4-amino-m-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 Apr. 23, 2007 to Apr. 30, 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: The OECD Guidance document for the conduct of skin absorption studies No. 28

Deviations:

no

GLP compliance:

no

Remarks:

experiment was performed in the spirit of the principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-m-cresol

EC Number:

220-621-2

EC Name:

4-amino-m-cresol

Cas Number:

2835-99-6

Molecular formula:

C7H9NO

IUPAC Name:

4-amino-m-cresol

Constituent 2

Reference substance name:

Oxyrot

IUPAC Name:

Oxyrot

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

UNLABELED TEST MATERIAL
- Name of test material: 4-amino-m-cresol; Oxyrot (Code # A00018)
- TSIN: 23533
- Substance type: Pure active substance
- Physical state: Beige-brown crystalline powder
- Stability under test conditions: The substance is considered to be stable for more than 8 yrs, if stored dry and protected from light at room temperature.
- Stability in solutions: 4-amino-m-cresol was considered to be stable over a period of 7 d in DMSO (10% w/v). Linear degradation and a confirmed degradation were observed in Acetone/water 1:1 (10% w/v) and phosphate-buffer (0.3% w/v) solution respectively. A black precipitate was observed after 2 days in phosphate-buffer (0.3% w/v) solution.
- Solubility: Solubility was observed in following:
2 weight% in acetronitrile
0.6 weight% in water/2g/L in water
4 weight% in acetone/water 1:1
10 weight% in DMSO
5.5 weight% in ethanol
- Storage condition of test material: Test substance was stored at ambient temperature, protected from light.

RADIOLABELED TEST MATERIAL
- Name of test material: 4-Amino-3-methyl [ring-U-14C]phenol
- Physical state: Not reported
- Specific activity: 68.2 mCi/mmoL (gravimetric analysis); 68 mCi/mmoL (mass spectrometry)
- Locations of the label: [U-14C] uniform labeling 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

Radiolabelling:

yes

Remarks:

14C

Administration / exposure

Duration of exposure:

30 minutes

Doses:

400 mg of the formulation (100 mg/cm2) containing 0.5% 4-amino-cresol ( 0.5 mg of test substance/cm2)

Details on study design:

DOSE PREPARATION
- Method for preparation of dose formulation: The color cream formulation was diluted prior to the application to the skin samples. Dose formulation containing 0.5% test substance concentration was prepared by adding 2 g of Color cream formulation with 1% 4-amino-3-methyl-phenol with 60 µL of solution of partially radiolabeled 4-amino-3-methyl [ring-U-14] phenol and 2 g of Welloxon perfect 6%.
The detailed composition of Welloxon Perfect is provided in the study report (Annex IV).
- Method of storage: All samples (application formulation, rinsings, and receptor fluid fractions) were stored at -20°C, if not processed immediately.

APPLICATION OF DOSE: 400 mg of the oxidative hair dye test formulation containing 0.5% of test material was applied on 6 skin samples for 30 minutes.
TEST SITE:
- Area of exposure: 4cm2

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: The formulations were removed followed by extensive washing in five steps (2 x 4 mL of water, 1 x 4 mL of shampoo (Salon shampoo, Energy, Duft shampoo), 2 x 4 mL of water)
- Time after start of exposure: 30 minutes
SAMPLE PREPARATION AND SAMPLING PROCEDURE
- 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 diluted with 15 mL of scintillation cocktail.
- Receptor fluid: The receptor fluid was sampled after 16, 24, 40, 48, 64 and 72 h. An aliquot of the collected receptor fluid fraction (i.e. 5 mL) was transferred to scintillation vials and 15 mL of scintillation cocktail were added.
- Skin: The skin compartments were separated by ‘heat - method’. 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 15-60 seconds, the packed skin sample was removed from the hot surface and unwrapped. The upper skin was separated from the lower skin with forceps. After the separation of the upper skin from the lower skin each of the skin compartments were dissolved with 2 mL of aqueous KOH solution (10M) at 80°C overnight. 200 µL of the resulting solution were mixed with 200 µL of conc. HCl and 3 mL scintillation cocktail.
- Aluminum foil (used to wrap the skin prior to separation): After the separation of the skin compartments, the aluminum foil was dipped into 1 mL of aqueous solution of 5% methanol overnight. Thereafter, the aluminum foil was removed and the extract diluted with scintillation cocktail.
- Rinsings: 30 µL of the rinsings were diluted with scintillation cocktail.

SAMPLE STORAGE: 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 counter (LSC; Packard Tricarb 2250CA)
- Liquid scintillation counting results (cpm) converted to dpm as follows: The limit of detection and limit of quantification for test material was determined according L. A. Currie. Based on background value for this experiment of 145 cpm and a counting interval of 5 minutes, the following limits were calculated:
Limit of detection: 127.92 counts
Limit of quantification: 434.06 counts
With a typical detection efficiency of 90.6 % for 14C on liquid scintillation counter, these limits were transformed to:
Limit of detection: 28.24 dpm
Limit of quantification: 434.06 dpm
- Validation of analytical procedure: A calibration curve was established with aliquots of the solution of the radiolabeled test material. The calibration curve was linear (R2 =0.9966) up to 1,500,000 dpm.
- Limits of detection and quantification: With respect to the applied formulation containing 0.5 % of test material:
Absolute limit of detection: 1.27 ng of test material
Absolute limit of quantification: 4.32 ng of test material

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Pig skin from 2 males and 1 female pig weighing 91.5 - 120 kg bw was obtained from Butchery Kunzli, CH-1724 Praroman-Le-Mouret on different dates.
- Type of skin: Split thickness skin samples obtained from back and flanks of different pigs. The skin samples were composed of the stratum corneum, the stratum germinativum and part of the dermis containing blood vessels.
- Preparative technique: Dermatome
- Thickness of skin: 0.78 ± 0.06 mm (thickness determined by micrometer)
- Membrane integrity check: Yes, (measured by the penetration characteristics of tritiated water by scintillation counting of 1 h fractions over a time of 4 h)
- Storage conditions: - 20°C until use
- Justification of species, anatomical site and preparative technique: Pig skin from flank and back represents a good model for human skin and mimics the human conditions.

