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Description of key information

Study report: OECD test guideline 442C-DPRA: cysteine depletion positive [Seiwert, 2021]


Study report: Profiling using the OECD QSAR Toolbox; profiling results: negative [Schlecker, 2021]


Study report: OECD test guideline 442E - hCLAT: positive [Seiwert, 2021]

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
in silico (QSAR toolbox profiling)
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Principles of method if other than guideline:
For the prediction of chemically-induced skin sensitization, a QSAR Toolbox 4.4.1 query is conducted. The QSAR Toolbox 4.4.1 has nine profilers to predict skin sensitisation.
The profilers of the QSAR Toolbox 4.4.1 consider if a covalent binding of the target molecule and skin proteins is in principle possible, based on mechanistic or empiric reasons. If a covalent binding is possible based on the structure of the target molecule and a known reaction mechanism an alert for protein binding (skin sensitization) is reported.
GLP compliance:
no
Remarks:
not applicable for in silico method
Remarks on result:
no indication of skin sensitisation
Remarks:
No alerts for protein binding are found by the used QSAR Toolbox 4.4.1 profilers.

Profiler - QSAR Toolbox 4.4 alerts for skin sensitisation





























































QSAR Toolbox protein binding profiler



Result



within applicability domain of profiler



Protein binding by OASIS



No alert found


 



yes



Protein binding by OECD



No alert found


 



yes



Protein binding alerts for skin sensitisation by OASIS



No alert found


 



yes



Protein binding alerts for skin sensitisation according to GHS



No alert found


 



yes



Protein binding potency                   Cys (DPRA 13%)



DPRA less than 9% (DPRA 13%);  


DPRA less than 9% (DPRA 13%) >> No protein binding alert



yes



Protein binding potency                   Lys (DPRA 13%)



DPRA less than 9% (DPRA 13%);


DPRA less than 9% (DPRA 13%) >> No protein binding alert



yes



Protein binding                  potency GSH



Not possible to classify according to these rules (GSH)



no



Protein binding                  potency h-CLAT



No alert found


 



yes



 Keratinocyte gene expression


 



Not possible to classify according to these rules



no



Skin sensitisation for DASS


 



Negative



yes


Interpretation of results:
other: This QSAR can be used as part of a testing battery based on the OECD adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.
Conclusions:
According to the QSAR Toolbox 4.4.1 Molidustat-Na (CAS 1375799-59-9) no alerts for skin sensitisation are found by QSAR Toolbox 4.4.1 profilers.
No experimental toxicological data are available for Molidustat-Na (CAS 1375799-59-9). Human Health Hazard Part of the QSAR Toolbox 4.4.1.
Furthermore, a negative result was detected by the profiler “Skin sensitization for DASS”. The customized profiler “Skin sensitization for DASS” is developed for Defined approaches (DA) purposes and it is part of the “Skin sensitization for defined approaches” automated workflow. The profiler combines application of the endpoint specific “Protein binding alerts for skin sensitization by OASIS” profiler with the Autoxidation and Skin metabolism simulators. Thus, it can identify the presence or absence of protein binding alerts in the parent chemical and predicted metabolites.

Based on the absence of alerts for skin sensitisation by profiling a score of 0 is applied according to OECD test guideline 497 (June 2021).
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/09/2021 to 20/10/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
26 June 2020
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details of test system:
cysteine peptide, (Ac-RFAACAA-COOH)
lysine peptide (Ac-RFAAKAACOOH)
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in approximately 20 mL aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
- Preparation of the test chemical solutions
- Preparation of the positive controls, reference controls and co-elution controls:
Preparation of positive control and cysteine peptide depletion samples and co-elution controls:
Triplicate solutions each of the positive control and test item stock solutions were diluted
with the cysteine peptide stock solution to prepare solutions containing 500 µM cysteine and
5 mM of trans-cinnamaldehyde or 5 mM ofthe test item. For the co-elution control, buffer
solution was used instead of the cysteine stock solution.
Preparation of positive control and lysine peptide depletion samples and co-elution controls:
Triplicate solutions each of the positive control and test item stock solutions were diluted
with the lysine peptide stock solution to prepare solutions containing 500 µM lysine and
25 mM of cinnamaldehyde or 25 mM of the test item. For the co-elution control, buffer
solution was used instead of the lysine stock solution.

