Registration Dossier

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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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.
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)
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
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
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.
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
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.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 4 September, 2017 to 4 December, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Kurimate
- Physical state: Clear colorless liquid
- Storage condition of test material: room temperature, in a dark place, air in the headspace of the container was replaced with nitrogen gas
Test system:
human skin model
Remarks:
LabCyte EPI-MODEL24; reconstructed three-dimensional human epidermis
Source species:
other:
Cell type:
other:
Cell source:
other:
Vehicle:
unchanged (no vehicle)
Details on test system:
After receipt, this model was stored at room temperature unopened until culture. After opening, all the epidermis tissues were cultured.
Assay Medium was warmed for 30 minutes to 37℃ using a water bath. The warmed assay medium (0.5 mL/well) was added to the 1st row of each 24-well assay plate. Three epidermis tissues were used per test substance, positive control and negative control. The LabCyte EPI-MODEL 24 aluminum package was opened and culture inserts with epidermis tissues were picked up using sterile forceps. The culture inserts were transferred into the assay medium filled wells of the 1st row. The plate was incubated overnight (actual time: 21.75 hours) in a CO2 incubator.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 µL
- Concentration: undiluted test substance
Duration of treatment / exposure:
15 minutes (at room temperature)
Number of replicates:
Three epidermis tissues were used per test substance, positive control and negative control.
Details on study design:
RINSING
After applying for 15 minutes, the culture insert was picked up with sterile forceps. Any remaining liquid on the epidermis tissues in the culture inserts was discarded by tilting and then tapping. The epidermis tissues in the culture insert was rinsed with wash buffer from a poly wash bottle. The rinsing was repeated 15 times and all residual test substance or control substances were almost completely removed from the epidermis surface. Tapping was not performed for the final rinsing operation. Leftover wash buffer outside the culture insert was removed with a sterile cotton bud. The culture inserts were transferred to wells in the 3rd row of the same columns.
POST-INCUBATION (DAY 0 to 2)
Assay plates were allowed to stand in a CO2 incubator for 42.25 hours.
MTT ASSAY (DAY 2)
PREPARATION OF WELLS FOR MTT ASSAY
MTT medium was warmed for 30 minutes to 37ºC using a water bath. Assay plates were removed from a CO2 incubator. The warmed MTT medium (0.5mL/well) was added to three wells of the 4th row of each plate.
MTT ASSAY
After post-incubation, culture inserts were transferred to the 4th row of the same columns. Assay plates were allowed to stand in a CO2 incubator for 3 hours.
FORMAZAN EXTRACTION AND MEASUREMENT (DAY 2 to 3)
FORMAZAN EXTRACTION
The epidermis tissues were pinched from each culture insert with forceps and transferred into a 1.5 mL micro tube. Isopropanol (300μL) was added to the each micro tube to soak the entire epidermis tissue. The micro tubes were incubated in a refrigerator overnight (actual time: 20.33 hours) in order to completely extract pigments.
OPTICAL DENSITY MEASUREMENTS OF EXTRACTS
The micro tubes were shaken to mix the solution then 200 μL of the solution was transferred into each well of a 96-well plate. Optical density (OD) of each well was measured using a microplate reader (measurement wavelength: 570 nm and 650 nm) and
determine the measured OD by subtracting 650 nm OD from 570 nm OD.
CALCULATION OF TISSUE VIABILITY
Tissue viability was calculated using the equation below (BlankOD value of isopropanol only).
Measured OD = [SampleOD (570nm -650nm)-[BlankOD (570nm -650nm)]
Individual tissue viability (%) = [Individual measured OD (Test substance, positive control or negative control)] / [Mean measured OD (Negative control)]×100
Tissue viability (%) = [Mean measured OD (Test substance or positive control)] / [Mean measured OD (Negative control)] ×100
DATA EVALUATION
ACCEPTANCE CRITERIA
The in vitro skin irritation test is considered acceptable by the following criteria.
 Viability: 0.7 ≤ mean OD (A570/650) measured value for negative control ≤ 2.5.
 Positive control: mean tissue viability for 5% SLS (positive control) ≤ 40%
 SD: SD (negative control and positive control) of tissue viability of 3 identically treated replicates ≤ 18%
DECISION CRITERIA
Tissue viability classification:
 Tissue viability is ≤ 50% ; Irritant
 Tissue viability is > 50% ; Non Irritant
CRITERIA FOR RE-EXPERIMENT
Criteria for re-experiment are shown below; however, as none fell into the criteria, no re-experiment was performed in this study.
(1) When bacterial contamination is found.
(2) When deviating from the culture conditions described in the protocol due to an equipment failure.
(3) Apparent mistakes in the operation.
(4) Acceptability of the test does not meet the criteria.
(5) When the study director decides the necessity of re-experiment
(a) When a value obtained in the re-experiment is within 100 ± 20% of the data obtained in the first experiment, the data of the first experiment is accepted.
(b) When a value obtained in the re-experiment is not within 100 ± 20% of the data obtained in the first assay, the third determination is performed if possible, and the median value of the three values is accepted.
(c) When a value obtained in the re-experiment is not within 100 ± 20% of the data obtained in the first assay, the third determination is not possible, and the values are used as reference data. It is described in this final report.
Irritation / corrosion parameter:
% tissue viability
Value:
5.8
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid

 Table 1 Summary of tissue viability in the in vitro skin irritation test with Kurimate

 Treatment Mean tissue viability (percentage of control)
 Negative control 100
 Kurimate 5.8 
 Positive control 2.8

Appendix 1 Individual OD value, mean OD value and individual tissue viability in the in vitro skin irritation test with Kurimate

Treatment Individual OD (570/650 nm) Mean OD (570/650 nm) Individual tissue viability (percentage of control) Standard deviation (percentage)
 Negative control  1.2644  1.2601 100.3 3.9
   1.2094   96.0  
   1.3064   103.7  
 Kurimate  0.0629  0.0727 5.0 1.3
   0.0634   5.0  
   0.0918   7.3  
 Positive control  0.0339  0.0354 2.7 0.9
   0.0480   3.8   
   0.0244   1.9  

The OD values are corrected for BlankOD value (isopropanol only).

Interpretation of results:
other: UN GHS Category 1 or Category 2
Conclusions:
Kurimate is a skin irritant or a skin corrosive.
Executive summary:

The skin irritation of Kurimate was evaluated using a human three dimensional epidermal model (LabCyte EPI-MODEL24).

Kurimate was applied directly to the surface of epidermis tissues for 15 minutes. After a 42-hour post-incubation period, determination of the tissue viability was performed using MTT assay. A cutoff value of 50% tissue viability of the negative control value was considered and used to classify test substances as irritant or non-irritant. 

The tissue viability of the positive control was 2.8%. The absolute mean OD (570/650 nm) of the negative control was within criteria (0.7 ≤ mean OD ≤ 2.5). The standard deviation value of three tissue viabilities of the positive control and the negative control was less than 3.9 %, indicating that the test system functioned properly (Appendix 1).

The tissue viability in the in vitro skin irritation test with Kurimate is shown in Table 1.

