Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 June - 23 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines (2000), including the most recent revisions

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

EC Number:

816-314-3

Cas Number:

927881-65-0

Molecular formula:

C83H87N3O21

IUPAC Name:

Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

Test material form:

solid: particulate/powder

Details on test material:

White solid
Test item storage: At room temperature

Specific details on test material used for the study:

White solid
Test item storage: At room temperature
Batch: 902/6460031/D/13/1 (is same batch as batch E010017351 on Certificate of Analysis)

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Rabbit
Strain: New Zealand White
Condition: SPF-Quality
Source: Charles River France, L’Arbresle, France
Number of Animals: 3 Males.
Age at the Initiation of Dosing: Young adult animals (approximately 12 weeks old) were selected.
Weight at the Initiation of Dosing: 2694 to 2929g.

Animal Identification:
At study assignment, each animal was identified using an ear mark with indelible ink.
Environmental Acclimation:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
Housing:
On arrival and following assignment to the study, animals were housed individually in labeled cages with perforated floors equipped with water bottles. These housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals were kept were documented in the study records. Each cage was clearly labeled.
Environmental Conditions:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 20°C with an actual daily mean relative humidity of 56 to 95%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Food:
Pelleted diet for rabbits was provided once daily throughout the study. In addition, hay was available during the study period.
Water:
Municipal tap-water was freely available to each animal via water bottles.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

Animals were treated by instillation of, on average, 34.9 mg (range 34.8 – 35.3 mg) 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.

Duration of treatment / exposure:

1 hr.

Observation period (in vivo):

Observations were made 1, 24, 48 and 72 hours after instillation.

Number of animals or in vitro replicates:

3 males

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of eye irritation observed in the first animal.
Pre-emptive Pain Management:
One hour prior to instillation of the test item, buprenorphine (Buprenodale®) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test item, two drops of the topical anaesthetic 0.5% proparacaine hydrochloric ophthalmic solution (Alcaine eye drops®) were applied to both eyes.
Administration of Test item:
Animals were treated by instillation of, on average, 34.9 mg (range 34.8 – 35.3 mg) 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 50 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.
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. Immediately after fluorescein examination on Day 2, 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.
Justification of Route and Dose Level:
The ocular route was selected because the test item may accidentally come into contact with the eyes during manufacture, handling and/or use. The dose level was based on the test guidelines.
Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Postdose Observations:
Postdose observations were performed once daily throughout the study, the observation period was 72 hours.
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.
Body Weights:
Animals were weighed individually on Day 1 (predose) and on the day of the final observation
Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded.
Terminal Procedures:
After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: 260

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal: 273

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal: 274

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

iris score

Basis:

animal: 260

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

iris score

Basis:

animal: 273

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal: 274

Time point:

24/48/72 h

Score:

5

Reversibility:

fully reversible within: 24 hrs.

Remarks on result:

positive indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal: 260

Time point:

24/48/72 h

Score:

6

Reversibility:

fully reversible within: 24 hrs.

Remarks on result:

positive indication of irritation

Results 8

Irritation parameter:

conjunctivae score

Basis:

animal: 273

Time point:

24/48/72 h

Score:

2

Max. score:

6

Reversibility:

fully reversible within: 48 hrs.

Remarks on result:

positive indication of irritation

Results 9

Irritation parameter:

conjunctivae score

Basis:

animal: 274

Time point:

24/48/72 h

Score:

2

Max. score:

6

Reversibility:

fully reversible within: 48 hrs.

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

There was no evidence of ocular corrosion.

Other effects:

Coloration / Remnants:
Remnants of the test item were present in the eye on Day 1.
Toxicity / Mortality:
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Instillation of the test item resulted in effects on the iris and conjunctivae. Iridial irritation was observed in one animal and resolved within 24 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 24 hours in one animal and within 48 hours in the other two animals. Remnants of the test item were present in the eye on Day 1.

Executive summary:

Based on the maximum group mean of 7.7, PF-06460260 is classified as minimal irritant to the rabbit eye according to the Kay and Calandra classification system.

Based on these results, PF-06460260 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the:

• Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments).

• Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).