Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 18 - July 31, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

EC Number:

943-175-7

IUPAC Name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Mono-RHL EXP:
Mono-RHL EXP (also known as Rhamnolipid R1), Batch no. 15.6.1-A,
SEAC S Number S3295301, was supplied by the Sponsor and stored at ambient temperature.
The expiry date/retest date was April 2017. The Rha1-C10-C10 content was 51% (w/w). The
physical appearance was light brown crystalline lumps

EXP 1411-01
EXP 1411-01 (also known as Rhamnolipid sample EXP1411-01 – Batch E2.2 (Unilever ID3),
Batch no. E2.2, SEAC S Number S3289301, was supplied by the Sponsor and stored at
ambient temperature. The expiry date was January 2017. The Rha2-C10-C10 content was
57.6% (w/w). The physical appearance was light brown crystalline lumps

Test animals

Details on test animals or test system and environmental conditions:

Full-thickness human skin samples were obtained from four female donors aged 44 to
58 years old. All donors gave informed consent, prior to undergoing surgery, for their excised
skin to be used for scientific purposes. Two samples were obtained from donors attending
NHS Lothian. The skin was transferred to Charles River on ice, where it was cleaned of
subcutaneous fat and connective tissue using a scalpel. The skin samples were washed in cold. running water and dried using tissue paper. The skin samples were then cut into smaller
pieces, vacuum packed and placed into self sealing plastic bags. Two samples were obtained
frozen from Tissue Solutions (a tissue bank), these were not further processed on arrival.
All skin samples were stored in a freezer set to maintain a temperature of -20°C until used in
the study. The age and sex of the donor and site from which the skin was taken, were
recorded centrally and in the study records.

Administration / exposure

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Full-thickness human skin samples
- Ethical approval if human skin: All donors gave informed consent, prior to undergoing surgery, for their excised skin to be used for scientific purposes
- Preparative technique: The skin samples were washed in cold running water and dried using tissue paper. The skin samples were then cut into smaller pieces, vacuum packed and placed into self sealing plastic bags. Two samples were obtained frozen from Tissue Solutions (a tissue bank), these were not further processed on arrival.The thickness of the full-thickness membranes was measured using a micrometer. Split-thickness membranes were prepared by pinning the full-thickness skin, stratum corneum uppermost, onto a raised cork board and cutting at a setting equivalent to 200-400 μm depth using a Zimmer® electric dermatome. The membranes were then laid out onto aluminium foil and the thickness of the membranes measured using a micrometer
- Thickness of skin (in µm): 200-400 μm
- Membrane integrity check: Skin samples were allowed to equilibrate at 32°C ± 1°C for ca 30 min. PBS (1 mL) was then added to the donor chamber and the skin samples were allowed to equilibrate for a further ca 30 min. The electrical resistance was then measured using a Tinsley Databridge (Model: 6401) set at low voltage alternating current, 1000 Hz with a maximum voltage of 300 mV root-mean-squared (rms) in the parallel equivalent circuit mode. Any skin sample
exhibiting a resistance less than 10.9 kΩ was excluded from subsequent absorption
measurements. The PBS was removed from the skin surface, and then the skin was rinsed with water and dried with tissue paper.
- Storage conditions: The split-thickness membranes were stored in a freezer set to maintain a temperature of -20°C for a maximum of 2 months
- Justification of species, anatomical site and preparative technique:

PRINCIPLES OF ASSAY
- Diffusion cell: A static diffusion cell system (PermeGear Inc) was used
- Receptor fluid: Phosphate buffered saline containing sodium azide (0.01%, w/v) was used as the receptor fluid throughout the study. The receptor fluid was stored in a refrigerator set to maintain a
temperature of 4°C.
- Solubility of test substance in receptor fluid: Rha1-C10-C10 and Rha2-C10-C10 have water solubility >300 mg/mL. The water solubility of the test items are >13,500 times the maximum concentration of the test items in the aqueous receptor fluid. Therefore, the receptor fluid was not rate limiting for solubility
- Static system: Split-thickness skin was removed from a freezer set to maintain a temperature of -20°C and allowed to reach ambient temperature. The receptor chambers were connected to a circulating waterbath. Magnetic stirrer bars were placed in the receptor fluid chambers which were filled with receptor fluid. Sections of split-thickness skin (ca 1.5 x 1.5 cm) were cut and mounted in the diffusion cells between the donor and receptor chamber. The donor chamber was tightened into place with a clamp. Each cell was filled above the calibration line on the receptor fluid arm with receptor fluid and checked visually afterwards to confirm that no cells were leaking (leak test). No cells were found to be leaking. No air bubbles were present in the receptor fluid chamber.
- Test temperature: 32°C ± 1°C
-Internal standards: The internal standard, n-Dodecyl β-D-maltoside, Batch no. SLBL6210V was stored in a freezer set to maintain a temperature of -20°C. The purity of the internal standard was 100%.
- Humidity:
- Other:

