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

Endpoint summary

Description of key information

In silico, in chemico and in vitro solubility testing data are used in a weight-of-evidence approach. In addition, the Local Lymph Node Assay (LLNA) is added as key study.

- In silico: The Derek Nexus assessment triggered an alert for skin sensitization for JNJ-39125190-AAA (T003897) and predicted the substance to be skin sensitizing (Van Gompel J, 2020). 

- In chemico (OECD 442C): JNJ-39125190-AAA (T003897) was positive in the DPRA; however, assignment to a reactivity class was not made (Maire F, 2021).  

- In vitro (OECD 442D): The KeratinoSensTM assay yielded a positive result (Groot A, 2021).

Taking into account the positive in silico, in chemico and in vitro results and as it is not possible to determine the potency (Cat. 1A or 1B) based on non-animal testing approaches, as required in Annex VII, section 8.3, additional in vivo testing (Local Lymph Node Assay in
mice according to OECD 429) was performed to assess the potency of the test item (Schrouff F, 2021).

- In vivo (OECD 429): Based on the results of a Local Lymph Node Assay (LLNA), the test item was not regarded as skin sensitizer (van de Wiel S, 2021).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2020-11-17 to 2020-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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: Solubility of the test item in an appropriate solvent was assessed before performing the DPRA (Test Facility Study No. 20263901). The test item was soluble at 100 mM (stock solution) in acetonitrile (ACN), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), ethanol and methanol and showed no precipitation or phase separation in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) and ethanol in the SPCC and SPCL test conditions. In methanol, precipitation was observed after incubation in the SPCL test conditions.
Based on the solubility assessment the preferred solvent for the DPRA is acetonitrile
(ACN).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assays, 33.84 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1965 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the clear solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

OTHER SPECIFICS:
- Correction factor: 1.00

Details on the study design:

Skin sensitisation (In chemico test system): details on Study Design

TEST SYSTEM
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC, and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Batch SPCC: 111016HS-MHeW0920
- Batch SPCL: 020517HS-MHeW0920
- Storage: The peptides were stored in the freezer (≤ -15°C) for a maximum of 6 months.

EXPERIMENTAL DESIGN
TEST ITEM PREPARATION
see details under "Specific details on test material used for the study"

PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- SPCC stock solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.1 mg of SPCC in 20.16 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC reference control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
- SPCC calibration curve: A SPCC calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- SPCL stock solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 20 mg of SPCL in 38.61 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL reference control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
- SPCL calibration curve: A SPCL calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

SAMPLE INCUBATIONS
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.2 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC analysis, the samples were visually inspected for precipitation.

HPLC Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
- Alliance separations module 2695 (Waters, Milford, MA, USA)
- Dual λ absorbance detector 2487 (Waters)

System 2 (used for Lysine Reactivity Assay):
- Alliance separations module 2695 (Waters, Milford, MA, USA)
- Dual λ absorbance detector 2487 (Waters)

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r²>0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50 ±0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
- The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
% Peptide Depletion = [ 1 - (Peptide Peak Area in Replication injection at 220 nm / Mean Peptide Peak Area in Reference Controls at 220 nm) ] x 100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item.
Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
As the overlap in retention time between the test item and either of the peptides was incomplete, the Percent Peptide Depletion was estimated and used in the Cysteine 1:10 /Lysine 1:50 prediction model. However, assignment to a reactivity class was not made.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Cysteine reactivity assay: The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 69.9% ± 0.2%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Lysine reactivity assay: The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 58.2% ± 0.7%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

2.5 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

35 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: % Mean of SPCC and SPCL depletion

Value:

18.7 %

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 DPRA assay was successfully validated at the laboratory and can be used to support the discrimination between sensitisers and non-sensitisers.

ACCEPTANCE OF RESULTS - Cysteine reactivity assay
- The correlation coefficient (r2) of the SPCC standard calibration curve was 0.998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.515 ± 0.010 mM while the mean peptide concentration of Reference Controls C was 0.488 ± 0.002 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
- The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.1%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 37.62. The mean A220/A258 ratio ± 10% range was 33.86-41.38. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.


