[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin Corrosion In Vitro, Andres (2017)

Under the conditions of the study, the test material was considered to be non-corrosive to the skin.

Skin Irritation In Vitro, Andres (2018)

Under the conditions of the study the test material is considered as non-irritant to skin.

Eye Irritation In Vitro, Andres (2017)

Under the conditions of the study no prediction of eye irritation can be made.

Eye Irritation In Vitro, Geitlinger (2018) RhCE

Under the conditions of the study no prediction of eye irritation can be made.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 October 2017 to 26 October 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:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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-Kit
- Source: MatTek In Vitro Life Science Laboratories, Bratislava.
- Tissue batch number(s): 25848 (main test) and 25835 (additional test).
- Delivery date: 10. Oct. 2017 (main test) and 08. Aug. 2017 (additional test).

PRE-TESTS
- Assessment of Coloured or Staining Test Materials: It was tested whether the test material develops a colour without MTT addition. 25.0 mg of the test material were given in a test tube with 0.3 mL demineralised water and incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour. The resulting solution was colourless, therefore no binding capacity had to be tested.
- Assessment of Direct Reduction of MTT by the Test Material: The test material was also tested for the ability of direct MTT reduction. To test for this ability, 26.1 mg of the test material were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour. Untreated MTT solution was used as control. The MTT solution changed its colour to purple within 1 hour.
- Additional Test: Direct Reduction of MTT with freeze killed Tissues: As the test material had shown its ability to reduce MTT in the pre-test, it was necessary to perform a functional test with freeze killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues. Freeze killed tissues were prepared by placing untreated tissues in freezer (– 20 ± 5 °C) over night. Once killed, the tissues were stored indefinitely in the freezer. In addition to the normal test procedure described, the functional check employed two freeze-killed tissues treated with the MTT reducing test material (for 3 minutes and 1 hour experiment) and two untreated killed tissues (for 3 minutes and 1 hour experiment) that showed the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissues. Therefore, direct MTT reduction had taken place and data correction was necessary.

PREPARATIONS
- On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the assay medium directly before use.
- The tissue plate was brought out of the fridge 1 hour before the treatment.
- The assay medium was warmed in the water bath to 37 ± 1 °C.

MAIN TEST
- Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour (pre-incubation).
- For each experiment (“3 minutes” and “1 hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μL MTT solution. One additional plate was left empty. The plates were stored in the incubator at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for testing the test material.
- Defined amounts of the test material were applied together with 25 μL demineralised water. Main Test: 26.0 and 26.2 mg (3 minute incubation), 25.9 and 25.5 mg (1 hour incubation). Additional test: 26.9 and 27.1 mg (3 minute incubation), 26.9 and 25.3 mg (1 hour incubation).
- At the start of each experiment (application of negative controls), a stop watch was started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation: 37 ± 1 °C (with MTT)

NUMBER OF REPLICATE TISSUES: 2

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1 °C and 5.0 ± 0.5 % CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT solution for 3 hours at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- After this time, the MTT solution was aspirated and replaced by DPBS. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 hours at room temperature.
- Afterwards, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
- From each well, three replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.

EVALUATION
The photometric absorbance of the negative controls was considered as 100 %. For the mean of the 3 replicates of test item and positive control, tissue viability was calculated as % photometric absorbance compared to the negative control.

CALCULATIONS
Calculations were performed as follows:
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test material
(Corrected OD value of negative control corresponds to 100 % viability)

% Viability = [ODcorrected if the test material or positive control/ OD corrected of mean negative control]·100

- Calculation for Additional Test (Correction)
OD Test Material (corrected) = OD Test Material (Main Test) - (mean OD test material with freezekilled tissues – mean OD negative control with freeze-killed tissues)

PREDICTION MODEL / DECISION CRITERIA
- Corrosivity was assessed using the following:
STEP 1
% tissue viability after 3 min. incubation time < 50 % of negative control = corrosive to skin
% tissue viability after 3 min. incubation time ≥ 50 % of negative control and % tissue viability after 1 h incubation time < 15 % of negative control = corrosive to skin
% tissue viability after 3 min. incubation time ≥ 50 % of negative control and % tissue viability after 1 h incubation time ≥ 15 % of negative control = non-corrosive to skin

STEP 2 (for substances/mixtures identified as corrosive in STEP 1)
% tissue viability after 3 min. incubation time < 25 % of negative control = GHS Skin Corrosive Sub-category 1A
% tissue viability after 3 min. incubation time ≥ 25 % of negative control = GHS Skin Corrosive Sub-categories 1B or 1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25.3 - 27.1 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration: 8 M

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

106.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

101.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MEASURED VALUES OF THE MAIN TEST
- As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate, this gave a mean of 0.039. The mean absorbance of isopropanol was subtracted from the values of the test material, positive control and negative control. The corrected mean and relative standard deviation (RSD) of the tissue replicates were also calculated. Results can be seen in Table 1.

