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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
4jul2003 / 1dec2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, no restrictions, fully adequate for assessment.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003
Reference Type:
other: Addendum to study
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine
EC Number:
219-145-8
EC Name:
N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine
Cas Number:
2372-82-9
IUPAC Name:
N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine
Details on test material:
- Name of test material (as cited in study report): N,N-bis(3-aminopropyl)laurylamine, in a form of hydrochloride salt, which was converted to the free amine prior to use.
- Analytical purity: 98.38%
- Lot/batch No.: 3494-134
- Radiochemical purity (if radiolabelling): 98.3%
- Specific activity (if radiolabelling): 25.7 mCi/mol
- Locations of the label (if radiolabelling): alpha-position of dodecyl moiety with respect to tertiary amine function
- Storage condition of test material: -20 ˚C, in the dark
Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
human
Sex:
not specified

Administration / exposure

Type of coverage:
open
Vehicle:
water
Duration of exposure:
24 hours
Doses:
6.4 µl (10 µl/cm2)
No. of animals per group:
11 skin samples
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: at the end of exposure period, the underside of the skin was washed with the receptor fluid (receptor wash) to remove the test material which was not collected in 22-24 hours receptor fluid sample. The material on the surface of the skin was washed with ca. 10 ml of ca. 2% soap solution and the skin was dried with tissue swabs. The stratum corneum was removed with 24 successive strips.
- Time after start of exposure: 24 hours

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting (all liquid samples) and combustion/liquid scintillation analysis for all remaining samples (skin and stratum corneum tape strips)

Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human (aged 24-66 years old) from routine surgery
- Ethical approval if human skin: yes
- Type of skin: 4 breast and 3 abdomen
- Thickness of skin (in mm): full thickness: 1.18-1.71, split thickness 0.39-0.4
- Membrane integrity check: yes
- Storage conditions: at ca. -20˚C until required, thawed before use.

PRINCIPLES OF ASSAY
- Diffusion cell: flow through
- Receptor fluid: tissue culture medium with bovine cerum albumin (ca. 5% w/v)
- Solubility of test substance in receptor fluid: very high
- Flow-through system: an authomated flow-through diffusion cell apparatus (Scott/Dick, University of Newcastle-upon-Tyle, UK)
- Test temperature: 32±1°C
- Occlusion: none

Results and discussion

Absorption in different matrices:
- Skin wash: 58.78%
- Tissue swab: 14.54%
- Cell wash: 5.09%
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): < 0.01% (< 0.01% receptor fluid and <0.01% receptor rinse).
- Stratum corneum: (i.e tape strips) 22.82%.
- Exposed skin: 0.91%
- Unexposed skin: < 0.01%
Total recovery:
102.14%
 
Percutaneous absorption
Dose:
6.4 µl
Parameter:
percentage
Absorption:
0.92 %
Remarks on result:
other: 24 hrs
Remarks:
The absorption and dermal delivery were <0.01% and 0.92% of the applied dose, respectively. The steady state flux calculated form the cumulative absorption in 0-24 hr corresponded to 0.3793 ng equiv./cm2/h (0.0004% absorption/hr).

Applicant's summary and conclusion

Conclusions:
Percutaneous absorption = 0.92% after 24 hours.
Considering the steady state flux value of 0.0004% per hour it seems both unreasonable and scientifically unjustifiable to include the stratum corneum value of 23% in the dermal absorption.
Executive summary:

OECD Guideline for Testing of Chemicals Draft New Guideline 428 Skin Absorption: In Vitro Method (2002)

OECD Environmental Health and Safety Publications Series on Testing and Assessment No 28 Draft Guidance Document for the Conduct of skin absorption Studies (2002)

An automated flow-through diffusion cell apparatus (Scott/Dick University of Newcastle-upon-Tyne, UK) was used The flow-through cells were placed m a steel manifold heated via a circulating water bath to maintain the skin surface temperature at 32±1°C.  The cells were connected to multi-channel peristaltic pumps from their afferent ports, with the receptor fluid effluent dropping via fine bore tubing into scintillation vials on a fraction collector The receptor chamber volume was 0.25 ml The peristaltic pumps were adjusted to maintain a flow rate of 1.5 ml/h

Sections of split-thickness human skin membrane, ca 1.5 x 1.5 cm, were cut out, positioned on the receptor chamber of the diffusion cell, containing a magnetic stirring flea, and the donor chamber was tightened into place with screws The cells were then placed in the heated manifold and connected to the peristaltic pump The Variomag magnetic stirrer was switched on to mix the contents of the receptor chamber An equilibration period of ca 15 mm was allowed while receptor fluid was pumped through the receptor chambers at ca 1.5 ml/hr The effluent was then collected for ca 30 mm and retained as blank samples for use in the barrier integrity assessment The barrier integrity assessment was performed using tritiated water (250 μl, equivalent to ca 100,000 dpm) which was applied to the surface of each skin sample Penetration of tritiated water was assessed by collecting hourly fractions for 2 hours and then analysing the fractions by liquid scintillation counting Any human skin samples exhibiting a Permeability coefficient (Kr) greater than 2 5E-03 cm/hr were excluded from subsequent absorption measurements At the end of the 2 hour period, residual tritiated water was removed from the skin surface by rinsing with water (ca 2 ml) and drying with tissue swabs. An equilibration period of ca 120 mm was allowed prior to collection of the pre-dose sample, which was collected for ca 30 min.

Following the collection of the pre-dose sample, 6.4 μl of the test substance in water formulation was applied to the stratum corneum of each of 11 skin samples. The donor chambers were left open to the atmosphere

Receptor fluid was collected in hourly fractions from 0-6 hrs post dose and then in 2 hr fractions from 6-24 his post dose. All receptor fluid samples were mixed with ca 10 ml scintillant and then analysed by LSC At 24 his post dose each diffusion cell was disconnected from the receptor fluid pump lines. The underside of the skin was washed (receptor rinse) with receptor fluid (ca 1.5 ml), which was collected and analysed by LSC. The exposed skin surface was washed (skin wash) with ca 10 ml of a 2% v/v soap solution. The skin wash was collected and analysed by LSC. The donor and receptor chambers were dismantled and the skin removed The chambers were washed with ethanol/water (cell wash). The solvent was mixed and left to extract the test item for ca 30 min. Aliquots of cell wash were taken for LSC analysis. The stratum corneum was removed with 24 successive strip tapes. The skin under the cell flange (unexposed skin) was cut off from the exposed skin with scissors The stratum corneum tape strip and skin samples were placed into separate Combustocones® for subsequent combustion/LSC analysis.

Percutaneous absorption = 0.92% after 24 hours. .
Only less then 0.01% completely passed the skin and 0.92% of the applied dose passed the stratum corneum but remained fixed in the skin after 24 hours. This demonstrates a low dermal absorption as well as a low dermal mobility.

The mean mass balance was 102.14% of the applied dose. At 24 hrs post dose, 73.32% of the applied dose was washed off. A further 5.09% of the applied dose was contained in the cell wash. Radioactivity associated with the stratum corneum was 22.82%. The absorbed dose (0.0 1%) was made up from the receptor fluid (0.0 1%) and the receptor rinse (0.01%). Dermal delivery (0.92%) was made up from the absorbed dose, exposed skin (0.91%) and unexposed skin (0.01%).

It is remarked here that this dermal absorption study used a 1% concentration for 24 hours, well above the NOAEL level for irritation of 0.15% for 6 hours, and therefore likely overestimates dermal absorption for non-irritating concentrations.

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