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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: In Vitro Dermal Absorption Rate Testing of Certain Chemicals of Interest to the Occupational Safety and Health Administration. Federal Register: April 26, 2004 (Volume 69, Number 80)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetonitrile
EC Number:
200-835-2
EC Name:
Acetonitrile
Cas Number:
75-05-8
Molecular formula:
C2H3N
IUPAC Name:
acetonitrile
Details on test material:
- Name of test material (as cited in study report): acetonitrile
- Analytical purity: >99%
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
- Supplier: non-radiolabeled acetonitrile (logP = 0.447) Sigma-Aldrich (St. Louis, MO); radiolabeled test substance, [2-14C]acetonitrile, was prepared by Perkin-Elmer Life Sciences (Boston, MA)
- Specific activity (if radiolabelling): 5.22 uCi/mmole
- Locations of the label (if radiolabelling): [2-14C]acetonitrile
- Radiochemical purity (if radiolabelling): >99%
Specific details on test material used for the study:
- Name of test material (as cited in study report): acetonitrile
- Analytical purity: >99%
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
- Supplier: non-radiolabeled acetonitrile (logP = 0.447) Sigma-Aldrich (St. Louis, MO); radiolabeled test substance, [2-14C]acetonitrile, was prepared by Perkin-Elmer Life Sciences (Boston, MA)
- Specific activity (if radiolabelling): 5.22 uCi/mmole
- Locations of the label (if radiolabelling): [2-14C]acetonitrile
- Radiochemical purity (if radiolabelling): >99%
Radiolabelling:
yes
Remarks:
[2-14C]acetonitrile

Test animals

Species:
other: human cadaver skin
Sex:
not specified
Details on test animals or test system and environmental conditions:
Samples of human cadaver skin from the National Disease Research Interchange (NDRI, Philadelphia, PA) were stored frozen at approximately -20°C until prepared for use. Samples were removed from donors within 24 hours of death and used within three months. Skin specimens selected for use were identified using a unique code (e.g., HCFA-26A = Human, Caucasian, Female, Abdomen sample 26-A).

Administration / exposure

Type of coverage:
occlusive
Duration of exposure:
PERMEABILITY COEFFICIENT: until steady state was achieved
DETERMINATION OF SHORT-TERM ABSORPTION RATE: 10 and 60 MINUTES
Exposure area of 0.64 cm2.
Doses:
PERMEABILITY COEFFICIENT
- Protocol Group A: 1200 μL/cm2

DETERMINATION OF SHORT-TERM ABSORPTION RATE 10 T0 60 MINUTES
- Protocol Group B: 10 μL/cm2
- Protocol Group C: 10 μL/cm2
- Protocol Group D: 10 μL/cm2
(Termination times: 2 replicates at 10 minutes, 2 replicates at 60 minutes)
No. of animals per group:
PERMEABILITY COEFFICIENT: Six skin replicates representing three donors.
DETERMINATION OF SHORT-TERM ABSORPTION RATE: three experiments each with two replicates at 10 minutes and two replicates at 60 minutes from the same donor.
Control animals:
no
Details on study design:
DOSE PREPARATION
The non-radiolabeled test substance, which was liquid at room temperature, was spiked with radiolabeled acetonitrile.
The homogeneity and the amount of radiolabeled acetonitrile in the solution was determined by subjecting aliquots of the spiked solution to radioanalysis by liquid scintillation counting (LSC).
The concentration of acetonitrile in the prepared solution was taken as its density, 0.786 g/mL (786,000 μg/cm3).
The results of the homogeneity analysis were used to calculate the specific activity of radiolabeled acetonitrile in the prepared solution (μCi/mg).

PERMEABILITY COEFFICIENT EXPERIMENT:
An infinite dose of non-radiolabeled acetonitrile spiked with radiolabeled acetonitrile was applied to the epidermal surface, via the donor chamber, at a target rate of 1200 μL/cm2, to 6 skin replicates representing 3 human subjects, and the donor chamber opening was occluded with a rubber stopper and Parafilm®. Serial receptor fluid samples were taken at 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours post-application and analyzed for radioactivity by liquid scintillation counting. At the end of the 8-hour exposure, excess acetonitrile was removed from the donor compartment and the skin washed with a 2% Ivory® soap solution followed by rinsing with water. The remaining receptor fluid was removed and discarded and the receptor and donor chambers were filled with 0.9% saline and an end of experiment integrity asssessment was determined using EI.

SHORT-TERM EXPERIMENT:
Finite dose of non-radiolabeled acetonitrile spiked with radiolabeled acetonitrile (10 μL/cm2) was applied to the epidermal surface, via the donor chamber, to 12 skin replicates representing 3 human subjects, and the donor chamber opening was covered with an organic volatile trap. At the end of the required exposure interval (10 minute and 60 minutes), 6 replicates each were terminated. At termination, the volatile trap was removed and extracted with acetonitrile. The skin surface was washed with a 2% Ivory® soap solution, rinsed with water, and the receptor fluid was removed and retained for analysis. The receptor and donor chambers were filled with 0.9% saline and end of experiment integrity asssessment was taken using EI.

ANALYSIS
- Method type(s) for identification: Samples were analyzed in a Packard liquid scintillation counter. Samples were counted for 10 minutes or until 160,000 disintegrations were accumulated (0.5%, 2σ), whichever came first. The limit of detection (LOD) for the analysis of each sample was taken as twice the background disintegration rate obtained from analysis of appropriate blank samples.


Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human cadaver skin
- Ethical approval if human skin: yes
- Type of skin: abdominal region
- Preparative technique: Samples of human cadaver skin obtained from the abdominal region, which were maintained frozen, were thawed at room temperature. Full thickness skin was dermatomed using a Padgett Electro Dermatome® (Padgett Instruments, Kansas City, MO). The membrane was then placed onto an aluminum pan, with its identification written on the pan, and stored refrigerated at 0-10°C.
- Thickness of skin (in mm): The thickness of the prepared skins, as measured with a Mahr micrometer, ranged from 229 to 421 μm.
- Membrane integrity check: Membranes were removed from refrigeration storage and hydrated in 0.9% saline for approximately 15 minutes. Following hydration, the membrane was mounted onto the top of the receptor chamber, stratum corneum uppermost, which was maintained with 0.9% saline. The donor chamber was then clamped in place and filled with 0.9% saline. The membrane was then allowed to equilibrate for approximately 30 minutes. During equilibration, the in vitro cells were heated using a recirculating water bath system to yield a receptor fluid temperature of 32°C. Following equilibration, the integrity of each membrane was assessed by measurement of electrical impedance (EI) prior to application of the test substance. (4-5) Membranes with an EI of ≥17 kΩ were considered intact and retained for use on study. Saline in the donor and receptor chambers was removed prior to dosing, and the receptor chamber filled with fresh receptor fluid.
- Storage conditions: The membrane was then placed onto an aluminum pan, with its identification written on the pan, and stored refrigerated at 0-10°C.
- Justification of species, anatomical site and preparative technique: Dermal contamination is a potential route of human exposure. In vitro dermal techniques, which are required by the test rule described in the Federal Register dated April 26, 2004 (Volume 69, Number 80), have been shown to be a conservative model for predicting percutaneous absorption of various chemicals in vivo.

PRINCIPLES OF ASSAY
- Receptor fluid: The receptor chamber was filled with 0.9% saline and allowed to equilibrate for at least 15 minutes prior to dosing. For the purpose of ensuring sink conditions, acetonitrile was considered to be infinitely soluble in the saline receptor fluid maintained at 32°C.
- Solubility of test substance in receptor fluid: Acetonitrile was considered infinitely soluble in the 0.9% saline receptor fluid at 32°C.

Results and discussion

Percutaneous absorptionopen allclose all
Parameter:
percentage
Absorption:
0.2 %
Remarks on result:
other: 8 hours
Remarks:
Based on the slope at steady-state, the permeability coefficient was calculated to be 1.82 x 10-4 cm/h.
Parameter:
percentage
Absorption:
<= 0.7 %
Remarks on result:
other: 60 minutes
Remarks:
Following a 60-minute exposure to a finite application, the short-term penetration rate was calculated to be 66.0 μg equiv/cm2/h.
Remarks on result:
other: 10 minutes
Remarks:
Following a 10-minute exposure to a finite application the short-term penetration rate was calculated to be 375.6 μg equiv/cm2/h.

Any other information on results incl. tables

Based on the slope at steady-state (143.4 μg equiv/cm2/h) and the concentration of the applied solution of acetonitrile taken as its density (786,000 μg/cm3), the permeability coefficient was calculated to be 1.82 x 10-4 cm/h.

Following a 10-minute exposure to a finite application of [2-14C]acetonitrile, a total of 22.5 μg equivalents of acetonitrile were detected in the receptor fluid, with 18.3 μg equivalents in the skin. Based on the amount of acetonitrile in the receptor fluid and skin, an exposure area of 0.64 cm2, and an exposure time of 10 minutes (0.17 hours), the short-term penetration rate was calculated to be 375.6 μg equiv/cm2/h.

Following a 60-minute exposure to a finite application of [2-14C]acetonitrile, a total of 29.1 μg equivalents of acetonitrile were detected in the receptor fluid and 13.1 μg equivalents in the skin. Based on the amount of acetonitrile in the receptor fluid and skin, an exposure area of 0.64 cm2, and an exposure time of one hour, the short-term penetration rate was calculated to be 66.0 μg equiv/cm2/h.

Recovery of the applied radioactive acetonitrile, exclusive of experimental group, exceeded 96%. For the Kp experiment, the majority of the infinite dose was removed from the skin surface at the end of the 8-hour exposure, and only a small portion penetrated the skin and was recovered in the receptor fluid (0.24%). Following a 10- and 60-minute exposure, a major portion of the finite dose was recovered in the organic volatile trap (~96%), with only a minor portion accounted for in the receptor fluid (≤0.74%).

Applicant's summary and conclusion

Conclusions:
The results presented in this report confirm that in vitro dermal exposure to finite doses of acetonitrile are likely to result in low systemic availability, and that use of the permeability coefficient value is expected to generate an overestimate of systemic absorption in vivo, given the volatile character of acetonitrile and the conservative nature of the in vitro model.
Executive summary:

In a guideline (OECD 428 equivalent) and GLP study, the in vitro absorption of Acetonitrile through human cadaver skin was shown to be low. The permeability coefficient was calculated to be 1.82 x 10-4 cm/h. Short-term penetration rates were calculated to be 375.6 μg equiv/cm2/h (10 minute exposure) and 66.0 μg equiv/cm2/h (60 minute exposure).

For the Kp experiment, the majority of the infinite dose was removed from the skin surface at the end of the 8-hour exposure, and only a small portion penetrated the skin and was recovered in the receptor fluid (0.24%). Following a 10- and 60-minute exposure, a major portion of the finite dose was recovered in the organic volatile trap (~96%), with only a minor portion accounted for in the receptor fluid (≤0.74%).