Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from July 9, 2007 to October 5, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Benzophenone-3
IUPAC Name:
Benzophenone-3
Constituent 2
Reference substance name:
2-Hydroxy-4-methoxybenzophenone
IUPAC Name:
2-Hydroxy-4-methoxybenzophenone
Constituent 3
Chemical structure
Reference substance name:
Oxybenzone
EC Number:
205-031-5
EC Name:
Oxybenzone
Cas Number:
131-57-7
Molecular formula:
C14H12O3
IUPAC Name:
(2-hydroxy-4-methoxyphenyl)(phenyl)methanone
Test material form:
other: solid
Radiolabelling:
no

Test animals

Species:
other: pig skin
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
Test System
Porcine ears were obtained from a local slaughter-house (Schlachthof Brensbach) immediately after slaughter and before steam cleaning. The outer ear region was washed and cleaned with cold water. A 400 ± 80 µm thick skin layer was obtained using a dermatome. Skin pieces were stamped out and the hair was removed with a scissor. The skin samples were used immediately. The surface of the skin which was in contact with the test item during the permeation assay was 1 cm2.
Per diffusion cell the ear of one animal was used. Thus, each cell represents one donor.

Administration / exposure

Vehicle:
other: methanol
Duration of exposure:
24 hours
Doses:
at an amount of 10 µL/cm²,
(corresponding to about 0.2 mg/cm² BZ-3 for BARNE-40/42 and BARNE-41/43, respectively)
No. of animals per group:
using six different ear skin samples in each experiment with the exception of 4 for BARNE-40 and 2 for BARNE-41 in experiment III, respectively.
Control animals:
no
Remarks:
Benzoic acid and Basic red 12 are used as positive and negative control substances
Details on study design:
The test items were applied on each skin sample at an amount of 10 µL/cm² (corresponding to about 0.2 mg/cm² and 0.6 mg/cm² BZ-3 for BARNE-40/42 and BARNE-41/43, respectively), left on the skin for 24 hours and then washed off using 3 x 1 mL eluent mixture for BARNE-40 and BARNE-41, and 3 x 1 mL methanol for BARNE-42 and BARNE-43. However, as the recovery was detected to be low (< 85 %) the wash off procedure was enhanced and the extraction procedure of the remaining skin and the tape strips was prolonged to improve the recovery of the reference item.
Details on in vitro test system (if applicable):
In each experiment one test item was investigated in 6 replicates under dynamic conditions with the exception of 4 for BARNE-40 and 2 for BARNE-41 in experiment III, respectively. The receptor solution was pumped with a flow rate of 1 - 2 mL/h for the first 8 hours and afterwards with a flow rate of 0.5-1.2 mL/h. The receptor vessels were changed at the listed time points. The volume of each sample was determined by measuring the weight difference of the vessels used for sampling. After checking the skin integrity, the test items were left on the skin for 24 hours under non-occluded conditions. Then the respective test item was washed off with eluent mixture for BARNE-40 and BARNE-41 or methanol for BARNE-42 and BARNE-43. Diffusion of the test item into the receptor buffer was monitored for 24 hours after the start of treatment.
After the penetration experiment, the skin samples were stripped 10 fold (2x5) with a 1 cm2 piece of Tesa-film and extracted. The remaining skin samples were also be extracted. 20 % EtOH in PBS was used as receptor solution.
