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Dermal absorption

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dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2006-August 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
13 April 2004
not specified
according to guideline
other: Test Guidelines for in vitro assessment of dermal absorption and percutaneous penetration of cosmetic ingredients, Diembeck et al, 1999, Food and Chemical Toxicology, 37:191-205
not specified
according to guideline
other: Opinion of the Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers on "Basic Criteria for the in vitro Assessment of Dermal Absorption of Cosmetic Ingredients", SCCNFP/0750/03 final
Version / remarks:
20 October 2003
not specified
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: nanoform
Details on test material:
Particles of nanosilver from NanoAmor Inc., Houston, Texas were coated in 0.2 % polyvinylpyrrolidone. Silver nanoparticles were dissolved at a nominal concentration of 100 µg/L in moderately hard reconstituted water by constant stirring. The suspension was sonicated for 10 minutes, stirred at allowed for 5 minutes to settle before the top three quarters of the solution were drawn off into a clean beaker and stirred again. The manufacturer's description indicated a particle size of 35 nm and this was confirmed by transmission electron microscopy (TEM) images. The hydrodynamic diameter was 156 ± 2 nm by nanoparticle tracking analysis (NTA) indicating aggregation when dispersed in moderately reconstituted hard water (MRHW), with 11.3 ± 2.2 % of silver nanoparticles undergoing dissolution to silver ions.

Particles of citrate coated nanosilver were prepared by adding 1 mM trisodium citrate to deionised water containing 1 mM AgNO3 at 70°C and heating until translucent green (approximately 2.5 hours). The resulting nanoparticles were washed several times in deionised water and three aliquots of the suspesion were analysed. Nanoparticles were determined to be ~40 nm by TEM images, with a hydrodynamic diameter of 65 ± 10 nm by nanoparticle tracking analysis (NTA) and 8.9 ± 1.6 % of silver nanoparticles underwent dissolution to silver ions.

The size of the nanoparticles in suspesion were characterised using Transmission Electron Microscopy (TEM) and the extent of aggregation in moderately hard reconstituted water was determined by Nanoparticle Tracking Analysis (NTA), with the suspensions filtered for dissolution analysis.

Administration / exposure

Type of coverage:
other: test item in hydrophylic formulation
Duration of exposure:
- Nominal doses: 1.5 and 0.5% silver in formulation A and B, respectively, corresponding to 0.3 and 0.1 mg silver/cm²
- Actual doses:
°mean measured concentration in formulations: 15.07 (n=6, SD = 0.28) and 5.09 (n=5, SD= 0.034, 1 outlier excluded) mg Ag/g(formulation) = 1.51 and 0.51% silver in formulation A and B, respectively.
°actual mass of silver per skin: 0.33 and 0.11 mg silver for formulations A and B, respectively
- Actual doses calculated as follows: based on measured Ag concentrations
- Dose volume: 20 mg formulation/cm²
- Rationale for dose selection: concentration in formulation corresponds to dose of human applications
Details on study design:
- Method for preparation of dose suspensions: silver powder applied as formulation containing 1.5 (formulation A) and 0.5% (formulation B)
- Method of storage: n.a.


- Justification for use and choice of vehicle (if other than water): hydrophylic formulation
- Amount(s) applied (volume or weight with unit): 20 mg formulation per cm² corresponding to 0.3 mg silver/cm² and 0.1 mg silver/cm³ (nominal concentrations) for formulations A and B, respectively
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

