2-ethylhexyl 3,5,5-trimethylhexanoate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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.

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

Radiolabelling:

not specified

Species:

other: in vitro assessment

Details on test animals or test system and environmental conditions:

Not applicable for an in vitro assessment.

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

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.

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 %

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 %

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 %

Key result

Time point:

24 h

Dose:

Levels of eheh in the surface wipe

Parameter:

other: % of the applied dose

Absorption:

85.5 %

Key result

Time point:

24 h

Dose:

Levels of eheh in the epidermis

Parameter:

other: % of the applied dose

Absorption:

2.34 %

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.

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%.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

September 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted using a read across substance

Justification for type of information:

The read across justification is included as an attachment in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Time point:

24 h

Dose:

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

Parameter:

other: % of the applied dose of the test substance

Absorption:

2.6 %

Remarks on result:

other: based on 2-Ethylhexyl-2-ethaylhexanoate/Dragoxat EH/ 7425-14-1

Key result

Time point:

24 h

Dose:

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

Parameter:

other: % of the applied dose of the test substance

Absorption:

4.3 %

Remarks on result:

other: based on 2-Ethylhexyl-2-ethaylhexanoate/Dragoxat EH/ 7425-14-1

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 of the test substance

Absorption:

4.74 %

Remarks on result:

other: based on 2-Ethylhexyl-2-ethaylhexanoate/Dragoxat EH/ 7425-14-1

Key result

Time point:

24 h

Dose:

Levels of the test substance in the surface wipe

Parameter:

other: % of the applied dose of the test substance

Absorption:

85.5 %

Remarks on result:

other: based on 2-Ethylhexyl-2-ethaylhexanoate/Dragoxat EH/ 7425-14-1

Key result

Time point:

24 h

Dose:

Levels of the test substance in the epidermis

Parameter:

other: % of the applied dose of the test substance

Absorption:

2.34 %

Key result

Time point:

24 h

Dose:

Total absorbed dose (the test substance and the metabolite, 2eha

Parameter:

other: % of the applied dose of the test substance

Absorption:

7.34 %

Conclusions:

In an small scale assessment of the in vitro human skin adsorption and metabolism ,absorption and metabolismof the test item was predicted from the source. The results of this small scale assessment indicated that there was limited absorption and metabolism of the source substance from the applied 10% formulation (see read across justification in section 13).
The total absorbed dose (defined as levels in the epidermis, dermis and receptor phase) was 7.34 ± 0.97% of the applied source 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 test substance, 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%.

Reference 3

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013-04-12

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effect of the the test substance on the penetration of indomethacin through hairless rat skin (no further details provided).

GLP compliance:

not specified

Specific details on test material used for the study:

The test substance is reported as 'ethylhexyl isononanoate'. No further details are provided.

Radiolabelling:

not specified

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

Not applicable for an in vitro study

Type of coverage:

not specified

Vehicle:

not specified

Duration of exposure:

10 hours

Doses:

No details reported.

No. of animals per group:

Not applicable for an in vitro study

Control animals:

no

Remarks:

Not applicable.

Details on study design:

The effect of the the test substance on the penetration of indomethacin through hairless rat skin. No further details reported.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Not reported
- Type of skin: Hairless rat skin
- Preparative technique: Not reported
- Thickness of skin (in mm): Not reported
- Membrane integrity check: Not reported
- Storage conditions: Not reported
- Justification of species, anatomical site and preparative technique: Not reported

PRINCIPLES OF ASSAY
- Diffusion cell: Not reported
- Receptor fluid: Not reported
- Solubility od test substance in receptor fluid: Not reported
- Static system: Not reported
- Flow-through system: Not reported
- Test temperature: Not reported
- Humidity: Not reported
- Occlusion: Not reported
- Reference substance(s):
- Other:

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

No details reported.

Total recovery:

No details reported.

Key result

Time point:

10 h

Dose:

Permeation

Parameter:

amount

Absorption:

23 other: µg/cm2

Conversion factor human vs. animal skin:

Not reported.

This report also reviews the dermal penetration of additional alkyl esters as well as the test substance. These data are as follows:

No isopropyl myrisate was detected in the receptor fluid of flow-through diffusion cell in a human epidermis in vitro study (no further details reported). This suggests that isopropyl myristate is largely retained in the stratum corneum.

In a human volunteer in vivo study, isostearyl isostearate was also located at the surface of the stratum coneum. In this study, pre-denatured isostearyl isostearate, 7 µl/cm2 was applied (non-occlusive) to a 2 cm x 8 cm site on the ventral forearm of 14 human subjects. Three hours after application, the test-site was test stripped and attenuated total reflectance Fourier transform infrared sprectra measurements were determined.