MEASURED VALUES OF THE ADDITIONAL TEST
- As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate, this gave a mean of 0.037. The mean absorbance of isopropanol was subtracted from the values from the measured absorbances of the freeze-killed tissues. The corrected mean and relative standard deviation (RSD) of the freeze-killed tissue replicates were also calculated. Results can be seen in Table 2.
- The mean value of the test material (freeze-killed tissues) was subtracted from the mean value of the negative control (freeze-killed tissues). This difference was -0.009 (3 minutes) and 0.019 (1 hour). These values were subtracted from the absorbance value of the test material in the main test to achieve the corrected absorbance values.
- Corrected values can be seen in Table 3.

CORROSIVITY OF THE TEST MATERIAL
- The mean value of relative tissue viability of the test material was increased to 106.9 % after 3 minutes treatment. This value is above the threshold for corrosivity (50 %).
- After 1 hour treatment, the mean value of relative tissue viability of the test material was increased to 101.4 %, lying above the threshold for corrosivity (15 %). Therefore, the test material is considered as not corrosive to skin.

VALIDITY OF THE TEST
- The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.9 (3 minutes) and 1.8 (1 hour). The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15 %), was fulfilled, too. The mean value of relative tissue viability was 7.4 %.
- Values for negative control and for positive control were within the range of historical data of the test facility. Therefore the experiment is considered valid.

Table 1: Mean Absorbance Values of the Main Test

Designation	Exposure time
	3 minutes			1 hour
	Negative Control	Test Material	Positive Control	Negative Control	Test Material	Positive Control
Mean – blank (tissue 1)	1.888	1.999	0.411	1.822	1.621	0.129
Mean – blank (tissue 2)	1.921	2.056	0.442	1.709	1.996	0.134
Mean of the two tissues	1.905	2.027	0.426	1.765	1.808	0.131
RSD	1.2 %	2.0 %	5.1 %	4.5 %	14.7 %	2.9 %

 

Table 2: Mean Absorbance Values of the Additional Test

Designation	Exposure time
	3 minutes		1 hour
	Negative Control	Test Material	Negative Control	Test Material
Mean – blank (tissue 1)	0.090	0.077	0.072	0.085
Mean – blank (tissue 2)	0.087	0.084	0.068	0.094
Mean of the two tissues	0.089	0.080	0.070	0.089
RSD	1.9 %	6.2 %	4.4 %	7.1 %

 

Table 3: Mean Corrected Absorbance Values of the Experiment

Designation	Test Material Exposure Time
	3 minutes	1 hour
Mean – blank (tissue 1)	2.008	1.602
Mean – blank (tissue 2)	2.065	1.977
Mean of the two tissues	2.036	1.789
RSD	2.0 %	14.8 %

Table 4: Percentage Tissue Viabilities (%)

Exposure Time	Test Material	Positive Control
3 Minutes	106.9	22.4
1 Hour	101.4	7.4

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is considered to be non-corrosive to the skin.

Executive summary:

The potential of the test material to cause corrosion to the skin was determined in vitro, in accordance with the standardised guidelines OECD 431 and EU Method B 40bis, under GLP conditions using the Reconstructed Human Epidermis (RHE) Test Method.

One valid experiment was performed. In the pre-test the test material showed MTT-reducing properties. The probability to influence the photometric measurement had to be excluded. Therefore an additional test using freeze-killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues was performed. The result of the additional test showed that MTT reduction by the test material did not influence the result of the study.

In the main test two tissues of the human skin model EpiDerm were treated with the test material for 3 minutes and 1 hour, respectively. Demineralised water was used as negative control and 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting formazan solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.9 (3 minutes experiment) and 1.8 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 7.4 % for the 1 hour treatment. After 3 minutes treatment with the test material, the mean value of relative tissue viability was increased to 106.9 %. This value is above the threshold for corrosion potential (50 %). After 1 hour treatment, mean value of relative tissue viability was increased to 101.4 %. This value, too, is above the threshold for corrosion potential (15 %).

