Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restriction; the study was conducted according to GLPs and OECD Guideline 428.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Test substance (as cited in report): Methyl Isoamyl Ketone
-Synonyms: MIAK, 5-methyl-2-hexanone, 5-methylhexan-2-one
-Physical State and Appearance: Clear, colorless liquid
-Purity: 99.3%
-Source of test substance: Eastman Chemical Company, Kingsport, TN
-The structure of the test substance was confirmed using GC/MS.
-The purity of the test substance was determined by GC with FID and found to be 99.3 ± 0.0% prior to use.
-The purity of the test substance recovered from the donor chambers following the 7 hour skin absorption study was determined by GC/FID and was found to be 98.6 ± 0.5%. Based on comparison of the purity before study initiation and after completion of the study, the test substance was considered to be stable during the test.
Radiolabelling:
no

Test animals

Species:
human
Sex:
not specified
Details on test animals and environmental conditions:
Dermatomed sections of human cadaver skin were obtained from the National Disease Research Interchange, Philadelphia, PA. Sufficient medical history for each donor was obtained to ensure that the tissue had not been adversely affected by any clinical condition or disease that would alter the results of this study. Samples of dermatomed skin were stored frozen at -70°C.

Administration / exposure

Type of coverage:
other: Not strictly occlusive, but the donor chamber was filled with fluid (^3H20 or MIAK), so the tissue was completely covered during the study.
Vehicle:
unchanged (no vehicle)
Duration of exposure:
6 hours for determination of the permeability of ^3H20 and 7 hours for the permeability of MIAK
Doses:
300µl
No. of animals per group:
3 different human donors were used
Control animals:
yes
Remarks:
One of the three skin samples from each donor was used as a control, i.e., was not exposed to MIAK.
Details on study design:
Overall Study Design:
The study consisted of a three-phase experiment; phases 1 and 3 were used to determine the permeability of ^3H20 and phase 2 was used to determine the permeability of the test substance, MIAK.

Tissue Samples and Sample Preparation:
Skin samples from three different human donors were used; the experiment used nine diffusion cells containing three skin preparations originating from each of the three human donors. Skin samples were collected using a dermatome at a specified thickness of 200-500µm. Each skin specimen was placed with the external surface uppermost over the opening of the receptor chamber of a diffusion cell. A CAPFE™ O-ring was placed under the skin specimen, and the donor chamber clamped in place to secure the tissue.

Receptor Solution:
The receptor solution was Dulbecco’s phosphate buffered saline containing penicillin (100 units/ml), streptomycin (100 µg/ml) and amphotericin B as Fungizone™ (0.25 µg/ml).

During each phase of the experiment, the receptor chambers were filled with the buffered (pH 7.1) isotonic saline antibiotic-antimycotic solution, and stirred continuously with a stir bar throughout the study. Duplicate background (0 hour) samples were taken from the receptor chamber of each cell. 300 µl of ^3H20 (specific activity 4.34 µCi/gram) donor solution was applied to the donor chambers for phases 1 and 3, while 300 µL of the neat test substance was applied directly to the surface of two skin samples from each donor in phase 2. The remaining skin sample was exposed to only saline during phase 2. The diffusion cells were incubated at 30°C for 6 hours in phases 1 and 3 and for 7 hours in phase 2. At hourly intervals, samples were removed from the receptor chambers in all phases and at 0, 3, 6, and 7 hours from the cells designated as the saline control of phase 2. The test substance in the receptor solution in phase 2 was assayed by GC/FID. Phase 1 and 3 solutions were assayed by liquid scintillation spectrometry (LSS; Wallac Model 1409 liquid scintillation spectrometer and polyfluor liquid scintillant). The receptor chambers were refilled after each sampling.

After the first two phases, the donor and receptor chambers were emptied, rinsed three times with saline, and refilled with antibiotic-antimycotic saline. The cells were allowed to stir overnight, after which they were emptied, rinsed, and refilled with the receptor solution for phase 3. The rate of increase in the concentration of the test substance or ^3H2O in the receptor chamber of each cell was used to calculate a permeability constant (cm/hr) and an absorption rate (mg/cm^2/hr) for the test substance and ^3H2O.
Details on in vitro test system (if applicable):
Each skin specimen was placed with the external surface uppermost over the opening of the receptor chamber of a Franz-type diffusion cell. A CAPFE™ O-ring was placed under the skin specimen, and the donor chamber clamped in place to secure the tissue.

