Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Sept. 29, 2008 to Oct. 27, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Guidance document for the conduct of skin absorption studies
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Test Guidelines of the COLIPA Task Force for In Vitro Assessment of Percutaneous Absorption and Penetration of Cosmetic Ingredients.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
according to Swiss ordinance relating OECD principles of GLP

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(methoxymethyl)benzene-1,4-diamine
EC Number:
679-526-3
Cas Number:
337906-36-2
Molecular formula:
C8H12N2O
IUPAC Name:
2-(methoxymethyl)benzene-1,4-diamine
Constituent 2
Reference substance name:
MBB
IUPAC Name:
MBB
Details on test material:
UNLABELED TEST MATERIAL
- Name of test material: 2-Methoxymethyl-p-phenylenediamine (MBB) (Code # A003825)
- TSIN: 804025
- Substance type: Pure active substance
- Physical state: White crystalline powder
- Stability under test conditions: The test substance was considered to be stable for more than 5 yrs, if stored dry and protected from light at room temperature.
- Storage condition of test material: At room temperature, protected from light and moisture
- Solubility: The solubility of test substance in different solvents is a follows:
Water: > 10 weight% (pH: 9.4)

RADIOLABELED TEST MATERIAL (Source: product specifications provided by supplier of the test material)
- Name of test material: [Ring-U-14C] MEOME PPD
- Physical state: Solid
- Substance type: Pure active substance
- Specific activity: 2.11 GBq/ mmol; 57 mCi/ mmol ( mass spectroscopy)
- Locations of the label: [Ring-U-14C] uniform labeling of the aromatic ring system
- Stability under test conditions: Not reported
- Storage condition of test material: The test substance was stored at - 20°C in the absence of moisture, light and air.
Radiolabelling:
yes
Remarks:
14C

Administration / exposure

Duration of exposure:
30 min
Doses:
400 mg of the formulation (100 mg/cm2), containing 1.824% of 2-Methoxymethyl-p-phenylenediamine (1.824 mg of test material/cm2)
Details on study design:
DOSE PREPARATION
- Method for preparation of dose formulation: In order to achieve a concentration of 1.824% of 2-Methoxymethyl-p-phenylenediamine, 1.84 g of Color cream formulation containing 6 % 2-Methoxymethyl-p-phenylenediamine, 100 µL of the radiolabeled solution of 2-Methoxymethyl-p-phenylenediamine and 1.12 g of Color Cream formulation Blank were added to a glass beaker. The resulting suspension was mixed until a homogeneous cream was obtained. To this mixture, 2.95 g of Welloxon Perfect 6 % were added and mixed again with a spatula until a homogeneous cream was obtained.

- Method of storage: All samples (application formulation, rinsings, receptor fluid fractions) were stored at -20°C, if not processed immediately.

APPLICATION OF DOSE: 400 mg of test formulation containing 1.824% of test material in the presence of hydrogen peroxide and an equimolar amount of a reaction partner was applied on 12 skin samples in two independent experiments for 30 min.

