluid sampling: Receptor fluid aliquots were collected at 0.5, 1, 2, 4, 8 and 24 h post dose. All receptor fluid samples were stored in a freezer set to maintain a temperature of -20ºC, until analysis by LC-MS/MS.
- Terminal Exposure Procedures and Post Exposure Monitoring (24 h Post Dose):
At 24 h post dose, the exposure period was terminated for all skin samples by washing twice with 942 μL (300 μL/cm²) of a 2% (w/v) aqueous solution of sodium dodecyl sulphate, then rinsed twice with 942 μL (300 μL/cm²) of water. For each washing step the soap or water was aspirated with a pipette, and the skin was dried with a tissue swab. An additional tissue swab was used after the final rinse. The soap and water were retained in a single pooled sample (skin wash). The pipette tip was retained and the residues associated with it were analysed. The tissue paper swabs were retained for analysis. The sealing clamp was removed and the donor chamber removed. The donor chamber was transferred to pre-weighed pots containing a pre-weighed volume (40 mL) of methanol. The pots containing the donor chambers were sonicated for ca 10 min to extract the test item. The donor chambers were removed from the pots, and the solvent was retained for analysis. Skin was removed from the cell and dried with additional tissue swabs, which were then retained with the skin wash tissue swabs. The tissue swabs were extracted in 10 mL methanol. The skin under the cell flange (unexposed skin) and outside the donor chamber was cut away from the exposed skin with a scalpel. The stratum corneum of the exposed skin was removed with 20 successive D-Squame® discs. Epidermis was not removed during this process. The tape strips were grouped into 1-2, 3-5, 6-10, 11-15, and 16-20 strips. The epidermis was separated from the dermis of the exposed skin using heat separation. The exposed skin was placed onto cling film, epidermis side up, and the cling film wrapped over the skin. A 200 g weight, heated to ca 65°C, was placed onto the skin for ca 90 s. The epidermis was removed from the skin with a scalpel. The epidermis, dermis, unexposed skin and cling film were placed into individual vials. Methanol (5 mL) was added to each tape strip, epidermis, dermis, unexposed skin and cling film vial to extract the test item. These were retained for analysis. The receptor fluid in the receptor chamber was removed into bulk receptor vials and stored in a freezer set to maintain a temperature of -20°C for further analysis if required. The receptor chamber was rinsed with 4 aliquots (10 mL) of methanol. The methanol was
pooled as a single 40 mL sample into a weighed receptor wash pot.

Results and discussion

Absorption in different matrices:
The majority of the test item was removed at 24 hours post dose by washing the skin surface (93.73% of applied dose)

- Stratum corneum: 0.79 % of the applied dose (0.52 µg/cm²)
- Epidermis without stratum corneum: 0.57% (0.37 µg/cm²)
- Dermis: 0.35 % (0.23 µg/cm²)
- Total absorbed dose (amount that had penetrated through the skin into the receptor fluid): 0.72% (0.48 µg/cm²)
- Dermal delivery (total absorbed dose + dermis + epidermis (without stratum corneum)): 1.63% (1.08 µg/cm²)
Total recovery:
- Total recovery: 96.34 %
- Limit of quantification (LLOQ): 1 ng/mL
- Quantification of values below LOD or LOQ:
All cumulative receptor fluid values were calculated from data which included values less than the lower LLOQ). Any receptor fluid value below the LLOQ was assigned the nominal value of the LLOQ, representing the “worst-case” result for absorbed test item. Values below the LLOQ in the receptor fluid were observed up to 2-4 h post-dose. The solubility of the test item in the receptor fluid was not rate limiting for absorption.
Percutaneous absorption
Dose:
3% (w/w)
Parameter:
percentage
Absorption:
1.63 %
Remarks on result:
other: 24 hours
Remarks:
1.63 ± 1.02 % (n=12), corresponding to 1.08 ± 0.67 µg/cm² (n=12)

Any other information on results incl. tables

Table 1: Distribution/absorption of the test item 24 hours after application in a typical suncare formulation (3 % w/wa) to human split-thickness skin

Distribution

Fraction of applied dose

mean ± SD (n = 12)

[%]

Amount

mean ± SD (n = 12)

[µg/cm²]

Total dislodgeable dose

93.73 ± 5.48

61.79 ± 3.61

Stratum corneum

0.79 ± 0.46

0.52 ± 0.30

Epidermis (withoutstratum corneum)

0.57 ± 0.48

0.37 ± 0.32

Dermis

0.35 ± 0.41

0.23 ± 0.27

Total unabsorbed dose

94.71 ± 5.06

62.44 ± 3.34

Total absorbed dose

0.72 ± 0.63

0.48 ± 0.42

Dermal delivery

1.63 ± 1.02

1.08 ± 0.67

Mass balance

96.34 ± 4.57

63.51 ± 3.01

a: nominal concentration; a test item concentration of 3.23 % (w/w) was determined by LC-MS/MS

n: number of samples, SD: standard deviation

Total dislodgeable dose: donor chamber wash + skin wash + tissue swabs + pipette tips

Total unabsorbed dose: total dislodgeable dose +stratum corneum+ unexposed skin

Total absorbed dose: cumulative receptor fluid + receptor rinse + receptor chamber wash

Dermal delivery: total absorbed dose + dermis + epidermis (without stratum corneum);

Mass balance: total unabsorbed dose + epidermis (without stratum corneum) + dermis + total absorbed dose

Applicant's summary and conclusion