Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
Light cream flakes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Procter & Gamble ; n°batch I-38110
- Expiration date of the lot/batch: 14th January 2006
- Purity test date: No data
- Purity : 99.9% w/w

RADIOLABELLING INFORMATION
- Radiochemical purity: 96.9% (certificate of analysis)
- Specific activity: 10.5 mCi/mmmol ; 388.5 MBq/mmol
- Locations of the label: [14C]-1-naphthol (N°batch 020K9440/41)
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark
- Stability under test conditions: Yes
- Solubility and stability of the test substance in the solvent/vehicle: Yes
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

FORM AS APPLIED IN THE TEST Cream
Radiolabelling:
yes
Remarks:
14C-1-naphthol

Administration / exposure

Type of coverage:
occlusive
Vehicle:
other: Blank formulation (Nice-n EasyTM tint)
Duration of exposure:
30 mn
Doses:
- Nominal doses: 2% w/w (400 µg/cm²)
- Actual doses: No data
- Actual doses calculated as follows: Liquid Scintillation counting
- Dose volume: 20 mg/cm²
- Rationale for dose selection: The dose used in this study were designed to simulate predicted normal human exposure to the test material.
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: The formulation was applied following a 1:1 w/w mix with two different developers (a peroxide developer and a placebo developer), both resulting in an nomial final dose concenration of 2% 1-Naphthol
- Method of storage: No data

APPLICATION OF DOSE: in the glass diffusion cells

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Amount(s) applied (volume or weight with unit): ca. 990 - 995 mg of developer
- Purity: No data
- Homegeneity and radioactive content were confirmed (triplicate samples - 10 ml - were diluted with scintillation fluid and each dilution were analysed by liquid scintillation counting).

TEST SITE
- Preparation of test site: glass diffusion cells
- Area of exposure: 2.54 cm²
- Type of cover : unoccluded after the exposure (30 mn) and during 48 hours

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: the dose was washed from the surface of the skin using natural sponges soaked in 3% Teepol
- Time after start of exposure: 30 mn

SAMPLE COLLECTION/ PREPARATION
- All the sponges were combined and digested in Soluene 350
- Samples of the receptor fluid (0.5 ml) were taken at recorded intervals over a 48h period (0.5 ; 1 ; 2 ; 4 ; 6 ; 24 ; 29 and 48)
- Layers of stratum corneum removed using a tape stripping technique (at tne end of the experiment and after a new washing). The adhesive strips were soaked in Soluene 350.
- The donor Chamber was washed with ethanol and the sample analysed.

ANALYSIS
- Membrane Integrity : Yes (electrical resistane across the skin membrane).
- Test substance absorption : Yes
- Mass balance : yes by LSC

- Liquid scintillation counting results (cpm) converted to dpm as follows: 6 minutes or to a 0.5% standard deviation of the count
- Limits of quantification: 0.004 µg/ml (0.007 µg/cm²)

Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: surgery or post mortem
- Ethical approval if human skin: No data
- Type of skin: human dermatomed skin
- Preparative technique: six membranes were cut form the whole skin samples using an electric dermatome.
- Thickness of skin (in µm): 400
- Membrane integrity check: No data
- Storage conditions: -20°C on aluminium foil


PRINCIPLES OF ASSAY
- Diffusion cell: Yes (3.3 cm diameter)
- Receptor fluid: water
- Solubility od test substance in receptor fluid: Yes
- Static system: yes
- Flow-through system: No
- Test temperature: 32 +/-1°C
- Other: cells were selected such that each application (peroxide dilution or placebo dilution) was represented by 12 intact membranes from eight differents subjects.

Results and discussion

Absorption in different matrices:
Peroxide developer mix
The fastest rate of penetration of 1-Naphthol form the peroxide developer mix (0.562 µg/cm²/h) occurred 0.5 - 2 h after application. Between 2 and 6h, the rate slowly reduced as the reservoir remaining in the skin depleted, following the washing at 0.5h. After 6h the penetration process was effectively complete.
The mean penetrated amount of 1-naphthol at 0.5h was 0.045 %g/cm² (0.0004% of dose),which increased to 0.214 µg/cm² (0.053%) at 1h, 2.07 µg/cm² (0.518%) at 6h and was 3.43 µg/cm² (0.857%) at the end of the experiment (48h). The mean residual amount in the remaining epidermis/ dermis, after tape stripping to remove the stratum corneum, was 0.149 %g/cm² (0.037%), thus the mean systemically available proportion of the dose (amounts penetrated + remaining epidermis/dermis) was 3.58 µg/cm² (0.894% of the dose).
The mean proportion of the dose recovered from the tape strips, representing the stratum corneum, was 1.621 µg/cm² (0.405%), while 1-naphthol recovered from the flange area was 0.064 µg/cm² (0.016%).
The greatest proportion of the applied 1-naphthol was removed from the surface of the skin by the washing procedure at 0.5h (394 µg/cm² ; 98.5%) with only a further 0.710 µg/cm² (0.178%) being removed by the later procedure at 48h.
The overall mean recovery of 1-Naphthol during this experiment was 100%.

