2-Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-

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:

Oct 1999 to Mar 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

475207-59-1

Cas Number:

475207-59-1

IUPAC Name:

475207-59-1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan/Winkelmann
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 wk
- Weight at study initiation: males: 161 g (136-192 g), females: 133 g (113-156 g)
- Housing: individually in type II Makrolon cages
- Diet (e.g. ad libitum): pelletized food (Altromin 1324), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 50-70%
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: mixture of Pluronic F68 / propylene glycol / PEG 400 (15 : 42.5 : 42.5 w : w : w)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in a mixture of Pluronic F68 / propylene glycol / PEG 400 (15 : 42.5 : 42.5 w : w : w) at a temperature at 70-80°C and then cooled down to approx 37°C. The lower concentrations were diluted each of the next higher concentration at a temperature of 35-40°C.
The formulations and the vehicle solidified at room temperature.
The formulations were stored at room temperature and protected from light up to 8 days.

Dose volume: 5 ml/kg bw
administration at a temperature of approx 37°C

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1.5, 7, 35, 170 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

main and recovery groups: 10
toxicikinetic groups: 8 (controls: 6)

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: daily before administration and on the day of necropsy, once weekly during the recovery period

FOOD CONSUMPTION, WATER CONSUMPTION
- Yes

FOOD EFFICIENCY:
- no

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4 (main groups), week 5 and 8 (recovery groups)
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: leuco (differential: neurophiles, lymphocytes, monocytes, eosinophiles, basophiles, atypical), ery (incl red blood cell morphology), HB, HCT, MCV, MCH, MCHC, reticulocytes, thrombocytes, HQUICK

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4 (main groups), week 5 and 8 (recovery groups)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: ASAT, ALAT, APh, GLDH, LDH, CK, glucose, Cholesterol, triglycerides, creatinine, urea, total bilirubin, protein, Na, K, Ca, Cl, P

URINALYSIS: Yes
- Time schedule for collection of urine: 16 h during day 3/4 and 23/24 + day 29 and 49 in recovery groups
- Animals fasted: Yes
- Parameters examined: volume, density, pH, protein, glucose, blood, bilirubin, keto, ubg, creatinine, NAG, GGT, AAP, LDH, sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Liver enzymes: ECOD, EROD, ALD, EH, GS-T, GLU-T
- at the end of the study

Determination of fluoride in femurs and teeth
- at the end of the study

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
weighed: brain, heart, liver, spleen, kidneys, adrenals, testes, epididymides, thymus

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dose dependent symptoms of bad condition were observed beginning at doses of 7 mg/kg bw. The lowest dose group showed soft feces only. Most of the symptoms persisted during the recovery period.

Mortality:

mortality observed, treatment-related

Description (incidence):

15 animals (12 from the main groups, 3 from the kinetic groups) from the dose group 35 mg/kg bw, and 7 animals from the dose group 170 mg/kg bw died due to the treatment with BAY 54-9085. One animal from the dose group 1.5 mg/kg bw (kinetic group) died for unknown reason. in the dosed recovery groups three animals died or had to be killed in extremis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was lower in the 35 and 170 mg/kg groups, and did not reach the controls especially in males after recovery.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was decreased in the 35 and 170 mg/kg groups, and increased during the recovery period.

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

effects observed, treatment-related

Description (incidence and severity):

Water intake was decreased in the 35 and 170 mg/kg groups, and increased during the recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hb, erythrocytes, and the dependent parameters were decreased beginning at dose Ievels of 35 mg/kg bw without recovery. Reticulocytes were decreased at 7 mg/kg bw, and increased at 35 mg/kg bw and higher. No recovery effects were found. The number of thrombocytes decreased at 35 mg/kg bw and higher. This parameter was similar to controls after the recovery period.Additionally a deviation to the left in the white blood cell differential were found beginning at 35 mg/kg bw. No recovery effects were seen for this finding.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the clinico-chemical investigations a Iot of parameters were changed: increase of ASAT, ALAT (beginning at 1.5 mg), increase of Alkaline Phosphatase (beginning at 7 mg/kg, with decrease at 35 and 170 mg/kg), increase of GLDH and LDH at 35 and 170 mg/kg, increase of cholesterol and bilirubine beginning at 7 mg/kg bw, decrease of protein, calcium (beginning at 35mg/kg), and of phosphate beginning at 7 mg/kg bw. With the exception of cholesterol most of the a.m. parameters were changed to normal after the recovery period. The investigations of enzymes in the liver tissue showed no signs of induction, but decreasing effects.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urinalyses showed a Iot of changes also. Mostly the 35 and 170 mg/kg groups were affected. The recovery groups did not return to normal values in all parameters.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Reduced absolute organ weights were noted for brain, adrenals, heart, liver , spleen, thymus, kidneys, testes, and epididymides. Reduced relative organ weights were noted for brain, adrenals, heart, liver, thymus, kidneys, testes, and epididymides.
Most of the organ weights showed differences to the controls beginning at dose Ievels of 7 mg/kg bw. in the recovery groups these changes were still measurable.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The necropsy of the main groups revealed findings in the animals of the dose groups 35 and 170 mg/kg, mainly related to their poor general condition (e.g. retardation in size, skinny appearance). Treatment related gross lesions were found in the digestive tract, the kidneys, and the adrenal glands.

