2-ethoxyethyl ethyl carbonic acid ester

Endpoint summary

Description of key information

Skin Irritation in vitro, Warren (2019)

Under the conditions of this study, the test material was not irritating.

Skin Corrosion in vitro, Warren (2019)

Under the conditions of this study, the test material was not corrosive to the skin.

Eye Irritation in vitro, Henzell (2019)

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

Eye Irritation in vitro, Spohr (2019)

Under the conditions of this study, it can be stated that the test material does not need to be classified for eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2018 to 01 October 2018

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:

updated 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

23 July 2009

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 in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Source: EpiSkin Laboratories, Lyon, France
- Tissue lot number: 18-EKIN-039
- Delivery date: 25 September 2018

TEST FOR DIRECT MTT REDUCTION
- A test material may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test material is checked for the ability to directly reduce MTT according to the following procedure: 10 μL of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 3 hours. Untreated MTT solution was used as a control.
- If the MTT solution containing the test material turns blue/purple, the test material is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed.
- 10 μL of test material was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
- Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Colour Satisfactory: Yes
Agar Medium Colour Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.

MAIN TEST
- Application of Test Material (Day 1): 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm²) of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7-minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5 % CO2 in air for 42 hours.

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

MTT LOADING/ FORMAZAN EXTRACTION (DAY 3)
- Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5 % CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS (DAY 6)
- At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
- Quantitative MTT Assessment (Percentage Tissue Viability): For the test material the relative mean tissue viabilities obtained after the 15-minute exposure period followed by the 42-hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of the test material / mean OD570 of the negative control) x 100
- Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to the following:
Relative mean tissue viability is ≤ 50 %: Irritant (H314 or H315 Category 1 or 2)
Relative mean tissue viability is > 50 %: Non-irritant (Not classified for irritation)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL (26.3 μL/cm²)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5 % w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours followed by 3 hours with MTT

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.

ASSESSMENT OF THE COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test material was colourless. It was therefore unnecessary to run colour correction tissues.

MAIN TEST RESULTS
- The relative mean viability of the test material treated tissues was 97.3 % after a 15-minute exposure period and 42-hour post-exposure incubation period.
- It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

QUALITY CRITERIA
- The relative mean tissue viability for the positive control treated tissues was 5.2 % relative to the negative control treated tissues and the standard deviation value of the viability was 2.7 %. The positive control acceptance criteria were therefore satisfied.
- The mean OD570 for the negative control treated tissues was 0.706 and the standard deviation value of the viability was 8.3 %. The negative control acceptance criteria were therefore satisfied.
- The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 6.8 %. The test material acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Treatment	OD570 of Tissues	Mean OD570 of Triplicate Tissues	± SD of OD570	Relative Individual Tissue Viability (%)	Relative Mean Viability (%)	± SD of Relative Mean Viability (%)
Negative Control	0.642	0.706	0.059	90.9	100*	8.3
	0.720			102.0
	0.757			107.2
Positive Control	0.056	0.037	0.019	7.9	5.2	2.7
	0.036			5.1
	0.018			2.5
Test Material	0.742	0.687	0.048	105.1	97.3	6.8
	0.656			92.9
	0.663			93.9

OD = Optical Density

SD = Standard deviation

∗= The mean viability of the negative control tissues is set at 100 %

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was not irritating.

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.

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm.

The relative mean viability of the test material treated tissues was 97.3 % after the 15-minute exposure period and 42-hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was not irritating.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September 2018 to 21 September 2018

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: Method B.40bis of Commission Regulation (EC) No 440/2008

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

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Source: MatTek
- Tissue lot number(s): 28655
- Delivery date: 18 September 2018
- The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.

TEST FOR DIRECT MTT REDUCTION
- A test material may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT according to the following procedure: 50 μL of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
- If the MTT solution containing the test material turned blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured or if it becomes coloured when in wet or aqueous conditions. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed.
- 50 μL of test material was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.

