Diboron calcium tetraoxide

Endpoint summary

Description of key information

Skin corrosion/irritation

An in vitro EpiDerm™ human skin model was used to evaluate the skin corrosivity potential of the test item after treatment periods of 3 and 60 minutes. Duplicate tissues were treated with the test item for the 3 and 60-minute exposure periods, as well as negative and positive control groups. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading followed by MTT extraction. The optical density (OD) was measured at 570 nm (OD570). The acceptance criteria required for the test were satisfied and the test item was considered to be non-corrosive to the skin.

An in vitro EpiSkin™ reconstructed human epidermis model was used to evaluate the skin irritation potential of the test item after triplicate tissues were treated for 15 minutes and a post-exposure incubation period of 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading, followed by extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 570 nm. The acceptance criteria required for the test were satisfied and under the experimental conditions the test item was classified as non-irritant to the skin.

Eye irritation

An in vitro Bovine Corneal Opacity and Permeability (BCOP) test method was used to evaluate the potential of the test item to induce serious eye damage or to identify the test item as not requiring classification for eye irritation or serious eye damage. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. In runs 1 and 2 the test item was applied at a concentration of 20 % w/v in sodium chloride 0.9 % w/v for 240 minutes. In runs 3 and 4 the test item was applied neat for 240 minutes. In runs 5 and 6 the test item was applied at a concentration of 40 % w/v in sodium chloride 0.9 % w/v for 240 minutes. Negative and positive control items were tested concurrently. Under the conditions of the test, the overall conclusion is that the test item is given No Prediction under UN GHS classification.

An EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method was used to evaluate the potential for the test item not requiring classification and labelling for eye irritation or serious eye damage. Duplicate tissues were treated with the test item for an exposure period of 6 hours and a post-exposure incubation of 18 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading followed by MTT extraction. The optical density (OD) was measured at 570 nm (OD570). The relative mean viability of the test item treated tissues was 69.2 %. The criteria required for acceptance of results in the test were satisfied and the test item was classified as non-irritant under EU CLP and UN GHS No Category.

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:

4 December 2019 to 6 December 2019

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)

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, 18 December 2006, of the European Parliament and of the Council on REACH

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E00616-912
- Expiration date of the lot/batch: 18 September 2020
- Purity: 82 %

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human skin

Details on animal used as source of test system:

TEST SYSTEM:
- EpiDerm™ Reconstructed Human Epidermis Model Kit:
Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 03 December 2019
EpiDermTM Tissues (0.63cm2) lot number: 30838
Assay Medium lot number: 112819MJC

Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

Justification for test system used:

The purpose of this test was to evaluate the corrosivity potential of the test item 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 item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-TEST PROCEDURES:
- Assessment of Direct Test Item Reduction of MTT:
The MTT assay, a colourimetric 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. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

To identify this possible interference, 25 mg of the test item 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 item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT.

- Assessment of Colour Interference with the MTT Endpoint:
A test item 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 item is present in the tissues when the MTT viability assay is performed. 25 mg of test item 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 of Test Item and Rinsing:
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. 25 mg of the test item and 50 µL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact. 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. 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 item. 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. The 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.

- Absorbance/Optical Density Measurements:
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. The microplate reader upper limit of accuracy for measured absorbance of MTT Formazan at 570 nm (OD570), filter band pass 10 nm, was determined to be at an optical density of 2.6.

DATA EVALUATION:
- Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test item 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 test item)/(mean OD570 of negative control)] x 100

ACCEPTANCE CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved.

- Negative Control:
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be >=0.8 and <=2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.

- Positive Control:
Potassium hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-minute positive control is <15 %.

