Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February 2018 to 23 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: white powder
- Storage conditions: at room temperature

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™ Reconstructed Human Epidermis Model kit (EPI-200)
- Tissue lot number(s): 27958

TEST FOR MTT REDUCTION
A test material may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test material is present on the tissues when the MTT viability test is performed.
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.

ASSESSMENT OF COLOUR INTERFERENCE WITH MTT
The test material was checked for possible colour interference before the study was started. Approximately 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.

MAIN TEST

PRE-INCUBATION
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 1 hour at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM just before the test material was applied.

APPLICATION OF TEST MATERIAL AND CONTROLS
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 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 30.3 to 37.7 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 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.

ACCEPTABILITY CRITERIA
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 - 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:
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 item 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: 30.3 - 37.7 mg (with 25 µL Milli-Q water to increase tissue surface contact)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N
Duration of treatment / exposure:
3 minutes or 60 minutes
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
100
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute expoure
Value:
70
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
DIRECT MTT REDUCTION AND ASSESSMENT OF COLOUR INTERFERENCE
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.

TEST MATERIAL AND CONTROLS
Mean OD570 values and viabilities for the negative control, positive control and test material are given in Table 1.
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with test material compared to the negative control tissues was 100 and 70 % 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.

QUALITY CRITERIA
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.9 %. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was ≤ 10 %, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue

Exposure period

Mean OD570 of individual tissues

Mean OD570 of duplicate tissues

Standard deviation

Coefficient of Variation (%)

Relative Mean Viability (%)

Negative control

3 minutes

1.579

1.643

0.090

7.4

100

1.706

60 minutes

1.749

1.792

0.060

4.7

1.834

Positive control

3 minutes

0.153

0.149

0.005

5.0

9.1

0.146

60 minutes

0.135

0.142

0.009

8.3

7.9

0.148

Test material

3 minutes

1.707

1.643

0.090

7.5

100

1.579

60 minutes

1.178

1.246

0.096

10

70

1.314

OD = optical density

 

Applicant's summary and conclusion

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

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.bis under GLP conditions using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.

Under the conditions of the study the positive control had a mean relative tissue viability of 7.9 % 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 ≤10%, indicating that the test system functioned properly.

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 test material compared to the negative control tissues was 100 and 70 %, 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.

In conclusion, the test material was not corrosive in the in vitro skin corrosion test.