1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-06-05 to 2019-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

EC Number:

813-811-7

Cas Number:

253265-97-3

Molecular formula:

C11H13NO7

IUPAC Name:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: powder
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: I18IB2522
- Expiration date of the lot/batch: 17 March 2020 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8 °C)
- Stability under storage conditions: stable until retest date
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not applicable, the test item was applied directly on top of the cornea epithelial construct

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable, the test item was applied directly on top of the cornea epithelial construct
- Final preparation of a solid: The solid test item (74.0 and 78.5 mg) was applied directly on top of cornea epithelial construct.

OTHER SPECIFICS:
- The correction factor is not applicable for the EpiOcular test, therefore no correction was made for the purity of the test item.

Test animals / tissue source

Species:

human

Strain:

other: Reconstructed Human cornea-like epithelium

Details on test animals or tissues and environmental conditions:

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at least 50 mg solid (74.0 to 78.5 mg)

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

6 hours ± 15 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes

Number of animals or in vitro replicates:

2 tissues per test item together with a negative control and positive control

Details on study design:

RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE)
- Model used: RhCE tissue construct used, including batch number : The EpiOcular tissue, MatTek, Ashland MA, U.S.A.
- Batch/lot number: 27481 kit B
- On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium. Incubation environmental conditions are described below.

TEMPERATURE USED FOR TEST SYSTEM
- All incubations, with the exception of the post incubation for 25 minutes, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 -100% (actual range 65-98%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.7-37.5°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2percentage may occur due to opening and closing of the incubator door. Based on laborory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent
- The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 -3 hours at room temperature withgentle shaking.The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.
- Test for the interference with the MTT endpoint: A test item may interfere with the MTT endpoint if itis coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
- Test for the interference with the MTT endpoint: The test itemwas checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 50 mg of the test itemwas added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT.
- Test for colour interference by the test item: The test itemwas checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, approximately 50 mgof the test itemor 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, approximately 50 mg of the test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 -3 hours at room temperature with gentle shaking. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Centrifugation was not considered necessary. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

SCORING SYSTEM
- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60%.

Relative mean viability of 2 individual tissues after
6 hoursof exposure and post-exposure incubation: Prediction to be considered
≤60% of the mean viability of the negative controls No prediction can be made
> 60% of the mean viability of the negative controls No category

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: relative viability %

Value:

1.56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/-0.1

Results 2

Irritation parameter:

other: optical density (OD570)

Value:

0.026

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/-0.001

Other effects / acceptance of results:

- viability (percentage of control) (range):
negative control: 100 +/- 1.0 % (100-100 %)
positive control: 14.9 +/- 0.8 % (14.5 - 15.3%)
- coefficient of variation between tissue replicates
negative control: 1.0
positive control: 0.8
- mean optical density:
negative control: 1.687 +/- 0.012
positive control: 0.251 +/- 0.009

The test item was checked for possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test itemdid not interact with the MTT endpoint. The test itemwas checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0154 and 0.0006, respectively. Therefore it was concluded that the test item did not induce color interference. In addition, because no color change wasobserved in the presence of MTT it was concluded that the test itemdid not interact with the MTT endpoint.

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test itemcompared to the negative control tissues was 1.56%(1.51 and1.61%). Since the mean relative tissue viability for the test itemwas below 60% it is considered to be potentially irritant or corrosive to the eye.The positive control had a mean cell viability after 6 hours ± 15minutes exposure of 14.9%(15.3 and 14.5%). The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix 3). The standard deviation valuesof the percentage viability of twotissues treatedidentically were1%, 0.8% and 0.1% for the negative, positive control and the test item respectively, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Finally, it is concluded that this test is valid and that JNJ-26144612-AAA (T002632) no prediction about the category can be made in the EpiOcular™ test under the experimental conditions described in this report.