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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 428) performed under GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
adopted on 13th April 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
RCC Ltd., Itlingen, Swizerland

Test material

Constituent 1
Test material form:
solid - liquid: suspension
Remarks:
dispersion containing micronized ETH50 (non-nano form)
Details on test material:
Please refer to "Confidential details on test material".
Radiolabelling:
yes
Remarks:
(14C)

Test animals

Species:
other: human skin and rat skin

Administration / exposure

Type of coverage:
other: non-occluded
Vehicle:
other: Formulation with surfactant (Plantacare®2000 UP), thickener (xanthan gum) and emulsifier (propylene glycol) in water (MiliQ)
Duration of exposure:
24 hours
Doses:
- Nominal dose: 2000 µg/cm²
- Actual dose: 1912 µg/cm²
- Actual dose determined as follows: The final formulation had a total volume of 8 mL and a concentration of 90.88 mg [14C] test item/mL as determined by LSC
- Dose volume: 13 µL
- Rationale for dose selection: In accordance with the usual dermal application rate for sun creams and with the in vivo dermal absorption study (RCC Ltd / Study No. A22432).
No. of animals per group:
HUMAN SKIN: two females (7 skin samples)
RAT SKIN: two males (7 skin samples)
Control animals:
no
Details on study design:
DOSE PREPARATION
- Stock solution: About 16 mg [14C] test item were dissolved in 10 mL chloroform.
- Formulation Procedure: 1216 mg unlabeled item for dilution was placed into a flask. A volume of 8.5 mL stock solution containing 15 mg [14C] labeled test item was added and the test item was totally dissolved by addition of 300 mL chloroform. This procedure yielded [14C] labeled test item with a final specific radioactivity of 78 kBq/mg (2.11 μCi/mg). The major part of the solvent was removed by a rotatory evaporator and finally by a gentle stream of nitrogen. Prior to the micronization Plantacare 2000 and water (MilliQ) were added to a total weight of 2.54 g. After micronization the formulated test item was mixed with 0.6 % xanthan gum/propylene glycol and diluted with 6.38 g water (MilliQ). The pH value of the final formulation was adjusted to pH 6.5 with citric acid (20% aqueous solution).
- Method of storage: not specified

APPLICATION OF DOSE: by microsyringe onto the skin membrane

TEST SITE
- Preparation of test site: The skin samples were removed from the freezer and allowed to thaw at room temperature. The subcutaneous fat was carefully removed from the full thickness skin and pieces of about 4 x 5 cm were stretched evenly over a cork block, with stratum corneum uppermost. Skin sections of about 200 µm thickness were cut off from the top using a dermatome.
- Area of exposure: 0.64 cm² exposed to the donor chamber (skin sections of ca. 1.8 x 1.8 cm mounted in diffusion cells).
- Type of cover of donor chambers: application of the test item: by permeable tape (non-occluded conditions); test of skin membrane integrity: by adhesive tape (occluded conditions)

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: skin membrane surface was rinsed three times with about 0.5 mL of a mild shower gel solution (1 % in tap water) followed by one time with 0.5 mL water each
- Time after start of exposure: 24 hours

SAMPLE COLLECTION
- Rinse fractions: combined according to the individual cells
- Stratum corneum: tape stripping (3-5 times) combined to one specimen, aliquots were measured for radioactivity after solubilization
- Remaining skin membranes after stripping: digested in tissue solubilizer
- Cell wash: in 150 mL chloroform

ANALYSIS
- Method type(s) for identification: LSC or HPLC connected to radioactivity flow monitor
- Liquid scintillation counting results (cpm) converted to dpm as follows: All specimens were corrected for background by subtraction of appropriate blank values from specimen count rates to give net "dpm" per specimen. The penetration rate, i.e. the maximum flux, was calculated as the slope determined by linear regression of the cumulative penetration versus time curve at steadystate conditions. For the integrity check with tritiated water the permeability of the membrane was expressed by the permeability coefficient.
- Validation of analytical procedure: not specified
- Limits of detection and quantification: calculated according to Currie (1968)
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of human (cadaver) skin: Institut für Pathologie, Kantonsspital Basel, Basel, Switzerland
- Ethical approval if human skin: yes
- Type of skin: cadaver full thickness, upper leg dorsal, Caucasian donors
- Age of donors: 99 and 78 (females)
- Preparative technique: split thickness
- Thickness of skin: 0.2 mm
- Membrane integrity checks: performed
- Storage conditions: at about -20°C until preparation of the skin membranes