The amount of Benzophenone-3 was determined in the receptor solutions, in the pipette washing solutions, in the combined washing solutions used to remove the test item after 24 hours, in the stripping solutions, in the skin extracts, and in the solution removed from the donor chambers after 24 hours as well as in the reference solutions.
Controls with Benzoic acid and Basic red 12 were used to check the performance of the skin penetration system every 3-4 months. The data of the latest control run were included as annex in this final report.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
- Receptor fluid, receptor chamber, donor chamber (in vitro test system):
After the penetration experiment, the skin samples were stripped 10 fold (2x5) with a 1 cm2 piece of Tesa-film and extracted. The remaining skin samples were also be extracted. 20 % EtOH in PBS was used as receptor solution.
- Skin preparation:
Porcine ears were obtained from a local slaughter-house (Schlachthof Brensbach) immediately after slaughter and before steam cleaning. The outer ear region was washed and cleaned with cold water. A 400 ± 80 µm thick skin layer was obtained using a dermatome. Skin pieces were stamped out and the hair was removed with a scissor. The skin samples were used immediately. The surface of the skin which was in contact with the test item during the permeation assay was 1 cm2 . Per diffusion cell the ear of one animal was used. Thus, each cell represents one donor.
- Stratum corneum (in vitro test system): (i.e tape strips)
The stratum corneum was separated by tape stripping from the lower skin layers. The tape strips (2 x 5 strips per sample) were pooled and analyzed as well as the remaining skin compartments. Analysis for the presence of test article was carried out by means of HPLC. The LOD was defined as 2.0 ng/mL and the LLOQ was 2.5 ng/mL in 20 % EtOH in PBS and eluent mixture.
Total recovery:
The lower limit of quantitation (LLOQ) for the method is the lowest amount of reference item which can be reproducibly detected. A deviation up to 20 % was accepted. For both matrices used the lower limit of quantitation of test article is about 2.5 ng/mL in saline and in eluent mixture. The limit of detection (LOD) is about 2.0 ng/mL.
For the determination of LLOQ and LOD both matrices were used, which was developed under the RCC-CCR study number 1098101 (non-GLP study). Extracts from tape strips and skin were performed in eluent mixture. Blank extracts were examined for interfering with the reference item.
Percutaneous absorptionopen allclose all
Dose:
200 ±9.73 µg/cm2
Parameter:
percentage
Absorption:
4.01 %
Remarks on result:
other: 0-24 hour
Remarks:
BARNE-40 (2 % BZ-3, O/W): Bioavailable portion (receptor fluid + epidermis + dermis)4.01±2.01
Dose:
583 ±42.1 µg/cm2
Parameter:
percentage
Absorption:
3.14 %
Remarks on result:
other: 0-24 hrs
Remarks:
BARNE-41 (6 % BZ-3, O/W): Bioavailable portion (receptor fluid + epidermis + dermis)3.14±1.86
Dose:
201 ±10.1 µg/cm2
Parameter:
percentage
Absorption:
3.34 %
Remarks on result:
other: 0-24hrs
Remarks:
BARNE-42 (2 % BZ-3, W/O): Bioavailable portion (receptor fluid + epidermis + dermis)3.34±2.22
Dose:
612 ±37.5 µg/cm2
Parameter:
percentage
Absorption:
3.14 %
Remarks on result:
other: 0-24 hrs
Remarks:
BARNE-43 (6 % BZ-3, W/O): Bioavailable portion (receptor fluid + epidermis + dermis)3.14±3.41

