Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 October 2003 - 12 February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-1H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo-, tetradecyl ester
- HAEL Number: 2003-0090
- KAN: 632021-1
- CIN: 10084241
- SRID or Lot No.: DEV84241
- Physical state and appearance: brown powder
- Storage conditions of test material: at room temperature
- Purity determination: 96.5 weight percent
- Stability: stable
Specific details on test material used for the study:
- Stability under test conditions: The purity of the test substance was determined by HPLC with UV detection to be 96.5 % prior to use and 96.0 % at study termination. Based on these results, the test substance was considered to be stable during the test period.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-8 weeks of age
- Weight at study initiation: Males: 237.45 - 268.97g; Females: 178.33 - 205.59 g
- Housing: Animal Care International-accredited vivarium in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The rats were singly housed in suspended, stainless-steel mesh cages. No other study was housed in the same room as this study. Cages and racks were washed once a week. Absorbent paper, used to collect excreta, was changed daily.
- Diet: Certified Rodent Diet, meal, was available ad libitum.
- Water: Water was available ad libitum through an automatic watering system. The source of the water was the local public water system.
- Acclimation period: The animals were isolated upon arrival and allowed to acclimate for a period of five days prior to assignment to this study. Animals were judged to be healthy prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature: The study room was maintained at 21.1 - 23.9 °C except for excursions in room temperature up to 25.4 on Day 7. Since these excursions persisted for less than two hours, they were not outside protocol allowances and did not constitute a protocol deviation.
- Humidity: 38.3 - 59.8 %
- Photoperiod: A photoperiod of 12 hours of light from 6 a.m. to 6 p.m. was maintained.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
(Crl:CD(SD)IGS BR)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Test substance mixtures were prepared once and used within 34 days based on the stability of the mixture.
- Mixing appropriate amounts with (Type of food): The test substance was mixed with ground certified chow to yield concentrations of 0, 1.5, 4.5 and 15 mg/g.
- Storage temperature of food: The feed samples (control and test substance) were stored at room temperature throughout the duration of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance in test diets was determined by repeated analysis of 1.5 and 15.0 mg/g mixtures of the test substance. Mixtures were analyzed using HPLC/UV on 0, 10, 17, 24 and 34 days after test mixture preparation. Concentrations of the test substance were 1.53 ± 0.030, and 15.2 ± 0.63 mg/g (mean ± SD) prior to storage, and were 1.49 ± 0.006 and 14.8 ± 0.20 mg/g after thirty-four days of storage, indicating the test substance was stable for at least thirty-four days in a ground chow mixture.

Homogeneity of the test substance in test diets was evaluated by measuring the concentration of the test substance at three levels (top, middle, and bottom of the mixing container) of 1.5, 4.5 and 15.0 mg/g mixtures of the test substance. The analytical concentrations of the test substance in the top, middle, and bottom layers for the 1.5 mg/g mixture varied between 1.254 and 1.562 mg/g, with a standard deviation of 0.105, for the 4.5 mg/g mixture varied between 4.458 and 4.774 mg/g, with a standard deviation of 0.099, and for the 15.0 mg/g mixture varied between 14.75 and 16.15 mg/g, with a standard deviation of 0.45. Based on these results, the preparations were considered homogeneous.

