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EC number: 201-891-0 | CAS number: 89-25-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Dermal absorption
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:
- 2 005
- 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
- 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-2,4-dihydro-3H-pyrazol-3-one
- Test material form:
- solid: particulate/powder
Constituent 1
- 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 -20C 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 -20C 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.
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