Under the conditions of the study, the test material was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 December 2017 to 15 December 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 (incl. QA statement)

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-Kit
- Source: MatTek In Vitro Life Science Laboratories, Bratislava.
- Tissue batch number(s): EPI-200-SIT 25864
- Delivery date: 12. Dec. 2017

PRE-TESTS
- Assessment of Coloured or Staining Test Materials: It was tested whether the test material develops a colour without MTT addition. 25.7 mg of the test material were given in a test tube with 0.3 mL demineralised water and incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour. The resulting solution was colourless, therefore no binding capacity had to be tested.
- Assessment of Direct Reduction of MTT by the Test Material: The test material was also tested for the ability of direct MTT reduction. To test for this ability, 26.1 mg of the test material were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour. Untreated MTT solution was used as control. The MTT solution changed its colour into blue/purple within 1 hour. The test material showed the ability of direct MTT reduction, however, it was considered unnecessary to perform a functional test with freeze killed tissues, because this additional test was previously performed in another GLP-study at the testing facility. The result of the additional test showed, that the MTT reduction by the test material did not influence the result of the study.


PRE-INCUBATION OF TISSUES
- All working steps were performed under sterile conditions. For each treatment group (negative control, test material and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 0.5 % CO2 for 1 hour.
- After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 19 hours.

TREATMENT
- One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used as positive control; each tissue was treated with 30 µL 5 % SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used for treatment with the test material: The tissues were wetted with 25 µL DPBS buffer before applying the test material and spreading it to match the tissue size. 25.8, 25.3 and 25.6 mg of test material was added to tissues 1, 2 and 3, respectively.
- Tissues were dosed in one minute intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1 °C and 5.0 ± 0.5 % CO2.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation: 37 ± 1 °C

NUMBER OF REPLICATE TISSUES: 2

REMOVAL OF TEST MATERIAL AND CONTROLS
- 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one minute intervals.
- After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
- Then, the tissues were set in the incubator for 23 hours and 25 minutes at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours and 5 minutes for post-incubation at 37 ± 1 °C and 5.0 ± 0.5 % CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After a total incubation time of 42 hours and 30 minutes, a 24-well-plate was prepared with 300 µL freshly prepared MTT-solution in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- After this time, the MTT-solution was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times.
- At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
- After 2 hours, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
- From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.

EVALUATION
- The values of the 96-well-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).

CALCULATIONS
Calculations were performed as follows:
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the three relating tissues for controls and test material
(Corrected OD value of negative control corresponds to 100 % viability, OD = Optical Density)

% Tissue Viability = [OD replicate test material resp. positive control/ OD mean of negative control]·100


PREDICTION MODEL / DECISION CRITERIA
- Skin irritation potential was assessed using the following:
≤ 50 % of negative control = Corrosive/ Irritant to skin
>50 % of negative control = Non-irritant to skin

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25.8, 25.3 and 25.6 mg
- The tissues were wetted with 25 µL DPBS buffer before applying the test material.

NEGATIVE CONTROL
- Amount(s) applied: 30 μL

POSITIVE CONTROL
- Amount(s) applied: 30 μL
- Concentration: 5 %

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours and 30 minutes

Number of replicates:

3 tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

104.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MEASURED VALUES
- As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate. The mean OD570 value was 0.040.
- From the measured absorbance’s, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol. The mean of the three tissues was also calculated.

TISSUE VIABILITY
- For the test material and the positive control, the percentage values of tissue viability were calculated in comparison to the negative control.

ASSESSMENT AND VALIDITY
- The mean value of relative tissue viability of the test material was increased to 104.8 % after the treatment. This value is above the threshold for tissue viability (50 %). Therefore, the test material is considered as non-irritant to skin.
- Validity:
OD of negative control (must be ≥ 0.8 and ≤ 2.8) was found to be 1.5.
% tissue viability of positive control SDS (must be ≤ 20 % of negative control) was found to be 2.2 %.
SD of mean viability of the tissue replicates (%) (must be ≤ 18 %) was found to be 1.6 % (negative control), 0.2 % (positive control) and 6.6 % (test material).
All validity criteria were met.
Values for negative control and for positive control were within the range of historical data of the test facility.

