Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 28 July 2011 to 2 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was performed according to internationally recognised guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Radiolabelling:
yes

Administration / exposure

Type of coverage:
other: not applicable
Duration of exposure:
30 minutes
Doses:
- Formulation nominal concentration: 4%
- On-head concentration: 2%
- Dose rate: 20 mg/cm², corresponding to a nominal 400 µg/cm² of test item
No. of animals per group:
12 intact skin membranes
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: a vial containing dried [14C]-test item with an analysed activity of 20.8 MBq was transferred to an AtmosBag® where the internal environment was maintained with a flow of Argon gas. The container was sealed in the Argon atmosphere and placed in the freezer overnight. Once defrosted, the container was placed in the AtmosBag® and 1994 mg of the pre-formulation was added. The preparation was mixed in the AtmosBag® for approximately 20 minutes using a magnetic stirrer, producing the ‘Hair dye formulation’. The final weight of the ‘Hair dye formulation’ was recorded and used in all subsequent calculations. Three weighed aliquots with a volume of approximately 400 μL, were transferred to three vials after which, 30 glass beads (1.8-2 μm) were added. Immediately before dosing each batch of 4 cells, one vial was removed from the AtmosBag® and an equal weight (around 400 μL) of Developer "Hydrogen Peroxide 178914" was added and mixed by vortexing until visually homogeneous, producing the ‘Dose Preparation’.

APPLICATION OF DOSE: the dose was applied to the skin membranes and the weight of the applied dose recorded.


REMOVAL OF TEST SUBSTANCE
At the end of the 30 minute exposure period, the skin surface was washed with water (10 x 1270 μL) followed by 2% sodium dodecyl sulphate (SDS) in water (1 x 1270 μL). Between each set of washes the washing fluid was aspired three times. The skin surface was washed further with water (10 x 1270 μL), after which the skin surface was dried with cotton wool swabs. Following decontamination the cells were returned to the water bath for the remainder of the 24 hour run time.

SAMPLE COLLECTION
After the final 24 hour receptor fluid sample had been taken, the remaining fluid in the receptor chamber was discarded and the chamber rinsed with fresh receptor fluid which was also discarded.
The donor chamber was carefully removed and the underside wiped with a single sponge pre-wetted with 3%Teepol L® in water which was added to the wash sponges (below). The donor chamber was washed with ethanol and sonicated for 10 minutes to ensure thorough extraction. Subsamples were taken for analysis by LSC.

SAMPLE PREPARATION
The skin surface was washed with sponges soaked in 3% Teepol®L in water and further sponges pre-wetted with water. The stratum corneum was removed by a tape stripping process removing upto a maximum of 20 strips from each skin membrane. The flange skin was cut away from the dermis and the epidermis on the remaining skin disc was separated from the dermis using a heat separation technique.

ANALYSIS
The penetration process was monitored using [14C]-radiolabelled test item, which was incorporated into the formulation, prior to application.
The distribution of test item within the test system was measured and a 24 hour penetration profile was determined by collecting receptor fluid samples 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours following application. The samples were analysed by liquid scintillation counting (LSC):
- Counting period: 6 minutes or to a 0.5% standard deviation of the count (0.5 minutes for time critical quick checks)
- Scintillation fluid: Goldstar
- Model of LSC: Packard 3100 TR
Test materials were also analysed by HPLC. Specificity, linearity, accuracy and precision were shown to be acceptable.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human skin samples were obtained from a tissue bank
- Type of skin: from the abdomen of female donors (post-mortem, 64-85 years old)
- Preparative technique: skin membranes were cut from the samples using an electric dermatome
- Thickness of skin (in mm): 400 µm
- Membrane integrity check: yes (11.17-23.49 kΩ)
- Storage conditions: stored frozen, at approximately -20°C, on aluminium foil
- Justification of species, anatomical site and preparative technique: the application rates and exposure conditions were designed to simulate predicted normal human exposure to the test material

PRINCIPLES OF ASSAY
- Diffusion cell: static glass diffusion cell with an exposed membrane area of 2.54 cm2 and a volume of approximately 4.5 mL
- Receptor fluid: degassed phosphate buffered saline (PBS) receptor fluid
- Solubility od test substance in receptor fluid: not indicated
- Static system: yes
- Test temperature: 32 ± 1 °C
- Humidity: no data
- Occlusion: no