INCUBATION
- Incubation conditions: 500 uM cysteine and lysine peptide solutions were incubated in glass autosampler vials with 5 mM or 25 mM of the test item, respectively. The reaction solutions were incubated in the dark at 22.5-30 °C for 24 ± 2 h prior to initiation ofthe analysis run.
- Precipitation noted

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Calibration standards of both peptides were prepared in a solution of 20% acetonitrile:buffer using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide. The following calibration solutions were prepared from the peptide stock solution of each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A blank ofthe dilution buffer was also included in the standard calibration curve for both peptides. The blank is 25% acteonitrile:buffer solution with phosphate buffer (pH 7 .5) for the cysteine peptide and with ammonium acetate buffer (pH 10.2) for the lysine peptide without peptide.
- Verification of the suitability of the HPLC for test chemical and control substances:
Reference control A: For the verification ofthe HPLC system suitability (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=1 with 3-fold injections.
Reference control B: For the stability ofthe reference controls over time (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=6.
Reference control C1: Peptide stability control for the solvent used to dissolve the test item
and the positive control (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=3.
Reference control C2: Peptide stability control for the solvent used to dissolve the test item
(samples containing only 0.5 mM peptide dissolved in the appropriate peptide buffer and deionized water. n=3.
Co-elution control: Sample prepared of the respective peptide buffer and the test item or
the positive control without peptide. n=1, each.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm and 258 nm
The concentration of cysteine or lysine peptide was photometrically determined at 220 nm in
each sample by measuring the peak area (area under the cuiye, AUC) ofthe appropriate
peaks and by calculating the concentration ofpeptide using the linear calibration curve
derived from the standards.
The peptide depletion of a sample was calculated as follows:
Peptidpeak area in replicate injection
Percent peptide depletion = [1 - ------------------------------------------------------------------------] x 100
Mean peptide peak area in reference control C

Vehicle / solvent:
water
Positive control:
cinnamic aldehyde
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
lysine depletion
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Remarks on result:
other: co-elution peak with lysine peptide buffer
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
cysteine depletion
Value:
42.7 %
At concentration:
5 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Outcome of the prediction model:
moderate reactivity [in chemico]
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control:Yes: 60.8-100% for the cysteine peptide: 74.5%
- Acceptance criteria met for reference controls A to C:Yes:
A: 495 µM(CV 0.853%, n=3)
B: 490 µM (CV 0.880%,n=6)
C1: 491 µM 8CV 0.674%, n=3)
C2: 505 µM (CV 0.491%, n=3)
SD = 2.55 (n=3)
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): only for Cysteine
- Acceptance criteria met for variability between replicate measurements: Yes: <14.9 percent points for the cysteine depletion: 2.55 (test item; 1.32 (positive control)
- Range of historical values if different from the ones specified in the test guideline: Please refer to 'any other information on results incl. tables'





































































































































Historical Control Data for the positive control



 



Cysteine peptide concentration (µM)1



Cysteine depletion %



Lysine peptide concentration (µM)²



Lysine depletion %



Mean



134



72.8



254



49.2



SD



14.9



3.00



45.9



9.27



CV (%)



11.1



4.12



18.0



18.9



n



57



57



54



54



1 Samples prepared at a concentration of 500 µM (376 µg/mL)


2 Samples prepared at a concentration of 500 µM (388 µg/mL]



 



Historical control data reference control A



 



Cysteine peptide concentration (µM)1



Cysteine peptide Rt (min)



Lysine peptide concentration (µM)²



Lysine peptide Rt (min)



Mean



505



11.5



508



8.54



SD



9.57



0.0526



13.8



0.0692



CV (%)



1.90



0.456



2.71



0.810



n



57



57



54



57



Rt Retention time


1 Samples prepared at a concentration of 500 µM (376 µg/mL)


2 Samples prepared at a concentration of 500 µM (388 µg/mL)



 



Historical Control Data Reference Control B



 



Cysteine peptide concentration (µM)1



Cysteine peptide Rt (min)



Lysine peptide concentration (µM)²



Lysine peptide Rt (min)



Mean



493



11.5



500



8.55



SD



11.9



0.0529



14.0



0.0955



CV (%)



2.43



0.459



2.79



1.12



n



114



114



108



114



Rt Retention time


1 Samples prepared at a concentration of 500 µM (376 µg/mL)


2 Samples prepared at a concentration of 500 µM (388 µg/mL)



 


 


Individual achieved peptide depletion values in the presence of the positive control and the test item

















































Sample



Cysteine peptide depletion [%]



Mean Cysteine depletion [%]



SD Cysteine (p.p.)