Conclusions:

The effect of the the test substance on the penetration of indomethacin through hairless rat skin was examined. Approximately 23 µg/cm2 indomethacin permeated in 10 hours.

Executive summary:

The data suggests that the test substance is poorly absorbed by the dermal route ( a solution of the test substance and indomethacin was found to permeate approximatley 23 µg/cm2 in 10 hours in an in vitro rat skin study). This is considered to be a typical characteristic of alkyl esters, as supported by dermal penetration studies using isoprpyl myrisate and isostearyl isostearate. Similar results of low dermal absorption for other alkyl esters (namely, ispropyl myristate and isostearyl isostearate) are also reported in this document.

Isopropyl myrisate is largely retained in the stratum corneum. No isopropyl myrisate was detected in the receptor fluid of flow-through diffusion cell in a human epidermis in vitro study (no further details reported).

Isostearyl isostearate was also located at the surface of the stratum corneum in a human volunteer in vivo study. In this study, pre-denatured isostearyl isostearate, 7 µl/c m2 was applied (non-occlusive) to a 2 cm x 8 cm site on the ventral forearm of 14 human subjects. Three hours after application, the test-site was test stripped and attenuated total reflectance Fourier transform infrared sprectra measurements were determined.

Description of key information

In the absence of specific data, a default 100% oral absorption is assumed. This assumption is supported by data on cetyl oleate which showed an oral bioavailability of 75 % when fed to rats at 20 % in the diet (Cosmetic Ingredient Review "Amended Safety Assessment of Alkyl Esters as Used in Cosmetics", 2013, Washington DC).

Since the substance does not hydrolyze into ions and is not considered to be extremely water soluble or interact with cell surfaces, the bioavailability of the substance upon inhalation is considered to be rather low. Therefore, the 2 fold uncertainy factor applied in risk assessment for exposure estimation from inhalation exposure while using an oral NOAEL, is considered not adequate. Rather a ratio of 1 is already very conservative.

Data suggest that dermal absorbtion in alkyl esters is low; an in vitro absorption study using a chemical of similar physico-chemical properties (2-ethylhexyl-2-ethylhexanoate) to the test substance showed that total absorbed dose after a 24 hour exposure period was 7.34 ± 0.97% of the applied dose (including the hydrolyis product). Data from Cosmetic Ingredients Review (CIR) (2013) provides further support that alkyl esters have low dermal absorption rates. Studies reported in the CIR (2013) document that support this conclusion include an in vitro rat study with the test substance (reported as ethylhexyl isononanoate) in a solution with 1 % indomethacin, that was found to permeate approximatley 23 µg/cm2 in 10 hours (no further details provided). In addition, the CIR reports the results from a human flow-through diffusion cell in a human epidermis in vitro study using the alkyl ester, isopropyl myristate as a non-polar penetration enhancer. It was reported that isopropyl myristae was largely retained in the stratum corneum, and was not detected in the receptor fluid. A further study in the CIR reports that the alkyl ester isostearyl isostearate was also located at the surface of the stratum corneum in a human volunteer in vivo study (7 µl/c m2 was applied (non-occlusive) to a 2 cm x 8 cm site on the ventral forearm of 14 human subjects for three hours).

Based on these data, a low dermal absorption rate of the test substance is anticipated. Using the data from the dermal absorption study using the read-across chemical (2-ethylhexyl-2-ethylhexanoate), a dermal absorption rate of 8% is assumed as a worst case scenario.

Bioaccumulation in rodents is not expected due to efficient metabolic pathways (see below for further details).

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

8

Absorption rate - inhalation (%):

100

Additional information

2-Ethylhexyl 3,5,5-trimethylhexanoate is expected to be readily absorbed via oral (assumed 100%) but less via dermal route (assumed 8%) while inhalation is not applicable due to its high boiling point and low vapour pressure. 2-Ethylhexyl 3,5,5-trimethylhexanoate is rapidly distributed to blood and organs, where it is hydrolysed to form ethylhexyl alcohol and trimethylhexanoic acid, which are further metabolized to acetate moieties that enter the citric acid cycle and are metabolized to carbon dioxide, water, and energy.

The half-life of 2-ethylhexyl 3,5,5-trimethylhexanoate was estimated to be less than 24 hr, based on ester cleavage. The main route of excretion of metabolites is via exhaled air, faeces and urine. As a conclusion, the accumulation of 2-ethylhexyl 3,5,5-trimethylhexanoate or its metabolites is very unlikely to occur due to the efficient metabolic and excretion pathways.