2-methylenepropane-1,3-diyl diacetate

Endpoint summary

Description of key information

A determination of Skin Irritation Potential using the EpiSkinTM Reconstructed Human Epidermis Model has been conducted (Envigo Research Limited, 2016, BM93DN) according to OECD 439 test guideline. The test item was classified as non-irritant.

To evaluate the corrosivity potential of the test item an in vitro Skin Corrosion: MatTek EpiDerm a test ( Envigo Research Limited, 2017, PS57JG) according to OECD 431 test guideline was conducted. The test item was considered to be non-corrosive to the skin.

In the Bovine Corneal Opacity and Permeability Assay (BCOP) conducted according to OECD 437 no prediction of eye irritation could be made ( Envigo Research Limited, 2016, SR35YB).

In the Assessment of Eye Irritation using the Reconstructed Human Cornea-Like Epithelium (RhCE) Test method EpiOcularTM (OECD 492) no prediction of eye irritation could be made (Labcorp Early Development Laboratories Ltd. 2022, 8481151).

Considering the results of the both the OECD 492 and the OECD 437 the overall weight-of-evidence would support classification as Category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

THis study was conducted between 18 January 2017 and 26 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was assigned Reliability 1 as it was conducted to OECD TG 431 and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

EC No. 440/2008 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: 2-methylene-1, 3-propanediol diacetate (CAS: 377529-9) Batch: 20160308(-88) Purity: 99.7% Physical state/Appearance: Clear colorless liquid Expiry Date: 31 March 2019 Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium

Cell source:

other: Not specified as study used an EpiDerm™ Reconstructed Human Epidermis Model Kit

Source strain:

not specified

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recognised in vitro test for corrosivity

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 24 January 2017
EpiDermTM Tissues (0.63cm2) lot number : 23389
Assay Medium lot number : 011917TMB
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

Study Design
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT MTT Dye 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 formazan dye 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.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
50 µL 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 turns blue/purple relative to the control, the test item was presumed to have reduced the MTT.

Assessment of Color Interference with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main Test
Pre-Incubation The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 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. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period. When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS 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 inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates 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-labeled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

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 OD532 test item / Mean OD562 negative control x 100

Classification of corrosivity potential is based on relative viabilities for both exposure times according to the following table:

Relative mean tissue viability (% of negative control) EU CLP Regulation (EC) No 1272/2008 UN GHS UN packing group
3 min: < 50 H314 Category 1A I
3 min: ≥ 50 1 hour: < 15 H314 Category 1B or 1C II or III
3 min: ≥ 50 1 hour: ≥ 15 Not classified for corrosivity Non-Corrosive

Quality Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control
The absolute OD562 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 OD562 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 8.0N 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
- The test item was used as supplied (50µL)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Potassium Hydroxide
- Concentration (if solution): 8.0N

Duration of treatment / exposure:

Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 minutes, 60 minutes
• Negative Control: 3 minutes, 60 minutes
• Positive Control: 3 minutes, 60 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 Minute Exposure

Value:

86.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

4.3

Remarks on result:

other: Not corrosive to skin

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 Minute Exposure

Value:

ca. 78.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

3.5

Remarks on result:

other: Not corrosive to skin

Other effects / acceptance of results:

Quality Criteria
The mean OD562 for the negative control treated tissues was 1.806 for the 3-Minute exposure period and 1.832 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.5% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.749	1.806	0.081	4.5	100*
		1.863
	60 Minutes	1.935	1.832	0.146	8.0
		0.728
Positive Control	3 Minutes	0.085	0.078	0.011	na	4.3
		0.070
	60 Minutes	0.072	0.065	0.010	na	3.5
		0.058
Test Item	3 Minutes	1.616	0.555	0.086	5.5	86.1
		1.494
	60 Minutes	1.574	1.138	0.192	13.4	78.5
		1.302

OD =  Optical density

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

na = Not applicable

Interpretation of results:

other: Not Corrosive to skin

Remarks:

according to EU CLP and UN GHS criteria

Conclusions:

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

Executive summary:

The purpose of this test is 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.  The results are used to make a prediction of the corrosivity potential of the test item.

