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

Endpoint summary

Description of key information

A key skin irritation study was conducted under GLP conditions and in accordance with EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion) and OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

other justification

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 5 to September 17, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

New Zealand White rabbits were supplied by Harlan Ltd., UK.; acclimatisation period of at least 26 days; At the start of the study the animals were in the weight range of 2.77 to 3.0 kg and up to eight weeks old. Free access to mains drinking water and food was allowed through out the study. The temperature and relative humidity were set to achieve limits of 15 to 23 C and 40 to 70% respectively. The rate of air exchange was at least 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 g of the test material, moistened with 0.5 ml of reverse osmosis water was introduced under 2.5 cm X 2.5 cm cotton gauze patch

Duration of treatment / exposure:

A single animal received three treatments first with the patch removed at 3 mins, 1 hr and 4 hours as preliminary screening. . The patch was removed after four hours and any residual material removed from the test area by gentle swabbing with luke warm water. Blotted dry with absorbant paper.

Observation period:

Approximately one hour after removing the patch and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored.

Number of animals:

3

Details on study design:

The day before the test the three rabbits were clipped free of fur from the dorsal/flank area and inspected for gross abnormalities of the epidermis. Only animals with an intact healthy epidermis are selected for the study. A suitable site on the back of each rabbit was selected as test site and 0.5 g of test material moistened with 0.5 ml of distilled water was introduced under a 2.5 cm X 2.5 cm cotton gauze patch which was secured in position with surgical tape. After patch removal, the test sites were evaluated after 1 hour, 24, 48, and 72 hours according to the Draize classification scheme for determination of irritation.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1, 24,48 and 72 hr

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1, 24, 48 and 72 hrs

Score:

0

Max. score:

0

Irritant / corrosive response data:

The individual scores for erythema/eschar and oedema were all zero for all three animals for the complete 72 hour observation period. No evidence of skin irritation was noted during the study except for one animal with a very slight irritation noted at one hour after bandage removal.

Other effects:

No evidence of skin irritation was noted during the study except very slight erthyema in one animal one hour after removal of the bandage. There were no signs of systemic toxicity

Interpretation of results:

not irritating

Conclusions:

The test material produced a score of 0.0 and was classified as a non-irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted. The test material did not meet the classification as irritant or corrosive according to the EU labelling regulations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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.

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.

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.

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.

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

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 25 to 18 Sept. 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Follows GLP and recognized in vitro method

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability Assay

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 fluorescein in an isolated bovine cornea.

GLP compliance:

yes

Species:

other: bovine corneas

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 penicillin/streptomycin and sent on ice packs. The corneas are used within 4 hour of receipt. 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 chamber and the anterior chamber secured on top. The chambers were filled with Minimum Essential Medium Eagle (MEM) without phenol red, supplemented with 1 % fetal bovine serum and incubated at 32 +- 2 C for a minimum of 1 hour.

Vehicle:

physiological saline

Amount / concentration applied:

An aliquot of 750ug was administered as 20 % w/v suspension.

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 complete MEM containing phenol red and a final rinse with complete MEM without phenol red. Then the solution is replaced with fresh complete MEM.

Observation period (in vivo):

Opacity measurements performed immediately after exposure period ends.

Number of animals or in vitro replicates:

three corneas are tested.

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0

Positive controls validity:

not valid

Remarks on result:

other:

Remarks:

4 hours

Irritant / corrosive response data:

The results of the positive control fell within 2 standard deviations of the historical mean (within a range of 110.3 to 170.3 ) and the assay was considered valid.

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 and >25 as potential eye irritants.

Interpretation of results:

not irritating

Conclusions:

Based on an in vitro score of 0.0 and valid positive controls, the test material was classified according to this assay's classification scheme as negative for eye irritancy.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results of the key in-vivo study for skin irritation reported a score of 0.0 and was classified as a non-irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted. The test material did not meet the classification as irritant or corrosive according to the EU CLP regulations.

An in-vitro eye irritancy Bovine Corneal Opacity and Permeability Assay was completed and based on an in vitro score of 0.0 and valid positive controls, the test material was classified according to this assay's classification scheme as negative for eye irritancy. In addition, an in-vivo eye irritancy study was conducted according to OECD Guideline 405 (Acute Eye Irritation / Corrosion) and EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion). 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 EU CLP regulations.