2-methyl-p-phenylenediamine sulfate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug. 27, 2002 to Sept. 25, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes

Remarks:

according to OECD principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-p-phenylenediamine sulfate

EC Number:

210-431-8

EC Name:

2-methyl-p-phenylenediamine sulfate

Cas Number:

615-50-9

Molecular formula:

C7H10N2.H2O4S

IUPAC Name:

2-methylbenzene-1,4-diamine; sulfuric acid

Details on test material:

UNLABELED TEST MATERIAL
- Name of test material: pTD-sulfate (Code # SAT 010935)
- Substance type: Pure active substance
- Physical state: Beige powder
- Stability under test conditions: Solutions should be prepared only shortly before application
- Storage condition of test material: Ambient temperature, in dark
- Solubility in water: Good

LABELLED TEST MATERIAL
- Name of test material: 14C- Labeled pTD-sulfate (Code # SAT 020522)
- Substance type: Pure active substance
- Physical state: Beige powder
- Stability: Dec. 2003
- Storage condition of test material: In the freezer at approx. -20°C

Radiolabelling:

yes

Administration / exposure

Duration of exposure:

30 min

Doses:

- Nominal doses: 20 mg formulation containing 1.08 mg of the test substance/cm2; 14 µCi/cm2 or approx. 0.05 MBq/cm2.
- Actual doses: In this study two independent experiments were set up.
Experiment 1= 21.2 ± 2.6 mg of formulation (containing 1.15 mg ± 0.14 mg of test substance)
Experiment 2 = 22.6 ± 1.6 mg of formulation (containing 1.26 mg ± 0.09 mg of test substance)
Experiment 1+2= 21.9 mg of formulation (containing 1.20 mg ± 0.13 mg of test substance)
- Actual doses calculated as follows: Quantification of the actually applied mass was done by weighing. Samples of the formulations were taken during the application procedure. The specific radioactivity was determined and was used to calculate the actually applied dose of the test substance. Samples were dissolved in isopropanol.
- Dose volume: 20 mg of the formulation containing the test substance
- Rationale for dose selection: The dose was suggested by the sponsor.

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: 3 parts of preparation (50 mg of non-radiolabelled test substance, 4 mg of 14C-test substance, 5 mg sodium sulfite, 77.5 mg of 25 % ammonia, 34 mg of NaOH, 200 mg of deionized water, 27 mg of 1,3-Dihydroxybenzene, 102.5 mg of deionized water, 250 mg of Creme-Basis Bth 66) was mixed with 1 part of 250 mg of water for formulation. The final blending of the test substance/vehicle formulation with water was performed a few minutes before the first and not longer than 30 min before the last test substance application of each experiment.
- Method of storage: Not reported

APPLICATION OF DOSE: The test substance preparation was applied once topically to the horny layer of the skin in a nominal quantity of 20 mg, which corresponded to 1.08 mg of the test substance. The test substance solution was spread evenly on the skin with a pipette tip. REPLICATES: The test formulation was tested in 2 independent experiments with 6 replicates per experiment.

TEST SITE
- Preparation of test site: For details refer to 'skin preparation’ under ‘details on in vitro test system’ section below
- Area of exposure: 1.0 cm2

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: The test substance/vehicle formulation was removed by 5 rinsings with approx. 1.5 mL of a 0.01 % Tween 20 solution and 5 rinsings with approx. 1.5 mL deionized water. The skin was dabbed dry with a cellulose pad.
- Time after start of exposure: 30 min

