1-Pyrrolidinecarboxylic acid, 3-[2-[(ethoxycarbonyl)[5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl]-4-ethyl-, phenylmethyl ester, (3R,4S)-

Endpoint summary

Description of key information

The results from the Keratinosens assay and H-Clat indicated a negative result for skin sensitization by the test material. Therefore, based on this approach, the test material was not classified according to the GHS criteria.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20th September 2019 to 17th April 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: OECD 442E In Vitro Skin Sensitisation Assays addressing the key event on activation of Dendritic cells on the adverse outcome pathway for Skin Sensitistation Adopted: 25 June 2018

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol no. 158

GLP compliance:

no

Remarks:

The study has been conducted with a reputable lab for another international submission.

Type of study:

activation of dendritic cells

Details on the study design:

The h-CLAT is an in vitro assay which measures the changes in expression of cell surface markers CD54 (ICAM-1) and CD86 associated with the process of dendritic cell activation in the human acute monocytic leukemia cell line, THP-1. Dendritic cell activation is considered one of the key biological events in the adverse outcome pathway for skin sensitisation, where CD54 and CD86 are subsequently involved in dendritic cell migration to the lymph nodes and T-cell priming. THP-1 cells, seeded at a density of 2.0 x 10 6 cells/ml in culture medium in 24-well plate format, define the Test System. After treatment of the test item or control articles to the test system, the expression of cell surface markers are measured by flow cytometry following cell staining with flourescein isothiocyanate (FITC) labelled antibodies. Cytometry measurement, using propidium iodide (PI) staining, is conducted concurrently to assess whether upregulation of surface expression occurs at subcytotoxic concentrations.

Solubility Determination:
Prior to the preliminary dose range finding assay, the test article was tested in a solubility test to determine an appropriate solvent. The following observations were determined during the test. The test article was found to be soluble at 500 mg/mL in DMSO with approximately 1 minute of vortexing. The test article dilution appeared to be a cloudy, white, non-viscous suspension. The tubes were vortexed immediately prior to dosing and the cloudiness remained.

Dose Range Finding Assay:
A preliminary dose range finding assay was performed to determine the viability of the THP-1 cells after 24 +/- 0.5 hour exposure to 8 test article concentrations. The CV75, which is the calculated test article concentration leading to 75% cell viability was calculated for the test article.

Definitive assays:
Based on the results from the dose range finding assay, the doses were chosen for the test article for the definitive assay. At least 2 valid definitive trials were performed.
The positive control, 2,4-Dinitrochlorobenzene, was tested in the dose rang and definitive trials.

Evaluation of test results:
The relative fluorescence intensity was calculated for the test article and control treated cell population. The EC200 and EC150 values, which are the calculated test article concentrations leading to an RFI of 200 or 150 respectively, were calculated for the test article.

Positive control results:

For the positive control, 2,4-Dinitrochlorobenzene, RFI values of both CD86 and CD54 were predicted to be positive (CD86 RFI ≥150 and CD54, RFI ≥200) and cell viability was >50%.

Run / experiment:

other: Dose Range and Definitive Assay

Parameter:

other: CV75 µg/mL

Value:

1 000

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: µg/mL

Key result

Run / experiment:

other: B1 assay 01 Oct 2019

Parameter:

other: CD54 EC200 µg/mL

Value:

370

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

µg/mL

Key result

Run / experiment:

other: B1 assay 01 Oct 2019

Parameter:

other: CD86 EC150 µg/mL

Value:

1 000

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

µg/mL

Key result

Run / experiment:

other: B2 assay 08 Oct 2019

Parameter:

other: CD54 EC200 µg/mL

Value:

1 000

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

µg/mL

Key result

Run / experiment:

other: B2 assay 8 Oct 2019

Parameter:

other: CD86 EC150 µg/mL

Value:

1 000

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

µg/mL

Key result

Run / experiment:

other: B4 assay 15 Oct 2019

Parameter:

other: CD54 EC200 µg/mL

Value:

1 000

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

µg/mL

Key result

Run / experiment:

other: B4 assay 15 Oct 2019

Parameter:

other: CD86 EC150 µg/mL

Value:

1 000

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

µg/mL

Other effects / acceptance of results:

Definitive trial 3 did not meet assay acceptance criteria and therefore, was not considered a valid trial

The assay met acceptance criteria when:

The cell viability values of the solvent control was > 90%.