Results and discussion

Absorption in different matrices:
TEST ITEM PENETRATION:
The mean test item penetration rate was 0.046 μg/cm2/h between 0-0.5 hours. Following the 0.5 hour decontamination, penetration through human skin increased slightly to 0.065 μg/cm2/h between 0.5-2 hours before slowing to 0.006 μg/cm2/h between 2-24 hours. Between 0-24 hours, the penetration rate was, on average, 0.010 μg/cm2/h.
The amounts (mean ± SD) of test item that penetrated through human skin at 4, 8 and 12 hours were 0.161 ± 0.075 μg/cm2, 0.200 ± 0.083 μg/cm2 and 0.226 ± 0.094 μg/cm2, respectively. These respective amounts expressed as percentages of the applied dose were 0.040, 0.050 and 0.057%. The mean amount penetrated over the entire 24 hour exposure period was 0.272 ± 0.115 μg/cm2, corresponding to 0.068% of the applied dose.
Total recovery:
None of the 12 dosed cells were rejected.
Mean recovery of the applied test material was very good at 98.7%, with individual cell values ranging from 95.1% to 103% (n=12).
The amount (mean ± SD) of remaining test item that was removed by washing the surface of the skin 0.5 hours after application was 98.1 ± 2.70% (391 μg/cm2). Washing at 24 hours removed a further 0.529 ± 0.213% (2.11 μg/cm2). The mean amount of the dose present in the outer layers of the stratum corneum was 0.007 ± 0.005% of the applied dose (0.030 μg/cm2) with 0.028 ± 0.012% of the dose (0.111 μg/cm2) present in the remaining epidermis.
The amount (mean ± SD) recovered from the dermis was 0.010 ± 0.014% of the applied dose (0.039 μg/cm2).
The proportion (mean ± SD) of the applied dose of test item present in receptor fluid following the total 24 hour exposure was 0.068 ± 0.029%. This percentage equated to 0.272 μg/cm2.
The mean total non-absorbed dose (donor chamber, skin wash, stratum corneum and flange skin) represented 98.6% (393 μg/cm2) of the applied dose.
The mean total systemically available dose of (remaining epidermis, dermis and receptor fluid) was 0.106% of the test item applied dose corresponding to 0.422 μg/cm2.
Percutaneous absorptionopen allclose all
Dose:
2%
Parameter:
percentage
Absorption:
0.04 %
Remarks on result:
other: 4 hrs
Dose:
2%
Parameter:
percentage
Absorption:
0.05 %
Remarks on result:
other: 8 hrs
Dose:
2%
Parameter:
percentage
Absorption:
0.057 %
Remarks on result:
other: 12 hrs
Dose:
2%
Parameter:
percentage
Absorption:
0.068 %
Remarks on result:
other: 24

Any other information on results incl. tables

Analysis:

LSC analysis of the dose preparations confirmed that the dose preparations were homogeneous both prior to and following dosing. HPLC analysis confirmed that prior to the addition of developer 178914, the radiochemical purity of [14C]-test item when formulated was over 95% for a 24 hour period following application.

Table 1 : Summary of test item Penetration through Human Dermatomed Skin:

Application of Test Material and Actual Concentration of Dose Preparation

Mean Penetration Rates

Mean Amount and Percentage of Dose Penetrated

Time period (h)

Penetration rate (μg/cm²/h±SD)

Time (h)

Amount (μg/cm2±SD)

Percentage Penetrated (±SD)

2% Oxidative Hair Dye

(19.9 mg test item/g)

20 mg/cm2 (399μg ai/cm2)

Unoccluded

Total run time: 24 hour n = 12

0-0.5

0.046 ± 0.018

4

0.161 ± 0.075

0.040 ± 0.019

0.5-2

0.065 ± 0.040

8

0.200 ± 0.083

0.050 ± 0.021

2-24

0.006 ± 0.005

12

0.226 ± 0.094

0.057 ± 0.024

0-24

0.010 ± 0.005

24

0.272 ± 0.015

0.068 ± 0.029

 

 

LOQ

0.007

0.00

Table 2: Summary of test item Distribution in the test system:

Test compartment

N = 12

µg test item per cm²

% of applied dose

Mean

SD

Mean

SD

Donor chamber

0.060

0.030

0.015

0.008

Skin wash at 0.5 hours

391

10.8

98.1

2.70

Skin wash at 24 hours

2.11

0.848

0.529

0.213

Stratum corneum

0.030

0.022

0.007

0.005

Remaining epidermis

0.111

0.047

0.028

0.012

Dermis

0.039

0.057

0.010

0.014

Flange

0.038

0.024

0.009

0.006

Receptor fluid

0.272

0.115

0.068

0.029

Total non-absorbed

393

10.1

98.6

2.54

Systematically available

0.422

0.174

0.106

0.044

Total recovered

394

10.2

98.7

2.56

Systemically available = Sum of remaining epidermis, dermis and receptor fluid.

Total non-absorbed = Sum of donor chamber, skin wash, flange and stratum corneum.

Stratum corneum = Amount in tape strips.

Remaining epidermis = Epidermal tissue after tape stripping.

Applicant's summary and conclusion

Conclusions:
The results obtained in this study indicate that the test item at 2% in a typical oxidative hair dye formulation penetrated through human dermatomed skin at a very slow rate. The extent of test item penetration through human skin amounted to only 0.068% (0.272 ± 0.115 μg/cm2) of the applied dose, after 24 hours.
The mean total systemically available dose of test item (remaining epidermis plus dermis and receptor fluid) was 0.106% of the applied dose (corresponding to 0.422 μg/cm2).
Executive summary:

The test item was tested in an in vitro percutaneous penetration study through human dermatomed skin over 24 hours according to OECD TG guideline No. 428 (GLP, scored as validity 1 according to Klimisch criteria). The dose preparation (a typical oxidative hair dye formulation containing a nominal 4% test item which was mixed with peroxide developer (1:1 w/w)) was applied to the skin for 30 minutes to mimic in-use conditions. The mass balance and distribution of test item within the test system following the 24 hour run time was also determined.

The penetration process was monitored using [14C]-radiolabelled test item, which was incorporated into the formulation, prior to application. The receptor fluid was degassed phosphate buffered saline (PBS), in order to ensure that adequate sink conditions were maintained for this particular test material. The distribution of test item within the test system was measured and a 24 hour penetration profile was determined by collecting receptor fluid samples 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours following application. The samples were analysed by liquid scintillation counting (LSC).

The results obtained in this study indicate that the test item at 2% in a typical oxidative hair dye formulation penetrated through human dermatomed skin at a very slow rate. The extent of test item penetration through human skin amounted to only 0.068% (0.272 ± 0.115 μg/cm2) of the applied dose, after 24 hours.

The mean total systemically available dose of test item (remaining epidermis plus dermis and receptor fluid) was 0.106% of the applied dose (corresponding to 0.422 μg/cm2).