Registration Dossier

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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meeting generally accepted scientific principles, acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
The effects of zwitterionic surfactants on skin barrier function.
Author:
Ridout, G. et al.
Year:
1991
Bibliographic source:
Fundamental and Applied Toxicology 16: 41-50

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of skin permeation through and sequestration in isolated mouse skin in vitro by measurement of radiolabelled test material uptake.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N,N-Dimethyl-N-dodecylglycine (C12BET, dodecylbetaine)
- Physical state: liquid
- Analytical purity: 98.9 %
- Composition of test material, percentage of components: 1.0 % water
- Radiochemical purity (if radiolabelling): 98.9 %
- Specific activity (if radiolabelling): 52 mCi/mmol
- Other: prepared by treating methyl [carbonyl-14C]chloroacetate with N,N-dimethyldodecylamine. The resulting ammonium chloride compound was hydrolyzed using Bio-Rad AG 1-8x resin to give the final product.
Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
mouse
Strain:
other: hairless (HRS/hr hr)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 6-14 weeks

Administration / exposure

Type of coverage:
open
Vehicle:
other: phosphate buffered saline (PBS)
Duration of exposure:
24 hours in permeation experiment; 16 hours in pre-treatment experiment
Doses:
- Actual doses: 16 mM
- Dose volume: 0.5 mL
- Rationale for dose selection: the concentration was chosen because it represented a similar chemical dose in µmoles as for C16BET, to which permeation of C12BET was compared.
No. of animals per group:
Not applicable, in vitro: eight separate measurements of penetration (using skin from a minimum of four mice)
Control animals:
no
Remarks:
not applicable
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: dilution in PBS (pH 7.4)


APPLICATION OF DOSE:
0.5 ml of surfactant solution (spiked with 14C-labelled compound) in PBS was administered to the epidermal skin surface (0.95 cm²) exposed in the donor compartment of the diffusion cell.

VEHICLE
- Justification for use and choice of vehicle (if other than water): PBS is widely used as buffer in physiological systems


TEST SITE
- Preparation of test site: Immediately after euthanizing a hairless mouse by CO2 asphyxiation, full thickness dorsal skin was excised. Any subcutaneous fat adhering to the dermal surface was carefully removed. The skin was then mounted between the donor and the receptor compartments of glass permeation cells (Laboratory Glass Apparatus, Berkeley, CA). The accessible skin area for transport was 0.95 cm²; one mouse, therefore, typically provided sufficient skin for four cells.
The receptor compartment (volume = 3 ml) was perfused with pH 7.4 PBS at a rate of 3 mL/hr. The chamber temperature was regulated by a thermostat to maintain the skin surface temperature at 32 ± 1°C .
- Area of exposure: 0.95cm²
- Time intervals for shavings or clipplings: not applicable, in vitro


SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: not applicable, in vitro


SAMPLE COLLECTION
- Collection of perfusate samples: For 24 hours perfusate samples of 3 mL at each hour were collected on a fraction collector and then assayed for surfactant.
- Terminal procedure: At the end of the transport experiment, the skin membrane was washed quickly in fresh buffer solution and was then dissolved in Soluene (Packard, Downers Grove, IL) before liquid scintillation counting. In this way the amount of test substance sequestered within the skin during the penetration process was determined.


ANALYSIS
- Method type(s) for identification: Liquid scintillation counting (Searle Model 6880)


OTHER:
To assess the effects of surfactant pre-treatment on skin barrier function, hairless mouse skin was excised and mounted in the diffusion cell as described above. The receptor chamber was filled with PBS and maintained at 32 ± 1°C without perfusion. Non-Labelled surfactant solution (0.5 mL) was applied to the epidermal surface in the donor compartment for 16 hours (i.e. overnight). Concentrations applied were 16, 100 and 800 mM. At the end of the pre-treatment period, surfactant solution was removed and the skin gently washed three times with 0.5 ml of distilled water. The receptor compartment was drained, flushed with fresh PBS, and then perfused at 3 mL/hr. Nicotinamide solution (0.5 mL, concentration 100 µg/mL, spiked with 14C-labelled compound) was administered to the epidermal surface and hourly 3 ml samples of the receptor phase were collected for the subsequent 12 hours. Nicotimamide permeation was determined by analysing the fractions using liquid scintillation counting. Experiments were performed in quadruplicate. Parallel control measurements were performed in which the pre-treatment solution was PBS. Typically, skins from two mice were used such that half of the tissue from each mouse was pretreated with surfactant and the other half with PBS.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: hairless mice
- Type of skin: full thickness dorsal skin
- Preparative technique: excised after CO2 asphyxiation, any subcutaneous fat adhering to the dermal surface was carefully removed
- Thickness of skin (in mm): full thickness, not further specified
- Storage conditions: not stored


PRINCIPLES OF ASSAY
- Diffusion cell: glass permeation cells (Laboratory Glass Apparatus, Berkeley, CA)
- Receptor fluid: PBS pH 7.4
- Solubility of test substance in receptor fluid: yes, fully soluble
- Static system: donor compartment
- Flow-through system: receptor compartment, 3 ml/hr
- Test temperature: 32 ± 1°C
- Occlusion: no

Results and discussion

Absorption in different matrices:
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): 10.3 ± 3.5 % absorbed after 12 hours and 46.5 ± 8.2 % absorbed after 24 hours (corresponding to 220 ± 80 and 1010 ± 180 µg, respectively) (mean ± SD)
- Skin preparation (in vitro test system): approximately 25 % associated to the skin after 24 hours
Total recovery:
- Total recovery: nearly 75 %
- Recovery of applied dose acceptable: yes
Percutaneous absorptionopen allclose all
Dose:
16 mM
Parameter:
percentage
Absorption:
ca. 10.3 %
Remarks on result:
other: 12 hours
Remarks:
mouse skin
Dose:
16 mM
Parameter:
percentage
Absorption:
ca. 46.5 %
Remarks on result:
other: 24 hours
Remarks:
mouse skin

Any other information on results incl. tables

Cumulative penetration of Nicotinamide (mean±SD) in 12 hours following pretreatment of hairless mouse skin with C12BET:

Surfactant

Applied concentration (mM)

Normalized concentration (C*)

Cumulative % dose absorbed

Number of replicates

Control

0

0

0.32 ± 0.26

42

C12BET

16

10

5.1 ± 1.7

4

 

100

62.5

8.0 ± 3.1

4

 

800

500

33.0 ± 7.4

4

C* = applied concentration divided by the CMC (1.6 mM)

Control pre-treated with pH 7.4 PBS containing no surfactant

Conclusion:

Pre-treatment with C12BET at all concentrations significantly (p < 0.05) promotes nicotinamide penetration across hairless mouse skin when compared to the controls.

Applicant's summary and conclusion