MAIN TEST
PRE-INCUBATION
- The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-minute and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

APPLICATION
- Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-minute and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24-well plate was prepared for the MTT-loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-minute exposure period was returned to the incubator, while the other was being dosed for the 60-minute exposure. For the 60-minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 μL of the test material and 50 μL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5 % CO2) for the 60-minute exposure period. When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading.

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The 3 minute plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60-minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
- After the 3-hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24-well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.
- After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.

NUMBER OF REPLICATE TISSUES: 2

DATA EVALUATION
- Quantitative MTT Assessment (percentage tissue viability): The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3 and 60-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of the test material / mean OD570 of negative control) x 100
- Classification of corrosivity potential is based on relative viabilities for both exposure times according to the following:
STEP 1:
< 50 % viability after 3 min exposure: Corrosive
≥ 50 % viability after 3 min exposure AND < 15 % viability after 60 min exposure: Corrosive
≥ 50 % after 3 min exposure AND ≥ 15 % after 60 min exposure: Non-corrosive
STEP 2 for test materials identified as corrosive in step 1:
< 25 % viability after 3 min exposure: H314; Sub-category 1A
≥ 25 % after 3 min exposure: H314; Combination of sub-categories 1B-and-1C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied:50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes and 60 minutes

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 period

Value:

102.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

MAIN STUDY
The relative mean viabilities for test material treated tissues were as follows:
3 minute exposure: 102.8 %
60 minute exposure: 61.0 %

QUALITY CRITERIA
- The mean OD570 for the negative control treated tissues was 1.980 for the 3-minute exposure period and 2.076 for the 60-minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 3.5 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Treatment	Exposure Period (mins)	Mean OD570 of Individual Tissues	Mean OD570 of Duplicate Tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3	1.958	1.980	0.031	1.6	100*
		2.002
	60	1.871	2.076	0.289	13.9
		2.280
Positive Control	3	0.094	0.091	0.004	na	4.6
		0.088
	60	0.085	0.073	0.018	na	3.5
		0.060
Test Material	3	1.992	2.035	0.061	3.0	102.8
		2.078
	60	1.343	1.266	0.109	8.6	61.0
		1.189

OD = Optical Density

∗= The mean viability of the negative control tissues is set at 100 %

na = Not applicable

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was not corrosive to the skin.

Executive summary:

The skin corrosion potential of the test material was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, under GLP conditions.

The purpose of this test was to evaluate the corrosivity potential of the test material using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test material.

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

After a 3 minute exposure the mean percentage viability of the test material treated tissues was 102.8 %. After a 60 minute exposure the mean percentage viability of the test material treated tissues was 61.0 %.

The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was not corrosive to the 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:

06 November 2018 to 29 November 2018

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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- EpiOcular™ kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiOcular™ tissue consists of normal, human-derived epidermal 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 which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts.
- EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (28 November 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
- Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70 % isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50 % of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2) overnight (about 17 hours).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 μL (83.3 μL/cm² according to guideline)

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

- 12 minute post-soak immersion incubation
- 120 minutes post-treatment incubation
- 180 minutes with MTT

Number of animals or in vitro replicates:

2

Details on study design:

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST MATERIAL
- Test materials may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test material prior to conducting any assays with viable tissues. For this purpose approximately 50 μL of the test material were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test material will be presumed to have reduced the MTT.
- Since the MTT solution colour did not turn blue/purple, the test material is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
- Coloured test materials or test materials which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colourant bound to the tissue and would be extracted together with MTT. Therefore, each test material had to be checked for its colouring properties
- Since the test material was non-coloured, additional tests had to be performed to assess, if it becomes coloured after contact with water or isopropanol. For this purpose, 50 μL of the test material were added to 2.0 mL of isopropanol (glass tube) and shaken for 2 to 3 hours at room temperature. 2.0 mL of isopropanol was used as control. Additionally 50 μL of the test material were added to 1.0 mL of deionised water (glass tube) and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for at least 1 hour. 1 mL of deionised water was used as control.
- According to guideline the absorbance of all samples was measured in duplicates at 570 nm (OD570) with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.
- Since the test material did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.

