4-chloro-7H-pyrrolo[2,3-d]pyrimidine

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21-11-2018 to 27-12-2018

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-7H-pyrrolo[2,3-d]pyrimidine

EC Number:

628-079-2

Cas Number:

3680-69-1

Molecular formula:

C6H4ClN3

IUPAC Name:

4-chloro-7H-pyrrolo[2,3-d]pyrimidine

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13290
Expiry date: 31 January 2020 (expiry date)
Physical Description: White to off-white solid
Purity/Composition: 99.1%
Storage Conditions: At room temperature

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).

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.

Age at the Initiation of Dosing: Young adult animals (approximately 8 weeks old) were
selected.
Weight at the Initiation of Dosing: 157 to 178 g.

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 in which the animals were kept was 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 21 to 22°C with
an actual daily mean relative humidity of 26 to 51% (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:

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, three
additional groups were dosed at 300, 50 and 50 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:

50, 300 and 2000 mg/kgbw

No. of animals per sex per dose:

3/sex/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. Animals showing pain, distress or discomfort, which was considered not transient
in nature or is likely to become more severe, were sacrificed for humane reasons based on
OECD guidance document on the recognition, assessment, and use of clinical signs as
humane endpoints for experimental animals used in safety evaluation (ENV/JM/MONO/
2000/7).

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 or euthanized moribund after Day 1.

Terminal Procedures
All moribund animals and 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:

All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.
The oral LD50 value of the test item was ranked within the following ranges: 0-5, 5-50, 50-
300 or 300-2000 mg/kg b.w. or as exceeding 2000 mg/kg b.w. The LD50 cut-off value was
established based on OECD guideline 423. No statistical analysis was performed (The
method used is not intended to allow the calculation of a precise LD50 value).
The results were evaluated according to:
• Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the
United Nations (2017) (including all amendments).
• Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16
December 2008 on classification, labelling and packaging of items and mixtures
(including all amendments).

Results and discussion

Effect levels

Effect levels 1

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

200 mg/kg bw

Based on:

test mat.

Effect levels 2

Sex:

female

Dose descriptor:

LD50

Effect level:

> 50 - <= 300 mg/kg bw

Mortality:

At 2000 mg/kg, two animals were found dead on Day 2, no further mortality occurred.
At 300 mg/kg, two animals were found dead on Day 2 and one animal was killed in extremis
on Day 2.
At 50 mg/kg, no mortality occurred.

Clinical signs:

other: t 2000 mg/kg, lethargy, tremor, flat and hunched posture, uncoordinated movements, labored respiration, piloerection, pale appearance, hypothermia and/or ptosis were noted for the animals on Days 1 and/or 2. Additionally, clinical signs noted for the s

Gross pathology:

Abnormalities of the stomach (several black foci glandular mucosa) and body cavities
(watery-clear fluid thoracic cavity) were found in the animals that died or were sacrificed for
humane reasons during the study, at macroscopic post mortem examination. Advanced
autolysis was noted for the animals that were found dead during the study. Macroscopic post
mortem examination of the surviving animals at termination did not reveal any abnormalities.

Applicant's summary and conclusion

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

The oral LD50 value of PF-01323624 in Wistar Han rats was established to be within the
range of 50-300 mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 200
mg/kg body weight.
Based on these results:
• according to the Globally Harmonized System of Classification and Labelling of
Chemicals (GHS) of the United Nations (2017) (including all amendments), PF-01323624
should be classified as: Toxic if swallowed (Category 3) for acute toxicity by the oral
route.
• according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging
of items and mixtures (including all amendments), PF-01323624 should be classified as
Category 3 and should be labeled as H301: Toxic if swallowed.

Executive summary:

The objective of this study was to determine the potential toxicity of  PF-01323624, 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.

Initially,  PF-01323624 was administered by oral  gavage  to three female Wistar Han rats at

2000 mg/kg body weight. In a stepwise procedure three additional groups of three females

were dosed at 300, 50 and 50 mg/kg body weight. All animals were subjected to daily

observations and weekly determination of body weight. Macroscopic examination was

performed on the day of death or after terminal sacrifice (Day 15).

At 2000 mg/kg, two animals were found dead on Day 2, no further mortality occurred. At

300 mg/kg, two animals were found dead on Day 2 and one animal was killed in  extremis  on

Day 2. At 50 mg/kg, no mortality occurred.

At 2000 mg/kg, lethargy, tremor, flat and hunched posture, uncoordinated movements,

labored respiration, piloerection, pale appearance, hypothermia and/or  ptosis  were noted for

the animals on Days 1 and/or 2. Additionally, clinical signs noted for the surviving animal

consisted of flat gait on Days 3, 4 and 5, piloerection on days 5 and 6 and hunched posture on

Days 7 and 8. At 300 mg/kg, lethargy, flat and hunched posture, lateral recumbency,

uncoordinated movements, decreased  locomotor  activity, slow breathing, labored respiration,

piloerection, chromodacryorrhoea (nose, eye left and right) and/or  ptosis  were noted for the

animals on Days 1 and/or 2. At 50 mg/kg, lethargy, hunched posture, uncoordinated

movements, piloerection and/or  ptosis  were noted for the animals on Days 1 and/or 2.

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

to be similar to that expected for normal untreated animals of the same age and strain.

Abnormalities of the stomach (several black foci glandular  mucosa)  and body cavities

(watery-clear fluid thoracic cavity) were found in the animals that died or were sacrificed for

humane reasons during the study, at macroscopic post  mortem  examination. Macroscopic

post  mortem  examination of the surviving animals at termination did not reveal any

abnormalities.

The oral LD50 value of  PF-01323624 in Wistar Han rats was established to be within the

range of 50-300 mg/kg body weight.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 200

mg/kg body weight.

Based on these results:

• according to the Globally Harmonized System of Classification and Labelling of

Chemicals (GHS) of the United Nations (2017) (including all amendments),  PF-01323624

should be classified as: Toxic if swallowed (Category 3) for acute toxicity by the oral

route.

• according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging

of items and mixtures (including all amendments),  PF-01323624 should be classified as

Category 3 and should be labeled as H301: Toxic if swallowed.