Lysine peptide depletion [%]



Mean Lysine depletion [%]



SD Lysine (p.p.)



Positive Control



73.01



74.5



1.32



62.0²



61.9



0.690



74.71



61.2²



75.61



62.6²



Test item



39.9³



42.7



2.55



0.004



0.00



0.00



43.6³



0.004



44.7³



0.004



SD Standard deviation


p.p. percent points


1 Calculated against a cysteine mean reference control (C1) area of 3332805 (n=3)


2 Calculated against a lysine mean reference control (C1) area of 2813285 (n=3)


3 Calculated against a cysteine mean reference control (C2) area of 3424662 (n=3)


4 Calculated against a lysine mean reference control (C2) area of 2753807 (n=3)


 


 


Acceptance criteria of each peptide

















































 



Peptide



Standard linearity



Positive control depletion (p.p.)



Reference controls (mean peptide concentration/coefficient of variation)



SD test item depletion (p.p.)



Acceptance criteria



Cysteine



r² > 0.99



60.8 – 100


(SD < 14.9)



450 - 550 µM (CV < 15%)



SD < 14.9



 



Lysine



r² > 0.99



40.2 – 69.0


(SD < 11.6)



450 - 550 µM (CV < 15%)



SD < 11.6



Achieved results



Cysteine



r² > 0.9998



74.5


(SD, 1.32, n=3)



A: 495 µM (CV 0.853%, n=3)


B: 490 µM (CV 0.880%, n=6)


C1: 491 µM (CV 0.674%, n=3)


C2: 505 µM (CV 0.491%, N=3)



SD 2.55


(n=3)



 



Lysine



r² > 0.9997



61.9


(SD, 0.690, N=3)



A: 503 µM (CV 0.390%, n=3)


B: 511 µM (CV 1.42%, n=6)


C1: 507 µM (CV1.67%, n=3)


C2: 496 µM (CV 0.362%, n=3)



SD 0.00


(n=3)



SD Standard deviation


p.p. percent points



 


 


 

Interpretation of results:
other: This DPRA can be used as part of a testing battery based on the OECD adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.
Conclusions:
Solutions of Molidustat were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. A co-elution peak was obseryed in the lysine assay.
Since co-elution of the test item was observed with the lysine peptide the prediction was one only with the cysteine peptide. With moderate mean depletion ofthe cysteine peptide (42.7%) in the presence of the test item, Molidustat is therefore predicted by DPRA as positive and to be a potential skin sensitizer based on this assay.
Based on the results a score of 2 is applied according to OECD TG 497 (June 2021)
Executive summary:

In an in chemico skin sensitisation study performed according to OECD test guideline 442C (Direct Peptide Reactivity Assay) the reactivity of the test item Molidustat was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.


The test item was dissolved at 100 mM in a 1:1 mixture of water:acetonitrile. The positive control was cinnamaldehyde, and for each peptide, the analytical batch included co-elution control samples and three reference control samples.


Reactivity (%depletion) was determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by liquid chromatography with UV-detection.


Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the two relevant reference control C samples for Cysteine. Since co-elution of the test item was observed with the lysine peptide the prediction was done only with the cysteine peptide. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied and thus demonstrated the validity of the study.


Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with the cysteine peptides. As a result, the mean percent depletion values were calculated for the cysteine peptide. For the cysteine peptide, the mean depletion value was 42.7%.


Since the mean of the percent cysteine depletionwas greater than 23.09%, the test item was considered to have moderate peptide reactivity. Therefore, the DPRA prediction would be considered as positive and the test item may have the potential to cause skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)
Version / remarks:
June 2018
GLP compliance:
yes (incl. QA statement)
Type of study:
human Cell Line Activation Test (h-CLAT)
Details of test system:
THP-1 cell line [442E]
Details on the study design:
442E

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: On the day of the experiment (prior to start) the test item was stably suspended in culture medium to prepare a stock solution.
- Preparation of the test chemical serial dilutions: As tested by a solubility test, 5000 µg/mL in culture medium was used as highest test item concentration in the cytotoxicity test as recommended by the OECD 442E guideline.
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions. For the test item exposure for the main experiments, the highest dose solution calculated from the cytotoxicity test was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution.

