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Diss Factsheets

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Apr. 20, 2011 to May 19, 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study, followed guideline with deviation, GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
yes
Remarks:
(No reference substance was used to establish the validity of the method)
GLP compliance:
yes (incl. QA statement)
Remarks:
according to UK, US, Japan and OECD principles of GLP

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hexylpyrazole-3,4-diamine;sulfuric acid
EC Number:
696-231-5
Cas Number:
1361000-03-4
Molecular formula:
C9H18N4 x 0.5 H2SO4
IUPAC Name:
2-hexylpyrazole-3,4-diamine;sulfuric acid
Constituent 2
Reference substance name:
C6 Pyrazole hemisulfate
IUPAC Name:
C6 Pyrazole hemisulfate
Constituent 3
Reference substance name:
4,5-diamino-1-hexyl-1H-pyrazole hemisulfate
IUPAC Name:
4,5-diamino-1-hexyl-1H-pyrazole hemisulfate
Test material form:
solid: crystalline
Details on test material:
UNLABELLED TEST SUBSTANCE- Name of test material: 4,5-diamino-1-hexyl-1H-pyrazole hemisulfate (Code: A0021277)- TSIN: WR804146 - Substance type: Pure active substance- Physical state: White feathery crystals- Storage condition of test material: Ambient temperature (in dark)LABELLED TEST SUBSTANCE- Name of test material: [14C] n-Hexylpyrazole Hemisulfate ([14C]-C6P)- Substance type: Pure active substance- Specific activity: 59 mCi/mmol (2.18 GBq/mmol) (measured by mass spectroscopy)- Locations of the label: Ring-14C- Storage condition of test material: At - 20°C.
Radiolabelling:
yes
Remarks:
(14 C)

