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

Endpoint summary

Description of key information

In an in vivo skin irritation study conducted to OECD Guideline 404, there were no signs of toxicity or ill health in any rabbits during the observation period. No dermal reaction was observed in any animal throughout the duration of the study.

When tested in the Bovine Corneal Opacity and Permeability assay ( OECD Guideline 437) there were no indications of eye irritation potential. 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. In an eye irritation study with rabbits, performed according to OECD/EC test guidelines, limited irritation was observed which was fully reversible within 72 hours. Based on these results BMS-589154-01 does not have to be classified for eye irritation.

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:

5 September to 17 September, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

EPA 712-C-98-196

GLP compliance:

yes

Remarks:

OECD ENV/MC/CHEM(98017 and EC Directive 1999/11/EC

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd, Bicester, Oxon, England
- Age at study initiation: at least 8 weeks
- Weight at study initiation: 2.74kg to 3.00kg
- Housing: Individually in stainless steel cages with perforated floors.
- Diet: 125 gram of standard laboratory rabbit diet (STANRAB [P] SQC pellet) per day
- Water: ad libitum
- Acclimation period: at least 26 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 to 23°C
- Humidity (%): 40% to 70%
- Photoperiod (hrs dark / hrs light): 12 hrs of artificial light in each 24 hr period

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: additional site similarly treated with the exception of the test substance.

Amount / concentration applied:

500 mg applied to a site pre-wetted with reverse osmosis water

Duration of treatment / exposure:

4hr

Observation period:

on removal of the dressing and at 1hr, 24hr, 48hr and 72hr after the dressing was removed

Number of animals:

3

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Interpretation of results:

not irritating

Conclusions:

No dermal reaction was observed in any animal throughout the duration of the study.

The test material does not labeling per GHS or EU CLP.

Executive summary:

There were no signs of toxicity or ill health in any rabbits during the observation period.

No dermal reaction was observed in any animal throughout the duration of the study

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:

18 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

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)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

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

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.

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.

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

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

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.

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.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2016 - 04 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147.

Version / remarks:

November 2000, including the most recent partial revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Dtae certficate 03 November 2015

Specific details on test material used for the study:

Test item handling: Use amber glassware or wrap container in aluminum-foil
pH (1% in water, indicative range): 4.5 – 4.4 (determined by Charles River Den Bosch)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Charles River France, L’Arbresle, France.
- Age at study initiation: Animals used within the study were between 12 and 14 weeks old.
- Weight at study initiation: Body weights were between 2720 grams and 3366 grams.
- Housing: Individually housed in cages with perforated floors and shelters.
- Diet: Pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy) approximately 100 grams per day. Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

Deviations from the maximum level of daily mean relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

IN-LIFE DATES: From: 11 July 2016 to 04 August 2016

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

average 35.6 mg (range: 35.4 mg – 35.9 mg) (a volume of approximately 0.1 mL)

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance

Number of animals or in vitro replicates:

3 males

Details on study design:

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 3 weeks later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
One hour prior to instillation of the test item, buprenorphine 0.01 mg/kg bw was administered subcutaneous. Five minutes prior to instillation of the test item, two drops of the topical anesthetic alcaine 0.5% were applied to both eyes.
Animals were treated by instillation of the test item 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 fluorescein examination, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg bw and meloxicam 0.5 mg/kg bw were administered by subcutaneous injection.

REMOVAL OF TEST SUBSTANCE
Immediately after the 1 hour observation, the treated eye was rinsed with approximately 30-50 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.
After the 24 hour observation, the treated eye of the first animal was rinsed again to remove any visible residual test item. For reference control the other eye was also rinsed.

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- After the final observation, the first animal was euthanatized by intra-venous injection of Euthasol® 20%. The other two animals were removed from the study alive.
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance.
The irritation scores and a description of all other (local) effects were recorded.The irritation was assessed according to OECD 405.
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.

The results were evaluated according to:
•Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of theUnited Nations (2015) (including all amendments).
•Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16December 2008 on classification, labelling and packaging of items and mixtures(including all amendments).

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

mean

Remarks:

(animal #1,2,3)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

(animal #1,2,3)

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Remarks:

(mean)

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Remarks:

(mean)

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Remarks:

(mean)

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal:

Remarks:

#1 and #2 (mean)

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

(mean)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

There was no evidence of ocular corrosion.

Instillation of approximately 35.6 mg of BMS-589154-01 (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of very slight to moderate redness, chemosis and discharge. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals.
Iridial irritation was observed and resolved within 24 hours in all animals. No corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.

Other effects:

No staining of (peri) ocular tissues by the test item was observed. Remnants of the test item were present in the eye on Days 1, 2 and/or 3.
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

In an eye irritation study with rabbits, performed according to OECD/EC test guidelines, limited irritation was observed which was fully reversible within 72 hours. Based on these results BMS-589154-01 does not have to be classified for eye irritation.

Executive summary:

An acute eye irritation/corrosion study was performed with BMS-589154-01 in the rabbit according to OECD/EC guidelines and in compliance with GLP principles. Single samples of approximately 35.6 mg of BMS-589154-01 (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 after instillation. After installation, irritation of the conjunctivae was observed, which consisted of very slight to moderate redness, chemosis and discharge. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals. Iridial irritation was observed and resolved within 24 hours in all animals. No staining of (peri) ocular tissues by the test item was observed. Remnants of the test item were present in the eye on Days 1, 2 and/or 3.

Based on these results, BMS-589154-01 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) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In an in vivoskin irritation study conducted to OECD Guideline 404, there were no signs of toxicity or ill health in any rabbits during the observation period. No dermal reaction was observed in any animal throughout the duration of the study. Therefore the substance does not meet the criteria for classification as skin irritant as set out in 1272/2008/EC (amended).

When tested in the Bovine Corneal Opacity and Permeability assay (OECD Guideline 437) there were no indications of eye irritation potential. 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. In an eye irritation study with rabbits, performed according to OECD/EC test guidelines, limited irritation was observed which was fully reversible within 72 hours. Therefore the substance does not meet the criteria for classification as eye irritant as set out in 1272/2008/EC (amended).