2-(4-amino-2-nitroanilino)ethanol

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 5 December 2004 to 6 June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-amino-2-nitroanilino)ethanol

EC Number:

220-701-7

EC Name:

2-(4-amino-2-nitroanilino)ethanol

Cas Number:

2871-01-4

Molecular formula:

C8H11N3O3

IUPAC Name:

2-(4-amino-2-nitroanilino)ethanol

Test material form:

solid: particulate/powder

Remarks:

Crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by Procter&Gamble, Batch No. L35939
- Expiration date of the lot/batch: 8 July 2006
- Purity test date: 8 July 2003

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.5% (by HPLC method)
- Specific activity: 57 mCi/mmol ; 2.109GBq/mmol ; 10.7MBq/mg
- Locations of the label: ethanolamine-1.2-14C-
- Expiration date of radiochemical substance: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: 0.75mg/ml

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
58.6 mg HC Red 3 was weighed into a glass round bottomed vial and dissolved in an ethanolic solution containing 18MBq[14C]-HC Red 3. The solution was mixed then blown to dryness in a stream of nitrogen. 39.82 mg of water and 1898.76 mg"Loving Care" blank tint were added, and the solution mixed.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Hair dye cream formulation containing 3% HC Red 3 (Loving Care Tint used as formulation)

Radiolabelling:

yes

Administration / exposure

Type of coverage:

open

Vehicle:

other: Hair dye cream formulation : Loving Care Tint

Duration of exposure:

30 minutes

Doses:

- Nominal doses: 20 mg/cm2

No. of animals per group:

12 replicates were used

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: 58.6 mg HC Red 3 was weighed into a glass round bottomed vial and dissolved in an ethanolic solution containing 18MBq[14C]-HC Red 3. The solution was mixed then blown to dryness in a stream of nitrogen. 39.82 mg of water and 1898.76 mg"Loving Care" blank tint were added, and the solution mixed.
- Method of storage: Not specified

APPLICATION OF DOSE: Since the test material was a gel, the doses were applied to the skin and spread over the surface using small glass rods and the weight of the applied dose recorded after spreading

VEHICLE
- Justification for use and choice of vehicle (if other than water): to mimic a hair dye cream formulation
- Amount(s) applied (volume or weight with unit): 20mg/cm2 (600µg/cm2 of HC Red 3)

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: gently swabbing with a series of sponge pre-wetted with 3% Teepol
- Time after start of exposure: 0.5 hours (30minutes)

ANALYSIS
- Method type(s) for identification Liquid scintillation counting
- Liquid scintillation counting results (cpm) converted to dpm as follows: not specified

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human skin samples from female donors (post-mortem)
- Ethical approval if human skin: not specified
- Type of skin: Information not available
- Preparative technique: Extraneous tissue was removed from human whole skin samples obtained from post mortem. Skin membranes were cut from whole skin samples at a thickness setting of 400 µm using an electric dermatome
- Thickness of skin (in mm): 400 µm
- Membrane integrity check: Performed by measurement of the electrical resistance across the skin membrane
- Storage conditions: stored at -20 °C on aluminium foil


PRINCIPLES OF ASSAY
- Diffusion cell: glass diffusion cell
- Receptor fluid: containing 4% polyoxyethylene 20 oleyl ether solution in phosphate
- Solubility of test substance in receptor fluid: 0.75 mg/mL
- Static system: Yes
- Flow-through system: No
- Test temperature: 32±1°C
- Humidity: not specified
- Occlusion: No, skin samples were unoccluded
- Reference substance(s): not specified

Results and discussion

Absorption in different matrices:

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): The mean penetrated amount of HC Red 3 at 0.5 hours was 0.001µg/cm2 which increased to 0.006µg/cm2 at 1 hour, 0.036 µg/cm2 at 6 hours and was 0.436 µg/cm2 (0.072%) at the end of exposure. The mean residual amount in the remaining epidermis/dermis after tape strapping was 0.612 µg/cm2 (1,01%), thus the mean systemically available proprtion of the dose (amount penetrated + remaining epidermis.dermis) was 1.05µg/cm2.
- Stratum corneum (in vitro test system): (i.e tape strips) The mean proportion of the dose recovered from tape strips representing the stratum corneum, was 0.480 µg/cm2 (0.079%).

