Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform
Details on test material:
14C-D & C Red No. 9 having a specific acti.vity of 10.6 mCi/mmol. was supplied by Amersham Corporation. This material was incorporated into the
vehicles. All radioactive formulations were prepared by Dr. John Slattery, School of Pharmacy, University of Washington.

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.
Radiolabelling:
yes

Administration / exposure

Vehicle:
castor oil
Remarks:
and mineral oil, cream blusher and Talc
Duration of exposure:
48h
Doses:
VEHICLE CONCENTRATION DOSE
D&C Red No. 9 (14C/volume/skin area)

1. mineral oil 5 % 3 muCi/5 mul/cm2
2. castor oil 5 % 3 muCi/5 mul/cm2
3. cream blusher 0.5 % 1.5 muCi/25 mul/cm2
4. Talc 5 % 3 muCi/5 mul/cm2
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Human abdominal skin was obtained at autopsy from various local hospitals. The skin specimens were obtained within 24 hours of death and either used immediately or refrigerated for no more than 20 hours.
- Preparative technique: Prior to use, each specimen was gently washed under tap water to remove dried blood or other adherent material, then blotted dry and placed epidermal side down an absorbent paper towel. The subcutaneaus tissue and approximately 50% of the dermis was removed by shaving with a #ll scalpel blade. The specimen was then cut into multiple pieces of sufficient size to fit the 1.0 cm2 Franz diffusion cells. In a number of experiments the skin was split into epidermis and dermis so that the permeability of the epidermal layer anly to D & C Red Na. 9 could be assessed separately. This was done by placing full thickness skin between two aluminum blocks, preheated to 600C°, and leaving it for 2-3 minutes.
- preparations: epidermis only and full thickness skin
- Membrane integrity check: As a check on the integrity of the skin used in these experiments, the permeability of each specimen to tritiated water (3H20) was determined prior to the application of any of the test formulations.

PRINCIPLES OF ASSAY
The absorption of D & C Red No. 9 from the four test vehicles has been determined using the doses listed above. All vehicles were run on duplicate samples from each donor skin Following application of the test vehicles to the skin, the dermal bathing solution was removed at regular intervals and an aliquot analyzed for radioactive content. Sampling was done at 4, 8, and 12 hours after application and at 12 hour intervals thereafter until a steady-state was reached oruntil 48 hours post application.
Due to the low water solubility of D & C Red No. 9, the receptor solution in almost all cases was 6% Volpo 20 in distilled water rather than isotonic saline as is normally used.
A 5 ml aliquot of each sample was gelled by the addition of 10 ml Aquasol-2 (New England Nuclear Corp.) and counted in a Beckman Liquid Scintillation Spectrometer.
Immediately following the last sample the test vehicles were removed from the skin surface by serially washing with 50/50 v:v dimethylformamide/toluene or 50/50 DMF/6% Volpo (cream blush only). Each wash consisted of a one minute application of 0.5 ml of solvent, and the procedure was repeated four times. The first two washes and the last two washes were combined and an aliquot of each analyzed for radioactive content. Following washing the skin was removed from the cell and separated into epidermis and dermis by manually stripping the epidermis with forceps. Both the epidermis and dermis were then dissolvoed in Soluene (Packard) and analyzed for radioactive content.

Results and discussion

Absorption in different matrices:
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): receptor solution in almost all cases was 6% Volpo 20 in distilled water
- Skin preparation (in vitro test system): see above "details on in vitro test system"
Percutaneous absorptionopen allclose all
Time point:
48 h
Dose:
5 % (epidermis only, mineral oil, in triplicate)
Parameter:
percentage
Absorption:
0.024 %
Time point:
48 h
Dose:
5% (full skin, mineral oil, 4 experiments)
Parameter:
percentage
Absorption:
0.01 %
Time point:
48 h
Dose:
5% (castor oil, epidermis only, 4 experiments)
Parameter:
percentage
Absorption:
0.041 %
Time point:
48 h
Dose:
5% (castor oil, full skin, in triplicate)
Parameter:
percentage
Absorption:
0.027 %
Time point:
48 h
Dose:
0.5% (cream blush, epidermis only, in tripilicate)
Parameter:
percentage
Absorption:
0.088 %
Time point:
48 h
Dose:
0.5% (cream blush, full skin, in triplicate)
Parameter:
percentage
Absorption:
0.073 %
Time point:
48 h
Dose:
5% (Talc, epidermis only, twice)
Parameter:
percentage
Absorption:
0.024 %
Time point:
48 h
Dose:
5% (Talc, full skin, twice)
Parameter:
percentage
Absorption:
0.022 %

Applicant's summary and conclusion

Executive summary:

As can be seen above, the percutaneous absorption of D & C Red No. 9 through human skin is very low. With all four vehicles, total absorption was less than 0.1% of the applied dose and the maximum flux rate achieved was less than 0.1 mug/cm2/24 hours. Due to the small number of experiments run, it was impossible to determine whether the apparent differences between vehicles were significant.

The vast majority of the unabsorbed material remained on the surface of the skin and was found in the skin wash. This amount varied from 85-103 %. Very little colorant was found in the epidermis or dermis, less than 0.3% in all cases. Total drug recovery averaged 83-103% of the applied dose.