Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Year 2000-2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: FDA guideline study comparable to OECD Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: FDA/ American Association of Pharmaceutical Scientists (AAPS) guidelines
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Cold material: methyl dihydrojasmonate, lot HRE-07-1824, purity 97.5%, expiry 12/1/00, liquid material
Radiolabelled material: 0.2 mCi methyl dihydrojasmonate, [3-methylene-14C] in ethanol, lot 00711, specific activity 47.23 mCi/mmol, radiochemical purity 98.28%, prepared by Wizard Laboratories, Inc., Sacramento, CA 95691 USA.

Test vehicle was prepared as followed:
249.46 mg cold fragrance weight + 0.96 mg calculated radiolabelled fragrance weight (total fragrance weight = 250.42 mg) were dissolved in 25 ml total vehicle volume wihci correspond to a concentration of 1.002 % (w/v).
Radioactivity of test vehicle was determined as follwed: Five 20 µl samples were removed using a 25 µl Hamilton syringe, placed in separate scintillation tubes, and counted on a Wallac 1409 scintillation counter. Mean radiolable content: 1705.2 ± 6.6 dpm/µg
Radiolabelling:
yes

Test animals

Species:
human
Strain:
not specified
Sex:
female
Details on test animals and environmental conditions:
Full thickness human female breast and abdominal skin were obtain from cosmetic surgery.
Donors:
224 = female, aged 52, breast, surgical specimen
225 = female, aged 63, breast, surgical specimen
227 = female, aged 27, abdominal, surgical specimen
235 = female, aged 28, breast, surgical specimen.

Administration / exposure

Type of coverage:
other: non-occlusive
Vehicle:
ethanol
Duration of exposure:
48 hours
Doses:
The composition of the test vehicle was as follows:
- 249.46 mg cold fragrance weight,
- 0.96 mag calculated radiolabelled fragrance weight
- 25 ml total vehicle volume
- 1.002% fragrance in vehicle
Mean radiolable content was 1705.2 ± 6.6 dpm/µg

- Nominal doses: ~20 µl/cm2 of a 1 % solution in ethanol corrsponding to ~ 200 µg/cm2
- Actual doses: see "Remarks on results incl. tables"
- Actual doses calculated as follows: see "Remarks on results incl. tables"
- Dose volume: ~ 20 µl/cm2
- Rationale for dose selection:
No. of animals per group:
Skin from each human donor tested in each condition
Control animals:
no
Details on study design:
SKIN PREPARATION:
Full-thickness human female breast and abdominal skin, obtained from cosmetic surgery and stored at -20°C, was thawed for processing. Following
removal of the subcutaneous fat by blunt dissection, individual portions of skin were immersed in water at 60°C for 50 seconds. The skin was then pinned, dermis side down, on a cork board and the epidermis (comprising stratum corneum and viable epidermis) gently removed from the underlying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and taken up onto aluminium foil. The membranes were thoroughly dried and stored flat at -200C until used.
On the day of use, the epidermal membranes were floated onto water from the aluminium foil and taken up onto filter paper supports. The membranes were then mounted onto diffusion cells and trimmed to size. Four different individual donors were used. Details of the distribution of the donors
throughout the test groups are given in the application regimen (See "details on results incl. tables").

DIFFUSION CELLS:
The skin samples were mounted as a barrier between the halves of horizontal Franz-type diffusion cells, the stratum corneum facing the donor chamber. The cells were designed such that the area available for diffusion was about 1.0 cm2, the exact area being measured for each diffusion cell (See details on results incl. tables). The diffusion cells were immersed in a constant temperature water bath such that the receptor chambers were maintained at 37.0±0.5°C throughout the experiment. This ensured that the skin surface temperature was maintained at 32.0±1°C. The receptor chamber contents were continuously agitated by small PTFE-coated magnetic followers driven by submersible magnetic stirrers. The receptor chambers of the diffusion cells were initially filled with a known volume (See "details on results incl. tables") of water, capped, and allowed to equilibrate to the correct temperature.

