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

Endpoint summary

Description of key information

According to OECD 439:2015, under the test conditions applied, the test substance is considered NON IRRITANT.

According to OECD Guideline 437 the test item FMOC-HIS-AIB-OH TFA was identified as test chemicals inducing serious eye damage (UN GHS Category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/01/2019 - 15/03/2019

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 160004
Preparation date: 30/11/2016
Expiration date: 09/02/2020
Storage conditions: Refrigerated (2-8 °C)

Test system:

other: SKINETHIC RhE: RECONSTRUCTED HUMAN EPIDERMIS

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

other: sterile ddH2O

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE
- Tissue batch number(s): 19-RHE-032
- Expire: 11/03/2019
- Date of initiation of testing:28/01/2019
Inserts were tested by supplier for the absence of HIV-1 and HIV-2, hepatitis C and B and syphilis. They were certified bacteria and mycoplasma free.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1°C

At the end of the treatment the meshes were removed and each RHE was washed with 1 ml of D-PBS for 25 times at 5-8 cm of distance from the insert
- Modifications to validated SOP: SOPa-132 rev 1

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml solution
- Incubation time: 3 hours ± 5 min
- Wavelength: 570 ± 8 nm
- Linear OD range of spectrophotometer: the OD linearity range for the measuring device is reported in SOPa 132

QUALITY CONTROL OF THE TEST SYSTEM
- Viability: Acceptance criteria: OD values between 0.8 and 3. Result: 1.2 (CV = 7.5%). PASS.
- Barrier function: Acceptance criteria: 4 hours-10 hours upon treatment with 1% Triton X-100.
Result: 5.3 hours. PASS.
- Morphology: Acceptance criteria: evidence of multi-layered human epidermis-like structure (at least 4 viable cell layers). Result: 5.5 cell layers. PASS.
RHE inserts were employed in the test.

NUMBER OF REPLICATE TISSUES: triplicate

PRE-CHECK:
Pre-check for possible direct MTT reduction with test substance
Being the relative conversion of MTT by the tissue the parameter evaluated in this assay, it was therefore necessary to assess the non-specific reduction of MTT by the substance under test prior to proceed to the skin irritation analysis.
Briefly, in a 24-well plate 4 wells were filled with 0.3 ml of MTT 1 mg/ml in D-PBS without Ca2+/Mg2. Then 16 ± 2 mg of solid test substance or 16 μl of ddH2O as a negative control, were added to two wells each, and carefully mixed. The plate was incubated for 3 hours at 37 ± 1 °C, 5 ± 1 % CO2.
After incubation, the visual scoring of MTT interaction was performed as follows:
Negative control: yellow
- Test substance which did not interact with MTT: yellow
- Test substance that interacted with MTT: blue
The color in both wells treated with the test substance was yellow indicating that no interaction occurred between the substance and MTT.

Pre-check method for colouring test substance
To identify color interference, the test substance was evaluated for intrinsic color or ability to become colored in contact with water/isopropanol, simulating a humid environment.
Briefly, 16 ± 2 mg of solid test substance were added to 300 μl of water in 1 well of a 24 well plate, in duplicate.
The plate was shaken for 60 minutes at 500 rpm at room temperature.
At the end of the shaking period no change in color was observed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
The test substance was applied topically to RHE inserts, in triplicate for a contact time of 42 minutes at room temperature. Afterwards, test substance was removed from the RHE inserts, using D-PBS without Ca2+/Mg2+. RHE inserts were placed at 37 ± 1°C, 5 ± 1 % CO2 for 42 h. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.

DECISION CRITERIA :
- The test substance is considered to be irritant to skin if tissue viability ≤ 50 % after exposure and post-treatment incubation
- The test substance is considered to be non-irritant to skin if tissue viability > 50 % after exposure and post-treatment incubation

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μl of sterile ddH2O and 16 ± 2 mg of solid test substance, were applied to each insert, in triplicate.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μl ± 0.5 of Dulbecco's Phosphate Buffered Saline (D-PBS) without Ca2+/Mg2+

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μl ± 0.5
- Concentration (if solution): a 5% Sodium Dodecyl Sulphate solution in sterile ddH2O.

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 h at 37 ± 1°C, 5 ± 1 % CO2

Number of replicates:

3 replicates

Irritation / corrosion parameter:

% tissue viability

Value:

94.68

Vehicle controls validity:

other: sterile ddH2O

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for blank: yes
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for standard deviation of test substance: yes

Data elaboration for Standard procedure (A):

- Data elaboration was performed using the validated data form (mod 363A GxP).

- Optical density at 570 nm (OD570) was measured for every well of the 96-well plate.

- Mean blank = mean OD calculated from the 3 replicates of blank loaded in wells H1 to H3.

- ODXX570 - Mean Blank where “XX” indicates negative control (NC), positive control (PC) or test substance (TS).

- Mean ODxx570/tissue = mean (ODXX570 - Mean Blank) calculated for the respective tissue.

- Mean ODxx570 = mean (Mean ODxx570/tissue) calculated for each treatment solution (test substances (TS), positive (PC) and negative control (NC)

- Standard deviation = standard deviation of Mean ODxx570 . Note that SD is expressed as % in accordance to acceptance criteria.

- Viability (%) = (mean ODXX570 / mean ODNC570) X 100

- Variation coefficient= SD / Mean ODXX570

In the table below the so calculated data are summarized:

	Viability (%)	Variation Coefficient	Result
Positive control	1.60	19.33	IRRITANT
Test substance	94.68	1.17	NON IRRITANT

Interpretation of results:

GHS criteria not met

Conclusions:

According to OECD 439:2015, under the test conditions applied, the test substance is considered NON IRRITANT.

Executive summary:

The test substance “FMOC-HIS-AIB-OH TFA LS; N-[(9H-FLUOREN-9-YLMETHOXY)CARBONYL]-L-HISTIDYL-2-METHYLALANINE and TRIFLUOROACETIC ACID (1:1); CAS 1446013-08-6”, batch n° 160004 was subjected to skin irritation assay, conducted according to OECD 439:2015.

The test was carried out using reconstructed human epidermis (RHE), in triplicate. The exposure of the insert to the test substance was carried out for 42 min at room temperature.

After treatment the inserts were rinsed with D-PBS and post-incubated with growth medium for additional 42 hours at 37 ± 1°C, 5 ± 1 % CO2. Finally, inserts were incubated with MTT solution in order to evaluate cell viability which is a direct measure of the irritant potential of the test substance.

Under these conditions, the test substance “FMOC-HIS-AIB-OH TFA LS; N-[(9H-FLUOREN-9-YLMETHOXY)CARBONYL]-L-HISTIDYL-2-METHYLALANINE and TRIFLUOROACETIC ACID (1:1); CAS 1446013-08-6”, batch n° 160004 resulted NON-IRRITANT.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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).

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

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

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

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

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

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)