Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
Remarks:
Not specified in report
Qualifier:
according to
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:
according to
Guideline:
other: OECD Draft Guidance Document for the Conduct of Skin Absorption Studies. OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 28. (2002)
Qualifier:
according to
Guideline:
other: European Commission Guidance Document on Dermal Absorption. Sanco/222/2000 rev 6 (2002).
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Unlabeled test material : purity 99.5%, Haskell #26675

Radiolabel: purity 99.8%, purchased from Moravek Biochemicals, Inc., Haskell #22705-113

When mixed with technical 2-nitropropane, the radiochemical purity of [14C]2-nitropropane was 98.20%.

The chemical concentration for the prepared solution was taken as its density, 988,000 μg/ml.
The verified radiochemical concentration (specific activity) for the prepared solution was 0.0928 μCi/mg.
Radiolabelling:
yes
Remarks:
[14C]2-nitropropane

Test animals

Species:
human
Strain:
other: Caucasian
Sex:
male/female
Details on test animals and environmental conditions:
Samples of human cadaver skin from the National Disease Research Interchange (NDRI) 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
Vehicle:
unchanged (no vehicle)
Duration of exposure:
10 or 60 minutes
Doses:
Determining the Permeability Coefficient (Kp)- 1200 μL/cm2
Determining the Short-Term Absorption Rate, 10 and 60 minutes- 10 μL/cm2



Solubility of 2-Nitropropane in Receptor Fluid- 2-Nitropropane was determined to have a maximum solubility of 24,660 μg/mL in 0.9% saline fortified with 6% polyethoxyoleate (polyethylene glycol (PEG) 20 oleyl ether), which is 1451- fold the maximum solubility in water of 17 μg/ml.
No. of animals per group:
Determining the Permeability Coefficient (Kp)- Number of skin replicates: 6, representing 3 donors
Determining the Short-Term Absorption Rate, 10 and 60 minutes- Number of skin replicates: 4, representing a single unique donor/ protocol group x 3 groups
Control animals:
no
Details on study design:
Determining the Permeability Coefficient (Kp)
Protocol Group: A
Number of skin replicates: 6, representing 3 donors
Dose volume: 1200 μL/cm2.
Termination time: following steady-state determination

Following dose application, the donor chamber opening was occluded with Parafilm®. Serial receptor fluid samples, duplicate 50 μL aliquot, were taken at 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours post-dose. The volume of receptor fluid in the receptor chamber was maintained by the replacement of a volume of fresh receptor fluid, equal to the sample volume. The receptor chamber arm remained occluded with Parafilm® at all times other than at sampling. At the end of the exposure period, the receptor fluid was removed and discarded.


Determining the Short-Term Absorption Rate, 10 and 60 minutes

Protocol Group: B
Number of skin replicates: 4, representing a single unique donor
Dose volume: 10 μL/cm2
Termination times: 2 replicates at 10 minutes, 2 replicates at 60 minutes

Protocol Group: C
Number of skin replicates: 4, representing a single unique donor
Dose volume: 10 μL/cm2
Termination times: 2 replicates at 10 minutes, 2 replicates at 60 minutes

Protocol Group: D
Number of skin replicates: 4, representing a single unique donor
Dose volume: 10 μL/cm2
Termination times: 2 replicates at 10 minutes, 2 replicates at 60 minutes

Following dose application, the donor chamber opening was occluded with an organic volatile
trap containing Anasorb 747 (SKC Inc., Eighty Four, Pennsylvania). At the end of the exposure
period, the receptor fluid was removed and placed into a suitable container for analysis.

Details on in vitro test system (if applicable):
In Vitro Diffusion Cell Model- A static diffusion cell model was used for this study. The in vitro cells had an exposure area of 0.64 cm2 and a receptor fluid chamber volume of approximately 5 mL.

Preparation of Skin Membranes- Samples of human cadaver skin obtained from the abdominal region, which were maintained frozen, were thawed at room temperature. Full thickness skin was immersed in 60°C water for 45 seconds to 2 minutes and the epidermis was peeled away from the dermis. The human epidermal membrane was then placed onto an aluminum pan, with its identification written on the pan, and stored refrigerated at 0-10°C until readied for use. The thickness of the prepared skins, as measured with a Mahr micrometer (Mahr Federal Inc., Providence, Rhode Island),
ranged from 31 to 46 μm.