Administration / exposure

Duration of exposure:
30 minutes
Doses:
12.8 mg (at application rate of 20 mg/cm2) of each test formulations containing test substance at target concentration of 0.165, 0.5 and 1.5% w/w
Details on study design:
DOSE PREPARATION: C6P was incorporated into a typical oxidative hair dye formulation at approximately 3%, 1% and 0.33% (w/w) containing equimolar concentrations of a reaction partner (3-amino-2, 6-dimethylphenol), before mixing with peroxide developer (1:1, w/w).Therefore, the concentration of C6P in the final test preparations applied to the skin was:Test preparation 1: 1.5% w/w [14C]-C6P (concentration by radioactivity was 1.636%)Test preparation 2: 0.5% w/w [14C]-C6P (concentration by radioactivity was 0.577%)Test preparation 3: 0.165% w/w [14C]-C6P (concentration by radioactivity was 0.158%)APPLICATION OF DOSEEach test preparation was applied over the surface of the stratum corneum of twelve cells using a Rainin® positive displacement pipette set to deliver a target mass of 12.8 mg (at application rate of 20 mg/cm2).OBSERVATION PERIOD: 72 h post exposure.TEST SITE- Area of exposure: The surface area of exposed skin within the cells was 0.64 cm2.REMOVAL OF TEST SUBSTANCE- Washing procedures: At 30 min post exposure, each skin was washed with ten successive 320 μL (500 μL/cm2) aliquots of water using a pipette set to deliver 320 μL. Each aliquot was aspirated three times onto the skin surface prior to removal. The pipette tip was removed and replaced by a fresh pipette tip. A single 320 μL aliquot of sodium dodecyl sulphate (SDS) solution in water (2%, w/v) was applied to each skin sample. This was aspirated 3 times. This process was repeated again.- Time after start of exposure: 30 minSAMPLE PREPARATION - Receptor fluid: The receptor fluid was collected in 30 min fractions from 0 to 1 hours post dose and hourly fractions from 1 to 6 hours post dose and then in 2 hourly fractions from 6 to 72 hours post dose.- Skin wash (At 30 min post exposure): The skin sample was washed with water and SDS using pipette. The water and SDS solution were pooled in one skin wash vial per skin sample. Duplicate weighed aliquots (1 mL) were removed from each skin wash vial, mixed with scintillation fluid (10 mL) and analyzed by liquid scintillation counting.- Tissue swabs (At 30 min post exposure): Three tissue swabs used for drying of skin surface were placed into a single vial. Scintillation fluid (10 mL) and methanol (1 mL) were added to each tissue swab vial and the samples analyzed by liquid scintillation counting.- Pipette tip (At 30 min post exposure): The pipette tips were mixed with methanol (1 mL) and scintillation fluid (10 mL) and analyzed by liquid scintillation counting.- Receptor rinse (72 hours post dose): At 72 hours post dose, the diffusion cell was disconnected from the receptor fluid pump lines. The underside of the skin was washed (receptor rinse) with receptor fluid (1-2 mL), which was collected into vials. The receptor rinse represented the absorbed test substance, which was in the receptor chamber but had not been collected in the 70 to 72 hours receptor fluid fraction.- Donor and receptor chambers: The cell was dismantled and the skin sample removed. The donor and receptor chambers were transferred into pre-weighed pots (donor wash and receptor wash) containing a weighed volume of water (40 mL). This was left for a minimum of 30 min to extract the test substance, during which time the sample was sonicated for 10 min. The donor and receptor chamber were removed from the pot.- Stratum corneum: The stratum corneum was removed with 20 successive tape strips (3M Scotch™ Magic Tape) and individually placed into a scintillation vial containing methanol (1 mL) and scintillation fluid (10 mL). Where a small piece of epidermis was removed, the tape strip number was recorded or where epidermis was completely removed tape stripping ceased. Each tape was placed into a separate vial and analyzed by liquid scintillation counting.