Registration Dossier

Administrative data

Description of key information

Skin Corrosion

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

Skin Irritation

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Eye irritation (BCOP)

Under the conditions of this study the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made

Eye Irritation (RhCE)

Under the conditions of this study the test material is non-irritant in the EpiOcular™ test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 August 2017 to 18 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 Bis
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
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)
- Tissue Lot no.: 26767

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.8 to 37.5 °C
- Temperature of post-treatment incubation (if applicable): 37 °C (with MTT)

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. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. 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 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.


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 per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM 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 27.8 to 29.1 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 over night 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:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be < 15 %.
- In the range 20 to 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:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
- 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:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.
Sub categorisation:
Viability measured after 3-minutes and 1 hour:
- < 50 % after 3 minute exposure: corrosive
- ≥ 50 % after 3 minute exposure AND < 15 % after 1 hour exposure: corrosive
- ≥ 50 % after 3 minute exposure AND ≥ 15 % after 1 hour exposure: non-corrosive
For substances/mixtures identified as Corrosive:
- < 25 % after 3 minute exposure: Optional Sub-category 1A
- ≥ 25 % after 3 minute exposure: A combination of optional Sub-categories 1B and 1C

ANALYSIS: Calculation of Cell Viability
- Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
- The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
- The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT). The % Viability for each sample and positive control is calculated as follows: %Viability = (ODc/mean x ODlt_u+MTT) * 100
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 27.8 to 29.1 mg (skin was moistened with 25 µL Milli-Q water)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 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:
Two per exposure time
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
89
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
98
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. 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.
- Table 1 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test material 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 89 and 98 % 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.

ACCEPTANCE OF RESULTS
- 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. The mean relative tissue viability following the 1-hour exposure to the positive control was 9.2 %. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤ 20 %, indicating that the test system functioned properly.

Table 1: Mean Absorption in the in vitro Skin Corrosion Test with the test material

 

3 Minute application

1 Hour application

A (OD570)

B (OD570)

Mean (OD570) ± SD

A (OD570)

B (OD570)

Mean (OD570) ± SD

Negative control

2.039

2.414

2.226 ± 0.266

1.939

1.901

1.920 ± 0.027

Test material

1.783

2.179

1.981 ± 0.279

1.667

2.078

1.872 ± 0.290

Positive control

0.204

0.213

0.209 ± 0.007

0.223

0.130

0.179 ± 0.066

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0436). Isopropanol was used to measure the background absorption.

 

Table 2:Mean Tissue Viability in thein vitroSkin Corrosion Test with the test item

 

3-minute application

viability (percentage of control)

1-hour application

viability (percentage of control)

Negative control

100

100

Test material

89

98

Positive control

9.4

9.2

Table 3: Coefficient of Variation between Tissue Replicates

 

3-minute

1-hour

Negative control

16

2.0

Test material

18

20

Positive control

4.5

42

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

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 with the test material compared to the negative control tissues was 89 and 98 %, 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.

The positive control had a mean relative tissue viability of 9.2 % 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 limit2.8) and the laboratory historical control data range. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was20 %, indicating that the test system functioned properly.

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2017 to 25 September 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:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model (EPISKIN-SM, 0.38 cm^2)
- Tissue batch number(s): 17-EKIN-038
- Source: SkinEthic Laboratories, Lyon, France.

TEST FOR INTERFERENCE WITH MTT ENDPOINT
- 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.
- The test material was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model. Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Cesium tin tungsten oxide did not interfere with the MTT endpoint.

SET UP OF THE TEST SYSTEM
- On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37 °C.
- MTT concentrate (3 mg/mL in PBS) WAS diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
- Environmental conditions: All incubations, with the exception of the test material incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 – 100 % (actual range 34 – 94 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 - 46.4 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

NUMBER OF REPLICATE TISSUES: 3

APPLICATION OF THE TEST MATERIAL
- The test was performed on a total of 3 tissues per test material together with negative and positive controls. The skin was moistened with 10 µL Milli-Q water to ensure close contact of the test material to the tissue and the solid test material (11.3 to 12.7 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5 % SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 µL isopropanol.
- Tubes were stored refrigerated and protected from light for approximately 68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
- The mean relative tissue viability of the positive control should be ≤ 50 % relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
- The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

INTERPRETATION
- A test material is considered as an irritant (Category 2) in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤ 50 % of the mean viability of the negative controls.
- A test material is considered as a non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is > 50 % of the mean viability of the negative controls.

