Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate

Endpoint summary

Description of key information

Study report: OECD test guideline 439; result: not irritating [Wingenroth, 2021]

Study report: OECD test guideline 492; result: at least irritating to the eye [Leidenfrost, 2021]

Study report: OECD test guideline 437; result: No Category (not corrosive) [Leidenfrost, 2021]

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

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

Version / remarks:

28 July 2018

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

According to the OECD Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Skin Corrosion and Irritation the use of validated in vitro methods for the determination of skin corrosion /irritation is recommended.
Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. The test consists of a topical exposure of the neat substance to a human reconstituted epidermis model followed by
a cell viability test (MTT-assay).
There are several synonyms for artificial 3D-skin models like human skin recombinants, reconstructed human skin/epidermis, 3D human skin/epidermal equivalents, in vitro engineered skin/epidermal substitutes, artificial skin/epidermis. The 3D-skin used closely mimics the biochemical and physiological properties of the upper part of the human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (Henkel AG & Co, KGaA, Düsseldorf)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 42 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not reported
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: The concentration of formazan was measured by determining the OD of the isopropanol-extracts in duplicates at 570 nm in an automatic reader (EL808, Bio-Tek; 96 well format, 200 µL). Data acquisition and evaluation were performed with "Gen5" (software by Bio-Tek).
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Reliability of the test was previously confirmed by interlaboratory validation

NUMBER OF REPLICATE TISSUES: triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: Not necessary

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure and post-treatment incubation is less than 50%
- The test substance is considered to be non-irritant to skin if he viability after exposure and post-treatment incubation is greater than or equal to 50%

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): 0.9 % NaCl (not vehicle but solution for moistening


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS solution

Duration of treatment / exposure:

20 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

100

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for the negative control

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

1.29

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for the positive control

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

89.83

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

Interpretation of results:

GHS criteria not met

Conclusions:

Molidustat sodium micronized did not show a significant impact on cell viability and is thus
identified as non-irritant substance in this test model.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), Molidustat was applied to the three-dimensional human epidermis model tissue for an exposure period of 20 minutes in triplicates. 30 μL of physiological saline were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 30 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 20 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (physiological saline) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 20 minutes treatment with Molidustat compared to the negative control tissues was 89.83%. Since the mean relative tissue viability for the test substance was above 50%, Molidustat is identified to be not irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-07-27 to 2021-10-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

27 June 2018

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method (RhCE) and considerations regarding applicability: The EpiOcularTM EIT is an in vitro procedure allowing the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. It makes use of reconstructed human comea-like epithelium (RhCE) which closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: Reconstructed ocular tissue model containing normal human keratinocytes.
All cells used to produce EpiOcular™ are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions. ln all cases, consent was obtained by these institutions from the donor or the donors legal nest of kin, for use of the cells or derivatives of the tissue for research purposes. HIV-1: negative; HepB: negative, HepC: negative; Bacteria, yeast and other fungi: negative.
- RhCE tissue or hCE cell construct used, including batch number: Kerationcyte strain: 4F1188, Lot Number: 34923
Analysis for tissue function ality and quality wil be decribed under 'Any other information on materials and methods incl. tables'

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:

6 h treatment with 18 h post-treatment period

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

duplicates

Details on study design:

- Details of the test procedure used:
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier. RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s
medium (Lot No.: 34923; Testing date: 03 Aug 2021).

- Doses of test chemical and control substances used:
Treatment: Before treatment 20 µL PBS were applied to all EpiOcular™ tissue surface for an incubation time of 30 ± 2 minutes under SCC. Following 50 mg of the neat test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
The tissues were treated for 6 hours ± 15 min under SCC. After the end of treatment time the test item was removed by rinsing with PBS followed by a post-soak immersion period of 25 ± 2 min. in fresh medium. Post-treatment incubation: the tissues were incubated for 18 hours ± 15 min in fresh medium under SCC.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment ofpotential direct MTT-reduction of the test item. In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay
2. Assessment ofpotential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence oftest item color on OD measurement, a color control was used in the
main assay. The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color tums blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is > 0.08 a Color Control must be conducted.
If a test item is identified as producing both, color interference and direct MTT reduction, a third set of controls (Non-specific killed control NS KC) is required, additionally from the PG KC and PG CC controls. This is usually the case with darkly coloured test items interfering
with the MTT assay (e.g. blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT. Color Control and Killed Control were not required.

