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Dermal absorption

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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:
From Jan. 14, 2003 to Jan. 29, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed method comparable to guideline with deviations, GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
yes
Remarks:
(no reference substance was tested to establish the validity of method)
GLP compliance:
yes (incl. certificate)
Remarks:
according to the Swiss Ordinance based on OECD principles of GLP

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-Amino-6-chloro-4-nitrophenol, Chloro orange base (Code# A000119)
- TSIN: WR23214
- Substance type: Pure active substance
- Physical state: Orange powder
- Stability under test conditions: The substance is considered to be stable for more than 7 years if stored dry and protected from light and humidity at room temperature
- Stability in solution: The test substance has showed very good stability when tested in DMSO solution (approximately 10% w/v), acetone/water solution (1:1, 7% w/v) and water solution (approximately 0.05% w/v) over a period of 7 days
- Solubility: Solubility in different solvents is as follows:
0.07-0.24 % (pH 4.3) in water
8.7 weight% (pH 3.6) in acetone/water 1:1
> 10 weight% in DMSO
Radiolabelling:
no

Administration / exposure

Duration of exposure:
30 minutes
Doses:
78 mg of the formulation (99.4 mg/cm2), containing 0.39 mg (0.5%) 2-Amino-6-chloro-4-nitrophenol (=0.5 mg of test substance/cm2)
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: Not reported
- Method of storage: All samples (application formulation, rinsings, receptor fluid fractions and skin compartment extracts) were stored at - 20°C, if not processed immediately.

APPLICATION OF DOSE: The five skin samples were covered with 78 mg of hair dye formulation containing 0.5% test substance on 0.785 cm2 for 30 minutes in a single series. Additionally, formulation without test substance was applied to one skin sample (negative control). However for calculations, the mean value of 5 valid skin samples in contact with test substance in formulation was used.

TEST SITE
- Area of exposure: 0.785 cm2

REMOVAL OF TEST SUBSTANCE
- Washing procedures: The formulation was removed by extensive washing in five steps (2 x 0.5 mL water, 1x 0.5 mL shampoo, 2 x 0.5 mL water)
- Time after start of exposure: 30 minutes

SAMPLE COLLECTION AND PREPARATION
- Receptor fluid: The receptor fluid was sampled after 16, 24, 40, 48, 64 and 72 hours. The fractions collected were concentrated directly after the pump on previously activated HLB solid phase extraction cartridges from WatersAss. The cartridges were eluted with 2.0 mL of methanol. After evaporation to dryness the eluates were re-dissolved in 100 µL of the mobile phase, then 50 µL injected.
- Separation of skin compartments: At the end of the experiment the 0.785 cm2 treated area was separated from the flanged skin. The skin samples were wrapped in aluminium foil, covered with a 40 g weight and heated during 45-60 seconds at 80-90° C. Then, the epidermis was separated from the upper dermis with forceps and the two compartments were extracted for analysis.
- Extractions of the skin compartments:
Each skin sample was extracted with 3 mL ethyl acetate + 3 mL H2O bidest during 1 hour. After centrifugation 2.5 mL out of 3 mL were evaporated to dryness, re-dissolved in 100 µL of the mobile phase, then 10 µL of the epidermal and 50 µL of the upper dermal fractions were injected.
- Recoveries: Recoveries for receptor fluid and skin samples were determined by appropriate controls treated in the same way.
- Rinsings: 100 µL of each washing solution were diluted 100 times in the mobile phase, and then 20 µL were injected.
- Storage procedure: All samples (application formulation, rinsings, receptor fluid fractions and skin compartment extracts) were stored at -20°C, if not processed immediately.

ANALYSIS
- Method type(s) for identification: HPLC. An HPLC system (Alliance from Waters Associates) equipped with a 996 PDA detector and controlled by Millenium software was used for the analyses.

