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Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 8 October 2004 to 13 January 2005
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
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
Adopted the 13 April 2004
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1-phenyl-5-pyrazolone
EC Number:
201-891-0
EC Name:
3-methyl-1-phenyl-5-pyrazolone
Cas Number:
89-25-8
Molecular formula:
C10H10N2O
IUPAC Name:
5-methyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-one
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch No. 62
- Expiration date of the lot/batch: September 2005
- Purity test date: 31 August 2004

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98.6% (by high performance liquid chromatography, 4 May 2004)
- Specific activity: 1.96 CBq/mmol, 53 mCi/mmol
- Locations of the label: 1-[Benzene ring-U-14C]Phenyl-3-methylpyrazolone
- Expiration date of radiochemical substance: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: was stored at 4°C in the dark under nitrogen gas.
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: 0.2% (w/v) in water, 0.1g/mL in Ethanol and 5g/L in chloroform

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Four formulations were used :
Formulation 1: Formulation containing Phenylmethyl Pyrazolone at 0.17%, (w/w) and PPD of ca 0.31% (w/w) to which [14C]-Phenylmethyl Pyrazolone was added. This was labelled “A039 0.17 g% couplage”, batch no. 1016193.

Formulation 2: Formulation containing Phenylmethyl Pyrazolone at 0.17%, (w/w) to which [14C]-Phenylmethyl Pyrazolone was added. This was labelled “A039 0.17 g%”, batch no. 1016194.

Formulation 3: Placebo formulation. This was labelled “Placebo”, batch no. 1016195.

Formulation 4: Developer (hydrogen peroxide, ca 6%). This was labelled “Developer”, batch no.BA124


Preparation of Oxidative Formulation
All preparation procedures stated below, up until the addition of the Developer, were carried out in a nitrogen atmosphere.
Three dose vials were required to be prepared due to dosing constraints and the nature of the formulation. Each vial was used for separate dosing occasions at 1 h intervals.
Two vials containing [14C]-Phenylmethyl Pyrazolone were removed from ca -20C storage and allowed to reach ambient temperature. Formulation 1 was then added to each vial (499.11 mg and 500.18 mg) and the formulation was mixed by sonication and vortexing.
The contents of the vials were transferred to a single dose vial and mixed by sonication and vortexing. To determine the radioactive homogeneity and concentration, three weighed 20 µL aliquots were taken, transferred into 5 mL volumetric flasks and made up to the 5 mL volume with ethanol and mixed. Duplicate 125 µL aliquots were removed from each volumetric flask and scintillation fluid (ca 10 mL) added to each for analysis by liquid scintillation counting.
By radioactivity, the concentration of Phenylmethyl Pyrazolone in the formulation was calculated to be 0.52% (w/w). The concentration was 103.34% of the target concentration of 0.50% (w/w). The formulation was homogeneous with a coefficient of variance (CV) of 1.13%.

Preparation of Non Oxidative Formulation

All preparation procedures stated below, up until the addition of the degassed water, were carried out in a nitrogen atmosphere.
Two vials of [14C]-Phenylmethyl Pyrazolone were removed from ca -20C storage and allowed to reach ambient temperature. Formulation 2 (503.39 mg and 498.40 mg, respectively) was added to the vials. The dose was mixed by sonication and vortexing.
The contents of the vials were transferred to a single dose vial and mixed by sonication and vortexing. To determine the radioactive homogeneity and concentration, five weighed 20 µL aliquots were taken, transferred into 10 mL volumetric flasks and made
up to the 10 mL volume with ethanol and mixed. Duplicate 250 µL aliquots were removed from each volumetric flask and scintillation fluid (ca 10 mL) added to each for analysis by liquid scintillation counting.
By radioactivity, the concentration of Phenylmethyl Pyrazolone in the formulation was calculated to be 0.52% (w/w). The concentration was 104.73% of the target concentration of 0.50% (w/w). The formulation was homogeneous with a CV of 0.59%.
Radiolabelling:
yes

Test animals

Species:
other: Human Skin Samples
Strain:
other: Full-thickness human skin samples (6 breast, 1 abdomen) were obtained from patients (aged 19 to 56 years old)
Sex:
female

Administration / exposure

Type of coverage:
not specified
Vehicle:
other: used in formulations (see above)
Duration of exposure:
30 minutes
Doses:
0.25% test item
No. of animals per group:
12 skin samples in oxydative conditions and 8 skin samples in non oxydative conditions
Control animals:
no
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source: Plastic Surgery Unit, St Johns Hospital, West Lothian NHS Trust, Livingston, UK
- Age of skin donors: aged 19 to 56 years old
- Storage conditions: plastic bags and stored at ca -20°C until required.
- Ethical approval if human skin: Patents were informed consent for their skin to be taken for scientific research purposes
- Type of skin: Breast and abdomen

- Preparative technique: The skin was washed in cold running water and dried using “blue roll” tissue paper. The skin was then cut into smaller pieces (where appropriate), wrapped in aluminium foil, put into self sealing plastic bags and stored at ca -20°C until required. The age and sex of the donor and site from which the skin was taken were recorded.
When required, skin samples were removed from storage and allowed to thaw at ambient temperature. The thickness of the uncut skin membranes was measured using a micrometer (pocket thickness gauge, Mitutoyo Corp, Kanagawa, Japan). Split-thickness membranes were prepared by pinning the full-thickness skin, stratum corneum uppermost, onto a raised cork board and cutting at a setting equivalent to 200-500 µm depth using a Zimmer electric dermatome. The membranes were then laid out onto aluminium foil and the thickness of the membranes measured using a micrometer.

- Thickness of skin (in mm): 200-500 µm

- Membrane integrity check: The integrity of the skin was checked by determination of the permeability coefficient for tritiated water which was < 2.5 x 10-3 cm/h for all selected membranes.

PRINCIPLES OF ASSAY

- Flow-through system:
An automated flow-through diffusion cell apparatus (Scott/Dick, University of Newcastle-upon-Tyne, UK) was used . The flow-through cells were placed in a steel manifold heated via a circulating water bath to maintain the skin surface temperature at ca 32 degree Celsius. The cells were connected to multi-channel peristaltic pumps from their afferent ports, with the receptor fluid effluent dropping via fine bore tubing into scintillation vials on a fraction collector.
The surface area of exposed skin within the cells was 0.64 cm2. The receptor chamber volume was 0.25 mL. The peristaltic pumps were adjusted to maintain a flow-rate of ca 1.5 mL/h

- Test temperature: 32°C
- Humidity: not specified
- Occlusion: not specified
- Reference substance(s): not specified

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
Absorption - Study Oxydative Test :

At the end of the 0.5 h exposure period, 90.01% of the applied dose was removed during the washing process (79.54% in the 0.5 h skin wash, 10.14 % in the 0.5 h tissue swab and 0.33% in the pipette tips).

At 24 h post dose, ie after a 23.5 h monitoring period, 0.08% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 1.79% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 91.88% of the applied dose. The total unabsorbed dose was 94.54% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (2.57%) and unexposed skin (0.09%). Those amounts retained by the stratum corneum and unexposed skin at 24 h are not considered to be dermally absorbed and thus do not contribute to the systemic dose. The absorbed dose (0.13%) was made up from the receptor fluid (0.13%) and the receptor rinse (<0.01%). Dermal delivery (0.56%) was made up from the absorbed dose and exposed skin (0.43%).

Absorption Study – Non Oxidative Test Preparation

At the end of the 0.5 h exposure period, 92.05% of the applied dose was removed during the washing procedure (82.56% in the 0.5 h skin wash, 9.02% in the 0.5 h tissue swab and 0.47% in the pipette tips).

At 24 h post dose, ie after a 23.5 h monitoring period, 0.05% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 1.23% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 93.33% of the applied dose. The total unabsorbed dose was 95.87% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (2.48%) and unexposed skin (0.05%). The absorbed dose (1.21%) was made up from the receptor fluid (1.20%) and the receptor rinse (0.01%). Dermal delivery (2.92%) was made up from the absorbed dose and exposed skin (1.72%).

Percutaneous absorptionopen allclose all
Key result
Time point:
24 h
Dose:
0.25%
Parameter:
amount
Absorption:
0.31 other: µg/cm²
Remarks on result:
other: corresponding to 0.56%
Remarks:
For Oxydation Experiment
Key result
Time point:
24 h
Dose:
0.25%
Parameter:
amount
Absorption:
1.6 other: µg/cm²
Remarks on result:
other: corresponding to 2.92%
Remarks:
For Non-Oxydation Experiment

Any other information on results incl. tables

Table1 :Summary of the main results of the study

Formulation / Test Preparation

Oxidative

Non Oxidative

Target Phenylmethyl Pyrazolone Concentration in Formulation (%, w/w)

0.50

0.50

Actual Phenylmethyl Pyrazolone Concentration in Formulation (%, w/w)

0.52

0.52

Target Phenylmethyl Pyrazolone Concentration in Test Preparation (% w/w)

0.25

0.25

Actual Phenylmethyl Pyrazolone Concentration in Test Preparation (% w/w) Target application rate of Test Preparation (mg/cm2)

Actual application rate of Test Preparation (mg/cm2)

0.27

20.0

20.2

0.27

20.0

20.2

Phenylmethyl Pyrazolone(% Applied Dose)