SAMPLE PREPARATION AND SAMPLING PROCEDURE:
-Rinsings of skin: Rinsings from the test substance/vehicle formulation removal were collected and joined with the extract of the pad used for dabbing the skin dry after test substance removal. The pad was extracted with isopropanol.
- Receptor fluid: Samples of the receptor fluid were drawn through a side arm of the receptor chamber before the application of the test substance preparation and 0.5, 1, 2, 4, 6, 24, 29 and 48 h post application. The removed volume was replaced with fresh receptor fluid.
- Rinsing of donor and receptor chamber: 48 h post administration, the skin was rinsed again as described above under removal of test substance. The skin was again dabbed dry with a cellulose pad and the pad was extracted with isopropanol. Rinsings were collected and joint with the extract of the pad. The donor chamber was filled with PBS and the second skin integrity check was performed. The PBS was removed and the skin was dabbed dry again with a cellulose pad. Then the skin was removed from the penetration cell. After removal of the skin the donor and also the receptor chamber were rinsed respectively extracted with isopropanol. The rinsings of the donor chamber were added to the rinsings of the skin. The amount of 14C in the rinsings of the receptor chamber was added by calculation to the amount of 14C in the receptor fluid 48 h post administration.
- Skin: The stratum corneum was torn off by adhesive taps (3M) in order to determine the absorbed parts of the test substance, which would cast off with the stratum corneum in an in vivo situation and were considered to be not systemically available. Fifteen tapes were used. The first 10 tapes were dissolved individually in Soluene-350. Tapes 11 to 15 were dissolved together. The remaining deeper layers of the skin (the dermis plus most parts of the epidermis) contained the absorbed test substance. This remaining skin was dissolved in Soluene-350 also.

ANALYSIS
- Method type(s) for identification: Aliquots of receptor fluids, rinsings of dissolved test substance formulations, skin and tapes were incorporated into a liquid scintillator Ultimagold MV. 14C was determined in a liquid scintillation counter (Packard Tri-Carb 2200CA) with automatic external standardization and facilities to subtract background counting rates and to compute quench corrected decay rates.
- Liquid scintillation counting results (cpm) converted to dpm as follows: Decays per min (dpm) were calculated in the liquid scintillation counter from the counts per min (cpm):
Decays of 14C per min (dpm) = [cpm sample – cpm background] / Eff
Where,
cpm = counts per min
Eff = Efficiency of the counting.- Limits of detection and quantification: The detection limits were calculated in the following manner:
Detection limit in decays per min (DL) = (0.3 x cpm background ) / MEff
Where,
MEff = mean efficiency of the counting, i.e. typically 0.80
As the detection limits based on cpm’s were not easily accessible, they were transformed to ‘% of the applied dose’

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: The skin was obtained from one male (25 kg and 10 wks; Experiment 1) and one female (20 kg and 9 wks; Experiment 2) pig supplied by a local farmer.
- Type of skin: Dermatomed intact, non-scalded skin
- Preparative technique: The skin was cleaned with water. Subcutaneous fat was removed to a large extent. Hair was clipped by an electric hair clipper. The thickness of the skin was reduced by a Zimmer Air Dermatome set to 0.75 mm. Only skin without visible lesions was used.
- Thickness of skin : The thickness of the skin samples ranged from 0.56 to 0.75 mm. The actual thickness of the skin was determined with calipers (Oditest) immediately before insertion into the penetration cells.
- Membrane integrity check: The skin was checked by determination of the transdermal electrical resistance. The resistance (impedance) was measured with alternating current in situ. Gold plated electrodes in PBS-buffer above and below the skin were used. Only skin samples with a resistance in the cell of at least 10 kΩ were selected. The first check was performed after an accommodation phase of ca. half an hour, before the test substance application. A second check was performed at 48 h post application.
- Storage conditions: The obtained skin samples were deep frozen at approx. -20°C until use within 2 months. The skin samples were allowed to thaw at room temperature before application of the test substance.
- Justification of species, anatomical site and preparative technique: The pig skin was selected because it shares essential penetration characteristics with human skin.