Results and discussion

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.183%, w/v

Parameter:

amount

Absorption:

<= 0.36 mg cm-2 h-1

Remarks on result:

other: Rha1-C10-C10 in Test Preparation 5

Percutaneous absorption 2

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.202%, w/v

Parameter:

amount

Absorption:

<= 0.36

Remarks on result:

other: Rha2-C10-C10 in Test Preparation 5

Percutaneous absorption 3

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.349%

Parameter:

amount

Absorption:

<= 0.34 mg cm-2 h-1

Remarks on result:

other: Rha2-C10-C10 in Test Preparation 2

Percutaneous absorption 4

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.175%, w/v

Parameter:

amount

Remarks:

total absorbed dose

Absorption:

<= 0.36 mg cm-2 h-1

Remarks on result:

other: Rha2-C10-C10 Test preparaion 1

Percutaneous absorption 5

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.433%

Parameter:

amount

Absorption:

<= 0.36 mg cm-2 h-1

Remarks on result:

other: Rha2-C10-C10 in Test Preparation 3

Percutaneous absorption 6

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.405%

Parameter:

amount

Absorption:

<= 0.38 mg cm-2 h-1

Remarks on result:

other: Rha2-C10-C10 in Test Preparation 4

Any other information on results incl. tables

Rha2-C10-C10 in Test Preparation 1 (0.175%, w/v)

Test Preparation 1 (Tai Chi 20% Dirhamnolipid Replacement formulation) was applied to a of 12 samples of human skin obtained from 4 different donors. The overall recovery of Rha2-C10-C10 for all samples was within 100% ± 15% (acceptance criteria according to the SCCS guidance document) with the exception of Cell 62. The mass balance for Cell 62 was131.92% of the applied dose, which was outside this range, therefore, Cell 62 was excluded from mean and SD calculations. The following results are therefore, based on 11 samples of human skin.

The mean mass balance was determined to be 105.52% (SD: 4.96%) of the applied dose. At 30 min post application, 96.69% of the applied dose was removed by washing. The skin wash, tissue swab (this sample also included the 24 tissue swab) and tip samples contained 93.78%, 0.99% (most values <LLOQ) and 0.94% (all values <LLOQ), respectively. A further 0.99% (most values <LLOQ) of the applied dose was recovered from the donor chamber wash. Therefore, the total dislodgeable dose was 96.69% of the applied dose. The mean total unabsorbed dose was 102.21% of the applied dose. This consisted of the dislodgeable dose at 24 h, unexposed skin (0.93%, all values <LLOQ) and the test item associated with the stratum corneum (4.59%, most values <LLOQ). The epidermis (epidermis value is a sum of epidermis and cling film values) and dermis contained 1.85% (all values <LLOQ) and 0.93% (all values <LLOQ) of the applied dose, respectively. The total absorbed dose (0.53%) was the sum of the receptor fluid (0.12%, all values <LLOQ) and receptor chamber wash (0.42%, all values <LLOQ). Dermal delivery was 3.31% of the applied dose.

 

Rha2-C10-C10 in Test Preparation 2 (0.349%, w/v)

Test Preparation 2 (Tai Chi 40% Dirhamnolipid Replacement formulation) was applied to a total of 12 samples of human skin obtained from 4 different donors. The overall recovery of Rha2-C10-C10 for all samples was within 100% ± 15% (acceptance criteria according to the SCCS guidance document) with the exception of Cell 14. The mass balance for Cell 14 was 78.72% of the applied dose, which was outside this range, therefore, Cell 14 was excluded from mean and SD calculations. The following results are therefore, based on 11 samples of human skin.

The mean mass balance was determined to be 99.24% (SD: 6.69%) of the applied dose. At 30 min post application, 93.80% of the applied dose was removed by washing. The skin wash, tissue swab (this sample also included the 24 tissue swab) and tip samples contained 92.23%, 0.93% (all values >LLOQ with the exception of Cell 22) and 0.64% (all values >LLOQ with the exception of Cells 17 and 20), respectively. A further 0.57% (most values <LLOQ) of the applied dose was recovered from the donor chamber wash at 24 h. Therefore, the total dislodgeable dose was 94.36% of the applied dose. The mean total unabsorbed dose was 97.52% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.51%) and the test item associated with the stratum corneum (2.65%, most values <LLOQ).

