1-[2-[2-methoxy-4-(morpholin-4-yl)-5-nitroanilino]pyrimidin-4-yl]-3-phenyl-1H-pyrazole-4-carbaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-11-25 to 2020-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

June 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-[2-[2-methoxy-4-(morpholin-4-yl)-5-nitroanilino]pyrimidin-4-yl]-3-phenyl-1H-pyrazole-4-carbaldehyde

Cas Number:

1903010-39-8

Molecular formula:

C25H23N7O5

IUPAC Name:

1-[2-[2-methoxy-4-(morpholin-4-yl)-5-nitroanilino]pyrimidin-4-yl]-3-phenyl-1H-pyrazole-4-carbaldehyde

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: solid
- Appearance: orange powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: I19HD2158
- Expiration date of the lot/batch: 2020-09-01
- Physical Description: Orange powder
- Purity: 100.4%
- Purity test date: 2019-09-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in vehicle: not indicated


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: No, the solid test item was applied directly on top of the skin tissue. The test item was spread to match the size of the tissue.

OTHER SPECIFICS
- correction factor: 1

In vitro test system

Test system:

human skin model

Remarks:

model of human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: not applicable

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200) supplied by MatTek Corporation, Ashland MA, U.S.A
- Tissue batch number(s): 32126 Kit A and Kit B
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium (Dulbecco’s Modified Eagle’s Medium) (supplemented DMEM medium, serum-free supplied by MatTek Corporation) per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature during 3-minute exposure and 37°C during 1-hour exposure
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2 °C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline (PBS) (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 µL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- The DMEM medium was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Test for the interference with the MTT endpoint: A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
- Test for colour interference by the test item: the test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for reduction of MTT by the test item: the test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple colour change or a blue/purple precipitate was observed.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (OD (540-570 nm)<1.0-3.0>): 1.884 +/- 0.089
- Barrier function (ET-50 <4.77-8.72 hrs>): 6.4hrs
- Sterility: sterile

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non-corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.9 to 45.6 mg

NEGATIVE CONTROL (Milli-Q water)
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL (8N KOH)
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4 replicates per test item, negative control and positive control: 2 for the 3-minute exposure and 2 for the 1-hour exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Individual values: 102 and 103

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Individual values: 92 and 93

Results 3

Irritation / corrosion parameter:

other: optical denisty

Run / experiment:

3-minute application

Value:

1.749

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Individual values: 1.753 and 1.746; SD +/- 0.005

Results 4

Irritation / corrosion parameter:

other: optical density

Run / experiment:

1-hour application

Value:

1.645

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Individual values: 1.642 and 1.647; SD +/- 0.004

Results 5

Irritation / corrosion parameter:

other: coefficient of variation

Run / experiment:

3-minute application

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation / corrosion parameter:

other: coefficient of variation

Run / experiment:

1-hour application

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

3-minute application:
- viability (percentage of control) (range):
negative control: 100 (96 and 104)
positive control: 19 (17 and 22)
- coefficient of variation between tissue replicates
negative control: 7.3
positive control: 22
- mean optical density:
negative control: 1.708 +/- 0.092
positive control: 0.328 +/- 0.056

1-hour application:
- viability (percentage of control) (range):
negative control: 100 (103 and 97)
positive control: 7.3 (7.6 and 7.0)
- coefficient of variation between tissue replicates
negative control: 5.6
positive control: 8.0
- mean optical density:
negative control: 1.779 +/- 0.072
positive control: 0.130 +/-0.008

Results:
The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 102% (102% and 103%) and 92% (92% and 93%) respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range. The mean relative tissue viability following the 3-minute and 1-hour exposure to the positive control was 19% (17% and 22%) and 7.3%(7.6% and 7.0%), respectively. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 8%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.