L-Ascorbic acid, 2-(dihydrogen phosphate), sodium salt (1:3)

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: non Guideline, non GLP but good public available literature data

Data source

Reference

Reference Type:

publication

Title:

Skin Penetration and Retention of L-Ascorbic Acid 2-Phosphate Using Multilamellar Vesicles

Author:

Juno Yoo, Srinivasan Shanmugam, Chung-Kil Song, Dae-Duk Kim, Han-Gon Choi, Chul-Soon Yong, Jong-Soo Woo, and Bong Kyu Yoo

Year:

2008

Bibliographic source:

Arch Pharm Res Vol 31, No 12, 1652-1658, 2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Animals care and procedures were conducted according to the guidelines for animal use in toxicology (Society of Toxicology USP 1989) and the study protocol was approved by the Animal Care and Use Committee, College of Pharmacy, Yeungnam University.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

113170-55-1

EC Number:

601-239-9

Cas Number:

113170-55-1

IUPAC Name:

113170-55-1

Constituent 2

Reference substance name:

Magnesium Ascorbyl Phosphate

IUPAC Name:

Magnesium Ascorbyl Phosphate

Details on test material:

chemical were of analytical grade and used without further purification.

Radiolabelling:

no

Test animals

Species:

other: mouse: skin penetration in vitro

Strain:

other: hairless

Sex:

male

Details on test animals or test system and environmental conditions:

Seven-week-old male hairless mice (weighing 35 ± 3.0 g) were obtained from Orient Bio (Seoul, Korea) and housed in groups not exceeding six per cage and maintained under standard conditions. The acclimation period was two weeks before the experimental procedure with a dark/light cycle of 12
h/12 h at the temperature of 23±2oC. Food and tap water were available ad libitum during the acclimation period.

Administration / exposure

Type of coverage:

open

Vehicle:

water

Duration of exposure:

6h

Doses:

0.4 mL was applied on the skin and allowed to spread over the skin.

No. of animals per group:

not indicated

Control animals:

no

Details on study design:

See details on in vitro system.

Details on in vitro test system (if applicable):

Seven-week-old male hairless mice (weighing 35 ± 3.0 g) were obtained from Orient Bio (Seoul, Korea) and housed in groups not exceeding six per cage and maintained under standard conditions. The acclimation period was two weeks before the experimental procedure with a dark/light cycle of 12 h/12 h at the temperature of 23±2°C. Food and tap water were available ad libitum during the acclimation period. Mice were sacrificed and full-thickness skin was taken, followed by removal of subcutaneous fat. Approximately 2 cm2 of the trimmed skin was placed into Keshary-Chien diffusion cell with 1.77 cm2 diffusion area.
0.4 mL were applied on the skin and allowed to spread over the skin. Donor compartment (epidermal layer of the skin) remained unoccluded, but covered with a lid, and the receptor compartment (dermal layer of the skin) was perfused with distilled water maintaining temperature at 37°C. Serial sampling of the receptor compartment was performed at predetermined intervals (0.25, 0.5, 1, 2, 3, 4, 5, and 6 h). Because L-ascorbic acid-2-phosphate magnesium salt (A2P) becomes metabolized to L-ascorbic acid (AA) by alkaline phosphatases in the skin, we measured both A2P and AA simultaneously. Penetrated amounts of A2P and AA were plotted as a function of time. The flux for both A2P and AA were calculated by the slope of the linear portion of the curve, which was achieved in about 1 h. We also performed the skin penetration study with A2P dissolved into distilled water as a control.
The skin was homogenized in 2 mL of distilled water under ice bath for 10 min followed by centrifugation at 12000 g for 5 min. Clear supernatant (500 μL) was taken and mixed with 500 μL of extraction solvent (10% metaphosphoric acid : 20% trichloroacetic acid : distilled water = 1:2:7) for protein precipitation, and was subjected to vortex for 30 sec and centrifugation at 16000 g for 3 min. The clear supernatant thus obtained was used for skin retention study by using HPLC assay.
For skin penetration study, the concentrations of A2P and AA were assayed simultaneously by HPLC system (Shimadzu, Japan) equipped with Class VP computer software, LC 10 AD VP pump, and SPD 10A UV-VIS detector at 240 nm. Column used was Inertsil ODS-3 (4.6 × 150 mm, GL Science Inc, Japan) and mobile phase consisted of a mixture of 0.02 M phosphate buffer and methanol (85:15, v/v) adjusted to pH 2.3 with phosphoric acid. Flow rate was 0.8 ml/min and the injection volume of the sample was 20 mL. The validation of the HPLC assay conditions was performed by repeating five times a day for five consecutive days.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

The dermal penetration of L-ascorbic acid-2-phosphate magnesium salt (A2P) was determined to be 0.6926 nmole x cm2/h. With a molecular weight of 278.39 g/mole a dermal penetration of 192.89 ng x cm2/h is calculated. For the dermal penetration assay 0.4 ml were applied on a surface area of 2 cm2. As a worst case assumption 1 mg/cm2 was used for calculation. In conclusion 0.019%/h of L-ascorbic acid-2-phosphate magnesium salt (A2P) is resorbed via the skin. For a 8 h working day a dermal penetration of 0.16 % is calculated.

Total recovery:

not examined

Percutaneous absorption

Dose:

0.4 ml

Parameter:

percentage

Absorption:

0.2 %

Remarks on result:

other: 8 h

Remarks:

calculated for a 8 h working day (see above)

Conversion factor human vs. animal skin:

not applicable