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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 June 2018 to 21 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Normal human epidermal keratinocytes (NHEK)
Cell source:
foreskin from a single donor
Source strain:
other: Neonatal-foreskin tissue (NHEK)
Vehicle:
unchanged (no vehicle)
Details on test system:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SCT
Day of delivery: 19. Jun. 2018
Batch: 28625
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μL of test or reference substance
Duration of treatment / exposure:
3 min and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2
Number of replicates:
duplicates

Test system

Details on study design:
Demonstration of Proficiency
The validity of the skin corrosion study at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 12 proficiency chemicals (indicated by the OECD 431 guideline) were tested. All of the 12 proficiency chemicals were correctly categorized. Therefore, the proficiency of the skin corrosion study was demonstrated.

Performance of the study
Pre-Tests
(a) Nylon mesh compatibility
First, the test substance was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 50 μL of the liquid test substance were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 hour incubation at room temperature.
(b) Assessment of Coloured or Staining Test Substances
It was tested whether the test substance develops a colour without MTT addition. 50 μL of the test substance were given in a test tube with 0.3 mL demineralised water and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.
(c) Assessment of Direct Reduction of MTT by the Test Substance
The test substance was also tested for the ability of direct MTT reduction. To test for this ability, 50 μL of the test substance were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. Untreated MTT solution was used as control. The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction had not taken place and no data correction was necessary.

Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the assay medium directly before use. The tissue plate was brought out of the fridge 1 hour before the treatment. The assay medium was warmed in the water bath to 37 ± 1°C.

Description of the Method
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts con-taining the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour (pre-incubation). For each experiment (“3 minutes” and “1 hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μL MTT solution. One additional plate was left empty. The plates were stored in the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2. For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for testing the test substance. The liquid test substance was applied without preparation (50 μL). After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1°C and 5.0 ± 0.5% CO2, the inserts were removed from the plates using sterile forceps. The inserts were thor-oughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cel-lulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT solution for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2. After this time, the MTT solution was aspirated and replaced by DPBS. This was then aspi-rated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then covered with Parafilm® and shaken for 2 hours at room temperature. Afterwards, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, three replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.

Evaluation
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®). The photometric absorbance of the negative controls was considered as 100%. For the mean of the 3 replicates of test item and positive control, tissue viability was calculated as % photometric absorbance compared to the negative control.
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results
Calculations were performed as follows:
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test item
Note: Corrected OD value of negative control corresponds to 100 % viability
% Viability = [Corrected optical density of positive control or test substance / Corrected optical density of mean negative control] X 100

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
ca. 44.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h exposure
Value:
ca. 10.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corrosive
Other effects / acceptance of results:
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.6 (3 minutes) resp. 1.4 (1 hour). The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 6.7 %. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Any other information on results incl. tables

Results

Measured Values

As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate. The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD 570 nm)

Replicate

1

2

3

4

5

6

 

Mean 0.038

Absorbance

0.040

0.038

0.039

0.039

0.039

0.038

Replicate

7

8

9

10

11

12

Absorbance

0.039

0.038

0.038

0.038

0.038

0.037

The absorbance values of negative control, test substance and positive control are given in the following table:

Absorbance Values (OD 570nm)

Incubation

Negative Control

Test Substance

Positive Control

 

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Tissue 1

Tissue 2

 

3 min

1.614

1.634

0.672

0.759

0.344

0.312

1.557

1.638

0.714

0.768

0.361

0.308

1.571

1.608

0.719

0.770

0.365

0.314

 

1 h

1.295

1.389

0.167

0.187

0.140

0.129

1.439

1.404

0.177

0.189

0.110

0.137

1.386

1.415

0.174

0.189

0.125

0.130

 

From the measured absorbances, the mean absorbance of isopropanol was subtracted. The corrected mean and relative standard deviation (RSD) of the two tissues were also calculated.

Mean Absorbance Values of the 3 Minutes Experiment 

Designation

Negative Control

Test Substance

Positive Control

Mean – blank (tissue 1)

1.542

0.663

0.318

Mean – blank (tissue 2)

1.588

0.727

0.273

Mean of the two tissues

1.565

0.695

0.296

RSD

2.1%

6.5%

10.8%

Mean Absorbance Values of the 1 h Experiment 

Designation

Negative Control

Test Substance

Positive Control

Mean – blank (tissue 1)

1.335

0.134

0.087

Mean – blank (tissue 2)

1.364

0.150

0.094

Mean of the two tissues

1.350

0.142

0.090

RSD

1.5%

7.8%

5.5%

Comparison of Tissue Viability

For the test substance and the positive control, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative controls:

% Tissue Viability 

Test Substance

Positive Control

Incubation

44.4%

18.9%

3 min

10.5%

6.7%

1 h

Assessment and Validity

Corrosivity of the Test Substance

The mean value of relative tissue viability of the test substance was reduced to 44.4% after 3 minutes treatment. This value is below the threshold for corrosivity (50%). After 1 hour treatment, the mean value of relative tissue viability of the test substance was reduced to 10.5%, lying below the threshold for corrosivity (15%). Therefore, the test substance is considered as corrosive to skin.

 

Validity

The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.6 (3 minutes) resp. 1.4 (1 hour). The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 6.7 %. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

 

Discussion

The test substance is considered corrosive to skin. In the UN GHS System for skin corrosive substances, test substance is considered to be in subcategory 1B/C. After 3 minutes treatment, the mean value of relative tissue viability of the test substance was decreased to 44.4%. Thi svalue is well below the threshold for corrosivity (50%). After 1 hour treatment the mean value of relative tissue viability of the test substance was reduced to 10.5%. This value is well below the threshold for corrosivity (15%). The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues. The positive control has met the validity criterion too, thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Applicant's summary and conclusion

Interpretation of results:
other: Category 1B/1C (corrosive) based on EU CLP criteria
Conclusions:
Under the study conditions of Reconstructed Human Epidermis (RHE) test method, the test substance was considered to be corrosive to skin.
Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the test substance, C12-14 HEDMAC (39.6% active)using reconstructed human epidermis (RHE) test method, according to the OECD Guideline 431, in compliance with GLP. Two tissues of the human skin model EpiDermTM were treated with the test substance for 3 mins and 1 h, respectively. The test substance was applied to each tissue and spread to match the tissue size. Demineralised water was used as negative control, whereas 8 M KOH was used as positive control. After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After treatment with the negative control, the absorbance values were founf to be within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD after 3 mins and 1 h exposures were determined to be 1.6 and 1.4 respectively. The positive control showed clear corrosive effects for both treatment intervals. After 3 mins treatment with the test substance, the mean value of relative tissue viability was reduced to 44.4%, which is below the threshold for corrosion potential (50%). After 1 h treatment, mean value of relative tissue viability was reduced to 10.5 %, which too, is below the threshold for corrosion potential (15%). Therefore, under the study conditions of Reconstructed Human Epidermis (RHE) test method, the test substance was considered to be corrosive to skin (Laus 2018).