Any other information on results incl. tables

 

BARNE-40 (2 % BZ-3, O/W)

Amount of BZ-3 in

Expressed asµg/cm2of skin surface meanS.D. (n =9)

Expressed as % of dose meanS.D. (n =9)

Application volume

200

±

9.73

100

 

 

Receptor fluid

4.47

±

2.76

2.27

±

1.44

Stratum corneum(isolated by tape stripping)

1.03

±

0.683

0.524

±

0.353

Epidermis + Dermis (after 24 hours)

3.44

±

1.57

1.74

±

0.852

Washing solution (after 24 hours)

178

±

12.2

89.1

±

4.52

Recovery

187

±

9.80

93.6

±

3.88

Bioavailable portion (receptor fluid + epidermis + dermis)

7.91

±

3.73

4.01

±

2.01

 

BARNE-41 (6 % BZ-3, O/W)

Amount of BZ-3 in

Expressed asµg/cm2of skin surface meanS.D. (n =10)

Expressed as % of dose meanS.D. (n =10)

Application volume

583

±

42.1

100

 

 

Receptor fluid

6.92

±

2.07

1.20

±

0.401

Stratum corneum(isolated by tape stripping)

4.90

±

10.4

0.828

±

1.70

Epidermis + Dermis (after 24 hours)

11.3

±

11.7

1.94

±

1.91

Washing solution (after 24 hours)

512

±

41.9

87.8

±

5.72

Recovery

535

±

35.7

91.8

±

4.07

Bioavailable portion (receptor fluid + epidermis + dermis)

18.3

±

11.3

3.14

±

1.86

 

BARNE-42 (2 % BZ-3, O/W)

Amount of BZ-3 in

Expressed asµg/cm2of skin surface meanS.D. (n =10)

Expressed as % of dose meanS.D. (n =10)

Application volume

201

±

10.1

100

 

 

Receptor fluid

2.34

±

0.772

1.17

±

0.409

Stratum corneum(isolated by tape stripping)

1.31

±

2.50

0.637

±

1.21

Epidermis + Dermis (after 24 hours)

4.40

±

4.86

2.17

±

2.34

Washing solution (after 24 hours)

177

±

14.2

88.3

±

7.60

Recovery

185

±

10.3

92.3

±

5.27

Bioavailable portion (receptor fluid + epidermis + dermis)

6.74

±

4.63

3.34

±

2.22

 

BARNE-43 (6 % BZ-3, O/W)

Amount of BZ-3 in

Expressed asµg/cm2of skin surface meanS.D. (n =12)

Expressed as % of dose meanS.D. (n =12)

Application volume

612

±

37.5

100

 

 

Receptor fluid

5.89

±

3.11

0.970

±

0.548

Stratum corneum(isolated by tape stripping)

8.03

±

20.0

1.29

±

3.15

Epidermis + Dermis (after 24 hours)

13.4

±

20.5

2.17

±

3.22

Washing solution (after 24 hours)

543

±

64.5

88.8

±

9.58

Recovery

571

±

43.4

93.3

±

4.98

Bioavailable portion (receptor fluid + epidermis + dermis)

19.3

±

21.6

3.14

±

3.41

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, between around 3-4 % (between 7-18 µg/cm2) depending on the concentration of test article present in the test items BARNE-40, BARNE-41, BARNE-42 and BARNE-43 penetrated the skin samples during 24 hours and has to be considered as dermal bioavailable.
Executive summary:

This study was performed to obtain information about the percutaneous absorption and/or penetration properties of the test item in standard sunscreen formulations BARNE-40, BARNE-41, BARNE-42, BARNE-43 topically applied on viable porcine ear skin. Three independent experiments were performed with the test items BARNE-40, BARNE-41 and BARNE-42, respectively, using six different ear skin samples in each experiment with the exception of 4 for BARNE-40 and 2 for BARNE-41 in experiment III, respectively. Four independent experiments were performed with test item BARNE-43. The relevant compound in the test items was Benzophenone-3 (BZ-3).

For this study a total of 4 skin penetration experiments with four test items containing different amounts of reference item were performed. BZ-3 was detected in all samples relevant for dermal absorption, in the skin extracts as well as in the receptor solution samples increasing over the whole sampling time of 24 hours. Thus, BZ-3 is considered to have penetrated the skin.

It was shown that the penetration kinetics as well as the penetrated percentage of dose of BZ-3 is very similar in all the test items. BZ-3 was first detected after about 1 h. The amount penetrating the skin and entering the receptor fluid seemed nearly independent of the concentration of BZ-3 in the respective test item during an incubation time of up to 8 hours (exception BARNE-42). However, at 24 hours sampling the highest penetration into the receptor fluid was recorded for BARNE-41 and BARNE-43 and the differences to BARNE-40 and BARNE-42 was nearly 2.5 to 3 fold. Compared to the amount in the stratum corneum, a higher amount of BZ-3 remained in the epidermis and dermis (between 43.5-69.4 % of the penetrated amount) and is considered as bioavailable portion.

In each case, the highest amounts of BZ-3 in the different compartments were noted using the test items with the highest BZ-3 concentrations. With all test items there was no indication of saturation. No relevant differences were observed with respect to the

composition of the test items (i.e. O/W or W/O).