[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 April 2020 - 10 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The weight of evidence approach taken is explained in the provided endpoint summary.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

other: Weight of evidence

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2020 - 17 July 2020

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:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Justification for test system used:

According to OECD guideline 431

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm Reconstucted Human Epidermis
Tissue batch number: 30867
CoA signed: 20 May 2020


TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment / exposure: 37 °C, 5% CO2


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 item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentration: A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to usage.
Incubation time: 3-Minute and 60-Minute exposure periods
Spectrophotometer: 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:
Two replicate tissues



PREDICTION MODEL / DECISION CRITERIA
Viability Measured after Exposure Time Points: Prediction to be considered according to EU CLP Regulation (EC) No 1272/2008 UN GHS

STEP 1
< 50% after 3 min exposure : Corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure: Corrosive
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure: Non-corrosive

STEP 2 for test items identified as corrosive in step 1
< 25% 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:

For test item, positive and negative control: 50 µL applied

Duration of treatment / exposure:

3-Minute and 60-Minute exposure periods

Number of replicates:

Two replicate tissues for the test item, positive and negative control

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute (mean)

Value:

84.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute (mean)

Value:

62.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD570 for the negative control treated tissues was 2.005 for the 3-Minute exposure period and 2.062 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 2.9% 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.

The relative mean viabilities for each treatment group were as follows:

 

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.3	84.3
60 minute	100*	2.9	62.5

 

 

 

Mean OD570values and viabilities for the negative control, positive control and test item are given in Appendix 1 (appended to attached background material) and replicated in the table below:

 

Tissue	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 minutes	1.621	2.005	0.543	27.1	100*
		2.389
	60 minutes	2.030	2.062	0.045	2.2
		2.093
Positive Control	3 minutes	0.065	0.066	0.001	N/A	3.3
		0.066
	60 minutes	0.059	0.059	0.000	N/A	2.9
		0.059
Test Item	3 minutes	1.850	1.691	0.225	13.3	84.3
		1.532
	60 minutes	1.379	1.288	0.129	10.0	62.5
		1.197

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

In this study and under the experimental conditions reported the test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

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).

 

Methods

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-labeled 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).

 

Results

The relative mean viabilities for each treatment group were as follows:

 

Negative control

3 minutes - 100%*

60 minutes - 100%*

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

 

Positive control

3 minutes - 3.3%

60 minutes - 2.9%

 

Test item

3 minutes - 84.3%

60 minutes - 62.5%

 

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

 

Conclusion

In this study and under the experimental conditions reported: The test item was considered to be non-corrosive to the skin.

Cross-reference 2

Reason / purpose for cross-reference:

other: Weight of evidence

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2020 - 17 September 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)

Version / remarks:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as described in Commission Regulation (EC) No. 640/2012, of 06 July 2012 amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

EpiSkin TM Small / Human Epidermis (SM/13)
Origin: Adult donors

Justification for test system used:

According to OECD guideline 439

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin TM Small / Human Epidermis (SM/13)
- Tissue batch number: 20-EKIN-026,
- Assay medium Lot number: 20-ESSC-016
- Maintenance Medium lot number: 20-MAIN3-016
- Date received: 23 June 2020
- Date of initiation of testing: 23 June 2020

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm (without a reference filter)
- Linear OD range of spectrophotometer: see annex 2 appended to attached background material

NUMBER OF REPLICATE TISSUES: 3 replicates each for test item, positive control and negative control

ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
MTT Salt Metabolism, Cell Viability Assay
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. 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.

Test for Direct MTT Reduction
As specified, a test item 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 item was checked for the ability to directly reduce MTT according to the following procedure:
10 µL 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.

ASSESSMENT OF COLOR INTERFERENCE WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is colored or becomes colored 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 µL 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 color was made.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL (26.3 µL/cm^2) test item, no vehicle

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS), was used as supplied.

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: SDS 5% w/v The positive control was formulated within 2 hours of being applied to the test system.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

83.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

100.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

88.7

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 item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item
The individual and mean OD570 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Appendix 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Appendix 1 (appended to attached backgorund material).
The relative mean viability of the test item treated tissues was 88.7% 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.

Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 4.0% relative to the negative control treated tissues and the standard deviation value of the viability was 0.4%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.748 and the standard deviation value of the viability was 3.2%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 10.5%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply:
- EU CLP Not classified for Irritation.
- UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

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 colorimetric 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.  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-labeled 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-labeled 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 88.7% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

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

 

In this study and under the experimental conditions reported, the test item was classified as non-irritant.  The following classifications apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Cross-reference 3

Reason / purpose for cross-reference:

other: Weight of evidence

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 June 2020 - 01 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The weight of evidence approach taken is explained in the provided endpoint summary.

