2-[(2-nitrophenyl)amino]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 24 November 2004 to 15 December 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No historical data was given.

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:

yes

Remarks:

No historical data was given.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-nitrophenyl)amino]ethanol

EC Number:

225-555-8

EC Name:

2-[(2-nitrophenyl)amino]ethanol

Cas Number:

4926-55-0

Molecular formula:

C8H10N2O3

IUPAC Name:

2-[(2-nitrophenyl)amino]ethanol

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: Supplied by the sponsor, Lot No. 17
- Expiration date of the lot/batch: 11 December 2005
- Purity test date: 11 December 2002

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 99.9% (HPLC)
- Specific activity: 54mCi/mmol; 1.998GBq/mmol; 11.0MBq/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: solubility 0.780mg/ml

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
18.37g HC Yellow 2 was weighed into a glass scintillation vial and dissolved in an ethanolic solution containing 18MBq [14C]-HC Yellow 2. The solution was mixed then blown to dryness in a stream of nitrogen. 80.41mg of water and 1904.12mg Loving Care blank were then added, and the solution mixed to dissolve the HC Yellow 2.
To confirm homogeneity and radioactive content, triplicate samples (nominally 10mg) were diluted with scintillation fluid, then triplicate samples of each dilution were analysed by liquid scintillation counting.


FORM AS APPLIED IN THE TEST (if different from that of starting material)
The test item was used 1% in formulation "Loving Care"

Radiolabelling:

yes

Administration / exposure

Type of coverage:

open

Vehicle:

other: used in formulation "Loving Care"

Duration of exposure:

24 hours

Doses:

- Nominal doses: 1% in formualtion
- Dose volume: not specified
- Rationale for dose selection: The application rates and exposure conditions used in this study were designed to simulate predicted normal human exposure to the test material

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: 18.37g HC Yellow 2 was weighed into a glass scintillation vial and dissolved in an ethanolic solution containing 18MBq [14C]-HC Yellow 2. The solution was mixed then blown to dryness in a stream of nitrogen. 80.41mg of water and 1904.12mg Loving Care blank were then added, and the solution mixed to dissolve the HC Yellow 2.
To confirm homogeneity and radioactive content, triplicate samples (nominally 10mg) were diluted with scintillation fluid, then triplicate samples of each dilution were analysed by liquid scintillation counting.
- Method of storage: not specified

APPLICATION OF DOSE:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The application rates and exposure conditions used in this study were designed to simulate predicted normal human exposure to the test material
- Amount(s) applied (volume or weight with unit): dose rate of 20mg/cm2
- Concentration (if solution):200µg HC Yellow 2 /cm2
- Lot/batch no. (if required): not specified
- Purity: not specified

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: Yes
- Time after start of exposure: 0.5 hours

ANALYSIS
- Method type(s) for identification Liquid scintillation counting
- Validation of analytical procedure: not specified
- Limits of detection and quantification: LOQ : 0.003µg/mL

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Surgery or Post Mortem samples, from females
- Ethical approval if human skin: not specified
- Type of skin: Extraneous tissue
- Preparative technique: Skin membranes were cut from the whole skin samples at a thickness setting of 400μm using an electric dermatome. The membranes were given an identifying number and stored frozen at approximately –20ºC on aluminium foil.
- Membrane integrity check: Membrane integrity was determined by measurement of the electrical resistance across the skin membrane.

PRINCIPLES OF ASSAY
- Diffusion cell: glass diffusion cell used in this study
- Receptor fluid: Yes
- Solubility od test substance in receptor fluid: 0.78 µG/mL
- Static system: Yes
- Flow-through system: No
- Test temperature: 32±1 deg Celius
- Humidity: Not specifed
- Occlusion: No, unoccluded for the duration of the exposure period (48h)
- Reference substance(s): not specified
- Other: At recorded intervals (0.5, 1, 2, 4, 6, 24, 29, 48h), 0.5ml samples of the receptor fluid were taken for analysis.

Results and discussion

Total recovery:

- Total recovery: See table 2 below
- Recovery of applied dose acceptable: Yes
- Quantification of values below LOD or LOQ: LOQ : 0.003µg/mL

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

0.5 h

Dose:

1%

Parameter:

rate

Absorption:

0 mg cm-2 h-1

Percutaneous absorption 2

Key result

Time point:

6 h

Dose:

1%

Parameter:

rate

Absorption:

0 mg cm-2 h-1

Percutaneous absorption 3

Key result

Time point:

48 h

Dose:

1%

Parameter:

rate

Absorption:

0 mg cm-2 h-1

Percutaneous absorption 4

Key result

Time point:

1 h

Dose:

1%

Parameter:

amount

Absorption:

0.001 mg cm-2 h-1

Any other information on results incl. tables

Table1 :SUMMARY OF HC YELLOW 2 DISTRIBUTION IN THE TEST SYSTEM

 

 

