1-Pyrrolidinecarboxylic acid, 3-[2-[(ethoxycarbonyl)[5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl]-4-ethyl-, phenylmethyl ester, (3R,4S)-

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20th February 2020 to 16th March 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

GLP compliance:

no

Remarks:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Test type:

fixed dose procedure

Test material

Test material information

Constituent 1

Reference substance name:

benzyl (3R,4S)-3-{2-[(ethoxycarbonyl)[5-(4-methylbenzenesulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl}-4-ethylpyrrolidine-1-carboxylate

EC Number:

944-343-2

Cas Number:

2050038-81-6

Molecular formula:

C32H35N5O7S

IUPAC Name:

benzyl (3R,4S)-3-{2-[(ethoxycarbonyl)[5-(4-methylbenzenesulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl}-4-ethylpyrrolidine-1-carboxylate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

Animals are fasted the night prior to dose administration. Animals are weighed and administered a single dose of the test article by intragastric intubation by means of a ball tip gavage needle and a syringe.
After dosing, the animals are returned to their cages and supplied with feed and water ad libitum. Clinical observations are made at 30 minutes, 1, 2, 3, 4, 6 and 24 hours and daily from 2 - 14 days.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Hydroxypropylmethylcellulose

Doses:

Dose volume
10 mL/kg (all concentrations) of the test material
Dose levels:
Sighting Study:
300 mg/kg, 2000 mg/kg of the test material
Main Study: 2000 mg/kg of the test material

No. of animals per sex per dose:

1 sighting study
4 main study

Control animals:

not specified

Details on study design:

Sighting Study: The purpose of the sighting study is to determine the starting dose for the main study. The test article is administered to two animals in a sequential manner with at least 24 hours between the dosing of each animal. Animals are maintained for a period of at least 14 days. Dose levels are fixed at 5, 50, 300, and 2000 mg/kg. The first animal is dosed at a level expected to produce toxicity based on available in vivo or in vitro data; or at 300 mg/kg when no toxicity information is available. Depending on signs of toxicity (see flow charts in Annex 3, OECD 420 Acute Oral Toxicity - Fixed Dose Method) the next animal is dosed at the next higher or next lower dose level. Dosing continues until a dose level for the Main Test can be determined or death is seen at the lowest fixed dose.

Main Test: The main test dose is determined by the sighting study. A total of five animals are used for each dose level. This includes one animal from the sighting study and an additional four animals. The course of the study depends on the response of the animals at the dose level for the main test; either the testing is stopped and the appropriate hazard classification class is assigned; or the testing continues at a higher fixed dose level; or testing continues at a lower fixed dose level. If additional dose levels are tested, the time interval between them is determined by the onset, duration, and severity of toxic signs. After dosing, the animals are returned to their cages and supplied with feed and water ad libitum. Clinical observations are made at 30 minutes, 1, 2, 3, 4, 6 and 24 hours and daily from 2 - 14 days. The frequency is determined by the response of the test animals to the treatment. However, the duration of observation is not fixed rigidly. It is determined by the toxic reactions, rate of onset and length of recovery period, and may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear and the time of death are important, especially if there is a tendency for deaths to be delayed. All observations are recorded and individual records are maintained for each animal. Cageside observations include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic, and central nervous system, and somatomotor activity and behavior pattern. Particular attention is directed to observations of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. The time of death is recorded as precisely as possible. Moribund animals that are killed for humane reasons are considered in the same way as animals that died on test.
Animals are weighed weekly and at the end of the observation period and then are sacrificed by exsanguination under deep inhalation anesthesia. Changes in weights are calculated and recorded when survival exceeds one day. Any animal found dead is necropsied as soon as possible, but in no case later than 12 hours after discovery. A gross necropsy is performed on all animals whether found dead, sacrificed in extremis, or sacrificed at the end of the study and all gross pathological changes are recorded. An evaluation of acute toxicity data includes the relationship, if any, between the animals exposed to the test article and the incidence and severity of all abnormalities, including behavioral and clinical abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects.

