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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a guideline study

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
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

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):
SKIN PREPARATION
- Source of skin: human female, abdominal origin from cosmetic surgery
- Ethical approval if human skin: no data
- Type of skin: abdominal
- Preparative technique: dermatome
- Thickness of skin (in µm): 500
- Membrane integrity check: yes
- Storage conditions: - 20 °C
- Justification of species, anatomical site and preparative technique: The main gender-specific difference in abdominal skin is the higher number of hair follicles in male samples. Because it is important not to cut the hair follicles when preparing the subcutis, female hairless abdominal skin is preferable for in vitro studies.

Preparation of the skin
Transport and preparation of full-thickness skin
The skin was made available in accordance with the relevant statutory and contractual provisions. The excised skin from the surgery was cooled to 4 °C and its surface was dried before being transported. Care was taken to ensure that the subcutaneous fatty tissue does not get into contact with the surface of the skin. Once in the laboratory, the skin was separated from the subcutaneous fatty layer and then issued by Across Barriers with an encrypted identification number.
Encryption was performed in accordance with the Across Barriers SOP A 005. The encrypted identification numbers are always used to refer to the skin specimens in laboratory records and reports. Across Barriers assured to the hospital that information on the patients and the surgery can only be accessed via a file held by Across Barriers. The suitability of the skin biopsies was assessed according to the following criteria:
Inclusion criteria:
• No pathological findings
• Hospital must have patient’s consent that tissue may be used for scientific purposes
Exclusion criteria:
• Skin damage, strongly marked scarring or stretch marks
Working under controlled storage conditions (4 °C), the subcutaneous fatty layer was removed within two hours after reception of the abdominal skin

Dermatomization of the skin
The human skin was cut into 4 cm broad stripes. Subsequently skin sections with a thickness of approximately 500 μm were prepared from the full-thickness skin samples using an Aesculap GA 630 dermatome. The skin surface with intact Stratum corneum is directed towards the dermatome. The use of the dermatome is described in SOP G 004. Prior to use, the thickness of the dermatomized skin was checked with a Heidenhain thickness gage (SOP G 026) at five different places of each skin sample. The measured thickness was not exactly the 500 μm scheduled in the study plan, as it strongly depends on the skin’s elasticity and cannot be estimated prior to dermatomization. The slightly varying thickness, however, has no influence on the permeability of the marker compound Caffeine [Bock et al. 2002]. Because the stratum corneum represents the principle absorption barrier a significant alteration of the test compounds’ transport kinetics by remaining deeper skin layers is not expectable.