Reason / purpose for cross-reference:

other: Weight of evidence

Reason / purpose for cross-reference:

other: Weight of evidence

Reason / purpose for cross-reference:

other: Weight of evidence

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:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

Justification of the test method and considerations regarding applicability:
The EpiOcular™ EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular™ EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).


Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
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. The EpiOcular™ tissue mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The EpiOcular™ tissues are cultured on specially prepared cell culture inserts with a porous synthetic membrane through which nutrients can pass to the cells.
EpiOcularTM Human Corneal Model (0.6 cm2)
A Certificate of Analysis supplied by the supplier is given in Annex 3 (appended to attached background material).
Supplier : MatTek In Vitro Life Science Laboratories, Bratislava - Slovakia
Date received : 21 July 2020
EpiOcularTM Tissues Lot Number : 30668
Assay Medium Lot Number : 072020ISA

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL of test item

Duration of treatment / exposure:

30 ±2 minutes at 37 °C, 5% CO2

Duration of post- treatment incubation (in vitro):

120 ±15 minutes at 37 °C, 5% CO2

Number of animals or in vitro replicates:

Duplicate exposure

Details on study design:

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 : 2131496
Isopropanol (MTT) extractant)
Lot Number : 1863832


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 µL 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 Color Interference with the MTT endpoint
Colored test items or those which become colored after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, the test item was checked for its colorant properties.
Test items which absorb light and appear red, yellow, green or blue should be considered as intrinsic colorants. A test item which appears black may absorb light and should be considered as a colorant. Blue, purple and black test items may be directly tested on colorant 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.
All other intrinsically colored test items (e.g. red, yellow, green) have to be tested for their ability to significantly absorb light at the wavelength used for the MTT assay’s end-point measurement. 50 µL of the test item was added to 2 mL of isopropanol, incubated in 6 well plates, and placed on an orbital plate shaker for 3 hours at room temperature. Two 200 µL aliquots of isopropanol solutions and of pure isopropanol were transferred to a 96-well plate and the absorbance is measured with a plate reader at the MTT measurement wavelength. If, after subtraction of the OD for isopropanol, the OD of the test item solution is > 0.08 (approximately 5% of the mean viability of the negative control) the test item has to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls has to be performed.

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-labeled 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).

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 μL of test item was applied atop duplicate cultures for an exposure period of 30 ±2 minutes at 37 °C, 5% CO2 followed by rinsing, a post-treatment immersion and a post-treatment incubation (described below). 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).
If it was not possible to remove the visible test item completely, this was noted in the study file. 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-labeled 12-well plate for a 12 ±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-labeled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 120 ±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.
Each insert was removed from the 24-well plate after approximately 3 hours. The bottom of the insert was blotted on absorbent paper and transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol flowed into the insert on the tissue surface. The plates were sealed with a film sealer (between the plate cover and upper edge of the wells) or a standard plate sealer and stored overnight at 2 to 10 °C in the dark. At the end of the extraction period, each tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.

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-labeled 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. Servicing, calibration, pass band width and linearity range of the microplate reader is given in Annex 2 (appendedto attached background materiial).
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

Classification of eye irritancy potential is based on relative viability according to the following table:

Viability Measured after exposure Time Points Prediction to be considered according to EU CLP Regulation (EC) No 1272/2008 UN GHS
Mean Tissue Viability >60% No Category
Mean Tissue Viability ≤60% No prediction can be made*

*If the relative mean tissue viability is ≤60%, differentiation between EU CLP/UN GHS Category 1 and Category 2 is not possible. This is because, in the case of a true positive, this method cannot resolve between UN GHS Categories 1 and 2. Thus further information on serious eye damage/irritation will be required to decide on the final classification. This will be conducted outside the confines of this study.