- Coefficient of Variation:
In the range 20 and 100 % viability, the coefficient of variation between tissue replicates should be <=30 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg test item and 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact

NEGATIVE CONTROL
- Amount(s) applied: 50 µL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied: 50 µL of 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-Minute exposure period

Value:

97

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

4.3 %

Remarks on result:

other: Non-corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-Minute exposure period

Value:

104.5

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

3.8 %

Remarks on result:

other: Non-corrosive

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple, this was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become coloured, this was taken to indicate the test item did not have the potential to cause colour interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 2.162 for the 3-minute exposure period and 2.058 for the 60-minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 0.078 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: 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.

Item	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative control	3 minutes	2.179 2.145	2.162	0.024	1.1	100
Negative control	60 minutes	1.984 2.131	2.058	0.104	5.1	100
Positive control	3 minutes	0.104 0.081	0.093	0.016	Not applicable	4.3
Positive control	60 minutes	0.089 0.067	0.078	0.016	Not applicable	3.8
Test item	3 minutes	2.022 2.171	2.097	0.105	5.0	97.0
Test item	60 minutes	2.104 2.196	2.150	0.065	3.0	104.5

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test was to evaluate the corrosivity potential of the test item 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 item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Duplicate tissues were treated with the test item 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 item 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).

The data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Negative control (3 minute exposure): 100 %

Positive control (3 minute exposure): 4.3 %

Test item (3 minute exposure): 97.0 %

Negative control (6 minute exposure): 100 %

Positive control (6 minute exposure): 3.8 %

Test item (6 minute exposure): 104.5 %

The acceptance criteria required for the test were satisfied and the test item 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:

16 December 2019 to 20 January 2020

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E00616-912
- Expiration date of the lot/batch: 18 September 2020
- Purity: 82 %

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Details on animal used as source of test system:

TEST SYSTEM:
- EPISKIN™ Reconstructed Human Epidermis Model Kit:
Supplier: EpiSkin Laboratories, Lyon, France
Date received: 14 January 2020
EpiSkinTM Tissues (0.38cm2) lot number: 20-EKIN-003
Maintenance Medium lot number: 20-MAIN3-002
Assay Medium lot number: 20-ESSC-002

Justification for test system used:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EpiSkinTM 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 is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

The EpiSkinTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between irritating and non-irritating test items. Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-TEST PROCEDURES:
- Assessment of Direct Test Item Reduction of MTT:
The MTT assay, a colourimetric 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. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

To identify this possible interference, 10 mg of the test item 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 item turns blue/purple, the test item 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.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues. Water-killed tissues were prepared prior to the study by placing untreated EpiSkinTM tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5 % CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-35 to -10 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour in an incubator.

In addition to the normal test procedure, the MTT reducing test item was applied to three water-killed tissues and three water-killed tissues remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

- Assessment of Colour Interference with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is coloured or becomes coloured when in contact with water. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 mg of test item 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 item and each control item. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.

MAIN TEST:
- Application of Test Item and Rinsing (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 item for an exposure period of 15 minutes. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface ensuring uniform covering. 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, 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 item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. 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 item. 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.

- 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 (-35 to -10 ºC) 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 EpiSkinTM 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 2 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. The microplate reader upper limit of accuracy for measured absorbance of MTT Formazan at 570 nm (OD570), filter band pass 10 nm, was determined to be at an optical density of 2.6.

DATA EVALUATION:
- Quantitative MTT Assessment (Percentage Tissue Viability):
For the test item 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 test item)/(mean OD570 of negative control)] x 100

ACCEPTANCE CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved.

- Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is <=40 % relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is <=18 %.

- Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is >=0.6 and <=1.5, and the SD value of the percentage viability is <=18 %.

- Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is <=18 %.

DEVIATIONS FROM STUDY PLAN:
The following deviations from the study plan occurred; however, these deviations were considered to have not affected the integrity or validity of the study.

