Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 02 march 2005 to 12 sept 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, following the required guidelines without deviations.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
yes

Test animals

Species:
other: see "details on in vitro test system" below

Administration / exposure

Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human cadaver purchased from the National Disease Research Interchange (NDRI), Philadelphia, PA.
- Ethical approval if human skin: yes
- Type of skin: dermatomed sections of abdominal human cadaver.
- Preparative technique: As reported by the supplier, the most common technique to procure the dermatomed skin involved use of a blade
dermatome, which provides rapid recovery of grafts of uniform thickness. A rapidly oscillating side-to-side blade was advanced over the skin with
thickness and width settings adjusted by the recovery coordinator. After proper blade orientation, width guard and depth setting were confirmed and recovery was initiated.
- Thickness of skin: 200 - 400 µm
- Membrane integrity check: The integrity of each skin sample was evaluated by measuring its permeability to tritiated water over a period of 4 or 6
hours on Phase 1 of the experiments.
The permeability constant for 3H20 for each skin specimen was compared with the historical database of values generated in this laboratory. All skin specimens in this study were considered to be representative of normal human skin because all of the permeability constant values were less than 5.0 x 10-3 cm/hr.
In addition, the ratio of the 3H20 permeability constants for Phase 3 and Phase 1 were used to calculate a damage ratio for each cell tested in the
definitive study. This value was used to assess any effect of the test substance on the integrity of the skin sample.

- Storage conditions: Samples of dermatomed skin were stored frozen at -70 °C for less than 3 months until time of use to avoid decomposition.

PRINCIPLES OF ASSAY
- Diffusion cells: Dermatoned skin
- Receptor fluid: The receptor solution used in this study was Dulbecco's phosphate buffered saline (pH 7.1) containing Penicillin, 100 Units/mL, Streptomycin, 100 tg/mL, Amphotericin B as FungizoneTM, 0.25 µg/mL, and 6% (w/v) Volpo-20TM, (60 mg/mL, polyethylene glycol 20 oleyl ether, a
non-ionic surfactant) as described by Bronaugh and Stewart, 1984.

- Test temperature:incubation at 32°C for 6 hours
- Reference substance(s): phosphate buffered saline

Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
Absorption of Tritium (Phase 1):
The absorption rates for 3H20 for the nine skin specimens used in the definitive study are provided in Table 1. The absorption rates for 3H2O for the twelve skin specimens used in the short-term exposure experiments are provided in Table 5.
The result for all cells (mean ± SD) for absorption of 3H2O for Phase 1 of the definitive study was found to be 1.21 ± 0.57 mg/cm2/hr.
The result for all cells (mean ± SD) for absorption of 3H20 for Phase 1 of the short-term absorption studies was found to be 0.83 ± 0.42 mg/cm2/hr. Although the results for the short term studies are slightly lower, these values are considered comparable to the historical value of 1.64 ± 0.90 mg/
cm2/hr that has been derived from the results of 30 previous studies.

The 3H20 permeability constant results for some of the skin specimens received initially from the supplier were found to be greater than the
5.0 x 10-3 cmlhr criteria and had to be excluded from the studies. Because of these unexpected results, another shipment of skin specimens had to
be ordered which necessitated two preparations of the 14C-labeled test substance solutions to complete this work. In the end, permeability constant values for all Phase 1 cells for both the definitive and short-term experiments were less than the 5.0 x 10-3 cm/hr criteria, and thus considered
representative of normal human skin.

Absorption of Tritium (Phase 3):
The absorption rates for 3H2O for Phase 3 of the study and the damage ratios for all 9 cells are presented in Table 3.
The mean (± SD) absorption rate of tritiated water for control cells was found to be 1.21 ± 0.66 mg/cm2/hr; and the mean damage ratio was 1.03 ± 0.22.
The mean damage ratio for the six cells exposed to the test substance for Phase 2 was 1.24 ± 0.28. This value is within the range of damage ratios
presented by Dugard et al. (1984) of 1-2 for water; and is within the range of values collected in this laboratory for skin specimens exposed to
physiological saline. Therefore, exposure to the test substance for 6 hours produces similar damage ratios to those expected from exposure to
physiological saline or water.
Total recovery:
The percentages of total 14C-labeled test substance recovery from each test substance cell for the definitive study are shown in Table 4. The mean
(± SD) total 14C-labeled test substance recovery was 93.9% ± 2.8%. The percentages of total 14C-labeled test substance recovery for the 10 minute
short-term absorption experiments and for the 60 minute short-term absorption experiments are shown respectively table 7 and 8.

