Registration Dossier

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
Study period:
2008-07-21 to 2008-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Read-across from a study comparable to guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
other: European Commission, SCCPs (Scientific Committee on Cosmetic Products) guideline 2006
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
no

Test animals

Species:
other: in vitro test: Skin membranes from female human abdominal origin from cosmetic surgery were used
Details on test animals and environmental conditions:
IN VITRO TEST
Human female skin samples. The skin samples were excised during surgical operations. The skin was not removed to provide samples for these in vitro investigations. The hospital had the prior consent of the patients that the tissue could be used for scientific research.
Donors were 4 females, age 32 to 45 years old, body mass index (BMI) 21.5 to 29.1.

Administration / exposure

Type of coverage:
other: covered with Parafilm® over the period of 24 hours
Duration of exposure:
24 h
Doses:
- Doses: 30 μL of the test solution 10 % CAPB were applied at the diffusion skin area and homogenously spread over the skin surface.
Details on study design:
ANALYSIS
- The first step of the study was to establish an analytical HPLC method for quantification of Coco AAPB in the used acceptor media (KRB buffer). The HPLC method establishment was not carried out under GLP conditions. The validation of the method was investigated in accordance with the FDA Guideline „Bioanalytical Method Validation“ (2001) and it was performed under GLP conditions. System suitability, selectivity, linearity in KRB pH 7.4, accuracy and precision, quantification limit and stability of the test product in extraction and acceptor media were verified.

- Further on, an extraction method was established: Before starting the penetration studies an appropriate extraction method was established under non-GLP conditions. In order to detect a possible adsorption of the test compound to the Tape film or to the skin in the extraction matrix, test solution samples were incubated with Tape strips, Tape strips + Stratum corneum and deeper skin layers, respectively. For penetration experiments a scotch tape purchased from Beiersdorf (Germany, 19 mm wide, product number 57330) was used. The skin of one donor was used for the establishment of the extraction method. For this purpose a skin sample was stripped by means of the Saarbruecken model. Moreover, Tape strips without skin contact and cryosections of deeper skin layers (epidermis and dermis) were prepared. Two different concentrations of Coco AAPB (50 μg/mL and 5 μg/mL) were added to the samples, which subsequently were agitated for 1 hour with extraction matrix. Two solutions were tested as extraction media: water and Methanol/water 50:50 (v/v, %). Finally, the recovery of the test compound was determined by comparing the extracted and the initially applied amounts. An appropriate extraction medium for the scheduled in vitro penetration studies was defined in this experiment.

Details on in vitro test system (if applicable):
- Source of skin: human female, abdominal origin from cosmetic surgery
- Preparative technique: dermatome
- Thickness of skin (in µm): 500
- Membrane integrity check: yes
- Storage conditions: - 20 °C

PRINCIPLES OF ASSAY
- Diffusion cell: Franz diffusion cell, flow-through system, test temperature 32°C

Results and discussion

Total recovery:
Determination of Coco AAPB content in the test solution:
- The content of Coco AAPB was determined in triplicate in the test solution employed in the in vitro experiments. The mean concentration value was 106.73 μg/mL which corresponds to the recovery mean value of 106.39 ± 0.67 %. This complies with the acceptance value 100 ± 10 %.

Skin permeation and penetration
The calculated total recovery rate of Coco AAPB for the three different skin donors used was 98.55 %. The mean recovery values have varied from 95.00 % until 100.39 %, which complies with the acceptance criteria of 100 ± 15 %.
Percutaneous absorption
Dose:
1 mg/cm²
Parameter:
percentage
Absorption:
0 %
Remarks on result:
other: 48 h

Any other information on results incl. tables

HPLC method development and validation

The HPLC method was successfully developed and validated with regard to method selectivity, linearity, accuracy and precision in biological acceptor medium (KRB buffer). Furthermore, the test compound has demonstrated good stability over the period of 48 hours at RT and 4 ºC in the extraction medium employed for the penetration experiments (water). In addition, Coco AAPB has presented good stability in the acceptor medium in 32 ºC over the period of 48 hours, which covers the experiment duration.