After the 4 week recovery period signs of retardation were still present in males. Additionally, both males and females showed findings in the teeth which were not observed in animals of the main groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The histopathological investigations showed at 35 and 170 mg/kg bw beside signs due to poor general condition a Iot of degenerative changes in the adrenal glands, liver, stomach, duodenum, pancreas, kidneys, heart, testes, and ovaries, regenerative processes in the liver (bile duct proliferation), pancreas duodenum, and kidneys, cytotoxic effects in bone marrow, spleen, lymph nodes, thymus, and tongue. Rare findings were observed in the teeth beginning at 1.5 mg/kg and femoral bone beginning at 7 mg/kg.
Most of the findings could be shown to be reversible. However, after 4 weeks of recovery, bile duct proliferation in the liver as well as basophilic tubules and tubular dilation in the kidneys were observed with almost the same severity when compared to the 170 mg/kg group of the main study. Nearly all treated animals of the recovery groups showed still a prominent decrease in numbers of mast cells in tongue. Signs of retardation were also still present in the 170 mg/kg recovery group.

The degenerative processes on incisors were not reversible within 4 weeks but even more pronounced. Furthermore, osteolytic and/or osteodystrophic processes in the jawbone became evident in the 170 mg/kg recovery group.
Thickening of the growth plate of the femoral bone was also still present in some treated animals of the recovery group. Additionally, increased bone formation beneath the growth plate as weil as bone malformation or epiphysiolysis were observed in some 170 mg/kg treated animals.
Summarizing the above, 35 or 170 mg/kg BAY 54-9085 are not tolerated and end in increased mortality. Treatment related findings were also seen in the 7 mg/kg dose group. However, due to the findings in the teeth and in the bone marrow of some males dose group 1.5 mg/kg is also affected.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

ln ashes from femurs and teeth of treated males, a statistically highly significant, 30 - 35 % increase of fluoride concentrations was observed compared to corresponding controls. Whilst in ashes from femurs of control and treated females comparable fluoride concentrations were measured, in ashes from teeth of treated females a mild increase of fluoride concentrations compared to controls was observed. Taking into consideration that femurs and teeth had been taken from recovery groups, the initial effect immediately at the end of the treatment period may have been even more pronounced.

Effect levels

1

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable

Remarks:

no NOAEL identified

2

Dose descriptor:

LOAEL

Effect level:

1.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

1.5 mg/kg bw/day (actual dose received)

System:

musculoskeletal system

Organ:

bone marrow

tooth

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Toxicokinetic part:

 

Toxicokinetics showed no significant differences in exposure between male and female rats. However, females appear to have slightly higher plasma concentrations and AUCs than males in most cases. There is a moderate increase (approx. 60- 70%) in exposure from Day 1 to the last day for the 1.5 and 7 mg/kg doses. There was no increase for the 35 mg/kg and a decrease in exposure (approx. 40%) for the 170 mg/kg dose Ievel.

Approximately dose proportional increases in exposure were observed in the 1.5 - 7 mg/kg and 7 - 35 mg/kg dose ranges (approximately 6 fold in each range). Between the 35 and 170 mg/kg dose there is only a marginal increase in AUC for Day 1. The exposure on Day 21 was smaller for 170 than for 35 mg/kg. This may be due to reduced absorption related to the poor solubility of the drug.

Applicant's summary and conclusion

Conclusions:

BAY 54-9085 doses of 35 or 170 mg/kg were not tolerated and resulted in increased mortality. Treatment related findings were also seen in the 7 mg/kg group and due to findings in teeth and bone of some males, the dose 1.5 mg/kg was also affected. A NOAEL was not established.

Executive summary:

Groups of 10 male and 10 female Wistar rats were treated orally by gavage for 4 weeks according to EU Method B.7 at the following doses of BAY 54-9085: 0, 1.5, 7, 35, or 170 mg/kg. In addition, 10 male and 10 females were treated analogously with the test item at 0 and 170 mg/kg for 4 weeks and were observed for a subsequent 4-week treatment-free period for reversibility, continuation or delayed occurrence of possible toxic effects. Further animals were used for toxicokinetic assessment. Study parameters included: clinical signs, body weight, food consumption, water consumption, clinical chemistry, hematology, urinalysis, gross pathology, organ weights, liver enzyme analysis, histopathology, and toxicokinetics.