- Number of tissue replicates used per test chemical and controls (positive control, negative control): duplicates

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm; spectrophotometer (EL808, Bio-Tek; 96 well format, 200 µL)

- Description of the method used to quantify MTT formazan, if applicable:
Immediately after the end of post-treatment incubation the MTT reduction assay was performed. For viability testing the inserts were placed in new plates containing MTT solution (1 mg/mL in Maintenance medium at 37°C, or Assay medium for the color control inserts).
The tissues were incubated for 180 ± 10 min. under SCC. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
For interpretation of cell viability results the cut-off value distinguishing classified (irritant)
from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.8
- mean relative viability ofthe positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

- Complete supporting information for the specific RhCE tissue construct or hCE cells used: please refer to 'Any other infomation on materials and methods incl. tables'

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean

Value:

28

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Summary of results
Compound	% measure Cell Viability	OD mean (n=4)
test item	27.46	0.479
test item KC	n.a.	n.a.
NC KC	n.a.	n.a.
Test item CC	n.a.	n.a.
Positive control	12.72	0.221
Negative control	100	1.739
% FINAL viability of the test item *	27.56
% FINAL viability of the test item rounded	28
*= % viability test item – (% viability test item KC - % viability NC KC) - % viability test item CC + viability NS KC
n.a. = not applicable, because no KC and NC KC control were required

Interpretation of results:

other:

Remarks:

According to the Evaluation criteria of OECD test guideline 492 the test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) because the mean percent tissue viability after exposure and and post exposure incubation is less than or equal (≤) to 60%.

Conclusions:

In this study under the given conditions the test item showed irritant effects.

Executive summary:

In this study conducted according to OECD test guideline 492 (adopted June 27, 2018), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular™, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

The test item was applied topically to the EpiOcular™ tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After an 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with deionized water.

The test item showed no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol and after mixture with deionized water. Therefore, NSCliving was not determined.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (28 %).

The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.739). The mean relative tissue viability (% negative control) of the positive control was < 50% (12.72%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20%.

In this study under the given conditions the test item showed irritant effects.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), Molidustat was applied to the three-dimensional human epidermis model tissue for an exposure period of 20 minutes in triplicates. 30 μL of physiological saline were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 30 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 20 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (physiological saline) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 20 minutes treatment with Molidustat compared to the negative control tissues was 89.83%. Since the mean relative tissue viability for the test substance was above 50%, Molidustat is identified to be not irritating.

 

In a study conducted according to ICH Topic M3 R2: Nonclinical Safety Studies: Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (CPMP/ICH/286/95), June 2009 and EMA: Note for Guidance on Non-Clinical Local Tolerance Testing of Medicinal Products CPMP/SWP/2145/00, February 2001, male and female New Zealand White rabbits were exposed to a formulation of the test item intravenously. A single dose of the formulation as well as the control article was administered intravenously (ear vein, uncongested) and paravenously (hind leg, adjacent to Vena saphena) to 4 male and 4 female rabbits. The administration volume was 0.5 mL for the intravenous route and 1.0 mL for the paravenous route, respectively. The left side of each administration route was treated with the control article, the right side with the formulated drug substance. The respective injection sites were observed immediately, 2, 4 and 24 h post administration (p.a.). Additionally, clinical observations were performed twice daily. Body weight was recorded on Day 1, 3 and 8. Each 2 male and 2 female animals were subjected to necropsy on Day 3 and on Day 8, respectively. The intravenous injection sites as well as the paravenous (adjacent tissue to V. saphena) injection sites were collected and processed for histopathological evaluation.

Following local treatment of the animals with BAY 1053048 and the control article, no clinical findings of toxicological relevance were observed. Animals treated with the test-item and the control article showed slight/moderate reddening and swelling at the injection site and/or around the injection site. Hematoma were also present after intravenous (iv) and paravenous (pv) injection. The local clinical signs (injection site and surrounding) observed started during the injection and returned to normal until Day 6. Overall, there was no indication of drug-induced changes.

 

 

 

In this study conducted according to OECD test guideline 492 (adopted June 27, 2018), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular™, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

The test item was applied topically to the EpiOcular™ tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After an 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with deionized water.

The test item showed no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol and after mixture with deionized water. Therefore, NSCliving was not determined.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (28 %).

The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.739). The mean relative tissue viability (% negative control) of the positive control was < 50% (12.72%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20%.

In this study under the given conditions the test item showed irritant effects.

 

An in vitro study was performed to assess them corneal irritation and damage potential of Molidustat (20% in isotonic saline solution) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 2020.

 

The corneae were incubated with the test substance and controls for 4 h. After exposure the corneae were rinsed with phenol red containing MEM. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean corrected OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

 

A 20% dilution of the test substance in isotonic saline solution caused no increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was -1.5.

 

The positive control (Imidazole) increased the opacity and permeability of the corneae in this experiment (mean in vitro irritation score 107.8.

 

With the negative control (isotonic saline solution) neither an increase of opacity nor permeability of the corneae could be observed in the experiment - mean in vitro irritation score 2.0.

 

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution of Molidustat in isotonic saline solution is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the presented data Molidustat does not need to be classified as irritant to the skin but need to be classified irritant to the eye (Category 2) according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).