- Preparation of the positive controls: DMSO (dimethyl sulfoxide, CAS No. 67-68-5, Purity ≥99%) final concentration 0.2% in culture medium.
- Preparation of the solvent, vehicle and negative controls: Medium control: Culture medium; Solvent control for the test item: Culture medium

- Stable dispersion obtained: Test item was solved in vehicle
- Log Kow of the test chemical: 0.27

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 5000 µg/mL
- Solubility in incubation medium: fully soluble
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: Cytotoxic effects (threshold of cytotoxicity: <75%) were observed following incubation with the test item starting with the concentration of 78.1 µg/mL up to the highest tested concentration (5000 µg/mL) in both cytotoxicity tests. Precipitations were observed starting at a concentration of 1250 µg/mL. Based on the results of the cytotoxicity assays a CV75 of 60.88 µg/mL could be calculated in the first cytotoxicity assay and 54.76 µg/mL in the second cytotoxicity assay. The mean CV75 value of both cytotoxicity tests was calculated as 57.82 µg/mL.
- Final concentration range selected on basis of: The mean of the two CV75 values was used to determine the dose-range for the main experiments (h-CLAT). Eight concentrations (µg/mL) were used for the test item in the main experiments (h CLAT). The highest concentration used was 1.2 × mean CV75 with a serial dilution factor of 1.2.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Triplicates
- Number of repetitions: Two independent runs
- Test chemical concentrations: 19.4, 23.2, 27.9, 33.5, 40.2, 48.2, 57.8, 69.4 µg/mL
- Application procedure: Each volume (500 µL) of the dilutions of the test item and culture medium was added to the cells.
- Exposure time 24 ± 0.5 h
- Study evaluation and decision criteria used: At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:

(MFI of test item treated cells) - (MFI of test item treated isotype control cells)
RFI[%]= ----------------------------------------------------------------------------------------------------------------------------- x100
(MFI of solvent control cells) - (MFI of solvent isotype control cells)

MFI = Geometric Mean Fluorescence Intensity (GeoMean)

The cell viability from the isotype control cells, CD54 and CD86 cells is calculated according to the following equation:
(Number of living cells)
Cell Viability [%]=-----------------------------------------------------------×100
(Total Number of acquired cells)

Where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). A h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

- Description on study acceptance criteria: The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥150% and CD54 ≥200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥150% and CD54 ≥200%) and the cell viability should be >50% in at least one concentration of the two tested positive control concentrations.
• For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of <90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability is >90% (OECD 442E guideline).


SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): The passage numbers of the used THP-1 cells were 14 and 16 in the cytotoxicity tests and 20 and 22 in the h CLAT for runs 1 and 2, respectively. On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 µL with a cell density of 1.8-2 E+06 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.
- Incubation conditions: The cells are sub-cultured at least twice a week. The cell density should not exceed 1E+06 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Culture medium: RPMI 1640 Medium, GlutaMAXTM supplement including 25 mM HEPES, supplemented with 10% FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1 mM sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2-8 °C and used within one month.
- Washing conditions: Each test item-treated and not test item-treated cells were collected in sample tubes, centrifuged (approx. 250 x g, 5 min), washed twice (2-8°C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 min before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added in each sample tube.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal. The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
- Plate used: 245 well flat-bottom-plate
- Preparation for CD54 and/or CD86 expression measurements/cell staining: Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1). The triplicates of each test item-treated and not test item-treated cells were pooled 2xg, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2-8°C (on ice) for approx. 15 min. After blocking, the cells were centrifuged, and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2-8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min at 2-8 °C (on ice). After staining with the antibodies, the cells were washed twice (2-8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 min before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

DATA EVALUATION
- Cytotoxicity assessment: See sections above
- Prediction model used: See sections above
Vehicle / solvent control:
DMSO
Negative control:
other: Culture medium
Positive control:
dinitrochlorobenzene (DNCB) [442E]
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
RFI CD86>200 [442E]
Remarks:
at concentration 19.4 µg/mL
Value:
394.7 %
Cell viability:
60.27%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at every concentration the RFI for CD 86 was > 200 and the viability was > 50%; see also under 'Any other information on results incl. tables'
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
RFI CD86>200 [442E]
Remarks:
at concentration 19.4 µg/mL
Value:
324.1 %
Cell viability:
59 %
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at every concentration the RFI for CD 86 was > 200 and the viability was > 50%; see also under 'Any other information on results incl. tables'
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
RFI CD54>150 [442E]
Remarks:
for the concentration 19.4 µg/mL
Value:
2 431.1 %
Cell viability:
60.27%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at every concentration the RFI for CD 54 was > 150 and the viability was > 50%; see also under 'Any other information on results incl. tables'
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
RFI CD54>150 [442E]
Remarks:
for the concentration 19.4 µg/mL
Value:
2 437.1 %
Cell viability:
59%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at every concentration the RFI for CD 54 was > 150 and the viability was > 50%; see also under 'Any other information on results incl. tables'
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
CV75 [442D and 442E]
Value:
57.82 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