MEMBRANE INTEGRITY ASSESSMENT:
The integrity of each membrane was assessed prior to dosing with fragrance material. The permeation of tritiated water was determined by applying 500 µl of 10 µCi/ml tritiated H20 to the skin surface and removing a 200 µl sample from the receptor phase one hour later. The sample was counted using liquid scintillation counting (LSC). The skin surface was subsequently washed seven times and the receptor chambers three times with water, prior to refilling with the selected receptor medium (50/50 ethanol/water).
The results of the membrane integrity assessment are given in "details on results incl. tables".
No cell exhibited a water permeability greater than 0.005 cm/h and the average for each test group was not greater than 0.002 cm/h.

APPLICATION OF DOSE:
The target dose was 20 µl/cm2 (applied using a 25 µl Hamilton syringe), see "Details on results incl. tables"

DETERMINATION OF SKIN PENETRATION:
Following application of the vehicles, 200µl samples were taken (using a digital pipette) from each receptor chamber at 2, 8, 24, 36 and 48 hours.
Each sample was placed into scintillation fluid and analysed for 14C by LSC.

DETERMINATION OF SKIN DISTRIBUTION:
Following the 48 hour receptor phase sample the diffusion cells were dismantled and the epidermal membranes were secured onto a small disc of
thin plastic using cyanoacrylate adhesive. The fragrance remaining on the skin surface was removed by gentle wiping with a cotton bud. Each cotton
bud was extracted into 3 ml of methanol and a sample analysed for radiolabel content. Each epidermal membrane was tape stripped 10 times using
adhesive tape (D-Squame). The tape strips were grouped (placed in the same vial) as follows: strip 1, strips 2-3, strips 4-6 and strips 7-10. To each
group of tape strips, 1 ml of OptiSolve® was added and the vials shaken overnight using a Gyro Rocker (Stuart Scientific). Samples were subsequently analysed. The remaining samples of skin were placed into individual glass vials with 2 ml of OptiSolve® and shaken overnight prior to sampling. The diffusion cell donor chambers were washed with 15 ml methanol and a sample analysed. The filter paper supports were retained and two per fragrance were extracted into 3 ml of methanol and analysed as a precautionary measure to demonstrate that no adsorption of permeated fragrance had occurred.

ASSESSMENT OF EVAPORATIVE LOSS:
The evaporative loss of the fragrance material (in vehicle), under the same experimental conditions as the skin studies was assessed as follows. PTFE
(polytetrafluoroethylene) sheet, mounted in diffusion cells (at 32°C surfacetemperature), was dosed with 20 µl of 1% solutions of the fragrance materials in ethanol. Six cells were prepared. Following dosing, one cell was dismantled at each of the following timepoints: 1, 2, 4, 8, 24 and 48 hr. The PTFE sheet was removed and washed twice with methanol (10 ml then 5 ml). The donor chamber was washed with 10 ml methanol. A sample of each wash solution was submitted to analysis by LSC which allowed the total remaining radiolabel at each timepoint to be determined.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: 4 female donor
- Ethical approval if human skin: not provided in the study report
- Type of skin: breast of abdominal skin, surgical specimen
- Preparative technique & Storage conditions:
Full-thickness human female breast and abdominal skin, obtained from cosmetic surgery and stored at -20°C, was thawed for processing. Following removal of the subcutaneous fat by blunt dissection, individual portions of skin were immersed in water at 60°C for 50 seconds. The skin was then pinned, dermis side down, on a cork board and the epidermis (comprising stratum corneum and viable epidermis) gently removed from the underlying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and taken up onto aluminium foil. The membranes were thoroughly dried and stored flat at -200C until used. On the day of use, the epidermal membranes were floated onto water from the aluminium foil and taken up onto filter paper supports. The membranes were then mounted onto diffusion cells and trimmed to size. Four different individual donors were used. Details of the distribution of the donors throughout the test groups are given in the application regimen (See "details on results incl. tables").
- Membrane integrity check: yes
The integrity of each membrane was assessed prior to dosing with fragrance material. The permeation of tritiated water was determined by applying 500 µl of 10 µCi/ml tritiated H20 to the skin surface and removing a 200 µl sample from the receptor phase one hour later. The sample was counted using liquid scintillation counting (LSC). The skin surface was subsequently washed seven times and the receptor chambers three times with water, prior to refilling with the selected receptor medium (50/50 ethanol/water).
The results of the membrane integrity assessment are given in "details on results incl. tables".
No cell exhibited a water permeability greater than 0.005 cm/h and the average for each test group was not greater than 0.002 cm/h.