The mean tissue viability obtained after 15 minutes treatment with Kurimate compared to the negative control was 5.8%. It was concluded that Kurimate is irritant in the in vitro skin irritation test under the experimental conditions described in this report. These results indicate that kurimate is a skin irritant or a skin corrosive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 4 September 2017 to 4 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Kurimate
- CAS number: 1864727-96-7
- Analytical purity: 99.4%
- Physical state: Clear colorless liquid
- Lot number: S17-6-100
- Expiration date: One year from the shipping date (7 August 2017)
- Storage condition of test material: room temperature, air in the headspace of the container was replaced with nitrogen gas, in a dark place
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
BOVINE EYES
- Source: Fifteen eyes from cattle (26 to 29 months old) excised as soon as possible after slaughter at a slaughterhouse (Kumamoto Chuou Meat Center Co.,Ltd., 548, Subayashi, Toyono-machi, Uki-shi, Kumamoto 861-4307, Japan) were transported in a cooler with Hanks’ Balanced Salt Solution to the test facility. The bovine eyes were used on the day of receipt.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 μL
- Concentration: undiluted test substance
Duration of treatment / exposure:
Exposure period of 10 minutes (at 32 ± 1°C)
Duration of post- treatment incubation (in vitro):
2 hours (at 32 ± 1°C)
Number of animals or in vitro replicates:
Three corneas were selected for each group.
Details on study design:
PREPARATION OF CORNEAS
The corneal holder, MEM, and cMEM (MEM without phenol red) were warmed in a water bath or a low temperature incubator (set at 32°C) until use. The eyes were checked for unacceptable defects, such as opacity, scratches and neovascularization. There were no eyes exhibiting defects. Corneas were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling. Isolated corneas were mounted in corneal holders that consist of anterior and posterior compartments, which interface with the epithelial and endothelial sides of cornea, respectively. Both compartments of the corneal holder were filled with cMEM. The corneas were incubated for 1 hour in a low temperature incubator (set at 32°C, permissible range: 32°C ± 1°C, actual temperature: 31°C to 32°C). After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity was calculated according to the following equation.
Opacity = [(baseline LUX†) / (opacity of cornea LUX) – 0.9894] / 0.0251
†; Baseline LUX=1000 (The baseline was adjusted to1000 with cMEM.)
Corneas that had an initial opacity reading higher than 7 were not used. Nine corneas were chosen for the above experiment excluding those with high opacity.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The following operations were performed in a draft chamber. Three corneas were selected for each group. The medium was removed from the anterior compartment and 750 μL of the negative control, the test substance and the positive control were introduced into the anterior compartment through dosing holes on the top surface of the chamber, and the holes were sealed (in the closed-chamber method). The holder was maintained in a horizontal position to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated for 10 minutes in a low temperature incubator (set at 32°C, permissible range: 32°C±1°C, actual temperature: 31°C to 32°C).

WASHING CORNEAS
After incubation, the control substances and the test substance were removed and the epithelium was washed three times with MEM. The corneas were washed once again because some test substance still remained. The corneas were given a final rinse with cMEM and were incubated for 2 hours in a low temperature incubator (set at 32°C, permissible range: 32°C±1°C, actual temperature: 31°C to 32°C).

OPACITY MEASUREMENT AND PERMEABILITY DETERMINATIONS
After the incubation period, each cornea was visually observed (e.g., tissue peeling, residual test substance, non-uniform opacity patterns). Medium was removed from both compartments and replaced with fresh cMEM and opacity determinations were performed. The medium of anterior compartments was removed and replaced with 1 mL of fluorescein sodium salt solution. Corneas were incubated in a horizontal position for 90 minutes in a low temperature incubator (set at 32°C, permissible range: 32°C±1°C, actual temperature: 31°C to 32°C). After the incubation period, the medium in the posterior compartments of each holder was removed and placed into a sampling tube. The optical density at 490 nm (OD490) of each sampling tube was measured using a spectrophotometer.

CALCULATION OF IN VITRO IRRITANCY SCORE
OPACITY
The final opacity for each individual cornea was calculated by subtracting the initial opacity reading from the post-treatment reading. The mean of the final opacity value of each treatment group was calculated. To calculate the in vitro irritancy score, values obtained by subtracting the mean final opacity of the negative control from that of the test substance or positive control were used for the calculation.

PERMEABILITY
The each values (OD490) was corrected for background value (OD490 value of cMEM only):
Permeability (OD490) = Each value (OD490) – Mean negative control value (OD490)

IN VITRO IRRITANCY SCORE
IVIS = Mean opacity value + (15 × Mean permeability OD490 value)
Individual IVIS = Individual opacity value + (15 × individual permeability OD 490 value)

DATA EVALUATION
DECISION CRITERIA
IVIS cut-off values for identifying the test substance as inducing serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given below.
IVIS ≤ 3 was classified as UN GHS No Category,
IVIS > 3; ≤ 55 as No prediction can be made, and
IVIS > 55 as UN GHS Category 1.