- Exposed skin and unexposed skin (flange): The skin under the cell flange (unexposed skin) was cut away from the exposed skin using scissors. The epidermis was separated from the dermis of the exposed skin. The exposed skin was placed onto cling film, epidermis side up, and the cling film wrapped over the skin. A 200 g weight, heated to approximately 65°C, was placed onto the skin for approximately 90 sec. The epidermis was then removed from the skin with a scalpel using a peeling motion. The epidermis, dermis and unexposed skin were placed into individual vials containing solvable (1 mL). The skin samples were placed into a water bath at 65°C for approximately 2 h to aid solubilisation. Once the skin had dissolved, the samples were mixed with stannous chloride in ethanol (0.2 g/mL, 150 μL) and scintillation fluid (10 mL) and analyzed by liquid scintillation fluid. Stannous chloride was added to aid in the reduction in luminescence.ANALYSIS- Method type(s) for identification: Liquid scintillation counting (LSC)All samples, except for tritiated water samples, were counted for 5 min together with representative blanks using a liquid scintillation analyser (Packard 2100-TR) with automatic quench correction by external standard. Prior to analysis, samples were allowed to stabilize with regard to light and temperature.The tritiated water samples were treated as above, except that they were subject to liquid scintillation counting for 1 min only.LIMIT OF RELIABLE MEASUREMENT:- A limit of reliable measurement of 30 dpm above background was instituted in the test laboratory. Counts that were below 30 dpm above background represented a true value. - Data were recorded with values that were less than the limit of reliable measurement where the sample was added directly to scintillation fluid (10 mL) for analysis and not sub-aliquoted prior to analysis.- The instrumental equipment used to quantify radioactivity recorded data to a fraction of dpm and reported this value as a mean rounded value over the counting period (5 min). - For this study, a lowest detection value of 1 dpm has been set. Following values in pg equiv./cm2 were calculated from the detection of 1 and 30 dpm above background:i) Test Preparation 1 (1.5% w/w): 3879.7 pg equiv./cm2 (30 dpm above background); 129.3 pg equiv./cm2 (1 dpm above background)ii) Test Preparation 2 (0.5% w/w): 1313.1 pg equiv./cm2 (30 dpm above background); 43.8 pg equiv./cm2 (1 dpm above background)iii) Test Preparation 3 (0.165% w/w): 1316.1 pg equiv./cm2 (30 dpm above background); 43.9 pg equiv./cm2 (1 dpm above background)
Details on in vitro test system (if applicable):
SKIN PREPARATION- Source of skin: Five samples of full-thickness human skin (1 abdomen and 4 breasts) were obtained from patients aged 21 to 67 years old. Two samples were obtained from NHS Lothian, St. John’s Hospital, Livingston, UK. Three samples were obtained from Nottingham City Tissue Bank. - Type of skin: Split thickness skin membrane - Preparative technique: Electric dermatome (Zimmer®)- Thickness of skin: 540 - 1520 µm (full thickness skin); 390 – 400 µm (split thickness skin)- Membrane integrity check: Yes, by examining penetration characteristic of tritiated water. The percentage absorption of tritiated water was calculated for each skin sample from the 1 hour receptor fluid sample collected. Any human skin sample exhibiting a percentage absorption value greater than 0.6% was excluded from subsequent absorption measurements. - Storage conditions: The skin samples were stored at approximately -20˚C until they were used in the study.- Justification of species: Split thickness skin membrane is an acceptable test system for predicting absorption of a test substance. PRINCIPLES OF ASSAY- Diffusion cell: 12 automated flow-through diffusion cells per test formulation (made of Polytetraflouroethylene (Scott/Dick, University of Newcastle-upon-Tyne, UK))- Receptor fluid: Phosphate buffered saline supplemented with sodium azide (0.01%, w/v) was used as the receptor fluid throughout the study.- Solubility of test substance in receptor fluid: Test substance had solubility of 0.3 mg/mL in PBS with 0.25% ascorbic acid. For an application of 20 mg/cm2 over a 0.64 cm2 application area, for the highest concentration test preparation (1.5%, w/w), the application was 192 μg of C6P/0.64 cm2. If 100% was absorbed in 0.5 h (0.75 mL), then 192 μg/0.75 mL was 0.256 mg/mL of C6P in receptor fluid. Therefore, this receptor fluid was not rate limiting for solubility and was accepted for use.- Stability of test substance in receptor fluid: Not reported- Flow-through system: Yes, sections of split-thickness skin membrane, approximately 1.5 x 1.5 cm, were cut and positioned on the receptor chamber of the diffusion cell, which contained a magnetic stirrer bar. The donor chamber was tightened into place with screws and the prepared cells were then placed in the heated manifold and connected to the peristaltic pump. A Variomag magnetic stirrer was switched on to mix the contents of the receptor chamber. An equilibration period of approximately 15 min was allowed while receptor fluid was pumped through the receptor chambers at 1.5 mL/h ± 0.15 mL/h. The effluent was then collected for approximately 30 min and retained as blank samples for use in the tritiated water barrier integrity assessment.- Volume of receptor chamber: 0.25 mL- Test temperature: 32 ± 1°C- Occlusion: No