Results of the dose finding assay (cytotoxicity test) 1:




























































Test group



Concentration [µg/mL]



Microscopic evaluation / cytotoxicity



Flow cytometric evaluation /cell viability [%]



Medium control



-



no



96.45



Test item



39.1



no



78.04



78.1



no



73.29



156



no



68.99



313



yes



71.26



625



yes



59.20



1250P



n.d.



30.17



2500P



n.d.



32.29



5000P



n.d.



23.63



P = precipitation


n.d. = not determined


Results of the second cytotoxicity test for the test item:




























































Test group



Concentration [µg/mL]



Microscopic evaluation / cytotoxicity



Flow cytometric evaluation / cell viability [%]



Medium control



-



no



97.09



Test item



39.1



no



77.43



78.1



no



72.44



156



no



66.39



313



yes



69.10



625



yes



63.98



1250P



n.d.



42.01



2500P



n.d.



42.10



5000P



n.d.



38.90



P = precipitation


n.d. = not determined


Results of the first h-CLAT run for the test item

























































































 



Concentration [µg/mL]



RFI CD54 antibody [%]



RFI CD86 antibody [%]



Cell viability [%]



Medium control



-



100.0



100.0



98.23



DMSO control



-



100.0



100.0



97.97



Positive control (DNCB)



3.0



295.5*



404.7*



92.62



4.0



487.5*



365.1*



72.65



Test item



69.4



593.5*



323.7*



81.27



57.8



772.6*



365.4*



78.21



48.2



767.7*



374.6*



77.26



40.2



1295.2*



285.1*



69.94



33.5



1272.6*



381.7*



69.03



27.9



1974.2*



247.8*



60.95



23.2



1940.3*



292.2*



59.15



19.4



2437.1*



324.1*



59.00



* = RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥150% and CD54 ≥200%).


Results of the second h-CLAT run for the test item

























































































 



Concentration [µg/mL]



RFI CD54 antibody [%]



RFI CD86 antibody [%]



Cell viability [%]



Medium control



-



100.0



100.0



97.35



DMSO control



-



100.0



100.0



97.33



Positive control (DNCB)



3



417.5*



408.7*



93.01



4



639.7*



436.7*



87.90



Test item



69.4



528.9*



245.5*



78.55



57.8



735.6*



300.0*



70.30



48.2



1053.3*



327.8*



65.23



40.2



1220.0*



332.5*



64.59



33.5



1584.4*



392.3*



62.97



27.9



1944.4*



429.2*



59.78



23.2



2273.3*



421.5*



59.04



19.4



2431.1*



394.7*



60.27



* = RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥150% and CD54 ≥200%).


 


 


 

Interpretation of results:
other: This hCLAT can be used as part of a testing battery based on the OECD adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.
Conclusions:
In conclusion, the test item Molidustat activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation AOP.
Based on the results a score of 2 is applied according to OECD TG 497 (June 2021).
Executive summary:

In the present test conducted according to OECD test guideline 442E (2018) THP-1 Cells were exposed to Molidustat stably suspended in culture medium at concentrations of 0, 19.4, 23.2, 27.9, 33.5, 40.2, 48.2, 57.8, 69.4 µg/mL for 24 ± 0.5 h.


Cytotoxic effects (threshold of cytotoxicity: <75%) were observed following incubation with the test item starting with the concentration of 78.1 µg/mL up to the highest tested concentration (5000 µg/mL) in both cytotoxicity tests. Precipitations were observed starting at a concentration of 1250 µg/mL. Based on the results of the cytotoxicity assays a CV75 of 60.88 µg/mL could be calculated in the first cytotoxicity assay and 54.76 µg/mL in the second cytotoxicity assay. The mean CV75 value of both cytotoxicity tests was calculated as 57.82 µg/mL.


The relative fluorescence intensity (RFI) of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in all concentrations of both runs. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.