Table 1: Mean Absorbance Values

Designation	Negative Control	Test Material	Positive Control
Mean – blank (tissue 1)	1.523	1.588	0.031
Mean – blank (tissue 2)	1.494	1.657	0.033
Mean – blank (tissue 3)	1.474	1.462	0.036
Mean of the three tissues	1.497	1.569	0.033

 

Table 2: % Tissue Viability

Designation	Test Material	Positive Control
% Tissue viability (tissue 1)	106.1	2.1
% Tissue viability (tissue 2)	110.7	2.2
% Tissue viability (tissue 3)	97.7	2.4
% Tissue viability (mean)	104.8	2.2
± SD of mean tissue viability (%)	6.6	0.2

Interpretation of results:

other: Not classified in accordance with EU Criteria.

Conclusions:

Under the conditions of this study the test material is considered as non-irritant to skin.

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46., under GLP conditions.

One valid experiment was performed. Three tissues of the human skin model EpiDerm™ were treated with the test material for 60 minutes. The test material was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as the negative control and 5 % SDS solution was used as the positive control.

After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2 % (required: ≤20 %). The variation within the tissue replicates of negative, control, positive control and test material was acceptable (required: ≤ 18 %).

After the treatment, the mean value of relative tissue viability was increased to 104.8 %. This value is above the threshold for tissue viability (50 %). 

Under the conditions of this study the test material is considered as non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 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

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Species: Bos primigenius Taurus
- Source: Slaughterhouse
- Characteristics of donor animals: The cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1 % Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 673.0, 590.2 and 658.6 mg

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
- On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% foetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
- After the arrival of the corneas, they were examined and only corneas that were free from damage were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
- After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10)

POSITIVE CONTROL USED: Imidazole solution: 20 %

APPLICATION DOSE AND EXPOSURE TIME
- 673.0, 590.2 or 658.6 mg, for 4 hours at 32 ± 1 °C.

TREATMENT METHOD: Open chamber
- For each treatment group (negative control solution, test material and positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative and positive control solution and a defined amount of test material (673.0, 590.2 or 658.6 mg) were applied to each replicate.
- In order to apply the test material, the nut of the cornea holder was unscrewed to remove the glass disc. The test material could then be applied directly to the cornea using a weighing funnel. The test material was applied to the epithelium in such a manner that as much as possible of the cornea was covered with test material.

REMOVAL OF TEST SUBSTANCE
- Thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red was performed, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Baseline opacity was measured before treatment and the final opacity was measured after removal of the test or control material.
- Corneal permeability: After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- Calculation of Opacity:
The change of opacity value of each treated cornea with test material, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test material and positive control to obtain a corrected opacity.

Opacity = [(I0 / I) - b] / a

a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
I0 = the empirically determined illuminance through a cornea holder with windows and Medium, here: Io= 1053.69
I = the measured illuminance (unit: LUX)

- Calculation of Permeability:
The corrected OD492 value of each cornea treated with test material and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test material, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

- Calculation of IVIS (In Vitro Irritancy Score):
The IVIS of each replicate of the negative control was calculated from the following equation:

IVIS = opacity difference + (15 x corrected OD492 value)

The IVIS of each replicate of the positive control and of the test material was calculated from the following equation:

IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

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

VALIDITY CRITERIA:
According to the guideline, the test is considered as valid if:
- The positive control causes an IVIS that falls within two standard deviations of the current historical mean.
- The negative control has to show an IVIS ≤ 3.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

45.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

VALIDITY
- The IVIS of the negative control (HBSS) was 0.90, this was ≤ 3 and therefore valid.
- The IVIS of the positive control (20 % imidazole solution) was 92.36, this was within the range of 70.37 – 159.65 and therefore valid.
- Values for negative and positive controls were within the range of historical data of the test facility, the test system was therefore acceptable.

ASSESSMENT
- In the negative control, no signs of eye irritation were observed.
- The positive control induced serious eye damage, which would be classified as GHS category I.
- The test material showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 45.89.
- According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP. Further testing may be required.
- The experiment is considered sufficient, as the resulting classification of the test material was unequivocal, because all three replicates of the test material lead to the same assessment for the test material.

Table 1: Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score*
Negative control	0.49	0.0271	0.90
Positive control	72.16	1.3470	92.36
Test material	8.92	2.4641	45.89

*Calculated using the negative control mean opacity and mean permeability values for the positive control and test material.

