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Description of key information

Based on the results of an in vitro skin irritation assay using human skin, the test item is not considered to be irritating to the skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 2017 to 30 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: 99.98% (LC area percentage)
- Impurity: Unknown 0.02%
- Supplier: JAPAN FINECHEM COMPANY, INC.
- Lot no.: 7E18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Airtight container at room temperature

OTHER SPECIFICS:
- Appearance: white powder
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Purchased or derived from tissue obtained by MatTek Corporation from accredited institutions by one or multiple donors.
Source strain:
other: EpiDerm tissue (Lot number 25832, manufactured on July 24, 2017)
Justification for test system used:
EPI-200SIT is recommended in the test method
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST KIT
- Name: EPI-200SIT kit
- Manufacturer MatTek Corporation
- Receipt date July 27, 2017
- Components: EpiDerm tissue (Lot number 25832, manufactured on July 24, 2017); Nylon mesh; Assay medium (medium, Lot number 072017TMB); Phosphate buffered saline without Mg2+ and Ca2+ (PBS(—)) (Lot number 022217ZSB); 5% SDS solution
- Storage conditions: EpiDerm tissue and the medium were stored in a cold place in the cell experimental room (permissible range: 1 to 10°C). Nylon mech and PBS(-) were stored at room temperature in the cell experimental room.
- Quality of reconstructed human epidermis (RHE) model: Tissue viability and the barrier function test are within the acceptable ranges.

CULTURE CONDITION
- Incubator: CO2 incubator (MCO-18AIC, SANYO Eletric)
- Temperature: 37°C
- Humidity: Under humid condition
- CO2 concentration: 5%

BUFFER SOLUTION, MEDIUM CONTAINING MTT SOLUTION AND MTT EXTRACTION SOLVENT
- Buffer solution: Phosphate buffered saline without Ca2+ and Mg2+ (PBS(-))
- Medium containing MTT solution: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Lot# KN079, for research, DOJINDO Laboratories) was dissolved in PBS(-) to prepare 5 mg/mL MTT solution. This solution was diluted with the medium to prepare medium containing 1 mg/mL MTT solution (MTT medium). MTT medium was prepared just before use. MTT medium was put into a light-resistant container and stored at room temperature until use.
- MTT extraction solvent: 2-Propanol (Lot# DSE4889, Special grade, Wako Pure Chemical Industries)

PRELIMINARY TEST
- Test for reactivity with MTT: 25 mg of the test substance and 1 mL MTT medium were mixed, the mixture was incubated for 60 minutes. After incubation, the change in colour of the MTT medium was evaluated. As a result, the MTT medium was changed colour into yellow. It was judged that the test substance had no reactivity with MTT because the colour was not blue ore purple. Therefore, interference of the test substance with MTT was not conducted in the skin irritation test.