The concentration of the test substance in or feed was determined prior to use by HPLC/UV analysis. The mean concentrations of the test substance were 92.7, 103 and 103 % of the target concentrations of 1.5, 4.5 and 15.0 mg/g, respectively.
Duration of treatment / exposure:
Rats were given feed containing 0, 1.5, 4.5 or 15.0 mg of the test substance per gram of feed for 28 (male) or 29 (female) consecutive days.
Frequency of treatment:
Continuous in the feed
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 other: mg/g
Dose / conc.:
4.5 other: mg/g
Dose / conc.:
15 other: mg/g
No. of animals per sex per dose:
Five male and five female rats were assigned to each exposure group.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: A probe study was conducted using female rats. Animals were dosed for 7 consecutive days and observed daily for signs of toxicity. Body weights were measured on Days 0, 2, 5 and 7. Based on the results, the following dose levels were selected for the four-week study: 0, 1.5, 4.5 and 15.0 mg test substance per gram of feed.
- Rationale for animal assignment: Randomization - The test animals were culled from the stock population based on body weight and randomly assigned to groups using a stratified randomization program. The body weights of individual animals in the culled population did not vary more than 20 % from the mean for each sex.
Positive control:
Positive control studies previously conducted in the testing laboratory have demonstrated:
a) the ability of observational methods to detect major neurotoxic endpoints;
b) the sensitivity and reliability of activity measuring devices and testing procedures to detect increases and decreases in motor activity;
c) the ability to detect histopathologic changes in tissues from the central nervous system and peripheral nervous system.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cageside observations were conducted in the afternoon. Animals were observed for moribundity/mortality each weekday afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical (hands-on) examinations were conducted every morning, except for days on which the functional observational battery was conducted. Clinical observations included, but were not limited to, examination of the hair, skin, eyes, mucous membranes, motor activity, feces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behavior patterns.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured on Days -5, 0, 3, 7, 14, 21 and 28. Terminal body weights were measured after exsanguination, but prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Feed consumption was determined on Days 3, 7, 14, 21 and 28. Animals were fasted the day prior to necropsy.

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The day following fasting, animals were anesthetized with carbon dioxide and blood was collected from the posterior vena cava.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Hematology tests included: hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, red blood cell indices, prothrombin time, and platelet count. Slides containing blood smears were examined for were examined for differential white blood cell count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The day following fasting, animals were anesthetized with carbon dioxide and blood was collected from the posterior vena cava.
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Clinical chemistry tests included: alanine aminotransferase, sorbitol dehydrogenase, creatinine, urea nitrogen, glucose, total bilirubin, total protein, albumin, albumidglobulin ratio, total cholesterol, triglycerides, calcium, phosphorus, sodium, and potassium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Detailed functional observations of animals were conducted prior to study start, and on Days 8, 15, 22 and 27. If possible, these observations were made at approximately the same time each day. The FOB observations followed an acclimation to the home cage of at least 30 minutes after motor activity measurements. The observations were rated with a score of 1 indicating typical behavior and scores of 2-4 indicating levels of behavior different from the typical pattern. Descriptive categories of abnormal behavior were also noted.
- Dose groups that were examined: All
- Battery of functions tested: The animals were observed for: Severity and degree of lacrimation, salivation, or discharges; Piloerection and hair coat; Pupillary size; Exophthalmus; Mucous Membrane/Skin Color; Unusual respiration; Feces (amount and consistency); Urine (amount and color); Description of body position, coordination of movement, and gait abnormalities; Ranking of reactivity; Arousal level/state of alertness; Description, incidence, and severity of convulsions and tremors; Stereotypy and bizarre behavior

Additionally, prior to study start and on Day 27, the animals were observed for: Sensory function (vision and audition); Proprioceptive reflex; Forelimb and hindlimb grip strength

OTHER:
- Motor Activity Determination. Motor activity was measured on Day 27. Motor activity was monitored sequentially in two replicates because of the number of motor activity measurement systems available. All exposure groups were evaluated concurrently. Motor activity was measured in 10 minute intervals for a total of 60 minutes using an automated cage rack photobeam activity system (San Diego Instruments, San Diego, CA). To reduce variability during data collection, the animals were randomly placed into the motor activity units within 5 minutes of each other. Motor activity was measured in an isolated room within the vivarium. No entry to the room was allowed during the measurement period. Motor activity data were collected from the automated cage rack photobeam activity system (PAS) using a Compaq 386SX computer. The motor activity system is composed of individual animal enclosures equipped with three pairs of infrared beams and sensors per frame. Each frame is placed over a clear plastic animal cage; as the animal moves about the cage, the infrared beams are broken. The number of breaks of each beam in the frame is transmitted to a digital input/output interface and is then stored in files on the hard disc of the Compaq computer. The system distinguishes and records two types of horizontal movement: 1) simple motor activity (single beam break) and 2) ambulation (multiple beam breaks over the 60 minute time period). During this study, motor activity (single beam breaks) was compiled every ten minutes for one hour. The total number of ambulations and total motor activity were calculated for the entire one-hour period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Following exsanguination, the animals were weighed and necropsied. The following tissues were fixed in 10 % neutral buffered formalin: trachea, lungs, heart, stomach, duodenum, jejunum, ileum, cecum, colon, liver, salivary glands, kidneys, urinary bladder, adrenal glands, thyroid glands, thymus, spleen, sternum (with bone marrow), mesenteric lymph nodes, cervical lymph nodes, brain (including sections of medulldpons, cerebellar cortex, and cerebral cortex), sciatic nerve, cervical spinal cord, testes, epididymides, coagulating gland, prostate gland, seminal vesicles, ovaries, vagina, uterus, Fallopian tubes, and gross lesions.

