7-Cyclopentyl-N,N-dimethyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Endpoint summary

Description of key information

Primary skin irritation/corrosion study with LEE011 -A4 in the rabbit (4-hour semi-occlusive application) showed that the substance has non-irritating effect.

The LEE011-A4 is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2103 - March 2104

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Albino rabbit, New Zealand White, (SPF-Quality).

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

no

Amount / concentration applied:

Each animal was treated by dermal application of 0.5 grams of the test substance. The test substance was moistened with 0.6 mL of the vehicle and applied to the skin of one flank, using a metalline patch# of 2x3 cm.

Duration of treatment / exposure:

4 h

Observation period:

The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test substance.

Number of animals:

3

Irritation parameter:

overall irritation score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

0

Reversibility:

other: No skin irritation

Remarks on result:

no indication of irritation

Irritation parameter:

overall irritation score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

0

Reversibility:

other: No skin irritation

Remarks on result:

no indication of irritation

Irritation parameter:

overall irritation score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

0

Reversibility:

other: No skin irritation

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

There was no evidence of a corrosive effect on the skin.

Other effects:

No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

No skin irritation was caused by 4 hours exposure to LEE011-A4.

There was no evidence of a corrosive effect on the skin.

No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

other: No skin irritation was caused by 4 hours exposure to LEE011-A4..

Conclusions:

Based on the results LEE011-A4 does not have to be classified and has no obligatory labelling requirement for skin irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2017 - Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Species:

human

Strain:

other: EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek)

Details on test animals or tissues and environmental conditions:

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The solid test item (52.7 to 54.0 mg) was applied directly on top of the skin tissue.
LEE011-A4 was spread to match the size of the tissue.

Duration of treatment / exposure:

6 hours ± 15 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

The tissues were incubated for 180 ± 10 minutes at 37°C.

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively.

Details on study design:

At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with LEE011-A4 (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.
The test item could not be removed completely. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in
5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

Irritation parameter:

other: tissue viability

Run / experiment:

the mean tissue viability obtained after 6 hours ± 15 minutes treatment with LEE011-A4 compared to the negative control tissues

Value:

ca. 24.4

Negative controls validity:

valid

Remarks:

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with LEE011-A4 compared to the negative control tissues was 24.4%.

Positive controls validity:

valid

Remarks:

The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 12.5%.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with LEE011-A4 compared to the negative control tissues was 24.4%. Since the mean relative tissue viability for LEE011-A4 was below 60% it is considered to be potentially irritant or corrosive to the eye.

The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 12.5%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 15%, indicating that the test system functioned properly.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In conclusion, LEE011-A4 is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2017 - Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes were used as soon as possible after slaughter.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

No correction was made for the purity/composition of the test item.
A solubility test in physiological saline was performed based on visual assessment.
A 20% (w/v) suspension of the test item was prepared in physiological saline.
Any residual volumes were discarded.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 minutes at 32C.

Duration of post- treatment incubation (in vitro):

After the incubation the solutions
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:

First experiment: 9 (3 Negative control, 3 Positive control, 3 LEE011-A4)
Second experiment: 9 (3 Negative control, 3 Positive control, 3 LEE011-A4)

Details on study design:

The test consists of topical application of LEE011-A4 on the epithelium of the bovine cornea for 4 hours. The non-surfactant solid test item was applied as a 20% suspension. After exposure the corneas were thoroughly rinsed. 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.

Irritation parameter:

cornea opacity score

Run / experiment:

first experiment

Value:

>= 1.3 - <= 2.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

other: permeability of the corneas

Run / experiment:

first experiment

Value:

>= 0.058 - <= 0.132

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

in vitro irritation score

Run / experiment:

first experiment

Value:

>= 2.9 - <= 4.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

cornea opacity score

Run / experiment:

second experiment

Value:

>= 1.8 - <= 2.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

in vitro irritation score

Run / experiment:

second experiment

Value:

>= 2.4 - <= 3.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

other: permeability of the corneas

Run / experiment:

second experiment

Value:

>= 0.038 - <= 0.093

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

In the first experiment, 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-A4 showed opacity values ranging from 1.3 to 2.5 and permeability values ranging from 0.058 to 0.132. The corneas were clear after the 240 minutes of treatment with LEE011-A4. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 2.9 to 4.5 after

240 minutes of treatment with LEE011-A4.

Since the results for LEE011-A4 were spread over 2 categories (2.9, 2.9 and 4.5, respectively), the test was inconclusive and a repeat experiment was performed.

In the second test, the individual in vitro irritancy scores for the negative controls ranged from -0.7 to 1.4. The individual positive control in vitro irritancy scores ranged from 142 to 184. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with LEE011-A4 showed opacity values ranging from 1.8 to 2.3 and permeability values ranging from 0.038 to 0.093. The corneas were clear after the 240 minutes of treatment with LEE011-A4. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 2.4 to 3.5 after

240 minutes of treatment with LEE011-A4.

Interpretation of results:

other: Since both tests provided inconclusive results, no conclusion can be drawn for the eye irritation or serious eye damage of LEE011-A4.

Conclusions:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole)
was 162 and 158 in the first and second experiment, respectively and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The mean in vitro irritancy score was 3.4 and 3.1 after 240 minutes of treatment with the test item in the first and second experiment, respectively. The results for LEE011-A4 were spread over 2 categories in both experiments.
Since both tests provided inconclusive results, no conclusion can be drawn for the eye irritation or serious eye damage of LEE011-A4.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Skin irritation/corrosion study showed that the substance has non-irritating effect.

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with LEE011-A4 compared to the negative control tissues was 24.4%. Since the mean relative tissue viability for LEE011-A4 was below 60% it is considered to be potentially irritant or corrosive to the eye.