Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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:
other: U.S. EPA Health Effects Test Guidelines OPPTS 870.3050
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: not reported as such

Test animals

Species:
mouse
Strain:
other: Crl:CD1(ICR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 49 days of age
- Mean body weight at study initiation: males ranged from 29.81 - 30.33 g; females ranged from 24.0 - 24.4 g
- Housing: Male animals were housed individually in solid-bottom caging with bedding with Shepherd's™ Cob + PLUS™ as enrichment, while female animals were housed 2 per cage (when possible). Each cage rack contained only animals of one sex.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Mice were quarantined for 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle (fluorescent light)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionised water
Details on oral exposure:
The test substance was suspended in deionised water. Dose suspensions were prepared with a correction for purity. Dosing formulations of the test substance were prepared within the established range of stability (conducted concurrently with this study) and stored at room temperature until used.
Animals were dosed daily at approximately the same time (± 2 hours) by intragastric intubation at a dose volume of 10 mL/kg body weight for at least 28 days. The amount of test substance each mouse received was based on the most recently collected body weight and the preparation concentration. Control animals were dosed with deionised water at the same volume of 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of all dose formulations were collected near the beginning of the study to verify mixing uniformity and concentration (average of uniformity). Near the end of the study, samples of all dose formulations were taken to verify concentration. Room temperature stability to cover the dose concentration range was established. Formulation samples for concentration verification and homogeneity and stability were stored room temperature until analysis was conducted. Samples were initially diluted water or further diluted with the diluted control. The concentrations of two components in diluted samples were chosen for quantification and measured by high performance liquid chromatography with mass spectrometry detection (LC/MS).
Results: The test substance was homogeneously mixed, at the targeted concentrations, and stable under the storage conditions for the study. Test substance was detected but not quantifiable in the 0 mg/mL control sample; however, there was no impact on the study.
Duration of treatment / exposure:
The test substance was administered for a minimum of 28 consecutive days. Following the dosing period, 10 mice/sex/group (Main Study) were euthanized; the remaining 5 mice/sex/group (Recovery Animals, when possible) were euthanized following a 4-week non-dosing (recovery) period.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Suspensions were adjusted for purity and density.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Suspensions were adjusted for purity and density.
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Suspensions were adjusted for purity and density.
No. of animals per sex per dose:
Main Study: 0 (vehicle control), 30, 100, 300 mg/kg groups: 10 mice/sex/group
One Month Recovery: 0 (vehicle control), 30, 100, 300 mg/kg groups: 5 mice/sex/group
One Main study male (control) and one Main study male (30 mg/kg) were euthanized prior to scheduled sacrifice and were replaced with two respective one-month Recovery males to maintain an adequate number of animals designated for the Main study. One Recovery male (30 mg/kg) was euthanized prior to scheduled sacrifice.
Control animals:
yes, concurrent vehicle
Details on study design:
See Table 1 for Study Design
- Dose selection rationale: Dose selection was based on the adverse effects in males observed at 1000 mg/kg/day in a previous 7-day repeated-dose gavage study in mice. In the 7-day study, groups of 5 male mice were administered the test substance (mixed with deionised water) via intragastric intubation at 0, 30, 300, or 1000 mg/kg/day for 7 consecutive days at a dose volume of 10 mL/kg/day. There were treatment-related effects on mean liver weights at 300 mg/kg/day and above. In addition, there were correlated microscopic findings observed for the liver observed at 30 mg/kg/day and above. Liver effects included hepatocellular hypertrophy (≥ 30 mg/kg/ day) and hepatocellular degeneration/necrosis (1000 mg/kg/day). Test substance-related but nonadverse microscopic findings were observed in the teeth and femur of mice administered ≥ 30 mg/kg/day of the test substance. These changes included incomplete decalcification of enamel (incisor teeth) and bone trabeculae (femur).
- Rationale for animal assignment (if not random): Mice of each sex were selected for use on study based on adequate body weight gain and freedom from any clinical signs of disease or injury. They were distributed by computerized, stratified randomization into study groups, so that there were no statistically significant differences among group body weight means within a sex. The weight variation of selected mice did not exceed ± 20% of the mean weight for each sex. The first 10 animals in each group were designated for repeated-dose toxicity (Main Study) evaluation.
- Post-exposure recovery period in satellite groups: An additional 5 animals of each sex in each group were designated for one-month recovery evaluations.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations included: moribund or dead mice and abnormal behaviour and/or appearance.

GENERAL CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, approximately 2 hours (± 1 hour) post-dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, at every weighing (except necropsy and FOBs)
- Each mouse was individually handled and examined for abnormal behaviour and appearance. Detailed clinical observations in a standardized arena were also evaluated on all mice. The detailed clinical observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Once each week. All mice were weighed on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The amount of food consumed by each mouse over each weighing interval was determined by weighing each feeder at the beginning and end of the interval. From these measurements, mean daily food consumption over the interval was determined.