Results and discussion

Absorption in different matrices:
1. [14C]-C6P IN TEST PREPARATION 1 (1.5% w/w): - Skin wash (at 30 min post application): The majority of the applied dose (95.39%) was removed by washing at 30 min post application (94.24%, 0.85% and 0.30% was recovered in the skin wash, tissue swab and pipette tip, respectively). - Skin wash (at 72 hours post application): At 72 hours post dose, a further 0.60% of the applied dose was removed (donor chamber wash, 72 h skin wash, 72 h tissue swabs and 72 h pipette tips contained 0.13%, 0.37%, 0.08% and 0.02% of the applied dose, respectively).- Therefore, the total dislodgeable dose was 95.98% (317.16 μg equiv. /cm2) of the applied dose.- The mean total unabsorbed dose was 96.7% (319.51 μg equiv. /cm2) of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.01%) and the radioactivity associated with the stratum corneum (0.71%). The first five tape strips contained 0.47% of the applied dose. There was a steady decrease in the recovery of radioactivity associated with the stratum corneum with tape stripping. The tape strips 6-10, 11-15 and 16-20 contained a further 0.12%, 0.06% and 0.04%, respectively. Those amounts retained by the stratum corneum at 72 h are not considered to be dermally absorbed and thus do not contribute to the systemic dose.- The absorbed dose 1.12% (3.71 μg equiv. /cm2) was the sum of the receptor fluid, 1.07% (3.52 μg equiv. /cm2), receptor rinse (<0.01%) and receptor chamber wash (0.05%).- The epidermis and dermis contained 0.30% (0.99 μg equiv. /cm2) and 0.19% (0.63 μg equiv. /cm2) of the applied dose, respectively.- Dermal delivery: Dermal delivery 1.61% (5.32 μg equiv. /cm2) was the sum of the absorbed dose, epidermis and dermis.2. [14C]-C6P IN TEST PREPARATION 2 (0.5% w/w):- Skin wash (at 30 min post application): The majority of the applied dose (90.77%) was removed by washing at 30 min post application (89.57%, 0.95% and 0.26% was recovered in the skin wash, tissue swab and pipette tip, respectively).- Skin wash (at 72 hours post application): At 72 h post dose, a further 0.85% of the applied dose was removed (donor chamber wash, 72 h skin wash, 72 h tissue swabs and 72 h pipette tips contained 0.15%, 0.58%, 0.10% and 0.02% of the applied dose, respectively).Therefore, the total dislodgeable dose was 91.63% (109.86 μg equiv. /cm2) of the applied dose.- The mean total unabsorbed dose was 92.80% (111.27 μg equiv. /cm2) of the applied dose. This consisted of the dislodgeable dose, unexposed skin (<0.01%) and the radioactivity associated with the stratum corneum (1.17%). The first five tape strips contained 0.76% of the applied dose. There was a steady decrease in the recovery of radioactivity associated with the stratum corneum with tape stripping; tape strips 6-10, 11-15 and 16-20 contained a further 0.21%, 0.11% and 0.09%, respectively. Those amounts retained by the stratum corneum at 72 h are not considered to be dermally absorbed and thus do not contribute to the systemic dose.- The absorbed dose 0.85% (1.02 μg equiv./cm2) was the sum of the receptor fluid, 0.83% (0.99 μg equiv./cm2), receptor rinse (<0.01%) and receptor chamber wash (0.02%).- The epidermis and dermis contained 0.45% (0.54 μg equiv. /cm2) and 0.18% (0.21 μg equiv. /cm2) of the applied dose, respectively.- Dermal delivery: Dermal delivery 1.48% (1.77 μg equiv. /cm2) was the sum of the absorbed dose, epidermis and dermis.3. [14C]-C6P IN TEST PREPARATION 3 (0.165% w/w):- Skin wash (at 30 min post application): The majority of the applied dose (88.76%) was removed by washing at 30 min post application (87.38%, 1.08% and 0.31% was recovered in the skin wash, tissue swab and pipette tip, respectively). - Skin wash (at 72 hours post application): At 72 h post dose, a further 1.22% of the applied dose was removed (donor chamber wash, 72 h skin wash, 72 h tissue swabs and 72 h pipette tips contained 0.24%, 0.79%, 0.16% and 0.03% of the applied dose, respectively).Therefore, the total dislodgeable dose was 89.98% (28.72 μg equiv. /cm2) of the applied dose.- The mean total unabsorbed dose was 91.72% (29.28 μg equiv. /cm2) of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.01%) and the radioactivity associated with the stratum corneum (1.73%).The first five tape strips contained 1.21% of the applied dose. There was a steady decrease in the recovery of radioactivity associated with the stratum corneum with tape stripping; tape strips 6-10, 11-15 and 16-20 contained a further 0.26%, 0.17% and 0.10%, respectively. Those amounts retained by the stratum corneum at 72 h are not considered to be dermally absorbed and thus do not contribute to the systemic dose.- The absorbed dose, 0.81% (0.26 μg equiv. /cm2) was the sum of the receptor fluid, 0.80% (0.26 μg equiv./cm2), receptor rinse (<0.01%) and receptor chamber wash (<0.01%).- The epidermis and dermis contained 0.49% (0.16 μg equiv. /cm2) and 0.21% (0.07 μg equiv. /cm2) of the applied dose, respectively.- Dermal delivery: Dermal delivery 1.51% (0.48 μg equiv. /cm2) was the sum of the absorbed dose, epidermis and dermis.
Total recovery:
- Total recovery: The mean mass balance for [14C]-C6P from all three test preparations (1, 2 and 3) at 72 h post dose was 98.31% (324.83 μg equiv./cm2), 94.27% (113.04 μg equiv./cm2) and 93.23% (29.76 μg equiv./cm2) of the applied dose, respectively.- Recovery of applied dose acceptable: Yes- Results adjusted for incomplete recovery of the applied dose: No
Percutaneous absorptionopen allclose all
Dose:
12.8 mg (at application rate of 20 mg/cm2) of test preparation
Parameter:
percentage
Absorption:
1.12 %
Remarks on result:
other: 72 hours
Remarks:
Total absorption after 30 min exposure from test preparation 1 [1.5% (w/w)]
Dose:
12.8 mg (at application rate of 20 mg/cm2) of test preparation
Parameter:
percentage
Absorption:
0.85 %
Remarks on result:
other: 72 hours
Remarks:
Total absorption after 30 min exposure from test preparation 2 [0.5% (w/w)]
Dose:
12.8 mg (at application rate of 20 mg/cm2) of test preparation
Parameter:
percentage
Absorption:
0.81 %
Remarks on result:
other: 72 hours
Remarks:
Total absorption after 30 min exposure from test preparation 3 [0.165% (w/w)]