Establishment of the extraction method for penetration study

Two extraction media were investigated for the penetration studies: MeOH/water (50:50 v/v, %) and water. With water as extraction medium the mean recovery values of Coco AAPB have shown a variation from 84.08. % to 116.55 % whereas with MeOH/water (50:50 v/v, %) the mean recovery values of Coco AAPB have ranged from 93.35 % to 118.66 %. The results from both extraction media have demonstrated similar behaviour: the recovery values were relatively higher for the samples lower concentrated. The results from both media employed has complied to the limit of 100 ±20 % specified in the SCCP Guideline, however we have decided to perform the penetration experiments with water as a extraction medium, due to the fact that its seems to be more reproducible. The standard deviations (n=2) for the samples were generally lower for water in comparison to MeOH/water as extraction medium.

Permeation study

The results from the in vitro permeation of Coco AAPB from the test product through the three skin samples have demonstrated that the test compound amount in all receptor samples was below the LLOQ of the analytical method (0.987 μg/mL). Since no permeation of Coco AAPB into the receptor medium was detected, the Papp value is considered to be 0. Another fact which support this low permeation are the recovery values found for the test product, which remained on the skin surface. These values have varied in the 6 Franz-cells from92.93 % to 103.43 %, which demonstrated that the Coco AAPB has mostly remained on the skin surface for all the samples.

Penetration study

The mean amount of Coco AAPB removed from the skin surface (skin wash) ranged from 94.92 % to 100.15 % of the dose applied in each single Franz cell and from 95.00 % to 100.39 % for the mean value from each skin donor. This demonstrates that the Coco AAPB has mostly remained on the skin surface. The amounts in the receptor could not be quantified since it was below the analytical LLOQ.

The mean recovery in the two first tape strips was 0.17 % during all performed experiments. In the further 18 tape strips a mean recovery of 0.07 % was documented. The recovery values for the cryocuts have accounted 0.01 % only for one skin sample. For the other two skin donors Coco AAPB was not detectable in the samples with cryocuts (below the analytical LLOQ).

The mean absorbed dose of Coco AAPB, sum of the amounts found in the viable epidermis, dermis and receptor medium was 0.1 %, which has accounted only the dose absorbed in one skin sample.

The calculated total recovery rate of Coco AAPB for the three different skin donors used was 98.55 %. The mean recovery values have varied from 95.00 % until 100.39 %, which complies with the acceptance criteria of 100 ± 15 %.

Quality control of the utilized skin (MEA)

The transport rates of caffeine demonstrate that the utilized three human skin samples represent intact tight barrier properties, which are congruent to data obtained on other human skin biopsies under comparable study design.

Mean amount [μg/cm²] of Coco AAPB in the samples from the three in vitro experiments.

Cumulative amount [μg/cm2] of Coco AAPB in the samples

Sample

Skin sample 1

Skin sample 2

Skin sample 3

All skins used

Mean

SD

Mean

SD

Mean

SD

Mean

SD

dose applied

973.27

0.00

1042.10

10.42

1012.26

68.64

1009.21

43.78

receptor

0.00*

0.00*

0.00*

0.00*

0.00*

0.00*

0.00*

0.00*

skin wash

974.73

42.77

1040.59

30.24

961.78

94.27

992.37

61.28

2 Tape strips

0.72

0.35

1.76

2.30

0.76

0.43

1.08

1.18

18 Tape strips

0.37

0.53

1.67

2.36

0.00*

0.00*

0.68

1.33

cryocuts

0.00*

0.00*

0.00*

0.00*

0.13

0.18

0.043

0.075

*Values below the LLOQ of the analytical method of 0.987μg/mL.

 

Mean recovery rate and dose absorbed [%] of Coco AAPB in the samples from the three in vitro experiments.

Recovery and dose absorbed [%] of Coco AAPB in the samples

Sample

Skin sample 1

Skin sample 2

Skin sample 3

All skins used

Mean

SD

Mean

SD

Mean

SD

Mean

SD

receptor

0.00*

0.00*

0.00*

0.00*

0.00*

0.00*

0.00*

0.00*

skin surface

100.15

4.39

99.87

3.90

94.92

2.88

98.31

3.94

2 Tape strips

0.07

0.04

0.35

0.04

0.07

0.04

0.17

0.15

18 Tape strips

0.04

0.05

0.16

0.22

0.00

0.00

0.07

0.13

cryocuts

0.00*

0.00*

0.00*

0.00*

0.01

0.02

0.00

0.00

Dose absorbed

0.00*

0.00*

0.00*

0.00*

0.01

0.02

0.00

0.00

Total recovery

100.26

4.48

100.39

3.72

95.00

2.93

98.55

4.01

 *Values below the LLOQ of the analytical method of 0.987μg/mL.

