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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26th January 2012 to 24th February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD (Guidance Document No. 28 (2004). The Conduct of Skin Absorption Studies.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance Note No. 156 (2011). Dermal Absorption
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Scientific Committee on Consumer Safety (SCCS) (2010).
Qualifier:
according to guideline
Guideline:
other: COLIPA (1997). Cosmetic Ingredients: Guidelines for Percutaneous Absorption/Penetration. The European Cosmetic and Perfumery Association.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Mexoryl SBO
IUPAC Name:
Mexoryl SBO
Details on test material:
- Name of test material (as cited in study report): Mexoryl SBO
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Nme of substance: R0034833C (other names Mexoryl SBO, 79705)
- DTL test substance reference number: TS00194/001
- Source and lot/batch No.of test material: L'Oreal, Lot no 0147352
- Expiration date of the lot/batch: 29th August 2014
- Purity test date: Not stated

RADIOLABELLING INFORMATION
- Batch/Lot no: TS00194/002; Lot 001 = nominal 37 MBq (1 mCi)
Lot 002 = nominal 37 MBq (1 mCi)
Lot 003 = nominal 3.7 MBq (100 μCi)
- Radiochemical purity: 99.6%
- Specific activity: 57 mCi/mmol (2.11 GBq/mmol), 8.86 MBq/mg
- Locations of the label: alkyl carbon adjacent to the cylcopentane ring structure
- Expiration date of radiochemical substance: Not stated - determined by radiochemical purity

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
A pre-formulation (819115P) containing 2.361% of tetrahydrojasmonate was prepared for use to incorporate the radiolabelled active ingredient (DTL test substance reference number: TS00196/002/002).
A solution of radiolabelled material was prepared in a water/dipropylene glycol solution (70:30 v/v) and mixed gently for one minute until visually homogeneous.

- Final dilution of a dissolved solid, stock liquid or gel: The unlabelled material R0034833C (TS00194/001) was added (93.9 mg) to the radiolabelled stock solution prepared (detailed above). After mixing, 1886 mg of pre-formulation 819115P (TS00196/002/002) was added. Twenty glass beads (1.7-2 mm) were added to the formulation and the formulation was mixed by vortexing for two minutes, until a visually homogeneous emulsion was achieved.


FORM AS APPLIED IN THE TEST (if different from that of starting material) Homogenous cream as confirmed by macroscopic and microscopic examination performed immediately after preparation and at 24 hours following preparation


OTHER SPECIFICS:
- Homogeneity and radioactivity content confirmed by liquid scintillation analysis (LSC)following formulation and again post 24 hours.
- Radiochemical purity, stability and concentration of the formulation was confirmed by HPLC:
Radiolabelling:
yes
Remarks:
Information specified in Source of Test Material, Certificate of Analysis in the report

Administration / exposure

Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: National Disease Research Interchange (NDRI, Philadelphia, Pennsylvania, U.S.A.)
- Ethical approval if human skin:
- Type of skin:
- Preparative technique: Discs of approximately 3.3 cm diameter of prepared skin membrane were mounted, dermal side down, in diffusion cells held together with individually numbered clamps and placed in a water bath maintained at 32°C ± 1°C.
- Thickness of skin (in mm): 400 μm
- Membrane integrity check: assessed by measurement of the electrical resistance across the skin membrane.
- Storage conditions: stored frozen, at approximately -20°C, on aluminium foil until required for use
- Justification of species, anatomical site and preparative technique:

PRINCIPLES OF ASSAY
- Diffusion cell: static glass diffusion cell
- Receptor fluid: phosphate buffered saline (PBS) receptor fluid
- Solubility od test substance in receptor fluid: Freely soluble
- Static system: Yes
- Flow-through system: No
- Test temperature: 32°C ± 1°C.
- Humidity: Not measured
- Occlusion: No
- Reference substance(s): None
- Other:

Results and discussion

Dermal irritation:
not examined
Absorption in different matrices:
-Three of the 12 dosed cells were rejected (cells 17,18 and 19). The penetration profiles of these rejected cells suggested that the membranes had become damaged during the experiment.
The mean recovery of the applied test material was very good at 99.7 ± 5.31%, with individual cell values ranging from 90.7% to 110% (n=9).
The percentage (mean ± SD) of remaining tetrahydrojasmonate that was removed by washing the surface of the skin 24 hours after application was 94.1 ± 5.31% (76.8 ± 4.33 μg/cm2). The mean percentage of the dose present in the outer layers of the stratum corneum was 2.54 ± 1.31% of the applied dose (2.07 ± 5.31 μg/cm2) with 2.30 ± 1.12% of the dose (1.88 ± 0.914 μg/cm2) present in the remaining epidermis.
The percentage (mean ± SD) recovered from the dermis was 0.130 ± 0.098% of the applied dose (0.106 ± 0.080 μg/cm2).
The proportion (mean ± SD) of the applied dose of tetrahydrojasmonate present in receptor fluid following the total 24 hour exposure was 0.494 ± 0.520%. This percentage equated to 0.403 ± 0.424 μg/cm2.
The mean total non-absorbed dose (donor chamber, skin wash, stratum corneum and flange skin) was 96.8 ± 5.79% (79.0 ± 4.73 μg/cm2) of the applied dose.
The mean total systemically available dose of (remaining epidermis, dermis and receptor fluid) was 2.92 ± 1.52% of the tetrahydrojasmonate applied dose corresponding to 2.39 ± 1.24 μg/cm2.
Total recovery:
- Total recovery: 99.7 ± 5.31%,
- Recovery of applied dose acceptable: Yes
- Results adjusted for incomplete recovery of the applied dose: No
- Limit of detection (LOD): LOD is approximately half the LOQ value
- Quantification of values below LOD or LOQ:not included in means, SD's and totals and reported as
Percutaneous absorption
Key result
Time point:
24 h
Dose:
4% in atypical leave-on skin care formulation
Parameter:
percentage
Absorption:
0.494 %

Any other information on results incl. tables

The radiochemical purity of the combined isomers of [14C]-tetrahydrojasmonate was determined to be 100%.

Stability of the formulated test materials over a period of time longer than that used in this study was also be confirmed using the HPLC methodology in the report.

LSC analysis of subsamples of the tetrahydrojasmonate ‘Leave-on’ skin care formulation confirmed that:

• Subsamples taken from the formulation immediately following preparation showed 4.06% deviation between the replicates. Post 24 hour analysis showed 0.911% deviation between the replicates. These values demonstrated that the tetrahydrojasmonate ‘Leave-on’ skin care formulation was homogenous prior to application and remained homogenous for a period of time longer than that used in this study.

• The achieved concentration was 40.8 mg tetrahydrojasmonate/g (4.08% tetrahydrojasmonate) of formulation.

HPLC analysis of the tetrahydrojasmonate ‘Leave-on’ skin care formulation immediately after preparation, confirmed that the radiochemical purity of the formulated radiolabelled test material was 100%.

The analysis performed 24 hours post application was 100%. This confirmed that the [14C]-tetrahydrojasmonate ‘Leave-on’ skin care formulation was stable for at least a 24 hour period.

Applicant's summary and conclusion

Conclusions:
The results obtained in this study indicate that tetrahydrojasmonate at 4% in a typical leave-on skin care formulation penetrated through human dermatomed skin at a very slow rate. The extent of tetrahydrojasmonate penetration through human skin amounted to only 0.494 ± 0.520% (0.403 ± 0.424 μg/cm2) of the applied dose, after 24 hours.
The mean total systemically available dose of tetrahydrojasmonate (remaining epidermis plus dermis and receptor fluid) was 2.92 ± 1.52% of the applied dose (corresponding to 2.39 ± 1.24 μg/cm2).
Executive summary:

The purpose of this study was to determine the in vitro percutaneous penetration of tetrahydrojasmonate through human dermatomed skin over 24 hours, to aid the quantitative assessment of the risk arising from skin contact with a typical ‘Leave-on’ skin care formulation containing a nominal 4% (w/w) tetrahydrojasmonate. The dose preparation was applied to the skin for 24 hours to mimic in-use conditions. The mass balance and distribution of tetrahydrojasmonate within the test system following the 24 exposure period was also determined.

The study was conducted according to the OECD principles of Good Laboratory Practice and was performed following the OECD and SCCS guidelines and guidance documents for skin penetration studies.

