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Link to relevant study record(s)

Reference
Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
other:
Version / remarks:
FDA/AAPS guidelines (Skelly et al. 1987)
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Wizard Laboratories, West Sacramento, CA

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.14%
- Specific activity: 57.26 mCi/mmol
- Locations of the label: benzyl-14C
Radiolabelling:
yes
Species:
other: human skin samples
Details on test animals and environmental conditions:
Full thickness human skin samples were obtained from either the breast or abdomen of cosmetic surgery patients.
Type of coverage:
open
Vehicle:
ethanol
Duration of exposure:
48 hours
Doses:
- Nominal doses: 1%
Control animals:
no
Details on study design:
APPLICATION OF DOSE: 20 µL of a 1% solution in ethanol of radiolabeled test item was applied to the surface of the membrane

VEHICLE
- Amount applied: 20 µL

TEST SITE
- Preparation of test site: The epidermal membranes were mounted on filter paper supports and then placed onto diffusion cells and trimmed to size. The skin samples were mounted as a barrier between the halves of horizontal Franz-type diffusion cells, the stratum corneum facing the donor chamber.
- Area of exposure: about 1.0 cm²

SAMPLE COLLECTION
- Samples of the receptor fluid (50% ethanol/water) were harvested from the receptor chamber at 2, 8, 24, 36 and 48 h and analyzed by liquid scintillation chromatography. Post-incubation epidermal membranes were wiped with a cotton bud, and then tape stripped 10 times using adhesive tape (D-Squame).

SAMPLE PREPARATION
- Storage procedure:
- Preparation details:

ANALYSIS
- Method type for identification: Liquid scintillation counting

OTHER:
- Total radioactivity was accounted for by extruding and counting test item equivalents associated with membrane wipes, tape strips, digested remaining skin, and donor chambers. To assess evaporative loss, 20 µL of a 1% test item solution was applied to PTFC sheets mounted in diffusion cells (at 32 °C surface temperature). These sheets were then placed in chambers for 24 or 48 h. The PTFE sheet was then removed and washed twice with methanol (10 mL then 5 mL). The donor chamber was washed with 10 mL methanol. A sample of each wash solution was submitted to analysis by LSC which allowed the total remaining radiolabel at each timepoint to be determined. Radiolabel that was unaccounted for was considered to be associated with evaporative loss.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human, cosmetic surgery patients
- Type of skin: full thickness human skin samples, obtained from either the breast or abdomen
- Preparative technique: The epidermal membranes were mounted on filter paper supports and then placed onto diffusion cells and trimmed to size. The skin samples were mounted as a barrier between the halves of horizontal Franz-type diffusion cells, the stratum corneum facing the donor chamber. The cells were designed such that the area available for diffusion was about 1.0 cm².
- Thickness of skin: full thickness human skin samples
- Membrane integrity check: Membrane integrity was determined by applying 500 µL of 10 µCi/mL ³H20 to the skin surface and removing a 200 µL sample from the receptor phase one hour later. The skin surface was washed seven times and the receptor chambers three times with water, prior to refilling the selected receptor medium (50/50 ethanol/water). The sample was counted using a Wallae 1400 liquid scintillation counter (LSC) with 1400 DSA ver. 2.2 operating system, 152Eu external standard and ChemiStrip TM chemiluminescence subraction. The LSC uses the external standard to measure the counting efficiency for each sample and spectrum libraries to generate dpm values. The LSC generated disintegration per minute (dpm) values (5 min counts), which were transferred electronically to Excel worksheets for subsequent calculations. The experimental specific activity was 2198 dpm/ug (0.3% RSD, n = 5). Background activity (l0 dpm) was subtracted from all samples, with the lowest active sample at least 17-fold greater than background. No cell exhibited water permeability greater than 5 x 10³ cm/h and the average for each lest group was not greater than 2 x 10³ cm/h.
- Storage conditions: The skin surface temperature was maintained at 32 °C.

PRINCIPLES OF ASSAY
- Diffusion cell: Franz-type diffusion cells
- Receptor fluid: 50/50 ethanol/water
- Static system: No; Receptor chambers were continuously agitated by submersible magnetic stirrers
- Flow-through system: No
- Test temperature: Receptor chambers were maintained at 37 °C throughout the experiment. The skin surface temperature was maintained at 32 °C.
- Occlusion: Not specified
Absorption in different matrices:
- Skin preparation (in vitro test system):
Total recovery:
- Total recovery: 86 % at 48 h (including an evaporation loss of 19 %)
Key result
Time point:
24 h
Dose:
1%
Parameter:
percentage
Absorption:
42 %
Key result
Time point:
48 h
Dose:
1%
Parameter:
percentage
Absorption:
50 %

Following incubation of epidermal membranes with the radiolabeled test item, only 67% of the applied dose was accounted for by 48 h. At 24 and 48 h, 42% and 50%, respectively, of the applied close was recovered in the fluid retrieved from the receptor chambers. Distribution of remaining radiolabel in surface wipes, tape strips, remaining epidermis and the donor chamber surface accounted for an additional 17%. The chemical nature of the absorbed radiolabel was not characterized (i.e. parent test item or metabolites). Evaporative loss, estimated from direct application to PTFE sheets, was approximately 8% and 19% of the applied dose at 24 and 48 h, respectively.