Placebo developer mix
The fastest rate of penetration of 1-Naphthol from the placebo developer mix (0.624 µg/cm²/h) occured 0.5-1h after application. Between 1 and 6h, the rate slowly reduced as the reservoir remaining in the skin depleted, following the washing at 0.5h. After 6h the penetration process was effectively complete.
The mean penetrated amount of 1-Naphthol at 0.5h was 0.066 µg/cm² (0.017% of dose), which increased to 0.378 µg/cm² (0.095% of dose) at 1h, 2.22 µg/cm² (0.555%) at 6 hours and was 3.49 µg/cm² (0.873%) at the end of the experiment (48 hours). The mean residual amount in the remaining epidermis/dermis, after tape stripping to remove the stratum corneum, was 0.107 µg/cm² (0.027%), thus the mean systemically available proportion of the dose (amounts penetrated + remaining epidermis/dermis) was 3.60 µg/cm² (0.899% of the dose).
The mean proportion of the dose recovered from the tape strips, representing the stratum corneum, was 0.200µg/cm² (0.050%), while the amount of 1-naphthol recovered from the flange area was 0.042 µg/cm² (0.011%).
As with the peroxide mix, the greatest proportion of the applied 1-naphthol was removed from the surface of the skin by the washing procedure (402 µg/cm² ; 101%), with only a further 0.277 µg/cm² (0.069%) being removed by the later procedure at 48h.
The overall mean recovery of 1-naphthol during this experiment was 102%.
Percutaneous absorptionopen allclose all
Key result
Time point:
48 h
Dose:
2%
Parameter:
amount
Remarks:
the mean systemically available proportion of the dose
Absorption:
ca. 0.899 %
Time point:
30 min
Dose:
2%
Parameter:
percentage
Remarks:
% of the mean penetrated
Absorption:
ca. 0.017 %
Time point:
1 h
Dose:
2%
Parameter:
percentage
Remarks:
% of the mean penetrated
Absorption:
ca. 0.095 %
Time point:
6 h
Dose:
2%
Parameter:
percentage
Remarks:
% of the mean penetrated
Absorption:
ca. 0.555 %
Time point:
48 h
Dose:
2%
Parameter:
percentage
Remarks:
% of the mean penetrated
Absorption:
ca. 0.873 %

Applicant's summary and conclusion

Conclusions:
The fastest rate of penetration of 1-Naphthol from the placebo developer mix (0.624 μg/cm²/h) occured 0.5-1h after application. Between 1 and 6h, the rate slowly reduced as the reservoir remaining in the skin depleted, following the washing at 0.5h. After 6h the penetration process was effectively complete. The mean penetrated amount of 1-Naphthol at 0.5h was 0.066 μg/Cm² (0.017% of dose), which increased to 0.378 μg/cm² (0.095% of dose) at 1h, 2.22 μg/cm² (0.555%) at 6 hours and was 3.49 μg/cm² (0.873%) at the end of the experiment (48 hours). The mean residual amount in the remaining epidermis/dermis, after tape stripping to remove the stratum corneum, was 0.107 μg/cm² (0.027%), thus the mean systemically available proportion of the dose (amounts penetrated + remaining epidermis/dermis) was 3.60 μg/cm² (0.899% of the dose).
Executive summary:

The percutaneous absorption of 1-naphthol from a proprietary oxidative hair dye base containing 4% 1-naphthol was evaluated in an in vitro assay using human dermatomed skin. Prior to dosing, the formulation was mixed 1:1 with either A) a hydrogen peroxide developer solution to give a final concentration of 2% 1-naphthol or B) a placebo developer to give a final concentration of 2% 1-naphthol.

A dose of 20 mg of test formulation/cm2 skin (400 μg 1-naphthol /cm2 skin) was applied to the skin samples for 30 minutes followed by thorough rinsing with 3% Teepol®. Measurements of the 1-naphthol penetrating the skin into the receptor fluid were taken following the 30 minute exposure period and at set intervals during the 48 hour measurement period (1, 2, 4, 6, 24, 29 and 48 hours post application). At the end of the 48 hour measurement period, tape stripping was conducted and the levels of 1-naphthol in the tape strips and the remaining epidermis/dermis determined.

Results

a) In the oxidative formulation the amount considered absorbed was 3.58 ± 1.13 (range 1.31 to 5.46 μg/cm².

b) In the placebo, non-oxidative formulation the amount considered absorbed was 3.60 ± 1.80 (range 1.49 to 8.28) μg/cm² [0.899 ± 0.451 (range 0.373 to 2.07) % of the applied dose].