2-ethylhexyl 3,5,5-trimethylhexanoate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The read across justification is included in Chapter 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Small scale assessment of the in vitro human skin absorbtion and metabolism of 2-ethylhexyl-2-ethylhexanoate (eheh).
- Short description of test conditions: Study conducted following an agreed protocol and using the testing laboratories standard operating procedures. The study appears to have been audited bythe testing laboratories quality assurance department.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 2-ethylhexanoate

EC Number:

231-057-1

EC Name:

2-ethylhexyl 2-ethylhexanoate

Cas Number:

7425-14-1

Molecular formula:

C16H32O2

IUPAC Name:

2-ethylhexyl 2-ethylhexanoate

Radiolabelling:

not specified

Test animals

Species:

other: in vitro assessment

Details on test animals or test system and environmental conditions:

Not applicable for an in vitro assessment.

Administration / exposure

Type of coverage:

open

Vehicle:

other: O/W Emulsion base, Contained 23 % of the exipient Neutralol.

Remarks:

Reported as the 'placebo formulation'.

Doses:

- Nominal doses: The average applied doses (mean ± standard error, SE) were 5.00 ± 0.14 and 5.03 ± 0.12 mg/cm2 for the active and placebo formulations respectively.
- Rationale for dose selection: Not reported

No. of animals per group:

Not applicable for an in vitro assessment

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The formulations were used as supplied
- Method of storage: Room temperature

APPLICATION OF DOSE:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Justification not provided.
- Amount(s) applied (volume or weight with unit): The target application was 5 mg/cm2
- Lot/batch no. (if required): Lot U. 53398 C. Manufacture date: September 2003
- Purity: Contained 23 % of the excipient Neutralol.

TEST SITE
- Preparation of test site: Full-thickness human female breast and abdominal skin, obtained from cosmetic surgery, was prepared for use by removal of the subcutaneous fat by blunt dissection. Small sections of skin were then mounted onto diffusion cells and trimmed to size. Two different donors were used, each within three hours of excision.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: Not applicable for an in vitro study.

REMOVAL OF TEST SUBSTANCE
- The test substance was removed by gentle wiping with a dry cotton bud. The cotton bud wipes for each cell were placed into 20 ml glass vials and frozen pending analysis. The diffusion cells were then dismantled and each skin sample tape stripped 10 times using adhevide tape. The tape strips were grouped (strips 1-2, strips 3-5 and strips 6-10) and frozen pending analysis. The remaining samples of skin were then heat seperated (75 seconds at 60 degC) to produce epidermis and dermis samples that were placed into glass vials and frozen pending analysis.
- Time after start of exposure: 24 hours

SAMPLE PREPARATION
The skin surface wipe, tape strip groups, epidermis and dermis samples were extracted into methanol (3, 1.5, 1.5 and 2 ml respectively) with the aid of vortexing and sonication (3 x 15 minute sessions) followed by overnight shaking. Aliquots of sample extract then underwent a solid phase extraction (SPE) procedure and analysis.

ANALYSIS
- Method type(s) for identification: HPLC analysis following derivatization to a fluroescent ester.
- Validation of analytical procedure: Not reported.
- Limits of detection and quantification: The analytical assay was not capable of differentiating between the metabolite, 2-eha and the isomeric fatty acid, n-octanoic acid (OA). As the formulation base contained an ester of OA, a background signal was therefore produced for all cells. This background signal was quantified using data from the control cells and subtracted from the data for active (test substance formulation dosed) cells, enabling calculation of the 2-eha and eheh data.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Cosmetic surgery
- Ethical approval if human skin: Not reported
- Type of skin: Full thickness human female breast and abdominal tissue
- Preparative technique: Removal of the subcutaneous fat by blunt dissection and then mounted onto diffusion cells and trimmed to size. Skin was used within 3 hours of excision.
- Thickness of skin (in mm): Not reported.
- Membrane integrity check: The integrity of each membrane was assessed prior to dosing with formulation. The permeation of tritiated water was determined by applying 500 pl of 2 pCi/m1 3H20 to the skin surface and removing a 200 pl sample from the receptor phase twenty minutes later. The sample was counted using liquid scintillation counting (LSC). The skin surface was subsequently washed six times with water and the receptor chambers three times with HBSS, prior to refilling with 2% BSA in HBSS. The cells were then allowed to re-equilibrate to the correct temperature. None of the utilised cells exhibited a water permeability of greater than 2.5 x 10-3 c m/h. This permeability coefficient is an industry accepted standard.
cut-off point for demonstration of skin membrane integrity.
- Storage conditions: Not reported
- Justification of species, anatomical site and preparative technique: Human skin is capable of hydrolysing many esters, and it is possible that
the eheh may be hydrolysed to the fatty acid 2-ethylhexanoic acid (2-eha, CAS No. 149-57-5, molecular weight 144 Daltons). This study utilised fresh human skin in order to retain maximal metabolic activity comparable to the in vivo situation.

