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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics 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 is in compliance with the Environmental Protection Agency final rule (40 CFR Parts 9 and 799).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Objective of study:
absorption
Test guideline
Qualifier:
according to
Guideline:
other: The study is in compliance with the Environmental Protection Agency final rule (40 CFR Parts 9 and 799), In Vitro Dermal Absorption Rate Testing of Certain Chemicals of Interest to the Occupational Safety and Health Administration.
Deviations:
yes
Remarks:
Two minor protocol deviations were reported, neither of which had an adverse impact on any of the study data.
Principles of method if other than guideline:
The study evaluated the rate and amount of [^14C]-methyl isoamyl ketone absorbed across human skin after in vitro exposure, and evaluated the disposition of [^14C]-methyl isoamyl ketone in the various layers (stratum corneum, epidermis, and dermis) of human skin after in vitro exposure.

Human skin samples were incubated for 10 minutes or 1 hour with the epidermal surface exposed to [^14C]-methyl isoamyl ketone in a Bronaugh flow-through diffusion cell under occluded conditions. The amount of [^14C]-methyl isoamyl ketone absorbed across the skin into the receptor fluid and the disposition of [^14C]-methyl isoamyl ketone in the various skin layers following each incubation period was determined by liquid scintillation counting. Receptor fluid collected at 10 minutes and 1 hour was used to calculate the rate and amount of absorption.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Test substance name (radiolabelled): [^14C]-methyl isoamyl ketone
-Specific activity: 250 μCi/mL, 0.037 mCi/mmol
-Purity: ≥ 99%
-Lot 036K9422

-Test substance name (non-radiolabelled): methyl isoamyl ketone
-Molecular weight: 114.19 g/mol
-Lot 03906PB

Labeled and unlabeled methyl isoamyl ketone were mixed to prepare a [^14C]-methyl isoamyl ketone dosing solution. The [^14C]-methyl isoamyl ketone dosing solution was prepared as a neat solution (target radiolabel concentration of 25 μCi/mL). Actual radiolabel concentrations achieved in the dose solutions were 20.2 (for 1 donor) and 20.3 μCi/mL (for 2 donors).
Radiolabelling:
yes

Test animals

Species:
human
Sex:
male/female
Details on test animals and environmental conditions:
Skin was obtained from the abdominal region of human cadavers via an organ procurement agency.

Administration / exposure

Route of administration:
other: in vitro, dermatomed skin
Vehicle:
unchanged (no vehicle)
Details on exposure:
Tissue Incubation:
Two sets of skin disks were dosed with [^14C]-methyl isoamyl ketone dosing solution. Each set contained two skin disks from each donor for a total of six skin disks per incubation set. Skin disks with acceptable barrier function were dosed with 200 μL of the [^14C]-methyl isoamyl ketone dosing solution on the epidermal surface of each disk. The skin disks were incubated for 10 minutes or 1 hour under occluded conditions and the receptor fluid was collected as specified below.

Set 1: The skin disks were incubated for 10 minutes and the receptor fluid was collected at 10 minutes.

Set 2: The skin disks were incubated for 1 hour and the receptor fluid was collected at 1 hour.

At the end of the incubation period for each set, the skin surface was wiped with cotton swabs, washed twice with detergent and twice with water. After the last wash, the skin disks were removed from the diffusion cells and separated into component layers (stratum corneum, epidermis, and dermis). Each skin layer was digested in 1 mL of 1 N sodium hydroxide and retained for liquid scintillation counting. The sides of each diffusion chamber were also wiped with cotton swabs to obtain any [^14C]-methyl isoamyl ketone remaining in the diffusion chamber after each skin disk had been removed.
Duration and frequency of treatment / exposure:
Once for either 10 or 60 minutes.
Doses / concentrations
Remarks:
Doses / Concentrations:
20.2 μCi/mL (1 donor) and 20.3 μCi/mL (2 donors).
Details on study design:
Tissue Preparation:
Skin samples were obtained from the abdominal region of five human cadavers obtained through an organ procurement agency and were stored at –20°C prior to use. The skin was processed to remove the subcutaneous fat layer and cut with a dermatome (Padgett Dermatome, Model B, S/N 1-8314) to a split thickness of 290-400 μm. Skin disks were prepared from the dermatomed skin and used for the incubations on the same day. The disks were prepared using a cork-boring tool and stored in receptor fluid kept on wet ice until incubation. Five skin disks (16–20 mm in diameter) per donor were used for the incubations.

Receptor Fluid:
The receptor fluid flowing across the lower surface of the skin was Hanks’ balanced salt solution (Gibco, lot 1327456) containing 6% polyethoxyoleate (Sigma, lot 069H0140) and antibiotics (TorpedoTM Antibiotic Mix, In Vitro Technologies, lot C13046C). The pH of the receptor fluid was 7.40-7.45.

