Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
report date: 2005-06-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Guideline "In vitro Dermal Absorption Rate Testing of Certain Chemicals of Interest to the Occupational Safety and Health Administration", Federal Registrer: April26, 2004 (Volume 69, Number 80)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
yes

Test animals

Species:
human
Sex:
male/female

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
6 hours
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions:
FORMULATION OF [14C] 4-TERT-BUTYLPYROCATECHOL
Preparation of the Stock Solution I:
About 8 mg [14C] 4-tert-butylpyrocatechol were dissolved in 2.5 mL diethyl ether.
The concentration of radioactivity in the stock solutions was determined by Liquid Scintillation Counting (LSC).

Preparation of the Stock Solution II:
33.2 mg unlabeled 4-tert-butylpyrocatechol were dissolved in 1 mL ethanol.

Formulation of the administration solution:
A volume of the stock solution I (250 µL) containing 0.7 mg [14C] 4-tert-butylpyrocatechol and a volume of the stock solution II (100 µL) containing 3.32 mg unlabeled 4-ter-butylpyrocatechol were placed in a flask. The organic solvent was removed by a gentle stream of nitrogen.This dilution with unlabeled 4-tert-butylpyrocatechol yielded [14C] 4-tert-butylpyrocatechol with a new specific radioactivity of 775 kBq/mg. The dry test item was thereafter dissolved in 4 mL water (MilliQ) leading to the target concentration of 1 mg 4-tert-butylpyrocatechol / mL administration solution.

ANALYSIS
- Method type(s) for identification: The purity of the formulated test item was determined by HPLC at the time of each application. And the skin membrane rinse pools of each subgroup were analyzed by HPLC.
HPLC analysis were carried out on a Merck HPLC system of a solvent-pump (Merck-Hitachi L-7100), an Autosampler (Merck-Hitachi L-7200), an UV-detector (Merck-Hitachi L-4000). The radioactivity signal was monitored with a Radiomatic 500TR radioactivity flow monitor (Packard Instrument Company inc.) connected to a personal computer. The flow monitor operated with a 500 µL liquid cell and continuous mixing of the eluent with 3 mL/min scintillation liquid (Flo-Scint A13).
Column: Phenomenex Luna C18 5 µm, 250 mm x 4.6 mm
Mobile phase: Eluent A (Water MilliQ), Eluent B (Acetonitrile)
Flow: 1 mL/min
Gradient: 0 - 25 min, 40% B (isocratic)

- Limits of quantification: The limit of quantification (LOQ) in the perfusate was calculated according to Currie: LOQ accounted for 0.0019 - 0.0021 [µg 4-TBC equivalents/cm²]
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Human cadaver skin form Caucasian donors was obtained from "Institut für Pathologie, Kantonsspital Basel", Basel, Switzerland.
- Ethical approval: yes
- Type of skin: upper leg dorsal (Q1A1, Q1A1a, Q1A2, Q2A1) and abdominal (Q1A1a, Q1A2a, Q2A1)
- Thickness of skin: 200 µm
- Membrane integrity check: yes
- Storage conditions: Upon receipt the skin was stored at about -20°C until preparation of the skin membranes. All skin samples used were almost hair-free and in good condition as visually observed.
- Justification of species, anatomical site and preparative technique: The upper leg area for withdrawal of the skin samples is the common standard at the institute of pathology. To get skin samples of the abdominal area is very difficult due to ethical aspects. However the anatomical properties for the 200 µm skin membranes were equal for both used body areas.

PRINCIPLES OF ASSAY
An automated flow-through cell system was used. Seven flow-through diffusion cells (Aesculap) designated cell 1 through cell 7 were placed in one aluminium manifold (Permegear) connected to a water bath (Digitana Ltd.) to maintain the temperature of the skin membranes at 32°C. The diffusion cells of the second part of the system with the same setup were designated cell 8 through cell 14. Each diffusion cell consisted of a donor and receptor chamber. The area of skin membrane exposed to the donor chamber was 0.64 cm². The receptor chambers were connected to a multi-channel peristaltic pump, model IPC-16 (Ismatec Ltd.). The pump speed was adjusted to about 3 mL/h. During the given time intervals, the effluent from the cell was collected directly into vials on a fraction collector, model ISCO retriever IV (IG Instrumenten Gesellschaft).

EXPERIMENTAL PROCEDURE
Pieces of the skin membranes (approximately 1.8 x 1.8 cm) were cut and mounted in the diffusion cells between the donor and receptor chamber. The cells were placed in the manifolds and connected to the peristaltic pump. For an equilibration period of 1 hour, saline (0.9% NaCl w/v) was pumped through the receptor chamber at a flow rate of about 3 mL/h.