FOOD EFFICIENCY: Yes
- From the food consumption and body weight data, the mean daily food efficiency was calculated.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood:
Main study: test day 28 (males) and 29 (females).
Recovery mice: approximately one month after the final dose (test day 57, 29 days recovery).
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Not reported.
- How many animals: All Main Study animals (10 mice/sex/group), Recovery animals (5 mice/sex/group).
- Parameters in Table No. 2 were examined. For recovery rats haematology (excluding coagulation), with bone marrow smears and serum chemistry parameters were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Main study: test day 28 (males) and 29 (females).
Recovery rmice: approximately one month after the final dose (test day 57, 29 days recovery).
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Not reported.
- How many animals: All Main Study animals (10 rats/sex/group), Recovery animals (5 rats/sex/group).
- Parameters in Table No. 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during acclimation (baseline) and during week 4
- Dose groups that were examined: all animals during acclimation (baseline) and during week 4 on all surviving animals
- Battery of functions tested: Abbreviated Functional Observational Battery (FOB): Responses to the following manipulations were assessed in the open field arena: approach and touch, sharp auditory stimulus (e.g., clicker), righting reflex. The following were measured using a digital force gauge: grip strength (combined forelimb, and hindlimb). The following parameters were assessed at the end of the MA session: pupillary constriction, polyuria, diarrhea. The darkened MA room allowed the constriction of dilated pupils to be observed, in response to a beam of light. The cage boards below the MA cages were evaluated for the presence of polyuria or diarrhea. Motor Activity (MA): Animals were individually tested in one of 30 identical, automated activity monitors. The infrared monitoring device enabled measurement of 2 dependent variables: duration of movement, and number of movements. A continuous movement, regardless of its duration, was counted as one movement. The technical characteristics of the measurement system are such that duration of movement is analogous to “counts” in other types of devices that employ interruption of light beams. Duration of movement and number of movements were evaluated in 9 consecutive intervals of 10 minutes each as well as for the total 90-minute session.
Sacrifice and pathology:
SACRIFICE AND PATHOLOGY
Main study: All mice on test day 29 (males) and 30 (females).
Recovery mice: All rats approximately one month after the final dose, test day 57, 29 days recovery.
SACRIFICE: Isoflurane anesthesia and exsanguination.
GROSS PATHOLOGY: Yes, all main study and recovery mice. Examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
ORGAN WEIGHTS: Yes, see Tables 4 for organs weighed. Paired organs were weighed together. Group mean values and organ weight ratios (% body weight and % brain weight) were calculated
HISTOPATHOLOGY: Yes, see Table 5 for tissues collected.
All tissues collected from male and female mice in the control (Group 1, 0 mg/kg/day) and high-dose (Group 4, 300 mg/kg/day) groups were processed to slides and evaluated microscopically. Gross lesions from all mice were also processed and examined. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist. Liver and kidneys from all the groups were processed for microscopic evaluation. Based on the results of the microscopic evaluation from controls and high dose, only the liver was considered as a target organ in male mice. Therefore, liver (females) and kidneys (both sexes) from the animals in the 30 and 100 mg/kg/day groups were not evaluated microscopically.
Statistics:
See Table 6 for statistical methods.
Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
Three mice died prior to the scheduled terminal sacrifice (one control male and 2 males at 30 mg/kg/day). The cause of death in each case was dosing injury.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight parameters were minimally increased in male mice administered 100 or 300 mg/kg/day. At 300 mg/kg/day, liver weight increases were associated with microscopic evidence of minimal centrilobular hepatocellular hypertrophy. These changes were reversible, as there were no test substance-related microscopic or organ weight changes in the 300 mg/kg/day recovery group males. Increased liver weight and microscopic hepatocellular hypertrophy of the liver were likely adaptive changes occurring secondary to induction of metabolizing enzymes and were considered to be non adverse. Minimal increases in some liver weight parameters in females, and in kidney weight parameters in males and females, at 300 mg/kg/day were not associated with correlative clinical pathology or microscopic changes and were thus considered to be nonadverse.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lack of adverse effects at highest dose level tested.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL = 300 mg a.i. /kg body weight/day, the highest level tested for males and females.
Executive summary:

The test substance in the vehicle (deionised water), was administered orally by gavage once daily for a minimum of 28 consecutive days to 3 groups of mice (15/sex/group). Dosage levels were 30, 100, and 300 mg/kg/day. Suspensions were adjusted for purity and density. A concurrent control group received the vehicle on a comparable regimen. The dose volume was 10 mL/kg for all groups. Following approximately 28 days of dose administration, 10 mice/sex/group (Main Study) were euthanized; the remaining 5 mice/sex/group (Recovery Animals, when possible) were euthanized following a 4-week non-dosing (recovery) period. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Clinical pathology evaluations (haematology and serum chemistry) were performed on all mice, on the day of their scheduled necropsy, after the treatment (study week 4) and recovery (study week 8) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.
There was no test substance-related mortality observed on this study. There were no significant clinical observations noted throughout the study. There were no treatment-related effects noted for body weight or food consumption parameters for male or female mice at any level tested. There were no treatment-related effects observed for any neurotoxicity or clinical pathology endpoint (haematology, coagulation, and clinical chemistry) evaluated at any level tested. There were no adverse organ weight changes and no adverse gross or microscopic findings at any of the doses tested. Liver weight parameters were minimally increased in male mice administered 100 or 300 mg/kg/day. At 300 mg/kg/day, liver weight increases were associated microscopically with minimal centrilobular hepatocellular hypertrophy microscopically. These changes were reversible, as there were no test substance-related microscopic or organ weight changes in the 300 mg/kg/day recovery group males. Increased liver weight and microscopic hepatocellular hypertrophy of the liver were likely adaptive changes occurring secondary to induction of metabolizing enzymes and were considered to be nonadverse. Minimal increases in some liver weight parameters in females, and in kidney weight parameters in males and females, at 300 mg/kg/day were not associated with correlative clinical pathology or microscopic changes and were thus considered to be nonadverse.
Under the conditions of the study, the no-observed-adverse-effect level (NOAEL) was co
nsidered to be 300 mg a.i. /kg/day, the highest level tested, for males and females, based on a lack of adverse effects at this dose.