- Preparation of test site: n.a.
- Area of exposure:
°topical application to the horny layer of the skin, application area 1.0 cm²
°evenly spreading formulation using spatula, quantification of actually applied mass by weighing
°single application, formulation not washed off the skin before termination of the experiment
°24h continuous application (recommended in guidelines and considered worst-case condition in humans)
- % coverage: 100% of the 1 cm²
- Type of cover / wrap if used: n.a.
- Time intervals for shavings or clipplings:
°prior to skin layer specific analysis, the directly exposed skin area of 1 cm² (inner circular part) was excised out of the skin disks fixed within the diffusion cells
°stratum corneum of inner circular part of the skin torn off by adhesive tape (3M, No 850) to determine teh absorbed parts of the test substance which would cast off with the stratum corneum in an in vivo situation and is not considered to be sytemically available. 15 tapes used, 3 first tapes analysed individually, tapes 4-8 analysed together, tapes 9-15 analysed together
°remaining deeper layers of the circular skin (dermis plus parts of epidermis) contain the absorbed test substance
°after exising of the circular part, the outer ring-shaped skin remains. This cannot be tape-stripped since it dries during experiment and gets resistant to stripping. This ring-shaped skin is being analysed.


- Removal of protecting device: n.a.
- Washing procedures and type of cleansing agent:
°formulations were removed after 24h by 5 rinsings with ca. 1.5 mL 0.01% Tween 80 solution, then 5 rinsings with ca. 1.5 mL deionised water and finally by dabbing the skin dry with a cellulose pad
°skin removed from cell after rinsing procedure
- Time after start of exposure: removal after 24h continuous exposure.

- Collection of blood: n.a.
- Collection of urine and faeces: n.a.
- Collection of expired air: n.a.
- Terminal procedure: n.a.
- Analysis of organs: n.a.

- Storage procedure:
°treated samples were analysed directly or -if necessary- after dilution with distilled water
°samples prepared asap for analysis, light exposure avoided during storage at ambient temperature
- Preparation details:
°HNO3 (65%) used to extract/digest/dissolve/stabilize samples:
1/ dosing formulation: 3 mL HNO3+1 mL H2O2 added to 20 mg formulation, digest under UV, filling up to 25 mL with water
2/skin rinsings: 20 mL acidified with 0.2 mL HNO3
3/ cellulose pads: digested with 3 mL HNO3 + 1 mL H2O2, 90 min UV irradiation
4/ donor chamber: extraction with 11 mL 3% HNO3 on shaker
5/ receptor fluid: 0.6 mL acidified with 0.6 mL 3% HNO3
6/ receptor chamber: extraction with 7 mL 3% HNO3
7/skin: extraction with 3 mL HNO3+1mL H2O2, 90 min UV irradiation
8/ flange skin region: extraction with 3 mL HNO3+1mL H2O2, 90 min UV irradiation
9/ adhesive tapes: extraction with 3 mL HNO3+1mL H2O2, 90 min UV irradiation

- Method type(s) for identification: ICP-MS
- Liquid scintillation counting results (cpm) converted to dpm as follows: n.a.
- Validation of analytical procedure:
°ICP-MS analysis according to internal SOP/LC/ICP/003.
°validation according to 'Kalibrierung von Analysenverfahren', DIN 38402 part 51, May 1986
- Limits of detection and quantification:
°analytical LOQ: 0.03 µg Ag/L

°all glasswater (incl. diffusion cells) were cleaned with nitric acid before assaying by steam stripping or boiling to avoid silver leaching. Plastic equipment used whenever possible
Details on in vitro test system (if applicable):
- Source of skin: female pig skin (ca 12 weeks old, ca 25 kg body weight)
- Ethical approval if human skin: n.a.
- Type of skin:
°intact, not scalded skin, was taken shortly after exsanguination from the back of the pig. skin was cleaned with water. Subcutaneous fat was removed at large extent. Hair was clipped by and electric hair clipper with 0.1mm cutter head.
°skin thickness determined at 0.79 and 0.91 mm
- Preparative technique: intact, not scalded skin, was taken shortly after exsanguination from the back of the pig. skin was cleaned with water. Subcutaneous fat was removed at large extent. Hair was clipped by and electric hair clipper with 0.1mm cutter head.
- Thickness of skin (in µm): thickness of skin was reduced by Zimmer Air Dermatome, set to 0.75 mm. Only skin without visual lesion was further used. Actual thickness of skin was determined with callipers (Oditest) immediately before insertion into penetration cell.
- Membrane integrity check:
°skin integrity was checked by determining transcutaneous electrical resistance (TER; Pt electrode, isotonic phosphate buffer above and below skin; skin samples with TER>=7kOhm were accepted for use). First check after accomodation phase of ca 0.5h before test item application, 2nd check at 24h p.a.
°mean measured TER: 18.9 and 32.1 kOhm before test item administration, individual TER >=8.4 kOhm, mean TER increased during 24h experiment to >=21.8 kOhm
- Storage conditions: skin samples were deep frozen at ca -20°C until use within 3 months. Skin samples were thawed at room temperature before application of test substance.
- Justification of species, anatomical site and preparative technique: Pig skin was selected because it shares essential penetration characteristics with human skin. Pig provided by local farmer.

