1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo-1-piperidinyl)phenyl]-, ethyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: An in vitro Assay for Occular Irritancy, Fundamental and Applied Toxicology 18: 442-449, 1992

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The Utility of a two-test in vitro battery to access the Ocular Irritancy of drug intermediates. Development in Animal and Veterinary Sciences. 27. Proceedings of the 2nd World Congress on alternative and Animal use in the Life Sciences.

Principles of method if other than guideline:

The purpose of the study is to evaluate the potential ocular irritancy/toxicity of a test article as measured by the test article's ability to induce opacity and permeability to sodium fluorescein in an excised bovine cornea, in vitro.

GLP compliance:

yes

Remarks:

Ec Commission Directive 1999/11/EC and OECD ENV/MC/CHEM(98)17

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

EC Number:

700-890-7

Cas Number:

503614-91-3

Molecular formula:

C27 H28 N4 O5

IUPAC Name:

ethyl 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

Details on test material:

Crystalline Solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes were obtained as by-product of freshly slaughtered animals, transported in Hank's Balanced Salt Solution supplemented with 1% penicillin/streptomycin solution. The corneas are used within 4 hour of slaughter. The tissue surrounding the eyeball were carefully pulled away and the corneas were excised such that a 2 to 3 mm rim of sclera was present around the cornea. The corneas are mounted in corneal folders with the endothelial side against the O-ring of the posterior half of the holder. The chambers were filled with cMEM and incubated at 32 ± 2°C for 60± 5 min.

Test system

Vehicle:

physiological saline

Remarks:

20% (w/w) in 0.9% sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

An aliquot of 750ug was administered as 20 % w/v ( 200 mg/ml) suspension in saline.

Duration of treatment / exposure:

Corneas were exposed to the test material suspension for approximately four hours and then the corneas are removed from the incubator. The epithelial side of each cornea was rinsed three times with cMEM.

Observation period (in vivo):

Opacity measurements performed immediately after exposure period ends.

Number of animals or in vitro replicates:

Three corneas are tested with the test substance. Three negative controls (0.9% sodium chloride solution) and three positive controls (imidazole) were also used

Details on study design:

The study design requires determination of an opacity value and a permeability measurement. Positive and negative controls are used in the study. The positive controls use a 20 % (w/v) suspension of imidazole in complete MEM. The negative control use a 0.9% Saline. The corneas were exposed for approximately four hours and then the corneas are removed from the incubator. Then the opacity measurements are taken. Permeability measurements: Once opacity measurements are complete the medium was replaced with 1 ml of a 5 mg/ml fluorescein solution and incubated for another 90 minutes with the anterior chamber up. . The opacity density at 490 nm (OD 490) was determined If an OD 490 value was greater than 1.8, a 1:5 dilution of the sample in complete MEM was made. The dilution samples were then retested and final permeability measurements were made.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

3.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: standard deviation = 2.3

Results 2

Irritation parameter:

fluorescein leakage

Run / experiment:

mean

Value:

0.001

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: standard deviation = 0.003

Other effects / acceptance of results:

Positive control results: opacity value; 87.0 ±8.7, Permeability; 2.678 ± 0.578
Negative control results: opacity value; 2.0 ± 1.7, Permeability; 0.009 ± 0.001

Any other information on results incl. tables

The bovine corneal opacity and permeability assay uses an opacity measurement and a permeability measurement to determine a final in vitro score. The opacity measurement is determined by the change in opacity of each cornea by subtracting the calculated pre-treatment opacity reading from the final opacity reading. The corrected opacity value of each cornea was calculated by subtracting the average change in opacity of the negative control corneas from that of the treated corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity value of the treated corneas from each treatment condition. The permeability measurements: the corrected OD 490 is determined by subtracting the negative controls mean OD 490 from each of the treated cornea OD 490 value. The mean OD 490 of each treatment group was calculated by averaging the corrected OD 490 values. The formula used is: in vitro score = mean opacity value + ( 15 x mean OD 490 Value). The in vitro score is then compared to a classification system established based on studies with a wide range of test materials. The classification provides a good initial guide to interpret these in vitro data: an in vitro score from 0 to 25 is negative irritation and > 25.1 positve for irritation.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

Based on an in vitro score of 3.7 ± 2.3 and valid positive and negative controls, the test material was classified according to this assay's classification scheme as a negative potential eye irritant.

Executive summary:

The in vitro bovine corneal opacity and permeability assay was completed in 2002 in accordance with GLP and before the official adoption of the EU method B.47 method (2010) and OECD 437 (2009). However, the method used in 2002 is equivalent or similar to the current EU B.47 and OECD 437 method and considered as a Klimish 1.

.The bovine corneal opacity and permeability assay uses an opacity measurement and a permeability measurement to determine a final in vitro score. The opacity measurement is determined by the change in opacity of each cornea by subtracting the calculated pre-treatment opacity reading from the final opacity reading. The corrected opacity value of each cornea was calculated by subtracting the average change in opacity of the negative control corneas from that of the treated corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity value of the treated corneas from each treatment condition. The permeability measurements: the corrected OD 490 is determined by subtracting the negative controls mean OD 490 from each of the treated cornea OD 490 value. The mean OD 490 of each treatment group was calculated by averaging the corrected OD 490 values. The formula used is: in vitro score = mean corrected opacity value + ( 15 x mean corrected OD 490 Value). The in vitro score is then compared to a classification system. The classification provides a good initial guide to interpret these in vitro data: an in vitro score from 0 to 25 as negative, and < 25.1 as positive

Based on an in vitro score of 3.7 ± 2.3 and valid positive and negative controls, the test material was classified according to this assay's classification scheme as a negative potential eye irritant.