PRINCIPLES OF ASSAY
- Diffusion cell: Static Franz Diffusion Cells from PermeGear, USA type 4G-01-00-11 were used.
- Receptor fluid: The receptor fluid was Dulbecco's phosphate buffered saline, autoclaved and degassed by ultrasonication (Sigma D-5652, pH 7.35).
- Static system: The skin was clamped horizontally between the upper donor chamber and the lower receptor chamber with the horny layer facing the donor compartment, which was not occluded. The accessible area for application to the clamped skin was 1.0 cm2. The cell was made of glass. The volume of the receptor chamber was approx. 8 mL and was determined for each cell. Care was taken not to insert air bubbles into the receptor compartment. The cell was checked for air bubbles at each sampling time. The receptor fluid in the cell was stirred with a rotating magnetic bar. Removed volume of receptor fluid for sampling was replaced with fresh receptor fluid.
- Test temperature: 32.0 – 32.3°C in each experiment. The Franz cell had a water jacket and the temperature of it was controlled by a thermostat. The actual temperature in the thermostat was measured before the application of the test substance and at each sampling time. The difference between the temperature in the thermostat and the individual receptor chambers of the cells was 0.1 and 0.3°C.
- Occlusion: Non-occlusive exposure

Results and discussion

Absorption in different matrices:

- Rinsing solutions: 96.4% of dose (sum of rinsings of the skin after 30 min (95.8%) and 48 h (0.64%)). The majority of the applied test substance formulation was removed from the skin by rinsing 30 min after the application.
- Receptor fluid (Percutaneous penetration): The mean value for the % of dose collected in the receptor fluid was 0.89% of dose (10.7 µg tse/cm2). The cumulative penetration approached a plateau at the later sampling times, after approx. 1 d.
- Skin preparation (Absorption): The mean absorption in the dermis and residual epidermis was 0.83% of dose (10.1 µg tse/cm2). Low amounts of radioactivity were absorbed 48 h post administration in the dermis and the residual epidermis, including remaining hair stubs and shafts.
- Stratum corneum (Adsorption): (i.e tape strips): The mean adsorption of test substance to stratum corneum was 0.28% of dose (3.2 µg tse/cm2) at 48 h post administration. The adsorption of the test substance to the stratum corneum was determined by stripping it with adhesive tapes. Most of the radioactivity was detected in the first tape, and then a gradual decrease was noted.
The absorption was higher than the adsorption. It should be mentioned that the stratum corneum of the dry flange-region was more resistant against removal by tape stripping technique. Therefore, any adsorbed test substance in this flange region was taken as within the residual skin and absorbed. Due to this, overestimations probably occurred.

Total recovery:

- Total recovery: The mean recovery was 98.4% of dose.
- Recovery of applied dose acceptable: Yes, the mean balance was within the limits of 100 ± 15% as set in the study plan.
- Results adjusted for incomplete recovery of the applied dose: No
- Limit of detection (LOD): Each of the radioactivities of the various samples, except for samples of the receptor fluid before the test substance administration, were above the detection limit.The approx. detection limits were:
Receptor fluid: From 0.0005 to 0.001% of dose
Remaining skin: 0.0002 to 0.0005% of dose
Tapes: 0.0001 to 0.0002% of dose
Rinsings: 0.0004 to 0.01% of dose
- Quantification of values below LOD or LOQ: Not reported

Percutaneous absorption

Key result

Dose:

21.9 mg/cm2 of formulation containing 1.20 mg/cm2 test substance

Parameter:

percentage

Absorption:

1.72 %

Remarks on result:

other: 48 h

Remarks:

The above value of absorption corresponds to 20.8 µg tse/cm2. The value is for both experiments and represents the percent radioactivity in the receptor fluid and the percent radioactivity absorbed into the skin.

Any other information on results incl. tables

Skin thickness and integrity check:

-The mean skin thickness ranged from 0.56 to 0.75 mm.

- The measured transdermal electrical resistance for each cell was greater than 10 kΩ, with values ranging from 10.4 to 30.7 kΩ before test substance administration.

- During the 48 h exposure, the electrical resistance (and potentially the integrity) of the skin decreased in each experiment. The mean decrease of the electrical resistance during the 48 h test was 8.2 and 4.0 kΩ for Experiments 1 and 2, respectively.