- An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

- The water killed frozen tissues used for the MTT correction were removed and placed in the incubator to thaw. The general study plan states that the tissues will be thawed at room temperature and not in an incubator. The tissues thawed, used and worked adequately and showed no interference therefore weren’t required for qualitative correction.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg test item and 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis

NEGATIVE CONTROL
- Amount(s) applied: 10 µL of Dulbecco’s Phosphate Buffered Saline

POSITIVE CONTROL
- Amount(s) applied: 10 µL of sodium dodecyl sulphate

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-Minute exposure period and 42-hour post-exposure incubation period

Value:

98.1

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

5.6 %

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: The solution containing the test item was colourless, it was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.810 and the standard deviation value of the viability was 5.1 %. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.6 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.0 %. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 10.2 %. The test item acceptance criterion was therefore satisfied.

Item	Mean OD570 of triplicate tissues	± SD of OD570	Relative Mean Viability (%)	± SD of Relative Mean Viability (%)
Negative control	0.810	0.041	100	5.1
Positive control	0.045	0.008	5.6	1.0
Test item	0.794	0.082	98.1	10.2

Interpretation of results:

GHS criteria not met

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as non-irritant.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EpiSkin^TM 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 is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5 -dimethylthiazol-2 -yl]-2,5 -diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 98.1 % after the 15 minute exposure period and 42 hours post-exposure incubation period. The acceptance criteria required for the test were satisfied and under the experimental conditions reported, the test item was classified as non-irritant.

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:

12 December 2019 to 10 March 2020

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Historical Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160. Adopted July 6, 2018 Paris

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E00616-912
- Expiration date of the lot/batch: 18 September 2020
- Purity: 82 %

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
- Source of Bovine Eyes:
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 (per experiment). The corneas were prepared immediately on arrival.

Vehicle:

other: Runs 1 and 2: 20 % w/v concentration in a 0.9 % sodium chloride solution; runs 3 and 4: neat solid; runs 5 and 6: 40 % w/v concentration in a 0.9 % sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied runs 1, 2, 5 and 6: 0.75 mL/cornea
- Amount(s) applied runs 3 and 4: 0.6 g/cornea
- Concentration runs 1 and 2: 20 % w/v solution in sodium chloride 0.9 % w/v
- Concentration runs 3 and 4: Not applicable (neat solid)
- Concentration runs 5 and 6: 40 % w/v solution in sodium chloride 0.9 % w/v

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

Triplicates for each run

Details on study design:

STUDY DESIGN:
- Preparation of Corneas:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. 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 ºC 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. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

- Treatment of Corneas:
For runs 1, 2, 5 and 6 the EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

For runs 3 and 4 the EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.6 g of the solid test item was found to adequately cover the corneal surface. 0.75mL of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

At the end of the 4-hour exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed at least three 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.

- Application of Sodium Fluorescein:
Following the 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 (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost at 32 ± 1 ºC 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.

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. Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

- Visual Observation:
The condition of the cornea was visually assessed post treatment.

CRITERIA FOR AN ACCEPTABLE TEST:
For an acceptable test the following positive control criterion should be achieved. 20 % w/v Imidazole 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:

1

Value:

109.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

large standard deviation in opacity readings

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

32.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

4.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Irritation parameter:

in vitro irritation score

Run / experiment:

4

Value:

2.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

5

Value:

7.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Irritation parameter:

in vitro irritation score

Run / experiment:

6

Value:

14

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Corneal Epithelium Condition:
When the test item was applied as a 20 % w/v formulation the corneas had opaque patches/cloudiness post treatment. When the test item was applied neat the corneas had small areas of test item adherence post treatment. When the test item was applied as a slurry of 40 % w/v formulation the corneas had partial cloudiness post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Across the three corneas exposed to the test item a large standard deviation in opacity was noted in run 1 (74.7, 204.7, 31.7), and therefore, even though there appeared to be evidence of severe eye irritation in two of the three corneas, the discordant results could not be used as sufficient evidence to classify the test item (IVIS score 109.9). The results obtained in the second run show a lower prediction and overall lower IVIS value (IVIS score 32.2) than in the first run; however, there were still relatively large standard deviations between individual opacity readings (37.3, 16.3, 39.3) indicating that there may have been an issue with an uneven distribution of the test item when suspended at 20 % w/v in saline. Owing to the lack of reproducibility of responses over the first two BCOP runs it was difficult to make a conclusion regarding classification of the substance at this time.