ANALYSIS
- Calculation of Cell Viability: The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw): ODc = ODraw – ODbl
- The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
- The %Viability for each sample and positive control is calculated as follows: %Viability = (ODc/mean ODlt_u+MTT) x 100
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 11.3 to 12.7 mg (The skin was moistened with 10 µL Milli-Q water)

NEGATIVE CONTROL
- Amount(s) applied: 25 µL

POSITIVE CONTROL
- Amount(s) applied: 25 µL
- Concentration: 5 % in PBS
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
95
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD= 4.3 %
Other effects / acceptance of results:
- The test material was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because no color changes were observed it was concluded that the test material did not interact with the MTT endpoint.
- The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 95 %. Since the mean relative tissue viability for the test material was above 50 % it is considered to be non-irritant.
- The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 5.4 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8 %, indicating that the test system functioned properly.

Table 1: Summary of Tissue Viabilities

 

Mean tissue viability (percentage of control)

Standard deviation (percentage)

Negative control

100

7.2

Test Material

95

4.3

Positive control

5.4

1.4

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.
Executive summary:

An in vitro skin irritation test using a human skin model was carried out in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

This study tests the ability of the test material to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM™)). The possible skin irritation potential was tested through topical application for 15 minutes.

Skin tissue was moistened with 10 µL of Milli-Q water and at least 10 mg of the test material was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 95 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment it is considered to be non-irritant.

The positive control had a mean cell viability of 5.4 % after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8 %, indicating that the test system functioned properly.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

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

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:
07 August 2017 to 08 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Characteristics of donor animals: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 20 % (w/v)

VEHICLE
- Amount(s) applied: 750 µL
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes with sodium-fluorescein solution
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium) containing 1 % (v/v) L-glutamine and 1 % (v/v) Fetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS: 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 reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES: Three corneas were selected at random for each treatment group.

VEHICLE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: 20 % w/v imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of 20 % (w/v) suspension for 240 ± 10 minutes

TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (20 % (w/v) Imidazole solution) or 20 % (w/v) suspension of the test material was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C

REMOVAL OF TEST SUBSTANCE: After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test material on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = [((I0/I)-0.9894)/0.0251]
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability:
Application of Sodium Fluorescein: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

DECISION CRITERIA: The IVIS cut-off values for identifying the test materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were:
- In vitro score range: ≤ 3 = UN GHS No Category; > 3 but ≤ 55 = No prediction can be made; and >55 = UN GHS Category 1

ACCEPTABILITY OF THE ASSAY The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
25
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
- Table 1 summarises the opacity, permeability and in vitro irritancy scores of the test material and the controls.

- The corneas treated with the test material showed opacity values ranging from 12 to 38 and permeability values ranging from -0.004 to 0.003. The corneas were translucent with spot after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 12 to 38 after 240 minutes of treatment with the test material.

- The individual in vitro irritancy scores for the negative controls ranged from -0.6 to 2.8. The individual positive control in vitro irritancy scores ranged from 91 to 147. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
- The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 125 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.

- The test material induced ocular irritation through one endpoints (opacity), resulting in a mean in vitro irritancy score of 25 after 240 minutes of treatment.

Table 1:Summary of Opacity, Permeability and In Vitro Scores

Treatment

Mean Opacity

Mean Permeability

Mean IVIS

Negative control

0.7

-0.001

0.7

Positive control

104

1.390

125

Test material

25

0.001

25

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). Calculated using the negative control mean opacity and mean permeability values for the positive control and test material.

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of this study the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made
Executive summary:

The eye hazard potential of the test material was investigated in accordance with the standardised guideline OECD437, under GLP conditions.

