Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Principles of method if other than guideline:

The study was conducted to determine the skin permeation and distribution of the test material, applied in 70 % ethanol/ 30 % water at a target maximum in-use concentration of 1.5 %, under both un-occluded and occluded conditions, using human tissue in vitro.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-enecarbaldehyde

EC Number:

915-617-9

Molecular formula:

C13H22O2

IUPAC Name:

Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-enecarbaldehyde

Test material form:

liquid

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

other: in vitro study

Strain:

other: in vitro study

Sex:

not specified

Administration / exposure

Type of coverage:

other: un-occluded and occluded

Vehicle:

other: ethanol:water (70:30 v/v)

Duration of exposure:

24 hours

Doses:

- Actual dose: 5 μL/cm2 (equivalent to 75 μg/cm2)
- Rationale for dose selection: maximum in-use concentration of 1.5 %

No. of animals per group:

Twelve active dosed diffusion cells were prepared (using 6 donors) for each application condition (unoccluded and occluded) plus one unoccluded control cell.

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The HMPCC test vehicle was prepared by transferring 74.46 mg HMPCC (unlabelled), ~3 mL pre-mixed 70/30 (v:v) ethanol/water, and 560 μL 14C-HMPCC solution (containing 0.72 mg HMPCC) to a 5 mL volumetric flask. Water (240 μL) was then added followed by pre-mixed 70/30 (v/v) ethanol/water to volume. The final vehicle concentration was 1.504 % (w/v) HMPCC, with a measured density of 0.8748 g/mL at 25 ºC.

APPLICATION OF DOSE:
- The 1.5 % HMPCC solution was applied to the skin surface at a dose of 5.00 μL/cm2 using a Gilson M25 microman positive displacement pipette. Twelve replicates were dosed with HMPCC solution under in-use (unoccluded) conditions, and twelve replicates under occluded conditions. Permeation was measured at twelve time-points over 24 hours, using a pH 7.4 phosphate buffered saline (PBS) receptor phase. For cells in the occluded group, a greased glass coverslip was applied to the donor chamber immediately after dosing.
- Potential evaporative loss of the test material was estimated by measuring the loss from PTFE (polytetrafluoroethylene) sheets under the same (unoccluded) experimental conditions.

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 μL/cm2
- Concentration (if solution): 1.5 % in 70:30 (v/v) ethanol/water

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleaning agent: the HMPCC remaining on the skin surface was removed by gentle wiping with a dry cotton bud. The cotton bud wipe for each cell was extracted with acetonitrile (5 mL) and a sample (500 μL) analysed for 14C by LSC. Each epidermal membrane was tape stripped 10 times using adhesive tape. The tape strips were grouped (placed in the same vial) as follows: strip 1, strips 2-3, strips 4-6 and strips 7-10. Strips were dissolved in OptiSolve (1 mL) and a sample (400 μL) analysed for 14C with LSC. The remaining samples of skin (epidermis plus any remaining stratum corneum) were dissolved in OptiSolve (2 mL) and a sample (400 μL) analysed for 14C by LSC. The epidermal membrane filter paper supports (used in the diffusion cells) were extracted with acetonitrile (2 mL) and samples (0.5 mL) analysed for 14C by LSC. The diffusion cell donor chambers (and coverslips for occluded cells) were wiped using cotton buds to remove sealing grease, the grease dissolved with methanol (10 mL) and samples (0.5 mL) analysed for 14C by LSC. The donor chambers (and coverslips for occluded cells) were then washed with acetonitrile (15 mL) to recover any HMPCC that may have evaporated from the skin surface and condensed on the donor chamber. A sample (0.5 mL) of each donor chamber wash was analysed for 14C by LSC.
- Time after start of exposure: 24 hours

ANALYSIS
- Method type for identification: Liquid scintillation counting

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: full-thickness human female abdominal and breast skin obtained from cosmetic surgery
- Preparative technique: Following removal of the subcutaneous fat by blunt dissection, individual portions of skin were immersed in water at 60 ºC for 60 seconds. The epidermis (comprising stratum corneum and epidermis) was then gently removed from the underlying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and taken up onto aluminium foil. The membranes were thoroughly dried and stored flat at -20 ºC until used. On the day of use, the epidermal membranes were floated onto water from the aluminium foil and taken up onto 25 mm diameter filter paper supports. The membranes were then mounted onto diffusion cells. Six different donors were used.
- Membrane integrity check: the integrity of each membrane was assessed by the permeation of tritiated water by applying 500 μL of 2 μCi/mL 3-H2O to the skin surface and removing a 200 μL sample from the receptor phase one hour 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 PBS, prior to refilling with PBS. The ells were then allowed to re-equilibrate to the correct temperature and allow normal hydration to be achieved.
- Storage conditions: stored at -20 ºC and thawed at room temperature for processing

