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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June 2014 - 26 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance Document 28: Guidance Document For the Conduct of Skin Absorption Studies
Version / remarks:
5 March 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EFSA Panel on Plant Protection Products and their Residues (PPR); Guidance on Dermal Absorption
Version / remarks:
EFSA Journal 2012; 10(4):2665
Deviations:
yes
Remarks:
according to the EFSA guidance, if standard deviations are > 25% of mean, the preferred approach should be adding the standard deviations to the mean values
GLP compliance:
yes

Test material

Constituent 1
Test material form:
solid
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
Radiolabelled isomer 1: 2-(1,1-Dimethylpropyl)[carbonyls-14C]anthraquinone
- Radiochemical purity: 99.2%
- Specific activity: 59 mCi/mmol (211 μCi/mg), 2.18 GBq/mmol (7.78 MBq/mg)
- Locations of the label: carbonyl group

Radiolabelled isomer 2: 2-(1,2-Dimethylpropyl)[carbonyls-14C]anthraquinone
- Radiochemical purity: 99.7%
- Specific activity: 59 mCi/mmol (211 μCi/mg), 2.18 GBq/mmol (7.78 MBq/mg)
- Locations of the label: carbonyl group

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: radiolabelled samples: c.a. -20°C, cold sample: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: soluble in diisobutyl carbinol (DIBC) and Caromax 20LN


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test compound was applied to the skin at two concentrations, the solid pure substance as a mixture of the 2 isomers and formulation of the substance containing a mixture of the 2 isomers.
For the solid substance 498.4 mg of non-radiolabelled 2-amylanthraquinone (provided as a mixture of the of the 2 isomers) was added to 1.05 mg of [14C]-2-(1,2-dimethylpropyl)anthraquinone and 1.16 mg of [14C]-2-(1,1-dimethylpropyl)anthraquinone. The mixture was dissolved in acetonitrile and the solvent evaporated off to provide a solid racemic mixture at the desired specific activity. Following radiodilution the test compound was stored in a freezer at ca. 20ºC until the day of application to skin.
For the formulation 747.2 mg of non-radiolabelled 2-amylanthraquinone (provided as a mixture of the of the 2 isomers) was added to 1.32 mg of [14C]-2-(1,2-dimethylpropyl)anthraquinone and 1.76 mg of [14C]-2-(1,1-dimethylpropyl)anthraquinone. The mixture was dissolved in acetonitrile and the solvent evaporated off to provide a solid racemic mixture at the desired specific activity. Following radiodilution the test compound was stored in a freezer at ca. 20ºC until the day of application to skin. On the day of application to skin a 500.22 mg aliquot of the radiodiluted material was dissolved in 0.5044 g of Diisobutyl carbinol (DIBC) followed by 1.0024 g of Caromax 20LN.


Radiolabelling:
yes

Test animals

Species:
other: in vitro human skin from 4 donors

Administration / exposure

Type of coverage:
open
Vehicle:
other: mixture of Diisobutyl carbinol (DIBC) and Caromax 20LN (0.5:1 w/w)
Duration of exposure:
6 hours, with 24 hours sampling period
Doses:
- Actual doses: 4 mg solid substance with 10 µL water added to mimic sweat, or 15 µL formulation, applied to 1.77 cm2 exposed skin
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Dermatomed human skin (359 - 540 μm, obtained from Biopredic International)
- Type of skin: dermatomed
- Preparative technique: not specified
- Thickness of skin (in mm): 359 - 540 μm
- Membrane integrity check: yes
The skin was checked for physical damage prior to being mounted in the test system. The total weight of the cell was recorded, the receptor chamber filled with receptor fluid (((0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water (w/w/w/v)) and the weight recorded again. The system was allowed to warm to approximately 32 ± 2°C prior to application of 50 μL tritiated water (10.3 μCi/mL) onto the skin of each cell. Aliquots (2 x 50 μL) were removed from the receptor fluid chamber for analysis at 0, 0.5, 1 and 2 hours after application and replaced with an equal volume of receptor fluid. Each 50 μL aliquot was mixed with 2 mL Ultima Gold XR® (Perkin Elmer) scintillant, counted on a liquid scintillation counter and the rate of penetration determined from the counts. After the last sampling time the tritiated water was desorbed by rinsing with 0.9% saline. The skin was maintained for use by filling the receptor chamber with receptor fluid and the temperature generally kept at 32 ± 2°C. The skin sample was deemed acceptable if the rate of penetration through the sample was not greater than 2 μL/cm2/h.


PRINCIPLES OF ASSAY
- Diffusion cell: no
- Receptor fluid: 0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water (w/w/w/v)
- Solubility of test substance in receptor fluid: yes
Prior to conducting the main in vitro dermal penetration experiment the solubility of 2-amylanthraquinone in selected receptor fluids was assessed. The receptor fluids were spiked with [14C]-2-amylanthraquinone at a target concentration of 0.2 mg/mL and aliquots taken for liquid scintillation counting to determine the concentration.
- Static system: yes, Franz cells with a nominal 12 ml receptor chamber volume and 1.77 cm2 exposed skin area for dosing
- Flow-through system: no
- Test temperature: Receptor fluid temperature was recorded at each sampling time point and the temperature was maintained at 31.1 – 31.9°C
- Occlusion: no
- Reference substance: yes, radiolabelled testosterone at ca. 0.1 mg/cm2

Samples were taken from the receptor fluid at 8 sampling points over a 24 hour period:
0, 0.5, 1, 2, 4, 6 (taken prior to removal of the dose formulation), 8 and 24 hours after application.
Duplicate aliquots (2 x 50 μl) were removed for sampling and replaced with an equal volume of receptor fluid (0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water w/w/w/v).

