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Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate
EC Number:
875-892-5
Cas Number:
1375799-59-9
Molecular formula:
C13 H14 N8 O2 . Na
IUPAC Name:
Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate

In chemico test system

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

Results and discussion

In vitro / in chemico

Resultsopen allclose all
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'

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



 


 


 

Applicant's summary and conclusion

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.