PERFORMANCE OF THE EXPERIMENT
- After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 30 minutes.
- After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control materials were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
- At the end of the 30 minutes treatment time, the test material was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test material. Each test material utilised a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control materials were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beaker of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test material absorbed into the tissue.
- At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 (post-treatment incubation).

MTT ASSAY
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
- Since the test material was colourless inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2 mL isopropanol in each well so that isopropanol was flowing into the insert. The plates were sealed with parafilm and a standard plate sealer, and were immediately extracted. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The tissues were pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically with piercing.
- The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
- The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

DATA EVALUATION
1) The mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) The mean value of the two replicates for each tissue was calculated.
3) The mean ODBlk from each mean OD value of the same experiment was subtracted (blank corrected values).
4) The mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test material (TM) was calculated (ODTM, ODNC, ODPC).
5) The mean OD value of the negative control corresponds to 100 % viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100 % Viability

Calculations for Viability Tests only
1) The percent viability of each of the two relating tissues for each control and test material relative to the negative control (= 100%) was calculated:
Viability (%) = 100. [(ODTM/ODPC/ODNC) / meanODNC]
2) The difference of the viability between duplicate tissues was calculated. If the difference is > 20 % the test is considered as non-qualified.
3) The mean test material viability (TM viability) was calculated and the test material was classified according to the prediction model.

Prediction Model
- If the test material-treated tissue viability is > 60 % relative to the negative control treated tissue viability, the test material is identified as not requiring classification and labelling according to UN GHS (No Category).
- If the test material-treated tissue viability is ≤ 60 % relative to negative control treated tissue viability, no prediction can be made from this result in isolation and requires additional information for classification purposes.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60 ± 5 %, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

Acceptability of the Assay
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50 % of the negative control viability.
3) The difference of viability between the two relating tissues of a single test material is < 20 % in the same run (for positive and negative control tissues and tissues of test materials). This applies also to the freeze-killed tissues (materials and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
The positive and negative control data shall fall within the historical control data.

Irritation parameter:

other: Mean absorbance value

Run / experiment:

Test material mean

Value:

81.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The optical pre-experiment (colour interference pre-experiment) to investigate the test material’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.
- Optical evaluation of the MTT-reducing capacity of the test material with MTT-reagent did not show blue/purple colour. Therefore, an additional test with freeze-killed tissues was not necessary.

MAIN EXPERIMENT
- The mean absorbance value of the test material, corresponding to the relative cell viability, decreased to 81.32 % (threshold: ≤ 60 %), consequently the test material is considered as “No Category” according to UN GHS.
- Concerning acceptance criteria:
The negative control OD is > 0.8 and < 2.5 (2.167 and 2.243).
The tissue viability of the positive control is below 50 % of the negative control viability (42.88 %).
- The difference of viability between the two relating tissues of a single material is < 20 % (values between 1.46 p.p and 7.56 p.p) in the same run (for positive and negative control tissues and tissues of single test materials).

Table 1: Results of the Main Experiment

Test Group	Tissue No.	Well 1 [OD570]	Well 2 [OD570]	Mean [OD570] (Well 1 and well 2)	Mean [OD570] blank corr. (Well 1 and well 2)	Mean [OD570] of T1 and T2	Tissue viabil.* [%]	rel. viabil. of T1 and T2**	Diff. of viabil. between T1 and T2 [p.p.]
Blank		0.035	0.035	0.035
Negative Control	1	2.243	2.192	2.218	2.182	2.158	100.0	101.2	2.30
	2	2.169	2.167	2.168	2.133			98.8
Positive Control	1	1.062	1.021	1.042	1.007	0.925	42.88	46.7	7.56
	2	0.892	0.865	0.879	0.844			39.1
Test Material	1	1.828	1.783	1.806	1.770	1.755	81.32	82.1	1.46
	2	1.804	1.744	1.774	1.739			80.6

* Tissue viability = (100 × (meanOD of T1&T2)(test material/ positve control/ negative control)/ (meanOD of T1&T2)(negative control)

** Relative Tissue viability = (100 × (meanOD blank corrected)(test material/ positve control/ negative control)/ (meanOD of T1&T2)(negative control)

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study, it can be stated that the test material does not need to be classified for eye irritation.

