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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July - 7 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

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

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: The test system was isolated bovine cornea obtained as a by-product from freshly slaughtered animals .

Source/transport: Bovine eyes were obtained from the abattoir of J.W. Teruth & Sonsn INc., Baltimore, MD. The eyes were excised as soon after slaughter as possilbe and were held in HBSS on ice. Once the eyes were obtained, they were transported to IIVS. Immediately upon reciept of hte eyes to the lab, preparation of the corneas was initiated.

Test system

Vehicle:

other: deionized water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 uL

Duration of treatment / exposure:

Three corneas were incubated in the presence of the test article at 32 +/- 1 degree C for 4 hours. Three corneas were incubated in the presence of each control at 32 +/- 1 degrees C for 4 hours. After removal of the test or contorl article form the corneas, a final opacity was determined.

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

N/A

Number of animals or in vitro replicates:

Three corneas for the test article and three corneas for each control

Details on study design:

Preparation of Corneas:
All the eyes were carefully examined for defects and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS prior to mounting. Corneas were then mounted in corneal holders with the endothelial side agianst the O-ring of the posterior chamber. The anterior chamber was positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were filled with complete MEM. THe posterior champer was always filled first.

Test Item Preparation:
When appropritae, test articles wer diluted or suspended in either sterile deioniezd water or other Sponsor-directed solvent.

Treatment of Corneas and Opacity Measurements:
Solid materials were tested as a 20% dilution w//v in sterile deionized water. 750 uL of the test substance was introduced into the anterior chamber. Although it was understood that a 750 uL dose could not be achieved, the corneas were to be compltely covered with the test material. The holder was slightly rotated to ensure uniform distribution of the test substance over the cornea. The corneas were incubated in a horizontal position at 32 +/- 1 degree C for approximately 4 hours. The test substance was then removed and the epithelium washed at least 3 times with Complete MEM. Once the media was free of the test material, the corneas were given a final rinse with Complete MEM. If the test material could not be removed from the cornea a note was recorded in the raw data report. The anterior and posterior chambers were then refilled with fresh Complete MEM and an opacity measurement performed immediately.

Opacity Measurement:
The opacitometer determined the difference in light transmission between each treated or control cornea and an air-filled chamber, and a numerical opacity value was recorded.

Permeability Determinations and application of sodium fluorescein:
After the opacity measurement was performed, the medium was reomved from the anterior chamber only and replaced with 1 mL of a 5 mg/mL sodium fluorescein solution. After the addition of the fluorescein solution to the anterior chamber, the corneas were incubated in a horizontal position for apporximately 90 minutes at 32 +/- 1 degree C. The medium from the posterior chamber was removed at the completion of the incubation period, and 360 uL was transferred to the appropriate wells of a labeled 96-well plate. 360 uL of fresh Complete MEM were added to the wells designated as blanks. The optical density at 490 nm was determined using a spectrophotometer. Samples reading 1,500 and above were diluted to bring the reading within the linear range of the platereader and the plate was read again.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

136.3

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The positive control in vitro irritancy score was 89.8.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion the test material induced an IVIS >55 and was therefore clsaasified as GHS Category 1: irreversible effects on the eye.