N-[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]pyrimidin-2-yl]-6-methoxy-4-(morpholin-4-yl)benzene-1,3-diamine

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:  A Derek Nexus assessment yielded an alert for skin sensitization for JNJ-73848281-AAA (T003904) and predicted the substance to be plausible for skin sensitization.The structur alalert was anortho orpara amino-or hydroxy-aniline. DEREK predicted the substance to be a moderate sensitizerbased on ten closeststructurally related substances.

 

- In chemico (OECD 442C): In the solubility test for the DPRA,the test item was not soluble at 100 mM (stock solution) in any of the solvents and therefore cannot be tested.

 

- In vitro (OECD 442D): In the solubility assessment for the KeratinoSensTM assay, precipitation was observed at all tested concentrations after incubation and therefore concentrations up to and including the top-dose (1000 μM) may not be reliable. Even if a reliable negative result is obtained at lower concentrations, a second negative result in the DPRA and U-SENSTM assay cannot be obtained due to the limited solubility of the substance.

 

- In vitro (OECD 442E): In the solubility assessment for the U-SENSTM assay, precipitation was observed at a concentration of 50 μg/mL in 0.4% DMSO in medium which will likely predetermine the overall outcome of the assay as positive.

 

Based on the considerations above, the available non-animal assays will not provide sufficient evidence to be able to draw a negative conclusion for the skin sensitizing properties of JNJ-73848281-AAA (T003904). In case the overall outcome of the non-animal methods is inconclusive or positive for skin sensitization, no data will be available on potency required for classification and risk assessment. Furthermore, a DEREK assessment predicted it to be plausible that the substance is a skin sensitiser, which prompts the need to address this endpoint further and determine classification.
Since all studies were performed before the publication of the OECD 497 guideline (22 June 2021) and in accordance with point 8.3.2 of Annex VII of REACH, if in vitro/in chemico test methods are not applicable or adequate for classification and risk assessment of the substance, an in vivo test may be performed. Since it is concluded that it is not possible to address this endpoint with the available non-animal methods, it is considered justified to perform an in vivo study to determine the skin sensitizing properties (Gijsbrechts J, 2020).

 

- In vivo (OECD 429): Based on the results of a Local Lymph Node Assay (LLNA), the test item was regarded as skin sensitizer (Category 1B) (van de Wiel, 2021).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with point 8.3.2 of Annex VII of REACH, if in vitro/in chemico test methods are not applicable or adequate for classification and risk assessment of the substance, an in vivo test may be performed. Since it is concluded that it is not possible to address this endpoint with the available non-animal methods, it is considered justified to perform an in vivo study to determine the skin sensitizing properties of JNJ-73848281-AAA (T003904).

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-11-16 to 2020-12-15

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: I20AD0249
- Expiration date of the lot/batch: 2022-01-21 (retest date)
- Physical description: slightly yellow powder
- Purity test date: 2020/02/04
- Purity: 99.9% (w/w)

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: not indicated
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Final preparation of a solid: the test item was added to the vehicle

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

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: female (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-Quality from Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 19.6 to 25.6 g
- Housing: group housed in labeled polycarbonate cages (Makrolon MIII type; height 18 cm) containing sterilised wooden fibers as bedding material equipped with water bottles.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days before the start of treatment, under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (actual 22°C)
- Humidity (%): 40 to 70% (actual 42-51%)
- 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: 2020-11-16 To: 2020-12-14

Vehicle:

methyl ethyl ketone

Concentration:

0, 2, 25 and 35% (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; 25 % and 35%. 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 animals were approximately 10-11 weeks (at initiation of treatment) and 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 25% and 35%, no signs of systemic toxicity were noted and no irritation was observed and
therefore a 35% 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 at 25% and 35% between Days 1 and 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 immediately prior to 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. 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 dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
- No adjustment was made for specific gravity of the vehicle.
- Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, 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 (2011) (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 2007; EC-CLP 2008 EC Hazard statement
SI < 3 No 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 6 monthly HCA reliability checks 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

Variability:

± 0.1

Test group / Remarks:

0% (w/w) group

Key result

Parameter:

SI

Value:

2.3

Variability:

± 0.7

Test group / Remarks:

2% (w/w) group

Key result

Parameter:

SI

Value:

6.3

Variability:

± 1.3

Test group / Remarks:

25% (w/w) group

Key result

Parameter:

SI

Value:

13.1

Variability:

± 0.4

Test group / Remarks:

35% (w/w) group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 306 ± 30
2% w/w group: mean DPM ± SEM: 718 ± 200
25% w/w group: mean DPM ± SEM: 1928 ± 385
35% w/w group: mean DPM ± SEM: 3995 ± 122
DPM = Disintegrations per minute
SEM = Standard Error of the Mean