Any other information on results incl. tables

 

Table1:                       Summary of the cutaneous absorption of1-HEXYL-1H-PYRAZOLE-4,5-DIAMINE HEMISULFATE

Amount of1-HEXYL-1H-PYRAZOLE-4,5-DIAMINE HEMISULFATEin:

Concentration of

1-HEXYL-1H-PYRAZOLE-4,5-DIAMINE HEMISULFATEin typical hair dye formulation:

1.5 %

0.5 %

0.165 %

Recovery [µg/cm2]

Rinsing solution

(after 30 minutes)

311.41

±

10.39

107.39

±

4.07

27.89

±

0.81

Dislodgeable dose*

(after 30 minutes)

315.19

±

10.16

108.84

±

3.86

28.33

±

0.89

Stratum Corneum

(72 hours)

2.33

±

0.05

1.40

±

0.77

0.55

±

0.23

Epidermis

(72 hours)

0.99

±

0.44

0.54

±

0.18

0.16

±

0.08

Dermis

(72 hours)

0.63

±

0.24

0.21

±

0.10

0.07

±

0.03

Receptor fluid

(72 hours)

3.71

±

1.30

1.02

±

0.54

0.26

±

0.16

Total balance (recovery)**

324.83

±

10.21

113.04

±

4.06

29.76

±

0.69

 

% Applied dose**

Rinsing solution

(after 30 minutes)

94.24

±

3.14

89.57

±

3.39

87.38

±

2.55

Dislodgeable dose*

(after 30 minutes)

95.39

±

3.08

90.77

±

3.22

88.76

±

2.77

 Stratum Corneum

(72 hours)

0.71

±

0.23

1.17

±

0.64

1.73

±

0.65

Epidermis

(72 hours)

0.30

±

0.13

0.45

±

0.15

0.49

±

0.25

Dermis

(72 hours)

0.19

±

0.07

0.18

±

0.08

0.21

±

0.09

Receptor fluid

(72 hours)

1.12

±

0.39

0.85

±

0.45

0.81

±

0.50

Total balance (recovery)***

98.31

±

3.09

94.27

±

3.38

93.23

±

2.17

All values aremean ± SD, n=12

* The dislodgeable dose is the sum of the skin wash, tissue swab and pipette tips

** Corrected for individual applied dose

*** Total is corrected for losses on tips

 

Table 2: Summary of mean results of in vitro percutaneous

absorption of radiolabelled C6P through human skin (study # 77723)

Test Preparation

 

1

 

2

3

 

Target Concentration (%, w/w)

 

1.50

 

0.50

 

0.165

 

Concentration by Radioactivity (%, w/w)

 

1.636

 

0.577

 

0.158

 

Total no. of donors

 

5

 

5

 

5

 

Total no. of replicates dosed

 

12

 

12

 

12

 

30 min Dislodgeable Dose (% Applied Dose)

 

95.39

 

90.77

 

88.76

 

Total Dislodgeable Dose (% Applied Dose)

 

95.98

 

91.63

 

89.98

 

Unabsorbed Dose (% Applied Dose)

 

96.70

 

92.80

 

91.72

 

