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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:
06 Feb 2019 to 08 Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Physical Description: Light yellow solid
Purity/Composition: UVCB (100%)
Storage Conditions: At room temperature protected from light

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item:
Esacure 3644 was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, at least 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

Test for Reduction of MTT by the Test Item:
Esacure 3644 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.

Test System Set Up:
Tissues:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.


Figure 1: A Diagram of the Application (attached below)

DMEM (Dulbecco’s Modified Eagle’s Medium):
Supplemented DMEM, serum-free supplied by MatTek Corporation.

MTT medium:
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions:
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 62 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.2 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation:
No correction was made for the purity/composition of the test item. The solid test item was applied directly on top of the skin tissue. Esacure 3644 was spread to match the size of the tissue.

Application/Treatment of the Test Item:
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see figure 1). The plates were incubated for approximately 1 hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before Esacure 3644 was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Esacure 3644 and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 27.6 to 32.1 mg of the solid test item was added into the 6-well plates on top of the skin tissues.

For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.

After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

Cell Viability Measurement:
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The solid test item was applied undiluted (27.6 to 32.1 mg) directly on top of the tissue.
Duration of treatment / exposure:
3 minutes for two tissues
1 hour for two other tissues
Duration of post-treatment incubation (if applicable):
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2.
Number of replicates:
2 replicates for each exposure time

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability, percentage of control
Run / experiment:
3-minute application
Value:
87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
29
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability, percentage of control
Run / experiment:
1-hour application
Value:
77
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
13
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Esacure 3644 was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with Esacure 3644 and controls are presented in Appendix 1, Table 1. The individual OD570 measurements are presented in Appendix 2.

Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with Esacure 3644 compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Esacure 3644 compared to the negative control tissues was 87% and 77% respectively. Because the mean relative tissue viability for Esacure 3644 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Esacure 3644 is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range (See Appendix 3). The mean relative tissue viability following the 1-hour exposure to the positive control was 13%.

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤14% for the negative control and test item (Appendix 1, Table 3). For the positive control, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 36%. Since both individual tissues were clearly positive it was concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, Esacure 3644 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate Esacure 3644 for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of Esacure 3644 was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch M7-238-1807001 of Esacure 3644 was a light yellow solid. Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of Esacure 3644 was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 13% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤14% for the negative control and test item. For the positive control, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 36%. Since both individual tissues were clearly positive it was concluded that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Esacure 3644 compared to the negative control tissues was 87% and 77%, respectively. Because the mean relative tissue viability for Esacure 3644 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Esacure 3644 is considered to be not corrosive.

In conclusion, Esacure 3644 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.