Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 14 to September 05, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP, scientific design and reporting contributed to assessment

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 (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Radiolabelling:
yes
Remarks:
14-C

Test animals

Species:
other: in vitro in rat and human skin
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Skin membranes
Human skin was obtained from three female donors directly after abdominal surgery Donor 1 TNA17/05, 36 years of age, arrival at TNO on 28 June 2005 Donor 2 TNA18/05, 43 years of age, arrival at TNO on 1 July 2005 Donor 3 TNA 19/05, 30 years of age, arrival at TNO on 6 July 2005. Informed consent was provided by the skin donors. The transportation of the skin to the laboratory was carried out as soon as possible after dissection, while the skin was kept on ice in a plastic container. Upon arrival at the laboratory, subcutaneous fat was removed. After removal of the subcutaneous fat, the skin was dermatomed using a Dermatome 25 mm (Nouvag GmbH, Germany). Subsequently, the dermatomed skin strips were cut into smaller pieces of approximately 2 cm2. The thickness of the skin membranes was measured with a digimatic micrometer (Mitutoyo Corporation, Japan) and recorded. The mean thickness (n=6) were as follows:
Human donor 1: 0.397 ± 0.030 mm
Human donor 2:0.357 ± 0.013 mm
Human donor 3: 0.519 ±0.090 mm
Rat skin was obtained from three female rats (Sprague-Dawley, Charles River Deutschland, Sulzfeld, Germany) at ca 10-12 weeks of age After the sacrifice of the rats, the dorsal and flank skin of the animals was clipped free of fur by means of electric clippers. Skin discs with a diameter of ca 16 mm will be punched out and subcutaneous fat was carefully removed using scissors. The thickness of all skin membranes was measured with a digimatic micrometer (Mitutoyo Corporation, Japan) The mean thickness (n=6) were as follows:
Rat donor 1: 0.669± 0 047 mm
Rat donor 2: 0.755± 0 073 mm
Rat donor 3: 0.763± 0 089 mm
Flow-through diffusion cells:
The skin membranes were placed in 9 mm flow-through automated diffusion cells (PermeGear Inc,Riegelsville, PA, USA) The temperature of the skin surface was regularly checked during the course of the study and was found to be ca 32 °C, at ambient humidity. The receptor fluid was pumped at a speed of ca 1.6 mL/h. For human skin membranes, the receptor fluid consisted of a mixture of Dulbecco’s Minimum Eagle Medium (DMEM) and Ham’s FI2 culture medium (3.1) supplemented with Epidermal Growth Factor (hEGF, 10 µg/L), hydrocortisone (400 µg/L), gentamicin (50 mg/L), L-glutamine (2 mM), and Fetal Calf Serum (FCS, 10 %, v/v)
For rat skin membranes, the receptor fluid consisted of Minimal Essential Medium, supplemented with gentamicin (50 mg/L), L-glutamine (2 mM) and FCS (10 %, v/v). During the experiment, the receptor fluid was continuously gassed with ca 95% O2 and ca 5 % CO2.
Since test article is poorly soluble in water, FCS (10 %, v/v) was added to increase solubility. The actual solubility of test article in the receptor fluid was determined prior to the experiment. In addition, the solubility of test substance was tested in culture medium without FCS but containing 5 % Bovine Serum Albumin (BSA)
Receptor fluid solubility:
In order to test the solubility of test article in the receptor fluid, [14C]test article was mixed with non-radiolabeled test article. This mix was further diluted in ethanol to a concentration of 10.11 mg/mL. A volume of 45 µL (containing ca 450 µg test article) was added to a glass vial and the ethanol was evaporated under nitrogen gas. Subsequently, 5 mL receptor fluid (for human skin) containing either 10 % FCS or 5 % BSA was added (in triplicate) to a final concentration of ca 90 µg /mL. The vials were rocked on a roller platform for ca 1 h after which the vials will be left standing for ca 1h. Samples were collected from the upper, middle and lower level of each vial and analysed for the amount of radioactivity 
Based on the following calculations, the required solubility of test article in the receptor fluid is ca 8 µg/mL. the maximum amount of the active ingredient applied to the skin was ca 0.5 mg/cm2 (= ca 320 µg a i /skin membrane) Assuming that 100 % of the dose reaches the receptor fluid over 24 h, the required solubility of test article is 320 µg in 38 4 mL (24 × flow rate of ca 1 6 mL/h), i e ca 8.3 µg /mL.
Based on the solubility in the receptor fluid after a 1-hour incubation period on the roller platform, receptor fluid containing 10 % FCS was selected for the absorption study. The solubility was determined to be approx. 12 µg/mL (both in receptor fluid containing 10 % FCS and 5 % BSA). After an additional 1-hour stationary incubation period, solubility was found to be lower (ca 2 – 3.5 µg/mL probably caused by precipitation of the test substance during that period Based on this test, solubility in the receptor was judged to be sufficient during the entire absorption experiment


