tert-Butyl 4-({6-[7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}pyridin-3-yl)piperazine-1-carboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2017 - April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

tert-Butyl 4-({6-[7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}pyridin-3-yl)piperazine-1-carboxylate

EC Number:

810-753-4

Cas Number:

1374639-78-7

Molecular formula:

C28H38N8O3

IUPAC Name:

tert-Butyl 4-({6-[7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}pyridin-3-yl)piperazine-1-carboxylate

Test material form:

solid: bulk

Test animals / tissue source

Species:

cattle

Strain:

other: Bovine eyes were used as soon as possible after slaughter.

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

No correction will be made for the purity/composition of the test compound.
Since no workable suspension of LEE011-A3 in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. LEE011-A3 was weighed in a glass tube and applied directly on the corneas in such a way that the cornea was completely covered (324.0 to 331.7 mg).

Duration of treatment / exposure:

240 ± 10 minutes

Duration of post- treatment incubation (in vitro):

The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Number of animals or in vitro replicates:

3 corneas for each treatment group

Details on study design:

The medium from the anterior compartment was removed and 750 micro l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. LEE011-A3 was weighed in a glass tube and applied directly on the corneas in such a way that the cornea was completely covered (324.0 to 331.7 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform
distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar
opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany).
The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1C.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

>= -0.6 - <= 1.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Value:

>= -0.6 - <= 1.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from -1.2 to 0.8. The individual positive control in vitro irritancy scores ranged from 156 to 170. The corneas treated with the positive control were turbid after the 240 minutes of

treatment.

The corneas treated with LEE011-A3 showed opacity values ranging from -0.6 to 1.2 and permeability values ranging from 0.013 to 0.018. The corneas were clear after the 240 minutes of treatment with LEE011-A3. No pH effect of the test item was observed on the rinsing medium.

Hence, the in vitro irritancy scores ranged from -0.6 to 1.2 after 240 minutes of treatment with LEE011-A3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

LEE011-A3 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.1 after 240 minutes of treatment.
In conclusion, since LEE011-A3 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.