Interpretation of the test results on the fixed dose procedure are based on Annex 3 of OECD 420, Acute Oral Toxicity - Fixed Dose

Results and discussion

Preliminary study:

Animal 5002 was dosed at 300 mg/kg of the test material as there was no information available on toxicity. Starting approximately 30 minutes after dosing clinical signs of decreased activity, partial palprebal closure and piloerection were observed. At 1 hour post-dose, slow motion movement and pilo-erection were observed. At 3 hours, post-dose walking on toes was observed in addition. Clinical signs (decreased activity, slow movements) were still present at 6 hours post-dose. All clinical signs in animal 5002 were resolved 24 hours after dosing and the animal appeared normal. As Animal 5002 survived, Animal 5004 was dosed the next day at the next highest dose of 2000 mg/kg. Approximately threee hours after dosing clinical signs including decreased activity and pilo-erection were observed. At 4 hours post-dose only pilo-erection was observed. All clinical signs on animal 5004 were resolved 6 hours after dosing and the animal appeared normal. Based on these observations 2000 mg/kg was chosen for the main study with the test material.

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Mortality:

None

Clinical signs:

other: Two animals (5006 and 5010) showed clinical signs of walking on toes 1 hour (5010) and 2 hours (5006 & 5010) post-dose. Clinical signs were resolved 3 hours post-dose. Animals 5008 and 5012 showed no clinical signs. All five animals doses with 2000 mg/kg

Gross pathology:

There were no findings during gross necropsy in any of the main study animals at the end of study; all tissues appeared normal.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Using OECD 420 Fixed Dose Procedure (main test), five animals survived the dose of 2000 mg/kg of the test material. Therefore, the test item was defined to have an estimated LD50 > 2000 mg/kg and therefore classified as a Hazard Category of 5 according to Globally Harmonized System (GHS). However, since category 5 is not implementedin the EU, GHS criteria was considered to be not met.

Executive summary:

Objective
The purpose of the study is to assess the acute oral toxicity of a test article via oral administration. The fixed dose test procedure as described in OECD 420 is used. This procedure minimizes the number of animals used, allows the observation of signs of toxicity and provides information both for hazard assessment and for hazard classification purposes.

General Procedure
Where necessary, the test article is dissolved or suspended in a suitable vehicle. Aqueous solutions will be considered first, followed by consideration of a solution/suspension in vegetable oil. Animals are fasted the night prior to dose administration. Animals are weighed and administered a single dose of the test article by intragastric intubation by means of a ball tip gavage needle and a syringe.

Sighting Study:
Animal 5002 was dosed at 300 mg/kg of the test material as there was no information available on toxicity. Starting approximately 30 minutes after dosing clinical signs of decreased activity, partial palprebal closure and piloerection were observed. At 1 hour post-dose, slow motion movement and pilo-erection were observed. At 3 hours, post-dose walking on toes was observed in addition. Clinical signs (decreased activity, slow movements) were still present at 6 hours post-dose. All clinical signs in animal 5002 were resolved 24 hours after dosing and the animal appeared normal. As Animal 5002 survived, Animal 5004 was dosed the next day at the next highest dose of 2000 mg/kg. Approximately threee hours after dosing clinical signs including decreased activity and pilo-erection were observed. At 4 hours post-dose only pilo-erection was observed. All clinical signs on animal 5004 were resolved 6 hours after dosing and the animal appeared normal. Based on these observations 2000 mg/kg was chosen for the main study with the test material.

Main Test: Four additional animals (Animals 5006, 5008, 5010, and 5012) were dosed with the test material at 2000 mg/kg. Two animals (5006 and 5010) showed clinical signs of walking on toes 1 hour (5010) and 2 hours (5006 & 5010) post-dose. Clinical signs were resolved 3 hours post-dose. Animals 5008 and 5012 showed no clinical signs. All five animals doses with 2000 mg/kg of the test material (Animals 5004, 5006, 5008, 5010, and 5012) were observed as normal between 3 hours and the end of 14-day observation period and all animals gained weight over the course of the study. There were no findings during gross necropsy in any of the main study animals at the end of study; all tissues appeared normal.

Conclusions: Using OECD 420 Fixed Dose Procedure (main test), five animals survived the dose of 2000 mg/kg of the test material. Therefore, the test item was defined to have an estimated LD50 > 2000 mg/kg and therefore classified as a Hazard Category of 5 according to Globally Harmonized System (GHS).