 

Mortalities prior to the end of 4 weeks treatment in the main group included 3 males (including 2 animals that died during blood sampling) and 9 females at 35 mg/kg, and 3 males and 1 female at 170 mg/kg. In the kinetics group, 2 males and 1 female at 35 mg/kg and in the recovery group 1 female at 170 mg/kg died. One mortality, considered incidental, occurred in the toxicokinetic group treated at 1.5 mg/kg. Dose-dependent clinical signs of toxicity were noted at 7 mg/kg and above. The lowest dose group showed soft feces, only. Most of the symptoms persisted during the recovery period.

 

Food and water intake were decreased in the 25 and 125 mg/kg groups and increased during the recovery period. Body weight gain was lower in these groups and did not reach the controls, especially in males, after recovery.

 

In hematological investigations carried out in week 4, hemoglobin, erythrocytes and the dependant parameters were decreased, beginning at the dose level of 25 mg/kg. Reticulocytes were decreased at 5 mg/kg, but increased at 25 mg/kg and higher. No recovery effects were found. Thrombocytes decreased at 25 mg/kg and higher and this parameter was similar after the recovery period. In the white blood cell differential counts, more immature cell types were found beginning at 25 mg/kg and no recovery effects were seen for this finding.

 

In the clinico-chemical investigations, increases were seen in AST, ALT (beginning at 1.5 mg/kg), GLDH and LDH (at 35 and 170 mg/kg), cholesterol and bilirubin (beginning at 7 mg/kg). An increase was seen in alkaline phosphatase (ALP) at 7 mg/kg with decreases at 35 and 170 mg/kg. Decreases were also seen in protein, calcium (beginning at 35 mg/kg) and phosphate (beginning at 7 mg/kg). In the highest dosing group, all abnormalities recovered completely except GLDH (in the males) and cholesterol, which were only partially recoveredafter 4 weeks post dosing. In urine, increases were seen in protein, urine creatinine (in females only), NAG, and LDH, particularly at 35 and 170 mg/kg. Increases in GGT and ALP were observed in females and decreases in males. Of note, the serum creatinine was not statistically significantly increased, even at the highest doses. The test compound revealed slight decreasing effects on liver enzyme activities, which are more pronounced in the male rats. Partial recovery of protein, NAG and a near full recovery of the LDH were noted 4 weeks after discontinuation of test article.

 

Necropsy revealed findings in the 35 and 170 mg/kg groups, which were mainly related to the poor general condition of the animals (e.g. retardation in size, skinny appearance). Treatment related gross lesions were found in the digestive tract, kidney and adrenal glands. Some organ weight changes were observed, of note were increased weights of the adrenals.

 

Histopathological evaluation revealed treatment-related findings in the majority of animals treated at 35 or 170 mg/kg. The spectrum of histopathological findings was narrower for the 7 mg/kg and the 1.5 mg/kg doses. Overall, the changes were classified as degenerative in the adrenal glands, liver, stomach, duodenum, pancreas, kidneys, heart, and ovaries. Regenerative changes were observed in the liver (bile duct proliferation), pancreas, duodenum and kidneys. Necrosis was observed in the spleen, lymph nodes, and thymus. Hypocellularity was seen in the bone marrow and tongue. Effects in male reproductive organs included retardation in testes, epididymides, prostate and seminal vesicles. Unusual findings were noted in the teeth (alteration of the dentin composition) and proximal growth plate of the femoral bone (irregular thickening, hypocellularity of the bone marrow).

 

Most of the findings could be shown to be reversible. However, after 4 weeks of recovery, bile duct proliferation in the liver as well as basophilic tubules and tubular dilation in the kidneys were observed with almost the same severity when compared to the 170 mg/kg group of the main study. Nearly all treated animals of the recovery groups still showed a prominent decrease in number of mast cells in the tongue. The degenerative processes on incisors were not reversible within 4 weeks but even more pronounced. Furthermore, osteolytic and/or osteodystrophic processes became evident in the 170 mg/kg recovery group. Thickening of the growth plate of the femoral bone was also present in some treated animals of the recovery group. Additionally, increased bone formation beneath the growth plate and bone malformation or epiphysiolysis was observed in some 170 mg/kg animals. In ash from femurs and teeth of treated males, a statistically highly significant (30-35%) increase of fluoride concentrations was observed in comparison to the corresponding controls.

 

In summary, BAY 54-9085 doses of 35 or 170 mg/kg were not tolerated and resulted in increased mortality. Treatment related findings were also seen in the 7 mg/kg group and due to findings in teeth and bone of some males, the dose 1.5 mg/kg was also affected. A NOAEL was not established.