Executive summary:

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

This in vitro study was performed to assess the eye irritation potential of the test material by means of the Human Cornea Model Test. The test material did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 μL of the test material, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50 % viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20 % in the same run (for test material tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the test material. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60 % (determined value for the test material: 81.32 %).

Under the conditions of this study, it can be stated that the test material does not need to be classified for eye irritation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 October 2018

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)

Version / remarks:

2017

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
- Source: Bovine eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control item.

Details on study design:

PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

TREATMENT METHOD
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the test material or control item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.
At the end of the exposure period the test material and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

PERMEABILITY DETERMINATIONS
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.
No histopathology was required for this study.

DATA EVALUATION
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IN VITRO IRRITANCY SCORE
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

VISUAL OBSERVATION
The condition of the cornea was visually assessed post treatment and post incubation.

DATA INTERPRETATION
The test material was classified according to the following prediction model:
IVIS ≤ 3: No Category (UN GHS)
IVIS >3; ≤ 55: No prediction can be made (UN GHS)
IVIS > 55: Category 1 (UN GHS)

CRITERIA FOR AN ACCEPTABLE TEST
- For an acceptable test the following positive control criterion should be achieved: Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
- For an acceptable test the following negative control criteria should be achieved: Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values calculated from the previous 12 months data for this testing facility.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

5.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

A summary of the results can be seen in Table 1.

- Corneal Epithelium Condition: The corneas treated with the test material were slightly cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

- Criteria for an Acceptable Test:
The positive control In Vitro Irritancy Score was within the range of 29.6 to 65.1. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤ 2.3 and permeability ≤ 0.41. The negative control acceptance criteria were therefore satisfied.

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 investigated in a study which was conducted in accordance with the standardised guidelines OECD 437 and EU Method B.47, under GLP conditions.

During the study, the undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The corneas treated with the test material were slightly cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

The In Vitro irritancy score for the test material was determined to be 5.8. The positive control In Vitro Irritancy Score was within the range of 29.6 to 65.1. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 2.3 and permeability ≤ 0.41. The negative control acceptance criteria were therefore satisfied.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation in vitro, Warren (2019)

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).

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm.

The relative mean viability of the test material treated tissues was 97.3 % after the 15-minute exposure period and 42-hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was not irritating.

Skin Corrosion in vitro, Warren (2019)

The skin corrosive potential of the test material was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, 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 purpose of this test is to evaluate the corrosivity potential of the test material using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test material.

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

After a 3 minute exposure the mean percentage viability of the test material treated tissues was 102.8 %. After a 60 minute exposure the mean percentage viability of the test material treated tissues was 61.0 %.

The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was not corrosive to the skin.

Eye Irritation in vitro, Henzell (2019)

The eye irritation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 437 and EU Method B.47, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The corneas treated with the test material were slightly cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

The In Vitro irritancy score for the test material was determined to be 5.8. The positive control In Vitro Irritancy Score was within the range of 29.6 to 65.1. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 2.3 and permeability ≤ 0.41. The negative control acceptance criteria were therefore satisfied.

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

Eye Irritation in vitro, Spohr (2019)

The eye irritation potential of 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).

This in vitro study was performed to assess the eye irritation potential of the test material by means of the Human Cornea Model Test. The test material did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 μL of the test material, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50 % viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20 % in the same run (for test material tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the test material. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60 % (determined value for the test material: 81.32 %).

Under the conditions of this study, it can be stated that the test material does not need to be classified for eye irritation.

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 or eye irritation.