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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to internationally accepted protocols and guidelines and followed good laboratory practice standards/

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: SCCNFP in vitro assessment of percutaneous absorption guidelines (SCCNFP, 2003)
Principles of method if other than guideline:
Reference: SCCNFP (2003). The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers. Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients, SCCNFP/0750/03. SCCNFP, Brussels.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dimyrcetol
- Analytical purity: 98.73%
- Composition of test material, percentage of components: Dimyrcetol A, 2,6-dimethyl-7-octen-2-ol and Dimyrcetol B, 2,6-dimethyl-7-octen-2-ol formate ester
- Isomers composition: 42.47% Dimyrcetol A; 56.26% Dimyrcetol B
- Lot/batch No.: SM/7013009
- Stability under test conditions: determined during the study
- Other: Dimyrcetol (50g) was received July 9 2007 from International Flavors and Fragrances, 1515 Highway 36, Union Beach, NJ, 07735, USA.
Radiolabelling:
no

Test animals

Species:
human
Strain:
other: not applicable
Sex:
female
Details on test animals and environmental conditions:
Full-thickness human female abdominal skin was obtained from cosmetic surgery and stored at -20 deg C, and was subsequently thawed at room temperature for processing. Subcutaneous fat was removed by blunt dissection and individual portions of skin were immersed in 60 deg C water for 60 seconds. The epidermis, comprised of the 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 aluminum foil. The membranes were thoroughly dried and stored at -20 deg C until used.

On the day of use, epidermal membranes were floated onto water from the aluminum foil and placed onto 20 mm diameter filter paper supports. The membranes were then mounted onto diffusion cells. Four donors were used. All samples were abdominal skin from Caucasian females aged 49 to 65 years.

Administration / exposure

Type of coverage:
other: unoccluded
Vehicle:
other: 70/30 (v/v) ethanol/water
Duration of exposure:
24 hours
Doses:
A 6% solution of dimyrcetol (70/30 ethanol/water, v/v) was applied to each skin surface at a dose of 5.00 microliters/cm^2.
No. of animals per group:
Skin samples from 4 donors was used
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose solutions: Dimyrcetol (0.59923 g) was directly weighed into a tared 10 mL volumetric flask and made to volume with premixed 70/30 (v/v) ethanol/water. The mixture was tightly capped and mixed.

APPLICATION OF DOSE/REFERENCE COMPOUND
The dimyrcetol solution was applied using a Gilson M10 Microman positive displacement pipette. Twelve replicate skin samples were dosed with the dimyrcetol and 3 replicates received blank vehicle (with dimyrcetol). The diffusion cell donor chambers were not covered to mimic in-use (unoccluded) conditions.

The positive control, 0.4% benzoic acid (described below), was applied at 25.0 microliters/cm^2 and the diffusion cell donor chambers immediately occluded with greased coverslips. The dose was equivalent to 100 micrograms benzoic acid/cm^2.

VEHICLE
- Amount(s) applied (volume or weight with unit): 5.00 microliters/cm^2. The dose applied was equivalent to 296.2 micrograms/cm^2 dimyrcetol comprising 127.4 micrograms/cm^2 dimyrcetol A and 168.8 micrograms/cm^2 dimyrcetol B.
- Concentration (if solution): 6%

TEST SITE
- Preparation of test site: described above
- Area of exposure: approximately 1.2 cm^2, with exact areas measured for each individual cell

SAMPLE COLLECTION
200 microliter samples were taken from each receptor chamber for dimyrcetol-dosed cells at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after dosing. The liquid removed by each sample was replaced with fresh temperature equilibrated blank receptor medium.

SAMPLE PREPARATION
- Storage procedure: Each sample was placed into a 200 microliter glass vial (gold grade, Chromacol) and a cap with PTFE-lined septum applied prior to freezing the vial at -20 deg C pending analysis.

