Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
In vitro penetration and subchronic toxicity of alpha-Methyl-1,3-benzodioxole-5-propionaldehyde
Author:
Api, A. M., Lapczynski, A., Isola, D. A., Glenn Sipes, I.
Year:
2007
Bibliographic source:
Food und Chemical Toxicology 45 (2007) 702 -707

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other:
Version / remarks:
FDA/AAPS guidelines (Skelly et al. 1987)
Deviations:
no
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Wizard Laboratories, West Sacramento, CA

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.14%
- Specific activity: 57.26 mCi/mmol
- Locations of the label: benzyl-14C
Radiolabelling:
yes

Test animals

Species:
other: human skin samples
Details on test animals and environmental conditions:
Full thickness human skin samples were obtained from either the breast or abdomen of cosmetic surgery patients.

Administration / exposure

Type of coverage:
open
Vehicle:
ethanol
Duration of exposure:
48 hours
Doses:
- Nominal doses: 1%
Control animals:
no
Details on study design:
APPLICATION OF DOSE: 20 µL of a 1% solution in ethanol of radiolabeled test item was applied to the surface of the membrane

VEHICLE
- Amount applied: 20 µL

TEST SITE
- Preparation of test site: The epidermal membranes were mounted on filter paper supports and then placed onto diffusion cells and trimmed to size. The skin samples were mounted as a barrier between the halves of horizontal Franz-type diffusion cells, the stratum corneum facing the donor chamber.
- Area of exposure: about 1.0 cm²

SAMPLE COLLECTION
- Samples of the receptor fluid (50% ethanol/water) were harvested from the receptor chamber at 2, 8, 24, 36 and 48 h and analyzed by liquid scintillation chromatography. Post-incubation epidermal membranes were wiped with a cotton bud, and then tape stripped 10 times using adhesive tape (D-Squame).

SAMPLE PREPARATION
- Storage procedure:
- Preparation details:

ANALYSIS
- Method type for identification: Liquid scintillation counting

OTHER:
- Total radioactivity was accounted for by extruding and counting test item equivalents associated with membrane wipes, tape strips, digested remaining skin, and donor chambers. To assess evaporative loss, 20 µL of a 1% test item solution was applied to PTFC sheets mounted in diffusion cells (at 32 °C surface temperature). These sheets were then placed in chambers for 24 or 48 h. The PTFE sheet was then removed and washed twice with methanol (10 mL then 5 mL). The donor chamber was washed with 10 mL methanol. A sample of each wash solution was submitted to analysis by LSC which allowed the total remaining radiolabel at each timepoint to be determined. Radiolabel that was unaccounted for was considered to be associated with evaporative loss.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human, cosmetic surgery patients
- Type of skin: full thickness human skin samples, obtained from either the breast or abdomen
- Preparative technique: The epidermal membranes were mounted on filter paper supports and then placed onto diffusion cells and trimmed to size. The skin samples were mounted as a barrier between the halves of horizontal Franz-type diffusion cells, the stratum corneum facing the donor chamber. The cells were designed such that the area available for diffusion was about 1.0 cm².
- Thickness of skin: full thickness human skin samples
- Membrane integrity check: Membrane integrity was determined by applying 500 µL of 10 µCi/mL ³H20 to the skin surface and removing a 200 µL sample from the receptor phase one hour later. The skin surface was washed seven times and the receptor chambers three times with water, prior to refilling the selected receptor medium (50/50 ethanol/water). The sample was counted using a Wallae 1400 liquid scintillation counter (LSC) with 1400 DSA ver. 2.2 operating system, 152Eu external standard and ChemiStrip TM chemiluminescence subraction. The LSC uses the external standard to measure the counting efficiency for each sample and spectrum libraries to generate dpm values. The LSC generated disintegration per minute (dpm) values (5 min counts), which were transferred electronically to Excel worksheets for subsequent calculations. The experimental specific activity was 2198 dpm/ug (0.3% RSD, n = 5). Background activity (l0 dpm) was subtracted from all samples, with the lowest active sample at least 17-fold greater than background. No cell exhibited water permeability greater than 5 x 10³ cm/h and the average for each lest group was not greater than 2 x 10³ cm/h.
- Storage conditions: The skin surface temperature was maintained at 32 °C.