SKIN IRRITATION TEST
Triplicate tissues were used for the test substance, negative control substance and positive control substance, respectively. Duplicate tissues were used to check the tissue-binding of the test substance.
- Pre-incubation: In all plates, 0.9 mL of medium was added to each well of 6-well plate (Asahi Glass). Triplicate tissue insert ere placed in the upper wells of each plate. These plates were pre-incubated for 60 ± 5 minutes. Tissue inserts were transferred from the upper wells to the lower wells of the 6-well plate. All plates were pre-incubated for further 18 ± 3 hours in a CO2 incubator.
- Exposure of the test substance: 25 mg of the test substance and 30 µL of the control substances were applied onto each tissue surface at 1 minute interval. For the test substance, 25 µL of PBS(-) was added to each tissue surface just before the exposure. After the exposure, an nylon mesh was placed on each tissue surface to spread the control substances. After the last tissue was exposed, all plates were incubated for 35 ± 1 minutes. All plates were taken out of the incubator and place at room temperature until 60 ± 1 minutes was completed for the first exposed tissue.
- Rinsing and post-incubation: After the exposure, each tissue was rinsed 15 times with PBS(-). After the 15th rinse, tissue inserts were completely submerged 3 times in 150 mL of PBS(-). Finally, tissue inserts were rinsed once from inside and outside with PBS(-). Remaining PBS(-) was removed from inside and outside of tissue insert with a sterile cotton swab. Tissue inserts were transferred into the upper wells of new 6-well plates containing 0.9 mL of fresh medium. PBS(-) on the surface of the tissues were removed with a sterile cotton swab. The plates were incubated for 24 ± 2 hours. Tissue inserts were transferred from the upper wells to the lower wells of the 6-well plates containing 0.9 mL of fresh medium. Further, all plates were incubated for 18 ± 2 hours.
- MTT reaction and extraction: Tissue inserts were transferred into a 24-well plate (Corning) containing 0.3 mL/well of MTT medium and incubated for 180 ± 5 minutes. Medium was removed form all wells. PBS(-) was added to the outside of tissue insert and removed. This operation was repeated twice. All tissues were transferred into a new 24-well plate. 2 mL of 2-propanol was added to each well. The plate was put into a plastic bag, and extraction was performed wat room temperature for 2 hours or more using a plate shaker. The extracts were moved from the inside of tissue inserts to plate and mixed to obtain homogenous solutions.
- Measuring optical density (OD): 200 µL per well of the extract were transferred into a 96-well plate (Corning) (n=2). 200 µL per well of 2-propanol was used as blank (n=6). OD of each extract was measured spectrophotometrically using Multimode Microplate Reader (FLUOstar OPTIMA, BMG LABTECH) at 570 nm. The mean of blank OD was substracted from ODS of each tissue and the mean value was calculated in each tissue to obtain OD of each tissue. The cell viability of each tissue was calculated by the following formula:
Cell viability (%) = OD of each tissue / Mean OD of the negative control group x 100
The mean and standard deviation (SD) of cell viabilities in each treatment group were calculated by the cell viability of each tissue.

TISSUE-BINDING TEST
The tissue-binding test was carried out using the same procedure as described in the above the skin irritation test, except medium without MTT was used instead of MTT medium. After measuring of OD, the staining ratio was calculated by the following formula:
Staining ratio (%) = Mean OD of the test substance group (without MTT) / Mean OD of the negative control substance group (with MTT) x 100
The staining ratio was below 5%. Therefore, correction of OD was not conducted.

ACCEPTABLE CRITERIA OF THE TEST
- Mean of OD in the negative control substance group is ≥1.0 and ≤2.5
- Mean cell viability in the positive control substance is ≤20%
- SDs of cell viabilities in each treatment group used three tissue inserts are <18%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount of test item: 25 mL
- Amount of negative control: 30 µL
- Amount of positive control: 25 µL
Duration of treatment / exposure:
- After the last tissue was exposed, all plates were incubated for 35 ± 1 minutes.
- All plates were taken out of the incubator and placed at room temperature until 60 ± 1 minutes was completed for the first exposed tissue.
Duration of post-treatment incubation (if applicable):
- The plates were incubated for 24 ± 2 hours.
- Tissue inserts were transferred from the upper wells to the lower wells of the 6-well plates containing 0.9 mL of fresh medium. Further, all plates were incubated for 18 ± 2 hours.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
99.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
108.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
101.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
103.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Mean OD in the negative control substance was 1.787. The mean cell viability in the positive control substance was 1.3%. SDs of cell viabilities in the negative an the positive control substances, and the test substances were 3.7, 0.2 and 4.9%, respectively. These results indicated that the present study was appropriately performed.
Interpretation of results:
GHS criteria not met
Conclusions:
The substance was not considered to be irritating to the skin.
Executive summary:

In an in-vitro skin irritation study performed according to the OECD guideline No. 439, the EpiDermTM test method was employed. Human skin cells derived from reconstructed human epidermis tissue containing normal human keratinocytes were used for the test system. Sterile PBS was used a negative control and 5% (w/v) sodium dodecyl sulfate (SDS) in water was used as a positive control. Triplicate tissues were used for the test item, negative control substance and positive control substance, respectively. Duplicate tissues were used to check the tissue-binding of the test substance (tissue-binding test). During the pre-incubation, 0.9 mL of medium was added to each well of 6-well plate (upper wells) and the plates were pre-incubated for 60 ± 5 minutes. Tissue inserts were transferred from the upper wells to the lover wells of the 6-well plate. All plates were pre-incubated for further 18 ± 3 hours in a CO2 incubator. During the exposure period, the test item was applied onto each tissue surface at 1 minute interval. After the exposure, a nylon mesh was placed on each tissue surface to spread the control substances. After the last tissue was exposed, all plates were incubated for 35 ± 1 minutes. All plates were taken out of the incubator and placed at room temperature until 60 ± 1 minutes was completed for the first exposed tissue. During post-incubation, each tissue was rinsed with PBS(-).Tissue inserts were transferred into the upper wells of new 6-well plates containing 0.9 mL of fresh medium. The plates were incubated for 24 ± 2 hours. Tissue inserts were transferred from the upper wells to the lower wells of the 6-well plates containing 0.9 mL of fresh medium. Further, all plates were incubated for 18 ± 2 hours. During the MTT reaction and extraction, tissue inserts were transferred into a 24-well plate containing 0.3 mL/well of MTT medium and incubated for 180 ± 5 minutes. Medium was removed from all wells. PBS (-) was added to the outsides of tissue insert and removed. This operation was repeated twice. All tissues were transferred into a new 24-well plate. 2 mLof 2-propanol was added to each well. The plate was put into a plastic bag, and extraction was performed at room temperature for 2 h or more using a plate shaker. The extracts were moved from the inside of tissue inserts to plate and mixed to obtain homogeneous solutions. To measure the optical density (OD), 200 µL / well of the extracts were transferred into a 96-well plate. 2-propanol was used as blank. OD of each extract was measured spectrophotometrically using Multimode Microplate Reader (FLUOstar OPTIMA, BMG LAB lECH) at 570 nm. Cell viability (%) was calculated as a ration between the OD of each tissue and the mean OD of the negative control. As a result of the skin irritation test, the cell viability treated by DMPO was 103.2%, which exceeds the threshold cell viability therefore the test item is not considered to be irritating to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion: in vitro

In an in-vitro skin irritation study performed according to the OECD guideline No. 439, the EpiDermTM test method was employed. Human skin cells derived from reconstructed human epidermis tissue containing normal human keratinocytes were used for the test system. Sterile PBS was used a negative control and 5% (w/v) sodium dodecyl sulfate (SDS) in water was used as a positive control. Triplicate tissues were used for the test item, negative control substance and positive control substance, respectively. Duplicate tissues were used to check the tissue-binding of the test substance (tissue-binding test). During the pre-incubation, 0.9 mL of medium was added to each well of 6-well plate (upper wells) and the plates were pre-incubated for 60 ± 5 minutes. Tissue inserts were transferred from the upper wells to the lover wells of the 6-well plate. All plates were pre-incubated for further 18 ± 3 hours in a CO2 incubator. During the exposure period, the test item was applied onto each tissue surface at 1 minute interval. After the exposure, a nylon mesh was placed on each tissue surface to spread the control substances. After the last tissue was exposed, all plates were incubated for 35 ± 1 minutes. All plates were taken out of the incubator and placed at room temperature until 60 ± 1 minutes was completed for the first exposed tissue. During post-incubation, each tissue was rinsed with PBS(-).Tissue inserts were transferred into the upper wells of new 6-well plates containing 0.9 mL of fresh medium. The plates were incubated for 24 ± 2 hours. Tissue inserts were transferred from the upper wells to the lower wells of the 6-well plates containing 0.9 mL of fresh medium. Further, all plates were incubated for 18 ± 2 hours. During the MTT reaction and extraction, tissue inserts were transferred into a 24-well plate containing 0.3 mL/well of MTT medium and incubated for 180 ± 5 minutes. Medium was removed from all wells. PBS (-) was added to the outsides of tissue insert and removed. This operation was repeated twice. All tissues were transferred into a new 24-well plate. 2 mLof 2-propanol was added to each well. The plate was put into a plastic bag, and extraction was performed at room temperature for 2 h or more using a plate shaker. The extracts were moved from the inside of tissue inserts to plate and mixed to obtain homogeneous solutions. To measure the optical density (OD), 200 µL / well of the extracts were transferred into a 96-well plate. 2-propanol was used as blank. OD of each extract was measured spectrophotometrically using Multimode Microplate Reader (FLUOstar OPTIMA, BMG LAB lECH) at 570 nm. Cell viability (%) was calculated as a ration between the OD of each tissue and the mean OD of the negative control. As a result of the skin irritation test, the cell viability treated by DMPO was 103.2%, which exceeds the threshold cell viability therefore the test item is not considered to be irritating to the skin (CERI, 2017).

Justification for classification or non-classification

Based on the available data, classification for skin irritation is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

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