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
Study period:
29th March 2011 to 29th November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP study performed in compliance with appropriate guidelines.

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:
not specified
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
not specified
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 Repeated dose 28-day oral toxicity study in rodents
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese METI, MHLW and MOE Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CN-3384A
- Physical state: solid
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon., UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 184-230 g (males); 14-182 g (females)
- Fasting period before study:
- Housing: individually in solid floor polypropylene cages with stainless steel mesh lids
- Diet (e.g. ad libitum): Rodent 2014C Teklad Global Certified Diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: 20th May 2011 To: 1st July 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance samples were analysed by HPLC using the following conditions:

HPLC: Agilent Technologies 1200
Column: Phenogel 10µ (300 x 7.8 mm id)
Mobile phase: tetrahydrofuran
Flow rate: 1 mL/min
UV detector wavelength: 230 nm
Injection volume: 25 µL
Retention time: ~8 to 10 minutes
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 7 day range-finding test (Liwska and Mullee, 2011)
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
MORBIDITY/MORTALITY: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to 30 minutes, one hour and five hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 prior to dosing and then at weekly intervals and after terminal kill. Recovery group animals were also weighed on days 36 and 43.

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
- Time schedule for examinations: weekly

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: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 28 days for non-recovery animals and 42 days for recovery animals
- Anaesthetic used for blood collection: No
- Animals fasted: No
- Parameters checked: haemoglobin, haematocrit, erythrocyte count, total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), prothrombin time, activated partial thromboplastin time, platelet count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 28 days for non-recovery animals and 42 days for recovery animals
- Animals fasted: No
- Parameters checked: blood urea, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, glucose, triglycerides, total cholesterol, gamma glutamyltranspeptidase, total bilirubin, bile acids and creatine

URINALYSIS: Yes
- Time schedule for collection of urine: during final week of dosing for non-recovery animals and final week of treatment-free period for recovery animals.
- Parameters checked: volume, pH, glucose, bilirubin, specific gravity, protein, ketones, urobilinogen, blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before initial dosing and once weekly thereafter.
- Parameters measured: motor activity, forelimb/hindlimb grip strength, sensory reactivity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

- Thyroid hormone assessment: at termination, serum was taken from blood samples from each animal and analysed for Triiodothyronine (T3), Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH).
- Gross examination: Full external and internal examination of all animals.
- Organ weights: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate and seminal vesicles, spleen, testes, thymus, thyroid/parathyroid, uterus with cervix

HISTOPATHOLOGY: Yes
Adrenals, aorta (thoracic), bone and bone marrow, brain, caecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mammary gland, muscle (skeletal), oesphagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skin (hind limb), spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus with cervix, vagina
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.

Where appropriate, quantitative data were analysed by Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed homogeneity of means, the data were analysed by a stepwise (Dunnett) parametric or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
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
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study. No animals of either sex from all treatment groups showed clinical signs of treatment-related toxicity.

BODY WEIGHT AND WEIGHT GAIN
No significant differences were detected in mean body weights in animals of either sex throughout this study when treated animals were compared to controls. However, statistically significant differences in mean body weight gains were evident in females of all treatment groups during week 4. In the absence of a true dose related response and there being no actual mean body weight loss among treatment groups, when compared to the control, the intergroup differences were considered not to be treatment-related.

FOOD CONSUMPTION AND COMPOUND INTAKE/FOOD EFFICIENCY
No treatment-related effects were detected on mean food intake or mean food efficiency during the study when treated animals were compared to the controls.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
There were no treatment-related effects detected in mean daily water consumption. Males treated at 1000 mg/kg bw/day showed a slight increase in water consumption during the final two weeks of the treatment period. Observations of this nature are commonly observed following the oral administration of an unpalatable test substance preparation and in isolation are not considered to represent systemic toxicity.

HAEMATOLOGY
Non-recovery 1000 mg/kg bw males showed a statistically significant increase in mean erythrocyte count and a significant reduction in mean lymphocyte count. These individual mean values were within the normal ranges for rats of this strain and age used for the study. In addition, these differences were not found in any of the treatment groups. Therefore, the intergroup differences were not considered to be biologically relevant.

Non-recovery males from all treatment groups showed a statistically significant reduction in mean corpuscular haemoglobin concentration (MCHC). Both in the absence of a dose-related response, and as both haematocrit and haemoglobin values for all non-recovery male treatment groups were comparable to the control, the intergroup differences for MCHC were considered not biologically relevant.

Recovery 1000 mg/kg bw males showed a statistically significant reduction in mean activated partial thromboplastin time (APTT) whilst recovery 1000 mg/kg bw/day females showed statistically significant increases in mean corpuscular haemoglobin concentration (MCHC), mean total leucocyte count (WBC) and mean lymphocyte count (TLC). The reduction in male mean APTT was not a similar effect seen in any non-recovery treated males and, therefore, was considered not biologically relevant. Increases in female MCHC, mean WBC and mean TLC were considered not treatment-related as both the haematocrit and haemoglobin values for all recovery treatment group females were comparable to the controls, regarding MCHC. In the case of increased WBC and TLC, this could suggest an infection which is unrelated to treatment.

