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Toxicological information

Dermal absorption

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Administrative data

dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Reason / purpose:
reference to same study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:

Test material

Details on test material:
- Name of test material (as cited in study report): Uvinul A plus
- Lot/batch No.: 844-1101
- Radiochemical purity: 99.5%
- Specific activity: 6.78% MBq/mg (of the labeled test substance)
- Locations of the label: benzoic ester-ring-U-C14
- Solvent used: toluene

Test animals

Details on test animals and environmental conditions:
- Source: Abdominal skin samples from three human female donors, which were excised during a cosmetic surgery.
- Age at study initiation: 32, 34, 36 years old
- Weight at study initiation: BMI of 21.5, 24.1, 23.9

Administration / exposure

Type of coverage:
other: It is an in vitro study and thus, the coverage that was made was done by covering the Franz diffusion cells with parafilm during the 24 hours exposure period in order to avoid any changes in the test substance formulation.
other: As vehicle, a formulation of a cream with 10% active ingredient containing non-labelled Uvinul A Plus was added to the radioactive test substance, but before toluene was evaporated.
Duration of exposure:
24-hours exposure
18 mg ± 5 % of the test formulation (approximately 10 mg per cm2 of the skin) were applied by a weighing procedure of the diffusion cell to the diffusion skin area (and homogenously spread over the skin surface using a spatula and cotton swab). A calculated / theoretical test substance contents in µg which were applied are as follows:
Mean dose applied (on three skin specimen) to donor 311-01-0108: 1826.69 µg
Mean dose applied to donor 312-01-0108: 1754.63 µg
Mean dose applied to donor 313-01-0208: 1809.93 µg
The mean applied dose which was used for all skin specimens of all donors was 1801.31 µg.
No. of animals per group:
Human skin from three different donors was divided in a way that skin of each donor had triplicate or four fold skin specimens. The skin specimen was then internally encoded.
Control animals:
For the quality control of the human skin the permeability of Caffeine (10 mg/L in KRB at pH 7.4) was carried out on skin samples of the same donors.
Details on study design:
- Method for preparation of dose suspensions:
As vehicle for the radio-labelled test substance Uvinul A Plus a cream with 10% Uvinul A Plus was used. The radio-labelled test substance was added to reach a final concentration of approximately 3.7 kBq/mg. The test formulation was prepared one day before the start of the experiment and stored over the night at 4 °C.
115 μL of the radioactive test substance were placed in an open beaker in order to evaporate the solvent (toluene) in the sample. 3 g of formulation were added to the residual and both have been homogenized using Ultraturrax for 3-5 minutes. The test formulation was mixed again at least for 2 hours prior to the start of the in vitro experiment. Two aliquots of the test formulation (approximately 100 mg) before and after the in vitro permeability study were stored in the refrigerator and sent on dry ice to the sponsor for the radio-HPLC analysis.
18 mg ± 5 % of the test formulation (approximately 10 mg per cm2 of the skin) were applied by a weighting procedure of the diffusion cell to the diffusion skin area (and homogenously spread over the skin surface using a spatula and cotton swab).

See above (dose preparation).

At the end of the permeation experiments (24 hours exposure), the skin specimens were washed twice with a surfactant solution (Texapon N 70 diluted with water 1:140 (v/v, %), applied volume of the surfactant solution: 250 μL).

Samples were analyzed by the Liquid Scintillation counting

Details on in vitro test system (if applicable):
- Source of skin: From a hospital where 3 female donors agreed to donate skin samples for research, excised during their cosmetic surgery.
- Ethical approval if human skin: No data. It was written that the hospital had the prior consent of the patients that the tissue could be used for scientific research.
- Type of skin: Skin from the abdomen
- Preparative technique: The excised skin from the surgery was cooled to 4 °C and its surface was dried before being transported. Care was taken to ensure that the subcutaneous fatty tissue does not get into contact with the surface of the skin. Once in the laboratory, the skin was separated from the subcutaneous fatty layer and then an encrypted identification number issued by Across Barriers was assigned to each skin specimen.
- Thickness of skin (in mm): The skin was dermatomized to approximately 0.5 mm
- Membrane integrity check: The skin samples that were used were found to be intact by using the OECD marker substance - caffeine.
- Storage conditions and justification of the anatomical site and preparative technique:
After the subcutaneous fatty layer was removed (within two hours after reception of the abdominal skin), the residual full-thickness skin was stored at -20 °C. According to the OECD Guideline, storage of skin at -20 °C for a period of up to twelve months does not alter its permeability.
The skin was used immediately after thawing. Repeated freeze-thaw cycles were avoided.

- Diffusion cell: Cylindrical glass Franz cell
- Acceptor fluid: For the test substance- KRB (Krebs Ringer buffer) at pH 7.4 supplemented with 1 % BSA (bovine serum albumine). For the permeability assay of caffeine (carried out on skin samples of the same donors) the acceptor medium that was used was the KRB medium supplemented with 0.05% NaN3.
- The stirring speed and the temperature were set at 400 rotations per minute and 32 °C ± 2 °C, respectively. At each sampling point the stirring speed and temperature were documented.
- Sampling times: The permeation study was performed over a period of 24 hours. After 0, 2, 4, 6, 8, 20, 22 and 24 hours and after 0, 1, 2, 4, 6, 8, 22 and 24 hours in the first and second experiment, respectively. One sample with a volume of 300 μL was taken from the acceptor compartments and analyzed for the test substance content by LSC. The sampled amount was replaced with a fresh medium preheated to the temperature of 32 °C.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
- Receptor fluid (in vitro test system): 0.02 +/- 0.01% of radio-labelled test substance.
- Skin preparation (in vitro test system): The mean recovery of the test substance found in the "remaining skin" (extraction of rest of the skin after tape stripping) amounts to 0.11 ±0.05 %.
Stratum corneum (in vitro test system): The mean recovery in the two first tape strips (first pool) was 1.54 ± 1.12 % over all performed experiments. In the further 18 tape strips a mean recovery of 0.41 ± 0.30 % was determined.
- Other: In the skin wash procedure after the in vitro experiment the mean range was 96.95% to 103.73%
Total recovery:
- Total recovery:
The calculated mean total recovery rate for each of the three different skin donors used was between 98.85 % and 105.10 % of the dose applied. The overall mean of total recovery rate for the three different skin donors analyzed was 101.08 ± 10.06 %.
Percutaneous absorption
1801.31 µg
0.5 %
Remarks on result:
other: 24 hours
According to the SCCNFP/0750/03 opinion the requested value should be calculated from the measurments of the test substance in the "receptor fluid" (0.02%), the "remaining skin" (0.11%) and in addition the second pool of the tape stripping (0.41%).

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion