4-amino-3-nitrophenol

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2004-11-15 to 2005-01-25

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

Test material information

Constituent 1

Reference substance name:

4-amino-3-nitrophenol

EC Number:

210-236-8

EC Name:

4-amino-3-nitrophenol

Cas Number:

610-81-1

Molecular formula:

C6H6N2O3

IUPAC Name:

4-amino-3-nitrophenol

Test material form:

solid: particulate/powder

Remarks:

Dark red powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch No. 0508916
- Expiration date of the lot/batch: September 2005
- Purity test date: 31 August 2004

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.3% (by high performance liquid chromatography, 18 May 2004)
- Specific activity: 2.14 CBq/mmol, 58 mCi/mmol
- Locations of the label: 4-Amino-3-Nitro[ring-U-14C]phenol
- Expiration date of radiochemical substance: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored under argon gas at ca +4 °C in the dark.
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: Water: <1 g/litre (25°C); Ethanol: 0.1 g + 10 mL = soluble (25°C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The following formulations were used :
Formulation 1: Formulation containing 4-Amino-3-Nitrophenol at 2.73%, (w/w) to which [14C]-4-Amino-3-Nitrophenol was added. This was labelled “B051 Colox 2.73g%, batch no. 1036018.
Formulation 2: Placebo formulation. This was labelled “Placebo Colox”, batch no. 1036019.
Formulation 3: Developer (hydrogen peroxide, ca 6%). This was labelled “Developer”, batch no. BA 124.
Formulation 4: Formulation containing 4-Amino-3-Nitrophenol at 0.87% (w/w) to which [14C]-4-Amino-3-Nitrophenol was added. This was labelled “B051
0.87 g%”, batch no. 1036500.
Formulation 5: Placebo formulation. This was labelled “Placebo1036501”, batch no. 1036501
Formulation 6: Solvent, batch no. 16/11/2004

Preparation of Oxidative Formulation
All formulation 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]-4-Amino-3-Nitrophenol were removed from ca -20°C storage and allowed to reach ambient temperature. Formulation 1 (500.54 mg and 500.51 mg) was then added to each vial 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, six weighed 15 μL aliquots were taken 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 4-Amino-3-Nitrophenol in the formulation was calculated to be 3.02% (w/w). The concentration was 100.58% of the target concentration of 3.00% (w/w). The formulation was homogeneous with a CV of 2.26%.

Preparation of Semi-Permanent Formulation
All formulation procedures stated below, 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 of [14C]-4-Amino-3-Nitrophenol were removed from ca -20°C storage and allowed to reach ambient temperature. Solvent (17.96 mg and 19.24 mg) was added and mixed by vortexing followed by Formulation 4 (883.32 mg and 881.76 mg, respectively) 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 vortex mixing.
To determine the radioactive homogeneity and concentration, six weighed 15 μL aliquots were taken 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 4-Amino-3-Nitrophenol in the formulation was calculated to be 1.06% (w/w). The concentration was 105.96% of the target concentration of 1.00% (w/w). The formulation was homogeneous with a CV of 2.94%.

Radiolabelling:

yes

Test animals

Species:

other: Human Skin Samples

Strain:

other: Full-thickness human skin samples (4 breast, 3 abdomen) were obtained from patients (aged 27 to 58 years old)

Sex:

female

Administration / exposure

Type of coverage:

not specified

Vehicle:

other: used in formulations (see above)

Duration of exposure:

30 minutes

Doses:

Target 4-Amino-3-Nitrophenol Concentration in Test Preparation (% w/w): 1.5 in oxidative condition and 1.0% in Semi-Permanent condition.

No. of animals per group:

10 skin samples in oxydative conditions and 12 skin samples in Semi-Permanent 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 27 to 58 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°C. 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, 96.15% of the applied dose was removed during the washing process (93.61% in the 0.5 h skin wash, 2.24 % in the 0.5 h tissue swab and 0.30% in the pipette tips).
At 24 h post dose, ie after a 23.5 h monitoring period, 0.20% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 0.54% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 96.89% of the applied dose. The total unabsorbed dose was 98.70% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (1.60%) and unexposed skin (0.21%). 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 (0.59%) was made up from the receptor fluid (0.58%) and the receptor rinse (0.01%). Dermal delivery (0.92%) was made up from the absorbed dose and exposed skin (0.32%).
The total recovery, dislodgeable dose, unabsorbed dose, absorbed dose and dermal delivery were 327.61, 318.57, 324.61, 1.95 and 3.00 μg equiv./cm2,
respectively.
The lag time and steady state flux could not be accurately determined since the test preparation was removed from the skin surface at 30 min post dose.
The data shows that [14C]-4-Amino-3-Nitrophenol in oxidative test preparation was effectively removed from the skin surface by the washing procedure employed.

Absorption Study – Semi-Permanent Test Preparation
At the end of the 0.5 h exposure period, 95.41% of the applied dose was removed during the washing procedure (92.84% in the 0.5 h skin wash, 2.45% in the 0.5 h tissue swab and 0.12% in the pipette tips).
At 24 h post dose, ie after a 23.5 h monitoring period, 0.06% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 1.00% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 96.47% of the applied dose. The total unabsorbed dose was 97.81% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (1.21%) and unexposed skin (0.14%). The absorbed dose (0.23%) was made up from the receptor fluid (0.23%) and the receptor rinse (<0.01%). Dermal delivery (0.28%) was made up from the absorbed dose and exposed skin (0.05%).
The total recovery, dislodgeable dose, unabsorbed dose, absorbed dose and dermal delivery were 206.36, 202.95, 205.77, 0.49 and 0.59 μg equiv./cm2, respectively.
The lag time and steady state flux could not be accurately determined since the test preparation was removed from the skin surface at 30 min post dose.
The data shows that [14C]-4-Amino-3-Nitrophenol in semi-permanent test preparation was effectively removed from the skin surface by the washing procedure employed.

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

24 h

Dose:

1.5 %

Parameter:

amount

Absorption:

3 other: µg/cm2

Remarks on result:

other: corresponding to 0.92% For Oxydation Experiment

Percutaneous absorption 2

Key result

Time point:

24 h

Dose:

1.00%

Parameter:

amount

Absorption:

0.59 other: µg/cm2

Remarks on result:

other: corresponding to 0.28% For Semi-Permanent Experiment

Any other information on results incl. tables

 Summary of the mean results of the study:

 

Formulation / Test Preparation	Oxidative	Semi-permanent
Target 4-Amino-3-Nitrophenol Concentration in Formulation (%, w/w)	3.00	1.00
Actual 4-Amino-3-Nitrophenol Concentration in Formulation (%, w/w)	3.02	1.06
Target 4-Amino-3-Nitrophenol Concentration in Test Preparation (% w/w)	1.50	1.00
Actual 4-Amino-3-Nitrophenol Concentration in Test Preparation (% w/w)	1.61	1.06
Target Application Rate of Test Preparation (mg/cm2)	20.00	20.00
Actual Application Rate of Test Preparation (mg/cm2)	20.42	19.91
4-Amino-3-Nitrophenol (% Applied Dose)	(Mean ± SD)
DislodgeableDose	96.89 ± 2.97	96.47 ± 2.92
Unabsorbed Dose *	98.70 ± 2.44	97.81 ± 2.42
Absorbed Dose **	0.59 ± 0.47	0.23 ± 0.21
Dermal Delivery ***	0.92 ± 0.54	0.28 ± 0.27
Mass Balance	99.61 ± 2.03	98.09 ± 2.34
4-Amino-3-Nitrophenol (μgequiv/cm2)	(Mean ± SD)
DislodgeableDose	318.57 ± 11.54	202.95 ± 6.14
Unabsorbed Dose *	324.61 ± 12.77	205.77 ± 5.09
Absorbed Dose **	1.95 ± 1.50	0.49 ± 0.44
Dermal Delivery ***	3.00 ± 1.75	0.59 ± 0.58
Mass Balance	327.61 ± 11.59	206.36 ± 4.93

