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Dermal absorption

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Administrative data

dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-guideline study, scientifically acceptable, well documented, please refer to IUCLID section 13 for read across justification.

Data source

Reference Type:
Permeation study of five formulations of alpha-tocopherol acetate through human cadaver skin
Bibliographic source:
]. Cosmet. Sei., 56, 91-103

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Reference substance 001

Administration / exposure

Details on study design:
Three different solutions and two gel formulations, all containing 5 % alpha-tocopherol acetate, were used in this study. The solvents of the solutions were ethanol USP, isopropyl myristate, and light mineral oil. The gels consisted of 1% and 3% Klucel® (hydroxypropylcellulose), respectively. Gel formulations were prepared by dispersing Klucel® powder in ethanol USP and mixing by means of a magnetic stirrer to prepare Klucel® gel. Mixing con­ tinued until all particles were thoroughly wet. Alpha-tocopherol acetate was weighed accurately and dissolved separately in 95% ethanol USP. Then the alpha-tocopherol acetate solution was introduced dropwise to the Klucel® gel and continuously stirred for four hours at room temperature by means of a magnetic stirrer so that the proper concentration of the gel wa achieved.

Alpha-tocopherol and alpha-tocopherol acetate concentrations were determined by HPLC with a UV-detector according to the method described by Rangarajan and Zatz (2001, J Cosmet Sci, 52, 225). The chromatography was performed on a reverse-phase C 18. The detection wavelength was 285 nm. The mobile phase consisted of acetonitrile:water (96:4). The isocratic flow rate was changed to 2.0 ml/min to reduce the retention time. (The retention times were 10.3 and 12.4 minutes for alpha-tocopherol and for alpha-tocopherol acetate, respectively, compared to 13 and 16 minutes in the source paper.) For both analytes, the peak areas vs concentration (µg/ml) curves were linear in the range of 10-1000 µg/ml. The injection volume was 10 µl.

In vitro diffusion studies were carried out using a modified Franz diffusion cell apparatus with a diameter of 15 mm and a diffusional area of 1.76 cm2 . A Spectra/Por®7 regenerated cellulose membrane was inserted between the donor and the receiving compartment and secured in place by means of a pinch clamp. The membrane had a thickness of 60-65 µm and a molecular weight cutoff point of 1,000. The receiving compartment (volume 13.l ml) was filled with degassed ethanol USP and it was maintained at 37°C by means of a water bath circulator and a jacket surrounding the cell, resulting in a membrane surface temperature of 32°C. The receiving medium was continuously stirred by a Teflon™-coated magnetic stirrer, to avoid diffusion layer effects. A 1.00-ml sample of each ATA formulation was accurately measured and placed in the donor compartment and sealed with aluminum foil and parafilm. Aliquots of 0.5 ml were withdrawn from the receiving compartment at 15, 30, and 45 minutes and 1, 2, 3, and 4 hours using a microsyringe, and replaced immediately with an equal volume of degassed ethanol USP. All samples were trans ferred to 1.5-ml vials and diluted with degassed ethanol USP up to 1.0 ml before analysis by HPLC. The experiment was carried out in triplicate for each formulation. This method was applied to the ethanol solution, 1% Klucel® gel, and 3% Klucel® gel. Where appropriate, the cumulative amount released of ATA vs time data was analyzed and equation 5 was used to calculate the diffusion coefficients of ATA in the formulation. In cases where the slope of the M -vs-t plots was nearly constant, the diffusion coefficient of ATA in the formulation was not calculated exactly, but lower limits for the value were calculated instead using equation 6 with t = 4 hours.

The same apparatus and experimental procedure described in the previous section were used to perform cadaver skin permeability studies. The sampling times were 2, 4, 6, 8, 12, 24, 30, 36, and 48 hours. The experiment was carried out in triplicate for each formulation. Human cadaver skin from the back of Caucasian subjects was kindly donated by Novartis (Somerville, NJ). Cadaver skin was prepared by hydrating it in an isotonic phosphate-buffered solution for one hour at room temperature before it was placed between the donor and receiving compartments. The integrity of the skin was checked by visual inspection of the cumulative amount-vs-time plots. Experiments in which the amount of permeated ATA suddenly reached a plateau or jumped by large amounts were disregarded and repeated.

Permeability calculations were clone by nonlinear regression. Other statistical analyses were performed with the SPSS 10.1 for Windows 2000 version using one-way analysis of variance (ANOVA). Analyses were performed primarily to determine whether there were any significant differences in alpha-tocopherol acetate release among the different concentrations of Klucel® in the formulations and between each solution formulation.

Results and discussion

Applicant's summary and conclusion

Permeability studies performed on five different formulations of ATA show that iso­propyl myristate favors the highest permeability through human cadaver skin in the experimental conditions tested in this study. There was not a statistically significant difference between the permeabilities of the other formulation, suggesting that the formulation had relatively minor effects on the permeation of ATA. Further studies are necessary to confirm these findings in vivo.
Executive summary:
Alpha-tocopherol (AT) is the vitamin E homologue with the highest in vivo biological activity. For stability considerations, alpha-tocopherol is usually used as its prodrug ester, alpha-tocopherol acetate (ATA), which once absorbed into the skin is hydrolyzed to alpha­ tocopherol, the active form. The objective of this research was to characterize in vitro the permeation properties of ATA from various solutions and gel formulations. Permeation studies were conducted using modified Franz diffusion cells and human cadaver skin as the membrane. Specifically, 5% (w/w) alpha­ tocopherol acetate was formulated in the following vehicles: ethanol, isopropyl myristate, light mineral oil, 1% Klucel® gel in ethanol, and 3% Klucel® gel in ethanol (w/w). The receiver temperature was 37°C. Samples from the receiver were collected at 2, 4, 6, 8, 12, 24, 30, 36, and 48 hours and analyzed by HPLC for concentrations of alpha-tocopherol acetate and alpha-tocopherol. The permeabilities of ATA through human cadaver skin were 1.0 x 10-4 , 1.1 x 10-2 , 1.4 x 10-4 , 2.1 x 10-4 , and 4.7 x 10-4 cm/h for the ethanol solution, isopropyl myristate solution, light mineral oil solution, 1% Klucel® gel, and 3% Klucel® gel, respectively. The results showed that the formulation had relatively minor effects on the permeability coefficients of ATA through cadaver skin in all cases except for the isopropyl myristate solution.

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