Analysis:
Cotton swabs used to remove [^14C]-methyl isoamyl ketone from the skin surface or the sides of the diffusion chamber, and cotton swabs used in the washes were extracted with liquid scintillation cocktail. Liquid scintillation cocktail was added to the entire volume or an aliquot of each of the fractions (skin layer digests or receptor fluid collections). The extracted swab samples and the fractions mixed with liquid scintillation cocktail were placed in the dark at ambient temperature for at least 24 hours to allow chemiluminescence to decay and analyzed using a liquid scintillation counter (Beckman, Model LS5000TA, Serial No. 7040864).

Criteria for Data Acceptance:
Data were accepted since the data were obtained using skin disks with sufficient barrier function. Prior to the determination of the percutaneous absorption of methyl isoamyl ketone, the permeability of tritiated water was determined in all the human skin disks. All skin disks used in the experiments had to have tritiated water absorption within the limits of acceptance, or < 0.3% penetration following incubation of up to one hour.
Statistics:
The amount of [^14C]-methyl isoamyl ketone absorbed across the skin barrier was calculated and expressed as μg/hour/cm^2. The percent distribution of [^14C]-methyl isoamyl ketone in each of the skin layers was determined. The mass balance of the test substance was also calculated. All calculations, including mean and standard deviation values, were performed using Microsoft® Office Excel 2000.

Results and discussion

Any other information on results incl. tables

Percutaneous absorption of methyl isoamyl ketone:

The average absorption of methyl isoamyl ketone into the receptor fluid following 10-minute exposure was 0.683 ± 1.07 μg/cm^2; thus, the first short-term absorption rate was 4.10 ±6.42 μg/cm^2/hour. The average absorption of methyl isoamyl ketone into the receptor fluid following 1-hour exposure was 37.4 ± 65.2 μg/cm^2; thus the second short-term absorption rate was 37.4 ± 65.2 μg/cm^2/hour.

Cutaneous disposition of methyl isoamyl ketone:

Data are summarized in tables 1 and 2.

-The average amount of MIAK present in the stratum corneum was 67.6 ± 130 and 4.35 ± 3.85 μg/cm2 following exposure for 10 minutes and 1 hour, respectively.

-The average amount of MIAK present in the epidermis was 6.14 ± 7.72 and 3.38 ± 1.93 μg/cm2 following exposure for 10 minutes and 1 hour, respectively.

-The average amount of MIAK present in the dermis was 24.5 ± 22.7 and 12.3 ± 6.14 μg/cm2 following exposure for 10 minutes and 1 hour, respectively.

-The average amount of MIAK present in the water used for heat separation of epidermis and dermis was 36.6 ± 17.0 and 32.7 ± 35.0 μg/cm2 following exposure for 10 minutes and 1 hour, respectively.

Total absorbed and recovered methyl isoamyl ketone:

The total absorbed amount of methyl isoamyl ketone was estimated based on the amount of [^14C]-methyl isoamyl ketone in the stratum corneum, epidermis, dermis, the water used to separate dermis and epidermis, and the receptor fluid. The total amount of methyl isoamyl ketone absorbed was 135 ± 135 and 90.2 ± 107 μg/cm², following exposure for 10 minutes and 1 hour, respectively. This corresponded to 0.0529 ± 0.0527 and 0.0352 ± 0.0420% of the total applied dose, following exposure for 10 minutes and 1 hour, respectively.

The total amount of MIAK recovered from the incubations (mass balance) was estimated based on the amount in all the surface wash fractions, the various skin layers, the water used to separate dermis and epidermis, and the receptor fluid. The total recovery of MIAK was 90.9 ± 2.37 and 91.7 ± 3.47% of the total applied dose following exposure for 10 minutes and 1 hour, respectively.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: The amount of MIAK dosed onto skin disks appeared to simulate an infinite dose under study conditions. This was inferred based on observing that the total amount absorbed was considerably less than 1% of available material after 10 min or 1 hr of exposure
In this study, human skin disks were exposed in vitro to a simulated infinite dose of methyl isoamyl ketone. The total amount of MIAK absorbed was less than 1% after both 10 minute and one hour exposures.
Executive summary:

In a study designed to determine the in vitro absorption of MIAK by human skin disks after a single topical application of [^14C]- methyl isoamyl ketone, less than 1% was absorbed after both 10 minute and one hour exposures. Of the material absorbed, the majority was located in the stratum corneum after a 10-min exposure and in the dermis after a 1-hr exposure.  In this study, [^14C]- methyl isoamyl ketone was applied at a dose concentration of 20.2-20.3 μCi/mL to 6 tissue samples (two tissue samples per individual) for either 10 minutes or 1 hour, after which time radioactivity was measured in the skin layers and the surrounding receptor fluid. The short-term absorption rates for methyl isoamyl ketone were estimated to be 4.10 ± 6.42 μg/cm^2/hour (following 10-minute exposure) and 37.4 ± 65.2 μg/cm^2/hour (following 1-hour exposure). The total amount of methyl isoamyl ketone absorbed was determined to be 135 ± 135 and 90.2 ± 107 μg/cm^2, following exposure for 10 minutes and 1 hour, respectively. The amount absorbed and the amount recovered from the wash fractions accounted for 90.8 ± 2.37 and 91.7 ± 3.45% of the total applied dose following exposure for 10 minutes and 1 hour, respectively.