The eye hazard potential of the test material was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested through topical application toisolated bovine corneasfor approximately 240 minutes. The test material was applied as a 20 % (w/v) suspension (750 µl) directly on top of the corneas. 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 125 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. 

The test material induced ocular irritation through one endpoints (opacity), resulting in a mean in vitro irritancy score of 25 after 4 hours of treatment.

Under the conditions of this study the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 September 2017 to 15 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
2015
Deviations:
no
GLP compliance:
yes
Species:
human
Details on test animals or tissues and environmental conditions:
- The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialised medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.
- Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg:

Duration of treatment / exposure:
6 hours ± 15 minutes
Duration of post- treatment incubation (in vitro):
- Post-soak: 25 ± 2 minute immersion incubation
- Followed by: 18 hours ± 15 minutes
Number of animals or in vitro replicates:
2 replicates
Details on study design:
TEST FOR COLOUR INTERFERENCE WITH MTT ENDPOINT
- Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the tissues during the exposure. To assess the colour interference, approximately 50 mg of the test material or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0 °C in the dark. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. Furthermore, approximately 50 mg of the test material or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g.
- At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Centrifugation was not considered necessary.
- If after subtraction of the negative control, the OD for the test material solution is >0.08, the test material is considered as possibly interacting with the MTT measurement.

TEST FOR MTT REDUCTION
- To assess the ability of the test material to reduce MTT, approximately 50 mg of the test material was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0 °C in the dark. A negative control, 50 µL sterile Milli-Q water was tested concurrently. If the MTT solution colour turned blue / purple or if a blue / purple precipitate was observed the test material interacts with MTT. Only test materials which bind to the tissue after rinsing can interact with MTT in the main assay.
-In case the test material reacts with the MTT medium in addition to the normal procedure, two killed tissues treated with test material and two killed non-treated tissues must be used for the cytotoxicity evaluation with MTT.

APPLICATION/ TREATMENT
- The test was performed on a total of 2 tissues per test material together with a negative control and positive control. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.
- At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with the test material (6 hours ± 15 minutes at 37.0 ± 1.0 °C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test material. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37 °C.
CELL VIABILITY MEASUREMENT
- After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37 °C.
- After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 to 3 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test material was classified according to remaining cell viability following exposure of the test material.

ACCEPTABILITY CRITERIA
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
- The mean relative tissue viability of the positive control should be < 50 % relative to the negative control.
- The difference between the % tissue viabilities of the two identically treated replicates should be < 20.
- The non-specific MTT reduction should be ≤ 50 % relative to the negative control OD.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- The test material is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %. In this case no further testing in other test methods is required.
- The test material is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60 %.

ANALYSIS
- Calculation of Cell Viability: The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw): ODc = ODraw – ODbl. The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT). The % Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100
- MTT Interacting Test Materials: Nonspecific MTT reduction (NSMTT) will be calculated. NSMTT is the difference between the mean OD of the untreated freeze-killed tissues (ODkt_u+MTT) and test material treated freeze-killed tissues (ODkt_t+MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).
%NSMTT = [(ODkt_t+MTT – ODkt_u+MTT)/ mean ODlt_u+MTT] * 100
True tissue viability is calculated as the difference between the living test material treated tissues incubated with MTT medium (ODlt_t+MTT) and the difference between ODkt_t+MTT and ODkt_u+MTT. OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
%Viability = [OD/ mean ODlt_u+MTT] * 100
In case the %NSMTT ≤ 0.0, there is no need to correct for interference of the test material.
Irritation parameter:
other: Tissue Viability
Run / experiment:
Mean
Value:
77
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
INTERFERENCE WITH MTT ENDPOINT
- The test material was checked for possible direct MTT reduction by adding the test material to MTT medium. Because there were colour changes observed it was concluded that the test material did interact with the MTT endpoint.
- The test material was checked for colour interference in aqueous conditions. Addition of the test material to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.04 and 0.003, respectively. Therefore it was concluded that the test material did not induce colour interference.
- Since the test material did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, two freeze-killed tissues treated with test material and one freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test material was 2.5% of the negative control tissues. The ODs of the test material treated viable tissues was corrected using the OD of the freeze-killed tissues.