Any other information on results incl. tables

Table 1: Definitive Study: Phase 1 Summary - Tritium Absorption Rates 

Cell Number

Absorption Rate, mg/cm2/hour

1

0.443

2

0.514

3

0.611

4

1.99

5

1.54

6

1.83

7

1.37

8

1.14

9

1.42

Mean ±SD

1.21±0.57 (N=9)

Historical Mean SD

1.64 ±0.90 (N=239)

Table 2: Definitive Study: Phase 2 Summary - Hydroquinone monomethyl ether 

Cell Number

Absorption Rate, mg/cm2/hour

Permeability constant (cm/hr)

1*

N/A

N/A

2

0.260

7.52x10-3

3

0.300

8.66x10-3

4*

N/A

N/A

5

0.382

1.11x10-3

6

0.500

1.45x10-3

7*

N/A

N/A

8

0.243

6.93x10-3

9

0.268

7.65x10-3

Mean ±SD

0.326 ±0.099 (N=6)

9.39±3.10 x10-3

 * Control cell, not applicable

Table 3: Definitive Study: Phase 3 Summary - Tritium Absorption Rates and Damage Ratios 

Cell Number

Absorption Rate*mg/cm2/hour

Damage Ratio**

(Phase 3 3H2O/ Phase 1 3H2O)

1

0.569

1.28

2

0.813

1.58

3

0.931

1.52

 

 

 

4

1.89

0.950

5

1.81

1.17

6

2.34

1.28

 

 

 

7

1.17

0.855

8

1.03

0.899

9

1.38

0.968

 

 

 

*Absorption Rate

Mean ± SD, Control Cells (1, 4, and 7):1.21± 0.66

Mean ± SD, Test Cells(2, 3, 5, 6, 8,and 9):1.38±0.59

 

**Damage Ratio:

Mean ± SD, Control Cells (1, 4, and 7):1.03±0.22(N=3)

Mean ±SD, Test Cells (2, 3, 5, 6, 8, and 9):1.24±0.28(N=6)


Table 5: Short-term Skin Absorption Experiments: Phase 1 Summary - Tritium Absorption Rates

 

Cell Number for 10 minute exposures

Absorption Rate, mg/cm2/hour

6S

0.973

9S

0.655

1S

1.03

4S

1.35

7SS

0.458

8SS

0.558

Cell Number for 60 minute exposures

Absorption Rate, mg/cm2/hour

7S

0.578

8S

0.754

2S

1.73

3S

1.07

5S

0.425

6SS

0.359

Mean ± SD                     0.83 ±0.42(N=12)

Historical Mean ± SD      1.64 ± 0.90(N=239)

Applicant's summary and conclusion

Conclusions:
The absorption rate of the test substance through dermatomed human skin was found to be 0.33 ± 0.10 mg/cm2/hr, giving a corresponding
permeability constant of 9.39 ± 3.10 (x 10-3) cm/hr. The test substance, under the conditions of this study, did not cause damage to the skin.
Executive summary:

The rate of percutaneous absorption of the test substance through dermatomed sections of human skin was measured in vitro. An excess of the test substance was applied to sections of human skin contained in glass diffusion cells. The measured absorption rate (mean ± SD) was found to be 0.33±0.10 mg/cm2/hr and the mean permeability constant (mean ± SD) was determined to be 9.39 ± 3.10 (x 10-3) cm/hr.

The integrity of each skin specimen was determined by measuring its permeability to tritiated water (H2O) in Phase 1. The mean (± SD) 3H20 absorption rate for all skin specimens was 1.21±0.57 mg/cm2/hr, in close agreement with the historical values from this laboratory, and published values from other laboratories. The mean damage ratio, calculated from the rates of 3H20 before and after exposure of the skin to the test substance, was similar to the control (unexposed) values, indicating that exposure to the test substance for 6 hrs did not damage human skin.

The total recovery of the test substance was measured by determining the percentage 14C-labeledtest substance remaining in test system components for each test substance cell. The total mean (± SD) 14C-labeled test substance recovery was 93.9% ± 2.8%.

 

A separate determination was also conducted to measure the short-term absorption rates following 10 and 60 minute exposure times. The short-term skin absorption was based on the sum of the test substance concentration measured in the receptor chambers and the amount of absorbed test substance remaining in the skin, and was determined to be 283.0 ± 127.4 µg/cm2/hr following the 10 minute exposure and 223.0 ± 65.0 µg/cm2/hr following the 60 minute exposure.

The absorption rate of the test substance through dermatomed human skin is relatively low and was found to be 0.33 ± 0.10 mg/cm2/hr, giving a corresponding permeability constant of 9.39 ± 3.10 (x 10-3) cm/hr. The test substance, under the conditions of this study, did not cause damage to the skin.