Consequently, the test item was applied neat to the corneas. After applying the test item neat in runs 3 and 4 it would be expected that this method would have exhibited the most severe eye irritation response. However, the results did not align with this prediction and it was apparent that the irritation potential was reduced using this method (run 3 IVIS score 4.6 and run 4 IVIS score 2.1). However, the test item adhered to the corneas, which in turn could have affected the reliability of the opacity readings and consequently the IVIS score, although it is anticipated that this would result in a higher IVIS score. The lowered IVIS score in runs 3 and 4 implied that the test item had the potential to cause irritation with the presence of moisture (when suspended as a formulation), as seen in runs 1 and 2.

In runs 5 and 6 it became important to exhibit the irritation potential of the test item using a slurry/open chamber methodology. The application of the test item slurry to the cornea ensured that sufficient moisture was present to allow any potential chemical reaction to take place and most importantly ensure an even distribution of the formulation across the corneal surface. Also the benefit of this method was shown to prevent test item adherence at the washing phase, which in turn improved the reliability of the opacity readings (run 5 IVIS score 7.4 and run 6 IVIS score 14.0).

With the range of results obtained over the course of six BCOP runs, it was apparent that the most appropriate classification would be ‘No Prediction’ under UN GHS classification. As a neat substance, minimal irritation effects were observed (run 3 IVIS score 4.6 and run 4 IVIS score 2.1); however, the test item adhered to the corneas, potentially impacting the scores. Increased irritation effects were observed in runs 5 and 6, although these effects were close to the UN GHS IVIS classification threshold of 3. The results of runs 5 and 6 highlight potential increased irritation of the test item when in contact with moisture. Therefore, it was considered appropriate that further testing would be required to reach a classification using other appropriate testing methods.

Interpretation of results:

other: No prediction can be made

Conclusions:

Run 1:
Inconclusive - No conclusion can be made.

Runs 2, 3, 5 and 6:
No prediction of eye irritation can be made.

Run 4:
No category. Not requiring classification to UN GHS.

Under the conditions of the test, the overall conclusion is that the test item is given No Prediction under UN GHS classification.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage, as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS). Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

In runs 1 and 2 the test item was applied at a concentration of 20 % w/v in sodium chloride 0.9 % w/v for 240 minutes. In runs 3 and 4 the test item was applied neat for 240 minutes. In runs 5 and 6 the test item was applied at a concentration of 40 % w/v in sodium chloride 0.9 % w/v for 240 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).

Under the conditions of the test, the overall conclusion is that the test item is given No Prediction under UN GHS classification.

Summarised below are the In Vitro Irritancy Scores and conclusions of each run:

Run 1 - 20 % saline

Treatment	In Vitro Irritancy Score
Test Item	109.9
Negative Control	0.4
Positive Control	97.3

Run 2 - 20 % saline

Treatment	In Vitro Irritancy Score
Test Item	32.2
Negative Control	0.7
Positive Control	97.3

Run 3 - Neat

Treatment	In Vitro Irritancy Score
Test Item	4.6
Negative Control	1.4
Positive Control	97.6

Run 4 - Neat

Treatment	In Vitro Irritancy Score
Test Item	2.1
Negative Control	1.8
Positive Control	98.9

Run 5 - 40 % w/v saline

Treatment	In Vitro Irritancy Score
Test Item	7.4
Negative Control	1.8
Positive Control	98.9

Run 6 - 40 % w/v saline

Treatment	In Vitro Irritancy Score
Test Item	14.0
Negative Control	1.8
Positive Control	98.9

Run 1:

Inconclusive - No conclusion can be made.

