Pyrido[2,3-d]pyrimidin-7(8H)-one, 6-bromo-2-chloro-8-cyclopentyl-5-methyl-

Endpoint summary

Description of key information

A 7-day repeated dose study (Dunster J S, 2013) is available which is key study. The oral NOEL value of test substance is considered to be 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 May to 09 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately nine weeks old
- Weight at study initiation: the males weighed 361 to 387g, the females weighed 242 to 278g
- Housing: in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): free access to food(Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories UK, Oxon, UK)
- Water (e.g. ad libitum): free access to water
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From 8 June to 25 June 2013

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP.

VEHICLE
- Concentration in vehicle: 37.5, 125, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

seven consecutive days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
150, 500, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of previous toxicity work (Harlan Laboratories Ltd., Study Number 41300893)

No additional information

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1, 4 and 8.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded for each cage group for study Days 1 to 4 and Days 4 to 8.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No Data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 7).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters [Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices - mean corpuscular haemoglobin (MCH), - mean corpuscular volume (MCV), - mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), Differential leucocyte count - neutrophils (Neut), - lymphocytes (Lymph), - monocytes (Mono), - eosinophils (Eos), - basophils (Bas), Platelet count (PLT), Prothrombin time (CT)] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (Day 7).
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters [Urea Calcium (Ca++), Glucose Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+) Creatinine (Creat), Potassium (K+), Total cholesterol (Chol),Chloride (Cl-), Total bilirubin (Bili), Triglycerides (Tri)] were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

No additional information

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (On completion of the dosing period all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.)
HISTOPATHOLOGY: Yes (All tissues were despatched to the histology processing Test Site Propath UK Ltd. for processing (Principal Investigator: N Fower). Tissues from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 150 and 500 mg/kg bw/day dose group animals.)

Other examinations:

The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight Change, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no clinically observable signs of toxicity.
One male treated with 1000 mg/kg bw/day showed clinical signs of tiptoe gait, staining around the snout, decreased respiratory rate, pilo-erection, lethargy and hunched posture on Day 2. This animal was subsequently terminated on the same day. This death was attributable to inappropriate dosing technique and was considered unrelated to systemic toxicity.
One female treated with 1000 mg/kg bw/day and one female treated with 500 mg/kg bw/day showed increased salivation on either Day 6 or Day 7 only. Observations of this nature are commonly observed following oral administration of an unpalatable test item formulation and in isolation are considered not to be of toxicological significance.
There were no unscheduled deaths related to treatment with the test item.
One male treated with 1000 mg/kg bw/day was killed in extremis following severe clinical signs on Day 2. This death was attributable to inappropriate dosing technique and was considered unrelated to systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN:
There were no adverse effects on body weight change detected for treated animals when compared to controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
There were no adverse effects on dietary intake for treated animals when compared to controls.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Daily visual inspection of water bottles did not reveal any overt intergroup differences in water intake.

HAEMATOLOGY:
There were no toxicologically significant effects detected in the haematological parameters investigated.
Males treated with 1000 and 500 mg/kg bw/day showed a statistically significant (p<0.05) reduction in mean corpuscular volume. All individual values were within normal ranges for rats of the strain and age used, therefore the intergroup differences were considered not to be of toxicological importance.

CLINICAL CHEMISTRY:
There were no toxicologically significant effects detected in the blood chemical parameters investigated.
Females treated with 1000 and 500 mg/kg bw/day showed a statistically significant (p<0.05) increase in inorganic phosphorus. All individual values were within normal ranges for rats of the strain and age used, therefore the intergroup differences were considered not to be of toxicological importance. Females treated with 500 mg/kg bw/day also showed a statistically significant (p<0.05) increase in sodium concentration. All individual values were within normal ranges for rats of the strain and age used and in the absence of a similar effect at 1000 mg/kg bw/day the intergroup difference was considered not to be of toxicological importance.

ORGAN WEIGHTS:
There were no treatment-related changes detected in the organ weights recorded.

GROSS PATHOLOGY:
There were no treatment-related macroscopic abnormalities detected.
The interim death animal showed gaseous distension of the small and large intestines, a fluid filled thoracic cavity and a hole present in the oesophagus. This death was most probably attributable to inappropriate dosing technique. One control female showed the left horn of the uterus in two parts (detached from one another). In the absence of treatment this was considered to be a congenital abnormality.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic abnormalities detected.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Conclusions:

The oral administration of the test substance to rats by gavage, at dose levels of 150, 500 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) was therefore considered to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1 (reliable without restriction)

Additional information

A 7-day repeated dose study was conducted according to OECD 407 using rats (Dunster J S, 2013). Key study.

It is considered that the test substance did not result in any toxicologically significant effects when administered to rats by daily oral gavage for a period of up to 7 consecutive days at 150, 500 and 1000 mg/kg bw/day. The "No Observed Effect Level" (NOEL) was therefore considered to be 1000 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This study was conducted according to OECD 407 using rat.

Justification for classification or non-classification

Repeated dose toxicity: A 7-day repeated dose study on rats showed that the test substance did not result in any toxicologically significant effects when administered to rats by daily oral gavage for a period of up to 7 consecutive days at 150, 500 and 1000 mg/kg bw/day, resulted a NOEL value 1000 mg/kg bw/day.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.9.3 the test substance is not classfied for repeated dose toxicity endpoint.