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

Endpoint summary

Description of key information

Skin irritation

An in vitro skin corrosion study and an in vitro skin irritation study are used in a weight of evidence approach.

 

In a K1 in vitro skin corrosion study according to OECD Guideline 431 and EU Method B.40 BIS, T002632 was not corrosive to the skin based on the criteria of the CLP regulation (EC) No 1272/2008.

 

In addition, in a K1 in vitro skin irritation study according to OECD Guideline 439 and EU Method B.46, T002632 was not irritating to the skin based on the criteria of the CLP regulation (EC) No 1272/2008.

 

Eye Irritation:

An in vitro eye irritation study using the Bovine Corneal Opacity test method and an in vitro eye irritation study using the Reconstructed human Cornea-like Epithelium (RhCE) test method are used in a weight of evidence approach.

 

In a K1 Bovine Corneal Opacity and Permeability (BCOP) test, performed according to OECD Guideline 437, no prediction on the classification can be made according to the criteria of the CLP regulation (EC) No 1272/2008.

 

In addition, in a K1 Reconstructed human Cornea-like Epithelium (RhCE) test, performed according to OECD Guideline 492, no prediction on the classification can be made according to the criteria of the CLP regulation (EC) No 1272/2008.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-12-12 to 2018-12-18

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:

Adopted 28 july 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

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: I16FD3167
- Expiration date of the lot/batch: 2018-06-30 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C), in tightly closed container
- Stability under test conditions: not stable in water
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, the test item was applied directly on top of the skin tissue.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test item was applied directly on top of the skin tissue. The test item was spread to match the size of the tissue.

OTHER SPECIFICS:
- The correction factor was not applicable for the in vitro skin irritation test, therefore no correction was made for the purity/composition of the test compound.

Test system:

human skin model

Remarks:

of human-derived keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small model (EPISKIN-SMTM, 0.38 cm²) supplied by SkinEthic Laboratories, Lyon France
- Tissue batch number(s): 17-EKIN-050
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 22 hours at 37°C.
- The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item was added into 12-well plates on top of the skin tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.3°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline (PBS) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 66 hours. 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. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.
- Test for reduction of MTT by test item: A test item may interfere with the MTT endpoint if it is 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. The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 520042). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (≥ 19.5%): 22.5 +/- 0.3
- Barrier function (IC50 determination, ≥ 1.5 mg/mL): 2.1
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: - on blood of the same donors, the following was verified: the absence of HIV1 and 2 antibodies, the absence of hepatitis C antibodies, the absence of hepatitis B antigen HBs; - on epidermal cells of the same donors, the following was verified: the absence of bacteria, fungus and mycoplasma.
- Expiration date: 2017-12-18

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

INTERPRETATION
- A test item is considered to be irritant in the skin irritation test (category 2) if: the relative mean tissue viability of three individual tussues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered to be non-irritant in the in vitro skin irritation test (no category) if the relative mean tissue viability after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM : 25.7 to 30.6 mg; The skin was moistened with 5 μl Milli-Q water to ensure close contact of the test item to the tissue.

NEGATIVE CONTROL (PBS): 25 µL

POSITIVE CONTROL (5% SDS): 25 µL in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per test item together with negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: 92 to 103%

Remarks:

SD: +/-5.4

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

1

Value:

1.251

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: 1.185 to 1.324

Remarks:

SD: +/-0.069

Other effects / acceptance of results:

mean tissue viability (percentage of control):
negative control: 100 +/- 5.1
positive control: 6.6 +/- 2.2
mean optical density:
negative control: 1.282 +/- 0.065
positive control: 0.085 +/- 0.029

The test item was checked for possible direct MTT reduction and colour interference in the in vitro skin corrosion test using EpiDerm as a skin model (project 520042). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

Interpretation:
The viability of all replicates was within one category.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 98% (92 to 103%). Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 6.6% (4.1 to 8.3%). The absolute mean OD570 of the negative control tissues was within 0.6 and 1.5. The standard deviation values of the percentage viability of three tissues treated identically were 5.1%, 2.2% and 5.4% for the negative, positive control and the test item respectively, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-11-27 to 2017-12-01

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:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test) (Official Journal of the European Union No. L142, 31 May 2008)

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: I16FD3167
- Expiration date of the lot/batch: 2018-06-30 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C), in tightly closed container
- Stability under test conditions: Not stable in water
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, the test item was applied neat directly on top of the skin tissue.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test item (26.40 to 42.45 mg) was applied neat directly on top of the skin tissue. The test item was spread to match the size of the tissue.