Test Compartment	Amount Recovered (µg/cm²)
	Cell 30	Cell 31	Cell 32	Cell 33	Cell 34	Cell 35	Cell 36	Cell 37	Cell 38	Cell 41	Cell 42	Cell 45	Mean	SD	SEM	n
Flange	0.040	0.076	0.062	0.086	0.109	0.151	0.024	0.036	0.048	0.075	0.044	0.036	0.066	0.037	0.011	12
Donor Chamber	0.012	0.016	0.012	0.257	0.058	0.040	0.068	0.087	0.032	0.131	0.037	0.135	0.074	0.072	0.021	12
Skin Wash @ 0.5h	180	204	194	209	190	195	200	198	190	197	193	189	195	7.54	2.18	12
Skin Wash @ 48h	0.664	0.717	0.665	0.81	1.76	1.83	0.257	0.330	0.501	0.669	0.609	0.801	0.800	0.493	0.142	12
StratumCorneum	0.072	0.108	0.185	0.100	0.782	0.414	0.068	0.113	0.131	0.180	0.072	0.131	0.196	0.207	0.060	12
Remaining Epidermis/Dermis	0.194	0.153	0.088	0.179	0.215	0.277	0.075	0.098	0.050	0.296	0.201	0.127	0.163	0.079	0.023	12
Receptor Fluid	6.72	4.36	2.99	5.04	3.47	3.39	2.55	3.79	3.66	2.96	2.59	4.80	3.86	1.21	0.348	12
Systemically Available*	6.91	4.51	3.08	5.22	3.69	3.67	2.62	3.89	3.71	3.25	2.79	4.92	4.02	1.29	0.371	12
TOTAL	188	210	198	215	197	201	203	203	195	202	197	195	200	6.93	2.00	12

 

Test Compartment	Percent of Dose Recovered (%)
	Cell 30	Cell 31	Cell 32	Cell 33	Cell 34	Cell 35	Cell 36	Cell 37	Cell 38	Cell 41	Cell 42	Cell 45	Mean	SD	SEM	n
Flange	0.020	0.038	0.031	0.043	0.055	0.076	0.012	0.018	0.024	0.038	0.022	0.018	0.033	0.018	0.005	12
Donor Chamber	0.006	0.008	0.006	0.129	0.029	0.020	0.034	0.044	0.016	0.066	0.019	0.068	0.037	0.036	0.010	12
Skin Wash @ 0.5h	90.3	102	97.1	105	95.2	97.7	100	99.3	95.3	98.8	96.7	94.8	97.7	3.78	1.09	12
Skin Wash @ 48h	0.333	0.359	0.333	0.405	0.880	0.915	0.129	0.165	0.251	0.335	0.305	0.401	0.401	0.247	0.071	12
StratumCorneum	0.036	0.054	0.093	0.050	0.392	0.207	0.034	0.056	0.066	0.090	0.036	0.066	0.098	0.104	0.030	12
Remaining Epidermis/Dermis	0.097	0.077	0.044	0.090	0.108	0.139	0.038	0.049	0.025	0.148	0.101	0.063	0.082	0.039	0.011	12
Receptor Fluid	3.36	2.18	1.50	2.52	1.74	1.70	1.28	1.90	1.83	1.48	1.30	2.40	1.93	0.604	0.174	12
Systemically Available *	3.46	2.26	1.54	2.61	1.85	1.84	1.31	1.95	1.86	1.63	1.40	2.47	2.01	0.644	0.186	12
TOTAL	100	103	99.5	102	102	99.8	97.3	103	99.9	106	91.0	98.7	100	3.46	0.997	12

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

 

 

TABLE 2 :PENETRATION OF HC YELLOW 2 FROM THE FORMULATION

 

 

 

Cell No	Skin No	Skin Integrity (kW)	Penetration Rate (ug/cm²/h)
			0 - 0.5h	0.5 - 1h	1 - 6h	6 - 48h
Cell 30	573	13.68	0.190	2.87	0.838	0.020
Cell 31	573	18.46	0.020	0.798	0.494	0.032
Cell 32	573	16.25	0.010	0.316	0.375	0.021
Cell 33	573	12.77	0.042	1.39	0.677	0.018
Cell 34	575	16.25	0.016	0.643	0.419	0.022
Cell 35	575	21.11	0.078	0.804	0.420	0.020
Cell 36	576	17.14	0.174	1.38	0.271	0.007
Cell 37	576	13.33	0.262	1.91	0.409	0.012
Cell 38	576	13.07	0.487	2.60	0.310	0.009
Cell 41	577	22.44	0.011	0.229	0.417	0.017
Cell 42	577	25.27	0.014	0.237	0.292	0.022
Cell 45	578	10.94	0.159	1.39	0.680	0.011
Mean	-	-	0.122	1.22	0.467	0.018
SD	-	-	0.138	0.850	0.168	0.007
SEM	-	-	0.040	0.245	0.049	0.002
n	-	-	12	12	12	12

 

 

Applicant's summary and conclusion

Conclusions:

Under experimental condition of this study, the results obtained in this study indicated that the penetration of HC Yellow 2 through human skin is very slow, only 2.01% (4.02µg/cm2) of the applied HC Yellow 2 was regarded as systemically 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 Yellow 2 through human skin over a 48 hour exposure period with a formulation containing HC Yellow 2 at a nominal concentration of 1% according OECD Guideline 428 Method.

The penetration and distribution of HC Yellow 2 from a nominal 1%w/w formulation, has been measured in vitro through human skin, following the incorporation of [14C]-HC Yellow 2.

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 Yellow 2 content. Penetration rates and distribution of HC Yellow 2 in the test system were calculated.

The mean penetrated amount of HC Yellow 2 at 0.5h was 0.061µg/cm2 (0.031% of dose) which increased to 0.669µg/cm2 (0.335%) at 1h, 3.11µg/cm2 (1.56%) at 6h and was 3.86µg/cm2 (1.93%) at the end of the experiment (48h). The mean residual amount in the remaining epidermis/dermis was 0.163µg/cm2 (0.082%), thus the mean systemically available proportion of the dose was 4.02µg/cm2 (2.01% of the dose).

Under experimental condition of this study, te results obtained in this study indicate that the penetration of HC Yellow 2 through human skin is very slow, only 2.01% (4.02µg/cm2) of the applied HC Yellow 2 was regarded as systemically available. Washing the formulation from the skin surface at 0.5h removed the vast majority of the test substance.