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 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 562 nm (OD562). 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:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	4.3	86.1
60 minute	100*	3.5	78.5

*The mean viability of the negative control tissues is set at 100% Quality criteria:  The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

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

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jul 2016 - 26 Jul 2016

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:

To avoid cross contamination occurring with test items from other studies within the laboratory and incubator, the pre-incubation for these tissues was extended for approximately 24 hours. The study was started and completed within the manufacturer’s expiration date of the tissue and there for there was no biological risk to the tissues.

Deviations:

no

Remarks:

This deviation was considered to have not affected the integrity or validity of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

To avoid cross contamination occurring with test items from other studies within the laboratory and incubator, the pre-incubation for these tissues was extended for approximately 24 hours. The study was started and completed within the manufacturer’s expiration date of the tissue and there for there was no biological risk to the tissues.

Deviations:

no

Remarks:

This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20160308(-88)
- Physical Appearance: clear colourless liquid
- Expiration date of the lot/batch: 31 Mar 2019
- Purity: 99.7%
- Storage conditions: room temperature in the dark

NEGATIVE CONTROL – Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Source and lot/batch No.of test material: 1553513
- Expiration date of the lot/batch: 01 Mar 2019
- Purity: unknown
- Storage conditions: approx. 4 °C

POSITIVE CONTROL - Sodium Dodecyl Sulphate (SDS)
- Source and lot/batch No.of test material: 1377955
- Expiration date of the lot/batch: 30 Jun 2019
- Purity: 99.6%
- Storage conditions: room temperature

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Model comprised of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN reconstructed human epidermis model
- Tissue batch number(s): 16-EKIN-029
- Delivery date: 19 Jul 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Treatment time: 15 min
- Temperature of post-treatment incubation (if applicable): 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- MTT amount: 2 mL
- Incubation time: 3 h
- Incubation temperature: 37°C
- Spectrophotometer:
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: 3 per control group

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10µL

NEGATIVE CONTROL
- Amount(s) applied: 10µL of DBPS

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

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per control group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean test item

Value:

81.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean negative control

Value:

100

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean positive control

Value:

16.4

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

The relative mean viability of the test item treated tissues was 81.1% after the 15 min exposure period and 42 hours post-exposure incubation.

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:

supporting study

Study period:

Experimental start date 02 November 2021
Experimental completion date 05 November 2021

Reliability:

1 (reliable without restriction)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: MPDAc
Chemical Name: 2-methylene-1,3-propanediol diacetate
CAS Number: 3775-29-9
Batch Number: 20160308-88
Purity: 99.7%
Expiry Date: 31 March 2022
Appearance: Clear colourless liquid
Storage Conditions: Room temperature, in the dark

Species:

human

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μL of test item, positive and negative control.

Duration of treatment / exposure:

30 ±2 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Duplicate

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 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.
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 eye irritation potential was performed in parallel on viable and non-viable, freeze-killed, tissues.
This step employs freeze-killed tissues that possess negligible metabolic activity but absorb and bind the test item similar to viable tissues.
Freeze-killed tissues were prepared in-house (outside of the confines of the study) by placing untreated EpiOcularTM tissues in a freezer (-35 to -10 C) overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once killed, the tissues may be stored indefinitely in the freezer. Freeze-killed tissues were thawed for approximately 60 minutes at 37 ±2 °C, 5 ±1% CO2 in air before use.
Each MTT reducing test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues remained untreated (the untreated controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay was performed on the frozen tissues in parallel to the viable tissues.
If the interference by the test item was ≤ 60% of the negative control value, the net OD of the test item treated killed control may be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
Data were corrected as follows: True viability = Viability of treated tissue – direct MTT interference from test item = OD tvt – (mean OD tkt – mean OD ukt)
Key:
OD = optical Density at 570 nm
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues
If the interference by the test item was greater than 60% of the negative control value the test item may be considered incompatible with this test system.

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.
For non-colored test items, tests have to be performed to assess if they become colorants after contact with water or isopropanol. For this purpose 50 μL 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 μL of the liquid 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-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).

