2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(1H-imidazol-5-yl)propanoyl]amino]-2-methyl-propanoic acid Trifluoroacetic acid salt (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

START OF EXPERIMENT: June 24, 2020 - END OF EXPERIMENT: June 24, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(1H-imidazol-5-yl)propanamido]-2-methylpropanoic acid; trifluoroacetic acid

EC Number:

815-122-7

Cas Number:

1446013-08-6

Molecular formula:

C27H27F3N4O7

IUPAC Name:

2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(1H-imidazol-5-yl)propanamido]-2-methylpropanoic acid; trifluoroacetic acid

Test material form:

solid: particulate/powder

Details on test material:

Batch : 0020000580
Category of the test item: chemical product Galenic farm and colour: white powder
Expiration date: May 16, 2023
pH : not applicable (powder)
Stable under the storage and test conditions.

Specific details on test material used for the study:

Batch : 0020000580.
Category of the test item: chemical product Galenic farm and colour: white powder
Expiration date: May 16, 2023
pH : not applicable (powder)
Stable under the storage and test conditions.

Test animals / tissue source

Species:

other: bovine

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse of Sobeval Boulazac 24759 - France
- Characteristics of donor animals (e.g. age, sex, weight): calves aged less than 8 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): in a short time after slaughtering of the animals, and transported in a Hanks's buffered saline solution with antibiotic to the laboratory.
- Time interval prior to initiating testing:
- Indication of any existing defects or lesions in ocular tissue samples: At reception, the eyes were carefully examined under lighting and these showing a visible (scratches, pigmentation, neo-vascularization) defect were eliminated.
- Indication of any antibiotics used: yes
- Selection and preparation of corneas: For each selected eye, an incision with a scalpel was practiced at the level of the scierai ring by means of scissors.
Approximately 2 to 3 mm of scierai ring were left to facilitate the further handlings. Corneas were immersed in Hanks's medium at room temperature.
Then the corneas were used at receipt for the test.
Mounting of corneas: Corneas were deposited, endothelial side upwards, on the posterior part of cornea holders. Then the anterior part was firmly clamped in place with 3 screws. The anterior (epithelial side) and posterior (endothelial side) compartments were then filled (posterior chamber first), with pre­ warmed Eagle's Minimum Essential Medium (EMEM) without phenol, with a pipette, taking care to eliminate air bubbles. The watertightness was insured by toric caps. Each cornea was identified by the number of the corresponding holder.
Pre-incubation: As soon as the corneas were mounted, the holders were maintained at 32 ± 1°C for 1 hour (pre­ incubation) in a bain-marie, in horizontal position, immersed at the three-quarters of their height.
OPTO measure: After pre-incubation, compartments were emptied with the specific sucking up system and fresh EMEM pre-warmed at 32 ± 1°C was added in both compartments.
The opacity at t=0 (OPTO) was then determined.
Corneas showing a value of opacity greater than seven opacity units were discarded.
3 corneas were selected as negative control. The remaining corneas were then distributed into treatment and positive control groups.
Treatment of corneas: After OPTO reading, the anterior compartment was completely and carefully emptied (thanks to the specific sucking up system) and received the test item (which can be taken with a syringe): 750 µI is directly put on the cornea after having removed the window-locking ring with a metallic spanner and glass window from the anterior compartment.
After dosing, the glass window is replaced on the anterior compartment to recreate a closed system.
3 corneas received the test item, 3 corneas served as negative control and 3 corneas served as positive control.
A chronometer was set off for each series of three corneas.
The holders were then incubated1 in a bain-marie (32 ± 1°C) in vertical position (screw upwards) immersed in such a way that the test item remained in contact with the cornea.
The treatment lasted (time of exposure) was far 4 hours.
After the end of the exposure period, precisely signalled by the ringing of the chronometer, the test item and controls are removed from the anterior compartment and the epithelium washed at least three times (or until no visual evidence of test item can be observed) with EMEM containing phenol red.
Phenol red-containing EMEM is used for rinsing since a color change in the phenol red may be monitored to determine the effectiveness of rinsing acidic or alkaline materials.
The use of a cotton bud was necessary in order to remove the excess of test item on the inner side of the support.
After the last effective rinsing, corneas were given a final rinse with EMEM without phenol red, what allowed to make sure that the inside of support was cleared of any phenol red before the next measure of opacity.
The anterior compartment was then refilled with fresh EMEM without phenol red.
The glass window and the window locking ring were put on again carefully after rinsing.
At the end of exposure with the test item, the corneal opacity was directly measured (OPT2).