For the solvent control, RFI values of both CD86 and CD54 were less than the positive criteria (CD86 RFI < 150 and CD54 RFI < 200).

For the positive control (DNCB), RFI values of both CD86 and CD54 were predicted to be positive (CD86 RFI >= 150 and CD54 RFI >= 200), and cell viability was > 50%.

For the medium and solvent controls, the MFI ration of both CD86 and CD54 to isotype control was >105%.

The cell viability of the test article-treated cultures was > 50% in at least four doses.

All acceptance criteria for a valid assay were met for the definitive trials.

Interpretation of results:

GHS criteria not met

Conclusions:

The test article A1634356.0 was considered a non-sensitizer according to h-CLAT.

Executive summary:

The Human Cell Line Activation Test (h-CLAT) was used to assess the skin sensitisation potential of the test article by monitoring the upregulation of cell surface markers, CD54 and CD86 on the surface of human acute monocytic leukemia cell line, THP-1. The upregulation of CD54 and CD 86 in response to a skin sensitiser is correlated to dendritic cell actication, which is the third key event of the skin sensitisation pathway.

Based on the results from the dose range finding assay, the doses were chosen for the test article for the definitive assay. At least 2 valid definitive trials were performed.
The positive control, 2,4-Dinitrochlorobenzene, was tested in the dose rang and definitive trials.

The relative fluorescence intensity was calculated for the test article and control treated cell population. The EC200 and EC150 values, which are the calculated test article concentrations leading to an RFI of 200 or 150 respectively, were calculated for the test article. 

As the test article elicted an EC200 >1000 for CD54 and an EC150 >1000 for CD86, with cell viability ≥50% in at least two independent runs, A-1634356.0 is considered a non-sensitiser. 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th September 2018 to 6th December 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Qualifier:

according to guideline

Guideline:

other: Natsch et al (2008)

GLP compliance:

no

Remarks:

The study has been conducted with a reputable lab for another international submission.

Type of study:

activation of keratinocytes

Details on the study design:

Subculture of KeratinoSens Cells into 96-Well Plates
The KeratinoSens cells were subcultured into transparent Costar 96-well plates or white-walled Perkin Elmer plates when the flasks were approximately 60 to 90% confluent. The flasks were rinsed and trypsinized. The cells were resuspended in 5 mL of Assay Medium per flask. The concentration of cells in suspension was determined using a Coulter Counter. A cell suspension of 1.0 x 105 cells/mL in Assay Medium was prepared. One hundred μL of the cell suspension was added to all but one well designated as a blank well (H12). The stock cell suspension was mixed often to ensure a uniform distribution of cells into each well. Three white-walled and one transparent plate were seeded for each test article replicate set (definitive trials). The plates were incubated for approximately 24 hours at standard culture conditions. The cultures seeded into the clear plates were examined under a phase contrast microscope and evaluated for uniform seeding and confluence prior to treating the cells with the test article or positive control.

Solubility Determination
The solubility of the test article was tested in DMSO on the day of the initial definitive assay (at the highest 100X concentration of 200 mM). Poor solubility in DMSO was observed for test article A-1634356.0. An additional sample of A-1634365.0 was diluted in water to assess solubility in an alternative solvent system, however the test article was less soluble in water compared to DMSO. Thus, the test article A-1634356.0 was tested as a suspension in DMSO.

MTT Direct Reduction Test
The ability of the test article to directly reduce MTT was assessed at the same time of test article treatment in the definitive assays. A 1.0 mg/mL MTT solution was prepared by dissolving a 10 mg/mL stock solution of MTT into warm MTT Addition Medium. Approximately 100 μL of the 100X (200 mM) test article concentration in DMSO was added to 1 mL of the MTT solution and then incubated in the dark at 37ºC for one to three hours. One hundred μL of a negative control (e.g. DMSO) was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT. A-1634356.0 turned the MTT solution color blue/purple, therefore, A-1634356.0 does not directly reduce MTT. However, A-1634356.0 formed a white precipitate.