Epidermis (% Applied Dose)

 

0.30

 

0.45

 

0.49

 

Dermis (% Applied Dose)

 

0.19

 

0.18

 

0.21

 

Absorbed Dose (% Applied Dose)

 

1.12

 

0.85

 

0.81

 

Dermal Delivery (% Applied Dose)

 

1.61

 

1.48

 

1.51

 

Mass Balance (% Applied Dose)

 

98.31

 

94.27

 

93.23

 

30 min Dislodgeable Dose (µg equiv./cm2)

 

315.19

 

108.84

 

28.33

 

Total Dislodgeable Dose (µg equiv./cm2)

 

317.16

 

109.86

 

28.72

 

Unabsorbed Dose (µg equiv./cm2)

 

319.51

 

111.27

 

29.28

 

Epidermis (µg equiv./cm2)

 

0.99

 

0.54

 

0.16

 

Dermis (µg equiv./cm2)

 

0.63

 

0.21

 

0.07

 

Absorbed Dose (µg equiv./cm2)

 

3.71

 

1.02

 

0.26

 

Dermal Delivery (µg equiv./cm2)

 

5.32

 

1.77

 

0.48

 

Mass Balance (µg equiv./cm2)

 

324.83

 

113.04

 

29.76

 

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this in-vitro percutaneous absorption study through human skin, the amount of 4,5-diamino-1-hexyl-1H-pyrazole hemisulfate (C6P) considered to be systemically available from a oxidative hair dye formulations containing three different concentration of C6P were:1.5% w/w C6P: 3.71 µg/cm2 (1.12%) (receptor fluid: 1.07%, receptor rinse: <0.01% and receptor chamber wash: 0.05%) 0.5% w/w C6P: 1.02 µg/cm2 (0.85%) (receptor fluid: 0.83%, receptor rinse: <0.01% and receptor chamber wash: 0.02%)0.165% w/w C6P: 0.26 µg/cm2 (0.81%) (receptor fluid: 0.80%, receptor rinse: <0.01% and receptor chamber wash: <0.01%)
Executive summary:

Percutaneous absorption (in vitro) of [14C] 4,5-diamino-1-hexyl-1H-pyrazole hemisulfate ([14C]-C6P), in 3 different formulations, using human split thickness membranes was determined following the OECD Guideline 428.

Five human skin samples (1 abdomen and 4 breast) were obtained from female patients aged 21 to 67 years old (source: 2 samples were obtained from NHS Lothian, St. John’s Hospital, Livingston, UK and 3 samples were obtained from Nottingham City Tissue Bank). Split-thickness human skin membranes were mounted into flow-through diffusion cells.

Prior to treatment, a tritiated water barrier integrity test was performed and any human skin sample exhibiting absorption greater than 0.6% of the applied dose was excluded from subsequent absorption measurements.

[14C]-C6P was incorporated into a typical oxidative hair dye formulation atapproximately3%, 1% and 0.33% (w/w) containing equimolar concentrations of a reaction partner (3-amino-2, 6-dimethylphenol), before mixing with peroxide developer (1:1, w/w). Therefore, the concentrationof C6P in the final test preparations applied to the skin w:

Test preparation 1: 1.5% w/w [14C]-C6P (concentration by radioactivity was 1.636%)

Test preparation 2: 0.5% w/w [14C]-C6P (concentration by radioactivity was 0.577%)

Test preparation 3: 0.165% w/w [14C]-C6P (concentration by radioactivity was 0.158%)

12.8 mg of each test preparation was applied toskin membrane atapplication rate of 20 mg/cm2. Each test preparation was applied to a total of 12 skin samples obtained from 5 different donors. 30 minutes after application, the skin samples were washed offwater and then dried with tissue paper swabs.

Absorption was assessed by collecting receptor fluid in 30 min fractions from 0 to 1 hour post dose, then hourly fractions from 1 to 6 hours post dose and then in 2-hourly fractions from 6 to 72 hours post dose. At 72 hours post application, the underside of the skin was rinsed with receptor fluid. The skin was then removed from the flow-through cells, dried and the skin divided into exposed and unexposed skin (i.e. the area of skin under the cell flange). The stratum corneum from the exposed skin was removed by tape stripping. The radioactivity in different matrices was quantified by means of a liquid scintillation counter.