MAIN ASSAY
- Eye hazard potential is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test material compared to the negative control tissues was 77 %. Since the mean relative tissue viability for the test material was above 60 % the test material is considered to be non-irritant.
- The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 30 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated identically was less than 17 %, indicating that the test system functioned properly.

Table 1:Mean Absorption in the EpiOcular™ Test

 

A (OD570)

B (OD570)

Mean (OD570)± SD

Negative control

1.899

1.510

1.705 ± 0.275

Test material

1.275

1.339

1.307 ± 0.045

Positive control

0.454

0.570

0.512 ± 0.082

OD = optical density

SD = Standard deviation

1 The test material values are corrected for the non-specific MTT reduction.

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

Table 2: Mean Tissue Viability in the EpiOcular™ Test

 

Mean tissue viability (percentage of control)

Standard deviation
 (%)

Negative control

100

16

Test material

77

2.7

Positive control

30

4.8

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study the test material is non-irritant in the EpiOcular™ test.
Executive summary:

The eye hazard potential of the test material the test material was investigated in accordance with the standardised guideline OECD 492 under GLP conditions.

The test material was topically applied on the Reconstructed Human EpiOcular™ Model and the possible eye hazard potential of the test material was tested through topical application for 6 hours.

The test material (50.0 to 52.3 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test material and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 30 % after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated identically was less than 17 %, indicating that the test system functioned properly.

The test material did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, two freeze-killed tissues treated with test material and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test material was 2.5 % of the negative control tissues. The ODs of the test material treated viable tissues was corrected using the OD of the freeze-killed tissues.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test material compared to the negative control tissues was 77 %. Since the mean relative tissue viability for the test material was above 60 % after 6 hours ± 15 minutes treatment the test material is considered to be non-irritant.

Under the conditions of this study the test material is non-irritant in the EpiOcular™ test.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion

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 study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 with the test material compared to the negative control tissues was 89 and 98 %, 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.

The positive control had a mean relative tissue viability of 9.2 % 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 limit2.8) and the laboratory historical control data range. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was20 %, indicating that the test system functioned properly.

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

Skin Irritation

An in vitro skin irritation test using a human skin model was carried out in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

This study tests the ability of the test material to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM™)). The possible skin irritation potential was tested through topical application for 15 minutes.

Skin tissue was moistened with 10 µL of Milli-Q water and at least 10 mg of the test material was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 95 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment it is considered to be non-irritant.

The positive control had a mean cell viability of 5.4 % after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8 %, indicating that the test system functioned properly.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Eye Irritation (BCOP)

The eye hazard potential of the test material was investigated in accordance with the standardised guideline OECD437, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The eye hazard potential of the test material was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested through topical application toisolated bovine corneasfor approximately 240 minutes. The test material was applied as a 20 % (w/v) suspension (750 µl) directly on top of the corneas. 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 125 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. 

The test material induced ocular irritation through one endpoints (opacity), resulting in a mean in vitro irritancy score of 25 after 4 hours of treatment.

Under the conditions of this study the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made.

Eye Irritation (RhCE)

The eye hazard potential of the test material the test material was investigated in accordance with the standardised guideline OECD 492 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was topically applied on the Reconstructed Human EpiOcular™ Model and the possible eye hazard potential of the test material was tested through topical application for 6 hours.

The test material (50.0 to 52.3 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test material and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 30 % after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated identically was less than 17 %, indicating that the test system functioned properly.

The test material did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, two freeze-killed tissues treated with test material and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test material was 2.5 % of the negative control tissues. The ODs of the test material treated viable tissues was corrected using the OD of the freeze-killed tissues.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test material compared to the negative control tissues was 77 %. Since the mean relative tissue viability for the test material was above 60 % after 6 hours ± 15 minutes treatment the test material is considered to be non-irritant.

Under the conditions of this study the test material is non-irritant in the EpiOcular™ test.

Justification for classification or non-classification