8-((3R,4S)-4-ethylpyrrolidin-3-yl)-3h-imidazo[1,2-a]pyrrolo[2,3-e]pyrazine dihydrocloride

Endpoint summary

Description of key information

The results from the DPRA analysis and the Keratinosens assay indicated a positive result for skin sensitization by the test material. Therefore, based on this approach, the test material was classified according to the GHS criteria as a category 1 skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 12, 2018 - April 9, 2019

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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

no

Remarks:

The procedures used here were adapted from OECD guideline 442C (In ChemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Justification for non-LLNA method:

The Direct Peptide Reactivity Assay was used to assess the skin sensitizing potential of the test articles. Synthetic peptides containing cysteine or lysine were reacted with the test articles for 24± 2 hours. After the incubation period, the extent of peptide depletion was analyzed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection.

Details on the study design:

Test Article Preparation:
The test articles were prepared at 100 mM concentrations in an appropriate solvent. Calculations using the molecular weight and purity of the test article were performed to determine the appropriate amount of test article to weigh out in order to achieve approximately 3 mL of the 100 mM sample. On the day of testing, the test articles were weighed into prelabeled glass vials and stored at room temperature until time to perform the assay.

Test Article Solubility Test:
A solubility test was performed for the test articles in order to determine an appropriate solvent that completely dissolved each test article at a 100 mM concentration.The test articles was found to be soluble in water with brief vortexing.

Peptide Preparation:
Custom synthetic peptides containing cysteine or lysine as the reactive centers (with phenylalanine to aid in detection) were used in this assay. The purity of each peptide was at least 90%. Peptide samples were newly prepared for the sample set, and a single preparation of the peptide was used throughout the sample set. The cysteine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 7.5±0.1 phosphate buffer. The actual pH of the phosphate buffer on the day of testing was 7.5. The lysine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 10.2±0.1 acetate buffer. The actual pH of the acetate buffer on the day of testing was 10.1. The peptide solutions were gently mixed on the shaker.

Peptide Standards:
A set of serially diluted standards were prepared for each peptide. The top stock of the standards (0.534 mM) was prepared from the 0.667 mM peptide solution in acetonitrile. The remaining standards were prepared by serial dilution in dilution buffer (20% acetonitrile in phosphate buffer for the cysteine peptide or acetate buffer for the lysine peptide). Six standards were prepared at concentrations of 0.534-0.017 mM. A seventh standard was prepared containing only dilution buffer. Approximately 1 mL of each standard was pipetted into the appropriate prelabeled autosampler vials.

Controls:
The positive control used in this assay was cinnamic aldehyde prepared at a concentration of 100 mM. The positive control was reacted with the peptides in the same fashion as the test articles. There were three sets of reference controls of acetonitrile run at different points throughout the assay (reference controls A-C). Triplicate reference controls were also prepared for each solvent used in the assay. These controls consist of the solvent (acetonitrileor isopropanol) reacted with the peptide in the absence of test article. A coelution control was also prepared for the test article.The coelution control consisted of the test article without the peptide. The purpose of the coelution control was to determine if the test article elution from the HPLC column overlapped with the peptide elution.

Reaction Mixture Preparation:
Prior to testing, the test article was diluted in the appropriate solvent to yield a 100 mM test article concentration. The test article dilutions were mixed as determined during the solubility test (vortexing). The final dosing solutions was prepared for the test article, positive control, and reference control in the pre-labeled auto sampler vials. Triplicate samples were prepared for the test articles and controls. A single sample was prepared for each coelution control.

HPLC Set-up and Operation:
The separations module used in this assay was a Waters 2695 HPLC system. This system consisted of a solvent management system for the mobile phases and a sample management system for the test articles and controls. The HPLC system was coupled to a photodiode array detector set at 220 nm. The column used was a Zorbax SB-C18 column (Agilent) with dimensions of 2.1 mm x 100 mm x 3.5 micron. The column was primed for at least two hours before the start of the assay. To prime the column, equal parts of mobile phase A (0.1% trifluoroacetic acid in HPLC grade water) and mobile phase B (0.08% trifluoroacetic acid in HPLC grade acetonitrile) were passed through the column.

Once the column was primed for at least two hours, and the samples were prepared, the autosampler vials were placed into the designated locations of the separations module carousels. The samples were incubated in the dark at room temperature for 24±2 hours.

A gradient elution was used in this assay. The mobile phase changed from 10-25% acetonitrile over a 10 minute period to allow for sample separation and gradually elute most of the sample from the column. This was followed by a rapid increase to 90% acetonitrile to remove anything remaining on the column. The column was allowed to equilibrate back to initial specs for 7 minutes between injections.

The Empower3software was used to convert the absorbance data from the UV detector into chromatograms of intensity versus retention time for the samples and controls. At the end of the run, each chromatogram was integrated in order for the software to calculate the area under the peptide peak. Cysteine and lysine elute from the column at known times, so it was possible to determine which peaks in the chromatograms represented the peptides and use the areas under those peaks for the subsequent calculations.

Positive control results:

The positive control results for cinnamic aldehyde were a % mean peptide depletion of cysteine of 74.56 and lysine of 67.46.

Key result

Parameter:

other: % Mean Peptide Depletion of Cysteine

Value:

10.51

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Parameter:

other: % Mean Peptide Depletion of Lysine

Value:

13.08

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Parameter:

other: % Mean Peptide Depletion of Cysteine and Lysine

Value:

11.79

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

According to the results from the direct peptide reactivity assay, the test material was considered to be a sensitizer and therefore, met the GHS criteria for category 1 classification.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 18 - December 6, 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)

GLP compliance:

no

Remarks:

The study was also performed in compliance with OECD Guideline for the Testing of Chemicals No. 442D: In VitroSkin Sensitisation: ARE-Nrf2 Luciferase Test Method.

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The Induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE-ReporterCell Line KeratinoSens skin sensitization assay is a high-throughput cell-based in vitrotest to screen for the skin sensitization potential of chemicals.

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 as previously described. The cellswere 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 10^5 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 articles 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).

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. The test material did not turn the MTT solution color blue/purple, therefore, the test material does not directly reduce MTT.

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
The test article 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. 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.

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 tes 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:

34.28

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: IC50

Value:

390.73

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Imax

Parameter:

other:

Value:

45.95

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: CI Max

Value:

125

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

A test article was predicted to have sensitization potential if:
1) The EC1.5 value fell below 1,000 µM in at least 2 of 3 repetitions;
2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%; and
3) There was apparent overall dose response which was similar between repetitions.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

According to the current prediction model and according to GHS criteria, the test article is predicted to be a category 1 skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results from these two studies, the test material was considered to be a skin sensitizer and was classified as a GHS category 1 skin sensitizer.