(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 Jan 2019 to 07 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline 423. Acute Oral Toxicity - Acute Toxic Class Method, December 2001.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA Health Effects Test Guideline OPPTS 870.1100. Acute Oral Toxicity in Rodents, December 2002.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No 440/2008 Part B. Acute Oral Toxicity, Acute Toxic Class Method, May 2008.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF No.12-Nousan-8147

Version / remarks:

Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural
Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF), November 2000, including the most recent revisions.

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

EC Number:

950-860-4

Molecular formula:

C15H20N4O2

IUPAC Name:

(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

Test material form:

solid: bulk

Details on test material:

White Crystalline Solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as recognized by international guidelines as a recommended test system. The test method and number of animals were based on the test guidelines. The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study. Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

Husbandry
Housing
On arrival and following assignment to the study, animals were group housed (up to 3 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room(s) in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 21°C with an actual daily mean relative humidity of 36 to 53% (see deviations in Appendix 2). A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Experimental Design
The toxicity of the test item was assessed by stepwise treatment of groups of 3 females. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups. The first group was treated at a dose level of 2000 mg/kg. Based on the results, one additional group was dosed at 2000 mg/kg.

Administration of Test item
A single dose of test item was administered to the appropriate animals by oral gavage on Day 1, using a syringe with a plastic gavage cannula attached. The dose volume for each animal was based on the body weight measurement prior to dosing. A dose volume of 20 mL/kg body weight was used for each dose (see deviations in Appendix 2). The dosing formulations were stirred continuously during dose administration. Animals were deprived of food overnight (for a maximum of 20 hours) prior to dosing and until 3-4 hours after administration of the test item. Water was available.

Justification of Route and Dose Levels
The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item. The dose levels were based on the OECD test guidelines and were selected from the series 5 (lowest dose level), 50, 300 and 2000 (highest dose level) mg/kg body weight. The starting dose level should be the one that is likely to produce mortality in at least some of the animals and was selected based on available toxicity data of the test item.

Doses:

2000mg/kg for both groups of 3

No. of animals per sex per dose:

3 females per dose

Control animals:

no

Details on study design:

Rationale for Vehicle
Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw Data of these trials will be retained by the Test Facility.

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. The characterization of the test item was conducted in a sponsor or sponsor subcontractor quality environment.

Reserve Samples
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility. The sample will be destroyed after the expiry date.

Test and Reference Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were stirred until and during dosing. Any residual volumes were discarded.

Sample Collection and Analysis
Analysis of test item in vehicle for concentration, stability, homogeneity was not performed.

Terminal Procedures
All animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded.

Statistics:

In-life Procedures, Observations, and Measurements

Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity
twice daily, in the morning and at the end of the working day. Animals were not removed
from cage during observation, unless necessary for identification or confirmation of possible
findings.

Clinical Observations
Postdose Observations
Postdose observations were performed at periodic intervals on the day of dosing (at least three times) and once daily thereafter. The observation period was 14 days. All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate). Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

Body Weights
Animals were weighed individually on Day 1 (predose), 8 and 15. A fasted weight was recorded on the day of dosing. Terminal body weights were collected from animals found dead after Day 1.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

One female was found dead on Day 3 post-treatment. No further mortality occurred.

Clinical signs:

other: Lethargy, hunched posture, uncoordinated movements, piloerection, salivation, chromodacryorrhoea and ptosis were noted for the animals between Days 1 and 3. Labored respiration was noted for the animal found dead.

Gross pathology:

No toxicological significant abnormalities were found at macroscopic post mortem examination of the surviving animals. Abnormalities of the lungs (right cranial lobe reduced in size and contents hemorrhagic clotted blood) were found for the animal that died during the study. This finding raises some concerns (see conclusion below). Isolated dark red foci on the thymus were noted for one surviving female. Since this is occasionally seen among rats of this age and strain and it was found in one animal only, this was considered not toxicologically significant.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 value of PF-07094402 in Wistar Han rats was established to exceed 2000 mg/kg body weight. According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), PF-07094402 does not have to be classified and has no obligatory labelling requirement for acute oral toxicity. According to the OECD 423 test guideline, the LD50 cut-off value would be considered as 2000 mg/kg body weight based on the animal found dead. However, there are some concerns about the cause of death since labored respiration and abnormalities of the lungs were seen for this animal. This suggests that there was exposure of the lungs to test item formulation. Taken this concern and the response of the other animals into account, a LD50 cut-off value that exceeds 5000 mg/kg body weight might be a better representation of the hazard of the test item. Based on this evaluation and according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), PF-07094402 should not have to be classified and has no obligatory labelling requirement for acute oral toxicity.