PRINCIPLES OF ASSAY
- Diffusion cell: Six permeation chambers (Teflon-chambers) of medium size with 9.1 cm2 surface.
- 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 and 3% Ethanol
- Solubility of test substance in receptor fluid: 2.2 mg/mL (at pH 7.3)
- Stability of test substance in receptor fluid: Quantitative recovery after 3 days (1 mg/ml, in the presence of 0.3% ascorbic acid and 0.4% sodium sulfite)
- 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: 20.7 – 29.5%
- Occlusion: No
- Reference substance: 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): 91.86 ± 4.17% or 0.428 ± 0.021 mg/cm2; After the removal of applied test material with water and shampoo after 30 minutes, the recovered test material was found predominantly in the rinsing solutions.
- Receptor fluid: After 72 hours, 0.031 ± 0.017% or 0.142 ± 0.083 µg/cm2 of test material was found in the receptor fluid.
With respect to the receptor fluid samples, test material was detectable predominantly within the first fraction collected (fraction 0 - 16 hours). In the fractions collected after 24 hours, the detected amounts of test material were below the limit of detection/quantification thus indicating, that test material remaining on or in the skin after the washing step does not tend to migrate to deeper layers (no depot effect).
- Skin preparation: After 72 hours small amounts of test material was found in the upper skin (0.543 ± 0.124% or 2.442 ± 0.572 µg/cm2) and in the lower skin (0.037 ± 0.016% or 0.171 ± 0.075 µg/cm2) compartments.

Total recovery:

- Total recovery: The mass balance of the test substance resulted in values of 93.4 to 103.5% recovery for all 6 skin samples taken into consideration for the calculation of the mean values.
- Recovery of applied dose acceptable: Yes
- Results adjusted for incomplete recovery of the applied dose: No
- Limit of detection (LOD): 1.27 ng of 4-amino-m-cresol as the absolute limit of detection with respect to the applied formulation containing 0.5% of 4-amino-m-cresol. 
- 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 of formulation containing 0.5% of test material (0.5 mg /cm2)

Parameter:

percentage

Absorption:

0.031 %

Remarks on result:

other: 72 hours

Remarks:

Absorption in receptor fluid after 30 minutes exposure

Percutaneous absorption 2

Dose:

100 mg/cm2 of formulation containing 0.5% of test material (0.5 mg /cm2)

Parameter:

percentage

Absorption:

0.037 %

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, which resulted in 0.6 to 1.6% of the applied dose found after 4 hours in the receptor fluids for 6 skin samples (within the limit of acceptance of ≤ 2.0%).

Applicant's summary and conclusion

Conclusions:

A maximum amount of 0.313 ± 0.112 µg/cm2 of 4-amino-m-cresol (Oxyrot) was considered as biologically available when 100 mg/cm2 hair dye formulation containing 0.5 % of test material was applied to pig skin for 30 minutes, in the presence of reaction partner (n = 6; receptor fluid + lower skin: 0.142 µg/cm2 + 0.171 µg/cm2).

Executive summary:

The cutaneous absorption (in-vitro) of 0.5 % 4-amino-cresol (Oxyrot) in a typical oxidative hair dye formulation with hydrogen peroxide and a reaction partner 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. Pig skin from 2 males and 1 female pig weighing 91.5 - 120 kg was obtained 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 (6 skin samples) was monitored by examination of penetration characteristics using tritiated water, which resulted in 0.6 to 1.6% of the applied dose found after 4 hours in the receptor fluids for all skin samples (was within the limit of acceptance of ≤ 2.0%).

After checking the skin integrity, 400 mg of the typical hair dye formulation (100 mg/cm2), containing 0.5% test material (i.e. 0.5 mg/cm2) was applied to skin samples for 30 minutes. After completion of the exposure period, the samples were washed off with water and shampoo and the amount of test material 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 material concentration. At the termination of the experiment, the skin was heat-treated to separate the upper and lower skin compartments. The different matrices (rinsing solution, receptor fluid, lower skin and upper skin) were processed and analyzed by means of a scintillation counter to determine radioactive concentration. 

The majority of the test material was recovered in the rinsing solutions (91.86% or 0.428 ± 0.021 mg/cm2). Small amounts of test material were detected in the upper skin (0.543% or 2.442 ± 0.572 µg/cm2), in the lower skin (0.037% or 0.171 ± 0.075 µg/cm2) and in the fractions of the receptor fluid collected within 72 hours (0.031% or 0.142 ± 0.083 µg/cm2).

The mass balance (total recovery) was 93.4 to 103.5% of the dose applied to the pig skin samples.

With respect to the receptor fluid samples, test material was detectable predominantly within the first fraction collected (fraction 0 - 16 hours). In the fractions collected after 24 hours, the detected amounts of test material were below the limit of detection/quantification thus indicating, that test material remaining on or in the skin after the washing step does not tend to migrate to deeper layers (no depot effect).

Under the test conditions, a maximum amount of 0.313 ± 0.112 µg/cm2 of 4-amino-m-cresol (Oxyrot) was considered as biologically available when 100 mg/cm2 hair dye formulation containing 0.5 % of test material was applied to pig skin for 30 minutes, in the presence of reaction partner (n = 6; receptor fluid + lower skin: 0.142 µg/cm2 + 0.171 µg/cm2).

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