4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile bis sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 September - 30 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(6-{3,6-diazabicyclo[3.1.1]heptan-3-yl}pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile

Molecular formula:

C22H24N6O2.[2]H2O4S

IUPAC Name:

4-(6-{3,6-diazabicyclo[3.1.1]heptan-3-yl}pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile

Test material form:

solid: particulate/powder

Details on test material:

Batch (Lot) Number: 18-547.25-002
Physical Description: White powder
Storage Conditions: Kept in a room temperature area, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS:
Source: Jackson Laboratories, Bar Harbor, Maine.
Age at initiation of dosing: Approximately 8-9 weeks
Weight at initiation of dosing: 15.0 - 23.1 g
Housing: The animals were group-housed at receipt in solid bottom cages with nonaromatic bedding. Subsequently, the animals were individual housed.
Diet (e.g. ad libitum): PMI diet, ad lbitum.
Water (e.g. ad libitum): Ad lbitum.
Acclimation period: 8 - 15 days

ENVIRONMENTAL CONDITIONS:
Target Temperature (°C): 68 - 79 °F
Target Relative Humidity (%): 30% - 70%
Air changes (per hr): Ten or greater air changes per hour wiht 100% fresh air
Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.

IN-LIFE DATES:
From:

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

1, 4.5, 5, 10, 15, 25, and 45%.

No. of animals per dose:

Group 1: 2 mice: test material dose concentration: 1%, Group 2: 2 mice: test material dose concentration: 5%, Group 3: 2 mice: test material dose concentration 10%, Group 4: 2 mice: test material dose concentration: 25%, Group 5: 2 mice: test material dose concentration: 45%, Group 6: 5 mice: vehicle dose concentration 0%, Group 7: 5 mice: HCA dose concentration: 35%, Group 8: 5 mice: test material dose concentration: 4.5%, Group 9: 5 mice: test material concentration: 15%, Group 10: 5 mice: test material concentration: 45%.

Details on study design:

Phase 1: The test article was administered once daily for 3 days starting on Day 1 via dermal application to the outer ear using a micropipette.
Phase 2: The test article, positive control article, or vehicle were administered once daily (approximately ± 1 hour apart) for 3 days starting on Day 1 via dermal application to the outer ear using a micropipette. The cell proliferation marker article was administered once on Day 6 via IV injection.
For dermal administration, the vehicle, test article, and positive control article were withdrawn from stirred formulations.

Clinical observations:
Observations for clinical signs all animals were conducted daily starting on Day -1. Examinations on dosing days were performed approximately 2 to 4 hours postdose.

Body Weight:
Body weights for all animals were measured and recorded within 3 days of receipt, on Day 1 prior to dosing, and Day 6.

Study Termination:
At study termination, Phase 1 animals were euthanized by carbon dioxide inhalation followed by a Testing Facility SOP approved method to ensure death. The carcasses were discarded without further evaluation.

Terminal Procedures:
Postmortem study evaluations were performed on all Phase 2 animals at the scheduled terminal necropsy.

Tissue Sample Collection:
At the terminal necropsy, 5 hours following 3H methyl thymidine administration on Day 6, the auricular lymph nodes were collected from all surviving Phase 2 animals. The tissues were collected in a plastic container on wet ice and processed for radioanalysis.

Positive control substance(s):

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

Statistics:

The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group. A group pair-wise comparison (general ANOVA) was performed.

Results and discussion

Positive control results:

The positive control group (35% HCA) resulted in a sensitization index (SI) of 4.34 which is indicative of a sensitizer (SI ≥3). In addition, the SI correlated well with the mean dpm values, which were significantly increased in the 35% HCA group relative to the vehicle group.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.95

Test group / Remarks:

4.5%

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

15%

Results 3

Key result

Parameter:

SI

Value:

4.74

Test group / Remarks:

45%

Any other information on results incl. tables

Mortality: All animals survived to the scheduled necropsy except one animal in the positive control group (Animal No. 7503) administered 35% HCA that was euthanized in extremis on Day 3.

Radioactivity Analysis: The average radiochemical concentration of the formulation Group No. 6 was 2688505278 dpm/g.

Clinical Observations:

There were no significant findings or signs of local irritation in either the Irritation Screen or in the Main Study Phase for animals administered the vehicle or the test article. White discoloration of the skin (left and right ear) was observed in animals administered COM-1079 at 15 and 45%. Red discoloration of the skin (left and right ear), wet hair in the cervical region, and unkempt appearance was observed in all animals administered 35% HCA on Days 1 through 3. Animal No. 7503 (Group 7, 35% HCA) was observed with hunched posture and decreased activity on Day 3 and was euthanized in extremis.

Body Weight: There was no effect on body weight during the course of this study.

Lymph Node Radioanalysis: When COM-1079 formulations were compared to the vehicle the sensitization indices were 0.95, 1.00, and 4.74 for the formulations at 4.5%, 15%, and 45% for the test article groups, respectively.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

COM-1079 formulations were determined to not be sensitizers at concentrations of 4.5 and 15% in the Murine Local Lymph Node Assay. COM-1079 formulations were determined to be sensitizers at a concentration of 45% in the Murine Local Lymph Node Assay.