Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 1998)
Deviations:
yes
Remarks:
no analytical purity reported; the low and intermediate dose differ from each other for a 20 fold interval
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): only trade name given- Physical state: pale yellow slightly viscous liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Details on strain: Crl:CD (SD) BR- Source: Charles River (UK) Limited, Margate, Kent- Age at study initiation: approx. 6-8 weeks old- Weight at study initiation: males: 201 - 276 g; females: 148 - 203 g- Housing: in groups of up to four by sex in polypropylene grid-floor cages- Diet: pelled diet (Rodent 5LF2 (Certified) Diet, International Product Supplies Ltd., Northants, UK), ad libitum- Water: drinking water, ad libitum- Acclimation period: 13 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 21 ± 2- Humidity (%): 55 ± 15- Air changes (per hr): at least 15- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
dried, BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:- Formulations were prepared weekly and stored at aapprox. +4°C in the dark.VEHICLE- Name: dried Arachis oil BP- Concentration in vehicle: 1.25 mg/mL (low dose), 12.5 mg/mL (intermediate dose), and 250 mg/mL (high dose) - Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. The results show that the formulations were stable for at least fourteen days.Samples were taken of each test material formulation and were analysed for concentrations of the test substance by high performance liquid chromatography (HPLC) using an external standard technique (UV detector). The results indicate that the prepared formulations were within ± 10% of the nominal concentration.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:5, 50, and 1000 mg/kg bw/dayBasis:actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a range-finder study. 1 group of 6 rats (3 males and 3 female) was administered by gavage with 1000 mg/kg bw test substance, while control animals (3 males and 3 female) were treated in an identical manner with dried arachis oil BP. The oral route was selected as the most appropriate route of exposure, based on the physical prperties of the test material.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: immediately before dosing, and one hour after dosing; additionally on working days 5 hours after dosing- Parameters: overt signs of toxicity, ill-health or behavioural changeDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity.- Parameters: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, piloerection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.BODY WEIGHT: Yes - Time schedule for examinations: on Day 0, at weekly intervals thereafter, and at terminal sacrifice.FOOD CONSUMPTION:- Food consumption for each animal determined: Yes- Food consumption for each cage group: at weekly intervalsWATER CONSUMPTION: Yes - Time schedule for examinations: daily for each cage group (by visual inspection of the water bottles)OPHTHALMOSCOPIC EXAMINATION: Yes - Time schedule for examinations: pre-treatment and before termination of treatment (during week 12)- Dose groups that were examined: all control and high dose animals- Parameters: observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 1.0% "Mydriacyl" solution, a detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.HAEMATOLOGY: Yes - Time schedule for collection of blood: on Day 90- Anaesthetic used for blood collection: No- Animals fasted: No - How many animals: all surviving animals- Parameters: haemoglobin (Hb), erythrocyte count (RBC), haematocrit (Hct), erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), total leucocyte count (WBC), differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), platelet count (PLT), reticulocyte count (Retic) (cresyl blue stained slides prepared, but not assessed), prothrombin time (CT), activated partial thromboplastin time (APTT).CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: on Day 90- Animals fasted: No - How many animals: all surviving animals- Parameters: urea, glucose, total protein (Tot.Prot), albumin, albumin/globulin (A/G) ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili).URINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: during week 12 functional performances tests were performed on all surviving animals, together with an assessment of sensory reactivity to different stimuli.- Dose groups that were examined: all- Battery of functions tested: sensory activity / grip strength / motor activity - Motor activity was assessed by 20 purpose built 44 infra-red beam automated activity monitors. Evaluation period was 16 hours for each animal.- Grip strength (forelimb, hindlimb) was determined by means of an automated grip strength meter. Three consecutive trials were performed for each animal.- Sensory activity to auditory, visual and proprioceptive stimuli. Parameters: grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex, blink reflex.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes On completion of the dosing period all surviving animals were sacrificed and subjected to a full external and internal examination.The following organs were dissected free from fat and weighed before fixation: adrenals, brain, epididymes, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus.HISTOPATHOLOGY: Yes Samples from the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, ileum (including Peyer's patches), jejunum, rectum, epididymides,eyes, gross lesions, heart, kidneys, liver, lungs (with bronchi), trachea, lymph nodes (cervical and mesenteric), mammary glands, ovaries, uterus, muscle (skeletal), pancreas, pituitary, prostate, seminal vesicles, sciatic nerve, spinal cord (cervical, mid-thoracic and lumbar), skin (hind limb), spleen, oesophagus, salivary glands (submaxillary), stomach, testes, thymus, thyroid, urinary bladder.
