isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27-11-2018 to 18-12-2018

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

EC Number:

950-484-0

Molecular formula:

C9H17NO2*C4H6O4

IUPAC Name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13335
Expiry date: 31 January 2020 (expiry date)
Physical Description: White solid
Purity/Composition: 97.1%
Storage Conditions: At room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Animals: 6 Females (nulliparous and non-pregnant). Each dose group consisted of 3 animals.
Age at the Initiation of Dosing: Young adult animals (approximately 8-9 weeks old) were selected.
Weight at the Initiation of Dosing: 153 to 180 g.

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).

Animal Identification
At study assignment, each animal was identified using an ear mark and tail mark with
indelible ink.

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. These housing conditions were maintained unless deemed inappropriate by the Study
Director and/or Clinical Veterinarian. The room 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 40 to 53%. 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:

water

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 10 mL/kg body weight was used for each dose.
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:

2000 mg/kg bw

No. of animals per sex per dose:

3 per sex per dose

Control animals:

no

Details on study design:

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

Statistics:

No statistical analysis was performed

Results and discussion

Preliminary study:

not applicable

Effect levels

Effect levels 1

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Effect levels 2

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: Hunched posture, piloerection and/or salivation were noted for all animals on Day 1.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 value of PF-07097547-24 in Wistar Han rats was established to exceed 2000
mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed
5000 mg/kg body weight.
Based on these results, PF-07097547-24 does not have to be classified and has no obligatory
labelling requirement for acute oral toxicity according to the Globally Harmonized System of
Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all
amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging
of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to determine the potential toxicity of  PF-07097547-24, when

given by oral  gavage  at a single dose to rats of a single sex at one or more defined doses to

evaluate the potential reversibility of any findings.

The study was carried out in compliance with the guidelines described in:

• OECD No.423 (2001) "Acute Oral Toxicity, Acute Toxic Class Method".

• EC No 440/2008, part B: "Acute Oral Toxicity, Acute Toxic Class Method".

• EPA, OPPTS 870.1100 (2002), "Acute Oral Toxicity".

• JMAFF Guidelines (2000), including the most recent revisions.  

PF-07097547-24 was administered by oral  gavage  to two consecutive groups of three female

Wistar Han rats at 2000 mg/kg body weight. Animals were subjected to daily observations

and weekly determination of body weight. Macroscopic examination was performed after

terminal sacrifice (Day 15).

Hunched posture, piloerection and/or salivation were noted for all animals on Day 1.

The body weight gain shown by the animals over the study period was considered to be

normal.

No abnormalities were found at macroscopic post  mortem  examination of the animals.

The oral LD50 value of  PF-07097547-24 in Wistar Han rats was established to exceed 2000

mg/kg body weight.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed

5000 mg/kg body weight.

Based on these results,  PF-07097547-24 does not have to be classified and has no obligatory

labelling requirement for acute oral toxicity according to the Globally Harmonized System of

Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all

amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging

of items and mixtures (including all amendments).