Runs 2, 3, 5 and 6:

No prediction of eye irritation can be made.

Run 4:

No category. Not requiring classification to UN GHS.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 April 2020 to 29 May 2020

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)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E00616-912
- Expiration date of the lot/batch: 18 September 2020
- Purity: 82 %

Species:

human

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
- EpiOcularTM Human Corneal Model (0.6 cm2)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Date received: 21 April 2020
EpiOcularTM Tissues Lot Number: 30655
Assay Medium Lot Number: 042020ISA

- MTT-Solution:
MTT concentrate and MTT diluent were supplied as an MTT test kit (MTT-100). An MTT solution was prepared when required. MTT concentrate was diluted in MTT diluent (2 mL of concentrate to 8 mL diluent) to produce a 1.0 mg/mL MTT solution and used within 1 hour.

- Miscellaneous Assay Reagents:
DPBS (without Ca++ Mg++) Lot Number: 2113984
Isopropanol (MTT) extractant) Lot Number: 1863832

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

Duplicates

Details on study design:

PRE-TEST PROCEDURE:
- Assessment of Direct Test Item Reduction of MTT:
A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. Therefore, it was necessary to assess this ability of the test item to directly reduce MTT prior to conducting the assay. This property of the test item is only a problem, if at the time of the MTT test (after the test item has been rinsed off) there is still a sufficient amount of the test item present on (or in) the tissues. In this case the (true) metabolic MTT reduction and the (false) direct MTT reduction can be differentiated and quantified by the procedure described as follows:

50 mg of the test item was added to 1 mL of MTT solution and incubated at 37 °C, 5 % CO2 for 3 hours. A control (50 µL sterile water in MTT solution) was run concurrently. If the MTT solution turned blue/purple, the test item was presumed to have directly reduced the MTT.

- Assessment of Colour Interference with the MTT endpoint:
Coloured test items or those which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement if the colourant binds to the tissue and is extracted together with MTT. Therefore, the test item was checked for its colourant properties. Test items which absorb light and appear red, yellow, green or blue should be considered as intrinsic colourants. A test item which appears black may absorb light and should be considered as a colourant. Blue, purple and black test items may be directly tested on colourant controls without further tests because it is obvious that they can interfere with the blue/purple MTT product. Such test items should also be tested on killed controls because it may not be possible to assess their potential to directly reduce MTT. For non-coloured test items additional tests have to be performed to assess if they become colourants after contact with water or isopropanol. For this purpose 50 mg of the test item was added to 1.0 mL of water in a 6-well plate and the mixture was incubated in the dark at 37 ±1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air for at least 1 hour. Furthermore, 50 mg of the solid test item was added to 2 mL of isopropanol, the same amount as used for MTT extraction, incubated in 6 well plates, and placed on an orbital plate shaker for 3 hours at room temperature.

- Preparation and Pre-Incubation of EpiOcular Tissues:
Upon receipt of the EpiOcularTM tissues, the sealed 24-well plate and the assay medium were placed into the refrigerator (2 to 10 °C) until the equilibration step. The vial containing the MTT concentrate was placed in the freezer (-35 to -10 °C) and the MTT diluent placed in the refrigerator (2 to 10 °C). The positive control, methyl acetate, was stored at room temperature, in the dark. On the day of receipt the equilibration step (15 minutes at room temperature in the 24-well shipping container) was started. An appropriate volume of EpiOcular™ Assay medium was warmed to approximately 37 °C and 1 mL of the medium 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 ethanol soaked tissue paper. The sterile gauze was removed and each tissue 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 container 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 1 hour in Assay Medium. After 1 hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 to 24 hours).

MAIN TEST:
- Application of Test Item and Rinsing:
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++ Mg++ free DPBS to mimic the wet condition of the human eye. If the Ca++ Mg++ free DPBS was not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at 37 °C, 5 % CO2 for 30 ± 2 minutes.