PRINCIPLES OF ASSAY
- Diffusion cell: The skin samples were mounted as a barrier between the halves of horizontal Franz-type diffusion cells, the stratum corneum facing the donor chamber. The cells were designed such that the area available for diffusion was about 1.0 cm2, the exact area being measured for each diffusion cell (See details on results incl. tables). The diffusion cells were immersed in a constant temperature water bath such that the receptor chambers were maintained at 37.0±0.5°C throughout the experiment. This ensured that the skin surface temperature was maintained at 32.0±1°C. The receptor chamber contents were continuously agitated by small PTFE-coated magnetic followers driven by submersible magnetic stirrers. The receptor chambers of the diffusion cells were initially filled with a known volume (See "details on results incl. tables") of water, capped, and allowed to equilibrate to the correct temperature.
- Receptor fluid:
- Solubility od test substance in receptor fluid: > 500 mg/l
- Test temperature: skin surface: 32 °C . Receptor chambers: 37 °C
- Occlusion: non-occulsive
- Reference substance(s): none

Results and discussion

Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
See "details on results incl. tables" : Table 3: Distribution of fragrance material in all compartments at 48 hours (µg/cm2 and % of applied dose)
Total recovery:
65.8 ± 2.8% of the applied dose. Evaporative loss was identified (from PTFE sheets) as the main cause of this low recovery. Estimated loss through evaporation from the PTFE surface was 14% over 48 hours.
Percutaneous absorptionopen allclose all
Dose:
200 µg/cm2
Parameter:
percentage
Absorption:
ca. 2 %
Remarks on result:
other: 2 hours
Dose:
200 µg/cm2
Parameter:
percentage
Absorption:
ca. 11.5 %
Remarks on result:
other: 8 hours
Dose:
200 µg/cm2
Parameter:
percentage
Absorption:
ca. 30.8 %
Remarks on result:
other: 24 hours
Dose:
200 µg/cm2
Parameter:
percentage
Absorption:
ca. 40.1 %
Remarks on result:
other: 36 hours
Dose:
200 µg/cm2
Parameter:
percentage
Absorption:
ca. 45.9 %
Remarks on result:
other: 48 hours
Conversion factor human vs. animal skin:
Not applicable

Any other information on results incl. tables

The complete raw data for this study are contained in “attached background material” with corresponding graphs (permeation versus time, distribution in fct of compartments, etc.) and are summarized in the tables below (i.e. mean ± standard deviations of 11 independent measurements performed with all the skin samples provided by 4 donors). Data are expressed as the amount (µg/cm2) and % of applied dose recovered from the receptor phase and the different compartments of the epidermal membrane.

 

Table 1.   Application regimen

Cell n°

Cellvol(ml)

Cell area (cm2)

Applied vehiclevol(µl)

Vehicle dose (µl/cm2)

Methyldihydrojasmonatedose (µg/cm2)

Water permeability (cm/h)

Mean

3.3

1.18

23.5

20.0

200.1

0.0011

SD

0.32

0.11

1.7

0.6

6.0

0.000723

 

Table 2.   Permeation into receptor phase (µg/cm2 and % applied dose)

 

Cell n°

hours

2

8

24

36

48

µg/cm2

Mean

3.9

23.2

61.8

80.5

92.2

SD

5.1

20.6

23.0

24.3

25.4

% applied dose

Mean

1.95

11.5

30.8

40.1

45.9

SD

2.5

10

11

11.5

12.0

 

Table 3.   Distribution of fragrance material in all compartments at 48 hours (µg/cm2 and % of applied dose)

 

Cell n°

Surface wipe

Strip 1

Strips 2-3

Strips 4-6

Strips 7-10

Remaining epidermis

Receptor phase

Donor chamber

Total recovered

µg/cm2

Mean

14.0

0.968

0.970

0.711

0.421

2.38

92.2

20.2

132

SD

6.2

0.320

0.476

0.421

0.285

0.88

25.4

9.5

21

%applied dose

Mean

6.99

0.483

0.486

0.356

0.211

1.19

45.9

10.1

65.8

SD

3.04

0.157

0.244

0.215

0.144

0.44

12.0

4.7

9.7

 

Penetration into the skin at 48 hours was determined by measuring the levels of fragrance material within the upper stratumcorneum(tape strips) and the remaining lower stratumcorneumand epidermis. The skin was wiped prior to tape stripping to remove fragrance material remaining at the surface. The total recovery of label for each tape strip group was divided by the number of tape strips in that group to generate amounts per strip data. These data are also shown in Figure 4 (See background attached material). In each case the amount of fragrance material recovered decreased with skin depth. Although the contents of the first tape strip could be regarded as surface material, the general shape of these profiles is a reflection of the 'concentration gradient' of fragrance material within the epidermal membrane.