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

Test system:

human skin model

Remarks:

of human-derived non-transformed keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200) supplied by MatTek Corporation, Ashland MA, U.S.A
- Tissue batch number(s): Lot no.: 27631
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium (Dulbecco’s Modified Eagle’s Medium) (supplemented DMEM medium, serum-free supplied by MatTek Corporation) per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 3 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature during 3-minute exposure and 37°C during 1-hour exposure
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 45 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 37.3°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 per centage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline (PBS) (In vitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.
- Test for the interference with the MTT endpoint: A test item may interfere with the MTT endpoint if it is 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 colour interference by the test item: the test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 25 mg of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for reduction of MTT by the test item: the test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item was added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change was observed.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (OD (540-570 nm)<1.0-3.0>): 2.023 +/- 0.099
- Barrier function (ET-50 <4.77-8.72 hrs>): 7.54
- Sterility: Long term antibiotic and antimiycotic free culture: sterile

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non-corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative
control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amounts applied:
- TEST ITEM: The skin was moistened with 25 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 26.40 to 42.45 mg of the solid test item (with a glass weight boat) was added into the 6-well plates on top of the skin tissues.
- NEGATIVE CONTROL (Milli-Q water): 50 µL
- POSITIVE CONTROL (8N KOH): 50 µL

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4 tissues per test item, negative control and positive control: 2 for the 3-minute exposure and 2 for the 1-hour exposure

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 99 and 95%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour application

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 93% and 89%

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

3 minutes application

Value:

1.722

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 1.758 and 1.687

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

1 hour application

Value:

1.652

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 1.683 and 1.622

Irritation / corrosion parameter:

other: Coefficient of Variation

Run / experiment:

3-minutes application

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Coefficient of Variation

Run / experiment:

1-hour application

Value:

3.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

3-minute application:
- viability (percentage of control) (range):
negative control: 100 (102 and 98)
positive control: 15 (18 and 11)
- coefficient of variation between tissue replicates
negative control: 4.5
positive control: 35
- mean optical density:
negative control: 1.777
positive control: 0.260

1-hour application:
- viability (percentage of control) (range):
negative control:100 (101 and 99)
positive control: 11 (12 and 9.9)
- coefficient of variation between tissue replicates
negative control: 2.7
positive control: 18
- mean optical density:
negative control: 1.817
positive control: 0.200

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 97% (99 and 95%) and 91% (93 and 89%) respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean
relative tissue viability following 3-minute exposure to the positive control was 15% (18 and 11%) and 11% (12 and 9.9%) after 1 hour exposure. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 5%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that the test is valid and that the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.

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:

weight of evidence

Study period:

2019-06-05 to 2019-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 25 June 2018

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 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.

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.

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

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

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.

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-11 to 2017-12-14

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)

Version / remarks:

Adopted October 09, 2017

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: I16FD3167
- Expiration date of the lot/batch: 2018-06-30 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C), in tightly closed container
- Stability under test conditions: not stable in water
- Solubility and stability in the solvent/vehicle: not applicable, the test item was added pure on top of the corneas

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Since the test item interacts with water, the test item was used as delivered by the sponsor and added pure on top of the corneas.

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

Species:

other: bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible but within 4 hours after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 316.7 to 378.7 mg

NEGATIVE CONTROL
- Amount applied: 750 µL

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20% (w/v) imidazole solution

Duration of treatment / exposure:

Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

After 240 ± 10 minutes of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed after the incubation period of 90 minutes ± 5 minutes following the opacity measurement.