- Diffusion cell:
°static Franz Diffusion Cell (PermeGear, Bethlehem, USA), type 4F-01-00-11.28-08
°skin clamped horizontally between upper donor chamber and lower receptor chamber with horny layer facing donor compartment which was not occluded.
°accessible area for application: 1 cm²
°cell made of glass
°volume receptor chamber ca 8 mL
°cell has water jacked and temperature controlled by a thermostat (32.3 °C measured), temperature difference between thermostat and individual receptor chambers was 0.1-0.3°C
- Receptor fluid:
°isotonic phosphate buffer, pH 7.0 (3.63 g KH2PO4 + 7.13 g Na2HPO4*H2O per liter water. Other buffers like phosphate buffered saline were avoided to prevent precipitation of possibly formed silver ions by chloride
°receptor fluid degassed by ultrasonication
°no insertion of air bubbles in receptor compartment (check for air bubbles at each sampling)
°receptor fluid stirred with rotating magnetic bar
°samples drawn through a side arm of the chamber before application of the test substance formulation and at 2, 6 and 24 h p.a.. Removed volume replaced by fresh receptor fluid.
°after removal of skin, the receptor chamber was extracted. Test substance content in the rinsings of the receptor chamber was added by calculation to the mass of the test substance in teh receptor fluid after 24h p.a.
- Solubility od test substance in receptor fluid:
- Static system: yes (cfr. above)
- Flow-through system: n.a.
- Test temperature: 32.3 +/- 0.3 °C
- Humidity: n.a.
- Occlusion: n.a.
- Reference substance(s): not included
- Other:
°test substance formulations were tested in two experiments, with 3 replicates per experiment (screening study setup)