Specific radioactivity and homogeneity of the formulation:

- The actual specific radioactivity of the formulation: 27.25 ± 0.34 kBq/mg (Experiment 1); 28.73 ± 0.71 kBq/mg (Experiment 2) This value was determined by taking aliquots during the administration procedure.

- Homogeneity: 1.6% (Experiment 1: 4.9% with outliner); 3.7% (Experiment 2: 45.6% with outliner). The homogeneity was well below 10%, with the exception of 1 outlier in Experiment 2. 

Penetration rate (flux):5.1 µg tse . cm²/h (mean maximum flux at 0.5 to 1 h)

The penetration rate gained its maximum at 0.5 to 1 h post administration, due to rinsing after 30 min, and then decreased rapidly. A flux was already observed at 0.5 h post administration therefore the lag-time would be lower than 0.5 h.

Table 1: Mean flux times for the various time points

Time period (h)	Mean flux (µg/cm^2.h)
0-0.5	2.164
0.5-1	5.142
1-2	2.301
2-4	0.831
4-6	0.309
6-24	0.092
24-29	0.041
29-48	0.035

Table 2: Summary of the percutaneous penetration/dermal absorption of pTD-sulfate in a hair dye formulation without hydrogen peroxide.

Parameters:	µg/cm^2	% dose
Skin rinsings	-	96.4
Absorption	10.1	0.83
Adsorption	3.20	0.28
Penetration	10.7	0.89
Bioavailability	20.8	1.72
Mass balance	-	98.4

Applicant's summary and conclusion

Conclusions:

20.8 µg/cm2 (1.72% dose) of pTD-sulfate in a hair dye formulation without hydrogen peroxide was considered as biologically available (receptor fluid: 10.7 µg/cm2, remaining skin other than stratum corneum: 10.1 µg/cm2) in the cutaneous absorption (in vitro) study.

Executive summary:

Percutaneous penetration/dermal absorption(in vitro) study of test material using pig skin preparations was determined following OECD Guideline 428.

Two independent experiments with 6 integrity checked skin preparations per experiment were performed. Pig skin from 1 male and 1 female weighing 20-25 kg bw was obtained from a local farmer.

21.9 mg (actual) of the formulation, containing 1.20 mg/cm2 of test substance was applied to topically to the horny layer of the skin for 30 min and subsequently washed off with Tween 20 and deionized water. The experiments were performed in static penetration cells (Franz-cells) with an application area of 1.0 cm2.

At termination of penetration (48 h after application) the skin was rinsed again as described above and the stratum corneum was removed by repeated stripping with adhesive tapes to obtain the adsorbed test substance. The remaining skin was taken to determine the absorbed test substance.

The content of test material was determined in the rinsing solutions after 30 min and 48 h of application and in the receptor fluid at 0.5, 1, 2, 4, 6, 24, 29 and 48 h. The penetration was calculated from the [14C]-amount in the receptor fluid consisting of phosphate buffered saline.

Most of the radioactivity was detected in the first tape, and then a gradual decrease was observed with the number of tapes. The majority of the applied test substance formulation was removed from the skin by rinsing 30 min after the application. A low amount of radioactivity was absorbed 48 h post administration in the dermis and the residual epidermis, including remaining hair stubs and shafts.

 The mean absorption in different matrices was:

 Receptor fluid (Percutaneous penetration): 0.89% or 10.7 µg tse/cm2

Stratum corneum (Adsorption): 0.28% or 3.2 µg tse/cm2

Dermis, residual epidermis, including remaining hair stubs and shafts (Absorption): 0.83% or 10.1 µg tse/cm2

Rinsing solution: 96.4% dose (sum of rinsings of the skin after 30 min (95.8%) and 48 h (0.64%))

 The absorbed and penetrated quantities were defined as overall amount of bioavailable test substance.

20.8 µg/cm2 (1.72% dose) of pTD-sulfatein a hair dye formulation without hydrogen peroxide was considered as biologically available (receptor fluid: 10.7µg/cm2, remaining skin other than stratum corneum: 10.1 µg/cm2) in the cutaneous absorption (in vitro) study.

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