Bis(2-ethylhexyl) 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-03-12 to 2008-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to the OECD guideline and under GLP regulation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

other: cosmetic formulation

Details on test material:

Test item: Oxynex ST
Concentration of test item in test item formulation: 0.5%

Radiolabelling:

no

Test animals

Details on test animals or test system and environmental conditions:

no animals used. in vitro study

Administration / exposure

Type of coverage:

open

Vehicle:

other: cosmetic formulation

Duration of exposure:

24 hours

Doses:

Concentration of test item in formulation: 0.5%
Application rate: 5 µL formulation per cm2
Amount test item applied: 29.3 µg/cm2

No. of animals per group:

not applicable

Details on study design:

DOSE PREPARATION
- Test item formulation: standard cosmetic formulation with 0.5% test item
- Method of storage: tightly closed at 2-8°C

APPLICATION OF DOSE:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard cosmetic formulation is used as a vehicle as the test item is a cosmetic ingredient
- Amount(s) applied (volume or weight with unit): 5 µL/cm2
- Concentration (if solution): 0.5%


TEST SYSTEM
- fresh dermatomed pig skin from porcine ears
- Area of exposure: 1 cm2

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: test item was removed with extraction solution (water:methanol 40:60, v/v)
- Time after start of exposure: 24 hours

SAMPLE COLLECTION
- Washing solution
- Receptor fluid
- stratum corneum
- epidermis + dermis



ANALYSIS
- Method type for identification: LC-MS/MS
- Validation of analytical procedure:
- Limits of detection and quantification: LOD = 2.0 ng/mL in receptor solution and 1.5 ng/mL in extraction solution; LOQ = 5.13 ng/mL in receptor and extraction solution.

Details on in vitro test system (if applicable):

Dermatomed pig skin (thickness: 450 µm) was used for this in vitro study. The skin slices were prepared from porcine ears obtained from the slaughterhouse.

Results and discussion

Signs and symptoms of toxicity:

not specified

Remarks:

in vitro study

Dermal irritation:

not specified

Remarks:

in vitro study

Absorption in different matrices:

- Skin wash solution: 91.6%
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): 0.09% (based on LOD and LLOQ in receptor fluid)
- Skin preparation (in vitro test system): 1.11%
- Stratum corneum (in vitro test system): (i.e tape strips): 0.592 %

Total recovery:

- Total recovery: 93.4% +/- 6.67%
- Recovery of applied dose acceptable: >/= 80.0%

Percutaneous absorption

Dose:

29.3 µg/cm2

Parameter:

percentage

Absorption:

ca. 1.2 %

Remarks on result:

other: 24 h

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions used, the dermal absorption of the test item is 1.20 %

Executive summary:

This study was designed to examine the in vitro percutaneous absorption of the test item through viable porcine skin. The substance was tested as a standard cosmetic formulation at 0.5 %. Two independent experiments were performed on fresh dermatomed pig skin samples under static conditions with 6 diffusion cells per experiment. The thickness of the skin used was approximately 450 μm. The conductivity across the skin samples of each cell was determined before treatment and after the last sampling as a measure of skin integrity. Five (5) μL of the test item were applied on each skin sample, left on the skin for 24 hours and then washed off

using 9 x 1 mL extraction solution (water : methanol, 40 : 60, v/v). The receptor solution used was 5 % BSA (w/v)/ 20 % (v/v) EtOH in PBS. The solubility of the test item in 5 % BSA (w/v) in PBS (containing 20 % organic solvent) was investigated and shown to be 3.7 μg/mL. The dermal absorption was monitored over 24 hours under non-occluded static conditions. The stratum corneum was separated by tape stripping from the epidermis and dermis. The tape stripes (2 x 5 stripes per sample) were pooled as two batches and each extracted for analysis, respectively, as well as the remaining skin compartments. The various sample solutions from the skin dermal absorption assay were analysed by LC MS/MS for the presence of the test item. Not all chambers met the acceptance

criteria for the recovery and, therefore, 10 chambers were used for the analysis of the test item. The LOD in receptor solution was defined as 2.0 ng/mL and in extraction solution as 1.5 ng/mL. The LLOQ was 5.13 ng/mL in receptor solution and in extraction solution, respectively. After the application of a formulation containing 0.5 % of the test item for 24 hours, the vast majority of Oxynex® ST was detected in the washing solution. No test item was detected in the receptor fluid of any chamber with exception of

chamber 6 in the first and chamber 1 in the second experiment. However, for worst-case considerations the LOD or LLOQ in receptor solution were used for the calculations.

Under the reported conditions, the dermal absorption of the test item is 0.337 +/- 0.385 µg/cm² or 1.20 +/- 1.49 %.