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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:
14 August 2017 to 18 August 2017
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:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 Bis
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Tungsten Oxide (WO3), caesium and tin-doped
EC Number:
945-942-1
Molecular formula:
Cs0.29Sn0.04WO3
IUPAC Name:
Tungsten Oxide (WO3), caesium and tin-doped
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Dark blue powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue Lot no.: 26767

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.8 to 37.5 °C
- Temperature of post-treatment incubation (if applicable): 37 °C (with MTT)

TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
- The test material was checked for possible colour interference before the study was started. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, at least 25 mg of the test material 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 colour change was observed.

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
- The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, at least 25 mg of the test material or 50 µL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.


APPLICATION/TREATMENT OF THE TEST MATERIAL
- 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. The plates were incubated for approximately 2 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM just before the test material was applied.
- The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test material and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water to ensure close contact of the test material to the tissue and 27.8 to 29.1 mg of the solid test material 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.
REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. 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 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.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be < 15 %.
- In the range 20 to 100 % viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30 %.

INTERPRETATION
A test material is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
- In addition, a test material considered non-corrosive (viability ≥ 50 %) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test material is decreased below 15 %.
A test material is considered non corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.
Sub categorisation:
Viability measured after 3-minutes and 1 hour:
- < 50 % after 3 minute exposure: corrosive
- ≥ 50 % after 3 minute exposure AND < 15 % after 1 hour exposure: corrosive
- ≥ 50 % after 3 minute exposure AND ≥ 15 % after 1 hour exposure: non-corrosive
For substances/mixtures identified as Corrosive:
- < 25 % after 3 minute exposure: Optional Sub-category 1A
- ≥ 25 % after 3 minute exposure: A combination of optional Sub-categories 1B and 1C

ANALYSIS: Calculation of Cell Viability
- Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
- The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
- The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT). The % Viability for each sample and positive control is calculated as follows: %Viability = (ODc/mean x ODlt_u+MTT) * 100
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 27.8 to 29.1 mg (skin was moistened with 25 µL Milli-Q water)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N
Duration of treatment / exposure:
3 minutes of exposure and 1 hour of exposure
Duration of post-treatment incubation (if applicable):
Incubated for 3 hours with MTT
Number of replicates:
Two per exposure time

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
89
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
98
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test material did not interfere with the MTT endpoint.
- Table 1 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 89 and 98 % respectively. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1 hour treatment the test material is considered to be not corrosive.

ACCEPTANCE OF RESULTS
- 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. The mean relative tissue viability following the 1-hour exposure to the positive control was 9.2 %. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤ 20 %, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean Absorption in the in vitro Skin Corrosion Test with the test material

 

3 Minute application

1 Hour application

A (OD570)

B (OD570)

Mean (OD570) ± SD

A (OD570)

B (OD570)

Mean (OD570) ± SD

Negative control

2.039

2.414

2.226 ± 0.266

1.939

1.901

1.920 ± 0.027

Test material

1.783

2.179

1.981 ± 0.279

1.667

2.078

1.872 ± 0.290

Positive control

0.204

0.213

0.209 ± 0.007

0.223

0.130

0.179 ± 0.066

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0436). Isopropanol was used to measure the background absorption.

 

Table 2:Mean Tissue Viability in thein vitroSkin Corrosion Test with the test item

 

3-minute application

viability (percentage of control)

1-hour application

viability (percentage of control)

Negative control

100

100

Test material

89

98

Positive control

9.4

9.2

Table 3: Coefficient of Variation between Tissue Replicates

 

3-minute

1-hour

Negative control

16

2.0

Test material

18

20

Positive control

4.5

42

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Applicant's summary and conclusion

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of this study the test material is not corrosive in the in vitro skin corrosion test.
Executive summary:

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 Bis under GLP conditions.

The ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) was investigated. The possible corrosive potential was tested through topical application for 3 minutes and 1 hour.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test material was applied directly on top of the skin tissue. Milli-Q water and 8 N KOH served as the negative and positive control, respectively.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 89 and 98 %, respectively. Because the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, the test material is considered to be not corrosive.

The positive control had a mean relative tissue viability of 9.2 % 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 limit2.8) and the laboratory historical control data range. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was20 %, indicating that the test system functioned properly.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.