The epidermis (epidermis value is a sum of epidermis and cling film values) and dermis contained 1.02% (all values <LLOQ) and 0.45% (all values <LLOQ) of the applied dose, respectively. The total absorbed dose (0.25%) was the sum of the receptor fluid (0.06%, all values <LLOQ) and receptor chamber wash (0.19%, all values <LLOQ). Dermal delivery was 1.72% of the applied dose.

The distribution of Rha2-C10-C10, by mass, at 24 h post dose is shown in Table 4. The total absorbed dose and dermal delivery were 0.34 μg/cm2 and 2.37 μg/cm2, respectively, of Rha2-C10-C10.

Rha2-C10-C10 in Test Preparation 3 (0.433%, w/v)

Test Preparation 3 (Surflex 40% Dirhamnolipid Replacement #2 formulation) was applied to a total of 12 samples of human skin obtained from 4 different donors. The overall recovery of Rha2-C10-C10 for all samples was within 100% ± 15% (acceptance criteria according to the SCCS guidance document) with the exception of Cell 25 and Cell 26. The mass balance for Cell 25 and Cell 26 was 115.34% and 119.96% of the applied dose, which was outside this range, therefore, Cells 25 and 26 were excluded from the mean and SD calculations. The following results are therefore, based on 10 samples of human skin.

The mean mass balance was determined to be 104.53% (SD: 6.16%) of the applied dose. At 30 min post application, 100.10% of the applied dose was removed by washing. The skin wash, tissue swab (this sample also included the 24 tissue swab) and tip samples contained 98.89%, 0.68% (all values >LLOQ with the exception of Cells 31 and 35) and 0.53% (all values >LLOQ with the exception of Cells 35 and 71), respectively. A further 0.38% (most values <LLOQ) of the applied dose was recovered from the donor chamber wash at 24 h.

Therefore, the total dislodgeable dose was 100.48% of the applied dose. The mean total unabsorbed dose was 103.04% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.37%, all values <LLOQ) and the test item associated with the stratum corneum (2.18%, most values <LLOQ). The epidermis (epidermis value is a sum of epidermis and cling film values) and dermis contained 0.90% (all values <LLOQ) and 0.37% (all values <LLOQ) of the applied dose, respectively. The total absorbed dose (0.22%) was the sum of the receptor fluid (0.05%, all values <LLOQ) and receptor chamber wash (0.17%, all values <LLOQ). Dermal delivery was 1.49% of the applied dose.

The total absorbed dose and dermal delivery were 0.36 μg/cm2 and 2.48 μg/cm2, respectively, of Rha2-C10-C10.

Rha2-C10-C10 in Test Preparation 4 (0.405%, w/v)

Test Preparation 4 (Shadow 40% Dirhamnolipid Replacement #2 formulation) was applied to a total of 12 samples of human skin obtained from 4 different donors. The overall recovery of Rha2-C10-C10 for all samples was within 100% ± 15% (acceptance criteria according to the SCCS guidance document) with the exception of Cell 38. The mass balance for Cell 38 was 84.49%, which was outside this range, therefore, Cell 38 was excluded from mean and SD calculations. The following results are therefore, based on 11 samples of human skin.

The mean mass balance was determined to be 95.72% (SD: 9.70%) of the applied dose. At 30 min post application, 91.97% of the applied dose was removed by washing. The skin wash, tissue swab (this sample also included the 24 tissue swab) and tip samples contained 91.08%, 0.49% (some values <LLOQ) and 0.40% (some values <LLOQ), respectively. A further 0.38% (most values <LLOQ) of the applied dose was recovered from the donor chamber wash at 24 h. Therefore, the total dislodgeable dose was 92.35% of the applied dose.

The mean total unabsorbed dose was 94.43% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.36%, all values <LLOQ) and the test item associated with the stratum corneum (1.72%, all values <LLOQ). The epidermis (epidermis value is a sum of epidermis and cling film values) and dermis contained 0.71% (all values <LLOQ) and 0.36% (all values <LLOQ) of the applied dose, respectively. The total absorbed dose (0.22%) was the sum of the receptor fluid (0.05%, most values <LLOQ) and receptor chamber wash (0.16%, all values <LLOQ). Dermal delivery was 1.29% of the applied dose.

Rha2-C10-C10 in Test Preparation 5 (0.202%, w/v)

Test Preparation 5 (Shadow 40% Dirhamnolipid Replacement (Di:Mono 50:50) #2 formulation) was applied to a total of 12 samples of human skin obtained from 4 different donors. The overall recovery of Rha2-C10-C10 for all samples was within 100% ± 15% (acceptance criteria according to the SCCS guidance document) with the exception of Cell 53.