Results and discussion

Percutaneous absorptionopen allclose all
Dose:
Phase 1: ^3H2O 300 µl
Remarks on result:
other: Absorption (%) (upper value): 2.12 ± 1.01 mg/cm^2/hr. Although slightly higher, this value was comparable to the historical control (n=27) of 1.62 ± 0.89 mg/cm^2/hr.
Dose:
Phase 2: MIAK 300 µl
Remarks on result:
other: Absorption (%) (upper value): 0.21 ± 0.15 mg/cm^2/hr. Permeability constant for study: 2.61X10^-4 cm/hr. 6 skin specimens exposed to test substance; remaining 3 specimens exposed to physiological saline served as controls for damage ratio calculations.
Dose:
Phase 3: ^3H2O 300 µl
Remarks on result:
other: Absorption (%) (upper value): 2.35 ± 1.22 mg/cm2/hr. Although slightly higher, this value was comparable to the historical control (n=27) of 1.62 ± 0.89 mg/cm^2/hr.

Any other information on results incl. tables

The mean damage ratio for phase 2 was 1.02 ± 0.14 and 1.10 ± 0.12 for phase 3. The value in phase 2 was within the range of damage ratios presented by Dugard et al (1984) of 1-2 for water and within acceptable ranges of the testing facility. Exposure to the test substance for 7 hours produced similar damage ratios to those expected from exposure to physiological saline. 

If it is assumed that skin absorption in man is similar to that of this in vitro study, then the internal dose of MIAK from a 1 hour exposure would be 2.19 mg/kg if the area of the hands is approximately 720 cm^2 in an average adult (70 kg).

Applicant's summary and conclusion

Conclusions:
In an in vitro dermal absorption study, the mean absorption rate of methyl isoamyl ketone through dermatomed human skin specimens was 0.21 ± 0.15 mg/cm^2/hr with a corresponding permeability constant of (2.61 ± 1.81) x 10^-4 cm/hr. Based on criteria established by Marzulli et al. (1969), the test substance would be considered a “moderate” penetrant relative to other chemical species. These data can be used to estimate the uptake in man following dermal exposure assuming that skin absorption in vivo is similar to that measured in vitro. Assuming an adult weighs 70 kg, the internal dose from a 1-hr exposure to both hands would be 2.19 mg/kg. The mean damage ratio for this study was not different from that expected from exposure to physiological saline. It is concluded that, under the conditions used in this study, methyl isoamyl ketone did not cause significant damage to the skin and that MIAK is absorbed through the skin.
Executive summary:

In an in vitro dermal absorption study, methyl isoamyl ketone was applied to dermatomed skin specimens using a Franz-type diffusion cell and incubated for 7 hours at 30°C. The receptor chambers were filled with buffered isotonic saline containing an antibiotic-antimycotic solution, and stirred continuously with a stir bar throughout the study. 300 µl of ^3H20 (specific activity 4.34 µCi/gram) donor solution was applied in the donor chambers in two separate experiments (phases 1 and 3) and 300 µL of the neat test substance was applied directly to the surface of the skin in a separate phase (phase 2).  At hourly intervals, samples were removed from the receptor chambers and the test substance in the receptor solution was assayed by GC/FID. ^3H20 was assayed by liquid scintillation spectrometry. The mean absorption rate of methyl isoamyl ketone was 0.21 ± 0.15 mg/cm^2/hr with a permeability constant of 2.61X10^-4 cm/hr. The mean damage ratio was 1.02 ± 0.14, which was within the range of damage ratios presented in the published literature and the testing facility. It was concluded that exposure to methyl isoamyl ketone for 7 hours produced damage similar to that observed with physiological saline. Using the definitions suggested by Marzulli, Brown and Maibach (1969), MIAK would be classified as moderate with respect to its absorption through human skin.