In vitro irritancy score (IVIS) = opacity difference + (15 x corrected OD492 value)

Interpretation of results:

other: No prediction of eye irritation can be made.

Conclusions:

Under the conditions of the study no prediction of eye irritation can be made.

Executive summary:

The eye irritation potential of the test material was determined in accordance with the standardised guidelines OECD 437 and EU method B.47, under GLP conditions. The test material was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) Test Method.

One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old. The test material was brought onto the cornea of a bovine eye, which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test material was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test material, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as the negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 0.90. 20 % imidazole solution was used as the positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 92.36.

Under the conditions of this study, the test material showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 45.89.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP. Further testing may be required. Under the conditions of the study no prediction of eye irritation can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2017 to 16 November 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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
- EpiOcular™ (OCL-212-EIT Batch: 27013)
- The EpiOcular™ tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organised basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm².
- 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 (e.g. OECD).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 53.6 (tissue 1) and 52.9 mg (tissue 2).

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours and 5 minutes

Number of animals or in vitro replicates:

The test was performed in duplicate.

Details on study design:

PRE-TESTS
Assessment of Direct Reduction of MTT by the Test Material
- The test material was tested for the ability of direct MTT reduction. To test for this ability, 51.9 mg of the solid test material were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 µL of H2O demin. was used as negative control.
- The colour of the MTT turned blue/purple, the test material is presumed to have reduced the MTT.
- As the mean value of relative tissue viability of the test material was less than the tissue viability cut-off value of 60 % in the main test, no prediction can be made. Therefore a functional test with freeze-killed tissues is not necessary, as a false negative result is already excluded.

Assessment of Coloured or Staining Test Materials
- 53.6 mg of the test material were added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 3 hours at room temperature. Then, two 200 µL aliquots of the resulting solution and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm.
- After subtraction of OD for isopropanol, the OD of the test material solution was -0.001 (≤ 0.08). Therefore, the main test was performed without colourant controls.


MAIN TEST
Preparations
- On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use.
The assay medium was warmed in the water bath to 37 ± 1 °C.
- 6-well-plates were labelled with test material, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 –100 % relative humidity for 1 hour.
- After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.

Exposure and Post-Treatment
- After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 29 minutes. After that, 50 µL of the controls and defined amounts of the test material (53.6 and 52.9 mg) were applied in duplicate in one minute intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one minute intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
- After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours and 5 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
- After the post-treatment incubation, the MTT Assay was performed.

MTT Assay and Extraction
- A 24-well-plate was prepared with 300 µL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 190 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
- At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol flowed into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.

Measurement
- The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation.
- From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

EVALUATION
- The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).

CALCULATION
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test material
(Corrected OD value of negative control corresponds to 100 % viability)

% Viability = OD corrected of test material or positive control / OD corrected of mean negative control) ·100

DECISION CRITERIA
ASSESSMENT AND VALIDITY
Eye irritation is assessed using the following:
> 60 % = Non eye irritant
≤ 60 % = No prediction can be made, category 1 or 2.

Irritation parameter:

other: % Tissue Viability

Run / experiment:

Mean

Value:

47.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MEASURED VALUES
- As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The mean OD570 value was 0.038.
- From the measured absorbance’s, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol. The mean of the three tissues was also calculated.

TISSUE VIABILITY
- For the test material and the positive control, the percentage values of tissue viability were calculated in comparison to the negative control.
- After treatment with the test material, the mean value of relative tissue viability was reduced to 47.1 %.

Validity:
- OD of negative control (must be ≥ 0.8 and ≤ 2.8) was found to be 1.9.
- % mean relative viability of positive control (must be < 50 % of negative control) was found to be 31.7 %.
- Variation within replicates (must be < 20 %) was found to be 1.5 % (negative control), 4.0 % (positive control) and 3.5 % (test material).
- Values for negative control and for positive control were within the range of historical data of the test facility.
- The experiment is considered valid.

Table 1: Mean Absorbance Values

Designation	Negative Control	Positive Control	Test Material
Mean – blank (Tissue 1)	1.890	0.558	0.917
Mean – blank (Tissue 2)	1.861	0.533	0.852

 

Table 2: % Viability Positive Control and Test Material

Designation	Positive Control	Test Material
% Viability (Tissue 1)	29.7	48.9
% Viability (Tissue 2)	33.7	45.4
% Viability Mean	31.7	47.1

Interpretation of results:

other: No prediction of eye irritation can be made.

