2-Pyridinamine, 5-bromo-3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 to 12 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-bromo-3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-2-amine

EC Number:

700-798-7

Cas Number:

877399-00-3

Molecular formula:

C13H10BrCl2FN2O

IUPAC Name:

5-bromo-3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-2-amine

Details on test material:

- Purity: 99.7% area (HPLC)
- Batch No.: E010010557

Test animals

Species:

rat

Strain:

other: Crl:Wl(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10 weeks.
- Weight at study initiation: all animals within ± 20% of the sex mean.
- Fasting period before study:
- Housing: Group housing of 3 animals per sex in Macrolon cages with sterilized sawdust as bedding material and paper as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0ºC (actual range: 20.1 – 22.2ºC)
- Humidity (%): 40-70% (actual range: 43 - 81%)
- Air changes (per hr): Approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 2010-10-04 To: 2010-10-12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information from the sponsor.
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg body weight.
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
150, 300 and 1000 mg/kg
Basis:
nominal in diet

No. of animals per sex per dose:

3 males and 3 females per dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Mortality/Viability: At least twice daily.

Clinical signs: At least once daily, detailed clinical observations were made in all animals. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

Body weights: On Days 1, 4 and 7.

Food consumption: Over Days 1-4 and 4-7.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical laboratory investigations:
Blood samples were collected under anaesthesia using an isoflurane in nitrous oxide/oxygen combination immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. Animals were deprived of food overnight (for a maximum of 20 hours), but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.

Sacrifice and pathology:

Necropsy:
Animals surviving to the end of the observation period were deeply anaesthetized using an isoflurane in nitrous oxide/oxygen combination and subsequently exsanguinated and subjected to a full post mortem examination. Samples of the tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin.

Histotechnology:
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

Histopathology:
The following slides were examined by a pathologist: all tissues collected at the scheduled sacrifice from all group 1 and 4 animals; all tissues from animal No 20 which died spontaneously; all gross lesions.
Based on treatment-related changes in the thymus, liver and kidneys the histological examination was extended to those particular organs of all animals of groups 2 and 3 (males and females).
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Other examinations:

Organ Weights:
The organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy.

Statistics:

None stated

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Observations

Mortality:
One female at 300 mg/kg was found dead on Day 4 prior to dosing. No definite cause of death could be established for this rat histopathologically. The other animals in this dose group and all animals at 1000 mg/kg survived the scheduled study duration. Therefore this death was considered not to be related to treatment with the test substance.
No mortality occurred among the control animals and animals at 150 and 1000 mg/kg.

Clinical Signs:
Hunched posture and/or piloerection were recorded for all males and females at 1000 mg/kg from Day 4 and 3 onwards, respectively. One of these males showed lethargy on Day 7. Hunched posture was also shown by all surviving females at 300 mg/kg from Day 3 onwards.
The female at 300 mg/kg which died on Day 4 showed hunched posture, quick breathing and piloerection on the preceding day.
No clinical signs were shown by males at 150 and 300 mg/kg and in females at 150 mg/kg.

Body Weights:
All animals at 1000 mg/kg showed body weight loss between Days 1 and 4 (up to 9% from Day 1 values). Except for one male and female, body weights of these animals essentially recovered to control levels between Days 4 and 7.

Food Consumption:
Food consumption before or after correction for body weight was lower for males and females at 1000 mg/kg throughout the treatment period when compared to control animals. Females at 300 mg/kg also showed a lower food consumption before or after correction for body weight between Days 1 and 4, which essentially recovered to control levels between Days 4 and 7.
At 150 mg/kg, food consumption before or after correction for body weight was considered to have been unaffected by treatment.

Clinical Laboratory Investigations

Haematology:
The following changes in haematology parameters distinguished treated animals from control animals:
− Lower red blood cell counts in males at 1000 mg/kg,
− Lower haemoglobin levels in males at 1000 mg/kg,
− Lower reticulocyte counts in females at 1000 mg/kg,
− Higher platelet counts in males at 300 and 1000 mg/kg, and in females at 1000 mg/kg,
− Higher prothrombin time in males at 300 and 1000 mg/kg, and in females at 1000 mg/kg,
− Lower partial thromboplastin time in males and females at 1000 mg/kg.
All other changes remained within the range considered normal for rats of this age and strain.

