Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 20 - March 16, 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:
The fixed dose test procedure as described in OECD 420 was used.
Test type:
fixed dose procedure
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride
EC Number:
845-622-0
Cas Number:
2050038-84-9
Molecular formula:
C14H19Cl2N5
IUPAC Name:
12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride
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 least once during the first 30 minutes with special attention given during the first 6 hours and then at least daily for a period of 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
Main Study: 300 mg/kg
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 one animal at a time 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 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 Teat: The Main Test dose is determined by the Sighting Study. A total of 5 animals are used for each dose level. This includes the animal from the sighting study administered the same dose 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 least once during the first 30 minutes with special attention given during the first 6 hours and then at least daily for a period of 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 anesthesia with intraperitoneal ketamine (100 mg/ kg)/xylazine (10 mg/kg). 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 6002 was dosed at 300 mg/kg A-1335930.3 as there was no information available on toxicity. Starting approximately 5 minutes after dosing consumption of bedding was observed which might be a sign of distress or abdominal pain but might also be related to the stress caused by the procedure itself, especially considering the fasting and the single housing. Approximately 10 minutes post dose, swaggering gait/ ataxia were seen in this animal. Approximately 20 minutes post dose, animal 6002 was in sternal recumbency, followed by lateral recumbency approximately 25 minutes post dose. At 45 minutes post dose, animal 6002 showed abdominal pronounced breathing. 1 hour post dose, animal 6002 showed decreased activity, partial palpebral closure and a hunched posture. 2 hours post dose, piloerection and ataxia were observed in addition. Clinical signs ( decreased activity, slow movements) were still present at 6 hours post dose. All clinical signs in animal 6002 were resolved 24 hours after dosing and the animal appeared normal. Based on these observations 300 mg/kg was chosen for the main study.
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Mortality:
None
Clinical signs:
other: All four animals showed clinical signs of decreased activity and piloerection between 30 minutes and 6 hours post dose. Additional clinical signs were hunched posture (6004, 6010), swaggering gait/ ataxia (6010), slow motion movements (6004), sternal recu
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:
Category 4 based on GHS criteria
Conclusions:
The test material was evaluated for potential acute toxicity following oral administration. Using OECD 420 Fixed Dose Procedure (main test), five animals survived the dose of 300 mg/kg. Therefore, the test item was defined to have an estimated
LD50>300 mg/kg but < 2000 mg/kg and therefore classified as a Hazard Category of 4 according to Globally Harmonized System (GHS).