ANALYSIS
- Method: Samples were analysed by GC-MS. Separations were performed on a RTX-5MS, 0.25 mm ID, 0.25 micrometer film thickness, 15 m capillary column (Thames Restek, UK, Saunderton, UK). The injection volume was 1 microliter (splitless). The over program was as follows: initial, 40 deg C (1.2 minute hold), ramp 1; 14 deg C/min to 115 deg C, ramp 2; 50 deg C/min to 250 deg C (4 minute hold). Quantitative detection was by selective ion monitoring (SIM) on a DSQ dual stage quadrapole II: dimyrcetol A, 59 m/z; dimyrcetol B, 95 m/z.
- Limits of detection and quantification: 0.0085 and 0.0113 micrograms/mL for dimyrcetol A and B, respectively, in ethanol/phosphate buffered saline; 0.0127 and 0.0169 micrograms/mL for dimyrcetol A and B, respectively, in acetonitrile. Six level quadratic calibrations (0.02 to 4 micrograms/mL) were prepared. Injection reproducibility was assessed using five replicate injections at three calibration levels for each injection solvent.

MEMBRANE INTEGRITY ASSESSMENT
The integrity of each membrane was assessed prior to application of either test solutions of dimyrcetol or benzoic acid. Isotonic saline (0.9%, 1 mL) was applied to the skin surface and electrical resistance of each mounted membrane measured 10 minutes later using a Tinsley 1604 LCR databridge at 100 Hz, using the serial equivalent circuit mode. Skin surfaces were subsequently washed three times with water, carefully dried and receptor chambers emptied prior to refilling with EPBS. The cells were then allowed to re-equilibrate to the correct temperature and to allow normal hydration to be achieved.

DETERMINATION OF SKIN DISTRIBUTION
After 24 hours, excess dimyrcetol solution was removed from the diffusion cell donor chambers and placed into tightly capped glass vials for subsequent analysis. The skin samples were removed from the diffusion cells and placed onto small discs of plastic held in place with cyanoacrylate glue. Remaining test material on the skin samples was removed by gently wiping with dry cotton buds and these placed into 20 mL vials with 2 mL of acetonitrile. Each membrane sample was tape stripped 10 times using adhesive tape (D-Squame, CuDerm Inc., Dallas, TX USA). The tape strips were grouped by placing into the same vial as follows: strip 1, strips 2-3, strips 4-6 and strips 7-10. Acetonitrile (2 mL) was added to each group. Diffusion cell chambers were wiped with cotton buds and these placed into 20 mL vials. Donor chambers were then washed with 15mL of acetonitrile to recover any condensed test material. All vials were capped and stored at -20 deg C pending analysis.

ASSESSMENT OF EVAPORATIVE LOSSES
The potential loss due to evaporation was determined by assembling 5 diffusion cells and replacing skin membranes with thin PTFE sheeting. Receptor chambers were filled with water. Diffusion cells were immersed in the constant temperature water bath maintained at 37 deg C throughout the experiment. The 6% dimyrcetol solution was applied to the PFTE membranes in the same manner as with skin membranes. One PTFE diffusion cell was dismantled at 1, 2, 6, 12 and 24 hours. During the dismantling, PTFE sheets were removed and washed with 10 mL, then 5 mL of acetonitrile. The washings were combined in 20 mL vials. The diffusion chambers were washed with 15 mL of acetonitrile and the solution transferred to 20 mL vials. Vials were stored at -20 deg C pending analysis.

DATA CALCULATIONS
Data calculations were performed for each dimyrcetol component (A and B) and also for the combined. According to the SCCNFP guidelines, levels of dimyrcetol in the epidermis, plus any remaining stratum corneum after tape stripping, filter paper supports and receptor fluids were combined to produce a total absorbed dose.
Details on in vitro test system (if applicable):
PRINCIPLES OF ASSAY
- Diffusion cell: Horizontal, Franz-type
- Receptor fluid: Phosphate buffered saline (PBS, pH 7.4). 25/75 (v/v) ethanol/PBS (EPBS) was prepared by mixing 62.5 mL ethanol and 187.5 mL PBS in a 250 mL flask.
- Solubility of test substance in receptor fluid: The solubility of dimyrcetol in EPBS was found to be 342-400 micrograms/mL prior to performing the skin penetration studies.
- Static system: yes
- Test temperature: 32 +/- 1 deg C
- Occlusion: no
- Reference substance: Benzoic acid (min. 99.5%) obtained from Sigma (Poole, UK). Prepared at 4.00 mg/mL in 50/50 (v/v) ethanol/water and applied to skin samples in the same manner as the test substance. Diffusion cell donor chambers were immediately occluded using greased coverslips.
- Other:

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Total recovery:
See Table 1 below.
Percutaneous absorptionopen allclose all
Dose:
6% solution
Parameter:
percentage
Absorption:
2.5 %
Remarks on result:
other: 24 hours
Remarks:
Dimyrcetol A, applied dose 127 micrograms/cm^2
Dose:
6% solution
Parameter:
percentage
Absorption:
0.662 %
Remarks on result:
other: 24 hours
Remarks:
Dimyrcetol B, applied dose 169 micrograms/cm^2
Dose:
6% solution
Parameter:
percentage
Absorption:
1.45 %
Remarks on result:
other: 24 hours
Remarks:
Dimyrcetol, combined

Any other information on results incl. tables

Table 1: Skin absorption of dimyrcetol (% of applied dose, mean±SE, n=12) at 24 hours

Compartment

dimyrcetol A,

dose: 127mg/cm2

dimyrcetol B,

dose: 169mg/cm2

combined,

dose: 296mg/cm2

Wipe

0.166±0.013

0.096±0.007

0.126±0.010

Donor chamber

0.333±0.029

0.891±0.276

0.651±0.163

Strip 1

0.065±0.015

0.031±0.006

0.045±0.010

Strips 2-3

0.069±0.011

0.033±0.004

0.048±0.007

Strips 4-6

0.034±0.004

0.015±0.002

0.023±0.003

Strips 7-10

0.014±0.003

0.011±0.002

0.012±0.002

Epidermis

0.066±0.017

0.028±0.008

0.045±0.012

Filter paper

0.068±0.006

0.138±0.017

0.108±0.013

Permeated

2.36±0.19

0.496±0.034

1.30±0.10

Overall recovery

3.18±0.21

1.74±0.29

2.36±0.24

Stability of dimyrcetol solutions:

Dimyrcetol A appeared to permeate to a greater degree than dimyrcetol B. Approximately 20% of dimyrcetol B was observed to hydrolyze to dimyrcetol A in 24 hours from a solution containing 4 micrograms/mL (determined in a separate experiment). The rate of hydrolysis was not sufficient to fully explain the differences in permeation values.

Evaporative losses:

Very rapid evaporative losses were recorded, with <10% recovered for both components after 1 hour form diffusion cells equipped with PTFE discs. No dimyrcetol was detectable in these cells after 6 hours. Dimyrcetol B evaporated more rapidly than dimyrcetol A (8.97% recovery at 1 hour), but neither component was detectable on PTFE surfaces at 2 hours.

Benzoic acid permeation:

The reference compound, benzoic acid, displayed a mean maximal absorption rate of approximately 29.8 micrograms/cm^2/h. This measured value is within the range of measurements reported in a multi-centre study (Van de Sandt et al., 2004).

Applicant's summary and conclusion

Conclusions:
The total absorbed dose of dimyrcetol was 2.50 +/- 0.20% and 0.662 +/- 0.040% for components A and B, respectively, and a combined total of 1.45 +/- 0.10%. Overall recoveries of dimyrcetol were low with values 3.18 +/- 0.21%, 1.74 +/- 0.29% and 2.36 +/- 0.24% of the applied dose for dimyrcetol A, B and combined, respectively. Evaporative losses were rapid from the application surfaces with <10% recovered from PTFE surfaces after 1 hour. Evaporative losses explain the low recoveries reported.
Executive summary:

The in vitro percutaneous absorption of dimyrcetol under in-use (unoccluded) conditions at an application concentration of 6% (w/v) in 70/30 (v/v) ethanol/water was determined in the current study. The absorption of each component, dimyrcetol A (2,6-dimethyl-7-octen-2-ol) and dimyrcetol B (2,6-dimethyl-7-octen-2-ol formate ester) were separately measured and combined for total dimyrcetol absorption.

Component A was observed to more rapidly permeate human skin than component B. In addition, component B was found to more rapidly evaporate than component A. Hydrolysis of dimyrcetol B to A was also an issue, and may have contributed to the overall observed absorption values.

The levels of dimyrcetol in the epidermis (with any remaining in stratum corneum after tape stripping), filter paper membrane supports and receptor fluids combined gave total absorbed doses of 2.50 +/- 0.20% and 0.662 +/- 0.040% for components A and B, respectively, and a combined value of 1.45 +/- 0.10%. Overall recoveries of dimyrcetol were low with values 3.18 +/- 0.21%, 1.74 +/- 0.29% and 2.36 +/- 0.24% of the applied dose for dimyrcetol A, B and combined, respectively. Evaporative losses were responsible for the low recoveries recorded.