PRINCIPLES OF ASSAY
- Diffusion cell: Franz diffusion cell - The cylindrical glass Franz cell is a diffusion chamber comprising an upper and a lower part between which the skin specimen will be clamped. The two halves of the cell are held together by means of a ball-and-socket clamp. The lower (receptor) chamber has a volume of approximately 20 mL, while the volume of the upper (donor) chamber is variable. The skin specimens was punched out (area of approximately 4 cm²) immediately prior to insertion in the Franz cells. The skin is always inserted with the dermal layer oriented downwards so that the skin’s horny layer is uppermost. Franz diffusion cells with a diffusion area of approximately 3 cm² and an acceptor volume of approximately 20 mL were used. The stirring speed and the temperature was set at 400 rotation per minute and 32 °C ± 2 °C, respectively. At each sampling point the stirring speed and temperature were documented.
- Solubility od test substance in receptor fluid: checked
- Static system: no
- Flow-through system: yes
- Test temperature: 32°C
- Doses: 30 μL of the test solution 10 % CAPB were applied at the diffusion skin area and homogenously spread over the skin surface. In order to avoid any changes in the formulation, Franz diffusion cells were kept covered with Parafilm® over the period of 24 hours.
- Receptor fluid: Krebs Ringer bicarbonate buffer (KRB buffer) at pH 7.4
- Sampling times: The permeation study was performed over a period of 24 hours, after 0.5, 2, 4, 6, 20, 22, 23 and 24 hours. The samples with a volume of 200 μL were taken from the acceptor compartments and analyzed for the drug content by HPLC. The taken amount was replaced with a fresh medium preheated to the temperature of 32 °C. In total 6 samples were analyzed for each time point (3 donors, n=2).
- Penetration experiments with the test product:
-- After the incubation time of 24 hours the residual formulation from the skin surface was removed using small cotton pieces. The skin surface was washed twice with 500 μl of the extraction medium and dried with a cotton piece. The skin wash solution and cotton pieces were collected into one beaker and extracted using 2 mL of the extraction medium. After removing the formulation from the skin surface, the skin was partitioned into horizontal segments to determine the distribution profile of CAPB in the skin.
-- After the residual formulation on the skin surface (skin wash sample) was removed the concentration of the test substance in the skin layers was quantified. Two segmenting techniques, Tape stripping and Cryosectioning, were applied to separate different skin layers parallel to the upper surface of the sample and thus provide information on the extent to which the test compound has penetrated into the different skin areas. After removing the residual formulation, the skin biopsies were transferred into a stripping apparatus. Additionally to the laborarory SOP M 019 the skin sample may be placed onto the Styrofoam for the stripping procedure. The upper corneous layers (Stratum corneum) of the skin were stripped off using Tape film. The stripped skin area was 1.767 cm² ± 5 %. In total 20 tape strips were performed per each skin biopsy. The first two strips were separately analyzed due to potential contaminations by residual drug on the surface of the skin. The samples for 2 tapestrips and 18 tape-strips were extracted in 3 mL and 5 ml of the extraction medium respectively and quantified by HPLC. Water was used as an extraction medium. After stripping, the skin biopsies were punched out and frozen at –80 °C. The residual skin was cryo-sectioned to determine the amount of test compound, which had penetrated into the deeper skin layers. The skin biopsy was cut into surface parallel sections with a thickness of 25 μm. The surface area of a single cut was approximately 1.327 cm². All cuts were collected into one beaker. All samples were extracted in 3 mL of the extraction medium and quantified by HPLC. Water was used as extraction medium.

CALCULATIONS
- Apparent permeability coefficient (Papp): The apparent permeability coefficient (Papp) was calculated from the linear part of the slope of the cumulative transport of Caffeine. The lag phase was estimated out of the transport curves. The Papp was calculated according to Eq.
Papp = ( ΔQ/Δt) x (1/mo)x(1/A)X Vd [cmXSexp-1]
ΔQ/Δt = permeability rate (steady state transport rate) obtained from the profile of the transported amount of substrate versus time [s]. Calculated by the linear regression of time and concentration
A = area of the exposed skin [cm²]
mo = initial mass of test compound in the donor compartment [μg]
VD = donor volume [cm³]

- Mass recovery: Results of experiments with test item are presented as a mass balance including the amount and percentage of the active compound permeated, penetrated and remaining in the donor compartment after the experiments. Based on the results of the three compartments a mass
balance was determined according to Eq.
mRC = mA + mOSS + mOSC + mPSC + mPDS [μg]
mRC = mass of test compound in the three compartments after the experiment [μg]
mA = mean value of the amount of test compound permeated through the tissue into the acceptor medium [μg]
mOSS = mean value of the amount of remaining test compound on the skin in the test formulation after the expermient [μg]
mOSC = mean value of the amount of test compound content in two first tape strips [μg]
mPSC = mean value of the amount of test compound penetrated into the Stratum corneum (18 tape strips) [μg]
mPDS = mean value of the amount of test compound penetrated into the deeper skin layers [μg]

and Eq RC= (mRC/MDO) X 100 [%]
RC = total recovery of the active compound after the experiment [%]
mRC = mass of test compound in the three compartments after the experiment [μg]
mD0 = initial mass of test compound in the donor compartment [μg]
The overall mass recovery in the range of 100 ± 15 % should be reached according to SCCP.

- Dose absorbed: Dose absorbed was calculated according to the Eq.
DA = rA + rPDS [%]
DA = dose absorbed of test compound after the experiment [%]
rA = recovery of test compound permeated through the tissue into the acceptor medium in relation to the apllied formulation amount [%]
rPDS = recovery of test compound penetrated into the deeper skin layers (cryosections) in relation to the apllied formulation amount [%]

- Reference substance(s): caffeine
- Other:

Results and discussion

Absorption in different matrices:
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.
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 AAPBfor 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

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.