Total recovery:

- Total recovery: 98.7%
- Recovery of applied dose acceptable: Yes

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

48 h

Dose:

3%

Parameter:

amount

Absorption:

1.53 other: µg/cm2

Percutaneous absorption 2

Key result

Time point:

48 h

Dose:

3%

Parameter:

percentage

Absorption:

0.252 %

Any other information on results incl. tables

Table1 :SUMMARY OF HC Red 3 DISTRIBUTION IN THE TEST SYSTEM

 

 

Test Compartment	Amount Recovered (µg/cm²)
	Mean	SD	SEM	n
Flange	0.203	0.369	0.107	12
Donor Chamber	0.313	0.217	0.063	12
Skin Wash @ 0.5h	594	8.58	2.48	12
Skin Wash @ 48h	1.79	1.01	0.290	12
StratumCorneum	0.480	0.404	0.116	12
Remaining Epidermis/Dermis	0.612	0.355	0.103	12
Receptor Fluid	0.436	1.77	0.051	12
Systemically Available*	1.05	1.29	0.154	12
TOTAL	598	7.5	2.17	12

 

Test Compartment	Percent of Dose Recovered (%)
	Mean	SD	SEM	n
Flange	0.034	0.063	0.018	12
Donor Chamber	0.02	0.036	0.010	12
Skin Wash @ 0.5h	98.0	1.42	0.409	12
Skin Wash @ 48h	0.296	0.166	0.048	12
StratumCorneum	0.079	0.067	0.019	12
Remaining Epidermis/Dermis	0.101	0.059	0.017	12
Receptor Fluid	0.072	0.029	0.08	12
Systemically Available *	0.173	0.088	0.025	12
TOTAL	98.3	1.24	0.36	12

* Systemically Available = Sum of Remaining Epidermis and Receptor Fluid data

 

 

TABLE 2 :PENETRATION OF HC Red 3 FROM THE FORMULATION

Hours	Amount penetrated
	µg/cm2	% of applied dose
0.5	0.001	0.000
1	0.006	0.001
2	0.012	0.002
4	0.022	0.004
6	0.036	0.006
24	0.218	0.036
29	0.269	0.044
48	0.436	0.072

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Under experimental condition of this study, the results obtained in this study indicate that the penetration of HC Red 3 through human skin is very slow, only 0.25% (1.52μg/cm2) of the applied HC Red 3 was regarded as available. Washing the formulation from the skin surface at 0.5h removed the vast majority of the test substance.

Executive summary:

The purpose of this study was to determine the in vitro percutaneous penetration of HC Red 3 through human skin over a 48 hour exposure period with a formulation containing HC Red 3 at aconcentration of 3% according to OECD Guideline 428 Method.

The penetration and distribution of HC Red 3  has been measured in vitro through human skin, following the incorporation of [14C]-HC Red 3. The mixed formulation was applied to 12 human dermatomed skin membranes (nominally 400μm thick), mounted in glass diffusion cells, at a nominal rate of 20mg/cm2. After a contact period of 30 minutes, the dose was washed from the surface of the skin using natural sponges soaked in 3% Teepol. Samples of the receptor fluid (4% polyoxyethylene 20 oleyl ether solution in phosphate buffered saline) were taken at recorded intervals over a 48h period, during which time the applications remained unoccluded. At the end of the experiment, the surface of the skin was washed again and layers of stratum corneum removed using a tape stripping technique. The receptor fluid samples, sponges, tape strips, residual skin and donor chambers were analysed for radioactivity, which was representative of the HC Red 3 content. Penetration rates and distribution of the Test Item in the test system were calculated.

Under experimental condition of this study, the results obtained in this study indicate that the penetration of HC Red 3 through human skin is very slow, only 0.25% (1.52μg/cm2) of the applied HC Red 3  was regarded as available. Washing the formulation from the skin surface at 0.5h removed the vast majority of the test substance.