The results of in vitro percutaneous absorption of [14C]-C6P through human skinas follows:

Test preparation 1 (1.5% w/w): The majority of the applied dose (95.39%) was removed by washing at 30 min post application. At 72 hours post dose, a further 0.60% of the applied dose was removed. Therefore, the total dislodgeable dose was 95.98% (317.16 μg equiv. /cm2) of the applied dose. The mean total unabsorbed dose was 96.7% (319.51 μg equiv. /cm2) of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.01%) and the radioactivity associated with the stratum corneum (0.71%).

The absorbed dose 1.12% (3.71 μg equiv. /cm2) was the sum of the receptor fluid, 1.07% (3.52 μg equiv. /cm2), receptor rinse (<0.01%) and receptor chamber wash (0.05%).The epidermis and dermis contained 0.30% (0.99 μg equiv. /cm2) and 0.19% (0.63 μg equiv. /cm2) of the applied dose, respectively.


Dermal delivery 1.61% (5.32 μg equiv. /cm2) was the sum of the absorbed dose, epidermis and dermis.

The mean mass balance was 98.31% of the applied dose at 72 h post dose.

Test preparation 2 (0.5% w/w): The majority of the applied dose (90.77%) was removed by washing at 30 min post application. At 72 hours post dose, a further 0.85% of the applied dose was removed. Therefore, the total dislodgeable dose was 91.63% (109.86 μg equiv. /cm2) of the applied dose. The mean total unabsorbed dose was 92.80% (111.27 μg equiv. /cm2) of the applied dose. This consisted of the dislodgeable dose, unexposed skin (<0.01%) and the radioactivity associated with the stratum corneum (1.17%).

The absorbed dose 0.85% (1.02 μg equiv. /cm2) was the sum of the receptor fluid, 0.83% (0.99 μg equiv. /cm2), receptor rinse (<0.01%) and receptor chamber wash (0.02%). The epidermis and dermis contained 0.45% (0.54 μg equiv. /cm2) and 0.18% (0.21 μg equiv. /cm2) of the applied dose, respectively. Dermal delivery 1.48% (1.77 μg equiv. /cm2) was the sum of the absorbed dose, epidermis and dermis.

The mean mass balance was 94.27% of the applied dose at 72 h post dose.

Test preparation 3 (0.165% w/w): The majority of the applied dose (88.76%) was removed by washing at 30 min post application. At 72 hours post dose, a further 1.22% of the applied dose was removed. Therefore, the total dislodgeable dose was 89.98% (28.72 μg equiv. /cm2) of the applied dose. The mean total unabsorbed dose was 91.72% (29.28 μg equiv. /cm2) of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.01%) and the radioactivity associated with the stratum corneum (1.73%).

The absorbed dose, 0.81% (0.26 μg equiv. /cm2) was the sum of the receptor fluid, 0.80% (0.26 μg equiv./cm2), receptor rinse (<0.01%) and receptor chamber wash (<0.01%). The epidermis and dermis contained 0.49% (0.16 μg equiv. /cm2) and 0.21% (0.07 μg equiv. /cm2) of the applied dose, respectively. Dermal delivery 1.51% (0.48 μg equiv. /cm2) was the sum of the absorbed dose, epidermis and dermis.


The mean mass balance was 93.23% of the applied dose at 72 h post dose.

The amounts retained by the stratum corneum at 72 h are not considered to be dermally absorbed and thus do not contribute to the systemic dose.


Under the conditions of this in-vitro percutaneous absorption study through human skin, the amount of 4,5-diamino-1-hexyl-1H-pyrazole hemisulfate (C6P) considered to be systemically available(receptor fluid + receptor rinse + receptor chamber wash) from a oxidative hair dye formulations containing three different concentration of C6P were:

1.5% w/w C6P: 3.71 µg/cm2 (1.12% of the applied dose)

0.5% w/w C6P: 1.02 µg/cm2 (0.85% of the applied dose)

0.165% w/w C6P: 0.26 µg/cm2 (0.81% of the applied dose)

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