Test of Skin Membrane Integrity:
The integrity of the skin membranes was checked by applying 50 μL tritium water (about 200,000 dpm) to the skin membrane surface. The donor chamber was covered with adhesive tape (occluded conditions). The cumulative penetration was determined over 6 hours by collecting hourly fractions. The permeability coefficient (Kp) of each skin membrane was calculated for the 3 - 6 hours interval. Skin membranes with Kp > 2.5x10-3 cm/h were excluded from the subsequent experiment.
After 6 hours, adhesive tape was removed from the donor cell chamber and the cells left open overnight with the saline flowing through the receptor chamber.

APPLICATION OF DOSE
A 64 μL aliquot of the application solution was applied manually to each skin membrane preparation using a μL-syringe. The amount applied to each cell was checked by determination of the radioactivity content of three control doses, taken prior to the first, in the middle, and after the last administration for each dose group. The doses applied and the concentration of the application solution are given in the table below:

Objective Subgroup Applied dose Concentration
µg/cell µg/cm² Dpm/cell mg/cm3
Short term penetration Q1A1 64.4 100.6 2994982 1.006
Q1A1a 63.5 99.2 2953104 0.992
Q1A2 64.4 100.7 2996111 1.007
Q1A2a 63.4 99.1 2950257 0.991
Permeability constant Q2A1 64.1 100.01 2979176 1.000

Application of the Test Item:
Those cells with skin membranes of acceptable Kp values in the integrity test were arranged on one manifold and aliquots of 64 μl dose solution were applied to the surface of each of the skin membrane. The donor chambers were covered by an adhesive tape (occluded conditions). The receptor fluid, i.e. aqueous saline (0.9% NaCl w/v), was delivered at a flow rate of about 3 mL/h during the testing period. The perfusates were collected at ambient temperature in time intervals as follows:

Objective Subgroup Sampling interval
Short term penetration Q1A1 0 – 10 minutes
Q1A1a 0 – 10 minutes
Q1A2 0 – 60 minutes
Q1A2a 0 – 60 minutes
Permeability constant Q2A1 0 – 6 hours: 1 hour intervals (6 intervals)
0 – 24 hours: 2 hour intervals (9 intervals)

End of the experimental period:
After the corresponding exposure period the cover tape was removed from the donor chamber and retained for radiometry. Thereafter test item which was still present on the skin membrane was removed from the skin surface by rinsing three times with about 0.5 ml of a mild shower gel solution (Nivea Douche Fitness) (1 % in tap water) followed by three times with 0.5 ml water (MilliQ).
The skin membrane rinse was collected for each cell for the determination of radioactivity by Liquid Scintillation Counting (LSC). The skin membranes were then removed from the diffusion cell digested in tissue solubilizer (Solvable), and the radioactivity was determined by LSC.
Finally the cells were washed with water (150 ml) and the radioactivity in the cell wash was determined by LSC.

Results and discussion

Total recovery:
INTEGRITY TEST:
Prior to each individual application of [14C] 4-tert-butylpyrocatechol the integrity of the human skin membranes was checked using tritiated water. All skin membrane preparations were selected showing a permeability coefficient (Kp) of tritiated water < 2.5 cm/h x 10-3. For the first experiments of the short-term penetration test (Q1A1 and Q1A2) two and three cells, respectively, did not pass the integrity test, therefore additional replicates were performed designated as Q1A1a and Q1A2a.

SHORT TERM PENETRATION
After a 10-minute exposure with aqueous [14C] 4-tert-butylpyrocatechol solution only 0.04% of the applied dose penetrated through the human skin membrane. The bulk of applied test item could be removed from the skin membrane after the exposure period, accounting for 96.80% of the dose. Additional 2.14% and 0.27% of the dose were found in/on the skin membrane and in the cell wash. The total recovery amounted to 99.25% of the dose.

During the exposure time of 60 minutes a mean of 2.22% of the applied [14C] 4-tert-butylpyrocatechol penetrated through the human skin membranes. The variation of the different subjects ranged from 0.86% to 3.23% of the dose. Again the bulk of applied test item could be washed off after the end of the exposure period accounting for 85.18%. A somewhat higher amount, i.e. 6.30% of the dose, was found in/on the skin membranes after the washing procedure. The total recovery after 60 minutes of exposure accounted for 95.11% of the dose.

