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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:
02 August 2016 to 05 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Yellow powder
- Storage conditions of test material: At room temperature protected from light
- Test material handling: Use amber glassware or wrap container in aluminium foil
- Stable at higher temperatures: Yes, maximum temperature: 95 °C
Specific details on test material used for the study:
No correction was made for the purity/composition of the test material.

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, kit E and F)
- Tissue batch number(s): Lot no.: 24318
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Source: MatTek Corporation, Ashland MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

CELL CULTURE
- 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 supplemented DMEM medium (Dulbecco’s Modified Eagle’s Medium, serum-free).
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Environmental conditions: All incubations, with the exception of the test material incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 %, containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.

NUMBER OF REPLICATE TISSUES: 2 per exposure time

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. 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 was 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. A negative control, sterile Milli-Q water was tested concurrently. 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 medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM medium 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 25.7 to 29.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8 N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM medium 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 overnight at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test material was classified according to remaining cell viability following exposure of the test material with either of the two exposure times.

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

- Data evaluation and statistical procedures
A test material is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) 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:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.7 to 29.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL KOH
- Concentration (if solution): 8 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:
2 per exposure time

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 - 3 minutes of exposure
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 2 - 1 hour of exposure
Value:
96
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. As the solutions did not turn blue / purple nor was a blue / purple precipitate observed, it was concluded that the test material did not interfere with the MTT endpoint.
The mean absorption at 570 nm measured after treatment with the test material and controls are presented in Table 1.
Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments 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 100 and 96 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after 3 minutes of treatment and not below 15 % after 1 hour of treatment, the test material is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 9 %.
In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤8 %, 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

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean (OD570) ± SD

A (OD570)

B (OD570)

Mean (OD570) ± SD

Negative control

1.462

1.459

1.461 ± 0.002

1.657

1.563

1.610 ± 0.066

Test material

1.478

1.434

1.456 ± 0.031

1.612

1.485

1.548 ± 0.090

Positive control

0.138

0.169

0.154 ± 0.021

0.180

0.104

0.142 ± 0.054

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability

 

3-minute application viability (% of control)

1-hour application viability (% of control)

Negative control

100

100

Test material

100

96

Positive control

11

9

 

Table 3: Coefficient of Variation between tissue replicates

 

3 minute exposure

1 hour exposure

Negative control

0.2

5.6

Test material

3.0

7.9

Positive control

18

42

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 compared to the negative control tissues was 100 and 96 %, respectively. As 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 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤8 %, indicating that the test system functioned properly. The test was concluded to be valid.

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