Controls
Each assay plate included a range of doses of the positive control, Cinnamic Aldehyde. A 100X concentration of positive control was prepared by weighing an appropriate amount of Cinnamic Aldehyde into a pre-labeled conical tube and adding the necessary amount of DMSO to prepare a 64,000 μM dilution. The 64,000 μM dilution was further diluted 1:10 in DMSO to prepare a 6,400 μM 100X stock concentration. The final 1X concentrations of the positive control were 64, 32, 16, 8, and 4 μM. The solvent control for the test articles and the positive control was 1% DMSO in the dilution solvent (1% DMEM.) Each plate included a set of 6 solvent control wells.

Testing Concentrations
The test articles had defined molecular weights provided by the Sponsor, and were diluted based on molarity. The 100X stock dilution was prepared to a top concentration of 200 mM. The final 1X tested concentrations were 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μM.

Definitive Assays
A-1634356.0 was tested in three independent definitive assays. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). Each plate tested a range of 12 dosing concentrations for each test article. Each plate also included 5 wells designated for the positive control (tested over a range of 5 dosing concentrations), 6 wells designated as the DMSO solvent control, and 1 well which was left blank. After approximately 24 hours of incubation, the Assay Medium was removed from the cells. The plates were decanted and gently blotted on sterile paper towels. One hundred and fifty microliters of fresh pre-warmed 1% DMEM were added to all wells, including the blank. The plates were returned to the incubator until the dosing was initiated. Twelve decreasing doses were selected for the assay .For the positive control, 5 decreasing doses were prepared. For each experiment, the positive control (5 doses), and the solvent control, a 100X DMSO master plate was made, followed by a 4X Master Plate. When added to the 150 μL of 1% DMEM already in each well, the addition of the 50 μL 4X dose brought the final dose on the plates to 1X.

100X Master Plate Preparation
The 100X DMSO Master Plate was labelled with the test article and control concentrations according to the standard plate map (see Figure 1). The 100X top stock concentration (200 mM) of the test article was prepared and then serially diluted to make the 11 remaining doses. The 100X top stock for the positive control (6,400 μM) was prepared and then serially diluted to make the 4 remaining doses. The wells designated for the solvent control received DMSO. The well designated as blank was left empty.

4X Master Plate Preparation
The corresponding 4X Master Plate was similarly labeled and the 96-well plate was pre-filled with 300 μL of 1% DMEM in each well. The prepared dilutions from the 100X DMSO Master Plate were further diluted 25-fold by adding 12.5 μL each 100X sample (test article concentration, positive control concentration, or solvent control) to the 4X Master Plate.

Test Article Treatment
The resulting 4X Master Plate was then used to dose the replicate assay plates (transparent or white-walled) containing cells. A final 1X concentration was achieved for each dose by removing 50 μL from each well of the 4X Master Plate and adding the dose to the corresponding well in the 1X plates (already containing 150 μL of 1% DMEM in each well) . The blank well received 50 μL of 1% DMEM. When all of the plates were dosed, the plates were sealed with a plate sealer to avoid evaporation of volatile compounds and to avoid cross-contamination between wells. The plates were then incubated at standard culture conditions for 48±1 hours.

Visual Observations
After approximately 48 hours of post-treatment incubation, visual observations of the cultures were performed for the cytotoxicity plate and recorded. Precipitate formation precluded visual observations of cells dosed with the test article A-1634356.0 at the highest testing concentrations in all three trials.

Treatment Termination & Luciferase Induction Determination
After 48 ±1 hours of exposure, each white-walled culture plate was removed from the incubator and allowed to equilibrate to room temperature for at least 30 minutes. Once at room temperature, the treatment medium was decanted from each plate. The cultures were rinsed with 250 μL of CMF-DPBS (room temperature), the CMF-DPBS rinsate was decanted from the wells, and the plates were gently blotted onto paper towels. Fifty microliters of CMF-DPBS was added to each well followed by fifty microliters of ONE-Glo™ Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer. The plates were read within 45 minutes of addition of the ONE-Glo™ Reagent. The luminescence determination of each plate was performed by a Berthold Detection Systems luminometer initiated from an IBM-PC hosting the Windows-based Simplicity™ software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLUs).