The mass balance for Cell 53 was 79.85% of the applied dose, which was outside this range, therefore, Cell 53 was excluded from mean and SD calculations. The following results are therefore, based on 11 samples of human skin.

The mean mass balance was determined to be 95.75% (SD: 6.78%) of the applied dose. At 30 min post application, 88.25% of the applied dose was removed by washing. The skin wash, tissue swab (this sample also included the 24 tissue swab) and tip samples contained 86.75%, 0.79% (most values <LLOQ) and 0.71% (most values <LLOQ), respectively. A further 0.71% (all values <LLOQ) of the applied dose was recovered from the donor chamber wash at 24 h. Therefore, the total dislodgeable dose was 88.96% of the applied dose. The mean total unabsorbed dose was 93.22% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.71%, all values <LLOQ) and the test item associated with the stratum corneum (3.54%, all values <LLOQ). The epidermis (epidermis value is a sum of epidermis and cling film values) and dermis contained 1.42% (all values <LLOQ) and 0.71% (all values <LLOQ) of the applied dose, respectively. The total absorbed dose (0.41%) was the sum of the receptor fluid (0.09%, all values <LLOQ) and receptor chamber wash (0.32%, all values <LLOQ). Dermal delivery was 2.54% of the applied dose.

The total absorbed dose and dermal delivery were 0.36 μg/cm2 and 2.24 μg/cm2, respectively, of Rha2-C10-C10.

Rha1-C10-C10 in Test Preparation 5 (0.183%, w/v)

Test Preparation 5 (Shadow 40% Dirhamnolipid Replacement (Di:Mono 50:50) #2 formulation) was applied to a total of 12 samples of human skin obtained from 4 different donors. The overall recovery of Rha1-C10-C10 for all samples was within 100% ± 15% (acceptance criteria according to the SCCS guidance document), with the exception of Cell 53. The mass balance for Cell 53 was 84.22% of the applied dose, which was outside this range, therefore, Cell 53 was excluded from mean and SD calculations. The following results are therefore, based on 11 samples of human skin.

The mean mass balance was determined to be 101.07% (SD: 5.85%) of the applied dose. At 30 min post application, 92.28% of the applied dose was removed by washing. The skin wash, tissue swab (this sample also included the 24 tissue swab) and tip samples contained 90.53%, 0.91% (most values <LLOQ) and 0.84% (most values <LLOQ), respectively. A further 0.83% (all values <LLOQ) of the applied dose was recovered from the donor chamber wash at 24 h. Therefore, the total dislodgeable dose was 93.12% of the applied dose. The mean total unabsorbed dose was 98.10% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.83%, all values <LLOQ) and the test item associated with the stratum corneum (4.15%, all values <LLOQ). The epidermis (epidermis value is a sum of epidermis and cling film values) and dermis contained 1.66% (all values <LLOQ) and 0.83% (all values <LLOQ) of the applied dose, respectively. The total absorbed dose (0.48%) was the sum of the receptor fluid (0.10%, all values <LLOQ) and receptor chamber wash (0.38%, all values <LLOQ). Dermal delivery was 2.97% of the applied dose.

Applicant's summary and conclusion

Conclusions:

In conclusion, following topical application of Rha2-C10-C10 in Test Preparation 1 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.36 μg/cm2 (≤0.53% of the applied dose) and ≤ 2.24 μg/cm2 (≤3.31% of the applied dose), respectively.
Following topical application of Rha2-C10-C10 in Test Preparation 2 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.34 μg/cm2 (≤0.25% of the applied dose) and ≤2.37 μg/cm2 (≤1.72% of the applied dose), respectively.
Following topical application of Rha2-C10-C10 in Test Preparation 3 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.36 μg/cm2 (≤0.22% of the applied dose) and ≤2.48 μg/cm2 (≤1.49% of the applied dose), respectively.
Following topical application of Rha2-C10-C10 in Test Preparation 4 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.38 μg/cm2 (≤0.22% of the applied dose) and ≤2.26 μg/cm2 (≤1.29% of the applied dose), respectively.
Following topical application of Rha2-C10-C10 in Test Preparation 5 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.36 μg/cm2 (≤0.41% of the applied dose) and ≤2.24 μg/cm2 (≤2.54% of the applied dose), respectively.
Following topical application of Rha1-C10-C10 in Test Preparation 5 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha1-C10-C10 through human skin in vitro were ≤0.36 μg/cm2 (≤0.48% of the applied dose) and ≤2.24 μg/cm2 (≤2.97% of the applied dose), respectively.
Almost all receptor values were less than the lower limit of quantification (LLOQ). The majority of stratum corneum values were