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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-15 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 439 with a minor deviation: standard deviation between negative control tissues replicates was greater than the maximum acceptable variability. Considering the results obtained, this deviation was considered as without impact on the conclusion of the study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
yes
Remarks:
standard deviation between negative control tissues replicates was greater than the maximum acceptable variability
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
dated 23 July 2009
Deviations:
yes
Remarks:
standard deviation between negative control tissues replicates was greater than the maximum acceptable variability
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
23 October 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 182596
Purity: 89.8%
Name of test material (as cited in study report): (E,Z)-2,6-DIMETHYLOCTA-2,4,6-TRIENE
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 29 December 2016

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 16-RHE-130) were received on 13 December 2016.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA, batch No. 16 MPE 133) during 3 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 1mL of maintenance medium (Episkin SA, batch No. 16 MA 077).

TREATMENT
- The test item was applied, as supplied, at the dose of 16 µL, to the epidermal surface of 3 living skin models during 42 minutes at room temperature. To ensure a good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA.
- In the same experimental conditions, a positive control (5% SDS), and a negative control (DPBS – PAN BIOTECH GmbH - Batch No. 9510916) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBF1623V) in a 10 mL volumetric flask (QS 10 mL of distilled water). Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA.


REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (PAN BIOTECH GmbH, Batch No. 9510916). The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues. They were incubated for a 41-hour and 8-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measured OD was proportional to the number of living cells. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >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%. In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.
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
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
41-hour and 8-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2
Number of replicates:
3 replicates of living skin models

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
main test (duration of exposure: 42 minutes)
Value:
4.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 37.8°C and 36.9°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained after 2 hours and 47 minutes of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.000 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered to be not interfering with the MTT assay and there was no need to add non-specific coloration controls to the study.

MTT VIABILITY ASSAY RESULTS
The mean percent viability of the treated tissues was 4.4%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

ACCEPTANCE OF RESULTS:
- The standard deviation of the negative control group was 26.0%, instead of ≤ 18% as initially scheduled. Considering the results obtained, this deviation is considered as without impact on the conclusion of the study.

Any other information on results incl. tables

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

Skin

OD

Mean OD / disc (#)

Mean OD / product

Viability %

Mean viability %

Standard deviation (SD)

Negative control

1

2.015

1.964

1.574

124.8

100.0

26.0

1.908

1.969

2

1.513

1.610

102.3

1.632

1.685

3

1.063

1.147

72.9

1.190

1.188

Positive control

1

0.027

0.027

0.023

1.7

1.5

0.5

0.028

0.027

2

0.015

0.015

1.0

0.015

0.015

3

0.027

0.028

1.8

0.029

0.028

Test item

1

0.055

0.055

0.069

3.5

4.4

1.0

0.055

0.055

2

0.086

0.085

5.4

0.086

0.084

3

0.067

0.067

4.3

0.067

0.066

#: mean of 3 values (triplicate of the same extract)

OD: optical density; measured after a 1:2 dilution of the formazan extracts in isopropanol.

 

Acceptability criteria: SD ≤ 18%.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In accordance with Regulation EC No 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.
Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or Category 2 “Irritant”.
Executive summary:

An in vitro skin irritation test using the Reconstructed human Epidermis (SkinEthic RHE® model) was performed according to Guideline OECD 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 8-minute post-incubation period at 37°C, 5% CO2. 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.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent viability of the treated tissues was 4.4%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

Therefore, in accordance with Regulation EC No 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.

Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or Category 2 “Irritant”.