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 min

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 heating. The skin samples were wrapped in aluminium foil and put on a hot surface (80 to 90°C) upside down (i.e. lower skin upside) charged with a small weight of approx. 40 g. After 40-60 sec, 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.
- Aluminium foil (used to wrap the skin prior to separation): After the separation of the skin compartments, the aluminium foil was dipped into 1 mL of aqueous solution of 5% methanol (in water) overnight. Thereafter, the aluminium foil was removed and the extract diluted with scintillation cocktail.
- Rinsings: 15 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: The radioactive concentration of test substance in all samples formulation, rinsings, receptor fluid, extracts of separated skin compartments, spatula (used for application of final formulation) and aluminium foil (used to wrap the skin prior to separation). The radioactivity of each of the samples was quantified with a liquid scintillation counter (Packard Tricarb 2250CA)
- Limits of detection and quantification (cpm) converted to dpm as follows: The limit of detection and limit of quantification was determined according L. A. Currie
Based on the background value of 142 cpm (1st experiment) and 171 cpm (2nd experiment) and a counting interval of 30 min for this experiment limit of detection (LOD) and limit of quantification (LOQ) was calculated in cpm (counts per minute):
LOD: 1st Experiment: 306.21 counts; 2nd Experiment: 335.76 counts
LOQ: 1st Experiment: 974.39 counts; 2nd Experiment: 1064.15 counts
The LOD and LOQ were converted to dpm using a detection efficiency of 90.6% established for 14C on LSC (liquid scintillation counter)
LOD: 1st series: 11.27 dpm; 2nd series: 12.35 dpm
LOQ: 1stseries: 35.85 dpm; 2nd series: 39.15 dpm
- Absolute limit of detection and quantification: With respect to the applied formulation containing 1.824% of test material the absolute LOD and LOQ corresponded to:
LOD: 1st series – 1.12 ng of test material; 2nd series – 1.23 ng of test material
LOQ: 1st series –3.57 ng of test material; 2nd series – 3.90 ng of test material
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Pig skin from 3 female pigs weighing 80-220 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 (rear and front) 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: 0.79 ± 0.09 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: 12 diffusion cells (6 per experiment) (Teflon-chambers with 9.1 cm2 surface, in-house development)
- Receptor fluid: Physiological receptor fluid composed of 0.14 M NaCI, 2 mM K2HPO4, 0.4 mM KH2PO4, 100 IU penicillin/mL, 97 µg streptomycin/mL, pH = 7.3
- Solubility of test substance in receptor fluid: 307.85 mg/mL (pH =7.30)
- Stability of test substance in receptor fluid: 88% recovery after 3 d
- Flow-through system: Yes, with a constant flow of the receptor fluid of 5 mL/h
- Test temperature: 32 ± 2°C
- Relative humidity: 17.7 to 22.8% (first series), 18.0 to 23.0% (second series)
- Occlusion: No
- Reference substance(s): Mannitol
- Reliability check: Reliability of test system and reproducibility of the method was verified at appropriate time intervals using mannitol as a reference substance to measure the cutaneous absorption with random skin samples from each pig.
- Diffusion barrier: Stratum corneum was identified as principal diffusion barrier; the integrity of diffusion barrier was controlled in each experiment by tritiated H2O penetration characteristics.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
- Receptor fluid, receptor chamber, donor chamber: 1.341± 0.328 µg/cm2 or 0.077 ± 0.019% dose. The test material was detectable predominantly within the first fraction collected (fraction 0 - 16 h). Smaller amounts of test material were detectable in the proceeding fractions.
- Stratum corneum + upper stratum germinativum (upper skin): 7.177± 3.211 µg/cm2 or 0.411 ± 0.179% dose
- Lower stratum germinativum + upper dermis (lower skin): 0.219± 0.143 µg/cm2 or 0.013 ± 0.008% dose
- Rinsing solution: 1618.43 ± 43.40 µg/cm2 or 92.88 ± 1.59% dose
Total recovery:
- Total recovery: The mass balance of the test substance resulted in values of 98.47 ± 1.79% recovery for all 11 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) and quantification (LOQ): Theoretical limit of detection and quantification were:
Receptor fluid (1st series):
16 h fraction: LOD – 4.22 ng/cm2; LOQ – 13.10 ng/cm2
8 h fraction: LOD – 2.11 ng/cm2; LOQ – 6.55 ng/cm2
Receptor fluid (2nd series):
16 h fraction: LOD – 2.17 ng/cm2; LOQ – 6.89 ng/cm
28 h fraction: LOD – 1.09 ng/cm2; LOQ - 3.44 ng/cm2
Skin compartments:
Upper skin: LOD – 2.64 ng/cm2; LOQ – 8.19 ng/cm2
Lower skin: LOD – 1.36 ng/cm2; LOQ – 4.31 ng/cm2
- Quantification of values below LOD or LOQ: Not reported
Percutaneous absorptionopen allclose all
Dose:
100 mg/cm2 formulation containing 1.824% test material
Parameter:
percentage
Absorption:
0.077 %
Remarks on result:
other: 72 h
Remarks:
Absorption in receptor fluid after 30 min exposure
Dose:
100 mg/cm2 formulation containing 1.824% test material
Parameter:
percentage
Absorption:
0.013 %
Remarks on result:
other: 72 h
Remarks:
Absorption in lower skin after 30 min exposure
Dose:
100 mg/cm2 formulation containing 1.824% test material
Parameter:
percentage
Absorption:
0.411 %
Remarks on result:
other: 72 h
Remarks:
Absorption in upper skin after 30 min 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 0.73 ± 0.44% of the applied dose (n=19, water as vehicle).

Calibration curve: The calibration curve established with aliquots of the solution of the radiolabelled test material was linear up to 1,200,000 dpm.

Integrity check: The integrity of each skin preparation was demonstrated by examination of penetration characteristics with tritiated water resulting in 0.7 to 2.5% of the applied dose found after 4 h in the receptor fluids, which was within the limit of acceptance (≤ 2.0%) for 11 skin samples. The skin samples with skin integrity values above 2% of the applied dose (1 skin samples) were not within the limit of acceptance (vide supra) and were not taken into consideration for the calculation of the mean.