CONDITIONS OF HPLC DETECTION:
- Detector range: 200 to 700 nm
- Wavelength at which test substance was analyzed: 443 nm
- Column used: Chromolith 4.6 x 100 mm + precolonne (Waters)
- Mobile phase: 92% 5 mM KH2PO4, pH 7 with KOH + 0.2 mM heptasulfonic acid (HSS) + 8% methanol
- Flow rate: 0.4 to 1.5 mL/minute
- Validation of analytical procedure: A calibration curve was established with aliquots of the formulation containing 0.5% test substance. The calibration curve was linear (R2 =0.9996) up to 40 ng.
- Limits of quantification: With respect to the applied formulation containing 0.5% of test substance:
Absolute limit of quantification [LOQ]: 1.6 ng/injection

After consideration of the injection and extraction factors of the sample preparation procedure, as well as the transformation to amount per surface and recovery rate, this limit results in 8 ng/cm2 per fraction as upper limit per skin.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Skin samples from female pigs ("Schweizer Edelschwein") were obtained from Butchery Kunzli, CH-1724 Praroman-Le-Mouret, Switzerland.
- Type of skin: Split thickness skin samples obtained from back and flanks. The preparations were composed of the stratum corneum, the stratum germinativum and part of the dermis containing blood vessels.
- Preparative technique: Not reported
- Thickness of skin: 1 mm
- Membrane integrity check: Yes, by tritiated water treatment with scintillation counting of one hour fractions for four hours
- Storage conditions: Stored at -20° C until use
- Justification of species, anatomical site and preparative technique: Not reported

PRINCIPLES OF ASSAY
- Diffusion cell: Six permeation chambers (Frantz cell type, CVO Glassware Co., Berkeley/USA) mini size (glass-chambers with 0.785 cm2 surface) were used. At the beginning of the experiment, thawed skin samples were punched and fixed into the permeation chambers.
- Receptor fluid: Receptor fluid was composed of 0.14 M NaCl, 2 mM K2HPO4, 0.4 mM KH2PO4, 100 IU Penicillin/mL and 97 µg Streptomycin/mL
- Solubility of test substance in receptor fluid: 1.5 mg/mL
- Flow-through system: Yes, the experiment was performed using a flow through system with a constant flow of receptor fluid of 2.5 mL/hour.
- Test temperature: 32 ± 2˚C
- Relative humidity: 11.1 to 20.6%
- Occlusion: No
- Reference substance(s): Not reported
- Principle diffusion barrier: Stratum corneum was identified as the principal diffusion barrier, the integrity of which was controlled in each experiment by 3H2O penetration characteristics.

Results and discussion

Absorption in different matrices:
- Rinsing solution (after 30 minutes): 100.316 ± 1.354% or 498385 ± 6725µg/cm2 was found in rinsing solution.
Nearly the half of all fractions of test substance in the receptor fluid was below the limit of quantification of approximately 1.6 ng per injection. Taking all fractions of receptor fluid together, the maximum content of test substance was found to be:

- Receptor fluid (72 hours): 0.013 ± 0.002 or 63 ± 12 µg/cm2 of applied dose, which was certainly an overestimation.
Probably due to the overestimated quantity of test substance, there was no observable gradient of increasing quantities against the skin surface. Nevertheless, the skin may act as a depot for the test substance in this formulation during the 72 hours of the experiment.

- Skin preparations: After 72 hours, amounts of test substance found in the upper dermis was 0.008 ± 0.006% or 41 ± 29 µg /cm2 and in the epidermis was 0.028 ± 0.012% or 140 ± 59 µg /cm2 compartments.
Total recovery:
- Total recovery: 100.365 ± 1.351 % (or 498628 ± 6714 µg /cm2) of the dose applied to the pig skin was recovered
- Recovery of applied dose acceptable: Yes
Percutaneous absorptionopen allclose all
Dose:
78 mg of the formulation (99.4 mg/cm2), containing 0.5% test substance (=0.39 mg, 0.5 mg/cm2)
Parameter:
percentage
Absorption:
0.028 %
Remarks on result:
other: 72 hours
Remarks:
Absorption in epidermis after 72 hours exposure
Dose:
78 mg of the formulation (99.4 mg/cm2), containing 0.5% test substance (=0.39 mg, 0.5 mg/cm2)
Parameter:
percentage
Absorption:
0.008 %
Remarks on result:
other: 72 hours
Remarks:
Absorption in upper dermis after 72 hours exposure
Dose:
78 mg of the formulation (99.4 mg/cm2), containing 0.5% test substance (=0.39 mg, 0.5 mg/cm2)
Parameter:
percentage
Absorption:
0.013 %
Remarks on result:
other: 72 hours
Remarks:
Absorption in receptor fluid after 72 hours exposure

