2-Propenamide, N-[5-[[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-2-pyrimidinyl]amino]-4-methoxy-2-(4-morpholinyl)phenyl]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-07-30 to 2019-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Refer to read-across section 13.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenamide, N-[5-[[4-[4- [(dimethylamino)methyl]-3-phenyl- 1Hpyrazol-1-yl]-2-pyrimidinyl]amino]-4- methoxy-2-(4- morpholinyl)phenyl]-, methanesulfonate (1:1)

Cas Number:

2411549-88-5

IUPAC Name:

2-Propenamide, N-[5-[[4-[4- [(dimethylamino)methyl]-3-phenyl- 1Hpyrazol-1-yl]-2-pyrimidinyl]amino]-4- methoxy-2-(4- morpholinyl)phenyl]-, methanesulfonate (1:1)

Test material form:

solid

Remarks:

White solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: PP-1030B-9003
- Expiry date: 2019-10-12 (retest date)
- Physical appearance: White solid
- Purity: 100.5%
- Purity test date : 2019-05-08

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

FORM AS APPLIED IN THE TEST: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: 20 female (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-quality from Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 18.4 to 24.2 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 sawdust as bedding material (Lignocel S 8-15, 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. For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home), except when interrupted by study procedures/activities.
- Diet: ad libitum, pelleted rodent diet was provided throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum, Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Indication of any skin lesions: not indicated
Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C (actual daily mean temperature: 22 to 23°C)
- Humidity (%): 40 to 70% (actual daily mean relative humidity of 53 to 79%)
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 2019-07-24 To: 2019-08-19

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Merck, Darmstadt, Germany

Concentration:

Pre-screen: 10, 30% w/w , Main study: 2, 10, 30% w/w

No. of animals per dose:

Pre-screen: 2 females per group, 2 groups
Main study 5 females per group; 4 groups

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw Data of these trials will be retained by the Test Facility. There was no information available about the stability and solubility of the test item in vehicle
- 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; a 10% and 30% concentration. The highest concentration was the maximum 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 and that no clinical observations were recorded on Day 4 (see section on deviations). 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: - Irritation: very slight irritation was observed at 30% on Days 2 and 3; - Systemic toxicity: no signs of systemic toxicity were noted; - Ear thickness measurements: Increase in ear thickness remained below 25% for 10 and 30% test item concentrations. Therefore a 30% concentration was selected as highest concentration for use in the main study; - Erythema scores: 0 for all animals treated with 10%. 1 for both animals treated at 30 % on Days 2 and 3, 0 from Day 4.

MAIN STUDY
ANIMAL ASSIGNMENT
Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range will not be assigned to the study.
Replacement: Before the initiation of dosing, any assigned animals considered unsuitable for use in the study will be replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
Disposition: The disposition of all animals will be documented in the Study Files.

TREATMENT
INDUCTION days 1, 2, 3:
- 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 killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). 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 approximately 3 mL 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 (diameter 125 μm). 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 of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). 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 within 4 hours prior to each dosing.
- 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 to be dosed.

- 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. 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).

Positive control substance(s):

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

Statistics:

No statistics were performed.

Results and discussion

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 July 2019, 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 0.7, 2.2 and 6.4 respectively. An EC3 value of 12.8% 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 14.1, 17.3, 9.8, 17.8, 14.3 and 16.3%.
Based on these 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.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Variability:

± 0.2

Test group / Remarks:

0 % (w/w) group

Results 2

Key result

Parameter:

SI

Value:

1.6

Variability:

± 0.4

Test group / Remarks:

2 % (w/w) group

Results 3

Key result

Parameter:

SI

Value:

8.8

Variability:

± 1.5

Test group / Remarks:

10 % (w/w) group

Results 4

Key result

Parameter:

SI

Value:

11.6

Variability:

± 1.8

Test group / Remarks:

30 % (w/w) group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
0% w/w group: mean DPM ± SEM:/animal: 1187 ± 253
2% w/w group: mean DPM± SEM:/animal: 1944 ± 423
10% w/w group: mean DPM± SEM:/animal: 10408 ± 1813
30 %w/w group: mean DPM± SEM:/animal: 13720 ± 2174
DPM = Disintegrations per minute
SEM = Standard Error of the Mean

DETAILS ON STIMULATION INDEX CALCULATION:
The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

EC3 CALCULATION:
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 3.6% was calculated.

CLINICAL OBSERVATIONS:
- Skin reactions/irritations: 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 at 30% throughout the observation period, which did not hamper scoring of the skin reactions.
- Macroscopy of the auricular lymph nodes and surrounding area: all auricular lymph nodes of the animals of the experimental groups were slightly enlarged with exception of one animal in the 10% test item dose group, which was considered normal in size. The nodes of the control group were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Body weights: body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the control and test item dose groups was considered not toxicologically significant since the changes were slight in nature and no dose-related incidence was apparent.
- Signs of toxicity: no mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

Applicant's summary and conclusion

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 3.6% was calculated. According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), JNJ‐73841937‐ZCY (Lazertinib mesylate monohydrate) should be classified as skin sensitizer (Category 1B).