 

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

** Absorbed dose = receptor fluid + receptor rinse

*** Dermal Delivery = exposed skin + absorbed dose

 

Applicant's summary and conclusion

Conclusions:

Under the present experimental conditions, for [14C]-4-Amino-3-Nitrophenol in the oxidative test preparation, most of the applied dose was removed at 30 min post dose (96.15% of the applied dose). At 24 h post dose, a further 0.74% was removed. Therefore, the dislodgeable dose was 96.89% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 0.59% (1.95 μg equiv/cm2) and 0.92% (3.00 μg equiv/cm2) of the applied dose, respectively. Under the present experimental conditions, for [14C]-4-Amino-3-Nitrophenol in the semi-permanent test preparation, most of the applied dose was removed at 30 min post dose (95.41% of the applied dose). At 24 h post dose, a further 1.06% was removed. Therefore, the dislodgeable dose was 96.47% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 0.23% (0.49 μg equiv/cm2) and 0.28% (0.59 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 3.00 μg equiv./cm2 for the oxidative test preparation and 0.59 μg equiv./cm2 for the semi-permanent 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 test item 4-Amino-3-Nitrophenol in oxydative and semi-permanent formulations on human skin samples.

4-Amino-3-Nitrophenol was incorporated into a typical oxidative hair dye formulation at ca 3% (w/w) before mixing with developer (1:1, w/w), to give a final concentration of 4-Amino-3-Nitrophenol of ca 1.5% (w/w) (oxidative hair dye test preparation) . 4-Amino-3-Nitrophenol was incorporated into a typical semi-permanent hair dye formulation at ca 1% (w/w) (semi-permanent hair dye test preparation).

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. [14C]-4-Amino-3-Nitrophenol was applied in two test preparations (oxidative and semi-permanent) to human split-thickness skin membranes mounted in flow-through diffusion cells in vitro. Both oxidative and semi-permanent hair dye test preparations were applied at a target application rate for the formulation of ca 20 mg/cm2.

Absorption was assessed by collecting receptor fluid (PBS) samples hourly from 0-24 h post dose (flow rate 1.5 mL/h). At 30 min post dose, the skin was washed with water, sodium dodecyl sulphate (SDS) solution (2% w/v) and water again. The skin was then dried with tissue paper swabs. At 24 h post dose the underside of the skin was rinsed with receptor fluid. The skin was then removed from the flow-through cells, dried and the stratum corneum removed by tape stripping. The remaining skin was divided into exposed and unexposed skin. All liquid samples were analysed by liquid scintillation counting and the remaining samples were analysed by combustion/ liquid scintillation counting.

In conclusion, [14C]-4-Amino-3-Nitrophenol in oxidative and semi-permanent test preparations was applied topically to human skin in vitro. Under the present experimental conditions, for [14C]-4-Amino-3-Nitrophenol in the oxidative test preparation, most of the applied dose was removed at 30 min post dose (96.15% of the applied dose). At 24 h post dose, a further 0.74% was removed. Therefore, the dislodgeable dose was 96.89% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 0.59% (1.95 μg equiv/cm2) and 0.92% (3.00 μg equiv/cm2) of the applied dose, respectively. Under the present experimental conditions, for [14C]-4-Amino-3-Nitrophenol in the semi-permanent test preparation, most of the applied dose was removed at 30 min post dose (95.41% of the applied dose). At 24 h post dose, a further 1.06% was removed. Therefore, the dislodgeable dose was 96.47% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 0.23% (0.49 μg equiv/cm2) and 0.28% (0.59 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 3.00 μg equiv./cm2 for the oxidative test preparation and 0.59 μg equiv./cm2 for the semi-permanent test preparation.