PRINCIPLES OF ASSAY
- Diffusion cell: The skin samples were mounted as a barrier between the halves of greased horizontal Franz-type diffusion cells, with the stratum corneum facing the donor chamber. The area available for diffusion was about 1.0 cm2, with the exact area being measured for each diffusion cell. The diffusion cells were immersed in a constant temperature water bath such that the receptor chambers were maintained at 37.0±0.5°C throughout the experiment. This ensured that the skin surface temperature was maintained at 32.0±1°C. The receptor chamber contents were continuously agitated by small PTFE-coated magnetic followers driven by submersible magnetic stirrers. The receptor chambers of the diffusion cells were filled with a known volume of HBSS, capped, and allowed to equilibrate to the correct temperature.
- Receptor fluid: 2% bovine serum albumin (BSA, fraction V) in HBSS was prepared by dissolving 2 g BSA in 100 ml HBSS.
- Solubility of test substance in receptor fluid: This receptor medium was selected to ensure that skin viability was maintained, and also that adequate solubility for both the test substance and the metabolite was provided.
- Test temperature: 32.0±1°C.
- Humidity: Not reported

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): The majority of the recovered metabolite, e-eha was found in the receptor phase (12.1 ± 1.5 pg/cm2, equivalent to 4.30 ± 0.54% of the applied dose of eheh).
- Skin preparation (in vitro test system): The majority of the applied dose was recovered as unchanged ester in the surface wipe (85.5 ± 4.8% applied dose).Levels of unchanged test substance in the skin were highest in the epidermis (2.34 ± 0.42% applied applied dose), with much lower levels found in the dermis (0.262 ± 0.057% applied applied dose) and no unchanged test substance was found in the receptor phase.

Total recovery:

The overall recovery in all compartments was 95.0 ± 4.8% applied dose.
Total recovery of the applied excipient Neutralöl for the control formulation dosed cellswas 79.4 ± 1.0% applied dose.

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

24 h

Dose:

Absorbed dose for unchanged eheh (levels in the epidermis, dermis and receptor phase)

Parameter:

other: % of the applied dose

Absorption:

2.6 %

Percutaneous absorption 2

Key result

Time point:

24 h

Dose:

Levels of the metabolite, 2eha, in the receptor phase

Parameter:

other: % of the applied dose

Absorption:

4.3 %

Percutaneous absorption 3

Key result

Time point:

24 h

Dose:

Absorbed dose of the metabolite, 2-eha, in the epidermis, dermis and receptor phase

Parameter:

other: % of the applied dose

Absorption:

4.74 %

Percutaneous absorption 4

Key result

Time point:

24 h

Dose:

Levels of eheh in the surface wipe

Parameter:

other: % of the applied dose

Absorption:

85.5 %

Percutaneous absorption 5

Key result

Time point:

24 h

Dose:

Levels of eheh in the epidermis

Parameter:

other: % of the applied dose

Absorption:

2.34 %

Percutaneous absorption 6

Key result

Time point:

24 h

Dose:

Total absorbed dose (eheh and the metabolite, 2eha)

Parameter:

other: % of the applied dose

Absorption:

7.34 %

Conversion factor human vs. animal skin:

Not applicable.

Applicant's summary and conclusion

Conclusions:

The results of this small scale assessment indicated that there was limited absorption and metabolism of the test susbtance from the applied 10% formulation.
The total absorbed dose (defined as levels in the epidermis, dermis and receptor phase) was 7.34 ± 0.97% of the applied test substance. Of the total absorbed dose, 2.60 ± 0.44% applied dose was recovered as unchanged test substance and 4.74 ± 0.67% applied dose was as the hydrolysis product, 2-eha.
Addition of the absorbed doses for unchanged eheh, and the hydrolysis product, 2-eha, showed the total absorbed dose was 7.34 ± 0.97% of the applied dose.
The total recovery of applied test substance was 95.0 ± 4.8%.