Registration Dossier

Diss Factsheets

Toxicological information

Dermal absorption

Currently viewing:

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

Constituent 1
Chemical structure
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 absorptionopen allclose all
Key result
Time point:
0.5 h
Dose:
1%
Parameter:
rate
Absorption:
0 mg cm-2 h-1
Key result
Time point:
6 h
Dose:
1%
Parameter:
rate
Absorption:
0 mg cm-2 h-1
Key result
Time point:
48 h
Dose:
1%
Parameter:
rate
Absorption:
0 mg cm-2 h-1
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.