Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 November, 2017 to 18 January, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 431 without any deviation.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
(Adopted 29 July 2016).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: ˃ 99%
- Lot/batch No.of test material: S17-6-100
- Physical state: Colorless liquid

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light

In vitro test system

Test system:
human skin model
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
The EpiDermTM SCT (EPI-200) model consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis.

Culture conditions: 37ºC, 5% CO2, under humid condition

Assay medium: Assay Medium
MTT diluent: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ and Assay Medium used for diluting MTT
Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+
Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT), 1.0 mg / mL MTT diluent
Extracting agent: 2-propanol
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL

CONTROLS
- Control tissues were concurrently applied with 50 μL of sterile distilled water (NC) or with 50 μL of 8N Potassium hydroxide solution (PC).
Duration of treatment / exposure:
3 minutes and 60 ± 1 minutes
Number of replicates:
2

Test system

Type of coverage:
other: a nylon mesh was placed on each tissue surface to spread the liquid substances.
Details on study design:
EXPERIMENTAL PROCEDURE

To assess the ability of the test substance to directly reduce MTT a pretest was performed. Fifty microliters of the test substance and 1 mL of MTT medium were mixed, the mixture was incubated for 60 minutes. After the incubation, the change in color of the MTT medium was evaluated. As a result, the MTT medium was changed color into light yellow and the precipitation of the test substance was not observed. However, the precipitation of the test substance was observed in the preliminary test that was conducted in “In vitro Eye Irritation Test of Kurimate Using EpiOcularTMEIT (OCL-200)” (Study Number K10-0334) at the testing facility. The precipitation of the test substance was light purple. It was judged that the test substance had reactivity with MTT. Therefore, interference of the test substance with MTT (interference test) was conducted in the skin corrosion test.
To assess the compatibility of nylon mesh a pretest was performed. Fifty microliters of the test substance was dropped onto a nylon mesh on a glass slide. After 60 minutes at room temperature, the corrosion of the nylon mesh was evaluated microscopically. As a result, the corrosion was not observed. Therefore, the skin corrosion test was conducted using nylon meshes.

- Basic procedure:
Two tissues were treated with each, the test substance, the PC and the NC.
Pre-incubation of the tissues:
Tissue inserts were placed in a 6-well plate filled with 0.9 mL/well of the medium and incubated for 60 ± 5 minutes.

- Application of the test substance:
3-minute and 60-minute exposures were conducted.
At 3-minute exposure, after pre-incubation, the medium was not removed before the application.
At 60-minute exposure, after pre-incubation, the medium was removed from all wells and 0.9 mL/well of fresh medium was added before the application.
Fifty microliters of the test substance was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile distilled water (NC) or with 50 μL of 8N Potassium hydroxide solution (PC).
Immediately after the application, a nylon mesh was placed on each tissue surface to spread the substances over the tissue surface. The 45 second interval allowed sufficient time for both application and washing procedures at the end of the exposure period.
At 3-minute exposure, each plate was placed at room temperature until 3 minutes was completed for the first exposed tissue in each plate.
At 60-minute exposure, each plate was placed into the incubator until 60 ± 1 minutes was completed for the first exposed tissue in each plate.

- Removal of the test substance:
To remove the test substance, the tissues were rinsed approximately twenty times with sterile PBS. Inside and outside of the tissue inserts were wiped. The tissue inserts were placed into new 24-well plates filled with 300 μL/well of fresh medium. Remaining PBS was completely removed from the tissue surface.

- MTT incubation:
After the removal of the test substance, the medium was replaced by 0.3 mL/well MTT solution and the tissues were incubated in the incubator for 180 ± 5 minutes.
After incubation, MTT solution was removed from all wells. The outside of tissue inserts were washed three times with PBS. All tissues were transferred into a new 24-well plate. Two milliliters of 2-propanol was added to the tissue inserts. The plate was placed into a plastic bag, and extraction was performed at room temperature for 2 hours or more using a plate shaker. The extracts in the tissue inserts were transferred into the wells and homogenized.

- Optical Density measurements:
The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with 2-propanol in a microtiter plate.
.
- RhE tissue construct used, including batch number:
Tissue model: EpiDermTM SCT (EPI-200 SCT)
Tissue Lot Number: 27618
Supplier: MatTek Corporation

- Evaluation of results:
The corrosion potential of the test substance is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile distilled water.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
3 minutes
Value:
96.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
60 minutes
Value:
3.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Applicant's summary and conclusion

Interpretation of results:
other: Category 1B (Corrosive) or Category 1C (Corrosive) based on GHS criteria
Conclusions:
The test substance (Kurimate), was corrosive. The data obtained was clear, and the classification carried out accordingly was explicit.
Executive summary:

The potential corrosive effect of Kurimate was studied with EpiDermTMSCT (EPI-200 SCT), in compliance with OECD TG 431. According to study results, the test substance was classified as corrosive.