ACCEPTABILIITY 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 colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: mean tissue viability (%)

Run / experiment:

1

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: mean tissue viability (%)

Run / experiment:

2

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: mean tissue viability (%)

Run / experiment:

mean

Value:

1.2

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 item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

ASSESSMENT OF COLOR INTERFERENCE WITH THE MTT ENDPOINT
Absorbance measurement at 570 nm (OD570) was measured using the LabTech LT-4500 microplate reader and LT-com analysis software.
After subtraction of the OD for isopropanol, the OD of the test item solution was 0.005 which was < 0.080. Therefore the test item was not considered to have the potential to cause color interference and it was unnecessary to include color correction tissues in the assay.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
The individual and mean OD570 values and tissue viabilities and the difference in viability for the test item, negative control item and positive control item are given in Appendix 1 (appended to attached background material). The mean viabilities of the test item and positive control, relative to the negative control are also given in Appendix 1.
The relative mean viability of the test item treated tissues was 1.2% after a 30-Minute exposure period and 2-Hour post-exposure incubation period.
It was reported that not all of the test item could be removed from the tissue culture surfaces by the rinsing and post-exposure immersion procedures.

ACCEPTANCE CRITERIA
The relative mean tissue viability for the positive control treated tissues was 34.5% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was 2.407. The negative control acceptance criterion was therefore satisfied.
The difference in viability between the two relating tissues in each treatment group was <20%. This acceptance criterion was therefore satisfied.

Interpretation of results:

study cannot be used for classification

Conclusions:

Mean Tissue Viability was ≤ 60% and therefore no prediction can be made.
In the case of a true positive, this method cannot resolve between UN GHS Categories 1 and 2.

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 30 minutes.  At the end of the exposure period each tissue was rinsed before incubating for 120 minutes.  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-labeled 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 1.2%.  

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

 

Mean Tissue Viability was ≤ 60% and therefore no prediction can be made.

In the case of a true positive, this method cannot resolve between UN GHS Categories 1 and 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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:

updated 09 October 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Histological Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160

Version / remarks:

Adopted July 6, 2018 Paris

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

IUPAC Name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

Test material form:

liquid

Details on test material:

Molecular weight: 357.269
Appearance: light amber viscous liquid
Storage conditions: room temperature, in the dark

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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. The corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test material: 0.75 mL of the test item or control items were applied

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND 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 ºC for 75 minutes. At the end of the incubation period each cornea was examined for defects.
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 (Annex 2, appended to attached background material)

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

NUMBER OF REPLICATES
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.

NEGATIVE CONTROL USED
Identification: Sodium chloride 0.9% w/v
Lot: 19E28BB1B
Purity: 0.9%
Supplier: Baxter Healthcare SA
Expiry Date: 01 April 2022
Storage Conditions: Room temperature
The negative control item, sodium chloride 0.9% w/v, was used as supplied.

POSITIVE CONTROL USED
Identification: Ethanol
Batch: STBJ5270
Purity: >99.8%
Supplier: Sigma Aldrich
Expiry Date: 16 March 2025
The positive control item, ethanol, was used as supplied.

APPLICATION DOSE AND EXPOSURE TIME
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the
test item 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 10 minutes.

POST-INCUBATION PERIOD: yes

REMOVAL OF TEST SUBSTANCE
Number of washing steps after exposure period:
At the end of the exposure period the test item 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.

- POST-EXPOSURE INCUBATION:

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 ºC for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:

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.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of Labtech LT-4500 microplate reader
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 ºC for 90 minutes.
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-labeled 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-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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 item induced a response through only one of the two endpoints.

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.

Data Interpretation
The test item was classified according to the following prediction model:
IVIS UN GHS
≤ 3 No Category
>3; ≤ 55 No prediction can be made
> 55 Category 1

DECISION CRITERIA:
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.
This data range is presented in Appendix 3 (appended to attached background material).

Major Computerized Systems
The following computerized systems were used in the study:
Labtech LT-4500 microplate reader and LT-com software Version 7.0 (UK0324).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

14.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Corneal Opacity and Permeability Measurement
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 (appended to attached backgorund material).

Corneal Epithelium Condition
The condition of each cornea is given in Appendix 2 (appended to attached backgorund material).
The corneas treated with the test item were clear post treatment and partly cloudy 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 acceptance range. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.
Assay acceptance criteria are given in Appendix 3 (appended to attached backgorund material).

Any other information on results incl. tables

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

 

Treatment	In Vitro Irritancy Score
Test Item	14.2
Negative Control	0.8
Positive Control	59.9

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

According to UN GHS Classification for the test item Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine, no prediction for eye irritation can be made under the conditions of the test.

Executive summary:

Introduction

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.  

 

Method

The undiluted test item 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).  

 

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS	UN GHS
≤ 3	No Category
>3; ≤ 55	No prediction can be made
> 55	Category 1

 

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	14.2
Negative Control	0.8
Positive Control	59.9

 

Conclusion

According to UN GHS Classification for the test item Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine, no prediction for eye irritation can be made under the conditions of the test.