Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline study evaluating the methodology and practical aspects of how solubility parameters correlate with permeability perameters for predictive capabilities in an in-vitro skin permeability study.

Data source

Reference
Reference Type:
publication
Title:
Permeability of Commercial Solvents Through Lining Human Skin
Author:
Ursin C, Hansen CM, Van Dyk JW, et al
Year:
1995
Bibliographic source:
Am. Ind. Hyg. Assoc. J. 56:651-660 (1995)

Materials and methods

Principles of method if other than guideline:
The study evaluates the correlation between various solubility parameters and an in-vitro skin permeability assay.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Radiolabelling:
yes

Test animals

Species:
other: Human skin
Strain:
other: N/A
Sex:
female

Administration / exposure

Type of coverage:
other: In vitro study using human skin
Details on study design:
PERMEATION MEASUREMENTS: Permeation measurements were made using a Franz diffusion Cell. The area of exposed skin in the cell was 0.64 cm2. The skin was stretched onto the cell giving a final thickness of about 300-600 um. (Water temp: 37°C The receptor fluid was nourished isotonic salinesolution. A specail cap prevented build up in the challenge half-cell (containing the test substance). Because the solubility in water of the challenge solvent was >100 mg/L it was not necesssary to add solubilizing agent.
3H was run for two hour prior to the test solvent in order to calibrate the relative permeability of the skin samples and detect defective(lleaky specimens).
The test substance was measured in the receptor fluid by GC


VIABILITY EVALUATIONS: The skin specimens were incubated with BrdUrd (10 uM) in Bulbecco's Medium for 17 hours at 37C. The skin was then washed in isotonic phospate-buffered saline (PBS) at 37C for 1 minute and then slowly cooled to 4C. This was followed by incubation in 1% acetic acid at 4C for 48 C. After the treatment the epidermis was separated from the dermis with tweezers. The epidermis was then incubated in 4C ethanol for at least 1 hour. Specimens then were exposed to 20C 4M HCl for 10 minutes, followed by washing twice for 4 minutes in cold PBS. This was then followed by incubation for 30 minutes with (monoclonal mouse) anti-BrdUrd antibody (Dako M744) diluted 1:10 in PBS at 20C, Then the samples were incubated for 30 minutes with the secondary antibody (polyclonal rabbit antimouse antibody [dako F313] diluted 1:10 in PBS) conjugated with fluorescent tracer FITC. Counter staining was done with the red fluorescent propidium iodide.

The viability test was run on skin that had been exposed to the test substance for 2 hours as a mechanism to evaluate the affects the substance has on the viability of the skin.

ANALYSIS
- Method type(s) for identification: GC
- Limits of detection and quantification: 0.1 ppm


Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Human skin obtained from healthy females during plastic surgery of the breast.
- Type of skin: skin from breasts
- Preparative technique: Skin samples were stored in Dulbecco's medium (with gentamicin) at 4C until thinned (<= 48 hr). During thinning, most of the dermal tissue was removed from the epidermis using a Downs-Watson dermatome.
- Thickness of skin (in mm): The thickness was stretched "split-skin" was about 1 to 2 mm. The area of exposed skin in the cells was 0.64 cm2. The skin was stretched (area increase of about 3 fold) onto a cell giving a final thickness of about 300 to 600 micrometers (1 to 2 mm divided by 3).
- Storage conditions: Split skin samples were store in Delbecco's medium until used for permeation measurements (<= 10 days).


PRINCIPLES OF ASSAY
- Diffusion cell: Franz Diffusion Cell, two-chamber with water jacket and magnetic stirrer in a collection cell.
- Receptor fluid: Isotonic, nourished, saline solution (Delbecco's medium with gentamycin (ug/ml).
- Solubility of test substance in receptor fluid: The solubility in water of the test substance was >100 mg/l, so it was not necessary to add solubilizing agent as bovine serum albumin to the receptor fluid to enhance partition between the skin and the fluid.
- Flow-through system: 3H-water was run for 2 hours before the test substance in order to calibrate the relative permeability of the skin samples and to detect defective (leaky) specimens. 3H-water permeation was measured by removing 0.5 ml samples from the collection chamber at times 0.16, 0.33, 0.5, 1.0, 1.33, and 2 hours. The 3H-water was then removed and replaced with the challenge test substance.
- Test temperature: water temperature was 37C (giving the skin a temperature of about 34C).

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Percutaneous absorption
Remarks on result:
other:

Any other information on results incl. tables

    Permeability Constant   Permeability Constant  
Test Substance   Skin Donor #  Un-Normalized Normalized   % Error in Slope**
Propylene carbonate   12*  2.6  0.7  29.9
Propylene carbonate  12 0.3  0.2  10.4
Propylene carbonate 12*   3.5  1.0  9.7
 Mean +/- Std. Dev.  N/A  2.11 +/- 1.66  0.66 +/- 0.44

 N/A

* Defective specimens - 3H-water permeability constant > 25 g/m2h.

**This is a mesurement of the linearity of the steady state region. It is the sandard devaiation of the slope espressed as a percent. A value of "0" indicate perfect linearity.

The toxicity of propylene carbonate appeared to play some role in the permeability effects identified in this in-vitro human skin absorption study.

Applicant's summary and conclusion

Conclusions:
Propylene carbonate showed a small effect of the presence of 3H-water. An attempt to use Hanson solubility parameters to correlate skin permeabilities did not result in much predictive capability. Inclusion on the resulting factors related to the kinetic aspects of permeability may result in improved correlatability.