Pyrocatechol

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 mar 2005 to 03 aug 2005

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pyrocatechol

EC Number:

204-427-5

EC Name:

Pyrocatechol

Cas Number:

120-80-9

Molecular formula:

C6H6O2

IUPAC Name:

pyrocatechol

Radiolabelling:

yes

Remarks:

Catechol UL 14C (Sigma, St Louis, Missouri) Batch 99H9427. Purity >= 98%

Test animals

Species:

human

Sex:

male/female

Administration / exposure

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
FORMULATION OF [14C] CATECHOL
Purification of the radiolabeled test item:
The analysis of the radiochemical purity of the delivered [14C] Catechol dated from September 15, 2000. HPLC analyses revealed an actual purity below 95 %, therefore the [14C] labeled Catechol was purified by a solid phase extraction on ISOLUTE SPE C18 material (2 g/6 ml). The radiolabeled test item was applied as aqueous solution to the stationary phase, washed with water, and eluted with water/acetonitrile mixtures with increasing ratios of acetonitrile, i.e. 90:10, 80:20, 70:30, 60:40, 50:50, 100 v/v. The fractions with the radiochemical purities > 98 % were combined and the solvent was removed by rotatory evaporator.

Preparation of the Stock Solution I:
About 1.2 mg of the purified [14C] Catechol were dissolved in 8.9 ml water (MilliQ).
The concentration of radioactivity in the stock solutions was determined by LSC.

Preparation of the Stock Solution II:
29.3 mg unlabeled Catechol were dissolved in 1 ml water (MilliQ).

Formulation of the administration solution:
A volume of the stock solution I (8000 μl) containing 1.14 mg [14C] Catechol and a volume of the stock solution II (100 μl) containing 2.93 mg unlabeled Catechol were mixed and the solvent was removed by rotatory evaporator. This dilution with unlabeled Catechol yielded [14C] Catechol with a new specific radioactivity of 321 kBq/mg. The dry test item was thereafter dissolved in 4 ml water (MilliQ) leading to the target concentration of 1 mg Catechol /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.
HPLC analysis were carried out on a Merck HPLC system consisting of a solvent-pump (Merck-Hitachi L-7100 or L-7100A), 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 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 5µm, 250 mm x 4.6 mm
Mobile phase: Eluent A (Water MilliQ), Eluent B (Acetonitrile).
Flow: 1 ml/min
UV/VIS: 220 nm

- Limits of detection and quantification: The limit of quantification (LOQ) in the perfusate was calculated according to Currie.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human cadaver skin from Caucasian donors was obtained from the “Institut für Pathologie, Kantonsspital Basel”, Basel, Switzerland.
- Ethical approval if human skin: yes
- Type of skin: Uper leg dorsal (Q1A1, Q1A2 and Q2A1) and abdominal (Q2A3)
- Thickness of skin: 200 µm
- Membrane integrity check: yes
- Storage conditions: Upon receipt the skin was stored for up to 3 month 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. Due to ethical aspects the availability of skin sample from abdominal area is very limited. However the anatomical properties for the 200 μm skin membranes were equal for both used body areas.

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 > 2.5·10-3 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, to allow all tritiated water to be removed from the system.

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, placed in a LSC vial, 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 63.6 99.4 1225534 0.994
Q1A2 63.7 99.5 1226033 0.995
Permeability constant Q2A1 63.7 99.6 1227726 0.996
Q2A2* 64.7 101.1 1245644 1.011
Q2A3 63.3 98.9 1218736 0.989
* Subgroup Q2A2 had to be prematurely stopped due to technical error of the fraction collector.


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 (14C-catechol in MilliQ water) were applied to the surface of each of the skin membranes. 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 in time intervals as follows:

Objective Subgroup Sampling interval
Short term penetration Q1A1 0 – 10 minutes
Q1A2 0 – 60 minutes
Permeability constant Q2A1 0 – 6 hours : 1 hour intervals (6 intervals)
Q2A3 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 solution8 (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 LSC. The skin membranes were then removed from the diffusion cell, digested in tissue solubilizer (Solvable9), and the radioactivity was determined by Liquid Scintillation Counting (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] Catechol 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 ·10-3 cm/h.
The corresponding permeability constant Kp for Catechol was calculated to be 1.430·10-3 cm/h with a standard deviation of 40 % (ranged from 0.712 to 2.120·10-3 cm/h) for the subjects used (n=4).

SHORT TERM PENETRATION:
After a 10-minute exposure with aqueous [14C] Catechol 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 96.89 % of the dose. Additional 0.28% and 0.09 % of the dose were found in/on the skin membrane and in the cell wash, respectively. The total recovery amounted to 97.30 % of the dose.

During the exposure time of 60 minutes a mean of 0.22 % of the applied [14C] Catechol =penetrated through the human skin membranes. Again the bulk of applied test item could be washed off after the end of the exposure period accounting for 94 %. A somewhat higher amount, i.e. 1.49 % of the dose, was found in/on the skin membranes after the washing procedure. The total recovery after 60 minutes of exposure accounted for 96.14 % of the dose.

