Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
Reconstructed Human Epidermis Test Method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 3, 2016 - November 24, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl ethyl(phenyl)carbamate
EC Number:
213-796-1
EC Name:
Ethyl ethyl(phenyl)carbamate
Cas Number:
1013-75-8
Molecular formula:
C11H15NO2
IUPAC Name:
ethyl ethyl(phenyl)carbamate
Test material form:
liquid
Details on test material:
Colorless
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions:yes
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:NO

In vitro test system

Test system:
human skin model
Remarks:
SkinEthic RHE® model,Episkin SA, RHE/S/17
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Justification for test system used:
The SkinEthic™ RHE model can be use in this study according the OECD guideline for irritation testing (OECD No. 439).
Vehicle:
unchanged (no vehicle)
Remarks:
Test item was applied as supplied.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:Episkin SA, RHE/S/17
- Tissue batch number(s):16-RHE-122
- Delivery date: 22.11.2016
- Date of initiation of testing:22.11.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ROOM TEMPERATURE
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:25 x 1 mL of DPBS
-Observable damage in the tissue due to washing:NO

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:300 μL of MTT solution at 1 mg/mL
- Incubation time:3 HOURS at temperaturwe between 36.4-37.8ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength:570nm
- Linear OD range of spectrophotometer: 0- 2.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:Specification :O.D>0.7, Result: O.D.=1.2 (CV=4.6%)
- Barrier function:Specification: 4.0h<=ET50<=10.0h, Result:5.2h
- Morphology:Specification: Number of cell layers >=4, Result:6 cell layers
- Contamination:no

NUMBER OF REPLICATE TISSUES:3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- no interference

PREDICTION MODEL / DECISION CRITERIA
The test item is considered to be non irritant to skin: if the viability after 42 minutes of exposure and 42 hours of post-treatment incubationis > 50%.
The test item is considered to be irritant to skin: if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):16 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):16 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight):16 μL
- Concentration : 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
41 hours and 15 minutes
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
MEAN viability
Value:
1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.0%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: NO

- Colour interference with MTT:The direct interaction of MTT with the test item was checked by adding 16 μL of the test item to 300 μL of solution of MTT at 1 mg/mL. After 3 hours of incubation was observed yellow solution - no direct interference between test item and MTT.

In water: The coloration potential of the test item in water was checked by adding 16 μL of the test item to 300 μL of distilled water. A colorless solution was obtained after 3 hours of incubation between 36.4°C and 37.8°C, 5% CO2.

In isopropanol: The coloration potential of the test item in isopropanol was checked by adding 16 μL of the test item to 1.5 mL of isopropanol. A colorless solution was obtained after 2 hours of incubation at room temperature. No significant coloration appeared. Therefore the test item will not interfere with the MTT assay.

DEMONSTRATION OF TECHNICAL PROFICIENCY:YES

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: SD value of the % viability ≤ 21.3%,instead of ≤ 18% as initially scheduled. This is due to a mean optical density (OD) value of the replicate No.3 treated with the negative control, which is slightly above the maximal OD value of 1.5.
- Acceptance criteria met for positive control: SD value of the % viability ≤ 18%.
- Acceptance criteria met for variability between replicate measurements:SD value of the % viability ≤ 18%.

Any other information on results incl. tables

Treatment of the results

% viability= (OD test Item / OD negative control )* 100%

Table No.1: Results

 

SKIN

OD

Mean OD/disc

Mean OD/product

Viability

%

Mean viability %

SD

CRITERIA

 

Negative control

1

0.943

1.019

 

 

 

 

1.309

77.8

 

 

 

 

100.00

 

 

 

 

21.3

 

1.039

1.075

2

1.308

1.334

101.9

1.352

1.343

3

1.512

1.575

120.3

1.557

1.657

 

Positive control

4

0.012

0.013

 

 

 

 

0.013

1.0

 

 

 

 

1.0

 

 

 

 

0.1

 

 

 

 

Irritant

0.013

0.013

5

0.011

0.011

0.8

0.012

0.011

6

0.013

0.014

1.1

0.014

0.014

 

Test item

PH-16/0550

15

0.018

0.018

 

 

 

 

0.019

1.4

 

 

 

 

1.5

 

 

 

 

0.2

 

 

 

 

Irritant

0.019

0.017

16

0.026

0.023

1.8

0.027

0.016

17

0.009

0.017

1.3

0.014

0.027

OD-optical density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Positive control:-irritant, SD value ≤ 18%, test item-SD value ≤ 18%, Negative control:non irritant, OD value in the range: ≥ 0.8 and ≤ 3%, SD value 21.3% / (acceptable)
Conclusions:
The mean corrected percent viability of the treated tissues was 1.5%, therefore test item has to be considered as irritant to skin.
Executive summary:

In accordance with GLP study and OECD TG 439, this study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). The test item was applied at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41 hours and 15 minutes post-incubation period at 37°C, 5% CO2. Then the epidermis were put in contact with MTT solution. In the same condition a positive and negative control were carried out. To ensure a good contact with epidermis, all items were recovered with a nylon mesh. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The measured OD values were used to calculate a percentage of viability. The mean corrected percent viability of the treated tissues was 1.5%, therefore test item has to be considered as irritant to skin.