The levels of fragrance material found in surface wipe, donor chamber wash, tape strips and remaining epidermis are shown graphically in Figure 5 in “Attached background material”.

No significant radioactivity was found in the filter paper skin supports (two analyzed), indicating that adsorption onto the filter paper had not occurred. The level (µg/cm2, mean ± se) of fragrance material in all compartments at 48 hours is shown graphically in Figure 6 in attached background material. The recovery (% applied dose, mean ± se) of test substances in all compartments is shown in Figure 7.

Overall recovery was 65.8 ± 2.8. The low recovery was not unexpected as the evaporative loss from PTFE over 48 hours was significant, with corresponding recoveries 86% applied dose, indicating that the losses through evaporation from the PTFE surfaces were 19 and 14% applied fragrance respectively.

 

Table 4.   Recovery of fragrance material (% applied dose) from PTFE sheet and cell donor chamber

Time after application (h)

% applied fragrance recovered from PTFE and donor chamber

1

105.7

2

105.8

4

103.5

8

101.7

24

95.9

48

86.4

 

The measurement of evaporation from the PTFE surfaces may have underestimated the evaporation from skin due to surface tension effects. When fragrance was applied to a PTFE surface, the vehicle remained as a discrete drop, whereas it spread completely over the skin. The actual evaporation rate of test material would be expected to differ somewhat due to the different surface characteristics and the fact that evaporation was competing with skin absorption. However, the data suggest that the low mass balance data may be explained by evaporative loss of the fragrance material.

A number of experimental configurations, involving trapping of evaporating fragrance using AnasorbCSC sorbent tubes (SKC Limited, UK), were assessed in the attempt to directly measure the loss of fragrance from each cell through evaporation. However, it was found impossible to trap volatile material without causing occlusion and consequently significant modification to skin permeation such that it did not model the in-use situation.

Applicant's summary and conclusion

Executive summary:

Introduction.This study was designed to determine the in-vitro human skin permeation rate and distribution of 14C-radiolabelled fragrance material methyldihydrojasmonate, following application in vehicle, under non-occlusive conditions. The test material was used as 1% (w/v) solutions in ethanol, a realistic concentration according to uses concentrations in formulations applied to human skin. The study was conducted according to the FDA/ American Association of Pharmaceutical Scientists (AAPS) guideline, a guideline similar/equivalent to the OECD TG 428 “Skin Absorption: in vitro Method”.

 

Human epidermal membranes were used and membrane integrity was assessed by measuring the permeation rate of tritiated water. Permeation of the test substance was measured over 48 hours, following which the epidermal membranes were tape stripped 10 times and the radiolabel content of the strips and remaining epidermis determined. The volatility of liquid fragrance material led to loss from the surface of the epidermal membranes. This evaporative loss was estimated by measuring the loss fromPTFE (polytetrafluoroethylene) sheets for each of the three fragrance materials.

 

Results.The human skin permeation of the test substance was moderately high. Following 48 hours exposure, 45.9 ± 3.5% of the applied dose (mean ± standard error, se) had permeated into the receptor phase.

Levels of fragrance material in the 48 hour surface wipe and donor chamber wash were: 1.54 % of the applied dose for stratum corneum tape strips and 1.19 % of the applied dose for the remaining epidermis. Overall recovery fragrance material was 65.8 ± 2.8%.

The evaporation of the fragrance material from PTFE (polytetrafluoroethylene) was significant and evaporative loss was identified as the main cause of the low recovery of methyl dihydrojasmonate when applied to epidermal membranes for 48 hours.

 

Conclusions.The results of this study indicated that the peructaneous absorption level of methyl dihydrojasmonate was moderately high. The test substance permeated the skin fairly rapidly, but the permeation subsequently plateaued somewhat as the donor phase became depleted through both evaporation and loss due to permeation.