DETERMINATION OF THE PERMEABILITY CONSTANT (KP):
In the first experiment Q2A1, within an exposure period of 24 hours a mean of 26.65 % of the applied [14C] Catechol penetrated through human skin membranes. After a lag time of about 6 hours Catechol penetrated with a flux (penetration rate at steady state) of 1.425 μg/cm²/h.
This steady state was achieved, on average, between 10 and 24 hours after start of exposure. The steady state was selected based on each individual cell, and most of the cells showed the steady state in the above mentioned time range. Some cells showed an unusual increase between 20 and 24 hours. This was likely an artifact due to changes in the skin membrane and not the beginning of a steady state. However, this slight increase was not excluded form the Kp calculation and therefore the Kp value was based on a conservative approach. Based on the calculated flux of 1.425 μg/cm²/h and the concentration of Catechol in the administration solution of 996 μg/cm³ the permeability constant (Kp) of Catechol was calculated to be 1.430·10-3 [cm/h]. The calculated permeability constant (Kp) showed a standard deviation of 40 % (1.430·10-3 +/-0.579·10-3 cm/h) for the four subjects used.
At the end of the exposure period 46.10 % of the dose could be washed off by the skin membrane rinse. After skin membrane rinse 6.26 % of the applied dose still remained in/on the skin membrane and additional 1.03 % of the dose was found in the cell wash. However the total recovery accounted only for about 80 % of the dose. Due to the low recovery the experiment for determination of the permeability constant was repeated.

The second experiment, assigned as Subgroup Q2A2, had to be prematurely stopped due to a technical error of the fraction collector, and therefore a third experiment was performed assigned as Q2A3.

The results of the Subgroup Q2A3 confirmed the obtained results of the first experiment (Subgroup Q2A1). Within an exposure period of 24 hours a mean of 23.63 % of the applied [14C] Catechol penetrated through human skin membranes. After a lag time of about 6 hours Catechol penetrated with a flux of 1.235 μg/cm²/h. The permeability constant (Kp) was calculated to be 1.249·10-3 [cm/h] with a standard deviation of 38 % for the four subjects used. But again the total recovery accounted only for 71.17 % of the applied dose. Compared to the Subgroup Q2A1 it is obvious that the reduced total recovery of subgroup Q2A3 (-10 %) is in line with a reduced amount of radioactivity determined in the skin membrane rinse (-13 %). All other values were found on a comparable level. Since the feasibility of the skin rinse method was validated by the results of the short term experiments, it is assumed that the donor chamber was not completely closed by the used cover tape. However, the objective of this part of the experiment, i.e. determination of the permeability constant, was not influenced by the reduced recovery.

Applicant's summary and conclusion

Conclusions:

[14C] Catechol, applied as aqueous solution at a concentration of 1 mg/cm³, corresponding to an area concentration of 0.1 mg/cm², to human split thickness skin membranes, showed a moderate penetration through the skin membranes. After a lag time of 6 hours Catechol penetrated with a flux of 1.425 μg/cm²/h corresponding to a permeability constant (Kp) of 1.430·10-3 [cm/h].

Executive summary:

The percutaneous penetration of Catechol, i.e. 1,2-Benzenediol, formulated as an 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] Catechol 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 Catechol/cm². The receptor fluid, i.e. aqueous saline (0.9% NaCl w/v), was delivered at a flow rate of about 3 ml/h. 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 Catechol. For each objective, i.e. short term penetration (10 and 60 min) and determination of the permeability constant, at least 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

                                      Q1A2           0 – 60 minutes

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

                                      Q2A3           0 – 24 hours : 2 hour intervals (9 intervals)

The results of the short term experiments revealed that Catechol penetrated to a very low extent through human skin membranes within the first hour of exposure. Only 0.04 % and 0.22 % 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 97 % and 94 % of the dose. After skin membrane rinse 0.28 % and 1.49 % of the dose remained in/on the skin membranes after 10 and 60 minutes of exposure.

Within an exposure period of 24 hours, a mean of 26.65 % of the applied [14C] Catechol penetrated through human skin membranes. After a lag time of about 6 hours, Catechol penetrated with a flux (penetration rate at steady state) of 1.425 μg/cm²/h through human skin membranes. The corresponding permeability constant Kp for Catechol was calculated to be 1.430·10-3 cm/h with a standard deviation of 40 % (ranged from 0.712 to 2.120·10-3 cm/h) for the subjects used (n=4). HPLC analysis of the skin membrane rinse pools revealed that Catechol remained unchanged on the skin membranes during the exposure period.

In conclusion, Catechol, applied as an aqueous solution to human skin membranes, penetrated very moderately with a penetration rate of 1.425 μg/cm²/h after a lag time of 6 hours. The permeability constant Kp was calculated to be 1.430 x 10E-3 cm/h.