CLINICAL CHEMISTRY
Statistically significant mean increases in glucose, albumin/globulin ratio and chloride concentration together with statistically significant mean reduction in phosphorus were evident in 1000 mg/kg bw/day males. All non-recovery treatment group males showed a statistically significant mean reduction in triglyceride levels and 1000 mg/kg bw/day recovery males also showed a statistically significant mean increase in chloride concentration. Non-recovery 1000 mg/kg bw/day females showed a statistically significant mean increase in sodium and chloride concentrations. The effect on chloride concentration was also extended to 300 mg/kg bw/day females. The majority of individual values were within the normal ranges for rats of the strain and age used for this study and as such were considered not to be treatment-related.

Non-recovery males from all treatment groups showed a statistically significant increase in calcium concentration. An increasing dose related response was revealed. Since the values showed an increase and not a decrease and the recovery makes revealed normal levels of calcium when compared to controls, the intergroup differences of non-recovery males were not considered to be biologically relevant.

Recovery 1000 mg/kg bw/day males showed a statistically significant mean reduction in aspartate aminotransferase. Recovery females showed a mean reduction in alanine aminotransferase which was within normal ranges for rats of the strain and age used for this study. In the absence of similar effects in non-recovery animals at the end of the dosing period, the intergroup differences were not considered to be of toxicological significance.

URINALYSIS
No treatment-related effects were detected.

NEUROBEHAVIOUR
No treatment-related changes in behavioural parameters, functional performance or sensory reactivity were observed.

All inter- and intra-group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used in this study. Differences were either more predominate in the control, equivalent to the control, transient or not dose dependent and therefore considered not to be test treatment related.

A statistically significant increase in hind and fore limb grip strength was detected for males treated with 300 and 1000 mg/kg bw/day. In the absence of any supporting or associated effects of neurotoxicity, these increased findings were considered not biologically relevant.

All inter- and intra-group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used for this study and, therefore, are not found to be treatment-related.

ORGAN WEIGHTS
There were no toxicologically significant effects noted when assessing the absolute and relative organ weights.

A statistically significant mean reduction in adrenal weight, both absolute and relative to body weight, was detected for non-recovery 1000 mg/kg bw/day animals of either sex when compared to the controls. The effect also extended to 30 and 300 mg/kg bw/day non-recovery females. There was no evidence of a dose related response in non-recovery females or any histopathological correlates observed in the adrenals of treated animals of either sex. In addition, all individual absolute and the majority of relative values were found to be within normal ranges for rats of the strain and age used for this study. Therefore the significant mean reduction in adrenal weight changes were considered not to be toxicologically significant.

GROSS PATHOLOGY
No toxicologically significant macroscopic abnormalities were detected. One non-recovery control female had distended vagina with narrowed vaginal opening with off white fluid at necropsy. One recovery control male had small and flaccid testes and small epididymides at necropsy. These effects were seen in control animals and are therefore not treatment-related.

HISTOPATHOLOGY
There were no treatment-related microscopic abnormalities detected. All morphological changes were those that are commonly observed in laboratory-maintanied rats of the age and strain employed for this study. There were no differences in incidence or severity between control and treatment groups (non-recovery and recovery) that were considered to be of any toxicological significance.

OTHER FINDINGS
There were no treatment-related effects noted when assessing levels of T3, T4 and TSH. Non-recovery males showed a statistically significant mean increase in T4 levels. In the absence of a similar effect present in males treated with 1000 mg/kg bw/day, this finding is not considered to be treatment-related.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the oral administration of the test substance to rats by gavage at 30, 300 and 1000 mg/kg bw/day did not result in any treatement-related or toxicologically significant effects. The NOAEL for the test substance was determined to be 1000 mg/kg bw/day.
Executive summary:

In a GLP compliant study conducted in accordance with standardised guidelines OECD 407, EU Method B.7., EPA OPPTS 870.3050 and appropriate Japanese guidelines, the repeat oral toxicity of the test substance was determined.

The test substance was administered by oral gavage to three groups consisting of five male and five female rats for twenty-eight consecutive days at dose levels of 30, 300 or 1000 mg/kg bw/day. A control group of five males and five females was dosed with the vehicle alone. Two recovery groups (each of five males and five females) were also treated with either 1000 mg/kg bw/day or to the vehicle alone for twenty-eight consecutive days and were then maintained without treatment for an additional fourteen days.

Clinical signs, body weight changes, food and water consumption were monitored during the study. Haematology, blood chemistry and urinanalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

There were no unscheduled deaths observed or clinical signs of toxicity detected during the study. No treatment-related changes were observed in any of the parameters measured during the study.

Under the conditions of the test, the NOAEL for systemic toxicity was determined to be 1000 mg/kg bw/day.