ethyl N2-dodecanoyl-.sc.l.sc.-argininate hydrochloride

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 17, 2010 to November 5, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

According to guidelines and offical methods with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Environmental Health and Safety Publications, Series on Testing and Assessment Nº 28. Guidance document for the conduct of skin absorption studies (March 2004)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: COLIPA Guidelines for Percutaneous Absorption/Penetration (August 2007)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: SCCS/1358/10 Basic Criteria for the in vitro Assessment of Dermal Absorption of Cosmetic Ingredients, adopted by the SCCS during the seventh plenary meeting of 22 June 2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-630-6

EC Name:

-

Cas Number:

60372-77-2

Molecular formula:

Hill formula: C20H41N4O3Cl

IUPAC Name:

ethyl N2-dodecanoyl-l-argininate hydrochloride

Details on test material:

- Name of test material (as cited in study report): [Arginine-U-14C]LAE·HCl
- Physical state: White solid
- Lot/batch No.: 3766CJW008-10
- Expiration date of the lot/batch: June 30, 2011
- Analytical purity: c.a 99%
- Radiochemical purity (if radiolabelling): 98%
- Specific activity (if radiolabelling): 540 MBq/mmol (128 MBq/mg)
- Locations of the label (if radiolabelling): C
- Storage condition of test material: In freezer (<=-15ºC)

Specific details on test material used for the study:

Substance: L.A.E.
Purity: ca. 99%
Batch number: 3766CJW008-10

Radiolabelling:

yes

Remarks:

14C

Administration / exposure

Details on study design:

PREPARATION OF TEST SUBSTANCE SOLUTIONS
The test solutions of 0.4% [Arginine-U-14C]LAE·HCl were prepared by dissolving an appropriate amount of [Arginine-U-14C]LAE·HCl in MQ. The activity of this solution was determined by liquid scintillation counting and a final activity of 5.1 MBq/mL was achieved by dilution with MQ. The final concentration of [Arginine-U-14C]LAE·HCl was 4035 mg/L.

The test solution of 0.8% [Arginine-U-14C]LAE·HCl was prepared by dissolving an appropriate amount of [Arginine-U-14C]LAE·HCl in MQ. The activity of this solution was determined by liquid scintillation counting and a final activity of 10.2 MBq/mL was achieved by dilution with MQ. The final concentration was 7992 mg/L [Arginine-U-14C]LAE·HCl.

HOMOGENEITY AND STABILITY OF THE TEST SOLUTIONS
The homogeneity of the test solutions was checked by taking three samples (from the top, the bottom and the middle of the vial) and determining the activity of these samples in Ultima Gold LSC cocktail by liquid scintillation counting. The Relative Standard Deviation (RSD) of the three measurements should be < 3%, and it was 1.6% for the test solution of 0.4% and 1.4% for the test solution of 0.8%.

The stability of the test solutions for the duration of the study (24 hours) was checked by analyzing an aliquot of both test solutions with concentrations of 0.4% and 0.8% [Arginine-U- 14C]LAE·HCl immediately after preparation and after 24 h using the LC-UV-RAD method described in the study.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Biopredic International, Rennes, France
- Ethical approval if human skin: Human skin is a recommended test for skin absorption studies
- Type of skin: Human
- Preparative technique: Frozen dermatomed human skin discs
- Thickness of skin (in mm): c.a 0.5 mm
- Storage conditions: dry ice and stored at <=-15ºC

SKIN INTEGRITY TEST
Skin membranes were thawed, mounted in the diffusion cell and the skin integrity was tested by permeation of tritiated water. 200 μL saline containing tritiated water (3.7 kBq) were applied to the donor compartment of the flow-through cells. The receptor fluid consisted of saline and was collected every hour up to 3 hours after application. At the end of the experiment, the tritiated water remaining at the donor compartment was removed with a pipet and the skin was dried with two cotton swabs (Etos BV, Beverwijk, The Netherlands). The receptor fluid samples were analyzed by LSC.

PRINCIPLES OF ASSAY
- Receptor fluid: Saline supplemented with 0.5% BSA
- Solubility of test substance in receptor fluid: Soluble (94.5% recovery)
- Flow-through system: Receptor fluid was pumped at a flow rate of 1.5 ml/h
- Test temperature: 32±1ºC
- Humidity: ambient humidity
- Occlusion:No

Results and discussion

Absorption in different matrices:

The percentages absorbed were lower for the concentration of 0.8% compared to the concentration of 0.4% [Arginine-U-14C]LAE·HCl. The average percentage absorbed was 0.81 ± 0.78% for the 0.8% concentration and 2.1 ± 0.9% for the 0.4% concentration.

Total recovery:

- Total recovery: The recoveries obtained in the experiments are an average of 95.1 ± 2.6% for the 0.4% [Arginine-U-14C]LAE·HCl test solution and 95.3 ± 4.0% for the 0.8% [Arginine-U-14C]LAE·HCl test solution. The total recovery of radioactivity for the individual skin samples was 100 ±10%,
which is according to the OECD guidelines.
- Limit of detection (LOD): ±2.6 (0.4%); ±3.9 (0.8%);

Percutaneous absorption

Percutaneous absorption 1

Key result

Dose:

0.4%

Parameter:

percentage

Absorption:

>= 1.2 - <= 3 %

Remarks on result:

other: 24 h

Percutaneous absorption 2

Key result

Dose:

0.8%

Parameter:

percentage

Absorption:

>= 0.04 - <= 1.6 %

Remarks on result:

other: 24 h

Any other information on results incl. tables

Skin integrity test

The permeability coefficients for water for the skin discs used in this study were within the acceptability criteria (Kp ≤ 4.5 x 10-3 cm/h).

Stability of [Arginine-U-14C]LAE·HCl test solutions

No decrease in the peak area of [Arginine-U-14C]LAE·HCl and no formation of additional degradation products after 24 h was

observed, indicating that [Arginine-U-14C]LAE·HCl was stable for the duration of the experiment.

Applicant's summary and conclusion

Conclusions:

The percentage of [Arginine-U-14C]LAE·HCl absorbed was assessed in 10 skin discs of human donors according to OECD 428 Guideline. Results showed a lower at a concentration of 0.8% than at a concentration of 0.4%. The average percentage absorbed was 0.81 ±0.78% for the 0.8% concentration and 2.1±0.9% for the 0.4% concentration.

Executive summary:

According to the Guideline 428, the present study has been designed to provide information on absorption of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) applied to excised skin.

With that objective, dermal absorption of [Arginine-U-¹⁴C]LAE·HCl was investigated in human skin in vitro.

Human skin discs from 4 donors were exposed to a concentration of 0.4% (4035 mg/l) and human skin discs from 5 donors were exposed to a concentration of 0.8% (7992 mg/l) [Arginine-U-¹⁴C]LAE·HCl in water for 24 hours.

The integrity of each skin disc was checked by determination of the permeation of titrated water and was within the acceptability criteria.

[Arginine-U-¹⁴C]LAE·HCl test solutions (0.4 and 0.8%) were stable for the durationof the experiment (24 h) and [Arginine-U-¹⁴C]LAE·HCl test solutions in receptor fluid were also stable for 24 h.

The percentage of [Arginine-U-¹⁴C]LAE·HCl absorbed was lower at a concentration of 0.8% than at a concentration of 0.4%. The average percentage absorbed was 0.81 ±0.78% for the 0.8% concentration and 2.1±0.9% for the 0.4% concentration.