5-amino-o-cresol

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 16, 2007 to July 23, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Environmental Health and Safety Publications, series on testing and assessment No. 28. Guidance Document for the conduct of skin absorption studies. Paris, March 2004.

Deviations:

no

GLP compliance:

no

Remarks:

Performed in the spirit of the GLP

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-o-cresol

EC Number:

220-618-6

EC Name:

5-amino-o-cresol

Cas Number:

2835-95-2

Molecular formula:

C7H9NO

IUPAC Name:

5-amino-2-methylphenol

Constituent 2

Reference substance name:

4-Amino-2-hydroxytoluene

IUPAC Name:

4-Amino-2-hydroxytoluene

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

UNLABELED TEST MATERIAL
- Name of test material: 5-Amino-2-methylphenol; 4-Amino-2-hydroxytoluene; A027
- TSIN: WR23032
- Substance type: Pure active substance
- Physical state: Beige crystalline powder
- Storage condition of test material: At room temperature, protected from light and moisture

LABELED TEST MATERIAL
- Name of test material: 5-Amino-2-methyl[U-14C]phenol
- Physical state: Solid
- Specific activity: 411 µCi/mg (gravimetric analysis); 53 mCi/mmol (mass spectrometry)
- Locations of the label: [U-14C] Uniform labelling of the aromatic ring system
- Stability under test conditions: Not reported
- Storage condition of test material: At - 20°C in the absence of moisture, light and air.

Color cream formulation with 0.8 % 4-Amlno-2-hydroxytoluene
Identity: Color Cream Formulation
- Batch N°: DTF333052B
- Formula N°: 85707287
- Appearance of cream formulation: Beige cream
- Aggregate state at room temperature: Paste-like
- Storage: At room temperature
- Supplier: I. Pauli, RD-IPC, COSMITAL SA
- Preparation Date: 16.05.2007
Expiration Date: 16.09.2007

Radiolabelling:

yes

Remarks:

(14C)

Administration / exposure

Duration of exposure:

30 minutes

Doses:

400 mg (100 mg/cm2) of formulation containing 0.4 % of 4-Amino-2-hydroxytoluene (0.4 mg test substance /cm2)

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The color cream formulation with 0.8% test substance was diluted prior to the application to the skin samples. To achieve a concentration of 0.4 % of 4-Amino-2-hydroxytoluene, 2 g of Color cream formulation containing 0.8 % 4-Amino-2-hydroxytoluene and 60 µL of the radiolabelled solution of 4-Amino-2-hydroxytoluene were added to a glass beaker. The resulting suspension was mixed until a homogeneous cream is obtained. To this mixture, 2 g of Welloxon Perfect 6 % were added and mixed again with a spatula until a homogeneous cream was obtained.
The detailed composition of color cream formulation and Welloxon Perfect 6% is provided in the study report (Annex III and Annex IV respectively).
- 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 six skin samples were covered with 400 mg of an oxidative hair dye formulation containing 0.4 % of 4-Amino-2hydroxytoluene on 4 cm2 for 30 minutes in a single series.

TEST SITE
- Area of exposure: 4 cm2

REMOVAL OF TEST SUBSTANCE
- Washing procedures: The formulation was removed by extensive washing in five steps (2x 4.0 mL water, 1x 4.0 mL shampoo, 2 x 4.0 mL water)
- Time after start of exposure: 30 minutes

SAMPLE COLLECTION
- Spatula: After application of the final formulation, the spatula was dipped into 1 mL of ethanol and rinsed with 4 mL of water. This solution was analyzed for radioactivity.
- Receptor fluid: The receptor fluid was sampled after 16, 24, 40, 48, 64 and 72 hours.
- Rinsings: 15 µL of rinsing solution was used for analysis of test substance content.
- Skin extract: After disassembly of the skin samples from the Teflon chambers, the skin samples were wrapped in aluminum foil and put on a hot surface (80 to 90 °C) upside down (i.e. lower skin upside) charged with a small weight of approximately 40 g. After 15 to 60 seconds, the packed skin sample was removed from the hot surface and unwrapped. The upper skin was separated from the lower skin using forceps and the two compartments were extracted for analysis.