Membrane Equilibration and Assessment of Membrane Integrity- 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.

Receptor Fluid- The receptor chamber was filled with 0.9% saline fortified with 6% polyethoxyoleate (polyethylene glycol (PEG) 20 oleyl ether), and allowed to equilibrate for at least 15 minutes prior to dosing. Solubility of 2-nitropropane in the selected receptor fluid was confirmed prior to study start to ensure that the maximum possible concentration of the 2-nitropropane in the receptor fluid, based on the total amount of 2-nitropropane applied to the skin surface, did not exceed 10% of its solubility.

Results and discussion

Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
2-Nitropropane, Permeability Coefficient

Key observations of mean data:
• The integrity of human skin, as determined by EI, was affected by continuous exposure under occlusive conditions to 2-nitropropane. The ratio of the post-EI values to pre-EI values was 0.42. This decrease in EI, which occurred over the exposure phase, may have been due to a loss in barrier properties of the stratum corneum. Therefore, the permeability coefficient should be considered conservative.
• 2-Nitropropane was detectable in the receptor fluid at the 0.5-hour serial sampling timepoint (36.6 μg equiv/cm2); the final receptor fluid sample (8 hours) was 777.1 μg equiv/cm2.
• Steady state penetration of 2-nitropropane, which was represented by a minimum of 4 data points, had a slope of 118.9 μg equiv/cm2/h.
• At the end of the 8-hour exposure interval, .074% of the applied 2-nitropropane was detected in the receptor chamber demonstrating that the static diffusion cell receptor fluid (0.9% saline fortified with 6% PEG) offered sink conditions to 2-nitropropane.
• The permeability coefficient was calculated to be 1.19 x 10-4 cm/h, based on the slope at steady-state (118.9 μg equiv/cm2/h) and the concentration of the applied dose of 2- nitropropane taken as its density (988,000 μg/cm3).

2-Nitropropane, 10- and 60-Minute Short-Term Penetration Rates

Key observations of mean data:
• The integrity of human skin, as determined by EI, was affected slightly by either short-term exposure interval of 10 and 60 minutes under occlusive conditions to 2-nitropropane. The ratio of the post-EI values to pre-EI values for the 10-minute and 60-minute exposure groups were 0.69 and 0.83, respectively.
• Following a 10-minute exposure to a finite application of 2-nitropropane, a total of 16.3 μg equivalents of 2-nitropropane were detected in the receptor fluid, with 14.8 μg equivalents in the skin. Based on the amount of 2-nitropropane 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 285.9 μg equiv/cm2/h.
• Following a 60-minute exposure to a finite application of 2-nitropropane, a total of 39.5 μg equivalents of 2-nitropropane were detected in the receptor fluid and 3.22 μg equivalents in the skin. Based on the amount of 2-nitropropane 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 66.8 μg equiv/cm2/h.
Total recovery:
2-Nitropropane, Permeability Coefficient- Recovery of the applied radioactive dose was 98.4%.

2-Nitropropane, 10- and 60-Minute Short-Term Penetration Rates- Recovery of the applied radioactive dose was 78.2% and 100.9% for the 10- and 60-minute exposure groups, respectively.
Percutaneous absorptionopen allclose all
Dose:
5034 ug
Remarks on result:
other: 10 minute exposure
Remarks:
short term penetration rate is 285.9 ug equiv/cm2/h
Dose:
5034 ug
Remarks on result:
other: 60 minute exposure
Remarks:
short term penetration rate is 66.8 ug equiv/cm2/h
Conversion factor human vs. animal skin:
Not applicable

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Based on the slope at steady-state (118.9 μg equiv/cm2/h) and the concentration of the applied dose of 2-nitropropane taken as its density (988,000 μg/cm3), the permeability coefficient was calculated to be 1.19 x 10-4 cm/h.
Following a 10-minute exposure to a finite application of 2-nitropropane, a total of 16.3 μg equivalents of 2-nitropropane were detected in the receptor fluid, with 14.8 μg equivalents in the skin. Based on the amount of 2-nitropropane in th 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 285.9 μg equiv/cm2/h.
Following a 60-minute exposure to a finite application of 2-nitropropane, a total of 39.5 μg equivalents of 2-nitropropane were detected in the receptor fluid and 3.22 μg equivalents in the skin. Based on the amount of 2-nitropropane 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 66.8 μg equiv/cm2/h.
Executive summary:

None