Applicant's summary and conclusion

Conclusions:
The mean absorbed dose of Coco AAPB, sum of the amounts found in the viable epidermis, dermis and receptor medium was 0 %.
Executive summary:

The aim of the present study was to investigate the in vitro permeation and penetration of Coco AAPB at human skin of 3 different donors in vitro according to SCCP requirements and to OECD Guideline 428. The product provided by the customer was diluted with HBSS buffer to the concentration of 10 % Coco AAPB and the pH was adjusted to 6.5. This formulation was used as the test solution in all experiments. The first step of the study was to establish an analytical method for quantification of Coco AAPB. The described analytical method was developed to quantify the compound in the used biological acceptor and extraction media. The method was validated under GLP conditions for this purpose. At the same time the stability of Coco AAPB from the test solution was tested over 48 hours at 32 ± 2 °C, 25 ± 5 °C and 4 ± 2 °C in biological acceptor medium (KRB buffer) used for the permeation study. Furthermore the stability of the test compound was also investigated in the extraction medium employed in the penetration studies (water). At the beginning of the study, the Coco AAPB content in the test solution was determined by HPLC in triplicate. The permeability of Coco AAPB from the test solution on human skin was investigated on fresh human skin from 3 donors in two fold (n=2) for each donor (totalizing n=6). Dermatomized (to approximately 500 μm) skin was used. The skin thickness was measured immediately before performing the studies. 8 samples were taken from the acceptor medium during a period of 24 hours to obtain information about permeability of Coco AAPB. At the end of the permeation experiment the remaining Coco AAPB content in the test solution was determined. For that goal the test formulation left on the skin surface was collected with cotton swabs and transferred to the falcon tube with the extraction medium, this is the so-called wash procedure. After removing residual formulation, the concentration of Coco AAPB in the skin, in the Stratum corneum and deeper skin layers, was quantified. The upper corneous layer of the skin was stripped off and the residual skin was cryo-sectioned. After the in vitro study a mass recovery was carried out to determine the mass balance and local distribution of Coco AAPB in the different skin compartments. For that goal a quotient of total mass of Coco AAPB at the end of the study on the skin surface in test solution, in Stratum corneum, Epidermis/Dermis and acceptor compartment versus the applied amount of Coco AAPB in the formulation at the start of the study was calculated. Parallel to the in vitro studies with Coco AAPB on human skin, the permeability of Caffeine was carried out (MEA: multiple endpoint analysis) at a concentration of 10 mg·L-1 in Krebs-Ringer-buffer (KRB) at pH 7.4 (n=2 for 3 skin donor). Caffeine is a recommended marker molecule of the OECD Guideline for the quality control of human skin. The results of the Caffeine permeation were compared with the permeability coefficients of previous studies on historical human skin membranes of different origin at Across Barriers.

The HPLC method was successfully developed and validated with regard to method selectivity, linearity, accuracy and precision in biological acceptor medium (KRB buffer). Furthermore, the test compound has demonstrated good stability over the period of 48 hours at RT and 4 ºC in the extraction medium employed for the penetration experiments (water). In addition, Coco AAPB has presented good stability in the acceptor medium in 32 ºC over the period of 48 hours, which covers the experiment duration. The content of Coco AAPB was determined in triplicate in the test solution and mean value was 106.39 ± 0.07 %, which complies to the acceptance criteria of 100 ± 10 %. The results from the extraction method has shown that water was most suitable medium for performing the penetration experiments presenting recovery values which has ranged between 84.74 % and 116.55 %, which complies to the acceptance limit 100 ± 20 %. The permeated amounts of Coco AAPB through three different human skins have presented values in the receptor below the LLOQ of the validated analytical method of 0.987 µg/mL.

This low absorption was confirmed through the mass recovery calculation, where 98.55 % (mean value for 6 Franz cells) of surfactant were determined in the test solution which remained at the skin surface after 24 hours. The mass recovery calculations present amounts and percentages of compound, which permeated, penetrated and remained in the donor compartment. The mean amount of Coco AAPB removed from the skin surface (skin wash) ranged from 95.00 % to 100.39 % of the dose applied. The mean recovery in the two first tape strips was 0.17 % during all performed experiments. In the further 18 tape strips a mean recovery of 0.07 % was documented. The mean absorbed dose of Coco AAPB, sum of the amounts found in the viable epidermis, dermis and receptor medium was 0 %. The mean Apparent Permeability Coefficient (Papp) values measured for Caffeine in the present study for the three human skin samples are in good agreement with Papp values determined for a variety of skin specimens from different donors under comparable conditions.