EC3 CALCULATION
These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 6% was calculated.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: The very slight irritation of the ears as shown by the animals treated at 35% between Days 1 and 3 was considered not to have a toxicologically significant effect on the activity of the nodes.
White test item remnants were present on the dorsal surface of the ears of all animals at 25% and 35% 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.
- Macroscopy of the auricular lymph nodes and surrounding area: The auricular lymph nodes of the animals dosed with the vehicle and 2% test item were considered normal in size. The nodes of the animals dosed with 25% and 35% 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, 25 and 35% were 718, 1928 and 3995 DPM, respectively. The mean DPM/animal value for the vehicle control group was 306 DPM. The SI values calculated for the test item concentrations 2, 25 and 35% were 2.3, 6.3 and 13.1, respectively.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response
and an EC3 value (the estimated test item concentration that will give a SI =3) of 6% was calculated.
Based on these results:
• According to the recommendations made in the test guidelines (including all amendments), JNJ-73848281-AAA (T003904) would be regarded as skin sensitizer.
• According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), JNJ73848281-AAA (T003904) should be classified as skin sensitizer (Category 1B).
• According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), JNJ-73848281-AAA (T003904) should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

Solubility Assessment for the Non-animal Skin Sensitization Studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-04-21 to 2020-04-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline required

Principles of method if other than guideline:

The present study is used to assess the solubility of a test item in the DPRA, KeratinoSensTM and U-SENS assayTM. These data can be used to evaluate the compatibility of a test item in the non-animal methods.
The design of this study is based on the following study guidelines:
- OECD Guideline for the Testing of Chemicals, Guideline 442C. In Chemico Skin Sensitization: Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins (18 June 2019)
- OECD Guideline TG 442D. In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method.(Adopted June, 2018)
- EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™. (Adopted March, 2018)
- OECD 442E –Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)’ (25 June 2018)
- EURL ECVAM DB-ALM Protocol n° 183:U937 Cell Line Activation Test for Skin Sensitization (U-SENS™)(Adopted October, 2017)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: I20AD0249
- Expiry date: 21 January 2022 (retest date)
- Physical description: slightly yellow powder
- Purity test date: 2020/02/04
- Purity: 99.9% (w/w)

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: not indicated



OTHER SPECIFICS
- Correction factor: 1

Solubility Assessment DPRA
The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), ethanol and methanol. The test item was not soluble in any solvent. Solubility of the test item in SPCC and SPCL test conditions was not assessed, since the test item was not soluble in any solvent.

 

Solubility Assessment KeratinoSens^TM
The following solvents were evaluated: DMSO, Milli-Q water and ethanol. The test item was homogenously suspended only in DMSO at 100 mM (stock solution). The solubility of the test item in DMSO was evaluated in KeratinoSensTM test conditions at a concentration range of 7.8 – 1000 µM (2-fold dilution series). Upon preparation the test item showed moderate precipitation at concentrations of 500 and 1000 µM, slight precipitation at 250 µM and microscopic precipitation at 125 µM. After incubation the test item showed moderate precipitation at concentrations of 500 and 1000 µM, slight precipitation at 125 and 250 µM and microscopic precipitation at 63 µM.

 

Solubility Assessment U-SENS^TM
The following solvents were evaluated: RPMI medium and DMSO. The test item was homogenously suspended only in DMSO at 50 mg/mL. The solubility of the test item in DMSO was evaluated in U-SENSTM test conditions at a concentration of 200 µg/mL. The test item showed precipitation upon preparation and after incubation.

Conclusions:

In the solubility test for the DPRA the test item was insoluble at 100 mM (stock solution) in Acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), ethanol and methanol. Based on the current solubility assessment no solvent was compatible to obtain the required test item concentration in the DPRA.
In the solubility test for the KeratinosensTM the test item was homogenously suspended only in DMSO at 100 mM (stock solution). Upon preparation the test item showed moderate precipitation at concentrations of 500 and 1000 µM, slight precipitation at 250 µM and microscopic precipitation at 125 µM. After incubation the test item showed moderate precipitation at concentrations of 500 and 1000 µM, slight precipitation at 125 and 250 µM and microscopic precipitation at 63 µM. Based on the solubility assessment the preferred solvent for the KeratinosensTM is DMSO.
In the solubility test for the U-SENSTM the test item was homogenously suspended only in DMSO at 50 mg/mL. Upon preparation and after incubation the test item showed precipitation. Based on the solubility assessment the preferred solvent for the USENSTM is DMSO. But due to the precipitation observed in the test conditions, the outcome of the assay runs will likely be predetermined as positive and therefore a negative result is unlikely to be obtained.

Endpoint conclusion

Additional information:

In silico, in chemico and in vitro 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, 25 or 35% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methylethylketone).

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 auricular lymph nodes of the animals dosed with the vehicle and 2% test item were considered normal in size. The nodes of the animals dosed with 25% and 35% 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, 25 and 35% were 718, 1928 and 3995 DPM, respectively.

The mean DPM/animal value for the vehicle control group was 306 DPM. The SI values calculated for the test item concentrations 2, 25 and 35% were 2.3, 6.3 and 13.1, respectively.

These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 6% was calculated.

 

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 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.