Results of the cutaneous absorption: The results of the cutaneous absorption after 72 h with 10 pig skin samples treated with 1.824 mg of test material/cm2(1.824% of test material) in a typical hair dye formulation is provided in the below table:

Table 1: Summary of the cutaneous absorption of 1.824% 1,4-diamino-2-methoxymethyl-benzene in a typical oxidative hair dye formulation (Study # 65173)

Concentration of test substance

µg/ cm2(mean ±SD, n=11)

%* (mean ±SD, n=11)

Receptor fluid (72 h)

1.341± 0.328

0.077 ± 0.019

Lower skin (72 h)

0.219± 0.143

0.013 ± 0.008

Upper skin (72 h)

7.177± 3.211

0.411 ± 0.179

Rinsing solution (after 30 min)

1618.43 ± 43.4

92.88 ± 1.59

Total balance**

1712.17 ± 25.1

98.47 ± 1.79

*Corrected for individual applied dose

** Total was corrected for losses on tips

Applicant's summary and conclusion

Conclusions:
Under the assumption that a depot effect is absent, a mean amount of 1.56 μg/cm² of 2- Methoxy-Methyl-P-Phenylenediamine in a typical oxidative hair dye formulation with 1.824% dye and in the presence of reaction partners was detected as the mean bioavailable fraction (n=11, three donors; receptor fluid + lower skin; 1.341 μg/cm2 + 0.219 μg/cm2).
Although 3 donors instead of 4 were used only 1 SD (0.36 μg/cm2) was added to the mean, because the study was carried out with pig skin, which is known to be less variable compared to human skin (Ana M. Barbero, H. Frederick Frasch: Pig and guinea pig skin as surrogates for human in vitro penetration studies, A quantitative review; Toxicology in Vitro: Vol. 23, 1-13, 2009). Therefore the total bioavailable fraction used for the risk assessment is 1.92 μg/cm².
Executive summary:

Cutaneous absorption (in vitro) of 1.824% 2-Methoxymethyl-p-phenylenediamine (MBB) using pig skin preparations was determined following OECD Guideline 428 (Skin Absorption: In Vitro Method).

Two independent experiments with 6 diffusion cells/ experiment were performed. Pig skin from 3 female pigs, weighing 80-220 kg, was used in study. Pig skins were obtained from Butchery Kunzli, CH-1724 Praroman-Le-Mouret on different dates.

400 mg of the formulation (100 mg/cm2), containing 1.824% 2-Methoxymethyl-p-phenylenediamine (i.e. 1.824 mg/cm2) was applied to the 12 skin (6 per experiment) samples for 30 min and subsequently washed off with water and shampoo. The content of test substance was determined in the rinsing solutions and in the receptor fluid at 16, 24, 40, 48, 64 and 72 h. At termination of the experiment, the skin was heat-treated and the upper skin was separated from the lower skin. Both skin compartments were extracted separately and analyzed for the amount of test substance. The radioactivity in different matrices was quantified by means of a scintillation counter to determine total radioactive recovery.

The majority of the test substance was found in the rinsing solutions and was not absorbed into the skin during the application period of 30 min. Small amounts of test substance were found in the upper skin, in the lower skin and in the fractions of the receptor fluid collected within 72 h.

The absorption in different matrices were:

Receptor fluid, receptor chamber, donor chamber: 1.341± 0.328 µg/cm2 or 0.077 ± 0.019%

Stratum corneum + upper stratum germinativum (upper skin): 7.177± 3.211 µg/cm2 or 0.411 ± 0.179%

Lower stratum germinativum + upper dermis (lower skin): 0.219± 0.143 µg/cm2 or 0.013 ± 0.008%

Rinsing solution: 1618.43 ± 43.4 µg/cm2 or 92.88 ± 1.59%

Total recovery: 1712.17 ± 25.1 µg/ cm2 or 98.47 ± 1.79%

The mass balance of the test substance resulted in values of 95.2 to 101.0% recovery for all skin samples taken into consideration for the calculation of the mean values.

With respect to the receptor fluid samples,2 -Methoxy-Methyl-P-Phenylenediamine was detectable predominantly within the first fractions collected (fractions 0 -16 hours). Smaller amounts of the test substance were detected in the fractions collected after 16 hours. At the end of the experiment (after 72 hours) the amounts of 2 -Methoxy-Methyl-P-phenylenediamine detectable in the receptor fluid declined, thus indicating that 2 -Methoxy-Methyl-P-Phenylenediamine remaining on or in the skin after 72 hours does not show a tendency to migrate to deeper layers. This assumption is further supported by the fact that after 72 hours, relatively low amounts of 2- Methoxy-Methyl-P-Phenylenediamine can be found in the lower skin compartment compared to upper skin and the total amount in the receptor fluid after 72 hours. With such an absorption profile, the amounts of 2- Methoxy-Methyl-P-Phenylenediamine found in the upper skin are not considered as biologically available (no depot effect).

Under the assumption that a depot effect is absent, a mean amount of 1.56 μg/cm² of2- Methoxy-Methyl-P-Phenylenediaminein a typical oxidative hair dye formulation with1.824% dye and in the presence of reaction partners was detected as the mean bioavailablefraction (n=11, three donors; receptor fluid + lower skin; 1.341 μg/cm2 + 0.219 μg/cm2).