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:
other: Modern GLP study conducted in accordance with modern guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
(Serum chemistry did not include measurement of bile acids. The justification for the choice of vehicle was not included in the study report).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid
- Analytical purity: >99%.
- Lot/batch No.: E0011-039E
- Expiration date of the lot/batch: 30 October 2012
- Storage condition of test material: Room temperature in the dark.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 7 weeks old
- Weight at study initiation: At initiation of dosing, males weighed 217–267 g and females weighed 148–190 g.
- Fasting period before study: None.
- Housing: Animals were individually housed in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum): Ad libitum, except during functional observational battery and motor activity testing and during the period of fasting prior to blood collection for clinical pathology and scheduled necropsies.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: 15 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Mean daily temperatures ranged from 20.8–22.0 deg C.
- Humidity (%): Mean daily humidity ranged from 32.6–43.9%.
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.


IN-LIFE DATES: From: 19 March 2008 To: 30 April 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test material for each formulation was weighed into a tared mortar and ground to a fine powder with a pestle. The appropriate amount of ground test material was then weighed into a tared, calibrated storage container. Vehicle was then added to each container to bring the formulations to the calibration mark. Each formulation was homogenised using an electronic homogeniser for approximately five minutes until a uniform mixture was obtained.
The test material formulations were prepared approximately weekly as single formulations for each dosage level., divided into aliquots for daily dispensation and stored at room temperature, protected from light with the following exception. The 100 mg/ml formulation for the last week of dosing (Week 3) was prepared daily on the day of dosing. Daily aliqupts were homogenised for approximately five minutes prior to dosing. The test material formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Justification not explicitly provided in study report; however, corn oil is a very common/standard solvent vehicle for test materials that are insoluble in water.
- Concentration in vehicle: 10, 30 and 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg.
- Lot/batch no. (if required): 126K0117 and 117K0127
- Purity: Not stated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two sets of duplicate samples (1 ml each) for pre-initiation homogeneity determination were collected from the top, middle and bottom strata of formulations prepared at test material concentrations of 5 and 100 mg/ml. The formulations that remained after sampling were divided into aliquots representative of the volumes used for daily dispensation. Representative aliquots were stored at room temperature for 8 and 18 days and samples were collected from the top and bottom strata and analyzed to assess re-suspension homogeneity and stability. One set of samples was analyzed and the other was stored at approximately -20°C as reserve samples to be discarded upon the study director’s approval of the results.

In addition, two sets of duplicate samples (1 ml each) for concentration analyses were
collected for study Weeks 0 and 3 (first daily 100 mg/ml formulation) from the middle stratum of each dosing formulation (including the vehicle formulation administered to the control group). Two sets of duplicate samples (1 ml each) for concentration analyses were also collected from the middle stratum of the second daily 100 mg/ml formulation. One set of samples was analyzed and the other set was stored at approximately -20°C as reserve samples to be discarded upon study director approval of analytical results. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC using a validated method.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
other: Nominal gavaged dose
No. of animals per sex per dose:
Group 1, Control group (0 mg/kg/day) 10 animals per sex
Group 2, Low-dose group (100 mg/kg/day) 5 animals per sex
Group 3, Mid-dose group (300 mg/kg/day) 5 animals per sex
Group 4, High-dose group (1000 mg/kg/day) 10 animals per sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a previous 14-day oral range-finding study.
- Rationale for selecting satellite groups: To investigate the persistence, delayed occurrence and reversibility of potential test substance-related effects.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Not specified.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed twice daily: at the time of dose administration and approximately one to two hours following dose administration.


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded at least weekly.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. Food consumption for each animal was determined and mean daily diet consumption calculated as g food/animal/day was calculated.


FOOD EFFICIENCY: No.
- 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


WATER CONSUMPTION: No.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was sampled for haematology on the day of the scheduled necropsies (Weeks 4 and 6).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes
- How many animals: All animals.
- Parameters in List 1 were examined.


BLOOD COAGULATION: Yes
- Time schedule for collection of blood: Blood was collected for coagulation parameters at the time of euthanasia.
- Animals fasted: Yes
- How many animals: All animals.
- Parameters in List 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was sampled for clinical chemistry on the day of the scheduled necropsies (Weeks 4 and 6).
- Animals fasted: Yes
- How many animals: All animals.
- Parameters in List 2 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected overnight prior to the day of scheduled necropsy.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes.
- Parameters in List 3 were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational Battery (FOB) assessments were recorded prior to initiation of dosing and prior to dosing during study Week 3 (final week of dosing period).
- Dose groups that were examined: All animals.
- Battery of functions tested: Parameters in List 4 were assessed.


OTHER:
LOCOMOTOR ACTIVITY: Yes. Locomotor activity was assessed for all animals prior to the initiation of dosing and prior to dosing following functional observationsl battery assessment during study Week 3 (final week of the dosing period).
Locomotor activity was measured automatically using a computerised system linked to a series of infrared photobeams surrounding a clear, plastic rectangular cage. Data were collected in 5-minute epochs and the test session duration was 60 minutes. These data were compiled as six 10-minute subsessions. Data for ambulatory and total locomotor activity (e.g. including grooming) were reported.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)
Statistics:
Body weight, body weight change, food consumption, continuous functional observational battery (FOB), clinical pathology and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test.

RANOVA statistical analyses were conducted for locomotor activity counts.

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.) and the number of animals (N) used to calculate the mean. In addition, percent difference from the control group was calculated for body weights, clinical pathology parameters (excluding urinalysis) and organ weights.

Results and discussion

Results of examinations

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:
not examined
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:
no effects observed
Behaviour (functional findings):
no effects observed
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:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects of the test material on the animals were observed at any dose
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: No effects of the test material on the animals were observed at any dose

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral (gavage) administration of the substance to rats for 28 consecutive days did not result in toxicity in any of the evaluated parameters at dosage levels of 100, 300 and 1000 mg/kg/day. Therefore, the no-observed effect level (NOEL) and the no-observed adverse effect level (NOAEL) for this study is 1000 mg/kg/day, the highest dose tested.
Executive summary:

The test substance was studied for oral toxicity potential in a 28-day gavage study in Crl:CD(SD) rats. The study was performed to GLP and in accordance with OECD Test Guideline 407. Dosage levels were 0, 100, 300 and 1000 mg/kg/day. The low- and mid-dose groups comprised five male and five female rats each, while the control and high-dose groups each comprised 10 animals of each sex. Following the 28 days of dosing, five animals per sex per group were euthanised; the remaining five animals per sex from the control and high-dose groups were maintained for a 14-day recovery period before sacrifice. Parameters evaluated included mortality, clinical observations, body weight, food consumption, locomotor activity, functional observational battery of assessments for neurotoxicity, haematology, serum chemistry, urinalysis, organ weights, gross pathology and histopathology. No test substance-related adverse effects on any of the evaluated parameters were reported. The no-observed effect level (NOEL) and no-observed adverse effect level (NOAEL) for this study is 1000 mg/kg/day, the highest dose tested.