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

Skin irritation / corrosion

Administrative data

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

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

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:

Target Substance Molecular Formula: C27H27F3N4O7
Target Substance Molecular Weight: 576.51 g/mol
Batch Number: 160004
Physical form and colour: Solid, White powder
Storage: Store at 2÷8°C

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)

In vitro test system

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

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.