Administration / exposure

Type of coverage:
other: in vitro
Vehicle:
not specified
Duration of exposure:
24 hr
Doses:
10.1%: Amount test article in formulation [%]
human skin: 544.9, 548.0, 541.2 µg/cm2
rat skin: 535.9 µg/cm2
No. of animals per group:
6
Control animals:
no
Remarks:
in vitro assay
Details on study design:
Preparation B was made on 14 June 2005 (when the preparation of the complete formulation was tested using non-radiolabelelled homosalate). Preparation A and the complete formulation (including radiolabel) was made on 22 June 2005. The experimental phase (from date of isolation of skin membranes until recovery finishing the recovery) of this study was performed as follows: Human skin donor 1 from 28 until 29 June 2005, human skin donor 2 from 1 until 2 July 2005, human skin donor 3 from 6 until 7 July 2005. The experiments using rat skin (3 donors) were performed from 22 until 23 June 2005. Measurement radioactivity was finished by 11 July 2005.
Determination of recovery of the test substance:
Receptor fluid: The samples of the receptor fluid were collected in vials for immediate analysis by liquid scintillation counting.
Skin wash: The cotton swabs were pooled per skin membrane in glass vials and extracted using a weighed amount of ethanol for at least 24 h at room temperature.
Donor compartment: The donor compartment was washed two times with 1.0 mL ethanol.
Tape stripping: At 24 h post exposure, each skin was tape stripped using D-squame (Monaderm, Monaco) (10 times per skin membrane) Tape stripping was discontinued when the epidermis was visually damaged. The tape strip containing (pieces of) epidermis was pooled with the skin membrane.
Formulation preparation:
The complete dose formulation (that is including the radiolabel) was prepared one day prior to the start of the first experiment. All experiments were performed using the same formulation. Until use, the formulation was stored at ambient temperature and protected from light.

Details on in vitro test system (if applicable):
Flow-through diffusion cells
The skin membranes were placed in 9 mm flow-through automated diffusion cells (PermeGear Inc., Riegelsville, PA, USA). The temperature of the skin surface was regularly checked during the course of the study and was found to be ca 32 °C at ambient humidity. The receptor fluid was pumped at a speed of ca 1.6 mL/h. For human skin membranes, the receptor fluid consisted of a mixture of Dulbecco’s Minimum Eagle Medium (DMEM) and Ham’s F12 culture medium (3 1) supplemented with Epidermal Growth Factor (hEGF, 10 µg/L), hydrocortisone (400 µg/L), gentamicin (50 mg/L), L-glutamine (2 mM), and Fetal Calf Serum (FCS, 10 %, v/v)
For rat skin membranes, the receptor fluid consisted of Minimal Essential Medium, supplemented with gentamicin (50 mg/L), L-glutamine (2 mM) and FCS (10 %, v/v). During the experiment, the receptor fluid was continuously gassed with ca 95 % O2 and ca 5 % CO2.
Since homosalate is poorly soluble in water, FCS (10 %, v/v) was added to increase solubility. The actual solubility of homosalate in the receptor fluid was determined prior to the experiment. In addition, the solubility of homosalate was tested in culture medium without FCS but containing 5 % Bovine Serum Albumin (BSA).
In order to test the solubility of homosalate in the receptor fluid, [14C]homosalate was mixed with non-radiolabeled homosalate. This mix was further diluted in ethanol to a concentration of 10 - 11 mg/mL. A volume of 45 µL (containing ca 450 µg homosalate) was added to a glass vial and the ethanol was evaporated under nitrogen gas. Subsequently, 5 mL receptor fluid (for human skin) containing either 10 % FCS or 5 % BSA was added (in triplicate) to a final concentration of ca 90 µg/mL. The vials were rocked on a roller platform for ca 1 h after which the vials will be left standing for ca 1h. Samples were collected from the upper, middle and lower level of each vial and analysed for the amount of radioactivity