50 mg of test item was applied atop duplicate cultures for an exposure period of 6 hours ± 15 minutes at 37 °C, 5 % CO2 followed by rinsing, a post-treatment immersion and a post-treatment incubation. 50 µL of the negative and positive controls were similarly applied. At the end of the test item exposure period, the test item was removed by extensively rinsing the tissues with Ca++ Mg++ free DPBS at room temperature. Three clean beakers (glass or plastic with minimum 150 mL capacity), containing a minimum of 100 mL each of Ca++ Mg++ free DPBS were used per test item or control with each test item or control item utilizing a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. The tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent paper towel 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 inserts were then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent paper. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent paper (to break the surface tension). Note that it was not possible to remove all the visible test item completely. No further rinsing was performed.

After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labelled 12-well plate for a 25 ± 2 minutes immersion incubation (post-treatment immersion) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue. At the end of the post-treatment immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, the insert was blotted on absorbent paper, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 18 hours ± 15 minutes at 37 °C, 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 paper. The tissues were placed into the 24-well plate containing 0.3 mL of 1.0 mg/mL MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated at 37 °C, 5 % CO2 in air for 3 hours. A procedure was used which only extracted from beneath the tissue, since residual test item may remain on the tissue and could contaminate the isopropanol. Inserts were removed from the 24-well plate after approximately 3 hours. The bottom of the insert was blotted on absorbent paper and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol flowed into the insert. The plates were sealed with a film sealer (between the plate cover and upper edge of the wells) and shaken for 2 – 3 hours to extract the formazan.

- Absorbance/Optical Density Measurements:
At the end of the extraction period, using a pipette fitted with a 1000 µL tip, the extraction solution was forced vigorously up and down to thoroughly mix. The tissues and empty inserts were discarded.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. 200 µL of isopropanol alone was added to eight wells designated as ‘blanks’. All wells were examined and any air bubbles were removed. The absorbance at 570 nm (OD570) of each well was measured using the LabTech LT-4500 microplate reader and LT-com analysis software.

The plate reader LT-com analysis software was set to correct for blanks and calculate the mean OD570 values of the duplicate wells representing each tissue. The mean OD570 values of the duplicate tissues were manually calculated.

DATA EVALUATION:
The relative mean tissue viabilities were compared to the mean of the negative control (n=2) treated tissues. The relative mean tissue viabilities were calculated as follows:

Relative mean tissue viability = [(mean OD570 of test item)/(mean OD570 of negative control)] x 100

ACCEPTABILITY CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

1) The negative control OD is > 0.8 and < 2.8.
2) The mean relative viability of the positive control is below 50 % of the negative control viability for either the 30 minutes or 6 hours exposures.
3) The difference of viability between the two relating tissues of a single group of duplicate tissues is <20 % in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (test items and negative killed control) and the colourant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: % relative mean viability

Value:

69.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT Reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Assessment of Colour Interference with the MTT endpoint: The water and isopropanol solutions were colourless. It was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Item	Mean OD570 of duplicate tissues	Relative mean viability (%)	Difference in viability (%)
Negative control item	1.619	100	15.5
Positive control item	0.402	24.8	3.6
Test item	1.120	69.2	6.3

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test item treated tissues was 69.2 % after a 6-hour exposure period and 18-hour post-exposure incubation period. It was noted that not all the visible test item could be removed by rinsing. There was no indication that this residual test item interfered with the results of the assay. The test item was classified as non-irritant and the following classifications apply: EU CLP and UN GHS No Category.

Executive summary:

The purpose of this study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

Duplicate tissues were treated with the test item for an exposure period of 6 hours. At the end of the exposure period each tissue was rinsed before incubating for 18 hours. At the end of the post-exposure incubation period 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 duplicate 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).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 69.2 %. The criteria required for acceptance of results in the test were satisfied. The test item was classified as non-irritant and the following classifications apply: EU CLP and UN GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

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 and eye irritation or corrosion.