PRINCIPLES OF ASSAY
- Diffusion cell: greased (high vacuum grease) horizontal Franz-type diffusion cells
- Receptor fluid: phosphate buffered saline (pH 7.4)
- Solubility of test substance in receptor fluid: > 500 μg/mL at 25 ºC
- Test temperature: 32 ± 1 ºC (skin surface temperature)
- Occlusion: occlusion was achieved by application of a greased coverslip to the diffusion cell donor chamber immediately following dosing
- Reference substance(s): benzoic acid (0.4 % in 50/50 (v/v) ethanol/water)

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

After 24 hours exposure (test material dose 200.8 ug/cm2), 5.54 +/- 0.6 and 18.6 +/- 1.9 ug/cm2 test material (mean +/- standard error, SE) corresponding to 7.37 +/- 0.86 % and 24.8 +/- 2.5 % of the applied dose had permeated for the unoccluded (n = 11) and occluded groups (n = 12), respectively. One cell was rejected for the unoccluded group due to abnormal permeability at early timepoints.
The 24 hour surface wipes contained 43.4 +/- 2.8 % and 31.2 +/- 2.2 % of the applied dose and donor chamber wash/wipes 20.7 +/- 1.5 % and 16.8 +/- 0.8% for the unoccluded and occluded conditions, respectively. The stratum corneum tape strips contained 7.75 +/- 0.62 % and 6.20 +/- 0.61 % of the applied dose and the epidermis, plus any remaining stratum corneum after tape stripping, 6.06 +/- 0.60 % and 8.69 +/- 0.57 %, for the unoccluded and occluded conditions, respectively. Overall recoveries of the applied test material at 24 hours were good at 86.2 +/- 2.0% and 90.5 +/- 0.7 % under unoccluded and occluded conditions, respectively due to evaporative loss. Assessment of evaporation of the test material from PTFE sheet, under unoccluded conditions showed limited evaporative loss. By 24 hours, recovery was 94.7% of the applied dose. This indicated that approximately 5.0 % of the applied dose had evaporated over the 24 hours. The levels of the test material in the epidermis (plus any remaining stratum corneum after tape stripping) filter paper membrane support and receptor fluid were combined to produce total absorbed dose values of 14.3 +/- 0.9 % and 36.4 +/- 2.6 % under unoccluded and occluded conditions, respectively.

Total recovery:

- Total recovery: 86.2 ± 2.0 % and 90.5 ± 0.7 % of the applied dose under unoccluded and occluded conditions, respectively
- Recovery of applied dose acceptable: yes

Percutaneous absorption

Percutaneous absorption 1

Key result

Dose:

5 μL/cm2

Parameter:

percentage

Absorption:

36.4 %

Remarks on result:

other: 24 hours

Remarks:

occluded exposure

Percutaneous absorption 2

Dose:

5 μL/cm2

Parameter:

percentage

Absorption:

14.3 %

Remarks on result:

other: 24 hours

Remarks:

unoccluded exposure

Any other information on results incl. tables

For the reference permeant, benzoic acid, the maximum absorption rate was within the range reported in a published multi-centre study.

Applicant's summary and conclusion

Conclusions:

In the in vitro skin absorption study using human skin, performed at a dose level of 5 μL/cm2, the total absorption of the test substance over 24 hours was 14.3 and 36.4 % under un-occluded and occluded conditions, respectively.

Executive summary:

The dermal absorption of the test substance was investigated in a non-GLP in vitro study similar to OECD 428 using skin from human volunteers, under unoccluded and occluded conditions, using a protocol similar to OECD Guideline 428. A dose of 5 μL/cm² in ethanol/water solution (70:30 v:v) of ¹⁴C-labeled test substance was applied for 24 hours to the skin surface of 12 replicates for occluded and 12 replicates for unoccluded conditions, and the amount of the test substance which penetrated into the receptor fluid was evaluated by liquid scintillation counting. Under unoccluded conditions, 7.37 ± 0.86 % of the total dose of the test substance penetrated through skin into the receptor fluid, while 7.75 ± 0.62 % was recovered in stratum corneum (tape strips) and 6.06 ± 0.60 % was recovered in epidermis. Under occluded conditions, 24.8 ± 2.5 % of the applied dose penetrated into the receptor fluid after 24 hours, 6.20 ± 0.61 % of the applied dose was recovered in stratum corneum and 8.69 ± 0.57 % was recovered in epidermis. The total recovery was 86.2 ± 2.0 % and 90.5 ± 0.7 % under unoccluded and occluded conditions, respectively. The levels of the test material in the epidermis (plus any remaining stratum corneum after tape stripping) filter paper membrane support and receptor fluid were combined to produce total absorbed dose values of 14.3 +/- 0.9 % and 36.4 +/- 2.6 % under unoccluded and occluded conditions, respectively.