(Mean ± SD)

DislodgeableDose

91.88 ± 3.20

93.33 ± 2.30

Unabsorbed Dose *

94.54 ± 2.89

95.87 ± 1.57

Absorbed Dose **

0.13 ± 0.05

1.21 ± 0.22

Dermal Delivery ***

0.56 ± 0.44

2.92 ± 0.53

Mass Balance

95.09 ± 2.73

98.79 ± 1.73

Phenylmethyl Pyrazolone (µgequiv/cm2)

(Mean ± SD)

DislodgeableDose

50.85 ± 1.85

51.05 ± 1.20

Unabsorbed Dose *

52.32 ± 1.65

52.44 ± 0.87

Absorbed Dose **

0.07 ± 0.03

0.66 ± 0.12

Dermal Delivery ***

0.31 ± 0.24

1.60 ± 0.30

Mass Balance

52.63 ± 1.54

54.04 ± 1.01

 

*       Unabsorbed dose = dislodgeable dose + stratum corneum + unexposed skin

**       Absorbed dose = receptor fluid + receptor rinse

***       Dermal Delivery = exposed skin (except stratum corneum) + absorbed dose

Applicant's summary and conclusion

Conclusions:
Under the present experimental conditions, for [14C]-Phenylmethyl Pyrazolone in the non oxidative test preparation, most of the applied dose was removed at 30 min post dose (92.05% of the applied dose). At 24 h post dose, a further 1.28% was removed, therefore, the dislodgeable dose was 93.33% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 1.21% (0.66 µg equiv/cm2) and 2.92% (1.60 µg equiv/cm2) of the applied dose, respectively. The dermal absorption figure to be taken into consideration for the calculation of the margin of safety is 0.31 µg equiv./cm2 for the oxidative test preparation and 1.60 µg equiv./cm2 for the non oxidative test preparation.
Executive summary:

This GLP-compliant study was perform in accordance with OECD Guideline 428 method for Skin Absorption : In Vitro Method. The study was assessed to evaluate the Skin Absorption of the registered item 1-phenyl-3-methyl-5-pyrazolone in oxydative and non oxydative formulations on human skin samples.

Human breast and abdominal skin samples were obtained from seven different female donors subjected to plastic surgery. The skin was transferred stored on ice and kept frozen at –20°C until use. Skin samples were dermatomed (390-400 μm in thickness) and mounted in flow-through diffusion cells, using bovine serum albumin (5%, w/v) in calcium and magnesium free phosphate-buffered saline as the receptor fluid. Twenty-four diffusion cells were used in two separate experiments, and skin was maintained at approximately 32°C.

In a first experiment (oxidative conditions), a typical oxidative hair dye formulation containing 0.5% phenyl methyl pyrazolone associated with the primary intermediate para-phenylenediamine (PPD, 0.3%) was mixed with the developer (1:1, w/w) to yield a final target concentration of 0.25 % phenyl methyl pyrazolone. About twenty (20) mg/cm² of this mixture (corresponding to exactly 50.5 μg/cm² of phenyl methyl pyrazolone) was applied to the skin surface and left for 30 minutes. After this time period, the remaining formulation on the skin surface was removed using a standardized washing procedure, simulating use conditions. Twenty-four (24) hours after application, the percutaneous absorption of [14C]- phenyl methyl pyrazolone was estimated by measuring its concentration by liquid scintillation counting (following combustion for non-liquid samples) in the following compartments/samples: skin washes (dislodgeable dose), stratum corneum (isolated by tape strippings), living epidermis/dermis, unexposed skin and receptor fluid.

In a second experiment, a similar experimental procedure was applied to evaluate the percutaneous absorption of phenyl methyl pyrazolone in non-oxidative conditions, using a formulation without primary intermediate and mixed with water (1:1, w/w) to yield a final concentration of 0.25% phenyl methyl pyrazolone (about 20 mg/cm2 were applied, corresponding exactly to 50.5 μg/cm2).

Most of the phenyl methyl pyrazolone applied on the skin surface was removed with the skin washes (about 92% and 93% of the applied dose in oxidative and non-oxidative conditions, respectively), and the total recovery rate was about 95% and 96% in oxidative and non-oxidative conditions, respectively. The mean amounts of phenyl methyl pyrazolone considered as absorbed (dermal delivery) were estimated as follows (sum of amounts measured in living epidermis/dermis and receptor fluid): 0.31 ± 0.24 μg equiv/cm2 (0.56 ± 0.44% of the applied dose) and 1.60 ± 0.30 μg equiv/cm2 (2.92 ± 0.53% of the applied dose) in oxidative and non-oxidative conditions, respectively.