All acceptance criteria were met. The cell viability of the medium and DMSO control was >90%. For both medium and solvent control, the MFI ratio of CD86 and CD54 to isotype control was >105%. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥200% and CD86 ≥150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥200% and CD86 ≥150%) and the cell viability was >50%. For the test item, the cell viability was more than 50% in at least four tested concentrations in each run.


Therefore, the test item is considered positive for the third key event of the skin sensitisation AOP.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

In an in chemico skin sensitisation study performed according to OECD test guideline 442C (Direct Peptide Reactivity Assay) the reactivity of the test item Molidustat was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.


The test item was dissolved at 100 mM in a 1:1 mixture of water:acetonitrile. The positive control was cinnamaldehyde, and for each peptide, the analytical batch included co-elution control samples and three reference control samples.


Reactivity (%depletion) was determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by liquid chromatography with UV-detection.


Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the two relevant reference control C samples for Cysteine. Since co-elution of the test item was observed with the lysine peptide the prediction was done only with the cysteine peptide. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied and thus demonstrated the validity of the study.


Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with the cysteine peptides. As a result, the mean percent depletion values were calculated for the cysteine peptide. For the cysteine peptide, the mean depletion value was 42.7%.


Since the mean of the percent cysteine depletionwas greater than 23.09%, the test item was considered to have moderate peptide reactivity. Therefore, the DPRA prediction would be considered as positive and the test item may have the potential to cause skin sensitisation.


 


 


In the present test conducted according to OECD test guideline 442E (2018) THP-1 Cells were exposed to Molidustat stably suspended in culture medium at concentrations of 0, 19.4, 23.2, 27.9, 33.5, 40.2, 48.2, 57.8, 69.4 µg/mL for 24 ± 0.5 h.


Cytotoxic effects (threshold of cytotoxicity: <75%) were observed following incubation with the test item starting with the concentration of 78.1 µg/mL up to the highest tested concentration (5000 µg/mL) in both cytotoxicity tests. Precipitations were observed starting at a concentration of 1250 µg/mL. Based on the results of the cytotoxicity assays a CV75 of 60.88 µg/mL could be calculated in the first cytotoxicity assay and 54.76 µg/mL in the second cytotoxicity assay. The mean CV75 value of both cytotoxicity tests was calculated as 57.82 µg/mL.


The relative fluorescence intensity (RFI) of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in all concentrations of both runs. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.


All acceptance criteria were met. The cell viability of the medium and DMSO control was >90%. For both medium and solvent control, the MFI ratio of CD86 and CD54 to isotype control was >105%. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥200% and CD86 ≥150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥200% and CD86 ≥150%) and the cell viability was >50%. For the test item, the cell viability was more than 50% in at least four tested concentrations in each run.


Therefore, the test item is considered positive for the third key event of the skin sensitisation AOP.


 


Furthermore, in accordance to OECD test guideline 497 (2021) an in silico prediction performed with the OECD QSAR Toolbox 4.4.1, was conducted. In accordance to the integrated testing strategy the target compound was profiled for protein binding alerts. No structurally similar structure could be identified, thus, read-across to a source substance was not possible. Instead the profilers outcomes were directly used. As described in Annex 2 of the guideline the applicability domain for profilers takes into account three layers: the parametric layer, the structuralc layer and the mechanistic layer.


For the parametric layer physico-chemical parameters of the substance are used for which ranges of variation are given from calculated values (EpiSuite). If a substance falls in these ranges  the parametric layer is met. In case of Molidustat the parametric layer is met due to a calculated logKow of -4.42; molecular weight of 336.29 g/mol; water solubility of 1E+06 mg/L. These values are all in the range variation as depicted in OECD testguideline 497.


The structural layer is met because the substance is considered within the structural domain of the DASS AW, i.e. 100% of its ACF (atom centered fragments) belong to the correct fragments.


The mechanistic layer is out of the domain as the negative prediction with profilers only is not sufficient to exclude protein binding for the substance.


However, according to the defined approach as described in the test guideline 'Integrated testing strategy' (ITS) from the applied in vitro tests a score of 2 for each result can be defined due to the unequivocal results, from the in silico prediction a score of 0 is applied, thus, the final score of 4 results in a classification according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) of Category 1B.

Justification for classification or non-classification

According to the defined approach as described in the test guideline 'Integrated testing strategy' (ITS) from the applied in vitro tests a score of 2 for each result can be defined due to the unequivocal results, from the in silico prediction a score of 0 is applied, thus, the final score of 4 results in a classification according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) of Category 1B.