PRINCIPLES OF ASSAY
- Diffusion cell: Franz-type diffusion cells
- Receptor fluid: 50/50 ethanol/water
- Static system: No; Receptor chambers were continuously agitated by submersible magnetic stirrers
- Flow-through system: No
- Test temperature: Receptor chambers were maintained at 37 °C throughout the experiment. The skin surface temperature was maintained at 32 °C.
- Occlusion: Not specified

Results and discussion

Absorption in different matrices:
- Skin preparation (in vitro test system):
Total recovery:
- Total recovery: 86 % at 48 h (including an evaporation loss of 19 %)
Percutaneous absorptionopen allclose all
Key result
Time point:
24 h
Dose:
1%
Parameter:
percentage
Absorption:
42 %
Key result
Time point:
48 h
Dose:
1%
Parameter:
percentage
Absorption:
50 %

Any other information on results incl. tables

Following incubation of epidermal membranes with the radiolabeled test item, only 67% of the applied dose was accounted for by 48 h. At 24 and 48 h, 42% and 50%, respectively, of the applied close was recovered in the fluid retrieved from the receptor chambers. Distribution of remaining radiolabel in surface wipes, tape strips, remaining epidermis and the donor chamber surface accounted for an additional 17%. The chemical nature of the absorbed radiolabel was not characterized (i.e. parent test item or metabolites). Evaporative loss, estimated from direct application to PTFE sheets, was approximately 8% and 19% of the applied dose at 24 and 48 h, respectively.

Applicant's summary and conclusion

Conclusions:
Moderately high human skin permeation was observed. The in vitro human skin penetration studies showed 42% and 50% of the applied dose of the test item permeated into the receptor phase at 24 and 48 h, respectively.
Executive summary:

An in vitro dermal absorption studies according to the FDA/AAPS guidelines (Skelly et al., 1987) was performed with full thickness human skin samples. Twenty µL of a 1% solution in ethanol of radiolabeled test item (0.2 mCi benzyl-14C. specific activity of 57.26 mCi/mmol; radiochemical purity of 99.14%) was applied to the surface of the membrane. Receptor chambers were continuously agitated by submersible magnetic stirrers and maintained at 37 °C throughout the experiment. The skin surface temperature was maintained at 32 °C. Samples of the receptor fluid (50% ethanol/water) were harvested from the receptor chamber at 2, 8, 24, 36 and 48 h and analysed by liquid scintillation chromatography. Post-incubation epidermal membranes were wiped with a cotton bud, and then tape stripped 10 times using adhesive tape. Total radioactivity was accounted for by extruding and counting test item equivalents associated with membrane wipes, tape strips, digested remaining skin, and donor chambers. To assess evaporative loss, 20 µL of a 1% test item solution was applied to PTFC sheets mounted in diffusion cells (at 32 °C surface temperature). These sheets were then placed in chambers for 24 or 48 h. The PTFE sheet was then removed and washed twice with methanol (10 mL then 5 mL). The donor chamber was washed with 10 ml methanol. A sample of each wash solution was submitted to analysis by LSC which allowed the total remaining radiolabel at each timepoint to be determined. Radiolabel that was unaccounted for was considered to be associated with evaporative loss.

Following incubation of epidermal membranes with the radiolabeled test item, only 67% of the applied dose was accounted for by 48 h. At 24 and 48 h, 42% and 50%, respectively, of the applied close was recovered in the fluid retrieved from the receptor chambers. Distribution of remaining radiolabel in surface wipes, tape strips, remaining epidermis and the donor chamber surface accounted for an additional 17%. The chemical nature of the absorbed radiolabel was not characterized (i.e. parent test item or metabolites). Evaporative loss, estimated from direct application to PTFE sheets, was approximately 8% and 19% of the applied dose at 24 and 48 h, respectively.