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 study was completed for acute oral toxicity according to OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) and EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method). A key study was completed for the acute dermal toxicity according to OECD Guideline 402 (Acute Dermal Toxicity) and EU Method B.3 (Acute Toxicity (Dermal)). Both of these studies were completed under GLP. An acute inhalation study was attempted but generating a representative atmosphere of the test items as supplied was not feasible and therefore the study was not conducted. However, in view of the physical nature of the test item and its apparent low volatility it is considered unlikely to represent a significant hazard by the inhalation route.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th Sept. to 30th Sept. 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

With exception of an overnight fast before dosing and 4 hours after dosing, free access to mains drinking water and food was allowed. The temperature and relative humidity were set to achieve limits of 22 +- 3 C and 40 to 70% respectively. The lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness. Weight at start 92-148 grams and 5 to 7 weeks old at the start of the study. animals provided by Harlan Ltd, UK. animals acclimatised for 7 days prior to start of the study.

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose

Details on oral exposure:

Using all available information on the toxicity of the test material, 2000 mg/kg was chosen as the starting dose.
dose level: 2000 mg/kg
concentration: 200 mg/ml
dose volume 10 ml/kg bodyweight
3 fasted females per dose, followed by a group of 3 fasted males per dose per the test guidelines.

Doses:

Female: 2000 mg/kg bw
male: 2000 mg/kg bw

No. of animals per sex per dose:

Female: 2000 mg/kg bw; Number of animals: 3 then, 3 additional males rats at same dose level.

Control animals:

no

Details on study design:

All animals were dosed once only by gavage. The animals were observed for deaths or overt signs of toxicity soon after the final dose and frequently the first day and subsequently once daily for fourteen days. At the end of the observation period the animals were killed using ascending concentrations of carbon dioxide. All animals were subjected to gross pathological observations. This consisted of external examination and opening of the cranial, abdominal and thoracic cavities for examination of major organs. The appearances of any macroscopic abnormalities were recorded. No tissues were retained.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

2000 mg/kg bw; Number of animals: 6; Number of deaths: 0

Clinical signs:

other: Signs of toxicity related to dose levels: There were no signs of systemic toxicity. All animals showed expected gains in bodyweight over the study period.

Gross pathology:

Effects on organs:
No abnormalities were noted at necropsy.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute oral median lethal dose (LD50) of the test material in the Sprague-Dawley CD strain rat was estimated as being greater than 2000 mg/kg bodyweight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

It was considered inappropriate to formulate the test item using a suitable vehicle as this would not be representative of the hazard that the test item poses in everyday use. Therefore, attempts were made to generate an atmosphere of the test item as supplied.
Three generation trials were carried out in an attempt to generate a dust atmosphere with mass median aerodynamic diameters (MMAD) ranging from 1 to 4 µm (details of these trials are recorded in materials and methods). At approximately 5 mg/L the minimum MMAD generated was 8.91 µm with the percentage of particles less than 4 µm = 33.1%.
In view of the physical nature of the test item and its apparent low volatility it is considered unlikely to represent a significant hazard by the inhalation route.

GLP compliance:

yes (incl. QA statement)

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 28 June 2016 and 14 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

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 weighed out according to each animal’s individual body weight and moistened with arachis oil BP prior to application.
The absorption of the test item was not determined.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Five male and five female Wistar (RccHan:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were 8 to 12 weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.
The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24 Hour exposure period and in groups of four, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °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.

Type of coverage:

semiocclusive

Vehicle:

arachis oil

Remarks:

used only to moisten test material

Details on dermal exposure:

On the day before treatment the back and flanks of each animal were clipped free of hair.
Using available information on the toxicity of the test item, one male and one female rat were initially treated with the test item at a dose level of 2000 mg/kg.
The appropriate amount of test item, moistened with arachis oil BP, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self adhesive bandage. The animals were caged individually throughout the study. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item.
As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females. The animals were caged individually for the 24 Hour exposure period. After the 24 Hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item. These animals were returned to group housing for the remainder of the test period.

Duration of exposure:

24hrs

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days.
After removal of the dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored according to the following scale:

EVALUATION OF SKIN REACTIONS
-Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4

-Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and extending beyond the area of exposure) 4

Any other skin reactions, if present were also recorded.
Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
At the end of the study the animals were euthanatized by cervical dislocation confirmed by palpation and blink/pedal reflex. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

There were no deaths

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period.

Gross pathology:

No abnormalities were noted at necropsy

Interpretation of results:

GHS criteria not met

Conclusions:

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

Executive summary:

Introduction

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat.

Methods

Initially, two animals (one male and one female) were given a single, 24 hour, semi‑occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg body weight. Based on the results of the initial test, a further group of eight animals (four males and four females) was similarly treated. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Results

Mortality. There were no deaths.

Clinical Observations. There were no signs of systemic toxicity.

Dermal Irritation. There were no signs of dermal irritation.

Body Weight. Animals showed expected gains in body weight except for one female which showed body weight loss during the first week with expected gain in body weight during the second week.

Necropsy. No abnormalities were noted at necropsy.

Conclusion

The acute dermal median lethal dose (LD₅₀) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Justification for classification or non-classification

The acute oral median lethal dose (LD50) of the test material in the Sprague-Dawley CD strain rat was estimated as being greater than 2000 mg/kg bodyweight. The acute dermal median lethal dose (LD50) of the test material in the Wistar strain of rats was estimated at being greater than 2000 mg/kg bodyweight. Therefore the substance is not classified according to the EU CLP regulation.