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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2018 - January 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-ethoxy-2-oxoethyl) 3-ethyl 2-methylidenepropanedioate
EC Number:
Cas Number:
Molecular formula:
1-(2-ethoxy-2-oxoethyl) 3-ethyl 2-methylidenepropanedioate
Test material form:
Details on test material:
- Purity: 98%
- Batch: MM212_S4
- Fabrication date: 18/07/2018
- Stabilizers:
7 ppm Methanesulfonic acid;
2000-3000 ppm 2,3,5-trimetilhidroquinona / Hydroquinone;
15 ppm Sulphuric acid

Test animals / tissue source

not specified
Details on test animals or tissues and environmental conditions:
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra
- Age at study initiation: Between 1 to 5 years (the age of the animals was determined based on the teeth count and horn ring count in addition to the Horizontal Diameter of corneas and central corneal thickness)
- Transportation Condition: Transported (in a sealed plastic container) under cold condition in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL].

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 750 μL of test item was applied on each cornea.
- Concentration (if solution): The test item was tested undiluted.
Duration of treatment / exposure:
10 minutes (± 30 seconds) at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 hours ±10 minutes at 32 ± 1 °C
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
The eyes were used within 24 hours of slaughter. Eyes were examined prior to use and corneas free from any visible defects were used. Corneas possessing an opacity less than seven opacity units or equivalent for the opacitometer were used in the study.

A baseline reading for each cornea holder was taken with medium prior to loading of the cornea on to the cornea holder. Corneas, free from defects, were dissected so they had a 2-3 mm rim of sclera and were transferred to a container containing Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL].
Selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then placed on the top of the cornea and fixed in place. Both chambers were then filled to excess with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) (the posterior chamber filled first to allow the cornea to return to its natural concave position), ensuring no bubbles were present within the holders. The device was then equilibrated at 32 ± 1°C for at least one hour to allow the corneas to equilibrate with the medium and achieve normal metabolic activity, to the extent possible. 
Following equilibration, the medium was removed from both chambers and fresh pre-warmed phenol red free EMEM added to both chambers. A baseline opacity reading was then taken for each cornea.

NUMBER OF REPLICATES : 3 corneas/group

NEGATIVE CONTROL USED : 0.9% sodium chloride solution

POSITIVE CONTROL USED : Dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME : The anterior compartment received the test item or negative or positive control at a volume of 750 μL on the surface of the corneas. The corneas were incubated in a horizontal position for 10 minutes (± 30 seconds) at 32 ± 1 °C in the water-bath.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes (2 hours ± 10 minutes at 32 ± 1ºC.)

- Number of washing steps after exposure period: Several washes.
Note: Test item removal from the surface of cornea was not possible, hence the chamber was opened to get the access to the cornea. Test item was tried to be removed by gently flushing Eagle's Minimum Essential Medium with Phenol Red using 1 mL pipette and using Johnson’s ear bud but the test item could not to be removed from the surface of the cornea.
- POST-EXPOSURE INCUBATION: yes, after rinsing, the corneas were incubated for an additional period of approximately 2 hours ± 10 minutes at 32 ± 1ºC.

- Corneal opacity: Corneal opacity is measured quantitatively, as the amount of light transmission through the cornea, using an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490) : Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
mean/ 3 corneas incubated 2 hours
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
other: The increase in the IVIS score is due to the residual test item which could not be removed from the surface of the cornea, hence the test item could not be classified.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
The mean IVIS score for the corneas treated with MM212 was found to be 119.33.
The increase in the IVIS score is due to the residual test item which could not be removed from the surface of the cornea, hence the test item could not be classified.
Executive summary:

This study was conducted to evaluate the ocular irritancy of MM212 in compliance with OECD test guideline 437 for the BCOP test.

Three sets, each consisting of three corneas were tested. The first set was treated with 750 μL of normal saline (control), the second set was treated with 750 μL (undiluted) of dimethylformamide (positive control), and the third set was treated with 750 μL of MM212. Post application, corneas were incubated for approximately 10 minutes after which the test item was washed-off. The corneas were then incubated for approximately 2 h at 32 ± 1 ºC and at the end of incubation, opacity readings were taken.

The post opacity permeability reading was measured by applying 1 mL of fluorescein sodium solution (4 mg/mL) on to the anterior surface of the cornea followed by incubation for approximately 90 min at 32 ºC. At the end of incubation the Optical Density (OD) was measured at 490 nm from fluid collected from the posterior chamber.

The mean In-Vitro Irritancy Score (IVIS) of normal saline (control) and dimethylformamide (positive control) treated corneas were found to be 0.90 and 147.34 which confirmed the reliability of the test procedure. The IVIS score for the corneas treated with MM212 was found to be 119.33 (increase in the IVIS score is due to the residual test item which could not be removed from the surface of the cornea).

Based on results of this study, the test item could not be classified as per the OECD classification.