Conclusions:
Moderately high human skin permeation was observed. The in vitro human skin penetration studies showed 42% and 50% of the applied dose of the test item permeated into the receptor phase at 24 and 48 h, respectively.
Executive summary:

An in vitro dermal absorption studies according to the FDA/AAPS guidelines (Skelly et al., 1987) was performed with full thickness human skin samples. Twenty µL of a 1% solution in ethanol of radiolabeled test item (0.2 mCi benzyl-14C. specific activity of 57.26 mCi/mmol; radiochemical purity of 99.14%) was applied to the surface of the membrane. Receptor chambers were continuously agitated by submersible magnetic stirrers and maintained at 37 °C throughout the experiment. The skin surface temperature was maintained at 32 °C. Samples of the receptor fluid (50% ethanol/water) were harvested from the receptor chamber at 2, 8, 24, 36 and 48 h and analysed by liquid scintillation chromatography. Post-incubation epidermal membranes were wiped with a cotton bud, and then tape stripped 10 times using adhesive tape. Total radioactivity was accounted for by extruding and counting test item equivalents associated with membrane wipes, tape strips, digested remaining skin, and donor chambers. To assess evaporative loss, 20 µL of a 1% test item solution was applied to PTFC sheets mounted in diffusion cells (at 32 °C surface temperature). These sheets were then placed in chambers for 24 or 48 h. The PTFE sheet was then removed and washed twice with methanol (10 mL then 5 mL). The donor chamber was washed with 10 ml methanol. A sample of each wash solution was submitted to analysis by LSC which allowed the total remaining radiolabel at each timepoint to be determined. Radiolabel that was unaccounted for was considered to be associated with evaporative loss.

Following incubation of epidermal membranes with the radiolabeled test item, only 67% of the applied dose was accounted for by 48 h. At 24 and 48 h, 42% and 50%, respectively, of the applied close was recovered in the fluid retrieved from the receptor chambers. Distribution of remaining radiolabel in surface wipes, tape strips, remaining epidermis and the donor chamber surface accounted for an additional 17%. The chemical nature of the absorbed radiolabel was not characterized (i.e. parent test item or metabolites). Evaporative loss, estimated from direct application to PTFE sheets, was approximately 8% and 19% of the applied dose at 24 and 48 h, respectively.

 

Description of key information

In a combined OECD Guideline 422 repeat dose study (Takano, 2016) a NOEL of 100 mg/kg/day was obtained, indicating that oral uptake of the substance occurs.

In an in vitro study using human skin 50 % of the applied dose were absorbed, indicating that the substance has a high skin penetration potential.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

Oral absorption

The substance has a molecular weight < 500, which is favourable for uptake. The substance also has a logP that is favourable for absorption (logP 2.4).

An acute oral study (Mallory, 1985) indicates that uptake does occur, with some systemic signs being noted at 1600, 2000 and 2500 mg/kg and death occurring at 3200 and 5000 mg/kg. In a combined repeat dose study (Takano, 2016) a NOEL of 100 mg/kg/day was obtained, again indicating that oral uptake does occur. However, very little on the quantitative value of oral absorption can be inferred from these studies.

In the absence of quantitive data, but in light of the high dermal absorption, oral absorption is set to 100% for the purposes of risk assessment.

Dermal absorption

In an in vitro study using human skin 50% of the applied dose were absorbed. Therefore, 50% absorption following dermal exposure is assumed for the purposes of risk assessment.

Inhalation absorption

In the absence of quantitative information, complete absorption (100%) following inhalation is assumed for the purposes of risk assessment.

Distribution

It can be expected that once absorbed the substance will be widely distributed throughout the body.

Metabolism

There is no metabolism data available for this substance. Conjugation is likely to occur, which will increase the water solubility and make the substance easier to excrete. There is considered to be no potentential for bioaccumulation.

Elimination

Although there is no elimination data available for this substance, compounds with a molecular weight < 300 tend to be excreted in the urine.