Results and discussion

Signs and symptoms of toxicity:
not examined
in vitro system
Dermal irritation:
not examined
in vitro system
Absorption in different matrices:
- Non-occlusive cover + enclosure rinse: n.a.
- Skin rinsings:
°58-69% of applied test substance was removed by rinsings at study termination (24h p.a.)
°majority of silver detected in pads used to dry skin after washing
°sum of mean rinsings of skin 24h p.a. for formulation A (n=3) 60% and formulation B (n=3) 66.7%
- Skin test site: n.a.
- Skin, untreated site: n.a.
- Blood: n.a.
- Carcass: n.a.
- Urine: n.a.
- Cage wash + cage wipe: n.a.
- Faeces: n.a.
- Expired air (if applicable): n.a.
- Serial non-detects in excreta at termination: n.a.
- Receptor fluid, receptor chamber, donor chamber (in vitro test system):
°percutaneous penetration 24h p.a. was low in each experiment and replicate. Note that the contribution also originates from teh extract of the receptor chamber after dissembling the diffusion cell
°mean penetration during 0-24h p.a. for formulation A (n=3): 0.007% of applied dose or 0.0024 µg/cm²
°mean penetration during 0-24h p.a. for formulation B (n=3): 0.0014% of applied dose or 0.0015 µg/cm²
Note that these values were below the analytical LOQ and the LOD
- Skin preparation (in vitro test system):
°low absorbed amounts detected 24h p.a. in dermis and residual epidermis:
°mean absorption formulation A (n=3): 2.00% of applied dose or 6.53 µg/cm²
°mean absorption formulation B (n=3): 1.38% of applied dose or 1.52 µg/cm²
°mean amount of test substance in the flange region: 2.90% of formulation A, 2.23% of formulation B
- Stratum corneum (in vitro test system): (i.e tape strips)
°mean absorption formulation A (n=3): 17.0% of applied dose or 58.0 µg/cm²
°mean absorption formulation B (n=3): 18.5% of applied dose or 20.5 µg/cm²
-Bioavailability = mainly determined by the amount of silver detected in the dermis (and residual epidermis):
°formulation A (n=3): 2.00% of applied dose or 6.54 µg/cm²
°formulation B (n=3): 1.38% of applied dose or 1.52 µg/cm²
Total recovery:
mass balance: mean recovery of the test substance in the various samples of experiments with formulation B was within the limits of 100 +/- 15%, and slighly outside this interval for experiments with formulation A:
-formulation A: 81.9% mean total recovery
-formulation B: 88.8% mean total recovery
A more refined extraction procedure of the donor chamber and the cellulose pads might improve recovery.
Percutaneous absorptionopen allclose all
Time point:
24 h
Concentrate / Dilution:
0.5% silver in formulation
0 mg cm-2 h-1
Remarks on result:
other: assessed as 'bioavailability', driven by amount of test item in dermis (and residual epidermis)
Time point:
24 h
Concentrate / Dilution:
1.5% silver in formulation
ca. 0 mg cm-2 h-1
Remarks on result:
other: assessed as 'bioavailability', driven by amount of test item in dermis (and residual epidermis)

Any other information on results incl. tables

Table: mean percentage values of silver for Franz Diffusion Cell (% in respect to the initally applied amount)

  1,5% formulation0,5% formulation
Donor chamer (rinsings)60%66,70%
stratum corneum(adsorption, overall)17,0%18,5%
Dermis (and epidermis) 2,0%1,38%
Flange region 2,9%2,23%
Receptor chamber (percutaneous penetration)0,0007%0,0014%

Applicant's summary and conclusion

The dermal absorption & percutaneous penetration of silver is assessed in an in vitro assay (according to OECD428, GLP compliant). After application of silver (powder form, D50 7.02 µm) in a hydrophylic formulation (1.5 and 0.5% Ag) in a static Franz Diffusion cell for 24h, the percutaneous penetration is assessed at 0.0007-0.0014%. Percuteneous absorption is assessed as 0.000063-0.00027 mg Ag/cm²/h, which is mainly determined by the amount of silver detected in the dermis and residual epidermis.
Executive summary:

The dermal absorption/percutaneous penetration of silver was determined in vitro (Bornatowicz, 2006). Silver was tested as micro-sized powder in a hydrophilic formulation at 0.5 and 1.5% Ag. The test substances was assessed via determination of Ag using ICP-MS.

Three integrity checked dermatomed skin preparations of one young pig were used in each experiment. Skins were inserted in static penetration cells (Franz-cells) with an application area of 1.0 cm².

The test substance formations were applied topically to the horny layer of the skin in nominal quantities of 20 mg/cm². A non-occlusive exposure under temperature controlled conditions was performed and formulations were left on the skin for 24h.

24h after application, the stratum corneum was removed by repeated stripping (absorbed test substance). The remaining skin was taken to determine absorbed test substance. Penetration was calculated via the mass of test substance in the receptor fluid. The amount of bioavailable test substance is defined as sum of absorbed + penetrated test substance.

Most of the test substance is wiped off at the end of exposure (60-67%). Tape stripping removed a large amount of test substance from the upper layers of the skin with the amount of silver decreasing with later strippings suggesting a low level of Ag in the deeper layers of the stratum 5%, absorption 1.38-2%). A very low amount of silver penetrates the skin (0.0007-0.0014%), with the amount of test item in the receptor fluid being below the Limit of Quantification for both formulations. The bioavailability of silver was calculated at 1.38-2.0%, corresponding to 1.52-6.54 µg/cm².