(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th November 2019 to 5th February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion".

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines (2000)

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)

IUPAC Name:

(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: approximately 12-14 weeks old
- Weight at study initiation: 2620 to 2739 g
- Housing: animals were housed individually in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The room in which the animals were kept was documented in the study records.Each cage was clearly labeled.
- Diet: Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from
Granovit AG, Kaiseraugst, Swizerland) was provided once daily throughout the study. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was available during the study period.
- Water: tap-water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 20°C
- Humidity (%): 46 to 57%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod: 12-hour light/12-hour dark cycle

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

64.3 mg (range 63.9 – 64.6 mg) of the test item (a volume of approximately 0.1 mL)

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3 animals, 1 Female, 2 Males.

Details on study design:

Animals were treated by instillation of the test item (a volume of approximately 0.1 mL) in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control.
Immediately after the 1 hour observation, the treated eye was rinsed with approximately 100 mL tepid tap water, using a velocity of flow which did not affect the eye, to remove any visible residual test item. For reference control the other eye was also rinsed. Additional injections of buprenorphine 0.01 mg/kg were administered immediately after the 1-hour observation and at the end of the first day to reduce pain and distress.
Immediately after the 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
Immediately after fluorescein examination on Day 2, the treated eye of the sentinel animal was rinsed with approximately 50 mL tepid tap water, using a velocity of flow which did not affect the eye, to remove residual test item. For reference control the other eye was also rinsed. In order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5
mg/kg were administered by subcutaneous injection. Additional injections of buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg were administered immediately after the 48-hour observation to reduce pain and distress. Additional injections of buprenorphine 0.03 mg/kg were supplied to reduce pain and distress.
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Results 8

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Results 9

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.33

Max. score:

3

Reversibility:

fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Results 10

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Results 12

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Remarks on result:

positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on these results:
• According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), PF04965842-01 should be classified as: irritating to eyes (Category 2A).
• According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments),PF-04965842-01 should be classified as Irritating to eyes (Category 2) and labeled as H319: Causes serious eye irritation.
• Based on the maximum group mean Draize score of 23 and the group mean total score at 72 hours of 9,PF-04965842-01 would be classified as moderate irritant to the rabbit eye according to the Kay and Calandra classification system.

Executive summary:

The objective of this acute eye irritation study was to assess the possible irritation or corrosion potential when a single dose of PF-04965842-01 was placed in the conjunctival sac of the rabbit eye.
The study was carried out in compliance with the guidelines described in:
• OECD No.405 (2017) "Acute Eye Irritation / Corrosion".
• EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion".
• EPA, OPPTS 870.2400 (1998), "Acute Eye Irritation".
• JMAFF Guidelines (2000), including the most recent revisions.
Single samples of approximately 64.3 mg of PF-04965842-01 (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation. The corneal injury consisted of opacity and epithelial damage. The corneal injury resolved within 24 hours in one animal and within 48 hours in the two remaining animals. Slight dulling of the normal luster, noted for two animals, is a normal finding and therefore considered not toxicologically relevant. Iridial irritation was observed and completely resolved within 7 days in all animals. The irritation of the conjunctivae consisted of redness, chemosis and discharge. The irritation had completely resolved within 14 days in one animal and within 21 days in the other two animals. Reduced elasticity of the eyelids was noted for one  animal between 48 hours and 7 days.
There was no evidence of ocular corrosion. Remnants of the test item were present in the eye between 0 and 72 hours after installation of the test item. 

Based on these results:
• According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), PF04965842-01 should be classified as: irritating to eyes (Category 2A).
• According to the Regulation (EC) No 1272/2008 on classification, labelling and
packaging of items and mixtures (including all amendments), PF-04965842-01 should be classified as Irritating to eyes (Category 2) and labeled as H319: Causes serious eye irritation.
• Based on the maximum group mean Draize score of 23 and the group mean total score at 72 hours of 9, PF-04965842-01 would be classified as moderate irritant to the rabbit eye according to the Kay and Calandra classification system.