ACCEPTANCE OF RESULTS - Lysine reactivity assay
- The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9999. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.540 ± 0.001 mM while the mean peptide concentration of Reference Controls C was 0.525 ± 0.016 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
- The CV of the peptide areas for the nine Reference Controls B and C was 1.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 30.82. The mean A220/A258 ratio ± 10% range was 27.74-33.90. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Results cysteine reactivity assay for the test item:

Preparation of a 100 mM JNJ-39125190-AAA (T003897) stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the test item samples.

In the CC sample, no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 211549/A-cys samples, the mean SPCC A220/A258 area ratio was 36.82. Since this was within the 33.86-41.38 range, this again indicated that there was no co-elution of the test item with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 2.5% ± 1.3%.

Results lysine reactivity assay for the test item:

Preparation of a 100 mM JNJ-39125190-AAA (T003897) stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the test item samples.

In the CC sample, a peak was observed near the retention time of SPCL. The peak area at 220 nm in the CC sample was 23046 μAU which was about 2% of the SPCL peak area at 220 nm in the 211549/A-lys samples (i.e., 1046980, 1053925 and 1091420 μAU in 211549/A-lys-1, 211549/A-lys-2 and 211549/A-lys-3, respectively) and was therefore considered having a limited impact on the quantitation of SPCL in the 211549/A-lys samples. However, the peak area at 258 nm in the CC sample was 26542 μAU which was about 63% of the SPCL peak area at 258 nm in the 211549/A-lys samples (i.e., 42097, 42051 and 41945 μAU in 211549/A-lys-1, 211549/A-lys-2 and 211549/A-lys-3, respectively). This was resulting, for the 211549/A-lys samples, in a mean SPCL A220/A258 area ratio of 25.32. This mean SPCL A220/A258 area ratio was slightly outside the 27.74-33.90 range and was due to the test item-related peak which is overlapping incompletely (i.e., not co-eluting) with the SPCL signal at 258 nm. The Percent Peptide Depletion was therefore estimated.
The estimated Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the test item was 35.0% ± 1.5%

 

DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.
An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay, the test item showed 2.5% SPCC depletion while in the lysine reactivity assay, the test item showed 35.0% SPCL depletion (estimated value). The mean of the SPCC and SPCL depletion was 18.7% (estimated value) and as a result, the test item was considered to be positive in the DPRA. As the overlap in retention time between the test item and SPCL was incomplete, the SPCL depletion was estimated and used in the Cysteine 1:10 / Lysine 1:50 prediction model. However, assignment to a reactivity class was not made.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. JNJ-39125190-AAA (T003897) was positive in the DPRA. As an incomplete overlap in retention time between the test item and SPCL was observed at 258 nm, the SPCL depletion was estimated and used in the Cysteine 1:10 / Lysine 1:50 prediction model. However, assignment to a reactivity class was not made.

Reference 2

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted June, 2018

Deviations:

no

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

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

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)

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.

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

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

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.

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

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

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

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.

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.

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.

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.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2021-01-25 to 2021-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: M19LD3723
- Expiration date of the lot/batch: 2021-11-11 (retest date)
- Physical Description: white to yellow solid
- Purity: 103.6 %
- Purity test date: not indicated

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 the solvent/dispersant/vehicle/test medium: solubility in Dimethylformamide >200 g/L (20°C); stability is not indicated.


FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

OTHER SPECIFIC:
Correction factor: 1

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: 20 females (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-Quality from Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation: 20.4 to 26.1 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): ad libitum, tap water. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: at least 5 days before the commencement of dosing, under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (actual mean 22°C)
- Humidity (%): 40 to 70% (actual 38 to 42%)
- Air changes (per hr): at least 10 air changes/hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-01-27 To: 2021-02-15

Vehicle:

dimethylformamide

Concentration:

0, 2, 10 and 30% (w/w)

No. of animals per dose:

5 females per group; 4 groups

Details on study design:

RANGE FINDING TESTS:
- A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
- Two test item concentrations were tested; 10 % and 30%. The highest concentration was the highest concentration that could be prepared homogeneously.
- The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
- Animals were sacrificed after the final observation.
- Pre-screen test results: At 10% and 30%, no signs of systemic toxicity and no irritation were observed. Therefore, the 30% concentration was selected as highest concentration for the main study. White test item remnants were present on the dorsal surface of the ears of all animals on Days 1-3, which did not hamper scoring of the skin reactions.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
INDUCTION days 1, 2, 3
- Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.
- The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day for three consecutive days. The concentrations were stirred with a magnetic stirrer until dosing.
- The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

EXCISION OF THE NODES - day 6
- Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of ³H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
- After five hours, all animals were euthanized according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

TISSUE PROCESSING FOR RADIOACTIVITY - day 6
- Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - day 7
- Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL ProSafe+ as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item preparations (w/w) were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
- No adjustment was made for specific gravity of the vehicle.
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. No correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.