Prior to dosing, the membrane integrity was checked by measurement of electrical resistance. The doses were applied to the surface of 12 intact skin membranes (from 4 human donors) at a rate of 2 mg/cm2, corresponding to a nominal 80 μg/cm2 of tetrahydrojasmonate. At the end of the 24 hour exposure period a mass balance procedure was performed. The skin surface was washed with 2% sodium dodecyl sulphate (SDS) in water (2 x 762 μL) followed by water (2 x 762 μL). Between each set of washes the washing fluid was aspired with a pipette tip, after which the skin surface was dried with cotton wool swabs. The stratum corneum was removed by a tape stripping process removing a maximum of 20 strips from each skin membrane. The flange skin was cut away from the dermis and the epidermis on the remaining skin disc was separated from the dermis using a heat separation technique.

The application rates and exposure conditions used in this study were designed to simulate predicted normal human exposure to the test material.

The penetration process was monitored using [14C]-radiolabelled tetrahydrojasmonate, which was incorporated into the formulation, prior to application. The receptor fluid was phosphate buffered saline (PBS), in order to ensure that adequate sink conditions were maintained for this particular test material. The distribution of tetrahydrojasmonate within the test system was measured and a 24 hour penetration profile was determined by collecting receptor fluid samples 1, 2, 4, 8, 12, 16, 20 and 24 hours following application. The samples were analysed by liquid scintillation counting (LSC).

Results

LSC analysis of the dose preparation confirmed that the dose preparation was homogeneous both prior to and for a period of time beyond that used in the study.

HPLC analysis confirmed that prior to the addition of developer 178914 U, the radiochemical purity of [14C]-tetrahydrojasmonate when formulated was 95% for at least a 1 day period.

Tetrahydrojasmonate penetration

After a short lag phase of 2 hours, the mean tetrahydrojasmonate penetration rate was 0.018 μg/cm2/h between 2-24 hours. Between 0-24 hours, the overall penetration rate was, on average, 0.016 μg/cm2/h.

The amounts (mean ± SD) of tetrahydrojasmonate that penetrated through human skin at 4, 8 and 12 hours were 0.007 ± 0.008 μg/cm2, 0.038 ± 0.045 μg/cm2 and 0.098 ± 0.111 μg/cm2, respectively. These respective amounts expressed as percentages of the applied dose were 0.008, 0.047 and 0.120%. The mean amount that penetrated over the entire 24 hour exposure period was 0.403 ± 0.424 μg/cm2, corresponding to 0.494% of the applied dose.

Mass balance and tetrahydrojasmonate distribution

Three of the 12 dosed cells were rejected. The penetration profiles of these rejected cells suggested that the membranes had become damaged during the experiment.

The mean recovery of the applied test material was very good at 99.7 ± 5.54%, with individual cell values ranging from 90.7% to 110% (n=9).

The percentage (mean ± SD) of remaining tetrahydrojasmonate that was removed by washing the surface of the skin 24 hours after application was 94.1 ± 5.31% (76.8 ± 4.33 μg/cm2). The mean percentage of the dose present in the outer layers of the stratum corneum was 2.54 ± 1.31% of the applied dose (2.07 ± 1.07 μg/cm2) with 2.30 ± 1.12% of the dose (1.88 ± 0.914 μg/cm2) present in the remaining epidermis.

The percentage (mean ± SD) recovered from the dermis was 0.130 ± 0.098% of the applied dose (0.106 ± 0.080 μg/cm2).

The proportion (mean ± SD) of the applied dose of tetrahydrojasmonate present in receptor fluid following the total 24 hour exposure was 0.494 ± 0.520%. This percentage equated to 0.403 ± 0.424 μg/cm2.

The mean total non-absorbed dose (donor chamber, skin wash, stratum corneum and flange skin) was 96.8 ± 5.79% (79.0 ± 4.73 μg/cm2) of the applied dose.

The mean total systemically available dose of (remaining epidermis, dermis and receptor fluid) was 2.92 ± 1.52% of the tetrahydrojasmonate applied dose corresponding to 2.39 ± 1.24 μg/cm2.

Conclusion

The results obtained in this study indicate that tetrahydrojasmonate at 4% in a typical ‘Leave-on’ skin care formulation penetrated through human dermatomed skin at a very slow rate. The extent of tetrahydrojasmonate penetration through human skin amounted to only 0.494 ± 0.520% (0.403 ± 0.424 μg/cm2) of the applied dose, after 24 hours.

The mean total systemically available dose of tetrahydrojasmonate (remaining epidermis plus dermis and receptor fluid) was 2.92 ± 1.52% of the applied dose (corresponding to 2.39 ± 1.24 μg/cm2).

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