1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 June 2016 and 30 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

EC Number:

611-033-0

Cas Number:

536759-91-8

Molecular formula:

C22H20N4O6

IUPAC Name:

ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

powder; stored at room temperature in the dark

Specific details on test material used for the study:

Identification: BMS-589152-01
Batch: AAG8999N
Vendor lot number: 15600T0001
Purity: 100.1%
Physical state / Appearance: beige powder
Expiry Date: 27 December 2017
Storage Conditions: room temperature in the dark

For the purpose of the study the test item was used as supplied.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.99 or 3.53 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: the left eye was untreated and used as a control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1ml (76mg)
- Concentration (if solution): 100%, as supplied

Duration of treatment / exposure:

Single application, the test eye did not undergo any rinsing after application of the test item

Observation period (in vivo):

72hrs

Number of animals or in vitro replicates:

2 animals (male)

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item, which was found to weigh approximately 76 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale.

Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977).
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Results and discussion

In vivo

Results

Irritation parameter:

other: ocular irritation score

Basis:

mean

Time point:

other: 1hr

Score:

8

Max. score:

8

Reversibility:

fully reversible within: 72hrs

Remarks on result:

probability of mild irritation

Other effects:

-Ocular reactions
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48-Hour observations.
Both treated eyes appeared normal at the 72-Hour observation.

-Body Weight
No gain in body weight was noted in one animal and expected gain in body weight was noted in the other animal during the study.

Any other information on results incl. tables

Individual Scores and Individual Total Scores for Ocular Irritation are displayed in the table below:

Rabbit Number and Sex	75479Male				75487Male
	IPR= 0				IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	1	1	0	2	1	1	0
B = Chemosis	1	1	0	0	1	1	0	0
C = Discharge	1	0	0	0	1	0	0	0
Score (A + B + C) x 2	8	4	2	0	8	4	2	0
Total Score	8	4	2	0	8	4	2	0

IPR=Initial pain reaction

Individual Total Scores and Group Mean Scores for Ocular Irritation are displayed in the table below:

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
75479Male	8	4	2	0
75487Male	8	4	2	0
Group Total	16	8	4	0
Group Mean Score	8.0	4.0	2.0	0.0

Individual Body Weights and Body Weight Change are displayed in the table below:

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
75479Male	3.53	3.53	0.00
75487Male	2.99	3.00	0.01

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation.

Conclusion

The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to theGlobally Harmonized Systemof Classification and Labelling of Chemicals.