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 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).
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 L 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.
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.

Irritation parameter:

percent tissue viability 

Run / experiment:

Mean

Value:

38.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT Reduction
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using, non-viable, freeze-killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint
The test item did not become colored in either the water or isopropanol solutions. It was therefore unnecessary to run color correction tissues.

Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 31.7% 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 2.387. 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.

Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	2.390	2.387	100.1	100	0.3
	2.383		99.8
Positive Control Item	0.672	0.757	28.2	31.7	7.0
	0.841		35.2
Test Item	0.949	0.924	39.8	38.8	2.1
	0.899		37.7

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:

Introduction
The purpose of this study was to determine if the test item does not require 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.
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. As the test item had been shown to directly reduce MTT additional non-viable, freeze-killed, 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. After MTT-loading the tissues was placed into 2 mL of isopropanol for formazan extraction.
At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 L 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 viability of the test item treated tissues was 38.8%.
Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.
Conclusion
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

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jul 2017 - 27 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20160308(-88)
- Expiration date of the lot/batch:
- Physical Appearance: clear colourless liquid
- Purity: 99.7%
- Storage: room temperature in the dark
- Expiration date: 31 Mar 2019

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: 3
- Characteristics of donor animals - age: 12 – 60 months
- Storage, temperature and transport conditions of ocular tissue: eyes were excised after slaughter, placed in HBSS supplemented with penicillin (100 IU/ mL and streptomycin at 100µg/ mL), transported to the test facility over packs of ice on the same day of slaughter. Corneas prepared immediately on arrival. Isolated corneas were mounted in BCOP holders which were filled with EMEM without phenol red and incubated at 32 ± °C for 60 min.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

120 min at 32°C ±1°C

Number of animals or in vitro replicates:

3 per control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were excised after slaughter, placed in HBSS supplemented with penicillin (100 IU/ mL and streptomycin at 100µg/ mL), transported to the test facility over packs of ice on the same day of slaughter. Corneas prepared immediately on arrival. Isolated corneas were mounted in BCOP holders which were filled with EMEM without phenol red and incubated at 32 ± °C for 60 min.

QUALITY CHECK OF THE ISOLATED CORNEAS
Each cornea was examined, only corneas free of damage were used.

NUMBER OF REPLICATES:
3 per control group

NEGATIVE CONTROL USED
Identification: Sodium Chloride 0.9% w/v
Batch: 3011424
Purity: 0.9%
Expiry Date: 01 Jan 2017
Storage: room temperature in the dark

POSITIVE CONTROL USED
Identification: Ethanol
Batch: STBD7546V
Purity: >99.8%
Expiry Date: 01 Apr 2018
Storage: room temperature in the dark


APPLICATION DOSE AND EXPOSURE TIME
0.75ml, undiluted, applied under safety lightening, exposure for 10 min.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
After exposure time the test item and the control item were removed from the anterior chamber and the each cornea was rinsed 3 times with fresh EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION: The BCOP holders were incubated, anterior chamber facing forward, at 32 ± 1° for 120 min.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: optical density 492nm (OD492) was measured using the Anthos 2001 microplate reader.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Irritation parameter:

in vitro irritation score

Run / experiment:

mean test item

Value:

8.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

mean negative control

Value:

2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

mean positive control

Value:

42.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

No prediction of eye irritation can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The result of the BCOP test (IVIS = 8.6) is only slightly above the cut-off for ‘No Category’ which is an IVIS  of 3.0. A false positive rate of 69% has been published for the BCOP test method for identification of ‘No Category’ substances, so there is a possibility that the test article may be a  ‘No Category’ substance with respect to eye irritation potential. Data from the BCOP test method validation dataset also indicate that substances producing an IVIS of less than 20 are very unlikely to require classification as Category 1. Considering this the test article is either ‘No Category’ or ‘Category 2’.

The result of the OECD 492 (EpiOcular™) test is ‘no prediction can be made from this result therefore the overall weight-of-evidence would supports classification as Category 2.