Treatment Termination: Cytotoxicity Using the MTT Endpoint
A 0.59 mg/mL MTT solution was prepared in 1% DMEM and used within 2 hours. After 48 ±1 hours, the clear 96-well plates designated for the MTT endpoint were decanted and gently blotted on paper towels. No rinsing was performed. Two hundred μL of 1% DMEM containing 0.59 mg/mL MTT was added to each well. The plate was incubated with a plate seal at standard culture conditions for approximately 4 hrs.
After approximately 4 hours, the MTT solution was decanted, the plate was blotted, and 200 μL of 10% SLS was added to each well. The plate was covered with a plate seal and incubated at standard culture conditions overnight. After the overnight incubation, each plate was placed on a plate shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.

Data Analysis
For the test article, a copy of the standard data file, provided by Givaudan, was made (i.e. 3 trials, each with 4 plates per trial-3 plates for the luciferase induction and 1 plate for MTT). Raw data from the luminometer was transferred directly from the luminometer software into the designated Excel spreadsheet for luminescence analyses. Raw data from the Vmax was transferred into the designated Excel spreadsheet for cytotoxicity analyses.

The data file automatically calculated the gene induction and the wells with statistically significant induction over a given threshold (default value set to 1.5 = 50% enhanced gene activity). Furthermore the maximal induction (Imax), the concentration for maximal gene induction (CImax) and the EC1.5 value (concentration for induction above threshold), both with linear and log-linear extrapolation, was calculated similar to the LLNA (Local Lymph Node Assay). Relative survival (viability) was obtained by comparing the amount of MTT conversion by test article treated groups compared to the associated solvent treated group on the same plate. The Givaudan Excel file calculates an IC50 value for each test article. The IC50 is determined by averaging the viability percentage of each concentration for the 3 definitive assays and then calculating by linear interpolation the IC50 concentration which results in 50% reduced cell viability using the concentration and viability percentage below and above 50% viability. A summary of the results from the 3 definitive assays was calculated. The luciferase induction and cytotoxicity data were plotted on the graphs. For the luciferase induction, the fold gene induction (as compared to the solvent controls) was plotted over the test article concentration. For the cytotoxicity, the % viability (as compared to the solvent controls) was plotted over the test article concentration.

Positive control results:

The Mean EC 1.5 (μM) and Mean IC50 (μM) for the positive control (cinnamic aldehyde) were 9.27 and >64 respectively.

Key result

Run / experiment:

mean

Parameter:

other: EC 1.5

Value:

2 000

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: IC50

Value:

0.97

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: Imax

Value:

1.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: CI max

Value:

1 000

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

According to the current prediction model, the test article is predicted to be a non-sensitizer.

Executive summary:

The Induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE- Reporter Cell Line KeratinoSens skin sensitization assay is a high-throughput cell-based in vitro test to screen for the skin sensitization potential of chemicals. The KeratinoSens cells (Givaudan, Switzerland) were propagated as a reporter cell line. The KeratinoSens cells are transfected HaCaT keratinocytes that include a 56-base-pair insertion containing the ARE sequence from the AKR1C2 gene, a SV40 promoter, the luciferase gene in the vector pGL4 from Promega, and a stable insertion allowing for cells to be selected in the presence of Geneticin (G418). The signalling pathway with the repressor protein Keap1 and the transcription factor Nrf2, which binds to the antioxidant / electrophile response element (ARE / EpRE), was shown to be a valuable cellular endpoint to detect skin sensitizers in vitro1,2. The induction of luciferase directly indicates the activation of ARE-dependent genes. Additionally, the cytotoxicity of a test article was assessed using cell MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide). Cytotoxicity was determined by measuring the relative conversion of MTT in test article-treated cultures compared to the solvent control.

The experimental design of this study consisted of at least three valid definitive assays to determine the maximal induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concentration for a statistically significant induction of 50% above solvent controls), and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for each test article. The KeratinoSens cells were cultured in quadruplicate plates for 24 hours, treated with the test article for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity (1 plate). The procedures that were performed in this assay were a modification of the procedures previously described by Natsch, et al. (2008) and were performed similar to those procedures performed by the Institute for In Vitro Sciences, Inc. in the KeratinoSens ring-study 3. The study was also performed in compliance with OECD Guideline for the Testing of Chemicals No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method.
The Induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE- Reporter Cell Line KeratinoSens Assay was performed to determine the skin sensitization potential of the test article A-1634356.0 was tested in three definitive trials.

According to the current prediction model, the test article A-1634356.0 is predicted to be a non-sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results from the two studies, the test material was not considered to be a skin sensitizer and was not classified according to the GHS criteria.