Results and discussion

Absorption in different matrices:
- Cell wash: mean 0.58% (SD = 0.73) (solid), 26.52** (SD = 8.70) (formulation)
**The atypically high recovery of radioactivity in the cell wash is believed to be formulation/radiolabelled material remaining in the upper portion of the Franz cell (donor chamber). As the concentrations of radioactivity in the receptor fluid were low it is unlikely this is absorbed material.
- Skin wash: mean 95.66% (SD = 1.98) (solid), 18.73% (SD = 6.50) (formulation)
- Skin test site: mean 0.01% (SD 0.2) (solid), 3.35% (SD 5.12) (formulation)
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): mean: below or near the limit of detection (< 0.01%) (solid), 0.14% (SD = 0.07) (formulation)
- Stratum corneum (in vitro test system): (tape strips 3-20): solid: 0.57%, formulation: 37.33% (both values calculated according to the EFSA guidance by adding the standard deviations to mean values in case SD is ≥ 25% of mean))
Total recovery:
- Total recovery: mean 97.40% (SD = 1.78) (solid), 96.17% (SD = 2.32) (formulation)
- Recovery of applied dose acceptable: yes, above 95%
- Results adjusted for incomplete recovery of the applied dose: not applicable
- Quantification of values below LOD or LOQ: radioactivity with less than twice background counts were considered to be below the limit of accurate quantification.
Percutaneous absorptionopen allclose all
Key result
Time point:
24 h
Dose:
Solid substance
Parameter:
percentage
Remarks:
potentially absorbed (receptor fluid, skin, stratum corneum (strips 3-20)
Absorption:
0.25 %
Remarks on result:
other:
Remarks:
As reported in the study report
Key result
Time point:
24 h
Dose:
33.2% in formulation (calculated value)
Parameter:
percentage
Remarks:
potentially absorbed (receptor fluid, skin, stratum corneum (strips 3-20)
Absorption:
24.82 %
Remarks on result:
other:
Remarks:
As reported in the study report
Key result
Time point:
24 h
Dose:
Solid substance
Parameter:
percentage
Remarks:
Potentially absorbed (receptor fluid, skin, stratum corneum (tape strips 3-20))
Absorption:
1 %
Remarks on result:
other:
Remarks:
calculated according to the EFSA guidance, by adding standard deviations to mean values in case SD is ≥ 25% of mean and rounding the value up to one significant figure.
Key result
Time point:
24 h
Dose:
33.2% in formulation (calculated value)
Parameter:
percentage
Remarks:
potentially absorbed (receptor fluid, skin, stratum corneum (tapes 3-20)
Absorption:
51 %
Remarks on result:
other:
Remarks:
calculated according to the EFSA guidance, by adding standard deviations to mean values in case SD is ≥ 25% of mean, and rounding the value to two significant figures.

Any other information on results incl. tables

The solubility of [14C]-2-amylanthraquinone in the selected receptor fluid 0.9% saline + 5% Bovine serum albumin (BSA) + 5% 2-hydroxypropyl-β-cyclodextrin in water (w/w/w/v) was determined. The percentage variation in concentration before and after centrifugation was less than or equal to 7.1% and therefore the solubility of [14C]-2-amylanthraquinone in the receptor fluid was considered acceptable.

To determine the skin preparation integrity tritiated water was applied to the test vessels as described in section 2.3.2. Skin samples were deemed viable if the rate of penetration was not greater than 2 μL/cm2/h. The observed rate of penetration was in the range of 0.460 to 1.82 μL/cm2/h.

During this study the reference standard ([14C]-testosterone) produced a mean penetration rate of 0.0187 μg/cm2/h. Radioactivity recovered was greater than 97%. These results indicate the test system was functional throughout the duration of the study.

Applicant's summary and conclusion

Conclusions:
Dermal absoprtion of 2-amylanthraquinone in the form of a solid substance and a 33.2% formulation was studied in an in vitro dermal absorption test. The potentially absorbed amounts were calculated to be 1% and 51% for a solid substance and a formulation, respectively.
Executive summary:

In a GLP-compliant OECD Guideline 428 study, dermal penetration of 2 -amylanthraquinone was studied in an in vitro dermal absorption test using dermatomized human skin from 4 volunteers. The substance was applied undiluted (with 10 µL water added to mimic sweat), or as a formulation in a mixture of Diisobutyl carbinol (DIBC) and Caromax 20LN (0.5:1 w/w) (calculated concentration 33.2% w/w). The experiment was conducted in Franz cells, representing a static cell design, with a nominal 12 mL receptor chamber volume and 1.77 cm2 exposed skin area for dosing, with each skin sample tested in duplicate. Integrity of the human skin used in each test vessel was demonstrated to be suitable. Functionality of the test system was demonstrated with a  reference compound. After 6 hours exposure the skin was washed, and dermal penetration was determined after 24 hours sampling time. Skin fractionation was performed by tape stripping. The total radioactivity recovery was above 95% in both experiments. The potentially absorbed doses (based on the radioactivity in the receptor fluid, skin and stratum corneum (tapes 3 -20)) were reported to be 0.25% for the solid substance and 24.82% for the formulation. However, as standard deviations in most cases exceeded the 25% of the mean values, the dermal absorption values were recalculated by adding standard deviations to the mean values in accordance with the EFSA Guidance on dermal absorption (2012). This resulted in the potentially absorbed doses of 0.6% for the solid substance and 51.35% for the formulation, which were rounded up to 1 and 2 significant figures, respectively, in accordance with the EFSA Guidance on dermal absorption (2012). This resulted in dermal absoprtion values of 1% for pure substance and 51% for the formulation.