Clinical Biochemistry:
The following changes in clinical biochemistry parameters distinguished treated animals from control animals:
− Higher alkaline phosphatase activity (ALP) in females at 1000 mg/kg,
− Lower total protein levels in females at 1000 mg/kg,
− Lower albumin levels in females at 1000 mg/kg,
− Higher total bilirubin levels in females at 150, 300 and 1000 mg/kg,
− Lower urea levels in females at 1000 mg/kg,
− Higher creatinine level in males at 1000 mg/kg,
− Lower glucose levels in males at 1000 mg/kg,
− Lower cholesterol levels in males at 300 and 1000 mg/kg,
− Lower triglyceride levels in males at 300 and 1000 mg/kg, and in females at 1000 mg/kg,
− Higher chloride levels in males and females at 1000 mg/kg (with a dose-related trend at lower dosages towards an increase),
− Higher potassium levels in females at 1000 mg/kg,
− Lower calcium levels in males at 1000 mg/kg.
One male at 1000 mg/kg (no. 12) showed a lower total protein and albumin level, and higher bilirubin and urea level. Values of other males of this dose group were considered to be normal.

Pathology

Macroscopic Examination:
Macroscopic observations at necropsy that were considered to be related to treatment included:
− Enlarged liver two males each at 300 and 1000 mg/kg, and in two females at 1000 mg/kg,
− Reduced size of the thymus in two males and one female at 1000 mg/kg,
− Purple contents in the gastro-intestinal tract or ileum (with purple discolouration of the ileum wall) each in one male at 1000 mg/kg,
− Enlarged kidneys in two males at 1000 mg/kg,
− (Black-) brown discolouration of the (cortex of) kidneys in all males at 1000 mg/kg,
− Black-brown contents in the urinary bladder of two males at 1000 mg/kg,
− Black discolouration of the skin of the tail in two males at 1000 mg/kg.
The enlarged liver seen in one male and one female at 150 mg/kg had no microscopic correlate.

Organ Weights:
The following changes in mean organ weights and organ to body weight ratios were considered to be related to treatment:
− Higher liver weight and liver to body weight ratio in males and females at 150, 300 and 1000 mg/kg (with a dose-related trend; liver to body weight ratio 104% and 89% higher than controls for males and females at 1000 mg/kg respectively),
− Lower thymus weight and thymus to body weight ratio in males at 1000 mg/kg,
− Higher kidney weight and kidney to body weight ratio in males at 1000 mg/kg.
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Microscopic Examination:
Treatment-related microscopic findings were present in the kidneys, liver and thymus of both sexes.
Kidneys:
− tubular dilatation was present in both male and female Group 2 (male: 3/3 minimal; female: 2/3 minimal), Group 3 (male: 1/3 minimal, 2/3 slight; female: 2/3 slight) and Group 4 (male: 2/3 slight, 1/3 moderate; female: 3/3 minimal) rats. This was the microscopic correlate to the macroscopic enlarged kidneys.
− Tubular degeneration was present in male and female rats at increasing incidence and severity in Group 3 (male: 1/3 slight; female: 1/3 minimal) and Group 4 (male: 3/3 moderate; female: 1/3 minimal, 2/3 moderate) rats.
− Hyaline droplets were present in male rats at increased incidence and severity in Group 2 (1/3 minimal, 2/3 slight) and Group 3 (1/3 slight, 2/3 moderate) compared to Group 1 (2/3 minimal).
− Tubular basophilia, mainly in the cortico-medullary area, was present at increased severity in male and female Group 4 rats (male: 2/3 moderate, 1/3 marked; female: 3/3 moderate).
− Tubular vacuolation was present in female Group 2 (3/3 slight) and Group 3 (1/3 minimal, 1/3 slight) rats.
− Attenuated tubular epithelium was present at slight degree in 1/3 male and at minimal degree in 1/3 female Group 4 rat.
Liver:
− hepatocellular hypertrophy was present in both male and female Group 2 (male: 2/3 minimal; female 1/3 minimal), Group 3 (male: 3/3 minimal; female: 2/3 minimal) and Group 4 (male: 1/3 minimal, 1/3 slight, 1/3 moderate; female: 2/3 slight, 1/3 moderate) rats. This was the microscopic correlate to the macroscopic enlargement. The anatomic distribution of enlarged hepatocytes (hypertrophy) ranged from centrilobular to panacinar (involving the entire liver lobule).
Thymus:
− lymphoid atrophy was present in 2/3 male (1 minimal; 1 moderate) and 1/3 female (1/3 slight) Group 4 rats. This was the microscopic correlate to the macroscopic reduction in size.
− Lymphocytolysis was present in male and female Group 2 (male: 1/3 minimal; female: 2/3 minimal) and Group 3 (male: 2/3 minimal; female: 1/3 minimal) and in male Group 4 (1/3 minimal, 1/3 slight) rats.
All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

< 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The test substance has toxic potential when administered to rats by daily oral gavage for a period of up to 7 consecutive days at 150 mg/kg.

2

Dose descriptor:

LOAEL

Effect level:

< 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The test substance has toxic potential when administered to rats by daily oral gavage for a period of up to 7 consecutive days at 150 mg/kg.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It is considered that the test substance has toxic potential when administered to rats by daily oral gavage for a period of up to 7 consecutive days at 150 mg/kg.