- Criteria used to consider a positive response:
A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, including all amendments. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).
Classification of results: UN-GHS 2017; EC Hazard statement
SI < 3 Not a sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skin reaction
EC3 value ≤ 2%: sub-category 1A
EC3 value ≥ 2%: sub-category 1B

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In this study, performed in November 2020, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKCD3159 (Sigma- Aldrich, Steinheim, Germany).
Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO).
The SI values calculated for the item concentrations 5, 10 and 25% were 2.1, 3.6 and 9.0 respectively. An EC3 value of 8.0% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the last HCA reliability tests of the recent years were 16.3, 12.8, 9.0 and 10.9%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity. The raw data, study plan and report from this study are kept in the Charles River Den Bosch archives. The test described above was performed in accordance with Charles River Den Bosch Standard Operating Procedures and the report was audited by the QA-unit.

Key result

Parameter:

SI

Value:

1.5

Variability:

± 0.1

Test group / Remarks:

2% (w/w) group

Key result

Parameter:

SI

Value:

1.3

Variability:

± 0.1

Test group / Remarks:

10% (w/w) group

Parameter:

SI

Value:

1.4

Variability:

± 0.2

Test group / Remarks:

30% (w/w) group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 227 ± 14
2% w/w group: mean DPM ± SEM: 342 ± 20
10% w/w group: mean DPM ± SEM: 306 ± 17
30% w/w group: mean DPM ± SEM: 326 ± 54
DPM = Disintegrations per minute
SEM = Standard Error of the Mean

EC3 CALCULATION
Since there was no indication that the test item elicits a SI ≥3 when tested up to 30%, the test item was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3)(if any) exceeds 30%.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: No irritation was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all animals dosed at 10% and 30% between Days 1 and 4, which did not hamper scoring of the skin reactions.
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Moderate body weight loss noted for some animals across the dose groups, which was considered not toxicologically significant since there were no corroborative findings for these animals and no concentration-related incidence was apparent.
- Macroscopy of the auricular lymph nodes and surrounding area: The majority of auricular lymph nodes were considered normal in size, except for the nodes in three animals dosed at 10% and one animal dosed at 30%, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity measurements and SI values: Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 10 and 30% were 342, 306 and 326 DPM, respectively. The mean DPM/animal value for the vehicle control group was 227 DPM. The SI values calculated for the test item concentrations 2, 10 and 30% were 1.5, 1.3 and 1.4, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥3 when tested up to 30%, the test iteù was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3)(if any) exceeds 30%.
The six-month reliability check with alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Based on these results, the test item is not regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In silico data and in chemico and in vitro solubility testing data are used in a weight-of-evidence approach. The studies are discussed in detail in the Weight-of-Evidence justification attached to this endpoint summary.

In addition, the Local Lymph Node Assay (LLNA) is added as key study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 10 or 30% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-dimethylformamide). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in three animals dosed at 10% and one animal dosed at 30%, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 2,10 and 30% were 342, 306 and 326 DPM, respectively. The mean DPM/animal value for the vehicle control group was 227 DPM. The SI values calculated for the test item concentrations 2, 10 and 30% were 1.5, 1.3 and 1.4, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 30%, JNJ39125190-AAA (T003897) was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 30%.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In conclusion, it is determined that it is not possible to conclude on the skin sensitization endpoint with the available data and non-animal test methods, therefore in vivo testing (Local Lymph Node Assay (LLNA) in mice according to OECD 429) has been performed to assess the potency of the test item. Based on the EC₃ value calculated in the LLNA, according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), the test item is not regarded as skin sensitizer. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).