Test system

Vehicle:

physiological saline

Remarks:

0.9 % NaCI

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was tested diluted at 20 % with 0.9 % NaCI.
The process of dilution was performed extemporaneously in weight/volume with 0.9 % NaCI (Cooper batch 13NLP271).
Test item appearance after dilution: white opaque liquid.
The dilution of the test item was tested at room temperature and was mixed by vortex.

Duration of treatment / exposure:

4 hours

Details on study design:

NUMBER OF REPLICATES:
3 corneas received the test item, 3 corneas served as negative control and 3 corneas served as positive control. A single test run composed of 3 corneas was sufficient to tested the test item. The 3 results were unequivocal.
The test was judged in conformity.

NEGATIVE CONTROL USED
The negative control was a substance used to ensure that nonspecific changes in the test system can be detected and to provide a baseline for the assay endpoints.

The test was considered acceptable if the negative control (0.9% NaCl, Cooper batch 13NLP271) gives the opacity OP < 12 and the optical density OD < 0.140.
They were respectively: OP= 0.7 and OD= 0.026.

POSITIVE CONTROL USED
The positive control was a substance known to induce a positive response in each assay in order to verify the integrity of the test system and its correct conduct.
The test was considered acceptable if the positive control (Imidazole diluted at 20 %, Sigma batch: SLBT7469) gives an IVIS that fall within two standard deviations of the current historical mean (80.60< IVIS < 178.3).
It was 130.1 ± 8.0.

APPLICATION DOSE AND EXPOSURE TIME
The test item was tested diluted at 20 % with 0.9 % NaCI.
The process of dilution was performed extemporaneously in weight/volume with 0.9 % NaCI (Cooper batch 13NLP271).
Test item appearance after dilution: white opaque liquid.
The dilution of the test item was tested at room temperature and was mixed by vortex.

TREATMENT METHOD:
After OPTO reading, the anterior compartment was completely and carefully emptied and received the test item: 750 µI is directly put on the cornea after having removed the window-locking ring with a metallic spanner and glass window from the anterior compartment. After dosing, the glass window is replaced on the anterior compartment to recreate a closed system.
A chronometer was set off for each series of three corneas.
The holders were then incubated, in a bain-marie (32 ± 1°C) in vertical position (screw upwards) immersed in such a way that the test item remained in contact with the cornea.
The treatment lasted (time of exposure) was for 4 hours.
After the end of the exposure period, precisely signaled by the ringing of the chronometer, the test item and controls are removed from the anterior compartment and the epithelium washed at least three times (or until no visual evidence of test item can be observed) with EMEM containing phenol red.
Phenol red-containing EMEM is used far rinsing since a color change in the phenol red may be monitored to determine the effectiveness of rinsing acidic or alkaline materials.
The use of a cotton bud was necessary in order to remove the excess of test item on the inner side of the support.
After the last effective rinsing, corneas were given a final rinse with EMEM without phenol red, what allowed to make sure that the inside of support was cleared of any phenol red before the next measure of opacity.
The anterior compartment was then refilled with fresh EMEM without phenol red.
The glass window and the window locking ring were put on again carefully after rinsing.
At the end of exposure with the test item, the corneal opacity was directly measured (OPT2).

Measurement of the cornea opacity
Opacity was determined by the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (OP-KIT), resulting in opacity values measured on a continuous scale. The opacitometer has 2 compartments (control compartment and experimental compartment), each one having one light source and a photocell.
The light from a halogen lamp was sent through the control compartment (empty compartment without windows or liquid) to a photocell and compared to the light sent thought the experimental compartment which houses the compartment containing the cornea, to a photocell.
The difference in light transmission from the photocells was compared and a numeric opacity value was presented on a digital display.
Calibration of the opacitometer was systematically performed before using the opacitometer. It was performed with 3 specific calibrators before and after each reading of the opacity. Calibrators 1, 2 and 3 should result in opacity readings equal to their set values.
The values obtained were included between:
Calibrator 1: 71 to 78
Calibrator 2: 142 to 157
Calibrator 3: 213 to 236
During the test, a deviation to the study plan was notified: the values read for Calibrator 2 was outside the established limits. This deviation was considered without impact on the validity of the results by the study director.