- Organ Weights: The liver, kidneys, adrenal glands, spleen, thymus, heart, brain, testes, and epididymides were weighed. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
For the control and high-dose groups, all tissues were embedded in paraffin and sectioned at 5 µm. The sternum was decalcified prior to being embedded and sectioned. The lungs were sectioned along a plane to allow visual examination of the major bronchi and bronchioles. The resulting tissue sections were stained with hematoxylin and eosin (H&E) stains and examined for histopathology.
Statistics:
Mean values were calculated for body weight, body weight change, feed consumption, total motor activity, total ambulations, grip strength, FOB behavior scores, serum chemistries, hematology values, organ weights, and organ-to-body weight ratios. Body weight, body weight change, feed consumption, total motor activity, total ambulations, grip strength, FOB behavior scores, clinical pathology (where possible), and organ weight data were evaluated using Bartlett’s
test (p ≤ 0.01), one-way analysis of variance (ANOVA) (p ≤ 0.05), and Dunnett’s t-test (p ≤ 0.05) to indicate statistical significance (MINITAB Statistical Software, State College, PA).
If significant differences in total motor activity or total ambulations were seen, then the values for each ten minute interval were evaluated using Bartlett’s test (p ≤ 0.01), one-way analysis of variance (ANOVA) (p ≤ 0.05), and Dunnett’s t-test (p ≤ 0.05) to indicate statistical significance (MINITAB Statistical Software, State College, PA). When the variances of the means were not considered equal by the Bartlett’s test (p ≤ 0.01), the data were evaluated using a Kruskal-Wallis
H-test (p ≤ 0.05) followed by Mann-Whitney U-test (p ≤ 0.05) (MINITAB Statistical Software, State College, PA). Categorical FOB scores were evaluated using a binary logistic regression (p ≤ 0.05) or a nominal logistic regression (p ≤ 0.05) followed by two proportions test (p ≤ 0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related clinical abnormalities were observed for any dose group during the study.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences were noted in mean body weights or mean body weight gains among groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistically significant differences were noted in feed consumption for any group. Mean feed consumption data are presented in graphs 1 and 2 attached.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No significant differences were noted for hematology values for any group when compared to the respective control.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Among clinical chemistry parameters, a decrease in the mean albumin/globulin ratio value was observed for female rats in the 1.5 mg/g dose group. Since there was no dose-response, and these values were within the historical control range for the laboratory, this change was not considered to be biologically significant. No other changes in clinical chemistry were noted between any treated group and the respective control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The FOB data for this study were very consistent, and only small variances were noted in the results. Statistically (p ≤ 0.05) lower scores were noted for constricted pupil size on Day 22 for the 15.0, 4.5 and 1.5 mg/g male groups. This was because constricted pupil size on Day 22 for the male control group was abnormally high. No significant differences were noted in mean motor activity and mean total ambulations for any group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences were observed for the mean absolute spleen weights, which were heavier for the 1.5 and 15.0 mg/g female dose groups when compared to the female control rats. Statistically significant differences in relative (to body weight) organ weights were limited to an increased mean relative spleen weight for the 1.5 and 15.0 mg/g female groups. All of the mean absolute and relative organ weight values for these animals were within the historical control range for the laboratory. No significant increases in absolute or relative spleen weights were observed for the 4.5 mg/g female rats. No other statistically significant differences were observed for mean absolute or relative organ weight values.


Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross lesions observed at necropsy were limited to minimal to minor thymic hemorrhage; this was noted for one male rat per treatment group and a single female rat in the 15.0 mg/g group. A single 4.5 mg/g male rat had a raised area on the spleen. Minor to moderate red discoloration of the lungs was observed for two male rats from the 15.0 mg/g and one female rat from the 1.5 mg/g dose groups. Minimal to minor uterine hydrometra was observed for one rat each in the 0.0 mg/g and 1.5 mg/g groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Foci of myocarditis (minimal) were noted in a single 15.0 mg/g male rat. Hepatocellular degeneration (minimal) was observed for all 15.0 mg/g female rats and three 0.0 mg/g female rats. Minimal hepatocellular degeneration is quite common, as a background lesion, in this strain of rat. One 15.0 mg/g female rat and two 0.0 mg/g male rats had a focus of minimal nerve fiber degeneration in the sciatic nerve. Minimal interstitial pneumonitis was observed more frequently for the 15.0 mg/g female rats than the control rats. Interstitial pneumonitis was observed more frequently for control male rats than for animals in the 15.0 mg/g male group. The microscopic lesions noted above represent common findings in control rats and are not considered to be related to exposure to the test substance.
Histopathological findings: neoplastic:
not examined
Details on results:
Based on the amounts of feed consumed, these dietary concentrations produced dose levels of approximately 1176, 346, 117 and 0 mg/kg body weight/day for male rats, and 1247, 367, 128 and 0 mg/kg body weight/day for female rats.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 176 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no significant effects seen at the highest dose level
Key result
Dose descriptor:
NOAEL
Effect level:
1 247 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no significant effects seen at the highest dose level

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the no-observed-adverse effect-level (NOAEL) for the test substance was determined to be 1176 mg/kg body weight/day for male rats and 1247 mg/kg body weight/day for female rats when administered in the feed for 28 (male) or 29 (female) consecutive days.
Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 407 and EU Method B.7, the 4 week repeated dose toxicity of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester was investigated.

Groups of five male and five female Sprague-Dawley rats were fed diets containing 15.0 mg/g (Group 4), 4.5 mg/g (Group 3), 1.5 mg/g (Group 2), or 0.0 mg/g (Group 1) of the test substance in feed for 28 (male) or 29 (female) consecutive days. Animals were observed daily for clinical signs of toxicity. A functional observational battery (FOB) was performed prior to treatment and weekly thereafter to assess changes in neurobehavior. Motor activity was measured on Day 27. Body weights and feed consumption were measured weekly throughout the study. Based on the amounts of feed consumed, these dietary concentrations produced dose levels of approximately 1176, 346, 117 and 0 mg/kg body weight/day for male rats, and 1247, 367, 128 and 0 mg/kg body weight/day for female rats.

All animals survived to study termination. No test substance-related clinical abnormalities were noted during the observation period. During the FOB assessment, significantly (p ≤ 0.05) lower scores were noted for constricted pupil size on Day 22 for the 15.0, 4.5 and 1.5 mg/g male groups. This was because constricted pupil size on Day 22 for the male control group was abnormally high. No toxicologically significant changes were observed, No significant differences were noted in mean motor activity and mean total ambulations for any group. No significant differences were noted in mean body weights, mean body weight gains, or mean feed consumption values in any group.

At study termination, animals were anesthetized with carbon dioxide and blood was obtained from the posterior vena cava for hematology and clinical chemistry analyses. Fasted body weight and selected organ weights were measured at necropsy. Selected tissues were collected from all animals. All tissues collected from the 15.0 and 0.0 mg/g dose groups, and gross lesions from all dose groups, were examined microscopically. No significant differences were noted for hematology values in any group when compared to the respective control. For clinical chemistry parameters, decreases in mean albumin/globulin ratio values were observed for female rats in the 1.5 mg/g dose group, but were not considered to be biologically significant since no dose-response was observed. Changes in organ weights were limited to heavier (p ≤ 0.05) mean absolute and relative (to body weight) spleen weights for the 1.5 mg/g and 15.0 mg/g female dose groups when compared with the female control group. These differences did not fit a dose-response pattern. No test substance-related gross or microscopic lesions were noted for any group.

Based on the results of this study, the no-observed-adverse effect-level (NOAEL) for the test substance was considered to be 1176 mg/kg body weight/day for male rats and 1247 mg/kg body weight/day for female rats when administered in the feed for 28 (male) or 29 (female) consecutive days.