ACCEPTANCE CRITERIA
The test is considered acceptable if:
(1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
(2) The negative control responses should result in opacity and permeability values below the upper limit of the laboratory historical range.

CRITERIA FOR RE-EXPERIMENT
Criteria for re-experiment are shown below; however, as none fell into the criteria, no re-experiment was performed in this study.
(1) Data are not obtained or reliability of the data is not confirmed due to an equipment failure.
(2) Apparent mistakes in the operation.
(3) Acceptability of the test does not meet the criteria.
(4) IVIS values vary among 3 corneas; predictions from 2 of the 3 corneas do not correspond to the mean of all 3 corneas or predictions from 1 of the 3 corneas do not correspond to the mean of all 3 corneas and the difference from the cut-off threshold (55) exceeds 10.
(a) When the results of a second testing run correspond to the prediction of the first testing run (based upon the mean IVIS value), the prediction of the second testing run should be accepted.
(b) When the results of a second do not correspond to the prediction from the first testing run (based upon the mean IVIS value), a third testing run should be conducted.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean In Vitro Irritancy Score
Value:
39.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table1 Summary of opacity, permeability, and in vitro irritancy score (IVIS)

Treatment

Mean opacity

Mean permeability

Mean in vitro irritancy score

Negative control

0.1

0.0282

0.5 

Kurimate

12.5

1.8063

39.1 

Positive control

81.1

2.6470 

120.4 

Appemdix 1 Individual opacity, permeability, and in vitro irritancy scores (IVIS)

Treatment Eyes Nos. Opacity Pereability in vitro irriteancy score (IVIS)
Negative control 1  -0.5 0.0199  -0.2 
  3  0.7 0.0427  1.3 
   0.0 0.0221  0.4 
Kurimate 6  12.4 1.9797  41.6 
  7  12.1 1.8822  39.8 
  8  12.9 1.5571  35.7 
Positive control 9  65.3 1.4107  85.9 
  14  90.4 3.4951  142.3 
  15  87.8 3.0351  132.8 

Appendix 2 Historical control data for BCOP studies

 Treatment Negative control      Positive control
   Permeability  Opacity  in vitro irritancy score    (IVIS)
 Range  <0.0590  <4.5  61.7 to 169.1
 Mean  0.0124  0.8  115.4
 n  42  42  39

The above data range of the control substances were obtained by collecting all data over the period of December 2015 to July 2017.

Interpretation of results:
other: No prediction of eye irritation can be made
Conclusions:
Since Kurimate induced an IVIS > 3 and ≤ 55, no prediction could be made for eye irritation or serious eye damage.
Executive summary:

The opacity, permeability andin vitroirritancy scores (IVIS) of Kurimate and the control substances are shown in Table 1 and Appendix 1.

The eye irritancy potential of Kurimate was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test). The test method used isolated corneas from the eyes of freshly slaughtered cattle.  The corneas damaged by the test substance were assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a spectrophotometer, respectively.  Both measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the eye irritancy of the test substance. 

Negative control values for opacity and permeability were below the upper limit of the test facility historical range.  Therefore, the negative control did not induce irritancy on the corneas. The mean IVIS of the positive control (N,N-dimethylformamide) was 120.4 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Kurimate induced opacity and increased permeability.  This resulted in a mean IVIS of 39.1 after 10 minutes of treatment.  Since Kurimate induced an IVIS > 3 and ≤ 55, no prediction could be made for eye irritation or serious eye damage.

Endpoint:
eye irritation: in vitro / ex vivo
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. 492 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
(Adopted 28 July 2015).
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium.

