3-phenyl-1H-pyrazole-4-carbaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-11-23 to 2020-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Adopted June, 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155

Version / remarks:

KeratinoSens™ (Adopted March, 2018)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-1H-pyrazole-4-carbaldehyde

EC Number:

632-673-7

Cas Number:

26033-20-5

Molecular formula:

C10H8N2O

IUPAC Name:

3-phenyl-1H-pyrazole-4-carbaldehyde

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch (Lot) No.of test material: M19LD3723
- Expiration date of the lot/batch: 2021-11-11 (retest date)
- Physical Description: Light yellow powder
- Purity test date: not indicated
- Purity: 103.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: A separate solubility test was performed prior to the current study (Test Facility Study No. 20263901). In addition, a confirmatory solubility test was performed to confirm vehicle and precipitation.
Based on this study the test item was dissolved in DMSO to a final concentration of 200 mM (clear yellow solution).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: In the main experiments the test item was dissolved in DMSO at 200 mM (colourless to yellow solution). From this stock 11 spike solutions in DMSO were prepared (2-fold and 1.5-fold dilution series in respectively experiments 1 and 2 ).

OTHER SPECIFICS
- Correction factor: 1.00

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- A solubility test was performed as described in Specific details on test material used for this study
- The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test
concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (experiment 1) and 2000, 1333, 889, 593, 395, 263, 176, 117, 78, 52, 35 and 23 μM (experiment 2) (final concentration of DMSO was 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.
No precipitation was observed at the start and end of the incubation period in the 96-well plates.
Test item concentrations were used within 3 hours after preparation.


PREPARATION OF THE POSITIVE CONTROL
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described above, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The formulation of the positive control was used in studies performed concurrently.

PREPARATION OF THE SOLVENT CONTROL
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

BLANK
On each plate three blank wells were tested (no cells and no treatment).

TEST SYSTEM
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum
Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).
Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

ENVIRONMENTAL CONDITIONS
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 39-92%%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 33.3 - 37.3 °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 and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

EXPERIMENTAL DESIGN
- Two experiments were conducted
- Subculturing: Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock and were not cultured for more than 25 passages from the frozen stock (P+25).
- Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+5 in experiment 1 and P+7 in experiment 2.
- Treatment of cells: The medium was removed and replaced with fresh exposure medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2. Initially, experiment 1 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 2 valid experiments were performed.
- Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady- Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
- Cytotoxicity Assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant equal or above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

DATA EVALUATION AND STATISTICAL PROCEDURES
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is equal or higher than 1.5-fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the vehicle (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 was used for statistical analysis of the data. The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

DATA INTERPRETATION
A minimum of two experiments was conducted, in case of two not concordant results, a third experiment was performed.
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is equal or higher than (≥) 1.5-fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity ≥ 1.5-fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Negative results obtained with concentrations <1000 µM or 200 µg/mL and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

other: ethylene dimethacrylate glycol (EDMG)

Results and discussion

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.19 and the EC1.5 was 45 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.96 and the EC1.5 was 52 μM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

2.05

Cell viability:

81%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

673 µM

Cell viability:

30%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Cell viability:

50%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

50% toxicity was not reached and thus no IC50 could be calculated.

Results 4

Key result

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

315 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 5

Key result

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

2.73

Cell viability:

95%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 6

Key result

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

783 µM

Cell viability:

30%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 7

Key result

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Cell viability:

50%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

50% toxicity was not reached and thus no IC50 could be calculated.

Results 8

Key result

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

231 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The KeratinoSensTM assay was successfully implemented and validated, and lab proficiency has been shown by obtaining the expected KeratinoSensTM prediction for the 10 proficiency chemicals that are described in the OECD 442D guideline.

ACCEPTANCE OF RESULTS:
Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (45 µM and 52µM in experiments 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.19-fold and 2.96-fold in experiments 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (3.7% in experiments 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Test item results

Experiment 1:No precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed toxicity. The calculated IC30 was 673 μM and the calculated IC50 was 1000 μM.

A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 2.05 and the EC1.5 315 μM.

 

Experiment 2: No precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed toxicity. The calculated IC30 was 783 μM. Fifty percent toxicity was not reached and thus no IC50 could be calculated.

A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 2.73 and the EC1.5 231 μM.

 

Historical Control Data for the KeratinoSensTM Studies:

 

 

	Positive control
	EC1.5(µM)	Imax
Range (mean ± 2x SD)	-3.0 – 120	-5.65 – 12.15
Mean	58.5	3.25
SD	30.7	4.45
n	503	503

 

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2017 to November 2020.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, JNJ-39125190-AAA (T003897) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.