Results and discussion

Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
In human skin, the mean flux constant for the absorption of test substance was 0.058 µg /cm2/h. The mean total absorption was 1.1 % of the applied dose and the mean recovery was 92.4%.
In rat skin, the mean flux constant for the absorption of test substance was 0.807µg/ cm2/h. The mean total absorption was 8.7 % of the applied dose and the mean recovery was 93.1%.
Details see the following tabs in the field for additional results (table 1 and table 2).
Total recovery:
In human skin, the mean recovery was 92.4% of the applied dose.
In rat skin, the mean recovery was 93.1% of the applied dose.
Percutaneous absorptionopen allclose all
Dose:
10.1%- Amount test article in formulation[%]
Parameter:
percentage
Absorption:
1.1 %
Remarks on result:
other: 24 hr
Remarks:
human skin: 1.1% of applied dose
Dose:
10.1%- Amount test article in formulation[%]
Parameter:
percentage
Absorption:
8.7 %
Remarks on result:
other: 24 hr
Remarks:
rat skin: 8.7% of applied dose
Conversion factor human vs. animal skin:
Based on flux constants rat skin was ca. 14 times more permeable to test article than human skin. Based on total absorption, the difference in absorption between rat and human skin was about 8-fold.

Any other information on results incl. tables

Table 1 - Overview table of thein vitropercutaneous penetration of test article in a standard sunscreen formulation through human skin


Group

A

B

C

Amount test article in formulation[%]

10.1

10.1

10.1

Dose[µg/cm2]

544.9

548.0

541.2

n

6

6

6

Penetration into the receptor fluid after 24h

µg/cm

%of

dose

µg/cm

%of

dose

µg/cm

%of

dose

1.36

0.25

0.87

0.16

0.66

0.12

Flux constant [µg/cm2/h]

0.077

0.057

0.039

Lag time [h]

6.5

7.0

7.6

Total absorption[%of dose]

1.4

0.9

0.9

Table 2 - Overview table of thein vitropercutaneous penetration of test article in a standard sunscreen formulation through rat skin


Group

A

B

C

Amount test article in formulation[%]

10.1

10.1

10.1

Dose[µg/cm2]

535.9

535.9

535.9

n

6

6

6

Penetration into the receptor fluid after 24h

µg/cm

%of

dose

µg/cm

%of

dose

µg/cm

%of

dose

7.12

1.33

19.37

3.62

18.50

3.45

Flux constant [µg/cm2/h]

0.412

0.997

1.012

Lag time [h]

6.8

4.6

5.7

Total absorption[%of dose]

7.4

7.7

11.0

Applicant's summary and conclusion

Conclusions:
In summary, in human skin, the mean total absorption was 1.1 % of the applied dose and the mean recovery was 92.4%. In rat skin, the mean total absorption was 8.7 % of the applied dose and the mean recovery was 93.1%. Based on total absorption, the difference in absorption between rat and human skin was about 8-fold.
Executive summary:

The study was designed to examine the in vitro percutaneous absorption of test substance through viable rat and human skin membranes using [I4C]test article.Test substance was tested in a standard sunscreen formulation at a concentration of 10.1% (w/w).The contact time was 24 hours.The objective of the study was to determine the dermal absorption of substance by monitoring the compound-related radioactivity in the receptor fluid in time. In addition to the amount of [14C]test item in the receptor fluid, the unabsorbed dose (skin wash after 24 h exposure) and the residues remaining in the skin membranes and in the stratum corneum were determined. The study was performed in flow-through diffusion cells. The study was conducted to comply with OECD 428 guideline under the GLP regulations.

The ultra violet screen was examined for its in vitro percutaneous absorption through viable human and rat skin membranes in a standard sunscreen formulation at 10.1 % (w/w). The exposure time was 24 hours. In human skin, the mean flux constant for the absorption of test substance was 0.058µg /cm2/h. The mean total absorption was 1.1 % of the applied dose and the mean recovery was 92.4%. In rat skin, the mean flux constant for the absorption oftest substance was 0.807µg/ cm2/h.The mean total absorption was 8.7 % of the applied dose and the mean recovery was 93.1%. Based on flux constants rat skin was ca 14 times more permeable to test article than human skin. Based on total absorption, the difference in absorption between rat and human skin was about 8-fold.