PRINCIPLES OF ASSAY
- Diffusion cell: automated flow-through cell system
- Receptor fluid: 6 % (w/v) polyethoxyoleate (PEG 20 oleyl ether) dissolved in physiological saline (0.9% NaCl w/v)
- Flow-through system: Two system parts containing seven flow-through diffusion cells each were placed in one aluminum manifold connected to a water bath. Each diffusion cell consisted of a donor and receptor chamber, the latter connected to a multi-channel peristaltic pump. During the given time intervals, the effluent from the cell was collected directly into vials on a fraction collector.
- Flow rate: 3 mL/h during the testing period
- Collection of perfusate: 0 - 6 hours: 1 hour intervals (6 intervals), 6 - 24 hours: 2 hours intervals (9 intervals)
- Humidity: not specified
- Occlusion: application of the test item: by permeable tape (non-occluded conditions); test of skin membrane integrity: by adhesive tape (occluded conditions)
- Reference substance: no data

Results and discussion

Absorption in different matrices:
- Receptor fluid, receptor chamber, donor chamber: yes
- Skin preparation: (mean penetration): yes
- Stratum corneum: tape strips
Total recovery:
- Total recovery (mean): rat skin membrane: 95.48; human skin membrane: 91.83
- Recovery of applied dose acceptable: yes
- Results adjusted for incomplete recovery of the applied dose: no
Percutaneous absorptionopen allclose all
Dose:
1912 µg/cm²
Parameter:
percentage
Absorption:
0.1 %
Remarks on result:
other: 24 h
Remarks:
human skin, perfusate: cumulative mean recovery [% of dose]; remaining treated skin: 0.18 %
Dose:
1912 µg/cm²
Parameter:
percentage
Absorption:
0.12 %
Remarks on result:
other: 24 h
Remarks:
rat skin, perfusate: cumulative mean recovery [% of dose]; remaining treated skin: 4.95 %
Conversion factor human vs. animal skin:
human/rat ratio 1:1.2 (based on the flux values)

Any other information on results incl. tables

The penetration of [14C] test item through rat and human skin membrane was extremely low.

Rat skin membrane:

Only 0.12 % of the applied dose penetrated into perfusate during 24 hours of exposure. At the end of exposure the bulk of the applied test item could be washed off from the skin membranes, accounting for 70.57 % of the dose. After skin membrane rinsing 20.92 % of the dose remained in/on the skin membrane. The major part of this remaining test item was located in the stratum corneum (tape strips), accounting for 15.97 % of the dose, and 4.95 % of the dose was found in the remaining skin membrane after tape stripping, indicating that the applied test item entered the skin membrane but it did not penetrate through the membrane to a significant extent.

The mean flux value was calculated to be 0.209 µg/cm²/h. The flux value was about 3 times lower than the estimated penetration rate limit caused by the very limited solubility of the test item in the perfusate, i.e. 0.750 µg/cm²/h.

Human skin membrane:

The penetration of the test item resembles very closely to that observed in rat skin membrane. Within 24 hours of exposure only 0.10 % of the applied dose penetrated totally through human skin membranes into perfusate. Also for human skin membranes the bulk of applied test item could be washed off 24 hours after start of exposure, i.e. 73.18 % of dose. In stratum corneum 15.38 % of the dose was found and 0.18 % of the dose was found in the remaining skin membrane after tape stripping, which was significantly lower than observed in rat skin membranes. The mean flux value was calculated to be 0.178 µg/cm²/h.

The amount of test item in lower skin layers was significantly lower in human skin membranes as compared to rat skin membranes. Therefore the total absorption, based on the amount penetrated through the skin membrane (perfusate) and the amount measured in the remaining skin membrane layers after tape stripping, was 5.07 % and 0.28 % of the applied dose. Based on the flux values a human/rat ration of 1:1.2 was calculated for dermal penetration.

Applicant's summary and conclusion