SAMPLE PREPARATION
- Spatula: The water solution (in which the spatula dipped in ethanol was rinsed) was diluted with 15 mL of scintillation cocktail and the radioactivity was quantified using a scintillation counter.
- Receptor fluid: An aliquot of collected receptor fluid fraction (i.e. 5 mL) was mixed with 15 mL of scintillation cocktail for radioactivity analysis.
- Skin samples: The skin samples were processed as follows:
Extractions of the skin compartments: Each of the skin compartments was dissolved overnight in 2 mL aqueous KOH solution (10M) at 80°C. 200 µL of the resulting solution were mixed with 200 µL of concentrated hydrochloric acid and 3 mL of the scintillation cocktail. The radioactivity was quantified with a scintillation counter.
Extraction of the aluminium foil: After the separation of the skin compartments, the aluminium foil was dipped overnight into 1 mL of a solution containing 5% methanol in water. Thereafter, the aluminum foil was removed and the extract was diluted with scintillation cocktail. The radioactivity was quantified with a scintillation counter.
- Rinsings: 15 µL of each of the rinsing fractions were diluted with 3 mL scintillation cocktail and the radioactivity was quantified.
- Storage procedure: All samples (application formulation, rinsings, receptor fluid fractions and skin compartment extracts) were stored at -20°C, if not processed immediately. Samples used for the determination of the stability in receptor fluid were stored at room temperature until analysis.
ANALYSIS
- Method type(s) for identification: Liquid scintillation counting (LSC; Packard Tricarb 2250CA)
- Liquid scintillation counting results (cpm) converted to dpm as follows: The limit of detection and limit of quantification for test substance was determined according L. A. Currie. Based on background value for this experiment of 192 cpm and a counting interval of 30 minutes, the following limits were calculated:
Limit of detection [LOD]: 355.62 counts
Limit of quantification [LOQ]: 1124.48 counts
With a typical detection efficiency of 90.6 % for 14C on liquid scintillation counter, these limits were transformed to:
Limit of detection: 13.08 dpm
Limit of quantification: 41.37 dpm
- Validation of analytical procedure: A calibration curve was established with aliquots of the solution of the radiolabeled test substance. The calibration curve was linear (R2 =1) up to 715'000 dpm.
- Limits of detection and quantification: With respect to the applied formulation containing 0.4% of test substance:
Absolute limit of detection: 0.47 ng of test substance
Absolute limit of quantification: 1.49 ng of test substance

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Skin samples from 3 pigs weighing 91.5 – 106 kg were obtained from Butchery Kunzli, CH-1724 Praroman-Le-Mouret
- 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: Dermatome
- Thickness of skin (in mm): 0.94 ± 0.06 mm (measured with a micrometer)
- Membrane integrity check: Yes, by tritiated water with scintillation counting of 1 hour fractions over a time period of 4 hours.
- Storage conditions: Stored at -20° C until use
- Justification of species, anatomical site and preparative technique: Among several animal species, pig skin from flank and back represent a good model for human skin.

PRINCIPLES OF ASSAY
- Diffusion cell: Six permeation chambers (Teflon-chambers) of medium size with 9.1 cm2 surface were used. At the beginning of the experiment, thawed skin samples were mounted keeping dermal side down in permeation chambers.
- Receptor fluid: Receptor fluid was composed of 0.14 M NaCl, 2 mM K2HPO4, 0.4 mM KH2PO4, 100 IU Penicillin/mL, 76 IU Streptomycin/mL (pH 7.3)
- Solubility of test material in receptor fluid: 5.096 mg/mL (at pH= 7.3)
- Stability of test material in receptor fluid: > 99 % recovery after 3 days (1 mg/mL solution)
- Static system: No
- Flow-through system: Yes, the experiment was performed using a flow through system with a constant flow of receptor fluid of 5 mL/hour.
- Test temperature: 32 ± 2˚C
- Relative humidity: 25.7 to 52.6 %
- Occlusion: No
- Reference material: Yes, mannitol was tested to assess the performance and reliability of the test system.
- 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): 93.99 ± 2.00 % or 365.62 ± 8.45 µg/cm2; After the removal of applied test substance with water and shampoo after 30 minutes, the majority of test substance was found in the rinsing solutions.
- Receptor fluid: After 72 hours, 0.196 ± 0.063 % or 0.762 ±0.245 µg/cm2 of test substance had penetrated into the receptor fluid.
With respect to the receptor fluid samples, the test substance was detectable predominantly within the first fraction collected (fraction 0 - 16 hours). Smaller amounts of test substance were detectable in the later fractions. At the end of the experiment (after 72 hours) the amounts of test substance detectable in the receptor fluid declined to the limit of detection/limit of quantification thus indicating, that no further test substance remaining on or in the skin after 72 hours does migrate to deeper layers. With such an absorption profile, the amounts of test substance found in the upper skin are not considered as biologically available (no depot effect).
- Skin preparation: After 72 hours, small amounts of test was found in the upper skin (0.341 ± 0.121 % or 1.326 ± 0.471 µg/cm2) and in the lower skin (0.043 ± 0.023 % or 0.168 ± 0.090 µg/cm2) compartments.
Under the assumption that a depot effect is not present, a maximum amount of 0.930 ± 0.261 µg/cm2 of test substance in a typical oxidative hair dye formulation with 0.4 % dye and in the presence of a reaction partner is considered as biologically available (n=5, three donors; receptor fluid + lower skin; 0.762 µg/cm2 + 0.168 µg/cm2).

