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

Skin irritation / corrosion

Administrative data

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

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

Version / remarks:

28 July 2018

GLP compliance:

yes

Test material

Test material information

Constituent 1

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 vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Applicant's summary and conclusion

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.