Any other information on results incl. tables

- Skin integrity: The integrity of each skin preparation was determined by examination of penetration characteristics with tritiated water resulting in0.10 to 0.25% of the applied dose found after 4 hours in the receptor fluids, which was within the limit of acceptance (≤ 0.5%) for all skin samples.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this in vitro cutaneous absorption study of 2-Amino-6-chloro-4-nitrophenol in Color Fresh Liquid Neu 5/55 formulation, a maximum amount of 0.013% (an overestimated value) of applied test substance was found in receptor fluid after 72 hours of application. In addition, 0.008 ± 0.006% and 0.028 ± 0.012% of applied test substance was found in upper dermis and epidermis respectively.
The majority of test substance (100.31 ± 1.35%) was found in rinsing solutions after 30 sec exposure.
Executive summary:

The cutaneous absorption (in-vitro) of 0.5% 2-Amino-6-chloro-4-nitrophenol in acolor fresh liquidformulation was determined following methods comparable to the OECD Guideline 428 (Skin Absorption: In Vitro Method).

Pig skin samples (from back and flanks) were used as the test system. Skin samples from female pigs(“Schweizer Edelschwein”) wereobtained fromButchery Kunzli, CH-1724 Praroman-Le-Mouret, Switzerland.In total, 6 skin samples were used in the study (5 for test substance and 1 as negative control).

The integrity of each skin preparation was determined by examination of penetration characteristics with tritiated water. During this examination,0.10 to 0.25%of the applied dose was found in the receptor fluids after 4 hours, which was within the limit of acceptance (≤ 0.5%) for all skin samples. After checking the skin integrity,78 mg of thecolor fresh liquidformulation (99.4 mg/cm2); containing 0.5% test substance (i.e. 0.39 mg of test substance (0.5 mg/cm2)) was applied to 5 skin samples for 30 minutes.Additionally, formulation without test substance was applied to the one skin sample (negative control). For calculations of absorptions in different compartments, the mean value of 5 valid skin samples in contact with test substance in formulation was used. After completion of exposure period, the samples were washed off with water and shampoo and the content of test substance in the rinsing solutions was determined.

The receptor fluid was sampled after 16, 24, 40, 48, 64 and 72 hoursto determine the test substance concentration. At the termination of the experiment, the skin was heat-treated to separate the upper and lower skin compartments. Different matrices (rinsing solution, receptor fluid,upper dermis and epidermis)were processed and analyzed by means of HPLC (High performance Liquid Chromatography) to determine test concentrations.

The following amounts of test substance were found:

Rinsing solution (after 30 minutes): 100.316 ± 1.354% or 498385 ± 6725 µg/cm2

Nearly half of all fractions of test substance in the receptor fluid were below the limit of quantification of approximately 1.6 ng per injection. Taking all fractions of receptor fluid together, the maximum content of test substance was found to be 0.013 ± 0.002 or 63 ± 12µg/cm2 of applied dose, which was certainly an overestimation.Probably due to the overestimated quantity of test substance, there was no observable gradient of increasing quantities against the skin surface. The quantities found in the upper dermis and in the epidermis were 0.008 ± 0.006% or 41 ± 29 µg/cm2 and 0.028 ± 0.012% or 140 ± 59 µg/cm2 respectively.

Nevertheless, the skin may act as a depot for the test substance in this formulation during the 72 hours of the experiment.

The mass balance (total recovery) was 100.365 ± 1.351%(or 498628 ± 6714 µg/cm2)of the dose applied to the pig skin samples.

This cutaneous absorption (in vitro) study is classified as acceptable, and satisfies the guideline requirements of the OECD 428 method.