Conclusions:

Under the conditions of this study, no prediction of eye irritation can be made.

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 492, under GLP conditions using the Reconstructed human Cornea-like Epithelium (RhCE) test method.

One valid experiment was performed.

The test material was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test material was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as the negative control and methyl acetate was used as the positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.9. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 31.7 % (criterion limit < 50 % of negative control). Variation within tissue replicates for all control and test material treatments was acceptable (< 20 %).

After treatment with the test material, the mean value of relative tissue viability was 47.1 %. This value is less than the tissue viability cut-off value of 60 % and therefore no prediction can be made. According to the OECD Guideline 492, the EpiOcular™ Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.

Under the conditions of this study, no prediction of eye irritation can be made.

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion/Irritation

Skin Corrosion In Vitro, Andres (2017)

The potential of the test material to cause corrosion to the skin was determined in vitro, in accordance with the standardised guidelines OECD 431 and EU Method B 40bis, under GLP conditions using the Reconstructed Human Epidermis (RHE) Test Method. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

One valid experiment was performed. In the pre-test the test material showed MTT-reducing properties. The probability to influence the photometric measurement had to be excluded. Therefore an additional test using freeze-killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues was performed. The result of the additional test showed that MTT reduction by the test material did not influence the result of the study.

In the main test two tissues of the human skin model EpiDerm were treated with the test material for 3 minutes and 1 hour, respectively. Demineralised water was used as negative control and 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting formazan solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.9 (3 minutes experiment) and 1.8 (1 hour experiment). The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 7.4 % for the 1 hour treatment. After 3 minutes treatment with the test material, the mean value of relative tissue viability was increased to 106.9 %. This value is above the threshold for corrosion potential (50 %). After 1 hour treatment, mean value of relative tissue viability was increased to 101.4 %. This value, too, is above the threshold for corrosion potential (15 %).

Under the conditions of the study, the test material was considered to be non-corrosive to the skin.

Skin Irritation In Vitro, Andres (2018)

The skin irritation potential of the test material was investigated 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).

One valid experiment was performed. Three tissues of the human skin model EpiDerm™ were treated with the test material for 60 minutes. The test material was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as the negative control and 5 % SDS solution was used as the positive control.

After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2 % (required: ≤20 %). The variation within the tissue replicates of negative, control, positive control and test material was acceptable (required: ≤ 18 %).

After the treatment, the mean value of relative tissue viability was increased to 104.8 %. This value is above the threshold for tissue viability (50 %). 

Under the conditions of this study the test material is considered as non-irritant to skin.

Eye Irritation

Eye Irritation In Vitro, Andres (2017) BCOP

The eye irritation potential of the test material was determined in accordance with the standardised guidelines OECD 437 and EU method B.47, under GLP conditions. The test material was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) Test Method. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old. The test material was brought onto the cornea of a bovine eye, which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test material was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test material, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as the negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 0.90. 20 % imidazole solution was used as the positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 92.36.

Under the conditions of this study, the test material showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 45.89.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP. Further testing may be required. Under the conditions of the study no prediction of eye irritation can be made.

Eye Irritation in vitro, Geitlinger (2018) RhCE

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 492, under GLP conditions using the Reconstructed human Cornea-like Epithelium (RhCE) test method.

One valid experiment was performed.

The test material was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test material was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as the negative control and methyl acetate was used as the positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.9. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 31.7 % (criterion limit < 50 % of negative control). Variation within tissue replicates for all control and test material treatments was acceptable (< 20 %).

After treatment with the test material, the mean value of relative tissue viability was 47.1 %. This value is less than the tissue viability cut-off value of 60 % and therefore no prediction can be made. According to the OECD Guideline 492, the EpiOcular™ Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.

Under the conditions of this study, no prediction of eye irritation can be made.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin corrosion or irritation.

The BCOP and RhCE studies were each inconclusive. Both tests have good sensitivity/specificity to correctly identify no classification substances. Both studies returned high irritation scores (> 45) indicating at least eye irritation is likely but not excluding serious eye damage. Therefore, as a precautionary measure, the substance is classified as a category 1 eye irritant and is assigned the hazard statement H318: Causes serious eye damage.