Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD guideline 428 (draft) and in compliance to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
(Draft)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Non-radiolabelled test substance
Test substance : HC Blue No 2
EC number : 251-410-3
Batch number : 114B5
Purity : 98.7%

Radiolabelled substance
Test substance : [ring-U-14C]-HC Blue No 2
Batch number : CFQ13908 batch 1
Radiochemical purity : 98.1% (HPLC), 98.3% (TLC)
Radiolabelling:
yes

Administration / exposure

Duration of exposure:
30 minutes
Details on in vitro test system (if applicable):
The study was designed to examine the in vitro percutaneous absorption of the hair dye HC Blue No 2 through human dermatomed skin membranes using [14C]-HC Blue No 2. The compound was tested as a semi-permanent hair dye under 'in-use' conditions in one formulation with a target concentration of 2.8w/w% (actual concentration 2.7w/w%). An amount of 20 mg/cm2 of formulation was applied on the skin surface. The contact time was 30 minutes after which the skin was rinsed following a well-standardised washing procedure. The post exposure time was 23.5 hours. In addition to the radioactivity in the receptor fluid, the residues remaining in/on the skin membranes and in the stratum corneum (after 24 hours of application) was determined.

Human skin membranes were prepared from frozen abdominal skin samples obtained from four female donors. The tissue was obtained directly after surgery. After thawing of the skin, this was dermatomed to a recorded thickness of approximately 400um. The integrity of the skin membranes was established by determination of the permeation coefficient for tritiated water, which was <2.5x10-3 cm/hour for all selected skin membranes.

The study was performed in flow-through diffusion cells. The temperature of the cells was approximately 32 deg C at ambient humidity. The receptor fluid was pumped at a rate of ca. 1.6 mL/hour and consisted of phosphate buffered saline containing 0.01% sodium azide (w/v). The solubility of the test substance, HC Blue No 2, was 425 ug/mL, which is considered sufficient.

Results and discussion

Percutaneous absorption
Dose:
20 mg/cm2 of formulation containing 2.7w/w% test item
Parameter:
percentage
Absorption:
0.004 %
Remarks on result:
other: 24 hours
Remarks:
mean total absorption (dermal delivery)

Any other information on results incl. tables

The percutaneous absorption of HC Blue No 2 was evaluated on 8 skin preparations from 4 different donors. None of the cells was rejected. The results, obtained under the present experimental conditions, are summarised in the table below (mean +/- SD).

   % of dose applied  ugeq/cm2
 Skin wash  103.0 +/- 4.4  585.0 +/- 33.2
 Dislodgeable dose  103.1 +/- 4.4  585.0 +/- 33.2
 Receptor fluid  0.001 +/- 0.001  0.006 +/- 0.005
 Stratum corneum  0.007 +/- 0.005  0.039 +/- 0.027
 Skin (epidermis and dermis)  0.003 +/- 0.001  0.014 +/- 0.008
 Unabsorbed dose  103.1 +/- 4.4  585.0 +/- 33.1
 Absorbed dose  0.004 +/- 0.002  0.020 +/- 0.009
 Total recovery  103.1 +/- 4.4  585.1 +/- 33.2

Dislodgeable dose is defined as the amount of test substance removable from the application site (skin wash, cotton swabs and donor compartment wash). Unabsorbed dose is defined as the dislodgeable dose including the amount recovered in the stratum corneum. Absorbed dose (dermal delivery) is defined as the amount in the receptor fluid, the receptor compartment wash and skin membrane, excluding strips.

The mean recovery of HC Blue No 2 was 103.1%. Most of the HC Blue No 2 was recovered in the skin wash after 30 minutes of exposure. The skin wash represented 103.0% of the applied dose and the dislodgeable dose 103.1% of the dose applied. After 24 hours, 0.007% of the applied dose was retained in the stratum corneum; those amounts are not considered to percutaneously absorbed. At the end of the experiment, 0.003% of the dose applied was found in the skin (epidermis and dermis) showing the absence of skin storage.

Virtually no penetration of HC Blue No 2 into the receptor fluid after 24 hours was observed (0.006 ug/cm2, 0.001% of the dose applied). The mean total absorption (dermal delivery), defined as the compound related radioactivity present in the receptor fluid, the receptor compartment wash and the skin (excluding tape strips) was 0.004% of the applied dose.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, the mean recovery of HC Blue No2 was 103.1%. Most of the test item was recovered in the skin wash after 30 minutes of exposure. Virtually no penetration of the test item into the receptor fluid after 24 hours was observed (0.006 ug/cm2, 0.001% of the dose applied). The mean total absorption defined as the compound related radioactivity present in the receptor fluid, the receptor compartment wash and the skin (excluding tape strips) was 0.020 ug/cm2, 0.004% of the dose applied.