Statistics:
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney'U' test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivation was detected immediately after dosing for 1000 mg/kg bw/day females from Day 17 and for 1000 mg/kg bw/day males from Day 18 onwards. Hunched posture was observed for individual 1000 mg/kg bw/day females from Day 37 with isolated incidents of tiptoe gait also observed on Days 37 and 38 and exophthalmia present in two females from Day 75 and 76 onwards. Exophthalmia was also evident in two 50 mg/kg bw/day females from Day 81 and hunched posture was observed for another 50 mg/kg bw/day female from Day 81 onwards. No such observations were detected for animals of either sex treated with 5 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no treatment-related deaths during the 90-day study. One male treated with 5 mg/kg bw/day was found dead on Day 36. There was no evidence to indicate this death was attributable to test material toxicity, the most plausible cause of death was a likely association with gavage mis-administration. One male treated with 50 mg/kg bw/day was terminated on Day 53 following observations indicative of a mal-dose. Neither death was considered to be treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically adverse effect on body weight development was detected during the study. Females from all treatment groups showed low weight gains compared with controls during Week 9. However in isolation and in the absence of a convincing dose dependent trend, this was considered not to be toxicologically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no toxicologically important adverse effect on food consumption during the study. However, a slight reduction in food consumption was observed for all female test groups at Week 9 returning to similar levels to controls thereafter in consideration of which these effects were considered not to be of toxicological relevance.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no toxicologically important adverse effect on food efficiency during the study. However, a slight reduction in food efficiency was observed for all female test groups at Week 9 returning to similar levels to controls thereafter in consideration of which these effects were considered not to be of toxicological relevance
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ocular effects were detected.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes detected in the haematological parameters measured. Females from all treatment groups showed statistically significant reductions in leucocyte count, specifically in the lymphocyte fraction, but these were considered attributable to higher than expected control values and not a consequence of treatment. Furthermore, 50 mg/kg bw/day males showed a reduction in haemoglobin concentration, however, in the absence of a convincing dose response and minimal statistical significance achieved (p<0.05), this value was considered to have arisen fortuitously. The reduction in erythrocyte count detected for 1000 mg/kg bw/day males achieved a statistical significance of p<0.01, however, in isolation, it was considered unrelated to treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected for the blood chemical parameters investigated. Statistical analysis did not reveal any statistically significant intergroup differences.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioural assessment: Weekly open field arena observations confirmed the clinical signs of hunched posture, and exophthalmia detected for females treated with 1000 mg/kg bw/day from week 6 and for 50 mg/kg bw/day females from Week 12 onwards. Exophthalmia was also observed for 1000 and 50 mg/kg bw/day females from week 12 during the study and an incident of noisy respiration was seen for one 50 mg/kg bw/day female during the Week 12 assessment. No such effects were detected for males of the high and intermediate dose groups and for animals of either sex treated with 5 mg/kg bw/day.
Functional Performance Tests: No treatment-related changes were detected.
Sensory Reactivity Assessments: No treatment-related changes were detected.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were detected. Liver weight, relative to terminal bodyweight was elevated of about 13% for animals of either sex treated with 1000 mg/kg bw/day (p<0.01). The toxicological significance of this finding is doubtful because of the absence of supporting biochemical or histopathological changes and therefore suggests that the organ weight increase is not a direct consequence of treatment. No such observations were detected for animals of either sex treated with 50 or 5 mg/kg bw/day
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related macroscopic abnormalities were detected. The decedent 5 mg/kg bw/day male showed dark kidneys and dark foci present on the liver and lungs and pallor of the gastric epithelium. These observations are characteristic of autolytic post mortem changes. The male terminated on Day 53 showed a mass of food present on the right side of the sternum and a liver pallor. In the absence of histopathological data to suggest an effect of treatment, this was considered attributable to the administration procedure and not a toxic effect of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in any of the tissues examined. There were no histopathological indications as to the cause of death for the 5 mg/kg bw/day male found dead during the study, but histopathological findings for the 50 mg/kg bw/day male suggested the animal may have suffered internal trauma as a consequence of mal-dosing. In general, the contribution made by interim death animals to a group assessment of treatment-related pathology should be regarded with caution, since animals dying before termination of the dosing period would not have received their full exposure to the test material. Both of these animals were excluded from statistical analyses. A variety of spontaneous histopathological changes were encountered, some of which warrant specific comment as follows:
HEART: Focal myocarditis was observed in several control and treated animals and is a common background entity in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.
LIVER: Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.
SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.
KIDNEYS: Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance as the severities or frequencies reported in this study. Similarly focal corticomedullary mineralization is a commonly observed background condition amongst female rats.
OESOPHAGUS: Inflammatory infiltration of the peripheral oesophageal musculature was observed in a few animals. This is a relatively frequently observed consequence of trauma of gavage dosing.
LUNGS: A minimal, and rarely slight, severity of bronchus associated lymphoid tissue was reported for most animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis are also a commonly observed pulmonary change in laboratory maintained rats of this age and are not suggestive of significant respiratory disease. A rather greater prevalence than might normally be expected of alveolar macrophage accumulations was seen among male rats, particularly control animals.
PROSTATE: Interstitial chronic inflammatory cell infiltrates are a commonly observed background finding in laboratory maintained rats.
BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.
UTERUS: Dilatation of the uterine horns is a commonly observed cyclical condition in laboratory maintained female rats.
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence of severity between control and treatment groups, all were considered to be without toxicological significance.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
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: The NOAEL corresponds to the highest dose level tested.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL 1000 mg/kg bw/day (actual dose received) is assigned. The NOAEL corresponds to the highest dose level tested. No significant effects were observed.
Executive summary:

NOAEL 1000 mg/kg bw/day (actual dose received) is assigned. The NOAEL corresponds to the highest dose level tested. No significant effects were observed.