Total recovery:

- Total recovery: 97.40±1.76 of the dose applied to the pig skin was recovered
- Recovery of applied dose acceptable: Yes
- Results adjusted for incomplete recovery of the applied dose: The total recovery was corrected for the losses on tips.
- Limit of detection (LOD): 0.47 ng of test substance as the absolute limit of detection with respect to the applied formulation containing 0.4% test substance.
- Quantification of values below LOD or LOQ: To each value measured, the amount of dpm of the blank was subtracted (net dpm value). Net dpm values below or equal to the limit of detection, the theoretical calculated relative limit of detection were taken for the calculation of the mean.
Theoretical limit of detection (LOD) and limit of quantification (LOQ) are provided under ‘Any other information on materials and methods incl. tables’ section above.

Percutaneous absorption

Percutaneous absorption 1

Dose:

100 mg/cm2 formulation containing 0.4 % of 4-Amino-2-hydroxytoluene (0.4 mg /cm2)

Parameter:

percentage

Absorption:

0.196 %

Remarks on result:

other: 72 hours

Remarks:

Absorption in receptor fluid after 30 minutes exposure

Percutaneous absorption 2

Dose:

100 mg/cm2 formulation containing 0.4 % of 4-Amino-2-hydroxytoluene (0.4 mg /cm2)

Parameter:

percentage

Absorption:

0.043 %

Remarks on result:

other: 72 hours

Remarks:

Absorption in lower skin after 30 minutes exposure

Any other information on results incl. tables

Reference control:The mean value for the penetration of mannitol through pig skin preparations calculated for all samples was 0.74 ± 0.45% of the applied dose (n=14, water as vehicle).

Skin integrity: The integrity of each skin preparation was demonstrated by examination of penetration characteristics with tritiated water resulting in 0.6 to 2.5% of the applied dose found after 4 hours in the receptor fluids, which was within the limit of acceptance (≤ 2.0%) for all skin samples.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this in vitro cutaneous absorption study of 4-Amino-2-hydroxytoluene, a maximum amount of 0.930 ±0.261 µg/cm2 of test substance in a typical oxidative hair dye formulation containing 0.4 % 4-Amino-2-hydroxytoluene and in the presence of a reaction partner (Toluene-2.5-diamine) is considered as biologically available.
Receptor fluid + lower skin; 0.762 µg/cm2 + 0.168 µg/cm2 =0.930 µg/cm2

Executive summary:

The cutaneous absorption (in-vitro) of 0.4% 4-Amino-2-hydroxytoluene in a typical hair dye formulation with hydrogen peroxide and reaction partner (Toluene-2,5-diamine sulphate) was determined following methods according to the OECD Guideline 428 (Skin Absorption: In Vitro Method).

Pig skin samples (from back and flanks) were used as the test system.Skins from 3 pigs weighing 91.5 – 106 kg wereobtained from Butchery Kunzli, CH-1724 Praroman-Le-Mouret, Switzerland. Two skin samples were obtained from each pig. In total, 6 skin samples were used in the study.

The integrity of each skin preparation was determined by examination of penetration characteristics with tritiated water. During this experiment, 0.6 to 2.5% of the applied dose was found in the receptor fluids after 4 hours, which was within the limit of acceptance (≤ 2.0%) for all skin samples. After checking the skin integrity, 400 mg of the typical hair dye formulation (100 mg/cm2); containing 0.4% test substance (i.e. 0.4 mg/cm2) was applied to 6 skin samples for 30 minutes. 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 at 16, 24, 40, 48, 64 and 72 hour intervals to 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, lower skin and upper skin) were processed and analyzed by means of a scintillation counter to determine radioactive concentrations. 

The majority of the test substance was recovered in the rinsing solutions (93.99 ± 2.00 % or 365.62 ± 8.45 µg/cm2 ;). Small amounts of test substance were detected in the upper skin (0.341 ± 0.121 % or 1.326 ± 0.471 µg/cm2), in the lower skin (0.043 ± 0.023 % or 0.168 ± 0.090 µg/cm2) and in the fractions of the receptor fluid collected within 72 hours (0.196 ± 0.063 % or 0.762 ± 0.245 µg/cm2).

The mass balance (total recovery) was 97.40 ± 1.76% of the dose applied to the pig skin samples.

With respect to the receptor fluid samples, the test substance was detectable predominantly within the first fraction collected (fraction 0 - 16 hours). Smaller amounts of test substance were detectable in the later fractions. At the end of the experiment (after 72 hours) the amounts of test substance detectable in the receptor fluid declined towards detection limits thus indicating, that no further test substance remaining on or in the skin after 72 hours migrated to deeper layers. With such an absorption profile, the amounts of test substance found in the upper skin were not considered as biologically available (no depot effect).

Under the conditions of this in vitro cutaneous absorption study of 4-Amino-2-hydroxytoluene, a maximum amount of 0.930 ±0.261 µg/cm2 of test substance in a typical oxidative hair dye formulationcontaining0.4 % 4-Amino-2-hydroxytoluene and in the presence of a reaction partner (Toluene-2.5 -diamine) is considered as biologically available.

Receptor fluid + lower skin; 0.762 µg/cm2 + 0.168 µg/cm2 =0.930 µg/cm2

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