DETERMINATION OF THE PERMEABILITY CONSTANT (Kp)
Within an exposure period of 24 hours a mean of 90.28% of the applied [14C] 4-tert-butylpyrocatechol penetrated through human skin membranes. After a lag time of about 0.5 hours 4-tert-butylpyrocatechol penetrated with a flux (penetration rate at steady state) of 6.836 µg/cm²/h. This steady state was achieved, on average, between 1 and 10 hours after start of exposure. The variation of the individual subjects ranged from 1-8 up to 1-18 hours. After the steady state period more than 50% the applied dose were penetrated through the skin membranes and therefore a infinite dose situation was no longer given. Based on the calculated flux of 6.836 µg/cm²/h and the concentration of 4-tert-butylpyrocatechol in the administration solution of 993 µg/cm3 the permeability constant (Kp) of 4-tert-butylpyrocatechol was calculated to be 6.881 x 10-3 [cm/h]. The calculated permeability constant (Kp) showed a standard deviation of 31% for the four subjects used.



Percutaneous absorptionopen allclose all
Dose:
0.10 mg/cm²
Parameter:
percentage
Absorption:
0.04 %
Remarks on result:
other: 10 minutes
Dose:
0.10 mg/cm²
Parameter:
percentage
Absorption:
2.22 %
Remarks on result:
other: 60 minutes
Dose:
0.10 mg/cm²
Parameter:
percentage
Absorption:
90.28 %
Remarks on result:
other: 24 hours

Applicant's summary and conclusion

Conclusions:
[14C] 4-tert-butylpyrocatechol, applied as aqueous solution at an infinite dose of 1 mg/cm3 to human split thickness skin membranes, showed a fast penetration through the skin membranes. After a lag time of 0.5 hours 4-tert-butylpyrocatechol penetrated wih a flux of 6.836 µg/cm²/h corresponding to a permeability constant (Kp) of 6.881 x 10-3 [cm/h].
Executive summary:

The percutaneous penetration of 4 -tert-butylpyrocatechol (4 -TBC), formulated as aqueous solution, was determined in vitro using split-thickness skin membranes from human skin.

The skin membranes were set up in flow-through diffusion cells and [14C] 4 -tert-butylpyrocatechol was applied onto the skin membranes at a concentration of 1 mg/cm3. Aliquots of 64 µL of the administration solution were applied to a skin membrane area of 0.64 cm² corresponding to a infinite dose of 0.1 mg 4 -TBC/cm². The in vitro percutaneous penetration was investigated in two groups, i.e. Groups Q1 and Q2. The Group Q1 was designed to investigate the short term dermal penetration and Group Q2 was designed to determine the permeability constant (Kp) for dermal penetration of 4 -tert-butylpyrocatechol. For each objective, i.e. short term penetration (10 and 60 min) and determination of the permeability constant, duplicates of valid skin membranes from at least 3 different individuals were used. The integrity of each skin membrane preparation was checked with tritiated water.

Objective                      Subgroup       Sampling interval

Short term penetration    Q1A1              0 - 10 minutes

Q1A1a           0 - 10 minutes

Q1A2 0 - 60 minutes

Q1A2a 0 - 60 minutes

Permeability constant Q2A1 0 - 6 hours: 1 hour interval (6 intervals)

0 - 24 hours: 2 hours interval (9 intervals)

The results of the short term experiments revealed that 4 -TBC penetrated to a moderate extent through human skin membranes within the first hour of exposure. Only 0.04% and 2.22% of the applied dose penetrated through the skin membrane within 10 and 60 minutes of exposure, respectively. The bulk of test item could be washed off at the end of the exposure period amounting to 96.80% and 85.18% of the dose. After skin membrane rinse 2.14% and 6.30% of the dose remained in/on the skin membranes after 10 and 60 minutes of exposure.

Within a exposure period of 24 hours in mean 90.28% of the applied [14C] 4 -TBC penetrated through human skin membranes. After a lag time about 0.5 hours 4 -TBC penetrated with a flux (penetration rate at steady state) of 6.836 µg/cm²/h through human skin membranes. The corresponding permeability constant Kp for 4 -TBC was calculated to be 6.881 x 10 -3 cm/h with a standard deviation of 31% (range 4.75 to 9.01) for the subjects used (n = 4).

HPLC analysis of the skin membrane rinse pools revealed that 4 -TBC remained unchanged on the skin membranes during the exposure period.

In conclusion, 4 -tert-butylpyrocatechol, applied as aqueous solution to human skin membranes, penetrated very fast with a penetration rate of 6.836 µg/cm²/h after a lag time of 0.5 hours.

The permeability constant Kp was calculated to be 6.881 x 10 -3 cm/h.