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (314.6 to 337.9 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- umber of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (l = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device BASF-OP3.0) was calculated according to:
opacity = ((I0/I)-0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea before/after test item treatment.
The change of opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

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

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

Irritation parameter:

in vitro irritation score

Value:

39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range 33 to 42

Irritation parameter:

cornea opacity score

Value:

27

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range 23 to 33

Irritation parameter:

other: permeability

Value:

0.768

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: 0.510 to 1.240

Other effects / acceptance of results:

mean in vitro irritancy score (range):
negative control: 0.1 (-2.7 to 2.6)
positive control: 124 ( 110 to 139)

mean opacity scores (range):
negative control: 0.0 (-2.5 to 2.5)
positive control: 99 (83 to 118)

mean permeability scores (range):
negative control: 0.003 (-0.011 to 0.010)
positive control: 1.660 (1.391 to 1.807)

The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with the test item showed opacity values ranging from 23 to 33 and permeability values ranging from 0.510 to 1.240. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 33 to 42 after 240 minutes of treatment with the test item.

Interpretation:
The IVIS of all replicates was within one category.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The permeability of one of the negative control eyes was just beneath the lower limit of the historical control data range, but the mean permeability of the negative control eyes was within the historical control data range. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 124 (110 to 139) and within the historical positive control data range. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 39 (33 to 42) after 240 minutes of treatment.

Interpretation of results:

study cannot be used for classification

Conclusions:

Since the test item induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion/Irritation

Eurlings (2018) investigated skin corrosion according to the OECD Guideline 431 and EU Method B.40 BIS. After 3 minutes and 1 hour of exposure to 26.40 to 42.45 mg of test item, 97% and 91% mean tissue viability was observed, respectively. The mean optical density at 3 minutes and 1 hour was 1.722 and 1.652, respectively. Based on the results, the test item is not corrosive to the skin. 

 

In addition, Eurlings (2018) investigated skin irritation according to the OECD Guideline 439 and EU Method B.46. After 15 minutes of exposure to 25.7 to 30.6 mg of test item, 98% mean tissue viability and 1.251 mean absorption was observed. Based on the results, the test item is not irritating to the skin. 

 

Eye irritation:

Eurlings (2018) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 316.7 to 378.7 mg of test item was applied on the top of 3 corneas for 240 minutes. Both opacity and permeability were measured, and the resulting objective values were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The corneas treated with the test item showed opacity values ranging from 23 to 33 and permeability values ranging from -0.510 to 1.240. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. The test item induced ocular irritation through both endpoints, resulting in in vitro irritancy scores ranging from 33 to 42 after 240 minutes of treatment with the test item. Since the test item induced an IVIS>3 and≤55, no prediction on the classification can be made according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

 

In addition, Eurlings (2019) investigated eye irritation in an in vitro Reconstructed human Cornea-like Epithelium (RhCE) test. 74.0 to 78.5 mg of test item was applied on top of the corneal epithelial construct for 6 hours. 1.56 % mean tissue viability and 0.026 mean absorption was observed. Based on the results, no prediction about the category can be made. Since the mean relative tissue viability for the test item was below 60% it is considered to be potentially irritant or corrosive to the eye.

 

Justification for classification or non-classification

Skin Corrosion/Irritation:

According to the in vitro skin corrosion and skin irritation studies no skin irritation was observed for T002632. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP regulation (EC) No 1272/2008.

 

Eye irritation:

According to the Bovine Corneal Opacity and Permeabilty (BCOP) test and the Reconstructed human Cornea-like Epithelium (RhCE) test, no prediction on the classification of the test item can be made according to the criteria of the CLP regulation (EC) No 1272/2008.

However, since the test item induced ocular irritation through both endpoints (opacity and permeability) and since the mean relative tissue viability in the RhCE test was below 60% the test item is considered to be potentially irritant or corrosive to the eye. According to the precautionary principle, the preferred approach is to opt for a worst case classification as causing serious eye damage Cat. 1 (H318).