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

Assay medium: OCL-200-ASY/Assay Medium
MTT diluent: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ and OCL-200-ASY/Assay Medium used for diluting MTT
Pre-treatment / 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
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative 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 methyl acetate (PC).
Duration of treatment / exposure:
30 ± 2 minutes
Duration of post- treatment incubation (in vitro):
120 ± 15 minutes
Number of animals or in vitro replicates:
2
Details on study design:
EXPERIMENTAL PROCEDURE

To assess the ability of the test material to directly reduce MTT a pretest was performed. Fifty microliters of the test substance was added to 1 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 180 minutes.
As a result, the MTT medium was changed color into yellow and the precipitation of the test substance was observed. 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 eye irritation test.
-Basic procedure:
Two tissues were treated with each, the test substance, the PC and the NC.
Pre-incubation of the tissues:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 60 ± 5 minutes the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 - 24 hours.
-Pretreatment of the tissues:
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
-Application of the test substance:
Fifty microliters of the test material 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 methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 30 ± 2 minutes was completed.
-Removal of the test substance and postincubation period:
To remove the test substance, the tissues were washed with sterile PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium in order to remove residual test substance. After 12 ± 2 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 120 ± 15 minutes.
-MTT incubation:
After the post-incubation period, the assay medium was replaced by 0.3 mL/well MTT solution and the tissues were incubated in the incubator for 180 ± 10 minutes.
After incubation, the outside of tissue inserts was wiped. All tissues were transferred into new 6-well plates filled with 2 mL/well of 2-propanol. The plate was put into a plastic bag, and extraction was performed at room temperature for 2 hours or more using a plate shaker. The extracts were mixed to obtain homogeneous solutions.
-Optical Density measurements:
The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 8 microtiter wells filled with 2-propanol in a microtiter plate.
.
- RhCE tissue construct used, including batch number:
Tissue model: OCL-200 EIT
Tissue Lot Number: 20997
Supplier: MatTek Corporation

- Evaluation of results:
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile distilled water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.
Irritation parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
30 minutes
Value:
14.4
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
- Range of historical values if different from the ones specified in the test guideline: no
Interpretation of results:
other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that Kurimate shows a serious eye damage or an eye irritation potential in the EpiOcularTM in vitro eye irritation test under the test conditions chosen.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

With regards to the skin irritation/ corrosion endpoint:

·        Two in vitro skin irritation/corrosion tests were conducted with Kurimate using the EpiSkin Reconstituted Human Epidermis Model. When tested undiluted, Kurimate would be classified as skin irritation/corrosion category 1B or 1C according to the criteria in the protocol.

As a result, Kurimate would be classified for corrosion under EU CLP and UN GHS. 

·        The in vivo skin sensitising test (LLNA) shows evidence that the test substance has no sensitising properties on an in vivo test model. This supports that Kurimate would not be classified as the most severe category suggested by the two in vitro skin irritation/corrosion tests

Therefore, based on the results of the skin sensitising test stated above, the substance Kurimate is to be classified as a skin corrosion Category 1C.

With regards to the eye irritation/ corrosion endpoint:

·        Two in vitro eye irritation/corrosion tests were conducted with Kurimate using the BCOP and the RhCE models. According to one of these assays, the substance would be classified as irritant UN GHS Category 1 or 2 

·        Two in vitro skin irritation/corrosion tests were conducted as stated above. When tested undiluted, Kurimate was classified as skin irritation/corrosion Category 1C. Since Kurimate was corrosive at 100% in an in vitro human skin model, this supports the conclusion that Kurimate would have effects on the eyes and would likely be severe (irreversible effects on the eyes) as stated by the Regulation (EC) No 1272/2008 (CLP)article 3.3.2.3. : “[…] Skin corrosive substances shall be considered as leading to serious damage to the eyes as well (Category 1) […].”

This supports the conclusion to classify Kurimate as EDI 1.

Justification for classification or non-classification

Due to the results of the existing in vitro assays on the substance Kurimate (EC 946-283-2 - CAS 1864727-96-7) and the justification above based on the weight of evidence approach, conducting an in vivo assay is not justified to conclude on the skin irritation/corrosion and eye irritation/corrosion endpoints. The substance Kurimate (EC 946-283-2 - CAS 1864727-96-7) can be classified as SCI Category 1C and EDI Category 1, according to the CLP and GHS classification.