Measurements of the opacities (OPTO and OPT2)
The opacity of each cornea was measured at 2 times, just before treatment with the test item (measurement called OPTO) and immediately after the end of the exposure period (measurement called OPT2). The measurements were performed as follows: a holder without cornea or glass windows or liquid, was put in the control compartment of the opacitometer, the holder containing the cornea to be treated, was put in the experimental compartment, so, we obtained the opacity measure of the cornea compared to "vacuum".

Measurements of the corneal permeability
The permeability was determined by the amount of sodium fluorescein solution that penetrated all corneal cell layers. The assessment of this parameter was performed after the 2nd opacity measurement (OPT2).
Both compartments were emptied with the specific sucking up system starting by the anterior compartment. The posterior compartment was then filled with fresh EMEM and 1 ml of a sodium fluorescein solution 5 mg/ml was added in the anterior compartment.
The holders were put back at 32 ±1°C far 90 ± 5 min in vertical position (screws upwards).
At the end of incubation, the rest of fluorescein was removed from in the anterior compartment and the medium contained in the posterior compartment was then taken with a single-use syringe fitted with a long needle, then poured in an identified macrocuve (number of the corresponding holder).
The Optic Density (OD) of the different media was then measured with a spectrophotometer at 490 nm (software VISION lite™ version 2.2). For each cornea a value was obtained and therefore 3 values for the control and 3 values for the test item.
Dismantling, cleaning and disinfections of supports:
Each holder was dismantled. The aspect of the cornea was observed and visible modifications of the cornea were noted (oedema, colouring...).
AII the materials used were cleaned, disinfected, dried and incinerated for the single use material by a company designed by the test facility.
Expression and interpretation of the results:
For the test item and controls, means of each parameter and standard value (OPT2-OPTO difference and OD) were calculated.
The adjustment of values was done by subtracting the mean values obtained on the 3 "negative control" corneas.
An In Vitro Irritancy Score (IVIS) was then calculated with the mean adjusted values according to the formula:
IVIS = mean opacity value + (15 x mean permeability 0D490 value)
Mean ± standard value with one decimal
The decision criteria as indicated in the TG was used: IVIS cut-off values for identifying test item as inducing serious eye damage (UN GHS Category 1) and test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were

IVIS UNGHS
≤ 3 No Category
3< IVIS≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1 (support n. 9)

Value:

ca. 73.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Observations: Oedema + slight epithelium detachment Corneas colored in white

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2 (support n.2 )

Value:

ca. 68.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Observations: Oedema + slight epithelium detachment Corneas colored in white

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3 (support n. 16)

Value:

ca. 83.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Observations: Oedema + slight epithelium detachment Corneas colored in white

Any other information on results incl. tables

The result was reported in Appendix 1 of the study. In brief,

Test item	Tested concentration	IVIS 4 hours. Mean ±SD
FMOC-HIS-AIB-OHTFALS-Ref.NA-Batch :0020000580	diluted at 20 % with 0.9 % NaCI	75.0 ± 7.3

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the defined grading scale, the test item FMOC-HIS-AIB-OH TFA was identified as test chemicals inducing serious eye damage {UN GHS Category 1).

Executive summary:

The aim of the study was to assess quantitatively the irritant potential of a test item after application to the isolated calf cornea according to OECD Guideline 437. The assessment was based on the measurement of two parameters: the opacity and permeability of the cornea whose deteriorations reflected the damage of the tissue. The test item was let in contact with the isolated cornea for 4 hours. An In Vitro Irritancy score (IVIS) (4 hours. Mean±SD) of 75.0±7.3 was obtained. According to the defined grading scale, the test item FMOC-HIS-AIB-OH TFA was identified as test chemicals inducing serious eye damage (UN GHS Category 1).